-------
the highest levels of radioactivity were found to be in the blood, liver, and kidneys. The distribution of
radiolabeled DINP to other tissues after 7 days of exposure, was gastrointestinal (GI) tract (0.097%), fat
(0.053%), muscle (0.024%), and other organs (<0.009%). No differences in excretion were apparent in
either sex at either dose. In the single dose studies, 50 percent of the radioactivity was recovered in the
urine and the remainder in the feces at the low dose; whereas at the high dose, 35 to 40 percent of the
radioactivity was excreted in the urine and the remainder in the feces, suggesting an inverse relationship
between dose level and absorption. In repeated dose studies, rats were administered 50, 150, and 500
mg/kg-day [14C]DINP for 5 days, and excretion was evaluated (McKee et al.. 2002). In the repeated
dose studies, about 60 percent of the administered dose was excreted at all doses, suggesting an
elevation of esterase activity and more rapid conversion to monoester following repeated treatment
(Table 2-1). The elimination (half-life) of absorbed [14C]DINP was about 7 hours.
In another study by Clewell et al. (2013a). pregnant Sprague-Dawley (SD) rats received 50, 250, and
750 mg/kg-day of DINP from gestation day (GD) 12 to 19 via oral gavage. The percentage of DINP
absorbed following oral exposure was lower at the higher doses of 750 mg/kg-day compared to the 250
mg/kg-day group. Additionally, Clewell et al. (2013a) characterized the metabolite disposition of DINP
in the fetus and demonstrated that MINP and its oxidative metabolites along with its glucuronidated
form (MINP-Gluc) were all present in the fetal plasma, testes, and amniotic fluid. MINP-Gluc was
present at higher concentrations in the fetal plasma than the maternal plasma (in contradiction with what
was observed with the other metabolites), indicating potential placental transfer of MINP-Gluc, or, more
likely, that conjugation could occur in the fetus by phase II detoxification enzyme systems. Because
these metabolites were localized in maternal plasma and MINP was present at similar concentrations as
MCiOP, it was suggested that (1) urinary clearance of both MINP and MINP-Gluc is limited, and (2)
these metabolites were poor predictors of plasma and tissue disposition for DINP.
A summary of different metabolites found in human and rat urine after oral administration of DINP is
presented in Table 2-2.
Table 2-1. Absorption and Excretion Summary of DINP
Species
Dose
Source
Absorption
Reference
Human
1.28 mg/kg
Urine
44% over 48 hours
(Koch and Anserer,
2007)
Human
0.78 and 7.3
mg/kg
Urine
33 ± 6.4% over 48 hours
(Anderson et al.,
2011)
Rat
50 mg/kg
500 mg/kg
50-500 mg/kg
Urine
Urine
Estimated
urine + bile
49% over 72 hours
39% over 72 hours
75% over 72 hours
(McKee et al.,
2002)
50, 150, or 500
mg/kg-day for 5
days
Urine
Estimated
urine + bile
56-62% over 24 hours, 62-64%
over 72 hours
90% over 72 hours
Rat
(non-
pregnant)
Single dose of 300
mg/kg
Urine
Mono(carb oxy-i soocty 1 )phthal ate
(MciOP) 82%
Other metabolites 18%
(Silva et al., 2006)
Page 21 of 282
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Silva et al. (2006) administered a single oral gavage dose of 300 mg/kg DINP to non-pregnant SD rats
and quantified the metabolites in urine daily for 4 days. MciOP accounted for 82 percent of the
identified metabolites, and the other metabolites constituted 18 percent. This study characterized the
different co- and co-1-oxidation metabolites found in urine and found that MciOP was the major urinary
metabolite recovered, while MINP and DINP were not found in significant amounts in the urine.
Based on the available data, EPA assumes an oral absorption of 100 percent for the risk evaluation of
DINP.
Table 2-2. Metabolites of DINP Identified in Urine from Rats and Humans after Oral
Administration
Metabolite(s)
Abbreviation(s)
Reference(s) (Species)
Monoisobutyl phthalate
MINP
(Anderson et al., 2011) (human)
(Suzuki et al., 2012) (human)
(Koch and Anserer, 2007) (human)
(Calafat et al., 2006a) (rat)
Glucuronidated MINP
MINP-Gluc
(Clewell et al., 2013a) (rat)
[mono-(4-methyl-7-
carboxyheptyl) phthalate]
representing:
Mono(carboxyisooctyl) phthalate
[D4-7carboxy-MmeHP]
C02-MINP; MCIOP
(Anderson et al., 2011) (human)
(Koch and Anserer, 2007) (human)
[D4-mono-(4-methyl-7-
hydroxyoctyl) phthalate]
representing:
Mono(hydroxyisononyl)
phthalate
[70H-MmeOP]
for OH-MINP; MHINP
(Anderson et al., 2011) (human)
(Koch et al., 2012) (human)
(Koch and Anserer, 2007) (human)
(Silva et al., 2006) (rat)
[D4-mono-(4-methyl-7-
oxoocty 1 )phthal ate] repre senting:
Mono(oxoisononyl) phthalate
[7oxo-MmeOP] for Oxo-
MINP; MOINP
(Anderson et al., 2011) (human)
(Koch et al., 2012) (human)
(Koch and Anserer, 2007) (human)
(Silva et al., 2006) (rat)
Monocarboxylisononyl phthalate
cx-MINP
(Koch et al., 2012) (human)
Mono-carboxy-isooctyl phthalate
MCIOP (MCOP is sometimes
used to represent MCIOP)
(Silva et al., 2006) (rat)
Mono(carboxy-isoheptyl)
phthalate
MciHpP
(Silva et al., 2006) (rat)
Mono-(3-carboxypropyl)
phthalate
MCPP
(Calafat et al., 2006b; Calafat et al.,
2006a) (rat)
Mono-n-octyl phthalate
MnOP
(Calafat et al., 2006b) (rat)
Phthalic acid
PA
(McKee et al., 2002) (rat)
2.2 Inhalation Route
No controlled human exposure studies or in vivo animal studies are available that evaluate the ADME
properties of DINP for the inhalation route. Therefore, EPA is assuming 100 percent absorption via
inhalation. Similarly, ECHA concluded 75 percent absorption via inhalation for adults and 100 percent
for newborns and infants as a vulnerable subpopulation (ECHA 2013b; ECB, 2003).
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2.3 Dermal Route
In vivo and in vitro studies have shown that absorption of phthalates through rat and human skin
decreases as the length of the alkyl chain increases (Mint et al.. 1994; Elsisi et al.. 1989; Scott et al..
1987). Dermal absorption data specific to DINP are limited. EPA only identified one study directly
related to the dermal absorption of DINP (McKee et al.. 2002; Midwest Research Institute. 1983). In this
study, neat [14C]DINP at 50 mg/kg-day was applied to the freshly shaven backs (3 cm x 4 cm) of three
groups of male F344 rats as "conditioned skin," "non-conditioned skin," and "occluded" (styrofoam cup
lined with aluminum foil) (McKee et al.. 2002; Midwest Research Institute. 1983). Dermal absorption
was estimated to be 2 to 4 percent over 7 days, with an absorption rate of approximately 0.3 to 0.6
percent per day based on amount of applied dose recovered in urine, feces, and other tissues.
Additionally, radioactivity increased with time on skin: 0.12, 0.26, and 0.27 percent of the applied dose
following exposure of 1, 3, and 7 days, respectively. For all dermal absorption experiments with DINP,
material recovery fell within the Organisation for Economic Co-operation and Development (OECD)
156 (2022) Guidelines of 90 to 110 percent for non-volatile chemicals. The metabolic profile of
dermally absorbed DINP was similar to DINP metabolic profile from oral administration.
Although specific data on DINP dermal absorption in humans is lacking, several regulatory agencies
(e.g., Danish EPA, ECHA, NICNAS) recognize that absorption of phthalates would likely be lower in
human skin than through rat skin. This observation is based on data from in vitro migration studies
conducted with DEHP and other phthalates. Notably, other regulatory agencies (e.g., Australia
NICNAS, ECHA) have reached similar conclusions regarding the low dermal absorption of DINP
(ECHA 2013b; NICNAS. 2012).
As described further in the Environmental Release and Occupational Exposure Assessment for
Diisononyl Phthalate (DINP) (U.S. EPA. 2025f) and the Consumer and Indoor Exposure Assessment for
Diisononyl Phthalate (DINP) (U.S. EPA. 2025b). for the risk evaluation of DINP, EPA used data from
the in vivo dermal absorption study of DINP with rats (McKee et al.. 2002; Midwest Research Institute.
1983) to estimate dermal absorptive flux, which is used to calculate occupational and consumer dermal
exposure estimates.
2.4 Summary
Toxicokinetic data indicates that orally administered DINP is rapidly metabolized in the gut to MINP
and distributed via blood to major tissues, particularly the liver and kidneys. DINP metabolites were
excreted in urine and to a lesser extent in feces. Repeated dosing did not result in accumulation of DINP
and/or its metabolites in blood and tissues but did result in increased formation and elimination of the
monoester oxidation products.
Tissue distribution patterns of DINP revealed that absorption from the GI tract was rapid after both
single and repeated oral dosing. DINP is then primarily hydrolyzed in the GI tract after oral
administration. DINP translocated from the GI tract via the blood rapidly to liver and kidney. The
metabolic profile suggests that DINP is recovered primarily as oxidized products and phthalic acid and
very little as the parent or the metabolite MINP, suggesting that DINP is rapidly metabolized in the GI
tract to the corresponding monoester with a second hydrolysis step in liver to phthalic acid.
DINP is primarily eliminated in urine following oral exposures. Available studies have reported that
more than 90 percent of [14C] DINP was eliminated over 72 hours, with the majority through urine and
to a minor extent through feces(Anderson et al.. 2011; Koch and Angerer. 2007; Silva et al.. 2006;
McKee et al.. 2002). The total radioactivity recovered from the previously identified metabolites
Page 23 of 282
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combined was 33 ± 6.4 percent of the labeled DINP in urine over 48 hours. Metabolite half-lives were
estimated to be 4 to 8 hours with over 90 percent excreted in the first 24 hours of urine collection.
In contrast to absorption following oral exposure, dermal absorption of DINP in adult male F344 rats is
low, ranging from 2 to 4 percent of the applied dose when measured 7 days after application (McKeeet
al.. 2002). This finding agrees with data from other in vivo and in vitro studies that show absorption of
phthalates through rat and human skin decreases as the length of the alkyl chain increases. The dermally
absorbed fraction is distributed to multiple tissues, including skin, GI tract, muscle, fat, and liver. The
recovery of radioactivity in feces and the GI tract suggests excretion of DINP or its metabolites in the
bile, which in turn suggests that after dermal absorption, DINP undergoes a similar metabolic fate as
orally administered DINP.
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3 HAZARD IDENTIFICATION
EPA has developed detailed hazard characterization and mode of action (MOA) analysis for the effects
on fetal testicular testosterone and liver cancer, with an emphasis on liver effects leading to liver tumors.
Effects on fetal testicular testosterone are presented in Section 3.1.2.1. Non-cancer liver effects are
presented in Section 3.2, while liver cancer and EPA's MOA analysis of liver tumors is presented in
EPA's Cancer Raman Health Hazard Assessment for Diisononyl Phthalate (DINP) (U.S. EPA. 2025a).
The scientific MOA analysis is presented in accordance with EPA's Guidelines for Carcinogen Risk
Assessment (U.S. EPA. 2005a) and the IPCS Mode of Action Framework (IPCS. 2007) and includes a
description of the state of the science with regards to key events, pathways of toxicity and weight of
evidence following the modified Bradford Hill criteria. Other hazards considered by EPA—such as
kidney, neurotoxicity, cardiovascular health effects, immune system toxicity, musculoskeletal toxicity,
and gastrointestinal toxicity—are presented in Sections 3.3 through 3.8.
3.1 Developmental and Reproductive Toxicity
3.1.1 Summary of Available Epidemiological Studies
EPA reviewed and summarized conclusions from previous assessment conducted by Health Canada
(2018b) and systematic review articles by Radke et al. (2019b; 2018) that investigated the association
between DINP exposure and male and female development and reproductive outcomes. In the Health
Canada (2018b) assessment, there were no studies that evaluated the association between DINP and its
metabolites and reproductive outcomes such as altered male puberty, pregnancy complication and loss,
uterine leiomyoma, sexual dysfunction in females, and age at menopause. There was inadequate
evidence for the association between DINP and its metabolites and reproductive outcomes such as
altered female puberty, changes in semen parameters, sexual dysfunction in males, polycystic ovary
syndromes, and sex ratios. There was also no evidence for the association between DINP and its
metabolites and reproductive outcomes such as gynecomastia, endometriosis and adenomyosis. Overall,
Health Canada found that the evidence could not be established for the association between DINP and
its metabolites and any reproductive outcomes, such as altered fertility.
In the conclusions from the systematic review articles by Radke et al. (2018). examining the association
between DINP male reproductive outcomes the authors found moderate evidence linking DINP
metabolites to lower testosterone levels. However, they could not find clear evidence linking DINP and
male reproductive outcomes such as AGD, time until pregnancy in males, and sperm parameters due to a
combination of low exposure levels (i.e., poor sensitivity) and data availability (i.e., fewer accessible
studies). In terms of the association between female reproductive and developmental outcomes and
DINP, Radke et al. (2019b) found that the evidence was indeterminate.
EPA identified 11 new epidemiological studies published between 2018 and 2019 that were not
evaluated by Health Canada or Radke et al. (2019b; 2018). Eight of the available studies were of
medium quality and three were of low quality. Overall, conclusions of the 11 new studies were
consistent with that of Health Canada and the systematic review articles by Radke et al. EPA concluded
that the existing epidemiological studies do not support quantitative dose-response assessment, but
rather provide qualitative support as part of weight of scientific evidence. Further information on the 11
new studies identified by EPA can be found in Appendix D.
3.1.2 Summary of Laboratory Animals Studies
The developmental effects of exposure to DINP in experimental animal models have been evaluated as
part of several existing assessments. NTP-CERHR (2003). ECHA (2013b). EFSA (2019). Australia
Page 25 of 282
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NICNAS (2012), Health Canada (EC/HC.2015) and U.S. CPSC (2014. 2010) have all consistently
concluded that oral exposure to DINP can cause developmental toxicity in experimental animal models.
Oral exposure to DINP has been shown to cause skeletal and visceral variations, reduced pup body
weight gain, and effects on the developing male reproductive system consistent with a disruption of
androgen action. Effects on the developing male reproductive system and other developmental and
reproductive toxicity are discussed in Sections 3.1.2.1 and 3.1.2.2, respectively.
3.1.2.1 Developing Male Reproductive System
EPA has previously considered the weight of scientific evidence and concluded that oral exposure to
DINP can induce effects on the developing male reproductive system consistent with a disruption of
androgen action (see EPA's Draft Proposed Approach for Cumulative Risk Assessment of High-Priority
Phthalates and a Manufacturer-Requested Phthalate under the Toxic Substances Control Act (also
called "Draft Proposed Approach for CRA for Phthalates") (U.S. EPA 2023a)). Notably, EPA's
conclusion was supported by the Science Advisory Committee on Chemicals (SACC) (U.S. EPA
2023b). A summary of available studies evaluating effects on the developing male reproductive system
are provided in Section 3.1.2.1.1, while a brief MO A summary is provided in 3.1.2.1.2. Readers are
directed to see EPA's Draft Proposed Approach for CRA for Phthalates (U.S. EPA 2023a) for a more
thorough discussion of DINP's effects on the developing male reproductive system and EPA's MO A
analysis. Effects on the developing male reproductive system are considered further for dose-response
assessment in Section 4.
3.1.2.1.1 Summary of Studies Evaluating Effects on the Developing Male
Reproductive System
Available studies (including 16 studies of rats) evaluating the antiandrogenic effects of DINP on the
male reproductive system are summarized below in Table 3-1.
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Table 3-1. Summary of DINP Stut
ies Evaluating Effects on the Developing Male Reproductive System
Brief Study Description
NOAEL/
LOAEL
(mg/kg-day)
Effect at
LOAEL
Remarks
Pregnant SD rats (8/dose/ timepoint
evaluated) gavaged with 0 (corn oil
vehicle), 50, 250, 750 mg/kg-day
DINP (CASRN 68515-48-0) on'
GD12-19. Dams sacrificed on GDI9
(2 hours post-dosing) or GD20 (24
hours post-dosina) (Clewell et al..
2013a)
50/250
[ fetal testicular
testosterone and
testicular
pathology
(MNGs)
Maternal Effects
-1 (12%) absolute and relative maternal liver weight (>250 mg/kg-day)
Developmental Effects
- J, (50-65%) testicular testosterone on GD19 (>250 mg/kg-day)
- Testicular pathology on GD20 (f MNGs [>250 mg/kg-day], Leydig cell
aggregates [750 mg/kg-day])
Unaffected outcomes
- Maternal body weight gain; terminal maternal body weight; fetal body
weight; male AGD(GD20); testicular testosterone on GD20; seminiferous
tubule diameter on GD20
Pregnant SD rats (20-24 litters/dose)
fed diets containing 0, 760, 3,800, or
11,400 ppm DINP (CASRN 68515-
48-0) on GD12 through PND14
(equivalent to: 56, 288, 720 mg/kg-
day on GDI3-20 and 109, 555, 1,513
mg/kg-day on PND2-14). Dams
allowed to deliver pups naturally, and
pups sacrificed on PND49 or 50
(Clewell et al.. 2013b)
56/288
[ male pup body
weight on
PND14 and |
incidence of
MNGs on PND2
Maternal Effects
- J, body weight on GD20, PND2 and 14 (11,400 ppm)
- [ (30%) body weight gain on GD10-20 (11,400 ppm)
- J, food consumption on GD10-20 (11,400 ppm) and PND2-14 (>3,800
ppm)
Developmental Effects
- J, (10-27%) male pup weight on PND2 (720 mg/kg-day) and 14 (>288
mg/kg-day)
- Testicular pathology on PND2 (f Leydig cell aggregates (720 mg/kg-day),
MNGs (>288 mg/kg-day)
-| AGDon PND14 (720 mg/kg-day)
- J, (10%) absolute LABC weight on PND49-50 (720 mg/kg-day)
Unaffected outcomes
- Live pups/litter; testicular testosterone (PND49); PPS; AGD(PND2, 49);
NR (PND14, 49); absolute testis and epididymis weight (PND2, 49);
gubernacular cord length (PND49); male offspring body weight (PND49);
absolute testes, epididymis, SV, ventral prostate, glans penis, Cowpcr's
Glands weight (PND49); reproductive tract malformations (PND49) (e.g.,
hypospadias, exposed os penis, undescended testes, epididymal agenesis);
testicular pathology (PND49)
Pregnant Wistar rats (# of litters per
dose not stated) fed soy-free diets
containing 0, 40, 400, 4,000, or
20,000 ppm DINP (CASRN 28553-
None/ 2
[ male pup
AGD, J, pup
body weight, [
Maternal Effects
- Not examined or reported
Developmental Effects
- [ male/female body weight on PND1 (>2 mg/kg-day)
Page 27 of 282
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Brief Study Description
NOAEL /
LOAEL
(mg/kg-day)
Effect at
LOAEL
Remarks
12-0) from GDI5 through PND21 and
allowed to deliver pups naturally
[received doses, as estimated by
(EC/HC. 2015): 2. 20. 200. 1.000
ms/ks-davl (Lee et al.. 2006a)
female lordosis
quotient
- [ male AGDon PND1 (>2 mg/kg-day)
- [ frequency of mounts, intromissions, ejaculations in male rats (PNW 20)
(only at 2 mg/kg-day, no dose-response)
- [ Lordosis quotient of females in PNW 20 (>2 mg/kg-day)
Unaffected outcomes
- Serum testosterone and estradiol (PND7); serum testosterone, luteinizing
hormone, follicle stimulating hormone, estradiol (PNW 20)
Pregnant SD rats (5/dose) fed soy-free
diets containing 0, 400, 4,000, 20,000
ppm DINP (CASRN 28553-12-0) on
GDI5 through PND10 (equivalent to:
31, 307, 1,165 mg/kg-day on GDI 5-
20 and 66, 657, 2,657 mg/kg-day on
PND2-10) (Masutomi et al.. 2003)
66/657
[ male body
weight on
PND27
Maternal Effects
- [ body weight gain and food consumption between GDI5-20 & PND2-10
(20,000 ppm)
Developmental Effects
-[ body weight gain between PND2-10 (both sexes) (20,000 ppm)
- J, (18-43%) body weight on PND27 for males (>4,000 ppm) and females
(20,000 ppm)
- J, Absolute testes weight on PND27 (20,000 ppm)
- Testicular pathology on PND77 (20,000 ppm) {i.e., vacuolar degeneration
of Sertoli cells, degeneration of meiotic spermatocytes at stage XIV,
scattered cell debris in ducts of epididymis)
Unaffected outcomes
-Number of live offspring; pup body weight (PND2); AGD(PND2); pup
body weight gain (PND 10-21); PPS; vaginal opening; absolute testes
weight (PND77)
Pregnant Wistar rats gavaged with 0
(corn oil vehicle), 300, 600, 750, 900
mg/kg-day DINP (CASRN 28553-12-
0) on GD7 through PND17. Dams
sacrificed on GD21 (subgroup 1) or
allowed to give birth naturally and
offspring sacrificed on PND90
(subaroup 2) (Bobera et al.. 2016.
2011)
300/600
1 MNGs in fetal
testis and [
sperm motility
on PND90
Maternal Effects
- None
Developmental Effects
- Testis pathology on GD21 (f incidence of MNGs (>600 mg/kg-day);
enlarged diameter of seminiferous cords (>750 mg/kg-day); gonocytes with
central location in chords (>750 mg/kg-day))
- [ Testicular testosterone on GD21 (600 mg/kg-day, no dose-response)
- [ male pup body weight on PND 13 (900 mg/kg-day)
- J, male pup AGDon PND1 (900 mg/kg-day) and | male pup NR on
PND 13 (>750 mg/kg-day)
- J, sperm motility on PND90 (>600 mg/kg-day)
Unaffected Outcomes
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Brief Study Description
NOAEL /
LOAEL
(mg/kg-day)
Effect at
LOAEL
Remarks
- Maternal body weight and weight gain; gestation length, post-implantation
loss, litter size, sex ratio, perinatal loss; testicular testosterone production
(GD21); plasma testosterone and luteinizing hormone (GD21); fetal birth
weight; male and female body weight (PND90); absolute reproductive organ
weight (PND90) (e.g., testis, prostate LABC, SV, ovary, uterus); AGDor
NR (PND90); testis testosterone (PND90); SV, prostate, testis pathology
Pregnant Harlan SD rats (5-9/dose)
gavaged with 0, 500, 750, 1,000, or
1,500 mg/kg-day DINP (CASRNs
28553-12-0 and 68515-48-0 tested) on
GD14-18. Dams sacrificed on GD18,
approximately 2 hours post-dosing
(Hannas et al.. 2011)
None/ 500
[ fetal testicular
testosterone
production
Maternal Effects
- None
Developmental Effects
- [ (30-69%) ex vivo fetal testicular testosterone production (>500 mg/kg-
day, both CASRNs)
-I expression of St A R and Cvplla mRNA in fetal testes (>1,000 mg/kg-
day, both CASRNs)
Unaffected Outcomes
- Dam mortality; dam body weight gain; litter size
Pregnant Harlan SD rats gavaged with
0, 500, 750, 1,000, or 1,500 mg/kg-
day DINP (CASRNs 28553-12-0 and
68515-48-0 tested) on GD14-18.
Dams sacrificed on GDI8,
approximately 2 hours post-dosing
(Hannas et al.. 2012)
NOEL/ LOEL:
None/ 500
[ steroidogenic
gene expression
in the fetal testes
Maternal Effects
- None
Developmental Effects
- J, mRNA expression of StAR, Cvplla, Cypllbl, Cypllb2, Hsd3b,
Cypl 7al, Scarbl, lnsl3, Dhcr7'm the fetal testes (>500 mg/kg-day, both
CASRNs)
Unaffected Outcomes
- Dam mortality; dam body weight gain; litter size
Pregnant SD rats (5-8/dose) gavaged
with 0 (corn oil vehicle), 250, or 750
mg/kg-day DINP (CASRN not
reported) on embryonic days 13.5—
17.5. Dams sacrificed on embryonic
dav 19.5 (Adamsson et al.. 2009)
NOEL/ LOEL:
250/750
t GATA-4, lnsl3,
P450scc mRNA
in the fetal testes
Maternal Effects
- None
Developmental Effects
- | Testicular mRNA expression of GATA-4, Insl3, P450scc (750 mg/kg-
day)
Unaffected Outcomes
- Plasma corticosterone; litter size; sex ratio; fetal body weight; testicular
testosterone; testicular mRNA expression of Star, 3/3-HSD, Sl'-I: testicular
protein expression of StAR, P450scc, 3|3-HSD, androgen receptor; testicular
pathology
Page 29 of 282
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Brief Study Description
NOAEL /
LOAEL
(mg/kg-day)
Effect at
LOAEL
Remarks
Pregnant SD rats (14-19/dose)
gavaged with 0 (corn oil vehicle) or
750 mg/kg-day DINP (CASRN
68515-48-0) from GD14 through
PND3. Dams were allowed to give
birth naturally and mall offspring were
sacrificed between 3-7 months of age
(Grav et al.. 2000)
None/ 750
t male pup NR,
reproductive
malformations
Maternal Effects
- J, (10%) maternal weight gain to GD21
Developmental Effects
-1 percent of males with areolas (22.4%) on PND13
- Reproductive malformations at 3-7 months: permanent nipples in 2/52
males from 2 litters, small and atrophic testes in 1/52 males; flaccid, fluid-
filled in 1/52 males; unilateral epididymal agenesis with
hypospermatogenesis in 1/52 males
Unaffected outcomes
- Maternal mortality; maternal weight gain to PND3; male pup weight at
birth; PPS; absolute reproductive organ weight at 3-7 months {i.e., testes,
LABC, SV, glans penis, ventral prostate, epididymis, cauda epididymis,
caput-corpus epididymis); serum testosterone (3-7 months); male
AGD(PND2); reproductive malformations at 3-7 months (hypospadias,
cleft phallus, vaginal pouch, SV agenesis, undescended testes, testis
absent, abnormal gubernacular cord)
Pregnant Harlan SD rats (3-5/dose)
gavaged with 0 (corn oil vehicle) or
750 mg/kg-day DINP on GDI4-18.
Dams sacrificed on GDI8,
approximately 2 hours post-dosing.
Study completed over several blocks.
Block 1 and 5 tested CASRN 68515-
48-0, Block 7 tested CASRN 28553-
12-0 (Furr et al.. 2014)
None/ 750
[ fetal testicular
testosterone
production
Maternal Effects
- None
Developmental Effects
- [ (24-50% across Blocks 1, 5, and 7) ex vivo fetal testicular testosterone
production
Unaffected Outcomes
- Maternal weight gain, fetal viability (all blocks)
Pregnant Wistar rats (8/dose) gavaged
with 0 or 750 mg/kg-day DINP
(CASRN 28553-12-0) on GD7-21.
Dams sacrificed on GD21 (Borch et
al.. 2004)
None/ 750
[ fetal testicular
testosterone
content and
production
Maternal Effects
- Not examined or reported
Developmental Effects
-1 ex vivo fetal testicular testosterone production and testicular testosterone
content (magnitude of effect not reported, only presented graphically
Unaffected Outcomes
- Plasma testosterone and luteinizing hormone
Hershberger assay: Testosterone
propionate-treated (0.4 mg/kg-day)
castrated immature (7 week old) male
NA
NA
- [ absolute SV (>20 mg/kg-day) (lacked dose-response) and LABC (500)
weight
Unaffected Outcomes
Page 30 of 282
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Brief Study Description
NOAEL /
LOAEL
(mg/kg-day)
Effect at
LOAEL
Remarks
SD rats were administered DINP via
gavage at 0, 20, 100, or 500 mg/kg-
dav for 10 davs (Lee and Koo. 2007)
- Terminal body weight; absolute liver, kidney, adrenal; ventral prostate,
Cowpcr's gland; Glans penis weight
Pregnant SD rats (6/dose) gavaged
with 0 (corn oil vehicle), 10, 100,
500, 1,000 mg/kg-day DINP (CASRN
not provided) on GDI2-21. Dams
were allowed to give birth naturally
and then pups were sacrificed (Li et
aL 2015)
None/ 10
[ male pup body
weight and fetal
Leydig cell
aggregation
Maternal Effects
- None
Developmental Effects
- [ male pup body weight (>10 mg/kg-day) (lacked dose-response)
- [ testicular testosterone (1,000 mg/kg-day)
-1 testis dysgenesis (>100 mg/kg-day)
-1 incidence of MNGs (>100 mg/kg-day)
- Fetal Leydig cell aggregation (>10 mg/kg-day)
- [ testicular gene expression {Ins 13 (>10), Lhcgr (>500), Star (>500),
Cypllal (;>100), Hsci3bl (>100), Cypl7al (> 100), Hsd17b3 (1,000))
Unaffected outcomes
- Gestation length; number of dams delivering litters; pups per litter; sex
ratio; dam body weight; male AGD
Pregnant Harlan SD rats gavaged with
0, 500, 750, 1,000, or 1,500 mg/kg-
day DINP on GD14-18. Dams
sacrificed on GDI8, approximately 2
hours post-dosina (Grav et al.. 2021)
None/ 500
[ ex vivo fetal
testicular
testosterone
production & [
steroidogenic
gene expression
in the fetal testes
Developmental Effects
- [ (29-68%) ex vivo fetal testicular testosterone production (>500 mg/kg-
day)
- [ mRNA expression of NrObl, Star, Cypllal, Hsd3b, Cypl7al, Scarbl,
Insl3, Dhcr7, Cypllbl and Inha in the fetal testis (>500 mg/kg-day) and
Cypllb2 (>1,000), Lhcgr (>1,000), and RoxlO (>1,000)
Additional Comments
- Gray et al. (2021) summarizes combined testosterone data originally
reported in (Furr et al.. 2014) and (Hannas et al.. 2011)
Pregnant SD rats (6-7 per dose)
gavaged with 0, 1,000, or 1,500
mg/kg-day DINP (CASRN 68515-48-
0) on GDI4 through PND3. F1 males
were euthanized on PND220-240
(Grav. 2023)
None/ 1,000
t F1 male
offspring nipple
retention
Maternal Effects
- [ body weight gain (<5%) throughout DINP administration (>1,000
mg/kg-day)
Developmental Effects
- J, absolute AGDfor F1 males on PND2 (1,500 mg/kg-day)
- [ F1 male and female body weight on PND2 (body weight effect no longer
apparent by PND13) (1,500 mg/kg-day)
- | F1 male offspring nipple retention on PND13 and F1 adult males (-210
days of age) (>1,000 mg/kg-day)
Page 31 of 282
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Brief Study Description
NOAEL/
LOAEL
(mg/kg-day)
Effect at
LOAEL
Remarks
- [ absolute glans penis, LABC, seminal vesicle weight in F1 adult males
(-210 days of age) (1,500 mg/kg-day)
- Increased incidence of total reproductive tract malformations (>1,000
mg/kg-day) (observed malformations include seminiferous tubule
hypospermatogenesis/atrophy; bilateral fluid filled testis in 3 males of each
dose group, hypoplasia and epididymal atrophy in 1 male of high-dose
group; unilateral testis and gubernacular testis agenesis and undescended
atrophic epididymis in 1 male of high-dose group)
Unaffected outcomes
- No overt signs of maternal toxicity observed; survival at birth; litter sizes
at PND2; female AGDon PND2; timing of preputial separation; absolute
weight of Cowpcr's gland, epididymis, ventral prostate, testes, liver,
adrenal, kidney
Additional Comments
- Grav (2023) also reports a statistical analvsis of combined data from Grav
(2023) and Grav et al. (2000). which was a studv of similar desian and
tested a lower dose of DINP {i.e., 750 mg/kg-day). Results from this
statistical analysis of combined data are not reported in this table.
Pregnant SD rats (7-8 dams per dose)
gavaged with 0, 750 mg/kg-day DINP,
250 mg/kg-day DBP, or binary
mixture of 750 mg/kg-day DINP and
250 mg/kg-day DBP on GDI4-18.
Dams were euthanized 2-4 hours after
the final dose on GDI8 (Grav et al..
2024)
None/ 750
[ ex vivo fetal
testicular
testosterone
production and [
steroidogenic
gene expression
in the fetal testes
Developmental Effects
- Ex vivo fetal testis testosterone production was reduced 45%
- [ mRNA expression of Cypllbl, Cypllal, Cypllb2, Cypl7cil, Cyp51,
Ebp, Hmgcr, Hmgcsl, Hsd3b3, Ildil, Inhci, Insl3, Lhcgr, Mvd, Scarbl, Star.
TM7SF2. SUPERGENE. RhoxlO, TEST IN in fetal testis
Unaffected outcomes
- Maternal body weight or weight gain; litter size; fetal viability
Additional Comments
- Only results for the DINP component of the study are reported in this
table.
Abbreviations: [ = statistically significant decrease; | = statistically significant increase; ND = NOAEL or LOAEL not established; NOAEL = no
observed-adverse-effect-level; LOAEL = lowest-observed-adverse-effect level; GD = gestation day; PND = postnatal day; PNW = postnatal week;
AGD= anogenital distance; MNGs = multinucleated gonocytes
Page 32 of 282
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3.1.2.1.2 Mode of Action for Phthalate Syndrome
The proposed MOA for phthalate syndrome is shown in Figure 3-1. which explains the link between
gestational and/or perinatal exposure to DINP and effects on the male reproductive system in rats. The
MOA has been described in greater detail in EPA's Draft Proposed Approach for CRA for Phthalates
(U.S. EPA. 2023a) and is described briefly below.
Chemical Structure
and Properties
Phthalate
exposure during
critical window of
development
V
Metabolism to
monoester &
transport to fetal
testes
Cellular
Responses
r~
>=>
Unknown MIE
(not believed to be
AR or PPARa
mediated)
^ Key genes involved in the AOP \
for phthalate syndrome
Scarbl
Chcr7
Mvd
Eta3b
StAP
Ebp
Nsdhl
Insl3
Cypllol
Fdps
RGD1S64999 Lhcgr
Cypllbl
Hmgcr
rm7sf2
Irtha
Cypllb2
Hmgcsl
Cyp46al
NrObl
Cypl 7a 1
Hsd3b
Ldlr
Rhoxld
Cyp5t
Fid/1
Insigl
Wnt7a
>=>
Organ
Responses
Adverse Organism
Outcomes
Fetal Male Tissue
4- AR dependent
mRNA/protein
synthesis
¦=>
4> Testosterone
synthesis
4» Gene
expression
(INSL3, lipid
> metabolism,
cholesterol and
androgen synthesis
and transport) '
IN.
4- INSL3 synthesis
Fetal Leydig cell
Abnormal cell
apoptosis/
proliferation
(Nipple/areolae
retention, .J, AGD,
Disrupted testis
tubules, Leydig cell
clusters, MNGs,
agenesis of
reproductive tissues)
Suppressed
gubernacular cord
development
(inguinoscrotal phase)
Suppressed
gubernacular cord
development
(transabdominal
Phase)
0
4- Androgen-
dependent tissue
weights, testicular
pathology [e.g.,
seminiferous tubule
atrophy),
malformations (e.g.,
hypospadias), 4/
sperm production
Impaired
fertility
1}
Undescended
i-
testes
Figure 3-1. Hypothesized Phthalate Syndrome Mode of Action Following Gestational Exposure
Source: Figure taken directly from (U.S. EPA. 2023a) and adapted from (Conlev et aL 2021; Gray et al.. 2021;
Schwartz et al.. 2021; Howdeshell et al.. 2017).
AR = androgen receptor; INSL3 = insulin-like growth factor 3; MNG = multinucleated gonocyte; PPARa =
peroxisome proliferator-activated receptor alpha.
The MOA underlying phthalate syndrome has not been fully established; however, key cellular-, organ-,
and organism-level effects are generally understood (Figure 3-1). The molecular events preceding
cellular changes remain unknown. Although androgen receptor antagonism and peroxisome proliferator-
activated receptor alpha activation have been hypothesized to play a role, studies have generally ruled
out the involvement of these receptors (Foster. 2005; Foster et al.. 2001; Parks et al.. 2000).
Exposure to DINP during the masculinization programming window (i.e., GDI 5.5 -18.5 for rats; GDI 4-
16 for mice; gestational weeks 8-14 for humans) in which androgen action drives development of the
male reproductive system can lead to antiandrogenic effects on the male reproductive system (MacLeod
et al... 2010; Welsh et al... 2008; Carruthers and Foster. 2005). In vivo pharmacokinetic studies with rats
have demonstrated that monoester metabolites of DINP can cross the placenta and be delivered to the
target tissue, the fetal testes (Clewell et al.. 2013a; Clewell et al.. 2010). Consistent with the MOA
outlined in Figure 3-1, studies of DINP have demonstrated that exposure to DINP during the
masculinization programming window in rats can reduce mRNA levels of insulin-like growth factor 3
(INSL3), as well as genes involved in steroidogenesis in the fetal testes (Gray et al.. 2024; Gray et al..
Page 33 of 282
-------
2021; Li et al.. 2015; Hannas et al.. 2011; Adamsson et al.. 2009). Consistently, studies have also
demonstrated that exposure to DINP during the masculinization programming window can reduce fetal
testicular testosterone content and/or testosterone production (Gray et al.. 2024; Gray et al.. 2021; Li et
al.. 2015; Furr et al.. 2014; Clewell et al.. 2013a; Boberg et al.. 2011; Hannas et al.. 2011; Borch et al..
2004). Exposure to DINP during the masculinization programming window can also reduce male pup
anogenital distance (AGD) and cause male pup nipple retention (NR) (Gray. 2023; Boberg et al.. 2016;
Clewell et al.. 2013b; Gray et al.. 2000). which are two hallmarks of antiandrogenic substance; however
effects on AGDand NR are less consistently observed following oral exposure to DINP in rats (see
Sections 3.1.3.3 and 3.1.3.4 of (U.S. EPA. 2023a) for additional discussion). In contrast, exposure to
DINP generally does not induce severe reproductive tract malformations such as hypospadias and/or
cryptorchidism, but has been shown to cause epididymal agenesis, seminiferous tubule
hypospermatogenesis/atrophy, fluid filled testis, and other reproductive tract malformations at high
doses ranging from 750 to 1,500 mg/kg-day (Gray. 2023; Gray et al.. 2000). Further, a spectrum of other
effects consistent with phthalate syndrome, including increased numbers of multinucleated gonocytes
(MNGs) (Li et al.. 2015; Clewell et al.. 2013a; Clewell et al.. 2013b; Boberg et al.. 2011). fetal Ley dig
cell aggregation (Li et al.. 2015; Clewell et al.. 2013a; Clewell et al.. 2013b). and decrease sperm
motility (Boberg et al.. 2011) have also been observed following gestational exposure to DINP during
the critical window of development.
Based on available data, EPA previously concluded that the weight of scientific evidence demonstrates
that oral exposure to DINP can induce effects on the developing male reproductive system consistent
with a disruption of androgen action and the MOA outlined in Figure 3-1 (see EPA's Draft Proposed
Approach for CRA for Phthalates (U.S. EPA. 2023a)). Notably, EPA's conclusion was supported by the
SACC (U.S. EPA. 2023b).
3.1.2.2 Other Developmental and Reproductive Outcomes
EPA has evaluated several oral exposure studies, including two prenatal developmental studies of rats
(Waterman et al.. 1999; Hellwig et al.. 1997). a one-generation study of reproduction of rats (Waterman
et al.. 2000; Exxon Biomedical. 1996a). and a two-generation study of reproduction of rats (Waterman
et al.. 2000; Exxon Biomedical. 1996b). The Agency identified several studies published from 2015 to
2024 evaluating estrogenic potential (Sedha et al.. 2015). reproductive effects (Santacruz-Marquez et al..
2024; Chen et al.. 2022; Chiang et al.. 2020a. b; Chiang and Flaws. 2019). developmental effects
(Bhurke et al.. 2023; Laws et al.. 2023; Neier et al.. 2018). and metabolic effects (Neier et al.. 2019) of
DINP in mice and rats treated in the perinatal period. No studies of development are available for the
dermal or inhalation exposure routes. Available studies are summarized in Table 3-2 and discussed
further below.
Page 34 of 282
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Table 3-2. Summary of DINP Studies Evaluating Ef
'ects on Reproduction and Development
Brief Study Description
NOAEL/
LOAEL
(mg/kg-day)
Effect at
LOAEL
Remarks
Pregnant SD rats (23-25/dose) gavaged
with 0 (corn oil vehicle), 100, 500, 1,000
mg/kg-day DINP (CASRN 68515-48-0)
on GD6-15. Dams sacrificed on GD21
(Waterman et al.. 1999)
100/5 0017
t Skeletal
variations
Maternal Effects
- [ (13%) food consumption on GD6-9 (1,000 mg/kg-day)
- [ body weight gain on GD6-9, 6-15 (1,000 mg/kg-day)
Developmental Effects
- | incidence of rudimentary lumbar (>500 mg/kg-day), supernumerary
cervical ribs (1,000 mg/kg-day), renal pelves (1,000 mg/kg-day)
Unaffected Outcomes
- Maternal survival, clinical signs, resorptions, post-implantation loss, fetal
viability, fetal body weight, sex ratio, incidence of fetal malformations
Pregnant Wistar rats (10/dose) gavaged
with 0 (corn oil vehicle), 40, 200, 1,000
mg/kg-day DINP-1 (CASRN 68515-48-
0) on GD6-15. Dams sacrificed on GD20
(Hellwia et al.. 1997)
200/ 1,000
t Skeletal
variations
Maternal Effects
- [ food consumption (1,000 mg/kg-day)
- Clinical signs (vaginal hemorrhage and urine-smeared fur in one dam)
(1,000 mg/kg-day)
- | (13%) relative kidney weight (1,000 mg/kg-day)
Developmental Effects
- | skeletal variations (rudimentary cervical and accessory 14th ribs) (1,000
mg/kg-day)
Unaffected Outcomes
- Survival; maternal body weight; uterus weight; relative liver weight;
resorptions; post-implantation loss; number of live fetuses per dam; fetal
weight
Pregnant Wistar rats (10/dose) gavaged
with 0 (corn oil vehicle), 40, 200, 1,000
mg/kg-day DINP-2 (CASRN 28553-12-
0) on GD6-15. Dams sacrificed on GD20
(Hellwia et al.. 1997)
200/ 1,000
t Skeletal
variations
Maternal Effects
- Clinical signs (vaginal hemorrhage in one dam) (1,000 mg/kg-day)
Developmental Effects
- | skeletal variations (rudimentary cervical and accessory 14th ribs) (1,000
mg/kg-day)
Unaffected Outcomes
- Survival; food consumption; maternal body weight; uterus weight; relative
liver and kidney weight; resorptions; post-implantation loss; number of live
fetuses per dam; fetal weight
Page 35 of 282
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Brief Study Description
NOAEL /
LOAEL
(mg/kg-day)
Effect at
LOAEL
Remarks
Pregnant Wistar rats (10/dose) gavaged
with 0 (corn oil vehicle), 40, 200, 1,000
mg/kg-day DINP-3 (CASRN 28553-12-
0, resulting from a different production
line than DINP-2) on GD6-15. Dams
sacrificed on GD20 (Hellwia et al.. 1997)
200/ 1,000
t incidence
of skeletal,
visceral, and
soft tissue
variations
Maternal Effects
- [ food consumption (1,000 mg/kg-day)
- [ body weight gain from GD6-15 (1,000 mg/kg-day)
- | (11%) relative liver weight (1,000 mg/kg-day)
Developmental Effects
-1 skeletal retardations (unossified or incompletely ossified sternebrae)
(1,000 mg/kg-day)
- | soft tissue variations (hydroureter) (1,000 mg/kg-day)
- | skeletal variations (rudimentary cervical and accessory 14th ribs) (1,000
mg/kg-day)
Unaffected Outcomes
- Survival; clinical signs; uterus weight; resorptions; post-implantation loss;
number of live fetuses per dam; fetal weight
Male and female SD rats (30/sex/dose)
fed diets containing 0, 0.5, 1.0, 1.5%
DINP (CASRN 68515-48-0) starting 10
weeks prior to mating, through mating,
gestation, and lactation continuously for
one generation. Received doses in units
of mg/kg-day shown in Table 3-5.
(Waterman et al.. 2000; Exxon
Biomedical. 1996a)
None/ 377
[ F1 male
and female
body weight
on PND0,
14,21
Parental (PI) Effects
- J, PI body weight (both sexes) (>1.0%)
- [ PI food consumption (both sexes) (>1.0%)
-1 absolute and relative liver weight (both sexes) (>0.5%)
-1 absolute and/or relative kidney weight (both sexes) (>0.5%)
- | absolute testes, right epididymis, and ovary weight (1.5%)
Fertility Effects
- None
Offspring (Fl) Effects
- [ live births, [ PND4 survival, [ PND14 survival, [ viability at weaning (all
at 1.5%)
- [ male and female body weights on PND0, 1, 14, 21 (>0.5%)
Unaffected Outcomes
- Clinical signs (PI); survival (PI); reproductive indices (male mating,
male/female fertility, female fecundity, gestational indices); litter size;
number of live/dead offspring at birth; sex ratio
Male and female SD rats (30/sex/dose)
fed diets containing 0, 0.2, 0.4, 0.8%
DINP (CASRN 68515-48-0) starting 10
weeks prior to mating, through mating,
gestation, and lactation continuously for
two-generations. Received doses in units
None/133
| F1 and F2
male and
female body
weight on
PND7 and 21
Parental (PI. P2) Effects
- J, PI female body weight on PND14 and 21 (0.8%)
- [ P2 male and female body weight (>0.4%)
- [ PI female food consumption during lactational period (0.8%)
- [ P2 male and female food consumption during premating, gestation, and
lactational periods (0.8%)
Page 36 of 282
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Brief Study Description
NOAEL /
LOAEL
(mg/kg-day)
Effect at
LOAEL
Remarks
of mg/kg-day shown in Table 3-7.
(Waterman et al.. 2000; Exxon
Biomedical. 1996b)
- | relative and/or absolute liver weight for PI males and females (>0.4%) &
P2 males and females (0.8%)
-1 absolute kidney weight for PI males (>0.4%) and females (>0.2%) & P2
males (0.8%)
- | incidence of minimal to moderate cytoplasmic eosinophilia (both sexes in
PI and P2) (>0.2%)
-1 incidence of minimal to moderate dilation of the renal pelves for P2 males
(>0.4%)
Fertility Effects
- None
Offspring (Fl. F2) Effects
- J, F1 male and female offspring body weight on PND21 (>0.2%)
- J, F2 female offspring body weight on PND7 (>0.2%)
Unaffected Outcomes
- Clinical signs (PI, P2); survival (PI, P2); reproductive indices (male mating,
male/female fertility, female fecundity, gestational indices) (PI, P2); litter
size (Fl, F2); number of live/dead offspring at birth (Fl, F2); sex ratio (Fl,
F2)
Uterotropic Assay: 20 day old female
Wistar rats (6/group) were gavaged with
0 (untreated), 0 (corn oil vehicle), 276,
1380 mg/kg-day DINP (CASRN 68515-
48-0), or 40 pg/kg-day diethylstilbesterol
for 3 days. Animals sacrificed 24 hours
after dosine (Sedha et al.. 2015)
None/ 276
| body
weight gain
- [ body weight gain (>276 mg/kg-day)
- Positive control gave anticipated results
Unaffected Outcomes
- Uterine and pair ovary wet weight
Pubertal Assay: 20 day old female Wistar
rats were gavaged with 0 (untreated), 0
(corn oil vehicle), 276, 1380 mg/kg-day
DINP (CASRN 68515-48-0), or
diethylstilbesterol 6 pg/kg-day
diethylstilbesterol for 20 days starting on
PND21. Animals were sacrificed on
PND41 (Sedha etal.. 2015)
None/ 276
| body
weight gain
- [ body weight gain (>276 mg/kg-day)
- [ (10-28%) relative and absolute ovary weight (1,380 mg/kg-day)
- Positive control gave anticipated results
Unaffected Outcomes
- Absolute and relative uterine wet weight and vaginal weight; vaginal
opening
CD-I female mice (4-12/dose) were
gavaged with 0 (corn oil vehicle), 0.02,
200/ None
NA
Maternal Effects b
- None definitively related to treatment
Page 37 of 282
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Brief Study Description
NOAEL /
LOAEL
(mg/kg-day)
Effect at
LOAEL
Remarks
0.1, 20, or 200 mg/kg-day DINP
(CASRN not provided) for 10 days and
then mated with untreated males
immediately after, as well as 3 and 9
months post-dosina (Chiang and Flaws.
2019)
Developmental Effectsh
- None definitively related to treatment
Unaffected Outcomes (all timepoints. unless otherwise noted)
- Body weight; absolute ovary, uterine, liver weight; time to mating; fertility
index; gestational index; gestation length; litter size; pup weight; pup
mortality; estrous cyclicity (0, 9 months)
CD-I female mice (6-12/dose) were
gavaged with 0 (corn oil vehicle), 0.02,
0.1, 20, or 200 mg/kg-day DINP
(CASRN not provided) for 10 days and
outcomes evaluated immediately after
dosing, as well as 3-, 6-, and 9-months
post-dosina (Chiana et al.. 2020a)
200/ None
NA
Maternal Effects c
- None definitively related to treatment
Developmental Effects c
- None definitively related to treatment
Unaffected Outcomes (all timepoints. unless otherwise noted)
- Hormones (serum progesterone, estradiol, FSH, and inhibin B)
- Ovarian histopathology (total number of follicles)
CD-I female mice (6-12/dose) were
gavaged with 0 (corn oil vehicle), 0.02,
0.1, 20, or 200 mg/kg-day DINP
(CASRN not provided) for 10 days and
then mated with untreated males 12- or
15-months post-dosing. Outcomes were
evaluated at 12-, 15-, and 18-months
post-dosina (Chiana et al.. 2020b)
200/ None
NA
Maternal Effects c
- None definitively related to treatment
Developmental Effects c
- None definitively related to treatment
Unaffected Outcomes (12 and 15 months)
- Estrus cyclicity, time to mating, litter size, percent of females who gave
birth, average live litter weight, serum levels of testosterone, progesterone,
estradiol, FSH, or inhibin B, total number of ovarian follicles
Unaffected Outcomes (18 months)
- Total number of ovarian follicles, percent of follicle type (i.e., primordial,
primary, preantral, antral)
Female yellow agouti mice (resulting in
15-17 litters/dose) were fed diets of 0 or
75 mg/kg feed DINP (equivalent to 0 or
15 mg/kg-day) from 2 weeks prior to
mating through weaning (PND21) with
body and organ weights were collected
on PND21 (Neier et al.. 2018)
None/ 15
t maternal
body weight
gain; | pup
body weight;
t pup relative
liver weight
Maternal Effects
- | body weight gains
Developmental Effects (PND21)
-1 pup body weight (both sexes)
-1 pup relative liver weight (females)
Unaffected Outcomes (PND21)
- Number of live pups per litter; maternal body weight; pup hepatic
triglycerides; pup gonadal fat, brain, spleen, and kidney weights; pup liver
weight (male); pup Avv DNA methylation
Female yellow agouti mice
(17-21/group) were fed diets of 0 or 75
None/ 15
[ birth rates;
Maternal Effects
- [ birth rates
Page 38 of 282
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Brief Study Description
NOAEL /
LOAEL
(mg/kg-day)
Effect at
LOAEL
Remarks
mg/kg feed DINP (equivalent to 0 or 15
mg/kg-day) from 2 weeks prior to mating
through weaning (PND21). 1 male and
female pup/litter were allowed to recover
for 10 months (Neier et al.. 2019)
t pup liver
masses;
altered pup
body
composition;
i glucose
tolerance
Developmental Effects (PND21)
-1 liver masses (males)
- | body fat (females, longitudinal 2-8 months)
- [ lean mass percentage (females, longitudinal 2-8 months)
- [ glucose tolerance (females, longitudinal 2-8 months)
Unaffected Outcomes (at 2 and 8 months unless noted)
- Pup body weight across life course (PND21-10 months); pup physical
activity; pup food intake; pup energy expenditure; resting metabolic rate,
respiratory exchange rate, fat oxidation rate, glucose oxidation rate; pup
plasma adipokines
Pregnant female CD-I mice (25/group)
were administered 0 or 20 (ig/kg-day
DINP by pipetting DINP directly into the
mouth of the mice on GDI-7. Seven
mice from each treatment group were
allowed to deliver litters naturally, while
the remaining mice were euthanized on
GD7, 13, and 18 (6 mice/dose/time point)
(Bhurkc et al.. 2023)
None/ 0.02
i litter size, J,
gestation
length, I
fetal and pup
weight, and
other
placental
defects (i.e.,
perturbation
in placental
histopatholog
y)
Maternal Effects
- Study authors do not report examination of any maternal outcomes
Developmental Effects (PND21)
- [ average litter size (decreased from 16-11 in DINP group)
- [ fetal and placental weight on GDI3 and [ fetal weight on GDI8
- [ gestation length (by 20-24 hours) and [ pup weight on PND1
- [ mRNA expression of genes involved in decidualization and angiogenesis
in uterine tissue on GD7
- [ mRNA expression of genes involved in trophoblast differentiation and
glucose transporters in placental tissue on GDI3
Unaffected Outcomes
- # of implantation sites on GD7; sex ratio; serum estrogen; serum
progesterone
Adult female CD-I mice (12-14 mice
per dose) fed diets containing 0, 0.15, 1.5
and 1,500 ppm DINP continuously for 11
months (equivalent to 0.024, 0.24, 240
mg/kg-day) and then mated with
untreated males after 11 months of
exposure (Laws et al.. 2023). Estrus
cyclicity was evaluated after 1, 3, 5, 7,
and 11 months of phthalate exposure.
0.24/ 240
[ gestation
index and
birth rate
Maternal Effects
- None
Developmental Effects
- [ gestation index and birth rate (240 mg/kg-day)
Unaffected Outcomes
- Body weight and weight gain; estrous cyclicity; mating index, fertility
index, pregnancy rate, dystocia rate
Adult female CD-I mice fed diets
containing 0, 0.15, 1.5, and 1,500 ppm
DINP for 1 and 6 months (equivalent to
0.24/ 240
Altered
ovarian
follicles
Maternal Effects
- Maternal outcomes (body weight, food consumption, clinical signs) not
evaluated
Page 39 of 282
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Brief Study Description
NOAEL/
LOAEL
(mg/kg-day)
Effect at
LOAEL
Remarks
0, 0.024, 0.24, and 240 mg/kg-day)
(Santacruz-Marquez et al.. 2024)
Reproductive Effects
-1 percentage of primordial follicles & [ percentage of preantral and antral
follicles (1,500 ppm at 6 months)
- [ serum follicle stimulating hormone after 1 month (1.5 ppm) and | follicle
stimulating hormone after 6 months (1.5 ppm) (not dose-related)
- [ serum luteinizing hormone after 6 months (>1.5 ppm)
Unaffected Outcomes
- Ovarian follicles (1 month); serum progesterone, testosterone, estradiol (1
and 6 months); mRNA expression of steroidogenic genes, follicle stimulating
hormone receptor, luteinizing hormone receptor in ovary (1 and 6 months);
serum luteinizing hormone levels (1 month)
11 Waterman et al.. (1999) originally identified a developmental NOAEL of 500 mg/kg-dav DINP based on increased incidence of skeletal variations. However, a
re-analysis of the data by study sponsors using the generalized estimating equation approach to the linearized model supported a NOAEL of 100 mg/kg-day
DINP. Results from the statistical re-analvsis are reported in (NTP-CERHR. 2003).
h The study authors in Chiang (2019) reported several statistically significant findings as related to treatment with DINP; however. EPA considered these
differences to be spurious and incidental to treatment because they were unrelated to dose, transient, and/or not adverse. These significant differences included:
differences in estrous cyclicity at 20 and 100 (ig/kg-day and 200 mg/kg-day DINP and fewer pregnant females at 20 (ig/kg-day at 3 months post-dosing;
differences in estrous cyclicity at 100 (ig/kg-day and reduced time to mating at 100 (ig/kg-day to 200 mg/kg-day DINP and increased percent males in litters at
100 (ig/kg-day and 20 and 200 mg/kg-day DINP at 9 months post-dosing.
c The study authors of Chiang et al. (2020a. b) reported statistically significant findings related to exposure to DINP; however. EPA considered these effects to
be unrelated to dose, transient, and/or not adverse. These findings included: changes in the percent of type of follicle (i.e.. primordial, primary, preantral, antral)
at doses as low as 0.02 mg/kg-day (decreased percent antral follicles at 6 months, but no other timepoint), changes in serum levels of estradiol, progesterone,
and/or inhibin B, percent of female pups (12 months), and gestation length (12 months).
Page 40 of 282
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In the first study, which adhered to EPA section 798.4900 (40 CFR part 798, 1985), Waterman et
all 1999) gavaged pregnant SD rats (23-25 per dose) with 0, 100, 500, and 1,000 mg/kg-day DINP
(CASRN 68515-48-0) on GD6 through 15. Maternal toxicity was limited to the high-dose group and
included a reduction in maternal body weight gain on GD6 through 9 and 6 through 15 (magnitude of
effect not reported), and a 13 percent decrease in food consumption on GD6 through 9. Food
consumption and body weight gain significantly increased after cessation of exposure between GDI 8
through 21 and mean maternal body weight recovered to control levels by GD21. No treatment-related
effects on maternal survival, clinical signs, resorptions, post-implantation loss, fetal viability, sex ratio,
or fetal body weight were observed. No malformations were observed at any dose. Fetal effects were
limited to treatment-related increases in skeletal and visceral variations, including increased incidence of
renal pelves at 1,000 mg/kg-day, rudimentary lumbar ribs at 500 and 1,000 mg/kg-day, and
supernumerary cervical ribs at 1,000 mg/kg-day (Table 3-3). EPA identified a developmental NOAEL
of 100 mg/kg-day DINP based on increased incidence of skeletal variations at 500 mg/kg-day and above
and a maternal NOAEL of 500 mg/kg-day based on reduced maternal weight gain and food
consumption at 1,000 mg/kg-day DINP.
Type of Fetal Variation
0
(mg/kg-day)
100
(mg/kg-day)
500
(mg/kg-day)
1,000
(mg/kg-day)
Skeletal variations
16.4
15.0
28.3*
43.4**
Visceral variations
0.5
3.3
3.7
Ui
be
¦X-
Renal pelves
0.0
3.3
3.7
5.3*
Rudimentary lumbar ribs
3.5
4.7
18.1*
34.2**
Supernumerary cervical ribs
1.6
1.5
1.0
5.5*
11 Adapted from Tables 5 and 6 in (NTP-CERHR. 2003).
h * indicates P<0.05 and ** indicates p < 0.01. Skeletal variation data was re-analyzed by study sponsors using
the generalized estimating equation (GEE) approach to the linearized model to account for potential litter
effects. The statistical re-analvsis conducted bv studv sponsors is reported in (NTP-CERHR. 2003). Renal
pelves data could not be re-analyzed using the GEE methodology due to the zero incidence in the control. Renal
pelves data was re-analyzed using two approaches, including a nested analysis that considered litter effects and
by changing one control fetus to affected and using the GEE approach. Both approaches provided similar
results (significant increase at 1,000 mg/kg-day).
In a second prenatal study, Hell wig et al. (1997) gavaged pregnant Wistar rats (10 per dose) with 0, 40,
200, and 1,000 mg/kg-day DINP on GD6 through 15. Three different formulations of DINP were
evaluated, including: DINP-1 (CASRN 68515-48-0, purity >99%), commercially available with the
alcohol moiety consisting of roughly equivalent amounts of 3,4-, 4,6-, 3,6-, 3,5-, 4,5-, and 5,6-
dimethylheptanol-1; DINP-2 (28553-12-0), with at least 95% of the alcohol components as alkyl-
substituted octanol or heptanol derived from //-butene; and DINP-3 (28553-12-0), resulting from a
different production line from DINP-2, with main alcohol components synthesized from //-isobutene,
resulting in >60% alkyl-substituted hexanols. The studies were Good Laboratory Practice (GLP)-
compliant and generally adhered to EPA section 798.4900 (40 CFR part 798, 1992), with the exception
that 10 dams, instead of 20 were employed per dose group. For DINP-1, maternal toxicity was limited to
the high-dose group and included reduced food consumption (magnitude of effect not reported), clinical
signs (i.e., vaginal hemorrhage and urine smeared fur in one dam), and a 13 percent increase in relative
kidney (but not liver) weight. No treatment-related effects on maternal body weight, maternal survival,
resorptions, post-implantation loss, number of live fetuses per dam, or fetal weights were observed.
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Developmental effects were limited to the high-dose group and included a statistically significant
increase in the percent fetuses per litter with variations (35.3, 41.5, 29.5, and 58.4% across dose groups).
Variations showing dose-related increases included rudimentary cervical and accessory 14th rib(s), and
an apparent, non-statistically significant, increase in dilated renal pelves (Table 3-4). For DINP-2, there
was no statistically significant maternal toxicity that was treatment-related. One dam given 1,000
mg/kg-day DINP-2 had vaginal hemorrhage on GDI4 and 15. No effects on food consumption, maternal
body weight, maternal survival, relative liver or kidney weight, resorptions, post-implantation loss,
number of live fetuses per dam, or fetal weights were observed. Study authors state that "the only
substance-related fetal effect was an increased incidence of a skeletal variation [accessory 14th rib(s)]"
in the high-dose group, although the incidence of rudimentary cervical rib(s) also appeared slightly
increased (Table 3-4). Multiple malformations were observed in one high-dose fetus, including globular-
shaped heart, unilobular lung, hydrocephaly, dilation of aortic arch, and anasarca, which were regarded
as spontaneous and not treatment related by study authors.
For DINP-3, maternal toxicity was limited to the high dose group, and included reduced food
consumption (magnitude of effect not reported), decreased body weight gain from GD6 to 15, increased
(11%) relative liver weight, and a non-statistically significant increase (9%) in relative kidney weight.
No effects on maternal survival, resorptions, post-implantation loss, number of live fetuses per dam, or
fetal weights were observed. Developmental effects were limited to the high-dose group and included a
statistically significant increase in the percent fetuses per litter with variations (35.3, 29.6, 39.5, and
60.7% across dose groups), including increased incidences of skeletal retardations (unossified or
incompletely ossified sternebrae), skeletal variations (rudimentary cervical and/or accessory 14th rib [s])
and soft tissue variations (hydroureter, dilated renal pelvis) (Table 3-4). Additionally, study authors
attributed some soft tissue malformations (predominately affecting the urogenital tract) and skeletal
malformations (shortened and bent humerus and femur in a single fetus) in the high-dose group to be
treatment-related. Overall, similar developmental findings were observed for all three tested
formulations of DINP and support a developmental NOAEL of 200 mg/kg-day based on increased
skeletal and visceral variations at 1,000 mg/kg-day.
Table 3-4. Incidence of Visceral, Skeletal, and Soft Tissue Variations (I
ellwig et al..
L997)"
DINP
Formulation
Number of Fetuses Evaluated and
Type of Fetal Variation
Control
40
(mg/kg-day)
200
(mg/kg-day)
1,000
(mg/kg-day)
No. fetuses (litters) evaluated
135 (9)
116(9)
111(8)
131 (10)
DINP-1
Rudimentary cervical rib(s)
2(1)
1
11(5)
Accessory 14th rib
2(2)
37 (10)
Dilated renal pelvis
12(7)
11(4)
8(4)
22 (9)
No. fetuses (litters) evaluated
135 (9)
116(9)
135(10)
141 (10)
DINP-2
Rudimentary cervical rib(s)
1
4(2)
10(5)
Accessory 14th rib
1
4(4)
No. fetuses (litters) evaluated
135 (9)
138(10)
135 (9)
120 (9)
Rudimentary cervical rib(s)
2(1)
12(7)
Accessory 14th rib
9(5)
34 (8)
DINP-3
Sternebrae not ossified
6(3)
1
3(2)
26 (7)
Sternebrae incompletely ossified or
reduced in size
20 (7)
11(7)
16(6)
36(9)
Dilated renal pelvis
12(7)
15(8)
13(9)
20 (9)
Page 42 of 282
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DINP
Formulation
Number of Fetuses Evaluated and
Type of Fetal Variation
Control
40
(mg/kg-day)
200
(mg/kg-day)
1,000
(mg/kg-day)
Hydroureter
4(3)
5(3)
1
12(8)
"Table adapted from Tables 10, 12, and 14 in Hellwig et al. (1997).
DINP has also been evaluated in both one- and two-generation studies of reproduction, which were GLP
compliant and conducted in accordance with EPA Test Guidelines for Reproductive and Fertility Effects
(40 CFR part 798, 1985) (Waterman et al.. 2000; Exxon Biomedical 1996a. b). In the one generation
study, SD rats (30/sex/dose) were continuously administered dietary concentrations of 0, 0.5, 1.0, and
1.5 percent DINP (CASRN 68515-48-0) starting 10 weeks prior to mating, throughout mating, gestation,
and lactation. Mean received doses in units of mg/kg-day are shown in Table 3-5. PI males were
sacrificed following delivery of the last litter of F1 pups, while PI females were sacrificed at F1
weaning on postnatal day (PND) 21. No treatment-related clinical signs or effects on survival were
reported for PI males or females. Body weight was statistically significantly reduced in mid- and high-
dose males and females during the premating phase, and in mid- (5.3-15.3% decrease) and high-dose
(10.8-23.3% decrease) PI females during gestation and lactation. Similarly, food consumption was
significantly reduced in mid- (5.3-8.7% decrease) and high-dose (5.8-10.5% decrease) males and
females during the premating phase, and in mid- (16.7-27.4% decrease) and high-dose (11.6-42.2%
decrease) PI females during gestation and lactation.
Treatment with DINP had no significant effects on any reproductive indices, including male mating,
male/female fertility, female fecundity, or gestational indices. Mean litter size, mean number of live and
dead offspring, and sex ratio were unaffected by treatment with DINP. At the high dose, treatment with
DINP significantly reduced percent live births (95.2 vs. 98.2% in controls), PND4 survival (85.6 vs.
93.1% in controls), PND14 survival (92.7 vs. 98.5% in controls), and viability at weaning (87.2 vs.
93.9% in controls). Male and female F1 offspring body weight was significantly reduced in all treatment
groups on PND0 (7.9-11.5%) and continued to be reduced, although not always statistically
significantly, in all treatment groups for both sexes through PND21 (Table 3-6). Overall, this study
supports a developmental LOAEL of 377 mg/kg-day (no NOAEL identified), based on reduced F1
offspring body weight throughout the lactational period.
Table 3-5. Mean Measured Doses (mg/kg-day) from the One-Generation Study of DINP in SD
Rats (Waterman et al., 2000; Exxon Biomedical, 1996a) "
Dose (%)
Premating - Males
Premating - Females
Gestation
Postpartum
0.5
301-591
363-624
377-395
490-923
1.0
622-1,157
734-1,169
741-765
1,034-1,731
1.5
966-1,676
1114-1,694
1087-1,128
1,274-2,246
11 Adapted from Table 12 in Exxon Biomedical (1996a).
Page 43 of 282
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Table 3-6. F1 Offspring Postnatal Body Weight (Grams) from the One-Generation Study of
Reproduction in SD Rats (Waterman et al., 2000; Exxon Biomedical, 1996a) ahc
F1 Offspring
Group
Male
Female
PND0
PND1
PND4
PND7
PND14
PND21
PND0
PND1
PND4
PND7
PND14
PND21
0%
6.98
7.34
9.80
16.02
33.77
54.34
6.68
7.05
9.58
15.60
32.72
52.19
0.2%
6.49**
6.83
9.18
14.52
30.00**
48.94*
6.15**
6.52*
8.81
14.07
29.40**
47.77**
0.4%
6.42**
6.92*
9.12
14.00*
26.23**
39.93**
6.05**
6.49**
8.56*
13.24*
25.04**
38.13**
0.8%
6.27**
6.58**
8.19**
11.04**
20.18**
29.32**
5.91**
6.25**
7.84**
10.71**
19.31**
27.71**
Historical
Control
6.35-
7.02
6.68-
7.46
8.53-
11.43
13.64-
18.74
28.81-
36.73
44.89-
60.77
5.96-
6.74
6.30-
7.16
8.32-
11.05
13.33-
17.69
27.22-
35.74
42.39-
61.19
" Data from Table 4 in Waterman et al. (2000).
4 and '**' indicate the mean is significantly different from the control mean by p < 0.05 and p < 0.01, respectively.
c Historical control data reported to be from the laboratory conducting the study. Further details (e.g., number of studies data
collected from, timespan of studies) regarding the source of historical control data were not provided in Exxon Biomedical
(1996a).
In the two-generation study, SD rats (30/sex/dose) were continuously administered dietary
concentrations of 0, 0.2, 0.4, and 0.8 percent DINP (CASRN 68515-48-0) starting 10 weeks prior to
mating, throughout mating, gestation, and lactation continuously for two generations (Waterman et al..
2000; Exxon Biomedical 1996b). Mean received doses in units of mg/kg-day are shown in Table 3-7.
For the first parental generation (PI), no treatment-related clinical signs or effects on survival were
reported for PI animals. No significant effects on PI body weight were observed, except for a 6.7 to 7.8
percent decrease in high-dose female body weight on PND14 and 21. Food consumption was
significantly reduced (9%) for high-dose females during the postnatal phase of the study but was not
reduced for males or females during other phases of the study. For the second parental generation (P2),
no treatment-related clinical signs or effects on survival were reported. At the start of the premating
period (six weeks after weaning), mean body weights for mid and high dose males and females were
lower than control. Females in the high-dose group had consistently lower body weight gain compared
to the control group during the premating (statistically significant for first 2 weeks), gestation (not
significant), and lactational (significant for PND4-21) phases. Small (<8%), but statistically significant,
decreases in food consumption were observed in high-dose males and females during the premating
period and in high-dose females during gestation (13-16% decrease) and lactation (9% decrease). No
treatment-related effects on any reproductive indices were observed for either generations, including
male mating, male/female fertility, female fecundity, or gestational indices.
Similarly, gestation length, mean litter size, mean number of live and dead offspring, sex ratio, percent
live births, survival on PND1, 4, 7, 14, and 21, and viability at weaning were unaffected by treatment
with DINP for both the F1 and F2 generations. F1 and F2 offspring body weight was significantly
reduced throughout PND0 to 21 (Table 3-8). For F1 offspring, bodyweight was significantly reduced 6.8
percent for high-dose males on PND0; 10 to 15 percent for mid- and high-dose males and females on
PND7 and 14; and 8.9 to 19 percent for males and females on PND21 in all dose groups. For F2
offspring, bodyweight was significantly reduced 14 to 17 percent for mid- and high-dose females on
PND4; 14 to 19 percent for mid- and high-dose males and 10 to 21 percent for females in all dose
groups on PND7; 12 to 21 percent for mid- and high-dose males and females on PND14; and 12 to 22
percent for mid- and high-dose males and females on PND21. Study authors state that the observed body
weight changes were within historical control ranges from the laboratory conducting the study and that
effects on body weight at 0.2 and 0.4 percent DINP "seem unrelated to treatment." However, no
information regarding the source of the historical control data is provided (e.g., number of studies, years
study conducted, strain/species tested, and diet animals were maintained on were not reported), so it is
Page 44 of 282
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unclear if an appropriate historical control data set was used. Overall, this study suggests a
developmental LOAEL of 0.2 percent DINP (equivalent to approximately 133 mg/kg-day during
gestation) for decrements in F1 and F2 body weight during lactation.
Table 3-7. Mean Measured Doses (mg/kg-day) from the Two-Generation Study of DINP in SD
Dose
(%)
PI Generation
P2 Generation
Premating -
Males
Premating -
Females
Gestation
Postpartum
Premating -
Males
Premating -
Females
Gestation
Postpartum
0.2
118-212
145-215
139-153
159-350
114-264
140-254
133-153
174-395
0.4
236-126
278-425
274-301
347-731
235-523
271-522
271-307
348-758
0.8
477-852
562-830
543-571
673-1,379
467-1,090
544-1,060
544-577
718-1,541
° Adapted from Tables 12 and 32 in Exxon Biomedical (1996b).
Table 3-8. F1 and F2 Offspring Postnatal Body Weight (Grams) from the Two-Generation Study
of Reproduction in SD Rats (Waterman et al., 2000; Exxon Biomedical, 1996b) "bc
F1 Offspring
Group
Male
Female
PND0
PND1
PND4
PND7
PND14
PND21
PND0
PND1
PND4
PND7
PND14
PND21
0%
6.90
7.49
10.63
17.62
35.01
57.25
6.47
7.11
10.26
16.70
33.52
53.99
0.2%
6.78
7.39
10.26
16.44
33.28
51.40*
6.36
6.96
9.61
15.54
31.89
49.19*
0.4%
6.48
7.03
9.54
15.28**
30.43**
47.95**
6.16
6.67
9.24
14.21**
29.14**
45.63**
0.8%
6.43*
7.05
9.74
15.67*
29.66**
46.52**
6.08
6.70
9.36
15.03*
28.41**
44.68**
Histor-
ical
Control
6.35-
7.02
6.68-
7.46
8.53-
11.43
13.64-
18.74
28.81-
36.73
44.89-
60.77
5.96-
6.74
6.30-
7.16
8.32-
11.05
13.33-
17.69
27.22-
35.74
42.39-
61.19
F2 offspring
0%
6.67
7.30
10.63
18.08
37.09
62.34
6.44
7.10
10.48
17.47
35.89
59.37
0.2%
6.49
7.12
10.05
16.43
34.80
57.89
6.13
6.75
9.60
15.72*
33.64
55.50
0.4%
6.55
7.08
9.73
15.48**
32.51**
54.82**
6.11
6.59
9.05**
14.56**
31.22**
51.98**
0.8%
6.18
6.64
9.05
14.70**
29.88**
49.12**
5.92
6.41
8.68**
13.76**
28.20**
46.20**
Histor-
ical
Control
6.35-
7.02
6.68-
7.46
8.53-
11.43
13.64-
18.74
28.81-
36.73
44.89-
60.77
5.96-
6.74
6.30-
7.16
8.32-
11.05
13.33-
17.69
27.22-
35.74
42.39-
61.19
" Data from Tables 8 and 11 in Waterman et al. (2000).
b and '**' indicate the mean is significantly different from the control mean by p < 0.05 and p < 0.01, respectively.
c Historical control data reported to be from the laboratory conducting the study. Further details (e.g., number of studies data
collected from, timespan of studies) regarding the source of historical control data were not provided in (Exxon Biomedical
1996b).
Chiang and Flaws (2019) gavaged adult CD-I female mice (4-12 per group) with 0 (corn oil vehicle),
0.02, 0.1, 20, or 200 mg/kg-day DINP (CASRN not provided) for 10 days and then evaluated effects on
organ weight, estrous cyclicity, and mating behavior with untreated male mice immediately after dosing,
as well as 3 and 9 months post-dosing. Treatment with DINP had no effect on body weight, absolute
ovary, uterine or liver weight at any timepoint. Three months post-dosing, females treated with 0.02 and
200 mg/kg-day spent significantly less time in proestrus and more time in metestrus and diestrus.
However, no dose-related effects on estrous cyclicity were observed immediately following dosing or
nine months post-dosing and the effects observed at three months appeared slight (magnitude of effect
not reported) and of uncertain toxicological significance. No adverse, dose-related, effects on time to
Page 45 of 282
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mating, fertility index, gestational index, gestation length, the number of females able to produce pups,
litter size, pup weight on PND20, pup mortality, sex ratio were observed at any timepoint. Several
parameters were statistically significantly altered (e.g., fertility index deceased at 0.02 mg/kg-day at 3
months [but not at higher doses or other timepoints], number of females able to produce pups decreased
at 0.02 mg/kg-day at 3 months and 20 mg/kg-day at 9 months [but not at higher doses]); however, these
findings were of uncertain toxicological significance, given the non-monotonic dose relationship and the
lack of mechanistic data from other studies supporting an effect of DINP on these endpoints.
Two follow-up studies by Chiang et al. (2020a. b) further evaluated the effects of DINP on follicle
populations and hormone levels at timepoints where fertility was disrupted in the prior study (Chiang et
al.. 2020a) or at additional timepoints several months after exposure has ended (Chiang et al.. 2020b).
The exposure paradigm for both was similar to the prior Chiang and Flaws study (2019). where adult
female CD-I mice (at least 6/group; 12 in controls) were dosed orally via pipette beginning at PND39 or
40 with 0, 0.02, 0.1, 20, or 200 mg/kg-day DINP for 10 consecutive days. In Chiang et al. (2020a).
outcomes were evaluated in non-mated females immediately after dosing, as well as at 3, 6, and 9-
months and included ovarian histopathology (i.e.,% follicle types and total number of follicles) and
serum hormone levels (testosterone, progesterone, estradiol, FSH, and inhibin B). Similar to the results
of Chiang and Flaws (2019). there were no effects that exhibited a linear dose-response. There were no
changes in total follicle number at any timepoint. Although there were significant changes in the percent
of follicles of a given type, there was no dose-response, and the directionality and/or dose at which a
significant effect was observed differed across timepoints (e.g., the percent of primary follicles in the
200 mg/kg-day group was increased at 6 months, but there was no change at this dose for other
timepoints, and the percent was decreased at 0.02 mg/kg-day at 9-months). Changes in serum hormone
levels were observed including significant decreases in testosterone immediately after dosing at doses as
low as 0.02 mg/kg-day, as well as the three, 6-month timepoints at doses as low as 0.1 mg/kg-day, but
there was no dose-response in any data set. Changes in estradiol and progesterone were also observed,
but the directionality was not consistent across timepoints.
In Chiang et al. (2020b). two groups of mice were used. The first group was used for cyclicity
monitoring (14 days) followed by a breeding trial at 12 and 15 months and ovary and sera collection at
18 months post-dosing. The second group was used for ovary and sera collection at 12 and 15 months
post-dosing. Altogether, outcomes were evaluated 12, 15, or 18 months after dosing and included estrus
cyclicity (i.e., % time spent in proestrus estrus, metestrus, diestrus at 12 or 15 months); pregnancy loss,
fertility, and sex ratio of pups at 12 months; ovarian histopathology (i.e., ovarian follicle populations) at
12, 15, and 18 months; and hormone levels (serum testosterone, progesterone, and estradiol at 12, 15,
and 18 months). Similar to Chiang and Flaws (2019) and Chiang et al. (2020a). there were no effects
that exhibited a linear dose-response. There were no significant changes in the percent of time in any
particular phase of estrus at 12 or 15 months, aside from a non-significant increase in estrus at the 12-
month timepoint in the 0.02 mg/kg-day group. There were no changes in total follicle numbers at 12, 15,
or 18 months. Although there were significant changes in the number and percentages of various follicle
types at 15 and 18 months, there was no consistent dose-related trend. Changes in hormone levels were
only noted at 18 months and included decreases in testosterone and estradiol in the 0.1 mg/kg-day group,
and an increase in inhibin B in the 20 mg/kg-day group.
Overall, the studies by Chiang et al. (2020a. b; 2019) provide some evidence of effects of DINP on the
female reproductive system; however, the lack of a linear dose-response relationship and inconsistent
effects across evaluated timepoints makes it difficult to interpret whether or not the observed effects are
directly related to DINP exposure.
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Two perinatal exposure studies of DINP have also been conducted using the viable yellow agouti mouse
model (Neier et al.. 2019; Neier et al.. 2018). In in the first study, a/a dams were fed phytoestrogen-free
diets containing 0 of 75 mg/kg DINP (CASRN not reported) (equivalent to approximately 15 mg/kg-day
DINP) starting 2 weeks prior to mating with Av>/a males and continuing throughout gestation and
lactation until weaning on PND21 (Neier et al.. 2018). The exact number of mating pairs per treatment
group is not provided in the 2018 study; however, 15 to 17 litters were produced for the control and
DINP treatment groups. Treatment with DINP had no effect on maternal body weight at PND21,
offspring sex ratio, mean pups per litter, pup mortality through PND21, or pup genotype. Body weight
was significantly increased 10 to 20 percent for females (of genotypes a/a and Avy/a) and 15 percent for
males (of genotype Avy/a) on PND21 in the DINP treatment group. Treatment with DINP correlated to
increased relative liver weight for female pups. There was no change in absolute or relative liver weight
(males only), gonadal fat, brain, spleen, or kidney weights in pups. Additionally, no change in pup DNA
methylation was observed. In summary, treatment with DINP showed modest decreases in pup body
weights and increased relative liver weight (females only).
The Neier et al. (2019) study followed the same dosing scheme with viable yellow agouti mouse dams
exposed from 2 weeks prior to mating through PND21 in diet at dosages of 0 and 75 mg/kg feed DINP
(equivalent to 0 and 15 mg/kg-day). A total of 17 control pairs and 21 DINP pairs were dosed to produce
a minimum of 15 litters per treatment group. The largest male and female from each litter (10 per sex
per dose) were fed a phthalate-free diet until 10 months old; one male in the 15 mg/kg-day group died
during glucose gavage at 2 months. The DINP treatment group showed a decreased birth rate, and a non-
significant increase in liver masses in males at 10 months (9.1% control vs. 33% treated). Effects were
not reported in dams and pups body weights were not altered. The Neier et al. (2019) study also
evaluated metabolic effects through adulthood in mice exposed to DINP perinatally with evaluations at
2, 8, and 10 months. The DINP treatment group showed altered body fat, lean mass percentage in
females longitudinally; however, these effects were not significant when accounting for multiple
comparisons. DINP treated females showed a moderate reduction in glucose tolerance longitudinally
driven by decreased glucose tolerance at two months that improved slightly at eight months. There was
no change in pup body weight across life, physical activity, or food intake. Additionally, there was no
alteration in energy expenditure, resting metabolic rate, respiratory exchange rate, fat oxidation rate,
glucose oxidation rate, or plasma adipokines. Overall, treatment with DINP resulted decreased birth rate,
as well as modest alterations to female pup body composition and glucose tolerance, without
corresponding alterations to diet, physical activity, or other markers for metabolic activity.
Bhurke et al. (2023) dosed pregnant CD-I mice (25 per dose group) with 0 or 20 |ig/kg-day DINP by
pipetting DINP directly into the mouth of the mice from GDI through GD7. Seven mice from each
treatment group were allowed to deliver litters naturally, while the remaining mice were euthanized on
GD7, 13, and 18 (6 mice/dose/time point). Study authors did not report evaluating maternal outcomes
such as food consumption, body weight, weight gain, or clinical signs; however, given the low dose of
DINP utilized in the study, overt maternal toxicity is not expected. Treatment with DINP reduced litter
size from an average of 16 pups per litter for concurrent controls to 11 pups per litter for DINP treated
mice. Treatment with DINP had no effect on sex ratio or number of implantation sites. Decreased
mRNA expression of genes involved in decidualization (i.e., Hand2, Bmp2, Wnt4, and Cebpfi) and
angiogenesis (i.e., Epasl, Vegfa, Angptl, and Angp2) were observed in uterine tissue from mice treated
with DINP on GD7; however, no effect on serum estrogen or progesterone was observed. On GD13,
fetal and placental weight were significantly reduced, and histologic examination demonstrated
alterations in placental architecture, reflected by a disorganized junctional zone, with cells from the
junctional zone having infiltrated the labyrinth layer. The average area of the labyrinth was significantly
reduced, while area of the junctional zone was increased by exposure to DINP. In the placenta, mRNA
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expression of several genes involved in trophoblast differentiation (i.e., SytiA, SynB, and Geml) and
glucose transport (i.e., Slc2al, Slc2a3, Slc6al, Slc6a4, and GjbT) were downregulated following
exposure to DINP. Overall, study authors concluded that the placental effects observed following
exposure to DINP were the cause of fetal loss and reduced litter sizes. Although this study provides
some evidence that DINP can reduced litter size and cause placental abnormalities, several limitations,
particularly with regard to exposure methods, exist that reduce confidence in the study. Limitations
include the fact that only a single dose level was evaluated and was administered via pipetting, which is
a non-standard method of administration that contributes uncertainty related to the actual received doses.
Additionally, effects in Bhurke et al. are difficult to reconcile with guideline studies that did not observe
reductions in litter size or placental weight at doses greater than three orders of magnitude higher (e.g.,
one- and two-generation studies by Waterman et al. (2000)).
In Laws et al. (2023) adult female CD-I mice (12-14 mice per dose) were fed diets containing 0, 0.15,
1.5 and 1,500 ppm DINP continuously for 11 months and then mated them with unexposed males after
11 months. Estrus cyclicity was examined after 1, 3, 5, 7, and 11 months of exposure (via vaginal lavage
for 14 days at each timepoint). Study authors reported that these doses corresponded to mean received
doses of 0.024, 0.24, and 240 mg/kg-day based on an assumption that a 25 g mouse eats a dose of
approximately 5 grams of food per day. Treatment with DINP had no effect on body weight gain or food
consumption throughout the 11-month study. Treatment with DINP had no significant effects on the
amount of time mice spent in estrus or metestrus/diestrus after 1, 5, or 11 months of exposure. After 3
months of exposure, mice treated with 1.5 ppm DINP tended to spend more time in estrus and less time
in metestrus/diestrus, while after 7 months of exposure, mice treated with 0.15 ppm DINP tended to
spend less time in estrus and more time in metestrus/diestrus. However, the effect at both 3 and 7
months was not statistically significant, was not observed consistently across timepoints, and did not
occur in a dose-dependent manner (no effect was observed at 1,500 ppm at either timepoint). Therefore,
EPA did not consider the changes in estrous cyclicity to be related to treatment. After 11 months of
exposure, DINP treated females were mated with untreated males. Treatment with DINP had no effect
on mating index, pregnancy rate, fertility index, or dystocia rate. However, treatment with 1,500 ppm
DINP led to a dose-related decrease in gestation index and birth rate, supporting a LOAEL of
240 mg/kg-day and a NOAEL of 0.24 mg/kg-day. Notably, this study is limited by large dose spacing
between the mid- and high-dose groups (i.e., 4 orders of magnitude).
In another study by the same research group, Santacruz-Marquez et al. (2024) fed adult (33 days old)
female CD-I mice diets containing 0, 0.15, 1.5, and 1,500 ppm DINP for 1 and 6 months. Mean
achieved doses were reported to be 0, 0.024, 0.24, and 240 mg/kg-day DINP based on the previous study
from (Laws et al.. 2023). Body weight and food consumption were not evaluated as part of this study.
Treatment with DINP had no effect on ovarian follicles after 1 month. After 6 months, treatment with
1,500 ppm DINP increased the percentages of primordial follicles and decreased the percentage of
preantral and antral follicles. At 1 and 6 months, serum sex hormone levels were determined; however,
treatment with DINP had no effect on serum levels of progesterone, testosterone, or estradiol. Treatment
with 1.5 ppm DINP significantly decreased serum follicle stimulating hormone (FSH) levels after 1
month and increased FSH levels after 6 months; however, no effect on FSH levels were observed at
1,500 ppm. No effect on serum luteinizing hormone levels were observed after 1 month; however,
luteinizing hormone levels were significantly decreased at 1.5 and 1,500 ppm after 6 months. Expression
of genes that regulate FSH and luteinizing hormones in the pituitary were investigated. Treatment with
DINP had no effect on mRNA expression of Fshb or Lhb, while Nr5al and Cga mRNA was increased
at 0.15 ppm after 6 months; however, these change in gene expression did not occur in a dose-related
manner. Treatment with DINP similarly did not affect mRNA expression of steroidogenic genes (i.e.,
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Star, Hsd3bl, Hsdl7bl, Cypl9al), follicle-stimulating hormone receptor (Fshr), or luteinizing hormone
receptor (Lhcgr) in the ovary after 1 or 6 months.
Chen et al. (2022) gavaged (5 week old) female Kunming mice with 0, 2, 20, or 200 mg/kg-day DINP
for 14 days and then evaluated ovarian histopathology and serum levels of estradiol at the end of study.
Body weight and food consumption were not evaluated, nor were animals monitored for clinical signs.
Study authors report that DINP caused follicular granulosa cells of the ovary to become disorganized;
however, the doses at which this effect was observed is not stated. Serum estradiol levels were decreased
by approximately 15 percent at 20 mg/kg-day DINP and above, but these data are difficult to interpret
given that the time of outcome evaluation is likely to overlap with the onset of the first estrus in the
mice. The authors also reported mechanistic evidence consistent with increased oxidative stress,
increased apoptosis, and autophagy in ovarian tissue, as demonstrated by increased levels of apoptosis-
related proteins (caspase-8, casepase-3, Bax), decreased levels of the anti-apoptosis protein Bcl-2, and
increased levels of autophagy-related proteins (LC3-II/LC3-I, Beclin 1, Atg 5) at all doses of DINP.
Levels of malondialdehyde were increased at 200 mg/kg-day, while levels of glutathione (at 20 mg/kg-
day and above) and activities of glutathione peroxidase (at 2 mg/kg-day and above) and superoxide
dismutase (at 20 mg/kg-day and above) were decreased, suggesting increased oxidative stress in ovary
tissue. In vitro experiments with primary mouse ovarian granulosa cells were further conducted to
investigate the mechanisms of DINP in the ovary. Similar to the in vivo study, DINP was found to
induce apoptosis, autophagy, and oxidative stress in primary mouse ovarian granulosa cells. While these
data may suggest perturbation of ovarian tissue following exposure to DINP, there were several
limitations. For instance, no quantification of the histopathology data was provided (i.e., only single
representative images were provided, no incidence data, etc.,), and insufficient detail is provided on the
methods used to evaluate histopathology (e.g., information on the number of animals examined per
group). Other reporting deficiencies mentioned above contribute additional uncertainty. Altogether,
these limitations impact the ability to interpret the results of the study.
Sedha et al. (2015) investigated the estrogenic potential of DINP in a three-day uterotrophic assay and a
20-day pubertal assay. For the uterotrophic assay, 20-day old female Wistar rats (6/dose/group) were
gavaged with 0 (corn oil vehicle), 276, or 1380 mg/kg-day DINP (CASRN 68515-48-0) for three
consecutive days, while an additional group was treated with diethylstilbesterol (40 |ig/kg-day), which
served as the positive control. Body weight gain was reduced in both DINP treatment groups compared
to the control; however, treatment with DINP had no significant effect on uterine or paired ovary wet
weight, while the positive control increased ovary and uterus wet weight. For the pubertal assay, 20-day
old female Wistar rats were gavaged with 0 (corn oil vehicle), 276, or 1380 mg/kg-day DINP and
diethylstilbesterol (6 |ig/kg-day) from PND21 to sacrifice on PND41. Body weight gain was
significantly reduced in all DINP treatment groups compared to the control. Absolute and relative
uterine wet weight and vaginal weight were unaffected by treatment with DINP, while relative and
absolute ovary weight was significantly reduced 10 to 28 percent by treatment with 1380 mg/kg-day
DINP. Timing of vaginal opening was unaffected by treatment with DINP. Collectively, results from
these assays indicate that DINP lacks estrogenic potential in vivo.
3.1.2.3 Conclusions on Reproductive and Developmental Toxicity
EPA previously proposed a MOA for male reproductive effects in rodents due to antiandrogenic activity
of DINP as part of a proposed approach for cumulative risk assessment of phthalates (U.S. EPA 2023 a).
which was supported by the SACC (U.S. EPA 2023b). As outlined in Table 3-1, male reproductive
effects were observed in 16 rat studies with gestational or perinatal exposures. Collectively, these data
support EPA's conclusion that exposure of pregnant female rodents to DINP during gestation results in
effects on male offspring consistent with androgen insufficiency.
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Additional developmental studies in mice and rats were included in the data set covering a wide
developmental window. Available studies included a one-generation study of reproduction in rats
(Waterman et al.. 2000; Exxon Biomedical 1996a) and two-generation study of reproduction in rats
(Waterman et al.. 2000; Exxon Biomedical 1996bI and a uterotrophic assay in rats (Sedha et al. 2015).
along with multiple studies covering the pre-mating, gestation, and lactation periods. All studies were
limited to oral exposures in rodents.
The evidence for effects on the female endocrine system and reproduction is less clear than the evidence
supporting androgen insufficiency. The uterotrophic assay in rats showed decreased body weight gains,
but no change to uterine or paired ovary wet weight (Sedha et al. 2015). In the pubertal assay, absolute
and relative uterine wet weight and vaginal weight were unaffected by treatment with DINP, while
relative and absolute ovary weight was significantly reduced at the high dose (1,380 mg/kg-day DINP).
Sexual maturation (time to vaginal opening) was unaffected by treatment with DINP. In the study by
Chiang and Flaws (2019) in which adult CD-I female mice were administered DINP via oral gavage and
mated with untreated male mice, there were no adverse effects of treatment on body weight, weights of
the uterus or ovaries, time to mating, fertility index, gestational index, gestation length, the number of
females able to produce pups, litter size, pup weight on PND20, pup mortality, or sex ratio. Several
parameters were significantly different from controls (e.g., decreases in fertility index and number of
females able to produce pups and differences in estrous cycle; however, these findings were of uncertain
toxicological significance, given the findings were often transient, and the non-monotonic dose
relationship and the lack of mechanistic data from other studies supporting an effect of DINP on these
endpoints. These limitations were also apparent in the two follow-up studies by Chiang et al. (2020a. b),
which evaluated the effects of DINP on follicle populations and hormone levels at timepoints where
fertility was disrupted in the prior study or at additional timepoints several months after exposure has
ended. In contrast, Bhurke et al. (2023) report decreased litter size and gestation length in females CD-I
mice dosed with an extremely low dose of DINP (0.02 mg/kg-day). Santacruz-Marquez et al. (2024)
report evidence of altered ovarian follicles in female CD-I mice after 6 months of dietary exposure to
DINP, while Chen et al. (2022) reports that DINP can induce apoptosis, autophagy, and oxidative stress
in the ovaries of female Kunming mice. Laws et al. (2023) report that treatment with DINP does not
alter estrous cyclicity in female CD-I mice. Similarly, Santacruz-Marquez et al. (2024) found that
treatment with DINP had no effect on serum estradiol in female CD-I mice, while in contrast Chen et al.
(2022) reported that DINP slightly reduced serum estradiol levels in female Kunming mice.
Collectively, results from these assays generally indicate that DINP lacks estrogenic potential in vivo,
and the results of in vitro receptor-binding assays (Kruger et al. 2008; Takeuchi et al. 2005; Roy et al.
2004) are consistent with the lack of effects in the uterotrophic and female pubertal assays in Sedha et
al. (2015).
Skeletal variations (Waterman et al. 1999; Hellwig et al. 1997) and reduced body weights were
observed in rat pups across multiple studies (Setti Ahmed et al. 2018; Sedha et al. 2015; Waterman et
al. 2000; Exxon Biomedical. 1996a). Maternal body weights and food consumption were decreased in
several studies on rats (Setti Ahmed et al. 2018; Waterman et al. 1999; Hellwig et al. 1997). The one
generation reproduction study showed decreased live births and postnatal survival (Waterman et al.
2000; Exxon Biomedical. 1996a). Two studies of yellow agouti mice dosed with 15 mg/kg-day DINP
from 2 weeks prior to mating through lactation found increased pup body weights, altered body
compositions, and decreased glucose tolerances (Neier et al. 2019; Neier et al. 2018). as well as
decreased birth rates (Neier et al. 2019). Although these data show different effects in mice and rats, the
low number of studies in mice make it difficult to confidently determine species sensitivity.
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Oral exposure to DINP has consistently been shown to cause developmental effects in animal models as
illustrated by the studies described above and concluded by previous assessments by NTP-CERHR
(2003). ECHA (2013b). EFSA (20191 Australia NICNAS (2012), Health Canada (EC/HC. 2015) and
U.S. CPSC (2014. 2010). Therefore, EPA is considering developmental toxicity for dose-response
analysis in Section 4.
3.2 Liver Toxicity
The non-cancer health effects and carcinogenicity of DINP have been evaluated primarily in animal
toxicological studies; no human epidemiologic studies evaluating hepatic effects were identified by
EPA's review of existing assessments (primarily Health Canada (2018a)). Moreover, existing
assessments have consistently identified the liver as one of the most sensitive target organs following
oral exposure to DINP in experimental animal studies (ECCC/HC. 2020; EFSA. 2019; EC/HC. 2015;
ECHA 2013b: NICNAS. 2012; U.S. CPSC. 2010; EFSA 2005; ECB. 2003; NTP-CERHR. 2003; U.S.
CPSC. 2001).
EPA identified twenty-five animal toxicology studies that evaluated non-cancer effects on the liver
following intermediate (>1 to 30 days), subchronic (>30 to 90 days), or chronic (>90 days) oral
exposure to DINP, and two following intermediate dermal exposure to DINP. Available studies include:
12 intermediate oral studies (7 studies on rats, 4 studies on mice, 1 study on cynomolgus monkeys); 9
subchronic oral exposure studies (6 on rats, 1 on mice, 1 on beagle dogs, and 1 on marmosets); 4 chronic
2-year oral exposure studies (3 on rats and 1 on mice); one-generation and two-generation studies of
reproduction of rats that report non-cancer liver effects; and two intermediate dermal studies in mice.
More detailed information on the available studies is provided in Appendix B, including information on
individual study design.
Exposure to DINP resulted in adverse non-cancer effects on the liver across study designs. Adverse non-
cancer effects such as increased absolute and/or relative liver weight consistently coincided with
increased incidences of non-neoplastic lesions or changes in clinical chemistry parameters, indicative of
liver toxicity. Adverse non-cancer effects on the liver were primarily observed in rats and mice of both
sexes, although there was also evidence of hepatotoxicity from one study in beagles. Two studies in non-
human primates with dose ranges comparable to those in the rodent and beagle studies did not provide
evidence of non-cancer or pre-neoplastic effects on the liver following 14- (Pugh et al.. 2000) and 90-
day oral exposures to DINP (Hall et al.. 1999). Changes in liver weights, histopathology, and clinical
chemistry parameters in rodents coincided with mechanistic endpoints indicative of Peroxisome
proliferator activated receptor alpha (PPARa) activation, which is discussed further in EPA's Cancer
Human Health Hazard Assessment for Diisononyl Phthalate (DINP) (U.S. EPA. 2025a).
In general, intermediate (9 of the 12 studies) and subchronic duration studies (9 of 9) consistently
reported increases in absolute and/or relative liver weight, sometimes in parallel with exposure-related
histopathological effects on the liver (e.g., hepatocellular hypertrophy) or coinciding with increases in
liver enzymes (e.g., ALT, AST, ALP), suggesting impaired liver function. These effects were generally
dose-dependent, observed in both sexes, and in multiple species, including rats, mice, and beagle dogs.
One 13-week study in marmoset monkeys reported non-statistically significant increases in liver weight,
but there was no dose-response, and the authors attribute the lack of statistical significance to high
variability and small sample size (Hall et al.. 1999). More detailed study information for intermediate
and subchronic studies is available in Appendix B within TableApx B-l, and TableApx B-2,
respectively.
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Three chronic 2-year studies in rats (Covance Labs. 1998c; Lington et al.. 1997; Bio/dynamics. 1987)
and one in mice (Covance Labs. 1998b) consistently reported non-cancer liver effects, while all except
the Lington et al. (1997) study reported statistically significant increased incidences of liver tumors (i.e.,
hepatocellular adenomas and/or carcinomas). Non-cancer liver effects that were observed across these
four studies included consistent increases in liver weight that corresponded with histopathological
alterations (e.g., spongiosis hepatis, necrosis) and/or increases in serum enzyme levels or activity in both
sexes. An additional one- and two-generation study in rats by Waterman et al. (2000; Exxon Biomedical.
1996a) found increases in liver weight in the parental generation that coincided with minimal to
moderate cytoplasmic eosinophilia in the liver. More detailed study information for intermediate and
subchronic studies is available in Appendix B within TableApx B-l, and TableApx B-2.
The NOAEL and LOAEL for non-cancer hepatic effects in F344 rats in Lington et al. (1997) were 15
and 152 mg/kg-day, respectively; based on a statistically significant increase in the incidence of
spongiosis hepatis in mid-dose male rats that was accompanied by increased absolute and relative liver
weights and changes in serum enzyme activities (i.e., increased ALT and AST). These effects are also
the basis for the LOAEL of 359 mg/kg-day (NOAEL of 88 mg/kg-day) in the Covance study (1998c) of
F344 rats. The incidence of spongiosis hepatis was dose-related and significantly increased in male rats
exposed to DINP in both studies. Moreover, a Histopathology Peer Review and Pathology Working
Group (EPL. 1999) independently evaluated the liver slides from rats chronically treated with DINP and
confirmed that the incidence of spongiosis hepatis was increased in male rats in each study.
Bio/dynamics (1987) also reported a significant increase incidence of spongiosis hepatis in male SD rats
of the two highest dose groups, and dose-related trends in both males and females. Detailed information
on lesion incidence is available in Appendix B within Table Apx B-6.
Conclusions on Non-cancer Liver Toxicity
Collectively, intermediate, subchronic, and chronic studies of mice, rats, and beagles provide consistent
evidence that oral exposure to DINP can cause liver toxicity. The lowest non-cancer NOAEL identified
by EPA was 15 mg/kg-day based on increased liver weight, increase serum ALT and AST, and
increased incidence of non-neoplastic lesions (e.g., spongiosis hepatis, enlargement, and granular and
pitted rough changes in hepatocytes, central vein dilation, enlarged, discolored, congestion, oedema, and
narrowing sinusoidal with loose cytoplasm) in 2-year study of F344 rats (Lington et al.. 1997). EPA
further considers liver toxicity for dose-response assessment in Section 4.
EPA summarizes the liver cancer associated with exposure to DINP in a separate technical support
document, the Cancer Raman Health Hazard Assessment for Diisononyl Phthalate (DINP) (U.S. EPA.
2025a).
3.3 Kidney Toxicity
Kidney effects generally occur at higher doses than liver effects and occur inconsistently across study
designs and species (EFSA. 2019; EC/HC. 2015; ECHA. 2013b; NICNAS. 2012; U.S. CPSC. 2010;
EFSA. 2005; ECB. 2003; NTP-CERHR. 2003).
Humans
Although the systematic review process used by Radke et al. identified five epidemiological studies that
investigated the association between DINP and renal effects, the evidence was deemed inadequate.
Three of the five studies had critical deficiencies in exposure measurement, and the other two studies
were of low confidence and had evidence of selection bias and reverse causality (Radke et al.. 2019a).
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EPA did not identify any new epidemiologic studies that examine the association between DINP and its
metabolites and/or biomarkers of kidney injury.
Laboratory Animals
Many experimental animal studies have evaluated the kidney toxicity of DINP following oral exposure.
Studies have evaluated the effects on kidney function (i.e., urinalysis parameters, serum BUN levels),
kidney weight, and histopathology. Seventeen studies are available that provide data on
histopathological effects of the kidney, 16 of which also provide data on absolute and/or relative kidney
weights. Six studies report changes in indices of kidney function such as serum BUN levels or urinalysis
parameters. One study was available for the dermal exposure route (Hazleton Laboratories. 1969). No
studies were available for the inhalation exposure route.
Intermediate (>l to 30 Days) Exposure Studies: EPA identified five intermediate studies in rodent
models that provide data on the effects of DINP on the kidney (Ma et al.. 2014; Kwack et al.. 2010;
Kwack et al.. 2009; BIBRA. 1986; Bio/dynamics. 1982a). A study by Bio/dynamics (1982a) exposed
male Fischer 344 (F344) rats to 0 or 2 percent (equivalent to 1,700 mg/kg-day) DINP for one week via
feed and evaluated kidney weights and histopathology at study termination. Significant increases in
absolute (7.5% increase) and relative (12.2% increase) kidney weights were observed in rats exposed to
DINP. No abnormal histopathological findings were observed in the kidneys. Another study in F344 rats
reported similar findings (BIBRA. 1986). BIBRA (1986) administered 0, 0.6, 1.2, 2.5 percent DINP for
21 days (equivalent to 0, 639, 1,192, 2,195 mg/kg-day [males]; 0, 607, 1,198, 2,289 mg/kg-day
[females]) in the diet to male and female rats and evaluated kidney weights and histopathology at study
termination. Dose-related increases in relative kidney weights were observed in males and females at all
dose levels; the LOAEL was 639 and 607 mg/kg-day for males and females, respectively. No exposure-
related histopathological findings were observed in the kidneys.
Not all intermediate studies reported dose-related changes in kidney weights that coincide with other
effects of the kidney. Two studies in male SD rats reported no change in relative kidney weights and/or
no change in BUN or other urinalysis parameters (Kwack et al.. 2010; Kwack et al.. 2009). while
another in B6C3F1 mice reported changes in weights without a consistent dose-related trend (Hazleton
Labs. 1991a). The studies by Kwack et al. exposed male SD rats to 0 or 500 mg/kg-day DINP via
gavage for 14 days (Kwack et al.. 2010; Kwack et al.. 2009). while the Hazleton study (1991a) exposed
mice to 0, 3,000, 6,000, or 12,500 ppm DINP for 4 weeks (equivalent to 0, 635, 1,377, 2,689, or 6,518
mg/kg-day [males]; 0, 780, 1,761, 3,287, or 6,920 mg/kg-day [females]). In the Hazleton study (1991a).
significant increases in relative kidney weight were observed at the highest dose in males (6,518 mg/kg-
day) and females (6,920 mg/kg-day), but significant decreases were observed at lower dose-levels in
both sexes, which was also true for absolute kidney weights. Nevertheless, the increased relative kidney
weights coincided with significant increased serum BUN levels in high-dose males and increased
incidences of tubular nephrosis in all high-dose males and females, supporting an exposure-related effect
on the kidney (Hazleton Labs. 1991a). In a study in which male Kunming mice were exposed to 0.2, 2,
20 or 200 mg/kg-day DINP for 14 days via gavage, Ma et al. (2014) reported significantly increased
incidences in histopathologic lesions of the kidney, including large reduction in tubular space and
extreme edema of epithelial cells in the glomeruli in animals exposed to the highest dose of DINP.
However, this publication only described these findings qualitatively in text and did not include
quantitative data on incidence or severity.
New Literature: EPA identified three new studies published from 2015 through 2024 that provided data
on toxicological effects of the kidney following intermediate exposure to DINP (Gu et al.. 2021; Liang
and Yan. 2020; Neier et al.. 2018). The developmental exposure study by Neier et al. (2018) reported no
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change in absolute or relative kidney weights at PND21 in male and female yellow agouti (Avy) mice
offspring. In that study, dams were administered 0 or 75 ppm DINP in the diet (equivalent to 15 mg/kg-
day) beginning 2-weeks before mating and continuing through PND21.
In a dermal exposure study, Liang and Yan (2020) applied 0, 0.02, 0.2, 2, 20, or 200 mg/kg-day DINP to
the shaved skin on the backs of male Balb/c mice (6/group) for 28 days and evaluated kidney weights,
kidney histopathology, and markers of oxidative stress in kidney tissue at the end of the study (i.e., ROS
via DCF-DA assay, GSH content, MDA content, and DNA-protein crosslinks (DPCs)). Increased
relative kidney weight was observed only in the high dose group (i.e., 200 mg/kg-day), and there was no
change in body weight or absolute kidney weight. Histopathological lesions were reported in the
kidneys, including a "large reduction of tubular space and extreme edema of epithelial cells in the
glomeruli." Although representative images of the histopathological effects were provided, no
quantitative data was provided, which impacts the interpretation of these results. Gu et al. (2021)
evaluated the effects of lower doses of DINP on the kidney. Male ICR mice (8/group) were exposed to
0, 0.05, or 4.8 mg/kg-day DINP daily for 5 weeks and then the lipidomic profile of kidney tissues
collected at the end of the study, and kidney weights and markers of oxidative stress were measured. No
significant changes were observed on kidney weights, nor body weights of the mice exposed to DINP.
Subchronic (>30 to 90 days) Exposure Studies: EPA identified six dietary studies from existing
assessments that provide data on the toxicological effects of DINP on the kidneys following subchronic
oral exposure (Hazleton Labs. 1991b; Bio/dynamics. 1982b. c; Hazleton Labs. 1981; Hazleton
Laboratories. 1971; Hazleton Labs. 1971) and one gavage study in marmoset monkeys (Hall et al..
1999). These studies provided data across a range of species and strains as well as both sexes. Increases
in absolute and/or relative kidney weights and histopathological effects were reported in all of the
studies, (Hazleton Labs. 1991b; Bio/dynamics. 1982b. c; Hazleton Labs. 1981; Hazleton Laboratories.
1971; Hazleton Labs. 1971). albeit the effects were sometimes attributable to decreased body weight.
Dose-related increases in absolute and/or relative kidney weights sometimes corresponded with
increased incidences of histopathological lesions or altered urine chemistry, but these trends were not
consistent across all studies.
A study by Bio/dynamics labs (1982b) exposed F344 rats to 0, 0.1, 0.3, 0.6, 1.0, or 2.0 percent DINP for
13 weeks via feed (equivalent to 0, 77, 227, 460, 767, 1,554 mg/kg-day). Dose-dependent increases in
kidney weight were noted in males, where doses as low as 227 mg/kg-day DINP resulted in increased
absolute (9.7%) and relative (21.9%) weights. The increase in kidney weight was accompanied by a
dose-dependent increase in dark brown discoloration in the kidney from 460 mg/kg-day. A similar study
from Bio/dynamics labs (1982c) exposed Sprague Dawley rats to 0.3 or 1.0 percent DINP in the diet for
13 weeks (equivalent to 201 or 690 mg/kg-day [males]; 251 or 880 mg/kg-day [females]). The authors
reported dose-related increases in absolute and relative kidney weights in males and females that
corresponded with altered clinical chemistry parameters in males, most notably a dose-dependent
decrease in triglycerides and increased calcium in high-dose males. The LOEL was 201 or 251 mg/kg-
day for males or females, respectively.
These results were similar to three studies from Hazleton Labs (1991b. 1981. 1971). each using a
different strain of rats. Hazleton Laboratories (1971) reported increases in absolute (9.3-17.6%
increases) and relative (14.4-25.5% increases) kidney weight in male and female albino rats of the
highest dose group (500 mg/kg-day). In that study, animals were exposed to 0, 50, 150, 500 mg/kg-day
DINP for 13 weeks. Hazleton Labs (1991b) administered 0, 2,500, 5,000, 10,000, or 20,000 ppm DINP
via diet to CDF (F344)/CrlBr rats for 13 weeks (equivalent to 176, 354, 719, or 1,545 mg/kg-day
[males]; 218, 438, 823, or 1,687 mg/kg-day [females]). Dose-dependent increases in absolute and
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relative kidney weights were observed in both sexes, which coincided with a dose-related increase in
granular casts and regenerative /basophilic tubules in the kidneys, beginning at 354 mg/kg-day in males.
Hazleton Laboratories (1981) administered 0, 1,000, 3,000, or 10,000 ppm DINP to SD rats via feed for
13 weeks (equivalent to 0, 60, 180, and 600 mg/kg-day). A LOAEL of 60 mg/kg-day was identified
based on an increased incidence of kidney lesions (focal mononuclear cell infiltration and
mineralization) in exposed males. Absolute and relative kidney weights were also increased in males
and females exposed to 600 mg/kg-day. Absolute weights increased 20 percent in males and 10.8
percent in females, while relative weight increased 17.7 percent in males and 13.7 percent in females.
Although there is ample evidence that the kidney is a target organ for DINP in rodents, the evidence is
less consistent and less numerous across other species, including dogs, monkeys, and rabbits. Increased
kidney weights were observed in high-dose animals in a study of beagle dogs by Hazleton Laboratories
(1971). but were attributed to deceased body weight. In that study, animals were administered 0.125,
0.5, or 2 percent DINP in feed for 13 weeks (equivalent to 37, 160, or 2,000 mg/kg-day). The study also
reported increased incidences of tubular epithelial cell hypertrophy in high-dose (2,000 mg/kg-day)
males and females. Urinalysis parameters were comparable between control and test groups. In contrast,
a study in marmoset monkeys by Hall et al. (1999) did not observe any kidney effects. In that study,
male and female marmoset monkeys were exposed to 0, 100, 500, or 2,500 mg/kg-day DINP via gavage
for 13 weeks. No histological findings were exposure related, and there were no changes in kidney
weights. Similarly, no effects on the kidney were observed in a dermal study of New Zealand White
rabbits exposed to up to 2,500 mg/kg-day DINP for 6 weeks (Hazleton Laboratories. 1969).
New Literature: EPA identified one new study published from 2015 through 2024 that provided data on
toxicological effects of the kidney following subchronic exposure to DINP (Deng et al.. 2019). Deng et
al. (2019) exposed male C57BL/6 mice to 0, 0.15, 1.5 or 15 mg/kg-day DINP for 6 weeks via gavage.
The authors reported vacuoles and hyaline degeneration in the glomerulus of the kidney, as well as
smaller glomeruli and a thickened glomerular basement membrane. However, the authors do not specify
at which doses the effects were observed and only single images are provided.
Chronic (>90 days) Exposure: EPA identified five rodent studies from existing assessments that provide
information on the toxicological effects of DINP on the kidney, including four studies following chronic
oral exposure to DINP (CASRN 68515-48-0) (Covance Labs. 1998b. c; Lington et al.. 1997). or DINP
(CASRN 71549-78-5)(Bio/dvnamics. 1987). and one study following a one- or two-generation exposure
in SD rats (Waterman et al.. 2000). These studies provide data on absolute and/or relative kidney
weights, histopathology, and urinalysis measures that reflect kidney function (i.e., BUN levels).
Lington et al. (1997) and Covance Labs (1998c) evaluated kidney weights, urinalysis parameters, and
kidney histopathology in F344 rats following exposure to DINP for 2 years. Both studies observed
increases in kidney weights in the mid- and high-dose animals but reported inconsistent results for
urinalysis parameters and histopathology. Significant increases were observed in relative and absolute
kidney weights in males and females of the mid- and high-dose groups (i.e., 152 and 307 mg/kg-day
[males] or 184 and 375 mg/kg-day [females] at most time points (i.e., 6, 12, 18, and 24 months).
Moreover, relative kidney weight at study termination was increased 10 to 20 percent and 7 to 10
percent in males and females, respectively. In the 2-year study by Covance Labs (1998c). increased
relative kidney weights were observed in rats receiving dietary doses greater than 359 mg/kg-day for
males (>25% increase) and 442 mg/kg-day for females (>14% increase) at study termination. Kidney
weights in the recovery groups were comparable to the same-sex control values at the end of the 26-
week recovery period.
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In Lington et al. (1997). there were no exposure-related changes in serum chemistry parameters such as
blood urea nitrogen (BUN). Some of the urine chemistry parameters were affected by DINP exposure in
males. Increased urine volume, potassium, and glucose were observed in high-dose (307 mg/kg-day)
males at most time intervals; potassium and glucose levels were also increased in mid dose males.
Excretion of renal epithelial cells was increased in high-dose males at 6 months, but not at other
timepoints. No urinalysis changes were observed in females. In contrast, Covance Labs (1998c) reported
increases in serum urea (BUN) levels in males and females from the two highest dose groups at multiple
timepoints during the study including study termination (i.e., weeks 26, 52, 78, and 104). BUN was
increased up to 32 percent over controls in the mid-dose (359 mg/kg-day [male] or 442 mg/kg-day
[female]), and 50 percent over controls at the high dose (733 mg/kg-day [male] or 885 mg/kg-day
[female]).
In Covance Labs (1998c). exposure-related increases in the severity of tubule cell pigment occurred in
the kidneys of males exposed to 733 mg/kg-day DINP (Table 3-9). At study termination, a dose-related
increase was observed in the incidence and severity of mineralization of the renal papilla in males at 359
and 733 mg/kg-day DINP as well as in the recovery group. Increased severity of tubule cell pigment was
observed at the two highest dose groups in both sexes (Table 3-9).
Table 3-9. Incidence and Severity of Selected Non-neoplastic Lesions in the Kidneys of Male and
Female F344 Rats Fed DINP for 2 Years (Covance Labs, 1998c)
Dose Group
mg/kg-day (ppm)
Control
29 M / 36 F
(500)
88 M /109 F
(1,500)
359 M / 442 F
(6,000)
733 M/ 885 F
(12,000)
Recovery"
637 M / 774 F
(12,000)
Number M/F
examined6
36/37
35/38
39/40
31/33
27/32
29/34
Mineralization of renal papilla (males)
Minimal
6
11
9
6
2
0
Slight
0
0
0
24
1
2
Moderate
0
0
0
0
22
27
Total
6
11
9
30
25
29
Tubule cell pigment (males)
Minimal
24
21
18
0
0
0
Slight
10
12
21
23
7
26
Moderate
0
1
0
6
17
3
Moderately severe
0
1
0
2
3
0
Total
34
35
39
31
27
29
Tubule cell pigment (Females)
Minimal
22
27
34
4
0
1
Slight
14
10
5
27
21
33
Moderate
0
1
1
1
10
0
Moderately severe
0
0
0
1
1
0
Total
36
38
40
33
32
34
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Source: Table 10D on page 350 of Covance Labs (1998c)
M = Male; F = female
" The 12,000 ppm recovery group received 12,000 ppm DINP in the diet for 78 weeks, followed by a 26-week recovery
period during which the test animals received basal diet alone.
h Number examined at terminal sacrifice; does not include unscheduled deaths.
Bio/dynamics (1987) also conducted a 2-year chronic dietary study in rats, albeit of a different strain
(SD), and noted significant increases in absolute and relative kidney weights in high-dose males at both
the interim (19 and 25%, respectively) and terminal (13 and 12%) timepoints. Kidney weights of mid-
dose group males (271 mg/kg-day) were increased by 11 percent, although this was not a statistically
significant change. In high-dose females (672 mg/kg-day), increased relative kidney weights were
observed (20% increase) at interim sacrifice as well as terminal sacrifice (14% increase). Increased
incidence of medullary mineral deposits in the kidney were observed in high-dose males (25/70 treated
vs. 3/70 controls). However, in females, incidences of renal medullary mineral deposits at the high dose
(15/70) were comparable to controls (14/70). No histopathological evaluation was conducted on samples
from the low- or mid-dose groups, which limits the assessment of dose-dependency and effect levels.
Waterman et al. (2000) assessed the potential kidney toxicity of DINP in one- and two-generation
studies conducted in SD rats. In the one-generation study, absolute and relative kidney weights in both
sexes were significantly increased at all doses, except in high-dose PI females, and generally in a dose-
related fashion. In the two-generation study, absolute kidney weights of PI males and females were
increased over controls at all DINP treatment levels. Although decreased mean body weights and body
weight gains were also observed in PI males and females for all doses, the changes in kidney weight are
not solely attributable to changes in body weight. Increased incidence of minimal to moderate renal
pelvis dilation was observed in F2 males of the two highest dose groups (0.4 and 0.8%, equivalent to
741-796, 1,087-1,186 mg/kg-day). No changes were observed in the females; therefore, the authors
attributed the increased incidence of kidney lesions to induction of male rat-specific alpha 2u-globulin
(a2u-globulin).
In contrast to the studies in rats which consistently reported increases in relative and/or absolute kidney
weight, a study in male B6C3F1 mice reported decreased kidney weights (Covance Labs. 1998b). In that
study, male and female mice were exposed to 0, 1,500, 4,000, or 8,000 ppm DINP for 2 years via feed
(equivalent to 0, 276, 742, or 1,560 mg/kg-day). No effects were observed in females. In addition to the
weight changes in males, the authors reported significant increases in urine output, decreases in mean
urine osmolality; and decreased sodium, potassium, and chloride levels in male and female mice from
the 1,560 mg/kg-day dose group at 26, 52, 78, and 104 weeks. The study authors concluded that there
was no DINP-related change in glomerular filtration rate; however, they suggested that this pattern of
urinalysis findings may indicate a compromised ability to concentrate urine in the renal tubule
epithelium, as an increased incidence of chronic progressive nephropathy was observed in high-dose
females (1,888 mg/kg-day). The kidneys of 1,888 mg/kg-day females also had a granular pitted/rough
appearance. The effects of DINP on the kidney, including decreased kidney weights in males, were
partially attenuated in the recovery groups, which were evaluated 26 weeks after the end of exposure.
The reversibility of the kidney effects in the recovery groups was not as pronounced as that for liver
effects (Section 3.1). The incidences of chronic progressive nephropathy in female mice were
comparable to those of the control group upon termination, suggesting that nephropathy is reversible or
that exacerbation of this lesion halted when exposure to DINP was discontinued.
New Literature: EPA did not identify any new studies published from 2015 through 2024 that provided
data on toxicological effects of the kidney following chronic exposure to DINP.
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Mechanistic Information
EPA identified four in vivo studies that provide data that may inform mechanisms of action of the
observed nephrotoxic effects of DINP. Mechanisms evaluated include oxidative stress in mice and male
rat-specific a2u-globulin.
Ma et al. (2014) evaluated the contribution of oxidative stress to the aforementioned tissue lesions
observed in the kidneys of male Kunming mice, which were primarily observed at 200 mg/kg-day. In
that study, mice were exposed to 0.2, 2, 20, or 200 mg/kg-day DINP for 14 days via gavage, and
endpoints relevant to oxidative stress were evaluated in renal and hepatic tissue homogenates. Increases
in reactive oxygen species (ROS) and MDA, in parallel with decreases in glutathione (GSH) content,
were observed at 200 mg/kg-day DINP, indicative of oxidative stress. Some indices of oxidative stress
were observed at lower doses than those that resulted in kidney lesions. Indeed, the authors also reported
DNA-protein-crosslinks at 200 mg/kg-day and increases in 8-hydroxydeoxyguanosine (8-OH-dG) at 20
and 200 mg/kg-day, which indicate oxidative damage to DNA. Levels of interleukin (IL)-l and tumor
necrosis factor alpha (TNFa) were also increased at 20 and 200 mg/kg-day, which would be consistent
with enhancement of an inflammatory response. The authors also evaluated the effect of combined
exposure of 200 mg/kg-day DINP and melatonin (50 mg/kg-day). Mice exposed to 200 mg/kg-day
DINP plus 50 mg/kg-day melatonin showed glomerular cell proliferation and milt renal tubule epithelial
cell edema, and attenuated indices of oxidative stress (ROS, GSH, MDA, DNA-protein-crosslinks, and
cytokine levels). These data indicate that melatonin can attenuate the oxidative stress that results from
exposure to DINP in mice, but not fully attenuate damage to renal tissue, and support an MOA where
oxidative stress may contribute to the toxicological effects of DINP on the kidney.
Both Liang and Yan (2020) and Gu et al. (2021) also evaluated the effect of DINP on oxidative stress.
Liang and Yan (2020) applied 0, 0.02, 0.2, 2, 20, or 200 mg/kg-day DINP to the shaved skin on the
backs of male Balb/c mice (6/group) for 28 days. In kidney homogenates, levels of ROS, MDA, and
DPCs were significantly increased at 20 and 200 mg/kg-day, with concomitant decreases in GSH at the
same doses. These data are further described in Appendix B. Gu et al. (2021) evaluated the effect of
lower doses of DINP (i.e., 0.05 and 4.8 mg/kg-day) in kidney tissues of male ICR mice exposed daily to
DINP for 5 weeks. No changes in markers of oxidative stress were observed in the kidney, aside from a
significant increase in GSH content in the 4.8 mg/kg-day dose group. However, a limitation of the data
set is that it is difficult to interpret increases in GSH content without understanding changes in the ratio
of reduced to oxidized glutathione. Collectively, studies by Ma et al. (2014) and Liang and Yan (2020)
provide some evidence that DINP can induce ROS in the kidneys of male mice; however, ROS has not
been evaluated in the kidneys of female mice or other species such as the rat.
Caldwell et al. (1999) followed up on observations from Lington et al. (1997). that kidney tumors were
observed in male rats, but not female rats. The male-specific nature of the findings led them to evaluate
a mechanism of action involving the male rat-specific a2u-globulin. Tissue sections from male and
female F344 rats at the 12-month interim sacrifice were evaluated. In male rats, a dose-dependent
increase in a2u-globulin accumulation was observed in regions of the kidney where increased cell
proliferation was also observed. In parallel, tubular epithelial hypertrophy and tubular regeneration were
observed. a2u-globulin was not detected in the kidneys of female rats, and renal cell proliferation of
DINP-exposed female rats was comparable to controls. These results are consistent a mechanism where
a2u-globulin accumulation leads to kidney tissue damage, cell proliferation, and subsequent neoplastic
lesions of the kidney in male rats. The two-generation study by Waterman et al. (2000) also attributed
their observations of renal pelvis dilation in the kidney of F2 male rats to induction of a2u-globulin.
However, these effects are not regarded as relevant to humans (Swenberg and Lehman-Mckeeman.
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1999; U.S. EPA. 1991a). Kidney tumors and evidence for an a2u-globulin MOA are further discussed in
EPA's Cancer Raman Health Hazard Assessment for Diisononyl Phthalate (DINP) (U.S. EPA. 2025a).
Conclusions on Kidney Toxicity
Twenty studies in experimental animal models have evaluated toxicologic effects of DINP on the kidney
following intermediate, subchronic, developmental, or chronic exposure to DINP. Findings were similar
across study designs, including increased absolute and/or relative kidney weights, and observed in both
sexes, but these data predominantly reflect rat studies, and the toxicological effects of DINP on the
kidney is less certain in other species.
Increases in absolute and/or relative kidney weight have been observed primarily in rat studies across
multiple study designs and often coincide with increased incidences of non-neoplastic lesions of the
kidney or altered urinalysis parameters. Indeed, increased kidney weights were reported in two
intermediate studies in F344 rats (BIBRA. 1986; Bio/dynamics. 1982a). five subchronic studies in
various strains of rats (Hazleton Labs. 1991b; Bio/dynamics. 1982b. c; Hazleton Labs. 1981. 1971).
three chronic studies in rats (Covance Labs. 1998c; Lington et al.. 1997; Bio/dynamics. 1987) and one
developmental study in rats (Waterman et al.. 2000).
In the 2-year study conducted by Lington et al. (1997). increased relative kidney weights of male and
female rats were observed following exposure to dietary levels of 152 and 307 mg/kg-day (males) or
184 and 375 mg/kg-day (females). In the 2-year study reported by Covance Labs (1998c). increased
relative kidney weights occurred in rats receiving dietary doses greater than 359 mg/kg-day for males
and 442 mg/kg-day for females. Urinalysis findings from the chronic studies included significant
increases in urine output and corresponding decreases in electrolyte levels in high-dose males,
suggesting compromised ability to concentrate urine in the renal tubule epithelium. These effects
occurred at the same dosages that produced changes in kidney weights. In the Covance Labs (1998c)
study, serum urea levels (a marker of kidney toxicity) were significantly increased in rats exposed to 359
mg/kg-day and higher during the second half of the study. Increases in urine volume and kidney lesions
were observed in the recovery group exposed to 733 mg/kg-day.
In many of the chronic studies, effects on the kidney generally occurred at doses equivalent to those
where effects on the liver were observed in rats (Covance Labs. 1998c; Lington et al.. 1997) and mice
(Covance Labs. 1998b). Moreover, the LOAELs ranged from 152 to 923 mg/kg-day which reflect
effects on both the liver and kidneys, including increases in absolute and relative kidney weight as well
as histopathologic findings in the kidney in two chronic studies of male rats (Covance Labs. 1998c;
Lington et al.. 1997). The NOAEL in the Lington et al. study was 15 mg/kg-day (males) or 18 mg/kg-
day (females). However, in a third chronic exposure study in rats (Bio/dynamics. 1987). effects on the
kidney were observed, but not at the LOAEL, suggesting that the kidney may be less sensitive than the
liver to the effects of DINP.
The findings of increased kidney weight in rats were inconsistent with one study of mice, which
reported decreased absolute kidney weight in males (LOAEL = 276 mg/kg-day; NOAEL = 90 mg/kg-
day in males) (Covance Labs. 1998b). That study also reported chronic progressive nephropathy in
female mice of the high-dose group (1,888 mg/kg-day) but no effects in males (Covance Labs. 1998b).
The lack of coherence of effects (e.g., organ weight, histopathology data do not coincide in males or
females) is a limitation of this study.
The MOA of kidney toxicity is not currently known, and effects on the kidney are primarily observed in
one species (rats). Furthermore, kidney effects observed in the rat are less sensitive than effects on the
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liver and on developmental outcomes. EPA is considering kidney toxicity for dose-response analysis in
Section 4.
3.4 Neurotoxicity
Humans
Health Canada (2018a) evaluated multiple studies that investigated the association between DINP
exposure and several behavioral and neurodevelopmental outcomes, including mental and psychomotor
neurodevelopment, behavioral and cognitive functioning (i.e., autism spectrum disorders, learning
disabilities, attention-deficit disorder, and attention-deficit hyperactivity disorder), neurological
function, and gender-related play behaviors. Across available studies of DINP, Health Canada
determined that the level of evidence for association between DINP and its metabolites and neurological
effects could not be established.
Radke et al. (2020a) evaluated the association between DINP and neurodevelopment and found that
there was no clear association between DINP and neurodevelopment. Three research studies examined
the relationship between DINP and cognition; however, two of the studies found no relationship and one
revealed an inverse relationship. As a result, the evidence supporting the relationship between DINP and
cognition is deemed inconclusive. Because of the limited number of studies examining this relationship,
the evidence linking DINP to motor ability is regarded as weak. The data supporting the link between
boys' behavior and DINP found no increased odds of ADHD with DINP exposure, and the authors
considered the evidence preliminary. Because of the inconsistent reports about the relationship between
DINP and newborn neurobehavior, the evidence was considered indeterminate. The inconsistent nature
of the currently available research renders the evidence for a connection between DINP and
autism/social impairment as unclear.
New Literature: EPA identified eleven new studies (2 high quality and 9 medium quality) that evaluated
the association between urinary DINP and neurological effects. The first high-quality study, by Shin et
al. (2018). examined a subset of the of mother-child pairs from Markers of Autism Risk in Babies
Learning Early Signs (MARBLES) cohort to evaluate the association between exposure to DINP
metabolite (MCOP) and Autism spectrum disorder (ASD) and non-typical development (Non-TD).
Among mothers who did not take prenatal vitamins, prenatal MCOP exposure during mid to late
pregnancy was associated with higher risk of non-TD (vs. typical development) (MCOP RRR = 1.86
[95% CI: 1.01, 3.39]). Among mothers who did take prenatal vitamins, prenatal MCOP exposure during
mid-to-late pregnancy was associated with lower risk of autism spectrum disorder (vs. typical
development) (MCOP RRR = 0.49 [95% CI: 0.27, 0.88]). There was an association in multinominal
logistic regression of MCOP during 2nd trimester and ASD (vs. TD) among mothers who took prenatal
vitamins (RRR = 0.41 [95% CI: 0.21, 0.79]).
Another high-quality, cross-sectional study, by Jankowska et al. (2019b). conducted from a subset of the
Polish Mother and Child Cohort (REPRO PL), examined the association between Child behavioral and
emotional problems at age 7 years as well as child cognitive and psychomotor development and DINP
exposure. Negative associations in peer relationship problems were noted for sum DINP metabolites,
and lower Intelligence and Development Scales (IDS) scores were generally positively associated with
higher phthalate concentrations.
The first medium quality prospective analysis, by Balalian et al. (2019). of maternal prenatal and child
age 3, 5, and 7 postnatal DINP metabolite (MCOP) exposures with motor skills at age 11 year as
assessed by the short form of the BOT-2 were selected from participants in an ongoing longitudinal birth
cohort study of mothers and newborns conducted by the Columbia Center for Children's Environmental
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Health (CCCEH). MCOP measured at age 3 was inversely associated with BOT-2 total, fine motor, and
gross motor composite scores among boys. In linear regression models, a 1 log-unit increase in age 3
MCOP was associated with lower total (beta: -3.08 [95% CI: -5.35, -0.80]), fine motor (beta: -1.64
[95% CI: -3.16, -0.12]), and gross motor (beta: -1.44 [95% CI: -2.60, -0.28]) composite scores in
boys. Comparisons of the 4th vs. 1st quartiles of age 3 years MCOP was also associated with all three
outcomes in boys (Q4 vs. Ql) total composite score (beta: -7.47 [95% CI: -12.60, -2.34]); fine motor
composite score (beta: -4.18 [95% CI: -7.51, -0.85]); gross motor composite score (beta: -3.29 [95%
CI -6.06, -0.52]). No significant associations were found between MCOP at age 3 and outcomes in
girls. There were no significant association for sex differences at age 3. There were also no significant
associations between prenatal MCOP and outcomes in either girls or boys. There were no significant
associations between MCOP measured at ages 5 or 7 and outcomes in either girls or boys.
A medium quality study, by Li et al. (2019). used data from children in the Cincinnati Health Outcomes
and Measures of the Environment (HOME) cohort to analyze associations between DINP metabolites
(MCOP, MCNP) and child cognition measured at ages 5 and 8 years. The pattern of associations for
MCOP and MCNP measures was heterogeneous (p < 0.20 for MCNP), and no adjusted associations
reached significance. Associations between child IQ scores and urinary MCOP measured at different
ages were not statistically significant and were heterogeneous (positive and negative). For exposure at
age 3 years, when associations with several other phthalate metabolites were significantly inverse,
adjusted beta for MCOP was -1.2 (95% CI: -3.2, 0.9)
Another medium quality cohort study, by Tanner et al. (2020). examined mother-child pairs from the
Swedish Environmental Longitudinal Mother and Child, Asthma and Allergy (SELMA) study and the
association between prenatal urinary DINP metabolite (MHiDP, MCNP, MHiNP, MOiNP, MCiOP)
exposure and child IQ at age 7 years. Because this is a mixtures analysis, the DINP metabolites of
interest were not directly analyzed as they were only above the threshold of concern in sensitivity
analyses using positive weights.
A medium quality prospective cohort study, by Jankowska et al. (2019a). evaluated the association
between prenatal and postnatal (age 2 years) OH-MINP and child behavior, cognition, and psychomotor
development at age 7 years. The study included a subset of mother-child pairs from the Polish Mother
and Child Cohort. There were no statistically significant associations between prenatal or postnatal OH-
MINP and any of the study outcomes. There was also no clear pattern of associations with behavioral
outcomes, and associations with cognitive and psychomotor scores were generally weakly negative.
oxo-MINP was measured, but associations with outcomes were not analyzed, as detection rates were
less than 70 percent (56 and 65% for pre- and postnatal measures, respectively).
A medium quality cohort study by Hyland et al. (2019) analyzed associations between prenatal DINP
metabolites and neurodevelopment in live singletons in Center for the Health Assessment of Mothers
and Children of Salinas (CHAMACOS), a birth cohort of low-income Mexican American children in
Salinas, California. Associations between IQ scores and MCOP were shown only for combined sexes,
and not significant.
A medium quality longitudinal cohort study, by Jacobson et al. (2021). used data from the NYU
Children's Health and Environment Study, to evaluate urinary DINP metabolites (MCiOP, MINP) levels
in pregnant women and assessed the association with postnatal and postpartum depression following
delivery. There were no significant associations for the Edinburgh Postnatal Depression Scale (EPDS)
score or postpartum depression for sum DINP phthalates.
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A medium quality study by Dzwilewski et al. (2021) used data from a subset of participants in the
Illinois Kids Development Study (IKIDS) to evaluate associations between prenatal exposure to DINP
metabolites (MINP, MCOP, MONP), and infant cognition assessed at 7 to 8 months of age. The authors
presented results of analyses using the sum of 2 (DINP2) or 3 (DINP3) metabolites, and MONP
individually. Associations varied by infant sex and by the set of images used in testing. DINP2 was
associated with longer processing time for image set 2, and DINP3 with longer processing time among
males viewing image set 2. DINP2 and DINP3 had weak negative associations with visual recognition
memory (novelty preference). Urinary EDINP2 metabolites (MINP and MCOP) was associated with
significant increases in average information processing speed (run duration) among infants viewing
image set 2. DINP2 was also associated with a non-significant decrease in visual recognition memory
(novelty preference). Urinary EDINP3 metabolites (MINP, MCOP, and MONP) were associated with
significant increases in average information processing speed (run duration) among male infants viewing
image set 2. DINP3 was also associated with a non-significant decrease in visual recognition memory
(novelty preference) overall, while MONP was associated with a non-significant increase in novelty
preference among infants viewing image set 2. DINP3 was associated with a non-significant decrease in
visual attention (time to familiarization) for image set 2.
A medium quality case-cohort study by Kamai et al. (2021) nested in the Norwegian Mother and Child
Cohort (MoBa) analyzed the association between prenatal DINP measured in spot urines at about 17
weeks' gestation and ADHD at age 3 years. DINP was non-linearly associated with increased odds of
preschool ADHD. Results of multivariate logistic regression found an association between increasing
DINP quintile 2 vs. quintile 1, OR = 2.04 (95% CI: 1.2 to 3.33; includes adjustment for DEHP).
The final medium quality study, a population-based nested case-control study by Engel et al. (2018).
assessed the association of DINP metabolites and ADHD in children of at least 5 years of age of mothers
within the Norwegian Mother and Child Cohort (MoBa). The authors reported no association of ADHD
with sumDINP metabolites. In Bayesian logistic regression models, there was no association (OR= 0.85,
[95% CI: 0.61, 1.15]) with log sum of DINP and ADHD. Associations with individual DINP metabolites
were also not significant.
Laboratory Animals
A limited number of experimental animal studies have evaluated the neurotoxicity of DINP following
oral exposure. Existing assessments of DINP have not drawn human health hazard conclusions on the
neurotoxicity of DINP, but have evaluated effects on behavior, brain weight, and/or brain histopathology
(U.S. EPA. 2023c: ECCC/HC. 2020: U.S. CPSC. 2014: ECHA. 2013b: NICNAS. 2012: ECB. 2003).
Only three rodent studies (Boberg et al.. 2016: Ma et al.. 2015; Peng. 2015; Boberg et al.. 2011) are
available that are specifically designed to evaluate neurotoxicity. Remaining studies evaluated brain
weight and/or brain histopathology. These included three subchronic exposure duration studies and three
chronic studies, as well as six developmental exposure studies (i.e., one- or two-generation studies of
reproduction, perinatal, postnatal, or peri-and-postnatal exposure studies). No studies were available for
the dermal or inhalation exposure routes.
One developmental study in Wistar rats (Boberg et al.. 2011) reviewed in existing assessments (U.S.
CPSC. 2014; NICNAS. 2012) provides data on behavior, including an evaluation of learning and
memory following DINP exposure. Boberg et al. (2011) exposed pregnant Wistar rats to 300, 600, 750,
or 900 mg/kg-day DINP via oral gavage daily from GD7 through PND17 and evaluated several
neurobehavioral endpoints on male and female offspring at later timepoints. Behavioral examinations
included those of motor activity levels at PND27 through 28, Morris Water Maze (MWM) at 2 to 3
months of age, sweet preference at 4 months, and radial arm maze performance at 5 to 7 months of age.
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The MWM test is used to evaluate learning and memory. In this test, animals are placed in a circular
pool of water and required to escape from water onto a hidden platform using spatial memory. No
changes were observed in motor activity levels and radial arm maze performances in male or female
offspring exposed to DINP during development. An increase in saccharin intake in the sweet preference
test was observed in female offspring of the 750 mg/kg-day group; however, this effect was not dose-
dependent, and the study authors concluded that it may be a chance finding. In the MWM test, dose-
dependent improvements in swim length and latency were observed on the first day of memory testing,
with significantly shorter swim length and latency in the 900 mg/kg-day females. The study authors
asserted that performance in the MWM test is sexually dimorphic, and concluded that DINP affected
spatial learning, as female offspring performed better than controls and similarly to control males in the
MWM, indicating masculinization of behavior in DINP exposed females. However, the effects were no
longer apparent on the second day of memory testing or when the platform was moved to a new position
in the maze. Performance was unaffected by exposure to DINP in males. Notably, the male reproductive
parameters were affected at a lower dose than the apparent effects on learning in memory in females,
with increased MNGs and decreased sperm motility at 600 mg/kg-day and above, increased nipple
retention at 750 mg/kg-day and above, and decreased AGD at 900 mg/kg-day.
Several rodent studies were identified in existing assessments that provide data on absolute and/or
relative brain weight following exposure to DINP. These include three chronic studies (Covance Labs.
1998a. c; Lington et al.. 1997) and two developmental studies (Masutomi et al.. 2003). In general,
changes in absolute and/or relative brain weight were not observed or were only observed at the highest
doses tested in both males and females. No changes in brain index (i.e., relative brain weight) were
observed in male Kunming mice exposed to 1.5, 15, or 150 mg/kg-day DINP for 9 days via gavage
(Peng. 2015). Similarly, no changes were observed in relative and/or absolute brain weight of: B6C3F1
mice exposed to up to 8,000 ppm DINP in feed for two years (equivalent to 1,600 mg/kg-day) (Covance
Labs. 1998b); F344 rats exposed for 2 years to up to 12,000 ppm (equivalent to 733 mg/kg-day in males;
885 mg/kg-day in females) (Covance Labs. 1998c); or up to 0.6 percent (equivalent to 307 mg/kg-day in
males; 375 mg/kg-day in females) (Lington et al.. 1997). In contrast, changes in brain weight were
observed in one perinatal exposure study (Masutomi et al.. 2003). In Masutomi et al. (2003). maternal
SD rats were fed test diets containing 0, 400, 4,000, or 20,000 ppm DINP from GDI5 through PND10
(equivalent to 31, 307, or 1,164 mg/kg-day during gestation and 66, 657, or 2,657 mg/kg-day during
lactation). Significant decreases in absolute brain weight were observed in male (12.9%) and female
(11.1%) rat pups from the highest dose group at PND27, while significant increases in relative brain
weight were observed in males (53.5%) and females (46%), which likely reflects decreased terminal
body weight at PND27 in the highest dose group in both males and females. Body weight gain of male
and female pups was decreased as well.
Data from existing assessments on the histopathological effects on the brain following DINP exposure
have been reported. Identified literature includes one intermediate exposure duration study (Midwest
Research Institute. 1981) and three chronic studies (Covance Labs. 1998b. c; Lington et al.. 1997). In
general, there were no exposure-related histopathological findings in the 28-day exposure study by the
Midwest Research Institute (1981) nor in the chronic exposure studies in mice (Covance Labs. 1998b)
and rats (Covance Labs. 1998c; Lington et al.. 1997).
New Literature: Four new studies were identified by EPA that had not been reviewed in existing
assessments (Neier et al.. 2018; Setti Ahmed et al.. 2018; Ma et al.. 2015; Peng. 2015). which provide
data on neurobehavioral outcomes, brain weights, and brain histopathology following exposure to DINP.
Results of Ma et al. (2015) and Peng et al. (2015) were not fully evaluated in the 2020 Health Canada
Screening Assessment (ECCC/HC. 2020). and are therefore considered new literature.
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Two intermediate exposure duration studies in male Kunming mice (Ma et al.. 2015; Peng. 2015) are
available that provide data on behavior. Impaired learning and memory following DINP exposure was
observed consistently across the two intermediate studies. Peng et al. (2015) and Ma et al. (2015) have
similar study designs. Peng et al. (2015) exposed mice to 1.5, 15, or 150 mg/kg-day DINP daily via oral
gavage for 9 days, while Ma et al. (2015) exposed mice to 0.2, 2, 20, or 200 mg/kg-day DINP daily via
oral gavage for 14 days. In both studies, the authors evaluated the effect of DINP on learning and
memory using the MWM test. In both studies, escape latency (i.e., time it took mice to locate submerged
escape platform) was evaluated throughout the exposure period ("training period"), and memory was
evaluated on the last day of exposure ("probe trial") following one day of no testing (a "forget" period).
Each study also investigated the combined effect of DINP and an antioxidant; these endpoints are
discussed in the mechanistic section. Mice were euthanized 24 h after the last DINP exposure, at which
point brain tissue was harvested for histological examination as well as various non-apical measures of
oxidative stress and inflammation (discussed in mechanistic information section below).
In both Ma et al. (2015) and Peng et al. (2015) escape latency in the MWM test was reduced in each
exposure group at the end of the training periods compared to the first day. Escape latency was increased
in all groups exposed to DINP compared to controls, indicating impaired learning in DINP groups. Peng
et al. (2015) reported decreased retention time in the target quadrant in the MWM test during the probe
trial, indicative of impaired memory. Similarly, Ma et al. (2015) reported decreased time and number of
entries into the target quadrant in the MWM test during the probe trial, indicative of impaired memory.
In addition to MWM, Ma et al. (2015) conducted an open field test to evaluate locomotor activity.
Decreased time and number of entries into the central area were observed for mice exposed to 200
mg/kg-day DINP, which the authors attributed to anxiety-like behavior.
Four new rodent studies were identified that provide data on absolute and/or relative brain weight
following exposure to DINP, three of which were oral exposure studies. These include one intermediate
exposure duration study (Peng. 2015). and two developmental studies (Neier et al.. 2018; Setti Ahmed et
al.. 2018). In general, changes in absolute and/or relative brain weight were not observed, with the
exception of one study weight in yellow agouti (Avy) mice, where biologically significant (i.e., >10%
change) changes in brain weight were observed at the highest doses tested in male mice, which may be
exposure-related. No changes in brain index (i.e., relative brain weight) were observed in male Kunming
mice exposed to 1.5, 15, or 150 mg/kg-day DINP for 9 days via gavage (Peng. 2015). Ahmed et al.
(2018) observed similar results. In that study, pregnant Wistar rats (36 dams/group) were exposed to 0 or
380 mg/kg-day DINP via oral gavage beginning on GD8 and continuing up to PND30. Interim sacrifices
were conducted on PND7, PND15, and PND21. Brain weight was determined at interim and terminal
timepoints. No changes were observed in absolute brain weights (relative brain weights not reported) at
PND7, PND15, or PND30. Body weight was significantly reduced in pups exposed to DINP at PND15
and PND30. In contrast to the findings of Ahmed et al. (2018). a developmental study by Neier et al.
(2018) reported changes in relative brain weight in yellow agouti (Avy) mice fed diets containing 5 ppm
(equivalent to 15 mg/kg-day) DINP from 2 weeks prior to mating until weaning. The authors reported
absolute and relative brain weights in PND21 offspring. Decreased relative brain weights were observed
in PND21 males only, and no changes in absolute weights were observed. Increased terminal body
weights were observed for females, but not males, at PND21, indicating that brain weight is decreased in
males even when adjusted for body weight. Although it is likely this observation is exposure-related,
uncertainty exists due to the use of the yellow agouti (Avy) mouse model in the Neier study.
New data on the histopathological effects on the brain following DINP exposure have been reported.
Identified literature includes two intermediate exposure duration studies (Ma et al.. 2015; Peng. 2015).
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which both reported histopathological alterations in the pyramidal cells of the CAi region of the
hippocampus following intermediate exposure to DINP via gavage. Ma et al. (2015) reported damaged
pyramidal neurons in the 20 and 200 mg/kg-day dose groups. Peng et al. (2015) reported that with
increasing DINP exposure, the arrangement of hippocampal cells became more disordered, cells
swelled, and apical dendrites shortened or disappeared. Limitations of the histopathological data set
from both studies include qualitative presentation of data that lacks incidence or severity information.
Mechanistic Information
EPA identified five in vivo studies and one in vitro study that provide data that may inform mechanisms
of the observed neurological effects of DINP. Three of the in vivo studies investigated mechanisms
involving oxidative stress in mouse models (Duan et al.. 2018; Ma et al.. 2015; Peng. 2015). The
aforementioned studies by Peng et al. (2015) and Ma et al. (2015) exposed male Kunming mice to DINP
via oral gavage daily for 9 or 14 days and evaluated several endpoints related to oxidative stress. Both
studies observed increases in ROS, decreases in superoxide dismutase activity, decreases in GSH
content, increases in inflammatory cytokines, and increases in caspase-3 levels, activity, or staining
intensity at the highest dose (200 mg/kg-day) (Ma et al.. 2015) or two highest doses (15 and 150 mg/kg-
day) (Peng. 2015). Ma et al. also reported increases in DNA-protein-crosslinks at 200 mg/kg-day and
increases in 8-OH-dG at 20 and 200 mg/kg-day, indicating oxidative damage to DNA. Although Ma et
al. did not quantify histopathological changes observed in the hippocampus (Section 3.4), they
quantified immunohistochemistry staining of glial fibrillary acidic protein, in addition to caspase-3 in
the hippocampus CAi region and cerebral cortex. Staining intensity of caspase-3 and glial fibrillary
acidic protein was increased at 200 mg/kg-day in both regions of the brain and increased in the cerebral
cortex at the 20 mg/mg-day dose.
Both studies also evaluated the combined effects of the highest tested dose of DINP in addition to
vitamin E or melatonin (i.e., 150 mg/kg-day + 50 mg/kg-day vitamin E (Peng. 2015); or 200 mg/kg-day
+ 50 mg/kg-day melatonin (Ma et al.. 2015)). Mice exposed to 200 mg/kg-day DINP plus 50 mg/kg-day
melatonin had less caspase-3 and glial fibrillary acidic protein staining than DINP alone, indicating that
melatonin can rescue the increase in caspase-3 and glial fibrillary acidic protein expression that follows
DINP exposure. The addition of melatonin was also sufficient to attenuate the effects consistent with an
oxidative stress response (i.e., increases ROS, DNA-protein-crosslinks, 8-OH-dG, cytokines; decreases
in superoxide dismutase activity and GSH content), implying that DINP induces oxidative stress in the
cerebral cortex which contributes to neuronal damage (Ma et al.. 2015). Similarly, Peng et al (2015)
observed that combined exposure of DINP + vitamin E, which has antioxidant properties, attenuated
effects consistent with an oxidative stress response, implying that the observed effects were consequent
to a pro-oxidant cellular environment in the brain.
In Duan et al. (2018). specific pathogen-free male Balb/c mice were divided into several groups
designed to evaluate the impact of DINP on an allergic response to an ovalbumin (OVA) antigen. The
authors also investigated the modulatory effect of melatonin, which they state has antioxidant properties,
as well as the role of nuclear factor kappa B (NFkB) signaling and oxidative stress using an inhibitor of
NFkB, dehydroxymethylepoxyquinomicin (DHMEQ). DINP exposure exacerbated effects consistent
with an oxidative stress response in brain homogenates (i.e., increase in ROS levels and decreases in
superoxide dismutase activity). DINP also increased IL-1B and IL-17 levels in brain homogenates as
well as nerve growth factor staining in the prefrontal cortex; all of which were attenuated by the
combined exposure of DINP + melatonin or DINP + DHMEQ—suggesting that the inflammation is
mediated by a pro-oxidant environment and activation of NFkB signaling. Other endpoints in this study
included: brain histopathology of pyramidal cells in the prefrontal cortex, and immunohistochemistry
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staining in the prefrontal cortex for eosinophil cationic proteins, nuclear factor erythroid 2-related factor
2 (Nrf2), NFkB. Limitations include lack on quantitative results for histopathology.
The other identified study provides a diverse set of data evaluating sexually dimorphic gene expression
in relation to effects on sexual behavior in rodents (Lee et al.. 2006b). Lee et al. investigated the effects
of perinatal exposure to DINP on expression of sex-steroid-regulated genes in the hypothalamus of
offspring and sexual behaviors as adults. Pregnant rats were administered 40, 400, 4,000, or 20,000 ppm
DINP in the diet from GDI5 through PND21. At PND7, male and female pups were sacrificed, and the
hippocampus was dissected from brains to quantify expression of sexually dimorphic genes such as
granulin (grn) and pi 30. After maturation, the authors evaluated and sexual behaviors (e.g., lordosis,
copulatory behavior), reproductive endpoints (e.g., estrus cycles, serum levels of estradiol, LH, FSH);
these data are discussed in detail in Section 3.1. In male PND7 pups, there was no change in
hypothalamic grn expression, and a non-monotonic dose response was observed in pi30, but expression
was increased at all dose levels. In females, grn was increased in the 40 and 400 ppm, and 20,000 ppm
exposure groups, and no change was observed in pi30. While the increased pi30 expression in males
coincided with impaired male sexual behavior (i.e., decreased copulatory behavior), serum hormone
levels (i.e., testosterone, FSH, LH) were not changed. The authors suggest that DINP may act on regions
of the hypothalamus that alter sexual behavior, but not gonadotropin secretion, to influence sex-specific
adult behavior.
Conclusions on Neurotoxicity
Fifteen studies in experimental animal models have evaluated neurotoxicological endpoints (i.e.,
behavior, brain weight, or histopathology) following exposure to DINP. However, only three of these
were specifically designed to evaluate behavioral neurotoxicity, which typically may provide insight
into more sensitive effects of DINP and supplement the neurobehavioral data from the epidemiological
database.
Two intermediate duration exposure studies with similar designs in male Kunming mice (Ma et al..
2015; Peng. 2015) provide consistent evidence for impaired learning and memory following DINP
exposure for 9 or 14 days, with parallel perturbations in the pyramidal cells of the hippocampus at doses
up to 200 mg/kg-day. The developmental exposure study by Boberg et al. (2011) exposed rats to doses
up to 900 mg/kg-day from GD7 to PND17 and conducted behavioral examinations at later timepoints.
No evidence of impairment was observed in males or females (2-3 months for MWM; radial arm maze
performance at 5-7 months). One consideration regarding the study design in Boberg et al. (2011) is that
a considerable amount of time had elapsed between the cessation of exposure and time of outcome
evaluation, which could make it more difficult to detect an exposure-related effect (i.e., bias towards the
null), and this difference makes a direct comparison to the studies by Ma et al. (2015) and Peng (2015)
challenging. However, this design also helps determine the extent to which perinatal exposures influence
behavior later in life. Nevertheless, discordant results across these studies may reflect study design
differences that influence the degree to which the received dose influences the test animals. Moreover,
Ma et al. (2015) and Peng et al. (2015) exposed adult male Kunming mice and measured outcomes in
adults, while Boberg et al. (2011) exposed pregnant rats and evaluated outcomes in the offspring. In
addition to the inconsistent findings across study designs, a limitation of the behavioral data set is the
relative lack of studies that consider outcomes in both sexes—especially given the fact that performance
in the MWM test is sexually dimorphic.
Although histopathological alterations were observed in the pyramidal cells of the hippocampus in two
independent intermediate exposure duration studies by Ma et al., (2015) and Peng et al., (2015). these
studies were limited by the lack of quantitative data and were inconsistent with findings of the 28-day
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exposure study by the Midwest Research Institute (1981) as well as all the chronic exposure studies in
mice (Covance Labs. 1998b) and rats (Covance Labs. 1998c; Lington et al.. 1997). Strengths of the data
set include coherence with the behavioral data sets from the Ma et al. (2015) and Peng (2015) studies;
pyramidal cells of the hippocampus are involved in learning and memory, and the mechanistic data set
from these studies provides evidence of biological plausibility via a mechanism involving ROS damage
by DINP to the pyramidal neurons. Limitations of the data set include lack of quantitative results for
incidence and severity of histopathology effects and lack of chronic exposure studies with
histopathology of neural tissues.
Overall, available laboratory animal studies provide some evidence that DINP may cause behavioral
effects in rodents. Although some uncertainty exists, EPA considered neurotoxicity further for dose-
response analysis in Section 4. Specifically, neurobehavioral endpoints from Ma et al. (2015) and Peng
(2015) were further considered.
3.5 Cardiovascular Health Effects
Humans
Health Canada (2018a) evaluated multiple studies that investigated the association between phthalate
exposure and several cardiovascular outcomes and/or associated risk factors (i.e., cholesterol, diastolic
and systolic blood pressure, HDL-cholesterol, LDL-cholesterol, and blood glucose levels); however only
two studies directly looked at evidence of an association between DINP and/or its metabolites and
cardiovascular effects. A cross-sectional study of good quality by Trasande et al. (2014) looked at
albumin/creatinine ratio (ACR), a biomarker of endothelial dysfunction and increased risk of CVD in
children and adolescents found that there was inadequate evidence for an association between ACR and
MCOP in children and adolescents (Health Canada. 2018a).
New Literature: EPA identified three new medium quality studies that evaluated the association between
urinary DINP levels of metabolite and cardiovascular effects. The first medium quality study, a
prospective birth cohort study, by Heggeseth et al. (2019). used data from the Center for the Health
Assessment of Mothers and Children of Salinas (CHAMACOS) cohort to assess the association between
prenatal urinary DINP measurements and BMI trajectories throughout childhood. The authors did not
report any significant results; however, functional principal components analysis found that MCOP was
an explanatory variable in variation of BMI trajectories among girls.
Another medium quality study, a cross-sectional and longitudinal analysis, by Diaz Santana et al.
(2019). of participants from a nested case-control included using data from the Women's Health
Initiative (WHI) evaluated the association between overweight and obesity as well as weight change and
DINP exposure. The study found no significant results in cross-sectional analyses by quartile of
exposure. However, there was significant association across quartiles with MCOP and overweight as
well as obese women, with p-trend <0.001 and p-trend=0.001 respectively.
Finally a medium quality study, a longitudinal cohort study, by Zettergren et al. (2021). examined
associations between DINP metabolites (MHiNP, MOiNP, MCiOP) and obesity measures through age
24y in a subset of participants in the Swedish Abbreviation for Children, Allergy, Milieu, Stockholm,
Epidemiology (BAMSE) cohort. The study found significant associations between increases in DINP
metabolites at age 4y and obesity measures obtained at ages 8 years and above. Urinary MHiNP,
MOiNP, MCiOP and DINP measures were significantly associated with an increased odds of
overweight at ages 8, 16, and 24 years, and with higher BMI [beta = 1.60 (95% CI: 0.37-2.84), waist
circumference (beta = 4.42 [95% CI: 1.35-7.49]), body fat percent (beta = 2.65 [95% CI: 0.52-4.77]),
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and trunk fat percent (beta = 2.70 [0.33-5.07]) at 24 years. The cross-sectional association between
DINP metabolites and obesity at age 4 were not significant.
Laboratory Animals
A limited number of experimental animal studies have evaluated the cardiovascular effects of DINP
following oral exposure. Existing assessments of DINP have not drawn human health hazard
conclusions on the cardiotoxicity of DINP. Nevertheless, data are available on the effects of DINP on
blood pressure, heart rate, other indicators of adverse cardiac events, heart weight and/or heart
histopathology (U.S. EPA. 2023c: NICNAS. 2012; U.S. CPSC. 2010; ECB. 2003). Only one study was
available that was specifically designed to evaluate cardiotoxicity (Deng et al.. 2019). Remaining studies
evaluated heart weight and/or heart histopathology (Kwack et al.. 2009; Lington et al.. 1997;
Bio/dynamics. 1982b; Midwest Research Institute. 1981). No studies are available for the dermal or
inhalation exposure routes.
Three studies of varying study designs were identified that provide data on the effect of DINP exposure
on heart rate, blood pressure, or other indicators of adverse cardiac events, including levels of total
cholesterol and triglycerides. A subchronic duration study by Deng et al. (2019) investigated the
mechanisms associated with increased blood pressure following exposure to DINP. Groups of C57BL/6
mice were administered 0, 0.15, 1.5 or 15 mg/kg-day DINP via oral gavage daily for 6 weeks. At study
termination, systolic blood pressure, diastolic blood pressure, mean blood pressure, and heart rate were
measured. Additionally, blood samples were collected for measurements of serum nitric oxide levels and
levels of angiotensin converting enzyme (ACE), angiotensin-II type 1 receptor (AT1R), and endothelial
nitric oxide synthase (eNOS), were evaluated via immunohistochemistry staining. Increased systolic,
diastolic, and mean blood pressure was observed in mice of the two highest dose groups (1.5 and 15
mg/kg-day). Immunohistochemistry of the aorta showed increased staining intensity of ACE and AT1R
as well as decreased staining intensity of eNOS and nitric oxide. These latter endpoints are discussed
more in detail below under Mechanistic Information.
One additional study is available that provides data on changes in triglycerides and cholesterol following
intermediate duration exposure to DINP (Kwack et al.. 2009). Kwack et al. exposed male SD rats to 0 or
500 mg/kg-day DINP daily for 4 weeks via oral gavage and evaluated several cardiovascular outcomes
including serum levels of total cholesterol and triglycerides. Serum triglycerides were significantly
increased (50% increase compared to controls), while no change was observed in serum total
cholesterol.
Three studies were identified that provide data on the effect of DINP on heart weight, including one
intermediate exposure duration study in male SD rats (Kwack et al.. 2009). one intermediate study in
male and female F344 (Bio/dynamics. 1982b). and one chronic study in male and female F344 rats
(Lington et al.. 1997). In general, no statistically or biologically significant (i.e., >10% change)
exposure-related changes in absolute or relative heart weight were observed across studies.
Two studies were identified that report histopathology of the heart and/or aorta following exposure to
DINP. The subchronic study in male mice by Deng et al. (2019) also evaluated histopathology of the
heart and aorta. Lesions were observed in the high-dose group (15 mg/kg-day), including ventricular
wall thickening and cardiomyocyte hypertrophy. In contrast, the study by the Midwest Research
Institute (1981) did not observe discernable lesions in the heart at study termination. In this study, male
and female F344 rats were exposed to 0, 0.2, 0.67, or 2 percent DINP for 28-days via feed (estimated
doses: 0, 150, 500, 1,500 mg/kg-day [males]; 0, 125, 420, 1,300 mg/kg-day [females]). A limitation of
these studies is that histopathology was reported qualitatively.
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Table 3-10. Summary of Study Evaluating Cardiovascular Outcomes
Brief Study
Description
(Reference)
NOAEL /
LOAEL
(mg/kg-
day)
Effect at LOAEL
Comments
C57BL/6 mice
(males only); oral
gavage; 0, 0.15, 1.5,
15 mg/kg-day; 6
weeks; with or
without induction of
hypertension (Dens
et al.. 2019)
0.15/ 15
t in systolic, diastolic,
and mean blood
pressure; ventricular
wall thickening &
cardiomyocyte
hypertrophy;
immunohistochemistry
of aorta showed \ ACE
&AT1R& jeNOS&
NO.
15 ms/ks-dav: t Heart Rate and diastolic
blood pressure. Pathological changes in the
heart, aorta, and kidney
Kidnev histopatholoav (qualitative onlv):
Study authors also state that "DINP exposure
and DEXA treatment could both induce
vacuoles and hyaline degeneration in the
glomerulus as compared to the saline group.
EPA also found that DINP exposure resulted
in smaller glomeruli and a thickened
glomerular basement membrane, and that
ACEI effectively inhibited these lesions."
Doses at which this occurred are not stated.
Mechanistic Information
EPA identified one in vivo study (Deng et al.. 2019) that provides data that may inform mechanisms of
the observed cardiovascular effects of DINP. The mouse study by Deng et al. (2019) investigated
mechanisms associated with increased blood pressure following exposure to DINP. Groups of C57BL/6
mice were exposed to 0, 0.15, 1.5, or 15 mg/kg-day DINP daily for 6 weeks via gavage. Parallel groups
of mice also received a subcutaneous injection of 1 mg/kg-day dexamethasone to induce hypertension
and/or 5 mg/kg-day of an ACE inhibitor via gavage in addition to the highest dose of DINP. In addition
to the evaluations of blood pressure described above, the authors measured serum nitric oxide (NO)
levels and determined levels (i.e., staining intensity) of ACE, AT1R, and eNOS in the aorta via
immunohistochemistry staining. The authors observed increased staining intensity of ACE and AT1R as
well as decreased staining intensity of eNOS in the aorta using immunohistochemistry following
exposure to 1.5 or 15 mg/kg-day DINP (AT1R and eNOS) or all doses (ACE). Co-exposure of
15 mg/kg-day DINP and dexamethasone resulted in similar changes in expression of ACE, AT1R, and
eNOS. Co-exposure of dexamethasone +15 mg/kg-day DINP + the ACE-inhibitor did not fully
attenuate the changes. Serum levels of NO were decreased following DINP exposure (all doses) as well
as with co-exposure to dexamethasone and/or the ACE inhibitor. Given the aforementioned increases in
systolic, diastolic, and mean blood pressure, in mice of the two highest dose groups (1.5 and 15 mg/kg-
day), these results provide some evidence to support a mechanism whereby DINP acts through the ACE
pathway to increase blood pressure.
Conclusions on Cardiovascular Health Effects
The database of studies in experimental animals that has evaluated cardiovascular toxicity and
associated risk factors following exposure to DINP is limited and findings were generally inconsistent
across study designs and species. Only one subchronic study was available that was specifically
designed to evaluate cardiotoxicity (Deng et al.. 2019). Limitations of the study included failure to
consider both sexes and reporting deficiencies, including the qualitative reporting of histopathology
data. Nevertheless, the consistency across endpoints within Deng et al. (2019). including increased
blood pressure and histopathological effects in the aorta suggest that DINP may be toxic to the
cardiovascular system. Mechanistic data from the same study suggest the underlying mechanism for
these effects involves the ACE pathway.
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Overall, there is limited evidence that DINP can elicit cardiotoxicity in experimental laboratory animals.
Only one study in one species of one sex evaluates cardiovascular outcomes (Deng et al.. 2019).
Additionally, the clinical implications, or relevance to humans, is uncertain for cardiovascular effects of
DINP. Due to these limitations and uncertainty, EPA is not further considering cardiotoxicity for dose-
response analysis.
3.6 Immune System Toxicity
Humans
Health Canada (2018a) evaluated multiple studies that investigated the association between urinary
metabolite and immunological outcomes. Across available studies of DINP, Health Canada found that
there was limited or inadequate evidence for association between DINP and its metabolites and
immunological outcomes.
New Literature: EPA identified three new studies (two medium quality studies and one low quality) that
evaluated the association between DINP and its metabolites and immune/allergy outcomes. The first
medium quality study, a prospective birth cohort, by Soomro et al. (2018). of the Etude des
Determinants pre et postnatals du developpement de la sante de l'Enfant (EDEN) study measured one
maternal urinary DINP metabolite (MCOP) and its association with eczema diagnosed at ages 1-5 in
boys, and with elevated serum IgE at age 5 years. Results for the main effect association between
MCOP and elevated IgE were described only as not significant for MCOP. There were no significant
associations found with MCOP and elevated serum IgE (>60 IU/mL). However, multivariate logistic
regression of MCOP and odds of diagnosed eczema was only significant for age 5 (OR = 1.60 [95% CI:
1.16, 2.23]). There was a significant association found in multivariate logistic regression of MCOP and
association with early onset eczema (first 2 years of life) (OR = 1.29 [95% CI: 1.04, 1.60], p < 0.05) and
late-onset (age 3-5 years) eczema (OR = 1.63 [95% CI: 1.20, 2.21], p < 0.05). There was also a
significant association in Cox proportional hazard model of MCOP and ever diagnosed with eczema
(HR = 1.09 [95% CI: 0.95, 1.25], p = 0.05).
Another medium quality study, a cross-sectional study, by Ait Bamai et al. (2018). that used data from
Hokkaido study on Environment and Children's Health examined the association between DINP and
eczema within the past 12 months. Logistic regression of DINP (jag/g dust) exposure on eczema found
significant gene-environmental interaction with FLG mutation (OR total =1.17 [95% CI: 0.91, 1.52]; p
= 0.039). No other significant associations were found between eczema and DINP exposure.
Finally, a low quality cohort study by Wan et al. (2021). which used data from the Kingston Allergy
Birth Cohort (KABC), examined the association between skin prick testing and DINP exposure. The
authors did not find any statistically significant results in adjusted logistic regression models for DINP
exposure relation to allergic sensitization.
Laboratory Animals
A limited number of studies are available that have been evaluated for the toxicological effects of DINP
on the immune system. Available studies have provided data on the adjuvant properties of DINP; an
adjuvant is a substance that can enhance immune responsiveness without itself being an antigen. ECB
(2003) summarized the irritation and sensitization data and determined that DINP is a very slight skin
and eye irritant, with effects reversible in short time. The U.S. CPSC (2010) concluded that "/>; vivo
studies in guinea pigs suggest that DINP is not a skin sensitizer"; however, "/>; vivo studies in mice show
that DINP or other o-DAP's may augment an antigen mediated IL-4, IgE, and/or IgGl reaction." These
finding suggest that DINP may potentiate allergic and/or asthmatic responses.
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The database of studies from existing assessments that evaluate the immune adjuvant effects of DINP is
limited to two studies (Koike et al.. 2010; Imai et al.. 2006). which investigate the effects of DINP on
atopic dermatitis and skin sensitization.
Koike et al. (2010) investigated the effect of DINP on atopic dermatitis resulting from contact with a
dust mite allergen. Male NC/NgaTndCrlj mice were injected intradermally on the ventral side of their
right ears with saline or extract of the dust mite, Dermatophagoides pteronyssinus (Dp) on study days 0,
3, 5, 8, 10, 12, 15, and 17. On study days 2, 5, 9, and 16, DINP was administered via intraperitoneal
(i.p.) injection dose levels: 0, 0.15, 1.5, 15, or 150 mg/kg-day. The authors evaluated several endpoints
including histopathology of the ears, protein expression (from ear homogenates) of Thi-type vs. Tin-
type cytokines , as well as chemokines such as eotaxin, eotaxin-2, and thymic stromal lymphopoietin
(TSLP), via ELISA. DINP exposure significantly increased ear thickening and macroscopic features of
the ears from 4 and 6 days after the first injection of Dp. However, no dose-dependent effects of DINP
were observed. Animals exposed to 15 mg/kg-day DINP + Dp had more skin lesions when compared to
animals exposed to Dp or saline (no Dp). Histopathological evaluation of the ears showed that while Dp
had increased infiltration of eosinophils into the skin lesions when compared with saline controls, 15
mg/kg-day DINP + Dp potentiated the infiltration of eosinophils into the skin lesion (compared to Dp)
in parallel with increased mast cell degranulation. Alterations in cytokine levels were observed in the
ears of animals exposed to Dp (compared to saline), including increased IL-4, -5, and -13 and decreased
interferon-y (IFN-y). There was a decrease in expression of IFN-y, eotaxin and eotaxin-2, and increased
expression of TSLP were also observed in the ears of mice exposed to DINP, compared to those exposed
to Dp + vehicle. These data suggest that DINP aggravates allergic dermatitis-like skin lesions caused by
the Dp antigen. To evaluate the adjuvant capacity of DINP for immunoglobulin (Ig) production, the
authors also measured serum levels of anti-DP-IgGi, IgE, as well as histamine release. Intradermal
injection of Dp increased the levels of Dp-specific IgGl, total IgE, and histamine levels in serum
compared to saline alone. Exposure to DINP significantly increased histamine levels in serum compared
to saline alone. However, no significant changes in serum levels of Dp-specific IgGl, total IgE, or
histamine were observed in groups exposed to DINP compared to Dp. Collectively, these data support
the conclusion that DINP is not an adjuvant in an atopic dermatitis mouse model.
Imai et al. (2006) investigated whether different phthalate esters (including DINP) have adjuvant effects
on skin sensitization using FITC as a sensitizer. Female CD-I (ICR) and BALB/c mice were used for
this skin sensitization study. Experimental groups include having multiple phthalates mixed with
acetone at a 1:1 ratio and the control group with acetone alone. ICR mice were epicutaneously sensitized
with FITC dissolved in an acetone solution containing one of various phthalate esters, including DINP.
The applications on the forelimbs were repeated on day 7 and on day 14; ear thickness and ear swelling
were measured. There were no significant differences in ear thickness/swelling between the DINP
treated group compared to the acetone control group. Similar results with DINP were confirmed using
BALB/c mice. Twenty-four hours following skin sensitization, draining lymph node cells were
examined for FITC fluorescence by means of flow cytometry. Mice sensitized with FITC in acetone
containing DINP did not show consistent ear-swelling response. DINP also showed no significant
increase in the FITC-positive cell number in the draining lymph nodes. These data suggest that DINP
does not act as an adjuvant in a FITC skin sensitization model in mice.
New Literature: EPA identified two new studies that investigated the effects of DINP exposure on
atopic dermatitis (Wu et al.. 2015; Sadakane et al.. 2014).
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Wu et al. (2015) investigated the effects of DINP on allergic dermatitis in a FITC-induced allergic
dermatitis model and the role of oxidative stress and inflammatory factors in skin lesions of the model
mice and characterize the mechanism involved in the DINP. Additionally, uncovering the protective role
of melatonin (MT) on allergic dermatitis and exploring its mechanism as an antioxidant. Forty-nine male
Balb/c mice were divided randomly into seven groups: control, melatonin (30 mg/kg-day) 3 hours after
saline skin exposure, 0.5 percent FITC-sensitized group (FITC), 1.4 mg/kg-day) DINP skin
exposure+0.5 percent FITC-sensitized group (FITC + DINP1.4), 14.0 mg/kg-day DINP skin
exposure+0.5 percent FITC-sensitized group (FITC+DINP 14), 140.0 mg/kg-day DINP skin
exposure+0.5 percent FITC sensitized group (FITC+DINP 140), and MT (30 mg/kg-day) 3 hours after
140.0 mg/kg-day DINP skin exposure combined with 0.5 percent FITC sensitized group (FITC+DINP
140.0+MT). The mice were exposed for 40 days, then given saline or FITC on days 41 and 42.
Sensitization was terminated on day 47 to measure ear thickness. This experiment was terminated on
day 48 and blood samples were collected to measure IgE levels and immunohistochemistry were
conducted on the sections from the right ear for TSLP, p-STAT3, p-STAT5, p-STAT6, NF-kB, and p65.
Markers of oxidative stress—including ROS, MDA, GSH, along with cytokines, IL-4 and IFN-y—were
evaluated from the ear tissue.
The highest concentration of DINP (140 mg/kg-day) with FITC significantly increased the number of
infiltrating inflammatory cells when compared with the FITC exposed only group. Moreover, the
pathological alterations and the number of infiltrating inflammatory cells were alleviated in the
FITC+DINP 140+MT group as compared with the FITC+DINP 140 group. Ear swelling and bilateral
ear weight were significantly altered in all FITC-immunized groups. Dermal DINP exposure
significantly increased ear swelling and bilateral ear weight when compared to the group exposed to
FITC only, and this adverse effect was potentiated. Also, when MT was added, it diminished the DINP-
induced ear swelling and the bilateral ear weight when compared to the same concentration of DINP
without MT. FITC alone and all concentrations of FITC+DINP exposure significantly enhanced serum T
IgE levels, at all concentrations. The highest dose of DINP (140 mg/kg) exposure drastically elevated
serum T-IgE levels compared with the FITC-sensitization only group. Further, T-IgE levels in the FITC
+ DINP 140 group significantly decreased when compared to the FITC+DINP 140+MT group.
Compared with the FITC only group, co-exposure with any concentration of DINP induced a significant
increase in IL-4, IL-5, and a resulting skew in the ratio of IL-4 to IFN-y. These adverse effects
exacerbated by DINP were concentration-dependent. However, MT alleviated the DINP-induced effects,
suggesting that DINP is associated with Th2 cytokine expression by FITC-mediated allergic
inflammation. Their results of histopathological examinations and measurements of ear swelling as well
as immunological and inflammatory biomarkers (total-immunoglobulin IgE and Th cytokines) supported
their conclusion that high doses of DINP might aggravate atopic dermatitis.
Lastly, Sadakane et al. (2014) investigated the role of DEHP and DINP on atopic dermatitis at doses
lower than the NOAEL for chronic liver toxicity (i.e., 15 mg/kg-day). Herein, only results for DINP are
discussed. The study included a control and 4 experimental treatment groups, each of which included 12
male NC/Nga mice. Animals in the experimental groups were exposed to the allergen,
Dermatophagoides pteronyssinus (Dp), by subcutaneous injection of 5 mg of Dp dissolved in 10 mL of
saline in the ventral side of the right ear for 2 to 3 days a week (a total of 8 times) under anesthesia.
Animals in the experimental DINP groups were exposed to the allergen and treated with 0 (Dp +
vehicle), 6.6 |ig DINP/animal (Dp + DINP 6.6), 131.3 |ig DINP/animal (Dp + DINP 131.3), or 2,625 |ig
DINP/animal (Dp + DINP 2625). In the experimental groups, mice were orally administered DINP
dissolved in 0.1 mL of olive oil 5 days before the first injection of the allergen. Control group animals
(saline + vehicle and Dp + vehicle groups) were not exposed to DINP and were orally administered 0.1
mL of olive oil.
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Twenty-four hours following Dp injections, skin disease symptomatology and ear thickness were
evaluated and scored for symptoms of skin dryness and eruption, edema, crusting and erosion. Also, the
clinical scores of the Dp + DINP 6.6 and Dp + DINP 131.3 groups began increasing when compared
with the Dp + vehicle group from day 16, the Dp + DINP 131.3. The Dp + DINP 131.3 group had a
higher (not significant) wound score compared with the Dp + vehicle group while the Dp + DINP 2625
did not change. Statistical tests revealed no significant differences between DINP treated groups and the
control at any doses to contribute to ASDLs. The dorsal skin of the Dp-treated groups with or without
DINP exposure exhibited epidermal and dermal thickening, eosinophil accumulation and mast cell
degranulation. The eosinophil counts of both DP+DINP treatments increased but not significantly.
However, oral exposure to DINP did not increase the eotaxin levels. Exposure to DINP modestly
increased mean total IgE levels. The rank of mean skin scores with specific DINP doses (Dp + DINP
131.34 > Dp + DINP 6.64 > Dp + DINP2625 > Dp + vehicle) was found to be strongly positively
correlated with the number of eosinophils, the number of severely degranulated mast cells, and
moderately positively correlated with the total number of mast cells. Overall, this study provides some
evidence that DINP can aggravate the allergic response in animal allergic dermatitis models.
Mechanistic Information
EPA identified seven studies that describe the mechanism of action for the adverse immunological
effects of DINP (Yun-Ho et al.. 2019; Duan et al.. 2018; Kang et al.. 2017; Kang et al.. 2016; Chen et
al.. 2015; Koike et al.. 2010; Lee et al.. 2004).
Koike et al. (2010) evaluated the adjuvant effects of DINP on bone-marrow-derived dendritic cells or
splenocytes in vitro. Bone-marrow-derived dendritic cells and splenocytes were exposed to DINP for 24
hours at concentrations of 0 (control), 0.1, 1, 10, and 100 [xM. At 100 |iM, DINP exposure for 24 hours
led to significantly increased the production of Th2 chemokines, TARC/CCL17 and MDC/CCL22, in
bone-marrow-derived dendritic cells when compared with vehicle control. However, Thi cytokine IL-
12p40 was not detected in any bone-marrow-derived dendritic cell culture. Moreover, DINP also
significantly increased the expression of the chemokine receptors CCR7, CXCR4, MHC class II, CD80,
and CD86 on bone-marrow-derived dendritic cells compared with controls. DINP exposure for 24 hours
significantly increased IL-4 production from splenocytes compared with controls. After 72-hours of
exposure to DINP in the presence of Dp, there was a significant increase in proliferation of splenocytes
at 0.001 to 1 |iM and decreased proliferation at 10 |iM compared with controls. These results show that
DINP augmented IL-4 production and Dp-stimulated proliferation of splenocytes and suggest that DINP
can aggravate allergic dermatitis-like skin lesions through TSLP-related activation of dendritic cells and
by direct or indirect activation of other immune cells.
Kang et al. (2016) examined the effects of DINP exposure on the development of allergies and the
underlying mechanisms. Male Balb/c mice were gavaged with 2, 20, or 200 mg/kg-day DINP for 21
days, then sensitized with either saline or 0.5 percent FITC (in 1:1 acetone/DBP) on days 22 and 23 via
dermal application to shaved skin. On day 28, the mice received a 0.5 percent FITC challenge (or saline)
to the right ear, and saline or vehicle (1:1 acetone/DBP) to the left ear and the baseline ear thickness was
measured. On day 29, the study was terminated, and blood samples were collected to determine IgE
levels. Immunohistochemistry staining was performed on the sections from the right ear to visualize the
localization and staining intensity of TSLP, p-STAT3, p-STAT5, p-STAT6, NF-kB, and p65. The
authors also evaluated ROS, MDA, and GSH levels in the ear tissue as well as levels of the cytokines,
IL-4 and IFN-y. In mice administered DINP + FITC, there was an increase in the number of infiltrating
inflammatory immune cells in their ear tissue. Dose-dependent, significant increases in IL-4 and IL-5
were observed in all groups exposed to FITC + DINP. In contrast, there was a dose-dependent decrease
in IFN-y, which increased the IL-4/IFN-y ratio, showing DINP only increases Th2-specific cytokines.
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However, no significant pathological changes were observed in the ears of mice exposed to DINP alone,
but the ears of mice from the FITC only group showed inflammatory cell infiltration into the skin.
Additionally, to uncover the pathway of these adverse effects, treatment with FITC + 200 mg/kg-day
DINP, and pyrollidine dithiocarbamate, a well-known inhibitor of NF-kB, markedly reduced the ear
swelling when compared to the FITC + 200 mg/kg-day DINP group. Further, bilateral ear weight
decreased significantly when the FITC + DINP-immunized groups were treated with pyrollidine
dithiocarbamate. There was an increase in ROS and MDA levels and a decrease in GSH levels observed
in the FITC + 200 mg/kg-day DINP exposure group compared to FITC alone, but pyrollidine
dithiocarbamate reversed these effects. The adverse pathological effects observed in higher dose groups
were attenuated with pyrollidine dithiocarbamate treatment, which suggest that the effects are facilitated
by the NF-kB signaling pathway. Results support the conclusion that DINP aggravates FITC-induced
allergic contact dermatitis through exacerbating increased MDA and ROS accumulation and IL-4 and
IL-5 production, while also decreasing GSH and IFN-y, which then activates the NF-kB pathway.
Following activation, TSLP expression and activation is increased, causing increased production of
STATs 3, 5, and 6.
A subsequent study by Kang et al. (2017) expanded on the previously mentioned underlying
mechanisms of DINP and the role of transient receptor potential (TRP) cation channel, subfamily A,
member 1 (TRPA1) on the NF-kB pathway. In this allergic dermatitis model, male BALB/c mice were
gavaged with saline (control) or DINP (2, 20, 200 mg/kg-day) from days 1 to 21. On days 22 and 23,
mice were smeared with saline or 0.5 percent FITC on their backs to sensitize them, then on day 28,
mice are given saline or FITC on their right ear. Following sensitization, skin lesions showed enhanced
levels of IgGl, IL-6, IL-13, and TRPA1 expression with DINP potentiating these levels. To determine
the role of TRPA1 and NF-kB for allergic dermatitis, on days 22, 23, and 28, mice were injected with
HC-030031, a TRPA1 antagonist, and NF-kB inhibitor, pyrollidine dithiocarbamate. Blocking NF-kB
inhibited TRPA1 expression; however, TRPA1 antagonism did not have any effect on NF-kB or TSLP
expression. These findings suggest that TRPA1 is dependent on NF-kB activation and TSLP expression
for DINP aggravated allergic dermatitis.
Similarly, Lee et al. (2004) examined the effects of DINP on IL-4 production in CD4+ T-cells and the
associated mechanisms. BALB/c mice were injected with Keyhole limpet hemocyanin in alum adjuvant
twice at 7-day intervals while being intraperitoneally injected with 2 or 5 mg/kg of DINP every other
day. Lymph node cells were harvested and cultured from these mice after 7 days of treatment and used
to measure IL-4 and IFN-y. DINP was shown to enhance IL-4 production in lymph node cells, which
originated from CD4+ T-cells in a concentration dependent manner and increase IgE serum levels in
vivo. Additionally, DINP exposure also increased IL-4 gene promotion activity in Phorbol-12-myristate-
13-acetate stimulated EL4 T-cells. IL-4 gene promoter contains multiple binding sites to nuclear factor
of activated T-cells (NF-AT), and DINP was shown to potentiate IL-4 production via enhancing PI and
P4 binding site activity on NF-AT. These results support the conclusion that DINP augments the allergic
response of IL-4 production in CD4+T-cells via increased NF-AT binding activity.
Chen et al. (2015) investigated how DINP exposure during gestation and lactation affects the allergic
response of pups and the role of the PI3K/Akt pathway. Female Wistar rats are treated with 0, 5, 50, and
500 mg/kg-day from GD7 to PND21. On PND22, 23, and 37, pups were sensitized with ovalbumin.
Then, protein expression and production of cytokines associated with PI3K/Akt were measured. In the
50 mg/kg-day DINP group, pups displayed significantly increased lung resistance (RI) when compared
to the controls. Moreover, all DINP-treated groups had significantly increased eosinophil infiltration into
the airways when compared to the control group, as indicated by immunohistochemistry. Pups exposed
to 50 mg/kg-day DINP had increased Akt phosphorylation, NF-kB translocation, and increased Th2
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cytokine (IL-13) expression, while having decreased Thi cytokine (INF-r) expression, when compared
to the vehicle control group. These results suggest DINP aggravates the ovalbumin-induced response
and enhances expression of the PI3K/Akt pathway and NF-kB translocation.
Using a neuroinflammation mouse asthma model, Duan et al. (2018) exposed (via intraperitoneal
injection) groups of Balb/c mice (8 mice/group) to: 1) saline only (control); 2) Ovalbumin (OVA) only;
3) OVA and formaldehyde (1 mg/m3, 5h/day) exposure (OVA + FA group); 4) OVA and 20 mg/kg-
DINP (OVA + DINP); 5) OVA and formaldehyde (lmg/m3, 5h/day) and 20 mg/kg-day DINP (OVA +
FA + DINP); 6-9) melatonin (10 mg/kg-day) blocking groups (OVA + MT group, OVA + FA + MT
group, OVA + DINP + MT group, OVA + FA + DINP + MT group); 10-13) were
dehydroxymethylepoxyquinomicin (DHMEQ; a NF-kB inhibitor) (lOmg/kg-day) NF-kB blocking
groups (OVA + DHMEQ , OVA + FA + DHMEQ, OVA + DINP + DHMEQ, OVA + FA + DINP +
DHMEQ). Following 18 days of exposure and 7 days of sensitization, allergic asthma symptoms
(eosinophilic catatonic protein) levels and mucus secretion), markers of oxidative stress (ROS
fluorescence, superoxide dismutase, andNrf2 levels), cytokines (IL-ip and IL-17), and NF-kB signaling
were measured in the brain. Exposure to DINP increased eosinophilic catatonic protein levels and the
number of mucus secreting cells in the airway of the mice with OVA sensitization. Additionally, DINP
exposure increased levels of IL-ip, IL-17, and nerve growth factor levels in the brain and increased NF-
kB activation in the pre-frontal cortex. Moreover, DINP exposure increased ROS fluorescence in the
brain, Nrf2, and decreased superoxide dismutase. Results of this study indicate that DINP promotes
neuroinflammation through potentiating oxidative stress and NF-kB signal pathway activation in this
mouse asthma model.
Lastly, Yun-Ho et al. (2019) investigated the role of TLR4 and HMGB1 in DINP-induced asthma. In
this study, female C57BL/6 mice were intraperitoneally injected with 50 mg/kg-day DINP for a week to
sensitize them and then challenged with saline or DINP on days 19, 21, and 23. During the challenge,
mice were injected in their tail vein with either 3 mg/kg TAK-242 (TLR4 inhibitor) or 10 mg/kg anti-
HMGB1 antibody, respectively, on each day of the challenge. DINP significantly increased airway
hyperresponsiveness, number of infiltrating cells in bronchoalveolar fluid, numbers of inflammatory
cells in blood, pulmonary fibrosis, mucus production, Th2 cytokine production (IL-4, IL-5, IL-13), and
lung cell apoptosis. In contrast, adding the TLR4 inhibitor or anti-HMGBl antibody following DINP
exposure reduces airway hyperresponsiveness, reduced production of IL-4, IL-5, and IL-13 cytokines,
and number of inflammatory cells in the airway. Overall, this study provides evidence that HMGB1 and
TLR4 signaling pathways can contribute to DINP-induced asthma.
Conclusions on Immune System Toxicity
There are multiple animal toxicity studies that support the adjuvant effects of DINP exposure on the
immune response in dermatitis models and in vitro experiments (Koike et al.. 2010; Imai et al.. 2006).
Koike et al. (2010) stated that DINP exposure did not aggravate serum levels of IgGl, IgE, and
histamine levels in vivo. Further, Imai et al. (2006) concluded that DINP is not considered a skin
sensitizer based on no significant increase in the FITC-positive cell number in the draining lymph nodes.
Additionally, there were three new studies that all support that DINP aggravates atopic dermatitis via
causing oxidative stress and NF-kB cellular pathway activation (Kang et al.. 2016; Wu et al.. 2015;
Sadakane et al.. 2014). Similarly, EPA identified six mechanistic studies that support DINP enhancing
NF-kB signaling, TSLP transcription, NF-AT, PI3K/Akt, TLR4, and HMGB1 in allergic dermatitis,
atopic dermatitis, and asthma mouse models (Yun-Ho et al.. 2019; Duan et al.. 2018; Kang et al.. 2017;
Kang et al.. 2016; Chen et al.. 2015; Lee et al.. 2004). Overall, available studies provide evidence that
DINP augments the inflammatory responses in several sensitization models and the underlying
mechanisms. Specifically, there are several studies that demonstrate DINP's role in potentiating ROS
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production, TSLP transcription, PI3K/Akt, TLR4, andNF-KB pathway activation, and Th2 cytokine
production in allergic dermatitis, neuroinflammation, and asthma in animal models.
Although available studies of laboratory animals provide some evidence for immune adjuvant effects of
DINP in sensitized animals, EPA is not further considering these effects for dose-response assessment or
for use in extrapolating human risk. Available studies evaluate the adjuvant properties of DINP in
experimental rodent models pre-sensitized by exposure to other compounds (e.g., FITC, ovalbumin).
While these studies may be useful for hazard identification for a specific population (pre-sensitized
individuals), the fact that the outcome evaluated in these studies requires prior exposure to another
chemical precludes its broader applicability.
3.7 Musculoskeletal Toxicity
Humans
Four epidemiologic studies, three cross-sectional and one cohort study examined the association
between DINP urinary levels of metabolites and bone mineral density, Osteoporosis and Vitamin D in
adults; however the evidence was considered inadequate due to inconsistent results (Health Canada.
2018a).
New Literature: EPA did not identify any new epidemiologic studies that examine the association
between DINP and its metabolites and musculoskeletal toxicity.
Laboratory Animals
Hwang et al. (2017) was the only study that investigated the relationship between DINP and osteopenia,
which is characterized by bone loss and deterioration of bone structure leading to fractures. DINP (2, 20,
or 200 mg/kg-day) was administered via intraperitoneal injection to 8-week-old female C3H/HeN
ovariectomized (OVX) mice (5 animals/group), including: a sham-operated control group injected with
PBS; a vehicle treated OVX group injected with PBS; and three DINP groups of 2, 20, or 200 mg/kg-
day. The vehicle and DINP were administered for 6 weeks, and the body weights were recorded weekly.
There was significant increase in body weights of OVX mice compared to sham control mice 6 weeks
after OVX surgery. DINP also significantly increased body weight compared to sham control mice.
DINP-treated mice had significantly reduced uterus weight and decreased tibia and femur lengths. Tibia
weights were decreased in OVX mice and in the DINP-treated mice. However, no differences were
noted in femur weights among the groups. DINP treatment of the normal mice increased the inorganic
phosphorus release. Lactate dehydrogenase was unaffected by OVX or DINP treatments.
Further, tartrate-resistant acid phosphatase activity (bone resorption marker) was significantly increased
in both OVX mice and in the mice treated with 200 mg/kg-day DINP at a similar magnitude over
controls. Bone ALP activity was lower than sham controls in the OVX mice and in the DINP mice
treated with 2 and 20 mg/kg-day; however, bone ALP activity in mice treated with 200 mg/kg-day DINP
was comparable to sham controls, indicating that these decreases were not dose-related. Further, the
microarchitecture of the femur and tibia were affected by OVX and DINP. The bone volume, tissue
volume, bone volume/tissue volume ratio, bone surface, bone surface/tissue volume ratio, trabecular
thickness, and trabecular number were all reduced, while the trabecular pattern factor, structure model
index, and trabecular separation were increased in the DINP-treated mice, although these differences
were not as substantial as in the OVX mice compared to sham controls. Similarly, the bone mineral
density of the femur and tibia was dose-dependently decreased in the DINP-treated mice, but not
decreased to the extent noted in the OVX mice, compared to the sham controls. The authors concluded
that these results indicate that DINP contributes to an increased risk of osteopenia via destruction of the
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microarchitecture and enhancement of osteoclastic activity, although it is difficult to conclude as the
mechanism of action is currently unknown.
Conclusions on Musculoskeletal Toxicity
Four epidemiological studies and one study in experimental animals have provided data on the
associations between exposure to DINP and musculoskeletal outcomes such as osteoporosis or
osteopenia. The human evidence was considered inadequate due to inconsistent results across study
designs and not further evaluated by EPA. The animal evidence suggests that DINP can reduce bone
mineral density in female mice. Overall, there is limited evidence that DINP can elicit musculoskeletal
toxicity in experimental laboratory animals; only one study in one species of one sex evaluates
musculoskeletal outcomes. Additionally, the clinical implications, or relevance to humans, is uncertain
given the limitations of the epidemiologic database. Due to these limitations and uncertainty, EPA is not
further considering musculoskeletal toxicity for dose-response analysis.
3.8 Gastrointestinal System Toxicity
Humans
EPA did not identify any epidemiologic studies of the gastrointestinal system for DINP and/or its
metabolites.
Laboratory Animals
EPA identified three animal toxicology studies that provide data on the effects of DINP on the
gastrointestinal system (Chiu et al.. 2021; Chiu et al.. 2020; Setti Ahmed et al.. 2018). Setti Ahmed et al.
(2018) investigated the effects of DINP on development of the small intestine. Pregnant Wistar rats (36
per dose) were gavaged with 0 (corn oil vehicle) or 380 mg/kg-day DINP (CASRN 68515-48-0) from
GD8 through PND30. Treatment with DINP reduced maternal food consumption 14 to 39 percent
during gestation and 48 to 62 percent during lactation (PND1-21); however, it is unclear if reduced food
consumption led to reduced dam body weight, as this outcome was not reported. Pup body weight gain
was significantly reduced (54-56%) from PND15 to 30. Study authors report that pup small intestine
weight was significantly reduced 41 percent by treatment with DINP; however, there are apparent
discrepancies between the text and tabular organ weight data (unclear if a statistical analysis as done on
individual organs). Histologically, offspring small intestine (duodenal, jejunal and ileal samples) showed
villous atrophy following exposure to DINP; however, no incidence data is reported (only representative
photomicrographs are provided). Lactase, maltase, sucrase, and ALP activity in the duodenum, ilium,
and jejunum were also reported to be impacted by treatment with DINP on PND7, PND15, and PND30.
Although results from this study suggest that DINP has effects on the developing small intestine in
offspring exposed via maternal exposure during gestation and lactation, these effects may be related to
the substantial decreases in offspring body weight gain which may be secondary to decreased maternal
food consumption during gestation and lactation.
Chiu et al. (2020) evaluated the effect of DINP on the intestinal endocrine system including levels of
testosterone and estradiol in the colon as well as distal colon histopathology and gross measurements of
colon length and weight. Female CD-I mice (6 per group) were orally administered 0, 0.02, 0.2, 2, 20,
and 200 mg/kg-day DINP (CASRN 28553-12-0) for 10 to 14 days. There was no effect of DINP on
colon weight, length, or weight-to-length ratio. Increased damage to the colon was noted in animals
exposed to 0.02, 0.2, 2, and 200 mg/kg-day, but there was no dose-response and there are limitations in
the methods for histopathological quantification (noted below). The authors qualitatively attribute
changes at 0.02 and 0.2 mg/kg-day DINP to cellular infiltration and aberrant colon walls, and attribute
changes at 2 and 200 mg/kg-day to edema based on their scoring criteria, but quantitative information
{i.e., incidence data) are not reported. There were no significant or dose-related changes in testosterone
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in colon tissues. Although significant decreases in estradiol were observed in mice exposed to 0.2, 20,
and 200 mg/kg-day DINP, there was no dose-response. There were no other dose-related effects. Several
additional factors exist in Chiu et al. (2020) which decrease confidence in the study. These include the
method of test substance administration (i.e., pipetting chemical into the mouse of the mice) and
methodological deficiencies in the histopathologic quantification of the colon that impact the ability to
interpret the results. Indeed, incidence data was not provided, and information of tissue sampling (i.e.,
number of sites evaluated in a given sample, number of slices, thickness of slices, etc.) was not
provided. Overall, EPA did not consider this study further for dose-response assessment given the
limitations.
In a second study, adult (2 months of age) female CD-I mice (6 per group) were dosed with 0, 0.02,
0.02, 2, 20, and 200 mg/kg-day DINP (CASRN 28553-12-0) for 10 to 14 days (Chiu et al.. 2021). Mice
were administered DINP via pipetting the chemical into the mouth of the mouse, which raises
uncertainty around the received doses of DINP. Colon tissue samples from 6 animals in each treatment
group were pooled and 40 different cytokines were evaluated in the pooled samples using a cytokine
array. Treatment with DINP appeared to decrease protein levels of CXCL12 and depending on the dose
increased or decreased levels of IL-1RA; however, because samples were pooled for the array, no
statistical analysis could be performed. Further analysis of CSCL12 and IL-1RA protein levels in the
colon were evaluated via ELISA; however, no statistically significant effects were observed. Finally,
serum levels of estradiol were determined. Treatment with DINP significantly increased serum levels of
estradiol at 20 mg/kg-day, but not at any other doses. Additionally, this finding is somewhat inconsistent
with that of Chiu et al. (2020). where estradiol measured from colon samples was found to be decreased
at several doses. The inconsistent directionality of these effects increases uncertainty in the data set.
Mechanistic Information
Mechanistic data are limited to two studies from the same authors (Chiu et al.. 2021; Chiu et al.. 2020).
Chiu et al. (2020) also evaluated molecular mediators involved in the aforementioned effects of DINP
on the intestinal endocrine system, including gene expression of cell adhesion molecules (Zo-1, Zo-2,
Zo-3, Ocln, CI tin-1, Cldn-4), cytokines (IL-4, IL-5, IL-13, IL-17a, IL-6, Ifn-y, and Tnf), cell-cycle
regulators (Ccnci-2, Ccnbl, CCnd2, Ccnel, Cdk4, Cdknlci), apoptotic factors (Aifml, Bcl2, Bcl2110), and
cell proliferation (Ki67). Additionally, TUNEL staining was conducted to evaluate apoptosis, and
protein levels of the cell-adhesion molecule, sICAM-1, were quantified. In parallel with histopathologic
findings of the colon at 200 mg/kg-day (i.e., those attributed to edema), decreases in sICAM-1 levels
and gene expression of Zo-3 were observed. These data, albeit limited to a single study, may suggest that
DINP elicits effects on cell-adhesion molecules at the molecular level. Increased expression of '////'was
observed at 0.2 mg/kg-day DINP, but no other doses, and there were no corresponding changes in TNF-
a protein levels, implying that the '////'findings may be spurious. No other significant changes in gene
expression of cytokines were observed. Gene expression was significantly increased for the apoptotic
factors Aifml at 0.2 mg/kg-day (approximately 1.3-fold) and Bci2110 at 20 mg/kg-day (approximately 2-
fold), but the fold-increases were small, no other changes were observed at other doses, and TUNEL
staining did not reveal significant cell death. These data do not support involvement of apoptosis in the
effects of DINP on the gastrointestinal system. No changes were observed in expression of Ki67.
Chiu et al. (2021) evaluated the effects of DINP on gene and/or protein expression of specialized
epithelial cells of the colon and various immune factors. Treatment with DINP significantly increased
the level of Ki67 protein (a marker of cell proliferation) in the colons of mice in all treatment groups.
Notably. This differs from the findings of the prior publication from these authors, which reported no
changes in gene expression of Ki67; the inconsistency across studies decreases confidence in the data.
Treatment with DINP did not affect mRNA expression of leucine-rich repeat-containing G-protein
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coupled receptor (.Lgi'5), cluster of differentiation 24 antigen (Cd24a), chromogranin A (ChgA), villin 1
(Vill), which are markers for various cells in the colon; mRNA levels of several mucins (Mucl, Muc2,
Muc3a, Muc4), which are secreted from goblet cells to trap microbes; or alter mRNA expression of Toll-
like receptor 4 or 5 (Tlr4, Tlr5) in the colon. Treatment with DINP did increase colon mRNA levels of
lysozyme 1 (Lyzl) (a marker of Paneth cells) at 200 mg/kg-day DINP.
Conclusions on Gastrointestinal System Toxicity
EPA did not identify any epidemiologic studies of the gastrointestinal system for DINP and/or its
metabolites. Available animal evidence is limited to two studies. One study also identified
gastrointestinal effects, including reduced small intestine weight and villous atrophy in duodenum,
ileum, and jejunum, although these findings are likely related to decreased offspring body weight gain,
and secondary to decreased maternal food consumption (Setti Ahmed et al.. 2018). Chiu et al. (2020)
reported histopathological alterations in the colon of mice exposed to DINP for 10 days, but there was
no linear dose-response, and the histopathological evaluation had several methodological limitations that
decreased confidence and impacted the ability to interpret the results. Chiu et al. (2021) found that
exposure to DINP can increase Ki67, a marker for cell proliferation in the colon; however, the earlier
study by Chiu et al. (2020) did not observe any change in Ki67 expression. Additionally, other effects on
mRNA and cytokines in the colon were generally unaffected in Chiu et al. (2021). Overall, there is
limited evidence that DINP can elicit gastrointestinal toxicity in experimental laboratory animals.
Therefore, EPA is not further considering gastrointestinal system toxicity for dose-response analysis.
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4 DOSE-REPONSE ASSESSMENT
EPA is considering four non-cancer hazard endpoints related to liver, kidney, neurological and
developmental toxicity for dose-response analysis as described in the following sections. These hazard
endpoints were selected for dose-response analysis because EPA has the highest confidence in these
hazard endpoints for estimating risk to human health in the non-cancer sections. The effects for liver,
kidney, and developmental effects were consistently observed across multiple rodent species and
durations of exposure and occurred in a dose-related manner. EPA considered liver and developmental
effects observed in experimental animal models to be relevant for estimating risk to human health. Other
non-cancer hazard endpoints considered by EPA (i.e., cardiovascular toxicity (Section 3.5), immune
system toxicity (Section 3.6), musculoskeletal toxicity (Section 3.7) and gastrointestinal (Section 3.8)
were not considered for dose-response analysis due to limitations in the number of studies, unknown
MO A and uncertainties that reduce EPA's confidence in using these endpoints for estimating risk to
human health.
EPA considered two approaches, including a NOAEL/LOAEL approach, and benchmark dose modeling
for liver effects and benchmark dose modeling of developmental effects performed by NASEM (2017).
EPA considered NOAEL and LOAEL values from oral toxicity studies in experimental animal models.
Acute, intermediate, and chronic non-cancer NOAEL/LOAEL and BMDL values identified by EPA are
discussed further in Sections 4.1.1, 4.1.2, and 4.1.3, respectively. As described in Appendix F, EPA
converted oral PODs derived from animal studies to human equivalent doses (HEDs) using allometric
body weight scaling to the three-quarters power (U.S. EPA 2011b). In the absence of dermal toxicology
studies, EPA used the oral HED to assesses risks from dermal exposures. Differences in dermal and oral
absorption are corrected for as part of the dermal exposure assessment. In the absence of inhalation
studies, EPA performed route-to-route extrapolation to convert oral HEDs to inhalation human
equivalent concentrations (HECs) (Appendix F).
4.1 Selection of Studies and Endpoints for Non-cancer and Threshold
Cancer Health Effects
EPA considered the suite of oral animal toxicity studies for adverse liver, kidney, neurological and
developmental effects identified during hazard identification (Section 3) when determining non-cancer
PODs for estimating risks for acute, intermediate, and chronic exposure scenarios, as described in
Sections 4.1.1, 4.1.2, and 4.1.3, respectively. EPA assessed relevant non-cancer health effects in these
studies based on the following considerations:
• exposure duration;
• dose range;
• relevance (e.g., what species was the effect in, was the study directly assessing the effect, is the
endpoint the best marker for the toxicological outcome?);
• uncertainties not captured by the overall quality determination;
• endpoint/POD sensitivity; and
• total uncertainty factors (UFs).
The following sections provide comparisons of the above attributes for studies and hazard outcomes
relevant to each of these exposure durations and details related to the studies considered for each
exposure duration scenario.
4.1.1 Non-cancer Oral Points of Departure for Acute Exposures
EPA considered 15 developmental toxicity studies with endpoints relevant to acute exposure duration
(U.S. EPA. 1991b). summarized in Table 4-2. The endpoints considered relevant to acute exposure
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durations include skeletal and visceral variations, and effects on the developing male reproductive
system consistent with a disruption of androgen action during the critical window of male reproductive
development in rats. These studies were subjected to dose-response analysis to select the study and
endpoint most appropriate to derive the POD for acute hazard. The dose-response array for these studies
is depicted graphically in Figure 4-1. Although these studies entailed exposure durations that exceeded a
single day, EPA considered endpoints from these developmental toxicity studies for which there is
evidence that they can result from a single exposure day during a critical window of development during
gestation. For example, several studies have demonstrated that a single dose of DBP, which is
toxicologically similar to DINP (U.S. EPA 2023a. b), during the critical window of development {i.e.,
GD15.5-18.5) is sufficient to disrupt fetal testicular testosterone production and steroidogenic gene
expression. Although analogous single dose studies are not available for DINP, studies of DBP support
the conclusion that effects on the developing male reproductive system may occur following acute,
single dose exposures in rodent models (see Appendix C for further justification). Notably, the SACC
agreed with this scientific justification to use reduced fetal testicular testosterone and subsequent
downstream apical outcomes linked with this MOA to determine the acute duration POD (U.S. EPA
2024g).
In two prenatal developmental toxicity studies (Waterman et al.. 1999; Hellwig et al.. 19971 an
increased incidence of fetal skeletal variations (e.g., rudimentary/supernumerary cervical or lumbar ribs)
and urogenital variations (Hellwig et al.. 1997) were observed following exposure during GD6 to 15.
rudimentary/supernumerary cervical or lumbar ribs) and urogenital variations were observed following
exposure during GD6 through 15. However, the doses at which fetal visceral and skeletal variations
occurred (500 and 1,000 mg/kg-day) were higher than doses in other developmental toxicity studies in
which more sensitive effects of androgen insufficiency were observed. Therefore, EPA did not select
these studies and endpoints because they do not provide the most sensitive robust endpoint for an acute
POD.
The remaining 13 developmental toxicity studies considered by EPA resulted in effects on the
developing male reproductive system consistent with a disruption of androgen action during the critical
window of development. EPA identified this hazard in the Draft Proposed Approach for CRA for
Phthalates (U.S. EPA. 2023a) and concluded that the weight of scientific evidence indicates that DINP
can induce effects on the developing male reproductive system consistent with a disruption of androgen
action and rat phthalate syndrome. Notably, EPA's conclusion was supported by the SACC (U.S. EPA.
2023b). The exposure durations for these 13 studies ranged from initiation of dosing at implantation
through the day prior to expected parturition (i.e., GD7 to 21) as employed in most guideline studies, to
more narrow windows of exposure during gestation in which the phthalate-specific effects on male
rodent offspring are known to occur (e.g., GDI4 to 18) or extended to encompass the perinatal period
(e.g., GD14 to PND3). Observed effects included decreased steroidogenic gene expression in the fetal
testes, decreased fetal testicular testosterone, decreased male offspring AGD, increased male offspring
NR, effects on fetal Ley dig cells, increased incidence of MNGs, and decreased sperm motility. LOAELs
for these effects ranged from 100 to 1,165 mg/kg-day.
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IHEDLOAEL
AHEDNOAEL • Other Doses Tested
Q)
O
<4/ fetal testicular testosterone production; GD 14-18 ; rats; Hannas, 2011,
788239*
f MNGs (GD 21); >J,sperm motility (PND 90); GD 7-17; rats; Boberg, 2011,
806135°
•J, male pup body weight; 1" fetal Leydig cell clusters/aggregates; -J,
testicular mRNA levels of InsIB; GD 12-21; rats; Li, 2015, 2807612
•T" testicular mRNA levels of P450scc, GATA-4, and InsIB; GD 13.5-17.5; rats;
Adamsson, 2009, 679859
4> fetal testicular testosterone content and production; GD 7-21; rats; Borch,
2004, 673587
4- maternal body weight gain; ¦f male pup nipples/areola retention; testes
malformations; GD 14 - PND 3; rats; Gray, 2000, 678742
^ Fetal testis testosterone production; GD 14-18; rats; Furr, 2014, 2510906
•f incidence of rudimentary cervical & accessory lumbar ribs; urogenital &
skeletal variations; GD 6-15; rats; Hellwig, 1997, 674193
1s skeletal variations (total skeletal variations and rudimentary lumbar ribs);
GD 6-15; rats; Waterman, 1999, 680201
-J, male pup weight (PND 14), "T" MNGs (PND 2) in the testes; GD 12 - PND
14; rats; Clewell, 2013,1325348
¦f incidence of MNGs, .J, fetal testes testosterone (2 hrs post final dose); GD
12-19; rats; Clewell, 2013,1325350
¦ •
a •
10 100
HED (mg/kg-day)
1000
Figure 4-1. Dose-Response Array of Studies Considered for Deriving the Acute Duration Non-
cancer POD
Notes: | = statistically significant increase in response compared to controls; [ = statistically significant decrease
in response compared to controls; M = males; F= females; GD= Gestational Day; PND = Postnatal Day; MNGs =
multinucleated gonocytes; HED = human equivalent dose; NOAEL = no-observed-adverse-effect level; LOAEL
= lowest-observed-adverse-effect level.
11 Study included in NASEM (2017) meta-regression analysis and BMD modeling.
In 2017, NASEM (2017) assessed experimental animal evidence for effects on fetal testicular
testosterone following in utero exposure to DINP using the systematic review methodology developed
by the National Toxicology Program's (NTP) Office of Health Assessment and Translation (OHAT).
Based on results from four studies of rats (Li et al.. 2015; Boberg et al.. 2011; Hannas et al.. 2011;
Adamsson et al.. 20091 NASEM found high confidence in the body of evidence and a high level of
evidence that fetal exposure to DINP is associated with a reduction in fetal testosterone in rats. NASEM
further conducted meta-regression analysis and benchmark dose (BMD) modeling on decreased fetal
testicular testosterone production data from two medium-quality prenatal exposure studies of rats
(Boberg et al.. 2011; Hannas et al.. 2011). Testosterone data from Li et al. (2015) was not included in
the meta-regression and BMD modeling analysis because NASEM only included testosterone data
measured during the fetal lifestage (Li et al. evaluated testosterone on PND1), while data from Adamson
et al. (2009) was excluded because sufficient data were not reported to support its inclusion (i.e., the
exact number of litters per dose group was not report). NASEM found a statistically significant overall
effect and linear trends in logio(dose) and dose, with an overall large magnitude of effect (>50%) in its
meta-analysis for DINP (Table 4-1). Further BMD analysis determined BMDLs and BMDL40 values of
49 and 552 mg/kg-day, the 95 percent lower confidence limit of the BMD associated with a benchmark
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response (BMR) of 5 and 40 percent, respectively (Table 4-1). EPA has higher confidence in the
NASEM meta-analysis since it takes into account data from multiples studies.
Table 4-1. Summary of NASEM (2017) Meta-Analysis and BMD Modeling for Effects of DINP in
Fetal Testosterone (Using Metafor Version 2.0.0) "b
Database
Supporting
Outcome
Confidence
in Evidence
Evidence of
Outcome
Heterogeneity
in Overall
Effect
Model with
Lowest AIC
BMDs mg/kg-
day (95% CI)
BMD40 mg/kg-
day (95%, CI)
4 rat studies
High
High
I2 = 83%
Linear
quadratic
76 (49, 145)
701 (552, 847)
" R code suDDortina NASEM's meta-reeression and BMD analysis of DINP is Diibliclv available through GitHub.
b NASEM (2017) calculated BMD40s for this endooint because "previous studies have shown that reproductive-tract
malformations were seen in male rats when fetal testosterone production was reduced by about 40%."
In response to recommendations from the SACC, EPA conducted an updated meta-analysis and BMD
modeling analysis of decreased fetal rat testicular testosterone for DINP (U.S. EPA 2024g). Using the
publicly available R code provided by NASEM (https://github.com/wachiuphd/NASEM-2017-
Endocrine-Low-Dose). EPA applied the same meta-analysis and BMD modeling approach used by
NASEM, with the exception that the most recent Metafor package available at the time of EPA's
updated analysis was used (i.e., NASEM used Metafor Version 2.0.0, while EPA conducted the updated
meta-analysis with Metafor Version 4.6.0 and 2.0.0 so that results could be compared). EPA also
evaluated an additional BMR of 10 percent. Appendix G provides justification for the evaluated BMRs
of 5, 10, and 40 percent, while Appendix H provides a more detailed description of methods and results
for the updated analysis. Fetal rat testicular testosterone data from four studies was included in the
updated analysis, including new data from two studies (Gray et al.. 2024; Furr et al.. 2014). as well as
data from the two studies included in the 2017 NASEM analysis (Boberg et al.. 2011; Hannas et al..
2011). Overall, the meta-analysis conducted using Metafor Version 4.6.0 found a statistically significant
overall effect and linear trends in logio(dose) and dose, with an overall effect that is large in magnitude
(>50% change) (TableApx H-3). There was substantial, statistically significant heterogeneity in the
overall analysis (I2>80%). The statistical significance of these effects was robust to leaving out
individual studies. The linear-quadratic model provided the best fit (based on lowest AIC) (Table Apx
H-4). BMD estimates from the linear-quadratic model were 74 mg/kg-day [95% confidence interval: 47,
158] for a 5 percent change (BMR = 5%), 152 mg/kg-day [97, 278] for a 10 percent change (BMR =
10%), and 699 mg/kg-day [539, 858] for a 40 percent change (BMR = 40%) (Table_Apx H-4). Notably,
BMDs and BMD40 estimates calculated by NASEM (2017) and as part of EPA's updated analysis are
nearly identical (i.e., BMD5 values of 49 and 47 mg/kg-day; BMD40 values of 701 and 699 mg/kg-day).
One study (Li et al.. 2015) appears to demonstrate similar effects on male offspring at lower doses than
indicated in many of the other developmental toxicity studies. However, EPA did not consider this study
further as the sole study on which to derive the POD because several areas of uncertainty reduced EPA's
confidence in the results when considered independently from the other studies in the analysis. While
dose-dependent increases in testes dysgenesis and decreases in fetal testicular testosterone were noted,
this study had limited statistical power (n = 6). It is also unclear what the study authors considered the
broad description of "testes dysgenesis" to represent, although there is some indication that they are
referring to seminiferous tubule atrophy. Further, effects on male pup body weight were not dose-
related, with an essentially flat dose-response across doses spanning three orders of magnitude. A
similar flat dose-response was noted in the frequency distribution of cluster sizes of fetal Leydig cells,
and this endpoint is of uncertain adversity. Although this study supports EPA's conclusions regarding
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the endpoint for hazard identification, there is too much uncertainty in the dose-response in this study to
use it quantitatively for determination of the acute POD.
Two additional developmental toxicity studies not included among the four studies considered in the
meta-analysis by NASEM (Clewell et al.. 2013a; Clewell et al.. 2013b; Hamner Institutes for Health
Sciences. 2011) resulted in decreased fetal testosterone production and other effects on the developing
male reproductive system (i.e., increased incidence of MNGs) at similar doses (LOAELs from 250 to
307 mg/kg-day and NOAELs from 50 to 56 mg/kg-day) to the BMDLs of 49 and 47 mg/kg-day derived
from the NASEM meta-analysis and EPA's updated meta-analysis. Therefore, these studies support the
selection of the BMDLs of 49 mg/kg-day for the acute POD.
Although several other additional studies were identified for effects on the developing male reproductive
system and specifically for decreased fetal testicular testosterone, they were single doses studies (Gray
et al.. 2024; Furr et al.. 2014; Borch et al.. 2004; Gray et al.. 2000) with an identified LOAEL of 750
mg/kg-day. Similarly, several other studies identified effects on testosterone, steroidogenic gene
expression, male offspring nipple retention, and reproductive tract malformations and support LOAELs
of 500 to 1,000 mg/kg-day, but did not test sufficiently low doses to allow for the identification of a
NOAEL (Gray. 2023; Gray et al.. 2021). These studies are not very sensitive and support LOAELs
considerably higher than the LOAELs identified in the studies discussed above.
In a dietary study by Lee et al. (2006a). decreased male pup AGD was reported at the lowest dose tested,
40 ppm (estimated to be approximately 2 mg/kg-day). However, several factors reduce EPA's
confidence in this study and its results. First, study authors did not report dam body weight, food intake,
or calculate received doses in units of mg/kg-day, so there is uncertainty related to the achieved doses in
the study. Further, the effect of DINP on male pup AGD normalized to the cube root of bodyweight was
slight (overall magnitude of effect not reported), and treatment with DBP (a more potent antiandrogen
compared to DINP) at equivalent or higher doses had no effect on male pup AGD normalized to the
cube root of body weight. This calls into question the significances of the slight change in AGD
observed for DINP. Given these uncertainties, EPA does not consider the study by Lee et al. (2006a)
suitable for use as the acute POD.
Overall, the NASEM (2017) meta-analysis of fetal rat testicular testosterone supports a BMDLs of 49
mg/kg-day (HED of 11.6 mg/kg-day, which rounds to 12 mg/kg-day), while EPA's updated meta-
analysis supports a BMDLs of 47 mg/kg-day (HED of 11.1 mg/kg-day, which rounds to 11 mg/kg-day),
and the study by Clewell et al. (2013a) supports a NOAEL of 50 mg/kg-day (HED of 11.8 mg/kg-day,
which rounds to 12 mg/kg-day) based on reduced fetal testicular testosterone and increased incidence of
MNGs. HEDs were extrapolated from the BMDLs or NOAEL using allometric body weight scaling to
the three-quarters power (U.S. EPA. 2011b). EPA selected an HED of 12 mg/kg-day to use as the acute
exposure duration POD, which was extrapolated from the BMDLs from the NASEM (2017) meta-
analysis and the NOAEL from the study by Clewell et al. (2013a). A total uncertainty factor of 30 was
selected for use as the benchmark margin of exposure (based on an interspecies uncertainty factor (UFa)
of 3 and an intraspecies uncertainty factor (UFh) of 10). Consistent with EPA guidance (2022. 2002b.
1993). EPA reduced the UFa from a value of 10 to 3 because allometric body weight scaling to the
three-quarter power was used to adjust the POD to obtain a HED (see Appendix F). EPA considered
reducing the UFa further to a value of 1 based on apparent differences in toxicodynamics between rats
and humans. As discussed in Section 3.1.4 of EPA's Draft Proposed Approach for CRA for Phthalates
(U.S. EPA. 2023a). several explant (Lambrot et al.. 2009; Hallmark et al.. 2007) and xenograft studies
(van Den Driesche et al.. 2015; Spade et al.. 2014; Heger et al.. 2012; Mitchell et al.. 2012) using human
donor fetal testis tissue have been conducted to investigate the antiandrogenicity of mono-2-ethylhexyl
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phthalate (MEHP; a monoester metabolite of DEHP), DBP, and monobutyl phthalate (MBP; a
monoester metabolite of DBP) in a human model. Generally, results from human explant and xenograft
studies suggest that human fetal testes are less sensitive than rat testes to the antiandrogenic effects of
phthalates; however, effects on Sertoli cells and increased incidence of MNGs have been observed in
two human xenograft studies of DBP (van Den Driesche et al.. 2015; Spade et al.. 2014; Heger et al..
2012; Mitchell et al.. 2012). As discussed in EPA's draft approach document (U.S. EPA 2023a). the
available human explant and xenograft studies have limitations and uncertainties, which preclude
definitive conclusions related to species differences in sensitivity. For example, key limitations and
uncertainties of the human explant and xenograft studies include: small sample size; human testis tissue
was collected from donors of variable age and by variable non-standardized methods; and most of the
testis tissue was taken from fetuses older than 14 weeks, which is outside of the critical window of
development (i.e., gestational weeks 8-14 in humans). Therefore, EPA did not reduce the UFa.
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Table 4-2. Dose-Response Analysis of Selected Developmental Studies Considered for Deriving the Acute Non-cancer POD
Study Details
(Species, Duration, Exposure
Route/ Method, Doses [mg/kg-day])
Study POD/
Type
(mg/kg-
day)
Effect
HED
(mg/kg-
day)
HEC
(mg/m3)
[ppm]
Uncertainty
Factors d
Reference
Wistar-Imamichi rats GDI5 to
PND21; estimated doses (as reported
bv (EC/HC. 2015)) 0. 2. 20. 200.
1,000 mg/kg-day; (28 days)
LOEL=2
J.AGD& AGI, | in hypothalamic
granulin (grn, females) and pi30
(males) mRNA levels; reduced
lordosis quotient in females
0.473
2.57
[0.150]
UFa = 3
UFh= 10
UFl=10
Total UF = 300
(Lee et al.. 2006a)b
Pregnant SD rats; oral gavage (corn
oil); 0, 10, 100, 500, 1,000 mg/kg-
day; GDI 2-21
NOAEL =
10
[ male pup body weight; | fetal
Leydig cell clusters/aggregates; [
testicular mRNA levels for Ins 13
2.36
12.9 [0.75]
UFa = 3
UFH= 10
Total UF = 30
(Li et al.. 2015)11
Meta-regression and BMD modeling
of fetal testicular testosterone in rats
bmdl5 =
47
{ Fetal testicular testosterone
11.1
60.5 [3.53]
UFa = 3
UFH= 10
Total UF = 30
EPA updated meta-
analysis
Meta-regression and BMD modeling
of fetal testicular testosterone in rats
bmdl5 =
49
[ Fetal testicular testosterone
11.6
63.0 [3.68]
UFa = 3
UFH= 10
Total UF = 30
CNASEM. 2017)c
Pregnant SD rats; oral gavage; 0, 50,
250, 750 mg/kg-day; GD12-19
NOAEL =
50
t incidence ofMNGs, J, fetal testes
testosterone (2 hours post final
dose)
11.8
64.3 [3.76]
UFa = 3
UFH= 10
Total UF = 30
(Clewell et al..
2013a)
Pregnant SD rats; dietary; 0, 760,
3,800, 11,400 ppm (estimated: 56,
288, 720 mg/kg-day on GD13-20;
109, 555, 1,513 mg/kg-day on PND2-
14); GD12-PND14
NOAEL =
56
J, male pup weight (PND14), f
MNGs (PND2) in the testes
13.2
72.1 [4.21]
UFa = 3
UFH= 10
Total UF = 30
(Clewell et al..
2013b)
Pregnant SD rats; oral gavage; 0, 100,
500, 1,000 mg/kg-day; GD6-15
NOAEL =
100
t skeletal variations (total skeletal
variations and rudimentary lumbar
ribs)
23.6
129 [7.52]
UFa = 3
UFH= 10
Total UF = 30
(Waterman et al..
1999)
Pregnant Wistar rats; oral gavage; 0,
40, 200, 1,000 mg/kg-day; GD6-15
NOAEL =
200
t incidences of rudimentary cervical
and accessory lumbar ribs;
urogenital and skeletal variations
47.3
257 [15.0]
UFa = 3
UFH= 10
Total UF = 30
(Hellwia et al..
1997)
Pregnant Wistar rats; oral gavage
(corn oil); 0, 300, 600, 750, 900
mg/kg-day; GD7-17
NOAEL =
300
| MNGs (GD21); J,sperm motility
(PND90)
70.9
386 [22.6]
UFa = 3
UFH= 10
Total UF = 30
(Bobera et al..
2011)17
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Study Details
(Species, Duration, Exposure
Route/ Method, Doses [mg/kg-day])
Study POD/
Type
(mg/kg-
day)
Effect
HED
(mg/kg-
day)
HEC
(mg/m3)
[ppm]
Uncertainty
Factors d
Reference
Pregnant Harlan SD rats; Oral gavage
(corn oil); 0, 500, 750, 1,000, 1,500
mg/kg-day; GD14-18
LOAEL =
500
[ ex vivo fetal testicular testosterone
production
118
643 [37.6]
UFa = 3
UFh=10
UFl = 10
Total UF = 300
(Hannas et al..
2011)17
Pregnant Harlan SD rats; Oral gavage
(corn oil); 0, 500, 750, 1,000, 1,500
mg/kg-day; GD14-18
LOAEL =
500
[ ex vivo fetal testicular testosterone
production and [ steroidogenic gene
expression in the fetal testis
118
643 [37.6]
UFa = 3
UFh=10
UFl = 10
Total UF = 300
(Grav et al.. 2021)
Pregnant SD rats; oral gavage (corn
oil); 0, 750 mg/kg-day; GD14-18
LOAEL =
750
[ ex vivo fetal testis testosterone
production
111
965 [56.4]
UFa = 3
UFH= 10
UFl = 10
Total UF = 300
(Furr et al.. 2014)
Pregnant Wistar rats; oral gavage
(peanut oil); 0, 750 mg/kg-day; GD7-
21
LOAEL =
750
[ ex vivo fetal testicular testosterone
content and production
111
965 [56.4]
UFa = 3
UFh=10
UFl = 10
Total UF = 300
(Borch et al.. 2004)
Pregnant SD rats; oral gavage (corn
oil); 0, 750 mg/kg-day; GD14-PND3
LOAEL =
750
[ maternal body weight gain; f
male pup nipples/areola retention;
testes malformations (small,
atrophic, flaccid, fluid-filled,
azoospermia, epididymal agenesis)
111
965 [56.4]
UFa = 3
UFH= 10
UFl=10
Total UF = 300
(Grav et al.. 2000)
Pregnant SD rats; oral gavage (corn
oil); 0, 750 mg/kg-day; GD14-18
LOAEL =
750
[ ex vivo fetal testis testosterone
production and [ steroidogenic gene
expression in the fetal testis
111
965 [56.4]
UFa = 3
UFH= 10
UFl = 10
Total UF = 300
(Grav et al.. 2024)
Pregnant SD rats; oral gavaged (corn
oil) 0, 1,000, 1,500 mg/kg-day;
GDI4- PND3
LOAEL =
1,000
t F1 male offspring nipple retention
and | incidence of total
reproductive tract malformations in
F1 males
236
1287 [75]
UFa = 3
UFH= 10
UFl = 10
Total UF = 300
(Grav. 2023)
Pregnant SD rats; oral gavage (corn
oil); 0, 250, 750 mg/kg-day;
embryonic day 13.5-17.5
NOEL =
250
t testicular mRNA levels of
P450scc, GATA-4, Insl3
(Adamsson et al..
2009)17
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Study Details
(Species, Duration, Exposure
Route/ Method, Doses [mg/kg-day])
Study POD/
Type
(mg/kg-
day)
Effect
HED
(mg/kg-
day)
HEC
(mg/m3)
[ppm]
Uncertainty
Factors d
Reference
11 Study considered as part of NASEM analysis (NASEM. 2017). EPA did not consider this study (Li et al.. 2015) further as the sole study on which to derive the
POD because several areas of uncertainty (e.g., low statistical power with n=6, questionable dose-response and uncertain adversity among several endpoints)
reduced EPA's confidence in the results when considered independently from the other studies in a meta-analysis.
h Lee et al. (2006a) was not suitable for use to determine an acute POD due to uncertainties (e.g., reporting deficiencies for dam body weight and food
consumption for a dietary exposure study, and others described in the text).
CR code supporting NASEM's meta-regression and BMD analysis of DINP is publicly available through GitHub (https://github.com/wachiuphd/NASEM-2017-
Endocrine-Low-Dose).
J EPA used allometric body weight scaling to the three-quarters power to derive the HED. Consistent with EPA Guidance (U.S. EPA. 2011b). the interspecies
uncertainty factor (UFa), was reduced from 10 to 3 to account remaining uncertainty associated with interspecies differences in toxicodynamics. EPA used a
default intraspecies (UFh) of 10 to account for variation in sensitivity within human populations due to limited information regarding the degree to which
human variability may impact the disposition of or response to DINP. EPA used a LOAEL-to-NOAEL uncertainty factor (UFl) of 10 to account for the
uncertainty inherent in extrapolating from the LOAEL to the NOAEL.
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4.1.2 Non-cancer Oral Points of Departure for Intermediate Exposures
EPA considered 12 intermediate (>1 to 30 days) oral exposure studies (6 of rats and 6 of mice) of DINP
for establishing the intermediate duration POD (Table 4-3). Figure 4-2 depicts the dose-response array
for available studies. Ultimately, EPA selected the acute POD (12 mg/kg-day) and benchmark MOE
(total UF of 30) identified in Section 4.1.1 to evaluate risk from intermediate exposures {i.e., ranging
from 1 to 30 days) to DINP.
The acute POD is more sensitive than many of the intermediate HEDs based on liver, kidney, or
developmental toxicity in rodents. As can be seen from Table 4-3 and Figure 4-2, of the 12 intermediate
studies under consideration, 7 supported HEDs ranging from 15.6 to 401 (Kwack et al.. 2009; Kaufmann
et al.. 2002; Smith et al.. 2000; Hazleton Labs. 1991a; BIBRA. 1986; Bio/dynamics. 1982a; Midwest
Research Institute. 1981). These studies are less sensitive than the acute POD (HED of 12 mg/kg-day).
Further, several of these studies are limited by poor dose selection and did not test doses low enough to
support NOAEL identification (Hazleton Labs. 1991a; BIBRA. 1986; Midwest Research Institute. 1981)
or only tested a single high dose of DINP (Kwack et al.. 2009; Bio/dynamics. 1982a).
Five intermediate studies (Ma et al.. 2015; Peng. 2015; Ma et al.. 2014; Masutomi et al.. 2003; Smith et
al.. 2000) report HEDs based on NOAELs ranging from 2.0 to 10 mg/kg-day, indicating that they are
more sensitive than the HED that EPA selected for a POD. However, each of these studies had
uncertainties that reduced EPA confidence in their use for deriving a POD for intermediate duration
exposure.
Masutomi et al. (2003) supports a developmental NOAEL of 31 mg/kg-day (HED of 7.3 mg/kg-day)
based on reduced F1 male offspring body weight on PND27. However, this study is limited by its small
sample size (5 rats per dose group). Further, the biological significance of the effect on F1 male body
weight is unclear, as F1 male bodyweight was unaffected on PND2, and no effect on F1 male
bodyweight gain was observed from PND2 to PND10 or PND10 to PND21, and by PND77 F1 male
body weight had recovered to control levels. These limitations and uncertainties reduce EPA's
confidence in using the study by Masutomi et al. (2003) for the intermediate POD.
Three studies (Ma et al.. 2015; Peng. 2015; Ma et al.. 2014) reported treatment-related effects on
endpoints indicating oxidative stress, but it is unclear if the apparent effects on neurotoxicity (Ma et al..
2015; Peng. 2015) reported in Section 3.4, and the findings in the liver and kidney (Ma et al.. 2014)
reported in Section 3.2 and 3.3 can be directly attributed to the oxidative stress and inflammatory
responses observed in the studies. Although there is some evidence showing protective effects of
antioxidants in mitigating the effects of treatment with DINP, there is not enough data to determine the
link to the apparent effects on neurotoxicity and on the liver and kidneys. This limitation is due, in large
part, to the lack of quantitative data on the incidence or severity of the histopathology findings in the
brain, liver, and kidney. These data were only described qualitatively, with representative micrographs
of control and high dose groups presented as images, which precludes their usefulness to set a POD.
Additional limitations in the two neurotoxicity studies are described below.
In the two neurotoxicity studies (Ma et al.. 2015; Peng. 2015). male Kunming mice were administered
DINP by oral gavage at doses up to 200 mg/kg-day, followed by swim trials in the Morris Water Maze
to determine effects on learning and memory, along with measurements of oxidative stress and
histopathology evaluation of the brain. However, EPA identified several deficiencies in the study
methods and reporting. First, both studies only report mean escape latency of each swimming trial over
the 7-day acquisition phase but provide no measure of variability. Neither study conducted statistical
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analysis on escape latency times within a given trial, but instead conducted statistical analysis on the
average escape latency times across the 7 trials. Therefore, EPA is not able to determine whether there
is a significant interaction between treatment and time to determine if the learning curve was steeper in
the controls compared to the mice administered DINP. Second, path length provides another measure of
learning, with path length decreasing over the acquisition phase if learning is occurring. The North
American Free Trade Agreement (NAFTA) Technical Working Group on Pesticides (TWG) -
Developmental Neurotoxicity Study Guidance Document (U.S. EPA 2016) indicates that the mean path
length per trial should be reported, as this outcome is highly correlated with escape latency times. Both
studies (Ma et al.. 2015; Peng. 2015) report use of camera tracking and computer software (ANY-
Maze), which has the capabilities to determine path length. However, neither study reports the path
length numerically for the swimming trials, but instead only depict an image of the swim path for a
representative trial in the high dose and control groups. The lack of quantitative data on swim path
length precludes EPA's ability to discern whether any increase in swim time is due to actual deficits in
learning and memory, or if there is an increase in swim time due to general toxicity (i.e., swimming
more slowly). Neither study included performance controls.
Per the NAFTA guidance document, swim speed and cued-trials are two common performance controls
that can be used to rule out treatment-related visual and motor impairments that can confound
interpretation of cognitive deficits (e.g., longer latency times may be due to slower swim speeds, not
cognitive impairment). Third, for the probe trial, Ma et al. (2015) report both the target quarter retention
time and the number of entries into the target quadrant, which is consistent with the NAFTA guidance
document. There is a clear treatment related effect on target quadrant retention time; however, the
controls spent only -16 seconds in the target quadrant, which is only slightly above chance levels of 25
percent. NAFTA guidance states that controls must show an increase in percent time in the correct
quadrant that exceeds chance levels of 25 percent. For the probe trial by Peng (2015). target quadrant
retention time is reported, and controls spent approximately 25 seconds in the target quadrant, well
above chance levels of 25 percent, but the number of entries into the target quadrant was not reported.
Fourth, both of these studies (Ma et al.. 2015; Peng. 2015) reported alterations in pyramidal cells in
hippocampus at the high dose (150 and 200 mg/kg-day); however, no quantitative data were provided on
the incidence or severity of the histopathology findings; the data were only described qualitatively, with
representative micrographs of control and high dose groups presented as images. Taken together, these
uncertainties limit the utility of the neurological studies for use in determining an intermediate duration
POD.
Finally, one study (Smith et al.. 2000) provided a HED value for the NOAEL (10 mg/kg-day) in the
same range as the acute HED value (12 mg/kg-day). Smith et al. (2000) report treatment-related
increases in liver weights, hepatic peroxisomal beta oxidation (PBOX), and DNA synthesis,
accompanied by inhibition of gap junctional intercellular communication (GJIC), in male B6C3F1 mice
fed diets containing 6,000 ppm DINP (approximately 900 mg/kg-day) for up to 4 weeks. This study is
limited in dose selection, with only two treated groups with doses spanning a wide range between the
NOAEL in the low-dose group at 75 mg/kg-day and the LOAEL in the high dose at 900 mg/kg-day.
Therefore, EPA did not consider the dose selection to be refined enough or endpoints examined to be
comprehensive enough to establish a robust POD. However, the fact that the HED value from this study
aligns with the HED from the acute POD adds further support to EPA's selection of the acute POD to be
protective of intermediate exposure durations.
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>
-------
Table 4-3. Dose-Response Analysis of Select
ed Studies Considered for Deriving the Intermediate
Non-cancer POD
Target
Organ/
System
Study Details
(Species, Duration, Exposure
Route/ Method, Doses [mg/kg-day])
Study POD/
Type (mg/kg-
day)
Effect
HEP
(mg/kg)
HEC
(mg/m3)
[ppm]
Uncertainty
Factors"
Reference
Neurotoxicity
Kunming mice (males only); oral
gavage; 0, 1.5, 15, 150 mg/kg-day; 9
days
NOAF.L = 15
i body weight gain; impaired learning
& memory in Morris Water Maze;
oxidative stress & inflammation;
histopathological alterations in
pyramidal cells in hippocampus
1.99
10.9
[0.634]
UFa = 3
UFh=10
Total UF = 30
(Pens. 2015)
Neurotoxicity
Kunming mice (males only); oral
gavage; 0, 0.2, 2, 20, 200 mg/kg-day;
14 days
NOAF.L = 20
Histopathological alterations in
pyramidal cells; oxidative stress &
inflammation
2.66
14.5
[0.845]
UFa = 3
UFh=10
Total UF = 30
(Ma et al..
2015)
Liver and
Kidney
Kunming mice (males only); oral
gavage; 0, 0.2, 2, 20, 200 mg/kg-day;
14 days
NOAF.L = 20
Markers of oxidative stress (t ROS,
| GSH, t MDA, t 8-OH-dG) &
inflammation (f IL-1, f TNFa)
2.67
14.5
[0.845]
UFa = 3
UFh = 10
Total UF = 30
(Ma et al..
2014)
Developmental
Pregnant SD rats; dietary; 0, 400,
4,000, 20,000 ppm (estimated: 31-66,
307-657, 1,165-2,657 mg/kg-day);
GD15 to PND10
NOAF.L = 31
(males);
66 (females)
i male body weight on PND27
7.33
39.9 [2.33]
UFa = 3
UFh = 10
Total UF = 30
(Masutomi et
al.. 2003)
Liver
B6C3F1 mice (males only); dietary;
0, 500, 6,000 ppm (estimated: 75, 900
mg/kg-day); 2 and 4 weeks
NOEL = 75
Hepatic changes (t liver weight,
t PBOX, t DNA synthesis; inhibition
of GJIC)
9.97
54.3 [3.17]
UFa = 3
UFh = 10
Total UF = 30
(Smith et al..
2000)
Liver
B6C3F1 mice (both sexes); dietary;
0, 500, 1,500, 4,000, 8,000 ppm
(estimated: 117, 350, 913, 1,860
mg/kg-day [males]; 167, 546, 1,272,
2,806 mg/kg-day [females]); 1 or 4
weeks
NOAF.L=
117 (males)
t absolute and relative liver weight; t
peroxisomal volume, and peroxisomal
enzyme activity; t hepatocyte
proliferation in males
15.6
84.7 [4.95]
UFa = 3
UFh= 10
Total UF = 30
(Kaufmann et
al.. 2002)
Liver
F344 rats (males only); dietary; 0,
1,000, 12,000 ppm (estimated: 100,
1,200 mg/kg-day); 2 and 4 weeks
NOAF.L = 100
Hepatic changes (t liver weight,
t PBOX, t DNA synthesis; inhibition
of GJIC)
23.6
129 [7.52]
UFa = 3
UFh = 10
Total UF = 30
(Smith et al..
2000)
Liver &
Kidney
F344 rats (both sexes); dietary; 0, 0.2,
0.67, 2% (estimated: 150, 500, 1,500
mg/kg-day [males]; 0, 125, 420,
1,300 mg/kg-day [females]); 28 days
LOEL=
125 (females)
t hepatic catalase and carnitine
acetyltransferase activity
29.6
161 [9.39]
UFa = 3
UFh= 10
UFl = 10
Total UF =
300
(Midwest
Research
Institute. 1981)
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Target
Organ/
System
Study Details
(Species, Duration, Exposure
Route/ Method, Doses [mg/kg-day])
Study POD/
Type (mg/kg-
day)
Effect
HED
(mg/kg)
HEC
(mg/m3)
[ppm]
Uncertainty
Factors"
Reference
Liver
B6C3F1 mice (both sexes); dietary;
0,3,000,6,000, 12,500 ppm
(estimated: 635, 1,377, 2,689, 6,518
mg/kg-day [males]; 780, 1,761,
3,287, 6,920 mg/kg-day [females]); 4
weeks
LOAEL=
635 (males)
Enlarged and discolored livers;
t incidence of hepatocytomegaly
84.4
460 [26.8]
UFa = 3
UFh= 10
UFl = 10
Total UF =
300
(Hazleton Labs.
1991a)
Liver
SD rats (males only); oral gavage; 0,
500 mg/kg-day; 28 days
LOAEL = 500
i body weight gain; f relative liver
weight; clinical chemistry (f AST,
ALP, triglycerides)
118
643 [37.6]
UFa = 3
UFh= 10
UFl = 10
Total UF =
300
(Kwack et al..
2009)
Liver
F344 rats (both sexes); diet; 0, 0.6,
1.2, 2.5% (estimated: 639, 1,192,
2,195 mg/kg-day [males]; 607, 1,198,
2,289 mg/kg-day [females]); 21 days
LOAEL=
607 (females)
t absolute and relative liver weight;
|11- and 12-hydroxylase activity,
hypolipidemic effects
144
781 [45.6]
UFa = 3
UFh= 10
UFl = 10
Total UF =
300
(BIBRA. 1986)
Liver and
Kidney
F344 rats (males only); dietary; 0, 2%
(estimated: 1,700 mg/kg-day); 7 days
LOAEL=
1,700
t absolute and relative liver and kidney
weight, macroscopic liver
observations, changes in clinical
chemistry
402
2,187
[128]
UFa = 3
UFh= 10
UFl = 10
Total UF =
300
(Bio/dvnamics.
1982a)
" EPA used allometric bodv weieht scaline to the three-auarters rower to derive the HED. Consistent with EPA Guidance (U.S. EPA. 201 lb), the interspecies
uncertainty factor (UFA), was reduced from 10 to 3 to account remaining uncertainty associated with interspecies differences in toxicodynamics. EPA used a default
intraspecies (UFH) of 10 to account for variation in sensitivity within human populations due to limited information regarding the degree to which human variability may
impact the disposition of or response to DINP. EPA used a LOAEL-to-NOAEL uncertainty factor (UFL) of 10 to account for the uncertainty inherent in extrapolating
from the LOAEL to the NOAEL.
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4.1.3 Non-cancer Oral Points of Departure for Chronic Exposures
EPA considered four 2-year chronic dietary studies (3 of rats, 1 of mice), six 13-week subchronic dietary
studies (4 of rats, and 1 each of mice and beagles), a one-generation study of reproduction of rats, and a
two-generation study of reproduction of rats for establishing the chronic POD (Table 4-5). Across one-
and two-generation studies of reproduction, reduced offspring bodyweight was the most sensitive effect,
while liver and kidney toxicity were the most sensitive effects observed across chronic and subchronic
studies, and these effects were considered for establishing the chronic POD. Figure 4-3 depicts the dose-
response array for available studies.
Across the one- and two-generation studies of reproduction (Waterman et al.. 2000; Exxon Biomedical
1996a. b), both of which were GLP-compliant and adhered to available guidelines (40 CFR part 798,
section 798.4700), LOAELs for developmental effects were 377 mg/kg-day in the one-generation study
based on reduced male and female F1 offspring body weight on PND0, 14, and 21; and 133 mg/kg-day
in the two generation study based on reduced F1 and F2 offspring body weight on PND7 and 21. Neither
study tested sufficiently low doses to establish a developmental NOAEL. Further, there is some
uncertainty associated with the LOAEL from the two-generation study, as F1 offspring bodyweight
(both sexes) was reduced on PND21, while F2 offspring body weight was reduced only on PND7 for
females (Table 3-8). More consistent effects on F1 and F2 offspring body weight were observed in the
mid-dose group. These sources of uncertainty reduce EPA's confidence in using the LOAEL of 133
mg/kg-day from the two-generation study as a chronic POD. Further, EPA identified more sensitive
PODs based on liver toxicity from subchronic and chronic studies that tested lower doses of DINP and
allowed for the identification of a NOAEL.
Across the six available subchronic studies, the lowest LOAELs for each of the tested species were 160
mg/kg-day in beagles (NOAEL = 37 mg/kg-day; HED = 23) based on increased absolute and relative
liver weight and increase serum ALT (Hazleton Laboratories. 1971); 972 mg/kg-day in mice (NOAEL =
365; HED = 49 mg/kg-day) based on increased absolute and relative liver weight and histopathological
findings (e.g., necrosis) (Hazleton Labs. 1992); and 60 mg/kg-day in SD rats (no NOAEL identified;
HED = 14 mg/kg-day) based on increased incidence of histopathological lesions in the kidney of male
rats (i.e., focal mononuclear cell infiltration and mineralization) (Hazleton Labs. 1981). LOAELs based
on liver and kidney toxicity from the remaining three subchronic studies of rats were less sensitive and
ranged from 176 to 227 mg/kg-day (Hazleton Labs. 1991b; Bio/dynamics. 1982b. c). The study of
beagles was conducted prior to the establishment of GLP principles and OECD test guidelines, and
additionally only included four dogs per sex in each treatment group, so no statistical analysis was
performed due to the small sample size (Hazleton Laboratories. 1971). These limitations reduced EPA's
confidence in using the study to establish a chronic POD, and importantly, other subchronic and chronic
studies of rats provide more sensitive and health protective candidate PODs. Similarly, the one
subchronic study of mice (Hazleton Labs. 1992) provides a less sensitive candidate POD compared to
studies of rats. The lowest subchronic LOAEL of 60 mg/kg-day in rats comes from a study conducted
prior to the establishment of GLP principles and OECD test guidelines (Hazleton Labs. 1981). and did
not test sufficiently low doses to establish a NOAEL. Furthermore, EPA did not consider this study
sufficient for selection of a POD because it only reported effects on kidney in male rats which may be
related to a2u-globulin and not relevant for human health.
Across the four available 2-year dietary studies of rats and mice, the lowest LOAEL is 152 mg/kg-day
(NOAEL =15 mg/kg-day; HED = 3.5 mg/kg-day) from a 2-year dietary study of F344 rats (Lington et
al.. 1997; Bio/dynamics. 1986). The study by Lington et al. is GLP-compliant and received a high
overall study quality determination. Although the study does not explicitly state compliance with any
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testing guidelines, it generally follows the guidelines outlined by OECD Test Number 453 (Combined
Chronic Toxicity/Carcinogenicity Studies). At the LOAEL, a spectrum of dose-related effects consistent
with liver toxicity was observed in male and female rats, including treatment related increases in relative
liver weight, serum ALT, AST, and ALP, and histopathological findings (e.g., spongiosis hepatis,
sinusoid ectasia). One source of uncertainty associated with the findings of Lington et al. results from
spongiosis hepatis. The MOA underlying spongiosis hepatis is unknown but is not believed to be related
to peroxisome proliferation. Further, as discussed by ECHA (2013b). spongiosis hepatis has been
observed in the livers of some strains of rats and certain species of fish (e.g., medaka), but is less
common in mice, has not been observed in non-human primates or dogs, and with the exception of two
case reports, has not been described in humans. These findings raise some uncertainty as to the human
relevance of spongiosis hepatis (Karbe and Kerlin. 2002). However, spongiosis hepatis co-occurred with
other hepatic effects that are more clearly adverse and relevant for use in human health risk assessment
(e.g., increase liver weight, serum ALT, AST, ALP, focal necrosis). Further supporting use of the
LOAEL reported by Lington et al., similar hepatic effects (e.g., increased relative liver weight, serum
ALT, AST, and ALP, spongiosis hepatis, necrosis) have consistently been reported in two other chronic
dietary studies of DINP with F344 (Covance Labs. 1998c) and SD rats (Bio/dynamics. 1987). albeit at
slightly higher doses of DINP (Table 4-5).
Given the broad dose spacing between the NOAEL of 15 mg/kg-day and LOAEL of 152 mg/kg-day
identified in Lington et al. (1997). EPA attempted to refine the POD by conducting BMD modeling in
accordance with EPA's Benchmark Dose Technical Guidance (U.S. EPA. 2012). Endpoints modeled
included relative liver weight at terminal sacrifice (both sexes); serum ALT at 6-and 18-month sacrifices
(males only); incidence of focal necrosis in the liver (both sexes); incidence of spongiosis hepatis (males
only); and incidence of sinusoid ectasia (males only). For each endpoint, multiple BMRs were modeled.
BMD modeling results are presented in Appendix E, and results for representative BMRs are presented
in Table 4-4. For dichotomous endpoints, BMDLio values ranged from 8.6 mg/kg-day for spongiosis
hepatis to 125 mg/kg-day for focal necrosis in male rats. BMDLio values for spongiosis (8.6 mg/kg-day)
in the liver and sinusoid ectasia in the liver (14 mg/kg-day) were less than the study NOAEL of 15
mg/kg-day; however, BMD/BMDL ratios were greater than 3 (ranging from 3.7 to 8.9), indicating
model uncertainty. For continuous endpoints, the BMDLio was 85 mg/kg-day for increased relative liver
weights for males, while no models adequately fit relative liver weight data for female rats. For increase
in serum ALT at 6 and 18 months, BMDLioo values were 87 and 134 mg/kg-day, respectively. A BMR
of 100 percent was selected for this endpoint since 2 to 3 fold changes in ALT are generally considered
biologically significant and outside the range of normal variation (Hall et al.. 2012; U.S. EPA. 2002a).
However, there is some uncertainty related to the BMR selection, so EPA also presents BMDLisd values
in Table 4-4, which is consistent with EPA's Benchmark Dose Technical Guidance (U.S. EPA. 2012).
BMDLisd values for increased serum ALT at 6 and 18 months were 16 and 33 mg/kg-day, respectively.
Overall, calculated BMDLs shown in Table 4-4 ranged from 8.6 to 134 mg/kg-day, which is similar to
the study NOAEL and LOAEL values of 15 and 152 mg/kg-day. The wide variability in BMDLs and
uncertainty in several modelled outcomes (i.e., BMD/BMDL ratios greater than 3) reduce EPA's
confidence in using the BMD modeling results for establishing a POD.
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Table 4-4. Summary of B
MD Model Results from Lingt
on et al. (1997)
Endpoint
Sex
Selected Model
BMDisd /
BMDLisd
(mg/kg-day)
BMDio/
BMDLio
(mg/kg-day)
BMDioo/
BMDLioo
(mg/kg-day)
Dichotomous endpoints
Focal necrosis in the liver
Male
Logistic
-
159/125
-
Focal necrosis in the liver
Female
Log-Probit
-
222/34
-
Spongiosis hepatis in the
liver
Male
Log-Probit
-
32/8.6
-
Sinusoid ectasia in the liver
Male
Log-Probit
-
125/14
-
Continuous endpoints
Relative liver weight at
terminal sacrifice
Male
Linear, CV
242/196
106/85
-
Relative liver weight at
terminal sacrifice
Female
None selected;
LOAEL (184 mg/kg-
day) was used
"
"
"
Serum ALT at 6-month
sacrifice
Male
Linear
23/16
-
125/87
Serum ALT at 18-month
sacrifice
Male
Power
63/33
-
179/134
Overall, EPA selected the NOAEL of 15 mg/kg-day based on liver toxicity observed in a 2-year dietary
study of F344 rats (Lington et al.. 1997; Bio/dynamics. 1986) as the chronic POD for use in estimating
non-cancer risk from exposure to DINP in the risk evaluation of DINP. This POD represents the most
sensitive POD identified by EPA. Furthermore, the NOAEL of 15 mg/kg-day supports the suite of
effects liver occurring at 152 mg/kg-day in Lington et al. (1997). Consistently, other regulatory bodies
have selected the same chronic POD for use in quantifying risk from exposures to DINP (ECCC/HC.
2020; EFSA 2019; U.S. CPSC. 2014; ECFLA 2013b). EPA used allometric body weight scaling to the
three-quarters power to derive an HED of 3.5 mg/kg-day from the NOAEL of 15 mg/kg-day (U.S. EPA
2011b). A total uncertainty factor of 30 was selected for use as the benchmark margin of exposure
(based on an interspecies uncertainty factor (UFa) of 3 to account for intraspecies differences in
toxicodynamics and an intraspecies uncertainty factor (UFh) of 10 to account for variability in the
human population that might lead to increased susceptibility). Consistent with EPA guidance (2022.
2002b. 1993). EPA reduced the UFa from a value of 10 to 3 because allometric body weight scaling to
the three-quarter power was used to adjust the POD to obtain a HED, which accounts for interspecies
differences in toxicokinetics (see Appendix F).
As discussed further in Appendix I, EPA considered reducing the toxicodynamics component of the
UFa should be reduced from 3 to 1 based on differences in species sensitivity to the liver effects that
form the basis of the chronic POD. As described in EPA's Cancer Raman Health Hazard Assessment
for DiisononylPhthalate (DINP) (U.S. EPA. 2025a). the weight of evidence indicates that humans are
less sensitive than rodents to liver effects associated with PPARa activation, which could support a
reduction in the toxicodynamics component of the UFa from 3 to 1. However, the chronic POD of 3.5
mg/kg-day is based on a spectrum of liver effects, some of which are related to PPARa activation (e.g.,
t liver weight, hypertrophy, necrosis) and some of which are PPARa-independent (i.e., spongiosis
hepatis). Given that the chronic POD is based on liver effects that are both dependent and independent
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of PPARa and the uncertainty in mode of action associated with spongiosis hepatis, EPA concluded that
a reduction in the toxicodynamics component of the UFAfrom 3 to 1 is not warranted.
Overall, EPA considers the selected chronic POD most applicable to adults (16 years and older). Use of
this POD for characterization of risk to infants and children from chronic exposure to DINP may be
conservative and may not be relevant. As described further in Appendix I, the chronic POD is based on
liver effects dependent and independent of PPARa activation. As discussed in EPA's Cancer Human
Health Hazard Assessment for Diisononyl Phthalate (DINP) (U.S. EPA. 2025a). there is evidence to
suggest humans are less sensitive than rats to liver effects associated with PPARa activation, while the
PPARa-independent effects (i.e., spongiosi s hepatis) are most prevalent in the livers of aging rats.
o>
>
>
0)
c
-a
5
>
QJ
o
1" abs. and rel. liver wt; hepatocyte enlargement; other histopathology in
liver; 13 weeks; mice (M/F); Hazleton Laboratories, 2000,1987581
1s abs. and rel. liver wt. and ¦f hepatocyte hypertrophy; 13 weeks; rats
(M/F); Hazleton Laboratories, 1971,1987593
t abs. and rel. liver wt.; 4^ cholesterol (F only); 13 weeks; rats (M/F);
Bio/dynamics, 1982,679936
'f abs. and rel. liver wt.; T ALT activity; 13 weeks; dogs (M/F); Hazleton
Laboratories, 1971,1987591
1" absolute & relative liver weight; i" non-neoplastic lesions; 2 years; mice
(F); Covance Labs, 1998,1325481
1" absolute & relative liver weight; 1s non-neoplastic lesions; 1s liver
masses; 2 years; mice (M); Covance Labs, 1998,1325481
1s absolute & relative liver weight; one-generation developmental study; PI
generation; rats (M/F); Waterman, 2000, 680202
t1 absolute & relative liver weight; in serum ALT & AST; non-neoplastic
lesions; 2 years; rats (F); Covance Laboratories, 1998,680087
1s absolute & relative liver weight; in serum ALT & AST; 1" non-neoplastic
lesions; 2 years; rats (M); Covance Laboratories, 1998,680087
't serum ALT, AST & ALP; 1* spongiosis hepatis & focal necrosis (minimal-to-
slight); 2 years; rats (M); Bio/dynamics, 1987,679889
/T' absolute & relative liver weight; f in serum ALT & AST; non-neoplastic
lesions; 2 years; rats (M/F); Lington, 1997,1239588
^ abs. and rel kidney wt. accompanied by 4' in triglycerides and altered
urine chemistry; 13 weeks; rats (M/F); Biodynamics 1982, 679937
4/ absolute kidney weight; 2 years; mice (M); Covance Labs, 1998,1325481
1s absolute & relative kidney weight; one-generation study; PI generation;
rats (M/F); Waterman, 2000,680202
f absolute & relative kidney weight; T- non-neoplastic lesions; 2 years; rats
(M); Covance Laboratories, 1998, 680087
"I* absolute & relative kidney weight; ^ non-neoplastic lesions; 2 years; rats
(M/F); Lington, 1997,1239588
offspring body weight on PNDs 7 & 21; two-generation study; rats (M/F);
Waterman, 2000, 680202
4^ offspring body weight on PND 0,14, & 21; one-generation study; rats
(M/F); Waterman, 1999, 680201
IHED LOAEL A HED NOAEL • Other Doses Tested
•-
a-
&—¦—•—•
¦ •
10 100 1000
HED (mg/kg-day)
10000
Figure 4-3. Dose-Response Array of Studies Considered for Considered for Deriving the Chronic
Non-cancer POD
Notes: f = statistically significant increase in response compared to controls; J, = statistically significant decrease
in response compared to controls; M = males; F= females; PI = parental generation; PND = Postnatal Day; ALT
= alanine aminotransferase; AST= aspartate aminotransferase; ALP = alkaline phosphatase; HED = human
equivalent dose; NOAEL = no-observed-adverse-effect level; LOAEL = lowest-observed-adverse-effect level.
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Table 4-5. Dose-Response Analysis of Seleci
ted Studies Considered for Deriving the C
ironic >
on-cancer POD
Target Organ/
System
Study Details
(Species, Duration, Exposure
Route/ Method, Doses [mg/kg-
day])
Study POD/
Type
(mg/kg-day)
Effect
HEP
(mg/kg)
HEC
(mg/m3)
[ppm]
Uncertainty
Factors ab
Reference(s)
Liver and
Kidney
F344 rats (both sexes); dietary; 0,
0.03, 0.3, 0.6% (estimated: 15, 152,
307 mg/kg-day [males]; 18, 184, 375
mg/kg-day [females]); 2 years
NOAF.L=
15 (males)
18 (females)
t absolute and relative liver and
kidney weight; t in serum ALT and
AST; histopathological alterations
(e.g., spongiosis hepatis, focal
necrosis)
3.55
19.3 [1.13]
UFa = 3
UFh = 10
Total UF =
30
(Linetonet al.. 1997;
Bio/dvnamics. 1986)
Liver
SD rats (both sexes); dietary; 0, 500,
5,000, 10,000 ppm (estimated: 27,
271, 553 mg/kg-day [males]; 33,
331, 672 mg/kg-day [females]); 2
years
NOAF.L = 27
t serum ALT, AST, ALP (males);
histopathological findings in the
liver (i.e., minimal-to-slight focal
necrosis, spongiosis hepatis)
6.38
34.7 [2.03]
UFa = 3
UFl = 10
Total UF =
30
(Bio/dvnamics. 1987)
Liver and
Kidney
F344 rats (both sexes); dietary; 0,
500, 1,500, 6,000, 12,000 ppm
(estimated: 29, 88, 359, 733 mg/kg-
day [males]; 36, 109, 442, 885
mg/kg-day [females]); 2 years
NOAF.L =
88 (males)
109 (females)
t absolute and relative liver and
kidney weight; t in serum ALT,
AST, BUN; histopathological
findings in liver (e.g., spongiosis
hepatis) and kidney (e.g.,
mineralization of renal papilla,
pigment in tubule cells)
20.8
113 [6.61]
UFa = 3
UFh = 10
Total UF =
30
(Co vancc Labs.
1998c)
Liver and
Kidney
B6C3F1 mice (both sexes); dietary;
0, 500, 1,500, 4,000, 8,000 ppm
(estimated: 90, 276, 742, 1,560
mg/kg-day [males]; 112, 336, 910,
1,888 mg/kg-day [females]); 2 years
NOAF.L =
90 (males)
112 (females)
t absolute and relative liver weight,
histopathological changes in the
liver; J, body weight gain (females);
t incidence of liver masses and j
absolute kidney weight (males)
12.0
65.1 [3.80]
UFa = 3
UFh = 10
Total UF =
30
(Covancc Labs.
1998b)
Developmental
Two-seneration studv: SD rats
LOAEL = 133
| F1 and F2 offspring body weight
onPND7, 14, 21
31.4
171 [10.0]
UFa = 3
UFh = 10
UFl = 10
Total UF =
300
(Waterman et al..
(30/group) administered 0, 0.2, 0.4,
0.8% DINP in the diet continuously
starting 10 weeks prior to mating,
throughout mating, gestation and
lactation for two generations
2000; Exxon
Biomedical. 1996b)
Developmental
One generation studv: SD rats
LOAEL = 377
i male and female offspring body
weight on PND0, 14, and 21
89.1
485 [28.3]
UFa = 3
UFh = 10
UFL=10
(Waterman et al..
(30/group); administered 0, 0.5, 1.0,
1.5% DINP in diet continuously
starting 10 weeks prior to mating
2000; Exxon
Biomedical. 1996a)
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Target Organ/
System
Study Details
(Species, Duration, Exposure
Route/ Method, Doses [mg/kg-
day])
Study POD/
Type
(mg/kg-day)
Effect
HEP
(mg/kg)
HEC
(mg/m3)
[ppm]
Uncertainty
Factors ab
Reference(s)
and throughout mating, gestation
and lactation for one generation.
Total UF =
300
Liver
Beagle dogs (both sexes); dietary; 0,
0.125, 0.5, 2% (estimated: 37, 160,
2,000 mg/kg-day); 13 weeks
NOAF.L = 37
t absolute and relative liver weight;
t serum ALT
23.0
125 [7.32]
UFa = 3
UFh = 10
Total UF =
30 b
(Hazleton
Laboratories. 1971)
Liver
B6C3F1 mice (both sexes); dietary;
0, 1,500, 4,000, 10,000, 20,000 ppm
(estimated: 365, 972, 2,600, 5,770
mg/kg-day); 13 weeks
NOAF.L = 365
t absolute and relative liver weight;
liver histopathology (e.g., necrosis,
degeneration, hepatocyte
enlargement)
48.5
264 [15.4]
UFa = 3
UFh = 10
UFS= 10
Total UF =
300
(Hazleton Labs. 1992)
Liver and
Kidney
F344 rats (both sexes); dietary; 0,
0.1, 0.3, 0.6, 1.0, 2.0% (estimated:
77, 227, 460, 767, 1,554 mg/kg-
day); 13 weeks
NOAF.L = 77
t absolute and relative liver and
kidney weight; j cholesterol level
(females)
18.2
99.1 [5.79]
UFa = 3
UFh = 10
UFS= 10
Total UF =
300
(Bio/dvnamics.
1982b)
Liver and
Kidney
F344 rats (both sexes); dietary; 0,
2,500, 5,000, 10,000, 20,000 ppm
(estimated: 176, 354, 719, 1,545
mg/kg-day [males]; 218, 438, 823,
1,687 mg/kg-day [females]); 13
weeks
LOAEL =
176 (males)
218 (females)
t kidney and liver weights
41.6
226 [13.2]
UFa = 3
UFh= 10
UFS= 10
UFl = 10
Total UF =
3000
(Hazleton Labs.
1991b)
Liver and
Kidney
SD rats (both sexes); dietary; 0, 0.3,
1.0% (estimated: 201, 690 mg/kg-
day [males]; 251, 880 mg/kg-day
[females]); 13 weeks
LOAEL=
201 (males)
251 (females)
t absolute and relative liver and
kidney weight accompanied by j in
triglycerides and altered urine
chemistry
47.5
259 [15.1]
UFa = 3
UFh= 10
UFS= 10
UFl = 10
Total UF =
3000
(Bio/dvnamics. 1982c)
Kidney
SD rats (both sexes); dietary; 0,
1,000, 3,000, 10,000 ppm '
(estimated: 60, 180, 600 mg/kg-
LOAEL = 60
(males)
t incidence of histopathology
lesions in the kidney [i.e., focal
14.2
77.2 [4.51]
UFa = 3
UFh= 10
(Hazleton Labs. 1981)
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Target Organ/
System
Study Details
(Species, Duration, Exposure
Route/ Method, Doses [mg/kg-
day])
Study POD/
Type
(mg/kg-day)
Effect
HED
(mg/kg)
HEC
(mg/m3)
[ppm]
Uncertainty
Factors ab
Reference(s)
day); 13 weeks
mononuclear cell infiltration and
mineralization]; males only
UFS= 10
UFl = 10
Total UF =
3000
"EPA used allometric bodv weieht scaline to the three-auarters nowcr to derive the HED. Consistent with EPA Guidance (U.S. EPA. 201 lb), the interspecies
uncertainty factor (UFA), was reduced from 10 to 3 to account remaining uncertainty associated with interspecies differences in toxicodynamics. EPA used a default
intraspecies (UFH) of 10 to account for variation in sensitivity within human populations due to limited information regarding the degree to which human variability
may impact the disposition of or response to DINP. EPA used a LOAEL-to-NOAEL uncertainty factor (UFL) of 10 to account for the uncertainty inherent in
extrapolating from the LOAEL to the NOAEL.
b EPA considered applying a subchronic-to-chronic UF (UFS) of 10 for the intermediate (13-week) dog study under consideration for deriving a chronic POD. However,
retrospective analyses of 13-week and 1-year dog studies have shown that dog studies beyond 13-weeks do not have a significant impact on the derivation of chronic
PODs (Bishoo et al.. 2023; Dellarco et al.. 2010; Box and Smelmann. 2005). Therefore, a UFs was not used.
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4.2 Weight of Scientific Evidence
4.2.1 POD for Acute and Intermediate Durations
EPA has concluded that the HED of 12 mg/kg-day (based on BMDLs of 49 mg/kg-day and NOAEL of
50 mg/kg-day) from the NASEM (2017) meta-regression of reduced fetal testicular testosterone in rats
and the study by Clewell et al. (2013a) is appropriate for calculation of risks for acute and intermediate
exposure durations. A total UF of 30 was selected for use as the benchmark MOE (based on an
interspecies UF (UFa) of 3 and an intraspecies UF (UFh) of 10). Consistent with EPA guidance (2022.
2002b. 1993). EPA reduced the UFa from a value of 10 to 3 because allometric body weight scaling to
the three-quarter power was used to adjust the POD to obtain a HED (Appendix F). EPA has robust
overall confidence in the selected POD based on the following weight of scientific evidence:
• EPA has previously considered the weight of scientific evidence and concluded that oral
exposure to DINP can induce effects on the developing male reproductive system consistent with
a disruption of androgen action (see EPA's Draft Proposed Approach for CRA for Phthalates
(U.S. EPA. 2023a)). Notably, EPA's conclusion was supported by the SACC (U.S. EPA. 2023b).
• DINP exposure resulted in treatment-related effects on the developing male reproductive system
consistent with a disruption of androgen action during the critical window of development in 16
studies of rats (Section 3.1.2.1). Observed effects included: reduced mRNA expression of INSL3
and genes involved in steroidogenesis in the fetal testes; reduced fetal testes testosterone content
and/or production; reduced male pup anogenital distance; increased male offspring nipple
retention; increased incidence of MNGs and fetal Ley dig cell aggregation; and decreased sperm
motility in adult rats exposed perinatally to DINP.
• The selected POD is based on meta-regression analysis of fetal testicular testosterone data from
two studies of rats that supports a BMDLs of 49 mg/kg-day (Boberg et al.. 2011; Hannas et al..
2011). and a gestational study of rats that supports a NOAEL of 50 mg/kg-day based on
decreased fetal testicular testosterone and increased incidence of MNGs (Clewell et al.. 2013a).
• EPA's updated meta-regression and BMD modeling analysis of fetal testicular testosterone data
from four studies of rats supports a BMDLs of 47 mg/kg-day (Gray et al.. 2024; Furr et al.. 2014;
Boberg et al.. 2011; Hannas et al.. 2011). EPA's updated analysis, which integrates more fetal
testicular testosterone data, further support the selected POD.
• One additional developmental toxicity study (Clewell et al.. 2013b) resulted in increased MNGs
in the testis on PND2 and decreased male pup body weight on PND14 at similar doses (LOAEL
of 307 mg/kg-day and NOAEL of 56 mg/kg-day) to the BMDLs of 49 mg/kg-day derived from
the NASEM meta-analysis. This study supports the selection of the BMDLs of 49 mg/kg-day for
the acute and intermediate duration PODs.
• Other regulatory and authoritative bodies have also concluded that DINP is a developmental
toxicant and can induce effects on the developing male reproductive system consistent with a
disruption of androgen action and phthalate syndrome and that these developmental effects are
relevant for estimating human risk (Table 1-1) (ECCC/HC. 2020; EFSA. 2019; U.S. CPSC.
2014; ECHA. 2013b: NICNAS. 2012).
There are no studies conducted via the dermal and inhalation route relevant for extrapolating human
health risk. Therefore, EPA is using the oral HED of 12 mg/kg-day to extrapolate to the dermal route.
Differences in absorption will be accounted for in the dermal exposure estimates in the risk evaluation
for DINP.
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EPA is also using the oral HED of 12 mg/kg-day to extrapolate to the inhalation route. EPA assumes
similar absorption for the oral and inhalation routes, and no adjustment was made when extrapolating to
the inhalation route. For the inhalation route, EPA extrapolated the daily oral HEDs to inhalation HECs
using a human body weight and breathing rate relevant to a continuous exposure of an individual at rest.
Appendix F provides further information on extrapolation of inhalation HECs from oral HEDs.
Route-to-route extrapolation of the oral HED to an inhalation HEC and dermal HED results in additional
uncertainty. EPA cannot predict whether the assumptions regarding route extrapolation for the chosen
POD would lead to over- or underprediction of risk for the dermal and inhalation routes.
4.2.2 POD for Chronic Durations
EPA has concluded that the HED of 3.5 mg/kg-day (NOAEL of 15 mg/kg-day) from the 2-year dietary
study of F344 rats based on liver toxicity (Lington et al.. 1997; Bio/dynamics. 1986) is appropriate for
calculation of risk for chronic exposure durations. A total UF of 30 was selected for use as the
benchmark MOE (based on an interspecies UF (UFa) of 3 and an intraspecies UF (UFh) of 10).
Consistent with EPA guidance (2022. 2002b. 1993). EPA reduced the UFa from a value of 10 to 3
because allometric body weight scaling to the three-quarter power was used to adjust the POD to obtain
a HED (Appendix F). EPA has robust overall confidence in the selected POD based on the following
weight of scientific evidence:
• The NOAEL of 15 mg/kg-day (HED = 3.5 mg/kg-day) from the 2-year dietary study of F344 rats
(Lington et al.. 1997; Bio/dynamics. 1986) represents the most sensitive POD identified by EPA
across the 12 relevant studies subjected to dose-response analysis, including four 2-year chronic
dietary studies (3 of rats, 1 of mice), six 13-week subchronic dietary studies (4 of rats, and 1 each
of mice and beagles), a one-generation study of reproduction of rats, and a two-generation study
of reproduction of rats.
• This study received a high overall study quality determination and is GLP-compliant.
• At the LOAEL, a spectrum of dose-related effects consistent with liver toxicity was observed in
male and female rats, including treatment related increases in relative liver weight, serum ALT,
AST, and ALP, and histopathological findings (i.e., spongiosis hepatis, focal necrosis, sinusoid
ectasia).
• Given the relatively broad dose-spacing between the NOAEL (15 mg/kg-day) and the LOAEL
(152 mg/kg-day) in the principal study (Lington et al.. 1997; Bio/dynamics. 1986). EPA
attempted to refine the POD by conducting BMD modeling of relevant dose-related findings
showing a substantial increase in magnitude over controls, including: relative liver weight at
terminal sacrifice (both sexes); serum ALT at 6-and 18-month sacrifices (males only); incidence
of focal necrosis in the liver (both sexes); incidence of spongiosis hepatis (males only); and
incidence of sinusoid ectasia (males only). Calculated BMDLs ranged from 8.6 to 125 mg/kg-
day, which is similar to the study NOAEL and LOAEL values of 15 and 152 mg/kg-day. The
wide variability in BMDLs and uncertainty in several modelled outcomes (i.e., BMD/BMDL
ratios greater than 3) reduce EPA's confidence in using the BMD modeling results for
establishing a POD, and further affirm the use of the NOAEL for establishing the POD.
• The NOAEL of 15 mg/kg-day in Lington et al. (1997) also aligns with the BMD05 of 12 mg/kg-
day for one of the more sensitive endpoints in this study, spongiosis hepatis, determined by
CPSC (2010). However, EPA considers it more appropriate to use the NOAEL of 15 mg/kg-day
instead of the BMD05 of 12 mg/kg-day because the NOAEL supports the suite of effects on the
liver occurring at 152 mg/kg-day instead of being based on the single effect of spongiosis hepatis
with its associated uncertainty regarding human relevance.
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• The endpoints indicative of liver toxicity on which the POD is based were robust in that they
were observed across species and durations.
o The remaining three chronic studies in rodents (Covance Labs. 1998b. c; Bio/dynamics.
1987) reported similar findings of liver toxicity (e.g., increased liver weights; clinical
chemistry changes such as increased ALT, AST, ALP; and histopathology findings such
as liver necrosis and spongiosis hepatis), with similar but less sensitive NOAELs ranging
from 27 to 112 mg/kg-day.
o Similar findings indicative of liver toxicity were observed in the subchronic studies,
although at higher doses than observed in the chronic study by Lington et al. (1997). In
these subchronic studies, the lowest LOAELs for each of the tested species were: 160
mg/kg-day in beagles (NOAEL = 37 mg/kg-day; HED = 23) based on increased absolute
and relative liver weight and increase serum ALT (Hazleton Laboratories. 1971) and 972
mg/kg-day in mice (NOAEL = 365; HED = 49 mg/kg-day) based on increased absolute
and relative liver weight and histopathological findings (e.g., necrosis) (Hazleton Labs.
1992). LOAELs based on liver toxicity from the remaining three subchronic studies of
rats were less sensitive and ranged from 176 to 227 mg/kg-day (Hazleton Labs. 1991b;
Bio/dynamics. 1982b. c).
• Consistently, other regulatory bodies have selected the same chronic POD (NOAEL 15 mg/kg-
day) for use in quantify risk from exposures to DINP (Table 1-1) (ECCC/HC. 2020; EFSA.
2019; U.S. CPSC. 2014; ECHA. 2013b).
EPA considers this chronic POD most applicable for adults (16+ years). Use of this POD for
characterization of risk to infants and children from chronic exposure to DINP may be conservative and
may not be relevant. As described further in Appendix I, the chronic POD is based on liver effects
dependent and independent of PPARa activation. As discussed in EPA's Cancer Raman Health Hazard
Assessment for Diisononyl Phthalate (DINP) (U.S. EPA. 2025a). there is evidence to suggest humans
are less sensitive than rats to liver effects associated with PPARa activation, while the PPARa-
independent effects (i.e., spongiosis hepatis) are most prevalent in the livers of aging rats.
There are no studies conducted via the dermal and inhalation route relevant for extrapolating human
health risk. Therefore, EPA is using the oral HED of 3.5 mg/kg-day to extrapolate to the dermal route.
Differences in absorption will be accounted for in the dermal exposure estimates in the risk evaluation of
DINP.
EPA is also using the oral HED of 3.5 mg/kg-day to extrapolate to the inhalation route. EPA assumes
similar absorption for the oral and inhalation routes, and no adjustment was made when extrapolating to
the inhalation route. For the inhalation route, EPA extrapolated the daily oral HEDs to inhalation HECs
using a human body weight and breathing rate relevant to a continuous exposure of an individual at rest.
Appendix F provides further information on extrapolation of inhalation HECs from oral HEDs.
Route-to-route extrapolation of the oral HED to an inhalation HEC and dermal HED results in additional
uncertainty. EPA cannot predict whether the assumptions regarding route extrapolation for the chosen
POD would lead to over- or underprediction of risk for the dermal and inhalation routes.
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5 CONSIDERATION OF PESS AND AGGEGRATE EXPOSURE
5.1 Hazard Considerations for Aggregate Exposure
For use in the risk evaluation and assessing risks from other exposure routes, EPA conducted route-to-
route extrapolation of the toxicity values from the oral studies for use in the dermal and inhalation
exposure routes and scenarios. Health outcomes that serve as the basis for acute, intermediate, and
chronic hazard values are systemic and assumed to be consistent across routes of exposure. EPA
therefore concludes that for consideration of aggregate exposures, it is reasonable to assume that
exposures and risks across oral, dermal, and inhalation routes may be additive for the selected PODs in
Section 6.
5.2 PESS Based on Greater Susceptibility
In this section, EPA addresses subpopulations expected to be more susceptible to DINP exposure than
other populations. Table 5-1 presents the data sources that were used in the potentially exposed or
susceptible subpopulations (PESS) analysis evaluating susceptible subpopulations and identifies whether
and how the subpopulation was addressed quantitatively in the risk evaluation of DINP. EPA identified
a range of factors that may have the potential to increase biological susceptibility to DINP, including
lifestage, chronic liver or kidney disease, pre-existing diseases, physical activity, diet, stress, and co-
exposures to other environmental stressors that contribute to related health outcomes.
Regarding lifestage, exposure to DINP during the masculinization programming window (i.e., GD15.5-
18.5 for rats; GD14-16 for mice; gestational weeks 8-14 for humans) can lead to antiandrogenic effects
on the male reproductive system (MacLeod et al.. 2010; Welsh et al.. 2008; Carruthers and Foster.
2005). Animal studies demonstrating effects of DINP on male reproductive development and other
developmental outcomes provide direct evidence that gestation is a particularly sensitive lifestage. EPA
considered the sensitivity of this lifestage in its derivation of the POD for acute and intermediate
exposure duration based on reduced fetal testicular testosterone in rats after evaluation of the weight of
scientific evidence that DINP resulted in treatment-related effects on the developing male reproductive
system consistent with a disruption of androgen action during the critical window of development in 13
studies of rats. In humans, there is moderate evidence for the association between DINP and testosterone
and semen parameters, based on studies that found decreasing testosterone levels with increasing DINP
exposure (Radke et al.. 2018). Based on this evidence from animal and human studies, EPA has
identified two groups that may be more susceptible to DINP exposure due to lifestages:
• pregnant women/women of reproductive age, and
• male infants, male toddlers, and male children.
Animal evidence also demonstrates that the liver, kidneys, nervous system, cardiovascular system,
immune system, may be sensitive target organs. EPA is quantifying risks based on liver and
developmental toxicity in the risk evaluation of DINP, and determining risk based these endpoints is
protective of the other hazards which occur at higher doses.
Regarding the factor of co-exposure, studies have demonstrated that co-exposure to DINP and other
toxicologically similar phthalates (e.g., DEHP, DBP, BBP) and other classes of antiandrogenic
chemicals (e.g., certain pesticides and pharmaceuticals that are discussed more in (U.S. EPA. 2023a))
can induce effects on the developing male reproductive system in a dose-additive manner. EPA details
how it intends to evaluate risk to above-identified PESS from co-exposure to DINP and several other
toxicologically similar phthalates in its Draft Proposed Approach for CRA for Phthalates (U.S. EPA.
2023a).
Page 104 of 282
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The effect of other factors on susceptibility to health effects of DINP is not known; therefore, EPA is
uncertain about the magnitude of any possible increased risk from effects associated with DINP
exposure for subpopulations that may be relevant to other factors.
For non-cancer endpoints, EPA used a default value of 10 for human variability (UFh) to account for
increased susceptibility when quantifying risks from exposure to DINP. The Risk Assessment Forum, in
A Review of the Reference Dose and Reference Concentration Processes (U.S. EPA. 2002b). discusses
some of the evidence for choosing the default factor of 10 when data are lacking and describe the types
of populations that may be more susceptible, including different lifestages (e.g., of children and elderly).
U.S. EPA (2002b); however, did not discuss all the factors presented in Table 5-1. Thus, uncertainty
remains whether additional susceptibility factors would be covered by the default UFh value of 10
chosen for use in the risk evaluation of DINP.
Page 105 of 282
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Table 5-1. P
CSS Evidence Crosswalk for Biological Susceptibility Considerations
Susceptibility
Category
Examples of
Specific
Direct Evidence this Factor
Modifies Susceptibility to DINP
Indirect Evidence of Interaction with Target
Organs or Biological Pathways Relevant to
DINP
Susceptibility Addressed in
Risk Evaluation?
Factors
Description of Interaction
Key Citations
Description of
Interaction
Key Citation(s)
Embryos/
fetuses/infants
Direct quantitative animal
evidence for developmental
toxicity (e.g., increased skeletal
and visceral variations,
decreased live births, decreased
offspring body weight gain, and
decreased offspring survival
with increased severity in the
second generation).
(Hellwie et al..
1997)
(Waterman et al..
1999)
(Waterman et al..
2000)
(U.S. EPA.
2023a)
(U.S. EPA.
2023b)
Acute and intermediate duration
PODs for developmental
endpoints protective of effects
in offspring
Lifestage
There is direct quantitative
animal evidence for effects on
the developing male
reproductive system consistent
with a disruption of androgen
action.
Pregnancy/
lactating status
Rodent dams not particularly
susceptible during pregnancy
and lactation, except for effects
related to reduced maternal
weight gain, food consumption,
and increased organ weight
(liver and kidney), which
occurred at doses higher than
those that caused developmental
toxicity.
(Hellwie et al..
1997)
(Waterman et al..
1999)
Acute and intermediate duration
PODs for developmental
endpoints protective of effects
in dams
Page 106 of 282
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Susceptibility
Category
Examples of
Specific
Factors
Direct Evidence this Factor
Modifies Susceptibility to DINP
Indirect Evidence of Interaction with Target
Organs or Biological Pathways Relevant to
DINP
Susceptibility Addressed in
Risk Evaluation?
Description of Interaction
Key Citations
Description of
Interaction
Key Citation(s)
Lifestage
Males of
reproductive
age
Increased testes, right
epididymis, liver, and kidney
weights. There was also
decreased food consumption.
(Waterman et al..
2000; Exxon
Biomedical.
1996a)
Use of default 10 x UFH
Children
Reduced rodent offspring
body weight gain between PND1
to 21 was observed in one and
two-generation studies of
reproduction.
(Waterman et al..
2000; Exxon
Biomedical.
1996a, b)
Acute and intermediate duration
PODs for developmental
endpoints protective of effects
of offspring bodyweight gain
Use of default 10 x UFH
Elderly
No direct evidence identified
Use of default 10 x UFH
Pre-existing
disease or
disorder
Health
outcome/
target organs
No direct evidence identified
Several preexisting
conditions may contribute
to adverse developmental
outcomes (e.g., diabetes,
high blood pressure,
certain viruses).
Individuals with chronic
liver and kidney disease
may be more susceptible
to effects on these target
organs
Viruses such as viral
hepatitis can cause liver
damage.
CDC (2023e)
CDC (2023 g)
Use of default 10 x UFH
Page 107 of 282
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Susceptibility
Category
Examples of
Specific
Direct Evidence this Factor
Modifies Susceptibility to DINP
Indirect Evidence of Interaction with Target
Organs or Biological Pathways Relevant to
DINP
Susceptibility Addressed in
Risk Evaluation?
Factors
Description of Interaction
Key Citations
Description of
Interaction
Key Citation(s)
Pre-existing
disease or
disorder
Toxicokinetics
No direct evidence identified
Chronic liver and kidney
disease are associated with
impaired metabolism and
clearance (altered
expression of phase 1 and
phase 2 enzymes,
impaired clearance),
which may enhance
exposure duration and
concentration of DINP.
Use of default 10 x UFH
Smoking
No direct evidence identified
Smoking during
pregnancy may increase
susceptibility for
developmental outcomes
(e.g., early delivery and
stillbirths).
CDC (2023f)
Qualitative discussion in
Section 5.2 and this table
Lifestyle
activities
Alcohol
consumption
No direct evidence identified
Alcohol use during
pregnancy can cause
developmental outcomes
(e.g., fetal alcohol
spectrum disorders).
Heavy alcohol use may
affect susceptibility to
liver disease.
CDC (2023d)
CDC (2023a)
Qualitative discussion in
Section 5.2 and this table
Physical
activity
No direct evidence identified
Insufficient activity may
increase susceptibility to
multiple health outcomes.
Overly strenuous activity
may also increase
susceptibility.
CDC (2022)
Qualitative discussion in
Section 5.2 and this table
Page 108 of 282
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Susceptibility
Category
Examples of
Specific
Direct Evidence this Factor
Modifies Susceptibility to DINP
Indirect Evidence of Interaction with Target
Organs or Biological Pathways Relevant to
DINP
Susceptibility Addressed in
Risk Evaluation?
Factors
Description of Interaction
Key Citations
Description of
Interaction
Key Citation(s)
Sociodemo-
Race/ethnicity
No direct evidence identified
(e.g., no information on
polymorphisms in DINP
metabolic pathways or diseases
associated race/ethnicity that
would lead to increased
susceptibility to effects of DINP
by any individual group).
Qualitative discussion in
Section 5.2 and this table
graphic status
Socioeconomic
status
No direct evidence identified
Individuals with lower
incomes may have worse
health outcomes due to
social needs that are not
met, enviromnental
concerns, and barriers to
health care access.
ODPHP (2023b)
Sex/gender
No direct evidence identified
Use of default 10 x UFH
Nutrition
Diet
No direct evidence identified
Poor diets can lead to
chronic illnesses such as
heart disease, type 2
diabetes, and obesity,
which may contribute to
adverse developmental
outcomes. Additionally,
diet can be a risk factor for
fatty liver, which could be
a pre-existing condition to
enhance susceptibility to
DINP-induced liver
toxicity.
CDC (2023e)
CDC (2023b)
Qualitative discussion in
Section 5.2 and this table
Page 109 of 282
-------
Susceptibility
Category
Examples of
Specific
Factors
Direct Evidence this Factor
Modifies Susceptibility to DINP
Indirect Evidence of Interaction with Target
Organs or Biological Pathways Relevant to
DINP
Susceptibility Addressed in
Risk Evaluation?
Description of Interaction
Key Citations
Description of
Interaction
Key Citation(s)
Nutrition
Malnutrition
No direct evidence identified
Micronutrient malnutrition
can lead to multiple
conditions that include
birth defects, maternal and
infant deaths, preterm
birth, low birth weight,
poor fetal growth,
childhood blindness,
undeveloped cognitive
ability.
Thus, malnutrition may
increase susceptibility to
some developmental
outcomes associated with
DINP.
CDC (2021)
CDC (2023b)
Qualitative discussion in
Section 5.2 and this table
Genetics/
epigenetics
Target organs
No direct evidence identified
Polymorphisms in genes
may increase
susceptibility to liver,
kidney, or developmental
toxicity.
Use of default 10 x UFH
Toxicokinetics
No direct evidence identified
Polymorphisms in genes
encoding enzymes (e.g.,
esterases) involved in
metabolism of DINP may
influence metabolism and
excretion of DINP.
Use of default 10 x UFH
Page 110 of 282
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Susceptibility
Category
Examples of
Specific
Direct Evidence this Factor
Modifies Susceptibility to DINP
Indirect Evidence of Interaction with Target
Organs or Biological Pathways Relevant to
DINP
Susceptibility Addressed in
Risk Evaluation?
Factors
Description of Interaction
Key Citations
Description of
Interaction
Key Citation(s)
Built
environment
No direct evidence identified
Poor-quality housing is
associated with a variety
of negative health
outcomes.
ODPHP (2023a)
Qualitative discussion in
Section 5.2 and this table
Other
Social
enviromnent
No direct evidence identified
Social isolation and other
social determinants (e.g.,
decreased social capital,
stress) can lead to negative
health outcomes.
CDC (2023c)
ODPHP (2023c)
Qualitative discussion in
Section 5.2 and this table
chemical and
nonchemical
stressors
Chemical co-
exposures
Studies have demonstrated that
co-exposure to DINP and other
toxicologically similar
phthalates (e.g., DEHP, DBP,
BBP) and other classes of
antiandrogenic chemicals (e.g.,
certain pesticides and
pharmaceuticals - discussed
more in (U.S. EPA. 2023a)) can
induce effects on the developing
male reproductive system in a
dose-additive manner.
See (U.S. EPA.
2023a) and (U.S.
EPA 2023b)
Qualitative discussion in
Section 5.2 and this table and
will be quantitatively addressed
as part of the phthalate
cumulative risk assessment.
Page 111 of 282
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6 POINTS OF DEPARTURE USED TO ESTIMATE RISKS FROM
DINP EXPOSURE
After considering hazard identification and evidence integration, dose-response evaluation, and weight
of scientific evidence of POD candidates, EPA chose two non-cancer endpoints for the risk evaluation—
one for acute and intermediate exposure scenarios and a second one for chronic scenarios (Table 6-1).
HECs are based on daily continuous (24-hour) exposure, and HEDs are daily values.
For purposes of assessing acute/intermediate non-cancer risks, the selected acute/intermediate POD is
considered most applicable to women of reproductive age, pregnant women, male infants, and male
children. Use of this POD to assess risk for other age groups (e.g., adult males) is conservative. For
purposes of assessing chronic non-cancer risks, the selected POD is considered most applicable to adults
(16+ years). Use of this POD for characterization of risk to infants and children from chronic exposure
to DINP may be conservative and may not be relevant (discussed further in Appendix I).
Table 6-1. Non-cancer HECs and HEDs Used to Estimate Risks
Exposure
Scenario
Target Organ
System
Species
Duration
POD
(mg/kg-
day)
Effect
HED
(mg/kg-
day)
HEC
(mg/m3)
[ppm]
Benchmark
MOE
Reference
Acute,
Intermediate
Development
Rat
5-14 days
throughout
gestation
BMDL5
= 49°
NOAEL
= 50 b
| fetal
testicular
testosterone, t
incidence of
MNGs
12 c
63
[3.7]
UFa= 3
UFh=10
Total UF=30
(NASEM.
2017;
Clewell et
al.. 2013a)
Chronic
Liver
Rat
2 years
NOAEL
= 15
t liver weight,
t serum
chemistry,
histopathology
d e
3.5
19
[1.1]
UFa= 3
UFh=10
Total UF=30
(Lineton et
al.. 1997;
Bio/dvnami
cs. 1986)
BMDL = benchmark dose lower limit; HEC = human equivalent concentration; HED = human equivalent dose; MOE = margin
of exposure; NOAEL = no observable adverse effect level; POD = point of departure; UF = uncertainty factor;
" The BMDLs was derived bv NASEM (2017) throueh meta-reeression and BMD modeline of fetal testicular testosterone data
from two studies of DINP with rats (Bobere et al.. 2011; Hannas et al.. 2011). R code suDDortina NASEM's meta-reeression
and BMD analysis of DINP is Diibliclv available throueh GitHub.
b The NOAEL was derived from the eestational c\do sure studv conducted bv Clewell et al. (2013a). which suDDorts a NOAEL
of 50 mg/kg-day based decreased fetal testicular testosterone and increased incidence of MNGs.
c The BMDLs of 49 mg/kg-day and NOAEL of 50 mg/kg-day both support an HED of 12 mg/kg-day.
''Liver toxicity included increased relative liver weight, increased serum chemistry (i.e., AST, ALT, ALP), and histopathologic
findines (e.s.. focal necrosis, sooneiosis henatis)) inF344 rats followine 2 vears of dietary exposure to DINP (Lineton et al..
1997; Bio/dvnamics. 1986).
e The Lington study presents a portion of the data from a larger good laboratory practice (GLP)-certified study by
Bio/dvnamics (1986).
Page 112 of 282
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alkyl esters, C9-rich); CASRNs 28553-12-0 and 68515-48-0 [EPA Report], (EPA-740-R-21-
002). Washington, DC: Office of Chemical Safety and Pollution Prevention.
https://www.epa.gov/svstem/files/documents/2021-08/casrn-28553-12-0-di-isononyl-phthalate-
final-scope.pdf
U.S. EPA. (2022). ORD staff handbook for developing IRIS assessments [EPA Report], (EPA 600/R-
22/268). Washington, DC: U.S. Environmental Protection Agency, Office of Research and
Development, Center for Public Health and Environmental Assessment.
https://cfpub.epa.gov/ncea/iris drafts/recordisplav.cfm?deid=356370
U.S. EPA. (2023a). Draft Proposed Approach for Cumulative Risk Assessment of High-Priority
Phthalates and a Manufacturer-Requested Phthalate under the Toxic Substances Control Act.
(EPA-740-P-23-002). Washington, DC: U.S. Environmental Protection Agency, Office of
Chemical Safety and Pollution Prevention. https://www.regulations.gov/document/EPA-HQ-
QPPT-2022-0918-0009
U.S. EPA. (2023b). Science Advisory Committee on Chemicals meeting minutes and final report, No.
2023-01 - A set of scientific issues being considered by the Environmental Protection Agency
regarding: Draft Proposed Principles of Cumulative Risk Assessment (CRA) under the Toxic
Substances Control Act and a Draft Proposed Approach for CRA of High-Priority Phthalates and
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a Manufacturer-Requested Phthalate. (EPA-HQ-OPPT-2022-0918). Washington, DC: U.S.
Environmental Protection Agency, Office of Chemical Safety and Pollution Prevention.
https://www.regulations.gov/document/EPA-HQ-OPPT-2022-0918-0Q67
U.S. EPA. (2023c). Technical review of diisononyl phthalate (Final assessment). Washington, DC:
Office Pollution Prevention and Toxics, Data Gathering and Analysis Division and Existing
Chemicals Risk Assessment Division.
U.S. EPA. (2024a). Draft Meta-Analysis and Benchmark Dose Modeling of Fetal Testicular
Testosterone for Di(2-ethylhexyl) Phthalate (DEHP), Dibutyl Phthalate (DBP), Butyl Benzyl
Phthalate (BBP), Diisobutyl Phthalate (DIBP), and Dicyclohexyl Phthalate (DCHP).
Washington, DC: Office of Pollution Prevention and Toxics.
U.S. EPA. (2024b). Draft Non-cancer Human Health Hazard Assessment for Butyl benzyl phthalate
(BBP). Washington, DC: Office of Pollution Prevention and Toxics.
U.S. EPA. (2024c). Draft Non-cancer Human Health Hazard Assessment for Dibutyl Phthalate (DBP).
Washington, DC: Office of Pollution Prevention and Toxics.
U.S. EPA. (2024d). Draft Non-cancer Human Health Hazard Assessment for Dicyclohexyl Phthalate
(DCHP). Washington, DC: Office of Pollution Prevention and Toxics.
U.S. EPA. (2024e). Draft Non-cancer Human Health Hazard Assessment for Diethylhexyl Phthalate
(DEHP). Washington, DC: Office of Pollution Prevention and Toxics.
U.S. EPA. (2024f). Draft Non-cancer Human Health Hazard Assessment for Diisobutyl phthalate
(DIBP). Washington, DC: Office of Pollution Prevention and Toxics.
U.S. EPA. (2024g). Science Advisory Committee on Chemicals Meeting Minutes and Final Report No.
2024-2, Docket ID: EPA-HQ-OPPT-2024-0073: For the Draft Risk Evaluation for Di-isodecyl
Phthalate (DIDP) and Draft Hazard Assessments for Di-isononyl Phthalate (DINP). Washington,
DC: U.S. Environmental Protection Agency, Science Advisory Committee on Chemicals.
U.S. EPA. (2025a). Cancer Human Health Hazard Assessment for Diisononyl Phthalate (DINP).
Washington, DC: Office of Pollution Prevention and Toxics.
U.S. EPA. (2025b). Consumer and Indoor Exposure Assessment for Diisononyl Phthalate (DINP).
Washington, DC: Office of Pollution Prevention and Toxics.
U.S. EPA. (2025c). Data Quality Evaluation Information for Human Health Hazard Animal Toxicology
for Diisononyl Phthalate (DINP). Washington, DC: Office of Pollution Prevention and Toxics.
U.S. EPA. (2025d). Data Quality Evaluation Information for Human Health Hazard Epidemiology for
Diisononyl Phthalate (DINP). Washington, DC: Office of Pollution Prevention and Toxics.
U.S. EPA. (2025e). Non-cancer Human Health Hazard Assessment for Diisononyl Phthalate (DINP)
Washington, DC: Office of Pollution Prevention and Toxics.
U.S. EPA. (2025f). Occupational Exposure Assessment for Diisononyl Phthalate (DINP). Washington,
DC: Office of Pollution Prevention and Toxics.
U.S. EPA. (2025g). Risk Evaluation for Diisononyl Phthalate (DINP). Washington, DC: Office of
Pollution Prevention and Toxics.
U.S. EPA. (2025h). Systematic Review Protocol for Diisononyl Phthalate (DINP) Washington, DC:
Office of Pollution Prevention and Toxics.
Valles. EG: Laughter. AR; Dunn. CS: Cannelle. S: Swan son. CL; Cattlev. RC: Corton. JC. (2003). Role
of the peroxisome proliferator-activated receptor alpha in responses to diisononyl phthalate.
Toxicology 191: 211-225. http://dx.doi.org/10.1016/S0300-483X(03)00260-9
van Den Driesche. S: McKinnell. C: Calarrao. A: Kennedy. L; Hutchison. GR; Hrabalkova. L; Jobling.
MS: Macpherson. S: Anderson. RA; Sharpe. RM; Mitchell. RT. (2015). Comparative effects of
di(n-butyl) phthalate exposure on fetal germ cell development in the rat and in human fetal testis
xenografts. Environ Health Perspect 123: 223-230. http://dx.doi.org/10.1289/ehp. 1408248
Wan. Y; North. ML: Navaranian. G: Ellis. AK; Siegel. JA; Diamond. ML. (2021). Indoor exposure to
phthalates and polycyclic aromatic hydrocarbons (PAHs) to Canadian children: the Kingston
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allergy birth cohort.
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Waterman. SJ; Ambroso. JL; Keller. LH; Trimmer. GW; Nikiforov. AI; Harris. SB. (1999).
Developmental toxicity of di-isodecyl and di-isononyl phthalates in rats. Reprod Toxicol 13:
131-136. htto://dx.doi.org/10.1016/S0890-6238(99)00002-7
Waterman. SJ: Keller. LH: Trimmer. GW: Freeman. JJ; Nikiforov. AI: Harris. SB: Nicolich. MJ;
McKee. RH. (2000). Two-generation reproduction study in rats given di-isononyl phthalate in
the diet. Reprod Toxicol 14: 21-36. http://dx.doi.org/10.1016/S0890-6238(99)00067-2
Welsh. M: Saunders. PTK: Fisken. M: Scott. HM; Hutchison. GR: Smith. LB: Sharpe. RM. (2008).
Identification in rats of a programming window for reproductive tract masculinization, disruption
of which leads to hypospadias and cryptorchidism. J Clin Invest 118: 1479-1490.
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Wu. Z: Li. J: Ma. P: Li. B: Xu. Y. (2015). Long-term dermal exposure to diisononyl phthalate
exacerbates atopic dermatitis through oxidative stress in an FITC-induced mouse model.
Frontiers in Biology 10: 537-545. http://dx.doi.org/10.1007/sl 1515-015-1382-v
Yun-Ho. H: Lee. Y; Man-Jeong. P; Sung-Tae. Y. (2019). Inhibitions of HMGB1 and TLR4 alleviate
DINP-induced asthma in mice. Toxicology Research 8: 621-629.
Zettergren. A: Andersson. N: Larsson. K: Kull. I; Melen. E: Georgelis. A: Berglund. M: Lindh. C:
Bergstrom. A. (2021). Exposure to environmental phthalates during preschool age and obesity
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preliminary study. Fertil Steril 111: 112-121. http://dx.doi.org/10.1016/i.fertnstert.2018.09.009
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APPENDICES
Appendix A EXISTING ASSESSMENTS FROM OTHER REGULATORY AGENCIES OF
DINP
The available existing assessments of DINP are summarized in TableApx A-l, which includes details regarding external peer review, public
consultation, and systematic review protocols that were used.
Table Apx A-l. Summary of Peer-Review, Public Comments, and Systematic Review for Existing Assessments of DINP
Agency
Assessment(s) (Reference)
External
Peer-
Review?
Public
Consultation?
Systematic
Review
Protocol
Employed?
Remarks
U.S. EPA
(authors are
affiliated with the
U.S. EPA's Center
for Public Health
and Environmental
Assessment)
Phthcdate exposure and male
reproductive outcomes: A systematic
review of the human epidemiological
evidence (Radke et al.. 2018)
Phthalate exposure and female
reproductive and developmental
outcomes: A systematic review of the
human epidemiological evidence
(Radke et al.. 2019b)
Phthalate exposure and metabolic
effects: A systematic review of the
human epidemiological evidence
(Radke et al.. 2019a)
Phthalate exposure and
nenrodevelopment: A systematic
review and meta-analysis of human
epidemiological evidence (Radke et
al.. 2020a).
No
No
Yes
- Publications were subjected to peer-review
prior to being published in a special issue of
Environment International
- Publications employed a systematic review
process that included literature search and
screening, study evaluation, data extraction,
and evidence synthesis. The full systematic
review protocol is available as a supplemental
file associated with each publication.
U.S. EPA
Technical review of diisononvl
phthalate (Final assessment) (U.S.
EPA. 2023c)
No
Yes
No
- Technical review of DINP was reviewed by
two internal EPA reviewers, but was not
subjected to external peer-review
- Draft technical review of DINP was subjected
to a public review period. Public comments
available here:
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Agency
Assessment(s) (Reference)
External
Peer-
Review?
Public
Consultation?
Systematic
Review
Protocol
Employed?
Remarks
https: //www .re aulations. aov/docket/EPA-HO-
TRI-2022-0262/comments
U.S. CPSC
Toxicity review of Diisononyl
Phthalate (DINP) (U.S. CPSC. 2010)
Chronic Hazard Advisory Panel on
Phthalates and Phthalate Alternatives
(U.S. CPSC. 2014)
Yes
Yes
No
- Peer-reviewed by panel of four experts. Peer-
review report available at:
https: //www .cpsc. aov/s3 fs -public/Peer-
Re view-Report-Comments.pdf
-Public comments available at:
https: //www .cpsc. aov/chap
- No formal systematic review protocol
employed.
- Details regarding CPSC's strategy for
identifying new information and literature are
provided on paee 12 of (U.S. CPSC. 2014)
NASEM
Application of systematic review
methods in an overall strategy for
evaluating low-dose toxicity from
endocrine active chemicals (NASEM.
2017)
Yes
No
Yes
- Draft report was reviewed by individuals
chosen for their diverse perspectives and
technical expertise in accordance with the
National Academies peer review process. See
Acknow ledgements Section of (NASEM.
2017) for more details.
- Employed NTP's Office of Heath Assessment
and Translation (OHAT) systematic review
method
Health Canada
State of the science report: Phthalate
substance grouping 1,2-
Benzenedicarboxylic acid, diisononyl
ester; 1,2-Benzenedicarboxylic acid,
di-C8-10-branched alky I esters, C9-
rich (Diisononyl Phthalate; DINP).
Chemical Abstracts Service Registry>
Numbers; 28553-12-0 and 68515-48-0
(EC/HC. 2015)
Supporting Documentation;
Carcinogenicity of Phthalates - Mode
Yes
Yes
No (Animal
studies)
Yes
(Epidemiologic
studies)
- Ecological and human health portions of the
screening assessment report (ECCC/HC. 2020)
were subject to external review and/or
consultation. See paae 2 of (ECCC/HC. 2020)
for additional details.
- State of the science report (EC/HC. 2015) and
draft screening assessment report for the
phthalate substance group subjected to 60-day
public comment periods. Summaries of
received public comments available at:
https://www.canada.ca/en/health-
canada/ service s/chemical-
Page 131 of 282
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Agency
Assessment(s) (Reference)
External
Peer-
Review?
Public
Consultation?
Systematic
Review
Protocol
Employed?
Remarks
of Action and Human Relevance
(Health Canada. 2015)
Supporting documentation:
Evaluation of epidemiologic studies
on phthalate compounds and their
metabolites for hormonal effects,
growth and development and
reproductive parameters (Health
Canada. 2018b)
Supporting documentation:
Evaluation of epidemiologic studies
on phthalate compounds and their
metabolites for effects on behaviour
and neurodevelopment, allergies,
cardiovascular function, oxidative
stress, breast cancer, obesity, and
metabolic disorders (Health Canada.
2018a)
Screening Assessment - Phthalate
Substance Grouping (ECCC/HC.
2020)
substances/substance-aroupinas-
initiative/phthalate .html#a 1
- No formal systematic review protocol
employed to identify or evaluate experimental
animal toxicology studies.
- Details regarding Health Canada's strategy
for identifying new information and literature
are provided in Section 1 of (EC/HC. 2015)
and (ECCC/HC. 2020)
- Human epidemiologic studies evaluated using
Downs and Black Method (Health Canada.
2018a, b)
NICNAS
Priority existing chemical assessment
report no. 35: Diisononvl phthalate
(NICNAS. 2012)
No
Yes
No
- NICNAS (2012) states "The report has been
subjected to internal peer review by NICNAS
during all stages of preparation." However, a
formal external peer-review was not
conducted.
-NICNAS (2012) states "Applicants for
assessment are given a draft copy of the report
and 28 days to advise the Director of any
errors. Following the correction of any errors,
the Director provides applicants and other
interested parties with a copy of the draft
assessment report for consideration. This is a
period of public comment lasting for 28 days
Page 132 of 282
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Agency
Assessment(s) (Reference)
External
Peer-
Review?
Public
Consultation?
Systematic
Review
Protocol
Employed?
Remarks
during which requests for variation of the
report may be made." See Preface of
(NICNAS. 2012) for more details.
- No formal systematic review protocol
employed.
- Details regarding NICNAS's strategy for
identifying new information and literature are
provided in Section 1.3 of (NICNAS. 2012)
EC HA
Evaluation of New Scientific Evidence
Concerning DINP and DIDP in
Relation to Entry 52 of Annex XVII to
REACH Regulation (EC) No
1907/2006 (ECHA. 2013b)
Yes
Yes
No
- Peer-reviewed by ECHA's Committee for
Risk Assessment (ECHA. 2013a)
- Subject to 12-week public consultation
- No formal systematic review protocol
employed
- Details regarding ECHA's strategy for
identifying new information and literature are
provided on pases 14-15 of (ECHA. 2013b)
EFSA
Update of the Risk Assessment of Di-
butylphthalate (DBP), Butyl-benzyl-
phthalate (BBP), Bis(2-
ethylhexyl)phthalate (DEHP), Di-
isononylphthalate (DINP) and Di-
isodecylphthalate (DIDP) for Use in
Food Contact Materials (EFSA. 2019)
No
Yes
No
- Draft report subject to public consultation.
Public comments and EFSA's response to
comments are available at:
httDs://doi.ora/10.2903/sD.efsa.2019.EN-1747
- No formal systematic review protocol
employed.
- Details regarding EFSA's strategy for
identifying new information and literature are
provided on page 18 and Appendix B of
(EFSA. 2019)
NTP-CERHR
NTP-CERHR monograph on the
potential human reproductive and
developmental effects of di-isononvl
vhthalate (DINP) (NTP-CERHR.'
2003)
No
Yes
No
- Report prepared by NTP-CERHHR
Phthalates Expert Panel and was reviewed by
CERHR Core Committee (made up of
representatives of NTP-participating agencies,
CERHR staff scientists, member of phthalates
expert panel)
- Public comments summarized in Appendix
III of (NTP-CERHR 2003)
Page 133 of 282
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Agency
Assessment(s) (Reference)
External
Peer-
Review?
Public
Consultation?
Systematic
Review
Protocol
Employed?
Remarks
- No formal systematic review protocol
employed.
Page 134 of 282
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Appendix B SUMMARY OF LIVER TOXICITY STUDIES
This Appendix contains more detailed information on the available studies described in the liver toxicity
hazard identification (Section 3.2), including information on individual study design.
Humans
No epidemiologic studies were identified by Health Canada (2018a) or by Radke et al. that examined the
association between DINP and/or its metabolites and biomarkers of liver injury.
New Literature: EPA considered new studies published since Health Canada's assessment (Health
Canada. 2018a); however, no studies were identified that fall within this date range and evaluated liver
injury for DINP and/or its metabolites.
Laboratory Animals
Existing assessments have consistently identified the liver as one of the most sensitive target organs
following oral exposure to DINP in experimental animal studies (ECCC/HC. 2020; EFSA 2019;
EC/HC. 2015; ECHA. 2013b; NICNAS. 2012; U.S. CPSC. 2010; EFSA 2005; ECB. 2003; NTP-
CERHR. 2003; U.S. CPSC. 2001). Intermediate (>1 to 30 days), subchronic (>30 to 90 days) and
chronic (>90 days) exposure studies have reported significant liver effects. Available studies include: 11
intermediate oral studies (six studies on rats, four studies on mice, 1 study on cynomolgus monkeys);
nine subchronic oral exposure studies (six on rats, one on mice, one on beagle dogs, and one on
marmosets) and five chronic oral exposure studies (four on rats and one on mice) Available studies are
summarized in TableApx B-l, TableApx B-2, and TableApx B-6, and are discussed further below.
Considerations for Interpretation of Hepatic Effects: Consistent with previous guidance documents
(Hall et al.. 2012; U.S. EPA. 2002a). EPA considered hepatocellular hypertrophy and corresponding
increases in liver size and weight to be adaptive non-adverse responses, unless accompanied by
exposure-related, biologically significant changes in clinical markers of liver toxicity (i.e., decreased
albumin; or increased alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline
phosphatase (ALP), gamma glutamyltransferase, bilirubin, cholesterol) and/or histopathology indicative
of an adverse response (e.g., hyperplasia, degeneration, necrosis, inflammation). Further, phthalates,
including DINP, can induce peroxisome proliferation in the livers of mice and rats (Corton et al.. 2018;
Lapinskas et al.. 2005; Valles et al.. 2003). and EPA considered evidence supporting a role for PPARa
activation in peroxisome-induced hepatic effects of DINP. For purposes of identifying study NOAEL
and LOAEL values, effects consistent with peroxisome proliferation and PPARa activation were also
considered relevant for setting the LOAEL.
Intermediate (>l to 30 Days) Exposure Studies: EPA evaluated 12 intermediate exposure animal studies
from existing assessments that evaluated liver effects following oral exposure to DINP (Ma et al.. 2014;
Kwack et al.. 2010; Kwack et al.. 2009; Valles et al.. 2003; Kaufmann et al.. 2002; Pugh et al.. 2000;
Smith et al.. 2000; Htils AG. 1992; Hazleton Labs. 1991a; BIBRA. 1986; Bio/dynamics. 1982a;
Midwest Research Institute. 1981). The database includes seven studies in various strains of rat, four
studies in mice, and one study in monkeys. One intermediate dermal exposure study in female B6C3F1
mice was identified (Butala et al.. 2004). These studies provide data on relative/and/or absolute liver
weights, histopathology, hepatic enzyme levels and/or activity (e.g., AST, ALT, ALP), and other
parameters useful to determining the effects of DINP on the liver. These studies are summarized in
Table Apx B-l.
Eight of the available intermediate oral studies and one of the dermal exposure studies reported
increases in absolute and/or relative liver weights or incidences of hepatocyte proliferation or other
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nonneoplastic lesions following oral exposure to DINP (Ma et al.. 2014; Kwack et al.. 2009; Valles et
al.. 2003; Kaufmann et al.. 2002; Smith et al.. 2000; Htils AG. 1992; Hazleton Labs. 1991a; BIBRA.
1986; Bio/dynamics. 1982a). These observations sometimes coincided with increases in peroxisomal
volume, peroxisomal beta oxidation, and activity of enzymes such as palmitoyl-CoA oxidase, indicative
of PPARa activation, which is discussed in further detail in the mechanistic section.
The BIBRA (1986) study evaluated the ability of DINP to induce peroxisome proliferation in male and
female F344 rats fed 0, 0.6, 1.2, or 2.5 percent DINP in the diet for 21 days (equivalent to 0, 639, 1,192,
or 2,195 mg/kg-day [males] and 0, 607, 1,193, or 2,289 mg/kg-day [females]). Body weights were
significantly reduced in males (6-12% decrease) and in females (6-14% decrease) in a time- and dose-
dependent manner. Food intake was also significantly reduced (19-49%) in males and females.
Significant dose-dependent increases in absolute and relative liver weight were observed in males and
females beginning in animals from the low dose group (639 mg/kg-day in males; 607 mg/kg-day
females). The effects observed on liver weight were considered exposure-related even though terminal
body weights were significantly reduced in males and in females in a dose-dependent manner, and body
weight gain was reduced in animals at the highest dose level. In parallel with the increases in liver
weights, the authors reported dose-dependent increases in cyanide-insensitive palmitoyl-CoA oxidation
levels in males and females of the mid- and high-dose groups, dose-dependent increases in microsomal
protein levels of males and females (all dose levels) and increases in lauric acid 11- and 12-hydroxylase
activities in males of the low-dose group (639 mg/kg-day in males). Hydroxylase activities were
increased in high-dose females. The authors also reported decreases in total cholesterol in males
(9-24%) and females (14-24%), as well as dose-dependent decreases in serum triglycerides in males
(24-48%). However, dose-dependent increases in serum triglycerides (24-26%) were observed in
females. The inconsistency of effects between sexes is source of uncertainty in the data set. The authors
also examined liver tissue via electron microscopy and observed increases in peroxisome proliferation in
males and females from the highest exposure groups. However, these effects were not further
quantitatively described, which is another limitation of the data set.
Data from BIBRA (1986) were consistent with Kwack et al. (2009). In the Kwack study, male SD rats
were administered 0 or 500 mg/kg-day DINP daily via gavage for 4 weeks. Increased relative liver
weight (45%) was observed, which coincided with perturbations in several clinical chemistry
parameters. Increases were observed in the serum levels of AST (32%), ALP (260%), and triglycerides
(53%). The observed effects were considered adverse because the liver weight changes were
accompanied by clinical chemistry markers of hep at oxi city. Interestingly, these results were not wholly
consistent with a study by the same authors with a shorter exposure duration (Kwack et al.. 2010). In
that study, male SD rats were again administered to 0 or 500 mg/kg-day DINP daily via gavage for 2
weeks. Increases in AST levels (31%) and ALP (159%) were observed as well as increases in serum
triglycerides. There was no change in ALT levels and no significant change in relative liver weight.
Several other studies reported increases in relative and/or absolute liver weight with concomitant
changes in other hepatic endpoints in B6C3F1 mice (Valles et al.. 2003; Kaufmann et al.. 2002; Smith et
al.. 2000; Hazleton Labs. 1991a) and/or F344 rats (Smith et al.. 2000; Htils AG. 1992; Bio/dynamics.
1982a; Midwest Research Institute. 1981). following oral exposure to DINP.
Smith et al. (2000) evaluated liver weights in mice and rats following 2- or 4-week dietary exposure to
DINP. In rats, increased relative liver weights were observed after 4 weeks of exposure to 12,000 ppm
DINP (equivalent to 1200 mg/kg-day). In mice, increased liver weights were observed after 2- or 4-
weeks exposure to 6,000 ppm DINP (equivalent to 900 mg/kg-day). The LOEL in each species was the
high-dose of DINP (1,200 mg/kg-day for rats, 900 mg/kg-day in mice). Valles et al. (2003) reported
Page 136 of 282
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similar findings in male and female B6C3F1 mice fed diets containing 0, 150, 1,500, 4,000, or 8,000
ppm of DINP (CASRN 68515-48-0) for 2 weeks. Relative liver weight was significantly increased in
both sexes at the two highest dose groups and in females at the mid dose-group. The percent change in
relative liver weight for the high dose group was 37 percent in males and over 50 percent in females.
The other statistically significant increases in females were less than 10 percent over controls, while
relative liver weight in males of the 4,000 ppm increased by almost 17 percent.
Two other studies (Kaufmann et al.. 2002; Hazleton Labs. 1991a) reported similar findings at lower
doses after similar exposure durations (i.e., 4 weeks). In Kaufmann et al. (2002). male and female
B6C3F1 mice were exposed to 0, 500, 1,500, 4,000, or 8,000 ppm DINP in the diet for 4 weeks
(equivalent to 0, 117, 350, 913, 1860 mg/kg-day [males]; or 0, 167, 546, 1272, or 2806 mg/kg-day
[females]). Significant increases in absolute and relative liver weight were observed in males and
females, which corresponded with increased peroxisomal volume and peroxisomal enzyme activity
(cyanide-insensitive palmitoyl-CoA) at doses as low as 350 mg/kg-day in males or 546 mg/kg-day in
females. The LOEL/NOEL was 350/117 mg/kg-day in males and 546/167 mg/kg-day in females.
Hazleton Labs (1991a) reported similar LOEL values for liver effects in males (635 mg/kg-day) and
females (780 mg/kg-day). That study exposed male and female B6C3F1 mice to 0, 3000, 6000, or
12,500 ppm DINP in the diet for 4 weeks (equivalent to 0, 635, 1,377, 2,689, or 6,518 mg/kg-day
[males]; 0, 780, 1,761, 3,287, or 6,920 mg/kg-day [females]) and evaluated liver weights,
histopathology, and serum liver enzymes at study termination.
Increases in absolute and relative liver weights were observed in all male and female exposure groups
except the low dose, and increased ALT activity was observed in males and females from the high dose
only. Additional findings included enlarged and discolored livers, increased incidence of
hepatocytomegaly (all male dose groups; all female dose groups except low dose), and increased
incidence of coagulative necrosis and/or separate chronic inflammatory foci in high-dose males (6,518
mg/kg-day) and females (6,920 mg/kg-day) as well as females of the 3,287 mg/kg-day group. Similar
findings were reported in a study by Ma et al. (2014). which administered 0.2, 2, 20 or 200 mg/kg-day
DINP to male Kunming mice via oral gavage daily for 14 days. This study established a NOAEL at 20
mg/kg-day and a LOAEL at 200 mg/kg-day based on increased histopathologic lesions (reported
qualitatively only) of the liver, including central vein dilation, congestion, and narrowing of the sinusoid
with loose cytoplasm in animals exposed to the highest dose of DINP.
The findings that support liver toxicity in mice and the rat study by Smith et al. (2000) were consistent
with two additional rat studies. A study by the Midwest Research Institute (1981) fed male and female
F344 rats 0, 0.2, 0.67, or 2 percent DINP in the diet for 28 days (estimated doses: 0, 150, 500, 1,500
mg/kg-day [males]; 0, 125, 420, 1,300 mg/kg-day [females]). Increases in hepatic catalase and carnitine
acetyltransferase activity were observed in low dose males (150 mg/kg-day) and females (125 mg/kg-
day). Increases in absolute and relative liver weight were also observed in the mid dose males (500
mg/kg-day) and females (420 mg/kg-day) with no corresponding change in body weight. Additionally,
Bio/dynamics (1982a) administered 0 or 1,700 mg/kg-day DINP in the diet to male rats for 1 week and
then evaluated liver weight, general appearance (i.e., macroscopic observation), and clinical chemistry
parameters, including serum ALP at study termination. At study termination, the treated animals had
increased absolute and relative liver weight, as well as increased body weight, and the authors noted
slight congestion in all lobes of the liver in animals exposed to DINP. No statistically or biologically
significant changes were observed for serum ALP levels. A 14-day study by Hiils AG (1992) exposed
female F344 rats to 0, 25, 75, 150, or 1,500 mg/kg-day and then evaluated liver weights, clinical
chemistry parameters, and histopathology at study termination, as well as activities of several
microsomal enzymes. In general, effects were observed at the highest dose, including increases in
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absolute and relative liver weight, and increases in EROD. A dose-dependent increase was observed in
lauric acid hydroxylase, beginning at 25 mg/kg-day. Of note, this study was not reasonable available to
EPA, and data reported on this study reflect those reported by Health Canada's Hazard Assessment
(EC/HC. 2015).
Not all studies identified in existing assessments reported hepatic effects consistent with peroxisomal
beta-oxidation and/or PPARa activation. Indeed, one study in cynomolgus monkeys (Pugh et al.. 2000)
reported no effect on relative liver weights, histopathology, or serum chemistry parameters in monkeys
administered 0 or 500 mg/kg-day DINP daily via oral gavage for 14 days.
New Literature: EPA identified two new studies published between 2015 and 2024 that provided data on
toxicological effects of the liver following intermediate exposure to DINP via the oral route (Neier et al..
2018) or dermal route (Liang and Yan. 2020). The developmental exposure study by Neier et al. (2018)
evaluated absolute and relative liver weights as well as hepatic triglyceride levels in PND21 male and
female yellow agouti (Avy) mice. Dams were administered 0 or 75 ppm DINP in the diet (equivalent to
15 mg/kg-day) beginning 2-weeks before mating and lasting through PND21. Increased absolute
(27.6%) and relative (15.5%) liver weights were observed in exposed female offspring at PND21. No
significant changes were observed in males. No significant changes were observed in hepatic
triglyceride levels, suggesting that differences in liver weight were not attributed to increases in lipid
accumulation in the liver in this study.
Liang and Yan (2020) applied 0, 0.02, 0.2, 2, 20, or 200 mg/kg-day DINP to the shaved skin on the
backs of male Balb/c mice (6/group) for 28 days and evaluated liver weights, liver histopathology, and
markers of oxidative stress in liver tissue at the end of the study. Significant increases in relative liver
weight were observed in the 20 (7% increase) and 200 mg/kg-day groups (11% increase) and no
significant changes in absolute liver weight were observed, nor changes in body weight. The increased
liver weight corresponded with increases in histopathological findings in the 20 and 200 mg/kg-day
group including enlarged hepatocytes, broadened liver cords, and expanded central veins. However, the
histopathological data was not reported quantitatively, only single representative histological images
were provided, which limits the ability to interpret these results. Changes were observed in various
markers of oxidative stress, including ROS via the DCF-DA assay, DNA-protein-crosslinks (DPC),
glutathione content (but not ratio of GSHGSSH), and MDA. In general, a dose-dependent increase in
ROS and MDA and corresponding dose-dependent decrease in GSH content was observed in liver
samples, with statistically significant effects at 20 and 200 mg/kg-day. These data suggest a pro-oxidant
environment in the liver. Additionally, DPCs were increased at 200 mg/kg-day in liver. While these data
support that DINP negatively impacts the liver at doses as low as 20 mg/kg-day, the study has several
limitations that impact the ability to interpret the results, namely those of reporting deficiencies (e.g.,
qualitative reporting of histopathological data), and exposure methods characterization for the dermal
application of DINP.
As discussed in Section 2.3, dermal absorption of DINP is low (i.e., 2-4% over 7 days), which indicates
that the dose of absorbed DINP that caused oxidative stress in the study by Liang and Yan (2020) is
much lower than the dermally applied dose of 20 mg/kg-day. However, there several uncertainties
associated with the study by Liang and Yan (2020) that raise uncertainty with the actual received doses
in the study, as only nominal doses are provided. Liang and Yan state that 20 |iL of test solution
(concentration of applied test solution not provided) was applied evenly to a 2 cm2 area of exposed skin
on the center of the back of the mouse; however, additionally methodological details pertaining to how
DINP was dermally administered were not provided. For example, study authors do not provide
information relating to how hair was removed from the backs of mice and whether or not care was taken
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to avoid applying solutions of DINP to abraded skin, which would be expected to increase dermal
absorption; how frequently DINP solutions were applied and whether DINP was washed from the skin
at the application site between dermal applications; and whether skin was covered with a bandage to
help limit evaporation, as well as oral ingestion of DINP through grooming.
TableApx B-l. Summary of Liver Effects Reported in Animal Toxicological Studies Following
Intermediate Duration Exposure to DINP
Brief Study Description
(Reference)
NOAEL/
LOAEL
(mg/kg-day)
Effect at LOAEL
Remarks
Kunming mice (males
only); gavage; 0, 0.2, 2, 20,
200 mg/kg-day; 14 days
(Maetal.. 2014)
20/ 200
Markers of oxidative
stress (| ROS, j
GSH, |MDA, t 8-
OH-dG) and
inflammation (fIL-1,
t TNFa) at > 20
mg/kg-day
Other liver effects:
Liver histopathology: |
incidences of edema (20 mg/kg-
day); central vein dilation,
congestion, edema, & narrowing
sinusoidal with extremely loose
cytoplasm (200 mg/kg-day)
Considerations: Bodvweisht not
reported
Limitations: Histopatholosv
qualitative only (no incidence
data or statistical analysis); organ
weight and clinical chemistry not
evaluated
F344 rats (females only);
gavage; 0, 25, 75, 150,
1,500 mg/kg-day; 14 days
(Huls AG. 1992)
25 (LOEL)
t lauric acid
hydroxylase (dose-
dependent beginning
at 25 mg/kg-day)
Other liver effects: t absolute
and relative liver weight at
1,500 mg/kg-day; | liver
microsomal enzyme activities
(pentoxyresorufin O-desalkylase
(PROD) and lauryl-CoA
oxidase) at 1,500 mg/kg-day
F344 rats (both sexes);
dietary; 0, 0.2, 0.67, 2%
(estimated: 150, 500, 1,500
mg/kg-day [males]; 0, 125,
420, 1,300 mg/kg-day
[females]); 28 days
(Midwest Research
Institute, 1981)
ND/ 125
(females;
LOEL)
ND/ 150
(males;
LOEL)
t in hepatic catalase
and carnitine
acetyltransferase
activity
Other liver effects: t absolute
and relative liver weight (500
mg/kg-day [males]; 420 mg/kg-
day [females])
B6C3F1 mice (both sexes);
dietary; 0, 500, 1,500,
4,000, 8,000 ppm
(estimated: 117, 350, 913,
1,860 mg/kg-day [males]; 0,
167, 546, 1,272, 2,806
mg/kg-day [females]); 1 or
117/350
(males)
167/546
(female)
t absolute and
relative liver weight;
t peroxisomal
volume, and
peroxisomal enzyme
activity; | hepatocyte
proliferation in males
Other liver effects:
Liver histopathology: |
hepatocyte proliferation in
females at >1,272 mg/kg-day.
Considerations: Multiple zones
of the liver examined for
Page 139 of 282
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Brief Study Description
(Reference)
NOAEL/
LOAEL
(mg/kg-day)
Effect at LOAEL
Remarks
4 weeks (Kaufmann et al.,
2002)
quantitative measurement of
hepatocyte proliferation;
bodyweight not reported
SD rats (males only); oral
gavage; 0, 500 mg/kg-day;
28 davs (Kwack et al.,
2009)
ND/ 500
I body weight gain; |
relative liver weight;
clinical chemistry (|
AST, ALP,
triglycerides)
Considerations: 1 bodv weight
gain (-10%) in DINP exposed
mice
F344 rats (both sexes); diet;
0, 0.6, 1.2, 2.5% (estimated:
639, 1,192, 2,195 mg/kg-
day [males]; 607, 1,198,
2,289 mg/kg-day [females]);
21 davs (BffiRA. 1986)
ND/ 639
(males)
ND/ 607
(females)
t absolute and
relative liver weight
(absolute increase in
males: 136, 150, and
165%; relative
increase in males:
136, 173,232%;
absolute increase in
females: 124, 164,
and 198%; relative
liver weights in
females: 131, 175,
231%); t 11-and 12-
hydroxylase activity,
hypolipidemic effects
Considerations: Bodv weights
and food intake were
significantly reduced in males
(6-12%) and in females (6-14%
decrease). Food intake was also
significantly reduced (19-49%)
in males and females.
B6C3F1 mice (both sexes);
dietary; 0, 3,000, 6,000,
12,500 ppm (estimated: 635,
1,377, 2,689, 6,518 mg/kg-
day [males]; 780, 1,761,
3,287, 6,920 mg/kg-day
[females]); 4 weeks
(Hazleton Labs, 1991a)
ND/ 635
(males)
ND/ 780
(females,
LOEL)
Enlarged and
discolored livers; |
incidence of
hepatocytomegaly
Other liver effects:
t incidence of coagulative
necrosis and/or separate chronic
inflammatory foci
B6C3F1 mice (males only);
dietary; 0, 500, 6,000 ppm
(estimated: 75, 900 mg/kg-
dav); 2 or 4 weeks (Smith et
al.. 2000)c
75 (NOEL)/
900 (LOEL)
| in relative liver
weight at 4 weeks
Other liver effects: t PBOX, T
DNA synthesis; inhibition of
GJIC
Limitations: Bodvweight not
reported
F344 rats (males only);
dietary; 0, 1,000, 12,000
ppm (estimated: 100, 1,200
mg/kg-day); 2 or 4 weeks
(Smith et al., 2000)c
100
(NOEL)/
1,200 (LOEL)
| in relative liver
weight at 4 weeks
Other liver effects: t PBOX, T
DNA synthesis; inhibition of
GJIC
Considerations: Significant
increases in relative liver weight
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Brief Study Description
(Reference)
NOAEL/
LOAEL
(mg/kg-day)
Effect at LOAEL
Remarks
observed at 4-week but not 2-
week timepoint.
Limitations: Onlv male rats were
evaluated.
F344 rats (males only);
dietary; 0, 2% (estimated:
1,700 mg/kg-day); 7 days
(Bio/dvnamics, 1982a)
ND/ 1,700
t absolute and
relative liver weight;
macroscopic liver
observations; changes
in clinical chemistry
(I triglycerides)
Cynomolgus monkeys
(males only); 0, 500 mg/kg-
day; oral gavage; 14 days
(Push et al., 2000)
500/ND
No statistically or biologically
significant effects were observed
SD rats (male and female);
0 or 500 mg/kg-day;
savase; 14 davs (Kwack et
al.. 2010)
ND/ 500
t AST activity (31%),
t ALP (159%); t
serum triglycerides
Other liver effects: t liver
weights, altered serum
biochemistry, and altered
urinalysis
Considerations: No change in
serum ALT
11 Dose equivalent calculated from 75 mg DINP/kg chow/day based on the assumption that pregnant and nursing
female mice weigh approximately 25g and eat approximately 5 g chow/day.
h Data for the Huls AG studv (1992) were not reasonably available to EPA; data in this table reflect those
reported by Health Canada's Hazard Assessment (EC/HC. 2015).
c Smith et al. (2000) evaluated two isomers of DINP: DINP-1 (CAS 68515-48-0) and DINP-A (CAS 71549-78-
5). The DINP-A isomer is outside the scope of the hazard evaluation; all results herein refer to the DINP-1
isomer.
Snbchronic (>30 to 90 Days) Exposure Studies: EPA identified nine studies from existing assessments
that provide data on the toxicological effects of DINP on the liver following subchronic duration oral
exposure, including six studies in rats (Hazleton Labs. 1991b; BASF. 1987; Bio/dynamics. 1982b. c;
Hazleton Labs. 1981. 1971). one in mice (Hazleton Labs. 1992). one study in dogs (Hazleton
Laboratories. 1971). and one study in marmoset monkeys (Hall et al.. 1999). The available studies are
summarized in Table Apx B-2 and discussed further below. One dermal exposure study in New Zealand
white rabbits was also available (Hazleton Laboratories. 1969).
The lowest achieved dose across these rodent studies was 50 mg/kg-day and the highest was 5,770
mg/kg-day (Table_Apx B-2). All studies reported increases in absolute and/or relative liver weight,
sometimes in parallel with exposure-related histopathological effects on the liver (e.g., hepatocytic
hypertrophy), and sometimes coinciding with increases in liver enzymes (i.e., ALT, ALP), suggesting
impaired liver function. These data suggest that the liver is a target organ for DINP, which is consistent
with conclusions from previous assessments by regulatory agencies.
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Hazleton Laboratories (1971) reported increased absolute and relative liver weights in both sexes at 500
mg/kg-day as well as exposure-related changes in liver histopathology in males (hepatocytic
hypertrophy throughout the panlobular section). In that study, albino rats were exposed to 0, 50, 150, or
500 mg/kg-day DINP for 13 weeks via diet. Two additional dietary exposure studies in rats by Hazleton
Labs (1991b. 1981) reported increased liver weights, and increased incidences of histopathological
lesions or altered clinical chemistry parameters that suggest liver toxicity. Consistent with the earlier
Hazleton study (1971). Hazleton Labs (1991b) found evidence to suggest liver toxicity in F344 rats
exposed to 0, 2500, 5,000, 10,000 or 20,000 ppm DINP for 13 weeks via feed (equivalent to 0, 176, 354,
719, or 1,545 mg/kg-day [males]; 0, 218, 438, 823, or 1,687 mg/kg-day [females]). Increases in absolute
and relative liver weight were accompanied by hepatocellular enlargement in the highest treatment
group. The LOEL was 176 mg/kg-day in males and 218 mg/kg-day in females based on increased liver
weights.
Another study from Hazleton Labs (1981) administered 0, 1,000, 3,000, or 10,000 ppm DINP to male
and female albino rats for 13 weeks in feed (equivalent to 0, 60, 180, or 600 mg/kg-day). Exposure
related increases in absolute and relative liver weights were observed in males and females from the
high dose groups (absolute weights: 33% increase in males, 23.3% increase in females; relative liver
weights: 30.2% increase in males; 33.3% in females). Unlike the other Hazleton rat studies (1991b.
1971). exposure-related nonneoplastic lesions in the liver were not observed, although hepatocellular
degeneration was noted in two individual high-dose (600 mg/kg-day) males. Moreover, the authors note
that exposure-related changes in histopathology were limited to the kidneys of high dose males. Dose-
related decreases in several clinical chemistry parameters were observed in both sexes, including total
protein, globulin, and total bilirubin, apart from total bilirubin from males of the mid-dose group (180
mg/kg-day). The decrease in globulin levels reached statistical significance in mid- (180 mg/kg-day) and
high-dose (600 mg/kg-day) females. Decreased bilirubin reached statistical significance in high-dose
males.
Two similarly designed studies in rats from Bio/dynamics (1982b. c) also reported increased absolute
and/or relative liver weight at similar doses in parallel with changes in clinical chemistry parameters. In
the first Bio/Dynamics study, male and female F344 rats were administered 0, 0.1, 0.3, 0.6, 1.0, or 2.0
percent DINP in diet for 13 weeks (equivalent to 0, 77, 227, 460, 767, or 1,554 mg/kg-day)
(Bio/dynamics. 1982b). In the second study, male and female SD rats were administered 0.3 or 1.0
percent DINP in diet for 13 weeks (equivalent to 0, 201 or 690 mg/kg-day [males]; 0, 251 or 880 mg/kg-
day [females]) (Bio/dynamics. 1982c). In the first study, increased absolute and relative liver weights
and decreased cholesterol were observed in females exposed to 227 mg/kg-day (LOAEL)
(Bio/dynamics. 1982b). Other effects included increases in ALT in the two highest doses in males (767
or 1,554 mg/kg-day) and highest dose in females. In the second study, increased relative liver weight
and decreased serum triglyceride levels were observed in males exposed to doses as low as 201 mg/kg-
day and females exposed to 251 mg/kg-day (LOEL), as well as at higher doses. These changes were
accompanied by a 49 or 53 percent increase in ALP (in males or females, respectively) and 31 percent
increase in ALT (males) in rats from the high dose groups. In both studies, terminal body weight was
decreased by at least 10 percent in high-dose males and females. In the SD rat study, terminal body
weight was also reduced in the low dose animals by 24 percent (males; 201 mg/kg-day) or over 15
percent (females; 251 mg/kg-day) (Bio/dynamics. 1982c).
An additional study from BASF (1987) reported effects on clinical chemistry and other hepatic changes
related to hepatotoxicity with similar LOAELs to the Bio/dynamics studies. In that study, male and
female Wistar rats were fed 0, 3,000, 10,000, or 30,000 ppm DINP in the diet for 13 weeks (equivalent
to 0, 152, 512, 1,543 mg/kg-day [males]; 0, 200, 666, 2,049 mg/kg-day [females]). Decreased
Page 142 of 282
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triglyceride levels and peripheral fat deposits in hepatocytes were reported in low-dose male (152 mg-
kg-day) and female (200 mg/kg-day) rats. Increased absolute and relative liver weights were observed at
1,101 mg/kg-day [males] and 1,214 mg/kg-day [females]), which are doses much higher than those in
which increased liver weights were observed in the two Bio/dynamics studies (1982b. c). The BASF
study (1987) was not reasonably available to EPA in English; it was identified from Health Canada's
Hazard Assessment (EC/HC. 2015) and therefore is not further considered.
One subchronic duration study in mice provided evidence that the liver is a target of DINP (Hazleton
Labs. 1992). In that study, male and female B6C3F1 mice were administered 1,500, 4,000, 10,000, or
20,000 ppm DINP (equivalent to 365, 972, 2,600, or 5,770 mg/kg-day) in the diet for 13 weeks.
Increases in absolute and relative liver weight, as well as histopathologic effects such as hepatocyte
enlargement, liver degeneration, necrosis, and pigment in Kupffer cells as well as in the bile canaliculi
were observed in the 972 mg/kg-day group (LOAEL). One limitation of this study was the small sample
size, which results in limited statistical power to detect differences between treated groups and controls.
Not all studies have consistently demonstrated the liver toxicity of DINP. Indeed, studies in non-rodent
species, including one study in beagle dogs (Hazleton Laboratories. 1971) and one study in marmoset
monkeys (Hall et al.. 1999). have reported contrasting findings. In a study by Hazleton Laboratories
(1971). 0, 0.125, 0.5, 2 percent DINP was administered to beagles in the diet for 13 weeks (equivalent to
0, 37, 160, or 2,000 mg/kg-day). Increases in absolute and relative liver weights were observed at 160
mg/kg-day in males and 2,000 mg/kg-day in both sexes. Histopathologic changes were also observed,
including hepatocyte hypertrophy associated with decreased prominence of hepatic sinusoids at 2,000
mg/kg-day in both sexes. Serum ALT levels increased by 37 percent in males and 48 percent in females
from week 4 at 160 and 2,000 mg/kg-day. Dose-responsive increases in ALT levels were observed in
males (47, 32 and 60% increase) and females (48, 74, and 107% increase) at study termination.
Limitations of this study include the small sample size and lack of statistical analysis, which increase
uncertainty in the data from this study. Nevertheless, existing assessments of DINP have supported
NOAEL and LOAEL values of 37 and 160 mg/kg-day based on increased absolute and relative liver
weights accompanied with histopathological changes at the highest dose (2,000 mg/kg-day) tested
(EC/HC. 2015). or a LOAEL of 37 mg/kg-day with no NOAEL based on increase liver weight and
serum ALT (ECHA. 2013b; ECB. 2003). Additional limitations of this study include reporting
deficiencies, including the lack of statistical analyses and inconsistencies between text and tables. These
limitations increase uncertainty in the data from this study.
In contrast, a study in marmoset monkeys by Hall et al. (1999) did not observe any statistically
significant liver effects. In that study, male and female marmoset monkeys were administered 0, 100,
500, or 2,500 mg/kg-day DINP daily via oral gavage for 13 weeks. Exposure to DINP increased liver
weight in males, but the effect was not dose-dependent nor statistically significant at any dose, which the
authors attribute to low sample size and high variability.
New Literature: EPA did not identify any new studies published from 2015 through 2024 that provided
data on toxicological effects of liver following chronic exposure to DINP.
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TableApx B-2. Summary of Liver Effects Reported in Animal Toxicological Studies Following
Subchronic Exposure to DINP
Brief Study Description
(Reference)
NOAEL/
LOAEL
(mg/kg-day)
Effect at LOAEL
Comments
Beagle dogs (both sexes);
dietary; 0, 0.125, 0.5, 2%
(estimated: 37, 160, 2,000
mg/kg-day); 13 weeks
(Hazleton Laboratories,
1971)
37/ 160
t absolute and
relative liver weight;
t serum ALT
Other liver effects:
Hepatocytic hypertrophy
associated with decreased
prominence of hepatic sinusoids
at 2,000 mg/kg-day.
Hepatocytic cytoplasm varied
from fine granular to vacuolated
appearance.
Considerations: No NOAEL
established due to absence of
statistical analysis and some
inconsistencies in data reporting
(i.e., text and tables in the
study)
F344 rats (both sexes);
dietary; 0, 0.1, 0.3, 0.6, 1.0,
2.0% (estimated: 77, 227,
460, 767, 1,554 mg/kg-
day); 13 weeks
(Bio/dvnamics, 1982b)
77/ 227
t absolute and
relative liver weight;
I cholesterol
(females)
Other liver effects: t ALT
(males at >767 mg/kg-day and
females at 1,554 mg/kg-day); [
cholesterol (females at >227
1,554 mg/kg-day)
Considerations:
I bodyweight gains at 767
mg/kg-day (males only). [
terminal bodyweight (>10%) at
1,554 mg/kg-day (both sexes).
Wistar rats (both sexes);
dietary; 0, 3,000, 10,000,
30,000 ppm (estimated:
152, 512, 1,543 mg/kg-day
[males]; 200, 666, 2,049
mg/kg-day [females]); 13
weeks ((BASF, 1987) as
cited by Health Canada
(EC/HC. 2015))t?
ND/ 152
(males)
ND/ 200
(females)
Clinical chemistry
and liver changes
related to
hepatotoxicity
(I triglyceride level
and I peripheral fat
deposits in
hepatocytes)
Considerations:
I bodyweight for males at 152
and 1,543 mg/kg-day.
Insufficient information to
discern if reported bodyweight
was terminal or bodyweight
change.
F344 rats (both sexes);
dietary; 0, 2,500, 5,000,
10,000, 20,000 ppm
(estimated: 176, 354, 719,
1,545 mg/kg-day [males];
218, 438, 823, 1,687
mg/kg-day [females]); 13
ND/ 176
(males)
ND/ 218
(females)
t liver weights
Other liver effects:
Hepatocellular enlargement at
the highest dose.
Considerations:
I bodyweight gain at 1,545
mg/kg-day (both sexes). [
terminal bodyweight (>10%).
(Body weight gains were
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Brief Study Description
(Reference)
NOAEL/
LOAEL
(mg/kg-day)
Effect at LOAEL
Comments
weeks (Hazleton Labs,
1991b)
decreased in both sexes at 1,545
mg/kg-day, along with
decreases in terminal body
weight >10% relative to
controls).
SD rats (both sexes);
dietary; 0, 1,000, 3,000,
10,000 ppm (estimated: 60,
180, 600 mg/kg-day); 13
weeks Hazleton Labs
(1981)
LOEL= 180
I total protein and
globulin levels
(males)
Other liver effects: t liver
weights (high dose for both
sexes); [ total protein, and total
bilirubin
Considerations:
histopathological findings
limited to the kidney
SD rats (both sexes);
dietary; 0, 0.3, 1.0%
(estimated: 201, 690
mg/kg-day [males]; 251,
880 mg/kg-day [females]);
13 weeks (Bio/dvnamics,
1982c)
ND/ 201
(males; LOEL)
ND/ 251
(females;
LOEL)
I terminal body
weights in both
sexes; | absolute and
relative liver weight
accompanied by [ in
triglycerides.
Other liver effects: t ALP
(males & females) and | ALT
(males) from the high dose
groups
Considerations:
I terminal bodyweight by 24%
and 28% in 201 mg/kg-day and
690 mg/kg-day males,
respectively. [ terminal
bodyweight by >15% and 31%
in 251mg/kg-day and 880
mg/kg-day females,
respectively.
Albino rats (both sexes);
dietary; 0, 50, 150, 500
mg/kg-day; 3 months
(Hazleton Labs, 1971)
150 (NOEL)/
500 (LOEL)
t absolute and
relative liver weight
and | hepatocyte
hypertrophy
Considerations:
Slight non-significant [
bodyweight gain in 500 mg/kg-
day males. Bodyweight gain
similar across all female groups.
Terminal bodyweight within
10% of controls for all male and
female dose groups.
B6C3F1 mice (both sexes);
dietary; 0, 1,500, 4,000,
10,000, 20,000 ppm
(estimated: 365, 972,
2,600, 5,770 mg/kg-day);
13 weeks (Hazleton Labs,
1992)
365/972
t absolute and
relative liver weight;
hepatocyte
enlargement; other
histopathology in
liver [i.e., pigments
in Kupffer cells and
bile canaliculi, liver
degeneration/
necrosis]
Considerations: 1 bodvweisht
gain and [ terminal bodyweight
of males and females at 5,770
mg/kg-day.
Page 145 of 282
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Brief Study Description
(Reference)
NOAEL/
LOAEL
(mg/kg-day)
Effect at LOAEL
Comments
Marmoset (both sexes); 0,
100, 500, 2,500 mg/kg-day;
oral gavage; 13 weeks
(Hall et al.. 1999)
500/ND
I body weight and
body weight gain
Considerations: 1 relative liver
weight (males) but not dose-
dependent and did not reach
statistical significance
11 The BASF (1987) studv was onlv available in German; EPA reports its use based on Health Canada's human
health hazard assessment (EC/HC. 2015).
Chronic (>90 daysj Exposure: EPA identified five studies from existing assessments that provide
information on the toxicological effects of DINP on the liver, including two oral exposure studies
conducted in F344 rats (Covance Labs. 1998c; Lington et al.. 1997). one oral study in SD rats
(Bio/dynamics. 1987). one oral exposure study conducted in B6C3F1 mice (Covance Labs. 1998b). and
a combined one and two generation study in SD rats (Waterman et al.. 2000; Exxon Biomedical. 1996a.
b). No chronic exposure data on DINP are available for humans or other primates. Available studies are
summarized in TableApx B-6.
Two studies in F344 rats reported similar findings, most notably of nonneoplastic lesions of the liver
including spongiosis hepatis (Covance Labs. 1998c; Lington et al.. 1997). Lington et al. (1997)
administered 0, 300, 3,000, or 6,000 ppm DINP to F344 rats in the diet for up to 24 months,
corresponding to mean daily intakes of 0, 15, 152, or 307 mg/kg-day in males and 0, 18, 184, or 375
mg/kg-day in females, respectively. Male and female rats in the mid- and high-dose groups had
statistically significant increases in absolute and relative liver weights throughout the exposure period
and study termination, where relative weight increased 19 to 31 percent in males and 16 to 29 percent in
females. Increases in liver weight corresponded with increases in liver enzyme levels. In males, dose-
related increases of 1.5- to 3-fold were observed in ALP, AST, and ALT activities of mid- and high-dose
groups throughout the study. No significant differences were observed in females. Increased incidences
of several non-neoplastic histopathological lesions were observed in the liver at 18 months, including
minimal to slight centrilobular to midzonal hepatocellular enlargement in high-dose males (incidence:
9/10 vs. 0/10 in controls) and females (10/10 vs 0/10 in controls). At study termination (i.e., 24 months),
dose-related increases were observed in the incidence of focal necrosis, spongiosis hepatis, sinusoid
ectasia, hepatocellular enlargement, and hepatopathy associated with leukemia (Table Apx B-3).
The study authors did not report statistical significance for any of the observed lesions. EPA conducted
an independent review of the incidences of spongiosis hepatis and hepatopathy associated with leukemia
and determined that these histopathology findings were significantly increased in mid- (152 mg/kg-day)
and high-dose (307 mg/kg-day) male rats (Table Apx B-3). Additionally at the high dose in the males,
the incidences of sinusoid ectasia, hepatocellular enlargement, and focal necrosis were significantly
increased over controls. In females, dose-related increases in the incidence of focal necrosis,
hepatopathy associated with leukemia, and hepatocellular enlargement were noted at study termination.
The independent statistical analysis determined that the incidences of hepatocellular enlargement and
hepatopathy associated with leukemia were significantly increased in high-dose females. The NOAEL
and LOAEL for non-cancer hepatic effects in this study were 15 and 152 mg/kg-day, respectively; both
are based on a statistically significant increase in the incidence of spongiosis hepatis in mid-dose male
rats that was accompanied by increased absolute and relative liver weights and changes in serum
enzyme activities.
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TableApx B-3. Incidence of Selected Non-neoplastic Hepatic Lesions in F344 Rats Exposed to
DINP for 24 Months (Lington et al., 1997)
Dose Group
Lesion
mg/kg-day (ppm)
Control
15 M/18 F
152 M/184
307 M/375
(300)
(3,000)
(6,000)
Males17
Spongiosis hepatis
24/81
24/80
51/80*
62/80*
(29.6%)
(30%)
(63.8%)
(77.5%)
Hepatopathy
22/81
17/80
34/80*
33/80*
associated
(27.2%)
(21.3%)
(42.5%)
(41.3%)
with leukemia
Sinusoid ectasia
16/81
16/80
24/80
33/80*
(19.8%)
(20.0%)
(30.0%)
(41.3%)
Hepatocellular
1/81
1/80
1/80
9/80*
enlargement
(1.2%)
(1.3%)
(1.3%)
(11.3%)
Focal necrosis
10/81
9/80
16/80
26/80*
(12.3%)
(11.2%)
(20.0%)
(32.5%)
Females'1
Focal necrosis
13/81
11/81
19/80
21/80
(16.0%)
(13.6%)
(23.8%)
(26.3%)
Spongiosis hepatis
4/81
1/81
3/80
4/80
(4.9%)
(1.2%)
(3.8%)
(5.0%)
Sinusoid ectasia
9/81
4/81
6/80
10/80
(11.1%)
(4.9%)
(7.5%)
(12.5%)
Hepatocellular
1/81
0/81
0/80
11/80*
enlargement
(1.2%)
(0%)
(0%)
(13.8%)
Source: Table 7 in Lington et al. (1997)
M = male; F = female
"Number of animals with lesion/total number of animals examined. Percent lesion incidence in parentheses.
* Statistically significant at p < 0.05 when compared to the control incidence using Fischer's Exact test;
statistical analysis performed by EPA.
Another 2-year study in F344 rats with comparable dose levels to Lington et al. (1997) provided data to
support the liver toxicity of DINP (Covance Labs. 1998c). In that study, DINP was administered to rats
at dietary concentrations of 500, 1,500, 6,000, or 12,000 ppm (equivalent to average daily doses of 29,
88, 359, or 733 mg/kg-day in males, and 36, 109, 442, or 885 mg/kg-day in females for 104 weeks.
Additional groups of male and female rats were given 12,000 ppm (637 and 774 mg/kg-day,
respectively) for 78 weeks and received basal diet only for the remainder of the study (26 weeks) to
evaluate the reversibility of DINP toxicity (recovery group). Increased absolute and relative liver
weights were observed in the two highest dose groups in males and females at multiple timepoints
throughout the study as well study termination. Relative liver weights were increased 35 to 61 percent in
males and 26 to 71 percent in females. There were no significant changes in absolute liver weights in the
recovery group at the end of the 26-week recovery period, suggesting a reversibility of liver
enlargement. Significant increases in activities of serum enzymes (AST and ALT) were also observed in
both sexes at the two highest doses at weeks 52, 78, and study termination. Serum liver enzyme
activities were also increased in the recovery group. Increases in palmitoyl-CoA oxidase activity were
observed in high dose male and female rats, which is further discussed in the mechanistic section below.
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Histological evidence of liver toxicity was observed in parallel with increases in liver weight and
alterations in serum enzyme activity. Incidences of select non-neoplastic lesions from the Covance study
are summarized in TableApx B-4. A dose-responsive increase in the incidence of spongiosis hepatis
was observed at doses as low as 359 mg/kg-day in males. Other lesions observed in males, such as
cytoplasmic eosinophilia, diffuse hepatocellular enlargement, pigment, and individual cell degeneration
or necrosis were generally observed at higher doses, suggesting spongiosis hepatis was the most
sensitive histopathological response to DINP. EPA's independent review determined that diffuse
hepatocellular enlargement was significantly increased in high-dose males and females at study
termination.
Table Apx B-4. Incidence of Selected Hepatic Lesions in F344 Rats Exposed to DINP in the Diet
for 2 Years (Covance Labs, 1998c)
Dose Group mg/kg-day (ppm)
Lesion
29 Ml
88 Ml
359 M/
733 Ml
Recovery" 637
Control
36 F
109 F
442 F
885 F
M/774 F
(500)
(1,500)
(6,000)
(12,000)
(12,000)
Males
Spongiosis
5/55b
5/50
2/50
13/55*
21/55*
9/50
hepatis
(9.1%)
(10.0%)
(4.0%)
(23.6%)
(38.2%)
(18.0%)
Cytoplasmic
0/55
0/50
0/50
0/55
31/55*
0/50
eosinophilia
(0%)
(0%)
(0%)
(0%)
(56.4%)
(0%)
Diffuse
0/55
0/50
0/50
0/55
17/55*
0/50
hepatocellular
(0%)
(0%)
(0%)
(0%)
(30.9%)
(0%)
enlargement
Increased
1/55
0/50
1/50
0/55
7/55*
9/50
pigment
(1.8%)
(0%)
(2.0%)
(0%)
(12.7%)
(18.0%)
Individual cell
0/55
0/50
0/50
1/55
5/55*
0/50
degeneration/
(0%)
(0%)
(0%)
(1.8%)
(9.1%)
(0%)
necrosis
Females
Spongiosis
0/55
0/50
0/50
1/55
2/55
0/50
hepatis
(0%)
(0%)
(0%)
(1.8%)
(3.6%)
(0%)
Cytoplasmic
0/55
0/50
0/50
0/55
35/55*
0/50
eosinophilia
(0%)
(0%)
(0%)
(0%)
(63.6%)
(0%)
Diffuse
0/55
0/50
0/50
0/55
33/55*
0/50
hepatocellular
(0%)
(0%)
(0%)
(0%)
(60.0%)
(0%)
enlargement
Increased
7/55
8/50
9/50
5/55
16/55*
10/50
pigment
(12.7%)
(16.0%)
(18.0%)
(9.1%)
(29.1%)
(20.0%)
Individual cell
0/55
0/50
0/50
0/55
0/55
0/50
degeneration/
(0%)
(0%)
(0%)
(0%)
(0%)
(0%)
necrosis
Page 148 of 282
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Dose Group mg/kg-day (ppm)
Lesion
29 Ml
88 Ml
359 M/
733 Ml
Recovery" 637
Control
36 F
109 F
442 F
885 F
M/774 F
(500)
(1,500)
(6,000)
(12,000)
(12,000)
Source: Tables 10A and IOC in Covance Labs (1998c)
M = male; F = female
* = significantly different from control (p < 0.05) by Fisher's Exact test as performed by EPA.
11 The 12,000 ppm recovery group received 12,000 ppm DINP in the diet for 78 weeks, followed by a 26-
week recovery period during which the test animals received basal diet alone.
h Number of animals with lesion/number of animals with livers examined; percentage is given in parentheses.
Incidence is sum of lesions observed in unscheduled deaths and at terminal sacrifice.
A third study in rats by Bio/dynamics (1987) provided data on liver weights, histopathology, and effects
on clinical chemistry parameters following chronic exposure to DINP. In that study, male and female
SD rats were administered 0, 500, 5,000, or 10,000 ppm DINP in the diet for up to 2-years (equivalent to
0, 27, 271, or 553 mg/kg-day in males and 0, 33, 331, or 672 mg/kg-day in females). Increased absolute
and relative liver weights were observed in high-dose males and females at the 12-month interim
sacrifice and study termination; all increases were between 14 and 34 percent. In the mid-dose females,
there were non-significant increases in absolute (14%) and relative (11%) liver weight at interim
sacrifice and absolute liver weight (15%) at terminal sacrifice, and a significant increase in relative liver
weight (16%) at terminal sacrifice. In mid-dose males, a nonsignificant increase of 11 percent was seen
in the mid-dose group at interim sacrifice. Histopathological findings were observed at lower doses than
changes in liver weights. Increased incidences of spongiosis hepatis and minimal-to-slight hepatic focal
necrosis were observed in males from the mid-dose group (271 mg/kg-day). The increases in liver
weights and incidences of nonneoplastic lesions were attributed to the administration of DINP.
In parallel with increases in liver weight and histopathological findings, changes in clinical chemistry
parameters were observed. Serum ALT was significantly increased in high-dose males at interim
sacrifices on months 6, 12, and 18 by 292, 203, and 232 percent, respectively. A non-statistically
significant increase of 218 percent was observed in males at study termination (24 months). Serum ALP
was significantly increased at months 6 and 12 in high-dose males by 88 and 76 percent, respectively.
Non-significant increases in AST were observed in males from the mid and high dose groups. In
females, non-significant increases in AST (63%) and ALT (89%) were observed at 6 months. Serum
ALP was significantly increased in females of the high-dose group by 81 percent at 18 months, while a
non-significant increase of 38 percent was observed at study termination. No exposure-related changes
in serum ALP were observed at earlier timepoints in this group or in females of the low- or mid-dose
groups. The increased serum AST, ALT, and ALP in treated males were for the most part not
statistically significant; however, these findings were considered treatment-related due to the
consistency with which they were noted in the treated males at most timepoints. The increased ALP in
females of the high-dose group at month 18 and month 24 is considered treatment-related and adverse.
However, the increased AST and ALT values in females of the high-dose group at month 6 were not
considered treatment-related due to their isolated occurrence in only one animal at only one timepoint.
Moreover, data from this animal were considered to be statistical outliers via the Grubb's outlier test.
Overall, the Bio/dynamics study (1987) supports a NOAEL of 27 mg/kg-day in male rats based on
treatment related increases in histopathologic lesions (i.e., spongiosis hepatis, focal necrosis) and
increases in serum ALT, AST, and ALP at the LOAEL of 271 mg/kg-day.
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One chronic study in mice by Covance Labs (1998b) was identified from existing assessments. Covance
Labs exposed male and female B6C3F1 mice to 500, 1,500, 4,000, or 8,000 ppm DINP for at least 104
weeks. These concentrations corresponded to average daily doses of 0, 90, 276, 742, and 1,560 mg/kg-
day in males and 0, 112, 336, 910, and 1,888 mg/kg-day in females. Evidence of liver toxicity was
observed in treated animals of both sexes. At interim sacrifice, significant increases were observed in
relative liver weights in mid-dose males (742 mg/kg-day) and females (910 mg/kg-day) and in high-dose
males (1,560 mg/kg-day). At study termination, significant increases were observed in absolute
(13-33% increase) and relative (25-60% increase) liver weights in males exposed to 742 or 1,560
mg/kg-day DINP. Relative liver weight was also significantly increased 32 percent in the recovery
group. In females, increases in absolute liver weight (18-34% increase) and relative liver weight
(24-39%) were observed in females exposed to 910 or 1,888 mg/kg-day DINP, as well as in the
recovery groups. However, the responses were not statistically significant.
Exposure-related changes in serum chemistry profiles were also observed and supported the liver as a
target organ. AST and ALT activities were increased in high-dose males (1,560 mg/kg-day) and
recovery group males and females. Exposure-related increases in the serum levels of total protein,
albumin, and globulin were also observed in high-dose males. Increases in albumin and globulin were
also observed in recovery males.
Gross findings, including liver masses, occurred with greatest frequency at the 910 and 1,560 mg/kg-day
dose groups, as well as the recovery group. These masses corresponded to hepatocellular neoplasms or
involvement by lymphoma or histiocytic sarcoma and are discussed further in (U.S. EPA 2025a).
Increased incidences of several nonneoplastic lesions were observed in the livers of high-dose males and
females, including cytoplasmic eosinophilia, diffuse slight to moderate hepatocellular enlargement, and
slight to moderate pigment (Table Apx B-5). These changes were also observed in the recovery group,
but generally at lower incidences than in the high-dose groups. No other statistically significant or dose-
related nonneoplastic lesions of the liver were observed in the Covance study (1998b). Liver weights in
recovery group animals were comparable to those of controls, and histological evidence of liver
enlargement was not observed in the male or female recovery groups. The incidences of non-neoplastic
lesions in the recovery groups were decreased at study termination relative to the high-dose groups, but
in most cases were significantly greater than the control values. These data suggest that DINP-induced
liver toxicity was partially reversed in the recovery groups.
EPA identified a LOAEL value from the Covance (1998b) study of 742 mg/kg-day in males and 910
mg/kg-day in females based on increased incidence of liver masses in males, and increased absolute and
relative liver weights, and decreased absolute and relative kidney weights (Section 3.3). ANOAEL of
276 mg/kg-day in males or 336 mg/kg-day in females was identified based on non-cancer and cancer
effects.
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TableApx B-5. Incidence of Selected Non-neoplastic Lesions in B6C3F1 Mice Exposed to DINP in
the Diet for 2 Years (Covance Labs, 1998b)
Lesion
Dose Group
mg/kg-day (ppm)
Control
90 M
112 F
(500)
276 M
336 F
(1,500)
742 M
910 F
(4,000)
1,560 M
1,888 F
(8,000)
Recovery*
1,560 M
1,888 F
(8,000)
Males
Diffuse hepatocellular
enlargement
0/55°
(0%)
1/50
(2.0%)
1/50
(2.0%)
2/50
(4.0%)
45/55*
(81.8%)
10/50*
(20.0%)
Increased cytoplasmic
eosinophilia
0/55
(0%)
0/50
(0%)
0/50
(0%)
0/50
(0%)
52/55*
(94.5%)
10/50*
(20.0%)
Pigment
0/55
(0%)
0/50
(0%)
0/50
(0%)
0/50
(0%)
49/55*
(89.1%)
6/50*
(12.0%)
Females
Diffuse hepatocellular
enlargement
0/55
(0%)
0/51
(0%)
0/50
(0%)
1/50
(2.0%)
52/55*
(94.5%)
6/50*
(12.0%)
Increased cytoplasmic
eosinophilia
0/55
(0%)
0/51
(0%)
0/50
(0%)
0/50
(0%)
53/55*
(81.8%)
6/50*
(12.0%)
Pigment
1/55
(1.8%)
1/51
(2.0%)
2/50
(4.0%)
2/50
(4.0%)
41/55*
(74.5%)
3/50
(6.0%)
Source: Tables 11A and 11C in Covance Labs (1998b).
M = male; F = female
* = significantly different from control (p < 0.05) by Fisher's Exact test performed by Syracuse Research
Corporation.
" Number of animals with lesion/total number of animals examined; percent incidence of lesion in parentheses.
Incidences are sum of unscheduled deaths and lesions observed at terminal sacrifice.
b The 8,000 ppm recovery group received 8,000 ppm for 78 weeks, followed by a 26-week recovery period during
which the test animals received basal diet alone.
Waterman et al. (2000) assessed the potential toxicity of DINP in one- and two-generation studies
conducted in SD rats. In the one-generation study, male and female animals were administered 0.5, 1.0,
or 1.5 percent DINP in the diet for 10 weeks prior to mating and lasting throughout the mating period.
The females were subsequently exposed throughout gestation and lactation until PND21. Mean received
doses in units of mg/kg-day are shown in Table 3-5. Parental body weight gain was significantly
reduced at the 1.0 and 1.5 percent dose groups in both sexes during the premating phase and in females
during gestation and lactation. Absolute liver weights in both sexes were significantly increased at all
doses, except in PI females at the 1.5 percent level.
For the two-generation study, male and female SD rats were fed DINP at dietary concentrations of 0.0,
0.2, 0.4, or 0.8 percent for 10 weeks before mating and for an additional 7 weeks, through mating,
gestation, and lactation continuously for two-generations. Mean received doses in units of mg/kg-day
are shown in Table 3-7. Absolute liver weights of PI males and females were increased over controls at
all DINP treatment levels. Minimal to moderate increases in cytoplasmic eosinophilia were observed in
all males and females from all dose groups of parents in both generations.
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TableApx B-6. Summary of Liver Effects Reported in Animal Toxicological Studies Following
Chronic Exposure to DINP
Brief Study Description
(Reference)
NOAEL/
LOAEL
(mg/kg-day)
Effect at LOAEL
Remarks
F344 rats (both sexes); dietary;
0, 0.03, 0.3, 0.6% (estimated:
15, 152, 307 mg/kg-day
[males]; 18, 184, 375 mg/kg-
day [females]); 2 years
(Linston et al„ 1997)
15/152
(males)
18/184
(females)
t absolute and
relative liver weight;
t in serum ALT,
AST; | non-
neoplastic lesions
(e.g., focal necrosis,
spongiosis hepatis)
SD rats (both sexes); dietary; 0,
500, 5,000, 10,000 ppm
(estimated: 27, 271, 553 mg/kg-
day [males]; 33, 331, 672
mg/kg-day [females]); 2 years
(Bio/dvnamics, 1987)
GLP-compliant study, non-
guideline
27/271 (males)
t serum ALT, AST,
ALP (males); |
spongiosis hepatis; |
hepatic focal
necrosis
Other liver effects: t
absolute and relative liver
weight (both sexes); |
serum ALP (females); |
incidence of hepatocyte
necrosis at low- and high-
doses (males)
Considerations: 1
bodyweight gains in females
(672 mg/kg-day); no change
in terminal bodyweight in
males; | food consumption
for females at multiple
timepoints during study
(672 mg/kg-day)
Male and female SD rats
(30/sex/dose) fed diets
containing 0, 0.5, 1.0, 1.5%
DINP (CASRN 68515-48-0)
starting 10 weeks prior to
mating, through mating,
gestation, and lactation
continuously for one generation
(received doses in units of
mg/kg-day shown in Table 3-5)
(Waterman et al., 2000; Exxon
Biomedical, 1996a).
ND/ 301 (LOEL)
t absolute and
relative liver weight
for PI and P2 males
and females; f
incidence of minimal
to moderate
cytoplasmic
eosinophilia
Male and female SD rats
(30/sex/dose) fed diets
containing 0, 0.2, 0.4, 0.8%
DINP (CASRN 68515-48-0)
starting 10 weeks prior to
mating, through mating,
gestation, and lactation
continuously for two-
Page 152 of 282
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Brief Study Description
(Reference)
NOAEL/
LOAEL
(mg/kg-day)
Effect at LOAEL
Remarks
generations. Received doses in
units of mg/kg-day shown in
Table 3-7. (Waterman et al„
2000; Exxon Biomedical,
1996b).
B6C3F1 mice (both sexes);
dietary; 0, 500, 1,500, 4,000,
8,000 ppm (estimated: 90, 276,
742, 1,560 mg/kg-day [males];
112, 336, 910, 1,888 mg/kg-day
[females]); 2 years
Recovery study; 0, 1,377
[males]; 0, 1,581 [females]);
diet; 78 weeks, followed by 26
weeks recovery. (Covance
Labs. 1998b)
GLP-compliant and adhere to
EPA guidelines (40 CFR
798.330)
276/742
(males)
336/910
(females)
t absolute liver
weight,
histopathological
changes in the liver
and I body weight
gain) (females); (|
incidence of liver
masses (males)
Significant neoplastic
findings: t hepatocellular
carcinoma; | incidence of
total liver neoplasms
(combined carcinomas and
adenomas)
Considerations:
I mean bodyweights in
males (>742 mg/kg-day)
and females (>336 mg/kg-
day)
F344 rats (both sexes); dietary;
0, 500, 1,500, 6,000, 12,000
ppm (estimated: 29, 88, 359,
733 mg/kg-day [males]; 36,
109, 442, 885 mg/kg-day
[females]); 2 years
Recovery study: 0, 637 mg/kg-
day [males]; 0, 774 mg/kg-day
[females]); diet; 78-week
exposure, followed by 26 week
recovery period (Covance Labs,
1998c)
GLP-compliant and adhere to
EPA guidelines (40 CFR
798.330)
88/359
(males)
109/442
(females)
t absolute and
relative liver weight;
t in serum ALT and
AST;
histopathological
findings in liver
Significant neoplastic
findings
t incidence of mononuclear
cell leukemia; | in
hepatocellular carcinoma; |
in combined hepatocellular
carcinoma and adenoma
(See (U.S. EPA. 2025a) for
further discussion)
Limitations:
Did not report results of
statistical analyses of lesion
incidence data
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Appendix C FETAL TESTICULAR TESTOSTERONE AS AN
ACUTE EFFECT
No studies of experimental animal models are available that investigate the antiandrogenic effects of
DINP following single dose, acute exposures. However, there are studies of dibutyl phthalate (DBP)
available that indicate a single acute exposure during the critical window of development (i.e., GDI 4-
19) can reduce fetal testicular testosterone production and disrupt testicular steroidogenic gene
expression. Two studies were identified that demonstrate single doses of 500 mg/kg DBP can reduce
fetal testicular testosterone and steroidogenic gene expression. Johnson et al. (2012; 2011) gavaged
pregnant SD rats with a single dose of 500 mg/kg DBP on GDI9 and observed reductions in
steroidogenic gene expression in the fetal testes three (Cypl7al) to six (Cypllal, StAR) hours post-
exposure, while fetal testicular testosterone was reduced starting 18 hours post-exposure. Similarly,
Thompson et al. (2005) reported a 50 percent reduction in fetal testicular testosterone 1-hour after
pregnant SD rats were gavaged with a single dose of 500 mg/kg DBP on GDI9, while changes in
steroidogenic gene expression occurred 3 (StAR) to 6 (Cypllal, Cypl7al, Scarbl) hours post-exposure,
and protein levels of these genes were reduced 6 to 12 hours post-exposure. Additionally, studies by
Carruthers et al. (2005) further demonstrate that exposure to as few as two oral doses of 500 mg/kg DBP
on successive days between GDI5 to 20 can reduce male pup AGD, cause permanent nipple retention,
and increase the frequency of reproductive tract malformations and testicular pathology in adult rats that
received two doses of DBP during the critical window.
In summary, studies of DBP provide evidence to support use of effects on fetal testosterone as an acute
effect. However, the database is limited to just a few studies of DBP that test relatively high (500 mg/kg)
single doses of DBP. Although there are no single dose studies of DINP that evaluate antiandrogenic
effects on the developing male reproductive system, there are four studies that have evaluated effects on
fetal testicular testosterone production and steroidogenic gene expression following daily gavage doses
of 500 to 1,500 mg/kg-day DINP on GD14 to 18 (5 total doses) (Gray et al.. 2021; Furr et al.. 2014;
Hannas et al.. 2012; Hannas et al.. 2011)—all of which consistently report antiandrogenic effects at the
lowest dose tested (500 mg/kg-day).
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Appendix D SUMMARY OF EPIDEMIOLOGY STUDIES ON
REPRODUCTIVE OUTCOMES
Radke et al. (2018) report the results of a systematic review that evaluated the association between
DINP and male reproductive outcomes. In examining the relationship between DINP exposure and
AGD, the authors found that there is little evidence linking DINP to AGD. The combination of low
exposure levels (i.e., poor sensitivity) and data availability (i.e., fewer accessible studies) may account
for the weaker evidence of an association between AGDand DINP. When evaluating the relationship
between DINP exposure and sperm parameters, the author determined that the association was moderate
due to the morphology's consistency across studies. In examining the association between DINP and the
time until pregnancy in males, the authors did not report a relationship for DINP, and the evidence was
deemed inconclusive due to the small number of studies and narrow range of exposure. Finally, when
examining the relationship between DINP metabolite (MINP or MCiOP) exposure and testosterone, the
authors found that there is moderate evidence linking DINP metabolites to lower testosterone levels.
Another systematic review by Radke et al. (2019b) evaluated the association between DINP and female
reproductive and developmental outcomes and also found no clear evidence of association due to
inadequate sensitivity in the available data. When examining the relationship between DINP exposure
and pubertal development the authors found that there was no association linking DINP and pubertal
development and the strength of the evidence was deemed indeterminate. Study evaluations of the
relationship between DINP and a woman's time to pregnancy found that the evidence of an association
between fecundity and exposure to DINP was deemed indeterminate due to lack of the evidence of
relationship for the key fecundity outcomes. The authors also found that in studies that measured the
relationship between DINP and spontaneous abortion, there was no association between early loss and
total loss. Thus, the evidence for an association between DINP and spontaneous abortion was deemed
indeterminate. Finally, when evaluating the association between DINP and gestational duration, the
authors found slight evidence for the association between DINP exposure and preterm birth; however
while there was modest increase in the odds of preterm birth with higher DINP exposure the association
was not statistically significant. In summary there was indeterminate evidence linking DINP and female
reproductive and developmental outcomes.
EPA identified 11 new studies (8 medium quality and 3 low quality) that evaluated the association
between DINP metabolites and developmental and reproductive outcomes. The first medium quality
study, a longitudinal cohort study, by Berger et al. (2018). using data from Center for Health Assessment
of Mothers and Children of Salinas (CHAMACOS) cohort examined prenatal urinary DINP levels and
the association with timing of puberty milestones (thelarche, menarche, pubarche, gonadarche) in
children. The authors found an association between pubarche and menarche age increased in "normal"
weight girls per log2 increase in MCOP. The authors also found gonadarche and pubarche age decreased
in all obese boys. There was not significant a significant association between thelarche age increased in
all girls per log2 increase in MCOP.
A medium quality birth cohort study, by Philipat et al. (2019). Etude des Determinants pre et postnatals
du developpement et de la sante de l'Enfant (EDEN) cohort, evaluated associations between DINP
metabolites (MCOP, MCNP) and a set of outcomes measured at birth (birth weight, placental weight,
placental-to-birth weight ratio). MCNP and MCOP were both associated with lower placental-to-birth
weight ratio; MCNP was additionally associated with lower placental weight. MCOP was associated
with lower placental-to-birth weight ratio (PFR) in multipollutant elastic net penalized regression
models. MCOP was not associated with birth weight or placental weight based on elastic net regression
models.
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A medium quality cross-sectional pilot study, by Zota et al. (2019), included a racially diverse
population of premenopausal women within the Fibroids Observational Research on Genes and the
Environment (FORGE) study presenting to a university gynecology clinic and undergoing either
hysterectomy or myomectomy for symptomatic uterine fibroids to examine the potential associations
between urinary DINP biomarkers and two measures of fibroid burden (uterine volume and fibroid size).
Higher urinary concentrations of MCOP and MCNP were significantly associated with odds of greater
uterine volume. In multivariate logistic regression analyses, each log-unit increase in MCOP was
significantly associated with 2.1 (95% CI: 1.2-3.5) times increased odds of greater uterine volume, and
each log-unit increase in MCNP was associated with 2.8 (95% CI: 1.2-3.5) times increased odds of
greater uterine volume, p<0.05. Results from additional multivariate linear regression analyses of
urinary phthalate exposure on percent increase in uterine volume were positive but not significant.
Results from multivariate logistic regression analysis of urinary DINP exposure on odds of fibroid size
increase for MCOP were non-significant. Results from additional multivariate linear regression analyses
of urinary MCOP phthalate exposure on percent increase in fibroid size (cm) were also non-significant.
A medium quality cross-sectional study, by Chang et al. (2019). evaluated the association between sex
hormone levels (luteinizing hormone (LH), follicle-stimulating hormone (FSH), sex hormone binding
globulin (SHBG), inhibin B, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEA-
S), androstenedione (AD), estrone (El), estradiol (E2), total testosterone (TT), free testosterone (FT),
dihydrotestosterone (DHT), dihydrotestosterone/total testosterone ratio, estradiol/total testosterone ratio,
estradiol/estrone ratio), Oxidative stress/Inflammation [(malondialdehyde (MDA), inducible nitric oxide
synthetase (iNOS), 8-hydroxy-2'-deoxyguanosine (8-OHdG)] and benign prostatic hyperplasia (prostate
specific antigen (PSA), prostate volume) and DINP exposure. There were significant positive
associations between the outcomes, FSH, Inhibin B, DHEA, iNOS and MINP with regression
coefficients of 0.91 (95% CI: 0.85, 0.98), 0.90 (95% CI: 0.83, 0.97), 1.58 (95% CI: 1.40, 1.79) and 1.61
(95% CI: 1.29, 2.03) respectively, p < 0.05. Multivariate regression coefficients showed significant
results for FHS, Inhibin B, iNOS and DHEA, but showed nonsignificant results for LH, SHBG, DHEA-
s, AD, El, E2, TT, FT, DHT, MDA, 8-OHdG, PSA, and prostate volume.
A medium quality study, by Mustieles et al. (2019). used data from a small cohort of subfertile couples
in the Environment and Reproductive Health (EARTH) study to analyze the association between
paternal and maternal preconception urinary DINP metabolites (MCOP), as well as maternal prenatal
DINP metabolites, and measures of placental weight. The authors did not find any significant
association between paternal and maternal preconception urinary phthalates, as well as maternal prenatal
phthalates, and measures of placental weight and MCOP.
A medium quality cohort, by Machtinger et al. (2018). examined the association between urinary
concentrations of DINP with intermediate and clinical in vitro fertilization (IVF) outcomes. There was
an association (adjusted means) between urinary MCOP concentration and intermediate outcomes of
assisted reproduction (total oocytes and mature oocytes) [total oocytes T2 = 10.2 (95% CI: 9.3, 11.2), T2
vs. T1 < 0.05; mature oocytes T2 = 8.4 (95% CI: 7.6, 9.3) T2 vs. T1 < 0.05], However, there was no
significant association (adjusted means) between urinary MCOP concentration and intermediate
outcomes of assisted reproduction (fertilized oocytes, top quality embryos). While there was an
association (adjusted means) between urinary MINP concentration and intermediate outcomes of
assisted reproduction (total oocytes) [total oocytes T2 = 9.2 (95% CI: 8.2, 10.2), T2 vs. T1 < 0.05]; there
was not an association (adjusted means) between urinary MINP concentration and intermediate
outcomes of assisted reproduction (mature oocytes, fertilized oocytes, top quality embryos).
Associations between MOiNP or MONP and intermediate outcomes of assisted reproduction (total
oocytes, mature oocytes, fertilized oocytes, top quality embryos) and live birth following assisted
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reproduction were all non-significant for T2, T3 vs. T1 intermediate outcomes and for p-trend of live
birth.
A medium quality case-control study, by Lee et al. (2020). assessed the relationship between uterine
fibroids and DINP metabolite concentrations. The authors did not find any statistically significant
associations between uterine fibroids and DINP metabolite concentrations. The authors did find
associations between cases and controls for OH-MINP concentrations (p-value: 0.042) as mono(4-
methyl-7-hydroxyoctyl) phthalate (OH-MINP) concentrations were significantly higher in the cases than
controls, but it was not statistically significant.
A medium quality occupational short longitudinal study, by Henrotin et al. (2020). observed the three-
day changes in levels of total and free testosterone and oxidized MINP exposure in male factory
workers. A significant inverse association was found between the decrease in serum total testosterone
(TT) concentrations between T1 and T2 and an increase in urinary OXO-MINP. There were no
significant associations observed for total testosterone and models for OH-MINP, or CX-MINP. No
significant associations were noted for free testosterone and oxo-MINP, OH-MINP, or CX-MINP.
Bivariate analyses of sexual health scales (IIEF-5 and ADAM) between DINP exposed and non-exposed
groups: No association was observed between the level of urinary oxo-MINP concentrations and FSH,
LH, index of aromatase activity (ratio of total testosterone to estradiol (TT/E2). No association was
observed between the level of urinary OXO-MINP concentrations and bone turnover biomarkers (P1NP,
CTX).
The first low quality study, a case control study, by Durmaz et al. (2018). examined the association
between DINP metabolites (MINP, MHiNP, MOiNP, MCiOP) and serum luteinizing hormone (LH),
plasma follicle stimulating hormone (FSH) and serum estradiol in non-obese girls aged 4 to 8 years with
premature thelarche. DINP metabolites (MINP, MHiNP, MOiNP, MCiOP and their sum) measured in
spot urine samples were compared among cases and controls. Spearman correlations with uterine
volumes, ovarian volume and pubic hair growth varied but were largely weak, negative and/or not
significant, with some significant positive correlation for the association between MCiOP, MINP and
pubic hair growth, rho = 0.440, p = 0.002 and rho = 0.480, p = 0.000, respectively. Thyroid hormone
levels had largely negative Spearman correlations with DINP metabolites; however MCiOP had a
significant negative correlation with fT4 (rho = -0.335, p = 0.041). Spearman correlations between
DINP metabolites (MCiOP, MiNP, MHiNP, MOiNP, SumDiNP) and BMI and weight were positive and
significant.
A low quality case-control study, by Moreira Fernandez et al. (2019). of women in Brazil evaluated the
association between one DINP metabolite (MINP) and endometriosis. The authors found that there was
a positive but non-significant association for the relationship between MINP and endometriosis (OR=2.5
[95% CI: 0.46, 13.78]).
A final low quality study, a case-control study, by Liao et al. (2018). examined associations between
exposure to one DINP metabolite (MINP) measured in urine samples and recurrent pregnancy loss
among women in Taiwan. The MINP samples was below the limit of detection. The highest sample was
70.4 ng/mL in controls (detection rate 2.6%) and 1.43 ng/mL in cases (detection rate 2.9%).
Page 157 of 282
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Appendix E BENCHMARK DOSE ANALYSIS OF LINGTON ET AL.
(1997)
E.l Background
OCSPP requested that CPHEA run benchmark dose (BMD) models that are available in EPA's
Benchmark Dose Software version 3.3.2 (BMDS 3.3.2), to estimate risk from DINP for select endpoints
from a chronic exposure study (Lington et al.. 1997; Bio/dynamics. 1986) using specified benchmark
response (BMR) levels. The specific endpoints and BMRs provided by OCSPP for analysis are as
follows:
1. Liver weight relative to bodyweight at terminal sacrifice (males and females)
o BMR: 1 control SD, 5%, 10%, 25%
2. Serum ALT at 6- and 18-month sacrifices (males only)
o BMR: 1 control SD, 10%, 20%, 100% (i.e., 2x)
3. Incidence of focal necrosis in the liver (males and females)
o BMR: 5%, 10%
4. Incidence of spongiosis hepatis in the liver (males only)
o BMR: 5%, 10%
5. Incidence of sinusoid ectasia in the liver (males only)
o BMR: 5%, 10%
Although BMD and BMDL values are provided for all of the BMRs, this report provides detailed model
run outputs for only the models that were run using the standard BMRs generally recommended by EPA
for these endpoints, 10 percent relative deviation from the control mean (10% RD) for the dichotomous
endpoints and organ weight change and 1 standard deviation change from the control mean (1 SD).
Detailed modeling results for all standard non-cancer models are provided for all six endpoints using all
of the BMRs requested by OCSPP in separately delivered BMDS Excel output files.
E.2 Summary of BMD Modeling Approach
All standard BMDS 3.3.2 dichotomous and continuous models that use maximum likelihood (MLE)
optimization and profile likelihood-based confidence intervals were used in this analysis. Standard
forms of these models (defined below) were run so that auto-generated model selection
recommendations accurately reflect current EPA model selection procedures EPA's benchmark Dose
Technical Guidance (U.S. EPA. 2012). BMDS 3.3.2 models that use Bayesian fitting procedures and
Bayesian model averaging were not applied in this work.
Standard BMDS 3.3.2 Models Applied to Continuous Endpoints:
• Exponential 3-restricted (exp3-r)
• Exponential 5-restricted (exp5-r)
• Hill-restricted (hil-r)
• Polynomial Degree 3-restricted (ply3-r
• Polynomial Degree 2-restricted (ply2-r)
• Power-restricted (pow-r)
• Linear-unrestricted (lin-ur)
Standard BMDS 3.3.2 Models Applied to Dichotomous Endpoints:
• Gamma-restricted (gam-r)
• Log-Logistic-restricted (lnl-r)
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• Weibull-restricted (wei-r)
• Dichotomous Hill-unrestricted (dhl-ur)
• Logistic (log)
• Log-Probit-unrestricted (lnp-ur)
• Probit (pro)
General Model Options Used for Individual Endpoint Analyses:
• Risk Type: Extra Risk
• Preferred Continuous Endpoint BMRs
o Relative Liver Weight: 0.1 (10%)
o Serum ALT: 1 Standard Deviation (1 SD)
• Preferred Dichotomous Endpoint BMR: 0.1 (10%)
• Confidence Level: 0.95
• Background response: Estimated
• Model Restrictions: Restrictions for BMDS 3.3.2 models are defined in the BMDS 3.3.2 User
Guide and are applied in accordance with EPA BMD Technical Guidance (U.S. EPA. 2012).
Model Selection:
The preferred model for the BMD derivations was chosen from the standard set of dichotomous and
continuous models listed above. The modeling restrictions and the model selection criteria facilitated in
BMDS 3.3.2, and defined in the BMDS User Guide, were applied in accordance with EPA BMD
Technical Guidance (U.S. EPA. 2012) for non-cancer endpoints.
With respect to the continuous endpoints, responses were first assumed to be normally distributed with
constant variance across dose groups. If no model achieved adequate fit to response means (BMDS Test
4p > 0.1) and response variances (BMDS Test 2 p > 0.05) under that assumption, models that assume
normal distribution with non-constant variance, variance modeled as a power function of the dose group
mean (U.S. EPA. 2012). were considered. If no model achieved adequate fit to response means and
variances (BMDS Test 2 p > 0.05) under that assumption, a BMD/BMDL was not derived, and a
LOAEL was selected as POD for the endpoint.
E.3 Summary of BMD Modeling Results
TableApx E-l. Summary of Benchmark Dose Modeling Results from Selected Endpoints in Male
and Fema
e F344 Rats Following a 2-Year Exposure to DEN
P (Lington ei
al.. 1997)
Section
Endpoint
Sex
Selected
Modela
BMDio
(mg/kg-
day)
BMDLio
(mg/kg-day)
E.4
Continuous endpoints
E.4.1.1
Relative Liver weight at terminal sacrifice
Male
Linear, CV
106
85.0
E.4.1.2
Relative Liver weight at terminal sacrifice
Female
LOAEL (184 mg/kg-day)
E.4.2.1
Serum ALT at 6-month sacrifice
Male
Linear, NCV
12.5
8.68
E.4.2.2
Serum ALT at 18-month sacrifice
Male
Power, NCV
37.2
17.4
E.5
Dichotomous endpoints
Page 159 of 282
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Section
Endpoint
Sex
Selected
Model"
BMDio
(mg/kg-
day)
BMDLio
(mg/kg-day)
E.5.1.1
Focal necrosis in the liver
Male
Logistic
159
125
E.5.1.2
Focal necrosis in the liver
Female
Log-Probit
222
34.3
E.5.2
Spongiosis hepatis in the liver
Male
Log-Probit
31.9
8.57
E.5.3
Sinusoid ectasia in the liver
Male
Log-Probit
125
14.4
CV = constant variance model; NCV = non-constant variance model
" As described in Section 2, BMDs for non-cancer endpoints were derived from the standard set of models as defined in
EPA BMD technical guidance and as identified in BMDS 3.3.2 as defaults. Since the standard approach gave adequate
results for all endpoints, non-standard models were not considered for BMD derivations.
E.4 Continuous Endpoints
E.4.1 Relative Liver Weight - Terminal Sacrifice
E.4.1.1 Male F344 Rats
Table Apx E-2. Dose-Response Modeling Data for Relative Liver Weight at Terminal Sacrifice in
Male F344 Rats Following a 2-Year Exposure to
DINP (Lington et al.. 1997)
Dose (mg/kg-day)
Number per Group
Mean
Standard Deviation
0
61
0.032
0.006
15
54
0.034
0.008
152
50
0.038
0.008
307
51
0.042
0.008
Page 160 of 282
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TableApx E-3. Summary of Benchmark Dose Modeling Results for Relative Liver Weight at Terminal Sacrifice in Male F344 Rats
Following a 2-Year Exposure to DINP (Constant Variance) (Lington et al., 1997)
Models"
Restriction b
BMR = 10%
P Value
AIC
BMDS
Recommends
BMDS
Recommendation
Notes
BMR = 5%
BMR = 1 SD
BMR = 25%
BMD
BMDL
BMD
BMDL
BMD
BMDL
BMD
BMDL
Exponential 3
Restricted
116.26
95.59
0.3786
-1497.4
98773
Viable -
Alternate
Modeled control
response std. dev. >1.5
actual response std. dev.
59.51
48.93
248.94
206.95
272.19
223.80
Exponential 5
Restricted
79.84
36.41
0.3253
-1496.4
71899
Viable -
Alternate
37.70
16.38
218.32
131.93
248.11
147.52
Hill
Restricted
154.16
151.00
NA
-1488.6
14597
Questionable
Residual at control > 2
d.f.=0, saturated model
(Goodness of fit test
cannot be calculated)
85.09
83.34
303.22
296.39
340.22
333.23
Polynomial
Degree 3
Restricted
36.76
10.37
NA
-1495.3
18631
Questionable
BMD/BMDL ratio > 3
d.f.=0, saturated model
(Goodness of fit test
cannot be calculated)
16.01
4.92
272.09
29.48
283.55
31.16
Polynomial
Degree 2
Restricted
88.20
49.76
0.3087
-1496.4
03289
Viable -
Alternate
42.54
23.75
225.74
141.55
254.52
155.99
Power
Restricted
106.22
85.08
0.4626
-1497.8
97726
Viable -
Alternate
53.11
42.54
241.06
195.89
265.55
212.69
Linear
Unrestricted
106.44
84.96
0.4627
-1497.8
97925
Viable-
Recommended
Lowest AIC
50.59
42.54
241.50
195.75
266.10
211.11
AIC = Akaike information criterion; BMD = benchmark dose; BMDL = benchmark dose lower limit; NA = not applicable
" Selected Model (bolded and shaded gray); residuals for doses 0, 15, 152, and 307 mg/kg-day were -0.8549, 0.7132, 0.4739, and -0.2682, respectively.
b Restrictions defined in the BMDS 3.3 User Guide.
Page 161 of 282
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Model Summary with BMR of 0.1 Relative Deviation for the BMD
and 0.95 Lower Confidence Limit for the BMDL
0 0455334
U.U4J J JJt
H 041^334
¦
0.0375334
0.0335334
n rm ^^a
U.Uj 1 j j
0.0295334
-43 7 57 107 157 207 257 307
MG/KG-DAY
Frequentist Exponential Degree 3 Estimated Probability Frequentist Exponential Degree 5 Estimated Probability
Frequentist Hill Estimated Probability Frequentist Polynomial Degree 3 Estimated Probability
Frequentist Polynomial Degree 2 Estimated Probability Frequentist Power Estimated Probability
Frequentist Linear Estimated Probability • Data
Page 162 of 282
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Selected Frequentist Linear Model with BMR of 0.1 Added Risk for
the BMD and 0.95 Lower Confidence Limit for the BMDL
f) 044SQQ7
U.UttJ J J /
0 049SQQ7
U.Uft ujj/
U.U4UJ33 /
U.UjOj /
Ct n2CCQQ7
(
>
n 034^007
n n^?qqQ7
<
U.UjZ jjj /
C
n n^n^qQ7
~
U.UjU J" J /
n mcic;QQ7
0.0265997
-43 7 57 107 157 207 257 307
MG/KG-DAY
Estimated Probability Response at BMD • Data BMD BMDL
Page 163 of 282
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Results for Selected Model - Linear, CV (Unrestricted) - Rel. Dev., BMR = 0.1
User Input
Model Results
Benchmark Ddse
BMD
105.440033
BMDL
34.95359659
BMD'J
139.9032525
AIC
-1497.897925
Test 4 P-value
0.452657772
D.O.F.
2
Model Parameters
# of Parameters
3
Variable
Estimate
£
0.032S14937
beta
3.08295E-05
alpha
5.54312E-05
Goodness of Fit
Dose
Size
Estimated
Median
Cale'd
Median
Observed
Mean
Estimated
5D
Calcd
SD
Observed
5D
Scaled
Residual
0
61
0.032814937
0.032
0.032
0.00744522
O.OD6
0.006
-0.854892965
15
54
0.0332773S
0.034
0.034
0.00744522
O.OOS
O.OOS
0.713230418
152
50
0.037501021
0.038
0.038
0.00744522
0.008
O.OOS
0.47390353
307
51
0.042279594
0.042
0.042
0.00744522
O.OOS
0.008
-0.268185348
Likelihoods of Interest
Model
Log Likelihood*
# of Parameters
AIC
A1
752.7197303
5
-1495.43946
A2
755.9925165
8
-1495.98503
A3
752.7197303
5
-1495.43946
fitted
751.9489626
3
-1497.S9793
R
726.8720033
2
-1449.74401
Tests of Interest
Test
-2*LoEUikelihocd Ratio)
Test ^
p-value
1
58.24102629
6
<0.0001
2
6.545572396
3
0.0878S246
3
6.545572396
3
0.0S788246
4
1.541535303
2
0.46265777
Page 164 of 282
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E.4.1.2 Female F344 Rats
Table Apx E-4. Dose-Response Modeling Data for Relative Liver Weight at
Terminal Sacrifice in Female F344 Rats Following a 2-Year Exposure to
DINP (Lington et al., 1997)
Dose (mg/kg-day)
Number per Group
Mean
Standard Deviation
0
65
0.031
0.005
18
57
0.032
0.007
184
48
0.036
0.008
375
53
0.04
0.007
Page 165 of 282
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TableApx E-5. Summary of Benchmark Dose Modeling Results for Relative Liver Weight at Terminal Sacrifice in Female F344
Rats Following a 2-Year Exposure to DINP (Non-constant Variance) (Lington et al., 1997)
Standard Models "
Restriction 4
BMR = 10%
P Value
AIC
BMDS Recommends
BMDS Recommendation
Notes
BMR = 5%
BMR = 1 SD
BMR = 25%
BMD
BMDL
BMD
BMDL
BMD
BMDL
BMD
BMDL
Exponential 3
Restricted
143.27
118.57
0.2610
-1596.49
Questionable
Non-constant variance test
failed (Test 3 p-value < 0.05)
Modeled control response
std. dev. >|1.5| actual
response std. dev.
73.34
60.66
268.59
219.51
335.42
277.61
Exponential 5
Restricted
86.77
35.03
0.3336
-1596.24
Questionable
Non-constant variance test
failed (Test 3 p-value < 0.05)
39.99
15.51
199.97
114.18
309.91
167.83
Hill
Restricted
135.95
99.63
NA
-1592.96
Questionable
Non-constant variance test
failed (Test 3 p-value < 0.05)
d.f.=0, saturated model
(Goodness of fit test cannot
be calculated)
69.29
48.44
263.02
194.84
338.00
256.96
Polynomial Degree 3
Restricted
72.04
14.45
NA
-1594.31
Questionable
Non-constant variance test
failed (Test 3 p-value < 0.05)
BMD/BMDL ratio > 3
d.f.=0, saturated model
(Goodness of fit test cannot
be calculated)
31.23
6.76
207.53
28.21
350.14
44.06
Polynomial Degree 2
Restricted
91.72
58.72
0.3068
-1596.13
Questionable
Non-constant variance test
failed (Test 3 p-value < 0.05)
44.59
27.86
204.48
123.24
308.82
189.00
Power
Restricted
131.94
106.23
0.3428
-1597.04
Questionable
Non-constant variance test
failed (Test 3 p-value < 0.05)
65.97
53.08
257.01
205.66
329.86
265.74
Linear
Unrestricted
128.47
105.83
0.3429
-1597.04
Questionable
Non-constant variance test
failed (Test 3 p-value < 0.05)
62.63
53.11
256.89
204.62
329.42
264.54
AIC = Akaike information criterion; BMD = benchmark dose; BMDL = benchmark dose lower limit; NA = not applicable
"No selected model due to inadequate fit of constant or non-constant variance models.
4 Restrictions defined in the BMDS 3.3 User Guide.
Page 166 of 282
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Model Summary with BMR of 0.1 Relative Deviation for the BMD
and 0.95 Lower Confidence Limit for the BMDL
n HAA1 7A3
n f)4?1743
U.Utt l/tJ
n D401743
u.utui / to
n 03R1743
U.UJOl / to
n A3C1 "7/ia
n H3/11 7/1 3
0.0321743
D 0301743
u.uoui / to
n mai7A3
5 25 75 125 175 225 275 325 375
MG/KG-DAY
Frequentist Exponential Degree 3 Estimated Probability Frequentist Exponential Degree 5 Estimated Probability
Frequentist Hill Estimated Probability Frequentist Polynomial Degree 3 Estimated Probability
Frequentist Polynomial Degree 2 Estimated Probability Frequentist Power Estimated Probability
Frequentist Linear Estimated Probability • Data
Page 167 of 282
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E.4.2 Serum ALT - Male F344 Rats
E.4.2.1 6-Month Sacrifice
Table Apx E-6. Dose-Response Modeling Data for Serum ALT Levels in Male F344
Rats Following a 6-]V
onth Exposure to DI
VP (Lington et al., 1997)
Dose (mg/kg-day)
Number per Group
Mean
Standard Deviation
0
10
37
8
15
10
38
7
152
10
81
52
307
10
128
145
Page 168 of 282
-------
TableApx E-7. Summary of Benchmark Dose Modeling Results for Serum ALT Levels in Male F344 Rats Following a 6-Month
Models "
Restriction 4
BMR = 10%
P Value
AIC
BMDS Recommends
BMDS
Recommendation
Notes
BMR = 1 SD
BMR = 20%
BMR =100%
BMD
BMDL
BMD
BMDL
BMD
BMDL
BMD
BMDL
Exponential 3
Restricted
20.05
15.84
0.0692
382.00
Questionable
Goodness of fit p-
value <0.1
Modeled control
response std. dev.
>|1.5| actual response
std. dev.
40.15
28.50
38.35
30.29
CF
CF
Exponential 5
Restricted
CF
CF
CF
CF
Unusable
BMD computation
failed
124.58
27.19
CF
CF
CF
CF
Hill
Restricted
19.94
9.12
NA
382.16
Questionable
d.f.=0, saturated model
(Goodness of fit test
cannot be calculated)
34.15
16.39
CF
CF
123.97
90.11
Polynomial Degree 3
Restricted
40.68
11.16
NA
380.67
Questionable
BMD/BMDL ratio > 3
d.f.=0, saturated model
(Goodness of fit test
cannot be calculated)
55.33
20.32
56.49
22.31
134.04
98.56
Polynomial Degree 2
Restricted
13.99
0
0.1351
380.89
Unusable
BMD computation
failed; lower limit
includes zero
BMDL not estimated
26.33
14.94
27.79
16.84
132.49
87.19
Power
Restricted
18.76
9.26
0.2143
380.20
Viable - Alternate
32.59
16.63
33.74
18.51
131.87
91.22
Linear
Unrestricted
12.52
8.68
0.3050
379.03
Viable - Recommended
Lowest AIC
23.42
15.50
25.04
17.37
125.20
86.83
AIC = Akaike information criterion; BMD = benchmark dose; BMDL = benchmark dose lower limit; NA = not applicable; CF = computation failed
" Selected Model (bolded and shaded gray); residuals for doses 0,15, 152, and 307 were 0.5396, -0.7686, 0.1084, 0.0955, respectively.
4 Restrictions defined in the BMDS 3.3 User Guide
Page 169 of 282
-------
Model Summary with BMR of 0.1 Relative Deviation for the BMD
and 0.95 Lower Confidence Limit for the BMDL
1 39 9fiQQR
IjZ.ZDjjO
119 9fiQQR
11Z.ZD330
Q9 9f=iQQR9
79 9&QQR9
A
CO OCOOQO
32.269982
-43 7 57 107 157 207 257 307
MG/KG-DAY
Frequentist Exponential Degree 3 Estimated Probability Frequentist Hill Estimated Probability
Frequentist Polynomial Degree 3 Estimated Probability Frequentist Polynomial Degree 2 Estimated Probability
Frequentist Power Estimated Probability Frequentist Linear Estimated Probability
• Data
Page 170 of 282
-------
Frequentist Linear Model with BMR of 0.1 Added Risk for the
BMD and 0.95 Lower Confidence Limit for the BMDL
<
»
J.Z J.uu / 0^7
1 A3 QC7CQ
lUD.oD/oy
Oj.OU / uou
CO OC.~lC.OC.
bo.ob /bob
AO T
4j.OD / UOD
td QC7CQC
Zj.Ou / DoD
2 QC7COCQ
D.OD/Oojy
43
-16.13231
57 107 157 207 257 31
>7
MG/KG-DAY
Estimated Probability Response at BMD • Data BMD BMDL
Page 171 of 282
-------
Results for Selected Model - Linear, NCV (Unrestricted) - Rel. Dev., BMR = 0.1
Model Results
Benchmark Dose
BMD
12.51986155
BMDL
8.683091255
BMDU
12.77902268
AIC
379.0287425
Test 4 P-value
0.304955816
D.O.F.
2
Model Parameters
# of Parameters
4
Variable
Estimate
8
35.85553524
beta
0.286389228
rho
4.902699939
alpha
1.07545E-06
Goodness of Fit
Dose
Size
Estimated
Median
Calc'd
Median
Observed
Mean
Estimated
SD
Calc'd
SD
Observed
SD
Scaled
Residual
0
10
35.85553524
37
37
6.7074289
8
8
0.539568203
15
10
40.15137365
38
38
8.85168002
7
7
-0.768581876
152
10
79.38669783
81
81
47.0696879
52
52
0.108386302
307
10
123.7770281
128
128
139.825984
145
145
0.095505923
Likelihoods of Interest
Model
Log Likelihood*
# of Parameters
AIC
A1
-228.508524
5
467.017048
A2
-184.1836225
8
384.367245
A3
-184.3267829
6
380.653566
fitted
-185.5143713
4
379.028743
Page 172 of 282
-------
E.4.2.2 18-Month Sacrifice
TableApx E-8. Dose-Response Modeling Data for Serum ALT Levels in Male F344
Rats Following an 18-Month Exposure to
DINP (Lington et al.. 1997)
Dose (mg/kg-day)
Number per Group
Mean
Standard Deviation
0
9
42
10
15
10
39
7
152
10
69
39
307
10
128
126
Page 173 of 282
-------
TableApx E-9. Summary of Benchmark Dose Modeling Results for Serum ALT Levels in Male F344 Rats Following an 18-Month
Exposure to DINP (Non-constant Variance) (Lington et al., 1997)
Models "
Restriction 4
BMR=10%
P Value
AIC
BMDS
Recommends
BMDS Recommendation
Notes
BMR=1 SD
BMR=20%
BMR=100%
BMD
BMDL
BMD
BMDL
BMD
BMDL
BMD
BMDL
Exponential 3
Restricted
28.31
19.66
0.0433
371.30
Questionable
Goodness of fit p-value <0.1
Modeled control response std.
dev. >|1.5| actual response std.
dev.
56.70
37.76
52.87
37.61
191.28
143.00
Exponential 5
Restricted
103.76
21.91
NA
370.80
Questionable
BMD/BMDL ratio > 3; d.f.=0,
saturated model (Goodness of
fit test cannot be calculated)
113.99
40.10
113.67
39.87
154.96
134.70
Hill
Restricted
61.57
28.62
NA
371.00
Questionable
d.f.=0, saturated model
(Goodness of fit test cannot be
calculated)
CF
CF
82.15
46.68
182.90
133.66
Polynomial
Degree 3
Restricted
63.43
20.61
NA
370.94
Questionable
BMD/BMDL ratio > 3
d.f.=0, saturated model
(Goodness of fit test cannot be
calculated)
85.51
40.83
84.98
40.09
200.71
131.37
Polynomial
Degree 2
Restricted
29.49
14.27
0.0428
371.32
Questionable
Goodness of fit p-value <0.1
56.99
28.32
55.73
28.45
210.39
132.17
Powerc
Restricted
37.19
17.45
0.0925
370.04
Questionable
Goodness of fit p-value <0.1
62.51
33.36
59.71
33.45
179.20
134.31
Linear
Unrestricted
20.06
12.52
0.0655
370.67
Questionable
Goodness of fit p-value <0.1
40.61
24.79
40.11
25.04
200.56
125.22
AIC = Akaike information criterion; BMD = benchmark dose; BMDL = benchmark dose lower limit; NA = not applicable
" Selected Model is bolded and shaded gray; residuals for doses 0, 15, 152, and 307 were 0.7610, -0.6609, -0.2070, and 0.0131, respectively.
4 Restrictions defined in the BMDS 3.3 User Guide
c Despite p < 0.1, the Power model fit would pass at p > 0.05, the variance model passed p>0.05, and visual fit of model to data is still adequate for BMD calculation.
Page 174 of 282
-------
-43
Model Summary with BMR of 0.1 Relative Deviation for the BMD
and 0.95 Lower Confidence Limit for the BMDL
130
110
107 157
MG/KG-DAY
307
Frequentist Exponential Degree 3 Estimated Probability
Frequentist Hill Estimated Probability
Frequentist Polynomial Degree 2 Estimated Probability
¦Frequentist Linear Estimated Probability
Frequentist Exponential Degree 5 Estimated Probability
Frequentist Polynomial Degree 3 Estimated Probability
Frequentist Power Estimated Probability
O Data
Page 175 of 282
-------
Frequentist Power Model with BMR of 0.1 Added Risk for the
BMD and 0.95 Lower Confidence Limit for the BMDL
MG/KG-DAY
Estimated Probability Response at BMD • Data BMD BMDL
Page 176 of 282
-------
Results for Selected Model - Power, NCV (Restricted) - Rel. Dev., BMR = 0.1
User Input
Model
Data
Dependent
Variable
mg/kg-day
Independe
nt
Variable
Total # of
Observatio
n
4
Info
Model
Frequentist Power,
NCV
Dataset
Name
MaleF344Rats_S eru
m ALT 18mon
Formula
M[dose] = g + v *
dose A n
Var[i] = alpha *
mean[il A rho
Options
Risk
Type
Rel. Dev.
BMR
0.1
Confiden
ce Level
0.95
Distributi
on
Normal
Variance
Non-Constant
Model Results
Benchmark Dose
BMD
37.19126348
BMDL
17.45080887
BMDU
37.96112263
AIC
370.0444752
Test 4 P-value
0.092488008
D.O.F.
1
Model Parameters
# of Parameters
5
Variable
Estimate
8
39.8382544
V
0.019980069
n
1.464367921
rho
4.643124981
alpha
2.69559E-06
Goodness of Fit
Dose
Size
Estimated
Median
Calc'd
Median
Observed
Mean
Estimated
SD
Calc'd
SD
Observed
SD
Scaled
Residual
0
9
39.8382544
42
42
8.5216504
10
10
0.761030608
15
10
40.89222207
39
39
9.05422294
7
7
-0.66087743
152
10
71.14361683
69
69
32.7473294
39
39
-0.207000441
307
10
127.4742711
128
128
126.82257
126
126
0.013108871
Likelihoods of Interest
Model
Log Likelihood*
# of Parameters
AIC
A1
-217.2980126
5
444.596025
A2
-178.4089743
8
372.817949
A3
-178.6069741
6
369.213948
fitted
-180.0222376
5
370.044475
Page 177 of 282
-------
E.5 Dichotomous Endpoints
E.5.1 Focal Necrosis in the Liver
E.5.1.1 Male F344 Rats
Table Apx E-10. Dose-Response Modeling Data for Focal Necrosis of the Liver in Male
F344 Rats Following a 2-Year Exposure to DINP (Lington et al., 1997)
Dose (mg/kg-day)
Number per Group
Incidence
0
81
10
15
80
9
152
80
16
307
80
26
Page 178 of 282
-------
TableApx E-ll. Summary of Benchmark Dose Modeling Results for Focal Necrosis of the Liver in Male F344 Rats Following a 2-
Models "
Restriction 4
BMR = 10%
P Value
AIC
BMDS Recommends
BMDS Recommendation
Notes
BMR = 5%
BMD
BMDL
BMD
BMDL
Dichotomous Hill
Restricted
154.87
48.90
NA
305.83
Questionable
BMD/BMDL ratio > 3
d.f.=0, saturated model
(Goodness of fit test cannot be
calculated)
132.94
18.97
Gamma
Restricted
161.40
85.98
0.7925
303.85
Viable - Alternate
100.26
41.86
Log-Logistic
Restricted
160.91
78.23
0.7930
303.85
Viable - Alternate
100.39
37.06
Multistage Degree 3
Restricted
162.13
85.74
0.7420
303.89
Viable - Alternate
94.76
41.74
Multistage Degree 2
Restricted
162.13
85.74
0.7420
303.89
Viable - Alternate
94.76
41.74
Multistage Degree 1
Restricted
126.33
84.11
0.8212
302.17
Viable - Alternate
61.50
40.94
Weibull
Restricted
161.48
85.94
0.7832
303.86
Viable - Alternate
98.74
41.84
Logistic
Unrestricted
158.52
124.56
0.9417
301.90
Viable - Recommended
Lowest AIC
88.34
69.47
Log-Probit
Unrestricted
159.84
46.47
0.8230
303.83
Viable - Alternate
BMD/BMDL ratio > 3
104.60
12.63
Probit
Unrestricted
153.31
118.45
0.9368
301.91
Viable - Alternate
83.82
64.96
Quantal Linear
Unrestricted
126.33
84.11
0.8212
302.17
Viable - Alternate
61.50
40.95
AIC = Akaike information criterion; BMD = benchmark dose; BMDL = benchmark dose lower limit; NA = not applicable
" Selected Model is bolded and shaded gray; residuals for doses 0, 15, 152 and 307 were 0.2347, -0.2546, 0.0189 and 0.0007, respectively.
4 Restrictions defined in the BMDS 3.3 User Guide.
Page 179 of 282
-------
Model Summary with BMR of 10% Extra Risk for the BMD and 0.95
Lower Confidence Limit for the BMDL
CD
U
£
CD
¦g
u
£
0.5
0.45
n a
\J. *T
0.35
n 3 i
n 9^:
U.Zj
0.15
0.05
-43
57
107 157
mg/kg-day
207
257
307
Frequentist Dichotomous Hill
Estimated Probability
Frequentist Gamma Estimated
Probability
Frequentist Log-Logistic Estimated
Probability
• Frequentist Multistage Degree 3
Estimated Probability
Frequentist Multistage Degree 2
Estimated Probability
¦ Frequentist Multistage Degree 1
Estimated Probability
¦ Frequentist Weibull Estimated
Probability
¦ Frequentist Logistic Estimated
Probability
¦ Frequentist Log-Probit Estimated
Probability
¦ Frequentist Probit Estimated
Probability
Page 180 of 282
-------
Frequentist Logistic Model with BMR of 10% Extra Risk for the
BMD and 0.95 Lower Confidence Limit for the BMDL
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
^^Estimated Probability
Response at BMD
O Data
BMD
BMDL
57
107 157
mg/kg-day
207
257
307
Page 181 of 282
-------
Results for Selected Model - Logistic (Unrestricted) - Extra Risk, BMR = 0.1
User Input
Info
Model
Logistic
Dataset Name
Male F344 Rats-
Formula
P[dose] =
l/[l+exp(-a-b*de
Options
Risk Type
Extra Risk
Model Data
BMR
0.1
Dependent Variable
mg/kg-day
Confidence
Level
0.95
Independent Variable
Incidence
Total # of Observation
4
Background
Estimated
Model Results
Benchmark Dose
BMD
158.52
BMDL
124.56
BMDU
239.50
AIC
301.50
P-value
0.94
D.O.F.
2.00
Chi3
0.12
Slope Factor
158.52
Model Parameters
# of Parameters
2
Variable
Estimate
a
-2.0393
b
0.00426
Goodness of Fit
Dose
Estimated
Probability
Expected
Observed
Size
Seated
Residual
0
0.115134137
9.32586507
ID
81
0.2347
15
0.121808403
9.744672223
9
80
-0.2546
152
0.199154436
15.932354SS
16
80
0.0189
307
0.324963347
25.99710772
26
80
0.0007
Analysis of Deviance
Model
Log Likelihood
# of Parameters
Deviance
Test dX
P Value
Full Model
-14S.SS97738
4
-
NA
Fitted Model
-148.950072
2
0.12059642
2
0.9414837
Reduced Model
-156.0920707
1
14.4045939
3
0.0024031
Page 182 of 282
-------
User Input
Info
Model
Logistic
Dataset Name
Male F344 Rats-
Formula
P[dose] =
l/[l+exp(-a-b"dc
Options
Risk Type
Extra Risk
Model Data
BMR
0.1
Dependent Variable
mg/kg-day
Confidence
Level
0.95
Independent Variable
Incidence
Total it of Observation
4
Background
Estimated
Model Results
Benchmark Dose
BMD
158.52
BMDL
124.56
BMDU
239.50
AIC
301:90
P-value
0.94
D.O.F.
2.00
Chi3
0.12
Slope Factor
158.52
Model Parameters
# of Parameters
2
Variable
Estimate
a
-2.0393
b
0.00426
Goodness of Fit
Dose
Estimated
Probability
Expected
Observed
Size
Scaled
Residual
0
0.115134137
9.32586507
10
81
0.2347
15
0.121808403
9.744672223
9
SO
-0.2546
152
0..199154436
15.93235483
16
SO
0.0189
307
0.324963S47
25.99710772
26
80
0.0007
Analysis of Deviance
Model
Log Likelihood
# of Parameters
Deviance
Test cJX.
P Value
Full Model
-14S.S897738
4
-
NA
Fitted Model
-148.950072
2
0.12059642
2
0.9414837
Reduced Model
-156.0920707
1
14.4045939
3
0.0024031
Page 183 of 282
-------
E.5.1.2 Female F344 Rats
Table Apx E-12. Dose-Response Modeling Data for Focal Necrosis of the Liver in
Female F344 Rats Following a 2-Year Exposure to DINP (Lington et al., 1997)
Dose (mg/kg-day)
Number per Group
Incidence
0
81
13
18
81
11
184
80
19
375
80
21
Page 184 of 282
-------
TableApx E-13. Summary of Benchmark Dose Modeling Results for Focal Necrosis of the Liver in Female F344 Rats Following a 2-
Models"
Restriction b
BMR=10%
P Value
AIC
BMDS Recommends
BMDS Recommendation Notes
BMR = 5%
BMD
BMDL
BMD
BMDL
Dichotomous Hill
Restricted
179.57
19.90
NA
323.73
Questionable
BMD/BMDL ratio > 3
d.f.=0, saturated model (Goodness of
fit test cannot be calculated)
148.09
7.87
Gamma
Restricted
247.12
136.68
0.7185
320.19
Viable - Alternate
120.31
66.54
Log-Logistic
Restricted
239.78
125.46
0.7335
320.15
Viable - Alternate
113.58
59.43
Multistage Degree 3
Restricted
247.12
136.68
0.7185
320.19
Viable - Alternate
120.31
66.53
Multistage Degree 2
Restricted
247.12
136.68
0.7185
320.19
Viable - Alternate
120.31
66.54
Multistage Degree 1
Restricted
247.12
136.68
0.7185
320.19
Viable - Alternate
120.31
66.54
Weibull
Restricted
247.12
136.68
0.7185
320.19
Viable - Alternate
120.31
66.54
Logistic
Unrestricted
275.16
179.48
0.6509
320.39
Viable - Alternate
148.92
98.02
Log-Probit
Unrestricted
222.08
34.30
0.4809
322.03
Viable - Recommended
Lowest BMDL
BMD/BMDL ratio > 3
96.76
0.90
Probit
Unrestricted
271.03
173.31
0.6617
320.36
Viable - Alternate
144.53
93.23
Quantal Linear
Unrestricted
247.12
136.68
0.7185
320.19
Viable - Alternate
120.31
66.54
AIC = Akaike information criterion; BMD = benchmark dose; BMDL = benchmark dose lower limit; NA = not applicable
° Selected Model is bolded and shaded gray; residuals for doses 0, 18, 184 and 375 were 0.3259, -0.4779, 0.3508 and -0.1977, respectively.
h Restrictions defined in the BMDS 3.3 User Guide.
Page 185 of 282
-------
0.4
0.35
0.3
0.1
0.05
Model Summary with BMR of 10% Extra Risk for the BMD and 0.95
Lower Confidence Limit for the BMDL
Frequentist Dichotomous Hill
Estimated Probability
Frequentist Gamma Estimated
Probability
Frequentist Log-Logistic Estimated
Probability
• Frequentist Multistage Degree 3
Estimated Probability
Frequentist Multistage Degree 2
Estimated Probability
¦ Frequentist Multistage Degree 1
Estimated Probability
¦ Frequentist Weibull Estimated
Probability
¦ Frequentist Logistic Estimated
Probability
¦ Frequentist Log-Probit Estimated
Probability
¦ Frequentist Probit Estimated
Probability
0
-25 25 75 125 175 225 275 325 375
mg/kg-day
Page 186 of 282
-------
Female F344 Relative Liver Weight vs mg/kg-day; LogProbit model
with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence
Limit for the BMDL
Page 187 of 282
-------
Results for Selected Model - ^4)gPyqbi^ (L n restricted) - Extra Risk, BMR = 0.1
User Input
Info
Model
Log-Pro bit
Dataset
Name
Female F344 Rats - focal necrosis
Formula
Pfdose] =
g+(l-E) * KmMDOT[atb*LDSfDose))
Options
Risk Type
Extra Risk
BMR
0.1
Confidence
Level
0.95
Background
Estimated
Model Data
Dependent Variable
mg/kg-day
Independent Variable
Incidence
Total # of Observation
4
Model Results
Benchmark Dose
BMD
222.0606266
BMDL
34.300140B
BMDU
Infinity
AIC
322.0-314517
P-value
0.48 QUI 731
D.O.F.
1
Chi2
0.496782444
Model Parameters
# of Parameters
3
Variable
Estimate
Background (g)
0.147649782
a
-3.644150287
b
0.437272073
Goodness of Fit
Dose
Estimated
Probability
Expected
Observed
Size
Scaled
Residual
0
0.147649782
11.95963234
13
81
0.3258509
18
0.155022564
12.55682771
11
81
-0.477945
184
0.221220007
17.69760055
19
80
0.3508162
375
0.2723415S
21.7873264
21
80
-0.19773B
Analysis of Deviance
Model
Log Likelihood
# of Parameters
Deviance
Test .it
P Value
Full Modell
-157.7653174
4
-
NA
Fitted Model
-158.0157259
3
0.50081701
1
0.4791414
Reduced Model
-160.5735074
1
5.61638012
3
0.1318411
Page 188 of 282
-------
E.5.2 Spongiosis Hepatis in the Liver - Male F344 Rats
Table Apx E-14. Dose-Response Modeling Data for Spongiosis Hepatis of the Liver
in Male F344 Rats Following 2-Year Exposure to DINP (Lington et al., 1997)
Dose (mg/kg-day)
Number per Group
Incidence
0
81
24
15
80
24
152
80
51
307
80
62
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TableApx E-15. Summary of Benchmark Dose Modeling Results for Spongiosis Hepatis of the Liver in Male F344 Rats Following a
2-Year Exposure to DINP (Lington et al., 1997)
Models"
Restriction b
BMR = 10%
P Value
AIC
BMDS Recommends
BMDS Recommendation
Notes
BMR = 5%
BMD
BMDL
BMD
BMDL
Dichotomous Hill
Restricted
53.05
9.92
1
394.27
Viable - Alternate
BMD/BMDL ratio > 3
37.76
4.81
Gamma
Restricted
26.33
20.77
0.8496
390.93
Viable - Alternate
12.82
10.11
Log-Logistic
Restricted
30.45
11.96
0.7322
392.47
Viable - Alternate
17.20
5.67
Multistage Degree 3
Restricted
26.33
20.77
1
-9999
Unusable
AIC not estimated
12.82
10.11
Multistage Degree 2
Restricted
26.33
20.77
1
-9999
Unusable
AIC not estimated
12.82
10.11
Multistage Degree 1
Restricted
26.33
20.77
0.8496
390.93
Viable - Alternate
12.82
10.11
Weibull
Restricted
26.33
20.77
0.8496
390.93
Viable - Alternate
12.82
10.11
Logistic
Unrestricted
42.42
35.87
0.6349
392.50
Viable - Alternate
21.74
18.35
Log-Probit
Unrestricted
31.88
8.57
0.8137
392.37
Viable - Recommended
Lowest BMDL; BMD/BMDL
ratio > 3
20.08
4.03
Probit
Unrestricted
42.55
36.41
0.6037
392.70
21.70
18.55
Quantal Linear
Unrestricted
26.33
20.77
0.8496
390.93
12.82
10.11
AIC = Akaike information criterion; BMD = benchmark dose; BMDL = benchmark dose lower limit
" Selected Model isbolded; residuals for doses 0, 15, 152, and 307 were 0.1279, -0.1656, 0.0941, and -0.0539, respectively.
h Restrictions defined in the BMDS 3.3 User Guide
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Model Summary with BMR of 10% Extra Risk for the BMD and
0.95 Lower Confidence Limit for the BMDL
—1—
n q
U.J
n £
U.o
n 7
u. /
n F>
¦
u.o
n c:
n A
n 5
U.3
n i
n 1
U. -L
0
-43 7 57 107 157 207 257 307
MG/KG-DAY
Frequentist Dichotomous Hill Estimated Probability Frequentist Gamma Estimated Probability
Frequentist Log-Logistic Estimated Probability Frequentist Multistage Degree 3 Estimated Probability
Frequentist Multistage Degree 2 Estimated Probability Frequentist Multistage Degree 1 Estimated Probability
Frequentist Weibull Estimated Probability Frequentist Logistic Estimated Probability
Frequentist Log-Probit Estimated Probability Frequentist Probit Estimated Probability
Frequentist Quantal Linear Estimated Probability • Data
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Frequentist Log-Probit Model with BMR of 10% Extra Risk for the
BMD and 0.95 Lower Confidence Limit for the BMDL
0.1
0
57
107 157
MG/KG-DAY
207
257
Estimated Probability
Response at BMD
O Data
BMD
¦BMDL
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Results for Selected Model - LogProbit (Unrestricted) - Extra Risk, BMR = 0.1
Model
Info
Options
Data
Model
Log-Probit
Risk
Dependent
Dataset
Name
Male F344
Type
Extra Risk
Variable
mg/kg-day
Rats spongiosis
BMR
0.1
Independe
hepatis
Confiden
nt
P[dose] = g+(l-g) *
ce Level
0.95
Variable
Incidence
Formula
CumNorm(a+b*Log(
Dose))
Backgrou
nd
Estimated
Total # of
Observatio
n
4
Vlodel Results
Benchmark Dose
BMD
31.87966632
BMDL
8.566931336
BMDU
77.63938389
AIC
392.3657526
P-value
0.813651618
D.O.F.
1
Chi2
0.055562904
Model Parameters
# of Parameters
3
Variable
Estimate
Background (g)
0.288658724
a
-4.003497521
b
0.786242291
Goodness of Fit
Dose
Estimated
Probability
Expected
Observed
Size
Scaled
Residual
0
0.288658724
23.38135661
24
81
0.1279398
15
0.310314502
24.82516015
24
80
0.1656122
152
0.629151263
50.33210107
51
80
0.094143
307
0.780322211
62.4257769
62
80
-0.053889
Analysis of Deviance
Log
#of
Test
Model
Likelihood
Parameters
Deviance
d.f.
P Value
Full Model
-193.1328632
4
-
-
NA
Fitted Model
-193.1828763
3
0.10002618
1
0.7517982
Reduced Model
-222.4986873
1
58.6316221
3
0.7517982
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E.5.3 Sinusoid Ectasia in the Liver Male F344 Rats
Table Apx E-16. Dose-Response Modeling Data for Sinusoid Ectasia of the Liver
in Male F344 Rats Following a 2-Year Exposure to DINP (Lington et al., 1997)
Dose (mg/kg-day)
Number per Group
Incidence
0
81
16
15
80
16
152
80
24
307
80
33
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TableApx E-17. Summary of Benchmark Dose Modeling Results for Sinusoid Ectasia of the Liver in Male F344 Rats Following a 2-
Models"
Restriction b
BMR=10%
P Value
AIC
BMDS Recommends
BMDS Recommendation
Notes
BMR = 5%
BMD
BMDL
BMD
BMDL
Dichotomous Hill
Restricted
126.62
19.59
NA
374.75
Questionable
BMD/BMDL ratio > 3
d.f.=0, saturated model
(Goodness of fit test cannot
be calculated)
79.29
7.58
Gamma
Restricted
121.73
68.52
0.9441
372.76
Viable - Alternate
66.95
33.36
Log-Logistic
Restricted
122.39
58.96
0.9572
372.75
Viable - Alternate
69.06
27.93
Multistage Degree 3
Restricted
118.39
68.47
0.9930
370.77
Viable - Alternate
60.57
33.33
Multistage Degree 2
Restricted
118.39
68.47
0.9930
370.77
Viable - Alternate
60.57
33.33
Multistage Degree 1
Restricted
104.19
68.30
0.9746
370.80
Viable - Alternate
50.72
33.25
Weibull
Restricted
121.20
68.51
0.9372
372.76
Viable - Alternate
65.82
33.35
Logistic
Unrestricted
128.86
97.30
0.9836
370.78
Viable - Alternate
68.24
51.73
Log-Probit
Unrestricted
125.23
14.42
0.9911
372.75
Viable - Recommended
Lowest BMDL
BMD/BMDL ratio > 3
76.52
2.40
Probit
Unrestricted
125.62
93.71
0.9883
370.77
Viable - Alternate
65.79
49.29
Quantal Linear
Unrestricted
104.19
68.30
0.9746
370.80
Viable - Alternate
50.72
33.25
AIC = Akaike information criterion; BMD = benchmark dose; BMDL = benchmark dose lower limit; NA = not applicable
° Selected Model is bolded; residuals for doses 0, 15, 152 and 307 were -0.0075, 0.0082, -0.0013 and 0.0007, respectively.
h Restrictions defined in the BMDS 3.3 User Guide
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Model Summary with BMR of 10% Extra Risk for the BMD and 0.95
Lower Confidence Limit for the BMDL
n c:
n A
n 3
0.2 (
n 1
0
-43 7 57 107 157 207 257 307
mg/kg-day
^^—Frequentist Dichotomous Hill
Estimated Probability
Frequentist Gamma Estimated
Probability
Frequentist Log-Logistic Estimated
Probability
^^—Frequentist Multistage Degree 3
Estimated Probability
^^—Frequentist Multistage Degree 2
Estimated Probability
^^—Frequentist Multistage Degree 1
Estimated Probability
^^—Frequentist Weibull Estimated
Probability
^^—Frequentist Logistic Estimated
Probability
^^—Frequentist Log-Probit Estimated
Probability
^^—Frequentist Probit Estimated
Probability
Page 196 of 282
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Male F344 Relative Liver Weight vs mg/kg-day; LogProbit model
with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence
Limit for the BMDL
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Results for Selected Model - LogProbit (Unrestricted) - Extra Risk, BMR = 0.1
wwWvavwwvvvw-
User Input
Options
Info
Risk Type
Extra Risk
Model Data
Model
Log-Probit
BMR
0.1
Dependent Variable
rng/kg-day
Dataset Name
Sinusoid Ectasia -
Confidence
Level
0.95
Independent Variable
Incidence
Formula
Pjdose] = g+(l-g)
Total # of Observation
4
Background
Estimated
Model Results
Benchmark Dose
BMD
125.23
BMDL
14.42
BMDU
247.62
AIC
372.75
P-value
0.99
D.O.F.
1.00
Chi2
0.00
Model Parameters
# of Parameters
Variable
Estimate
g
0.197861854
a
-4.343490179
b
0.73743943
Goodness of Fit
Dose
Estimated
Probability
Expected
Observed
Size
Scaled
Residual
0
0.197861854
16.02681018
16
SI
-0.0075
15
0.199634872
15.97073978
16
SO
0.00S2
152
0.300063561
24.00543434
24
SO
-0.0013
307
0.412461541
32.99692324
33
SO
0.0007
Analysis of Deviance
Model
Log Likelihood
of Parameters
Deviance
Test slX.
P Value
Full Model
Full Model
-133.3755714
4
-
Fitted Model
Fitted Model
-183.3756339
3
0.00012493
1
Reduced Model
Reduced Model
-139.500S934
1
12.2506439
3
Page 198 of 282
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Appendix F CALCULATING DAILY ORAL HUMAN
EQUIVALENT DOSES AND HUMAN EQUIVALENT
CONCENTRATIONS
For DINP, all data considered for PODs are obtained from oral animal toxicity studies in rats, mice, or
beagles. Because toxicity values for DINP are from oral animal studies, EPA must use an extrapolation
method to estimate HEDs. The preferred method would be to use chemical-specific information for such
an extrapolation. EPA identified one study reporting a physiologically based pharmacokinetic model for
DINP based on humanized liver mice (Miura et al.. 2018). Since the study made use of genetically
modified animals and has not been validated by the Agency, it was not considered fit-for-purpose or
used to calculate HEDs. EPA did not locate other DINP information to conduct a chemical-specific
quantitative extrapolation. In the absence of such data, EPA relied on the guidance from U.S. EPA
(201 lb), which recommends scaling allometrically across species using the three-quarter power of body
weight (BW3/4) for oral data. Allometric scaling accounts for differences in physiological and
biochemical processes, mostly related to kinetics.
For application of allometric scaling in risk evaluations, EPA uses dosimetric adjustment factors
(DAFs), which can be calculated using EquationApx F-l.
EquationApx F-l. Dosimetric Adjustment Factor
U.S. EPA (2011b). presents DAFs for extrapolation to humans from several species. However, because
those DAFs used a human body weight of 70 kg, EPA has updated the DAFs using a human body
weight of 80 kg for the DINP risk evaluation (U.S. EPA. 2011a). EPA used the body weights of 0.025,
0.25, and 12 kg for mice, rats and dogs, respectively, as presented in U.S. EPA (2011b). The resulting
DAFs for mice, rats, and dogs are 0.133, 0.236, and 0.622, respectively.
Use of allometric scaling for oral animal toxicity data to account for differences among species allows
EPA to decrease the default intraspecies UF (UFa) used to set the benchmark MOE; the default value of
10 can be decreased to 3, which accounts for any toxicodynamic differences that are not covered by use
of BW3 4. Using the appropriate DAF from Equation Apx F-l, EPA adjusts the POD to obtain the HED
using Equation Apx F-2:
Equation Apx F-2. Daily Oral Human Equivalent Dose
Where:
DAF
BWa
BWh
Dosimetric adjustment factor (unitless)
Body weight of species used in toxicity study (kg)
Body weight of adult human (kg)
HEDDaiiy — PODDauy X DAF
Where:
HEDoaily
P ODDaily
DAF
Human equivalent dose assuming daily doses (mg/kg-day)
Oral POD assuming daily doses (mg/kg-day)
Dosimetric adjustment factor (unitless)
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For the risk evaluation of DINP, EPA assumes similar absorption for the oral and inhalation routes, and
no adjustment was made when extrapolating to the inhalation route. For the inhalation route, EPA
extrapolated the daily oral HEDs to inhalation HECs using a human body weight and breathing rate
relevant to a continuous exposure of an individual at rest, as follows:
EquationApx F-3. Extrapolating from Oral HED to Inhalation HEC
BWh
HECoaily, continuous ~ HEDjja(iy X (-
I IV^ * Lj L/Q
Where:
HECoaily, continuous ~ Inhalation HEC based on continuous daily exposure (mg/m3)
HEDoaiiy = Oral HED based on daily exposure (mg/kg-day)
BWh = Body weight of adult humans (kg) = 80
IRr = Inhalation rate for an individual at rest (m3/hr) = 0.6125
EDc = Exposure duration for a continuous exposure (hr/day) = 24
Based on information from U.S. EPA (201 la). EPA assumes an at rest breathing rate of 0.6125 m3/hr.
Adjustments for different breathing rates required for individual exposure scenarios are made in the
exposure calculations, as needed.
It is often necessary to convert between ppm and mg/m3 due to variation in concentration reporting in
studies and the default units for different OPPT models. Therefore, EPA presents all PODs in
equivalents of both units to avoid confusion and errors. Equation Apx F-4 presents the conversion of the
HEC from mg/m3 to ppm.
Equation Apx F-4. Converting Units for HECs (mg/m3 to ppm)
mg 24.45
X ppm = Y —5- x
m3 MW
Where:
24.45 = Molar volume of a gas at standard temperature and pressure (L/mol), default
MW = Molecular weight of the chemical (MW of DINP = 418.62 g/mol)
F.l DINP Non-cancer HED and HEC Calculations for Acute and
Intermediate Duration Exposures
The acute and intermediate duration non-cancer POD is based on a BMDLs of 49 mg/kg-day, and the
critical effect is decreased fetal testicular testosterone. The BMDLs was derived by NASEM (2017)
through meta-regression and BMD modeling of fetal testicular testosterone data from two studies of
DINP with rats (Boberg et al.. 2011: Hannas et al.. 2011). R code supporting NASEM's meta-regression
and BMD analysis of DINP is publicly available through GitHub). This non-cancer POD is considered
protective of effects observed following acute and intermediate duration exposures to DINP. EPA used
Equation Apx F-l to determine a DAF specific to rats (0.236), which was in turn used in the following
calculation of the daily HED using Equation Apx F-2:
mq mq
11.6 — = 49- — X 0.236
kg — day kg — day
Page 200 of 282
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EPA then calculated the continuous HEC for an individual at rest using EquationApx F-3:
mq mq 80 kq
63-0 —j = 11.6- x( ^ )
m kg day 0.6125 * 24 hr
hr
Equation Apx F-4 was used to convert the HEC from mg/m3 to ppm:
mq 24.45
3.68 ppm = 63.0 —- x
HH m3 418.62
F.2 DINP Non-cancer HED and HEC Calculations for Chronic Exposures
The chronic duration non-cancer POD is based on a NOAEL of 15 mg/kg-day, and the critical effect is
liver toxicity (i.e., increased relative liver weight, increased serum chemistry (AST, ALT, ALP),
histopathologic findings (e.g., focal necrosis, spongiosis hepatis) in F344 rats following 2 years of
dietary exposure to DINP (Lington et al.. 1997; Bio/dynamics. 1986). EPA used Equation Apx F-l to
determine a DAF specific to rats (0.236), which was in turn used in the following calculation of the daily
HED using Equation Apx F-2:
mq mq
3.54 — = 15- — x 0.236
kg — day kg — day
EPA then calculated the continuous HEC for an individual at rest using Equation Apx F-3:
mq mq 80 kq
19.3 -f = 3.54 x ( ^ )
m kg day 0.6125 * 24 hr
hr
Equation Apx F-4 was used to convert the HEC from mg/m3 to ppm:
mg 24.45
1.13 ppm = 19.3 —7 x ^ ^ ^
m3 418.62
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Appendix G CONSIDERATIONS FOR BENCHMARK RESPONSE
(BMR) SELECTION FOR REDUCED FETAL
TESTICULAR TESTOSTERONE
G.l Purpose
EPA has conducted an updated meta-analysis and benchmark dose modeling (BMD) analysis of
decreased fetal rat testicular testosterone. During the July 2024 SACC peer review meeting of the Draft
Risk Evaluation of Diisodecyl Phthalate (DIDP) and Draft Raman Health Hazard Assessment for
Diisononyl Phthalate (DINP), the SACC recommended that EPA should clearly state its rational for
selection of benchmark response (BMR) levels evaluated for decreases in fetal testicular testosterone
relevant to the single chemical assessments (U.S. EPA 2024g). This appendix describes EPA's rationale
for evaluating BMRs of 5, 10, and 40 percent for decreases in fetal testicular testosterone. (Note: EPA
will assess the relevant BMR for deriving relative potency factors to be used in the draft cumulative risk
assessment separately fi'om this analysis.)
G.2 Methods
As described in EPA's Benchmark Dose Technical Guidance (U.S. EPA. 2012). "Selecting a BMR(s)
involves making judgments about the statistical and biological characteristics of the dataset and about
the applications for which the resulting BMDs/BMDLs will be used." For the updated meta-analysis and
BMD modeling analysis of fetal rat testicular testosterone, EPA evaluated BMR values of 5, 10, and 40
percent based on both statistical and biological considerations.
In 2017, NASEM (2017) modeled BMRs of 5 and 40 percent for decreases in fetal testicular
testosterone. NASEM did not provide explicit justification for selection of a BMR of 5 percent.
However, justification for the BMR of 5 can be found elsewhere. As discussed in EPA's Benchmark
Dose Technical Guidance (U.S. EPA. 2012). a BMR of 5 percent is supported in most developmental
and reproductive studies. Comparative analyses of a large database of developmental toxicity studies
demonstrated that developmental NOAELs are approximately equal to the BMDLs (Allen et al.. 1994a.
b; Faustman et al.. 1994).
EPA also evaluated a BMR of 10 percent as part of the updated BMD analysis. BMD modeling of fetal
testosterone conducted by NASEM (2017) indicated that BMDs estimates are below the lowest dose
with empirical testosterone data for several of the phthalates (e.g., DIBP). As discussed in EPA's
Benchmark Dose Technical Guidance (U.S. EPA. 2012) "For some datasets the observations may
correspond to response levels far in excess of a selected BMR and extrapolation sufficiently below the
observable range may be too uncertain to reliably estimate BMDs/BMDLs for the selected BMR."
Therefore, EPA modelled a BMR of 10 percent because data sets for some of the phthalates may not
include sufficiently low doses to support modeling of a 5 percent response level.
NASEM (2017) also modeled a BMR of 40 percent using the following justification: "previous studies
have shown that reproductive-tract malformations were seen in male rats when fetal testosterone
production was reduced by about 40% (Gray et al.. 2016; Howdeshell et al.. 2015)."
Further description of methods and results for the updated meta-analysis and BMD modeling analysis
that evaluated BMRs of 5, 10, and 40 percent for decreased fetal testicular testosterone are provided in
EPA's Draft Meta-analysis and Benchmark Dose Modeling of Fetal Testicular Testosterone for Di(2-
Page 202 of 282
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ethylhexyl) Phthalate (DEHP), Dibutyl Phthalate (DBF), Butyl Benzyl Phthalate (BBP), Diisobutyl
Phthalate (DIBP), andDicyclohexylPhthalate (DCHP) (U.S. EPA. 2024a).
G.3 Results
BMD estimates, as well as 95 percent upper and lower confidence limits, for decreased fetal testicular
testosterone for the evaluated BMRs of 5, 10, and 40 percent are shown in TableApx G-l. BMDs
estimates ranged from 8.4 to 74 mg/kg-day for DEHP, DBP, DCHP, and DINP; however, a BMDs
estimate could not be derived for BBP or DIBP. Similarly, BMDio estimates ranged from 17 to 152 for
DEHP, DBP, DCHP, DIBP and DINP; however, a BMDio estimate could not be derived for BBP.
BMD40 estimates were derived for all phthalates (i.e., DEHP, DBP, DCHP, DIBP, BBP, and DINP) and
ranged from 90 to 699 mg/kg-day.
In the mode of action (MOA) for phthalate syndrome, which is described elsewhere (U.S. EPA. 2023a)
and in Section 3.1.2 of this document, decreased fetal testicular testosterone is an early, upstream event
in the MOA that precedes downstream apical outcomes such as male nipple retention, decrease
anogenital distance, and reproductive tract malformations. Decreased fetal testicular testosterone should
occur at lower or equal doses than downstream apical outcomes associated with a disruption of androgen
action. Because the lower 95 percent confidence limit on the BMD, or BMDL, is used for deriving a
point of departure (POD), EPA compared BMDL estimates at the 5, 10, and 40 percent response levels
for each phthalate (DEHP, DBP, DCHP, DIBP, BBP, DINP) to the lowest identified apical outcomes
associated with phthalate syndrome to determine which response level is protective of downstream
apical outcomes.
Table Apx G-l provides a comparison of BMD and BMDL estimates for decreased fetal testicular
testosterone at BMRs of 5, 10, and 40 percent, the lowest LOAEL(s) for apical outcomes associated
with phthalate syndrome, and the POD selected for each phthalate for use in risk characterization. As
can be seen from Table Apx G-l, BMDL40 values for DEHP, DBP, DIBP, BBP, DCHP, and DINP are
all well above the PODs selected for use in risk characterization for each phthalate by 3x (for BBP) to
25.4x (for DEHP). Further, BMDL40 values for DEHP, DBP, DIBP, BBP, and DCHP, but not DINP, are
above the lowest LOAELs identified for apical outcomes on the developing male reproductive system.
These results clearly demonstrate that a BMR of 40 percent is not appropriate for use in human health
risk assessment.
As can be seen from Table Apx G-l, BMDL10 values for DBP (BMDL10, POD, LOAEL = 20, 9, 30
mg/kg-day, respectively) and DCHP (BMDL10, POD, LOAEL = 12, 10, 20 mg/kg-day, respectively) are
slightly higher than the PODs selected for use in risk characterization and slightly less than the lowest
LOAELs identified based on apical outcomes associated with the developing male reproductive system.
This indicates that a BMR of 10 percent may be protective of apical outcomes evaluated in available
studies for both DBP and DCHP. BMDL10 values could not be derived for DIBP or BBP (Table Apx
G-l). Therefore, no comparisons to the POD or lowest LOAEL for apical outcomes could be made for
either of these phthalates at the 10 percent response level.
For DEHP, the BMDL10 is greater than the POD selected for use in risk characterization by 5X
(BMDL10 and POD = 24 and 4.8 mg/kg-day, respectively) and is greater than the lowest LOAEL
identified for apical outcomes on the developing male reproductive system by 2.4X (BMDL10 and
LOAEL = 24 and 10 mg/kg-day, respectively). This indicates that a BMR of 10 percent for decreased
fetal testicular testosterone is not health protective for DEHP. For DEHP, the BMDL5 (11 mg/kg-day)
is similar to the selected POD (NOAEL of 4.8 mg/kg-day) and the lowest LOAEL identified for apical
outcomes on the developing male reproductive system (10 mg/kg-day).
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G.4 Weight of Scientific Evidence Conclusion
As discussed elsewhere (U.S. EPA. 2023a). DEHP, DBP, BBP, DIBP, DCHP, and DINP are
toxicologically similar and induce effects on the developing male reproductive system consistent with a
disruption of androgen action. Because these phthalates are toxicologically similar, it is more
appropriate to select a single BMR for decreased fetal testicular testosterone to provide a consistent
basis for dose response analysis and for deriving PODs relevant to the single chemical assessments. EPA
has reached the conclusion that a BMR of 5 percent is the most appropriate and health protective
response level for evaluating decreasedfetal testicular testosterone when sufficient dose-response data
are available to support modeling of fetal testicular testosterone in the low-end range of the dose-
response curve. This conclusion is supported by the following weight of scientific evidence
considerations.
• For DEHP, the BMDLio estimate is greater than the POD selected for use in risk characterization
by 5x and is greater than the lowest LOAEL identified for apical outcomes on the developing
male reproductive system by 2.4 x. This indicates that a BMR of 10 percent is not protective for
DEHP.
• The BMDLs estimate for DEHP is similar to the selected POD and lowest LOAEL for apical
outcomes on the developing male reproductive system.
• BMDLio estimates for DBP (BMDLio, POD, LOAEL = 20, 9, 30 mg/kg-day, respectively) and
DCHP (BMDLio, POD, LOAEL = 12, 10, 20 mg/kg-day, respectively) are slightly higher than
the PODs selected for use in risk characterization and slightly less than the lowest LOAELs
identified based on apical outcomes associated with the developing male reproductive system.
This indicates that a BMR of 10 percent may be protective of apical outcomes evaluated in
available studies for both DBP and DCHP. However, this may be a reflection of the larger
database of studies and wider range of endpoints evaluated for DEHP, compared to DBP and
DCHP.
• NASEM (2017) modeled a BMR of 40 percent using the following justification: "previous
studies have shown that reproductive-tract malformations were seen in male rats when fetal
testosterone production was reduced by about 40% fGrav et al.. 2016; Howdeshell et al.. 2015)."
However, publications supporting a 40 percent response level are relatively narrow in scope and
assessed the link between reduced fetal testicular testosterone in SD rats on GDI8 and later life
reproductive tract malformations in F1 males. More specifically, Howdeshell et al. (2015) found
reproductive tract malformations in 17 to 100 percent of F1 males when fetal testosterone on
GDI8 was reduced by approximately 25 to 72 percent, while Gray et al. (2016) found dose-
related reproductive alterations in F1 males treated with dipentyl phthalate (a phthalate not
currently being evaluated under TSCA) when fetal testosterone was reduced by about 45 percent
on GD18. Although NASEM modeled a BMR of 40 percent based on biological considerations,
there is no scientific consensus on the biologically significant response level and no other
authoritative or regulatory agencies have endorsed the 40 percent response level as biologically
significant for reductions in fetal testosterone.
• BMDL40 values for DEHP, DBP, DIBP, BBP, DCHP, and DINP are above the PODs selected for
use in risk characterization for each phthalate by 3x to 25,4/ (Table Apx G-l). BMDL40 values
for DEHP, DBP, DIBP, BBP, and DCHP, but not DINP, are above the lowest LOAELs
identified for apical outcomes on the developing male reproductive system. These results clearly
demonstrate that a BMR of 40 percent is not health protective.
Page 204 of 282
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TableApx G-l. Comparison of BMD/BMDL Values across BMRs of 5%, 10%, and 40% with PODs and LOAELs for Apical
Outcomes
for DEHP, DBP, DIBP, BBP, DCHP, and DINP
Phthalate
POD (mg/kg-day) Selected for use
in Risk Characterization
(Effect)
Lowest LOAEL(s)
(mg/kg-day) for Apical
Effects on the Male
Reproductive System
BMDS
Estimate"
(mg/kg-day)
[95% CI]
BMDio
Estimate"
(mg/kg-day)
[95% CI]
BMD40
Estimate"
(mg/kg-day)
[95% CI]
Reference For Further
Details on the Selected
POD and Lowest
Identified LOAEL
DEHP
NOAEL = 4.8
(t male RTM in F1 and F2 males)
10 to 15
(NR, | AGD, RTMs)
17 [11, 31]
35 [24, 63]
178 [122, 284]
(U.S. EPA. 2024e)
DBP
BMDL5 = 9
(J, fetal testicular testosterone)
30
(t Testicular Pathology)
14 [9, 27]
29 [20, 54]
149 [101,247]
(U.S. EPA. 2024c)
DIBP
BMDL5 = 24
(J, fetal testicular testosterone)
125
(t Testicular Pathology)
_b
55 [NA, 266f
279 [136, 517]
(U.S. EPA. 2024f)
BBP
NOAEL = 50
(phthalate syndrome-related effects)
100
(IAGD)
_b
_b
284 [150, 481]
(U.S. EPA. 2024b)
DCHP
NOAEL = 10
(phthalate syndrome-related effects)
20
(t Testicular Pathology)
8.4 [6.0, 14]
17 [12, 29]
90 [63, 151]
(U.S. EPA. 2024d)
DINP
BMDL5 = 49
(J, fetal testicular testosterone)
600
(J, sperm motility)
74 [47, 158]
152 [97, 278]
699 [539, 858]
(U.S. EPA. 2025e)
AGD = anogenital distance; BMD = benchmark dose; BMDL = lower 95% confidence limit on BMD; CI = 95% confidence interval; LOAEL = lowest-observed-
adverse-effect level; NOAEL = no-observed-adverse-effect level; POD = point of departure; RTM = reproductive tract malformations
11 The linear-quadratic model provided the best fit (based on lowest AIC) for DEHP, DBP, DIBP, BBP, DCHP, and DINP.
h BMD and/or BMDL estimate could not be derived.
Page 205 of 282
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Appendix H UPDATED META-ANALYSIS AND BMD MODELING
OF FETAL TESTICULAR TESTOSTERONE
H.l Purpose
EPA has conducted an updated meta-analysis and benchmark dose modeling (BMD) analysis of
decreased fetal rat testicular testosterone. During the July 2024 Science Advisory Committee on
Chemicals (SACC) peer-review meeting of the draft risk evaluation of diisodecyl phthalate (DIDP) and
draft human health hazard assessments for diisononyl phthalate (DINP), the SACC recommended that
EPA should conduct a new BMD modeling analysis that should consider new experimental studies (U.S.
EPA. 2024g). This appendix describes EPA's updated meta-analysis and BMD modeling analysis of
fetal testicular testosterone for DINP.
H.2 Methods
In 2017, NASEM demonstrated the utility of meta-analysis and meta-regression to summarize several
outcomes from experimental animal studies (NASEM. 2017). The 2017 NASEM analysis included
reduced fetal testicular testosterone, reduced male anogenital distance (AGD), and increased incidence
of hypospadias in rodents following oral exposure to DEHP, DBP, BBP, DIBP, and DINP. Boxes 3-3
and 3-4 in (NASEM. 2017) provide detailed descriptions of the meta-analysis approach employed by
NASEM. Briefly, NASEM conducted meta-analyses using the Metafor (Version 2.0.0) meta-analysis
package for R (https://wviechtb.github.io/metafor/index.html). which employs a standard random effects
model using the Restricted Maximum Likelihood Estimate. The meta-analyses conducted by NASEM
focused on the dose-response relationship and employed three models, including the linear, log-linear,
and linear-quadratic models. For the linear and linear-quadratic models, BMD values were estimated
based on benchmark response (BMR) levels of 5 and 40 percent (BMR selection rationale is provided in
Appendix G). R code used by NASEM to conduct all meta-analyses is publicly available
(https://github.com/wachiuphd/NASEM-2017-Endocrine-Low-Dose).
As part of its updated analysis, EPA used a similar meta-analysis and BMD modeling approach as
employed by NASEM (2017). with several notable differences. First, EPA used the most recent version
of the R Metafor package (Version 4.6.0) available at the time of the updated analysis, while NASEM
used Metafor Version 2.0.0. However, EPA also conducted the updated analysis with Metafor Version
2.0.0 so that results from the two different versions of Metafor could be compared. Similar to the
NASEM approach, EPA's updated meta-analysis focused on the dose-response relationship and
employed the linear, log-linear, and linear-quadratic models. Another notable difference between the
NASEM analysis and EPA's updated analysis is that EPA evaluated BMRs of 5, 10, and 40 percent,
while NASEM evaluated BMRs of 5 and 40 percent (BMR selection rationale is provided in Appendix
G). As part of the updated meta-analysis, EPA utilized all of the same fetal rat testicular testosterone
data included in the original NASEM (2017) analysis, as well as new fetal rat testosterone data
identified through the 2024 TSCA literature update for DINP, as described in the systematic review
protocol for DINP (U.S. EPA. 2025h). EPA also considered new literature identified outside of the 2019
TSCA literature searches that was identified through the literature searches conducted in support of
EPA's Draft Proposed Approach for Cumulative Risk Assessment of High-Priority Phthalates and a
Manufacturer-Requested Phthalate under the Toxic Substances Control Act (U.S. EPA. 2023 a).
Consistent with the meta-analysis and BMD modeling approach employed by NASEM (2017). new fetal
rat testicular testosterone data were included in the updated meta-analysis if the following criteria were
met:
Page 206 of 282
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• Study conducted with pregnant rats (all strains considered relevant, including Sprague-Dawley,
Wistar, Long Evans, F344, etc.). For the updated analysis, studies of mice were excluded,
because rats are considered for the more sensitive species.
• Study exposed rats via the oral route.
• Study measured fetal testis testosterone content or ex vivo fetal testicular testosterone production.
Studies measuring only serum or plasma testosterone not included. Studies measuring
testosterone at non-fetal lifestages were excluded. Studies measuring testosterone production
following stimulation with luteinizing hormone were excluded.
• Study should include an exposure that covers the male programming window (defined by
NASEM as GDI6-18).
• Study fully reports data (i.e., mean, standard deviation or standard error, and sample size) to
support inclusion in the meta-analysis.
H.3 Results
In 2017, NASEM included fetal rat testicular testosterone data from two studies (Boberg et al.. 2011;
Hannas et al.. 2011) as part of its meta-analysis and BMD modeling analysis for DINP. Fetal
testosterone data from Boberg et al. and Hannas et al. was included as part of EPA's updated analysis.
EPA identified new fetal rat testicular testosterone data from two studies (Gray et al.. 2024; Furr et al..
2014). which was included as part of the updated meta-analysis and BMD modeling analysis for DINP.
Table Apx H-l provides an overview of the four studies included in the updated analysis.
EPA identified testosterone data from six other studies of DINP (Gray. 2023; Gray et al.. 2021; Li et al..
2015; Clewell et al.. 2013a; Clewell et al.. 2013b; Adamsson et al.. 2009). Testosterone data from these
studies was not included in the updated analysis for various reasons. Studies by Li et al. (2015) and
Adamson et al. (2009). which were previously considered by NASEM (2017). were excluded because Li
et al. evaluated testicular testosterone on PND1 (not a fetal lifestage), while Adamson et al. did not
sufficiently report data to support its inclusion (i.e., the exact number of litters per dose group was not
report, the number of litters were dose group was reported as a range). Studies by Gray et al. (2023;
2021) were not included because these publications re-report fetal testosterone data previously reported
by the same research group in publications by Hannas et al. (2011) and Furr et al. (2014). which are both
studies included in the updated analysis. Testosterone data from Clewell et al. (2013b) was not included
because testicular testosterone was measured in F1 males on PND49 (not a fetal lifestage). Finally,
testosterone data from Clewell et al. (2013a) was not included because of data reporting limitations (data
reported as percent control with number of litters per dose group reported; however, no measure of
variability provided [i.e., standard deviation or standard error]).
EPA conducted the updated meta-analysis using random effects models, as implemented in the R
Metafor package. Metafor versions 2.0.0 and 4.6.0 were used so that results could be compared.
Additionally, the updated analysis included a sensitivity analysis to determine if the meta-analysis was
sensitive to leaving out results from individual studies. In 2017, NASEM did not conduct a sensitivity
analysis because there were too few studies available to do so.
Page 207 of 282
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TableApx H-l. Summary of Studies Included in EPA's Meta-analysis and BMD Modeling
Analysis for D
[NP
Reference
(TSCA
Study
Quality
Rating)
Included in NASEM
Meta-analysis and
BMD Modeling
Analysis?
Brief Study Description
Measured Outcome
(Hannas et
al.. 2011)
(Medium)
Yes
Pregnant SD rats (5-9
dams/group) gavaged with 0,
500, 750, 1,000, 1,500 mg/kg-
day DINP onGD14-18
Ex vivo fetal testicular
testosterone production (3-
hour incubation) on GDI8
(Bobere et al.,
2011)
(Medium)
Yes
Pregnant Wistar rats (9-10
dams/group) gavaged with 0,
300, 600, 750, 900 mg/kg-day
DINP on GD7-21.
Ex vivo fetal testicular
testosterone production and
testes testosterone on GD21
(Furr et al.,
2014)
(High)
No
Pregnant SD rats (3-5
dams/group) gavaged with 0,
750 mg/kg-day DINP on
GD14-18 (Block 1).
Ex vivo fetal testicular
testosterone production (3-
hour incubation) on GDI8
No
Pregnant SD rats (3-5
dams/group) gavaged with 0,
750 mg/kg-day DINP on
GD14-18 (Block 5).
No
Pregnant SD rats (3-5
dams/group) gavaged with 0,
750 mg/kg-day DINP on
GD14-18 (Block 7).
(Grav et al..
No
Pregnant SD rats (4 dams/group)
gavaged with 0, 750 mg/kg-day
DINP on GD14-18 (Block 166).
Ex vivo fetal testicular
testosterone production (3-
hour incubation) on GDI8
2024)
(Medium)
No
Pregnant SD rats (3-4
dams/group) gavaged with 0,
750 mg/kg-day DINP on
GD14-18 (Block 167).
Overall meta-analyses and sensitivity analyses results obtained using Metafor Versions 2.0.0 and 4.6.0
are shown in TableApx H-2 and TableApx H-3, respectively. A comparison of BMD estimates
obtained by NASEM (2017) and as part of EPA's updated analysis are shown in TableApx H-4.
Additional meta-analysis results (i.e., forest plots) and BMD model fit curves are shown in FigureApx
H-l through FigureApx H-4. For meta-analyses conducted using both versions of Metafor, there was a
statistically significant overall effect and linear trends in logio(dose) and dose, with an overall effect that
is large in magnitude (>50% change). For both meta-analyses, there was substantial, statistically
significant heterogeneity in all cases (I2> 40% for Metafor v.2.0.0; I2> 50% for Metafor v.4.6.0). The
statistical significance of these effects was robust to leaving out individual studies for analyses
conducted with both versions of Metafor. Although there was substantial heterogeneity, standard
deviation of the random effect (tau) was less than the estimated size of the effect at higher doses.
Page 208 of 282
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Therefore, the heterogeneity does not alter the conclusion that gestational exposure to DINP reduces
fetal testicular testosterone in the rat.
For meta-analyses conducted using both versions of Metafor, the linear-quadratic model provided the
best fit {i.e., had the lowest AIC) (TableApx H-4). BMD estimates from the linear-quadratic model
were 79 mg/kg-day [95% CI: 52, 145] for a 5 percent change (BMR = 5%), 160 mg/kg-day [108, 262]
for a 10 percent change (BMR = 10%), and 715 mg/kg-day [584, 842] for a 40 percent change (BMR =
40%) when Metafor Version 2.0.0 was used. Similarly, BMD estimates were 74 mg/kg-day [47, 158] for
a 5 percent change (BMR = 5%), 152 mg/kg-day [97, 278] for a 10 percent change (BMR = 10%), and
699 mg/kg-day [539, 858] for a 40 percent change (BMR = 40%) when Metafor Version 4.6.0 was used.
Notably, Metafor versions 2.0.0 and 4.6.0 provided similar BMDs (79 vs. 74 mg/kg-day), BMDio (160
versus 152 mg/kg-day), and BMD40 (715 vs. 699 mg/kg-day) estimates for the best fitting, linear-
quadratic model (Table Apx H-4), and these results are similar to those obtained in the 2017 NASEM
meta-analysis (i.e., BMD5 and BMD40 estimates of 76 and 701 mg/kg-day, respectively, based on the
best fitting linear quadratic model).
Table Apx H-2. Updated Overall Meta-Analyses and Sensitivity Analyses of Rat Studies of DINP
and Fetal Testosterone (Metafor Version 2.0.0)
Analysis
Estimate
Beta
CI,
Lower
Bound
CI,
Upper
Bound
P value
Tau
I2
P value for
Heterogeneity
AICs
Primary Analysis
Overall
intrcpt
-58.82
-73.97
-43.67
2.76E-14
25.23
79.73
1.78E-10
162.76
Trend in loglO(dose)
loglO(dose)
-124.31
-186.04
-62.59
7.91E-05
14.10
53.75
3.50E-03
148.19
Linear indoselOO
doselOO
-7.37
-8.49
-6.26
1.21E-38
11.37
44.83
2.33E-02
150.63
Linear Quadratic in dose 100
doselOO
-6.45
-9.98
-2.92
3.441 • 04
11.57
44.90
2.32E-02
145.92*
Linear Quadratic in dose 100
I(dosel00A2)
-0.10
-0.44
0.25
5.87E-01
11.57
44.90
2.32E-02
145.92
Sensitivity analysis
Overall minus Boberg et al.
2011
intrcpt
-62.16
-80.41
-43.90
2.50E-11
25.21
85.62
8.04E-10
82.20
Overall minus Hannas et al.
2011b
intrcpt
-49.60
-63.79
-35.41
7.35E-12
17.31
57.55
3.22E-03
121.50
Overall minus Furr et al. 2014
intrcpt
-62.58
-81.06
-44.11
3.15E-11
27.43
79.60
3.95E-09
135.63
Overall minus Gray et al. 2024
intrcpt
-59.12
-76.62
-41.61
3.62E-11
27.72
82.87
4.19E-11
145.49
* Indicates lowest AIC.
Page 209 of 282
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TableApx H-3. Updated Overall Meta-Analyses and Sensitivity Analyses of Rat Studies of DINP
Analysis
Estimate
Beta
CI,
Lower
Bound
CI,
Upper
Bound
P value
Tau
I2
P value for
Heterogeneity
AICs
Primary analysis
Overall
intrcpt
-58.82
-73.97
-43.67
2.76E-14
25.23
79.73
1.78E-10
162.76
Trend in loglO(dose)
loglO(dose)
-124.31
-186.04
-62.59
7.91E-05
14.10
53.75
3.50E-03
148.19
Linear indoselOO
dose100
-7.72
-9.73
-5.71
5.36E-14
27.81
82.93
1.54E-14
157.08
Linear Quadratic in dose 100
dose100
-6.83
-11.16
-2.51
1.97E-03
16.65
62.81
3.49E-04
146.87*
Linear Quadratic in dose 100
I(dosel00A2)
-0.07
-0.49
0.36
7.56E-01
16.65
62.81
3.49E-04
146.87
Sensitivity analysis
Overall minus Boberg et al.
2011
intrcpt
-62.16
-80.41
-43.90
2.50E-11
25.21
85.62
8.04E-10
82.20
Overall minus Hannas et al.
2011b
intrcpt
-49.60
-63.79
-35.41
7.35E-12
17.31
57.55
3.22E-03
121.50
Overall minus Furr et al.
2014
intrcpt
-62.58
-81.06
-44.11
3.15E-11
27.43
79.60
3.95E-09
135.63
Overall minus Gray et al.
2024
intrcpt
-59.12
-76.62
-41.61
3.62E-11
27.72
82.87
4.19E-11
145.49
* Indicates lowest AIC.
TableApx H-4. Comparison of Benchmark Dose Estimates for DINP and Fetal Testosterone in
Rats
Analysis
BMR
BMD
CI, Lower Bound
CI, Upper Bound
2017 NASEM analysis using Metafor Version 2.0.0
(as reported in Table C6-16 of NASEM, 2017)
Linear in dose 100
5%
68
59
80
Linear in dose 100
40%
676
588
795
Linear Quadratic in dose 100*
5%
76
49
145
Linear Quadratic in dose 100*
40%
701
552
847
Updated analysis using Metafor Version 2.0.0 including study by Furr et al. (2014) & Gray et al. (2024)
Linear in dose 100
5%
70
60
82
Linear in dose 100
10%
143
124
168
Linear in dose 100
40%
693
602
816
Linear Quadratic in dose 100*
5%
79
52
145
Linear Quadratic in dose 100*
10%
160
108
262
Linear Quadratic in dose 100*
40%
715
584
842
Page 210 of 282
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Analysis
BMR
BMD
CI, Lower Bound
CI, Upper Bound
Updated Analysis using Metafor Version 4.6.0 including study by Furr et al. (2014) & Gray et al. (2024)
Linear in dose 100
5%
66
53
90
Linear in dose 100
10%
136
108
185
Linear in dose 100
40%
662
525
895
Linear Quadratic in dose 100*
5%
74
47
158
Linear Quadratic in dose 100*
10%
152
97
278
Linear Quadratic in dose 100*
40%
699
539
858
* Indicates model with lowest AIC.
Page 211 of 282
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Rat DINP All Doses
Study and animal group
Dose (mg/kg-d) Estimate [95% CI]
Boberg et al. 2011 Wrstar rat.1
Boberg et al. 2011 Wistar rat.2
Hartnas et al. 2011 b Sprague Dawley rats.1
Boberg et al. 2011 Wrstar rat.3
Boberg et al. 2011 Wistar rat.4
Boberg et al. 2011 Wistar rat.S
Boberg et al. 2011 Wistar rat-6
Fuit et al. 2014 Sprague-Dawley rat: Study 1
Furr et al. 2014 Sprague-Dawley rat: Study 2
Fuit et al. 2014 Sprague-Dawley rat Study 3
Gray et a). 2024 Sprague-Dawley rati
Gray et al. 2024 Sprague-Dawley rat.2
Hartnas et al. 2011 b Sprague Dawley rats.2
Boberg et al. 2011 Wistar rat7
Boberg et al. 2011 Wistar rat.8
Hannas et al. 2011b Sprague Dawley rats.3
Hannas et al. 2011 b Sprague Dawley rats.4
300 -69.31 [-170.38, 31.75]
300 -13.84 [ -34.54. 6.87]
500 -35.67 [ -48.54, -22.80]
600 -128.251-234.31, -22.19]
600 -68.20 [-123.71, -12.68]
750 -109.08 [-225.45, 7.28]
750 -34.55 [-84.83, 15.73]
750 -27.30[-43.16,-11.44]
750 -48.38 [ -75.01, -21.76]
750 -70.25 [ -89.42, -51.08]
750 -60.95 [ -83.26, -38.63]
750 "59.70 [-100.14, -19.27]
750 -59.27 [ -89.36, -29.17]
900 -134.05 [-248.84, -19.26]
900 -44.56 [-64.50,-24.62]
1000 -85.40 [-101.45, -69.34]
1500 -118.511-145.21,-91.81]
RE Model
(12=79.7%)
-58.82 [ -73.97, -43.67]
-3D0
-200
-100
100
Fetal testes T log(Ratio of mean)
FigureApx H-l. Meta-analysis of Studies of DINP and Fetal Testosterone in Rats (Metafor
Version 2.0.0)
Page 212 of 282
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c
71
0)
E
O
tr
S>
s
tfll
&
03
Rat DINP
o -
Log-linear model
o
o
T-
\
T — ^
1
flB 1
r T
r "^5
O
CD
1
100
1000
DINP Dose mg/kg-d
10000
i
E
o
g
la
(T
CT
5
tfl
Q
O
O -
Rat DINP
Linear model
r SBMD(-11)=143[124, 168]
€)=6&.6lte0.4. 81.91
BMD(-51 )=693[602, 816]
~t 1—r—r"
n 1 1—i—|
10000
100
1000
DINP Dose mg/kg-d
CO o —
o
Rat DINP
Linear-quadratic model
1
aj
a
£ « _jt?
CO i >
!£5.1)=7
MD(-11)=160[108, 262] BMD(-51)=715[5841 842]
3.6 [52, 1451
100
~i 1 1 1 1 r—i—i—|
1000
~1 1 1 1 1 1 1—I—I
10000
DINP Dose mg/kg-d
FigureApx H-2. Benchmark Dose Estimates from Rat Studies of DINP and Fetal Testosterone
(Metafor Version 2.0.0)
Page 213 of 282
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Rat DINP All Doses
Study and animal group
Dose (mg/kg-d)
Estimate [95% CI]
Boberg et a!. 2011 Wistar rat.1
Boberg et al. 2011 Wislar rat.2
Hannas et al. 2011b Sprague Dawley rats.1
Boberg et ai. 2011 Wistar rat.3
Boberg et al. 2011 Wislar rat.4
Boterg et al. 2011 Wistar rat.5
Boberg et al. 2011 Wistar rat.6
Fuit et al. 2014 Sprague-Dawley rat Sluidy 1
Furr et al. 2014 Sprague-Dawley rat Study 2
Furr et al. 2014 Sprague-Dawley rat Study 3
Gray et al. 2024 Sprague-Dawley rat.1
Gray et al. 2024 Sprague-Dawley rat.2
Hannas et al. 2011b Sprague Dawley rats.2
Boterg et al. 2011 Wistar rat.7
Boberg et al. 2011 Wistar rat.8
Hannas et al. 2011b Sprague Dawley rats.3
Hannas et al. 2011b Sprague Dawley rats.4
-aee—i
| y.Q
5O0
\ 6Q0
-600
^S0
-^se-i
'50
I 750
750
750
H 750
I ¦ 1 750
1 900
900
1000
1500
-69.31 [-170.38, 31.75]
-13.84 [-34.54, 6.87]
-35.67 [ -48.54. -22,80]
-128.25 [-234.31,-22.19]
-68.20[-123.71,-12.68]
-109.08 [-225.45, 7.28]
-34.55 [-84.83, 15.73]
-27.30 [-43.16, -11.44]
-48.38 [-75.01,-21.76]
-70.25 [-89.42, -51.08]
-60.95 [ -83.26, -38.63]
-59.70 [-100.14, -19.27]
-59.27 [ -89.36, -29.17]
-134.05 [-248,84, -19-26]
-44.56 [ -64.50, -24.62]
-85.40 [-101.45, -69.34]
-118.51 [-145.21, -91.81]
RE Model
(12=79.7%)
-58.82 [ -73.97, -43.67]
-300
-200 -100 0
Fetal testes T log[Ratio of mean)
100
Figure Apx H-3. Meta-analysis of Studies of DINP and Fetal Testosterone in Rats (Metafor
Version 4.6.0)
Page 214 of 282
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c
o
CD
o
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t—
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4—
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o
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CO
o
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o
CO
CO
I
u_
Rat DINP
^ Log-linear model
^ -r*^. -T-
1
1
< r *
100
1000
DINP Dose mg/kg-d
10000
§
E
o
o
¦¦1=1
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£
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Rat DINP
Linear model
_ j
D(-11)=1
2.7, 89 8
36(108,185] BMD(-51 )=
l :
=662[525, 895]
o
o
l#=66'.4[!
100
1000
DINP Dose mg/kg-d
10000
c
03
'D
E
o
o
"cS
o
I
¦a
o
o
o
O
o
cj
C*>
Rat DINP
Linear-quadratic model
i-V1D(-11)=1
. 5f46.6, 158
52[97.2, 278] BMD(-51
!' : :
(i
)=099[5
39, 858]
gjU=74.
100
1000
DINP Dose mg/kg-d
10000
FigureApx H-4. Updated Benchmark Dose Estimates from Rat Studies of DINP and Fetal
Testosterone (Metafor Version 4.6.0)
Page 215 of 282
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Appendix I CONSIDERATION OF APPLICABILITY OF CHRONIC
POD AND BENCHMARK MOE FOR DIFFERENT
LIFESTAGES
Background
As described Section 4.2.2, EPA selected a chronic POD of 3.5 mg/kg-day (based on a NOAEL) from
the 2-year dietary study of F344 rats based on liver toxicity (Lington et al.. 1997; Bio/dynamics. 1986)
to calculate risk for chronic exposure durations. A total UF of 30 was selected for use as the benchmark
MOE (based on an interspecies UF (UFa) of 3 and an intraspecies UF (UFh) of 10). Consistent with
EPA guidance (2022. 2002b. 1993). EPA reduced the UFa from a value of 10 to 3 because allometric
body weight scaling to the three-quarter power was used to adjust the POD to obtain a HED (Appendix
F).
During the SACC peer-review and the public comment period for the draft DINP hazard assessment,
EPA received comments (see EPA-HQ-QPPT-2018-0096 and EPA-HQ-OPPT-2024-0069) suggesting
the following:
• The toxicodynamics portion of the interspecies UF (UFa) of 3 should be reduced to 1 based on
toxicodynamic differences between rats and humans, with rats being more sensitive that humans
to liver toxicity associated with PPARa activation.
• The POD based on chronic liver toxicity is not appropriate for characterizing risk from chronic
exposure to infants and children because spongiosis hepatis is lesion prevalent in aging rats.
EPA addresses these points raised by stakeholders below.
Consideration of the Toxicodynamics Component of the interspecies UF (UFa)
• EPA considered whether the toxicodynamics component of the UFa should be reduced from 3 to
1 based on differences in species sensitivity to the liver effects that form the basis of the chronic
POD.
• As described in EPA's Cancer Raman Health Hazard Assessment for Diisononyl Phthalate
(DINP) (U.S. EPA. 2025a). the weight of evidence indicates that humans are less sensitive than
rodents to liver effects associated with PPARa activation, which could support a reduction in the
toxicodynamics component of the UFa from 3 to 1.
• However, the chronic POD of 3.5 mg/kg-day is based on a spectrum of liver effects, some of
which are related to PPARa activation (e.g., | liver weight, hypertrophy, necrosis) and some of
which are PPARa-independent (i.e., spongiosis hepatis).
• The mode of action underlying spongiosis hepatis is unknown but is not believed to be related to
peroxisome proliferation. Further, as discussed by ECHA (2013b). spongiosis hepatis has been
observed in the livers of some strains of rats and certain species of fish (e.g., medaka), but is less
common in mice, has not been observed in non-human primates or dogs, and with the exception
of two case reports, has not been described in humans. These findings raise some uncertainty as
to the human relevance of spongiosis hepatis (Karbe and Kerlin. 2002).
• Spongiosis hepatis is considered independent of mononuclear cell leukemia (MNCL). This is
based on the Pathology Working Groups re-analysis of liver histopathology data from Lington et
al. (1997) and Covance Labs (1998c) two-year dietary studies of DINP with F344 rats that
demonstrated that MNCL and spongiosis hepatis co-occurred in rats only about 50% of the time
(EPL. 1999).
Page 216 of 282
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• Given that the chronic POD is based on liver effects that are both dependent and independent of
PPARa and the uncertainty in mode of action associated with spongiosis hepatis, EPA concluded
that a redaction in the toxicodynamics component of the UFa from 3 to 1 is not warranted.
Applicability of the Chronic POD to Adults
• The chronic POD of 3.5 mg/kg-day is based on a spectrum of liver effects, some of which are
related to PPARa activation (e.g., | liver weight, hypertrophy, necrosis) and some of which are
considered PPARa-independent (i.e., spongiosis hepatis).
• EPA considers the chronic POD applicable for characterization of risk from exposure to DINP
for male andfemale adult workers, consumers and members of the general population that may
be exposed to DINP through TSCA releases. It plausible that these populations may be exposed
chronically to DINP through work, regular contact with consumer products and/or articles
containing DINP, or through TSCA releases of DINP to the environment.
Applicability of the Chronic POD to Infants and Children
• As discussed above, humans are less sensitive than rodents to liver effects associated with
PPARa activation, while spongiosis hepatis is most common in the livers of aging rats. Given
that spongiosis hepatis is common in aging rats, the applicability of using this lesion to
characterizing risk to infants and children is questionable.
• EPA considered whether gestational and/or perinatal exposure to DINP might result in increased
incidence of spongiosis hepatis later in life. Of the gestational/perinatal studies listed in Table
3-1, four studies evaluated liver outcomes in adult SD and Wistar rat following gestational
and/or perinatal exposure to DINP (Gray. 2023; Clewell et al.. 2013b; Boberg et al.. 2011; Gray
et al.. 2000). All four studies evaluated liver weight in F1 offspring between approximately 3.5
to 8 months of age; however, none of the available studies evaluated liver histopathology
precluding conclusions pertaining to the effect of gestational/perinatal exposure on incidence of
spongiosis hepatis later in life for this study type.
• EPA also considered whether continuous exposure to DINP for two-generations (including
gestational/perinatal exposures for F1 and F2 offspring) may increase the incidence of spongiosis
hepatis. In an initial dose-range finding one-generation study of DINP with SD rats,
histopathologic examinations were not included (Waterman et al.. 2000; Exxon Biomedical.
1996a). In a subsequent two-generation study of SD rats (Waterman et al.. 2000; Exxon
Biomedical. 1996b). liver histopathologic examinations were included for PI and P2 adults. No
significant increase in spongiosis hepatis was observed in male or female PI or P2 rats. These
findings indicate that gestational/perinatal exposure to DINP may not significantly increase the
incidence of spongiosis hepatis later in life. However, some uncertainty remains, as spongiosis
hepatis was observed in F344 rats after up to two-years of oral exposure in the study by Lington
et al. (1997). while liver histopathology in PI and P2 SD rats in the two-generation study was
examined after exposure to DINP for approximately 15 to 24 weeks of exposure to DINP. Given
that spongiosis hepatis is most prevalent in aging rats, the two-generation study may not have
examined liver histopathology in old enough rats to detect this lesion.
• Given that spongiosis hepatis is most prevalent in aging rats, use of the chronic POD of 3.5
mg/kg-day to assess risk from chronic exposure DINP for infants and children may be
conservative and may not be relevant.
• EPA considered whether other candidate intermediate and chronic PODs may be more
appropriate for assessing risk to infants and children from chronic exposure to DINP.
Page 217 of 282
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As discussed in Sections 4.1.1 and 4.1.2, EPA selected an acute/intermediate POD of 12 mg/kg-
day based on reduced fetal testicular testosterone. Use of this POD to calculate chronic risks
from exposure to DINP for infants and children would result in MOEs above the benchmark of
30 for all consumer exposure scenarios discussed in Section 4.3.3 of the risk evaluation of DINP
(U.S. EPA 2025g).
Of the candidate PODs listed in Table 4-5, the most sensitive candidate POD most directly
applicable to the infant and children lifestages is a LOAEL of 133 mg/kg-day (HED of 31
mg/kg-day) based on reduced F1 and F2 male and female offspring body weight on PND7, 14,
and 21 in a two-generation study of SD rats (Waterman et al.. 2000; Exxon Biomedical 1996b).
Given that no NOAEL could be identified, this study supports a benchmark MOE of 300, based
on an intraspecies UF (UFh) of 10, interspecies UF (UFa) of 3, and a LOAEL-to-NOAEL UF
(UFl) of 10. Given the additional UFl of 10, this candidate POD and benchmark (HED of 31 and
benchmark MOE of 300) would lead to nearly identical risk conclusions for infants and children
as were obtained using the current chronic POD based on liver toxicity (HED of 3.5 and
benchmark MOE of 30).
The magnitude of the effect of DINP on offspring body weight was relative small at the LOAEL,
with statistically significant decreases of 8.9% for F1 females on PND21 to 10% for F1 males on
PND21 and F2 males on PND7 (Waterman et al.. 2000; Exxon Biomedical. 1996b). Given the
magnitude of the effect, a full UFl of 10 for this LOAEL may be over-conservative.
To refine the LOAEL based on reduced F1 and F2 offspring bodyweight from the two-
generation study of reproduction, EPA conducted benchmark dose (BMD) modeling of F1 and
F2 male and female offspring body weights on PND7, PND14, and PND21. BMD modeling
results are provided in Appendix J. The lowest BMDLs derived was 65 mg/kg-day (HED of 15
mg/kg-day) based on reduced F1 male body weight on PND21 (Table Apx J-l). Consistent with
U.S. EPA guidance (U.S. EPA. 2022. 2002b. 1993). since the LOAEL was refined to a BMDLs,
the UFl of 10 was no longer necessary.
Overall, this analysis supports an HED of 15 mg/kg-day and a total UF of 30, based on an
intraspecies UF (UFh) of 10, and an interspecies UF (UFa) of 3. This HED is less sensitive than
the acute/intermediate POD of 12 mg/kg-day and total UF of 30. Use of this HED to calculate
chronic risks from exposure to DINP for infants and children would result in MOEs above the
benchmark of 30 for all consumer exposure scenarios discussed in Section 4.3.3 of the risk
evaluation of DINP (U.S. EPA 2025 g).
Page 218 of 282
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Appendix J BENCHMARK DOSE MODELING OF PUP
BODYWEIGHT DATA FROM WATERMAN ET AL.
(2000)
The two-generation study of reproduction of DINP with SD rats by Waterman et al. supports a LOAEL
of 133 mg/kg-day based on reduced F1 and F2 male and female offspring body weight on PND7, 14,
and 21 (Waterman et al.. 2000; Exxon Biomedical. 1996b). The magnitude of the effect of DINP on
offspring body weight was relative small at the LOAEL, with statistically significant decreases of 8.9%
for F1 females on PND21 to 10% for F1 males on PND21 and F2 males on PND7 (Waterman et al..
2000; Exxon Biomedical. 1996b). EPA conducted benchmark dose (BMD) modeling of decreased F1
and F2 male and female body weight on PND7, PND14, and PND21 to refine the LOAEL.
The BMD modeling for continuous data was conducted with EPA's BMD software (BMDS 3.3.2). All
standard BMDS 3.3.2 continuous models that use maximum likelihood (MLE) optimization and profile
likelihood-based confidence intervals were used in this analysis. Standard forms of these models
(defined below) were run so that auto-generated model selection recommendations accurately reflect
current EPA model selection procedures EPA's benchmark Dose Technical Guidance (U.S. EPA. 2012).
BMDS 3.3.2 models that use Bayesian fitting procedures and Bayesian model averaging were not
applied in this work.
Standard BMDS 3.3.2 Models Applied to Continuous Endpoints:
• Exponential 3-restricted (exp3-r)
• Exponential 5-restricted (exp5-r)
• Hill-restricted (hil-r)
• Polynomial Degree 3-restricted (ply3-r)
• Polynomial Degree 2-restricted (ply2-r)
• Power-restricted (pow-r)
• Linear-unrestricted (lin-ur)
EPA evaluated benchmark response (BMR) levels of 1 control standard deviation (1 SD) and 5%
relative deviation. Model fit was judged consistent with EPA's benchmark Dose Technical Guidance
(U.S. EPA. 2012). An adequate fit was judged based on the %2 goodness-of-fit p-value (p > 0.1),
magnitude of the scaled residuals in the vicinity of the BMR, and visual inspection of the model fit. In
addition to these three criteria forjudging adequacy of model fit, a determination was made as to
whether the variance across dose groups was constant. If a constant variance model was deemed
appropriate based on the statistical test provided in BMDS (i.e., Test 2; p-value > 0.05 [note: this is a
change from previous versions of BMDS, which required variance p-value > 0.10 for adequate fit]), the
final BMD results were estimated from a constant variance model. If the test for homogeneity of
variance was rejected (i.e., p-value < 0.05), the model was run again while modeling the variance as a
power function of the mean to account for this nonconstant variance. If this nonconstant variance model
did not adequately fit the data (i.e., Test 3; p-value < 0.05), the data set was considered unsuitable for
BMD modeling. Among all models providing adequate fit, the lowest BMDL was selected if the
BMDLs estimated from different adequately fitting models varied >3-fold; otherwise, the BMDL from
the model with the lowest AIC was selected.
If no model adequately fit the data set using the approach described above, EPA removed the highest
dose group and modelled the data again using the approach described above.
Page 219 of 282
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TableApx J-l summarizes BMD modeling results for reduced F1 and F2 male and female bodyweight
on PND7, PND14, and PND21, while more detailed BMD model results for F1 and F2 offspring are
provided in Appendices J.l and J.2, respectively.
Table Apx J-l. Summary of BMD Model Results for Reduced F1 and F2 Offspring Bodyweight
Waterman et al., 2000)
Data Set
BMR
Best-Fit Model
(Variance)
BMD
(mg/kg-
day)
BMDL
(mg/kg-
day)
Notes
Appendix
Containing
Results
F1 Males PND7
5%
-
-
-
No models adequately fit the data
set
J.l.l
F1 Males PND14
5%
Linear
(Constant)
106
87
Adequate fit with highest dose
group removed
J.l.2
F1 Males PND21
5%
Exponential 3
78
65
Adequate fit with highest dose
group removed
J.l.3
F1 Females PND7
5%
-
-
-
No models adequately fit the data
set
J.l.4
F1 Females PND14
5%
Linear
(Constant)
106
87
Adequate fit with highest dose
group removed
J.l.5
F1 Females PND21
5%
Exponential 3
(Constant)
83
69
Adequate fit with highest dose
group removed
J.l.6
F2 Males PND7
5%
-
-
-
No models adequately fit the data
set
J.2.1
F2 Males PND14
5%
-
-
-
No models adequately fit the data
set
J.2.2
F2 Males PND21
5%
Exponential 3
(Constant)
118
102
J.2.3
F2 Females PND7
5%
-
-
-
No models adequately fit the data
set
J.2.4
F2 Females PND 14
5%
Linear
(Constant)
104
85
Adequate fit with highest dose
group removed
J.2.5
F2 Females PND21
5%
Exponential 3
(Constant)
111
98
J.2.6
J.l F1 Offspring Bodyweight
J.l.l F1 Male Offspring Bodyweight on PND7
Table Apx J-2. F1 Male Offs
)ring Bodyweight on PND7
Dietary
Dose (%)
Received Dose
(mg/kg-day)
N
Mean
SD
Statistical
Significance
Location of Data
0
0
97
17.62
2.35
Table 8 in (Waterman et
al.. 2000); Table 19 in
0.2
139
95
16.44
2.85
0.4
274
90
15.28
3.19
*
(Exxon Biomedical,
1996b)
0.8
543
94
15.67
1.74
*
Page 220 of 282
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Table Apx J-3. BMD Model Result
s for
Male Offspring Bodyweight on PND7 (All Dose Groups Included)
Models "
Restriction b
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Exponential 3
Restricted
Constant
1 SD
749.8948
556.508
0.000257
1799.008897
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
BMD higher than maximum dose
BMDL higher than maximum dose
Exponential 5
Restricted
Constant
1 SD
-
-
-
-
Unusable
BMD computation failed
Hill
Restricted
Constant
1 SD
-
-
-
-
Unusable
BMD computation failed
Polynomial
Degree 3
Restricted
Constant
1 SD
754.8555
570.8735
0.0001778
1799.746131
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
BMD higher than maximum dose
BMDL higher than maximum dose
Polynomial
Degree 2
Restricted
Constant
1 SD
762.1407
570.6021
0.0001776
1799.748087
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
BMD higher than maximum dose
BMDL higher than maximum dose
Power
Restricted
Constant
1 SD
755.59
570.8596
0.0001778
1799.746106
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
BMD higher than maximum dose
BMDL higher than maximum dose
Linear
Unrestricted
Constant
1 SD
755.5899
570.8596
0.0001778
1799.746106
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
BMD higher than maximum dose
BMDL higher than maximum dose
Exponential 3
Restricted
Constant
5%
231.2154
175.338
0.000257
1799.008897
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
Exponential 5
Restricted
Constant
5%
127.1849
61.77582
NA
1787.534509
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Hill
Restricted
Constant
5%
134.7184
109.4018
0.3035716
1785.534556
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Polynomial
Degree 3
Restricted
Constant
5%
245.5176
189.1077
0.0001778
1799.746131
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
Page 221 of 282
-------
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Polynomial
Degree 2
Restricted
Constant
5%
247.7713
189.0559
0.0001776
1799.748087
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
Power
Restricted
Constant
5%
245.745
189.102
0.0001778
1799.746106
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
Linear
Unrestricted
Constant
5%
245.745
189.0999
0.0001778
1799.746106
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
Exponential 3
Restricted
Non-
constant
1 SD
1013.702
851.8207
0.0009372
1794.28836
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
BMD higher than maximum dose
BMDL higher than maximum dose
|Residual at control| > 2
Exponential 5
Restricted
Non-
constant
1 SD
-
-
-
-
Unusable
BMD computation failed
Hill
Restricted
Non-
constant
1 SD
-
-
-
-
Unusable
BMD computation failed
Polynomial
Degree 3
Restricted
Non-
constant
1 SD
-
-
-
-
Unusable
BMD computation failed
Polynomial
Degree 2
Restricted
Non-
constant
1 SD
-
-
-
-
Unusable
BMD computation failed
Power
Restricted
Non-
constant
1 SD
952.6842
705.9678
0.0008987
1794.372126
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
BMD higher than maximum dose
BMDL higher than maximum dose
|Residual at control| > 2
Linear
Unrestricted
Non-
constant
1 SD
1002.876
708.8889
0.0009355
1794.291951
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
BMD higher than maximum dose
BMDL higher than maximum dose
|Residual at control| > 2
Page 222 of 282
-------
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Exponential 3
Restricted
Non-
constant
5%
302.3829
197.5137
0.0009372
1794.28836
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
|Residual at control| > 2
Exponential 5
Restricted
Non-
constant
5%
128.6863
61.77299
0.0226984
1787.534509
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
Hill
Restricted
Non-
constant
5%
133.935
107.1847
0.0300325
1787.050504
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
Polynomial
Degree 3
Restricted
Non-
constant
5%
-
-
-
-
Unusable
BMD computation failed
Polynomial
Degree 2
Restricted
Non-
constant
5%
243.9276
243.2026
<0.0001
1799.962907
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
Power
Restricted
Non-
constant
5%
272.7174
210.5346
0.0008987
1794.372126
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
|Residual at control| > 2
Linear
Unrestricted
Non-
constant
5%
282.7436
211.2587
0.0009355
1794.291951
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
|Residual at control| > 2
AIC = Akaike information criterion; BMD = benchmark dose; BMDL = benchmark dose lower limit; NA = not applicable
" Selected Model (bolded and shaded gray).
4 Restrictions defined in the BMDS 3.3 User Guide.
Page 223 of 282
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Table Apx *
-4. BMD Model Results for F1 Ma
e Offspring Bodyweight on P
VD7 (Highest
Dose Group Removed)
Models "
Restriction b
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Exponential 3
Restricted
Constant
1 SD
328.1537
251.186
NA
1387.511903
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
BMD higher than maximum dose
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Exponential 5
Restricted
Constant
1 SD
332.9178
148.3848
NA
1389.511903
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
BMD higher than maximum dose
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Hill
Restricted
Constant
1 SD
330.0178
158.5132
NA
1389.511903
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
BMD higher than maximum dose
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial
Degree 2
Restricted
Constant
1 SD
326.1608
252.4184
0.99564
1385.511933
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
BMD higher than maximum dose
Power
Restricted
Constant
1 SD
326.461
252.4097
NA
1387.511906
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
BMD higher than maximum dose
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Linear
Unrestricted
Constant
1 SD
327.042
252.3835
0.9839528
1385.512308
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
BMD higher than maximum dose
Exponential 3
Restricted
Constant
5%
104.6753
76.73575
NA
1387.511903
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Exponential 5
Restricted
Constant
5%
106.1192
51.88479
NA
1389.511903
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Hill
Restricted
Constant
5%
105.3628
48.60752
NA
1389.511903
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial
Degree 2
Restricted
Constant
5%
104.1664
81.52328
0.99564
1385.511933
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Power
Restricted
Constant
5%
104.1108
81.52331
NA
1387.511906
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Linear
Unrestricted
Constant
5%
103.184
81.54133
0.9839528
1385.512308
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Exponential 3
Restricted
Non-
constant
1 SD
328.1538
251.1877
0.0035502
1387.511903
Questionable
Goodness of fit p-value <0.1
BMD higher than maximum dose
Page 224 of 282
-------
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Exponential 5
Restricted
Non-
constant
1 SD
328.4096
251.1132
NA
1389.511903
Questionable
BMD higher than maximum dose
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Hill
Restricted
Non-
constant
1 SD
280.8265
147.1053
NA
1383.011249
Questionable
BMD higher than maximum dose
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial
Degree 2
Restricted
Non-
constant
1 SD
326.2536
252.4134
0.0142597
1385.511903
Questionable
Goodness of fit p-value <0.1
BMD higher than maximum dose
Power
Restricted
Non-
constant
1 SD
326.445
252.4109
0.0035502
1387.511908
Questionable
Goodness of fit p-value <0.1
BMD higher than maximum dose
Linear
Unrestricted
Non-
constant
1 SD
327.042
252.3792
0.0142568
1385.512308
Questionable
Goodness of fit p-value <0.1
BMD higher than maximum dose
Exponential 3
Restricted
Non-
constant
5%
104.6752
76.73574
0.0035502
1387.511903
Questionable
Goodness of fit p-value <0.1
Exponential 5
Restricted
Non-
constant
5%
104.7656
51.88902
NA
1389.511903
Questionable
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Hill
Restricted
Non-
constant
5%
100.4919
46.75259
NA
1383.011249
Questionable
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial
Degree 2
Restricted
Non-
constant
5%
103.9411
81.52232
0.0142597
1385.511903
Questionable
Goodness of fit p-value <0.1
Power
Restricted
Non-
constant
5%
104.1388
81.52362
0.0035502
1387.511908
Questionable
Goodness of fit p-value <0.1
Linear
Unrestricted
Non-
constant
5%
103.184
81.54133
0.0142568
1385.512308
Questionable
Goodness of fit p-value <0.1
AIC = Akaike information criterion; BMD = benchmark dose; BMDL = benchmark dose lower limit; NA = not applicable
" Selected Model (bolded).
4 Restrictions defined in the BMDS 3.3 User Guide.
Page 225 of 282
-------
J.1.2 F1 Male Offspring Bodyweight on PND14
Table Apx J-5. F1 Male Offs
pring Bodyweighi
on PND
[4
Dietary
Dose (%)
Received Dose
(mg/kg-day)
N
Mean
SD
Statistical
Significance
Location of Data
0
0
97
35.01
3.94
Table 8 in (Waterman et
al.. 2000); Table 19 in
(Exxon Biomedical,
1996b)
0.2
139
94
33.28
4.82
0.4
274
90
30.43
4.36
*
0.8
543
92
29.66
2.55
*
Page 226 of 282
-------
Table Apx J-6.1
IMD Mode
Results for F1 Male Of
'spring I
lodyweight on PND14 (All Dose Groups Included)
Models "
Restriction b
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Exponential 3
Restricted
Constant
1 SD
385.4421
321.5715
0.002458
2108.61844
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
Exponential 5
Restricted
Constant
1 SD
236.4395
146.3635
NA
2100.601621
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Hill
Restricted
Constant
1 SD
227.8099
158.521
NA
2100.601621
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Polynomial Degree 3
Restricted
Constant
1 SD
398.0597
341.1634
0.0011488
2110.139761
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
Polynomial Degree 2
Restricted
Constant
1 SD
464.2469
322.2132
<0.0001
2114.546805
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Power
Restricted
Constant
1 SD
402.9291
341.0565
0.0011577
2110.124342
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
Linear
Unrestricted
Constant
1 SD
403.2053
341.0542
0.0011578
2110.124084
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
Exponential 3
Restricted
Constant
5%
158.7313
135.2375
0.002458
2108.61844
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Exponential 5
Restricted
Constant
5%
139.8526
99.88388
NA
2100.601621
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Hill
Restricted
Constant
5%
139.6717
116.9422
NA
2100.601621
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Polynomial Degree 3
Restricted
Constant
5%
169.2801
147.7802
0.0011488
2110.139761
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Polynomial Degree 2
Restricted
Constant
5%
201.8421
140.6115
<0.0001
2114.546805
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Power
Restricted
Constant
5%
171.1518
147.8322
0.0011577
2110.124342
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Linear
Unrestricted
Constant
5%
171.3268
147.8477
0.0011578
2110.124084
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Page 227 of 282
-------
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Exponential 3
Restricted
Non-
constant
1 SD
513.9093
419.4072
0.064527
2093.256241
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
Exponential 5
Restricted
Non-
constant
1 SD
356.4487
256.1837
NA
2091.771992
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Hill
Restricted
Non-
constant
1 SD
388.605
251.3757
NA
2091.771992
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Polynomial Degree 3
Restricted
Non-
constant
1 SD
555.656
544.2749
0.0030411
2098.557523
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
BMD higher than maximum dose
BMDL higher than maximum dose
|Residual at control| > 2
Polynomial Degree 2
Restricted
Non-
constant
1 SD
519.0093
437.047
0.0520345
2093.686597
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
Power
Restricted
Non-
constant
1 SD
518.6872
437.0171
0.0150328
2095.687521
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
Linear
Unrestricted
Non-
constant
1 SD
489.2304
426.7211
0.0361383
2094.415703
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
Exponential 3
Restricted
Non-
constant
5%
172.1766
147.9772
0.064527
2093.256241
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
Exponential 5
Restricted
Non-
constant
5%
178.7382
144.6059
NA
2091.771992
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Hill
Restricted
Non-
constant
5%
174.3258
141.3404
NA
2091.771992
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Polynomial Degree 3
Restricted
Non-
constant
5%
237.7356
232.8004
0.0030411
2098.557523
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
Page 228 of 282
-------
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
|Residual for Dose Group Near BMD| > 2
|Residual at control| > 2
Polynomial Degree 2
Restricted
Non-
constant
5%
183.4337
160.7366
0.0520345
2093.686597
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
Power
Restricted
Non-
constant
5%
183.4279
160.7333
0.0150328
2095.687521
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
Linear
Unrestricted
Non-
constant
5%
177.9682
157.9424
0.0361383
2094.415703
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
AIC = Akaike information criterion; BMD = benchmark dose; BMDL = benchmark dose lower limit; NA = not applicable
" Selected Model (bolded).
4 Restrictions defined in the BMDS 3.3 User Guide.
Table Apx J-7.1
IMD Model Results for F1IV
ale Offs
pring Bodyweight on PND14 (
lighest Dose Group Removed)
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Exponential 3
Restricted
Constant
1 SD
264.5474
219.5794
NA
1633.027224
Questionable
d.f.=0, saturated model (Goodness of fit test
cannot be calculated)
Exponential 5
Restricted
Constant
1 SD
264.5445
219.5679
NA
1635.027224
Questionable
d.f.=0, saturated model (Goodness of fit test
cannot be calculated)
Hill
Restricted
Constant
1 SD
168.7185
149.9951
NA
1633.027224
Questionable
d.f.=0, saturated model (Goodness of fit test
cannot be calculated)
Polynomial Degree
2
Restricted
Constant
1 SD
265.6614
221.2077
NA
1633.027546
Questionable
d.f.=0, saturated model (Goodness of fit test
cannot be calculated)
Power
Restricted
Constant
1 SD
264.7751
252.1797
NA
1633.027224
Questionable
d.f.=0, saturated model (Goodness of fit test
cannot be calculated)
Linear
Unrestricted
Constant
1 SD
262.5855
211.4703
0.2824621
1632.18243
Viable -
Recommended
Lowest AIC
Exponential 3
Restricted
Constant
5%
140.1249
88.30174
NA
1633.027224
Questionable
d.f.=0, saturated model (Goodness of fit test
cannot be calculated)
Page 229 of 282
-------
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Exponential 5
Restricted
Constant
5%
140.1246
85.99834
NA
1635.027224
Questionable
d.f.=0, saturated model (Goodness of fit test
cannot be calculated)
Hill
Restricted
Constant
5%
139.1467
124.0619
NA
1633.027224
Questionable
d.f.=0, saturated model (Goodness of fit test
cannot be calculated)
Polynomial Degree
2
Restricted
Constant
5%
140.7787
90.98064
NA
1633.027546
Questionable
d.f.=0, saturated model (Goodness of fit test
cannot be calculated)
Power
Restricted
Constant
5%
140.1461
90.99624
NA
1633.027224
Questionable
d.f.=0, saturated model (Goodness of fit test
cannot be calculated)
Linear
Unrestricted
Constant
5%
105.765
87.06488
0.2824621
1632.18243
Viable -
Recommende
d
Lowest AIC
AIC = Akaike information criterion; BMD = benchmark dose; BMDL = benchmark dose lower limit; NA = not applicable
" Selected Model (bolded).
4 Restrictions defined in the BMDS 3.3 User Guide.
Page 230 of 282
-------
Frequentist Linear Model with BMR of 0.05 Added Risk for the BMD
and 0.95 Lower Confidence Limit for the BMDL
41
39
-26
24
37
35 (
33
31
29
97
z /
25
Estimated Probability
Response at BMD
O Data
BMD
BMDL
74
124
mg/kg-day
174
224
274
User Input
Info
Model
frequentist Linear
Model Restriction
Unrestricted
Dataset Name
Waterman-2OO0_F1-BW-M-PND14_HDD
User notes
[Add user notes here]
Dose-Response Modi
M[dose] = q + bTdose
Variance Model
Var[i] = alpha
Model Options
BMR Type
Rel. Dev.
BMRF
0.05
Tail Probability
-
Confidence Level
0.05
Distribution Type
Normal
Variance Type
Constant
Model Data
Dependent Variable
mg^kg-day
Independent Variable
g
Total # of Observation
3
Adverse Direction
Downward
Page 231 of 282
-------
Model Results
Benchmark Dose
BMD
105.7650391
EiMDL
87.06487914
BMDU
135.5891999
AIC
1632.18243
Test 4 P-value
0.282462111
D.O.F.
1
Model Parameters
# of Parameters
3
Variable
Estimate
Std Error
Lower Conl
Upper Conl
9
35.198387
0.40748413
34.39973
35.33704
beta
-0.016639897
2.33E-03
-0.02121
-0.01207
alpha
19.09161088
3.08E+01
-41.1776
79.36081
Goodness of Fit
Dose
Size
Estimated
Calc'd
Observed
Estimated
Calc'd
Observe
Scaled
Median
Median
Mean
SD
SD
d SD
Residual
0
97
35.198387
35.01
35.01
4.369395
3.94
3.94
-0.424635
139
94
32.8854413
33.28
33.28
4.369395
4.82
4.82
0.8754962
274
90
30.6390552
30.43
30.43
4.363395
4.36
4.36
-0.453301
Likelihoods of Interest
Model
Loq Likelihood"
#of
Parameters
AIC
A1
-812.5136122
4
1633.027
A2
-810.5814201
6
1633.163
A3
-812.5136122
4
1633.027
fitted
-813.0912149
3
1632.182
R
-836.4720145
2
1676.944
' Includes additive constant of -258.22173. This constant wa
Tests of Interest
Test
2'Log(Likeliho
od Ratio)
Test df
p-value
1
51.78118886
4
<0.0001
2
3.864384145
2
0.14483
3
3.864384145
2
0.14483
4
1.155205454
1
0.282462
constant was not included in the LL derivation prior to BMDS 3.0.
J.1.3 F1 Male Offspring Bodyweight on PND21
Table Apx J-8. F1 Male Offs
)ring Bodyweight on PND21
Dietary
Dose (%)
Received Dose
(mg/kg-day)
N
Mean
SD
Statistical
Significance
Location of Data
0
0
96
57.25
6.73
Table 8 in (Waterman et
al.. 2000); Table 19 in
0.2
139
94
51.4
8.52
*
0.4
274
90
47.95
7.94
*
(Exxon Biomedical,
1996b)
0.8
543
92
46.52
5.15
*
Page 232 of 282
-------
Table Apx J-9. BMP Model Results for F
Male Offspring Bodyweight on PND21 (All Dose Groups Included)
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Exponential 3
Restricted
Constant
1 SD
367.0454
306.7887
<0.0001
2545.274942
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
|Residual at control| > 2
Exponential 5
Restricted
Constant
1 SD
177.2542
125.8513
NA
2530.405182
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Hill
Restricted
Constant
1 SD
173.107
125.1314
NA
2530.405182
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial Degree 3
Restricted
Constant
1 SD
393.4128
333.2567
<0.0001
2548.05993
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
|Residual at control| > 2
Polynomial Degree 2
Restricted
Constant
1 SD
399.7122
333.1663
<0.0001
2548.089361
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
|Residual at control| > 2
Power
Restricted
Constant
1 SD
392.6991
333.2785
<0.0001
2548.05962
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
|Residual at control| > 2
Linear
Unrestricted
Constant
1 SD
392.6991
333.2779
<0.0001
2548.05962
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
|Residual at control| > 2
Exponential 3
Restricted
Constant
5%
132.7414
113.5851
<0.0001
2545.274942
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Exponential 5
Restricted
Constant
5%
70.4488
37.76684
NA
2530.405182
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
BMDL 3x lower than lowest non-zero dose
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Hill
Restricted
Constant
5%
82.09051
35.8347
NA
2530.405182
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
BMDL 3x lower than lowest non-zero dose
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Page 233 of 282
-------
Models "
Restriction b
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Polynomial Degree 3
Restricted
Constant
5%
147.4963
127.677
<0.0001
2548.05993
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Polynomial Degree 2
Restricted
Constant
5%
149.6593
127.6308
<0.0001
2548.089361
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Power
Restricted
Constant
5%
147.2538
127.6716
<0.0001
2548.05962
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Linear
Unrestricted
Constant
5%
147.2538
127.6716
<0.0001
2548.05962
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Exponential 3
Restricted
Non-
constant
1 SD
477.7192
377.3672
0.004205
2538.203338
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Exponential 5
Restricted
Non-
constant
1 SD
213.5096
138.4185
NA
2531.26037
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Hill
Restricted
Non-
constant
1 SD
209.6099
138.4402
NA
2531.26037
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial Degree 3
Restricted
Non-
constant
1 SD
503.5426
406.2491
0.0019455
2539.744912
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Polynomial Degree 2
Restricted
Non-
constant
1 SD
-
-
-
-
Unusable
BMD computation failed
Power
Restricted
Non-
constant
1 SD
497.1371
407.0933
0.001981
2539.708698
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Linear
Unrestricted
Non-
constant
1 SD
503.0601
407.0398
0.0019937
2539.695929
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Page 234 of 282
-------
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Exponential 3
Restricted
Non-
constant
5%
146.3514
123.8637
0.004205
2538.203338
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Exponential 5
Restricted
Non-
constant
5%
74.63699
40.39051
NA
2531.26037
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
BMDL 3x lower than lowest non-zero dose
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Hill
Restricted
Non-
constant
5%
83.13899
35.5995
NA
2531.26037
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
BMDL 3x lower than lowest non-zero dose
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial Degree 3
Restricted
Non-
constant
5%
162.0372
138.6099
0.0019455
2539.744912
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Polynomial Degree 2
Restricted
Non-
constant
5%
-
-
-
-
Unusable
BMD computation failed
Power
Restricted
Non-
constant
5%
160.2951
138.658
0.001981
2539.708698
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Linear
Unrestricted
Non-
constant
5%
161.2557
138.8609
0.0019937
2539.695929
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
AIC = Akaike information criterion; BMD = benchmark dose; BMDL = benchmark dose lower limit; NA = not applicable
" Selected Model (bolded and shaded gray).
4 Restrictions defined in the BMDS 3.3 User Guide.
Page 235 of 282
-------
Table Apx J-10. BMD Model Results for F
Male Offspring Bodyweight on PND2:
(Highest Dose Group Removed)
Models a
Restriction b
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Exponential 3
Restricted
Constant
1 SD
222.0347
179.7111
0.3426081
1945.657114
Viable -
Recommended
Lowest AIC
Exponential 5
Restricted
Constant
1 SD
203.5517
138.8042
NA
1948.756466
Questionable
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Hill
Restricted
Constant
1 SD
204.7198
138.6643
NA
1946.756466
Questionable
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Polynomial Degree 2
Restricted
Constant
1 SD
227.9437
187.1874
0.2467275
1946.098206
Viable -
Alternate
Power
Restricted
Constant
1 SD
226.9683
187.1713
0.2469453
1946.096969
Viable -
Alternate
Linear
Unrestricted
Constant
1 SD
226.9683
187.1707
0.2469453
1946.096969
Viable -
Alternate
Exponential 3
Restricted
Constant
5%
78.14108
64.98007
0.3426081
1945.657114
Viable -
Recommended
Lowest AIC
Exponential 5
Restricted
Constant
5%
59.6461
34.40916
NA
1948.756466
Questionable
BMDL 3x lower than lowest non-zero dose
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Hill
Restricted
Constant
5%
58.25307
28.57678
NA
1946.756466
Questionable
BMDL 3x lower than lowest non-zero dose
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Polynomial Degree 2
Restricted
Constant
5%
83.8116
70.57765
0.2467275
1946.098206
Viable -
Alternate
Power
Restricted
Constant
5%
83.48213
70.56802
0.2469453
1946.096969
Viable -
Alternate
Linear
Unrestricted
Constant
5%
83.48212
70.56801
0.2469453
1946.096969
Viable -
Alternate
AIC = Akaike information criterion; BMD = benchmark dose; BMDL = benchmark dose lower limit; NA = not applicable
" Selected Model (bolded).
4 Restrictions defined in the BMDS 3.3 User Guide.
Page 236 of 282
-------
Frequentist Exponential Degree 3 Model with BMR of 0.05 Added Risk for the
BMD and 0.95 Lower Confidence Limit for the BMDL
Estimated Probability
Response at BMD
Data
BMD
BMDL
Estimated Probability
Response at BMD
O Data
BMD
BMDL
User Input
Info
Model
frequentist Exponential degree 3
Model Restriction
Restricted
Dataset Name
Waterman-2000_F1-BW-M-PN021_HDD
User notes
[Add user notes here]
Dose-Response Modi
M[dose] = a1 enpf±11 (b1 dosef d)
Variance Model
Var[i] = alpha
Model Options
BMR Type
Rel. Dev.
BMRF
0.05
Tail Probability
-
Confidence Level
0.95
Distribution Type
Normal
Variance Type
Constant
Model Data
Dependent Variable
mg^kg-day
Independent Variable
g
Total # of Observation
3
Adverse Direction
Downward
59
54
49
T
44
39
-26 24 74 124 174 224 274
mg/kg-day
Page 237 of 282
-------
Model Results
Benchmark Dose
BMD
78.14108041
BMDL
64.8800676
BMDU
110.6809379
AIC
1945.657114
Test4P-ualue
0.342608058
D.O.F.
1
Model Parameters
# of Parameters
4
Variable
Estimate
Std Error
Lower Conl
Upper Conl
a
56.93026038
0.73570373
55.4883
58.37223
b
0.000656418
7.38E-05
0.0005
0.000813
d
Bounded
NA
NA
NA
log-alpha
4.087321065
8.45E-02
3.922396
4.253446
Goodness of Fit
Dose
Size
Estimated
Calc'd
Observed
Estimated
Calc'd
Observe
Scaled
Median
Median
Mean
3D
3D
d 3D
Residual
0
96
56.330261
57.25
57.25
7.721129
6.73
6.73
0.4057425
133
94
51.9657463
51.4
51.4
7.721129
8.52
8.52
-0.710403
274
90
47.5588648
47.35
47.35
7.721129
7.34
7.34
0.4805819
Likelihoods of Interest
#of
Model
Loq Likelihood"
Parameters
AIC
A1
-363.3782332
4
1346.756
A2
-366.6852344
6
1345.37
A3
-369.3782332
4
1346.756
fitted
-969.8285572
3
1945.657
R
-1000.384573
2
2004.769
' Includes additive constant of -257.30279. This constant was not included in the LL derivation prior to BMDS 3.0.
Tests of Interest
Test
2"Log(Likeliho
od Ratio)
Test df
p-value
1
67.33868874
4
<0.0001
2
5.385397686
2
0.067678
3
5.385337686
2
0.067678
4
0.900647851
1
0.342608
J.1.4 F1 Female Offspring Bodyweight on PND7
Table Apx J-ll. F1 Female Offspring Bodyweight on PND7
Dietary
Dose (%)
Received Dose
(mg/kg-day)
N
Mean
SD
Statistical
Significance
Location of Data
0
0
96
16.7
2.15
Table 8 in (Waterman et
al.. 2000); Table 19 in
(Exxon Biomedical,
1996b)
0.2
139
94
15.54
2.79
0.4
274
95
14.21
3.21
*
0.8
543
97
15.03
1.72
*
Page 238 of 282
-------
Table Apx J-12. BMP Model Results for F1 Female Offspring Bodyweight on PND7 (All Dose Groups Included)
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Exponential 3
Restricted
Constant
1 SD
1
873.3122
<0.0001
1820.613354
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
BMD higher than maximum dose
BMDL higher than maximum dose
|Residual at control| > 2
Exponential 5
Restricted
Constant
1 SD
-
-
-
-
Unusable
BMD computation failed
Hill
Restricted
Constant
1 SD
-
-
-
-
Unusable
BMD computation failed
Polynomial
Degree 3
Restricted
Constant
1 SD
1
871.7201
<0.0001
1821.327416
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
BMD higher than maximum dose
BMDL higher than maximum dose
[Residual at control| > 2
Polynomial
Degree 2
Restricted
Constant
1 SD
1
880.3641
<0.0001
1821.326564
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of tit p-value <0.1
[Residual for Dose Group Near BMD| > 2
BMD higher than maximum dose
BMDL higher than maximum dose
[Residual at control| > 2
Power
Restricted
Constant
1 SD
1
877.7875
<0.0001
1821.326396
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
[Residual for Dose Group Near BMD| > 2
BMD higher than maximum dose
BMDL higher than maximum dose
[Residual at control| > 2
Linear
Unrestricted
Constant
1 SD
1
877.7877
<0.0001
1821.326396
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of tit p-value <0.1
[Residual for Dose Group Near BMD| > 2
BMD higher than maximum dose
BMDL higher than maximum dose
[Residual at control| > 2
Exponential 3
Restricted
Constant
5%
254.4842
186.3013
<0.0001
1820.613354
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of tit p-value <0.1
[Residual for Dose Group Near BMD| > 2
[Residual at control| > 2
Exponential 5
Restricted
Constant
5%
126.7009
67.24957
NA
1803.662419
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Page 239 of 282
-------
Models "
Restriction b
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Hill
Restricted
Constant
5%
134.2006
107.7629
0.0243488
1801.662519
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Polynomial
Degree 3
Restricted
Constant
5%
269.2419
201.2969
<0.0001
1821.327416
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
|Residual at control| > 2
Polynomial
Degree 2
Restricted
Constant
5%
271.806
201.4052
<0.0001
1821.326564
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
|Residual at control| > 2
Power
Restricted
Constant
5%
271.0633
201.3112
<0.0001
1821.326396
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
|Residual at control| > 2
Linear
Unrestricted
Constant
5%
271.0633
201.3093
<0.0001
1821.326396
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
|Residual at control| > 2
Exponential 3
Restricted
Non-
constant
1 SD
1183.284
1008.202
<0.0001
1818.804639
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
BMD higher than maximum dose
BMDL higher than maximum dose
|Residual at control| > 2
Exponential 5
Restricted
Non-
constant
1 SD
-
-
-
-
Unusable
BMD computation failed
Hill
Restricted
Non-
constant
1 SD
-
-
-
-
Unusable
BMD computation failed
Polynomial
Degree 3
Restricted
Non-
constant
1 SD
1171.144
705.7232
<0.0001
1818.783138
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
BMD higher than maximum dose
BMDL higher than maximum dose
|Residual at control| > 2
Polynomial
Degree 2
Restricted
Non-
constant
1 SD
948.1995
666.0418
<0.0001
1821.732072
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
BMD higher than maximum dose
BMDL higher than maximum dose
|Residual at control| > 2
Power
Restricted
Non-
constant
1 SD
1097.551
771.3424
<0.0001
1818.870369
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
BMD higher than maximum dose
BMDL higher than maximum dose
|Residual at control| > 2
Page 240 of 282
-------
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Linear
Unrestricted
Non-
constant
1 SD
1171.145
775.6192
<0.0001
1818.783138
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
BMD higher than maximum dose
BMDL higher than maximum dose
|Residual at control| > 2
Exponential 3
Restricted
Non-
constant
5%
358.6459
208.7593
<0.0001
1818.804639
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
|Residual at control| > 2
Exponential 5
Restricted
Non-
constant
5%
134.2801
67.24804
<0.0001
1803.662419
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
Hill
Restricted
Non-
constant
5%
131.732
99.5847
<0.0001
1800.18868
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
Polynomial
Degree 3
Restricted
Non-
constant
5%
320.1139
225.7198
<0.0001
1818.783138
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
|Residual at control| > 2
Polynomial
Degree 2
Restricted
Non-
constant
5%
284.8783
199.4796
<0.0001
1821.732072
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
|Residual at control| > 2
Power
Restricted
Non-
constant
5%
305.2412
224.6379
<0.0001
1818.870369
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
|Residual at control| > 2
Linear
Unrestricted
Non-
constant
5%
320.1141
225.718
<0.0001
1818.783138
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
|Residual at control| > 2
AIC = Akaike information criterion; BMD = benchmark dose; BMDL = benchmark dose lower limit; NA = not applicable
" Selected Model (bolded).
4 Restrictions defined in the BMDS 3.3 User Guide.
Page 241 of 282
-------
Table Apx J-13. BMD Model Results for F
Female Offspring Bodyweight on P>
D7 (Highest Dose Group Removed)
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Exponential 3
Restricted
Constant
1 SD
298.5781
236.6102
NA
1390.26048
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
BMD higher than maximum dose
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Exponential 5
Restricted
Constant
1 SD
299.0891
146.5579
NA
1392.26048
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
BMD higher than maximum dose
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Hill
Restricted
Constant
1 SD
302.2253
153.6427
NA
1392.26048
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
BMD higher than maximum dose
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Polynomial Degree 2
Restricted
Constant
1 SD
296.9767
237.1552
0.7810776
1388.337717
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
BMD higher than maximum dose
Power
Restricted
Constant
1 SD
297.812
237.8871
NA
1390.26048
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
BMD higher than maximum dose
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Linear
Unrestricted
Constant
1 SD
301.1549
236.8887
0.7646431
1388.350114
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
BMD higher than maximum dose
Exponential 3
Restricted
Constant
5%
104.5492
69.92462
NA
1390.26048
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Exponential 5
Restricted
Constant
5%
105.0005
53.80445
NA
1392.26048
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Hill
Restricted
Constant
5%
107.5409
51.41115
NA
1392.26048
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Polynomial Degree 2
Restricted
Constant
5%
92.13786
74.34106
0.7810776
1388.337717
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Power
Restricted
Constant
5%
103.7932
74.58938
NA
1390.26048
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Linear
Unrestricted
Constant
5%
92.11699
74.3079
0.7646431
1388.350114
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Exponential 3
Restricted
Non-
constant
1 SD
298.5781
236.6077
0.0001469
1390.26048
Questionable
Goodness of tit p-value <0.1
BMD higher than maximum dose
Exponential 5
Restricted
Non-
constant
1 SD
299.0267
146.5157
NA
1392.26048
Questionable
BMD higher than maximum dose
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Page 242 of 282
-------
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Hill
Restricted
Non-
constant
1 SD
224.0718
143.2736
NA
1379.84887
Questionable
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Polynomial Degree 2
Restricted
Non-
constant
1 SD
296.842
237.7491
0.0001469
1390.26048
Questionable
Goodness of fit p-value <0.1
BMD higher than maximum dose
Power
Restricted
Non-
constant
1 SD
297.812
237.8872
0.0001469
1390.26048
Questionable
Goodness of fit p-value <0.1
BMD higher than maximum dose
Linear
Unrestricted
Non-
constant
1 SD
301.1549
236.888
0.0007097
1388.350114
Questionable
Goodness of fit p-value <0.1
BMD higher than maximum dose
Exponential 3
Restricted
Non-
constant
5%
104.5494
69.92475
0.0001469
1390.26048
Questionable
Goodness of fit p-value <0.1
Exponential 5
Restricted
Non-
constant
5%
104.9473
53.80475
NA
1392.26048
Questionable
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Hill
Restricted
Non-
constant
5%
109.5812
47.40981
NA
1379.84887
Questionable
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Polynomial Degree 2
Restricted
Non-
constant
5%
102.5201
74.54359
0.0001469
1390.26048
Questionable
Goodness of fit p-value <0.1
Power
Restricted
Non-
constant
5%
103.7928
74.58906
0.0001469
1390.26048
Questionable
Goodness of fit p-value <0.1
Linear
Unrestricted
Non-
constant
5%
92.11699
74.3079
0.0007097
1388.350114
Questionable
Goodness of fit p-value <0.1
AIC = Akaike information criterion; BMD = benchmark dose; BMDL = benchmark dose lower limit; NA = not applicable
" Selected Model (bolded).
4 Restrictions defined in the BMDS 3.3 User Guide.
Page 243 of 282
-------
J.1.5 F1 Female Offspring Bodyweight on PND14
Table Apx J-14. F1 Female Offspring Bodyweight on PND14
Dietary
Dose (%)
Received Dose
(mg/kg-day)
N
Mean
SD
Statistical
Significance
Location of Data
0
0
96
33.52
3.7
Table 8 in (Waterman et
al.. 2000); Table 19 in
(Exxon Biomedical,
1996b)
0.2
139
93
31.89
4.57
0.4
274
94
29.14
4.5
*
0.8
543
97
28.41
3.1
*
Page 244 of 282
-------
Table Apx J-15. BMD Model Results for F
Female Offspring Bodyweight on PN
)14 (All Dose Groups Included)
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Exponential 3
Restricted
Constant
1 SD
405.3895
336.35
0.0030515
2146.282814
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
Exponential 5
Restricted
Constant
1 SD
246.5212
153.6476
NA
2138.698573
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Hill
Restricted
Constant
1 SD
239.1764
160.0051
NA
2138.698573
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Polynomial Degree 3
Restricted
Constant
1 SD
426.8875
356.1585
0.0014995
2147.703832
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Polynomial Degree 2
Restricted
Constant
1 SD
421.1136
356.0104
0.0015043
2147.697443
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Power
Restricted
Constant
1 SD
422.8253
356.0833
0.0015054
2147.696003
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Linear
Unrestricted
Constant
1 SD
422.8253
356.0834
0.0015054
2147.696003
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Exponential 3
Restricted
Constant
5%
159.7284
135.3876
0.0030515
2146.282814
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Exponential 5
Restricted
Constant
5%
140.9941
99.25743
NA
2138.698573
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Hill
Restricted
Constant
5%
140.5682
116.8619
NA
2138.698573
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Polynomial Degree 3
Restricted
Constant
5%
173.9302
148.0675
0.0014995
2147.703832
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Polynomial Degree 2
Restricted
Constant
5%
171.739
148.1089
0.0015043
2147.697443
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Power
Restricted
Constant
5%
172.391
148.2262
0.0015054
2147.696003
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Linear
Unrestricted
Constant
5%
172.391
148.2262
0.0015054
2147.696003
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Exponential 3
Restricted
Non-
constant
1 SD
470.6668
375.5155
0.0139371
2144.223772
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
Exponential 5
Restricted
Non-
constant
1 SD
276.0887
150.6508
NA
2139.677365
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Page 245 of 282
-------
Models "
Restriction b
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Hill
Restricted
Non-
constant
1 SD
276.738
167.5475
NA
2139.677365
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Polynomial Degree 3
Restricted
Non-
constant
1 SD
502.5831
396.2205
0.0086929
2145.167866
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
Polynomial Degree 2
Restricted
Non-
constant
1 SD
488.4594
397.0909
0.0089226
2145.115705
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
Power
Restricted
Non-
constant
1 SD
486.0556
397.154
0.0089092
2145.118707
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
Linear
Unrestricted
Non-
constant
1 SD
488.4594
397.0906
0.0089226
2145.115705
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
Exponential 3
Restricted
Non-
constant
5%
166.6613
140.7766
0.0139371
2144.223772
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
Exponential 5
Restricted
Non-
constant
5%
148.574
102.5272
NA
2139.677365
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Hill
Restricted
Non-
constant
5%
146.8637
119.7526
NA
2139.677365
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Polynomial Degree 3
Restricted
Non-
constant
5%
183.2216
153.8292
0.0086929
2145.167866
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
Polynomial Degree 2
Restricted
Non-
constant
5%
179.7093
154.0859
0.0089226
2145.115705
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
Power
Restricted
Non-
constant
5%
179.3159
154.1033
0.0089092
2145.118707
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
Linear
Unrestricted
Non-
constant
5%
179.7093
154.0868
0.0089226
2145.115705
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
Page 246 of 282
-------
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
AIC = Akaike information criterion; BMD = benchmark dose; BMDL = 1
" Selected Model (bolded).
4 Restrictions defined in the BMDS 3.3 User Guide.
benchmark dose lower limit; NA = not applicable
Table Apx J-16. BMP Model Results for F1 Female Offspring Bodyweight on PND14 (Highest Dose Group Removed)
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Exponential 3
Restricted
Constant
1 SD
268.1257
255.3466
NA
1629.716555
Questionable
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Exponential 5
Restricted
Constant
1 SD
268.1257
223.1241
NA
1631.716555
Questionable
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Hill
Restricted
Constant
1 SD
255.0912
150.8382
NA
1631.716555
Questionable
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial
Degree 2
Restricted
Constant
1 SD
266.4022
223.9918
NA
1629.821805
Questionable
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Power
Restricted
Constant
1 SD
268.2731
223.9493
NA
1629.716555
Questionable
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Linear
Unrestricted
Constant
1 SD
266.8633
214.6232
0.2713215
1628.926609
Viable -
Recommended
Lowest AIC
Exponential 3
Restricted
Constant
5%
141.6317
88.39683
NA
1629.716555
Questionable
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Exponential 5
Restricted
Constant
5%
141.6317
88.39683
NA
1631.716555
Questionable
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Hill
Restricted
Constant
5%
140.3123
116.9823
NA
1631.716555
Questionable
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial
Degree 2
Restricted
Constant
5%
130.2598
90.61312
NA
1629.821805
Questionable
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Power
Restricted
Constant
5%
141.6808
91.03917
NA
1629.716555
Questionable
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Linear
Unrestricted
Constant
5%
105.6747
86.89817
0.2713215
1628.926609
Viable -
Recommended
Lowest AIC
AIC = Akaike information criterion; BMD = benchmark dose; BMDL = benchmark dose lower limit; NA = not applicable
" Selected Model (bolded).
4 Restrictions defined in the BMDS 3.3 User Guide.
Page 247 of 282
-------
Frequentist Linear Model with BMR of 0.05 Added Risk for the BMD
and 0.95 Lower Confidence Limit for the BMDL
User Input
Info
Model
frequentist Linear
Model Restriction
Unrestricted
Dataset Name
Waterman-2OO0_F1-BW-F-PND14_HDD
User notes
[Add user notes here]
Dose-Response Modi
M[dose] = q + bTdose
Variance Model
Var[i] = alpha
Model Options
BMR Type
Rel. Dev.
BMRF
0.05
Tail Probability
-
Confidence Level
0.05
Distribution Type
Normal
Variance Type
Constant
Model Data
Dependent Variable
mg^kg-day
Independent Variable
g
Total # of Observation
3
Adverse Direction
Downward
Page 248 of 282
-------
Model Results
Benchmark Dose
BMD
105.6747086
ElMDL
86.89616003
BMDU
135.7553767
AIC
1628.326609
Test 4 P-value
0.271321544
D.O.F.
1
Model Parameters
# of Parameters
3
Variable
Estimate
Std Error
Lower Conl
Upper Conl
Q
33.70965857
0.33867194
32.92828
34.43104
beta
-0.015949729
2.25E-03
-0.02037
-0.01153
alpha
18.11691691
2.76E+01
-35.9638
72.19765
Goodness of Fit
Dose
Size
Estimated
Median
Calo'd
Median
Ubserved
Mean
Estimated
SD
Calc'd
SD
Observe
d SD
Scaled
Residual
0
36
33.7096586
33.52
33.52
4.256397
3.7
3.7
-0.436582
139
93
31.4926462
31.89
31.89
4.256397
4.57
4.57
0.900278
274
34
23.3394328
29.14
29.14
4.256397
4.5
4.5
-0.454275
Likelihoods of Interest
Model
Log Likelihood"
#of
Parameters
AIC
A1
-810.8582776
4
1629.717
fi2
-808.3435504
6
1628.699
A3
-810.8582776
4
1629.717
fitted
-811.4633043
3
1628.927
R
-834.5184646
2
1673.037
" Includes additive constant of -260.0536. This constant was not included in the LL derivation prior to BMDS 3.0.
Tests of Interest
Test
2"Log(Likeliho
od Ratio)
Test df
p-value
1
52.33782852
4
<0.0001
2
5.017454444
2
0.081372
3
5.017454444
2
0.081372
4
1.210053412
1
0.271322
J.1.6 F1 Female Offspring Bodyweight on PND21
Table Apx J-17. F1 Female Offspring Bodyweight on PND21
Dietary
Dose (%)
Received Dose
(mg/kg-day)
N
Mean
SD
Statistical
Significance
Location of Data
0
0
96
53.99
6.17
Table 8 in (Waterman et
al.. 2000); Table 19 in
(Exxon Biomedical,
1996b)
0.2
139
93
49.19
7.54
*
0.4
274
94
45.63
7.31
*
0.8
543
97
44.68
5.68
*
Page 249 of 282
-------
Table Apx J-]
8. BMD Model Resu
ts for F1 Female Offspring Bodyweight on PND21 (All Dose Groups Included)
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Exponential 3
Restricted
Constant
1 SD
393.9975
327.3418
0.0001081
2545.025349
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
|Residual at control| > 2
Exponential 5
Restricted
Constant
1 SD
192.929
180.0446
NA
2530.760975
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Hill
Restricted
Constant
1 SD
185.2249
144.9092
NA
2530.760975
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial
Degree 3
Restricted
Constant
1 SD
418.2624
352.1953
<0.0001
2547.393332
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Polynomial
Degree 2
Restricted
Constant
1 SD
455.8233
346.4451
<0.0001
2547.998204
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Power
Restricted
Constant
1 SD
417.4673
352.1606
<0.0001
2547.392998
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Linear
Unrestricted
Constant
1 SD
417.4673
352.1597
<0.0001
2547.392998
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Exponential 3
Restricted
Constant
5%
145.0004
123.2199
0.0001081
2545.025349
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Exponential 5
Restricted
Constant
5%
89.14845
44.68508
NA
2530.760975
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
BMDL 3x lower than lowest non-zero dose
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Hill
Restricted
Constant
5%
100.591
81.65762
NA
2530.760975
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial
Degree 3
Restricted
Constant
5%
159.5909
137.3508
<0.0001
2547.393332
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Polynomial
Degree 2
Restricted
Constant
5%
172.5487
135.1877
<0.0001
2547.998204
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Page 250 of 282
-------
Models "
Restriction b
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Power
Restricted
Constant
5%
159.3053
137.2556
<0.0001
2547.392998
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Linear
Unrestricted
Constant
5%
159.3053
137.2556
<0.0001
2547.392998
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Exponential 3
Restricted
Non-
constant
1 SD
439.3371
347.1973
0.0002438
2545.398207
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Exponential 5
Restricted
Non-
constant
1 SD
192.929
146.2211
0.9722902
2530.760975
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Hill
Restricted
Non-
constant
1 SD
184.4397
140.2982
NA
2532.759768
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial
Degree 3
Restricted
Non-
constant
1 SD
466.1738
375.9204
<0.0001
2547.335844
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Polynomial
Degree 2
Restricted
Non-
constant
1 SD
465.929
375.9273
<0.0001
2547.335807
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Power
Restricted
Non-
constant
1 SD
465.6475
375.9364
<0.0001
2547.335844
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Linear
Unrestricted
Non-
constant
1 SD
462.4941
375.9167
<0.0001
2547.346425
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Exponential 3
Restricted
Non-
constant
5%
150.8913
127.0587
0.0002438
2545.398207
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Exponential 5
Restricted
Non-
constant
5%
89.14798
44.68552
0.9722902
2530.760975
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
BMDL 3x lower than lowest non-zero dose
Hill
Restricted
Non-
constant
5%
100.5987
73.2584
NA
2532.759768
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Page 251 of 282
-------
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial
Degree 3
Restricted
Non-
constant
5%
165.868
141.7386
<0.0001
2547.335844
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Polynomial
Degree 2
Restricted
Non-
constant
5%
165.8318
141.7415
<0.0001
2547.335807
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Power
Restricted
Non-
constant
5%
165.7909
141.7454
<0.0001
2547.335844
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Linear
Unrestricted
Non-
constant
5%
165.5491
141.7212
<0.0001
2547.346425
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
AIC = Akaike information criterion; BMD = benchmark dose; BMDL
" Selected Model (bolded).
4 Restrictions defined in the BMDS 3.3 User Guide.
= benchmark dose lower limit; NA = not applicable
Table Apx J-19. BMD Model Resuli
ts for F1 Female Offspring Bodyweight on PND21 (Highest Dose Group Removed)
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Exponential 3
Restricted
Constant
1 SD
224.9896
182.5315
0.6656393
1909.657582
Viable -
Recommended
Lowest AIC
Exponential 5
Restricted
Constant
1 SD
217.6212
139.8341
NA
1911.470837
Questionable
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Hill
Restricted
Constant
1 SD
207.4601
190.2318
NA
1913.470837
Questionable
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial Degree
2
Restricted
Constant
1 SD
228.849
188.8653
0.5274787
1909.870083
Viable -
Alternate
Power
Restricted
Constant
1 SD
228.9162
188.8651
0.5274824
1909.870076
Viable -
Alternate
Linear
Unrestricted
Constant
1 SD
228.9162
188.8645
0.5274824
1909.870076
Viable -
Alternate
Exponential 3
Restricted
Constant
5%
83.05437
69.15258
0.6656393
1909.657582
Viable -
Recommended
Lowest AIC
Page 252 of 282
-------
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Exponential 5
Restricted
Constant
5%
73.45746
43.55987
NA
1911.470837
Questionable
BMDL 3x lower than lowest non-zero dose
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Hill
Restricted
Constant
5%
89.14739
39.34772
NA
1913.470837
Questionable
BMDL 3x lower than lowest non-zero dose
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial Degree
2
Restricted
Constant
5%
88.0599
74.44438
0.5274787
1909.870083
Viable -
Alternate
Power
Restricted
Constant
5%
88.08571
74.44467
0.5274824
1909.870076
Viable -
Alternate
Linear
Unrestricted
Constant
5%
88.08571
74.44467
0.5274824
1909.870076
Viable -
Alternate
AIC = Akaike information criterion; BMD = benchmark dose; BMDL = benchmark dose lower limit; NA = not applicable
" Selected Model (bolded).
4 Restrictions defined in the BMDS 3.3 User Guide.
Page 253 of 282
-------
Frequentist Exponential Degree 3 Model with BMR of 0.05 Added
Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
63
58
Estimated Probability
Response at BMD
O Data
BMD
BMDL
mg/kg-day
User Input
Info
Model
frequentist Exponential degree 3
Model Restriction
Restricted
Dataset Name
Waterman-2000_F1-BW-F-PND21_HDD
User notes
[Add user notes here]
Dose-Response Modi
M[dose] = a1 ewp(±11 (b1 dose)* d)
Variance Model
Var[i] = alpha
Model Options
BMR Type
Rel. Dev.
BMRF
0.05
Tail Probability
-
Confidence Level
0.95
Distribution Type
Normal
Variance Type
Constant
Model Data
Dependent Variable
mg/kg-day
Independent Variable
g
Total # of Observation
3
Adverse Direction
Downward
Page 254 of 282
-------
Modal Results
Benchmark Dose
BMD
83.05437022
BMDL
63.15258454
BMDU
125.1576767
AIC
1303.657582
Test4P-value 1
0.665633238
D.O.F.
1
Model Parameters
# of Parameters
4
Variable
Estimate
Std Error
Lower Conf
Upper Conf
a
53.37735537
0.66438723
52.5746
55.18131
b
0.000617587
7.50E-05
0.000471
0.000765
d
Bounded
NA
NA
NA
log-alpha
3.888828168
8.41E-02
3.724062
4.053535
Goodness of Fit
Dose
Size
Estimated
Calc'd
Observed
Estimated
Calc'd
Observe
Scaled
Median
Median
Mean
SD
3D
d 3D
Residual
0
36
53.877356
53.33
53.33
6.383535
6.17
6.17
0.1570638
133
33
43.4457855
43.13
43.13
6.383535
7.54
7.54
-0.352314
274
34
45.4304573
45.63
45.63
6.383535
7.31
7.31
0.1335623
Likelihoods of Interest
#of
Model
Loq Likelihood"
Parameters
AIC
A1
-351.7354183
4
1311.471
A2
-343.6122154
6
1311.224
A3
-351.7354183
4
1311.471
fitted
-351.828731
3
1309.653
R
-382.4232261
2
1368.858
' Includes additive constant of -260.0596. This constant was not included in the LL derivation prior to BMDS 3.0.
Tests of Interest
Test
2"Log(Likeliho
od Ratio)
Test df
p-value
1
65.63402137
4
<0.0001
2
4.24640576
2
0.113648
3
4.24640576
2
0.113648
4
0.186745415
1
0.665639
J.2 F2 Offspring Bodyweight
J.2.1 F2 Male Offspring Bodyweight on PND7
Table Apx J-20. F2 Male Offspring Bodyweig
it on PNE
17
Dietary
Dose (%)
Received Dose
(mg/kg-day)
N
Mean
SD
Statistical
Significance
Location of Data
0
0
87
18.08
3.18
Table 11 in (Waterman
et al.. 2000); Table 39
in (Exxon Biomedical,
1996b)
0.2
133
79
16.43
2.34
0.4
271
83
15.48
2.90
*
0.8
544
72
14.70
3.00
*
Page 255 of 282
-------
Table Apx J-21. BMD Model Results for F2 Ma
e Offspring Bodyweight on P
ND7 (All Dose Groups Included)
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Exponential 3
Restricted
Constant
1 SD
461.6142
366.133
0.046876
1597.673704
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Exponential 5
Restricted
Constant
1 SD
330.5471
198.1884
NA
1595.553206
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Hill
Restricted
Constant
1 SD
333.4077
190.6706
NA
1595.553206
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial
Degree 3
Restricted
Constant
1 SD
478.1074
390.0737
0.0254152
1598.898024
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Polynomial
Degree 2
Restricted
Constant
1 SD
497.7536
388.9764
0.0246232
1598.961341
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Power
Restricted
Constant
1 SD
480.7334
389.9598
0.0254369
1598.896317
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Linear
Unrestricted
Constant
1 SD
480.7334
389.9593
0.0254369
1598.896317
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Exponential 3
Restricted
Constant
5%
132.6866
108.0924
0.046876
1597.673704
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Exponential 5
Restricted
Constant
5%
63.67049
38.79517
NA
1595.553206
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
BMDL 3x lower than lowest non-zero dose
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Hill
Restricted
Constant
5%
67.92507
29.57314
NA
1595.553206
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
BMDL 3x lower than lowest non-zero dose
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial
Degree 3
Restricted
Constant
5%
145.4626
121.4488
0.0254152
1598.898024
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Polynomial
Degree 2
Restricted
Constant
5%
151.0412
121.1417
0.0246232
1598.961341
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Power
Restricted
Constant
5%
146.213
121.4082
0.0254369
1598.896317
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Linear
Unrestricted
Constant
5%
146.213
121.4085
0.0254369
1598.896317
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Exponential 3
Restricted
Non-
constant
1 SD
461.6142
366.1331
0.0929516
1597.673704
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
Exponential 5
Restricted
Non-
constant
1 SD
349.6853
202.3994
0.9340853
1595.262507
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Page 256 of 282
-------
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Hill
Restricted
Non-
constant
1 SD
358.4047
195.4941
NA
1597.255666
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial
Degree 3
Restricted
Non-
constant
1 SD
480.7334
389.9548
0.0540526
1598.896317
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
Polynomial
Degree 2
Restricted
Non-
constant
1 SD
480.7723
389.9577
0.0219198
1600.896392
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
Power
Restricted
Non-
constant
1 SD
480.7334
389.9598
0.0540526
1598.896317
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
Linear
Unrestricted
Non-
constant
1 SD
480.7334
389.9598
0.0540526
1598.896317
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
Exponential 3
Restricted
Non-
constant
5%
132.6866
108.0924
0.0929516
1597.673704
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
Exponential 5
Restricted
Non-
constant
5%
60.71248
36.62418
0.9340853
1595.262507
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
BMDL 3x lower than lowest non-zero dose
Hill
Restricted
Non-
constant
5%
60.89926
26.79399
NA
1597.255666
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
BMDL 3x lower than lowest non-zero dose
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial
Degree 3
Restricted
Non-
constant
5%
146.213
121.4083
0.0540526
1598.896317
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
Polynomial
Degree 2
Restricted
Non-
constant
5%
146.2209
121.4081
0.0219198
1600.896392
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
Power
Restricted
Non-
constant
5%
146.213
121.4082
0.0540526
1598.896317
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
Linear
Unrestricted
Non-
constant
5%
146.213
121.4083
0.0540526
1598.896317
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
AIC = Akaike information criterion; BMD = benchmark dose; BMDL = benchmark dose lower limit; NA = not applicable
" Selected Model (bolded).
4 Restrictions defined in the BMDS 3.3 User Guide.
Page 257 of 282
-------
Table Apx J-22. BMD Model Resu
ts for F2 Male Offspring Bodyweight on PN
D7 (Highest
)ose Group Removed)
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Exponential 3
Restricted
Constant
1 SD
294.8547
224.4383
0.4014984
1230.413076
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
BMD higher than maximum dose
Exponential 5
Restricted
Constant
1 SD
320.706
136.3415
NA
1233.709242
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
BMD higher than maximum dose
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Hill
Restricted
Constant
1 SD
1144.681
140.9213
NA
1233.709242
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
BMD/BMDL ratio > 3
BMD higher than maximum dose
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial
Degree 2
Restricted
Constant
1 SD
295.3284
229.5497
0.3312634
1230.653194
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
BMD higher than maximum dose
Power
Restricted
Constant
1 SD
294.8149
229.5624
0.3312941
1230.653074
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
BMD higher than maximum dose
Linear
Unrestricted
Constant
1 SD
294.8149
229.5624
0.3312941
1230.653074
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
BMD higher than maximum dose
Exponential 3
Restricted
Constant
5%
88.39555
69.21579
0.4014984
1230.413076
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Exponential 5
Restricted
Constant
5%
63.28412
27.97896
NA
1233.709242
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
BMDL 3x lower than lowest non-zero dose
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Hill
Restricted
Constant
5%
91.79895
60.42561
NA
1233.709242
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial
Degree 2
Restricted
Constant
5%
93.72347
74.7514
0.3312634
1230.653194
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Power
Restricted
Constant
5%
93.56592
74.75603
0.3312941
1230.653074
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Linear
Unrestricted
Constant
5%
93.56592
74.7555
0.3312941
1230.653074
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Exponential 3
Restricted
Non-
constant
1 SD
311.7912
232.4983
0.2051934
1231.574104
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
BMD higher than maximum dose
Exponential 5
Restricted
Non-
constant
1 SD
-
-
-
-
Unusable
BMD computation failed
Page 258 of 282
-------
Models "
Restriction b
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Hill
Restricted
Non-
constant
1 SD
460.2859
144.0986
NA
1233.969021
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
BMD/BMDL ratio > 3
BMD higher than maximum dose
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial
Degree 2
Restricted
Non-
constant
1 SD
299.217
233.5823
0.1368367
1232.182261
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
BMD higher than maximum dose
Power
Restricted
Non-
constant
1 SD
296.895
231.7951
0.1215844
1232.365876
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
BMD higher than maximum dose
Linear
Unrestricted
Non-
constant
1 SD
310.049
236.4869
0.1647878
1231.898816
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
BMD higher than maximum dose
Exponential 3
Restricted
Non-
constant
5%
89.0521
69.69518
0.2051934
1231.574104
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Exponential 5
Restricted
Non-
constant
5%
71.81779
16.30153
NA
1233.969021
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
BMD/BMDL ratio > 3
BMDL 3x lower than lowest non-zero dose
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Hill
Restricted
Non-
constant
5%
49.78012
3.140952
NA
1233.969021
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
BMD/BMDL ratio > 3
BMDL 3x lower than lowest non-zero dose
BMDL 10 x lower than lowest non-zero dose
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial
Degree 2
Restricted
Non-
constant
5%
93.50841
74.58525
0.1368367
1232.182261
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Power
Restricted
Non-
constant
5%
93.43043
74.09874
0.1215844
1232.365876
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Linear
Unrestricted
Non-
constant
5%
94.37421
75.34447
0.1647878
1231.898816
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
AIC = Akaike information criterion; BMD = benchmark dose; BMDL = benchmark dose lower limit; NA = not applicable
" Selected Model (bolded).
Page 259 of 282
-------
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
b Restrictions defined in the BMDS 3.3 User Guide.
Page 260 of 282
-------
J.2.2 F2 Male Offspring Bodyweight on PND14
Table Apx J-23. F2 Male Offspring Bodyweig
it on PNE
•14
Dietary
Dose (%)
Received Dose
(mg/kg-day)
N
Mean
SD
Statistical
Significance
Location of Data
0
0
87
37.09
4.68
Table 11 in (Waterman
et al.. 2000); Table 39
in (Exxon Biomedical,
1996b)
0.2
133
82
34.80
3.47
0.4
271
83
32.51
4.85
*
0.8
544
72
29.88
4.00
*
Page 261 of 282
-------
Table Apx J-24
. BMD Model Results for F2 Male
Dffspring Bodyweight on PN
)14 (All Dose Groups Included)
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Exponential 3
Restricted
Constant
1 SD
310.9715
256.7945
0.3516382
1869.101249
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Exponential 5
Restricted
Constant
1 SD
254.0299
185.7105
0.6721636
1869.190026
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Hill
Restricted
Constant
1 SD
249.6883
186.3998
NA
1871.010944
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial Degree 3
Restricted
Constant
1 SD
325.1459
278.5947
0.2210705
1870.029491
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Polynomial Degree 2
Restricted
Constant
1 SD
346.8172
271.7199
0.0449369
1873.031951
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Power
Restricted
Constant
1 SD
324.2721
278.6708
0.2211645
1870.028641
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Linear
Unrestricted
Constant
1 SD
324.2721
278.6708
0.2211645
1870.028641
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Exponential 3
Restricted
Constant
5%
126.8871
109.2117
0.3516382
1869.101249
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Exponential 5
Restricted
Constant
5%
96.50161
68.47018
0.6721636
1869.190026
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Hill
Restricted
Constant
5%
110.4933
66.3493
NA
1871.010944
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial Degree 3
Restricted
Constant
5%
138.8628
121.73
0.2210705
1870.029491
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Polynomial Degree 2
Restricted
Constant
5%
151.2085
119.1812
0.0449369
1873.031951
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Power
Restricted
Constant
5%
138.5227
121.772
0.2211645
1870.028641
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Linear
Unrestricted
Constant
5%
138.5227
121.772
0.2211645
1870.028641
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Exponential 3
Restricted
Non-
constant
1 SD
316.2307
261.0941
0.4073079
1870.545289
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Exponential 5
Restricted
Non-
constant
1 SD
258.9523
190.356
NA
1872.748917
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Hill
Restricted
Non-
constant
1 SD
304.6504
178.2839
0.2920302
1871.859155
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Polynomial Degree 3
Restricted
Non-
constant
1 SD
335.0496
281.3713
0.233684
1871.656489
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Polynomial Degree 2
Restricted
Non-
constant
1 SD
334.8946
281.3551
0.233678
1871.65654
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Power
Restricted
Non-
constant
1 SD
334.8787
281.3555
0.2336786
1871.656535
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Page 262 of 282
-------
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Linear
Unrestricted
Non-
constant
1 SD
335.0402
281.3703
0.2336844
1871.656485
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Exponential 3
Restricted
Non-
constant
5%
127.2935
110.1911
0.4073079
1870.545289
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Exponential 5
Restricted
Non-
constant
5%
105.3862
68.36967
NA
1872.748917
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Hill
Restricted
Non-
constant
5%
119.3041
59.85716
0.2920302
1871.859155
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Polynomial Degree 3
Restricted
Non-
constant
5%
139.3297
122.4641
0.233684
1871.656489
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Polynomial Degree 2
Restricted
Non-
constant
5%
139.318
122.4646
0.233678
1871.65654
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Power
Restricted
Non-
constant
5%
139.3187
122.4652
0.2336786
1871.656535
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Linear
Unrestricted
Non-
constant
5%
139.3322
122.4638
0.2336844
1871.656485
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
AIC = Akaike information criterion; BMD = benchmark dose; BMDL = benchmark dose lower limit; NA = not applicable
" Selected Model (bolded).
4 Restrictions defined in the BMDS 3.3 User Guide.
Table Apx J-25. BMP Model Results for F2 Male Offspring Bodyweight on PND14 (Highest Dose Group Removed)
Models a
Restriction b
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Exponential 3
Restricted
Constant
1 SD
257.3344
202.0522
NA
1465.131238
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Exponential 5
Restricted
Constant
1 SD
257.0731
136.7782
NA
1467.131238
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Hill
Restricted
Constant
1 SD
256.2765
139.8734
NA
1467.131238
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Polynomial Degree 2
Restricted
Constant
1 SD
258.8061
206.1186
0.9358909
1463.137708
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Power
Restricted
Constant
1 SD
257.9058
206.0472
0.9425274
1463.136435
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Linear
Unrestricted
Constant
1 SD
257.9058
206.0469
0.9425274
1463.136435
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Page 263 of 282
-------
Models a
Restriction b
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Exponential 3
Restricted
Constant
5%
107.5215
84.93791
NA
1465.131238
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Exponential 5
Restricted
Constant
5%
107.9591
60.49319
NA
1467.131238
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Hill
Restricted
Constant
5%
109.2554
57.44817
NA
1467.131238
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Polynomial Degree 2
Restricted
Constant
5%
110.1849
89.62363
0.9358909
1463.137708
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Power
Restricted
Constant
5%
109.6875
89.65173
0.9425274
1463.136435
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Linear
Unrestricted
Constant
5%
109.6875
89.65173
0.9425274
1463.136435
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Exponential 3
Restricted
Non-
constant
1 SD
257.3345
202.0522
0.6887133
1465.131238
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Exponential 5
Restricted
Non-
constant
1 SD
256.8383
136.6679
NA
1467.131238
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Hill
Restricted
Non-
constant
1 SD
154.4324
139.7685
NA
1468.970755
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Polynomial Degree 2
Restricted
Non-
constant
1 SD
257.9056
206.047
0.9204987
1463.136435
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Power
Restricted
Non-
constant
1 SD
257.9058
206.0471
0.9204987
1463.136435
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Linear
Unrestricted
Non-
constant
1 SD
257.9058
206.0471
0.9204987
1463.136435
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Exponential 3
Restricted
Non-
constant
5%
107.5217
84.93756
0.6887133
1465.131238
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Exponential 5
Restricted
Non-
constant
5%
108.3242
60.0712
NA
1467.131238
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Hill
Restricted
Non-
constant
5%
130.7914
124.4926
NA
1468.970755
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Page 264 of 282
-------
Models a
Restriction b
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Polynomial Degree 2
Restricted
Non-
constant
5%
109.6871
89.65201
0.9204987
1463.136435
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Power
Restricted
Non-
constant
5%
109.6875
89.65172
0.9204987
1463.136435
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Linear
Unrestricted
Non-
constant
5%
109.6875
89.65172
0.9204987
1463.136435
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
AIC = Akaike information criterion; BMD = benchmark dose; BMDL = benchmark dose lower limit; NA = not applicable
" Selected Model (bolded).
4 Restrictions defined in the BMDS 3.3 User Guide.
Page 265 of 282
-------
J.2.3 F2 Male Offspring Bodyweight on PND21
Table Apx J-26. F2 Male Offspring Bodyweig
it on PNE
•21
Dietary
Dose (%)
Received Dose
(mg/kg-day)
N
Mean
SD
Statistical
Significance
Location of Data
0
0
87
62.34
7.68
Table 11 in (Waterman
et al.. 2000); Table 39
in (Exxon Biomedical,
1996b)
0.2
133
79
57.89
6.56
0.4
271
82
54.82
7.45
*
0.8
544
72
49.12
7.38
*
Page 266 of 282
-------
Table Apx 3-21. BMP Model Results
for F2 Male Offspring Bodyweight on PND21 (All Dose Groups Included)
Models "
Restriction1
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS Recommends
BMDS Recommendation Notes
Exponential 3
Restricted
Constant
1 SD
285.4789
242.8247
0.6383876
2182.321925
Viable - Recommended
Lowest AIC
Exponential 5
Restricted
Constant
1 SD
253.8703
187.7986
0.6513583
2183.628498
Viable - Alternate
Hill
Restricted
Constant
1 SD
253.3356
185.5706
0.6705813
2183.605229
Viable - Alternate
Polynomial Degree 3
Restricted
Constant
1 SD
304.7243
263.0126
0.4122808
2183.196407
Viable - Alternate
Polynomial Degree 2
Restricted
Constant
1 SD
Unusable
BMD computation failed
Power
Restricted
Constant
1 SD
304.1596
262.977
0.4124379
2183.195645
Viable - Alternate
Linear
Unrestricted
Constant
1 SD
304.1596
262.977
0.4124379
2183.195645
Viable - Alternate
Exponential 3
Restricted
Constant
5%
117.5098
102.4965
0.6383876
2182.321925
Viable - Recommended
Lowest AIC
Exponential 5
Restricted
Constant
5%
99.05525
70.38601
0.6513583
2183.628498
Viable - Alternate
Hill
Restricted
Constant
5%
97.87309
69.56036
0.6705813
2183.605229
Viable - Alternate
Polynomial Degree 3
Restricted
Constant
5%
129.4509
114.4269
0.4122808
2183.196407
Viable - Alternate
Polynomial Degree 2
Restricted
Constant
5%
253.9388
248.7375
<0.0001
2200.267654
Questionable
Goodness of fit p-value <0.1
|Residual for Dose Group Near BMD| > 2
|Residual at control| > 2
Power
Restricted
Constant
5%
129.1878
114.3972
0.4124379
2183.195645
Viable - Alternate
Linear
Unrestricted
Constant
5%
129.1878
114.3972
0.4124379
2183.195645
Viable - Alternate
AIC = Akaike information criterion; BMD = benchmark dose; BMDL = benchmark dose lower limit; NA = not applicable
" Selected Model (bolded).
4 Restrictions defined in the BMDS 3.3 User Guide.
Page 267 of 282
-------
Frequentist Exponential Degree 3 Model with BMR of 0.05 Added
Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
User Input
Info
Model
frequentist EHponential degree 3
Model Restriction
Restricted
Dataset Name
Waterman-200C_F2-BW-M-PND21
User notes
[Add user notes here]
Dose-Response Modi
M[dose] = a" ewp(±11 (b1 dosefd)
Variance Model
Var[i] = alpha
Model Options
BMR Type
Rel. Dev.
BMRF
0.05
Tail Probability
-
Confidence Level
0.95
Distribution Type
Normal
Variance Type
Constant
Model Data
Dependent Variable
mg/kg-day
Independent Variable
g
Total # of Observation
4
Adverse Direction
Downward
Page 268 of 282
-------
Model Results
Benchmark Dose
BMD
117.509799
BMDL
102.4965127
BMDU
154.9025277
AIC
2192.921925
Test4P-value 1
0.638387563
2
Model Parameters
# of Parameters
4
Variable
Estimate
Std Error
Lower Conf
Upper Conf
a
61.91561838
0.63620624
60.66868
63.16256
b
0.000436502
3.83E-05
0.000361
0.000512
d
Bounded
NA
NA
NA
log-alpha
3.963128954
7.91E-02
9.90818
4.118078
Goodness of Fit
Dose
Size
Estimated
Calc'd
Observed
Estimated
Calc'd
Observe
Scaled
Median
Median
Mean
3D
3D
d 3D
Residual
0
87
61.9156184
62.34
62.34
7.254083
7.68
7.68
0.5456745
133
79
58.4234682
57.89
57.89
7.254089
6.56
6.56
-0.653641
271
82
55.0080945
54.82
54.82
7.254083
7.45
7.45
-0.234801
544
72
48.8285521
49.12
49.12
7.254083
7.38
7.38
0.3409138
Likelihoods of Interest
#of
Model
Log Likelihood"
Parameters
AIC
A1
-1087.712153
5
2185.424
A2
-1086.591076
8
2189.182
A3
-1087.712153
5
2185.424
fitted
-1088.160969
3
2182.322
R
-1145.233171
2
2294.466
" Includes additive constant of -294.06033. This constant was not included in the LL derivation prior to BMDS 3.0.
Tests of Interest
Test
2"Log(Likeliho
od Ratio)
Test df
p-value
1
117.2841904
6
<0.0001
2
2.2421544
9
0.529693
3
2.2421544
3
0.523693
4
0.897619428
2
0.638388
J.2.4 F2 Female Offspring Bodyweight on PND7
Table Apx J-28. F2 Female Offspring Bodyweight on PND7
Dietary
Dose (%)
Received Dose
(mg/kg-day)
N
Mean
SD
Statistical
Significance
Location of Data
0
0
84
17.47
2.88
Table 11 in (Waterman
et al.. 2000); Table 39
in (Exxon Biomedical,
1996b)
0.2
133
80
15.72
2.22
*
0.4
271
73
14.56
3.03
*
0.8
544
78
13.76
2.49
*
Page 269 of 282
-------
Table Apx J-29. BMP Model Results for F2 Female Offspring Bodyweight on PND7 (All Dose Groups Included)
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Exponential 3
Restricted
Constant
1 SD
386.1792
315.1079
0.017201
1522.616108
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Exponential 5
Restricted
Constant
1 SD
231.33
156.711
NA
1518.490529
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Hill
Restricted
Constant
1 SD
229.2439
155.0224
NA
1518.490529
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial Degree 3
Restricted
Constant
1 SD
412.9036
343.1368
0.0067363
1524.491032
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Polynomial Degree 2
Restricted
Constant
1 SD
410.103
343.3094
0.0067351
1524.491367
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Power
Restricted
Constant
1 SD
411.6254
343.2307
0.0067387
1524.490298
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Linear
Unrestricted
Constant
1 SD
411.6254
343.2304
0.0067387
1524.490298
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Exponential 3
Restricted
Constant
5%
115.3871
96.82845
0.017201
1522.616108
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Exponential 5
Restricted
Constant
5%
64.38746
36.81541
NA
1518.490529
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
BMDL 3x lower than lowest non-zero dose
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Hill
Restricted
Constant
5%
70.58941
30.0512
NA
1518.490529
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
BMDL 3x lower than lowest non-zero dose
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial Degree 3
Restricted
Constant
5%
129.7898
110.6013
0.0067363
1524.491032
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Polynomial Degree 2
Restricted
Constant
5%
128.9872
110.6659
0.0067351
1524.491367
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Power
Restricted
Constant
5%
129.4239
110.6332
0.0067387
1524.490298
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Linear
Unrestricted
Constant
5%
129.4239
110.6331
0.0067387
1524.490298
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
Exponential 3
Restricted
Non-
constant
1 SD
404.4602
319.4208
0.0179951
1524.23099
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
Exponential 5
Restricted
Non-
constant
1 SD
246.4343
160.2465
NA
1520.19568
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Page 270 of 282
-------
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Hill
Restricted
Non-
constant
1 SD
333.6476
128.9798
0.0948987
1520.984929
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
Polynomial Degree 3
Restricted
Non-
constant
1 SD
432.9737
348.2139
0.0073081
1526.033221
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
Polynomial Degree 2
Restricted
Non-
constant
1 SD
430.775
348.2967
0.0073205
1526.029846
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
Power
Restricted
Non-
constant
1 SD
432.128
348.2421
0.0073146
1526.031449
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
Linear
Unrestricted
Non-
constant
1 SD
430.8024
348.2957
0.0073205
1526.029846
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
Exponential 3
Restricted
Non-
constant
5%
116.9971
97.77591
0.0179951
1524.23099
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
Exponential 5
Restricted
Non-
constant
5%
58.3255
36.26001
NA
1520.19568
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
BMDL 3x lower than lowest non-zero dose
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Hill
Restricted
Non-
constant
5%
80.94619
21.80169
0.0948987
1520.984929
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
BMD/BMDL ratio > 3
BMDL 3x lower than lowest non-zero dose
Polynomial Degree 3
Restricted
Non-
constant
5%
131.5494
111.7242
0.0073081
1526.033221
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
Polynomial Degree 2
Restricted
Non-
constant
5%
131.2504
111.7513
0.0073205
1526.029846
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
Power
Restricted
Non-
constant
5%
131.4056
111.7371
0.0073146
1526.031449
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
Linear
Unrestricted
Non-
constant
5%
131.2537
111.7512
0.0073205
1526.029846
Questionable
Non-constant variance test failed (Test 3 p-value < 0.05)
Goodness of fit p-value <0.1
AIC = Akaike information criterion; BMD = benchmark dose; BMDL = benchmark dose lower limit; NA = not applicable
" Selected Model (bolded).
4 Restrictions defined in the BMDS 3.3 User Guide.
Page 271 of 282
-------
Table Apx J-30. BMP Model Results for F2 Female
Offspring Bodyweight on PND7 (Highesi
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Exponential 3
Restricted
Constant
1 SD
248.5057
193.4175
0.4944471
1151.359205
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Exponential 5
Restricted
Constant
1 SD
242.0645
134.3484
NA
1152.89237
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Hill
Restricted
Constant
1 SD
242.3856
136.5245
NA
1154.89237
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial Degree 2
Restricted
Constant
1 SD
251.9734
200.3953
0.3876062
1151.638809
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Power
Restricted
Constant
1 SD
251.6715
200.4059
0.3876267
1151.638744
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Linear
Unrestricted
Constant
1 SD
251.6715
200.4059
0.3876267
1151.638744
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Exponential 3
Restricted
Constant
5%
75.22937
60.2912
0.4944471
1151.359205
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Exponential 5
Restricted
Constant
5%
59.12399
30.58226
NA
1152.89237
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
BMDL 3x lower than lowest non-zero dose
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Hill
Restricted
Constant
5%
59.35102
23.61017
NA
1154.89237
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
BMDL 3x lower than lowest non-zero dose
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial Degree 2
Restricted
Constant
5%
80.64244
65.91709
0.3876062
1151.638809
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Power
Restricted
Constant
5%
80.55384
65.91988
0.3876267
1151.638744
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Linear
Unrestricted
Constant
5%
80.55384
65.91988
0.3876267
1151.638744
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Exponential 3
Restricted
Non-
constant
1 SD
248.5057
193.4175
0.7807922
1151.359205
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Exponential 5
Restricted
Non-
constant
1 SD
241.742
134.4873
NA
1154.89237
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Hill
Restricted
Non-
constant
1 SD
238.6763
135.7701
NA
1156.864312
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial Degree 2
Restricted
Non-
constant
1 SD
255.768
198.732
0.3164059
1153.868058
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Power
Restricted
Non-
constant
1 SD
251.6715
200.4059
0.6789445
1151.638744
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Dose Group Removed)
Page 272 of 282
-------
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Linear
Unrestricted
Non-
constant
1 SD
251.6715
200.4059
0.6789445
1151.638744
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Exponential 3
Restricted
Non-
constant
5%
75.22939
60.29121
0.7807922
1151.359205
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Exponential 5
Restricted
Non-
constant
5%
59.83184
29.8113
NA
1154.89237
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
BMDL 3x lower than lowest non-zero dose
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Hill
Restricted
Non-
constant
5%
62.43963
22.61827
NA
1156.864312
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
BMDL 3x lower than lowest non-zero dose
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial Degree 2
Restricted
Non-
constant
5%
80.99489
65.37813
0.3164059
1153.868058
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Power
Restricted
Non-
constant
5%
80.55385
65.91989
0.6789445
1151.638744
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Linear
Unrestricted
Non-
constant
5%
80.55385
65.91989
0.6789445
1151.638744
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
AIC = Akaike information criterion; BMD = benchmark dose; BMDL = benchmark dose lower limit; NA = not applicable
" Selected Model (bolded).
4 Restrictions defined in the BMDS 3.3 User Guide.
Page 273 of 282
-------
J.2.5 F2 Female Offspring Bodyweight on PND14
Table Apx J-31. F2 Female Offspring Bodyweight on PND14
Dietary
Dose (%)
Received Dose
(mg/kg-day)
N
Mean
SD
Statistical
Significance
Location of Data
0
0
84
35.89
4.12
Table 11 in (Waterman
et al.. 2000); Table 39
in (Exxon Biomedical,
1996b)
0.2
133
85
33.64
3.66
0.4
271
73
31.22
4.81
*
0.8
544
78
28.20
3.32
*
Page 274 of 282
-------
Table Apx J-32. BMP Model Results
for F2 Female Offspring Bodyweight on P>
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Exponential 3
Restricted
Constant
1 SD
266.1234
225.7549
0.5462158
1798.066503
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Exponential 5
Restricted
Constant
1 SD
228.0086
173.9014
NA
1800.857021
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Hill
Restricted
Constant
1 SD
227.3191
173.3892
NA
1800.857021
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Polynomial Degree 3
Restricted
Constant
1 SD
284.1348
246.7919
0.3186125
1799.14458
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Polynomial Degree 2
Restricted
Constant
1 SD
394.9127
386.7879
0.0005599
1810.761746
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Goodness of fit p-value <0.1
|Residual at control| > 2
Power
Restricted
Constant
1 SD
282.7796
246.879
0.3192175
1799.140786
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Linear
Unrestricted
Constant
1 SD
282.7795
246.879
0.3192175
1799.140786
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Exponential 3
Restricted
Constant
5%
115.6055
100.6113
0.5462158
1798.066503
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Exponential 5
Restricted
Constant
5%
109.0935
70.33997
NA
1800.857021
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Hill
Restricted
Constant
5%
109.7149
68.27143
NA
1800.857021
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Polynomial Degree 3
Restricted
Constant
5%
126.7898
112.8911
0.3186125
1799.14458
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Polynomial Degree 2
Restricted
Constant
5%
-
-
-
-
Unusable
BMD computation failed
Power
Restricted
Constant
5%
126.2408
112.9247
0.3192175
1799.140786
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Linear
Unrestricted
Constant
5%
126.2408
112.9244
0.3192175
1799.140786
Questionable
Constant variance test failed (Test 2 p-value < 0.05)
Exponential 3
Restricted
Non-
constant
1 SD
282.6982
236.805
0.7259715
1798.334396
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Exponential 5
Restricted
Non-
constant
1 SD
248.9075
183.6547
NA
1801.693906
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Hill
Restricted
Non-
constant
1 SD
248.7313
183.1052
NA
1801.693906
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
D14 (All Dose Groups Included)
Page 275 of 282
-------
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Polynomial Degree 3
Restricted
Non-
constant
1 SD
287.3642
251.4546
0.2714972
1800.301513
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Polynomial Degree 2
Restricted
Non-
constant
1 SD
304.509
258.5391
0.4623337
1799.236843
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Power
Restricted
Non-
constant
1 SD
304.4458
258.5427
0.4623842
1799.236625
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Linear
Unrestricted
Non-
constant
1 SD
303.9056
258.5765
0.46257
1799.235821
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Exponential 3
Restricted
Non-
constant
5%
115.0306
101.7281
0.7259715
1798.334396
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Exponential 5
Restricted
Non-
constant
5%
105.1363
70.99505
NA
1801.693906
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Hill
Restricted
Non-
constant
5%
105.4723
69.52159
NA
1801.693906
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
d.f.=0, saturated model (Goodness of fit test cannot
be calculated)
Polynomial Degree 3
Restricted
Non-
constant
5%
126.3314
112.0662
0.2714972
1800.301513
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Polynomial Degree 2
Restricted
Non-
constant
5%
127.4205
114.2369
0.4623337
1799.236843
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Power
Restricted
Non-
constant
5%
127.4102
114.2391
0.4623842
1799.236625
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
Linear
Unrestricted
Non-
constant
5%
127.3599
114.2466
0.46257
1799.235821
Questionable
Non-constant variance test failed (Test 3 p-value <
0.05)
AIC = Akaike information criterion; BMD = benchmark dose; BMDL = benchmark dose lower limit; NA = not applicable
" Selected Model (bolded).
4 Restrictions defined in the BMDS 3.3 User Guide.
Page 276 of 282
-------
Table Apx J-33. BMP Model Results for F2 Female Offspring Bodyweight on PND14 (Highest Dose Group Removed)
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Exponential 3
Restricted
Constant
1 SD
241.9772
189.9277
NA
1385.405068
Questionable
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Exponential 5
Restricted
Constant
1 SD
241.5843
135.662
NA
1387.405068
Questionable
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Hill
Restricted
Constant
1 SD
199.0526
181.6711
NA
1387.405068
Questionable
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial Degree 2
Restricted
Constant
1 SD
245.1661
193.9458
NA
1385.410712
Questionable
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Power
Restricted
Constant
1 SD
242.448
193.9561
NA
1385.405068
Questionable
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Linear
Unrestricted
Constant
1 SD
241.8307
193.8945
0.9404642
1383.410646
Viable -
Recommended
Lowest AIC
Exponential 3
Restricted
Constant
5%
107.1502
81.01798
NA
1385.405068
Questionable
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Exponential 5
Restricted
Constant
5%
107.515
62.22856
NA
1387.405068
Questionable
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Hill
Restricted
Constant
5%
122.8124
99.34293
NA
1387.405068
Questionable
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial Degree 2
Restricted
Constant
5%
108.6735
85.47927
NA
1385.410712
Questionable
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Power
Restricted
Constant
5%
106.6836
85.49744
NA
1385.405068
Questionable
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Linear
Unrestricted
Constant
5%
104.2095
85.49203
0.9404642
1383.410646
Viable -
Recommended
Lowest AIC
AIC = Akaike information criterion; BMD = benchmark dose; BMDL = benchmark dose lower limit; NA = not applicable
" Selected Model (bolded).
4 Restrictions defined in the BMDS 3.3 User Guide.
Page 277 of 282
-------
Frequentist Linear Model with BMR of 0.05 Added Risk for the BMD
and 0.95 Lower Confidence Limit for the BMDL
41
39
User Input
Info
Model
frequentist Linear
Model Restriction
Unrestricted
Dataset Name
Waterman-2000_F2-BW-F-PND14_HDD
User notes
[Add user notes here]
Dose-Response Modi
M[dose] = q + bl'dose
Variance Model
Var[i] = alpha
Model Options
BMR Type
Rel. Dev.
BMRF
0.05
Tail Probability
-
Confidence Level
0.95
Distribution Type
Normal
Variance Type
Constant
Model Data
Dependent Variable
mg/kg-day
Independent Variable
g
Total # of Observation
3
Adverse Direction
Downward
Page 278 of 282
-------
Model Results
Benchmark Dose
BMD
104.2094736
BMDL
85.43203164
BMDU
134.3933288
AIC
1383.410646
Test4P-value 1
0.940464162
1
Model Parameters
# of Parameters
3
Variable
Estimate
Std Error
Lower Conf
Upper Conf
9
35.30399974
0.41409874
35.09238
36.71562
beta
-0.01722684
2.46E-03
-0.02205
-0.01241
alpha
17.35537836
2.74E+01
-36.3137
71.02445
Goodness of Fit
Dose
Size
Estimated
Calo'd
Observed
Estimated
Calc'd
Observe
Scaled
Median
Median
Mean
SD
3D
d SD
Residual
0
84
35.9039997
35.89
35.89
4.165379
4.12
4.12
-0.030733
133
85
33.61283
33.64
33.64
4.165379
3.66
3.66
0.0601286
271
73
31.2355262
31.22
31.22
4.165373
4.81
4.81
-0.031343
Likelihoods of Interest
tt of
Model
Log Likelihood"
Parameters
AIC
A1
-688.7025338
4
1385.405
A2
-685.7506247
6
1383.501
A3
-688.7025338
4
1385.405
fitted
-688.7053228
3
1383.411
R
-711.0548619
2
1426.11
' Includes additive constant of -222.38313. This constant was not included in the LL derivation prior to BMDS 3.0.
Tests of Interest
Test
2"Log(Likeliho
od Ratio)
Test df
p-value
1
50.60867432
4
<0.0001
2
5.303818186
2
0.05224
3
5.303818186
2
0.05224
4
0.005578071
1
0.340464
J.2.6 F2 Female Offspring Bodyweight on PND21
Table Apx J-34. F2 Female Offspring Bodywe
ight on PND21
Dietary
Dose (%)
Received Dose
(mg/kg-day)
N
Mean
SD
Statistical
Significance
Location of Data
0
0
84
59.37
7.70
Table 11 in (Waterman
et al.. 2000); Table 39
in (Exxon Biomedical,
1996b)
0.2
133
79
55.50
6.36
0.4
271
73
51.98
7.48
*
0.8
544
78
46.20
6.50
*
Page 279 of 282
-------
Table Apx J-35. BMP Model Results for F2 Female Offspring Bodyweight on PND21 (All Dose Groups Included)
Models "
Restriction 4
Variance
BMR
BMD
BMDL
P Value
AIC
BMDS
Recommends
BMDS Recommendation Notes
Exponential 3
Restricted
Constant
1 SD
272.4073
233.1442
0.891522
2118.778944
Viable -
Recommended
Lowest AIC
Exponential 5
Restricted
Constant
1 SD
254.4618
189.4706
0.9922188
2120.549389
Viable -
Alternate
Hill
Restricted
Constant
1 SD
254.5222
188.6821
NA
2122.549294
Questionable
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial Degree 3
Restricted
Constant
1 SD
291.846
253.7424
0.643646
2119.430507
Viable -
Alternate
Polynomial Degree 2
Restricted
Constant
1 SD
291.2796
253.7626
0.6435126
2119.430921
Viable -
Alternate
Power
Restricted
Constant
1 SD
291.7664
253.7459
0.6436504
2119.430493
Viable -
Alternate
Linear
Unrestricted
Constant
1 SD
291.7664
253.7459
0.6436504
2119.430493
Viable -
Alternate
Exponential 3
Restricted
Constant
5%
111.0568
97.60821
0.891522
2118.778944
Viable -
Recommended
Lowest AIC
Exponential 5
Restricted
Constant
5%
100.7087
71.77118
0.9922188
2120.549389
Viable -
Alternate
Hill
Restricted
Constant
5%
100.4403
69.74917
NA
2122.549294
Questionable
d.f.=0, saturated model (Goodness of fit test cannot be
calculated)
Polynomial Degree 3
Restricted
Constant
5%
122.8587
109.5126
0.643646
2119.430507
Viable -
Alternate
Polynomial Degree 2
Restricted
Constant
5%
122.6421
109.5202
0.6435126
2119.430921
Viable -
Alternate
Power
Restricted
Constant
5%
122.8279
109.514
0.6436504
2119.430493
Viable -
Alternate
Linear
Unrestricted
Constant
5%
122.8279
109.514
0.6436504
2119.430493
Viable -
Alternate
AIC = Akaike information criterion; BMD = benchmark dose; BMDL = benchmark dose lower limit; NA = not applicable
" Selected Model (bolded).
4 Restrictions defined in the BMDS 3.3 User Guide.
Page 280 of 282
-------
Frequentist Exponential Degree 3 Model with BMR of 0.05 Added
Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
69
64
Estimated Probability
Response at BMD
O Data
BMD
BMDL
mg/kg-day
User Input
Info
Model
frequentist Exponential degree 3
Model Restriction
Restricted
Dataset Name
Waterman-2000_F2-BW-F-PND21
User notes
[Add user notes here]
Dose-Response Modi
M[dose] = a1 ewp(±11 (b1 dose)* d)
Variance Model
Var[i] = alpha
Model Options
BMR Type
Rel. Dev.
BMRF
0.05
Tail Probability
-
Confidence Level
0.95
Distribution Type
Normal
Variance Type
Constant
Model Data
Dependent Variable
mg/kg-day
Independent Variable
g
Total # of Observation
4
Adverse Direction
Downward
Page 281 of 282
-------
Model Results
Benchmark Dose
BMD
111.0567927
BMDL
37.60820616
BMDU
155.3477035
AIC
2118.778344
Test4P-value 1
0.831522023
D.O.F.
2
Model Parameters
# of Parameters
4
Variable
Estimate
Std Error
Lower Conf
Upper Conf
a
53.17778148
0.82138338
57.35333
80.33583
b
0.000481885
3.83E-05
0.000387
0.000537
d
Bounded
NA
NA
NA
log-alpha
3.830718337
7.38E-02
3.734238
4.047141
Goodness of Fit
Dose
Size
Estimated
Calc'd
Observed
Estimated
Calc'd
Observe
Scaled
Median
Median
Mean
3D
3D
d 3D
Residual
0
84
53.1777815
59.37
59.37
8.996144
7.7
7.7
0.2518119
133
73
55.8520022
55.5
55.5
6.336144
6.36
6.36
-0.13311
271
73
52.2155847
51.98
51.98
8.338144
7.48
7.48
-0.287682
544
78
46.0233081
46.2
46.2
6.396144
6.5
6.5
0.2147138
Likelihoods of Interest
tt of
Model
Log Likelihood"
Parameters
AIC
A1
-1056.274647
5
2122.543
A2
-1054.042132
8
2124.0S4
A3
-1056.274647
5
2122.543
fitted
-1056.383472
3
2118.773
R
-1118.934618
2
2241.869
" Includes additive constant of -288.5467. This constant was not included in the LL derivation prior to BP-IDS 3.0.
Tests of Interest
Test
2'LogfLikeliho
od Ratio)
Test df
p-value
1
123.7843717
6
<0.0001
2
4.465030273
3
0.215431
3
4.465030273
3
0.215431
4
0.229650264
2
0.831522
Page 282 of 282
-------