United States Environmental Protection 1=1 m m Agency EPA/690/R-11/026F Final 5-11-2011 Provisional Peer-Reviewed Toxicity Values for Diphenylamine (CASRN 122-39-4) Superfund Health Risk Technical Support Center National Center for Environmental Assessment Office of Research and Development U.S. Environmental Protection Agency Cincinnati, OH 45268 ------- AUTHORS, CONTRIBUTORS, AND REVIEWERS CHEMICAL MANAGER J. Phillip Kaiser, PhD National Center for Environmental Assessment, Cincinnati, OH DRAFT DOCUMENT PREPARED BY ICF International 9300 Lee Highway Fairfax, VA 22031 PRIMARY INTERNAL REVIEWERS Ambuja Bale, PhD, DABT National Center for Environmental Assessment, Washington, DC Q. Jay Zhao, PhD, MPH, DABT National Center for Environmental Assessment, Cincinnati, OH This document was externally peer reviewed under contract to Eastern Research Group, Inc. 110 Hartwell Avenue Lexington, MA 02421-3136 Questions regarding the contents of this document may be directed to the U.S. EPA Office of Research and Development's National Center for Environmental Assessment, Superfund Health Risk Technical Support Center (513-569-7300). ------- TABLE OF CONTENTS COMMONLY USED ABBREVIATIONS iii BACKGROUND 1 DISCLAIMERS 1 QUESTIONS REGARDING PPRTVS 1 INTRODUCTION 2 REVIEW OF POTENTIALLY RELEVANT DATA (CANCER AND NONCANCER) 4 HUMAN STUDIES 11 Oral Exposures 11 Inhalation Exposures 11 Chronic-duration Studies 11 ANIMAL STUDIES 13 Oral Exposures 13 Short-term Studies 13 Sub chronic-duration Studies 14 Chronic-duration Studies 18 Developmental Studies 25 Reproductive Studies 26 Inhalation Exposures 28 OTHER STUDIES (SHORT-TERM TESTS, OTHER EXAMINATIONS) 28 DERIVATION 01 PROVISIONAL VALUES 40 DERIVATION OF ORAL REFERENCE DOSES 41 Derivation of Subchronic Provisional RfD (Subchronic p-RfD) 41 Derivation of Chronic RfD (Chronic RfD) 41 DERIVATION OF INHALATION REFERENCE CONCENTRATIONS 41 CANCER WEIGHT-OF-EVIDENCE (WOE) DESCRIPTOR 41 DERIVATION OF PROVISIONAL CANCER POTENCY VALUES 42 Derivation of Provisional Oral Slope Factor (p-OSF) 42 Derivation of Provisional Inhalation Unit Risk (p-IUR) 42 MODE-OF-ACTION DISCUSSION 42 APPENDIX A. PROVISIONAL SCREENING VALUES 44 APPENDIX B. DATA TABLES 49 APPENDIX C. BENCHMARK DOSE CALCULATIONS FOR THE SCREENING SUBCHRONIC p-RlD 60 APPENDIX D. REFERENCES 71 li Diphenyl amine ------- COMMONLY USED ABBREVIATIONS BMC benchmark concentration BMD benchmark dose BMCL benchmark concentration lower bound 95% confidence interval BMDL benchmark dose lower bound 95% confidence interval HEC human equivalent concentration HED human equivalent dose IUR inhalation unit risk LOAEL lowest-observed-adverse-effect level LOAELadj LOAEL adjusted to continuous exposure duration LOAELhec LOAEL adjusted for dosimetric differences across species to a human NOAEL no-ob served-adverse-effect level NOAELadj NOAEL adjusted to continuous exposure duration NOAELhec NOAEL adjusted for dosimetric differences across species to a human NOEL no-ob served-effect level OSF oral slope factor p-IUR provisional inhalation unit risk p-OSF provisional oral slope factor p-RfC provisional reference concentration (inhalation) p-RfD provisional reference dose (oral) POD point of departure RfC reference concentration (inhalation) RfD reference dose (oral) UF uncertainty factor UFa animal-to-human uncertainty factor UFC composite uncertainty factor UFd incomplete-to-complete database uncertainty factor UFh interhuman uncertainty factor UFl LOAEL-to-NOAEL uncertainty factor UFS subchronic-to-chronic uncertainty factor WOE weight of evidence 111 Diphenyl amine ------- FINAL 5-11-2011 PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR DIPHENYLAMINE (CASRN 122-39-4) BACKGROUND A Provisional Peer-Reviewed Toxicity Value (PPRTV) is defined as a toxicity value derived for use in the Superfund Program. PPRTVs are derived after a review of the relevant scientific literature using established Agency guidance on human health toxicity value derivations. All PPRTV assessments receive internal review by a standing panel of National Center for Environment Assessment (NCEA) scientists and an independent external peer review by three scientific experts. The purpose of this document is to provide support for the hazard and dose-response assessment pertaining to chronic and subchronic exposures to substances of concern, to present the major conclusions reached in the hazard identification and derivation of the PPRTVs, and to characterize the overall confidence in these conclusions and toxicity values. It is not intended to be a comprehensive treatise on the chemical or toxicological nature of this substance. The PPRTV review process provides needed toxicity values in a quick turnaround timeframe while maintaining scientific quality. PPRTV assessments are updated approximately on a 5-year cycle for new data or methodologies that might impact the toxicity values or characterization of potential for adverse human health effects and are revised as appropriate. It is important to utilize the PPRTV database (http://hhpprtv.ornl.gov) to obtain the current information available. When a final Integrated Risk Information System (IRIS) assessment is made publicly available on the Internet (www.epa.gov/iris), the respective PPRTVs are removed from the database. DISCLAIMERS The PPRTV document provides toxicity values and information about the adverse effects of the chemical and the evidence on which the value is based, including the strengths and limitations of the data. All users are advised to review the information provided in this document to ensure that the PPRTV used is appropriate for the types of exposures and circumstances at the site in question and the risk management decision that would be supported by the risk assessment. Other U.S. Environmental Protection Agency (EPA) programs or external parties who may choose to use PPRTVs are advised that Superfund resources will not generally be used to respond to challenges, if any, of PPRTVs used in a context outside of the Superfund program. QUESTIONS REGARDING PPRTVS Questions regarding the contents and appropriate use of this PPRTV assessment should be directed to the EPA Office of Research and Development's National Center for Environmental Assessment, Superfund Health Risk Technical Support Center (513-569-7300). 1 Diphenyl amine ------- FINAL 5-11-2011 INTRODUCTION Diphenylamine is produced in the United States in fairly large amounts and is primarily used in dye manufacturing and as a nitrocellulose explosive and celluloid stabilizer (Hazardous Substances Data Bank [HSDB], 2010). In the analytical chemistry field, it is used in the detection of nitrates, chlorates, and other oxidizing substances. It is also used as a rubber antioxidant and accelerator, solid rocket propellant, fungicide and herbicide, storage preservative for apples, topical application in antiscrewworm mixtures in the pharmaceutical industry, and as a stabilizer for formaldehyde copolymers, epoxy resins, polyvinyl chloride, and polyoxyethylene. A foliar application of 1% diphenylamine in dust formation reportedly has decreased injury caused by ozone to various leaves of plants, including apple, bean, muskmelon, and petunia (HSDB, 2010). HSDB (2010) reports that diphenylamine is used as a potential model to research human bilateral disorder (an autosomally inherited kidney disease) by using the chemical to study polycystic kidneys in animals. The empirical formula for diphenylamine is C12H11N (see Figure 1). A table of the physicochemical properties is provided below (see Table 1). Figure 1. Diphenylamine Structure Table 1. Physicochemical Properties Table (Diphenylamine)3 Property (Unit) Value Boiling point (°C) 302 Melting point (°C) 52.9b Density (g/cm3) 1.16 Vapor pressure (Pa at 25°C) 0.089 pH (unitless) NA Solubility in water (mg/L at 20°C) 53 Vapor density (air =1) 5.82 Molecular weight (g/mol) 169.23 aValues from HSDB unless otherwise specified; http://toxnet.nlm.nih.gov/cgi-bin/sis/htiiilgen7HSDB bValues from ChemlD Plus Advanced; http://chem.sis.nlm.nih.gov/chemidplus/ProxyServlet?objectHandle=DBMaint&actionHandle=default&nextPage=jsp /chemidheavy/ResultScreen.jsp&ROW_NUM=0&TXTSUPERLISTID=0000122394 NA = not available 2 Diphenylamine ------- FINAL 5-11-2011 2 A chronic oral Reference Dose (RfD) of 2.5 x 10" mg/kg-day for diphenylamine is included in the IRIS database (U.S. EPA, 2010a). This value is based on decreased body-weight gain and increased liver and kidney weights in dogs in a 2-year feeding study (Thomas et al., 1967a). However, no Reference Concentration (RfC) or cancer assessment is available on IRIS (U.S. EPA, 2010a). Diphenylamine is not included on the Drinking Water Standards and Health Advisories List (U.S. EPA, 2009). No RfD or RfC values have been reported in the HEAST (U.S. EPA, 2010b). The CARA list (U.S. EPA, 1994a) has a Health and Environmental Effects Profile (HEEP) for diphenylamine (U.S. EPA, 1985). The toxicity of diphenylamine has not been reviewed by ATSDR (2008) or the World Health Organization (WHO, 2010). CalEPA (2008) has not derived toxicity values for exposure to diphenylamine. The American Conference of Governmental Industrial Hygienists (ACGIH) has set an 8-hour time-weighted average (TWA) of 10 mg/m3 for occupational exposures to diphenylamine (ACGIH, 2010) based on industrial poisoning symptoms such as bladder and skin problems, abnormal heartbeat, and high blood pressure. This value is also the recommended exposure limit (REL) set by the National Institute of Occupational Safety and Health (NIOSH, 2005) and the personal exposure limit (PEL) set by the Occupational Safety and Health Administration (OSHA, 2010). The HEAST (U.S. EPA, 2010b) does not report any cancer values for diphenylamine. The International Agency for Research on Cancer (IARC, 2010) has not reviewed the carcinogenic potential of diphenylamine. Diphenylamine is not included in the 11th Report on Carcinogens (NTP, 2005). CalEPA (2008) has not prepared a quantitative estimate of carcinogenic potential for diphenylamine. ACGIH has classified diphenylamine as a Group A4 carcinogen—Not Classifiable as a Human Carcinogen (ACGIH, 2010). Literature searches were conducted on sources published from 1900 through April 2011 for studies relevant to the derivation of provisional toxicity values for diphenylamine, CAS Number 122-39-4. Searches were conducted using EPA's Health and Environmental Research Online (HERO) database of scientific literature. HERO searches the following databases: AGRICOLA; American Chemical Society; BioOne; Cochrane Library; DOE: Energy Information Administration, Information Bridge, and Energy Citations Database; EBSCO: Academic Search Complete; GeoRef Preview; GPO: Government Printing Office; Informaworld; IngentaConnect; J-STAGE: Japan Science & Technology; JSTOR: Mathematics & Statistics and Life Sciences; NSCEP/NEPIS (EPA publications available through the National Service Center for Environmental Publications [NSCEP] and National Environmental Publications Internet Site [NEPIS] database); PubMed: MEDLINE and CANCERLIT databases; SAGE; Science Direct; Scirus; Scitopia; SpringerLink; TOXNET (Toxicology Data Network): ANEUPL, CCRIS, ChemlDplus, CIS, CRISP, DART, EMIC, EPIDEM, ETICBACK, FEDRIP, GENE-TOX, HAPAB, HEEP, HMTC, HSDB, IRIS, ITER, LactMed, Multi-Database Search, NIOSH, NTIS, PESTAB, PPBIB, RISKLINE, TRI, and TSCATS; Virtual Health Library; Web of Science (searches Current Content database among others); World Health Organization; and Worldwide Science. The following databases outside of HERO were searched for risk assessment values: ACGIH, ATSDR, CalEPA, EPA IRIS, EPA HEAST, EPA HEEP, EPA OW, EPA TSCATS/TSCATS2, NIOSH, NTP, OSHA, and RTECS. A number of unpublished studies with diphenylamine were located in a formal government risk assessment document (European Communities, 2008), where they are reported only as summaries, although full references are given. Given that these are acknowledged in the 3 Diphenylamine ------- FINAL 5-11-2011 assessment as full reports and not, for example, as abstracts from a scientific conference, all listed therein are considered as relevant and valid for use in this assessment. It is also acknowledged here that these report summaries are evaluated in this assessment as to their accuracy and internal consistency. REVIEW OF POTENTIALLY RELEVANT DATA (CANCER AND NONCANCER) Table 2 provides an overview of the relevant database for diphenylamine and includes all potentially relevant repeated short-term-, subchronic-, and chronic-duration studies. No-observed-adverse-effect levels (NOAELs), lowest-observed-adverse-effect levels (LOAELs), and benchmark dose lower limits/benchmark concentration lower limits (BMDLs/BMCLs) are provided in human equivalent doses/human equivalent concentrations (HEDs/HECs) for comparison except that oral noncancer values are not converted to HEDs and are identified in parentheses as (Adjusted). Developmental studies are not adjusted for continuous exposure. Principal studies are identified. Entries for the principal studies are bolded. The phrase, "statistical significance" used throughout the document, indicates a /7-value of <0.05. 4 Diphenylamine ------- FINAL 5-11-2011 Table 2. Summary of Potentially Relevant Data for Diphenylamine (CASRN 122-39-4) Notes3 Category Number of Male/Female, Strain Species, Study Type, Study Duration Dosimetryb Critical Effects NOAEL" BMDL/ BMCL" LOAEL" Reference (Comments) Human 1. Oral (mg/kg-day)b Subchronic None Chronic None Developmental None Reproductive None Carcinogenic None 2. Inhalation (mg/m3)b Subchronic None Chronic/ Carcinogenic 220 male cases and 440 male controls, occupational case- control study at rubber and tire factories Exposure concentration not measured Statistically Significantly elevated (p < 0.05) odds ratios for bladder cancer compared to expected rates for milling (OR = 1.91) and calendar operation (OR = 2.21) jobs Not Determinable Not run Not Determinable Checkoway et al. (1981) Effects from many chemicals used in the industry; effects specific to diphenylamine could not be separated. Developmental None Reproductive None Carcinogenic 33,815 males, retrospective cohort study of cancer mortality in the British rubber industry Exposure concentration not measured Significant (p < 0.001) excess of cancer deaths; lung cancer and stomach cancers most common cancer types Not Determinable Not run Not Determinable Parkes et al. (1982) Effects from many chemicals used in the industry; effects specific to diphenylamine could not be separated. 5 Diphenyl amine ------- FINAL 5-11-2011 Table 2. Summary of Potentially Relevant Data for Diphenylamine (CASRN 122-39-4) Notes3 Category Number of Male/Female, Strain Species, Study Type, Study Duration Dosimetryb Critical Effects NOAEL" BMDL/ BMCL" LOAEL" Reference (Comments) Animal 1. Oral (mg/kg-day)b Short-term Female (number not specified), Sprague- Dawley rat, diet, 3- 6 weeks 0 or 2451° Gross cysts in the corticomedullary region of the kidney and other morphological changes Not Determinable Not run 245 ld Eknoyan et al. (1976) NPR Short-term 6/6, Fischer rat, gavage, 28 days 0, 111, 333, or 1000e Decreased body-weight gain; increased liver, spleen, and kidney weights; anemia; histopathological changes in forestomach, renal tubules, and bone marrow lllf Not run 1000f Yoshida et al. (1989) NPR Subchronic 4/4, beagle dog, gelatin capsule, 90 days 0, 10,25, or 50e Treatment-related changes not observed in either sex 50f Not run Not Determinablef Krohmer (1992a) NPR Subchronic 10/10, rat (strain not reported), diet, 90 days Males: 0, 9.8, 25, 78,236, or791c Females: 0,12, 32, 96, 303, or 978c Increased mean relative liver, spleen, kidney, and body weight Males: 78d Females: 12d Not run Males: 236d Females: 32d Dow Chemical Company (1958); LOAEL identified by causing 10% increase in relative liver weight in female rats considered to be biologically significant. NPR Subchronic 10/10, Sprague-Dawley rat, diet, 90 days Males: 0, 9.6, 96, 550, or 1200e Females: 0,12, 110,650, or 1300e Increased absolute and relative liver weights; decreased hematocrit; and histopathological changes in kidneys, spleen, and liver Female: 12f Male: 96f Not run Female: 110f Male: 550f Krohmer (1992b); LOAEL identified by causing statistically significant increase in relative and absolute liver weight 6 Diphenyl amine ------- FINAL 5-11-2011 Table 2. Summary of Potentially Relevant Data for Diphenylamine (CASRN 122-39-4) Notes3 Category Number of Male/Female, Strain Species, Study Type, Study Duration Dosimetry6 Critical Effects NOAEL" BMDL/ BMCL" LOAEL" Reference (Comments) PS/NPR Subchronic 15/15, CD-I mouse, diet, 90 days Males: 0,1.7, 94, 444, or 926c Females: 0, 2.1, 107, 555, or 1101c Splenic effects (i.e., hemosiderosis and congestion), increased relative and absolute liver weight, increased absolute kidney weight (males only) and relative kidney weight (females only); decreased ovary weight in female mice Female: 2.1d Male: 94d 11.51 for increased incidence of spleen hemosid- erosis in male mice. Female: 107d Male: 444d Botta (1992); NOAEL and LOAEL identified for increased incidence of splenic effects (i.e., hemosiderosis and congestion). IRIS, 1993 Chronic 2/2, purebred beagle dog, 2 years 2.5,25,250 Decreased body-weight gain; increased liver and kidney weights 2.5 Not run 25 Thomas et al. (1967a); NOAEL and LOAEL formerly identified in the IRIS review of Diphenylamine. NPR Chronic 4/4, beagle dog, gelatin capsule, 52 weeks 0, 10,25, orl00e Changes in clinical chemistry (bilirubin, BUN) and hematological (platelet) parameters 10f Not run 25f Botta (1994a) Chronic 2/2, beagle dog, diet, 2 years Males: 2.1, 21, or 208c Females: 1.9,19, or 185c Severely inhibited growth; reduced hemoglobin levels; increased liver weight; possibly increased kidney and spleen weights; some hemosiderosis of the spleen, kidney, and bone marrow Not determinable Not run Not determinable DeEds (1963a); Because raw data were not provided in the study report and the use of a control group was not specified, a LOAEL and a NOAEL cannot be determined from this study. Chronic 20/20, albino rat (strain not specified), diet, 734 days Males: 0, 0.72, 7.2, 72, 362, or 723c Females: 0.82, 8.2, 82,410, or 820c Depressed growth; reduced feed consumption (>10%); moderate anemia; increased incidence of cystic dilated renal tubules and chronic interstitial nephritis Not determinable Not run Not determinable DeEds (1963b); Study authors reported that kidney lesions were not related to diphenylamine exposure. 7 Diphenyl amine ------- FINAL 5-11-2011 Table 2. Summary of Potentially Relevant Data for Diphenylamine (CASRN 122-39-4) Notes3 Category Number of Male/Female, Strain Species, Study Type, Study Duration Dosimetry6 Critical Effects NOAEL" BMDL/ BMCL" LOAEL" Reference (Comments) NPR Chronic Male (number not specified), Sprague- Dawley rat, diet, 76 weeks 500e Focal proliferation of distal tubular and collecting duct cells at Week 5; cystic dilatations with focal necrosis in collecting ducts at Week 10 Not determinedf Not run Not determinedf Evan et al. (1978); Study authors reported that study was not designed for toxico logical purposes. Chronic 0/6, albino rat (strain not specified), 226 days 0,21,82,410, 820, or 1230c Dose-related decrease in final body weight; pigmentation in liver, spleen, adrenals, heart, and kidneys; kidney cysts 82d Not run 410dfor microscopic changes in the kidney. Thomas et al., 1957 NPR Chronic/ Carcinogenicity 10/10, Sprague-Dawley rat, diet, 2 years Males: 0,8.1,29, 150, or 300e Females: 0, 7.5, 25, 140, or290e Changes in hematological parameters (hematocrit, hemoglobin, erythrocytes); histopathological changes in the spleen, liver, and kidneys; no treatment-related increase in tumors was observed 7.5f Not run 25f Botta (1994b) Chronic/ Carcinogenicity 20/20, albino Slonaker- Addis rat, diet, 2 years Males: 0, 0.72, 7.2, 72, 362, or 723c Females: 0, 0.82, 8.2, 82,410, or 820c HED males: 0, 0.21,2.1,21,104, or 207 HED females: 0, 0.21,2.1,21,107, or 213 Decreased body weight and increased incidence and severity of chronic nephritis Males: 72d Females: 82d Not run Males: 361.5d Females: 410d Thomas et al. (1967b); according to study authors, tumor incidence was due to senility of the animals and not related to treatment of the compound. Chronic/ Carcinogenicity 0/20, Sprague-Dawley rat, gavage, single dose, observed for up to 6 months 1.7C; HED: 0.44 No results specific to diphenylamine exposure presented 1.7 Not run None Griswold et al. (1966) NPR Chronic/ Carcinogenicity 60/60, CD-I mouse, diet, 78 weeks Males: 0, 73, 370, or 760e Females: 0, 91, 460, or 940e Decreased body-weight gain; decreased survival; hematological changes (MCV, MCH, hematocrit, erythrocytes), gross and microscopic pathological alterations (spleen, liver, kidneys, urinary bladder); tumors comparable between groups N/A Not run N/A Botta (1994c) 8 Diphenyl amine ------- FINAL 5-11-2011 Table 2. Summary of Potentially Relevant Data for Diphenylamine (CASRN 122-39-4) Notes3 Category Number of Male/Female, Strain Species, Study Type, Study Duration Dosimetryb Critical Effects NOAEL" BMDL/ BMCL" LOAEL" Reference (Comments) NPR Chronic/ Carcinogenicity 150/150, CD-I albino mouse, diet, 92 weeks Males: 0, 8.7, 17, or 43 Females: 0, 9.3, 18, or 45 HED males: 0, 1.3,2.6, or 6.6 HED females: 0, 7.1,14, or 35 No effects noted (tumor or nontumor), including in timing or incidence of tumors N/A Not run N/A Fordetal. (1972); Only abstract was available for this study. NPR Chronic/ Carcinogenicity 125 (sex not specified), NRMI outbred albino mouse, gavage, dosed 78 days over the 18-month study duration 300e HED: 6.2 Tumor incidence reported to be unrelated to diphenylamine exposure and comparable to controls N/A Not run N/A Holmberg et al. (1983) Developmental 0/16, New Zealand white rabbit, gavage, GDs 7- 19 0, 33, 100, or 300 No maternal or developmental effects Maternal and developmental: 300d Not run Not Determinable Edwards et al. (1983b) NPR Developmental 0/25, Sprague-Dawley rat, gavage, GDs 6-15 0, 10, 50, or 100e Maternal: increased spleen weights, enlarged spleens, and blackish-purple colored spleen Developmental: no effects Maternal: 50' Developmental: ^100f Not run Maternal: 100' Developmental : Not Determinablef Rodwell (1992) NPR Reproductive 28/28, Sprague-Dawley rat, diet, two generations Males: 0,40, 115, or 399e Females: 0,46, 131, or448e Parental: gross pathology changes in spleen and microscopic changes in kidneys, liver and spleen Developmental: decreased F2 body weight Reproductive: decreased litter size in F1 and F2 generations Parental (males and females, respectively): <40f and <46f Developmental: 46f Reproductive: 131f Not run Parental (Males: 115f Females: 131f respectively): Developmental : 131f Reproductive: 448f Rodwell (1993) Reproductive 3/12, Slonaker-Addis rat, diet, two generations Males: 0, 90, 226, or451c Females: 0,101, 252, or 503c Dose-dependent decrease in litter size in F1 and F2 generations; in second mating, number of pups per litter at birth significantly lower compared to controls at all 3 dose levels; no firm conclusions possible Not Determinable Not run Not Determinable Thomas et al. (1967c) Study authors concluded that results were not conclusive. 9 Diphenyl amine ------- FINAL 5-11-2011 Table 2. Summary of Potentially Relevant Data for Diphenylamine (CASRN 122-39-4) Notes3 Category Number of Male/Female, Strain Species, Study Type, Study Duration Dosimetryb Critical Effects NOAEL" BMDL/ BMCL" LOAEL" Reference (Comments) 2. Inhalation (mg/m3)b Subchronic None Chronic None Developmental None Reproductive None Carcinogenic None aNotes: IRIS = utilized by IRIS, date of last update; PS = principal study, NPR = not peer reviewed, N/A= not applicable. bDosimetry: NOAEL, BMDL/BMCL, and LOAEL values are converted to adjusted daily dose, human equivalent dose (HED in mg/kg-day) or human equivalent concentration (HEC in mg/m3) units. All exposure values of long-term exposure (4 weeks and longer) are converted from a discontinuous to a continuous (weekly) exposure. Values for inhalation (cancer and noncancer) and oral (cancer only) are further converted to an HEC/HED. Values from animal developmental studies are not adjusted to a continuous exposure. HED = NOAELadj x (body weight animal body weight human)0 25 cAdjusted Daily Dose (ADD) = administered dose x food consumption per day x (1 body weight) x (days dosed total days). ''NOAEL/LOAEL determined from study report. eDose conversion presented by European Communities (2008). fNOAEL/LOAEL reported by European Communities (2008). 10 Diphenyl amine ------- FINAL 5-11-2011 HUMAN STUDIES Oral Exposures The effects of oral exposure to diphenylamine on humans have not been evaluated in subchronic- or chronic-duration, developmental, reproductive, or carcinogenicity studies. Inhalation Exposures The effects of chronic-duration and carcinogenic exposure to various amines, including diphenylamine, on humans were explored in two occupational studies (Checkoway et al., 1981; Parkes et al., 1982). While a specific exposure pathway was not identified by Checkoway et al. (1981) or Parkes et al. (1982), it may be inferred that because the exposure was in an occupational setting, the primary route of exposure was most likely via inhalation pathway along with dermal exposure. Effects of inhalation exposure to diphenylamine on humans have not been evaluated in sub chronic-duration, developmental, or reproductive studies. Chronic-duration Studies In a case-control study, Checkoway et al. (1981) investigated the association between certain jobs and work areas in rubber and tire factories and bladder cancer. All subjects were hourly workers from five rubber factories in Akron, Ohio. Bladder cancer cases (232 total; 220 men and 12 women) were identified from a hospital record review or from death certificates obtained for mortality studies. A control:case ratio of 2:1 was employed; controls from the hourly paid workers were matched to cases based on company, sex, race, and year of birth (± 2 years). Investigators gathered work histories and grouped workers into 21 job title groups based upon similarity of materials and machinery used. Exposure to specific chemicals was not measured. The mean exposure duration among subjects diagnosed with bladder cancer was approximately 25 years compared to the mean exposure duration of 24 years in the corresponding control group. In order to estimate the risk of bladder cancer by job type, study authors calculated odds ratios (ORs) and performed chi-squared (x ) tests of association using the methods of Mantzel and Haenzel. Student's ^-tests for paired samples were used to compare years and ages of initial hire, and x tests for linear trend were used to examine dose-response relationships. Investigators limited analysis to males due to the small number of female bladder cancer cases (220 males compared to 12 females). Among males from the 113 cases identified using death certificates, 107 cases with reliable histological classification were established from hospital listings. Transitional cell carcinoma was observed in 88 (82%) of the 107 cases; other notable observations included squamous cell carcinoma (4/107), adenocarcinoma (1/107), allantoid papilloma (7/107), and undifferential carcinoma not otherwise specified (7/107). When odds ratios were calculated by the 21 different job types, only those job types suspected as having the highest exposure—milling (OR = 1.91) and calendar operation (OR = 2.21)—had statistically significantly elevated (p < 0.05) odds ratios compared to expected rates. The work group for final inspection of tires also had a weak association with bladder cancer risk (OR = 1.49). An apparent increase in relative risk was observed in the milling and calendar operation jobs with increased exposure duration, although tests for linear trend were not significant. However, a similar association was not observed in the final inspection of the tires job category. 11 Diphenylamine ------- FINAL 5-11-2011 When one company was analyzed separately, it appeared that the bladder cancer risk for the three job titles presented above was localized to this particular plant. The study authors stated that milling and calendar operations involve handling and heating uncured stock, which could lead to exposure from volatilized rubber chemicals. Diphenylamine is a common rubber chemical that may produce aromatic amines, but there are other carcinogenic chemicals produced during the process as well. The study authors concluded that association between certain job types and bladder cancer must be viewed with caution because the effects of the complex chemical mixture used in the rubber industry could not be separated. Furthermore, the study authors stated that their analysis was limited because confounding factors such as effects of smoking, coffee consumption, and other lifestyle factors were not considered. Because of the limitations of this study, a NOAEL or LOAEL cannot be identified. In a retrospective cohort study, Parkes et al. (1982) investigated the association between death from cancer and working in the British rubber industry. The study included 33,815 men employed in 13 tire or rubber manufacturing plants in Scotland and England, divided into 3 cohorts (I, II, and III) based upon the date they began work in the industry. Subjects were first employed between 1946 and 1960 and were followed until 1975. Investigators required that all workers had worked continuously for a minimum of 1 year in the industry and also required that the workers had survived for a minimum of 9 years after the first year of employment in order to allow for a long latency period for cancer. Factory records and a subsequent screening of the Office of Population Censuses and Surveys in England were used to ascertain current or former workers' status at the end of the study period. Details were collected from death certificates, with deaths coded according to the International Classification of Diseases (ICD). Jobs were placed in 1 of 10 categories based on similar characteristics. The expected numbers of deaths were calculated using national age-specific mortality rates for England and Scotland. Ap-value was calculated for the difference between observed and expected deaths. Standardized mortality ratios (SMRs) were calculated when appropriate. The follow-up rate was high (98.5%); 4882 men were identified as dead, and 28,410 were traced as alive at the end of the study. Investigators determined a significant (p < 0.001) overall excess of cancer deaths with a total of 1359 recorded deaths compared to 1221 expected deaths. However, Cohorts I (men entering the industry between 1946 and 1950) and III (men entering the industry between 1956 and 1960) were the only cohorts showing excess deaths. The largest excess was seen in the general rubber goods sector of Cohort I (SMR = 144). Furthermore, specific occupations in the industry, including component building, preparation, assembly, vulcanizing, curing, molding press, autoclave, pan, inspection, painting, trimming, site workers, internal transport, and general truck drivers, were primarily responsible for the statistically significant (p < 0.05 to p < 0.001) increase in deaths. The most common causes of death were lung cancer (observed: 638, expected: 517\p< 0.01) and stomach cancer (observed: 183; expected: 141. 8;^<0 .01). For lung cancer, almost the entire excess came from Cohort I, partially due to the small numbers in Cohorts II and III. For stomach cancer, most of the excess also came from Cohort I. Although the study authors noted that the smaller number of deaths precludes any meaningful examination by occupation, statistically significant excesses occurred in the following job categories: compounding, weighing, mixing, reforming, and washing (p < 0.01); component building, preparation, and assembly (p < 0.05); finished goods, packaging, and dispatch (p < 0.01); and site workers, internal transport, and general truck drivers (p < 0.001). Bladder cancer was only slightly associated with work in the tire sector of Cohort I 12 Diphenylamine ------- FINAL 5-11-2011 (nonsignificant), mainly in jobs involving heating of the rubber product. The study authors noted that it is likely that only men entering the industry before 1950 were exposed to known bladder carcinogens that were later withdrawn from production. There were also statistically significant (p < 0.05 top < 0.001) excesses in esophageal cancer in all three cohorts, although these cancers were less common than lung and stomach cancers. This study did not collect information on smoking and other lifestyle choices and, thus, did not include these confounding factors in the overall analysis. In addition, the investigators did not calculate social class-specific mortality, which would have helped control for potential bias introduced by class. This study is also limited by the inability to separate the effects of exposure to a number of different chemicals in the industry. Because of the limitations of this study, a NOAEL or LOAEL cannot be identified. ANIMAL STUDIES As described above, a number of unpublished studies with diphenylamine were located in a formal government risk assessment document (European Communities, 2008), where they are reported as thorough descriptive summaries along with the full reference to the complete report. It is clear that these summaries have been prepared from existing full reports and not abstracts only as may exist, for example, from a scientific conference. Given that these summaries are acknowledged and utilized in the European assessment, all those listed are considered as relevant and valid for use in this assessment. The adjusted doses listed in these summaries were provided in the former risk document (European Communities, 2008) and were not calculated. Oral Exposures The effects of oral exposure to diphenylamine on animals have been evaluated in 2 short-term studies (Eknoyan et al., 1976; Yoshida et al., 1989), 4 subchronic-duration studies (Krohmer, 1992a,b; Dow Chemical Company, 1958; Botta, 1992), 12 chronic-duration and/or carcinogenicity studies (Botta, 1994a,b,c; DeEds, 1963a,b; Evan et al., 1978; Ford et al., 1972; Griswold et al., 1966; Holmberg et al., 1983; Thomas et al., 1967a,b; Thomas et al., 1957), 2 developmental toxicity studies (Edwards et al., 1983b; Rodwell, 1992), and 2 reproductive toxicity studies (Rodwell, 1993; Thomas et al., 1967c). Short-term Studies In a peer-reviewed study to evaluate renal function and histopathology as a consequence from exposure to diphenylamine, Eknoyan et al. (1976) administered 2.5% diphenylamine in the diet for 3-6 weeks to female Sprague-Dawley rats (number not specified) that weighed between 70 and 90 g. The estimated average daily converted dose for diphenylamine is 2451 mg/kg-day with control animals receiving diet (Wayne Rat Chow, Allied Mills, Inc., Chicago, IL) only. It is unclear whether this study complied with Good Laboratory Practice (GLP). The study authors found gross cysts in the corticomedullary region of approximately 10% of all kidneys and morphological alterations in the kidneys of all experimental animals. The most common change was cystic dilatation of the collecting ducts, although the study authors occasionally noted proteinaceous casts in the dilated tubules. Some animals had focal dilatation of the cortical collective ducts and distal tubules. The duration of the diphenylamine treatment affected the severity and extent of these morphological changes. There was a significant but temporary drop (p-value not reported) in maximal urine osmolality 1 week after the beginning of 13 Diphenylamine ------- FINAL 5-11-2011 treatment and at 2 weeks when this measure was decreased to 1401 ± 96 milliosmoles (mOsm) compared to 3224 ± 144 mOsm before treatment. The osmolality remained steady for the rest of the treatment period. Micropuncture studies revealed focal regions of dilated tubules in treated rats, although gross cysts were not apparent on the kidney surface. The study authors concluded that, based on these results, the nephron (specifically the terminal portion of the collecting duct) is the site most likely affected by diphenylamine exposure to at least temporarily decrease the ability to concentrate urine in treated rats. Based on the results presented above, a LOAEL of 2.5% (2451 mg/kg-day) is identified in this study for significant reduction in maximal urine concentrating ability and for the appearance of gross cysts in the corticomedullary region of the kidney. A NOAEL cannot be identified in this study. This study is limited by the use of a small number of animals per investigated outcome and the use of a single dose of diphenylamine in feed. In a 28-day unpublished study, Yoshida et al. (1989) examined the oral toxicity of diphenylamine in Fischer rats. The full study is not available for review. A summary of the study is available on page 78 of the European Union Risk Assessment Report on diphenylamine (Yoshida et al. 1989 as discussed in European Communities, 2008). In a guideline conform oral 28 day study Fischer rats of both sex received 111, 333 and 1000 mg/kg bw/d diphenylamine by gavage. Thirty-six animals were divided into 6 groups of equal numbers, 4 groups being usedfor treatment and the remainder for investigation of recovery. Inhibition of body weight gain, increase of liver, spleen and kidney weights and anemia were observed in the high dose group in both sexes. The same group demonstrated mucosal hyperplasia in the forestomach, dilatation, degeneration or necrosis of renal tubules in the corticomedullary junction and hyperplasia in the bone marrow histopathologically. Slight increase [s] of spleen, liver and kidney weights as well as slight degeneration of renal tubules were evident in several animals of the 333 mg/kg bw/d dose group. Repair of histopathologic lesions and anemia occurred after 14 days. As there were no toxic effects in the low dose group a NOAEL of 111 mg/kg bw/d was derived under these experimental conditions (Yoshida et al. 1989). No other information or statistical analysis is available. A LOAEL of 1000 mg/kg-day was identified in the European Union Risk Assessment Report on diphenylamine (Yoshida et al. 1989 as discussed in European Communities, 2008; Table 4.1.2.6). Subchronic-duration Studies Krohmer (1992a) conducted a 90-day unpublished study examining the oral toxicity of diphenylamine in purebred beagle dogs (four/sex/group; Krohmer 1992a as discussed in European Communities, 2008). The full study is not available for review. A study summary is available on page 81 of the European Union Risk Assessment Report on diphenylamine (European Communities, 2008). Groups of four pure-bred beagle dogs of each sex received technical-grade diphenylamine (purity, > 99%) in gelatin capsules at doses of 0, 10, 25 or 50 mg/kg bw per day for 90 days. They were observedfor deaths, clinical signs, body weight, food consumption, ophthalmological, haematological, clinical chemical and urinary 14 Diphenylamine ------- FINAL 5-11-2011 parameters, organ weights, and gross and histopathological appearance. There were no deaths, and no treatment-related changes were seen in any of the above parameters. Statistically significant increases were seen, however, in some clinical chemical parameters including albumin content, the albumin:globulin ratio in males, and bilirubin content in females at the high dose. These effects may have been incidental. The NOAEL was 50 mg/kg bw/day, the highest dose tested. An unpublished study by Dow Chemical Company (1958) examined the effects of dietary subchronic-duration exposure to diphenylamine on rats. Ten rats (strain not reported) per sex per group were administered doses of 0-, 0.01%- (100-ppm), 0.03%- (300-ppm), 0.1%- (1000-ppm), 0.3%- (3000-ppm), or 1%- (10,000-ppm) diphenylamine (purity not reported) neat in the diet for 90 days. Rats were 45 days old at the time of the first dose. Information about adherence to GLP guidelines is not provided in the study report. Rats were housed two per cage, and food (Purina lab chow pellets) and water were available ad libitum. Appropriate body weight data and food consumption data for dose conversion were not provided in the study. Therefore, average values provided for the available rat strains by EPA (1994b) for body weight (0.235 kg for males and 0.1728 kg for females) and food consumption (0.0212 kg for males and 0.0174 kg for females) are used in the dosimetric calculation. Adjusted daily doses were 0, 9.8, 25, 78, 236, or 791 mg/kg-day for males and 0, 12, 32, 96, 303, or 978 mg/kg-day for females, respectively. Rats were weighed twice weekly during the first 28 days of the study and once per week thereafter. Food consumption was recorded for the first month of the study. Study authors observed animals for clinical signs of toxicity, and dead or moribund animals were autopsied. Hematological values were obtained at necropsy from five females at the 0, 303 and 978 mg/kg-day dose levels. The study authors recorded lung, heart, liver, kidneys, spleen, and testes weights at study termination. These organs, as well as the pancreas and adrenals, were also examined histologically. Use of statistical methods was not reported, and it appears that this report was not peer reviewed. The Dow Chemical Company (1958) reported no evidence of critical effects in males in the 96 and 303 mg/kg-day dose groups. It is unclear if this study was performed according to GLP standards. The study authors noted the appearance of weakness of the hind legs in both sexes at the 1.0%-dose level after 50 days of treatment. Additionally, growth retardation was noted in both sexes with the effect being more pronounced in male rats. The study authors also reported an elevated level of mortality in male rats (further details not provided), but they attributed this finding mainly to upper respiratory infections. Average hemoglobin values of female rats decreased slightly in a dose-related manner (see Table B.l). Table B.2 provides data for body weights at necropsy and relative organ weights for male and female rats. Average relative liver weight was statistically significantly (p < 0.01) increased in females at all dose levels and in males at doses >78 mg/kg-day of diphenylamine. Average relative spleen weight was statistically significantly increased (p < 0.01) in females at doses >32 mg/kg-day of diphenylamine and in males at the highest dose level. Average relative kidney weights were increased in both males and females treated with 1.0% diphenylamine (791 mg/kg-day for males and 978 mg/kg-day for females). The study authors reported a brown coloration of tissues in females at the three highest dose levels and in males at the two highest dose levels. The study authors reported central lobular necrosis in the livers, increased interstitial nephritis in the kidneys, and marked congestion in the spleens of both sexes at the highest dose level. Incidence data for these changes were not available in the study report. Based on these results, LOAELs of 15 Diphenylamine ------- FINAL 5-11-2011 0.03% (32 mg/kg-day) in female rats and 0.3% (236 mg/kg-day) in male rats are identified in this study because of a 10%-increase in relative liver weight, considered to be biologically significant. NOAELs of 0.01% (12 mg/kg-day) and 0.1% (78 mg/kg-day) are identified from this study in female and male rats, respectively. Krohmer (1992b) conducted a 90-day unpublished study examining the oral toxicity of diphenylamine in Sprague-Dawley rats. The full study is not available for review. A study summary is available on pages 79-80 of the European Union Risk Assessment Report on diphenylamine (European Communities, 2008). Groups of 10 male and 10 female Sprague Daw ley rats received technical-grade diphenylamine in the diet at 0, 150, 1500, 7500, or 15000 ppm for 90 days, equal to doses of 0, 9.6, 96, 550, and 1200 mg/kg bw per day in males and 0, 12, 110, 650, and 1300 mg/kg bw per day in females. The frequency of darkening of the urine increased with dose, starting with one female at 1500 ppm and 100% of rats at 15000 ppm. Haematological measures indicated decreased erythrocyte counts and haemoglobin values, which were statistically significantly different from those of controls in animals at 7500 and 15,000 ppm at termination. The haematocrit values were statistically significantly lower than those of controls in females at the three highest doses. Small, statistically significant increases in alkaline phosphatase activity, albumin content, and albumin:globulin ratio in males and glucose and albumin content and albumin:globulin ratio in females were observed at 7500 and 15000 ppm. The cholesterol concentration increased with dose in females and was statistically significantly different from that of controls at the three higher doses. In males, the absolute and relative weights of the liver and spleen increased with dose and were statistically significantly raised at 7500 and 15000 ppm; the relative weights of the kidney and gonad also increased with dose and were also statistically significant at the two higher doses. In females, the absolute and relative weights of the liver increased with dose, and the change in relative weights was statistically significant at doses > 1500 ppm. The kidneys were dark in animals of each sex at 7500 and 15000 ppm, and about 60% of the females at the high dose had dark and/or enlarged livers. The spleens of both males andfemales at the two higher doses were congested. Histopathological examination revealed an increased incidence of haematopoiesis and pigment in the liver, haematopoiesis, haemosiderosis, and congestion in the spleen, and pigmented kidneys in animals of each sex at 7500 and 15000 ppm. The spleens of all females at 1500 ppm also showed an increase from minimal to slight haematopoiesis and haemosiderosis. The NOAEL was 150 ppm, equal to 12 mg/kg bw per day, on the basis of increased clinical signs of toxicity, clinical chemical changes, organ weights, and gross and histopathological appearance. A LOAEL of 110 mg/kg-day and NOAEL of 12 in females was identified in the European Union Risk Assessment Report on diphenylamine (Krohmer 1992b as discussed in European Communities, 2008; Table 4.1.2.6). The subchronic-duration study by Botta (1992) is selected as the principal study for the derivation of a subchronic p-RfD. Botta (1992) conducted a 90-day unpublished, nonpeer-reviewed study examining the effects of technical-grade diphenylamine on Swiss-derived CD-I mice. Fifteen mice per sex per dose received 0-, 10-, 52-, 260-, or 16 Diphenylamine ------- FINAL 5-11-2011 5200-ppm diphenylamine (purity not given) in feed. Adjusted daily doses provided in the principal study (Botta, 1992) are 0, 1.7, 94, 444, or 926 mg/kg-day for males and 0, 2.1, 107, 555, or 1101 mg/kg-day for females. This study adhered to GLP guidelines. All animals were observed for clinical signs daily, including mortality/moribundity checks twice daily. Body weight and food consumption were recorded weekly, and ophthalmology examinations were conducted prior to treatment initiation and during the final week of the study. Food consumption was recorded throughout the study period. A complete postmortem examination was performed on all mice that died or were sacrificed in a moribund state. After 90 days of dietary exposure to diphenylamine, remaining animals were fasted overnight, final body weight was recorded, and mice were euthanized in a CO2 chamber. Animals were bled for clinical chemistry analyses. Selected organs (liver, kidneys, heart, spleen, pituitary, brain/brainstem, testes/epididymides, ovaries, and adrenals) were weighed, and the terminal organ and body weights were used to calculate relative organ-/body-weight ratios. Tissues were collected and prepared for histopathological examination. Statistical analysis (Dunnett's Test) was performed on body weight, food consumption, hematology, and organ-weight data. Six unscheduled deaths occurred during the study (Botta, 1992); there were three female deaths in the control group and three male deaths in the high-dose group. It was not explained by the study authors if these deaths were treatment related. Slight treatment-related effects were observed at 525 ppm (94 mg/kg-day for males and 110 mg/kg-day for females) with the appearance of brownish-yellow pigment and extra-medullary hematopoiesis in the liver. At the third highest dose group (94 mg/kg-day) in females and the fourth highest dose group (560 mg/kg-day) in males, there were statistically significant increased incidences of congestion and hemosiderosis in the spleen. As the dosage was increased, not only were the incidence and severity of the lesions in the spleen and liver increased, but pigment in kidneys, increased cellularity in the bone marrow, and cystitis (in the 5250-ppm group [926 mg/kg-day for males and 1101 mg/kg-day for females]) were also present. Treatment-related increases in relative organ weights were seen in the liver and spleen in the highest dose groups (2625 [444 mg/kg-day for males and 555mg/kg-day for females] and 5250 ppm [926 mg/kg-day for males and 1101 mg/kg-day for females]). There were also increases in absolute liver weight observed in males which were statistically significant at the second highest dose (444 mg/kg-day) tested and were both biologically (>10% increase) and statistically significant at the highest dose (926 mg/kg-day) group. Absolute kidney weight was biologically significantly increased at the highest dose (926 mg/kg-day) in males coupled with increased relative kidney weight in female mice in the highest dose (1101 mg/kg-day) group, which was biologically significant. Relative ovary weight was statistically significantly decreased at the middle (107 mg/kg-day) and high (1101 mg/kg-day) dose group in female mice. Table 2 presents data for the effect on diphenylamine exposure on organ and body weight. Hematology parameters showing treatment- related effects included statistically significant decreases in red blood cell (RBC) count and hematocrit (data not shown). Also, statistically significant increases in mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) in the two highest dose groups were observed (data not shown). The highest dose group also showed a marked increase in reticulocytes (data not shown). In the 525-ppm group (94 mg/kg-day for males and 110 mg/kg-day for females), there was a statistically significant increase in MCHC (data not shown). Based on these results, LOAELs of 107 mg/kg-day in female mice and 444 mg/kg-day in male rats are identified in this study by causing statistically significant increased incidences of congestion and hemosiderosis in the spleen. NOAELs of 17 Diphenylamine ------- FINAL 5-11-2011 2.1 mg/kg-day and 94 mg/kg-day are identified from this study in female and male rats, respectively. The data for these effects (i.e., congestion and hemosiderosis in the spleen) are further evaluated with the BMDS modeling program for determination of a POD for the screening subchronic p-RfD in Appendices A and C. Chronic-duration Studies IRIS (U.S. EPA, 2010a) has provided an RfD. However, subsequent to the IRIS file, additional studies that may be relevant have been discovered. These studies are summarized in this PPRTV assessment. These additional studies do not provide a lower or more credible POD than the study formerly used by IRIS (i.e., Thomas et al., 1967a). In the Thomas et al 1967a study, groups of sixteen beagle dogs (two per sex per group) were treated orally with diphenylamine in concentrations of 0.01% (2.5 mg/kg bw/d), 0.1% (25 mg/kg bw/d), and 1.0% (250 mg/kg bw/d) in the diet for 2 years. Mid- and high-dose animals developed marked growth retardation after 1 year. A dose-dependent anemia was seen in the same treatment groups, being pronounced in the high-dose group and moderate in the mid-dose group. After 2 years, the erythrocytes of the dogs on the 1.0%-diet showed a moderate decreased resistance to hypotonicity. Liver function as a result of sulfobromophthalein testing at Days 618 to 627 indicated a moderate degree of liver damage in the 1.0%-group. These animals also showed an increased organ weight of the liver with perilobular fatty changes and increased lipid content, a mild haemosiderosis of the spleen, kidneys, and bone marrow, and a slight increase in kidney weight (Thomas et al., 1967a). A LOAEL of 25 mg/kg-day was identified by IRIS for decreased body-weight gain and increased liver and kidney weights. A NOAEL was identified to be 2.5 mg/kg-day. Botta (1994a) conducted a 52-week unpublished study examining the oral chronic toxicity of diphenylamine in beagle dogs (four/sex/group; Botta 1994a as discussed in European Communities, 2008). The full study is not available for review. The study summary available on pages 90-91 of the European Union Risk Assessment Report on diphenylamine (European Communities, 2008) is presented below. Four beagles of each sex received diphenylamine (purity > 99%) by gelatin capsule at a dose of 0, 10, 25, or 100 mg/kg bw per day for 52 weeks and were observedfor clinical signs, body weight, food consumption, ophthalmological, haematological, clinical chemical and urinary end-points, organ weights, and gross and histopathological appearance. No treatment-related clinical signs were seen at termination. One dog at the intermediate dose and two at the high dose had greenish hair. There were no deaths or treatment-related effects on body weight, food consumption, or ophthalmological parameters. Haematological examination revealed decreased mean erythrocyte counts (by 11% in comparison with controls), haemoglobin (9.3%), andhaematocrit (8.7%) in males at the high dose; smaller decreases in these parameters were found in females. The platelet count increased with dose in males at the 13-, 26-, 39-, and 52-week evaluation periods, becoming statistically significant at the intermediate and high doses. There was a dose-related increase in mean total bilirubin concentration, which was statistically significant for animals at the intermediate and high doses throughout the study, in animals at the low dose at week 26, and in females only at week 39. The mean cholesterol concentration appeared to increase with dose at all evaluation periods but was statistically significantly increased only in males at the high dose at week 13 (by 18 Diphenylamine ------- FINAL 5-11-2011 68%) and in females at the high dose at week 39 (by 37%). The blood urea nitrogen concentration was decreased in females at the intermediate (by 16%) and high doses (by 20%) at week 52. The mean absolute and relative weights of the liver and thyroid appeared to increase with dose in males, but only the mean absolute liver weight of males at the high dose was statistically significantly increased. The mean absolute and relative weights of the thyroid decreased with dose in females but did not reach statistical significance at any dose. There were no treatment-related gross or histopathological changes. The NOAEL for toxicity was 10 mg/kg bw per day on the basis of small clinical chemical changes. A LOAEL of 25 mg/kg-day based on "small clinical changes" was identified in the European Union Risk Assessment Report on diphenylamine (Botta 1994a as discussed in European Communities, 2008; Table 4.1.2.6). In a published summary of chronic-duration toxicity studies on diphenylamine, DeEds (1963a,b) administered diphenylamine in feed to dogs and rats over a 2-year period. It is unclear whether either of these studies was GLP compliant. Raw data were not provided for either of these studies, and details regarding experimental methods were limited. In the dog study (DeEds, 1963a), groups of two beagles per sex were fed diets of 0.01%- (100-ppm), 0.1%- (1000-ppm), or 1%- (10,000-ppm) diphenylamine (purity = 99.9%) over 2 years. Adjusted daily doses calculated using default data for body weight (U.S. EPA, 1994b) and food consumption (U.S. EPA, 1988) are 2.1, 21, or 208 mg/kg-day for males and 1.9, 19, or 185 mg/kg-day for females. Treated animals were examined for changes in growth, changes in hematological parameters, and changes in liver and kidney function. At study termination, animals were sacrificed and received a histopathological examination. Use of a control group was not specified in the study report. Mortality data were not presented in the study report. Growth in dogs treated with 1%-diphenyl amine was severely inhibited up to Day 400. (An outbreak of distemper after 400 days made further interpretation of growth difficult.) Between Days 724 and 731, dogs treated with 1%-diphenyl amine experienced reduced hemoglobin levels and RBC counts, as well as crenated red blood cells that were fragile. Liver function tests revealed that retention of sulphobromophthalein may have been increased in dogs exposed to l%>-diphenylamine. Histopathological examination revealed no parasites, tumors, or other indications of disease. Liver weight (no data regarding absolute or relative weight) was increased at 1%-diphenyl amine due to marked fatty change measured in extractable lipids and intracellular bilirubin present in moderate amounts. Changes in RBC fragility in the 1 %>-dose group were reflected by some hemosiderosis of the spleen, kidney, and bone marrow. The small numbers of animals made organ-weight data difficult to interpret, but the study authors noted that there might have been increased kidney and spleen weights in dogs treated with l%>-diphenylamine in diet. Because raw data were not provided in the study report and the use of a control group was not specified, firm conclusions regarding the toxic effect of diphenylamine exposure in dogs cannot be made from the study results. Hence, a LOAEL and a NOAEL cannot be determined from this study. In the rat study (DeEds, 1963b), groups of 20 albino weanling rats per sex were given feed containing 0-, 0.001%>- (10-ppm), 0.01%>- (100-ppm), 0.1%>- (1000-ppm), 0.5%- 19 Diphenylamine ------- FINAL 5-11-2011 (5000-ppm), or 1%- (10,000-ppm) diphenylamine for 734 days. Adjusted daily doses calculated using default data for body weight (U.S. EPA, 1994b) and food consumption (U.S. EPA, 1988) are 0, 0.72, 7.2, 72, 362, or 723 mg/kg-day for males and 0.82, 8.2, 82, 410, or 820 mg/kg-day for females. Treated animals were examined for growth and food consumption, survival, and changes in hematological parameters. At study termination, animals were sacrificed and received a histopathological examination. Survival was not reduced in any group. Results indicated that growth inhibition did not occur at or below 0.1%, but at higher levels, growth was depressed. Because data tables were not provided, percent change in growth between treated groups and the control group cannot be determined. Food consumption was reduced by more than 10% in animals fed 1%-diphenylamine. At 1 %-diphenylamine, animals experienced moderate anemia, indicated by reduced hemoglobin and RBC levels and an increase in circulating normoblasts (RBC precursors). Histopathological examination revealed that treated animals had cystic dilated renal tubules and chronic interstitial nephritis; however, these lesions were not different from controls at 0.01%) and lower. The lesions were much more severe in the 0.1%>- and 0.5%>-groups compared to controls. The study authors stated that, based on a detailed record of pathological changes (data not presented) in various groups, the lesions were not related to diphenylamine exposure. Because data tables were not provided in the study report, firm conclusions regarding the toxic effect of diphenylamine exposure in rats cannot be made from the study results. Hence, a LOAEL and a NOAEL cannot be determined from this study. Evan et al. (1978) conducted a 76-week study examining the oral toxicity of diphenylamine in male Sprague-Dawley rats (number not specified). The full study is not available for review. A summary of the study available on page 79 of the European Union Risk Assessment Report on diphenylamine (European Communities, 2008) is presented below. In a study male Sprague Daw ley rats were given 1.0% (500 mg/kg bw/d) diphenylamine in food up to 76 weeks. After 2 to 6 weeks the animals developed polyurea with diluted urine. The first histological change was noted after 5 weeks, and was described as focal proliferation of distal tubular and collecting duct cells. Focal areas of medullary tubuli appeared thickened because several cells were layered upon each other. By week 10 collecting ducts showed cystic dilatations with focal necrosis and increasing cast material in the duct lumina. As the study was not designed for toxicologic purposes, a NOAEL was not derived. Thomas et al. (1957) published a peer-reviewed study examining the effects of diphenylamine exposure to rats for 226 days (approximately 32 weeks). The study authors did not report whether this study was conducted according to GLP guidelines. Six albino female rats (strain not specified) per sex per group were administered 0-, 0.025 (250-ppm), 0.1%>- (1000-ppm), 0.5%)- (5000-ppm), 1.0%- (10,000-ppm), or 1.5%- (15,000-ppm) diphenylamine (purity not reported) in feed for 226 days. Adjusted daily doses calculated using default data for body weight (U.S. EPA, 1994b) and food consumption (U.S. EPA, 1988) are 0, 21, 82, 410, 820 for male rats, or 1230 mg/kg-day for female rats. No details regarding husbandry of rats were provided. Body weights were recorded at study termination, and food consumption was recorded (further details not provided). Animals were sacrificed at study termination and 20 Diphenylamine ------- FINAL 5-11-2011 necropsied. Microscopic examinations were performed on liver, kidney, spleen, adrenal, and heart tissue. The study authors did not specify any statistical tests used to analyze the data. Thomas et al. (1957) reported no mortalities with the exception of one rat at the 0.10%-dose level that was removed from the study on Day 156 due to a severe respiratory infection. The study authors noted a dose-related decrease in final average body weight and stated that food consumption did not vary by more than 7.5% in all treated groups from control values (see Table B.3). The study authors observed a drop in the growth curve of rats in the 1.50%-dose group during the first few days of the study due to temporary lack of food intake. Enlargement of the kidneys was observed in rats in the 1.50%-diphenylamine dose group (30% larger than controls). Microscopic examination of sections of liver, kidney, spleen, adrenals, and heart revealed pigment deposition possibly due to blood destruction, along with multiple cystic structures in the kidneys. Microscopic examination also revealed brown pigmentation of Kupffer cells in the liver and in the tubular epithelial cells of the kidneys, particularly in the epithelial cells of the proximal convoluted tubules. This pigmentation was noted in rats administered >0.50%-diphenylamine, and the amount of pigmentation appeared to be correlated with the amount of diphenylamine consumed. The study authors also recorded the presence of cysts in the kidneys of rats at the highest dose level. A LOAEL of 0.50% (410 mg/kg-day) is identified in this study for microscopic changes in the kidney. A NOAEL of 0.10%) (82 mg/kg-day) is also identified. In a chronic-duration toxicity/carcinogenicity study, Botta (1994b) administered diphenylamine to Sprague-Dawley rats (10 animals per sex per dose group) in the diet for 2 years at concentrations of 0, 200, 750, 3750, or 7500 ppm in males and 0, 150, 500, 2500, or 5000 ppm in females. These concentrations are equivalent to average daily doses of 0, 8.1, 29, 150, or 300 mg/kg-day in males and 0, 7.5, 25, 140, or 290 mg/kg-day in females, as calculated by European Communities (2008). It is unclear whether this study is GLP compliant, although it appears that is has been peer reviewed. The full study is not available for review. The study review as provided in the European Union Risk Assessment Report (European Communities, 2008; pages 91-92) is summarized below. Sprague-Dawley rats received diphenylamine (>99.0%) at dose levels of 0, 200, 750, 3750 or 7500ppm in males (equal to 8.1, 28.8, 146.7, or 302.1 mg/kg/day) and 0, 150, 500, 2500, or 5000 ppm in females (equal to 7.5, 24.9, 137.8, or 286.1 mg/kg/day) in the diet for 2 years. A one year interim sacrifice of 10 animals per sex per dose group was used. There was no treatment related mortality noted, however, the study was terminated early due to increased mortality in the control and low dose animals. No effects were noted in ophthalmic examinations. The only treatment related clinical observation was a greenish tint to the hair coat in the urogenital or ventral cervical area which was assumed to be due to an... oxidative product of the interaction of the test article or a metabolite with urine or feces in the high mid and high dose groups. Systemic toxicity was noted at the high mid and high dose groups in both sexes as decreased mean body weights and body weight gains (statically significant). Food consumption was increased in the same dose groups; however, due to food spillage, when food consumption values exceeded two standard deviations from the mean, they were not included in calculation of the group mean food consumption. Treatment related effects were noted in 21 Diphenylamine ------- FINAL 5-11-2011 hematology involving red cell elements mainly in the high mid and high dose groups. Increases in albumin levels, decreases in globulin levels and increased albumin/globulin ratios were noted but, the biological relevance to these changes is unknown since there was no related pathology. Some slight transient effects were also noted in alkaline phosphatase and total bilirubin also in serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT). Urinalysis did not reveal any specific treatment related effects except a slight increase in ketones in the high dose due to incomplete or partial interference of the test article causing a false positive reading. There was an increase in spleen weights in both sexes in the high mid and high dose groups at the interim sacrifice and terminal sacrifice. Gross necropsy observations revealed a roughened surface to the kidneys in the high dose groups. Treatment related non-neoplastic observations were splenic congestion, increased hemosiderosis and hematopoiesis in the spleen, pigment deposits in the kidneys, and increased hematopoiesis in the liver in the high mid and high dose groups. No treatment related increase in any tumor type or site was seen in either sex at any dose level. Methemoglobin was not measured in this study. For chronic toxicity the NOEL was 28.8 mg/kg/day in males and 24.9 mg/kg/day in females and the LOEL was 146.7 mg/kg/day in males and 137.8 mg/kg/day in females based on reduced mean body weight and body weight gains, changes in hematological parameters, splenic and kidney lesions and increased clinical signs of toxicity. There was no evidence of carcinogenicity. A LOAEL of 25 mg/kg-day based on haematological and histological effects and NOAEL of 7.5 in female rats were identified in the European Union Risk Assessment Report on diphenylamine (Botta 1994b as discussed in European Communities, 2008; Table 4.1.2.6). Thomas et al. (1967b) published a peer-reviewed 2-year study examining the chronic toxicity and carcinogenicity of diphenylamine administered in the diet to weanling albino Slonaker-Addis rats. Twenty rats per sex per group were administered 0-, 0.001%- (10-ppm), 0.01%- (100-ppm), O.P/o- (1000-ppm), 0.5%- (5000-ppm), or 1.0%- (10,000-ppm) diphenylamine (purity 99.9%) in feed with water provided ad libitum. Adjusted daily doses calculated using default data for body weight (U.S. EPA, 1994b) and food consumption (U.S. EPA, 1988) are 0, 0.72, 7.2, 72, 362, or 723 mg/kg-day for males and 0, 0.82, 8.2, 82, 410, or 820 mg/kg-day for females. Rats were housed five per cage. The study authors did not report whether this study adhered to GLP guidelines. Rats were weighed once per week during the first 5 months of the study and approximately once per month thereafter. The study authors recorded food consumption in 6- to 8-day intervals. All survivors at study termination were necropsied beginning on Day 734. Hematology data were collected only from select rats in the 1.0%-dose group. The Duncan method was used to test statistical difference in data between the treated and control groups. Thomas et al. (1967b) generally reported 2-4 mortalities by Day 240 in males and females due to respiratory infection (see Table B.4). The study authors reported depressed average weights of females receivings 0.1%, which appears to be considerably less than 10% from control rats, and males receiving > 0.5%, which appears to be somewhat greater than 10% (only graphical data provided). Average food consumption during the first 240 days was 22 Diphenylamine ------- FINAL 5-11-2011 significantly decreased for both sexes in the two highest dose groups. Hemoglobin and RBC counts of albino male and female Slonaker-Addis rats at the 1.00% dose level were decreased from controls but not significantly (see Table B.5). The study authors reported that diphenylamine-related lesions were limited to the urinary tract. Lesions included chronic nephritis, tubular cysts, and epithelial hyperplasia/metaplasia (see Table B.6) and were distinguishable from those of control rats at the >0.5%-diphenylamine dose levels. Irregularly dilated kidneys were occasionally filled with proteinaceous fluid or with iron-positive pigment resulting from blood-derived masses. Morphological changes were not observed in the glomeruli and were rarely observed in the proximal convoluted tubules. Mild epithelial hyperplasia or squamous metaplasia of the epithelial linings of the renal pelvis and bladder were observed, along with an accumulation of masses of pigment and protein in these organs. Interstitial lymphocyte accumulation and scarring were also noted, along with renal cystic changes. Frequent accumulation of neutrophils in the pigment-filled cysts was also reported. Severe inflammation was not observed in the absence of marked cystic changes. Based on the increased incidence of rats having a severity score for chronic nephritis "two plus" as well as the increase (significance not indicated) of the mean severity score over the controls (in Table 6 of the Thomas et al. 1967b study), the 0.5%-dose group appears to be a clear effect level. In contrast, the "break-point" for cystic change was not clear, but it was reported by the study authors to occur at the 0.1%-dose group. Tumor incidence was reported by the study authors to be due to senility of the test rats and not due to exposure of diphenylamine. Adenomatous hyperplasia of the adrenal medulla, three of which were malignant, was reported to be the most commonly occurring tumor (see Table B.7). In addition, cystic dilation of the peripancreatic lymph node and occurrence of eosinophilic granuloma of the gastric submucosa were also frequently observed. The study authors also reported senile pancreatic and testicular atrophy as well as chronic respiratory disease. Although urinary tract lesions were observed in treated animals, the incidences were comparable to those noted in the concurrent control group (see Table B.6). Lesions included cystic dilation of renal tubules and interstitial inflammation (see Table B.6) and were distinguishable from those of control rats at the >0.5%-diphenylamine dose levels. Based on these results, a LOAEL of 0.5% is identified for decreased body weight and increased incidence and severity of chronic nephritis in both female (410 mg/kg-day) and male (362 mg/kg-day) rats in this study. It should also be noted, however, that the decreases in weight were accompanied by significant decreases in food consumption. A NOAEL of 0.1% (82 mg/kg-day for females and 72 mg/kg-day for males) is also identified. Griswold et al. (1966) published a peer-reviewed study on the mortality and carcinogenicity effects of a single maximum tolerated oral dose to each of 50 different chemicals in 20 female Sprague-Dawley rats. Evaluation was at 6 months after the single dose. Diphenylamine was administered intragastrically in sesame oil at 300 mg/rat. The adjusted daily doses calculated using default data for body weight (U.S. EPA, 1994b) and food consumption (U.S. EPA, 1988) is 1.7 mg/kg-day. A separate group of animals (n = 89) served as a control group, receiving sesame oil alone. Dimethylbenzanthracene, known to cause mammary gland tumors in this species under these time constraints (6 months), was used as the positive control. While purity of the chemicals tested in this study was not reported, the study authors stated that 23 Diphenylamine ------- FINAL 5-11-2011 chemicals were checked for purity, and when necessary, purified using appropriate techniques. Rats were housed five per cage and supplied food and water ad libitum. It is unclear whether this study adhered to GLP guidelines. Two animals of the 20 administered diphenylamine died by 30 days postadministration with no others dying between 30 days and termination of the experiment at 6 months. Mortality was also observed among the controls, with 5/89 dying before the 6-month sacrifice. Histological descriptions of tissues that were considered to be neoplastic or suggestive of preneoplasia were listed in Table 2 of this study (including hyperplasia or metaplasia of the mammary glands, tubo-ovarian disease, lung lesions, as well as any other miscellaneous diseased tissues found during the course of the examinations). Tissues in which disease entities were not directly suggestive were not included in this study report table. As no mention of diphenylamine is made in this table, it is presumed that no such effects were seen with this compound. In a chronic-duration toxicity/carcinogenicity study (Botta, 1994c), CD-I mice (60 sex/group) were administered diphenylamine (>99%) in the diet at levels of 0, 525, 2625, or 5250 ppm (males: 73, 370, and 760 mg/kg/day; females: 90, 460, or 940 mg/kg/day) for 78 weeks. There was a significant treatment-related increase in overall mortality in the 2625- and 5250-ppm groups. The increased mortality was due to cystitis in males and amyloidosis in females. A greenish staining of the hair was the most frequently observed clinical sign, with some of the 525-ppm group and essentially all of the 2625- and 5250-ppm groups affected by the end of the study. Mean body-weight gain was significantly decreased in the 5250-ppm group males at the majority of the time points in the study. Haematological examination revealed decreased mean erythrocyte counts (by 11% in comparison with controls), haemoglobin (9.3%), and haematocrit (8.7%) in males at the high dose; smaller decreases in these parameters were found in females. Changes in the hematology parameters indicate that the chemical produced a regenerative anemia in the 2625- and 5250-ppm group males and females. On gross examination at the interim and terminal necropsies, the liver and spleen of the 2625- and 5250-ppm group animals were dark and enlarged. The absolute and relative weights of the liver and spleen were also increased in these animals. On histopathology at the interim and terminal necropsies, the 525-ppm group and above had increased incidence of hemosiderosis and congestion in the spleen; also, the 2625- and 5250-ppm groups had increased incidences and/or severity of hematopoiesis in the spleen and liver, and pigment in the reticuloendothelial cells of the liver. Pigment was also observed in the convoluted epithelial cells of the kidney of these groups at terminal necropsy. The incidence of pyelonephritis in the 5250-ppm group males was marginally increased. There were increased incidences of cystitis and dilatation of the urinary bladder and balanoposthitis in the penis and preputial area of the 2625- and 5250-ppm groups at both of the necropsies. For the 5250-ppm group females, the incidence of amyloidosis was increased in the thyroid, adrenals, kidneys (also in the 2625-ppm group), stomach, small intestines, ovaries, and uterus. For chronic toxicity, the LOAEL is identified to be 525 ppm (73 mg/kg/day for males and 91 mg/kg/day for females) based on histopathological lesions in the spleen. Because 525 ppm was the lowest dose tested, a NOAEL cannot be identified from this study. There was no evidence of carcinogenicity. Ford et al. (1972; available as abstract only) conducted a 92-week study examining the chronic oral toxicity and carcinogenicity of diphenylamine in CD-I albino mice. The full study is not available for review. A summary of the abstract available on page 94 of the European 24 Diphenylamine ------- FINAL 5-11-2011 Union Risk Assessment Report on diphenylamine (European Communities, 2008) is presented below. Adjusted daily doses calculated using default data for body weight (U.S. EPA, 1994b) and food consumption (U.S. EPA, 1988) are 0, 8.7, 17, and 43 mg/kg-day for male mice, or 9.3, 18, and 45 mg/kg-day for female mice. Charles River CD-I Albino mice of both sexes (150 per group) were fed diets containing 0, 0.005, 0.01, and 0.025% diphenylamine over a time period of 92 weeks. No effects of diphenylamine exposure were noted on the nature and incidence of histopathological changes in particular the times of appearance and incidence of tumors. Holmberg et al. (1983) conducted an 18-month study examining the chronic oral toxicity and carcinogenicity of diphenylamine in outbred albino mice. The full study is not available for review. A summary of the abstract available on page 90 of the European Union Risk Assessment Report on diphenylamine (European Communities, 2008) is presented below. NMRI outbred Albino mice, 8 weeks old, were administered with 300 mg/kg bw/d diphenylamine in soy bean oil by gavage once a week during 18 months (78 times). A positive control group receiving dimethynitrosamine (DMNA) (15 mg/kg bw/d) was equally investigated. The diphenylamine treatment group consisted of 125 mice and the control group of 30 animals. Sacrifices were at 25 and 52 weeks. Total observation time lasted 126 weeks. There were no changes in the frequency of tumors as compared to the vehicle treated controls. In the diphenylamine group 22.9% of the animals developed tumors. The most common tumor type\s] were lymphomas (8.3%) and alveolar adenomas (16.5). The tumor incidence in the vehicle control group were 22.2% with 11.1% lymphomas and 11.1% alveolar adenomas. As a non neoplastic alteration an increasedfrequency of lymphohistiocytic nephritis occurred in diphenylamine treated animals. The results on tumor morphology and incidence did not reveal a diphenylamine related change of tumors compared to vehicle treated controls. A LOAEL or NOAEL was not identified for this study in the European Union Risk Assessment Report on diphenylamine (Holmberg et al. 1983 as discussed in European Communities, 2008). Developmental Studies Based on a range-finding preliminary study (Edwards et al., 1983a), study authors administered 0, 33, 100, or 300 mg/kg-day of diphenylamine (99.9% pure) via gastric intubation in 1%-methyl cellulose to groups of 16 pregnant female rabbits per dose from gestation days (GDs) 7-19 (Edwards et al., 1983b). Females had been mated with males and injected with luteinizing hormone to promote ovulation; the day of mating was considered GD 0. Dose volumes were adjusted according to body weights. Environmental conditions and feeding methods were the same as described in the preliminary study. All animals were observed daily for signs of toxicity and were weighed on GDs 1, 7, 9, 11, 15, 20, 24, and 29. Food intake was measured during periods between weighing. After sacrifice on GD 29, dams were dissected and examined pathologically, with special attention paid to ovaries and uteri. The investigators counted the number of corpora lutea, the number and distribution of live fetuses, and the number and distribution of embryonic/fetal deaths. Embryonic/fetal deaths were classified as early with only placenta seen at termination, late with both embryonic and placental remnants seen at termination, and abortion with only implantation scars seen at termination. Fetuses were 25 Diphenylamine ------- FINAL 5-11-2011 weighed and examined for abnormalities in the brain and skeleton. Those showing visible abnormalities were examined more thoroughly for histopathological changes. The study authors calculated group means for litter size and various litter parameters (as percentages). Kruskal-Wallis and Jonckheere tests were used to analyze nonparametric data. All treated animals, but particularly those dosed with 100 or 300 mg/kg-day, experienced green discoloration of the urine. Data on animals that died or were removed from the study for welfare reasons (number not specified) were not considered. Mean food consumption was reduced at 300 mg/kg-day (statistical significance not reported). Animals dosed with 300 mg/kg-day experienced lower weight gain compared to controls. Mean body weight gain was slightly reduced at 33 mg/kg-day, but animals treated with 100 mg/kg-day gained more weight compared to controls (see Table B.8). The study authors stated that weight gain and weight loss were not treatment related. There were no statistically significant (p < 0.05) or dose-related differences in litter size or pre- and postimplantation losses among groups. Fetal weight was slightly lower than controls at all doses, but the differences were not statistically significant (see Table B.9). The four fetal malformations (two in the controls, one at 33 mg/kg-day, and one at 100 mg-kg/day) were reportedly unrelated to treatment with diphenylamine. Skeletal variations were not significantly different among groups (see Table B.9). Based on these results, a maternal and developmental NOAEL of 300 mg/kg-day is identified based on this study. A LOAEL cannot be identified in this study due to lack of maternal and developmental effects at the administered doses. Rodwell (1992) conducted an unpublished teratology study examining the developmental effects of diphenylamine on Sprague-Dawley rats. The full study is not available for review. The study as summarized in the European Union Risk Assessment Report on diphenylamine (European Communities, 2008; page 98) is presented below. In an unpublished teratology study by Rodwell, 1992, pregnant female Sprague-Dawley rats (25/group) received diphenylamine (99.9%) in corn oil by oral gavage at dose levels of 0, 10, 50, or 100 mg/kg bw/dfrom gestation day six through gestation day 15 inclusive; dams were sacrificed on gestation day 20. None of the rats died during the study. Maternal toxicity was evidenced by increased splenic weights, enlarged spleens and blackish-purple colored spleen in the dams at 100 mg/kg bw/d. The maternal toxicity NOAEL was 50 mg/kg bw/d and the LOAEL was 100 mg/kg bw/d. No developmental toxicity was seen at any dose level. The developmental toxicity NOAEL was equal to or greater than 100 mg/kg bw/d, the highest tested dose (citedfrom EPA RED report, 1998 and JMPR report 1998; the original study was not available). Reproductive Studies In an unpublished two-generation reproductive study, Rodwell (1993) administered diphenylamine (99.8% purity) to Sprague-Dawley rats (28/sex/group) in the diet for 90 days at concentrations of 0, 500, 1500, or 5000 ppm. These concentrations are equivalent to average daily doses of 0, 40, 115, or 399 mg/kg-day in F0 males and 0, 46, 131, or 448 mg/kg-day in F0 females, as calculated in the European Union Risk Assessment Report on diphenylamine (Rodwell 1993 as discussed in European Communities, 2008). It is unclear whether this study is GLP compliant, although it appears that is has been peer reviewed. The full study is not 26 Diphenylamine ------- FINAL 5-11-2011 available for review. The study as summarized in the European Union Risk Assessment Report on diphenylamine (European Communities, 2008; page 96) is presented below. Compound- related systemic toxicity was observed in a dose related manner among both sexes and generations at all dose levels. In general, females were more affected than males and F1 animals were more affected than F0 animals. Clinical signs (bluish colored fluid in the cage and bluish colored staining of the coat in both sexes, and swelling of mammary gland(s) or palpable lateral-ventral masses, primarily in females) were evident at 5000ppm. Body weight was decreased at 1500 and 5000 ppm. At 5000 ppm, there was a 6-9% decrease in body weight values, as compared to control, for F0 males, 5-8% for F0 females, 22-28% for F1 males, and 11-23% for F1 females. At 1500 ppm, there was a 5-8% decrease in body weight values from controls for F0 females, 7-9% for F1 males, and 5% for F1 females. Food consumption (g/animal/day) was also decreased at 1500 and 5000 ppm. Kidney, spleen, and liver appeared to be the target organs as evidenced by weight differences from control at 5000 ppm in males and at 1500 and 5000 ppm in females and gross and microscopic findings at all dose levels in both sexes. Gross findings included enlarged and blackish-purple spleens. Microscopic findings included brown pigment in the proximal convoluted tubules of the kidney, hepatocytic hypertrophy, brown pigments in the Kupffer cells of the liver, congestion and hemosiderosis of the spleen. The systemic toxicity NOAEL was less than 500 ppm (40 mg/kg-bw/d in males and 46 mg/kg-bw/d in females). The LOAEL was less than or equal to 500 ppm based on gross pathological findings in the spleen (enlarged, discolored), and on microscopic findings in the kidney (brown pigment in the proximal convoluted tubule), liver (hepatic hypertrophy and brown pigment in the Kupffer cells), and spleen (congestion and hemosiderosis). Developmental toxicity was observed at 1500 and 5000 ppm, as evidenced by significantly decreased body weight for F1 pups at 5000 ppm throughout lactation (11-25% less than control), for F2 pups at 5000 ppm from LD 4 through 12 LD 21 (10%-29% less than control), andfor F pups at 1500 ppm on LD 14 (10%) and LD 21 (12%). The developmental toxicity NOAEL was 500ppm (46 mg/kg-bw/dfor maternal animals) and the LOAEL was 1500 ppm (131 mg/kg bw/dfor maternal animals) based on decreased F2 pup body weight in late lactation. Reproductive toxicity was noted as smaller litter sizes at birth (significant for the F2 litters) in both generations at 5000 ppm. The reproductive toxicity NOAEL was 1500 ppm (131 mg/kg-bw/dfor maternal animals) and the LOAEL was 5000 ppm (448 mg/k- bw/dfor maternal animals), based upon decreased litter size in both generations (citedfrom EPA RED report, 1998 and JMPR report, 1998; original publication not available). Thomas et al. (1967c) also published a peer-reviewed study examining the reproductive effects of diphenylamine on weanling Slonaker-Addis rats. Twelve, 5-week-old female and three male rats per group were administered concentrations of 0-, 0.1%- (1000-ppm), 0.25%- (2500-ppm), or 0.5%- (5000-ppm) diphenylamine (purity 99.9%) in feed for approximately 65 days and were mated at 100 days of age. Adjusted daily doses calculated using default data for body weight (U.S. EPA, 1994b) and food consumption (U.S. EPA, 1988) are 0, 90, 226, or 451 mg/kg-day for males and 0, 101, 252, or 503 mg/kg-day for females. (Average daily doses displayed in Table B.10 represent an average of the doses between males and females.) The 27 Diphenylamine ------- FINAL 5-11-2011 study authors did not report whether this study followed GLP guidelines. Rats (F0) were housed four females and one male per cage during mating, and males were rotated once per week to a cage of four different females for 3 weeks. When the first litter of offspring was weaned, a second round of mating was performed in this same manner. First generation (Fl) offspring were also mated in the same manner described above to produce a second generation (F2). The study authors reported a general trend of decreasing litter size with increasing dose in the first and second mating of the F0 generation (see Table B.10). The number of pups per litter at birth in the first F0 mating was significantly (p < 0.05) lower in the 0.5%-dose group compared to the control, while in the second mating, the number of pups per litter at birth was significantly (p < 0.05 or p < 0.01) lower compared to the concurrent control group for all three dose groups (see Table B. 10). The number of pups per litter at birth and weaning in Fl animals exposed to 0.1%-diphenylamine was significantly (p < 0.01) lower than the control group; however, a similar effect was not observed in Fl animals exposed to 0.25%- and 0.5%- diphenylamine (see Table B. 10). The study authors reported that average weights of the second-generation weanling pups were comparable to the control group for both males and females exposed to 0.1%- and 0.25%-diphenylamine, while weanling weights were significantly (p < 0.01) lower in the 0.5%-dose group compared to the concurrent controls. The study authors concluded that because weanling weight loss was not observed at the 0.1%- and 0.25%-dose levels, the weight loss observed in the 0.5%-dose group could not be firmly attributed to diphenylamine exposure. Because the study results are not conclusive, a LOAEL or NOAEL cannot be identified for this study. Inhalation Exposures The effects of inhalation exposure to diphenylamine on animals have not been evaluated in subchronic- or chronic-duration, developmental, reproductive, or carcinogenic studies. OTHER STUDIES (SHORT-TERM TESTS, OTHER EXAMINATIONS) A few studies pertaining to the mutagenicity, genotoxicity, clastogenecity, cytotoxicity, acute toxicity via oral and intraperitoneal (ip) exposure, and metabolism/toxicokinetics are available for diphenylamine (see Table 3). Mutagenicity studies (Mobil Oil Corporation, 1994a; Litton Bionetics, 1994; Braun et al., 1977; Probst et al., 1981a; Zeiger et al., 1988; Babish et al., 1983; Dolara et al., 1993) on diphenylamine conducted using a traditional or modified Ames assay in Salmonella typhimurium strains TA97, TA98, TA100, TA1000, TA1535, TA1537, TA1538, G46, C3076, and D3052 were all negative in the presence and absence of S-9 activation. Similarly, mutagenicity studies (Probst et al., 1981a) in E. coli strains WP2 and WP2uvrA were also negative. The murine lymphoma assay, unscheduled DNA synthesis in rat hepatocytes, DNA single strand breaks (SSBs) in rat hepatocytes and V79 cells, and sister chromatid exchanges (SCEs) in human lymphocytes were all negative as well when tested using various doses/concentrations of diphenylamine, either alone (Mobil Oil Corporation, 1994b; Probst et al., 1981b; Gorsdorf et al., 1988) or in a mixture (Dolara et al., 1993). Cytogenicity studies examining increases in micronucleated cells following diphenylamine exposure were mostly negative (Mobil Oil Corporation, 1994c; Dolara et al., 1994). A study by Dolara et al. (1993) reported that slight cytogenicity was noted following treatment with diphenylamine based on nonsignificant increases in polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs) ratios. One cytotoxicity test by Masubuchi et al. (2000) stated that diphenylamine was cytotoxic in vitro at concentrations ranging from 10 to 500 [xM in cultured 28 Diphenylamine ------- FINAL 5-11-2011 male Wistar rat hepatocytes. Genotoxicity results were equivocal when determined in various test systems. Ardito et al. (1996) reported that diphenylamine was weakly genotoxic in human lymphocytes, while Lodovici et al. (1997) reported that diphenylamine produced free radicals capable of inducing genetic damage to the cell in livers of Wistar rats dosed with 0.14-mg/kg-day diphenylamine for 10 days. Contrary to these results, von der Hude et al. (1988) reported that diphenylamine-related genotoxicity was not noted in an SOS chromotest conducted using E. coli PQ37. Acute oral toxicity studies (Lenz and Carlton, 1990a,b,c) indicated that acute exposures to diphenylamine via gavage were more toxic to hamsters when compared to rats and gerbils, with toxicity primarily noted in the kidneys. A single study (Lenz and Carlton, 1990d) in hamsters following ip administration with diphenylamine also suggested that kidneys were the target organ. A metabolism and toxicokinetic study by Alexander et al. (1965) reported that major metabolites in rats, rabbits, and humans were 4-hydroxydiphenylamine and 4,4'-dihydroxydiphenylamine. Toxicokinetic studies in rats indicated that [14C] diphenylamine was rapidly metabolized and excreted in the urine and bile. Metabolism studies in female NMRI mice suggested that iV-hydroxyl-derivate of diphenylamine may be a potential metabolite of diphenylamine (Appel et al., 1987). Gutenmann and Lisk (1975a,b) conducted metabolism studies in a Holstein cow and fresh rumen and concluded that conjugates of diphenylamine were not observed in the hydrolyzed urine from the cow, or in the rumen. However, the study authors stated that microsomal hydroxylation of diphenylamine is possible in bovine systems, but these were not detected due to the limited analytical techniques employed in their test system. Crocker et al. (1972) conducted a mechanistic study focused on the kidneys of fetal rats and their dams that were administered diphenylamine from several different commercial sources. The study authors also chromatographically isolated and tested the effects of major diphenylamine contaminants in pregnant rats and in their offspring. Commercial preparations of diphenylamine were administered in the feed of a group of pregnant Sprague-Dawley rats from GD 14 to term at high doses (1.5% or 2.5%, or about 2143 or 3571 mg/kg-day) to ensure that effects were seen. A total of three contaminants chromatographically isolated from aged preparations of diphenylamine, as well as purified diphenylamine itself, were dissolved in 70%) ethanol and fed by gastric tube at doses estimated at about 0.05 mg/day to another group of pregnant rats for 7 days prior to delivery. Control groups of pregnant rats received either no diphenylamine in their food or 70% ethanol. Live offspring were removed from their mothers shortly after birth, and histology was performed upon the kidneys of both parent and offspring. Results indicated that cystic lesions were confined to the proximal nephron in newborn rats and were in contrast with the localization in the collecting ducts after long-term feeding of diphenylamine to adult rats. Variations observed in the incidence and severity of the cystic changes appeared to depend upon the commercial source of the diphenylamine. Renal lesions were also produced in newborn offspring of pregnant rats fed microgram quantities of one of the components isolated from diphenylamine, whereas none were seen after feeding of chromatographically pure diphenylamine. In a GLP-compliant preliminary range-finding study, Edwards et al. (1983a) administered 0, 200, 400, or 600 mg/kg-day of diphenylamine (99.9% pure) in 1%> methyl-cellulose via 29 Diphenylamine ------- FINAL 5-11-2011 gavage to groups of six pregnant female New Zealand white rabbits per day for 13 days. Rabbits were obtained from Cheshire Rabbit Farms, Ranch Rabbits, and Buxted Rabbit Co., Ltd. (England) and were between 12 and 20 weeks of age upon arrival. Animals were housed individually in metal cages in a room kept at 18 ± 3°C with a relative humidity of 55 ± 5%. Artificial lighting supplemented natural light between the hours of 7:00 am and 9:00 pm. Tap water and food (SDS Rabbit Diet) were provided ad libitum. Contaminant analysis indicated that no nonnutrient substance in feed/water or nutrient levels in the feed would interfere with the test system. Animals were observed daily for clinical signs of toxicity and other changes. All animals were weighed prior to treatment and every day during treatment. One animal was sacrificed early after persistent weight loss. All treated animals exhibited green discoloration of the urine. Food consumption was slightly reduced, and body weight was lowered in all dose groups, although recovery was seen in the 400-mg/kg-day group by the end of the study. Autopsy revealed no treatment-related macroscopic changes. The study authors did not report whether statistical tests were performed on the preliminary study data. It is unclear whether this study was peer reviewed. 30 Diphenyl amine ------- FINAL 5-11-2011 Table 3. Other Studies Test Materials and Methods Results Conclusions References Mutagenicity Ames Salmonella (strain: TA1535) assay without metabolic activation; diphenylamine in DMSO at doses of 3, 5, 10, 15, 20, 30, 50, or 75 ng/^1 per bacterial plate incubated for 48 hr at 37°C Negative; reversion frequencies not substantially different from solvent or spontaneous controls Diphenylamine was not mutagenic inTA1535 in the absence of metabolic activation Mobil Oil Corporation (1994a) Mutagenicity Murine lymphoma mutagenesis assay with S-9 activation (at doses ranging from 0.58—0.007-nl/mL diphenylamine); S-9 fraction obtained from male S-D rats induced with Aroclor 1254; without S-9 activation (at doses ranging from 0.075-0.0056 iil/mL); counted induced forward mutations from the heterozygote (TK+/-) to the homozygote (TK-/-) in a matrix cytotoxic to TK +/-; cells exposed for 180 minutes and given 48 hours to express mutation before cloning Toxicity not associated with dose; no significant increase in mutagenic frequency in those with acceptable (>_10%) total growth, with or without activation Diphenylamine was not mutagenic in the murine lymphoma assay Mobil Oil Corporation (1994b) Mutagenicity Ames Salmonella (strains TA1535, TA1537, TA1538, TA98, and TA100) assay with and without S-9 activation; S-9 fraction obtained from male S-D rat liver induced with Aroclor 1254; diphenylamine in DMSO at doses of 0.1, 1, 10, 100, or 500 |ig per bacterial plate; incubated for 48 hours at 37°C Diphenylamine toxic to all strains at 500 |ig: mutagenicity results negative with and without activation Diphenylamine was not mutagenic in TA1535, TA1537, TA1538, TA98, or TA100, both with and without metabolic activation. Litton Bionetics (1994) Mutagenicity Host-mediated assay with Salmonella typhimurium strain TA1950; male NMRI mice injected via ip with 2 mL of bacterial culture; diphenylamine dissolved inNaCl administered simultaneously to mice via gavage at doses ranging from 1450—2900-^moles/kg diphenylamine; replicate experiments were performed No mutagenic activity observed in mice injected with TA1950 and treated with diphenylamine Diphenylamine was not mutagenic to mice injected with Salmonella typhimurium strain TA1950 Braunetal. (1977) 31 Diphenyl amine ------- FINAL 5-11-2011 Table 3. Other Studies Test Materials and Methods Results Conclusions References Mutagenicity Ames Salmonella assay (TA1538) with and without S-9 activation treated with 100-|ig diphenylamine; incubation time unreported No mutagenic activity observed following treatment with diphenylamine in the presence and absence of S-9 activation Diphenylamine was not mutagenic to TA1538 Ferretti et al. (1977) Mutagenicity Modified Ames assay using Salmonella strains G46, TA1535, TA1000, C3076, TA1537, D3052, TA1538, and TA98, and E. coli strains WP2 and WP2 uvrA" with and without S-9 metabolic activation; S-9 fraction obtained from Aroclor 1254- treated rats; diphenylamine concentration range not specified; chemical incorporated into 4-gradient plates to give a 10-fold concentration range per plate No mutagenic activity observed in any of the Salmonella or the E. coli strains, both in the presence and absence of S-9 activation Diphenylamine was not mutagenic Probst etal. (1981a) Mutagenicity Unscheduled DNA synthesis assay using Fischer-344 rat hepatocytes exposed to 100-nmoles/mL diphenylamine for a 5- hour incubation period No unscheduled DNA synthesis observed in treated rat hepatocytes Diphenylamine was not mutagenic Probst etal. (1981b) Mutagenicity Ames assay using Salmonella strains TA97, TA98, TA100, and TA1535 exposed to concentrations of 0-, 1-, 3-, 10-, 33-, 66-, 100-, 166-, or 333-^g/plate diphenylamine and incubated for 2 days; strains first tested without metabolic activation; subsequently tested with S-9 activation if results from the first assay were positive No mutagenic activity observed in any of the Salmonella strains tested Diphenylamine was not mutagenic Zeiger et al. (1988) Mutagenicity Alkaline elution assay used to detect DNA single strand breaks in V79 cells and rat hepatocytes; doses not specified Positive results observed in rat hepatocytes only No firm conclusions presented; results are presented in an abstract; complete article is not available Gorsdorf et al. (1988; abstract) 32 Diphenyl amine ------- FINAL 5-11-2011 Table 3. Other Studies Test Materials and Methods Results Conclusions References Mutagenicity Ames assay using Salmonella strain TA100 exposed to concentrations of 15-, 21-, 29.3-, 41-, 57.3-, or 80-ng/plate diphenylamine with and without S-9 activation following an incubation for 18 and 48 hours; modified liquid suspension assay used to test for mutagenicity of the aqueous extract from sterilized nipples No dose-related increase in revertants with or without S-9 activation; tests of mutagenicity in the aqueous extract positive (significant increases in reversions with and without activation in a number of different samples) Diphenylamine was not mutagenic in TA100 when tested via the standard Ames assay; positive mutagenicity with the aqueous extract may be related to dimethyl- and diethylnitrosamines (as opposed to diphenylamine), which were readily detected in the extract Babishetal. (1983) Mutagenicity/Clastogenicity Mixture of 15 pesticides, including 50-ppb diphenylamine, tested using the standard plate test (Ames) with and without metabolic activation (S-9) at concentrations up to 500 |ig/platc in Salmonella strains TA1537, TA1538, TA98, TA100, TA1535, and TA102 Human lymphocytes tested for sister chromatid exchanges (SCEs) after incubation with 0.1-20 ng/mL of the mixture for 72 hours at 37°C (cells stained with acridine orange and observed with fluorescent microscope) Micronucleus assay using Wistar rat bone marrow (rats had been administered 1, 10, or 100 ng/kg of the mixture) in which polychromatic erythrocytes (PCEs) and normochromatic erythrocytes were (NCEs) counted with a fluorescent microscope Concentrations of up to 500 ng/plate of pesticide mixture not mutagenic in Ames assay Small but statistically significant increase in SCEs compared to controls from dose of 1 ng/mL of the mixture Nonsignificant increase in the ratio between PCEs and NCEs in rat bone marrow (indicating slight toxicity) The study authors concluded that the mixture did not have "appreciable genotoxic activity" in these test systems; they noted that nonmutagenic pesticides might have interfered with the DNA interaction of a genotoxic pesticide, such as diphenylamine, inhibiting its mutagenic potential Dolara et al. (1993) 33 Diphenyl amine ------- FINAL 5-11-2011 Table 3. Other Studies Test Materials and Methods Results Conclusions References Clastogenicity Micronucleus bone marrow assay; rats (strain not specified); 10 M/10F treated dermally for 90 days, 5 days/week, with 0-, 500-, or 2000-mg/kg-day diphenylamine; PCEs and NCEs scored for micronuclei; cytotoxicity also recorded Ratio means of PCEs and NCEs not significantly different between treated and untreated groups; cytotoxicity not a factor in micronucleus induction evaluation; percentage of micronucleated PCEs and NCEs not different between treated animals and negative controls Diphenylamine did not significantly increase micronucleated cells Mobil Oil Corporation (1994c) Clastogenicity Human lymphocytes incubated with 1- 20 iig/mL of a mixture of 15 pesticides, including 14.4%-diphenylamine (incubation period not specified); followed "standard" methods with Giemsa staining and classified for cytogenic abnormalities (e.g., altered chromosome numbers, gaps, deletions) No significant changes in chromosome number; dose- dependent increase in the frequency of nonsynchronous centromeric separations that disappeared after benomyl was removed from the mixture The mixture was not genotoxic in the absence ofbenomyl Dolara et al. (1994) Cytotoxicity Freshly obtained male Wistar rat hepatocytes incubated with or without oligomycin and 0-, 10-, 25-, 50-, 100-, 250-, or 500-|iIVI diphenylamine dissolved methanol (incubation time unreported); fructose added after 90 minutes of treatment with diphenylamine; samples of suspension removed after 60 minutes to analyze cellular ATP content Pseudoenergetic mitochondrial swelling and uncoupling of mitochondrial oxidative phosphorylation, depleting cellular ATP Diphenylamine was a cytotoxic agent under the experimental conditions tested in this study Masubuchi et al. (2000) Genotoxicity Human lymphocytes obtained from whole blood, exposed to diphenylamine at concentrations of 0.6, 6, or 60 ng/mL and incubated for up to 48 hours with and without metabolic activation (rat liver induced with phenobarbital and benzoflavone, or S-9 fraction); frequency of SCEs analyzed Significant (p < 0.05) increase in SCEs in cells treated with 6 |ig/mL compared to controls without activation; similar increase not seen with or without activation with S-9 after 4 hours of incubation Diphenylamine was reported to be a weak, direct-acting genotoxic agent. Ardito et al. (1996) 34 Diphenyl amine ------- FINAL 5-11-2011 Table 3. Other Studies Test Materials and Methods Results Conclusions References Genotoxicity 6 male Wistar rats administered 0.14- mg/kg-day diphenylamine dissolved in corn oil for 10 days; animals sacrificed and livers examined for elevated levels of 8-OH-2-deoxyguanosine (8-OH-2DG) Significantly (p < 0.05) increased levels of 8-OH-2DG in rat liver DNA relative to controls In vivo, diphenylamine produced free radicals capable of inducing genetic damage to cells Lodovici et al. (1997) Genotoxicity SOS chromotest using /•'. coli PQ37 with and without S-9 metabolic activation; S-9 fraction obtained from Aroclor 1254- idnuced Wistar rats and Syrian hamsters; culture incubated for 2 hours No genotoxicity observed following exposure to diphenylamine Diphenylamine was not genotoxic von der Hude et al. (1988) Acute Oral Toxicity 10 male Syrian hamsters per dose group administered 400-, 600-, or 800-mg/kg-day diphenylamine via gavage for 3 days; 10 control animals dosed with peanut oil; moribund animals during the study euthanized and necropsied; surviving animals euthanized 24 hours after final dose and necropsied 4 mortalities at 400 mg/kg-day, and 100% mortality at 600 and 800 mg/kg-day; 9 incidences of splenomegaly at 400 mg/kg-day; 2 incidences of brown kidneys, 6 incidences of yellow-brown papilla, and 1 incidence of gastric ulcers at 600 mg/kg-day; 3 incidences of brown kidneys, and 5 incidences of yellow-brown papilla at 800 mg/kg-day; 4 incidences at 400 mg/kg-day, 7 incidences at 600 mg/kg-day, and 6 incidences at 800 mg/kg-day of total renal papillary necrosis Hamsters were more sensitive than rats (see below) to nephrotoxic effects of diphenylamine Lenz and Carlton (1990a) Acute Oral Toxicity 10 male Sprague-Dawley rats per dose group administered 400-, 600-, or 800- mg/kg-day diphenylamine via gavage for 3 days; 10 control animals dosed with peanut oil; moribund animals during the study euthanized and necropsied; surviving animals euthanized 24 hours after final dose and necropsied No mortalities at any dose; 1 incidence of bilateral renal cortical pallor at necropsy at 600 mg/kg-day; 2 incidences of renal papillary necrosis at 800 mg/kg-day Rats were less sensitive than hamsters (see above) to nephrotoxic effects of diphenylamine Lenz and Carlton (1990b) 35 Diphenyl amine ------- FINAL 5-11-2011 Table 3. Other Studies Test Materials and Methods Results Conclusions References Acute Oral Toxicity 10 male Mongolian gerbils per dose group administered 400-, 600-, or 800-mg/kg-day diphenylamine via gavage for 3 days; 10 control animals dosed with peanut oil; moribund animals during the study euthanized and necropsied; surviving animals euthanized 24 hours after final dose and necropsied No mortalities at any dose; no gross or microscopic lesions at any dose Diphenylamine did not demonstrate nephrotoxic effects in male Mongolian gerbils Lenz and Carlton (1990c) Acute Oral Toxicity Male Syrian hamsters (8/dose group) administered diphenylamine in peanut oil at 0, 200, 400, or 600 mg/kg-bw via gavage; kidneys removed and the papilla, outer medulla and the cortex analyzed for GSH levels; all groups dosed at 7 hours and hamsters from each dose group sacrificed at 8.00 or 11.00 hours to ensure that the cardiac variation on tissue GSH concentrations was observable A dose-dependent statistically significant reduction in cortical GSH observed 1 hour after diphenylamine administration; no significant changes in the outer medullar or the papilla cortical GSH levels (reduced by 53% compared to controls) in the cortex at 200 mg/kg at 4 hours; GSH in the cortex significantly reduced at 400 and 600 mg/kg at 4 hours The study authors concluded that the reduced cortical GSH and papillotoxicty are mechanistically unrelated and that the papillotoxicity of diphenylamine is mediated by mechanisms other than oxidative cell injury Lenz (1996) Intraperitoneal Injection 10 male Syrian hamsters per group administered 400-, 600-, or 800-mg/kg-day diphenylamine through ip injection for 3 days; 5 control animals dosed with peanut oil alone; animals euthanized 24 hours after final dose 5 mortalities at 600 mg/kg-day and 4 at 800 mg/kg-day; 1 incidence each of pale renal cortex at 600 and 800 mg/kg-day; 1 incidence at 600 mg/kg-day and 2 incidences at 800 mg/kg-day of brown kidneys; 4 incidences at 600 mg/kg-day and 2 incidences at 800 mg/kg-day of yellow- brown papilla; 5 incidences at 600 mg/kg-day and 4 incidences at 800 mg/kg-day of total renal papillary necrosis Diphenylamine demonstrated nephrotoxic effects in male Syrian hamsters Lenz and Carlton (1990d) 36 Diphenyl amine ------- FINAL 5-11-2011 Table 3. Other Studies Test Materials and Methods Results Conclusions References Range-finding-short term study 6 pregnant New Zealand White rabbits administered 0, 200, 400, or 600 mg/kg-day of diphenylamine in 1%- methyl-cellulose via gavage for 13 days All animals exhibited green discoloration of the urine; food consumption and body weight reduced at all doses; no treatment- related macroscopic changes Diphenylamine caused reduction in food consumption and body weight as well as green discoloration of the urine Edwards et al. (1983a) Mechanistic Pregnant Sprague-Dawley rats (number not specified) administered 2143- or 3571-mg/kg-day commercial preparation of diphenylamine via feed from GD 14 to term; pregnant Sprague-Dawley rats (number not specified) administered 0.05- mg/day three contaminants from aged diphenylamine in 70%-ethanol 7 days prior to delivery via gastric tube; control group (number not specified) received 70%- ethanol via gastric tube Cystic lesions confined to the proximal nephron in newborn rats in contrast with the localization in the collecting ducts after long- term feeding of diphenylamine to adult rats; variations in incidence and severity of the cystic changes dependent upon the commercial source of the diphenylamine; renal lesions also produced in newborn offspring of pregnant rats fed microgram quantities of one of the components isolated from diphenylamine Diphenylamine contaminants produced renal lesions in pups Crocker et al. (1972) Metabolism/T oxicokinetic Male white rats treated with 5-mg diphenylamine in propylene glycol via ip injection and urine collected 24 hours after treatment; cannulated bile duct from anaesthetized male white rats treated with 2-mg diphenylamine in 50%-aq. ethanol injected into the femoral vein and bile collected for 6 hours and examined for metabolites; 1 male rabbit fed 1 oral dose of a suspension of 5-g diphenylamine in divided doses of 1 g over 9 days and urine collected for analysis; 2 human subjects given 100 mg of diphenylamine orally and urine collected for 24 hours; 1 cat administered 1-mM/kg aqueous suspension of diphenylamine and blood collected for methaemoglobin determination; 1 rat given a single dose of 3-mg [14C]diphenylamine Major metabolites in rats, rabbits, and humans: 4-hydroxydiphenylamine and 4,4'-dihydroxy diphenylamine; O-sulphate and O-glucuronide (as the tri-O-acetylmethyl ester) conjugates of 4-hydroxydiphenylamine in rabbit urine; considerable methaemoglobin formation in cat; [14C] diphenylamine rapidly metabolized and excreted in rats based on excretion in the urine and bile; 75% of ip dose excreted in the urine at 48 hours; 25% of the iv dose excreted in the bile after 6 hours Major metabolites in rats, rabbits and humans are 4- hydroxydiphenylamine and 4, 4'- dihydroxy diphenylamine; diphenylamine was rapidly excreted through both urine and bile Alexander et al. (1965) 37 Diphenyl amine ------- FINAL 5-11-2011 Table 3. Other Studies Test Materials and Methods Results Conclusions References via ip injection and urine collected for 24 hours; cannulated bile ducts from male rats injected with 5-mg/kg [14C]diphenylamine in 50% aqueous ethanol via iv and bile samples collected at 1-hour intervals Metabolism/T oxicokinetic Female NMRI mice pretreated with 0.1%- phenobarbital in the drinking water for 5 days; liver microsomes from mice extracted and treated with 1-mM diphenylamine Rate of metabolism approximately 40-nMol compound/mL at 10 minutes and 55-nMol compound/mL at 30 minutes (inferred from Figure 3 in article) iV-hydroxyl-derivate of diphenylamine may be a potential metabolite of diphenylamine Appel et al. (1987) Metabolism/T oxicokinetic Holstein cow fed 5 ppm (based on daily ration of 22.7 kg) of pure recrystallized diphenylamine (in acetone) in grain for 4 days; milk sampled in the morning and evening before, during, and after dosing; total daily urine and manure samples also collected and analyzed No diphenylamine detected in milk or urine; small amounts found in the feces (fecal excretion = 1.4% or 6.2 mg) No conjugates of diphenylamine were observed in the hydrolyzed urine; the study authors stated that microsomal hydroxylation of diphenylamine is possible in bovine systems; however, the analytical techniques used in the study were not capable of identifying such hydrolyzed derivatives of diphenylamine Gutenmann and Lisk (1975a) 38 Diphenyl amine ------- FINAL 5-11-2011 Table 3. Other Studies Test Materials and Methods Results Conclusions References Metabolism/T oxicokinetic 1 mL of diphenylamine in acetone (500 |ig/mL) solution mixed with 100 mL of fresh filtered rumen fluid and later analyzed for diphenylamine using electron affinity gas chromatography; 5-ppm diphenylamine incubated with fresh beef liver fraction for 30 minutes at 37°C and analyzed using gas chromatography Diphenylamine stable in rumen fluid for 23 hours; roughly 50% diphenylamine lost during the 30 minutes of incubation with beef liver fraction No conjugates of diphenylamine were observed in the rumen; the study authors stated that microsomal hydroxylation of diphenylamine is possible in bovine systems; however, the analytical techniques used in the study were not capable of identifying such hydrolyzed derivatives of diphenylamine Gutenmann and Lisk (1975b) 39 Diphenyl amine ------- FINAL 5-11-2011 DERIVATION OF PROVISIONAL VALUES Table 4 presents a summary of noncancer reference values. Table 5 presents a summary of cancer values. IRIS data are indicated in the table if available. Table 4. Summary of Noncancer Reference Values for Diphenylamine (CASRN 122-39-4) Toxicity Type" (units) Species/Sex Critical Effect p-Reference Value POD Method POD UFC Principal Study Screening Subchronic p-RfD (mg/kg-day) Mouse/M+F Increased incidences of splenic hemosiderosis and splenic congestion 2 x 10~2 NOAEL 2.1 100 Botta (1992) Chronic RfD (IRIS) (mg/kg-day); 1993 Dog/M+F Decreased body weight; increased liver and kidney weights 2.5 x 10~2 NOAEL 2.5 100 Thomas et al. (1967a) "3 Subchronic p-RfC (mg/m ) None None None None None None None Chronic p-RfC (mg/m3) None None None None None None None aAll reference values obtained from IRIS are indicated with the latest review date. Table 5. Summary of Cancer Reference Values for Diphenylamine (CASRN 122-39-4) Toxicity Type Species/Sex Tumor Type Cancer Value Principal Study p-OSF None None None None p-IUR None None None None 40 Diphenyl amine ------- FINAL 5-11-2011 DERIVATION OF ORAL REFERENCE DOSES Derivation of Subchronic Provisional RfD (Subchronic p-RfD) No subchronic p-RfD can be derived because a nonpeer-reviewed study is selected as the principal study. However, derivation of a screening value is provided in Appendix A. Derivation of Chronic RfD (Chronic RfD) A chronic oral RfD of 2.5 x 1CT2 mg/kg-day is included in the IRIS database (U.S. EPA, 2010a) based on a 2-year chronic-duration toxicity study in male and female beagle dogs (eight/sex/group) by Thomas et al. (1967a). The critical end point identified in the IRIS database is decreased body-weight gain and increased liver and kidney weights in beagle dogs exposed to diphenylamine in diet. A no-observed-effect-level (NOAEL) of 2.5 mg/kg-day and a lowest-effect-level (LEL) of 25 mg/kg-day were identified in the IRIS database, and the NOEL served as a point of departure (POD) for chronic RfD derivation. A composite uncertainty factor of 100 (10 for interspecies extrapolation and 10 for intraspecies extrapolation) was used to derive the chronic RfD. The IRIS value was last revised on April 1, 1993. DERIVATION OF INHALATION REFERENCE CONCENTRATIONS No published studies investigating the effects of subchronic- or chronic-duration inhalation exposure to diphenylamine in animals were identified. Chronic occupational studies in humans (Checkoway et al., 1981; Parkes et al, 1982) examined the effects of mixtures of chemicals, which precludes their use in the derivation of a chronic p-RfC for diphenylamine. Overall, the lack of toxicity studies for diphenylamine via inhalation exposure precludes the derivation of subchronic- and chronic-duration inhalation toxicity values. CANCER WEIGHT-OF-EVIDENCE (WOE) DESCRIPTOR Table 6 identifies the cancer weight-of-evidence (WOE) descriptor for diphenylamine. 41 Diphenylamine ------- FINAL 5-11-2011 Table 6. Cancer WOE Descriptor for Diphenylamine Possible WOE Descriptor Designation Route of Entry (Oral, Inhalation, or Both) Comments "Carcinogenic to Humans" N/A N/A No strong human cancer data are available. "Likely to Be Carcinogenic to Humans" N/A N/A No strong animal cancer data are available. "Suggestive Evidence of Carcinogenic Potential" N/A N/A Chronic-duration toxicity/carcinogenicity studies (Botta, 1994b,c; Thomas et al., 1967b; Griswold et al., 1966; Ford et al., 1972; Holmberg et al., 1983) in various strains of rats and mice showed no increase in tumor incidence. Tumors that were observed were reportedly unrelated to diphenylamine exposure. "Inadequate Information to Assess Carcinogenic Potential" Selected Oral Available information is inadequate to assess carcinogenic potential. "Not Likely to Be Carcinogenic to Humans" N/A N/A No strong evidence of noncarcinogenicity in humans or animals is available. N/A = not applicable. DERIVATION OF PROVISIONAL CANCER POTENCY VALUES Derivation of Provisional Oral Slope Factor (p-OSF) The lack of carcinogenic activity in five chronic-duration toxicity/carcinogenicity studies (Botta, 1994a,b; Thomas et al., 1967b; Ford et al., 1972; Holmberg et al., 1983) following exposure to diphenylamine via the oral exposure route precludes the derivation of an oral slope factor. Derivation of Provisional Inhalation Unit Risk (p-IUR) The lack of quantitative data on the carcinogenicity of diphenylamine via the inhalation exposure route precludes the derivation of an inhalation unit risk. MODE-OF-ACTION DISCUSSION The Guidelines for Carcinogenic Risk Assessment (U.S. EPA, 2005) define mode of action as the following: ".. .a sequence of key events and processes starting with the interaction of an agent with a cell, proceeding through operational and anatomical changes, and resulting in cancer formation.. .There are many examples of possible modes of carcinogenic action, such as mutagenicity, mitogenesis, inhibition of cell death, cytotoxicity with reparative cell proliferation, and immune suppression" (p. 1-10). Studies exploring the mutagenic potential of diphenylamine reported that it was not mutagenic to various Salmonella typhimurium or E. coli strains tested using either the traditional or modified Ames assay (Mobil Oil Corporation, 1994c; Litton Bionetics, 1994; Braun et al., 42 Diphenylamine ------- FINAL 5-11-2011 1977; Probst et al., 1981a; Zeiger et al., 1988; Babish et al., 1983; Dolara et al., 1993). Genotoxicity results for diphenylamine were equivocal and ranged from the production of free radicals capable of genotoxic effects in rat liver cells to weakly genotoxic in human lymphocytes to nongenotoxic in an SOS chromotest in E. coli PQ37 (Ardito et al., 1996; Lodovici et al., 1997; von der Hude et al., 1988). Studies examining the ability of diphenylamine to cause unscheduled DNA synthesis, DNA single strand breaks, SCE, and increases in micronucleated cells were all negative (Mobil Oil Corporation, 1994b,c; Probst et al., 1981b; Gorsdorf et al., 1988; Dolara et al., 1993, 1994). While these results are limited to select test systems, they suggest that diphenylamine is unlikely to be mutagenic or genotoxic in the test systems examined. In addition, the lack of carcinogenic activity following oral exposure in two animal strains (rat and mouse) precludes a more detailed discussion regarding diphenylamine carcinogenic mode of action. 43 Diphenylamine ------- FINAL 5-11-2011 APPENDIX A. PROVISIONAL SCREENING VALUES For reasons noted in the main PPRTV document, it is inappropriate to derive a provisional subchronic p-RfD for diphenylamine. However, information is available for this chemical which, although insufficient to support derivation of a provisional toxicity value, under current guidelines, may be of limited use to risk assessors. In such cases, the Superfund Health Risk Technical Support Center summarizes available information in an Appendix and develops a "screening value." Appendices receive the same level of internal and external scientific peer review as the PPRTV documents to ensure their appropriateness within the limitations detailed in the document. Users of screening toxicity values in an appendix to a PPRTV assessment should understand that there is considerably more uncertainty associated with the derivation of an appendix screening toxicity value than for a value presented in the body of the assessment. Questions or concerns about the appropriate use of screening values should be directed to the Superfund Health Risk Technical Support Center. DERIVATION OF SCREENING PROVISIONAL ORAL REFERENCE DOSES Derivation of Screening Subchronic Provisional RfD (Screening Subchronic p-RfD) The 90-day toxicity study in the mouse (Botta, 1992) is selected as the principal study for the derivation of the screening subchronic p-RfD. The critical effect is increased incidences of splenic hemosiderosis and congestion in male and female CD-I mice. This unpublished study has not been peer reviewed, but the study was performed according to GLP principles. The study seems sufficient for the derivation of a screening subchronic p-RfD based on the number of animals and presentation of information. Details are provided in the "Review of Potentially Relevant Data" section. The number of potential toxicity end points that were examined include body and organ weights, clinical hematology, and histopathology. This study provides the most sensitive toxicological end point and the lowest POD for developing a screening subchronic p-RfD value. As detailed in the section "Review of Potentially Relevant Data," statistically significant changes included increased incidences of splenic hemosiderosis and congestion in the three highest dose groups in female mice and in the two highest dose groups in male mice. Because these two end points (i.e., splenic hemosiderosis and congestion) were the most sensitive effects reported in this study, all of the common dichotomous models available in the EPA's Benchmark Dose Software (BMDS, version 2.1.2) were fit to the data (see Table B. 11). After completion of BMD modeling, the output was reviewed for elimination of models that failed acceptability criteria (see Appendix C for BMD modeling results). BMD methods for choosing adequate model fit are described in Appendix C. For increased incidence of splenic congestion, the Quantal-Linear model was considered the best fit and produced a BMDio and BMDLio of 22 and 14 mg/kg-day for both male and female mice, respectively. For the increased incidence of splenic hemosiderosis, the Quantal- Linear model in male mice produced a BMDio and BMDLio of 18 and 12 mg/kg-day, respectively. The data for increased incidence of splenic hemosiderosis in female mice are not amenable to BMD modeling because there are no data at the low-response range, which is necessary for BMD modeling. Because the data for increased incidence of splenic hemosiderosis 44 Diphenylamine ------- FINAL 5-11-2011 in female mice did not provide adequate model fit, the NOAEL of 2.1 mg/kg-day for this parameter in female mice was considered as an alternate POD. Although splenic effects were the most sensitive data from the principal study (Botta, 1992), there were also significant changes in organ weights (i.e., kidneys, liver, spleen, and ovaries) that could be modeled by BMD for consideration of a POD because the LOAEL for some of these changes are within 10-fold of the LOAEL of 107 mg/kg-day for female mice splenic effects identified from the Botta, 1992 study. However, some of the data for these organ- weight changes (i.e., kidneys, liver, spleen, and ovaries) do not have a clear dose response, which is necessary for BMD modeling. The only organ-weight data that could be modeled were increases in both relative and absolute liver weight in male mice. For increased absolute liver weight in male mice, the Linear model was considered the best fit and produced a BMDio and BMDLio of 456 and 349 mg/kg-day, respectively. For increased relative liver weight in male mice, the Linear model was considered the best fit and produced a BMDio and BMDLio of 401 and 331 mg/kg-day, respectively. Both, the modeling results and methods are presented in Appendix C. Alternate PODs for the effects (e.g., decreased relative ovaries) that could not be modeled would be their respective NOAELs listed in Table A.l, which besides for relative ovary weight changes, are not lower than the BMDL values for splenic hemosiderosis and congestion. Specifically, a NOAEL of 2.1 mg/kg-day for a 17% decrease in relative ovary weights is identified from Table B. 12. After reviewing all of the modeling results listed in Appendix C, the most sensitive BMDLio is 11.51 mg/kg-day for increased incidence of splenic hemosiderosis in male mice. However, whereas the selection of this BMDLio from the splenic hemosiderosis data set as the POD would protect against both splenic congestion and organ-weight changes in male and female mice, it may not confer protection against the more sensitive endpoint of splenic hemosiderosis in female mice. The POD for increased incidence of splenic hemosiderosis in female mice is a NOAEL of 2.1 mg/kg-day. This NOAEL is identical to that indentified for decreased ovary weights in female mice. The selection of increased incidence of splenic hemosiderosis as the critical effect is supported by the observation that the spleen appears to be a target organ of diphenylamine toxicity. Specifically, not only did diphenylamine cause splenic hemosiderosis and congestion in both sexes of mice but also biologically and statistically significantly increased absolute and relative spleen weight in both sexes of mice as mentioned in the section "Review of Potentially Relevant Data." This information provides greater support for spleen being the target organ of diphenylamine toxicity rather than ovary, in which only relative ovary weights were affected by diphenylamine exposure with no accompanying histological data. Therefore, the NOAEL of 2.1 mg/kg-day based on increased incidence of splenic hemosiderosis in female mice (Botta, 1992) was chosen as the POD to derive a screening subchronic p-RfD. BMD input data for these splenic data are presented in Table B. 11. The curve and BMD results are provided in Appendix C. 45 Diphenylamine ------- FINAL 5-11-2011 Table A.l. Potential PODs from the Botta (1992) CD-I Mouse Study for Derivation of a Screening Subchronic p-RfD POD POD Critical Effect Sex Method (mg/kg-day) Comments Increased incidence of Male BMDL 14 splenic congestion Increased incidence of Male BMDL 12 splenic hemosiderosis Increased incidence of Female BMDL 14 splenic congestion Increased incidence of Female NOAEL 2.1 Data were not splenic hemosiderosis amenable to BMD modeling. Chosen as the POD. Decreased absolute gonad Male NOAEL 926 No statistically weights significant effect at any dose tested. Increased absolute kidney Male NOAEL 444 Data were not amenable weights to BMD modeling. Increased absolute liver Male BMDL 349 weight Increased absolute spleen Male NOAEL 94 Data were not amenable weight to BMD modeling. Decreased relative gonad Male NOAEL 926 No statistically weights significant effect at any dose tested. Increased relative kidney Male NOAEL 926 No biologically weights significant effect (>10% change) at any dose tested. Increased relative liver Male BMDL 331 weight Increased relative spleen Male NOAEL 94 Data were not amenable weight to BMD modeling. Decreased absolute ovary Female NOAEL 1101 No statistically weights significant effect at any dose tested. Increased absolute kidney Female NOAEL 1101 No biologically weights significant effect (>10% change) at any dose tested. Increased absolute liver Female NOAEL 555 Data were not amenable weight to BMD modeling. Decreased relative ovary Female NOAEL 2.1 Data were not amenable weights to BMD modeling. Increased absolute spleen Female NOAEL 107 Data were not amenable weight to BMD modeling. Increased relative kidney Female NOAEL 555 Data were not amenable weights to BMD modeling. 46 Diphenyl amine ------- FINAL 5-11-2011 Table A.l. Potential PODs from the Botta (1992) CD-I Mouse Study for Derivation of a Screening Subchronic p-RfD Critical Effect Sex POD Method POD (mg/kg-day) Comments Increased relative liver weight Female NOAEL 555 Data were not amenable to BMD modeling. Increased relative spleen weight Female NOAEL 555 Data were not amenable to BMD modeling. Adjusted points for daily exposure: The following dosimetric adjustments were made for each dose in the principal study for diet treatment in adjusting for daily exposure. Dosimetric adjustment for 2.1 mg/kg-day is presented below. (DOSEadj) = DOSEbotta, 1992 x [conversion to daily dose] = 2.1 mg/kg-day x (days of week dosed ^ 7) = 2.1 mg/kg-day x (7 -h 7) = 2.1 mg/kg-day The screening subchronic p-RfD for diphenylamine based on a NOAELadj of 2.1 mg/kg-day in female CD-I mice (Botta, 1992) is derived as follows: Screening Subchronic p-RfD = NOAELadj UF = 2.1 mg/kg-day ^ 100 = 2 x 10 2 mg/kg-day Table A.2 summarizes the uncertainty factors for the screening subchronic p-RfD for diphenylamine. 47 Diphenylamine ------- FINAL 5-11-2011 Table A.2. Uncertainty Factors for the Screening Subchronic p-RfD of Diphenylamine UF Value Justification UFa 10 A UFa of 10 is applied for interspecies extrapolation to account for potential toxicokinetic and toxicodynamic differences between mice and humans. There are no data to determine whether humans are more or less sensitive than mice in Botta (1992) to subchronic oral toxicity of diphenylamine. ufd 1 A UFd of 1 is applied because the database includes two acceptable developmental toxicity studies (Edwards et al., 1983b; Rodwell, 1992) via the oral exposure route. In addition, one acceptable two-generation reproductive toxicity study (Rodwell, 1993) via oral exposure was also located, lending support for a relatively complete database for diphenylamine. UFh 10 A UFh of 10 is applied for intraspecies differences to account for potentially susceptible individuals in the absence of information on the variability of response to humans. ufl 1 A UFl of 1 is applied for using a POD based on a NOAEL identified for increased incidence of splenic hemosiderosis in female mice from a subchronic-duration study by Botta (1992). UFS 1 A UFS of 1 is applied because results from a subchronic-duration study (Botta, 1992) were utilized as the principal study. UFC <3000 100 48 Diphenyl amine ------- FINAL 5-11-2011 APPENDIX B. DATA TABLES Table B.l. Average Hematological Values of Female Rats Following Dietary Exposure to Diphenylamine for 90 Daysa'b Endpoint Exposure Group (Average Daily Dose [mg/kg-day]) 0 0.3% (302) 1.0% (1007) Sample size 5 5 5 Hematocrit 50 46 (92%) 45 (90%) Hemoglobin (g/100 mL) 14.3 13.7 (96%) 12.1 (85%) Total leukocytes 19.68 18.33 (93%) 19.29 (98%) Differential count % Neutrophils 18.2 17.2 (95%) 13.8 (76%) % Lymphocytes 80.4 82.2 (102%) 85.4 (106%) % Monocytes 0.8 0.2 (25%) 0.6 (75%) % Eosinophils 0.6 0.4 (67%) 0.2 (33%) 'Dow Chemical Company (1958). bValues represent the mean (% of controls). 49 Diphenyl amine ------- FINAL 5-11-2011 Table B.2. Final Average Body Weight and Relative Organ Weights of Male and Female Rats Following Dietary Exposure to Diphenylamine for 90 Daysa'b Endpoint Exposure Group (Average Daily Dose [mg/kg-day]) Males 0 0.01% (9) 0.03% (27) 0.1% (90) 0.3% (271) 1.0% (902) Sample size 10 6 8 9 5 5 Necropsy body weight (g) 275 268 (97%) 269 (98%) 273 (99%) 256 (93%) 216 (79%)** Lung (g/100 g-bw) 0.61 0.61 (100%) 0.60 (98%) 0.55 (90%) 0.59 (97%) 0.66 (108%) Heart (g/100 g-bw) 0.34 0.36 (106%) 0.34 (100%) 0.38(112%) 0.37 (109%) 0.36 (106%) Liver (g/100 g-bw) 2.92 2.92 (100%) 2.88 (99%) 3.12 (107%)* 3.37 (115%)** 4.54 (155%)** Kidney (g/100 g-bw) 0.76 0.77 (101%) 0.77 (101%) 0.77 (101%) 0.76 (100%) 0.88 (116%)** Spleen (g/100 g-bw) 0.30 0.27 (90%) 0.28 (93%) 0.28 (93%) 0.28 (93%) 0.54 (180%)** Testes (g/100 g-bw) 0.94 1.00 (106%) 1.00 (106%) 1.02 (109%) 1.06 (113%) 1.19 (127%)** Females 0 0.01% (10) 0.03% (30) 0.1% (101) 0.3% (302) 1.0% (1007) Sample size 9 8 8 10 9 8 Necropsy body weight (g) 165 178 (108%) 172 (104%) 181 (110%) 162 (98%) 141 (85%)** Lung (g/100 g-bw) 0.75 0.78 (104%) 0.71 (95%) 0.74 (99%) 0.80 (107%) 0.80 (107%) Heart (g/100 g-bw) 0.41 0.38 (93%) 0.40 (98%) 0.39 (95%) 0.42 (102%) 0.43 (105%) Liver (g/100 g-bw) 2.93 3.13 (107%)* 3.22 (110%)** 3.22 (110%)** 3.73 (127%)** 4.60 (157%)** Kidney (g/100 g-bw) 0.82 0.80 (98%) 0.83 (101%) 0.80 (98%) 0.87 (106%) 1.02 (124%) Spleen (g/100 g-bw) 0.27 0.32(119%) 0.34 (126%)** 0.34 (126%)** 0.39 (144%)** 0.63 (233%)** aDow Chemical Company (1958). bValues represent the mean (% of controls). *p < 0.05, test name not provided. **p < 0.01, test name not provided. 50 Diphenyl amine ------- FINAL 5-11-2011 Table B.3. Average Final Body Weight and Food Consumption of Albino Rats Following Dietary Exposure to Diphenylamine for 226 Daysa'b Endpoint Exposure Group (Average Daily Dose [mg/kg-day]) 0 0.025% (21) 0.10% (82) 0.50% (410) 1.00% (820) 1.50% (1230) Sample size 6 6 6 6 6 6 Body weight (g) 233 226 (97%) 222 (95%) 207 (89%) 200 (86%) 174 (75%) Food intake (g/rat/day) 11.21 11.39 (102%) 11.26(100%) 10.37 (92.5%) 10.96 (98%) 11.45 (102%) aThomas et al. (1957). bValues represent the mean (% of controls). Table B.4. Survival and Food Consumption of Albino Male and Female Slonaker-Addis Rats Following Dietary Exposure to Diphenylamine for 2 Years" Endpoint Exposure Group (Average Daily Dose [mg/kg-day]) Males 0 0.001% (1) 0.01% (7) 0.10% (72) 0.50% (362) 1.00% (723) Survival13 Day 240 20 18 (90%) 18 (90%) 20 (100%) 17 (85%) 18 (90%) Day 640 13 12 (92%) 15 (115%) 15 (115%) 12 (92%) 13 (100%) Day 734 13 12 (92%) 15 (115%) 14 (108%) 12 (92%) 10 (77%) Food consumption (g/rat/day)c 11.65 11.47 (98%) 11.68(100%) 11.82(101%) 10.86* (93%) 9.99* (86%) Females 0 0.001% (1) 0.01% (8) 0.10% (82) 0.50% (410) 1.00% (820) Survival13 Day 240 20 17 (85%) 19 (95%) 19 (95%) 18 (90%) 16 (80%) Day 640 14 12 (86%) 18 (129%) 17 (121%) 12 (86%) 12 (86%) Day 734 11 9 (82%) 15 (136%) 11 (100%) 9 (82%) 11 (100%) Food consumption (g/rat/day)c 9.44 9.55 (101%) 9.57 (101%) 9.25 (98%) 8.77* (93%) 8.12* (86%) aThomas et al. (1967b). bValues represent number of survivors (% of control). 0Values represent the mean (% of control). *p < 0.01. Duncan method. 51 Diphenyl amine ------- FINAL 5-11-2011 Table B.5. Average Hematological Values of Albino Male and Female Slonaker-Addis Rats Following Dietary Exposure to Diphenylamine for 2 Yearsa'b Endpoint Exposure Group (Average Daily Dose [mg/kg-day]) Males 0 1.00% (723) Hemoglobin (g/100 mL) Day 126° 13.85 12.67 Day 182°'d 13.45 12.67 Day 230e 13.45 12.60 Day 267 13.95 13.65 Day 360 14.25 12.69 Day 463 14.70 13.66 Red Blood cells (105/mm3) Day 126° 9.30 8.39 Day 182°'d 8.96 8.04 Day 230e 9.23 8.50 Day 267 9.32 8.33 Day 360 8.49 7.58 Day 463 8.96 7.88 Females 0 1.00% (820) Hemoglobin (g/100 mL) Day 16 lf 14.24 14.19 Day 264 13.48 12.33 Day 371s 14.37 13.44 Day 463 14.05 13.20 Red cells (105/mm3) Day 16 lf 9.02 8.11 Day 264 8.81 - Day 371s 9.01 7.94 Day 463 8.29 8.37 "Thomas et al. (1967b). bValues represent the mean; n = 2 unless otherwise noted. °n = 3. dDay 177 eDay 231 f« = 4. gDay 369 for 1%-diphenylamine group, for 1%-diphenylamine group. for 1%-diphenylamine group. 52 Diphenyl amine ------- FINAL 5-11-2011 Table B.6. Urinary Tract Lesions of Albino Male and Female Slonaker-Addis Rats Following Dietary Exposure to Diphenylamine for 2 Years"' Endpoint Exposure Group (Average Daily Dose [mg/kg-day]) 0.001% 0.01% 0.1% 0.5% 1.0% Males 0 (1) (7) (72) (362) (723) Kidney Chronic nephritis 13 12 14 13 11 10 Tubular cysts 13 12 14 13 11 10 Bladder Epithelial hyperplasia or metaplasia 0 1 1 0 1 5 0.001% 0.01% 0.1% 0.5% 1.0% Females 0 (1) (8) (82) (410) (820) Kidney Chronic nephritis 10 10 14 15 10 8 Tubular cysts 10 10 14 15 10 8 Bladder Epithelial hyperplasia or metaplasia 1 0 0 0 0 1 aThomas et al. (1967b). bValues represent number of animals observed with lesions and include animals that exhibited negligible incidences of lesions. Animals that died before Day 640 were not autopsied. 53 Diphenyl amine ------- FINAL 5-11-2011 Table B.7. Benign and Malignant Tumor Incidence in Male and Female Albino Slonaker-Addis Rats Following Dietary Exposure to Diphenylamine for 2 Yearsa'b Endpoint Exposure Group (Average Daily Dose [mg/kg-day]) 0.001% 0.01% 0.1% 0.5% 1.0% Males 0 (1) (7) (72) (362) (723) Adrenal Medulla Adenomatous hyperplasia0 8 0 8 5 4 5 Adenocarcinoma - 1 1 - - - Cortical Adenocarcinoma - 1 - - - - Pheochromocytoma0 - 1 1 - - - Abdomen Lipoma0 1 - - 1 - - Mammary Adenofibroma0 1 2 1 0 0 0 Pituitary Adenoma0 1 - - - - - Thyroid Adenoma0 - - 1 - - - Liver Hemangioepithelioma - - - - - 1 Hepatoma - - - 1 - - Lung Lymphosarcoma 1 - - - - - Pancreas Adenocarcinoma - - - 1 - - 0.001% 0.01% 0.1% 0.5% 1.0% Females 0 (1) (8) (82) (410) (820) Adrenal Medulla Adenomatous hyperplasia0 5 3 2 6 1 3 Adenocarcinoma 1 - - - - - Mammary Adenofibroma0 2 5 6 5 2 0 Pituitary Adenoma0 - 1 3 1 1 1 Adenocarcinoma - 1 - - - - Lung Adenocarcinoma - - - - 1 - Uterus Leiomyoma0 - 1 - - - - Ovary Granulosa cell0 1 - - - - - Vulva Squamous cell carcinoma 1 - - - - - aThomas et al. (1967b). bValues represent number of animals observed with lesions; tumors were malignant unless otherwise noted. °Tumors were benign. 54 Diphenyl amine ------- FINAL 5-11-2011 Table B.8. Survival, Body Weight, Body Weight Change, and Food Consumption in Rabbits Dosed Orally with Diphenylamine from GDs 7-19a'b Endpoint Exposure Group (Average Daily Dose [mg/kg-day])c 0 33 100 300 Survival 13/16 14/16 15/18d 15/16 Body weight Day 1(g) 3421.9 3382.9 (99%) 3305.8 (97%) 3406.2 (100%) Body weight Day 29 (g) 4101.2 3962.9 (97%) 4065.0 (99%) 3963.2 (97%) Body-weight change on Day 29 relative to start of treatment on Day 7 (g) 488.9 405.8 (83%) 535.0 (109%) 389.4 (80%) Food consumption Days 1-6 (g/animal/day)e 200.7 199.3 (99%) 191.3 (95%) 193.8 (97%) Food consumption Days 7-8 204.5 201.5 (99%) 208.5 (102%) 166.5 (81%) Food consumption Days 9-10 194.5 193.0 (99%) 194.0 (100%) 163.5 (84%) Food consumption Days 11-14 195.5 184.0 (94%) 199.8 (102%) 161.5 (83%) Food consumption Days 15-19 210.2 174.0 (83%) 220.2 (105%) 170.6 (81%) Food consumption Days 20-23 199.8 178.8 (89%) 210.3 (105%) 171.8 (86%) Food consumption Days 24-28 184.6 156.6 (85%) 168.0 (91%) 162.4 (88%) aEdwards et al. (1983). bValues represent the mean (% of controls). °The study authors reported average daily dose as the exposure group in the study results, includes 2 animals culled prior to start of treatment and then replaced. eFood consumption means calculated for less than indicated number of animals when overt diet wastages recorded. 55 Diphenyl amine ------- FINAL 5-11-2011 Table B.9. Fetal Weight, Developmental Malformations, and Anomalies in Fetuses of Rabbits Dosed Orally with Diphenylamine from GDs 7-19a'b Endpoint Exposure Group (Average Daily Dose [mg/kg-day])c 0 33 100 300 Number fetuses/total litters examined 92/12 98/12 89/12 115/13 Mean fetal weight (g) 46.6 42.8 (92%) 43.2 (93%) 42.5 (91%) Mean % of fetuses with malformations 4.0 0.9 (23%) 1.0 (25%) 0.0 (0%) Number fetuses with malformations 2 1 (50%) 1 (50%) 0 (0%) Mean % of fetuses with anomalies in gross autopsy 0.8 1.7 (213%) 2.1 (263%) 1.6 (200%) Number fetuses with anomalies in gross autopsy 1 1 (100%) 2 (200%) 2 (200%) Mean % of fetuses with skeletal anomalies 20 16 (80%) 12 (60%) 13 (65%) Number fetuses with skeletal anomalies 22.8 17.9 (79%) 12.4 (54%) 11.8(52%) 'Edwards et al. (1983). Values represent the mean (% of controls). °The study authors reported average daily dose as the exposure group in the study results. 56 Diphenyl amine ------- FINAL 5-11-2011 Table B.10. Reproductive Data of Albino Male and Female Slonaker-Addis Rats Following Dietary Exposure to Diphenylaminea'b Endpoint Exposure Group (Average Daily Dose [mg/kg-day])c 0 0.1% (95) 0.25% (237) 0.5% (473) First Mating"1 At birth No. litters 12 10 10 10 No. pups per litter 8.3 9.0 6.8 6.3* At weaning No. litters 10 10 9 8 No. pups per litter 7.5 8.3 7.1 6.3 Weight at weaning (g) Males 29.6 31.9 29.9 26.9 Females 27.8 28.7 27.8 25.8 Second Mating® At birth No. litters 9 12 12 11 No. pups per litter 9.6 7.3* 7.3* 6.6** At weaning No. litters 9 11 12 11 No. pups per litter 9.3 7.1 7.3 6.6 Weight at weaning (g) Males 28.3 37.0 35.5 28.2 Females 27.5 36.0 34.4 28.2 Second Generationd At birth No. litters 10 11 12 11 No. pups per litter 8.6 5.8** 7.3 7.0 At weaning No. litters 10 11 12 11 No. pups per litter 8.5 5.7** 7.0 6.6 Weight at weaning (g) Males 31.0 31.1 30.0 24.9** Females 30.6 31.2 29.3 24.8** aThomas et al. (1967c). bValues represent the mean. 0 Adjusted Daily Doses shown as averages of values of males and females. dNumber of mated females = 12. "Number of mated females = 12, except in the 95-mg/kg-day group where number of females =11. *p < 0.05, Duncan method. **p < 0.01, Duncan method. 57 Diphenyl amine ------- FINAL 5-11-2011 Table B.ll. Incidence of Splenic Hemosiderosis and Congestion in the Mouse After Dietary Administration of Diphenylamine in a 90-Day Subchronic-Duration Toxicity Study" Adjusted Dose Group Number Incidence of Splenic Incidence of (mg/kg-day) of Mice Hemosiderosis Splenic Congestion Males 0 15 0 0 1.7 14 0 0 94 15 4 2 444 15 15* 15* 926 15 15* 15* Females 0 15 0 0 2.1 15 0 0 107 15 12* 6* 555 15 15* 14* 1101 15 15* 15* aValues obtained from Botta (1992). *p < 0.05 by Fisher's Exact Test. 58 Diphenyl amine ------- FINAL 5-11-2011 Table B.12. Relative and Absolute Organ Weights and Body Weight of Male and Female CD-I Mice Following Dietary Exposure to Diphenylamine for 90 Days" Endpoint Exposure Group (Adjusted Daily Dose [mg/kg-day]) Males 0 ppm 10 ppm (1.7) 525 ppm (94) 2625 ppm (444) 5250 ppm (926) Sample size 15 15 15 15 12 Necropsy body weight (g) 31.7±1.9 32.7±3.9 32.6±2.9 31.9±2.3 31.4±1.9 Absolute gonad weights (g) 0.401±0.057 0.374±0.084 0.405±0.055 0.394±0.046 0.395±0.043 Absolute kidney weights (g) 0.592±0.083 0.585±0.110 0.640±0.055 0.611±0.054 0.636±0.090 Absolute liver weight (g) 1.341±0.091 1.415±0.154 1.443±0.152 1.521±0.180** 1.669±0.173** Absolute spleen weight (g) 0.083±0.029 0.074±0.013 0.095±0.019 0.132±0.043** 0.242±0.062** Relative gonad weights (g/100 g-bw) 1.266±0.145 1.159±0.286 1.249±0.177 1.238±0.158 1.258±0.100 Relative kidney weights (g/100 g-bw) 1.869±0.211 1.808±0.343 1.973±0.179 1.918±0.165 2.024±0.244 Relative liver weight (g/100 g-bw) 4.248±0.340 4.349±0.348 4.441±0.404 4.765±0.393** 5.309±0.362** Relative spleen weight (g/100 g-bw) 0.262±0.086 0.229±0.043 0.292±0.060 0.416±0.144** 0.774±0.204** Females 0 ppm 10 ppm (2.1) 525 ppm (107) 2625 ppm (555) 5250 ppm (1101) Sample size 12 15 15 15 15 Necropsy body weight (g) 26.7±2.5 25.5±2.3 26.8±2.2 25.3±2.5* 25.7±1.5** Absolute ovary weights (g) 0.041±0.009 0.033±0.009 0.031±0.009 0.032±0.008 0.030±0.009 Absolute kidney weights (g) 0.419±0.033 0.414±0.040 0.416±0.051 0.419±0.041 0.459±0.060 Absolute liver weight (g) 1.251±0.162 1.159±0.112 1.159±0.127 1.229±0.175 1.502±0.153** Absolute spleen weight (g) 0.079±0.018 0.076±0.017 0.098±0.016 0.163±0.046** 0.276±0.088** Relative ovary weights (g/100 g-bw) 0.154±0.034 0.128±0.030 0.115±0.036** 0.129±0.030 0.116±0.031* Relative kidney weights (g/100 g-bw) 1.576±0.160 1.637±0.196 1.559±0.191 1.664±0.136 1.784±0.204* Relative liver weight (g/100 g-bw) 4.681±0.443 4.562±0.372 4.329±0.335 4.849±0.471 5.836±0.462** Relative spleen weight (g/100 g-bw) 0.295±0.055 0.297±0.054 0.367±0.062 0.642±0.176** 1.068±0.313** aBotta (1992); data are presented as means ± standard deviation. *p < 0.05 by Dunnet's test. **p < 0.01 by Dunnet's test. 59 Diphenyl amine ------- FINAL 5-11-2011 APPENDIX C. BENCHMARK DOSE CALCULATIONS FOR THE SCREENING SUBCHRONIC p-RfD Modeling Procedure for Dichotomous Data The BMD modeling of dichotomous data was conducted with EPA's BMDS (version 2.1.2). For these data, all of the dichotomous models (i.e., Gamma, Multistage, Logistic, Log-logistic, Probit, Log-probit, Weibull, and Quantal-linear models) available within the software were fit using a default BMR of 10% extra risk. Adequacy of model fit was judged based on the % goodness-of-fitp-value (p > 0.1), magnitude of scaled residuals in the vicinity of the BMR, and visual inspection of the model fit. Among all models providing adequate fit, the BMDL from the model with the lowest AIC was selected as a potential POD from which to derive the screening subchronic p-RfD. Modeling Procedure for Continuous Data The BMD modeling of continuous data was conducted with EPA's BMDS (version 2.1.2). For these data (e.g., increased relative liver weight), all continuous models available within the software were fit using a default BMR of 10% extra risk. An adequate fit was judged based on the % goodness-of-fit p-v alue (p> 0.1), magnitude of the scaled residuals in the vicinity of the BMR, and visual inspection of the model fit. In addition to these three criteria forjudging adequacy of model fit, a determination was made as to whether the variance across dose groups was homogeneous. If a homogeneous variance model was deemed appropriate based on the statistical test provided in BMDS (i.e., Test 2), the final BMD results were estimated from a homogeneous variance model. If the test for homogeneity of variance was rejected (p < 0.1), the model was run again while modeling the variance as a power function of the mean to account for this nonhomogeneous variance. If this nonhomogeneous variance model did not adequately fit the data (i.e., Test 3; p-v alue < 0.1), the data set was considered unsuitable for BMD modeling. Among all models providing adequate fit, the lowest BMDL was selected if the BMDLs estimated from different models varied greater than 3-fold; otherwise, the BMDL from the model with the lowest AIC was selected as a potential POD from which to derive the screening subchronic p-RfD. 60 Diphenyl amine ------- FINAL 5-11-2011 INCREASED INCIDENCE OF SPLENIC HEMOSIDEROSIS IN MALE CD-I MICE TREATED WITH DIPHENYLAMINE FOR 90 DAYS (Botta, 1992) All dichotomous models available in BMDS (version 2.1.2) were fit to the incidence data of splenic hemosiderosis in male CD-I mice treated with diphenylamine for 90 days (Botta, 1992; Table B.l 1). In the absence of a mechanistic understanding of the biological response to a toxic agent, data from exposures much higher than the study LOAEL do not provide reliable information regarding the shape of the response at low doses. Such exposures, however, can have a strong effect on the shape of the fitted model in the low-dose region of the dose-response curve. Thus, if the lack of fit is due to characteristics of the dose-response data for high doses, then the EPA Benchmark Dose Technical Guidance Document allows for data to be adjusted by eliminating the high-dose group (U.S. EPA, 2000, 052150). Because the focus of BMD analysis is on the low dose and response region, eliminating high-dose groups is deemed reasonable. For increased incidence of splenic hemosiderosis in male CD-I mice, all modeling results shown are without the highest dose groups being included in the analysis (see Table C.l and Figure C.l). As assessed by the % goodness-of-fit statistic and visual inspection, only the Quantal-linear model adequately fit the data (see Table C. 1 and Figure C. 1). Estimated doses associated with 10% extra risk and the 95% lower confidence limit on these doses (BMDio values and BMDLio values, respectively) were 18 and 12 mg/kg-day. Table C.l. BMD Dose-Response Modeling Results Based on the Increased Incidence of Splenic Hemosiderosis in Male CD-I Mice Treated with Diphenylamine for 90 Days Modeld %2/>-value AIC BMD10 BMDL10 Gamma3 1 21.398 75 25 Multistage13 0.9995 19.428 54 19 Logistic 1 21.398 88 49 Log-logistic0 1 21.398 86 41 Probit 1 21.398 82 44 Log-probitc 1 21.398 80 40 Weibulf 1 21.398 68 23 Quantal-linear 0.4172 23.581 18 12 "Restrict power >1. bRestrict betas >0; degree of polynomial = 2; lowest degree polynomial with an adequate fit reported. °Slope restricted to >1. dAll models besides Quantal-linear failed based on visual inspection due to S-shaped curves. 61 Diphenylamine ------- FINAL 5-11-2011 Quantal Linear Model with 0.95 Confidence Level Quantal Linear BMD Lower Bound 0.8 0.6 0.4 0.2 BMDL BMD 0 50 100 150 200 250 300 350 400 450 dose 13:35 11/01 2010 Figure C.l. Dose-Response Modeling for Increased Incidence of Splenic Hemosiderosis in Male CD-I Mice Treated with Diphenylamine for 90 Days 62 Diphenylamine ------- FINAL 5-11-2011 INCREASED INCIDENCE OF SPLENIC CONGESTION IN MALE CD-I MICE TREATED WITH DIPHENYLAMINE FOR 90 DAYS (Botta, 1992) All dichotomous models available in BMDS (version 2.1.2) were fit to the incidence data of splenic congestion in male CD-I mice treated with diphenylamine for 90 days (Botta, 1992; Table B. 11). For these data, all modeling results shown are without the highest dose groups being included in the analysis (see Table C.2 and Figure C.2). As assessed by the % goodness- of-fit statistic and visual inspection, only the Quantal-linear model adequately fit the data (see Table C.2 and Figure C.2). Estimated doses associated with 10% extra risk and the 95% lower confidence limit on these doses (BMDio values and BMDLio values, respectively) were 22 and 14 mg/kg-day. Table C.2. BMD Dose-Response Modeling Results Based on the Incidence of Splenic Congestion in Male CD-I Mice Treated with Diphenylamine for 90 Days Modeld %2 /j-value AIC BMD10 BMDL10 Gamma3 1 15.78 89 50 Multistage13 1 14.381 68 34 Logistic 1 15.78 92 62 Log-logistic0 1 15.78 92 61 Probit 1 115.78 91 57 Log-probitc 1 15.78 90 61 Weibulf 1 15.78 85 14 Quantal-linear 0.01362 21.801 22 14 "Restrict power >1. bRestrict betas >0; degree of polynomial = 2; lowest degree polynomial with an adequate fit reported. °Slope restricted to >1. dAll models besides Quantal-linear failed based on visual inspection due to S-shaped curves. 63 Diphenylamine ------- FINAL 5-11-2011 Quantal Linear Model with 0.95 Confidence Level Quantal Linear BMD Lower Bound 1 0.8 0.6 0.4 0.2 0 BMDL BMD 0 50 100 150 200 250 300 350 400 450 dose 13:39 11/01 2010 Figure C.2. Dose-Response Modeling for Increased Incidence of Splenic Congestion in Male CD-I Mice Treated with Diphenylamine for 90 Days 64 Diphenylamine ------- FINAL 5-11-2011 INCREASED INCIDENCE OF SPLENIC CONGESTION IN FEMALE CD-I MICE TREATED WITH DIPHENYLAMINE FOR 90 DAYS (Botta, 1992) All dichotomous models available in BMDS (version 2.1.2) were fit to the incidence data of splenic congestion in female CD-I mice treated with diphenylamine for 90 days (Botta, 1992; Table B. 11). For these data, all modeling results shown are without the highest dose groups being included in the analysis (see Table C.3 and Figure C.3). As assessed by the % goodness- of-fit statistic and visual inspection, the Gamma, Multistage, Weibull, and the Quantal-linear models adequately fit the data (see Table C.3 and Figure C.3). Of these models, the Quantal- linear model was the best fit based on the lowest AIC and provided BMDio values and BMDLio values of 22 and 14 mg/kg-day, respectively. Table C.3. BMD Dose-Response Modeling Results Based on the Incidence of Splenic Congestion in Female CD-I Mice Treated with Diphenylamine for 90 Days Modeld %2 /j-value AIC BMD10 BMDL10 Gamma3 0.9386 31.745 28 14 Multistage13 0.9276 31.832 23 14 Logistic 0.0374 39.753 83 51 Log-logistic0 0.9965 31.552 41 10 Probit 0.038 39.442 84 57 Log-probitc 1 31.538 41 24 Weibull3 0.933 31.783 26 14 Quantal-linear 0.9846 29.843 22 14 "Restrict power >1. bRestrict betas >0; degree of polynomial = 2; lowest degree polynomial with an adequate fit reported. °Slope restricted to >1. dAll models besides Gamma, Multistage, Weibull, Quantal-linear failed based on visual inspection due to S-shaped curves. 65 Diphenylamine ------- FINAL 5-11-2011 Quantal Linear Model with 0.95 Confidence Level Quantal Linear BMD Lower Bound 1 0.8 0.6 0.4 0.2 0 BMDL BMD 0 100 200 300 400 500 dose 13:42 11/01 2010 Figure C.3. Dose-Response Modeling for Increased Incidence of Splenic Congestion in Female CD-I Mice Treated with Diphenylamine for 90 Days 66 Diphenylamine ------- FINAL 5-11-2011 INCREASED RELATIVE LIVER WEIGHT OF MALE CD-I MICE TREATED WITH 1)1 I'lll \VI AMI\I FOR 90 DAYS (Botta, 1992) All available continuous models in BMDS (version 2.1.2) were fit to the increased relative liver-weight data from male CD-I mice exposed to diphenylamine for 90 days (Botta, 1992; Table B. 12). As assessed by the % goodness-of-fit statistic and visual inspection, the Linear and Power models provided the best fit models (see Table C.4 and Figure C.4). Estimated doses associated with 10% extra risk and the 95% lower confidence limit on these doses (BMDio values and BMDLio values, respectively) were 401 and 331 mg/kg-day. Table C.4. BMD Modeling Results on Increased Relative Liver Weight in Male CD-I Mice Exposed to Diphenylaminefor 90 Days Model Test 2 Test 3 X p-Value AIC BMD10 BMDL10 Males Linear 0.9522 0.9522 0.8569 -69.348 401 331 Polynomial 0.9522 0.9522 0.6834 -67.355 409 332 Power 0.9522 0.9522 0.8569 -69.348 401 331 Hill 0.9522 0.9522 0.6808 -67.348 401 331 67 Diphenylamine ------- FINAL 5-11-2011 Polynomial Model with 0.95 Confidence Level Polynomial 5.6 5.4 5.2 5 4.8 4.6 4.4 4.2 4 BMDL BMD 0 200 400 600 800 dose 08:40 03/01 2011 Figure C.4. Dose-Response Modeling for Increased Relative Liver Weight in Male CD-I Mice Treated with Diphenylamine for 90 Days 68 Diphenylamine ------- FINAL 5-11-2011 INCREASED ABSOLUTE LIVER WEIGHT OF MALE CD-I MICE TREATED WITH 1)1 I'lll \VI AMI\I FOR 90 DAYS (Botta, 1992) All available continuous models in BMDS (version 2.1.2) were fit to the increased absolute liver-weight data from male CD-I mice exposed to diphenylamine for 90 days (Botta, 1992; Table B.12). As assessed by the % goodness-of-fit statistic and visual inspection, the Linear, Power, and Polynomial models provided the best fit (see Table C.5 and Figure C.5). Estimated doses associated with 10% extra risk and the 95% lower confidence limit on these doses (BMDio values and BMDLio values, respectively) were 456 and 349 mg/kg-day. Table C.5. BMD Modeling Results on Increased Absolute Liver Weight in Male CD-I Mice Exposed to Diphenylamine for 90 Days Model Test 2 Test 3 X p-Value AIC BMD10 BMDL10 Males Linear 0.13 0.13 0.4815 -195.704 456 349 Polynomial 0.13 0.13 0.4815 -195.704 456 349 Power 0.13 0.13 0.4815 -195.704 456 349 Hill 0.13 0.13 0.2989 -193.754 422 169 69 Diphenylamine ------- FINAL 5-11-2011 Polynomial Model with 0.95 Confidence Level Polynomial 1.8 1.7 1.6 1.5 1.4 1.3 BMDL BMD 0 200 400 600 800 dose 08:53 03/01 2011 Figure C.5. 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