PUBLIC RELEASE DRAFT

May 2024

EPA Document# EPA-740-D-24-009

May 2024

Office of Chemical Safety and
Pollution Prevention

Draft Cancer Human Health Hazard Assessment for Diisononyl

Phthalate (DINP)

Technical Support Document for the Draft Risk Evaluation

CASRNs: 28553-12-0 and 68515-48-0

(Representative Structure)

xvEPA

United States

Environmental Protection Agency

May 2024


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TABLE OF CONTENTS

1	INTRODUCTION	6

2	GENOTOXICITY AND MUTAGENICITY	7

3	CANCER HAZARD IDENTIFICATION AND CHARACTERIZATION	10

3.1	Human Evidence	10

3.2	Animal Evidence	10

3.2.1	Liver Tumors	10

3.2.1.1 Conclusions on Liver Tumors	11

3.2.2	Mononuclear Cell Leukemia	14

3.2.2.1 Conclusions on Mononuclear Cell Leukemia	15

3.2.3	Kidney Tumors	17

3.2.3.1 Conclusions on Kidney Tumors	19

3.2.4	Other Tumors	21

4	POSTULATED MODE OF ACTION FOR LIVER TUMORS IN RATS AND MICE	24

4.1	Postulated Mode of Action in Rats and Mice	24

4.1.1	Key Event 1: PPAR.a Activation	25

4.1.2	Key Event 2: Alterations in Cell Growth Pathways	26

4.1.3	Key Event 3: Perturbation of Cell Growth and Survival	26

4.1.4	Key Event 4: Selective Clonal Expansion of Preneoplastic Foci	27

4.1.5	Modulating Factors	28

4.2	Dose-Response Concordance of Key Events with Tumor Response	28

4.3	Temporal Association of Key Events with Tumor Response	32

4.4	Strength, Consistency, and Specificity of Association of Tumor Response with Key Events.... 32

4.5	Biological Plausibility and Coherence	33

4.6	Other Modes of Carcinogenic Action	33

4.7	Uncertainties and Limitations	35

4.8	Weight of Scientific Evidence: Cancer Classification	36

4.9	Human Relevancy	37

5	CONCLUSIONS AND NEXT STEPS	38

REFERENCES	39

Appendix A PATHOLOGY WORKING GROUP REVIEW FOR SPONGIOSIS HEPATIS

AND MNCL (EPL, 1999)	45

LIST OF TABLES

Table 2-1. Summary of Genotoxicity Studies of DINP	7

Table 3-1. Incidences of Neoplastic Lesions in the Livers of Male and Female F344 Rats Exposed to

DINP for 24 Months (Lington et al., 1997; Bio/dynamics, 1986)	 11

Table 3-2. Incidence of Liver Tumors in Male and Female F344 Rats Exposed to DINP in the Diet for 2

Years (Covance Labs, 1998b)	12

Table 3-3. Incidence of Neoplastic Lesions in the Liver of Male and Female SD Rats Exposed to DINP

in the Diet for 2 Years (Bio/dynamics, 1987)a	13

Table 3-4. Incidence of Liver Tumors in Male and Female B6C3F1 Mice Exposed to DINP in the Diet
for 2 Years (Covance Labs, 1998a)	13

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Table 3-5. Incidence of MNCL in F344 Rats Exposed to DINP for 2 Years (Lington et al., 1997;

Bio/dynamics, 1986)	 14

Table 3-6. Incidence of MNCL in F344 Rats Exposed to DINP in the Diet for 2 Years (Covance Labs,

1998b 	15

Table 3-7. MNCL as a Cause of Unscheduled Death in F344 Rats Exposed to DINP in the Diet

(Covance Labs, 1998b)	15

Table 3-8. Incidence of Kidney Tumors in Male F344 Rats Exposed to DINP in the Diet for 2 Years

(Covance Labs, 1998b)	18

Table 3-9. Incidence of Kidney Tumors in F344 Rats Exposed to DINP for 2 Years (Lington et al.,

1997; Bio/dynamics, 1986)	 19

Table 3-10. Incidence of Tumors in Pancreas, Testes, and Uterus in SD Rats Exposed to DINP for 2

Years (Bio/dynamics, 1987)	23

Table 4-1. Dose-Response Concordance for PPAR.a MOA in Rats	30

Table 4-2. Dose-Response Concordance for PPAR.a MOA in Mice	31

Table 4-3. Summary of Active ToxCast Assays for DINP	34

LIST OF APPENDIX TABLES

TableApx A-l. Incidence of MNCL and Selected Hepatic Lesions at Terminal Sacrifice (104 Weeks)
in the Lington et al. (1997) Study in F344 Rats as Determined by the PWG (EPL, 1999)

	46

Table Apx A-2. Incidence of MNCL and Selected Hepatic Lesions at Terminal Sacrifice (104 Weeks)

in the Covance Labs (1998b) Study in F344 Rats as Determined by the PWG (EPL, 1999)

	46

Table Apx A-3. Comparison of Spongiosis Hepatis with MNCL as Determined by the PWG (EPL,

1999)	47

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ABBREVIATIONS AND ACRONYMS

a2u-globulin Alpha 2u-globulin

AhR	Aryl hydrocarbon receptor

ALP	Alkaline phosphatase

ALT	Alanine aminotransferase

AST	Aspartate aminotransferase

CAR	Constitutive androstane receptor

CASRN	Chemical abstracts service registry number

CPSC	Consumer Product Safety Commission (U.S.)

DINP	Diisononyl phthalate

DNA	Deoxyribonucleic acid

ECB	European Chemicals Bureau

ECHA	European Chemicals Agency

EFSA	European Food Safety Authority

EPA	Environmental Protection Agency (U.S.)

F344	Fischer 344 (rat)

GLP	Good Laboratory Practice

IARC	International Agency for Research on Cancer

KE	Key event

LOAEL	Lowest-ob served-adverse-effect level

MNCL	Mononuclear cell leukemia

MOA	Mode of action

NF-kB	Nuclear factor kappa B

NICNAS	National Industrial Chemicals Notification and Assessment Scheme

NOAEL	No-observed-adverse-effect level

OCSPP	Office of Chemical Safety and Pollution Prevention

OEHHA	Office of Environmental Health Hazard Assessment (California)

OPPT	Office of Pollution Prevention and Toxics

POD	Point of departure

PPARa	Peroxisome proliferator-activated receptor alpha

PWG	Pathology Working Group

PXR	Pregnane X receptor

ROS	Reactive oxygen species

SACC	Science Advisory Committee on Chemicals

SD	Sprague-Dawley (rats)

TSCA	Toxic Substances Control Act

U.S.	United States

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ACKNOWLEDGMENTS

This report was developed by the United States Environmental Protection Agency (U.S. EPA or the
Agency), Office of Chemical Safety and Pollution Prevention (OCSPP), Office of Pollution Prevention
and Toxics (OPPT).

Acknowledgements

The Assessment Team gratefully acknowledges the participation, input, and review comments from
OPPT and OCSPP senior managers and science advisors and assistance from EPA contractors SRC, Inc.
(Contract No. 68HERH19D0022). Special acknowledgement is given for the contributions of technical
experts from EPA's Office of Research and Development (ORD) and Office of Pesticide Programs
(OPP), including Christopher Corton and Gregory Akerman for their technical review of EPA's cancer
mode of action analysis.

As part of an intra-agency review, the draft DINP Risk Evaluation was provided to multiple EPA
Program Offices for review. Comments were submitted by EPA's Office of Air and Radiation (OAR),
Office of Children's Health Protection (OCHP), Office of General Counsel (OGC), ORD, and Office of
Water (OW).

Docket

Supporting information can be found in the public docket, Docket ID (EPA-HQ-OPPT-2018-0436).
Disclaimer

Reference herein to any specific commercial products, process or service by trade name, trademark,
manufacturer, or otherwise does not constitute or imply its endorsement, recommendation, or favoring
by the United States Government.

Authors: John Allran, Anthony Luz, Ashley Peppriell, Myles Hodge, Sailesh Surapureddi, Christelene
Horton, Collin Beachum (Branch Chief)

Contributors: Amy Benson, Susanna Wegner, Abhilash Sasidharan

Technical Support: Mark Gibson, Hillary Hollinger

This report was reviewed and cleared by OPPT and OCSPP leadership.

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1 INTRODUCTION

On May 24, 2019, the United States Environmental Protection Agency (U.S. EPA or the Agency)
received a request, pursuant to 40 CFR 702.37, from ExxonMobil Chemical Company, through the
American Chemistry Council's High Phthalates Panel (ACC H	), to conduct a risk evaluation

for diisononyl phthalate (DINP) (CASRNs 28553-12-0 and 68515-48-0) (Docket ID: EPA-HO-OPPT-
2018-0436). EPA determined that these two CASRNs should be treated as a category of chemical
substances as defined in 15 U.S.C § 2625(c). On August 19, 2019, EPA opened a 45-day public
comment period to gather information relevant to the requested risk evaluation. The Agency reviewed
the request (along with additional information received during the public comment period) and assessed
whether the circumstances identified in the request constitute conditions of use under 40 CFR 702.33,
and whether those conditions of use warrant inclusion within the scope of a risk evaluation for DINP.
EPA determined that the request meets the applicable regulatory criteria and requirements, as prescribed
under 40 CFR 702.37. EPA granted the request on December 2, 2019, and published the draft and final
scope documents for DINP in August 2020 and 2021, respectively (	)2\, 2020).

Following publication of the final scope document, one of the next steps in the Toxic Substances
Control Act (TSCA) risk evaluation process is to identify and characterize the human health hazards of
DINP and conduct a dose-response assessment to determine the toxicity values to be used to estimate
risks from DINP exposures. This technical support document summarizes the cancer hazards associated
with exposure to DINP. Non-cancer hazards associated with exposure to DINP are summarized in a
separate technical support document, the Draft Non-cancer Human Health Hazard Assessment for
Diisononyl Phthalate (DINP) (	24).

The carcinogenicity of DINP has been evaluated in existing assessments by Health Canada, U.S.
Consumer Product Safety Commission (U.S. CPSC), European Chemicals Agency (ECHA), Australia
National Industrial Chemicals Notification and Assessment Scheme (NICNAS), and California's Office
of Environmental Health Hazard Assessment (OEHHA) (ECCC/HC. 2020; EC/HC. * , a I) \ :01
Tomar et at.. 2013; NICNAS. 2012; U.S. CPSC. 2010; ] U' _WV; 1 i 1"X. 2001). To date, DINP
has been classified as a carcinogen by California OEHHA and is listed under California's Proposition 65
as a carcinogen (OEHHA. JO I l'< nnar et at.. 2013). Other authoritative agencies have not classified
DINP as a carcinogen or evaluated DINP quantitatively for carcinogenic risk to human health.

This technical support document summarizes the available evidence for the carcinogenicity of DINP, the
majority of which comes from experimental animal models. The remainder of this document is
organized as follows:

•	Section 2 summarizes available genotoxicity data for DINP.

•	Section 3 summarizes available human and animal evidence for the carcinogenicity of DINP.

•	Section 4 summarizes available liver tumor data and postulated mode of action (MOA) for liver
tumors in rodents.

•	Section 5 summarizes EPA's conclusions and next steps.

•	Appendix A summarizes the results of a Pathology Working Group's review for spongiosis
hepatis and mononuclear cell leukemia .

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2 GENOTOXICITY AND MUTAGENICITY

The genotoxicity of DINP has been evaluated in several existing assessments, which have consistently
concluded that DINP is not genotoxic nor is it likely to be genotoxic (ECCC/HC. 2020; EC/HC. 2015;
ECU.A. 2013; NU ^	i 2010; EFSA. 2005; ECB. 1WV; 1 i VSC. 20011 EPA

reviewed available genotoxicity studies of DINP that were cited in existing assessments (Table 2-1) and
considered newer studies published between 2014 and 2019. No new genotoxicity studies of DINP were
identified.

The mutagenic and genotoxic potential of DINP has been evaluated in 20 studies (Table 2-1). Available
studies include two in vivo micronucleus tests in rodents, one in vitro chromosomal aberration assay,
two in vitro mouse lymphoma assays, five bacterial reverse mutation assays, one in vitro unscheduled
DNA synthesis assay, and nine in vitro cell transformation assays. No evidence of mutagenic activity
was observed in five bacterial reverse mutation assays or two in vitro mouse lymphoma assays (with or
without metabolic activation). DINP did not induce chromosomal aberrations in Chinese hamster ovary
cells in vitro, cause unscheduled DNA synthesis in primary rat hepatocytes, or induce clastogenic effects
or micronuclei formation in vivo in studies of mice or rats. Of the nine available in vitro transformation
assays, only one study reported a positive result for transformation in Balb/c-3T3 A31 mouse cells in the
absence of metabolic activation (Microbiological Associates. 1982c).

Consistent with the conclusions of existing assessments of DINP, available studies that evaluated the
mutagenic and genotoxic potential of DINP are consistently negative. Therefore, EPA considers the
weight of scientific evidence to indicate that DINP not likely to be genotoxic or mutagenic.

Table 2-1. Summary of Genotoxicity Studies of DINP

Test
Tvpc

Test System
(Species/Strain/Sex)

Dose/Duration

Metabolic
Activation

Result

Rct'crcnce(s)

Chromosomal aberrations - in vivo

Micronucleus
(bone marrow)
(Adhered to
OECD 474)

Male CD-I mice

Oral (gavagc) doses of
0,500, 1,000, or 2,000
mg/kg-day for 2 days;
sacrificed on day 3

Not applicable

Negative for
micronuclei

(McLCee et aL

2000)

Chromosomal
aberrations in
femoral bone
marrow cells

Male F344 rats

Oral (gavage) doses of
0, 0.5, 1.7, or 5.0
mL/kg-day for 5 days

Not applicable

Negative for
micronuclei

(Microbiological

Associates. 1982b)

( luoniosonial jIvitjIioiis in vitro

Chromosomal
aberrations

Chinese hamster
ovary cells

0, 40, 80, or 160 jig/mL
for 3 hours (with
activation) or 20 hours
(without activation)

± Aroclor-
induced rat
liver S9

Negative for

chromosomal

aberrations

(McKee et aL,
2000)

(iono mulJlions in vitro

Mouse
lymphoma
mutation assay

L5178Y+/- mouse
lymphoma cells

0, 0.001,0.01,0.1, 1.0,
10, 100 nL/mL (±S9)

± Aroclor-
induced rat
liver S9

Negative for
mutagenicity

i iG Mason

ResearshinstiM^

1982a)

Mouse
lymphoma
mutation assay

L5178Y+/- mouse
lymphoma cells

1.5-8 nl/ml (-S9);
0.05-0.6 nL/mL (+ S9)

± Aroclor-
induced rat
liver S9

Negative for
mutagenicity

(Barber et al., 2000)

Bacterial
reverse

mutation assay

S. typhimurium strains
TA 98, TA 100,

0.1,0.5,2.5,5, 10
|u L/plate

± Aroclor-
induced rat
liver S9

Negative for
mutagenicity

(EG&G Mason
Research Institute,
1982b)

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Test
Tvpc

Test System
(Species/Strain/Sex)

Dose/Duration

Metabolic
Activation

Result

Ret'erence(s)



TA 1535, TA 1537,
TA 1538









Bacterial
reverse

mutation assay

S. typhimurium strains
TA 98, TA 100,
TA 1535, TA 1537

0, 100,333, 1,000,
3,333, 10,000 (ig/plate

± Aroclor
1254-induced
rat or hamster
liver S9

Negative for
mutagenicity

CZeiaer et al. 1985)

Bacterial
reverse

mutation assay

S. typhimurium strains
TA 98, TA 100,
TA 1535, TA 1537

20-5,000 (ig/plate

± Aroclor-
induced rat
liver S9

Negative for
mutagenicity

rCBA!

1986) as reported
bv ECB C2003)la

Bacterial
reverse

mutation assay
(plate

incorporation
assay)

S. typhimurium strains
TA 98, TA 100,
TA 1535, TA 1537,
TA 1538

0.5-5,000 (ig/plate

± Aroclor-
induced rat
liver S9

Negative for
mutagenicity

CMcKee et al..

2000)

Bacterial
reverse

mutation assay
(pre-incubation
;iss;iy)

S. typhimurium strains
TA 98, TA 100,
TA 1535, TA 1537

20-5,000 (ig/plate

± Aroclor-
induced rat
liver S9

Negative for
mutagenicity

CMcKee et al..

2000)

( )iIkt uonolo\icil\ ;iss;i\ s

Unscheduled
DNA synthesis

R;il Ik'|XUoc\ lc
primary culture

ii. (i 625, 1 \\ 1 5. 5 ii.

\o

\o increase in
unscheduled
DNA
synthesis

i in Bionetics.

10.0 jiL/mL

b)

In vitro cell
transformation

Balb/c-3T3 A31
mouse cells

125-3,750 nL/mL

No

No significant
increase in
transformed
foci

CLitton Bionetics,
1985)

In vitro cell
transformation

Balb/c-3T3 A31
mouse cells

2.5-254.5 jig/mL

No

No significant
increase in
transformed
foci

CLitton Bionetics,
1981)

In vitro cell
transformation

Balb/c-3T3 A31
mouse cells

0.0326-3,260 ^g/mL

No

No significant
increase in
transformed
foci

CLitton Bionetics,
1982a)

In vitro cell
transformation

Balb/c-3T3 A31
mouse cells

0.125-3.750 jiL/mL

No

No significant
increase in
transformed
foci

CBarber et al, 2000)

In vitro cell
transformation

Balb/c-3T3 A31
mouse cells

0.1-1 \\L!raL

± rat liver S9

No significant
increase in
transformed
foci

CMicrobioloaical
Associates, 1982a)

In vitro cell
transformation

Balb/c-3T3 A31
mouse cells

0.03-1 (iL/mL

No

No significant
increase in
transformed
foci

CMicrobioloaical
Associates. 1982c)

In vitro cell
transformation

Balb/c-3T3 A31
mouse cells

0.01-1.0 nL/mL

No

No significant
increase in

CMicrobioloaical
Associates. 1981)

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Test
Tvpc

Test System
(Species/Strain/Sex)

Dose/Duration

Metabolic
Activation

Result

Ret'erence(s)









transformed
foci



In vitro cell
transformation

Balb/c-3T3 A31
mouse cells

0.03-1 (iL/mL

No

Positive
(significant
increase in
transformed
foci)

(Microbiological

Associates, 1982d)

In vitro cell
transformation

Balb/c- 3T3 mouse
cells co-cultured with
transformed cloned
cells (strain 4-1-1)

5-5,000 ng/mL

No

No increase in
proliferation
rate of Balb/c
3T3 cells

(Fushiwaki et aL,

2003)

" Studv reports were not reasonably available to EPA. Information is as reported by ECB (2003).

234

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3 CANCER HAZARD IDENTIFICATION AND
CHARACTERIZATION

This section summarizes available human (Section 3.1) and animal evidence (Section 3.2) for the
carcinogenicity of DINP. Section 3.2 discusses evidence for mononuclear cell leukemia (MNCL),
kidney tumors, and other tumors observed in experimental animal models. Evidence for liver tumors in
rodents and EPA's mode of action (MOA) analysis for liver tumors is provided in Section 4.

3.1	Human Evidence

No epidemiologic studies were identified by Health Canada (2018) that examined the association
between DINP and its metabolites and biomarkers of cancer.

EPA identified two new medium quality studies that evaluated exposure to DINP and cancer. The first
medium quality study, a case-control analysis by Parada et al., 2018 (2018) with a mortality follow-up
component among women in the Long Island Breast Cancer Study Project, evaluated breast cancer
mortality among cases with spot urine sample collected 3 months after breast cancer diagnosis. Inverse
associations were observed between urine levels of two DINP metabolites {i.e., MCNP and MCOP) and
breast cancer for single quintiles, but the associations were not statistically significant.

The second medium quality study, a nested case-control study by Reeves et al. (2019) of the Women's
Health Initiative prospective cohort, investigated the association between incident breast cancer cases in
postmenopausal women and DINP. The authors found no significant association with one urinary DINP
metabolite {i.e., MCOP) and breast cancer in analysis using either ln-transformed or quartile exposure
variables (adjusted odds ratio in models using ln-MCOP = 1.02; 95% CI: 0.90-1.16]). Findings were
similar in models stratified by estrogen/progesterone receptor status and body mass index.

3.2	Animal Evidence

Four 2-year dietary studies evaluating the carcinogenicity of DINP in rodent models are available,
including three studies of male and female Fischer 344 (F344) and Sprague-Dawley (SD) rats (Covance
Labs. 1998b; Lington et al. \ , i namics. 1987) and one study of male and female B6C3F1 mice
(Covance Labs. 1998a). Available studies have been discussed extensively in existing assessments of
DINP. No new carcinogenicity studies of DINP with experimental laboratory animals were identified by
EPA.

Across available studies, statistically significant increases in liver tumors, MNCL, and kidney tumors
have been reported. Non-statistically significant increases in tumors in the testes, uterus, and pancreas
have also been reported. Evidence for liver tumors, MNCL, kidney tumors, and other tumors is
discussed in Sections 3.2.1 through 3.2.4.

3.2.1 Liver Tumors

The Draft Non-cancer Human Health Hazard Assessment for Diisononyl Phthalate (DINP) (
2024) describes the non-cancer liver effects observed following exposure to DINP in experimental
animal models. Notably, many of the non-cancer liver effects observed in rodents following oral
exposure to DINP comprise a suite of effects that may represent a progression from non-cancer to cancer
{e.g., increased liver weight, increased serum levels of ALT, AST, and ALP, histopathologic lesions
such as hepatocellular hypertrophy and focal necrosis).

DINP has been evaluated for carcinogenicity in two 2-year dietary studies of F344 rats (Covance Labs.
1998b; Lington et al.. 1997). one 2-year dietary study of SD rats (Bio/dynamics. 1987). and one 2-year

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dietary study of B6C3F1 mice (Covance Labs. 1998a). Statistically significant increased incidences of
tumors in the liver were reported in three out of four of the chronic 2-year studies (see Table 3-1 through
Table 3-4). In one study, no statistically significant increases in neoplastic nodules and/or hepatocellular
carcinomas were observed in male or female F344 rats treated with up to 307 to 375 mg/kg-day DINP
for two-years (Table 3-1)—although hepatocellular cancer was observed in 3 out of 80 males from the
high-dose groups compared to 0 out of 80 in controls (Lington et ai. 1997; Bio/dynamics. 1986).

Two other studies of F344 and SD rats by Covance Labs (1998b) and Bio/dynamics (1987).
respectively, included higher doses than Lington et al. (1997). and reported significant increases in
hepatocellular adenoma and/or carcinoma (Table 3-2 and Table 3-3). Increased incidence of
hepatocellular carcinoma (males only), and adenomas or carcinomas combined (both sexes) were
observed in male and female F344 rats treated with up to 733 to 885 mg/kg-day DINP for 2 years
(Covance Labs. 1998b) (Table 3-2). In the second study, hepatocellular carcinomas were significantly
increased in high-dose female SD rats treated with 672 mg/kg-day DINP for 2 years, while no
significant increase in neoplastic nodules or hepatocellular carcinomas were observed in male SD rats
treated with up to 553 mg/kg-day DINP for 2 years (Table 3-3) (Bio/dynamics. 1987).

Finally, in a 2-year chronic study of DINP with B6C3F1 mice, the incidence of carcinomas was
significantly increased in males at 1,560 mg/kg-day and females at 910 mg/kg-day and above, while the
combined incidence of hepatocellular adenomas and carcinomas were significantly increased in both
males (>742 mg/kg-day) and females (>336 mg/kg-day) (Table 3-4) (Covance Labs. 1998a).

3.2.1.1 Conclusions on Liver Tumors

Collectively, available studies provide consistent evidence that chronic oral exposure to DINP can cause
treatment-related liver tumors in both sexes of several strains of rats {i.e., F344 and SD) and mice
(B6C3F1). EPA further considers the weight of evidence for liver carcinogenesis and its underlying
MOA in Section 4.

Table 3-1. Incidences of Neoplastic Lesions in the Livers of Male and Female F344 Rats Exposed

to DINP for 24 Months (Lington et a

1997; Bio/dynamics, 1986)

Lesion

Dose Group
mg/kg-day (ppm)

Control

15M/18F
(300)

152 M /184 F
(3,000)

307 M / 375 F
(6,000)

Males'

Neoplastic nodules

3/81 (3.7%)

1/80(1.3%)

1/80(1.3%)

1/80(1.3%)

Hepatocellular cancer

0/81 (0%)

0/80 (0%)

0/80 (0%)

3/80 (3.8%)

Neoplastic nodules or cancer (combined)

3/81(3.7%)

1/80(1.3%)

1/80(1.3%)

4/80 (5.0%)

Ivimik-s

Neoplastic nodules

0/80 (0%)

2/81 (2.5%)

0/80 (0%)

1/80(1.3%)

Hepatocellular cancer

1/81 (1.2%)

0/81 (0%)

0/80 (0%)

1/80(1.3%)

Neoplastic nodules or cancer (combined)

1/81 (1.2%)

2/81 (2.5%)

0/80 (0%)

2/80 (2.5%)

Source: Table 8 in Liimton et al. (1997)

M = male; F = female

a Number of animals with lesion/ total number of animals examined. Percent lesion incidence in parentheses. No
statistically significant increases in hepatocellular nodules and/or cancer was observed in either sex.

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Table 3-2. Incidence of Liver Tumors in Male and Female F344 Rats Exposed to DINP in the Diet

^	/tTkiS. T «Ts if*® £X 1 «Tsl"&fD 1 QQfiKW ^

Lesion

Dose Group
mg/kg-dav (ppm)

Control

29 M / 36 F
(500)

88 M / 109 F
(1,500)

359 M / 442 F
(600)

733 M / 885 F
(12,000)

MiiL-s

Hepatocellular adenoma

4/65& (6%)

3/50 (6%)

2/50 (4%)

6/65 (9%)

6/65 (15%)

Hepatocellular carcinoma

1/65 (2%)

0/50 (0%)

0/50 (0%)

1/65 (2%)

12/65* (18%)

Adenoma or carcinoma (combined)

5/65 (8%)

3/50 (6%)

2/50 (4%)

7/65 (11%)

18/65* (28%)

Females

Hepatocellular adenoma

0/65 (0%)

1/49 (2%)

0/50 (0%)

1/65 (2%)

3/65 (5%)

Hepatocellular carcinoma

1/65 (2%)

0/49 (0%)

0/50 (0%)

1/65 (2%)

5/65 (8%)

Adenoma or carcinoma (combined)

1/65 (2%)

1/49 (2%)

0/50 (0%)

2/65 (3%)

8/65* (12%)

Source: U.S. CPSC ("2001): Table IX-1 foe. 68s); text rases 68-71 and AoDcndix B.

M = male; F = female

* = statistically significant at p < 0.05 by one or more of the following: Fisher's Exact test, Poly-3, Logistic Regression, or
Life Table analysis.

" Where results are of borderline significance or greater, level of statistical significance computed by logistic regression is
given. Significance value for trend is given in the column for the control group. Significance values for these findings
calculated using different statistical tests are given in Appendix B, section A. Analysis of individual animal data as
performed bv the National Toxicoloev Proeram (TJ.S. CPSC. 2001).

h Number of animals with neoplasm/ total number of animals examined. Percent tumor incidence in parentheses. Based on
extraction and analysis of individual animal data as reported in U.S. CPSC ("2001). Overall incidence for control. 6.000 DDin
and 12,000 ppm groups (n = 65) includes incidence data for unscheduled deaths, interim sacrifice at week 78 and terminal
sacrifice. Overall incidence for the remaining groups includes incidence data for unscheduled deaths and terminal sacrifice.

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313	Table 3-3. Incidence of Neoplastic Lesions in the Liver of Male and Female SD Rats Exposed to

314	DINP in the Diet for 2 Years (Bio/dvnamics. 1987 V	

Lesion

Dose Group
mg/kg-dav (ppm)

Control

27 M / 33 F
(500)

271 Ml 331 F
(5,000)

553 M / 672 F
(10,000)

Males

Hepatocellular carcinoma

2/70 (2.9%)

2/69 (2.9%)

6/69 (8.7%)

4/70 (5.7%)

Neoplastic nodulc(s)''

2/70 (2.9%)

5/69 (7.2%)

6/69 (8.7%)

5/70 (7.1%)

Ivmak-s

Hepatocellular carcinoma

0/70 (0%)t

0/70 (0%)

5/70 (7.1%)

7/70 (10%)*

Neoplastic nodule(s)

1/70 (1.4%)

1/70 (1.4%)

5/70 (7.1%)

2/70 (2.9%)

Source: AroendixK. Fieure 1. dd. 11 (m 426 of the studv rcoort PDF) (Bio/dvnamics. 1987).

* Statistically significant (p < 0.05) from the control group by a two-tailed Fisher's exact test
f Statistically significant trend (p < 0.05) based on a Chi-square contingency trend test calculated for this review.

"Data in this table indicate all animals assessed for histopathology throughout the study; i.e., including the interim
sacrifice, the terminal sacrifice, and unscheduled deaths. For late-developing tumors (hepatocellular carcinoma, pancreatic
islet cell tumors, testicular interstitial cell tumors), statistical analysis was performed excluding animals that died or were
sacrificed up to 12 months, leaving n = 57, 57, 59, 59 in males and n = 59, 56, 60, 59 in females in the control, low-, mid-
and high-dose groups, respectively.

h Pathology report does not define this lesion further, which is a reporting deficiency that reduces the ability to compare
results of Bio/dynamics (1987) to those of other studies which report incidences of hepatocellular adenomas, carcinomas,
and adenomas or carcinomas, combined.

315

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Table 3-4. Incidence of Liver Tumors in Male and Female B6C3F1 Mice Exposed to DINP in the

Lesion

Dose Group
mg/kg-dav (ppm)

Control

90M/112F
(500)

276 M / 336 F
(1,500)

742 M / 910 F
(600)

1,560 M / 1,888 F
(12,000)

Males

Hepatocellular adenoma

10/706 (14%)

7/67 (10%)

8/66 (12%)

15/65 (23%)

13/70 (19%)

Hepatocellular carcinoma

10/70 (14%)

8/67 (12%)

10/66 (15%)

17/65 (26%)

20/70* (29%)

Adenoma or carcinoma (combined)

16/70 (23%)

13/67 (19%)

18/66 (27%)

28/65* (43%)

31/70* (44%)

Females

Hepatocellular adenoma

2/70 (3%)

4/68 (6%)

5/68 (7%)

4/67 (6%)

18/70* (26%)

Hepatocellular carcinoma

1/70 (1%)

2/68 (3%)

5/68 (7%)

7/67* (10%)

19/70* (27%)

Adenoma or carcinoma (combined)

3/70 (4%)

5/68 (7%)

10/68* (15%)

11/67* (16%)

33/70* (47%)

Source: U.S. CPSC (2001) Table IX-6 (rase 73) and Aroendix B.

M = male; F = female

* = significant from the control at p < 0.05 by logistic regression analysis

" Where results are of borderline significance or greater, level of statistical significance computed by logistic regression is
given. Significance value for trend is given in the column for the control group. Significance values for these findings
calculated usins different statistical tests are siven in Aroendix B. section B (U.S. CPSC. 2001).
h Number of animals with tumor/total number of animals examined. Percent tumor incidence in parentheses.

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3,2,2 Mononuclear Cell Leukemia

MNCL has been observed in F344 rats in two 2-year dietary studies (Covance Labs. 199\h. 1 mgton et
ai. 1997; Bio/dynamics. 1986). In contrast, MNCL has not been observed in SD rats in a 104 week
study (Bio/dynamics. 1987) nor in B6C3F1 mice exposed to DINP for at least 104 weeks (Covance

Labs. 1998a).

Lington et al. (1997) reported the incidence data for MNCL. The incidence of MNCL was statistically
significantly increased in the mid- and high-dose groups for both sexes when compared with the
concurrent control groups (Table 3-5). MNCL was detected in 41, 35, 60, and 64 percent of males and
27, 25, 38, and 54 percent of females in the control, low-, mid-, and high-dose groups, respectively. As
reported by the study authors, MNCL has a significant increasing trend over time and was the most
common cause of unscheduled deaths and/or morbidity. In many of the treated rats, MNCL was detected
at a very early stage and was limited to an increase in the mononuclear cells in the hepatic sinusoids.

Table 3-5. Incidence of MNCL in F344 Rats Exposed to DINP for 2 Years (Lington et ai.. 1997;
Bio/dynamics. 1986)	

Lesion

Dose Group
(mg/kg-day) (ppm)

Control

15 Male / 18 Female
(300)

152 Male / 184 Female
(3,000)

307 Male/375 Female
(6,000)

Males"

33/81 (41%)

28/80 (35%)

48/80* (60%)

51/80* (64%)

Females0

22/81 (27%)

20/81 (25%)

30/80* (38%)

43/80* (54%)

Source: Table 8 in Lington et al. (1997)

" Number of animals with lesion/ total number of animals examined. Percent lesion incidence in parentheses.
* Statistically significant at p < 0.05 when compared to the control incidence using Fisher's Exact test; statistical analysis
performed bv Lington et al. 0 997).

In a study by Covance Labs (1998b). the incidences of MNCL in male and female rats receiving the
6,000 and 12,000 ppm concentrations of DINP in the diet were significantly increased with statistically
significant dose-related trends (Table 3-6). The incidences of MNCL in the recovery groups were also
significantly greater than in the controls. There is some evidence that the onset of MNCL was earlier in
treated males. MNCL was first detected in the 6,000 ppm group via an unscheduled death at study day
352. In comparison, MNCL was first detected in the control group at an interim sacrifice at day 549.
Decreases in hemoglobin concentration and red blood cell numbers and a statistically significant
increase in mean spleen weight in both male and female rats were correlated with the incidence of
MNCL. Between 31 and 60 percent of unscheduled deaths in the study were attributable to MNCL
(Table 3-7), demonstrating that this lesion is life-threatening in rats treated with DINP.

A Histopathology Peer Review and a Pathology Working Group (PWG) review (EPL. 1999) was
conducted on selected lesions of the liver and spleen observed in F344 rats in the 2-year bioassays
reported by Lington et al. (1997) and Covance Labs (1998b). The PWG review evaluated the
significance of spongiosis hepatis, foci of cellular alteration, primary hepatocellular neoplasms in the
liver, and the significance of MNCL. Notably, the results of the Histopathology Peer Review and PWG
(EPL. 1999) generally confirmed the original findings of the study pathologists), including incidence of
MNCL in F344 rats in both studies. PWG findings are further discussed in Appendix A.

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Table 3-6. Incidence of MNCL in F344 Rats Exposed to DINP in the Diet for 2 Years (Covance
Labs. 1998h)gftt'	



Dose Group
mg/kg-day (ppm)

Sex

Control

29 M / 36 F

88 M /109 F

359 M / 442 F

733 M / 885 F

High-Dose /
Recovery''
637 M/ 774 F (12,000)



(500)

(1,500)

(6,000)

(12,000)

Males

22/65 (34%)

23/50 (46%)

21/50 (42%)

32/65* (49%)

30/65* (46%)

31/50*^(62%)

Females

17/65 (26%)

16/49 (33%)

9/50(18%)

30/65* (46%)

29/65* (45%)

24/50*d (48%)

Source: U.S. CPSC (2001) text rases 68-71 and Aroendix B.

M = male; F = female

* = statistically significant at p < 0.05 by one or more of the following: Fisher's Exact test, Poly-3, Logistic Regression, or
Life Table analysis.

" Analysis of individual animal data as performed by the National Toxicology Program and reported in the text and
Appendix B of U.S CPSC (2001).

b The high-dose/recovery group received 12,000 ppm for 78 weeks, followed by a 26-week recovery period during which the
animals received basal diet alone.

c Number of animals with neoplasm/ total number of animals examined. Percent tumor incidence in parentheses. Based on
extraction and analvsis of individual animal data as rcDortcd in U.S. CPSC (2001). Overall incidence for control. 6.000 DDin
and 12,000 ppm groups (n = 65) includes incidence data for unscheduled deaths, interim sacrifice at week 78, and terminal
sacrifice. Overall incidence for the remaining groups includes incidence data for unscheduled deaths and terminal sacrifice.
d Statistical significant at p < 0.05 by Fisher's Exact test conducted by Syracuse Research Corporation.

Table 3-7. MNCL as a Cause of Unscheduled Death in F344 Rats Exposed to DINP in the Diet

'Covance

Labs. 1998b)

Sex

Dose Group
mg/kg-day (ppm)

Control

29 M / 36 F

88 M /109 F

359 M / 442 F

733 M / 885 F

Recovery"
637 M / 774 F
(12,000)



(500)

(1,500)

(6,000)

(12,000)

Males

H22b (32%)

8/23 (35%)

7/21 (33%)

16/32 (50%)

18/30 (60%)

14/31 (45%)

Females

7/17(41%)

5/16(31%)

3/9 (33%)

12/29 (41%)

13/30 (43%)

12/24 (50%)

Source: Compiled from incidence data and death comments in Table 10E (rases 365 and 381) in Covance Labs (\,998b).
M = male; F = female

" The high-dose/recovery group received 12,000 ppm for 78 weeks, followed by a 26-week recovery period during which
test animals received basal diet alone.

h Number of deaths attributed to MNCL/total number of deaths; percentage of deaths attributable to MNCL in parentheses.

3.2.2.1 Conclusions on Mononuclear Cell Leukemia

The incidence of MNCL was significantly elevated in male and female F344 rats exposed to DINP in
the diet when compared to study control animals in two independent carcinogenicity studies (Covance
Labs. 1998b; Limetom et al. 1997). In Lington et al. (1997). incidences of MNCL were statistically
significantly increased at 152 and 307 mg/kg-day in the males (60 to 64 percent in treated rats versus 41
percent in concurrent controls) as well as in the females at 184 and 375 mg/kg-day (38 to 54 percent in
treated rats versus 27 percent in concurrent controls). In the 2-year study in F344 rats conducted by
Covance Labs (1998b). incidences of MNCL were significantly increased at 359 and 733 mg/kg-day in
the treated males (46 to 62 percent incidence) compared to concurrent controls (34 percent incidence) as
well as in the treated females at 442 and 885 mg/kg-day (45 to 48 percent) compared to concurrent
controls (26 percent incidence). Inconsistent with findings from the two chronic studies of F344 rats,
MNCL was not observed in male or female SD rats treated with up to 553 to 672 mg/kg-day DINP for 2

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years (Bio/dynamics. 1987) or male and female B6C3F1 mice treated with up to 1,560 to 1,888 mg/kg-
day DINP for two years (Covance Lai 3a).

MNCL is a spontaneously occurring neoplasm of the hematopoietic system that reduces lifespan and is
one of the most common tumor types occuring at a high background rate in the F344 strain of rat
(Thomas et ai. 2007). Historical control data from NTP have demonstrated an increase in the
spontaneous background incidence of MNCL in untreated male and female F344 rats from 7.9 and 2.1
percent in males and females, respectively, in 1971 to 52.5 and 24.2 percent in males and females,
respectively, from 1995 through 1998 (Thomas et ai. 2007). Spontaneous incidence of MNCL in other
strains of rat appear to be rare. Brix et al. (2005) report the incidence of MNCL in female Harlan SD rats
to be 0.5 percent in NTP 2-year studies. Further, MNCL does not appear to occur naturally in mice
(Thomas et al.. 2007).

Given the high and variable background rate of MNCL in F344 rats, it is important to consider
concurrent control data, historical control data, and time to onset of MNCL to assist in determining
whether observed increases in MNCL are treatment-related.

EPA acknowledges that MNCL has a high background incidence in F344 rats as is noted by concurrent
control incidence of 26 to 41 percent in the two studies described above (Covance Labs. 1998b; Lington
etai. 1997). The incidence of MNCL was significantly elevated in male and female rats exposed to
DINP in the diet when compared to concurrent controls in these studies; however, no historical control
data from the performing laboratories were provided. EPA's Guidelines for Carcinogen Risk Assessment
(2005) state that the most relevant historical control data comes from the same laboratory and supplier
and are within 2 to 3 years of the study under review, and that other historical control data should be
used with extreme caution. Lack of relevant laboratory historical control data for incidence and time to
onset of MNCL make it challenging to determine if the increase in MNCL observed in high-dose F344
rats treated with DINP, which was statistically significant compared to concurrent controls, is treatment-
related and is a source of uncertainty.

The limited information available indicates that time to onset of MNCL was shorter in DINP-treated
animals compared to concurrent controls. In Lington et al. (1997). the study authors reported that MNCL
has a significant increasing trend over time and was the most common cause of unscheduled deaths
and/or morbidity. In many of the treated rats, MNCL was detected at a very early stage but was limited
to an increase in the mononuclear cells in the hepatic sinusoids. Similar to the Lington study, in the 2-
year study in rats conducted by Covance Labs (1998b). there is some evidence that the onset of MNCL
was earlier in treated males, with the first detected in the 359 mg/kg-day group via an unscheduled death
at study day 352 compared to the first detected in the control group at an interim sacrifice at day 549.

Another source of uncertainty is lack of MO A information for induction of MNCL in F344 rats. The
MO A for induction of MNCL in F344 rats is unknown. Lack of MO A information makes it difficult to
determine human relevancy. There is additional uncertainty related to the human correlate to MNCL in
F344 rats. Some researchers have suggested that based on the biological and functional features in the
F344 rat, MNCL is analogous to large granular lymphocyte (LGL) in humans (Caldwell et i 9;
Caldwell. 1999; Reynolds and Foon. 1984). There are two major human LGL leukemias, including
CD3+ LGL leukemia and CD3- LGL leukemia with natural killer cell activity (reviewed in (Maronpot
et al.. 2016; Thomas et al.. 2007). Thomas et al. (2007) contend that MNCL in F344 rats shares some
characteristics in common with aggressive natural killer cell leukemia (ANKCL) in humans, and that
ANKCL may be a human correlate. However, Maronpot (2016) point out that ANKCL is extremely rare
with less than 98 cases reported worldwide, and its etiology is related to infection with Epstein-Barr

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virus, not chemical exposure. This is in contrast to MNCL in F344 rats, which is a more common form
of leukemia and is not associated with a viral etiology. However, under EPA's Guidelines for
Carcinogen Risk Assessment (	2005). site concordance is not always assumed between

animals and humans.

EPA considers the available data inadequate for delineation of a plausible sequence of events leading to
development of MNCL in rats exposed to DINP. Therefore, the significance of MNCL and its biological
relevance for human cancer risk remains uncertain. Other regulatory agencies have also considered the
human relevance of MNCL. Generally, other agencies such as Australia NICNAS (2012)1 Health
Canada (EC/HC. 2015).2 U.S. CPSC (JO 10),3 and EC HA (2013)4 have concluded that MNCL observed
in F344 rats is not human relevant or has unclear human relevance and refrained from using MNCL to
predict cancer risk in humans. In contrast, California OEHHA ("Tomar et at.. ^ ) lists MNCL in F344
rats as one of the tumor types to support the Proposition 65 listing of DINP; however, OEHHA does not
appear to draw any specific conclusions related to the MOA underlying MNCL or its human relevance.

Overall, considerable scientific uncertainty remains. Therefore, EPA did not consider it appropriate to
derive quantitative estimates of cancer hazard for data on MNCL from these two studies in F344 rats.

3.2.3 Kidney Tumors

Statistically significant increased incidence of kidney tumors have been observed in one 2-year dietary
study of F344 rats ("Covance Labs. 1998b). Malignant renal tubule cell carcinomas were detected in two
high-dose (733 mg/kg-day) male rats and four males treated with 637 mg/kg-day DINP for 78 weeks
followed by a 26-week recovery period (Table 3-8). However, incidence of renal tubular carcinomas
only reached statistical significance in the recovery group.

1	Australia NICNAS concluded "In rat carcinogenicity studies, increased incidences of MCL, kidney and liver neoplasia were
observed. MCL was observed in DINP toxicological studies with Fischer 344 rats but not with Sprague Dawley rats. MCL is
a common neoplasm in Fischer 344 rats with no comparable tumour type in humans and its increased incidence after chronic
exposure to some substances is a strain-specific effect (Caldwell .1.999). Therefore. MCL observed in Fischer 344 rats is not
regarded as relevant to humans" (p. 49 of (NICNAS. 2012V).

2	Health Canada concluded "Mononuclear cell leukemia of the spleen was also reported in Fischer rats. However, this type of
lesion is likely specific to aging rats of this strain and is unlikely to be relevant to humans (Health Canada 2015d)." (p. 95 of

(Health Canada. 20.1.5)).

3	U.S. CPSC concluded "Elevated incidence of MNCL is a common finding in chronic studies in Fischer rats. Due to its high
background rate, MNCL is often considered to be of uncertain relevance in the evaluation of the cancer hazard in humans.
Furthermore, no hematopoietic neoplasms were found in Sprague-Dawley CD rats treated with DINP-A (Bio/dvnamics.
1986) or in mice treated with DINP-1 (Caldwell. .1.999). Therefore, MNCL will not be used to predict cancer risk in humans"
(P- 82 of (U.S. CPSC. 2010V).

4	ECHA concluded "With regard to MNCL, the review by (Thomas et at. 2007') suggests that unlike previously thought there
might be a human counterpart to MNCL in rats. The probability that the MNCL seen in the Exxon and Aristech studies
would be a result of chance findings seems low. Nevertheless, the increased incidences of MNCL remain difficult to interpret
in the light of the high and variable background incidences and the unclear relevance to humans. DINP is not genotoxic, and
it is argued (Caldwell. .1.999) that MNCL follows a threshold mode of action. The available information does not allow to
draw definite conclusions on the matter. However, as a reasonable approach it would be possible to conclude that the MNCL
findings further strengthen the selected NOAELs for repeated dose toxicity (15 and 88 mg/kg bw/day). Since such conclusion
would not influence the outcome of the current risk assessment, the endpoint is not taken further to the risk characterization
step" (p. 98 of (ECHA. 20.1.3)).

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Table 3-8. Incidence of Kidney Tumors in Male F344 Rats Exposed to DINP in the Diet for 2
Years (C'ovance Labs. 1998bYbc	

Lesion

Dose Group
mg/kg-day (ppm)

Control

29 M / 36 F
(500)

88 M /109 F
(1,500)

359 M / 442 F
(6,000)

733 M / 885 F
(12,000)

High-Dose/ Recovery
637 M / 774 F
(12,000)

Renal tubular
carcinoma

0/65
(0%)

0/55
(0%)

0/55
(0%)

0/65
(0%)

2/65
(3.1%)

4/50*
(8.0%)

Source: U.S. CPSC (2001) text pages 68-71 and Appendix B.

* = statistically significant at p<0.05 by one or more of the following: Fisher's Exact test, Poly-3, LogisticRegression,
or Life Table analysis.

a Analysis of individual animal data as performed by the National Toxicology Program and reported in the textand
Appendix B of U.S. CPSC (2001)

b The high-dose/recovery group received 12,000 ppm for 78 weeks, followed by a 26-week recovery periodduring
which they received basal diet alone.

c Number of animals with neoplasm/ total number of animals examined. Percent tumor incidence in parentheses. Based
on extraction and analvsis of individual animal data as reported in U.S. CPSC (2001)

Overall incidence for control, 6,000 ppm and 12,000 ppm groups (n = 65) includes incidence data for unscheduled
deaths, interim sacrifice at week 78 and terminal sacrifice. Overall incidence for the remaining groups includes
incidence data for unscheduled deaths and terminal sacrifice.

Lington et al. (1997) reported the incidence data for selected transitional cell carcinomas, transitional
cell adenomas, and tubular cell carcinomas and adenomas in the kidney (Table 3-9). Renal tubular cell
carcinomas were observed in one male in the low-dose group and two males in the high-dose group and
renal transitional cell carcinoma was observed in three male rats in the mid-dose group. However,
neither tumor type was statistically significantly increased. Further, no preneoplastic renal lesions were
detected in rats of either sex and no neoplastic lesions were detected in the kidneys of female rats.

Kidney tumors have not been observed in male or female SD rats treated with up to 553 to 672 mg/kg-
day DINP for 2 years (Bio/dynamics. 1987) or male and female B6C3F1 mice treated with up to 1,560
to 1,888 mg/kg-day DINP for 2 years (Covance Labs. 1998a).

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Table 3-9. Incidence of Kidney Tumors in F344 Rats Exposed to DINP for 2 Years (Lington et ai.

1997; Bio/dynamics. 1986) 	

Lesion

Dose Group
mg/kg-day (ppm)

Control

15M/18F
(300)

152 M /184 F
(3,000)

307 M / 375 F
(6,000)

Milk's

Transitional cell carcinoma

0/81 (0%)

0/80 (0%)

3/80 (3.8%)

0/80 (0%)

Transitional cell adenoma

0/81 (0%)

0/80 (0%)

0/80 (0%)

0/80 (0%)

Tubular cell carcinoma

0/81 (0%)

1/80(1.3%)

0/80 (0%)

2/80 (2.5%)

Tubular cell adenoma

0/81 (0%)

0/80 (0%)

0/80 (0%)

0/80 (0%)

Iv mules

1 liinsilional cell ciiicmoniii

ii. SI (ii",,)

(1 S 1 (II",,)

(I Si) ((

(I Si) (0",,)

Transitional cell adenoma

0/81 (0%)

0/81 (0%)

0/80 (0%)

0/80 (0%)

Tubular cell carcinoma

0/81 (0%)

0/81 (0%)

0/80 (0%)

0/80 (0%)

Tubular cell adenoma

0/81 (0%)

0/81 (0%)

0/80 (0%)

0/80 (0%)

Source: Table 8 in Liimton et al. (1997)

M = male; F = female

a Number of animals with lesion/ total number of animals examined. Percent lesion incidence in parentheses.
b Statistically significant at p < 0.05 when compared to the control incidence using Fisher's Exact test;statistical
analysis performed by Liimton et al. (1997).

3.2.3.1 Conclusions on Kidney Tumors

Two tumor types have been reported in the kidneys of male F344 rats following chronic oral exposure to
DINP, including renal transitional cell carcinomas and renal tubule cell carcinomas.

Renal transitional cell carcinoma, an uncommon tumor type in rats, has been reported in two out of four
rodent carcinogenicity studies. Lington et al. (1997) report transitional cell carcinoma in 3/80 mid-dose
(151 mg/kg-day) male F344 rats. However, the response was not statistically significant and did not
occur in a dose-dependent manner (not observed in high-dose males [307 mg/kg-day]). Similarly, in a
study conducted by Covance Labs (1998b). transitional cell carcinoma was detected in 1/65 male F344
rats treated with 359 mg/kg-day DINP; however, the response was not statistically significant and did
not occur in high-dose (733 mg/kg-day) or high-dose recovery (637 mg/kg-day) males. Renal
transitional cell carcinoma was not reported in male SD rats treated with up to 553 mg/kg-day DINP
(Bio/dynamics. 1987) or male B6C3F1 mice treated with up to 1,560 mg/kg-day DINP (Covance Labs.
1998a). and has not been reported in female mice or rats at any dose. Given the lack of dose-response
and statistical significance across available studies, the low incidence of renal transitional cell
carcinomas observed in male F344 rats is considered to be of uncertain toxicological significance.

Renal tubule cell carcinomas have also been reported in two of four rodent carcinogenicity studies. In
the study conducted in F344 rats by Covance Labs (1998b). renal tubule cell carcinoma was observed in
2/65 high-dose (733 mg/kg-day) males and 4/50 recovery high-dose (637 mg/kg-day) males compared
to 0/65 in the control group. The response in recovery males was statistically significant relative to the
control group. In the Lington et al. (1997) study, a non-statistically significant increase in renal tubule
cell carcinoma was observed in 1/80 low-dose (15 mg/kg-day), 0/80 mid-dose (152 mg/kg-day), and

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2/80 high-dose (307 mg/kg-day) male F344 rats. Renal tubule cell carcinomas were not observed in SD
rats treated with up to 533 mg/kg-day DINP (Bio/dynamics. 1987) or in male B6C3F1 mice treated with
up to 1,560 mg/kg-day DINP (Covance Labs (1998a)). No preneoplastic or neoplastic lesions were
observed in female rats or mice at any dose.

The male rat specific alpha 2u-globulin (a2U-globulin) MOA has been implicated as being causative of
renal tubule cell carcinomas. U.S. EPA (1991)5 and I ARC (1995) 6 have published related criteria for
establishing an a2u-globulin MOA for this tumor type. EPA does not consider kidney tumors arising
through a a2U-globulin MOA to be human relevant (	). Data are available to support

many, but not all of, the EPA and IARC criteria for an a2u-globulin MOA. The three specific criteria for
establishing an a2u-globulin MOA include demonstration (1) that renal tubule cell carcinomas only occur
in male rats, (2) immunohistochemical evidence, and (3) histological evidence. In the case of DINP,
these three requisites have been met across four chronic studies: kidney tumors were only observed in
male rats, and the weight of evidence indicates that DINP is not genotoxic. Much of the additional
evidence supporting a a2U-globulin MOA comes from Caldwell et al.'s (1999) retrospective evaluation
of archived kidney tissue taken from the 12-month interim sacrifice from the chronic rat study
conducted by Lington et al. (1997). Caldwell et al. report a dose-dependent increase in the accumulation
of a2u-globulin and increased droplet size in the kidneys of high-dose male (but not female) rats. Cell
proliferation measured via immunohistochemical staining for proliferating cell nuclear antigen in kidney
sections was not statistically significantly elevated in high-dose males (125 percent of controls) or
females (112 percent of control).

Photomicrographs for proliferating cell nuclear antigen and a2u-globulin staining showed foci of
proliferating cells and a2U-globulin accumulating in proximal tubule cells of the P2 segment; however,
some cell proliferation was also observed in PI and P3 cells. Histopathologic re-analysis of kidney
sections showed a dose-dependent increase in minimal tubular regeneration (incidence 6/9, 10/10, 9/10,
and 10/10 in control, low-, mid-, and high-dose males, respectively) and minimal tubular epithelial
hypertrophy (0/9, 0/10, 10/10, and 9/10 in control, low-, mid-, and high-dose males, respectively).
Tubular epithelial hypertrophy was not observed in control or high-dose females; however, minimal
tubular regeneration was observed in 1/10 high-dose female. Collectively, Caldwell et al. concluded that
findings were consistent with an a2u-globulin MOA.

Additional histopathological findings consistent with an a2u-globulin MOA have been noted. For
example, a dose-related increase in incidence of mineralization of renal papilla was reported in the
kidneys of male, but not female, F344 rats in the chronic study conducted by Covance Labs (1998a).

Generally, EPA's three primary criteria for establishing an a2u-globulin MOA have been met. However,
data are not available to inform all of the IARC criteria and several findings raise uncertainty. First,
reversible binding of DINP to a2u-globulin has not been demonstrated. Additionally, chronic exposure to

5	EPA criteria include (1) an increase in number and size of hyaline (protein) droplets in kidney proximal tubule cells of
treated male rats; (2) immunohistochemical evidence of Ofeu-globulin accumulating protein in the hyaline droplets; and (3)
histopathological evidence of kidney lesions associated with ofeu-globulin nephropathology. The Agency also acknowledges
additional information that may be useful for the analysis that are consistent with IARC criteria (e.g., chemical is negative for
genotoxicity, reversible binding of chemical to Ofeu-globulin, sustained cell division in the proximal tubule of the male rat).

6	IARC criteria include (1) tumors occur only in male rats, (2) acute exposure exacerbates hyaline droplet formation, (3) 
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DINP has been shown to increase absolute and relative kidney weight in both male and female rats
(Covance Labs. 1998b; Limetom et A s , <'< » aamics. 1987) as well as cause a significant dose-
related increase in chronic progressive nephropathy in female mice (Covance Labs. 1998a); however,
this lesion was not elevated in the high-dose recovery group females, indicating its reversibility. These
kidney effects cannot be explained by an a2U-globulin MOA.

Other agencies have evaluated the renal tubule cell carcinoma MOA. The U.S. CPSC (2010).7 Australia
NICNAS (2012),8 and EC HA (2013) 9 have all concluded that the renal tubule cell carcinomas observed
in male rats occur through an a2u-globulin MOA that is not relevant for use in human health risk
assessment. Although Health Canada (EC/HC. 2015)10 concluded that certain effects observed in the
kidneys of female rats and mice cannot be explained by an a2U-globulin MOA, Health Canada
considered the kidney tumors in rodents to be of little or unclear relevance to humans. In contrast,
California OEHHA concluded that "a2U-globulin accumulation in the renal tubules of male rats do not
explain the renal tubule carcinomas observed in DINP-exposed rats" and that renal tubule cell
carcinomas were one of the tumor types listed to support the Proposition 65 listing of DINP (Tomar et
al. 2013).

Although some uncertainty remains, much of the available literature supports an a2u-globulin MOA to
explain the incidences of renal tubule cell carcinomas observed in male rats exposed to DINP. EPA does
not consider kidney tumors arising through a ocu-globulin MOA to be human relevant (IJ..S J. 1.1A.
Therefore, EPA did not consider it appropriate to derive quantitative estimates of cancer hazard for data
on kidney tumors observed in these studies.

3,2,4 Other Tumors	

The carcinogenicity of DINP was investigated in a Good Laboratory Practice (GLP)-compliant 2-year
dietary study in SD rats by Bio/dynamics (1987). Incidence data for select histopathological
observations and results from statistical analyses are provided in Table 3-10. In addition to findings in
the liver and kidney previously discussed, tumors were noted in the pancreas, testes, and uterus.
However, for these organs histopathologic examination was only conducted on control and high-dose
rats.

Pancreatic islet cell adenomas (8/70 treated vs 6/70 controls) and carcinomas (4/70 treated vs 1/70
controls) were observed at a slightly higher incidence in the high-dose males compared to controls, and
the nonsignificant incidences of pancreatic tumors were considered to be within the range of normal

7	The U.S. CPSC concluded "A small number of renal tubular cell carcinomas were observed only in males exposed to 1.2
percent DINP. Furthermore, there is experimental evidence that these tumors arose by a mechanism involving the
accumulation of a2u-globulin (Caldwell et al. 1999). a2u-Globulin is a protein that is specific to the male rat. Renal tubular
cell tumors induced by this mechanism are not considered relevant to human risk assessment (Schaeffer 1991)" (p.81 of (U.S.
CPSC 2010))

8	Australia NICNAS concluded "kidney tumours in male rats appear consistent with a specific gender- and species-specific
alpha 2(i-globulin accumulation mechanism that is not regarded as relevant to humans" (p. 49 of (NICNAS. 20.1.2)).

9	ECHA concluded "The available new information on the carcinogenicity of DINP further supports the conclusions of the
EU Risk Assessment concerning renal tumors (EC 2003a). These neoplasms are assumed to have modes of actions which are
not considered to be relevant for humans (alpha-2u-globulin)" (p. 98 of (ECHA. 20.1.3)).

10	Health Canada concluded "Renal tubular cell carcinomas were also reported in one chronic study in rats. It has been
suggested that the mechanism responsible for these tumours was related to accumulation of a2u-globulin, a protein specific to
the male rat (Health Canada 2015d). While this type of neoplastic lesion has not been observed in female rats, increased
kidney weights accompanied by histopathological changes were noted in female rats exposed for 2 years (Covance Labs.
1998b) and treatment-related nephropathy was noted in female mice in another chronic study conducted by the same author
(Covance Labs. 1998a). Those kidney effects cannot be explained by an a2u-globulin mode of action. Overall, findings in the
kidneys of rodents could be considered of little or unclear relevance to humans" (p. 95 of (EC/HC. 20.1.5)).

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biological variation. Furthermore, in the females, pancreatic islet cell adenomas were only observed in
one high-dose and one control animals, and no pancreatic islet cell carcinomas were noted in females.

In the testes, incidences of interstitial cell hyperplasia were significantly increased at the high-dose
(22/70) compared to controls (4/70) and were also reported to exceed historical controls. Testicular
interstitial cell tumors was increased at the high-dose (7/70) compared to controls (2/70); however, the
increase in tumors was not statistically significant and was reported to be within the range of historical
controls.

Similarly, in the uterus, incidence of endometrial hyperplasia was significantly increased at the high-
dose (13/69) compared to controls (2/70). Endometrial adenocarcinoma was observed in 2/69 females at
the high-dose compared to 0/70 controls; however, the increase in tumors was not statistically
significant.

It is plausible that the significantly increased incidences of hyperplasia noted in the testes and uterus at
the high-dose are proliferative responses that can lead to the slight (not significant) increases in
testicular and uterine tumors. However, the fact that the incidences of these tumors is low and, for the
testes data, within the range of historical controls, there is not strong evidence of a carcinogenic
response. Furthermore, the lack of examination of the low- and mid-dose groups limits the examination
of dose-dependency for the cancer incidence in these organs and may miss low-dose effects on any
hormonally-influenced tumors or receptor-mediated carcinogenicity. Finally, tumors in the testes and
uterus were not noted in other chronic studies of DINP in rodents. Overall, there is too much uncertainty
for EPA to consider using these data to derive quantitative estimates of cancer risk.

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Table 3-10. Incidence of Tumors in Pancreas, Testes, and Uterus in SD Rats Exposed to DINP for 2 Years (Bio/dvnamics. 1987)"

Observation

Dose Group
mg/kg-dav (ppm)

Males

Females

0

27 (500)

271 (5,000)

553 (10,000)

0

33 (500)

331 (5,000)

672 (10,000)

Pancreas

No. examined

70

0

0

70

69

0

0

70

Pancreatic islet cell adenoma

-

6

-

-

8

1

-

-

1

Pancreatic islet cell carcinoma

-

1

-

-

4

0

-

-

0

1 oslcs

No. examined

69

0

0

70

N/A

N/A

N/A

N/A

Interstitial cell hyperplasia

Total

4

-

-

22*

-

-

-

-

Unilateral

3

-

-

9

-

-

-

-

Bilateral

1

-

-

13

-

-

-

-

Interstitial cell tumors

Total

2

-

-

7

-

-

-

-

Unilateral

2

-

-

6

-

-

-

-

Bilateral

0

-

-

1

-

-

-

-

I lU'US

\o c\;iniiiK-d

\ A

\.A

\ A

\ A

70

0

0

69

Endometrial hyperplasia

-

-

-

-

-

2

-

-

13*

Endometrial adenocarcinoma

-

-

-

-

-

0

-

-

2

* p < 0.05 based on a two-tailed Fisher's exact test calculated for this review.

" Data in this table indicate all animals assessed for histopathology throughout the study; that is, including the interim sacrifice, the terminal sacrifice, and unscheduled
deaths. For late-developing tumors (pancreatic islet cell tumors, testicular interstitial cell tumors), statistical analysis was performed excluding animals that died or were
sacrificed up to 12 months, leaving n = 57, 57, 59, 59 in males and n = 59, 56, 60, 59 in females in the control, low-, mid- and high-dose groups, respectively. Data from
Appendix K of (Bio/dvnamics, 1987).

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4 POSTULATED MODE OF ACTION FOR LIVER TUMORS IN
RATS AND MICE

As described in Section 3.2.1, available studies provide consistent evidence that chronic oral exposure to
DINP can cause treatment-related hepatocellular adenomas and/or carcinomas in male and female F344
and SD rats and male and female B6C3F1 mice. EPA further considers the weight of evidence for liver
carcinogenesis and its underlying MOA in Sections 4.1 through 4.9.

4.1 Postulated Mode of Action in Rats and Mice

Studies have demonstrated that DINP can activate peroxisome proliferator-activated receptor alpha
(PPARa) in hepatocytes and cause hepatocellular adenomas and carcinomas in mice and rats. Existing
assessments of DINP by U.S. CPSC (:01 I, ;.Q10). Health Canada (ECCC/HC. 2020; EC/HC. 2015;
Health Canada.: ), ECHA (20131 and NICNAS (2012) have postulated that DINP causes liver
tumors in rats and mice through a PPARa MOA. PPARa is a nuclear receptor that controls transcription
of genes involved in fatty acid P-oxidation and peroxisome proliferation. PPARa activation in
hepatocytes in rodent models can cause hepatocellular cancer through a non-genotoxic MOA that
involves activation of Kupfer cells. Activated Kupfer cells secrete cytokines such as TNFa, IL-la, and
IL-ip that influence hepatocyte growth and fate. As discussed by Corton et al. (2018; 2014). studies
have demonstrated that Kupffer cell activation following PPARa activation plays a crucial role in
several tumor precursor effects. These effects include increased DNA synthesis and cell proliferation in
both normal and preneoplastic hepatocytes, as well as suppression of apoptosis. Altered cell growth and
survival can facilitate clonal expansion of initiated cells leading to the selective clonal expansion of
preneoplastic foci cells and ultimately tumor formation.

The PPARa MOA for liver tumorigenesis considered by EPA is described further by Corton et al.
(2018; 2014). Consistent with U.S. EPA Guidelines for Carcinogen Risk Assessment (	)05)

and the IPCS Mode of Action Framework (IPCS. 2007). EPA further evaluated the postulated PPARa
MOA for liver tumors, as well as evidence for other plausible MO As for DINP.

The PPARa MOA includes the following sequence of key events (KEs):

•	KE1: activation of PPARa in hepatocytes;

•	KE2: alterations in cell growth pathways (e.g., Kupfer cell activation leading to increased
cytokine (e.g., TNFa, IL-la, IL-ip) secretion;

•	KE3: perturbation of cell growth and survival (i.e., increased cell proliferation and inhibition of
apoptosis); and

•	KE4: selective clonal expansion of preneoplastic foci cells leading to the apical outcome,
hepatocellular adenomas, and carcinomas.

Several modulating factors associated with the PPARa MOA have also been proposed, including
increases in reactive oxygen species (ROS) and activation of nuclear factor kappa B (NF-kB) (Corton et
al.. 2018). These modulating factors are not considered necessary to induce liver tumorigenesis but may
modulate the dose-response behavior or the probability of inducing one or more KEs (Corton et al..
2014).

Evidence for each KE (Sections 4.1.1 to 4.1.4) and EPA's analyses of dose-response (Section 4.1.5);
temporality (Section 4.3); strength, consistency, and specificity (Section 4.4); biological plausibility and
coherence (Section 4.5); other carcinogenic MOAs (Section 4.6); uncertainties and limitations (Section
4.7); weight of scientific evidence for liver tumors (Section 4.8) are presented below.

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4,1,1 Key Event 1: PPARa Activation

PPARa activation can be assessed using trans-activation assays or by measuring specific events
associated with PPARa activation, such as increased expression of genes involved in beta oxidation or
peroxisome proliferation, increased activity of palmitoyl-CoA oxidase, increased peroxisomal beta
oxidation (PBOX), and/or peroxisome proliferation in hepatocytes. Activation of PPARa in hepatic cells
by DINP has been consistently demonstrated in five in vivo studies of mice and four in vivo studies of
rats. No evidence of PPARa activation in hepatic cells was observed in two in vivo studies of monkeys.
Additionally, four in vitro studies investigating PPARa activation are available. Available data for KE1
are discussed further below.

Evidence from In Vitro Studies

Four in vitro studies of DINP are available that consistently demonstrate that rat and mouse hepatocytes
are more sensitive to PPARa activation compared to human and monkey hepatocytes. Bendford et al.
(1986) demonstrated that in vitro treatment of primary rat hepatocytes isolated from adult Wistar rats
with concentrations of MINP ranging from 0.1 to 0.5 mM for 3 days caused large (up to approximately
750 percent) dose-dependent increases in palmitoyl-CoA oxidation and laurate hydroxylation activity.
Comparatively, smaller (approximately 200 to 300 percent) increases in palmitoyl-CoA oxidation and
laurate hydroxylation activity were observed in primary marmoset hepatocytes under similar
experimental conditions. Hasmall et al. (1999) demonstrated that treatment of primary rat hepatocytes
isolated from male F344 rats with 250 and 500 |iM (but not 750 |iM) DINP can induce increases in
PBOX activity. In contrast, no increase in PBOX was noted in primary human hepatocytes treated with
up to 750 |iM DINP under similar experimental conditions. Similarly, Shaw et al. (2002) report dose-
related induction of PBOX activity in primary rat hepatocytes isolated from male F344 rats treated with
150 to 250 |iM MINP, however, PBOX activity was not increased in primary human hepatocytes treated
with up to 250 |iM MINP under similar experimental conditions. Finally, Bility et al. (2004)
demonstrated that mouse PPARa is more inducible and activated at lower concentrations compared to
human PPARa in mouse 3T3-L1 fibroblasts transfected with a plasmid encoding mouse or human
PPARa luciferase reporter (lowest activation concentration: 3 and 10 |iM for mouse and human,
respectively; maximal fold-induction: 27.1 and 5.8 for mouse and human, respectively).

Evidence from In Vivo Studies of Rats

Three studies of rats provide consistent evidence of treatment-related increases in PPARa activation
following oral exposure to DINP. Smith et al. (2000) reported treatment-related increases in hepatic
PBOX in male F344 rats fed diets containing up to 12,000 ppm DINP (approximately 1,200 mg/kg-day)
for 2 or 4 weeks; however, no change was observed in the low-dose group (approximately 100 mg/kg-
day). Similarly, BIBRA (1986) reported increased hepatic cyanide-insensitive palmitoyl-CoA oxidation
levels and hepatic lauric acid 11- and 12-hydroxylase activities in male and female F344 rats treated
with high-doses of DINP for 21-days (biomarkers of PPARa activation increased in males and females
starting at 639 and 1,198 mg/kg-day, respectively). Finally, cyanide-insensitive palmitoyl-CoA oxidase
activity was increased in the livers of male and female F344 rats treated with 733 (males) to 885
(females) mg/kg-day DINP after 1,2, 13, and 104 weeks of exposure to DINP, as well as for females
treated with 442 mg/kg-day DINP for 104 weeks (CovamceLahh h)08b). In contrast, no evidence of
peroxisome proliferation (evaluated via electron microscopy) was reported in hepatocytes from male or
female F344 rats treated with up to 307 (males) to 375 (females) mg/kg-day DINP for 2 years (Lington
et al. 1997).

Evidence from In Vivo Studies of Mice

Five studies of mice provide consistent evidence of treatment-related increases in PPARa activation
following oral exposure to DINP. Smith et al. (2000) reported treatment-related increases in hepatic

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PBOX in male B6C3F1 mice fed diets containing up to 6,000 ppm DINP (approximately 900 mg/kg-
day) for 2 or 4 weeks; however, no change was observed in the low-dose group at either timepoint
(approximately 75 mg/kg-day). In a second study, Kaufmann et al. (2002) reported dose-related
increases in the number and volume of peroxisomes and hepatic cyanide-insensitive palmitoyl-CoA
oxidation activity in male B6C3F1 mice after 4 weeks at doses as low as 117 mg/kg-day, while similar
changes were observed in female mice starting at 546 mg/kg-day DINP. Similarly, Valles et al. (2003)
reported treatment related increases in hepatic palmitoyl-CoA oxidase activity in male and female
B6C3F1 mice treated with diets containing 4,000 to 8,000 ppm DINP (approximately 600 to 1,200
mg/kg-day) for 2 weeks. In a study by Hazleton Labs (1992). large (albeit not always statistically
significant), dose-related, increases in hepatic cyanide-insensitive palmitoyl CoA oxidation were
observed in male and female B6C3F1 mice treated with 365 and 2,600 mg/kg-day DINP for 4, 31, and
91 days. Similarly, large increases in hepatic cyanide-insensitive palmitoyl-CoA oxidation activity were
observed in male and female B6C3F1 mice treated with 1,560 (males) to 1,888 (females) mg/kg-day
DINP for 79 and 105 weeks (Covance Labs. 1998a).

Evidence from In Vivo Studies of Monkeys

Two studies have evaluated biomarkers of PPARa activation in monkeys. Oral (gavage) exposure to
DINP had no effect on PBOX in male cynomolgus monkeys treated with 500 mg/kg-day DINP for 14-
days (Push et al.. 2000). Similarly, no effect on cyanide-insensitive palmitoyl CoA oxidase activity or
cytochrome P450 concentration and lauric acid 11- and 12-hydroxylase activities in hepatic microsomes
were observed in male and female marmosets gavaged with up to 2,500 mg/kg-day DINP for 13 weeks
(Hall et a )J.

4.1.2	Key Event 2: Alterations in Cell Growth Pathways	

EPA identified one in vivo study of mice investigating alterations in cell growth pathways. No in vivo
studies of rats or monkeys for KE2 were identified. Ma et al. (2014a) administered DINP via oral
gavage to male Kunming mice at 0, 0.2, 2, 20, and 200 mg/kg-day DINP daily for 14 days and then
determined TNFa and IL-1 in liver homogenates. IL-1 and TNFa content was significantly increased at
20 and 200 mg/kg-day. However, this study is limited by the fact that study authors do not identify the
specific IL-1 subtypes evaluated (e.g., IL-la vs. IL-ip).

4.1.3	Key Event 3: Perturbation of Cell Growth and Survival

Evidence of increased cell proliferation comes from five in vivo studies of mice, two in vivo studies of
rats, one in vivo study of monkeys, and two in vitro studies of primary rat and human hepatocytes.
Across in vivo studies of mice and rats, an acute cell proliferative response in the liver is consistently
observed. In contrast, cellular proliferation in the liver is not sustained chronically in either species.
However, as discussed by Corton et al. (2018). PPARa activators tend to "produce transient increases in
replicative DNA synthesis during the first few days or weeks of exposure followed by a return to
baseline levels." Therefore, lack of a sustained proliferative response is consistent with the proposed
MO A. No evidence of replicative DNA synthesis was observed in one in vivo study of monkeys. In the
two in vitro studies, DINP consistently suppressed apoptosis and increased replicative DNA synthesis in
rat, but not human hepatocytes. Available data for KE3 is discussed further below.

Evidence from In Vitro Studies

Two in vitro studies are available that consistently demonstrate that DINP can suppress apoptosis and
increase replicative DNA synthesis in rat but not human hepatocytes. Hasmall et al. (1999) treated
primary rat hepatocytes obtained from male F344 rats and primary human hepatocytes with 250 to 750
|iM DINP. Treatment with DINP increased replicative DNA synthesis, suppressed apoptosis, and
suppressed TGFP1-induced apoptosis in rat but not human hepatocytes. Similarly, Shaw et al. (2002)

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treated primary rat hepatocytes obtained from male F344 rats and primary human hepatocytes with 150
to 250 |iM DINP and observed treatment-related suppression of apoptosis and increased replicative
DNA synthesis in rat but not human hepatocytes.

Evidence from In Vivo Studies of Rats

Two studies of rats have evaluated cell proliferation in the liver following oral exposure to DINP. In
both studies, bromodeoxyuridine (BrdU) was administered to rats via osmotic minipumps and cell
proliferation was evaluated via BrdU labeling. No in vivo studies of rats have evaluated effects on
hepatocyte apoptosis. Smith et al. (2000) reported treatment-related increases in hepatocellular
replicative DNA synthesis in male F344 rats fed diets containing 12,000 ppm DINP (approximately
1,200 mg/kg-day) for 2 or 4 weeks; however, no change was observed in the low-dose group
(approximately 100 mg/kg-day). In the second study, increased hepatocellular replicative DNA
synthesis was observed in male and female F344 rats after 1 week of dietary exposure to 733 (males) or
885 (females) mg/kg-day DINP, but not after 2, 13, or 104 weeks of exposure (Covance Labs K^Sb).

Evidence from In Vivo Studies of Mice

Five studies have evaluated cell proliferation (measured via BrdU labeling in all five studies) and/or
apoptosis in the liver following oral exposure to DINP. Valles et al. (2003) fed female B6C3F1, SV129,
and Ppara-mx\\ mice diets containing 8,000 ppm DINP (approximately 1,200 mg/kg-day) for 1 week
and observed increased hepatocellular replicative DNA synthesis in B6C3F1 and SV129 mice, but not
Ppara-null mice. Smith et al. (2000) report treatment-related increases in hepatocellular replicative
DNA synthesis in male B6C3F1 mice fed diets containing up to 6,000 ppm DINP (approximately 900
mg/kg-day) for 2 but not 4 weeks. Further, no change in replicative DNA synthesis was observed in the
low-dose group at either timepoint (approximately 75 mg/kg-day). Two other studies reported no
increase in hepatocellular replicative DNA synthesis in the livers of male or female B6C3F1 mice dosed
with 2,600 mg/kg-day DINP for 4, 31, and 91 days (Hazleton Labs. 1992) or 1,560 (males) to 1,888
(females) mg/kg-day DINP for 79 and 105 weeks (Covance Labs. 1998a).

In another study, Kaufmann et al. (2002) evaluated hepatocellular replicative DNA synthesis and
apoptosis (via TUNEL staining) in male and female B6C3F1 mice administered 117 to 2,806 mg/kg-day
DINP for 1 or 4 weeks. Dose-related increases in hepatocellular replicative DNA synthesis were
observed in male and female mice after 1 week at doses as low as 116 (male) to 1,272 (female) mg/kg-
day; however, no significant changes in females were noted after 4 weeks at doses as high as 2,806
mg/kg-day, while significant increases in males after 4 weeks were observed at doses as low as 117
mg/kg-day but without a clear dose-response relationship. In males, apoptosis was increased after 1
week in the high-dose group (1,860 mg/kg-day). At 4 weeks, apoptosis appeared reduced in all treatment
groups for males; however, the effect was not statistically significant. No clear treatment-related effects
on apoptosis were observed for females at either timepoint.

Evidence from In Vivo Studies of Monkeys

Treatment with DINP had no effect on replicative DNA synthesis (measured via proliferating cell
nuclear antigen [PCNA] immunohistochemistry) in male cynomolgus monkeys treated with 500 mg/kg-
day DINP for 14 days (Push et al. 2000).

4.1.4 Key Event 4: Selective Clonal Expansion of Preneoplastic Foci

EPA identified no in vitro or in vivo studies of DINP that evaluated KE4. Further, hepatocellular
hyperplasia, which may provide some evidence of expansion of preneoplastic foci, has not been reported
in any short-term, subchronic, or chronic studies of DINP.

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4,1,5 Modulating Factors

EPA identified no studies evaluating activation of NF-kB in the liver.

Two studies provide data on the relationship between oxidative stress and DINP following in vivo
exposures in male Kunming mice (Ma et ai. 2014b) or in vitro investigations in human hepatic eel 1 -
types (Gutierrez-Garria et al.: ). Available studies provide evidence that DINP can induce ROS in
the liver.

Ma et al. (2014b) exposed male Kunming mice to DINP via oral gavage daily for 14 days and evaluated
several endpoints related to oxidative stress in homogenized hepatic tissue. Indices of oxidative stress
were generally observed at the same doses that resulted in histopathological lesions of the liver, although
quantification of the tissue sections was not performed. Dose-dependent increases in ROS and increases
in malondialdehyde were observed, reaching significance at 200 mg/kg-day. In parallel, decreases in
glutathione content occurred at 200 mg/kg-day DINP, indicative of oxidative stress. The authors also
reported DNA-protein-crosslinks and increases in 8-hydroxydeoxyguanosine at 200 mg/kg-day, which
indicate oxidative damage to DNA.

An in vitro study in HepG2 cells by Gutierrez-Garcia et al. (2019) evaluated the potential for DINP to
elicit oxidative stress and investigated a mechanism involving sirtuins (srts), which are a group of
mitochondrial NAD+-dependent histone deacetylases. Increases in ROS were observed at the highest
concentration tested in parallel with increases in lysine acetylation and dose-dependent reductions in
expression of several sirtuin genes {i.e., Sirtl, Sirt2, Sirt3, Sirt5), as well as decreases in sirtuin protein
levels. Although the data does not directly provide evidence that ROS is a modulating factor within the
PPARa activation MOA for hepatic tumors, considered more broadly, it does suggest that DINP can
induce ROS in hepatocytes.

4.2 Dose-Response Concordance of Key Events with Tumor Response

Dose-Response Concordance: Rats

As discussed in Sections 4.1.1 through 4.1.4, data from in vivo rat studies is limited to KE1, KE3, and
the apical outcome, hepatocellular adenomas and/or carcinomas. No data is available for KE2 or KE4.
Available data used by EPA for its dose-response concordance analysis of the PPARa MOA in rats is
presented in Table 4-1.

Although limited, there is some evidence to demonstrate that KE1 occurs at lower doses than KE2 and
the apical outcome, liver tumors. For KE1, three studies report consistent dose-related increases in
several biomarkers of PPARa activation {i.e., increased PBOX, lauric acid 11- and 12-hydroxylase,
palmitoyl-CoA oxidase activity) (Smith et al.. 2000; Covance Labs. 1998K i'M., \ \ S6). The lowest
dose at which PPARa activation was reported in rats is 442 mg/kg-day, following 104 weeks of
exposure to DINP (Covance Labs. 1998b). For KE3, one study reports a dose-related increased in
hepatocellular replicative DNA synthesis at very high doses of DINP {i.e., 1,200 mg/kg-day) after 2 and
4 weeks of exposure (Smith et al.. 2000). A second study, which only evaluated hepatocellular
replicative DNA synthesis at a single dose {i.e., 733 (males) to 885 (females) mg/kg-day), reports
increased hepatocellular replicative DNA synthesis and palmitoyl-CoA oxidase activity after 1 week of
exposure (Covance Labs. 1998b). Statistically significant dose-related increases in hepatocellular
carcinomas and/or combined adenomas and carcinomas have been observed in two studies of rats at
doses at low as 672 to 885 mg/kg-day (Covance Labs. 1998b; Bio/dynamics. 1987). In the study of F344
rats by Covance Labs (1998b). increased hepatic palmitoyl-CoA oxidase activity (KE1) was observed in
female (but not male) rats at lower doses than which adenomas and carcinomas were observed after 104
weeks of treatment {i.e., 442 vs. 885 mg/kg-day for tumors), providing evidence of concordance.

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Overall, there is some evidence to support dose-response concordance for KE1, KE3, and hepatocellular
adenomas and/or carcinomas. However, no data are available for KE2 or KE4, or apoptosis (part of
KE3) in rat hepatocytes, which prevents a complete analysis of dose-response concordance across all
KEs in the postulated MOA.

Dose-Response Concordance: Mice

As discussed in Sections 4.1.1 through 4.1.4, data from in vivo mouse studies is limited to KE1, KE2,
KE3, and the apical outcome, hepatocellular adenomas and/or carcinomas. No data is available for KE
4. Available data considered by EPA for its dose-response concordance analysis of the PPARa MOA in
mice is presented in Table 4-2.

Although limited, available data indicate the KE1, KE2, and KE3 occur in mice at lower doses than
hepatocellular adenomas and/or carcinomas, providing some evidence of concordance. However,
concordance across KE1, KE2, and KE3 is less apparent. As can be seen from Table 4-2, the lowest
dose at which biomarkers of PPARa activation were increased was 117 mg/kg-day for male mice after 4
weeks of exposure (Kaufmann et at.. 2002); for KE2 increased TNFa and IL-1 in liver homogenate has
been observed at doses as low as 20 mg/kg-day (Ma et at.. 2014a); for KE3 increased DNA synthesis
has been reported at doses as low as 116 mg/kg-day in male mice (Kaufmann et at.. 2002); and
hepatocellular adenomas and carcinomas have been observed at doses as low as 336 mg/kg-day in
female mice. However, there are several sources of uncertainty related to KE2 data from Ma et al.
(2014a). First, Ma et al. evaluated DINP exposure with Kunming mice, while other studies of DINP
were performed with B6C3F1 mice, and it is unclear if there is a strain difference in sensitivity or if
studies testing lower doses of DINP with B6C3F1 mice would produce similar results. Additionally, Ma
et al. report increased IL-1 in liver homogenate, but do not differentiate between cytokine subtypes (e.g.,
IL-1 a, IL-ip). Another limitation of the available dataset is that PBOX is generally not considered as
sensitive of a biomarker as other measures of PPARa activation, especially compared to measures of
PPARa-inducible genes.

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Table 4-1.

)ose-Response Concordance for PPARa M<

3 A in Rats

Dose
(mg/kg-
day)

KE 1

(Sex; Dose in mg/kg-dav; Timcpoint)

KE 2

KE3

(Sex; Dose in mg/kg-dav; Timcpoint)

KE 4

Hepatocellular Tumors

1-200

NC - PBOX (M; 120; 2, 4 wks)"



NC - DNA synthesis (M; 120; 2, 4
weeks)"



NC - Neoplastic nodules, hepatocellular cancer,
or combined (M/F; 15-184; 104 weeks/
NC - Adenomas, carcinomas, combined (M/F,
29-109; 104 weeks)6

NC - Neoplastic nodules, carcinoma (M/F; 27-
33; 104 weeks)8

201-400

NC - PP (M/F; 307-375; 2 yrs)rf







NC - Neoplastic nodules, hepatocellular cancer,
or combined (M/F; 307-375; 104 weeks/
NC - Adenomas, carcinomas, combined (M/F,
359-442; 104 weeks)6

NC - Neoplastic nodules, carcinoma (M/F; 271-
331; 104 weeks)8

401-600

| Palm CoA (F (not M); 442; 104 (but not
1,2, or 13) wks)6

-

-

-

-

601-1,000

NC - Palm CoA (M/F;607-639; 3 weeks)8
t 11/12 H-lase (M, notF); 639; 3 weeks)8
t Palm CoA (M/F; 733-885; 1, 2, 13, 104
weeks)6



t DNA synthesis (M/F; 733-885; 1
week (but not 2, 13, 104 weeks)6



t Carcinoma (F (not M); 672; 104 weeks)8
t Carcinoma (M (notF); 733-885; 104 weeks)6
t Combined adenoma and carcinoma (M/F); 733-
885; 104 weeks)b

1,001-1,400

t Palm CoA (M/F; 1,192-1,198; 3 weeks)8
t 11/12 H-lase (M, notF); 1,192; 3 weeks)8
t PBOX (M; 1,200; 2, 4 weeks)"



| DNA synthesis (M; 1,200; 2,4
weeks)"





1,401-2,000



-

-

-

-

2,001-2500

t 11/12 H-lase (M/F; 2,195-2,289; 3 weeks)

C

-

-

-

-

" (Smith et al. 2000)

6 (Covance Labs. 1998b)
c (BIBRA. 1986)
d (Lington et al. 1997)

8 (Bio/dvnamics. 1987)

11/12 H-lase = lauric acid 11- and 12-hydroxylase; F = female; M = male; NC = no significant change; Palm CoA: cyanide-insensitive palmitoyl-CoA oxidation; PBOX
= peroxisomal beta-oxidation; PP = peroxisomal proliferation
indicates no experimental evidence is available

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846 Table 4-2. Dose-Response Concordance for PPARg MOA in Mice	

Dose
(mg/kg-day)

KE 1

(Sex; Dose in mg/kg-day; Timcpoint)

KE 2

KE3

(Sex; dose in mg/kg-day; timcpoint)

KE 4

Hepatocellular Tumors

1-200

NC - PBOX (M; 75; 2,4 weeks)"
t PP & Palm CoA (M (but not F); 117;
4 weeks)6

NC - TNFa (M,
0.2-2, 2 weeks/
t TNFa (M, 20-
200, 2 weeks/

NC - DNA synthesis (M; 75; 2, 4 weeks)"
| DNA synthesis (M (but not F); 116-167; 1, 4
weeks)*

NC - Apoptosis (M/F; 116-167; 1, 4 weeks)*



NC - Adenomas or
carcinomas (M/F; 90-112, 2
yrs/

201-400

t PP & Palm CoA (M; 350; 4 weeks)*
t Palm CoA (M/F; 365; 4, 31, 91
days/



| DNA synthesis (M; 337-350; 1, 4 weeks)*
NC - Apoptosis (M; 337-350; 1, 4 weeks)*



t Combined adenomas &
carcinomas (F (but not M);

336, 2 yrs/

401-600

| PP & Palm CoA (F; 546; 4 weeks)6
| Palm CoA (M/F; 600; 2 weeks)8

-

NC - DNA synthesis (F; 520-546; 1, 4 weeks)*
NC - Apoptosis (F; 520-546; 1, 4 weeks)*

-

-

601-800

-

-

-

-

t Combined adenomas &
carcinomas (M; 742, 2 yrs/

801-1,000

t PBOX (M; 900; 2,4 weeks)"
t PP & Palm CoA (M; 913; 4 weeks)*



t DNA synthesis (M; 75; 2 (not 4) weeks)"
| DNA synthesis (M; 901-913; 1,4 weeks)*
NC - Apoptosis (M; 901-913; 1,4 weeks)*



t Carcinomas and combined
adenomas & carcinomas (F;
910, 2 yrs/

1,001-1,400

| Palm CoA (M/F; 1,200; 2 weeks)8
t PP & Palm CoA (F; 1,272; 4 weeks)*



| DNA synthesis (F; 1200; 1 week)8
t DNA synthesis (F; 1272-1278; 1 (but not 4)
weeks)*

NC - Apoptosis (F; 1272-1278; 1, 4 weeks)*





1,401-2,000

t PP & Palm CoA (M; 1860; 4 wks)*
t Palm CoA (M/F; 1,560-1,888; 79,
105 wks/



| DNA synthesis (M; 1766-1860; 1, 4 weeks)*
NC-DNA synthesis (M/F; 1,560-1,888; 79, 105
weeks/

t Apoptosis (M; 1,766-1,860; 1 (but not 4) weeks)*



t Adenomas and/or
carcinomas (M/F; 1,560-
1,888, 2 yrs/

2,001-3,000

t Palm CoA (M/F; 2600; 4, 31, 91
days/

t PP & Palm CoA (F; 2806; 4 weeks)*



| DNA synthesis (F; 2593-2806; 1 (but not 4)
weeks)*

NC - DNA synthesis (M/F; 2,600; 4, 41, 91 days/
NC - Apoptosis (F; 2,593-2,806; 1, 4 weeks)*





" (Smith et al. 2000)

* (Kaufmann et al.. 2002)
c (Hazleton Labs, 1992)
d (Covance Labs. 1998a)

' (Valles et al. 2003)
f (Ma et al. 2014a)

t = significant increase; { = significant decrease; 11/12 H-lase = lauric acid 11- and 12-hydroxylase; F = female; M = male; NC = no significant change; Palm CoA:
cyanide-insensitive palmitoyl-CoA oxidation; PBOX = peroxisomal beta-oxidation; PP = peroxisomal proliferation
indicates no experimental evidence is available

847

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4.3	Temporal Association of Key Events with Tumor Response

In rats, it is clear that KE1 and KE3 precede tumor formation, however, the temporal sequence of KE1
and KE3 cannot be established (Table 4-1). Biomarkers of PPARa activation (KE1) and hepatic cell
proliferation (KE3) are both increased as early as 1 week following oral exposure to DINP (Covance
Labs. 1998b); however, no studies are available that evaluate either KE at early timepoints.
Comparatively, liver neoplasms were first detected during an interim sacrifice on study week 79 in a
study of F344 rats by Covance Labs (1998b) (albeit without a clear dose-relationship; adenomas
detected in one control male and one high-dose female; carcinoma detected in one high-dose male).

In mice, it is clear that KE1, KE2, and KE3 precede tumor formation; however, the temporal sequence
of KE1, KE2, and KE3 cannot be established (Table 4-2). Biomarkers of PPARa activation (KE1) are
significantly increased in one study as early as 4 days after oral exposure (Hazletom Labs. 1992). while
KE2 is measured in only a single study that reports increases in TNFa and IL-1 in liver homogenate
after 14 days (Ma et al. 2014a). and hepatic cell proliferation (KE3) is increased after 1 week of oral
exposure to DINP (Kaufmann et al.. 2002). However, no studies are available that evaluate any of these
KEs at earlier timepoints. Comparatively, in the available 2-year bioassay of mice (Covance Labs.
1998a). hepatocellular adenomas and carcinomas were first detected on study days 167 and 366,
respectively, in a single high-dose male at each timepoint (as reported by (	3. 2001)).

4.4	Strength, Consistency, and Specificity of Association of Tumor
Response with Key Events

Available in vivo studies of mice and rats and in vitro studies of rat and mouse hepatocytes provide
remarkably consistent evidence that DINP can activate PPARa (KE1).There is also consistent evidence
that DINP can cause acute proliferative cellular responses in the livers of rats and mice in vivo and rat
hepatocytes in vitro (KE3). In contrast, cellular proliferation in the liver is not sustained chronically in
either species. As discussed by Corton et al. ( ), PPARa activators tend to "produce transient
increases in replicative DNA synthesis during the first few days or weeks of exposure followed by a
return to baseline levels." Chronic or sustained proliferative responses for potent PPARa activators tend
to be much lower compared to acute proliferative responses. Comparatively, DINP is a relatively weak
PPARa activator and low levels of chronic hepatic cell proliferation may be difficult to detect over
variable background levels. Therefore, lack of a detectable sustained proliferative response is consistent
with the proposed MOA for a weak PPARa activator such as DINP. Further adding to the strength of
evidence, KE1 and KE3 have been observed in studies of differing design and originating from different
laboratories, with hepatic effects such as increases in relative liver weight and hepatocellular
hypertrophy observed in short-term, subchronic, and chronic studies of rats and mice. These effects,
although not KEs in the PPARa MOA, are frequently observed following PPARa activation and
subsequent peroxisome proliferation.

A notable inconsistency in the database stems from an unexplained difference in sensitivity across sexes
in mice. In the 2-year bioassay of mice, liver tumors were observed at doses as low as 335 mg/kg-day in
female mice and 742 mg/kg-day in male mice (Covance La 8a), indicating female mice are more
sensitive than males. In contrast, other studies have demonstrated that PPARa activation (KE1) and
cellular proliferation (KE3) occur at lower doses in male mice compared to females (Kaufmann et al..
2002). This apparent inconsistency cannot be explained.

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4.5	Biological Plausibility and Coherence

Extensive evidence exists to support the hypothesis that chronic PPARa activation can lead to
alterations in cell growth pathways, perturbations of cell growth and survival, and selective clonal
expansion of preneoplastic foci cells leading to hepatocellular tumorigenesis in rodents (reviewed in
(Gorton et al.. 2018; Gorton et al. )). This proposed MOA for DINP-induced liver tumors in rats
and mice is consistent with available data, indicating biological plausibility. Available data from mice
and rats demonstrate PPARa activation after short-term (several days to weeks) oral exposure to DINP
that can be sustained with chronic exposure (Covance Labs. 1998a. b). Although studies also
demonstrate that oral exposure to DINP can cause acute hepatic cell proliferative responses, other
studies demonstrate that oral exposure to DINP does not cause chronic proliferative response in the liver
of mice or rats. As discussed by Gorton et al. (2018) chronic or sustained proliferative responses for
potent PPARa activator are much lower compared to acute proliferative responses. Comparatively,

DINP is a relatively weak PPARa activator and low levels of chronic hepatic cell proliferation may be
difficult to detect over variable background levels.

4.6	Other Modes of Carcinogenic Action

This section summarizes evidence for other modes if carcinogenic action in the liver for DINP.
Ppara-Null Mice

Valles et al. (2003) conducted a series of short-term (1- to 3-week) studies in which male and female
B6C3F1, wild-type SV129, and Ppara-mx\\ mice were exposed to DINP. Repeated dose studies well-
established that in response to exposure to DINP, male and female B6C3F1 wild-type show
hepatotoxicity. Across these studies, dose-dependent increases in relative liver weight that were
dependent on PPARa were generally observed; however, in one study of older (30-week) female Ppara-
null mice, PPARa-independent increases in relative liver weight has also been observed, (these increases
were specific for older female mice; younger female or older male Ppara-null mice did not exhibit any
changes in liver to body weight ratios after exposure to DINP), thereby hinting at the possibility of
PPARa-independent mechanisms being at play in the liver under certain conditions. Unique gene
expression changes in older Ppara-null female mice have been identified in expression arrays, like
testosterone hydroxylase (Cyp2d9). Cyp2d9 is down-regulated by DINP in wild-type mice, but Cyp2d9
was up-regulated in Ppara-null mice. The relevance of these subtle PPARa-independent effects to
hepatocarcinogenesis is not known, but Ppara-null mice are resistant to the carcinogenicity of a
prototypical PPARa activator (Peters et al.. 1997). It is important to note that most of the studies
conducted by Valles et al. support the hypothesis that PPARa plays a dominant role in mediating the
carcinogenic effects of DINP in the liver.

Other Nuclear Receptors

Constitutive androstane receptor (CAR), pregnane X receptor (PXR), and aryl hydrocarbon receptor
(AhR) are known to play a role in liver homeostasis and disease. Although their precise role, if any, in
liver tumorigenesis in response to chronic exposure to DINP has not yet been established. In addition to
PPARa, DINP has been shown to activate multiple nuclear receptors that may play a role in liver
tumorigenesis. Several studies have demonstrated that DINP can activate CAR, which is a nuclear
receptor with an adverse outcome pathway with KEs like those of PPARa and has been implicated in
hepatic carcinogenesis in rodents (Felter et al.. 2018). DeKeyser et al. (2011) used transactivation and
mammalian two-hybrid assays in COS-1 cells to demonstrate that DINP is a strong activator of human
CAR variant 2 (hCAR2). Furthermore, DINP induced expression of CYP2B6, one of the primary target
genes of CAR, in primary human hepatocytes. In a subsequent study by the same research group,
Laurenzana et al. demonstrates that MINP, metabolite of DINP, can also activate hCAR2 (Laurenzana et

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ai. 2016). Additionally, in vitro studies have also shown that DINP /MINP can activate human PXR
(Laurenzana et al. 2016; Dekevser et ai. ) as well as mouse and human PPAR gamma, although
the degree of PPAR. gamma activation was greater for the mouse receptor than for the human receptor
under the conditions of the study (Bilitv et al.. 2004). DINP has also been shown to promote and induce
tumorigenesis in a variety of cell types through AhR-mediated genomic and nongenomic pathways
(Wane et al.. 2012). DINP induces several changes in rodent liver consistent with PPARa activation
(Lauren zan a et al.. 2016). DINP induces some of these liver changes independently of PPARa activation
as shown in Ppara-null mice (Valles et al.. 2003).

DINP has also been evaluated in 442 high-throughput assays as part of EPA's Toxicity ForeCaster
(ToxCast) program. Curated high-throughput screening data for DINP accessed through the National
Toxicology Program's Integrated Chemical Environment (ICE) indicated that DINP was inactive in the
majority of tested assays and active in only seven assays (Table 4-3). Consistent with available
literature, DINP was active in two assays for PXR activation. However, DINP was inactive in assays for
other nuclear receptors {i.e., CAR, AhR, PPARa, PPARy) and other assays of PXR {i.e.,
TOX21_PXR_Agonist, TOX21_PXR_viability) and these results are inconsistent with available
literature.

Table 4-3. Summary of Active ToxCast Assays for DINP"

ToxCast Assay

Mode of Action

AC50/LOEC
(jiM)

BSK_SAg_Eselectin_up

Cancer - KCC6: Chronic Inflammation, CardioTox
- Endothelial Injury/Coagulation

0.2

BSK_CASM3C_TissueFactor_down

AcuteTox - Immune and Inflammatory Response,
CardioTox - Endothelial Injury/Coagulation

0.2

ATGPXRECISup

Cancer - KCC8: Receptor Mediated Effects

1.2

ATG PXR TRANS up

Cancer - KCC8: Receptor Mediated Effects

1.7

BSKKF3 CTIL1 adown



4

NV SENZhBACE



8.7

ACE A ER AUC viability

AcuteTox - Cytotoxicity, Cancer - KCC10: Cell
Proliferation/Death/Energetics

38.8

AC50 = concentration at which 50% maximum activity is observed; LOEC = lowest-observed-effect-
concentration

a Data accessed through NTP's Integrated Chemical Environment in February 2024.

Gap Junction Intercellular Communication

Gap junctional intercellular communication (GJIC) is the only portal by which multicellular organisms
mediate the intercellular exchange of cellular signal factors from the interior of one cell to that of
neighboring cells (Loewenstein. 1987; Pitts and Finbow. 1986). GJIC is considered to play a crucial role
in the maintenance of homeostasis, and in turn, aberrant GJIC is likely to be involved in carcinogenesis,
given that cancer cells do indeed behave as if they have dysfunctional GJIC and are dissociated from the
homeostasis maintained by the organism. Inhibition of GJIC has been proposed as a non-genotoxic
carcinogenic mechanism (Yamasaki et al.. 1995; Yamasaki. 1995). Aberrant GJIC has been known as a
non-genotoxic event that is important for carcinogenesis. This is based on the observation that many
non-genotoxic tumor-promoting agents inhibit GJIC (Klaunie et al.. 2003). Several tumor types,
including hepatocellular carcinomas, have been shown to demonstrate inhibited GJIC (Trosko et al..

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1990c; Trosko et al. 1990a. h; l'iosko and Chang. 1989). DINP is shown to inhibit hepatic GJIC, and
the inhibition of GJIC has been proposed as a non-genotoxic carcinogenic mechanism, in rodents
exposed to DINP for 2 or 4 weeks (Smith et al.. 200^\ I ko et al.. 1990c; Trosko et al.. 1990b).

Cytotoxicity and Regenerative Proliferation

Cytotoxicity followed by regenerative proliferation is an established nongenotoxic MO A (Felter et al..
2018). There is some limited evidence that DINP may act through a cytotoxic MOA. The KEs for
establishing a cytotoxic MOA are (1) the chemical is not DNA reactive; (2) evidence of cytotoxicity by
histopathology (e.g., the presence of necrosis and/or increased apoptosis); (3) evidence of toxicity by
increased serum enzymes indicative of cellular damage that are relevant to humans; (4) presence of
increased cell proliferation as evidenced by increased labeling index and/or increased number of
hepatocytes; (5) demonstration of a parallel dose response for cytotoxicity and formation of tumors; and
(6) reversibility upon cessation of exposure (Felter et al.. 2018). As discussed in Section 2 as well as
below in the genotoxicity section, EPA considers DINP not likely to be genotoxic or mutagenic. Four
studies have provided quantitative liver histopathology with clear evidence of lesions consistent with
cytotoxicity, namely focal necrosis, including three 2-year bioassay studies in rats (Covance Labs.
1998b; Lington et al.. 1997; Bio/dynamics. 1986). one 13-week study in mice (Hazleton Labs. 1992).
and one 4-week study in mice (Hazleton Labs. 1991). In Lington et al (1997). a significant dose-related
increased incidence of focal necrosis was observed in male rats, and the Bio/dynamics study (1987)
reported increased incidence of focal necrosis in males of the mid-dose group, with no clear dose-
response. In the rat study by Covance Labs (1998b). individual cell degeneration/necrosis was
significantly increased in males of the high-dose group. However, not all chronic studies reported this
lesion. The 2-year study in mice by Covance Labs (1998a) did not observe focal necrosis or apoptosis,
even with a study design that included higher doses.

As mentioned above in Section 4.1.3, DINP has been shown to elicit acute proliferative responses in
mouse hepatocytes in vivo and in vitro. Hyperplasia has not been observed in hepatic tissues, suggesting
against regenerative proliferation. Increases in periportal hepatocellular replicative DNA synthesis have
been reported in mice and rats following exposure to 12,000 ppm DINP for 2 or 4 weeks (Smith et al..
2000). consistent with increases in hepatocyte proliferation observed in two other mouse studies at doses
ranging from 150 to 8,000 ppm for 1 to 4 weeks (Valles et al.. 2003; Kaufmann et al.. 2002) or in rats up
to 855 mg/kg-day DINP for up to 104 weeks (Covance Labs. 1998b). Two in vitro studies (Shaw et al..
2002; Hasmall et al.. 1999) reported increased replicative DNA synthesis and suppressed apoptosis in rat
hepatocytes at doses of DINP ranging from 150 to 750 |iM, The available data do not consistently
support the various KEs in the MOA for cytotoxicity, suggesting other MO As are at play.

4.7 Uncertainties and Limitations

There are several limitations and uncertainties associated with the available dataset for the postulated
PPARa MOA. First, no data is available for KE2 and KE4 for rats or mice, with the exception of a
single study of mice that reported increased TNFa and IL-1 (KE2) in liver homogenate (Ma et al..
2014a). However, that study is limited in that it evaluated a single duration of exposure (14 days) and
did not distinguish between IL-1 subtypes (i.e., IL-la, IL-ip). Lack of data for KE2 and KE4 is a data
gap, which reduces EPA's confidence in the postulated PPARa MOA.

For KE3, only one in vivo study of mice (and none of rats) is available that examined apoptosis in the
liver (Kaufmann et al.. 2002). In the available study, apoptosis was significantly increased after one
week of exposure to DINP and was unaffected after 4 weeks. This is inconsistent with the postulated
MOA, in which suppression of apoptosis is anticipated. However, this uncertainty is somewhat
addressed by the two available in vitro studies of rat hepatocytes that report consistent, dose-related,

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increases in PPARa activation (KE1), increases in replicative DNA synthesis (KE3) and suppression of
apoptosis (KE3) in hepatocytes following exposure to DINP (Shaw et al. 2002; Hasmall et ah. 1999).

Most of the available data for KE1 and KE3 comes from in vivo studies of rats and mice; however,
available studies are of variable design and in some instances employ large dose spacing, which makes
comparisons across studies difficult. Although it is clear that KE1 and KE2 occur at lower doses and
earlier than the apical outcome, liver tumors, providing some evidence of dose-response and temporal
concordance, concordance between KEs could not be established, which reduces EPA's confidence in
the postulated PPARa MOA.

Another uncertainty stems from an unexplained difference in sensitivity across sexes in B6C3F1 mice.
In the 2-year bioassay of B6C3F1 mice, liver tumors were observed at doses as low as 335 mg/kg-day in
female mice and 742 mg/kg-day in male mice (Covance Labs. 1998a). In contrast, other studies have
demonstrated that PPARa activation and proliferative DNA synthesis occur at lower doses in male
B6C3F1 mice compared to females (Kaufmann et al.. 2002). This inconsistency further reduced EPA's
confidence in the postulated PPARa MOA.

Despite remaining uncertainties, there is strong evidence to support the postulated PPARa MOA.
Available evidence indicates that DINP is not genotoxic (Section 2). Furthermore, other potential modes
of carcinogenic action, such as activation of CAR, PXR, and AhR, as well as cytotoxicity and
regenerative proliferation are also non-genotoxic threshold MO As. Finally, as discussed further below in
Section 4.8, the chronic non-cancer point of departure (POD) identified in EPA's Draft Non-cancer
Human Health Hazard Assessment for Diisononyl Phthalate (DINP) (	2024) will adequately

account for all chronic toxicity, including carcinogenicity and activation of PPARa (KE1), which could
potentially result from exposure to DINP.

4.8 Weight of Scientific Evidence: Cancer Classification

Under the Guidelines for Carcinogen Risk Assessment (	2005). EPA reviewed the weight of

evidence and determined that DINP is Not Likely to be Carcinogenic to Humans at doses below levels
that do not result in PPARa activation (KE1). This classification was based on the following weight of
scientific evidence considerations:

•	DINP exposure resulted in treatment related PPARa activation (KE1) in male mice at doses
greater than or equal to 117 mg/kg-day (Kaufmann et al.. 2002) and female rats at doses greater
than or equal to 442 mg/kg-day (Covance Labs. 1998b).

•	DINP exposure resulted in treatment related liver tumors (adenomas and/or carcinomas
combined) in female mice at doses greater than or equal to 336 mg/kg-day DINP (Covance Labs.
1998a) and female rats at doses greater than or equal to 672 mg/kg-day DINP (Bio/dynamics.
1987).

•	Available MOA data for liver tumors in mice and rats support the proposed PPARa MOA.

•	Limited data are available that indicate a role for other non-genotoxic, threshold, MO As,
including activation of other nuclear receptors (e.g., CAR, PXR, AhR, PPARy), inhibition of
GJIC, and cytotoxicity and regenerative proliferation.

•	There is no evidence for mutagenicity.

Further, the non-cancer chronic POD (NOAEL/LOAEL of 15/152 mg/kg-day based on non-cancer liver
effects (see Draft Non-cancer Human Health Hazard Assessment for Diisononyl Phthalate (DINP) (
E 24)) will adequately account for all chronic toxicity, including carcinogenicity, which could
potentially result from exposure to DINP. In one study of male mice (Kaufmann et al.. 2002).
biomarkers of PPARa activation were significantly increased at 117 mg/kg-day, which is less than the

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chronic LOAEL of 152 mg/kg-day based on non-cancer liver effects. Although, the study by Kaufman
et al. did not test sufficiently low doses to establish a NOAEL for PPARa activation, other studies of
mice have established a NOAEL of 75 mg/kg-day for PPARa activation (Smith et al.. 2000). Therefore,
the non-cancer chronic POD of 15 mg/kg-day is considered protective of PPARa activation.

4.9 Human Relevancy

Several panels have been convened to address the human relevancy of liver tumors in rodents occuring
through a PPARa MOA (Felter et al. 2018; Gorton et al.. 2014). These panels have generally concluded
that the PPARa MOA is not relevant to humans or unlikely to be relevant to humans based on
qualitative and quantitative differences between species. Nevertheless, uncertainty and differing
scientific opinions on the human relevance of the PPARa MOA for liver tumorigenesis remain, despite
the related efforts of previous panels and workshops.

Several authoritative agencies have evaluated the role of PPARa and peroxisome proliferation in
inducing hepatocellular tumors in rodents following chronic exposure to DINP. Australia NICNAS
(2 ) and U.S. CPSC (2010) concluded that liver tumors in rodents observed following exposure to
DINP are not likely to be human relevant, while ECHA (2013) and Health Canada (EC/HC. 2015)
concluded that liver tumors in rats are of unclear human relevance. However, none of these agencies
quantitatively evaluated DINP for carcinogenic risk to humans.

As discussed further in EPA's Draft Non-cancer Human Health Hazard Assessment for Diisononyl
Phthalate (DINP) (	2024). not all of the non-cancer liver effects observed in rodents are

consistent with PPARa activation (e.g., spongiosis hepatis). Furthermore, the non-cancer chronic POD
(NOAEL/LOAEL of 15/152 mg/kg-day) that is based on non-cancer liver toxicity will adequately
account for all chronic toxicity, including carcinogenicity, which could potentially result from exposure
to DINP.

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5 CONCLUSIONS AND NEXT STEPS

DINP has been evaluated for carcinogenicity in two 2-year dietary studies of F344 rats (Covamce Labs.
1998b; Lington et ai. 1997). one 2-year dietary study of SD rats (Bio/dynamics. 1987). and one 2-year
dietary study of B6C3F1 mice (Covamce Labs. 1998a). Across available studies, treatment-related
hepatocellular adenomas and carcinomas have consistently been observed in F344 and SD rats as well as
B6C3F1 mice. Existing assessments of DINP by U.S. CPSC (2014. 2010). Health Canada (ECCC/HC.
2020; EC/HC. 2015; Health Canada.: ), ECHA (2013). and NICNAS (2012) have postulated that
DINP causes liver tumors in rats and mice through a PPARa MOA. Consistent with EPA Guidelines for
Carcinogen Risk Assessment (U.S. EPA. 2005) and the IPCS Mode of Action Framework (IPCS. 2007).
EPA further evaluated the postulated PPARa MOA for liver tumors, as well as evidence for other
plausible MO As for DINP.

Although some uncertainties remain, there is strong evidence to support the postulated, non-genotoxic,
PPARa MOA. Under the Guidelines for Carcinogen Risk Assessment (	s05). EPA

determined that DINP is Not Likely to be Carcinogenic to Humans at doses below levels that do not
result in PPARa activation (KE1). Further, the non-cancer chronic POD (NOAEL/LOAEL of 15/152
mg/kg-day based on non-cancer liver effects; see EPA's Draft Non-cancer Human Health Hazard
Assessment for Diisononyl Phthalate (DINP) (	324)) will adequately account for all chronic

toxicity, including carcinogenicity, which could potentially result from exposure to DINP. Therefore, the
non-cancer chronic POD of 15 mg/kg-day is considered protective of PPARa activation and
carcinogenicity.

EPA is soliciting comments from the Science Advisory Committee on Chemicals (SACC) on charge
questions and comments from the public for an upcoming SACC meeting.

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Appendix A PATHOLOGY WORKING GROUP REVIEW FOR
SPONGIOSIS HEPATIS AND MNCL (EPL. 1999)

A Histopathology Peer Review and a Pathology Working Group (PWG) review (EPL. 1999) was
conducted on selected lesions of the liver and spleen observed in F344 rats in the 2-year bioassays
reported by Lington et al. (1997) and Covance Labs (1998b). The PWG review evaluated the
significance of spongiosis hepatis, foci of cellular alteration, primary hepatocellular neoplasms in the
liver, and the significance of MNCL. The peer and PWG reviews were conducted in accordance with
EPA Pesticide Regulation Notice 94-5 that describes the procedure to be followed for submission of
pathology re-reads to the Agency (EPL. 1999).

Spongiosis Hepatis

Induction of spongiosis hepatis, also referred to as cystic degeneration by some authors, is of interest
because it appears to be the most sensitive non-neoplastic response in rats chronically exposed to DINP
(Covance Labs. 199Kb; 1 iiieton et al.. 1997). However, questions have arisen regarding the relationship
of this lesion to other pathological processes occurring in animals treated with DINP that may not be
relevant to humans, including peroxisome proliferation and MNCL. Although a few differences were
noted, the Histology Peer Review and the PWG review of lesions in the liver and spleen generally
confirm the incidence data reported by the original study pathologists. The incidences of spongiosis
hepatis in the Lington et al. (1997) and Covance Labs (1998b) studies as determined by the PWG are
shown in TableApx A-l and TableApx A-2.

The PWG noted that spongiosis hepatis might be found as an independent lesion or within foci of
cellular alteration or hepatocellular neoplasms. In the reviewed studies, spongiosis hepatis was
diagnosed whenever it occurred, regardless of relationship to other hepatic changes that were also
present. This method of diagnosis differs from some standard pathology guidelines, which recommend
that spongiosis hepatis not be diagnosed separately when it occurs within foci or tumors. The PWG
concluded that the method of diagnosis used in the DINP rat studies made interpretation of spongiosis
hepatis as a treatment-related effect difficult. As noted in EPL (1999). some differences were noted in
the pathology protocols for the two studies which may have affected the reported incidences. These
differences include the number of sections taken from the liver in each study and the protocol for
examination of the spleen. These differences make the direct comparison of the results from Lington et
al. (1997) and Covance Labs (1998b) difficult and may account for the greater incidence of foci of
cellular alteration and foci of spongiosis hepatis observed by Lington et al. (1997).

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TableApx A-l. Incidence of MNCL and Selected Hepatic Lesions at Terminal Sacrifice (104

Lesion

Dose Group mg/kg-day (ppm)

Control

15M/18F
(300)

152 M /184 F
(3,000)

307 M / 375 F
(6,000)

Males

MNCL

32/81

27/80

48/80

49/80

Hepatocellular adenoma

3/81

1/80

2/80

1/80

Hepatocellular carcinoma

0/81

1/80

0/80

3/80

Eosinophilic foci

58/81

50/80

46/80

52/80

Basophilic foci

53/81

62/80

48/80

42/80

Spongiosis hepatis

22/81

24/80

51/80

62/80

Ivnuik-s

MNCL

22/81

21/81

29/80

41/80

Hepatocellular adenoma

0/81

4/81

0/80

2/80

Hepatocellular carcinoma

1/81

0/81

0/80

1/80

Eosinophilic foci

59/81

47/81

42/80

32/80

Basophilic foci

72/81

64/81

64/80

55/80

Spongiosis hepatis

4/81

1/81

3/80

4/80

Source: Modified from data in Table 6 in EPL (1999)
M = male; F = female

TableApx A-2. Incidence of MNCL and Selected Hepatic Lesions at Terminal Sacrifice (104

Lesion

Dose Group mg/kg-dav (ppm)

Control

29 M / 36 F
(500)

88 M /109 F
(1,500)

359 M / 442 F
(6,000)

733 M / 885 F
(12,000)

Recovery
637 M / 773 F
(12.000)

M;ik-s

MNCL

21/55

23/50

21/50

32/55

28/55

30/50

Hepatocellular
adenoma

2/55

4/50

1/50

4/55

7/55

6/50

Hepatocellular
carcinoma

1/55

0/50

0/50

3/55

11/55

3/50

Eosinophilic foci

22/55

14/50

16/50

15/55

10/55

12/50

Basophilic foci

40/55

34/50

33/50

28/55

27/55

25/50

Spongiosis hepatis

6/55

6/50

3/50

18/55

26/55

10/50

IvilKlk'S

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Lesion

Dose Group mg/kg-dav (ppm)

Control

29 M / 36 F
(500)

88 M /109 F
(1,500)

359 M / 442 F
(6,000)

733 Ml 885 F
(12,000)

Recovery
637 M / 773 F
(12,000)

MNCL

17/55

16/50

9/50

28/55

28/55

24/50

Hepatocellular
adenoma

1/55

1/50

0/50

1/55

1/55

1/50

Hepatocellular
carcinoma

0/55

0/50

0/50

1/55

6/55

2/50

Eosinophilic foci

10/55

5/50

7/50

7/55

0/55

4/50

Basophilic foci

37/55

32/50

31/50

18/55

5/55

13/50

Spongiosis hepatis

0/55

0/50

0/50

1/55

2/55

0/50

Source: Modified from data in Tables 9 and 10 in EPL (EPL, 1999)
M = male; F = female

Examination of Co-occurrence of MNCL and Spongiosis Hepatis

It has been suggested that the occurrence of spongiosis hepatis in rats exposed to DINP is a consequence
of MNCL (EPL. 1999). To address this possibility, the PWG examined the co-occurrence of spongiosis
hepatis and MNCL in the study by Lington et al. (1997) and Covance Labs (1998b). A comparison of
the numbers of animals with spongiosis hepatis with and without MNCL diagnosed by the study
pathologist did not support the conclusion that spongiosis hepatis is a consequence of MNCL as shown
in TableApx A-3. Although approximately half of the rats with spongiosis hepatis also had MNCL,
spongiosis hepatis was also observed in the absence of MNCL in the remainder of the affected animals.

Table Apx A-3. Comparison of Spongiosis Hepatis with MNCL as Determined by the PWG (EPL.

1999)	

Sex

Dose Group
(ppm)

Total with Spongiosis
Hepatis

Spongiosis Hepatis
without MNCL

Spongiosis Hepatis
with MNCL

( onipiiiison ol\laUi from l.intJlon cUil ( )

F

0

4

1

3

F

300

1

1

0

F

3,000

3

0

3

F

6,000

4

1

3

M

0

24

16

8

M

300

24

12

12

M

3,000

54

17

37

M

6,000

66

27

39

( oni|\inson ol\lal;i from ( owiikv 1 .iihs ( )

F

0

0

0

0

F

500

0

0

0

F

1,500

0

0

0

F

6,000

1

0

1

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Sex

Dose Group
(ppm)

Total with Spongiosis
Hepatis

Spongiosis Hepatis
without MNCL

Spongiosis Hepatis
with MNCL

F

12,000

2

0

2

F

12,000 recovery

0

0

0

M

0

5

1

4

M

500

5

4

1

M

1,500

2

1

1

M

6,000

14

8

6

M

12,000

21

11

10

M

12,000 recovery

9

5

4

Source: Modified from data in Tables 11 and 12 in EPL (1999)
M = male; F = female

1447

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