xvEPA EPA Document# EPA-740-R-25-008 January 2025 United States Office of Chemical Safety and Environmental Protection Agency Pollution Prevention Cancer Human Health Hazard Assessment for Diisononyl Phthalate (DINP) Technical Support Document for the Risk Evaluation CASRNs: 28553-12-0 and 68515-48-0 (Representative Structure) January 2025 ------- TABLE OF CONTENTS 1 INTRODUCTION 5 2 GENOTOXICITY AND MUTAGENICITY 6 3 CANCER HAZARD IDENTIFICATION AND CHARACTERIZATION 9 3.1 Human Evidence 9 3.2 Animal Evidence 9 3.2.1 Liver Tumors 9 3.2.1.1 Conclusions on Liver Tumors 10 3.2.2 Mononuclear Cell Leukemia 13 3.2.2.1 Conclusions on Mononuclear Cell Leukemia 14 3.2.3 Kidney Tumors 17 3.2.3.1 Conclusions on Kidney Tumors 18 3.2.4 Other Tumors 20 4 POSTULATED MODE OF ACTION FOR LIVER TUMORS IN RATS AND MICE 23 4.1 Postulated Mode of Action in Rats and Mice 23 4.1.1 Key Event 1: PPARa Activation 24 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 27 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 32 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 36 5 CONCLUSIONS 38 REFERENCES 39 APPENDICES 46 Appendix A PATHOLOGY WORKING GROUP REVIEW FOR SPONGIOSIS HEPATIS AND MNCL (EPL, 1999) 46 LIST OF TABLES Table 2-1. Summary of Genotoxicity Studies of DINP 6 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) 11 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) 12 Page 2 of 49 ------- 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) 12 Table 3-5. Incidence of MNCL in F344 Rats Exposed to DINP for 2 Years (Lington et al., 1997; Bio/dynamics, 1986) 13 Table 3-6. Incidence of MNCL in F344 Rats Exposed to DINP in the Diet for 2 Years (Covance Labs, 1998b) 14 Table 3-7. MNCL as a Cause of Unscheduled Death in F344 Rats Exposed to DINP in the Diet (Covance Labs, 1998b) 14 Table 3-8. Incidence of Kidney Tumors in Male F344 Rats Exposed to DINP in the Diet for 2 Years (Covance Labs, 1998b) 17 Table 3-9. Incidence of Kidney Tumors in F344 Rats Exposed to DINP for 2 Years (Lington et al., 1997; Bio/dynamics, 1986) 18 Table 3-10. Incidence of Tumors in Pancreas, Testes, and Uterus in SD Rats Exposed to DINP for 2 Years (Bio/dynamics, 1987) 22 Table 4-1. Dose-Response Concordance for PPARa MOA in Rats 30 Table 4-2. Dose-Response Concordance for PPARa 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) 47 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) 48 Table Apx A-3. Comparison of Spongiosis Hepatis with MNCL as Determined by the PWG (EPL, 1999) 49 KEY ABBREVIATIONS AND ACRONYMS a2u-globulin Alpha 2u-globulin AhR Aryl hydrocarbon receptor ALP Alkaline phosphatase ALT Alanine aminotransferase AST Aspartate aminotransferase BrdU Bromodeoxyuridine 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.) (or the Agency) F344 Fischer 344 (rats) GJIC Gap junctional intercellular communication GLP Good Laboratory Practice GSH Glutathione Page 3 of 49 ------- I ARC International Agency for Research on Cancer IL Interleukin KE Key event LOAEL Lowest-ob served-adverse-effect level LOEC Lowest-ob served-effect concentration MINP Monoisononyl phthalate 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 NTP National Toxicology Program OCSPP Office of Chemical Safety and Pollution Prevention OEHHA Office of Environmental Health Hazard Assessment (California) OPPT Office of Pollution Prevention and Toxics PBOX Peroxisomal beta oxidation 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) Srts Sirtuins TSCA Toxic Substances Control Act U.S. United States Page 4 of 49 ------- 1 INTRODUCTION On May 24, 2019, the United States Environmental Protection Agency (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 HPP. 2019). to conduct a risk evaluation for diisononyl phthalate (DINP) (CASRNs 28553-12-0 and 68515-48-0) (Docket ID: EPA-HO-QPPT-2018- 0436). EPA determined that these two CASRNs should be treated as a category of chemical substances as defined in 15 U.S.C. section 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 (1) whether the circumstances identified in the request constitute "conditions of use" under 40 CFR 702.33, and (2) 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. The Agency granted the request on December 2, 2019, and published the draft and final scope documents for DINP in August 2020 and 2021, respectively (U.S. EPA 2021. 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 Non-cancer Raman Health Hazard Assessment for Diisononyl Phthalate (DINP) (U.S. EPA. 2025). 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. 2015; ECHA. 2013; Tomar et al.. 2013; NICNAS. 2012; U.S. CPSC. 2010; ECB. 2003; U.S. CPSC. 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. 2013; Tomar et al.. 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. Page 5 of 49 ------- 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; ECHA. 2013; NICNAS. 2012; U.S. CPSC. 2010; EFSA. 2005; ECB. 2003; U.S. CPSC. 2001). EPA reviewed available genotoxicity studies of DINP that were cited in existing assessments (Table 2-1) and considered newer studies published between 2014 and 2024. 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 is not likely to be genotoxic or mutagenic. Table 2-1. Summary of Genotoxicity Studies of DINP Test Type Test System (Species/Strain/Sex) Dose/Duration Metabolic Activation Result Reference(s) Chromosomal aberrations - in vivo Micronucleus (bone marrow) (Adhered to OECD 474) Male CD-I mice Oral (gavage) doses of 0,500, 1,000, or 2,000 mg/kg-day for 2 days; sacrificed on day 3 Not applicable Negative for micronuclei (McKee 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) Chromosomal aberrations - in vitro Chromosomal aberrations Chinese hamster ovary cells 0, 40, 80, or 160 ng/mL for 3 hours (with activation) or 20 hours (without activation) ± Aroclor- induced rat liver S9 Negative for chromosomal aberrations (McKee et al.. 2000) Gene mutations - 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 (EG&G Mason Research Institute. 1982a) Mouse lymphoma mutation assay L5178Y+/— mouse lymphoma cells 1.5-8 jil/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) Page 6 of 49 ------- Test Type Test System (Species/Strain/Sex) Dose/Duration Metabolic Activation Result Reference(s) TA 1535, TA 1537, and TA 1538 Bacterial reverse S. typhimurium strains TA 98, TA 100, 0, 100,333, 1,000, 3,333, 10,000 (ig/plate ± Aroclor 1254-induced Negative for mutagenicity (Zeiaer et al.. 1985) mutation assay TA 1535, and TA 1537 rat or hamster liver S9 Bacterial reverse mutation assay S. typhimurium strains TA 98, TA 100, TA 1535, and TA 1537 20-5,000 (ig/plate ± Aroclor- induced rat liver S9 Negative for mutagenicity IYBASF. 1995. 1986) as reported by ECB (2003)]17 Bacterial S. typhimurium strains 0.5-5,000 (ig/plate ± Aroclor- Negative for (McKee et al.. reverse mutation assay TA 98, TA 100, TA 1535, TA 1537, induced rat liver S9 mutagenicity 2000) (plate and TA 1538 incorporation assay) Bacterial reverse S. typhimurium strains TA 98, TA 100, 20-5,000 (ig/plate ± Aroclor- induced rat Negative for mutagenicity (McKee et al.. 2000) mutation assay TA 1535, and TA liver S9 (pre-incubation 1537 assay) Other genotoxicity assays Unscheduled Rat hepatocyte 0, 0.625, 1.25,2.5,5.0, No No increase in (Litton Bionetics. DNA synthesis primary culture 10.0 |iL/mL unscheduled DNA synthesis 1982b) In vitro cell Balb/c-3T3 A31 125-3,750 nL/niL No No significant (Litton Bionetics. transformation mouse cells increase in transformed foci 1985) In vitro cell Balb/c-3T3 A31 2.5-254.5 |ig/mL No No significant (Litton Bionetics. transformation mouse cells increase in transformed foci 1981) In vitro cell Balb/c-3T3 A31 0.0326-3,260 yg/mL No No significant (Litton Bionetics. transformation mouse cells increase in transformed foci 1982a) In vitro cell Balb/c-3T3 A31 0.125-3.750 nL/mL No No significant (Barber et al.. 2000) transformation mouse cells increase in transformed foci In vitro cell Balb/c-3T3 A31 0.1-1 nL/mL ± rat liver S9 No significant (Microbiological transformation mouse cells increase in transformed foci Associates. 1982a) In vitro cell transformation Balb/c-3T3 A31 mouse cells 0.03-1 |iL/mL No No significant increase in transformed foci (Microbiological Associates. 1982c) In vitro cell transformation Balb/c-3T3 A31 mouse cells 0.01-1.0 jiL/mL No No significant increase in (Microbiological Associates. 1981) Page 7 of 49 ------- Test Type Test System (Species/Strain/Sex) Dose/Duration Metabolic Activation Result Reference(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). Page 8 of 49 ------- 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 MO A analysis for liver tumors is provided in Section 4. 3.1 Human Evidence EPA reviewed conclusions from previous assessments conducted by Health Canada (2018) that investigated the association between exposure to DINP and cancer. Additionally, EPA also evaluated new epidemiologic studies published after the Health Canada (2018) assessment (i.e., published 2018- 2019) to determine if newer epidemiologic studies would provide useful information for evaluating exposure-response relationship. The Agency 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) 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., mono-(carboxynonyl) phthalate [MCNP] and mono-(carboxyoctyl) phthalate [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% confidence interval: 0.90-1.16). Findings were similar in models stratified by estrogen/progesterone receptor status and body mass index. Health Canada (2018) also evaluated the relationship between phthalates and breast cancer. However, no epidemiologic studies were identified by Health Canada that examined the association between DINP and its metabolites and biomarkers of breast cancer. 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.. 1997; Bio/dynamics. 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. Statistically non-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 Section 3.2.1 through Section 3.2.4. 3.2.1 Liver Tumors The Non-cancer Raman Health Hazard Assessment for Diisononyl Phthalate (DINP) (U.S. EPA. 2025) 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 Page 9 of 49 ------- 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 alanine aminotransferase [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.. 19971 one 2-year dietary study of SD rats (Bio/dynamics. 1987). and one 2-year 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 2 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 al.. 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. Page 10 of 49 ------- 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/dvnamics, 1986) Lesion Dose Group mg/kg-day (ppm) Control 15M/18F (300) 152 M /184 F (3,000) 307 M / 375 F (6,000) Males17 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%) Females'1 Neoplastic nodules 0/81 (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 Lington et al. (1997) M = male; F = female " 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 were observed in either sex. 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)a& 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) Males Hepatocellular adenoma 4/65& (6%) 3/50 (6%) 2/50 (4%) 6/65 (9%) 10/65 (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 (t>g. 68); oo. 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 Dcrformcd b\ the National Toxicology Program (NTP) (U.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. Page 11 of 49 ------- 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)" Lesion Dose Group mg/kg-day (ppm) Control 27 M / 33 F (500) 271 M/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 nodule(s) h 2/70 (2.9%) 5/69 (7.2%) 6/69 (8.7%) 5/70 (7.1%) Females 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: Appendix K, Figure 1, pp. 11 (pp. 426 of the study report PDF) (Bio/dynamics, 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. b 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. 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) Lesion Dose Group mg/kg-day (ppm) Control (%) 90M/112F (500) 276 M / 336 F (1,500) 742 M / 910 F (6,000) 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 (d. 73) and AoDcndix 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). b Number of animals with tumor/total number of animals examined. Percent tumor incidence in parentheses. Page 12 of 49 ------- 3.2.2 Mononuclear Cell Leukemia MNCL has been observed in F344 rats in two 2-year dietary studies (Covance Labs. 1998b; Lington et al.. 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 al., 1997; Bio/dynamics, 1986) Lesion Dose Group (mg/kg-day) (ppm) Control 15M/18F (300) 152 M /184 (3,000) 307 M / 375 F (6,000) Males17 33/81 (41%) 28/80 (35%) 48/80* (60%) 51/80* (64%) Females'1 22/81 (27%) 20/81 (25%) 30/80* (38%) 43/80* (54%) Source: Table 8 in Lington et al. (1997) M = male; F = female " Number of animals with lesion/ total number of animals examined. Percent lesion incidence in parentheses. * Statistically significant at p < 0.05 when compared to the control incidence using Fisher's Exact test; statistical analysis performed by Lington et al. (1997). 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 PWG (EPL. 1999) generally confirmed the original findings of the study pathologist(s), including incidence of MNCL in F344 rats in both studies. PWG findings are further discussed in Appendix A. Page 13 of 49 ------- Table 3-6. Incidence of MNCL in F344 Rats Exposed to DINP in the Diet for 2 Years (Covance Labs. 1998b) "bc Sex 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 / Recovery6 637 M/ 774 F (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*'# (48%) Source: U.S. CPSC (2001) text vv. 68-71 and Aoocndix 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. 11 Analysis of individual animal data as performed by NTP and reported in the text and Appendix B of U.S CPSC (2001). h 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 analysis 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. d Statistically 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 (500) 88 M /109 F (1,500) 359 M / 442 F (6,000) 733 M / 885 F (12,000) Recovery " 637 M / 774 F (12,000) 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 (pp. 365 and 381) in Covance Labs (1998b). M = male; F = female 11 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; Lington 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 (60-64% in treated rats vs. 41% in concurrent controls) as well as in the females at 184 and 375 mg/kg-day (38-54% in treated rats vs. 27% 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 (46-62% Page 14 of 49 ------- incidence) compared to concurrent controls (34% incidence) as well as in the treated females at 442 and 885 mg/kg-day (45-48%) compared to concurrent controls (26%). 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 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). MNCL is a spontaneously occurring neoplasm of the hematopoietic system that reduces lifespan and is one of the most common tumor types occurring at a high background rate in the F344 strain of rat (also referred to as Fisher rat leukemia because it is so common) (Thomas et al.. 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 al.. 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). The F344/N strain of rat was used in NTP 2-year chronic and carcinogenicity bioassays for nearly 30 years (King-Herbert et al.. 2010; King-Herbert and Thayer. 2006). However, in the early 2000s NTP stopped using the F344/N strain of rat in large part because of high background incidence of MNCL and testicular Ley dig cell tumors that confounded bioassay interpretation. NTP subsequently replaced the F344 strain of rats with the Harlan SD strain (King-Herbert et al.. 2010; King-Herbert and Thayer. 2006). 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 et al.. 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 two to three 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 Page 15 of 49 ------- 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 al.. 1999; 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 et al. (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 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 (U.S. EPA. 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 (2010),3 and ECHA (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 al.. 2013) 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, the Science Advisory Committee on Chemicals (SACC) recommended that "the observation of an increased incidence of MNCL in a chronic bioassay employing the Fisher 344 rat should not be considered a factor in the determination of the cancer classification..." and "Most Committee members agreed that given the material presented in a retrospective review, MNCL and Ley dig Cell Tumors, among other tumor responses in F344 rat carcinogenicity studies lack relevance in predicting human carcinogenicity (Maronpot et al., 2016)." (U.S. EPA. 2024). Consistent with the recommendations of the 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. 1999). Therefore, MCL observed in Fischer 344 rats is not regarded as relevant to humans" (p. 49 of (NICNAS. 2012)). 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. 2015)). 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/dynamics. 1986) or in mice treated with DINP-1 (Caldwell. 1999). Therefore, MNCL will not be used to predict cancer risk in humans" (p. 82 of (U.S. CPSC. 2010)). 4 ECHA concluded, "With regard to MNCL, the review by (Thomas et al„ 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. 1999) 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 2013)). Page 16 of 49 ------- SACC, EPA is not further considering MNCL as a factor in the determination of the cancer classification for DINP. 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. Table 3-8. Incidence of Kidney Tumors in Male F344 Rats Exposed to DINP in the Diet for 2 Years (Covance Labs, 1998b) ahc 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 vv. 68-71 and AtroendixB. * = statistically significant at p < 0.05 by one or more of the following: Fisher's Exact test, Poly-3, LogisticRegression, or Life Table analysis. 11 Analysis of individual animal data as performed by NTP and reported in the textand Appendix B of U.S. CPSC (2001). h 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 analysis 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). Page 17 of 49 ------- Table 3-9. Incidence of Kidney Tumors in F344 Rats Exposed to DINP for 2 Years (Lington et al., 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) Males17 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%) Females'1 Transitional cell carcinoma 0/81 (0%) 0/81 (0%) 0/80 (0%) 0/80 (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 Lington et al. (1997) M = male; F = female 11 Number of animals with lesion/ total number of animals examined. Percent lesion incidence in parentheses. h Statistically significant at p < 0.05 when compared to the control incidence using Fisher's Exact test; statistical analysis performed by Lington 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) reported transitional cell carcinoma in 3 out of 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 out of 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 with F344 rats by Covance Labs (1998b). renal tubule cell carcinoma was observed in 2 out of 65 high-dose (733 mg/kg-day) males and 4/50 recovery high-dose (637 mg/kg-day) males compared to 0 out of 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 statistically non-significant increase in renal tubule cell carcinoma was observed in 1 out of 80 low-dose (15 mg/kg-day), 0 out of 80 mid-dose Page 18 of 49 ------- (152 mg/kg-day), and 2 out of 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 the International Agency for Research on Cancer (IARC) (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 (U.S. EPA. 1991). 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% of controls) or females (112% 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 repair (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 repair was observed in 1 out of 10 high-dose females. 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 Obu-globulin accumulating protein in the hyaline droplets; and (3) histopathological evidence of kidney lesions associated with obu-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 Obu-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) ctu- globulin accumulates in hyaline droplets, (4) intermediate lesions include granular casts and linear papillary mineralization, (5) absence of hyaline droplets and other histopathological changes in female rats and mice, and (6) negative for genotoxicity. Additional supporting evidence includes (1) reversible binding of chemical to Obu-globulin, (2) increased sustained cell proliferation in proximal tubule (P2 segment), and (3) dose-response relationship between hyaline droplet severity and renal tumor incidence. Page 19 of 49 ------- DINP has been shown to increase absolute and relative kidney weight in both male and female rats (Covance Labs. 1998b; Lington et al.. 1997; Bio/dynamics. 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 ECHA (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. 2Q15)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 (Tomaret 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 a2u-globulin MOA to be human relevant (U.S. EPA. 1991). Therefore, EPA did not consider it appropriate to derive quantitative estimates of cancer hazardfor data on kidney tumors observed in these studies and did not farther consider kidney tumors as a factor in the determination of the cancer classification for DINP. This conclusion was supported by the SACC. In its final report to EPA, the SACC states "The Agency has provided substantial evidence that the kidney tumors produced by DINP are due to a 2u-globulin MOA and correctly classified them as not relevant to humans" (U.S. EPA. 2024). 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. 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-globiilin accumulation mechanism that is not regarded as relevant to humans" (p. 49 of (NICNAS. 2012)). 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. 2013)). 111 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. 2015)). Page 20 of 49 ------- 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 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 of SD rats, 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 out of 69 females at the high-dose compared to 0 out of 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; however, the background rate of interstitial cell tumors is high in male F344 rats making it difficult to detect treatment-related increases in this tumor type in this strain of rat (King-Herbert et al.. 2010; King-Herbert and Thayer. 2006). Overall, there is too much uncertainty for EPA to consider using these data to derive quantitative estimates of cancer risk. Page 21 of 49 ------- Table 3-10. Incidence of Tumors in Pancreas, Testes, and Uterus in SD Rats Exposed to DINP for 2 Years (Bio/dynamics, 1987)" Observation Dose Group mg/kg-day (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 Testes 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 - - - - Uterus No. examined N/A N/A N/A N/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, and 59 in males and n = 59, 56, 60, and 59 in females in the control, low-, mid- and high-dose groups, respectively. Data from Appendix K of (Bio/dynamics, 1987). Page 22 of 49 ------- 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 and its metabolite monoisononyl phthalate (MINP), 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 (2014. 2010), Health Canada (ECCC/HC. 2020; EC/HC. 2015; Health Canada. 2015). ECHA (2013). and NICNAS (2012) have postulated that DINP causes liver tumors in rats and mice through a PPARa MOA. In contrast, California OEHHA has concluded that "PPARa activation may not be causally related to DINP-induced liver tumors in rats and mice" (OEHHA. 2013; Tomar et al.. 2013). 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) and Klaunig et al. (2003). 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) and Klaunig et al. (2003). Consistent with EPA's 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 and its metabolite MINP. 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 might modulate the dose-response behavior or the probability of inducing one or more KEs (Corton et al.. 2014V 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 Page 23 of 49 ------- 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. 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 six 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, six 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 and two in vitro studies of MINP, a metabolite 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 albino rats with concentrations of MINP ranging from 0.1 to 0.5 mM for 3 days caused large (up to -750%) dose-dependent increases in palmitoyl-CoA oxidation and laurate hydroxylation activity. Comparatively, smaller (-200-300%) increases in palmitoyl-CoA oxidation and laurate hydroxylation activity were observed in primary hepatocytes from marmoset monkeys 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. 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). Laurenzana et al. (2016) demonstrated that MINP can activate human PPARa in COS-1 cells transfected with a human PPARa luciferase reporter. Briefly, transfected cells were treated with 0, 0.1, 1, 10, or 100 |iM MINP for 24 hours. An approximate two-fold increase in MINP stimulated receptor activation was observed at the highest concentration. Finally, Kamenduliz et al. (2002) demonstrated that three different isomers of MINP (MINP-1, MINP-2, MINP-3) can increase PBOX in primary hepatocytes isolated from female B6C3F1 mice and male F344 rats treated with concentrations of each isomer ranging from 10 to 300 |iM for 72-hours, but not in primary hepatocytes isolated from male Syrian golden hamsters, male cynomolgus monkeys, or human donor liver tissue. 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 (-1,200 mg/kg-day) for 2 or 4 weeks; however, no change was observed in the low-dose group (-100 mg/kg-day). Similarly, BIBRA (1986) reported increased hepatic cyanide-insensitive palmitoyl-CoA oxidation levels and hepatic lauric Page 24 of 49 ------- acid 11- and 12-hydroxylase activities, as well as marked to very marked increases in hepatic peroxisomes (evaluated via transmission electron microscopy), 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 (Covance Labs. 1998b). In contrast, no evidence of peroxisome proliferation (evaluated via transmission electron microscopy) was reported in hepatocytes from male or female F344 rats treated with up to 307 (males) or 375 mg/kg-day DINP (females) for 2 years (Lington et al.. 1997). Evidence from In Vivo Studies of Mice Six 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 PBOX in male B6C3F1 mice fed diets containing up to 6,000 ppm DINP (-900 mg/kg-day) for 2 or 4 weeks; however, no change was observed in the low-dose group at either timepoint (-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 for 2 weeks (equivalent to approximately 600 to 1,200 mg/kg-day). 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). ToxStrategies (2024) reported microarray data from archived mouse liver tissue samples from a study originally conducted in 1999 at the Chemical Industry Institute of Toxicology (CUT). Briefly, wild-type male mice (12-weeks of age) were orally gavaged with either vehicle control, 50 mg/kg-day WY14643 (WY), 1000 mg/kg-day DINP (low-dose), or 2000 mg/kg-day DINP (high-dose) for 1 or 3 weeks. RNA was isolated from archived samples and analyzed by Affymetrix GeneTitan array analyzer. A total of 33, 43, and 330 genes were differentially expressed in low-dose, high-dose, and WY groups of compared to controls. In the low- and high-dose DINP groups, the top up-regulated gene was Cyp4al4, which is a target gene of PPARa, and is involved in fatty acid metabolism. Other significantly up- regulated genes in both DINP treatment groups were involved in lipid and fatty acid metabolism, and PPARa activation, including Cyp4alO, Cyp4a31, Vcminl, Acotl/2, and Accial/2. Only five gene sets were enriched in both DINP dose groups, with the top gene sets enriched in both DINP treatment groups being related to fatty acid metabolism, protein localization, and organic hydroxy compound metabolic process. Comparatively, 58 gene sets were enriched in the WY group, with gene sets mostly related to fatty acid and lipid metabolism, peroxisome activity, and PPAR signaling. Overall, PPARa was the top (WY and high-dose DINP groups) or only (low-dose DINP group) transcriptional regulator predicted for all three treatment groups. However, there were some sources of uncertainty that may impact the interpretation of the results. For instance, the ToxStrategies report noted uncertainty pertaining to the duration of exposure; ToxStrategies indicated that conflicting durations of 1 and 3 weeks were listed in the original study. Additionally, a considerable length of time had elapsed between the original study and the microarray analysis by ToxStrategies, although the authors did evaluate mRNA quality and Page 25 of 49 ------- excluded low quality samples. Despite these and other uncertainties in study design, this study provides evidence that the primary response in the liver following 1 or 3 weeks of exposure to high doses of DINP (i.e., 1,000 and 2,000 mg/kg-day) are related to PPARa activation. Consistent with DINP being a weaker PPARa activator compared to WY, the transcriptomic response in the livers of DINP treated mice was lower than that of the potent PPARa activator, WY. 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 (Pugh 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 al.. 1999). 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 were significantly increased at 20 and 200 mg/kg-day. However, the study authors do not identify the specific IL-1 subtypes evaluated (e.g., IL-la vs. IL-ip), which is an important consideration when interpreting these results. 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 has been consistently observed. In contrast, cellular proliferation in the liver is not sustained chronically in either species. However, as discussed by Corton et al. (2018). weak 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 MOA. 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 are 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 TGFpi-induced apoptosis in rat but not human hepatocytes. Similarly, Shaw et al. (2002) 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 Page 26 of 49 ------- replicative DNA synthesis in male F344 rats fed diets containing 12,000 ppm DINP (-1,200 mg/kg-day) for 2 or 4 weeks; however, no change was observed in the low-dose group (-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. 1998b). 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-null mice diets containing 8,000 ppm DINP (-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 (-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 (-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 (Pugh 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. 4.1.5 Modulating Factors EPA identified no studies evaluating activation of NF-kB in the liver. Three studies provide data on the relationship between oxidative stress and DINP following in vivo exposures in male Kunming mice (Ma et al.. 2014b) and male Balc/c mice (Liang and Yan. 2020) or in vitro investigations in human hepatic cell-types (Gutierrez-Garcia et al.. 2019). Available studies provide evidence that DINP can induce ROS in the liver. Page 27 of 49 ------- 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 20 mg/kg-day DINP and above, 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. Liang and Yan (2020) exposed male Balb/c mice dermally to 0, 0.02, 0.2, 2, 20, and 200 mg/kg-day DINP for 28 days and then evaluated several endpoints related to oxidative stress in homogenized liver tissue. Dose-dependent increases in ROS and increases in malondialdehyde were observed at 20 mg/kg- day DINP and above, while levels of glutathione (GSH) decreased at 20 mg/kg-day DINP and above. Additionally, increased DNA-protein-crosslinks were observed at 200 mg/kg-day, which indicate oxidative damage to DNA. These data are consistent with increases in oxidative stress at doses of 20 mg/kg-day and above. In parallel, significant increases in relative liver weight were observed at 20 mg/kg-day DINP and above, which may have been associated with activation of PPARa; however, no specific biomarkers of PPARa activation were evaluated in this study. As discussed in the Non-cancer Raman Health Hazard Assessment for Diisononyl Phthalate (DINP) (U.S. EPA. 2025). dermal absorption of DINP is low (i.e., 2-4% over 7 days), which suggests that the dose of absorbed DINP that caused oxidative stress in the study by Liang and Yan (2020) is much lower than the dermally applied dose of 20 mg/kg-day. However, there are several sources of uncertainty associated with the study by Liang and Yan (2020) with regard to the actual received doses in the study, as only nominal doses are provided. Liang and Yan state that 20 |iL of test solution (concentration of applied test solution not provided) was applied evenly to a 2 cm2 area of exposed skin on the center of the back of the mouse, however, additionally methodological details pertaining to how DINP was dermally administered were not provided. For example, study authors do not provide information relating to how hair was removed from the backs of mice and whether or not care was taken to avoid applying solutions of DINP to abraded skin, which would be expected to increase dermal absorption; how frequently DINP solutions were applied and whether DINP was washed from the skin at the application site between dermal applications; and whether skin was covered with a bandage to help limit evaporation, as well as oral ingestion of DINP through grooming. 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, SirtS) 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. Page 28 of 49 ------- 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 reported consistent dose-related increases in several biomarkers of PPARa activation (i.e., increased PBOX, lauric acid 11- and 12-hydroxylase, and palmitoyl-CoA oxidase activity) (Smith et al.. 2000; Covance Labs. 1998b; BIBRA. 1986). 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 reported 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 and 885 mg/kg-day in males and females, respectively), reported 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. 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, 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 KE4. 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 al.. 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 al.. 2014a); for KE3 increased DNA synthesis has been reported at doses as low as 116 mg/kg-day in male mice (Kaufmann et al.. 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 data set 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. Page 29 of 49 ------- Table 4-1. )ose-Response Concordance for PPARa M< 3 A in Rats Dose (mg/kg- day) KE 1 (Sex; Dose in mg/kg-day; Timepoint) KE 2 KE3 (Sex; Dose in mg/kg-day; Timepoint) KE 4 Hepatocellular Tumors 1-200 NC - PBOX (M; 120; 2, 4 weeks)a NC - DNA synthesis (M; 120; 2, 4 weeks)" NC - Neoplastic nodules, hepatocellular cancer, or combined (M/F; 15-184; 104 weeks) d NC - Adenomas, carcinomas, combined (M/F, 29-109; 104 weeks)b NC - Neoplastic nodules, carcinoma (M/F; 27- 33; 104 weeks)e 201-400 NC - PP (M/F; 307-375; 2 years) d NC - Neoplastic nodules, hepatocellular cancer, or combined (M/F; 307-375; 104 weeks) d NC - Adenomas, carcinomas, combined (M/F, 359-442; 104 weeks)b NC - Neoplastic nodules, carcinoma (M/F; 271- 331; 104 weeks)e 401-600 | Palm CoA (F (not M); 442; 104 (but not 1,2, or 13) weeks)b - - - - 601-1,000 NC - Palm CoA (M/F;607-639; 3 weeks)c t 11/12 H-lase (M, not F); 639; 3 weeks)c t Palm CoA (M/F; 733-885; 1, 2, 13, 104 weeks)* t DNA synthesis (M/F; 733-885; 1 week (but not 2, 13, 104 weeks)b t Carcinoma (F (not M); 672; 104 weeks)e t Carcinoma (M (not F); 733-885; 104 weeks)b 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)c t 11/12 H-lase (M, not F); 1,192; 3 weeks)c t PBOX (M; 1,200; 2, 4 weeks)a | 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 t PP (M/F; 2,195-2,289; 3 weeks)c - - - - 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 ° (Smith et al.. 2000) h (Covance Labs. 1998b) <¦ (BIBRA. 1986) d (Lineton et al.. 1997) e (Bio/dvnamics. 1987) Page 30 of 49 ------- Table 4-2. Dose-Response Concordance for PPARg MOA in Mice Dose (mg/kg-day) KE 1 (Sex; Dose in mg/kg-day; Timepoint) KE 2 KE3 (Sex; dose in mg/kg-day; timepoint) KE 4 Hepatocellular Tumors 1-200 NC - PBOX (M; 75; 2,4 weeks)" t PP & Palm CoA (M (but not F); 117; 4 weeks)b NC - TNFa (M, 0.2-2, 2 weeks)f t TNFa (M, 20- 200, 2 weeks)f NC - DNA synthesis (M; 75; 2, 4 weeks) ° t DNA synthesis (M (but not F); 116-167; 1, 4 weeks) b NC - Apoptosis (M/F; 116-167; 1, 4 weeks)b NC - Adenomas or carcinomas (M/F; 90-112, 2 years) d 201-400 t PP & Palm CoA (M; 350; 4 weeks)6 t Palm CoA (M/F; 365; 4, 31, 91 days)c | DNA synthesis (M; 337-350; 1, 4 weeks)b NC - Apoptosis (M; 337-350; 1, 4 weeks)b t Combined adenomas & carcinomas (F (but not M); 336, 2 years) d 401-600 | PP & Palm CoA (F; 546; 4 weeks)b | Palm CoA (M/F; 600; 2 weeks)e - NC - DNA synthesis (F; 520-546; 1, 4 weeks)b NC - Apoptosis (F; 520-546; 1, 4 weeks)b - - 601-800 t Combined adenomas & carcinomas (M; 742, 2 years) d 801-1,000 t PBOX (M; 900; 2,4 weeks)a t PP & Palm CoA (M; 913; 4 weeks)b t gene expression (M; 1,000; 1 or 3 weeks)g t DNA synthesis (M; 75; 2 (not 4) weeks) ° | DNA synthesis (M; 901-913; 1,4 weeks)b NC - Apoptosis (M; 901-913; 1,4 weeks)b t Carcinomas and combined adenomas & carcinomas (F; 910, 2 years) d 1,001-1,400 | Palm CoA (M/F; 1,200; 2 weeks)e t PP & Palm CoA (F; 1,272; 4 weeks)b | DNA synthesis (F; 1200; 1 week)e t DNA synthesis (F; 1272-1278; 1 (but not 4) weeks)b NC - Apoptosis (F; 1272-1278; 1, 4 weeks)b 1,401-2,000 t PP & Palm CoA (M; 1,860; 4 weeks)b t Palm CoA (M/F; 1,560-1,888; 79, 105 weeks) d t gene expression (M; 1,000; 1 or 3 weeks)g | DNA synthesis (M; 1766-1860; 1, 4 weeks)b NC - DNA synthesis (M/F; 1,560-1,888; 79, 105 weeks) d t Apoptosis (M; 1,766-1,860; 1 (but not 4) weeks)b t Adenomas and/or carcinomas (M/F; 1,560- 1,888, 2 years) d 2,001-3,000 t Palm CoA (M/F; 2600; 4, 31, 91 days) c t PP & Palm CoA (F; 2806; 4 weeks)6 | DNA synthesis (F; 2593-2806; 1 (but not 4) weeks)b NC - DNA synthesis (M/F; 2,600; 4, 41, 91 days)c NC - Apoptosis (F; 2,593-2,806; 1, 4 weeks)b indicates no experimental evidence is available; t = significant increase; j = 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 ° (Smith et al.. 2000) b (Kaufmann et al.. 2002) c (Hazleton Labs. 1992) d (Covance Labs. 1998a) e (Valles etal.. 2003) ^(Ma et al.. 2014a) g(ToxStrateeies. 2024) Page 31 of 49 ------- 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 one 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 (Hazleton 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 (U.S. CPSC. 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. (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." 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. 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 (Corton et al.. 2018; Corton et al.. 2014)). 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 Page 32 of 49 ------- studies demonstrate that oral exposure to DINP does not cause chronic proliferative response in the liver of mice or rats. As discussed by Corton 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 of carcinogenic action in the liver for DINP. Ppara-NuU 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-mxW 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- mxW 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-mxW 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-mxW 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-mxW mice. The relevance of these subtle PPARa-independent effects to hepatocarcinogenesis is not known, but Ppara-mxW 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, a metabolite of DINP, can also activate hCAR2 (Laurenzana et al.. 2016). Additionally, in vitro studies have also shown that DINP and MINP can activate human PXR (Laurenzana et al.. 2016; Dekevser et al.. 2011) 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 (Wang et al.. 2012). DINP induces several changes in rodent liver consistent with PPARa activation (Laurenzana et al.. 2016); notably, DINP induces some of these liver changes independently of PPARa activation as shown in Ppara-mxW mice (Valles et al.. 2003). Page 33 of 49 ------- 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 (NTP) 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, and 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 AC50fl / 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 ATGPXREC ISup 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 ACEA ER AUC viability AcuteTox - Cytotoxicity, Cancer - KCC10: Cell Proliferation/Death/Energetics 38.8 AC50 = concentration at which 50% maximum activity is observed; LOEC = lowest-observable-effect concentration; LOEC = lowest-observed-effect concentration 11 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 (Klaunig et al.. 2003). Several tumor types, including hepatocellular carcinomas, have been shown to demonstrate inhibited GJIC (Trosko et al.. 1990c; Trosko et al.. 1990a. b; Trosko 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.. 2000; Trosko et al.. 1990c; Trosko et al.. 1990b). Additionally, DINP has been shown to inhibit GJIC in vitro. Kamenduliz et al. (2002) demonstrate that three different MINP isomers (MINP-1, MINP-2, MINP-3) can inhibit GJIC in primary hepatocytes isolated from female B6C3F1 mice and male F344 rats treated with concentrations of each isomer ranging from 50 to 300 |iM for 72 hours—but not in primary hepatocytes isolated from male Syrian golden hamsters, male cynomolgus monkeys, or human donor liver tissue. Page 34 of 49 ------- Cytotoxicity and Regenerative Proliferation Cytotoxicity followed by regenerative proliferation is an established nongenotoxic MOA (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 while 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 data set 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 and 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 1 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, 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 al.. 1999). Page 35 of 49 ------- 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. 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 Non-cancer Raman Health Hazard Assessment for Diisononyl Phthalate (DINP) (U.S. EPA. 2025) 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 (U.S. EPA. 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 (doses >117 mg/kg-day) (Kaufmann et al.. 2002) and female rats (doses >442 mg/kg-day (Covance Labs. 1998b). • DINP exposure resulted in treatment-related liver tumors (adenomas and/or carcinomas combined) in female mice (doses >336 mg/kg-day DINP) (Covance Labs. 1998a) and female rats (doses >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 Non-cancer Human Health Hazard Assessment for Diisononyl Phthalate (DINP) (U.S. EPA. 2025)) 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 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 occurring through a PPARa MOA (Felter et al.. 2018; Corton 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 Page 36 of 49 ------- 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 (2012) 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 Non-cancer Raman Health Hazard Assessment for Diisononyl Phthalate (DINP) (U.S. EPA 2025). 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. Page 37 of 49 ------- 5 CONCLUSIONS DINP has been evaluated for carcinogenicity in two 2-year dietary studies of F344 rats (Covance Labs. 1998b; Lington et al.. 19971 one 2-year dietary study of SD rats (Bio/dynamics. 1987). and one 2-year dietary study of B6C3F1 mice (Covance 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. 2015). 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 (U.S. EPA. 2005). EPA determined that DINP is Not Likely to be Carcinogenic to Ramans 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 Non-cancer Raman Health Hazard Assessment for DiisononylPhthalate (DINP) (U.S. EPA. 2025)) 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. 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Environ Mol Mutagen 7: 213-232. http://dx.doi.org/10.1002/em.28600702Q9 Page 45 of 49 ------- APPENDICES 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, which 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. 1998b; Lington 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). Page 46 of 49 ------- 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) 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 Females 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 Page 47 of 49 ------- TableApx 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) Dose Group mg/kg-day (ppm) Lesion Control 29 M / 36 F 88 M/109 359 M / 442 F 733 M / 885 F Recovery 637 M / 773 F (12,000) (500) F (1,500) (6,000) (12,000) Males 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 1/55 0/50 0/50 3/55 11/55 3/50 carcinoma 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 Females 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 0/55 0/50 0/50 1/55 6/55 2/50 carcinoma 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 (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 Table Apx 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. Page 48 of 49 ------- TableApx 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 Comparison of data from Lington et al. (1997) 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 Com parison of data from Covance Labs (1998b) F 0 0 0 0 F 500 0 0 0 F 1,500 0 0 0 F 6,000 1 0 1 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 Page 49 of 49 ------- |