United States
Environmental Protection
1=1 m m Agency
EPA/690/R-09/069F
Final
9-30-2009
Provisional Peer-Reviewed Toxicity Values for
Tris(2-chloroethyl)phosphate (TCEP)
(CASRN 115-96-8)
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
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Commonly Used Abbreviations
BMD
Benchmark Dose
FEL
frank effect level
IRIS
Integrated Risk Information System
IUR
inhalation unit risk
LOAEL
lowest-observed-adverse-effect level
LOAELadj
LOAEL adjusted to continuous exposure duration
LOAELhec
LOAEL adjusted for dosimetric differences across species to a human
NOAEL
no-ob served-adverse-effect level
NOAELadj
NOAEL adjusted to continuous exposure duration
NOAELrec
NOAEL adjusted for dosimetric differences across species to a human
NOEL
no-ob served-effect level
OSF
oral slope factor
p-IUR
provisional inhalation unit risk
p-OSF
provisional oral slope factor
p-RfC
provisional inhalation reference concentration
p-RfD
provisional oral reference dose
RfC
inhalation reference concentration
RfD
oral reference dose
UF
uncertainty factor
UFa
animal to human uncertainty factor
UFC
composite uncertainty factor
UFd
incomplete to complete database uncertainty factor
UFh
interhuman uncertainty factor
UFl
LOAEL to NOAEL uncertainty factor
UFS
subchronic to chronic uncertainty factor
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PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
TRIS(2-CHLOROETHYL)PHOSPHATE (CASRN 115-96-8)
Background
On December 5, 2003, the U.S. Environmental Protection Agency's (U.S. EPA) Office of
Superfund Remediation and Technology Innovation (OSRTI) revised its hierarchy of human
health toxicity values for Superfund risk assessments, establishing the following three tiers as the
new hierarchy:
1) U.S. EPA's Integrated Risk Information System (IRIS).
2) Provisional Peer-Reviewed Toxicity Values (PPRTVs) used in U.S. EPA's Superfund
Program.
3) Other (peer-reviewed) toxicity values, including
~ Minimal Risk Levels produced by the Agency for Toxic Substances and Disease
Registry (ATSDR),
~ California Environmental Protection Agency (CalEPA) values, and
~ U.S. EPA Health Effects Assessment Summary Table (HEAST) values.
A PPRTV is defined as a toxicity value derived for use in the Superfund Program when
such a value is not available in U.S. EPA's IRIS. PPRTVs are developed according to a Standard
Operating Procedure (SOP) and are derived after a review of the relevant scientific literature
using the same methods, sources of data, and Agency guidance for value derivation generally
used by the U.S. EPA IRIS Program. All provisional toxicity values receive internal review by
two U.S. EPA scientists and external peer review by three independently selected scientific
experts. PPRTVs differ from IRIS values in that PPRTVs do not receive the multiprogram
consensus review provided for IRIS values. This is because IRIS values are generally intended
to be used in all U.S. EPA programs, while PPRTVs are developed specifically for the Superfund
Program.
Because new information becomes available and scientific methods improve over time,
PPRTVs are reviewed on a 5-year basis and updated into the active database. Once an IRIS
value for a specific chemical becomes available for Agency review, the analogous PPRTV for
that same chemical is retired. It should also be noted that some PPRTV documents conclude that
a PPRTV cannot be derived based on inadequate data.
Disclaimers
Users of this document should first check to see if any IRIS values exist for the chemical
of concern before proceeding to use a PPRTV. If no IRIS value is available, staff in the regional
Superfund and Resource Conservation and Recovery Act (RCRA) program offices are advised to
carefully review the information provided in this document to ensure that the PPRTVs used are
appropriate for the types of exposures and circumstances at the Superfund site or RCRA facility
in question. PPRTVs are periodically updated; therefore, users should ensure that the values
contained in the PPRTV are current at the time of use.
It is important to remember that a provisional value alone tells very little about the
adverse effects of a chemical or the quality of evidence on which the value is based. Therefore,
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users are strongly encouraged to read the entire PPRTV document and understand the strengths
and limitations of the derived provisional values. PPRTVs are developed by the U.S. EPA
Office of Research and Development's National Center for Environmental Assessment,
Superfund Health Risk Technical Support Center for OSRTI. Other U.S. EPA programs or
external parties who may choose of their own initiative to use these PPRTVs are advised that
Superfund resources will not generally be used to respond to challenges of PPRTVs used in a
context outside of the Superfund Program.
Questions Regarding PPRTVs
Questions regarding the contents of the PPRTVs and their appropriate use (e.g., on
chemicals not covered, or whether chemicals have pending IRIS toxicity values) may be directed
to the U.S. EPA Office of Research and Development's National Center for Environmental
Assessment, Superfund Health Risk Technical Support Center (513-569-7300), or OSRTI.
INTRODUCTION
Tris(2-chloroethyl)phosphate (TCEP) is a clear organic liquid that is primarily used in
industry as a flame retardant and fire-resistant cellulose resin plasticizer (HSDB, 2008). Figure 1
shows the chemical structure of TCEP. There is no RfD or RfC for TCEP on IRIS (U.S. EPA,
2008), the Drinking Water Standards and Health Advisories list (U.S. EPA, 2006), or in the
Health Effects Assessment Summary Tables (HEAST; U.S. EPA, 1997). The CARA list
(U.S. EPA, 1991, 1994) does not include any documents pertaining to TCEP. ATSDR (2008)
has not prepared a toxicological profile for TCEP. The World Health Organization (WHO)
(1998) prepared an Environmental Health Criteria document for TCEP, but it does not derive
toxicity values. CalEPA (2008a,b) has not assessed the noncancer toxicity of TCEP.
Occupational exposure limits for TCEP have not been derived by the American Conference of
Industrial Hygienists (ACGIH) (2008), the National Institute for Occupational Safety and Health
(NIOSH) (2008), or the Occupational Safety and Health Administration (OSHA) (2008).
Figure 1. Chemical Structure of Tris-(2-chloroethyl) Phosphate
A cancer assessment for TCEP is not available on IRIS (U.S. EPA, 2008) or in the
Drinking Water Standards and Health Advisories list (U.S. EPA, 2006) or HEAST (U.S. EPA,
1997). NTP (1991) assessed the carcinogenicity of TCEP, concluding that there was clear
evidence of carcinogenic activity in male and female rats based on increases in the incidence of
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renal tubule adenomas, and equivocal evidence of carcinogenicity in mice based on increased
incidences of renal tubule cell neoplasms (males) and Harderian gland adenomas (females).
However, TCEP is not included in the 11th Report on Carcinogens (NTP, 2005). CalEPA (2006)
included TCEP in its list of Chemicals Known to the State to Cause Cancer or Reproductive
Toxicity based on a positive finding of carcinogenicity; however, CalEPA (2008b) has not
prepared a quantitative estimate of carcinogenic potential. The International Agency for
Research on Cancer (IARC) (1990, 1999) classified TCEP in Group 3: "The agent or mixture is
not classifiable as to its carcinogenicity to humans."
Literature searches were conducted from the 1960s through July 2009 for studies relevant
to the derivation of provisional toxicity values for TCEP. Databases searched include
MEDLINE, TOXLINE (with NTIS), BIOSIS, TSCATS/TSCATS2, CCRIS, DART, GENETOX,
HSDB, RTECS, Chemical Abstracts, and Current Contents (last 6 months).
REVIEW OF PERTINENT DATA
Human Studies
No human studies involving oral or inhalation exposure that could be used in
dose-response assessment of TCEP were located.
Animal Studies
Oral Exposure
Subchronic Studies—Groups of F344/N rats (10/sex) were administered TCEP
(approximately 98% pure) in corn oil by gavage at doses of 0, 22, 44, 88, 175, or 350 mg/kg-day,
5 days/week, for 16 weeks (Matthews et al., 1990; NTP, 1991). Animals in the two highest dose
groups accidentally received twice the intended dose for three consecutive days during the fourth
week of dosing. Following observation of clinical signs in female rats (convulsions, salivation,
gasping, and lack of coordination) that did not occur in male rats, dosing was suspended for the
fourth scheduled day of dosing during the fourth week and then resumed on the fifth day. Body
weight and clinical observations were made prior to the test and at weekly intervals. Serum
cholinesterase was assessed from blood drawn at terminal sacrifice. No other clinical chemistry
or urinalysis are assessed in this study. Matthews et al. (1990) reported that technical difficulties
prevented planned assessment of sperm morphology. All animals were necropsied and
pathologic examinations of major tissues and organs were made for animals in the control and
two highest dose groups. Additional examination of brain tissue was made for females
administered 88 mg/kg-day. Due to the observation of damage in the hippocampal area of the
brain during histological evaluation, neuronal damage in the hippocampus was evaluated in a
blind study by a pathologist.
Two female rats in each of the two highest dose groups (175 and 350 mg/kg-day) died
during the episode of overdosing, and two more females in the second highest group died
because of gavage errors (Matthews et al., 1990; NTP, 1991). There was also additional
mortality attributable to TCEP treatment at doses of 175 (one male) and 350 mg/kg-day (four
males and three females). Final mean body weights were generally similar among dosed and
control male rats—although the final mean body weight of surviving high-dose females was
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about 20% greater than that of controls. Serum cholinesterase (as a measure of neurotoxicity)
was significantly1 decreased only in female rats treated with doses of 175 (25% decrease relative
to controls) or 350 mg/kg-day (41% decrease relative to controls). Absolute liver weights were
significantly increased compared to controls in TCEP-treated males at 175 and 350 mg/kg-day
(7.5%) and 14.6%>, respectively) and in females receiving 44-350 mg/kg-day (12.3—83,6%>) (see
Table 1). Absolute kidney weights were significantly increased at 350 mg/kg-day in males
(21.9% relative to controls) and in females receiving 44-350 mg/kg-day (7%—45.6% relative to
controls) (see Table 1). Relative (i.e., organ-to-body-weight ratio) liver and kidney weights were
significantly increased compared to controls in TCEP-treated males at 350 mg/kg-day (19.9%
Table 1. Absolute and Relative Liver and Kidney Weights of F344/N Rats Given
TCEP by Gavage for 16 Weeksa'b
Male
Dose (mg/kg-day)
0
22
44
88
175
350
Absolute liver weight
(g)
13.4 ±
0.27
13.5 ±
0.74
13.2 ±
0.33
13.2 ±
0.40
14.4 ±
0.3 ld
15.7 ±
0.50d
Relative liver weight
(mg/g)c
37.1 ±
0.64
36.8 ±
1.55
37.4 ±
0.60
38.2 ±
1.23
39.3 ±
1.43
44.5 ±
0.29 d
Absolute kidney weight
(g)
1.28 ±
0.03
1.25 ±
0.03
1.30 ±
0.03
1.28 ±
0.03
1.32 ±
0.04
1.56 ±
0.07d
Relative kidney weight
(mg/g)c
3.54 ±
0.08
3.42 ±
0.07
3.68 ±
0.04
3.68 ±
0.04
3.65 ±
0.09
4.42 ±
0.07d
Female
Absolute liver weight
(g)
6.10 ±
0.14
6.34 ±
0.19
6.85 ±
0.14d
6.52 ±
0.08d
7.56 ±
0.37d
11.2 ±
1.34 d
Relative liver weight
(mg/g)c
32.0 ±
0.57
33.9 ±
0.73d
36.2 ±
0.58 d
35.3 ±
0.30d
38.0 ±
0.60 d
48.2 ±
2.49 d
Absolute kidney weight
(g)
0.71 ±
0.01
0.72 ±
0.02
0.76 ±
0.01d
0.76 ±
0.01d
0.83 ±
0.02 d
1.04 ±
0.07d
Relative kidney weight
(mg/g)c
3.69 ±
0.04
3.83 ±
0.06
4.03 ±
0.04 d
4.10 ±
0.07d
4.18 ±
0.06 d
4.51 ±
0.06d
aNTP (1991); Matthews et al. (1993)
bMean ± standard error; n = 10 for all groups except 22 mg/kg-day males (n = 9), 175 mg/kg-day and 350
mg/kg-day males (n = 4), 22 mg/kg-day and 175 mg/kg-day females (n = 8), 350 mg/kg-day females (n =
5), where noted
Defined as organ-weight to body-weight ratio
Significantly different (p < 0.05) from the control (0 mg/kg-day) group by Dunn's or Shirley's test
and 24.9%), respectively) (see Table 1). Relative liver weights were significantly increased in
females receiving 22-350 mg/kg-day (5.9-50.6%) relative to controls), and relative kidney
weights were significantly increased in females receiving 44-350 mg/kg-day (9.2-22.2% relative
1 Use of the terms "significant" and "significantly" throughout this document refer to statistical significance
(p < 0.05).
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to controls) (see Table 1). There were no apparent liver or kidney histological changes
accompanying these organ weight changes. Lesions potentially related to TCEP treatment were
found in the hippocampal region of in the brain. The lesion was primarily characterized by a loss
of CA1 pyramidal neurons (which are involved in learning, memory, and spatial navigation) and
was sometimes accompanied by mineralization and microgliosis (i.e., the presence of microglia
in neuronal tissue). These lesions were observed in 8 out of 10 and 10 out of 10 females at the
175 and 350 mg/kg-day doses, respectively, with a dose-related increase in severity. Only 2 out
of 10 males receiving 350 mg/kg-day were affected, suggesting a greater sensitivity among
females. The incidences of brain lesions of females treated with 88 mg/kg-day were not reported
(although presumably 0 out of 10 based on the dose selection rationale for the chronic 2-year
study), and the incidences were 0 out of 10 for both control males and females. Histopathology
was not evaluated in the other male dose groups. Neuronal necrosis was also observed in the
thalamus of females receiving 350 mg/kg-day (data not shown; no incidence reported). Due to
the fact that only relative liver weight was increased at the lowest dose tested (22 mg/kg-day), as
well as the absence of serum liver enzyme alterations, the liver effect at this dose is not
considered to be biologically meaningful. Thus, 22 mg/kg-day is identified as a NOAEL. A
LOAEL of 44 mg/kg-day is identified from this subchronic study as the dose at which both
absolute and relative liver and kidney weights in females were significantly increased. A FEL of
175 mg/kg-day is established due to the observation of treatment-related mortality at this and the
highest dose. It is uncertain whether deaths that were not due to the overdose episode were
directly related to brain lesions or to other functional derangements.
Groups of B6C3Fi mice (10/sex) were administered TCEP (approximately 98% pure) in
corn oil by gavage at doses of 0, 44, 88, 175, 350, or 700 mg/kg-day, for 5 days/week, for
16 weeks (Matthews et al., 1990; NTP, 1991). Body weight and clinical observations were made
prior to testing and at weekly intervals. Serum cholinesterase was assessed from blood drawn at
terminal sacrifice. No other clinical chemistry or urinalysis are assessed in this study. Sperm
counts and morphology were assessed for all males that survived to terminal necropsy. All
animals were necropsied and pathologic examinations of major tissues and organs were made for
controls and mice exposed to 700 mg/kg-day. Kidneys in mice receiving 44, 88, 175, and
350 mg/kg-day were also examined.
As in the rat study, mice in the two highest dose groups (350 and 700 mg/kg-day)
accidentally received twice the intended dose for 3 consecutive days during the fourth week of
dosing, received no treatment on the fourth day, and then resumed dosing on the fifth day of that
week. Although all female mice in the 350 mg/kg-day dose group appeared to be uncoordinated
and two males in the 350-mg/kg-day group had convulsions and labored breathing, there were no
other clinical signs and no mortality because of the dosing error (Matthews et al., 1990;
NTP, 1991). There were no TCEP-related effects on survival, body weight, or serum
cholinesterase in mice of either sex. The authors reported that sperm count was significantly
reduced in males treated with 700 mg/kg-day (data not shown). In females, absolute and relative
liver weight was significantly increased compared to controls at 175-700 mg/kg-day. The
increases in liver weight occurred in the apparent absence of histopathological changes in the
700 mg/kg-day group (the 175 and 350 mg/kg-day dose groups were not examined). Relative
kidney weight was significantly decreased in male mice treated with doses of 175 mg/kg-day and
higher. Pathology findings were limited to mild enlargement of the nuclei of renal epithelial
cells in all high-dose mice of both sexes. These lesions were observed primarily in the proximal
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convoluted tubules of the inner cortex and outer stripe of the outer medulla (data not shown).
The NOAEL and LOAEL for this study are 88 and 175 mg/kg-day, respectively, and are based
on increased absolute and relative liver weight in females and decreased relative kidney weight
in males.
Chronic Studies—Groups of F344 rats (60/sex/group) were administered TCEP
(approximately 98% pure) in corn oil by gavage at doses of 0, 44, or 88 mg/kg-day, 5 days/week,
for 104 weeks (NTP, 1991; Matthews et al., 1993). Animals were observed twice daily for
morbidity and mortality, and clinical signs were recorded monthly. Body weights were recorded
weekly for the first 13 weeks, then monthly, and at 3-4 week intervals for the last 3 months.
Groups of 10 rats/sex/dose were sacrificed for interim evaluation (organ weights for brain, liver
and kidney; hematology2, clinical chemistry3 and histological examination of all animals) after
66 weeks of treatment. Gross necropsy was performed on all animals that died or were
sacrificed at the end of the study, and the weights of liver, kidney, and brain were recorded. A
comprehensive histological examination was conducted for all groups.
There were no treatment-related effects on body weight or clinical signs (NTP, 1991;
Matthews et al., 1993). In the 104-week study, survival was reduced in high-dose females
starting on about Week 70 of the study. Survival in high-dose males was also reduced—but only
during the last month of the study. The Kaplan-Meier survival percentages4 estimated by NTP
(1991) were 78, 68, and 51% for 0, 44, and 88 mg/kg-day males, respectively, and were 66, 71,
and 37%) for 0, 44, and 88 mg/kg-day females, respectively. The differences in survival between
high-dose and control animals is marginally significant for males (p = 0.043) and highly
significant for females (p = 0.008) based on pair-wise comparisons. Females that died early
frequently had brain lesions (see below), while males did not.
At the 66-week interim sacrifice, absolute liver and kidney weights were significantly
increased compared to controls in TCEP-treated males at 88 mg/kg-day (20.1%> and 13.8%>,
respectively) but not in females (see Table 2). Relative liver weight in males was also
significantly increased compared to controls (6.6%>) at 44 mg/kg-day (see Table 2). Relative
liver and kidney weights were significantly increased compared to controls in high-dose males
(18.8%o and 12.2%>, respectively) (see Table 2). No liver or kidney pathological changes are
noted. Decreased serum alkaline phosphatase (ALP) and serum alanine transferase (ALT) was
also observed in high-dose (88 mg/kg-day) females but not males (magnitude not reported; data
not shown). Lesions that were considered to be treatment-related but not statistically significant
at the 66-week interim sacrifice included an adenoma of the renal tubule in one 88 mg/kg-day
male and degenerative lesions of the brain (focal lesions in the cerebellum and thalamus) in three
88 mg/kg-day females (NTP, 1991; Matthews et al., 1993). The authors characterized the brain
lesions observed at the interim as necrosis of the neuropil with accumulation of inflammatory
cells, reactive gliosis, and endothelial hypertrophy and hyperplasia.
hematocrit, hemoglobin, erythrocytes, leucocytes with differential, mean cell volumes, mean cell hemoglobin,
mean cell hemoglobin concentration, and reticulocyte count.
3Blood urea nitrogen, serum glucose, creatinine, alkaline phosphatase, serum cholinesterase, cholesterol, sorbitol
dehydrogenase, alanine aminotransferase and aspartate aminotransferase.
4Survival rates adjusted for gavage deaths, accidents, and interim sacrifice
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Histological evaluations conducted at the end of the 104-week duration revealed treatment-
related hyperplastic and neoplastic changes in the kidney, thyroid neoplasms, mononuclear cell
leukemia, and degenerative lesions in the brain (females only). Table 3 summarizes the
incidences of brain lesions. The renal tubule hyperplasia and neoplasms occurred in the cortex.
The hyperplasia was focal and multifocal in nature and was characterized by stratification of the
epithelial cells with partial to complete obliteration of the lumen. The renal tubule cell
hyperplasia may likely be preneoplastic in nature. Similar to the subchronic study, due to the
fact that only relative liver weight was increased at the lowest dose tested in the 66-week study
(44 mg/kg-day), as well as the absence of serum liver enzyme alterations, the magnitude of the
liver effect at this dose is not considered to be biologically meaningful. Thus, 44 mg/kg-day is
identified as a NOAEL. A LOAEL of 88 mg/kg-day is identified from the 66-week interim
time-point in this chronic study as the dose at which both absolute and relative liver and kidney
weights in males were significantly increased. For the 104-week duration, a FEL of 88 mg/kg
day is identified for significantly decreased survival in females.
Table 2. Absolute and Relative Liver and Kidney Weights of F344/N Rats
Given TCEP By Gavage for 66 Weeksa'b
Male
Dose (mg/kg-day)
0
44
88
Absolute liver weight (g)
14.9 ±0.84
16.2 ±0.33
17.9 ± 0.35 d
Relative liver weight (mg/g)c
31.9 ± 1.11
34.0 ± 1.55 d
37.9 ± 0.50d
Absolute kidney weight (g)
1.52 ±0.06
1.60 ±0.03
1.73 ± 0.03 d
Relative kidney weight (mg/g)c
3.28 ±0.12
3.37 ±0.06
3.68 ± 0.06d
Female
Absolute liver weight (g)
8.86 ±0.26
8.62 ±0.20
9.13 ±0.26
Relative liver weight (mg/g)c
31.1 ±0.96
32.0 ±0.77
32.8 ±0.70
Absolute kidney weight (g)
0.87 ±0.04
0.88 ±0.03
0.92 ±0.02
Relative kidney weight (mg/g)c
3.03 ±0.14
3.26 ±0.13
3.31 ±0.12
aNTP (1991); Matthews et al. (1993)
bMean ± standard error; n = 10 for all groups except control (0 mg/kg-day) males and 88
mg/kg-day females (n = 9)
Defined as organ-weight-to-body-weight ratio
Significantly different (p < 0.05) from the control (0 mg/kg-day) group by Dunn's or Shirley's
test
Table 4 summarizes the incidences of neoplastic lesions observed after the 104-week
duration. There was a significant increase in the incidence of renal tubule cell adenomas in the
high-dose male rats. Smaller, but still significant, increases were also seen for renal adenomas
and thyroid follicular cell tumors (adenomas or carcinomas) in female rats and mononuclear cell
leukemia in both sexes. NTP (1991) considered the increased renal tubular cell adenomas to be
especially noteworthy due to low spontaneous occurrence of renal tubular cell neoplasms in
F344/N rats. The incidences of thyroid tumors and mononuclear cell leukemia in the high-dose
groups were near the upper limit of the range of historical control values. NTP (1991) concluded
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that there was "clear evidence of carcinogenic activity for male and female F344/N rats receiving
tris(2-chloroethyl) phosphate as shown by increased incidences of renal tubule adenomas.
Thyroid follicular cell neoplasms and mononuclear cell leukemia in male and female rats may
have been related to chemical administration."
Table 3. Incidence of Nonne
oplastic Brain Lesions in F344/N Rats Given TCEP
jy Gavage for 2 Years3
Dose (mg/kg-day, 5/7 days/wk)
0
44
88
Incidence of Lesions (%)
Male
Brain Stem Hemorrhage
0/50 (0)
0/49 (0)
1/50 (2)
Pigment, hemosiderin
1/50 (2)
0/49 (0)
0/50 (0)
Cerebrum Gliosis, focal
0/50 (0)
0/49 (0)
1/50 (2)
Hemorrhage
0/50 (0)
1/49 (2)
1/50 (2)
Pigment, hemosiderin
0/50 (0)
0/49 (0)
1/50 (2)
Pons Hemorrhage
0/50 (0)
0/49 (0)
3/50 (6)
Female
Brain Stem Gliosis, focal
1/50 (2)
0/50 (0)
15/50 (30)b
Hemorrhage
1/50 (2)
0/50 (0)
12/50 (24)b
Mineralization
0/50 (0)
0/50 (0)
7/50 (14)b
Necrosis
0/50 (0)
0/50 (0)
1/50 (2)
Pigment, hemosiderin
1/50 (2)
0/50 (0)
17/50 (34)b
Cerebrum Gliosis
0/50 (0)
0/50 (0)
19/50 (38)b
Hemorrhage
1/50 (2)
0/50 (0)
17/50 (34)b
Mineralization
0/50 (0)
0/50 (0)
15/50 (30)b
Pigment, hemosiderin
0/50 (0)
0/50 (0)
22/50 (44)b
Pons Hemorrhage
0/50 (0)
1/50 (2)
0/50 (0)
aNTP (1991); Matthews et al. (1993)
V<0.01
Groups of B6C3Fi mice (60/sex/dose) were administered TCEP (approximately
98% pure) in corn oil by gavage at doses of 0, 175, or 350 mg/kg, 5 days/week, for 104 weeks
(NTP, 1991; Matthews et al., 1993). Animals were observed twice daily for morbidity and
mortality, and clinical signs were recorded monthly. Body weights were recorded weekly for the
first 13 weeks, then monthly and at 3-4 week intervals for the last 3 months. Groups of
10 mice/sex/dose were sacrificed for interim evaluation (organ weights for brain, liver and
kidneys; hematology5, clinical chemistry6, and histological examination of control and high-dose
animals) after 66 weeks of exposure. Gross necropsy was performed on all animals that died or
were sacrificed at the end of the study, and liver, kidneys, and brain weights were recorded. A
comprehensive histological examination was conducted for all controls and high-dose animals,
and sections from the Harderian gland, kidney, livers, lung, and stomach were also evaluated for
low-dose mice.
There were no significant treatment-related effects on mouse survival, body weight, or
clinical signs (NTP, 1991; Matthews et al., 1993). At interim sacrifice after 66 weeks of
hematocrit, hemoglobin, erythrocytes, leucocytes with differential, mean cell volume, mean cell hemoglobin, mean
cell hemoglobin concentration, and reticulocyte count.
6Blood urea nitrogen, serum glucose, creatinine, alkaline phosphatase, serum cholinesterase, cholesterol, sorbitol
dehydrogenase, alanine aminotransferase, and aspartate aminotransferase.
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Table 4. Incidence of Treatment-Related Hyperplasia, Adenomas, and Carcinomas in
F344 Rats Given TCEP by Gavage for 2 Years3
Dose (mg/kg-day)
0
44
88
Male
Kidney Renal Tubule Cell
Hyperplasia
0/50 (0%)
2/50 (4%)
24/50 (48%)b
Adenoma
1/50 (2%)
5/50 (10%)
24/50 (48%)b
Carcinoma
1/50 (2%)
0/50 (0%)
1/50 (2%)
Adenoma or Carcinoma
2/50 (4%)
5/50 (10%)
25/50 (50%)b
Historical Control Incidence (adenoma or carcinoma)
12/2142 (0.6 ± 0.9%); range = 0-2%
First Incidence of Adenoma or carcinoma (days)
729
729
575
Thyroid Follicular Cell
Hyperplasia
0/50 (0%)
0/48 (0%)
0/50 (0%)
Adenoma
1/50 (2%)
2/48 (4%)
3/50 (6%)
Carcinoma
0/50 (0%)
0/48 (0%)
2/50 (4%)
Adenoma or Carcinoma
1/50 (2%)
2/48 (4%)
5/50 (10%)
Historical Control Incidence (adenoma or carcinoma)
51/2106 (2.4 ± 2.3%); range = 0-10%
First Incidence of Adenoma (days)
729
574
674
First Incidence of Carcinoma (days)
Not applicable
Not applicable
696
Mononuclear Cell Leukemia
5/50 (10%)
14/50 (28%)°
13/50 (26%)°
Historical Control Incidence
321/2149 (14.9 ± 10.8%); range = 0-44%
First Incidence (days)
539
620
584
Female
Kidney Renal Tubule Cell
Hyperplasia
0/50 (0%)
3/50 (6%)
16/50 (32%)b
Adenoma
0/50 (0%)
2/50 (4%)
5/50 (10%)b
Carcinoma
0/50 (0%)
0/50 (0%)
0/50 (0%)
Adenoma or Carcinoma
0/50 (0%)
2/50 (4%)
5/50 (10%)b
Historical Control Incidence (adenoma)
1/2144 (0.1 ± 0.3%); range = 0-2%
First Incidence of Adenoma (days)
Not applicable
729
729
Thyroid Follicular Cell
Hyperplasia
1/50 (2%)
0/50 (0%)
1/50 (2%)
Adenoma
0/50 (0%)
1/50 (2%)
1/50 (2%)
Carcinoma
0/50 (0%)
2/50 (4%)
3/50 (6%)c
Adenoma or Carcinoma
0/50 (0%)
3/50 (6%)
4/50 (8%)°
Historical Control Incidence (adenoma or carcinoma)
34/2107 (1.6 ± 1.6%); range=0-6%
First Incidence of Adenoma or carcinoma (days)
Not applicable
697
718
Mononuclear Cell Leukemia
14/50 (28%)
16/50 (32%)
20/50 (40%)b
Historical Control Incidence
329/2150 (15.3 ± 10.6%); range = 0-38%
First Incidence (days)
335
561
469
aNTP (1991); Matthews et al. (1993)
Significantly different (p < 0.01) from control by pair-wise comparison (kidney and thyroid) or life table test
(leukemia)
Significantly different (p < 0.05) from control by pair-wise comparison (kidney and thyroid) or life table test
(leukemia)
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Table 5. Incidence of Selected Renal Tubule Cell Lesions in B6C3Fi Mice Given TCEP
by Gavage for 2 Years3
Dose (mg/kg-day)
0
175
350
Male
Karyomegaly
2/50
16/50°
39/50°
Hyperplasia
1/50
0/50
3/50
Adenoma
1/50
1/50
3/50
Adenocarcinoma
0/50
0/50
1/50
Female
Karyomegaly
0/50
5/49b
44/50°
Hyperplasia
0/50
1/49
2/50
Adenoma
0/50
1/49
0/50
Adenocarcinoma
0/50
0/49
0/50
aNTP (1991); Matthews et al. (1993). Incidences hyperplasia, adenoma and adenocarcinoma are for original and
step-sections combined
Significantly different (p < 0.05) from control by logistic regression tests
Significantly different (p < 0.01) from control by logistic regression tests
treatment, there were no significant treatment-related effects on organ weights, hematology,
clinical chemistry, or histopathology. Table 5 summarizes the incidences of nonneoplastic and
neoplastic changes in the kidney. The most prominent renal lesion was renal tubular cell
karyomegaly (nuclear enlargement), which was significantly increased at both doses in both
sexes in a dose-related manner. This lesion was minimal in severity and was observed primarily
in the proximal convoluted tubules of the inner kidney cortex and outer stripe of the medulla.
Low incidences of hyperplasia and renal tumors (see below) are also reported in the kidney.
Based on renal tubule cell karyomegaly, this study identifies a LOAEL of 175 mg/kg-day. A
NOAEL is not identified.
None of the neoplastic lesions identified in mice following terminal necropsy and
pathologic evaluation of tissues could unequivocally be attributed to treatment with TCEP
NTP, 1991; Matthews et al., 1993). Initially, renal tubule adenomas were observed in one
control male, one high-dose male, and one low-dose female, and an adenocarcinoma was
observed in one high-dose male. Due to the rare occurrence of renal tubule neoplasms in
B6C3Fi mice, additional step sections were evaluated, yielding final incidences of 1 out of 50,
1 out of 50, and 3 out of 50 for renal tubular adenomas in 0-, 175-, and 350-mg/kg male mice,
respectively (see Table 5). The difference from controls in the high-dose group is not
statistically significant. Besides the kidneys, increases in tumors or precursor lesions were also
seen in the Harderian gland and the liver. In the Harderian gland, there was an increase in the
incidence of combined adenomas and carcinomas in female mice. Incidences were 3 out of 50,
8 out of 60 and 10 out of 60 for 0, 175, and 350 mg/kg, respectively (statistically significant
trend; incidence at the high dose was significantly greater than the control incidence). With
regard to the liver, there was an increase in the incidence of eosinophilic foci in high-dose males.
Incidences were 0 out of 50, 3 out of 50, and 8 out of 50 in 0-, 175-, and 350-mg/kg males,
respectively. Although there is a significant trend for increased adenomas in male mice, there
are no TCEP-related effects on the incidences of basophilic or clear cell foci, and no significant
increases in the incidences of adenoma, carcinoma, or combined adenoma and carcinoma of the
liver. Given that the development of eosinophilic, basophilic, and clear-cell foci are considered
to be precursors to liver neoplasms, NTP (1991) concluded that the biological importance of the
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increase in eosinophilic foci in the absence of the development of neoplastic change is uncertain;
they noted that there was "equivocal evidence of carcinogenic activity for male B6C3Fi mice as
shown by an increased incidence of renal tubular cell neoplasms." Additionally, "equivocal
evidence of carcinogenic activity for female B6C3Fi mice was shown by an increased incidence
of harderian gland adenomas."
In another carcinogenicity study, groups of ddY mice (50/sex/group) were fed TCEP in
their diet at concentrations of 0, 0.012, 0.06, 0.3, or 1.5% for 18 months (Takada et al., 1989).
This study is written in Japanese, but the information reported here is based on the abstract,
tables and figures, all of which are reported in English. Based on measured data for body weight
and food consumption, the dietary concentrations used in the study were equivalent to doses of 0,
9.3, 46.6, 232.8, or 1687.5 mg/kg-day for males, and 0, 10.7, 53.3, 266.7, or 1875.0 mg/kg-day
for females. Survival, body weight, and food consumption were monitored throughout the study.
It is not clear whether clinical chemistry, urinalysis, or hematology were endpoints in this study.
A comprehensive evaluation of tissues and organs appears to have been conducted for all dose
groups.
The major findings of this study are summarized in Table 6. Mortality (death or
moribund sacrifice) was higher in males fed 1687.5 mg/kg-day and in females fed 266.7 or
1875.0 mg/kg-day TCEP than in the control or lower-dose groups (Takada et al., 1989). The
early morbidity and mortality may have been associated with neoplastic changes, as the
incidence of animals with tumors (including those that died, were sacrificed, or survived to the
end of the study) was significantly increased above control values in males fed
1687.5 mg/kg-day and in females fed 266.7 or 1875.0 mg/kg-day. Body weight was
significantly decreased (compared to control values) throughout the study (approximately
29-31%) in both males and females fed 1687.5 and 1875.0 mg/kg-day TCEP, respectively,
although food consumption was unaffected by addition of TCEP to the diet. Tumors potentially
related to treatment were seen in the kidneys, liver, forestomach, and hematopoietic system. In
males fed 1687.5 mg/kg-day TCEP, there were statistically significant increases in the incidences
of renal cell adenoma, carcinoma and adenoma plus carcinoma combined. These tumors were
also seen in females at the same dietary concentration, albeit at low incidences that did not
approach statistical significance. In males fed 232.8 or 1687.5 mg/kg-day TCEP, statistically
significant increases in hepatocellular adenoma and combined hepatocellular adenoma and
carcinoma were seen. A low incidence of these tumors was also seen in the 1875.0-mg/kg-day
females. Statistically significant increases of combined forestomach tumors (papillomas and
squamous cell carcinomas) and leukemia (specific type not identified) were observed in female
mice but not in males. There are no treatment-related tumors in any other organs or tissues. The
accessible parts of the report provided few data on nonneoplastic lesions. Renal tubular cell
enlargement was apparently observed, as Figure 8 of the report is an image of enlarged nuclei
from renal tubular epithelium taken from a male mouse fed 232.8 mg/kg-day TCEP for 79
weeks. No further information on this endpoint is provided.
Reproductive/Developmental Studies—The National Toxicology Program assessed the
reproductive/developmental toxicity of TCEP in CD-I mice using the "Reproductive Assessment
by Continuous Breeding" (RACB) protocol (Gulati et al., 1991). The RACB protocol consists of
four sequentially executed tasks consisting of (1) dose range-finding, (2) continuous breeding,
(3) identification of the affected sex, and (4) assessment of the fertility of F1 offspring. Figure 2
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illustrates a flow diagram of the RACB protocol. For the range-finding task, mice (8/sex/dose)
were administered TCEP in corn oil by gavage at doses of 0, 87.5, 175, 350, 700, or
1000 mg/kg-day for 14 consecutive days. Based on a single treatment-related mortality at the
high dose, and the lack of treatment-related effects on body weight and food consumption, doses
of 75, 350, and 700 mg/kg-day were selected for the continuous breeding phase of the study.
In the continuous breeding phase (Task 2), groups of male and female mice
(40 untreated/control pairs and 20 pairs/dose) were housed together and received TCEP (purity
>98% in corn oil) by gavage at doses of 0, 75, 350, or 700 mg/kg-day for 98 consecutive days
(Gulati et al., 1991). Endpoints for this task include clinical signs, parental body weight, average
water consumption, fertility, litters/pair, live pups/litter, proportion of pups born alive, sex of live
pups, and pup weights at birth. There was no significant treatment-related mortality or clinical
Table 6. Effects in ddY Mice Fed TCEP in the Diet for 18 Months3
Dietary Concentration (%)
Variable
0
0.012
0.06
0.3
1.5
Male
Estimated Body Weight (kg)b
0.058
0.058
0.058
0.058
0.040
Estimated Food Consumption (kg/day)b
0.0045
0.0045
0.0045
0.0045
0.0045
Estimated Dose (mg/kg-day)c
0
9.3
46.6
232.8
1687.5
% Mortality (includes moribund sacrifice)
40
36
44
42
62
% with Tumors
68
78
80
83
©
o
o
Renal Cell Adenoma
0/50
0/49
0/49
2/47
9/50e
Renal Cell Carcinoma
2/50
0/49
2/49
3/47
32/50e
Renal Cell Adenoma+Carcinoma
2/50
0/47
2/49
5/47
4 l/50e
Hepatocellular Adenoma
3/50
4/49
3/49
10/47d
16/50d
Hepatocellular Carcinoma
1/50
1/49
4/49
2/47
3/50
Hepatocellular Adenoma+Carcinoma
4/50
5/49
7/49
12/47e
19/50e
Leukemia
7/50
4/50
6/49
4/47
4/50
Forestomach Papilloma +Squamous Cell
Carcinoma
0/50
0/49
1/49
2/47
2/50
Female
Estimated Body Weight (kg)b
0.045
0.045
0.045
0.045
0.032
Estimated Food Consumption (kg/day)b
0.004
0.004
0.004
0.004
0.004
Estimated Dose (mg/kg-day)c
0
10.7
53.3
266.7
1875.0
% Mortality (includes moribund sacrifice)
34
38
48
56
64
% with Tumors
61
57
66
80d
82d
Renal Cell Adenoma
0/49
0/49
0/50
0/49
2/50
Renal Cell Carcinoma
0/49
0/49
0/50
0/49
1/50
Renal Cell Adenoma+Carcinoma
0/49
0/49
0/50
0/49
3/50
Hepatocellular Adenoma
0/49
0/49
0/50
0/49
2/50
Hepatocellular Carcinoma
0/49
0/49
0/50
0/49
0/50
Hepatocellular Adenoma+Carcinoma
0/49
0/49
0/50
0/49
2/50
Leukemia
1/49
3/49
6/50
9/49d
9/50d
Forestomach Papilloma+Squamous Cell
Carcinoma
0/49
0/49
0/50
1/49
7/50d
"Takada et.al. (1989)
bEstimated from graphs shown in study Figures 3 and 4
Estimated dose = (% diet x 10000 x estimated food consumption)/estimated body weight
Statistically significant difference from controls (p < 0.05)
eStatistically significant difference from controls (p < 0.01)
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NECROPSY
Sperm Morphology
Vaginal Cytology
NECROPSY
Spaim Morphology
Vaginal Cytology
NECROPSY
Sparm Morphology
Vaginal Cytology
TASK 3
Datarmlnatlon ol
Aftaetad Sax
(F» Qanaratlon)
TASK 4
Offspring Aaaaasmant
Pi Qanaratlon
(Control 4 1 dosad group)
TASK 4
Offspring Assassmant
F. Qanaratlon
(Control & 3 doaed groups)
TASK 1
Dosa Finding Study
(14 days treatment)
TASK 2
Continuous Breading Phase
F« Qanoratkxn
(1 ark 4-14 waaks + Holding Parted)
optional optional optional
Hlatopathology Hlatopathology Histopatftology
Homtona Assays Honnona Assays Hormona Assays
Figure 2. Reproductive Assessment by Continuous Breeding Protocol (Gulati et al., 1991)
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signs. Male and female body weights were within 10% of the control values in all test groups
throughout the study. Significant decreases in fertility (both litter production and number of live
pups per litter) were observed at 350 and 700 mg/kg-day (see Tables 7 and 8). The few dams in
the 700-mg/kg-day group that did produce second and third litters took significantly longer to do
so than dams in the control or lower-dose groups (see Table 8).
Table 7. Fertility of Breeding Pairs of Swiss CD-I Mice Given TCEP by Gavage
for 98 Days (Task 2)a
Litter
Number of Fertile Pairs/Number of Cohabiting Pairs
Dose Group (mg/kg-day)
0
175
350
700
1
37/38
18/19
18/18
18/18
2
37/38
18/19
18/18
12/18b
3
37/38
18/19
16/18
2/18b
4
37/38
17/19
16/18
0/18b
5
35/38
17/19
13/18b
0/18b
"Gulatietal (1991)
bp < 0.05
Table 8. Effects of TCEP (Given By Gavage) on Reproductive Performance of Swiss
CD-I Mice During Continuous Breeding (Task 2)a
Dose (mg/kg-day)b
Variable
0
175
350
700
Average Litters per Pairc,d
4.9 ±0.0 (37)
4.9 ±0.1 (18)
4.5 ±0.2 (18)'
1.8 ±0.2 (18)'
Live Pups per Litter°'d
Male
6.4 ±0.3 (37)
6.1 ±0.3 (18)
5.1 ±0.4 (18)'
3.9 ±0.3 (18)'
Female
6.3 ±0.3 (37)
6.1 ±0.3 (18)
5.0 ±0.2 (18)'
4.6 ±0.5 (18)'
Combined
12.7 ±0.5 (37)
12.1 ±0.4 (18)
10.1 ±0.5 (18)'
8.6 ±0.6 (18)'
Cumulative Days to Litter6
2nd litter
40.8 ±0.3 (37)
40.9 ±0.5 (18)
48.1 ±4.7 (18)
65.9 ±6.4 (12)'
3rd Litter
61.9 ±0.3 (37)
60.8 ±0.5 (18)
61.8 ± 1.2(16)
102.5 ± 14.5 (2)g
aGulati et al. (1991)
bOnly pairs surviving to the end of Task 2 were included for statistical evaluation
°Mean ± standard error (number of fertile pairs)
dEach dose is compared to the control group with Shirley's test when a trend is present (p < 0,1 from Jonckhere's
trend test), otherwise Dunn's test is used
"Mean ± standard error (number of dams)
{p < 0.05
gThere were too few animals to conduct statistics
Because of the effects on fertility noted in Task 2, a 1-week crossover mating trial
(Task 3) was performed to determine which sex had been affected by treatment in Task 2
(Gulati et al., 1991). Three groups of 20 breeding pairs were mated as follows: untreated males
were paired with untreated females, untreated males were paired with high-dose
(700 mg/kg-day) females, and untreated females were paired with high-dose (700 mg/kg-day)
males. The endpoints evaluated for this task included sperm morphology, sperm count, vaginal
cytology, weight of reproductive organs, and pathologic examination of reproductive and major
organs (in 10 randomly selected high-dose and control mice of each sex). There were no
TCEP-related effects on mortality or clinical signs. Effects on fertility were noted in the group
with high-dose males bred to untreated females (1 out of 18 pregnancies versus 12 out of
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20 pregnancies for untreated pairs) and in the group with high-dose females bred to untreated
males (7.2 ± 0.9 live pups/litter versus 10.3 ± 0.7 live pups/litter from untreated pairs). There
were no treatment-related effects on vaginal cytology or estrous cycling. Sperm effects in the
males at the 700 mg/kg-day dose include significant decreases in mean concentration of sperm
(810.8 ± 76.8 per mg caudal tissue versus 1223 ± 68.7 in controls) and percent motile sperm
(35.0 ± 8% versus 77.8 ±1.6 % in controls), as well as a significant increase in the percentage of
abnormal sperm (31.5 ± 3.1% versus 9.1 ± 0.59% in controls). The only remarkable
treatment-related histological finding is an increase in the incidence of minimal to mild
cytomegaly of the renal tubule cells (10 out of 12 males and 5 out of 13 females7) in
TCEP-treated mice compared with controls (0 out of 10 males and 0 out of 12 females). No
treatment-related lesions were found in the brain or ovaries. These results show that both sexes
are affected by TCEP, with the males being relatively more sensitive (larger effect at the same
dose), and the consequence on male fertility likely being due to an effect on sperm.
In Task 4, members of the last litter born to each pair in Task 2 were allowed to reach
sexual maturity, and then they were paired individually with a member of the opposite sex from
a separate litter within the same treatment group (Gulati et al., 1991). These pups received the
same control or TCEP exposure as their parents. The high-dose (700 mg/kg-day) group was
excluded from this phase of the experiment due to an insufficient number of pups. Pairs were
mated at approximately 74 days of age and assessed for the same endpoints as in Task 2 (clinical
signs, parental body weight, average water consumption, fertility, litters/pair, live pups/litter,
proportion of pups born alive, sex of live pups, pup weights at birth, vaginal cytology 12 days
before sacrifice and epididymal sperm count and morphology). F1 mice were sacrificed at the
end of Task 4, and the presence of morphological and histopathological changes in reproductive
organs was assessed. Mating and fertility indices in the control and treated groups were similar,
but there was a statistically significant decrease in the number of live F2 pups/litter in the
350 mg/kg-day group (7.6 ±1.1 versus 11.4 ± 0.5 in controls), specifically males (3.4 ± 0.6
versus 6.4 ± 0.6 in controls). Epididymal sperm count, sperm motility, and the incidence of
abnormal sperm were unaffected by TCEP treatment up to the 350 mg/kg-day level in F1 males.
There were no apparent effects on estrous cycling or on the average estrous cycle length in
treated F1 females. There were no remarkable findings upon histopathologic examination.
Based on the findings of all tasks (Gulati et al., 1991), the NOAEL for reproductive
toxicity in Swiss CD-I mice is 175 mg/kg-day. The LOAEL is 350 mg/kg-day and is based on
decreased fertility (decreased number of consecutive litters produced, average litters per pair, and
number of live pups per litter), which is likely due, at least in part, to observed effects on sperm
count, sperm motility, and sperm morphology.
The developmental toxicity of TCEP (purity unknown) dissolved in olive oil was
assessed in groups of Wistar rats (23-30 pregnant females/dose) treated by gavage at doses of 0,
50, 100, or 200 mg/kg-day on Days 7 through 15 of gestation (Kawashima et al., 1983)8. Half of
the dams were sacrificed on Day 20 of gestation and their uterine contents were examined. The
remaining dams were allowed to deliver their litters, and their pups were observed (up to
7The tissues examined (reported in Tables 3—8 and 3-9 of Gulati et al., 1991) include those from an additional two
males and three females that died, or were sacrificed, during Task 2.
8This report is written in Japanese. The account of this study presented here is based on the abstract and tables that
are reported in English.
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10 weeks) for physical and neurobehavioral development. The neurobehavioral testing includes
assessments of spontaneous motor behavior (ambulation, rearing behavior, defecation) and
performance in a water maze.
There was no mortality at doses of 100 mg/kg-day or less, but 7 out of 30 dams treated at
200 mg/kg-day died; food consumption was markedly reduced in this group, and piloerection
and general signs of weakness were evident (Kawashima et al., 1983). No effects on body
weight, food consumption, or general appearance were reported in dams treated with doses of
50 or 100 mg/kg-day. There were no treatment-related effects on the mean number of corpora
lutea, number of implants, implants per corpora lutea or kidney weights among the 15 dams per
group killed at term. There were no treatment-related effects on the mean number of live fetuses,
sex ratio, fetal body weight, or fetal mortality (including early or late resorptions). No fetuses
with malformations were observed in any treatment group, and there were no treatment-related
effects on skeletal variations or ossification. Among dams that delivered and reared their pups,
there were no treatment-related effects on the number of implantation sites, number of births or
any indicator of fetal or pup survival. The only significant effect on spontaneous motor activity
was a decrease in rearing among male—but not in female pups born to dams treated with
200 mg/kg-day (9.8 ± 5.6 seconds compared with 19.3 ± 9.4 seconds in controls). There were no
treatment-related effects on ambulation or defecation variables. With regard to performance in a
water maze (an assessment of memory and learning ability where the test animal has to swim
through a maze to find a platform), the only significant effect is an increased time required to
find the platform in the fourth of four trials among high-dose males (72.7 seconds versus
35.4 seconds in control males). There were no treatment-related differences in the first three
trials for males, or in any of the four trials for females at any treatment level. There are no
differences between treatment groups of either sex with regard to the number of errors made.
Based on these findings, 100 mg/kg-day is aNOAEL for maternal toxicity and developmental
toxicity. The highest dose tested, 200 mg/kg-day, is a FEL for maternal mortality.
Inhalation Exposure
Few chronic or subchronic toxicity studies of TCEP conducted by the inhalation route of
exposure were located. In a study from the Russian literature, (Shepelskaya and Dyshinevich,
1981) exposed male rats continuously to TCEP in air (no further details of exposure conditions)
at concentrations of 0, 0.5, or 1.5 mg/m3 for 4 months, and then they mated the animals to naive
females. Results are presented here as reported in the English abstract of the report and as
discussed by Gulati et al. (1991). There are significant decreases in litter size, and increases in
both pre- and postimplantation loss among females mated to males exposed to 1.5 mg/m3. It is
reported that fetal weight and crown-rump length were significantly decreased in pups born to
dams mated with males exposed to 0.5 mg/m3. Effects on the testes were also observed, but the
nature of these effects and the concentrations at which they were observed cannot be determined
from the report.
Other Studies
Acute/Short-term Studies
No treatment-related effects on spontaneous behavior, memory, or learning were
observed when mice were exposed to low doses of TCEP (0.4-40 mg/kg-day) as a single gavage
dose during the critical period for brain development (Postnatal Day 10), and then tested as
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adults at 2-4 months of age (Eriksson et al., 2004). No other information on study design or
findings was presented.
Genotoxicity
The overall weight-of-evidence for the mutagenicity of TCEP is negative. As discussed
below, some equivocal results have been obtained, but the majority of studies have yielded
negative findings. With one exception, all studies that conducted Ames tests for TCEP reported
entirely negative results. TCEP was not mutagenic in Salmonella typhimurium strains TA97a,
TA98, TA100, TA102, TA104, TA1535, TA1537, or TA1538 when tested with or without S9 in
studies by Simmon et al. (1977), Haworth et al. (1983), Kubo et al. (2002), and Follmann and
Wober (2006). One other study reported negative results in most strains (TA98, TA100,
TA1537, and TA1538), but it did obtain weak positive results for TCEP in TA1535—only in the
presence of S9 (Nakamura et al., 1979).
TCEP was not mutagenic in V79 Chinese hamster lung fibroblasts, but it induced sister
chromatid exchange (SCE) in the same cell line when tested without S9 (Sala et al., 1982).
TCEP was considered to produce equivocal findings in an evaluation of SCE in Chinese Hamster
Ovary (CHO) cells, with positive results in one trial and negative results in a second conducted
under the same conditions (Galloway et al., 1987). TCEP did not produce any change in
chromosomal aberrations in CHO cells incubated with or without S9 (Galloway et al., 1987).
TCEP did not induce DNA strand breaks in V79 cells in the Comet assay—with or without
metabolic activation (Follmann and Wober, 2006). TCEP gave a negative result for cell
transformation in C3H10T1/2 cells but produced transformation in Syrian hamster embryonic
cells (Sala et al., 1982).
An in vivo assay for micronucleus production in Chinese hamsters produced equivocal
results (Sala et al., 1982). TCEP yielded negative results in a w/w+ bioassay9 for somatic cell
damage that was conducted with Drosophila melanogaster (Vogel and Nivard, 1993).
DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC ORAL RfD
VALUES FOR TRIS(2-CHLOROETHYL)PHOSPHATE
There are no human studies that can be used to quantitatively assess oral or inhalation
exposure to TCEP. The oral toxicity database for animals is fairly complete, and Table 9
summarizes the available studies.
Subchronic p-RfD
Subchronic rat studies show significant increases in absolute and relative liver and kidney
weights in the absence of frank effects (i.e., mortality) at lower dose levels of TCEP
(Matthews et al., 1990; NTP, 1991). Additional support for the consideration of liver and kidney
weight changes as critical effects is that significant increases also occurred in rats following a
short-term (16-day) TCEP dosing regimen (NTP, 1991), as well as in mice following a
subchronic (16-week) dosing regimen (Matthews et al., 1990; NTP, 1991). From these
9 This assay monitors genetic damage resulting from a loss of heterozygosity and the formation of white spots in the
eyes of adult females.
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Table 9. Summary of Oral Noncancer Dose-Response Information for TCEP
Species
(n/sex/group)
Exposure
NOAEL
(mg/kg-day)
LOAEL
(mg/kg-
day)
Duration-
adjusted"
LOAEL
(mg/kg-
day)
Responses at the
LOAEL
Comments
Reference
Subchronic Studies
Rat, F344/N
(10/sex/dose)
0, 22, 44, 88, 175,
or 350 mg/kg-day
via gavage in corn
oil 5 days/week for
16 weeks.
22
44
31.4
Increased absolute and
relative liver and kidney
weight in females.
A FEL of 175 mg/kg-
day was established for
mortality in both sexes.
Matthews et al., 1990; NTP,
1991
Mouse, B6C3Fi
(10/sex/dose)
0, 22, 44, 88, 175,
or 350 mg/kg-day
via gavage in corn
oil 5 days/week for
16 weeks.
88
175
125
Increased absolute and
relative liver weight in
females; decreased
relative kidney weight in
males.
Matthews et al., 1990; NTP,
1991
Chronic Studies
Rat, F344/N
(50/sex/dose)
0, 44, or 88 mg/kg-
day via gavage in
corn oil 5
days/week for 104
weeks.
44
88
62.9
Brain lesions (cerebrum,
pons, and brain stem) in
females; reduced survival
in females; renal
hyperplasia in males and
females.
NTP, 1991; Matthews et al.,
1993
Rat, F344/N
(10/sex/dose)
0, 44, or 88 mg/kg-
day via gavage in
corn oil 5
days/week for 66
weeks (interim
evaluation).
44
88
62.9
Increased absolute and
relative liver and kidney
weights in males.
NTP, 1991; Matthews et al.,
1993
Mouse, B6C3Fi
(10/sex/dose)
0, 175, or 350
mg/kg-day via
gavage in corn oil
5 days/week for
104 weeks.
Not defined
175
125
Enlargement of nuclei in
renal tubule cells
(karyomegaly).
NTP, 1991; Matthews et al.,
1993
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Table 9. Summary of Oral Noncancer Dose-Response Information for TCEP
Species
(n/sex/group)
Exposure
NOAEL
(mg/kg-day)
LOAEL
(mg/kg-
day)
Duration-
adjusted"
LOAEL
(mg/kg-
day)
Responses at the
LOAEL
Comments
Reference
Reproductive/Developmental Toxicity Studies
Rat, Wistar (20-30
dams/dose)
0, 50, 100, 200
mg/kg-day via
daily gavage in
olive oil on GD 7-
15
100 (maternal
and neuro-
developmental)
200 (FEL)
200 (FEL)
Maternal mortality.
Kawashima et al., 1983
Mouse,
Swiss CD-I
(20 pairs/dose)
0, 175, 350 or 700
mg/kg-day via
daily gavage (prior
to mating, through
mating, gestation,
lactation for up to 5
litters and two
generations)
175
350
350
Decreased fertility (#
consecutive litters
produced, mean
litters/pair, mean live
pups/litter) likely due to
adverse effects on sperm
count, motility and
morphology.
Gulatietal., 1991
aAdjusted for continuous exposure
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subchronic studies, a NOAEL of 22 mg/kg-day and a LOAEL of 44 mg/kg-day are identified for
increased absolute and relative liver and kidney weights in female F344/N rats given TCEP by
gavage 5 days/week for 16 weeks (Matthews et al., 1990; NTP, 1991). All available continuous
models in the U.S. EPA Benchmark Dose Software (BMDS) version 2.1 beta were applied to the
female absolute and relative liver and kidney-weight data. Due to the lack of a dose-response
relationship, absolute and relative liver and kidney-weight data in male rats were not suitable for
BMD modeling. For females, BMD modeling was performed using the doses administered in
the study before duration adjustment. For absolute and relative liver-weight data, as well as
absolute kidney-weight data, no adequate model fits were achieved with all of the dose groups—
even when the highest two dose groups were dropped from the analysis. For a dose-dependent
increase in relative kidney weight in female rats treated with TCEP for 16 weeks, a default
benchmark response (BMR) of 1 standard deviation (SD) from the control mean was used in
modeling this endpoint. A BMDLisd of 9.66 mg/kg-day was calculated and identified as the
point of departure (POD) for the subchronic p-RfD derivation. Details of the BMD modeling
and plots of the models are presented in Appendix A.
The 16-week subchronic rat study involved exposure by oral gavage 5 days/week.
Therefore, the BMDLisd was duration adjusted to 6.9 mg/kg-day for continuous exposure
(5/7 days). This duration adjusted BMDLisd (BMDLisd[adj]) was divided by a composite UF of
300 to derive a subchronic p-RfD for TCEP as follows:
Subchronic p-RfD = BMDLiSd[adj] UF
= 6.9 mg/kg-day -^300
= 0.02 or 2 x 10~2 mg/kg-day
The composite UF of 300 is composed of the following:
• UFh: A factor of 10 is applied for extrapolation to a potentially susceptible human
subpopulation because data for evaluating susceptible human response are
unavailable.
• UFa: A factor of 10 is applied for animal-to-human extrapolation because data for
evaluating relative interspecies sensitivity are unavailable.
• UFl: A factor of 1 is applied because the POD is based on a BMDL.
• UFd: The existing database for TCEP consists of subchronic and chronic studies in
rats and mice, a reproductive study in mice, and a developmental study in rats that
included neurodevelopmental endpoints. However, a partial factor of 3 (i.e., 10°5)
is applied for database inadequacies, including lack of a comprehensive
neurotoxicity study in rats (in light of brain lesions in the subchronic and chronic rat
studies) and lack of a multigenerational reproduction study.
Confidence in the principal subchronic rat study (NTP, 1991) is medium. The study is a
comprehensive investigation of toxicity in male and female rats. However, deaths due to gavage
and dosing errors may have interfered with study findings. Confidence in the database is
medium. The database for TCEP includes subchronic and chronic studies in rats and mice, a
reproductive study in mice, and a developmental study in rats. However, the database is missing
a full neurotoxicity assessment (suggested by hippocampal lesions in the subchronic and chronic
rat studies) and a multigenerational assessment of reproduction. Overall confidence in the
subchronic p-RfD is medium.
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Chronic p-RfD
Chronic studies exist in two laboratory animal species (F344/N rats and B6C3F1 mice)
given TCEP by gavage 5 days/week for 66 or 104 weeks (Matthews et al., 1993; NTP, 1991). A
high incidence of brain lesions (in the cerebrum, pons, and brain stem) and significantly reduced
survival in female rats (with brain lesions observed in many of these animals) occurred after
treatment with 88 mg/kg-day for 104 weeks. Renal tubular hyperplasia seen in association with
renal tumors in both males and females in the 104-week study duration was may likely be
preneoplastic in nature.
Similar to the subchronic study, significantly increased absolute and relative liver and
kidney weights were the most sensitive effects occurring in the absence of frank effects
following 66 weeks of TCEP exposure. However, at the interim 66-week sacrifice, increased
absolute and relative liver and kidney weights were only observed in male rats at 88 mg/kg-day
(LOAEL). This dose is twice that of the LOAEL of 44 mg/kg-day identified for the similar liver
and kidney effects observed only in female rats from the subchronic study (Matthews et al.,
1990; NTP, 1991). The only change in serum enzymes from any group tested are decreases in
serum ALP and ALT in high-dose female rats in the 66-week study, which is not indicative of
biological and/or functional liver alterations. Likewise, no accompanying liver or kidney
pathological changes were observed in 175 and 350 mg/kg-day rats or 700 mg/kg-day mice
(>44 mg/kg-day for kidney) exposed subchronically or 88 mg/kg-day rats and mice exposed
chronically. The explanation for the discrepancy between significantly increased absolute and
relative liver and kidney weights between the subchronic study (treatment-dependent effects
observed only in female rats at a LOAEL of 44 mg/kg-day) and the 66-week study (duration-
dependent effects observed only in male rats at a LOAEL of 88 mg/kg-day) is unclear because
these two studies were performed concurrently by the same laboratory.
The LOAEL of 44 mg/kg-day for increased absolute and relative liver and kidney weights
in female rats from the 16-week subchronic study is more sensitive than the LOAEL of
88 mg/kg-day for the same endpoints in male rats from the 66-week study. In light of the
available data on TCEP, and because the shorter exposure duration provided the most sensitive
response, the BMDLiSd[adj] of 6.9 mg/kg-day for increased relative kidney weight from the
subchronic 16-week study serves as the POD for deriving the chronic p-RfD value. The
subchronic BMDLiSd[adj] was divided by a composite UF of 1000 to derive a chronic p-RfD for
TCEP, as follows:
Chronic p-RfD = Subchronic BMDLiSd[adj] ^ UF
= 6.9 mg/kg-day ^ 1000
= 0.007 or 7 x 10 3 mg/kg-day
The composite UF of 1000 is composed of the following:
• UFh: A factor of 10 is applied for extrapolation to a potentially susceptible human
subpopulation because data for evaluating susceptible human response are
unavailable.
• UFa: A factor of 10 is applied for animal-to-human extrapolation because data for
evaluating relative interspecies sensitivity are unavailable.
• UFl: A factor of 1 is applied because the POD was based on a BMDL.
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• UFs: A partial factor of 3 (i.e., 10°5) is applied. A full factor of 10 for extrapolation
from a subchronic to chronic exposure duration is not warranted because the
available data suggests that severity of the critical effects (i.e., increased absolute
and relative liver and kidney weights in female rats) did not increase from a 16
week exposure compared with a 66 week exposure duration.
• UFd: The existing database for TCEP consists of subchronic and chronic studies in
rats and mice, a reproductive study in mice, and a developmental study in rats that
included neurodevelopmental endpoints. However, a partial factor of 3 (i.e., 10°5)
is applied for database inadequacies, including lack of a comprehensive
neurotoxicity study in rats (in light of brain lesions in the subchronic and chronic rat
studies) and lack of a multigenerational reproduction study.
Confidence in the principal study is medium. The NTP study employs appropriate dose
levels, uses sufficient numbers of animals, is comprehensive in scope, and includes a 66-week
interim sacrifice as well as an evaluation over the lifespan of the animal. However, the
significantly increased mortality in male and female rats in the high-dose group complicates the
dose-response assessment of noncancer effects after a 104-week exposure duration. Confidence
in the database is medium. However, the database is missing a full neurotoxicity assessment
(suggested by brain lesions in the subchronic and chronic rat studies) and a multigenerational
assessment of reproduction. Overall confidence in the chronic p-RfD is medium.
FEASIBILITY OF DERIVING PROVISIONAL SUBCHRONIC AND CHRONIC
INHALATION RfC VALUES FOR TRIS(2-CHLOROETHYL)PHOSPHATE
No subchronic or chronic toxicity studies of inhaled TCEP were located. An
inadequately reported developmental toxicity study from the Russian literature (Shepelskaya and
Dyshinevich, 1981) was located, but it does not provide an adequate basis for derivation of
p-RfC values.
PROVISIONAL CARCINOGENICITY ASSESSMENT
FOR TRIS(2-CHLOROETHYL)PHOSPHATE
Weight-of-Evidence Descriptor
Under the 2005 Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005), the
available evidence suggests that TCEP is "Likely to be Carcinogenic to Humans" based on
(1) clear evidence of renal tubule cell adenomas in male and female F344/N rats, (2) suggestive
evidence of renal tubule cell adenomas in B6C3Fi mice, (3) clear evidence of renal tubule
adenomas and carcinomas in male ddY mice, (4) the rarity of spontaneous renal tubule cell
tumors in F344/N rats and B6C3Fi mice, (5) suggestive evidence of hepatocellular adenomas in
two strains of male mice, (6) suggestive evidence of leukemia in female ddY mice and in rats of
both sexes, (7) suggestive evidence of Harderian gland adenomas in female B6C3Fi mice, and
(8) equivocal evidence of thyroid follicular cell carcinoma in female F344/N rats.
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Chronic toxicity studies with male and female F344/N rats (NTP, 1991) and with male
ddY mice (Takada et al., 1989) yielded clear evidence of a dose-related significant increase in
renal tubular cell adenomas in response to oral (gavage) administration of TCEP for 2 years
(male and female F344N rats) and following dietary administration for 18 months (male ddY
mice). In the Takada et al. (1989) study, the incidence of renal cell carcinomas in male mice was
also significantly increased. Evidence for TCEP-induced mononuclear cell leukemia in male and
female F344/N rats was considered equivocal in the (NTP, 1991) study because although the
incidences of this cancer were significantly elevated in both sexes, they were within the range of
incidences observed for historical controls. A significantly increased incidence of leukemia was
also observed among female—but not male—ddY mice in the Takada et al. (1989) dietary study,
but no historic control values are presented. The incidences of thyroid follicular cell carcinoma
and combined carcinoma plus adenoma were significantly increased in female—but not male—
rats (NTP, 1991). However, this result provides only equivocal evidence of carcinogenicity due
to a low incidence in high-dose rats (8%) that only marginally exceeded the upper limit of the
historical control range for this neoplasm (6%) in NTP corn oil gavage studies. Results from the
NTP (1991) mouse bioassay provide no clear evidence of carcinogenicity. Nonsignificant
increases in renal tubule cell adenomas or carcinomas in male mice, and in Harderian gland
adenomas and carcinomas in female mice, were considered by NTP (1991) to provide equivocal
evidence of carcinogenicity. In the NTP (1991) study, there is a significant trend for increased
hepatocellular adenomas. In the Takada et al. (1989) study, the incidences of hepatocellular
adenomas and combined adenomas plus carcinomas are significantly elevated in males exposed
to estimated dietary doses of approximately 233 and 1688 mg/kg-day. A significant increase in
forestomach papillomas and squamous cell carcinomas is also documented in female ddY mice
(Takada etal. 1989).
Mode-of-Action Discussion
The U.S. EPA (2005) Guidelines for Carcinogen Risk Assessment defines mode of action
as "a sequence of key events and processes, starting with the interaction of an agent with a cell,
proceeding through operational and anatomical changes and resulting in cancer formation."
Toxicokinetic processes leading to the formation or distribution of the active agent (i.e., parent
material or metabolite) to the target tissue are not part of the mode of action. Examples of
possible modes of carcinogenic action include mutagenic, mitogenic, antiapoptotic (inhibition of
programmed cell death), cytotoxic with reparative cell proliferation and immunologic
suppression.
Ames tests are primarily negative in five studies reported by different investigators. One
study reported a weakly positive result for one strain in the presence of S9 (Nakamura et al.,
1979), but these results are not replicated in studies reported by four other groups of investigators
(Simmon et al., 1977; Haworth et al., 1983; Kubo et al., 2002; Follmann and Wober, 2006).
TCEP did not induce mutation in V79 cells (Sala et al., 1982). Most tests for other types of
genetic damage (DNA strand breaks, clastogenic effects) were negative as well. The
overwhelmingly negative response in the Ames tests conducted for TCEP is consistent with a
general observation that the majority of chemical carcinogens that act on the kidney test negative
in mutation tests with Salmonella typhimurium (Gold et al., 1993; Dybing and Sanner, 1999).
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A potential mode of action for the development of renal tumors in male rats is one
associated with the accumulation of a-2u-globulin10. However, several lines of evidence show
that this mode of action is not relevant to TCEP. First, both male and female rats had
significantly increased incidences of the renal tumors, and male ddY mice also exhibited an
increased incidence of renal cell tumors (Takada et al., 1989). When cancer develops due to a-
2u-nephropathy, the male rat is the only sex and species affected. Second, none of the renal
findings typically associated with a-2u-globulin formation were observed in the study; neither
hyaline droplets nor tubular casts are reported in the kidneys of either sex. Finally, TCEP was
used as a negative control in a recent study investigating the mechanism of compound-mediated
induction of a-2u-globulin formation in male F344N rats (Pahler et al., 1999). In that study,
TCEP did not induce a-2u-globulin formation in circumstances that were positive for other
compounds.
Proliferative and preneoplastic lesions were found in association with the renal tumors in
rats (hyperplasia) and mice (karyomegaly) and with the liver tumors in mice (eosinophilic foci)
(NTP, 1991). There was no evidence of degenerative lesions in either organ (NTP, 1991). The
continuum of cellular changes (hyperplasia, renal tubular cell enlargement, and
adenoma/carcinoma) observed with rats and mice are similar to that which has been observed in
the expression of renal tubular cell cancers in humans (Beckwith, 1999). However, beyond the
general association with known proliferative and preneoplastic lesions, there are no data
available outlining specific potential key events in the mode of action for TCEP-induced tumors
in the kidney or in other organs.
Quantitative Estimates of Carcinogenic Risk
Oral Exposure
The three data sets that were considered to derive an oral slope factor for TCEP are
(1) the combined incidence of renal tubule cell adenomas and carcinomas in male F344/N rats
(NTP, 1991; Table 10), (2) the combined incidence of renal tubule cell adenomas and
carcinomas in female F344/N rats (NTP, 1991), and (3) the combined incidence of renal tubule
cell adenomas and carcinomas in male ddY mice (Takada et al., 1989; Table 11). Tables 4 and 6
summarize these data . The incidences of other tumor types were lower and considered
equivocal evidence of carcinogenicity, so those tumor types are not further considered. BMD
modeling was conducted for male F344/N rats and male ddY mice. Renal tumor incidence in
female rats is considerably lower than in males, so this data set is not modeled. The data
modeled for male F344/N rats and ddY mice are summarized in Tables 10 and 11, including the
calculation of human equivalent doses (HED). Appendix B presents the details of BMD
modeling.
10Some compounds that induce tumors in the kidneys of male rats act through a mechanism involving the a-2u
globulin protein—a component that is not produced in the kidneys of female rats or in other species (including
humans). Therefore, kidney tumors that are known to occur as a consequence of a-2u-globulin nephropathy in male
rats are not considered relevant to the assessment of carcinogenic potential in humans.
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Table 10. Dose-Response Data for the Combined Incidence of Renal Tubular Cell
Adenomas and Carcinomas in Male F344/N Ratsa Given TCEP by Gavage
Animal Dose (mg/kg-day,
adjusted to 7d/wk)
Human Equivalent Doseb
(mg/kg-day)
Incidence
0
0
2/50
31.4
8.8
5/50
62.9
17.7
25/50
aNTP (1991)
bHuman Equivalent Dose = animal dose x (animal bw/human bw)0 25, where animal body weights = 0.439 kg
(control), 0.434 kg (low dose) and 0.438 (high dose), and human body weight = 70 kg
Table B-l and Figure B-l of Appendix B shows model predictions for the combined
incidence of renal tubular cell adenomas and carcinomas in male F344/N rats. The 2-degree
multistage cancer model provides the best fit to the data on combined incidence of renal tubular
cell carcinomas and adenomas in male F344/N rats, yielding a BMDLi0[hed] value of
5.41 mg/kg-day (human-equivalent dose). Model predictions for the combined incidence of
renal tubular cell adenomas and carcinomas in male ddY mice are shown in Table B-2 and
Figure B-2 of Appendix B. The 2-degree model also yielded the best fit to the data on combined
incidence of renal tubular cell adenomas and carcinomas in male ddY mice, with a BMDLi0[hed]
value of 21.45 mg/kg-day (human-equivalent dose).
Table 11. Dose-Response Data for the Combined Incidence of Renal Tubular Cell
Adenomas and Carcinomas in Male ddYY Mice3 Given TCEP by Gavage
Animal Dose (mg/kg-day,
adjusted to 7d/wk)
Human Equivalent Doseb
(mg/kg-day)
Incidence
0
0
2/50
9.3
1.5
0/47
46.6
7.4
2/49
232.8
37.1
5/47
1687.5
261
41/50
"Takadaetal. (1989)
bHuman Equivalent Dose = animal dose x (animal bw/human bw)0 25, where animal body weights = 0.045 kg (all but
highest dose) and 0.040 kg (highest dose), and human body weight = 70 kg
In the absence of a defined mode of action for TCEP, a linear low-dose extrapolation is
applied. Using the lower BMDLi0[hed] of 5.41 mg/kg-day for the combined incidence of renal
tubular cell adenomas and carcinomas in male F344/N rats (NTP, 1991; Matthews et al., 1993)
as the POD, a p-OSF for TCEP is calculated as follows:
p-OSF = 0.1 BMDLio[hed]
= 0.1 ^ 5.41 mg/kg-day
= 0.02 or 2 x 10~2 (mg/kg-day)"1
The oral slope factor for TCEP should not be used with exposures exceeding the POD
(BMDLio[hed] =5.4 mg/kg-day) because at exposures above this level, the fitted dose-response
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model better characterizes what is known about the carcinogenicity of TCEP. Table 12 shows
the doses associated with specific levels of cancer risk based on the p-OSF estimated herein.
Table 12. Doses of TCEP Associated with Some Specific Levels of Cancer Risk
Risk Level
Dose (mg/kg-day)
icr4
5 x 10"3
10"5
5 x icr4
icr6
5 x 10"5
Inhalation Exposure
There are no inhalation studies that address the carcinogenic potential of inhaled TCEP.
Therefore, it is not possible to derive a p-IUR for TCEP.
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Air Contaminants. Part Z, Toxic and Hazardous Substances. Online, http://www.osha.gov/pls/
oshaweb/owadi sp. show_document?p_table=ST AND ARD S&p_id=9992.
Pahler, A., K. Blumbach, J. Herbst and W. Dekant. 1999. Quantitation of o^-globulin in rat
kidney cytosol by capillary electrophoresis. Anal. Biochem. 267(1):203-211.
Sala, M., Z.G. Gu, G. Moens and I. Chouroulinkov. 1982. In vivo and in vitro biological effects
of the flame retardants tris(2,3-dibromopropyl) phosphate and tris(2-chlorethyl)orthophosphate.
Eur. J. Cancer Clin. Oncol. 18(12): 1337-1344.
Shepelskaya, N.R. and N.E. Dyshinevich. 1981. Experimental study of the gonadotoxic effect
of tris(chloroethyl)phosphate. Gig. Sanit. 6:20-21.
Simmon, V.F., K. Kauhanen and R.G. Tardiff 1977. Mutagenic activity of chemicals identified
in drinking water. Dev. Toxicol. Environ. Sci. 2:249-258.
Takada, K., K. Yasuhara, Y. Nakaji et al. 1989. Carcinogenicity study of tris(2-chloroethyl)
phosphate in ddY mice. J. Toxicol. Pathol. 2(2):213-222.
U.S. EPA. 1991. Chemical Assessments and Related Activities (CARA). Office of Health and
Environmental Assessment, Washington, DC. April.
U.S. EPA. 1994. Chemical Assessments and Related Activities (CARA). Office of Health and
Environmental Assessment, Washington, DC. December.
U.S. EPA. 1997. Health Effects Assessment Summary Tables. FY-1997 Update. Prepared by
the Office of Research and Development, National Center for Environmental Assessment,
Cincinnati, OH, for the Office of Emergency and Remedial Response, Washington, DC. July
1997. EPA/540/R-97/036. NTIS PB 97-921199.
28
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U.S. EPA (2000c) Benchmark dose technical guidance document [external review draft]. Risk
Assessment Forum, Washington, DC; EPA/630/R-00/001. Online.
http://www.epa.gov/nceawwwl/pdfs/bmds/BMD-External_10_13_2000.pdf.
U.S. EPA. 2005. Guidelines for Carcinogen Risk Assessment. U.S. Environmental Protection
Agency, Risk Assessment Forum, Washington, DC. EPA/630/P-03/001B. Online.
http://www.thecre.com/pdf/20050404 cancer.pdf.
U.S. EPA. 2006. Drinking Water Standards and Health Advisories. Office of Water,
Washington, DC. Online, http://www.epa.gov/waterscience/criteria/drinking/dwstandards.pdf.
U.S. EPA. 2008. Integrated Risk Information System (IRIS). Online. Office of Research and
Development, National Center for Environmental Assessment, Washington, DC. Online.
http://www.epa.gov/iris/.
Vogel, E.W. and M.J. Nivard. 1993. Performance of 181 chemicals in a Drosophila assay
predominantly monitoring interchromosomal mitotic recombination. Mutagenesis. 8(1):57—81.
WHO (World Health Organization). 1998. Flame retardants: Tris(chloropropyl)phosphate and
Tris(2-chloro ethyl)phosphate. Environmental Health Criteria 209. Online, http://www.mche
m. org/documents/ehc/ehc/ehc209. htm
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APPENDIX A. DETAILS OF BENCHMARK DOSE MODELING FOR THE
PROVISIONAL RfDs
Model Fitting Procedure for Continuous Data
The BMD modeling for continuous data (i.e., relative kidney weight changes) was
conducted with the U.S. EPA's BMD software (BMDS version 2.1 beta). The original data were
modeled with all the continuous models available within the software employing a BMR of
1 SD. An adequate fit was judged based on three criteria: (1) the goodness-of-fit p value
(p > 0.1), (2) magnitude of scaled residuals in the vicinity of the BMR, and (3) visual inspection
of the model fit. In addition to the three criteria forjudging the adequate model fit, whether the
variance needed to be modeled, and if so, how it was modeled also determined final use of the
model results. If a constant variance model was deemed appropriate based on the statistical test
provided in the BMDS (i.e., Test 2), the final BMD results were estimated from a constant
variance model. If the test for constant variance was rejected (p < 0.1), 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; />value
< 0.1), 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 models
varied > 3-fold; otherwise, the BMDL from the model with the lowest AIC was selected as a
potential POD from which to derive an RfD.
Model Predictions for Relative Kidney Weight in Female F344/N Rats Given TCEP by
Gavage for 16 Weeks
All available continuous models in the BMDS (version 2.1 beta) have been fit to the
relative kidney-weight data from the subchronic study (Matthews et al., 1990; NTP, 1991) (see
Table Al). BMD modeling has been performed using the doses administered in the study before
duration adjustment. A default BMR of 1 SD from the control mean was used in the BMD
modeling because no specific criteria on the magnitude of change of relative kidney weight that
would be considered biologically significant of changes could be identified. Due to the lack of a
dose-response relationship, relative kidney-weight data in male rats are not suitable for BMD
modeling. For relative kidney-weight data in females, only the Hill model run with constant
variance met the goodness of fitp value >0.1 criteria and the scaled residual criteria for
assessing adequate model fit, and Test 2 (p = 0.4089) also indicated that using a constant
variance model was appropriate for modeling these data (see Table A-l, Figure A-l). Visual
inspection of the dose-response curve suggested that the dose-response relationship is better
characterized in the low-dose region. Thus, the highest dose was removed from the analysis for
biological considerations, which significantly improved model fit (Figure A-2). Thus, the
estimated BMDisd and BMDLisd based on relative kidney-weight data are 22.74 and
9.66 mg/kg-day, respectively.
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Table A-l. BMD Modeling Results Based on Relative Kidney-Weight Data from Female
F344/N Rats Given TCEP By Gavage for 16 Weeks
Model
Test 2
Test 3
Goodness of fit p value
AIC
bmd1sd
BMDL1sd
All Doses
Lineara'b
0.4546
0.3421
0.0056
-117.304
96.14
65.93
Polynomiala'b
0.4546
0.3421
0.0056
-117.304
96.14
65.93
Powerb,c
0.4546
0.3421
0.0056
-117.304
96.14
65.93
Hillbc
0.4546
0.3421
0.1494
-124.579
17.24
9.06
5 Doses (without the highest dose group)
Lineara'd
0.4089
0.4089
0.0060
-105.457
68.36
51.47
Polynomiala'd
0.4089
0.4089
0.0060
-105.457
68.36
51.47
Powerc'd
0.4089
0.4089
0.0060
-105.457
68.36
51.47
Hillcd
0.4089
0.4089
0.4770
-113.407
22.74
9.66
aRestrict betas > 0
bNonconstant variance
°Restrict power > 1
dConstant variance
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Hill Model with 0.95 Confidence Level
c
a
8
q:
c
11:10 07/31 2000
Figure A-l. Dose-Response Modeling of Relative Kidney Weight (All Dose Groups) in
Female F344/N Rats Given TCEP By Gavage for 16 Weeks
Hill Model. (Version: 2.14; Date: 06/26/2008)
Input Data File: C:\USEPA\BMDS21Beta\Data\lHilTCEHil.(d)
Gnuplot Plotting File: C:\USEPA\BMDS21Beta\Data\lHilTCEHil.plt
Fri Jul 31 11:10:17 2009
BMDS Model Run
The form of the response function is:
Y[dose] = intercept + v*doseAn/(k^n + doseAn)
Dependent variable = Mean
Independent variable = Dose
Power parameter restricted to be greater than 1
The variance is to be modeled as Var(i) = exp(lalpha + rho * ln(mean(i)))
Total number of dose groups = 6
Total number of records with missing values = 0
Maximum number of iterations = 25 0
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Hill
4.6
4.4
4.2
4
3.8
3.6
BMDL
BMD
0 50 100 150 200 250 300 350
Dose
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Default Initial Parameter Values
lalpha = -3.61439
rho =
intercept =
v =
n =
k =
0
3. 69
0. 82
0.207813
2 62
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -n
have been estimated at a boundary point, or have been specified by
the user,
lalpha
rho
intercept
and do not appear in the correlation matrix )
lalpha
1
-1
0.08
0.33
0.31
rho
-1
1
-0.082
-0.33
-0.31
intercept
0.08
-0.082
1
0.12
0.55
0.33
-0.33
0.12
1
0.84
k
0.31
-0.31
0.55
0.84
1
Interval
Variable
Limit
lalpha
0.592029
rho
12.6014
intercept
3.77384
v
1.24726
n
k
195.926
Parameter Estimates
Estimate Std. Err.
-10.2762 5.54513
4.77951 3.99085
3.69228 0.0416158
0.915182 0.16943
1 NA
101.282 48.289
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
-21.1445
-3. 04242
3.61071
0.583105
6.63697
NA - Indicates that this parameter has hit a bound
implied by some inequality constraint and thus
has no standard error.
Table of Data and Estimated Values of Interest
Dose
Obs Mean
Est Mean Obs Std Dev Est Std Dev Scaled Res.
0
22
44
10
8
10
10
3.69
3.83
4.03
4.1
3. 69
3.86
3.97
4.12
0.13
0.17
0.13
0.22
0.133
0.148
0.158
0.173
-0.0541
-0.49
1.21
-0.325
33
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FINAL
9-30-2009
175 8 4.18 4.27 0.17 0.189 -1.38
350 5 4.51 4.4 0.13 0.203 1.19
Model Descriptions for likelihoods calculated
Model A1: Yij = Mu(i) + e(ij)
Var{e(ij)} = SigmaA2
Model A2: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma(i)^2
Model A3: Yij = Mu(i) + e(ij)
Var{e(ij)} = exp(lalpha + rho*ln(Mu(i)))
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e(i)
Var{e(i)} = Sigma^
Likelihoods of Interest
Model
A1
A2
A3
fitted
R
Log (likelihood)
69.858620
72 .204625
69.952821
67.289523
39.035206
# Param's
7
12
8
5
2
AIC
-125.717241
-120.409251
-123.905642
-124.579046
-74.070412
Explanation of Tests
Test 1: Do responses and/or variances differ among Dose levels?
(A2 vs. R)
Test 2: Are Variances Homogeneous? (A1 vs A2)
Test 3: Are variances adeguately modeled? (A2 vs. A3)
Test 4: Does the Model for the Mean Fit? (A3 vs. fitted)
(Note: When rho=0 the results of Test 3 and Test 2 will be the same.)
Tests of Interest
Test -2*log(Likelihood Ratio) Test df p-value
Test 1 66.3388 10 <.0001
Test 2 4.69201 5 0.4546
Test 3 4.50361 4 0.3421
Test 4 5.3266 3 0.1494
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose levels
It seems appropriate to model the data
The p-value for Test 2 is greater than .1. Consider running a
homogeneous model
The p-value for Test 3 is greater than .1. The modeled variance appears
to be appropriate here
The p-value for Test 4 is greater than .1. The model chosen seems
to adeguately describe the data
34
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9-30-2009
Benchmark Dose Computation
Specified effect =
Risk Type =
Confidence level =
BMD =
BMDL =
Estimated standard deviations from the control mean
0.95
17.2397
9.05952
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9-30-2009
Hill Model with 0.95 Confidence Level
Hill
4.3
4.2
4.1
4
3.9
3.8
3.7
3.6
BMDL
BMD
0
20
40
60
80
100
120
140
160
180
11:18 07/31 2009 uose
Figure A-2. Dose-Response Modeling of Relative Kidney Weight (Without the Highest
Dose Group) in Female F344/N Rats Given TCEP by Gavage for 16 Weeks
Hill Model. (Version: 2.14; Date: 06/26/2008)
Input Data File: C:\USEPA\BMDS21Beta\Data\lHilTCEHil.(d)
Gnuplot Plotting File: C:\USEPA\BMDS21Beta\Data\lHilTCEHil.plt
Fri Jul 31 11:18:58 2009
BMDS Model Run
The form of the response function is:
Y[dose] = intercept + v*doseAn/(k^n + doseAn)
Dependent variable = Mean
Independent variable = Dose
rho is set to 0
Power parameter restricted to be greater than 1
A constant variance model is fit
Total number of dose groups = 5
Total number of records with missing values = 0
Maximum number of iterations = 25 0
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
36
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FINAL
9-30-2009
Default Initial Parameter Values
alpha = 0.0279122
rho =
intercept =
v =
n =
k =
0
3. 69
0.49
1.47172
54.45
Specified
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -rho
have been estimated at a boundary point, or have been specified by
the user,
alpha
intercept
v
n
k
and do not appear in the correlation matrix )
alpha intercept v n
2 . 3e-009
-1.3e-008
3.3e-008
-1.7e-008
2.3e-009
1
-0.59
0.19
0.35
-1. 3e-008
-0.59
1
-0.75
0.37
3. 3e-008
0.19
-0.75
1
-0.41
-1. 7e-008
0.35
0.37
-0.41
1
Interval
Variable
Limit
alpha
0. 035433
intercept
3.7854
v
0.670228
n
4.84442
k
47.6277
Estimate
0.0251533
3.68803
0.483805
2.19378
31.5522
Parameter Estimates
Std. Err.
0.00524483
0. 0496782
0. 0951159
1.35239
8.20197
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
0.0148736
3.59066
0.297381
-0.456867
15.4766
Table of Data and Estimated Values of Interest
Dose
0
22
44
88
175
10
8
10
10
Obs Mean
3. 69
3. 83
4.03
4.1
4.18
Est Mean Obs Std Dev Est Std Dev Scaled Res.
3.69
3.84
4.01
4.13
4.16
0.13
0.17
0.13
0.22
0.17
0.159
0.159
0.159
0.159
0.159
0.0393
-0.16
0.31
-0.513
0.342
Model Descriptions for likelihoods calculated
37
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FINAL
9-30-2009
Model A1: Yij = Mu(i) + e(ij)
Var{e(ij)} = SigmaA2
Model A2 : Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma(i)^2
Model A3: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma^
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e(i)
Var{e(i)} = Sigma/S2
Likelihoods of Interest
Model
A1
A2
A3
fitted
R
Log(likelihood)
61.956501
63.945662
61.956501
61.703602
42.726595
# Param's
6
10
6
5
2
AIC
-111.913002
-107.891324
-111.913002
-113.407204
-81.453190
Explanation of Tests
Test 1: Do responses and/or variances differ among Dose levels?
(A2 vs. R)
Test 2: Are Variances Homogeneous? (A1 vs A2)
Test 3: Are variances adeguately modeled? (A2 vs. A3)
Test 4: Does the Model for the Mean Fit? (A3 vs. fitted)
(Note: When rho=0 the results of Test 3 and Test 2 will be the same.)
Tests of Interest
Test
Test 1
Test 2
Test 3
Test 4
-2*log(Likelihood Ratio) Test df
42.4381
3.97832
3.97832
0.505798
p-value
<.0001
0.4089
0.4089
0. 477
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose levels
It seems appropriate to model the data
The p-value for Test 2 is greater than .1. A homogeneous variance
model appears to be appropriate here
The p-value for Test 3 is greater than .1. The modeled variance appears
to be appropriate here
The p-value for Test 4 is greater than .1. The model chosen seems
to adeguately describe the data
Benchmark Dose Computation
Specified effect = 1
Risk Type = Estimated standard deviations from the control mean
38
-------
Confidence level
BMD
BMDL
FINAL
9-30-2009
0.95
22 .7443
9.65587
39
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FINAL
9-30-2009
APPENDIX B. DETAILS OF BENCHMARK DOSE MODELING
FOR THE PROVISIONAL ORAL SLOPE FACTOR
Model-Fitting Procedure for Cancer Incidence Data
The model fitting procedure for dichotomous cancer incidence data is as follows. The
multistage-cancer model in the U.S. EPA BMDS is fit to the incidence data using the extra risk
option. The multistage-cancer model is run for all polynomial degrees up to n-1 (where n is the
number of dose groups including control). Adequate model fit is judged by three criteria:
goodness-of-fit/?-value (p > 0.1), visual inspection of the dose-response curve, and scaled
residual at the data point (except the control) closest to the predefined BMR. Among all the
models providing adequate fit to the data, the lowest BMDL is selected as the POD when the
difference between the BMDLs estimated from these models are more three-fold; otherwise, the
BMDL from the model with the lowest AIC is chosen. In accordance with U.S. EPA (2000)
guidance, benchmark doses (BMDs) and lower bounds on the BMD (BMDLs) associated with an
extra risk of 10% are calculated.
Model Predictions for Renal Tubular Cell Neoplasms (Combined Adenoma and
Carcinoma) in Male F344/N Rats (NTP, 1991)
Model predictions for the combined incidence of renal tubular cell carcinomas and
adenomas in male F344/N rats are shown in Table B-l and Figure B-l. The 2-degree multistage
cancer model provides adequate fit, yielding a BMDLio value of 5.41 mg/kg-day
(human-equivalent-dose) and a p-OSF of 0.018 (mg/kg-day)"1.
Table B-l: Model Predictions for Renal Tubular Cell Neoplasms (Combined
Adenoma and Carcinoma) in Male F344/N Rats (NTP, 1991)
Model
Degrees
of
Freedom
x2
x2
Goodness
of Fit p-
Valueb
AIC
BMDiohed
(mg/kg-
day)
BMDLio hed
(mg/kg-day)
Cancer
slope factor
Multistage-Cancer
(1-degree)0
1
7.46
0.0063
130.95
4.03
2.94
0.034028
Multistage-Cancer
(2-degree)c
1
1.85
0.1734
124.63
7.55
5.41
0.018481
40
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FINAL
9-30-2009
Multistage Cancer Model with 0.95 Confidence Level
0.7
Multistage Cancer
Linear extrapolation
0.6
0.5
0.4
0.3
0.2
0.1
0
BMD
BMD
0
2
4
6
8
10
12
14
16
18
Dose
14:03 01/30 2009
Figure B-l. Fit of the 2-Degree Multistage Cancer Model to Data on the Combined
Incidence of Renal Tubular Cell Adenomas and Carcinomas in Male F344/N Rats
(NTP, 1991)
BMDs and BMDLs indicated are associated with an extra risk of 10%, and are human-equivalent doses in units of
mg/kg-day
41
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Model Predictions for Renal Tubular Cell Neoplasms (Combined Adenoma and
Carcinoma) in Male ddY Mice (Takada et al., 1989)
Model predictions for the combined incidence of renal tubular cell adenomas and
carcinomas in male ddY mice are shown in Table B-2 and Figure B-2. The 2-degree multistage
cancer model provides adequate fit, yielding a BMDLio value of 21.45 mg/kg-day
(human-equivalent-dose) and a p-OSF of 0.005 (mg/kg-day)"1.
Table B-2: Model Predictions for Renal Tubular Cell Neoplasms (Combined
Adenoma and Carcinoma) in Male ddY Mice (Takada et al., 1989)
Model
Degrees
of
Freedom
x2
x2
Goodness
of Fit
p-\ alueb
AIC
BMDiohed
(mg/kg-day)
BMDLiohed
(mg/kg-day)
Cancer slope
factor
Multistage-Cancer
(l-degree)°
3
6.53
0.0886
124.02
19.27
14.92
0.00670225
Multistage-
Cancer (2-
degree)c
2
1.95
0.3775
121.49
42.48
21.45
0.00466094
Multistage-Cancer
(3-degree)°
2
1.95
0.3775
121.49
42.48
21.23
0.00471001
Multistage-Cancer
(4-degree)°
2
1.95
0.3775
121.49
42.48
21.18
0.00472245
42
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FINAL
9-30-2009
Multistage Cancer Model with 0.95 Confidence Level
1
Multistage Cancer
Linear extrapolation
0.8
0.6
0.4
0.2
0
BMDL
BMD
0 50 100 150 200 250
Dose
14:17 01/30 2009
Figure B-2. Fit of the 2-Degree Multistage Cancer Model to Data on the Combined
Incidence of Renal Tubular Cell Adenomas and Carcinomas in Male ddY Mice
BMDs and BMDLs indicated are associated with an extra risk of 10%, and are human-equivalent doses in units of
mg/kg-day
43
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