United States Environmental Protection 1=1 m m Agency EPA/690/R-12/022F Final 11-05-2012 Provisional Peer-Reviewed Toxicity Values for Nitrofen (CASRN 1836-75-5) 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 Chris Cubbison, PhD National Center for Environmental Assessment, Cincinnati, OH DRAFT DOCUMENT PREPARED BY ICF International 9300 Lee Highway Fairfax, VA 22031 PRIMARY INTERNAL REVIEWERS Ghazi Dannan, PhD National Center for Environmental Assessment, Washington, DC Dan D. Petersen, PhD, 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). li Nitrofen ------- TABLE OF CONTENTS TABLE OF CONTENTS iii COMMONLY USED ABBREVIATIONS iv BACKGROUND 1 DISCLAIMERS 1 QUESTIONS REGARDING PPRTVs 1 INTRODUCTION 2 REVIEW OF POTENTIALLY RELEVANT DATA (CANCER AND NONCANCER) 3 HUMAN STUDIES 9 Oral Exposures 9 Inhalation Exposures 9 Other Exposures 9 ANIMAL STUDIES 9 Oral Exposures 9 Inhalation Exposures 32 OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS) 32 DERIVATION 01 PROVISIONAL VALUES 38 DERIVATION OF ORAL REFERENCE DOSES 38 Derivation of Subchronic Provisional RfD (Subchronic p-RfD) 38 Derivation of Chronic Provisional RfD (Chronic p-RfD) 40 DERIVATION OF INHALATION REFERENCE CONCENTRATIONS 42 CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR 42 DERIVATION OF PROVISIONAL CANCER POTENCY VALUES 43 Derivation of Provisional Oral Slope Factor (p-OSF) 43 Derivation of Provisional Inhalation Unit Risk (p-IUR) 44 APPENDIX A. PROVISIONAL SCREENING VALUES 45 APPENDIX B. DATA TABLES 46 APPENDIX C. BMD OUTPUTS 57 APPENDIX D. REFERENCES 65 in Nitrofen ------- COMMONLY USED ABBREVIATIONS BMC benchmark concentration BMCL benchmark concentration lower bound 95% confidence interval BMD benchmark dose 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 POD point of departure p-OSF provisional oral slope factor p-RfC provisional reference concentration (inhalation) p-RfD provisional reference dose (oral) 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 iv Nitrofen ------- FINAL 11-5-2012 PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR NITROFEN (CASRN 1836-75-5) 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 flittp://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 (http://www.epa.eov/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 Nitrofen ------- FINAL 11-5-2012 INTRODUCTION Nitrofen is a selective pre- and postemergent herbicide that is currently banned in the United States. All products were voluntarily cancelled in the United States by the manufacturer on September 15, 1983 (U.S. EPA, 1998). The empirical formula for nitrofen is C12H7CI2NO3 (see Figure 1). A table of physicochemical properties is provided below (see Table 1). In this document, "statistically significant" denotes a/rvalue of <0.05. ci^q—0—^ y—no2 CI Figure 1. Nitrofen Structure Table 1. Physicochemical Properties Table (Nitrofen)3 Property (Unit) Value Boiling point (°C at 0.25 mm Hg)b 180-190 Melting point (°C)b 70-71 Density (g/cm3 at 83°C) 1.80 Vapor pressure (mPa at 40°C)b 1.06 pH (unitless) Not available Solubility in water (mg/L at 22°C) 0.7-1.2 Relative vapor density (air =1) Not available Molecular weight (g/mol) 284.1 Flash point (°C)° >200 Octanol/water partition coefficient (log Kow unitless)b 5.534 aWHO, 1996 (unless otherwise noted). hHSDB, 2011. CIPCS, 1999. No Reference Dose (RfD), Reference Concentration (RfC), or cancer assessment for nitrofen is included in the IRIS database (U.S. EPA, 2012) or on the Drinking Water Standards and Health Advisories List (U.S. EPA, 2006). No RfD or RfC values were reported in the HEAST (U.S. EPA, 1997). The CARA list (U.S. EPA, 1991a, 1994a) does not include a Health and Environmental Effects Profile (HEEP) for nitrofen. The toxicity of nitrofen has not been reviewed by ATSDR (2009) or the World Health Organization (WHO, 2011). CalEPA (2007) has derived a no-significant-risk level of 9 (j,g/day for exposure to nitrofen. No occupational exposure limits for nitrofen have been derived by the American Conference of Governmental Industrial Hygienists (ACGIH, 2011), the National Institute of Occupational Safety and Health (NIOSH, 2010), or the Occupational Safety and Health Administration (OSHA, 2006). 2 Nitrofen ------- FINAL 11-5-2012 The HEAST (U.S. EPA, 1997) did not report a cancer weight-of-evidence classification or an oral slope factor for nitrofen. The International Agency for Research on Cancer (IARC; 2011) states that there is "sufficient evidence" that technical grade nitrofen is carcinogenic to animals and classifies nitrofen in Group 2B (Possibly Carcinogenic to Humans). The 12th Report on Carcinogens (NTP, 2011) states that nitrofen is "Reasonably Anticipated to be a Human Carcinogen" based on "Sufficient Evidence of Carcinogenicity in Experimental "3 Animals." CalEPA (2008) has derived an inhalation unit risk of 0.000023 per (J,g/m , an inhalation slope factor of 0.082 per mg/kg-day, and an oral slope factor of 0.082 per mg/kg-day based on the carcinogenic potential for nitrofen. Literature searches were conducted on sources published from 1900 through November 2011, for studies relevant to the derivation of provisional toxicity values for nitrofen, CAS No. 1836-75-5. 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 health-related values: ACGM, AT SDR, CalEPA, EPA IRIS, EPA HEAST, EPA HEEP, EPA OW, EPA TSCATS/TSCATS2, NIOSH, NTP, OSHA, and RTECS. REVIEW OF POTENTIALLY RELEVANT DATA (CANCER AND NONCANCER) Table 2 provides an overview of the relevant database for nitrofen and includes all potentially relevant repeated short-term-, subchronic-, and chronic-duration studies. Principal studies are identified. The phrase "statistical significance" used throughout the document indicates ap-value of <0.05. 3 Nitrofen ------- FINAL 11-5-2012 Table 2. Summary of Potentially Relevant Data for Nitrofen (CASRN 1836-75-5) Category Number of Male/Female, Strain Species, Study Type, Study Duration Dosimetry3 Critical Effectsb NOAELa BMDL/ BMCLa LOAEL' Reference (Comments) Notes'1 Human 1. Oral (mg/kg-day) None 2. Inhalation (mg/m ) None Animal 1. Oral (mg/kg-day)a Subchronic 25/0 per dose, Sprague-Dawley rat, diet, 7 days/week, 13 weeks 10/10 per dose, Wistar-derived rat, diet, 7 days/week, 13 weeks 0, 7, 37, 186 (Adjusted) Male: 0, 9, 46, 230, 1152, 4608 (Adjusted) Female: 0, 10, 51,256, 1282, 5128 (Adjusted) Decreased body-weight gain in males at 186 mg/kg-day; clinical chemistry changes (decreased glucose and increased total protein, albumin, globulin, and cholesterol) at 186 mg/kg-day in males; Increased relative weights of the male liver, kidneys, and testes at >37 mg/kg-day; histopathological effects in male liver at >37 mg/kg-day (number of animals affected not reported) Increased mortality in males at >1152 mg/kg-day and in females at >1282 mg/kg-day; decreased body weight in males at >1152 mg/kg-day and in females at 1282 mg/kg-day; increased relative liver weight in males at >46 mg/kg-day and in females at all doses; increased relative kidney weight in males at >230 mg/kg-day and in females at 1282 mg/kg-day; increased relative heart and spleen weight in males at 1152 mg/kg-day and in females (heart only) at 1282 mg/kg-day; increased relative testes weight at >230 mg/kg-day; histopathological liver effects in males at >1152 mg/kg-day and females at >1282 mg/kg-day None Not run Not run 37 10 O'Hara et al. (1983) Ambrose et al. (1971a) 4 Nitrofen ------- FINAL 11-5-2012 Table 2. Summary of Potentially Relevant Data for Nitrofen (CASRN 1836-75-5) Category Number of Male/Female, Strain Species, Study Type, Study Duration Dosimetry3 Critical Effectsb NOAELa'c BMDL/ BMCLa LOAELac Reference (Comments) Notes'1 5/5 per dose, Osborne-Mendel rat, diet, 7 days/week, 6 weeks followed by 2 weeks observation Male: 0, 66, 117, 207, 369, 656 (Adjusted) Female: 0, 71, 126, 224, 398, 709 (Adjusted) Decreased body-weight gain in males at >207 mg/kg-day and in females at >126 mg/kg-day 71 Not run 126 NCI (1978a) noncancer results 5/5 per dose, Fischer F344 rat, diet, 7 days/week, 4 weeks followed by 2 weeks observation Male: 0, 453, 667, 978, 1437, 2102 (Adjusted) Female: 0, 512, 753, 1104, 1623, 2373 (Adjusted) Decreased body-weight gain in both males and females at all doses; increased observation of arched backs and decreased survival in high-dose males and females None Not run 453 NCI (1979a) noncancer results 5/5 per dose, B6C3FJ mouse, diet, 7 days/week, 6 weeks followed by 2 weeks observation Male: 0, 241, 428, 760, 1353, 2408 (Adjusted) Female: 0, 260, 462, 822, 1463, 2605 (Adjusted) Decreased body-weight gain in males at 428 and 760 mg/kg-day and in females at 462 and 822 mg/kg-day; dose-dependent increase in mortality (data not reported) Not derived Not run Not derived NCI (1978b) noncancer results 5/5 per dose, B6C3Fi mouse, diet, 7 days/week, 4 weeks followed by 2 weeks observation Male: 0, 142, 307, 661, 1672, 3069 (Adjusted) Female: 0, 153, 332,715, 1808, 3320 (Adjusted) Mortality at highest dose for both males and females; rough hair and arched backs in males at >1672 mg/kg-day and in females at >1808 mg/kg-day; mottled kidneys in high- dose females 661 Not run 1672 NCI (1979b) noncancer results 1/1 per dose, mongrel dog, diet, 7 days/week, 4 weeks Male: 0, 169, 421, 1053 (Adjusted) Female: 0, 123, 308, 771 (Adjusted) Decreased food consumption and body weight in all dogs (severity of decrease not known, as data not reported) Not derived Not run Not derived Ambrose et al. (1971b) 5 Nitrofen ------- FINAL 11-5-2012 Table 2. Summary of Potentially Relevant Data for Nitrofen (CASRN 1836-75-5) Category Number of Male/Female, Strain Species, Study Type, Study Duration Dosimetry" Critical Effectsb NOAELa'c BMDL/ BMCLa LOAELac Reference (Comments) Notes'1 Chronic 25/25 per dose, Wistar-derived albino rat, diet, 7 days/week, 97 weeks Male: 0, 1.09, 11.5, 116 (Adjusted) Female: 0, 1.17, 12.2, 127 (Adjusted) Poor survival in males and females at all doses (including controls); increased relative kidney and liver weight in males at 116 mg/kg-day; decreased relative splenic weight in females at 127 mg/kg-day Not derived Not run Not derived Ambrose et al. (1971c) 2/2 per dose, purebred beagle dog, diet, 7 days/week, 2 years 0,0.36,3.9,38 (Adjusted) Increased relative liver weights 3.9 Not run 38 Ambrose et al. (1971d) 50/50 per dose (20/20 in controls), Fischer F344 rat, diet, 7 days/week, 78 weeks with an additional 26 weeks of untreated observation Male: 0,51.75, 109.23 (HED) Female: 0, 52.03, 108.85 (HED) None 105.85 Not run None NCI (1979c) Noncancer results 50/50 per dose (20/20 in controls), Osborne- Mendel rat, diet, 7 days/week, 78 weeks with 32 additional weeks of untreated observation (4 weeks in high-dose males) Male: 0,31.38, 70.35 (HED) Female: 0, 21.88, 46.97 (HED) Decreased survival in males and females at all doses None Not run 21.88 NCI (1978c) Noncancer results Reproductive and Developmental6 0/13 (8 at highest dose), Sprague-Dawley CD rat, gavage, GDs 8-16, offspring observed through PNDs 133-161 0, 0.46,1.39, 4.17,12.5 Diaphragmatic hernias in pups at >1.39 mg/kg-day; hyperactivity (transient) on PNDs 17 and 24 at >1.39 mg/kg-day (returned to normal by PND 45); decreased Harderian gland weight at >4.17 mg/kg-day; hydronephrosis at >4.17 mg/kg-day; decreased pup survival at >4.17 mg/kg-day on PNDs 1,2, and 6 0.46 0.29 1.39 Ostby et al. (1985) PS, PR 6 Nitrofen ------- FINAL 11-5-2012 Table 2. Summary of Potentially Relevant Data for Nitrofen (CASRN 1836-75-5) Category Number of Male/Female, Strain Species, Study Type, Study Duration Dosimetry" Critical Effectsb NOAELa'c BMDL/ BMCLa LOAELac Reference (Comments) Notes'1 Carcinogenic 50/50 per dose (20/20 in controls), Osborne- Mendel rat, diet, 7 days/week, 78 weeks with 32 additional weeks of untreated observation (4 weeks in high-dose males) Male: 0,31.38, 70.35 (HED) Female: 0, 21.88, 46.97 (HED) Pancreatic carcinoma, lymphoma, ovarian granulose cell tumor in females at 46.97 mg/kg-day NA 24.1 NA NCI (1978c) 50/50 per dose (20/20 in controls), Fischer F344 rat, diet, 7 days/week, 78 weeks with an additional 26 weeks of untreated observation Male: 0,51.75, 109.23 (HED) Female: 0, 52.03, 108.85 (HED) None NA Not run NA NCI (1979c) 50/50 per dose (20/20 in controls), B6C3Fi mouse, diet, 7 days/week, 78 weeks with an additional 12 weeks of untreated observation Male: 0,60.70, 128.26 (HED) Female: 0, 65.38,137.68 (HED) Hepatocellular carcinomas in male and female mice at all doses; hemangiosarcomas in males at 128.26 mg/kg-day NA 2.6 NA NCI (1978d) PS 50/50 per dose (20/20 in controls), B6C3F, mouse, diet, 7 days/week, 78 weeks with an additional 13 weeks of untreated observation Male: 0, 70.66, 147.03 (HED) Female: 0, 76.47, 160.09 (HED) Hepatocellular adenomas and carcinomas (combined) in males at >70.66 mg/kg-day; hepatocellular carcinomas in males at >70.66 mg/kg-day; hepatocellular adenomas and carcinomas (combined) in females at >76.47 mg/kg-day; hepatocellular carcinomas in females at 160.09 mg/kg-day NA 6.7 NA NCI (1979d) 7 Nitrofen ------- FINAL 11-5-2012 Table 2. Summary of Potentially Relevant Data for Nitrofen (CASRN 1836-75-5) Number of Male/Female, Strain Species, Study Type, BMDL/ Reference Category Study Duration Dosimetry3 Critical Effectsb NOAELa'c BMCLa LOAEL"* (Comments) Notes'1 2. Inhalation (mg/m3) None aDosimetry: NOAEL, BMDL/BMCL, and LOAEL values are converted to an adjusted daily dose (ADD in mg/kg-day) for oral noncancer effects. Values for oral carcinogenic effects are converted to a human equivalent dose (HED in mg/kg-day). All long-term exposure values (4 wk and longer) are converted from a discontinuous to a continuous (weekly) exposure. Values from animal developmental studies are not adjusted to a continuous exposure. Adjusted daily dose (ADD) = dose (ppm) x food consumption per day x (l -f- body weight) x (days dosed total days) (Ambrose et al., 1971a,b,c,d) Human equivalent dose (HED) = dose (ppm) x food consumption per day x (1 -f- body weight) x (days dosed ^ total days) x (body weight animal ^ body weight human)0 25 (NCI, 1978c,d, 1979c,d). bFor studies with a BMDL listed, the critical effect used as the POD is listed first. °NOAEL and LOAEL values are determined from the data by the PPRTV authors. dNotes: IRIS = utilized by IRIS, date of last update; PR =peer reviewed; PS = principal study; NPR = not peer reviewed; NA = not applicable. "Additional reproductive and developmental studies are summarized in Table 3. 8 Nitrofen ------- FINAL 11-5-2012 HUMAN STUDIES Oral Exposures No studies were identified. Inhalation Exposures No studies were identified. Other Exposures Studies evaluating the effects of nitrofen following dermal exposure are reported in the literature in a foreign language and are summarized in the review provided by Burke Hurt et al. (1983). According to these studies, skin and eye irritation were the only effects related to dermal exposure by humans to nitrofen. ANIMAL STUDIES Oral Exposures The effects of oral exposure of animals to nitrofen have been evaluated in 7 sub chronic-duration (Ambrose et al., 1971a,b; NCI, 1978a,b, 1979a,b; O'Hara et al., 1983; see Table 2), 2 chronic-duration (Ambrose et al., 1971c,d; see Table 2), 44 reproductive and developmental (see Table 3), and 4 carcinogenic (NCI, 1978c,d, 1979c,d) studies. Subchronic-duration Studies O'Hara et al. (1983) conducted a published, peer-reviewed subchronic-duration oral study in which male Sprague-Dawley (COBS-CD(S-D)BR) rats were administered nitrofen (95.7% purity, impurities unknown) for 95 days before mating with untreated females. The study also evaluated male reproductive effects. Compliance with good laboratory practice (GLP) is unknown. Males received 0-, 100-, 500-, or 2500-ppm nitrofen in their food for 95 days prior to cohabitation and then 0-, 6-, 30-, or 155-mg/kg nitrofen in corn oil in 10-mL/kg volumes via gavage during cohabitation with females, up to 10 days until mating. Control rats were fed normal diet or a gavage of corn oil only. The study authors reported the averaged daily doses of nitrofen were 0, 7, 37, and 186 mg/kg-day before mating and during cohabitation. Both sexes were of equal age and were obtained from Charles River Breeding Farms (Kingston, NY). Unmated male and female rats were housed in stainless steel cages with wire mesh floors and fronts. After mating, suspected pregnant female rats were placed in larger cages (17"W x 10"D x 7.5"H) lined with bedding. Cages were inspected daily for births after 20 days of gestation. Environmental conditions were described by a temperature of 23 ± 2°C, relative humidity of 50 ± 15%, and a 12-hour light/12-hour dark cycle. Animals were fed Purina Rodent Laboratory Chow (No. 5001-Meal) and water ad libitum. Researchers observed male rats twice daily to monitor for any ill health, morbidity, mortality, or reactions to nitrofen treatment (O'Hara et al., 1983). Male rats were weighed 1 week before administering the first dietary nitrofen dose and then weighed every week for the first 13 weeks (91 days) of the 95-day premating period. Mean body weights and food consumption were averaged weekly within dosed male study groups. After a maximum of 10 days of cohabitation with females, male rats were fasted overnight, and blood was removed from the orbital sinus for hematological and chemical analyses. Male rats were then sacrificed by exsanguination, necropsied, and examined thoroughly for gross abnormalities. Male kidneys, liver, and testes were weighed and then reported as percentage of total body weight. These organs were then fixed in 10%-buffered formalin, sectioned in 5-[j.m slices, and stained with 9 Nitrofen ------- FINAL 11-5-2012 hematoxylin and eosin for histopathological examination. Pregnant females were observed at different days of gestation, and offspring were observed twice daily for 13 weeks after birth. Pregnant dams were weighed on gestation days (GDs) 0, 6, 10, 16, and 21 and on lactation days (LDs) 0, 4, 10, 15, and 21. Three days after their expected delivery date, females were sacrificed and then grossly examined for lesions in their abdominal region and for the number of implantation sites present in their uterine horns. Subchronic data were analyzed using Sheffe's Multiple Range Test. No effect on food consumption was observed in males from any dose group (O'Hara et al., 1983). At the highest dose, a decrease in body-weight gain in males was seen throughout the first 13 weeks of exposure to nitrofen. Males exposed to 2500-ppm (186-mg/kg-day) nitrofen for 15 weeks exhibited statistically significant differences in blood glucose (86% of control value), total protein (113% of control value), albumin (111% of control value), globulin (116% of control value), and cholesterol levels (215% of control value) (see Table B. 1). Male organ weights were increased in the liver (absolute—2500 ppm [186 mg/kg-day]; relative—>500 ppm [37 mg/kg-day]), kidneys (relative—>500 ppm [37 mg/kg-day]), and the testes (absolute and relative—>500 ppm [37 mg/kg-day]) (see Table B.2). Gross necropsy revealed enlarged (6/25) and darkened (3/25) livers at 2500 ppm (186 mg/kg-day). The same dose resulted in slight-to-marked hydrotrophy of the liver and increased cytoplasmic basophilia of the male centribular hepatocytes (no further details reported). At 500 ppm (37 mg/kg-day), slight-to-very slight effects were seen in the hepatocytes of males (no further details reported). While histopathological data are discussed and pictures provided to illustrate the effects, no effort was made by the study authors to quantify the histopathological effects of nitrofen to male livers. No effects on gestation, fertility, litter size, weight, or sex ratio were seen in any dose group. Based on increased relative weights of the male liver, kidney, and testes, a LOAEL of 500 ppm (37 mg/kg-day) and a NOAEL of 100 ppm (7 mg/kg-day) are assigned for subchronic-duration oral exposure to nitrofen. In a published subchronic-duration study, Ambrose et al. (1971a) investigated the oral toxicity of nitrofen (95% purity) in Wistar-derived albino rats. Impurities consisted of 3% p-chloronitrobenzene, 1% dichlorophenol, and 1% unknown. It is unclear whether the study was conducted in compliance with GLP. The study authors administered 0-, 100-, 500-, 2500-, 12,500-, or 50,000-ppm nitrofen in commercial Purina Laboratory Chow to groups of 10 male and 10 female rats ad libitum for 13 weeks. Adjusted daily doses are 0, 9, 46, 230, 1152, and 4608 mg/kg-day for males and 0, 10, 51, 256, 1282, and 5128 mg/kg-day for females. Animals were obtained from Albino Farms in Red Bank, NJ. The study authors measured animal weights weekly and food consumption over 3 days at the end of the first month of exposure and at study termination. At study termination, the study authors performed hematological examinations and urinary analyses on five rats/sex/dose. Additionally, at study termination, the study authors weighed the heart, spleen, kidneys, liver, and testes of surviving rats and performed histopathological examinations on the bladder, lung, small and large intestines, stomach, pancreas, adrenals, brain, pituitary, thyroid, bone marrow, skeletal muscle, skin, heart, spleen, kidneys, liver, and testes. The statistical methods were not reported. All rats administered 50,000-ppm (4608-mg/kg-day males; 5128-mg/kg-day females) nitrofen experienced mortality in the first week (Ambrose et al., 1971a). For rats administered 12,500-ppm nitrofen (1152-mg/kg-day males; 1282-mg/kg-day females), 4/10 males and 4/10 females experienced mortality before study termination. No significant mortality or 10 Nitrofen ------- FINAL 11-5-2012 changes in food consumption or growth were observed in rats administered 0-, 100- (9-mg/kg-day males; 10-mg/kg-day females), or 500-ppm nitrofen (46-mg/kg-day males; 51-mg/kg-day females). Significantly reduced body weight was observed in male and female rats administered 12,500-ppm (1152-mg/kg-day males; 1282-mg/kg-day females) nitrofen and male rats administered 2500-ppm nitrofen (230-mg/kg-day). Values from hematological examinations and urinary analyses were not significantly different from the control. Organ weights following exposure to nitrofen are reported (see Table B.3). A dose-dependent increase in relative liver weight was observed in both sexes at all dose groups, except for male rats administered 100-ppm nitrofen (9-mg/kg-day males; 10-mg/kg-day females). Female rats administered 12,500-ppm nitrofen (1282-mg/kg-day) had increased relative organ weights for the heart and kidney. Males administered 12,500-ppm nitrofen (1152-mg/kg-day) had significantly increased relative organ weights for the heart, spleen, kidney, liver, and testes. Males from the 2500-ppm dose group (230-mg/kg-day) had significantly increased relative organ weights for the liver, kidney, and testes. Edema, glycogen granules located on cell peripheries, cytoplasmic swelling, and liver nuclei possessing prominent nucleoli were observed in male and female rats administered >12,500-ppm (1152-mg/kg-day males; 1282-mg/kg-day females) nitrofen. Based on increased relative liver weights in females, a LOAEL of 100 ppm (10 mg/kg-day) is determined; due to effects being seen at all doses in females, aNOAEL is precluded. The National Cancer Institute (NCI) (1978a) performed a peer-reviewed subchronic-duration study to investigate the effects of nitrofen in rats. It is unclear whether the studies are GLP compliant. Technical-grade nitrofen (with a manufacturer's estimated purity of 87% and gas-liquid chromatography-estimated purity of greater than 80%) was administered in the feed to Osborne-Mendel rats. A total of at least five impurities were detected and consisted of xylene, dichlorphenol, p-chloronitrobenzene, and multiple chloronitrodiphenyl ethers. The percentages of the impurities were not reported. The rats were obtained from the Battelle Memorial Institute (Columbus, OH). The basal laboratory diet for all animals consisted of 2% Duke's® corn oil added to Wayne Lab-Blox® meal (Allied Mills, Inc.). Food and water were supplied ad libitum. For the duration of the experiment, rats were individually housed in steel and wire-mesh cages. Temperature was maintained at 20-24°C, and relative humidity was maintained at 45—55%. Twelve-hour fluorescent light/dark cycles were provided. All animals were weighed immediately prior to the start of the experiment. The subchronic toxicity test was initially performed in rats to help determine the maximum tolerated dose of nitrofen that would be administered in the chronic-duration study (NCI, 1978a). Nitrofen mixed with a small amount of corn oil was administered in the feed to Osborne-Mendel rats (5/sex/dose group) at concentrations of 0, 1000, 1780, 3160, 5620, or 10,000 ppm. Adjusted daily doses are calculated using default data for body weight (U.S. EPA, 1994b) and food consumption (U.S. EPA, 1988) and are 0, 66, 117, 207, 369, and 656 mg/kg-day for males and 0, 71, 126, 224, 398, and 709 mg/kg-day for females. Test diets were administered for 6 weeks; after dosing, there was a 2-week observation period in which all test animals were fed the basal diet. To select the initial high doses for the chronic-duration study, the study authors examined two criteria—mortality and retardation in body-weight gain (expressed as a percentage of the weight gain of the control animals); no other endpoints were evaluated. No mortality was observed in any test group. Body-weight gain retardation was 10% and 25% in the 3160-ppm (207 mg/kg-day) and 5620-ppm (369 mg/kg-day) groups, respectively, for males, and 17% and 26% in the 1780-ppm (126 mg/kg-day) and 3160-ppm groups 11 Nitrofen ------- FINAL 11-5-2012 (224 mg/kg-day), respectively, for females. Effects on body-weight gain at 10,000 ppm (656 mg/kg-day) in males and at >5620 ppm (398 mg/kg-day) in females were not reported. However, it is assumed that nitrofen had an effect on body-weight gain at these levels as well as the concentrations estimated to induce 20% body-weight gain retardation were selected as the initial high doses for the chronic-duration study—4600 ppm for males and 2600 ppm for females. Based on decreased body-weight gain in female rats, a LOAEL of 1780 ppm (126 mg/kg-day) and aNOAEL of 1000 ppm (71 mg/kg-day) are determined. NCI (1979a) performed an additional peer-reviewed sub chronic-duration study to investigate the effects of nitrofen in a different strain of rats. It is unclear whether the study is GLP compliant. Technical-grade nitrofen (purity not specified) was administered in feed to Fischer F344 rats. The rats (4 weeks old) were obtained from A. R. Schmidt (Madison, WI) and Laboratory Supply Company, Inc. (Indianapolis, IN). The basal laboratory diet for all animals consisted of Wayne Lab-Blox® meal (Allied Mills, Inc.). Food and acidulated water (pH 2.5) were provided ad libitum. Animals were grouped and distributed among cages, where the average body weight per cage was approximately equal for the particular species and sex. Animals were housed by sex in groups of four in polycarbonate cages suspended from aluminum racks. Temperature was maintained at 22-26°C, and relative humidity was maintained at 45-55%. Fluorescent light was provided for 8 hours per day. All animals were weighed immediately prior to the start of the experiment. Subchronic toxicity tests were initially performed in rats to help determine concentrations of nitrofen that would be administered in the chronic-duration studies (NCI, 1979a). Nitrofen was administered in the feed to Fischer F344 rats (5/sex/dose group) at concentrations of 0, 6800, 10,000, 14,670, 21,560, or 31,530 ppm. Adjusted daily doses are calculated using default data for body weight (U.S. EPA, 1994b) and food consumption (U.S. EPA, 1988) and are 0, 453, 667, 978, 1437, and 2102 mg/kg-day in males and 0, 512, 753, 1104, 1623, and 2373 mg/kg-day in females. Test diets were administered for a total of 4 weeks; after dosing, there was a 2-week observation period in which all test animals were fed the basal diet. Two times per week, individual body weights and food consumption data were reported. At the end of the study, animals were euthanized and necropsied. The study gives no indication that a histopathological examination was performed. No statistical tests were reported. Table B.4 shows survival, mean body-weight change, and incidence of arched back in rats following exposure to nitrofen (NCI, 1979a). The results indicated decreased body-weight gain in both sexes at all doses. An increase in arched backs and a decrease in survival in males and females of the highest dose group were observed compared to controls. No other endpoints were reported. For this study, a LOAEL of 6800 ppm (453 mg/kg-day) is determined based on decreased body-weight in male rats. Because treatment-related effects were seen at the lowest dose, derivation of a NOAEL is not feasible. NCI (1978b) evaluated the effects of subchronic-duration exposure to nitrofen in mice. Purity and GLP compliance are the same as reported in NCI (1978a). Experimental design was identical to that reported for NCI (1978a) with a few exceptions. B6C3Fi mice were obtained from Charles River Breeding Laboratories, Inc. (Wilmington, MA). Mice were housed by sex in groups of 10 in polypropylene cages with solid bottoms and filter tops. Nitrofen was mixed with a small amount of corn oil and administered in the feed to mice (5/sex/dose group) at concentrations of 0, 1780, 3160, 5620, 10,000, or 17,800 ppm. Adjusted daily doses are 12 Nitrofen ------- FINAL 11-5-2012 calculated using default data for body weight (U.S. EPA, 1994b) and food consumption (U.S. EPA, 1988) and are 0, 241, 428, 760, 1353, and 2408 mg/kg-day for males and 0, 260, 462, 822, 1463, and 2605 mg/kg-day for females. The study authors reported retardation in body-weight gain at concentrations of 3160 and above (428 mg/kg-day males; 462 mg/kg-day females) but noted that observations were not clearly dose related (NCI, 1978b). In the 3160 ppm group (428 mg/kg-day males; 462 mg/kg-day females), males experienced a body-weight-gain reduction of 12%, while females showed an 8%-reduction. At 5620 ppm (760 mg/kg-day males; 822 mg/kg-day females), body-weight-gain reduction increased to 37% in males and 40% in females. No further results for body weight were reported. In both sexes, mortality increased with concentration (data not reported). No other endpoints were reported. For males and females, the study authors selected the initial high dose for the chronic-duration study as 3550 ppm. Because complete data for body weight and mortality were not available and the study authors noted that effects on body weight were not clearly dose related, it is not feasible to develop a LOAEL or NOAEL for this study. NCI (1979b) conducted an additional study evaluating the effects of subchronic-duration exposure to nitrofen in mice. GLP compliance and compound purity are unknown. Experimental design was identical to that reported for NCI (1979a) with some exceptions. B6C3Fi mice (4 weeks old) were obtained from Charles River Breeding Laboratories, Inc. (Wilmington, MA). Animals were housed by sex in groups of five in polycarbonate cages suspended from aluminum racks. Mice were exposed to 0-, 1180-, 2550-, 5500-, 13,900-, or 25,520-ppm nitrofen in the feed. Adjusted daily doses are calculated using default data for body weight (U.S. EPA, 1994b) and food consumption (U.S. EPA, 1988) and are 0, 142, 307, 661, 1672, and 3069 mg/kg-day for males and 0, 153, 332, 715, 1808, and 3320 mg/kg-day for females. Table B.5 provides survival, mean body-weight change, and observations of abnormal clinical signs in mice following exposure to nitrofen (NCI, 1979b). Survival was decreased in the highest dose group for both sexes. Abnormal clinical signs were observed in all animals of both sexes at the two highest doses (13,900 [1672 mg/kg-day males; 1808 mg/kg-day females] and 25,520 ppm [3069 mg/kg-day males; 3320 mg/kg-day females]). Males and females in these groups had arched backs and rough hair. Females in the 25,520 ppm group (3320 mg/kg-day) also experienced mottled kidneys. Mean body-weight change results were comparable to controls for both sexes at all doses. No other effects were reported. For this study, the LOAEL and NOAEL are 13,900 ppm (1672 mg/kg-day) and 5500 ppm (661 mg/kg-day), respectively, for observations of abnormal clinical signs in male mice. Ambrose et al. (1971b), in a published subchronic-duration study, investigated the oral toxicity of nitrofen (95% purity) in mongrel dogs. Impurities consisted of />chloronitrobenzene (3%>), dichlorophenol (1%), and unknowns (1%). Dogs were obtained from Medical College of Virginia, Central Animals Facilities. It is unknown whether the study was conducted in compliance with GLP. The study authors administered 0-, 4000-, 10,000-, or 25,000 ppm nitrofen in basal diet to 1 male and 1 female dog per dose for 4 weeks. Adjusted daily doses are calculated using default data for body weight (U.S. EPA, 1994b) and food consumption (U.S. EPA, 1988) and are 0, 169, 421, and 1053 mg/kg-day for males and 0, 123, 308, and 771 mg/kg-day for females. The basic diet consisted of 87% ground basal diet, 12% corn oil, 13 Nitrofen ------- FINAL 11-5-2012 and 1% USP cod liver oil. The study authors measured animal weights weekly and food consumption daily. Decreases in food consumption and body weight were observed in all dogs administered nitrofen in basal diet (data not reported). Due to small sample sizes and the absence of data for critical effects, neither a LOAEL nor a NOAEL can be determined for this study. Chronic-duration Studies Parallel to the subchronic-duration study in rats, Ambrose et al. (1971c) published a chronic-duration study investigating the oral toxicity of nitrofen (95% purity) in Wistar-derived albino rats. Impurities consisted of />chloronitrobenzene (3%), dichlorophenol (1%), and unknown (1%). It is unknown whether the study was conducted in compliance with GLP. The study authors administered 0-, 10-, 100-, or 1000 ppm nitrofen in commercial Purina Laboratory Chow to groups of 25 male and 25 female rats ad libitum for 97 weeks. Adjusted daily doses are calculated by averaging the body-weight data measured at intervals (1, 3, 6, 13, 26, 52, 78, and 96 weeks) for each dose level in the study and using default data for food consumption (U.S. EPA, 1988). These values are 0, 1.09, 11.5, and 116 mg/kg-day for males and 0, 1.17, 12.2, and 127 mg/kg-day for females. Animals were obtained from Albino Farms in Red Bank, NJ. The study authors measured animal weights weekly and food consumption over 3 days at the end of Months 1, 3, 6, and 12. The study authors performed hematological examinations and urinary analyses on 5 rats/sex/dose every 3 months. Additionally, at study termination, the study authors performed histological examinations; weighed the heart, spleen, kidneys, liver, and testes of surviving rats; and calculated organ-to-body weight ratios. The statistical methods were not reported. Poor survival was observed in male and female rats in all dose groups, including controls, after 65 weeks, forcing the study to end at 97 weeks (see Table B.6). No explanation was given for the decreased rate of survival. Significantly reduced growth was observed irregularly in male and female rats administered 100- (11.5-mg/kg-day males; 12.2-mg/kg-day females) and 1000-ppm (116-mg/kg-day males; 127-mg/kg-day females) nitrofen at weekly intervals throughout the study (see Table B.6). No significant changes in food consumption were observed. The study authors noted that values from hematological examinations and urinary analyses were not significantly different from the control (data not reported). Male rats administered 1000-ppm (116-mg/kg-day) nitrofen had increased organ-to-body-weight ratios in the kidney and liver (see Table B.7). Decreased relative splenic weight was seen in females at 1000 ppm (127 mg/kg-day) (see Table B.7). No histopathological findings in any of the dose groups differed from those found in the control groups (data not reported). Given the low rate of survival seen at all doses, derivation of a NOAEL or LOAEL is not feasible. NCI (1978c) conducted a study to evaluate the effects of nitrofen in the Osborne-Mendel rat. GLP compliance is unknown. Technical-grade nitrofen (with a manufacturer's estimated purity of 87% and gas-liquid chromatography estimated purity of greater than 80%) was administered in the feed to Osborne-Mendel rats. A total of at least five impurities were detected and consisted of xylene, dichlorphenol, />chloronitrobenzene, and multiple chloronitrodiphenyl ethers. The percentages of the impurities were not reported. The rats were obtained from the Battelle Memorial Institute (Columbus, OH). The basal laboratory diet for all animals consisted of 2% Duke's® corn oil added to Wayne Lab-Blox® meal (Allied Mills, Inc.). Food and water were supplied ad libitum. For the duration of the experiment, rats were individually housed in 14 Nitrofen ------- FINAL 11-5-2012 steel and wire-mesh cages. Temperature was maintained at 20-24°C, and relative humidity was maintained at 45-55%. Twelve-hour fluorescent light/dark cycles were provided. Nitrofen was administered at concentrations of 0 (20/sex); initial low and high concentrations of 2300 ppm and 4600 ppm, respectively, for males (50/dose); and initial low and high concentrations of 1300 ppm and 2600 ppm, respectively, for females (50/dose) (NCI, 1978c). Rats were treated for 78 weeks. At Week 46, study authors determined that the male high-dose rats were not tolerating 4600 ppm, so the dose was decreased to 2300 ppm (resulting in a time-weighted average of 3627 ppm over the 78 weeks). Adjusted daily doses (HEDs) are calculated using body-weight data as reported by the study authors and default data for food consumption (U.S. EPA, 1988) and are 0, 105 (31.38), and 241 (70.35) mg/kg-day for males and 0, 83 (21.88), and 183 (46.97) mg/kg-day for females. Control and low-dose males and all female rats were observed (untreated; fed basal diet and corn oil mixture) for an additional 32 weeks. Male high-dose rats were observed (untreated) for 4 weeks after treatment before being sacrificed at Week 83 of the study. All animals were observed daily for mortality (NCI, 1978c). Study authors recorded body weights, food consumption, and data regarding appearance, behavior, signs of toxic effects, and incidence, size, and location of tissue masses on a weekly basis for the first 10 weeks, then once per month for the remainder of the study. This study focused primarily on the possible carcinogenic potential of nitrofen in rats and did not report organ weights or results for serum or clinical chemistry. Observation and palpation were used to find tissue masses. All animals in the study were necropsied. Study authors performed gross and microscopic examination of major tissues, organs, and gross lesions. Microscopic examination was completed for the following: skin, subcutaneous tissue, lungs and bronchi, trachea, bone marrow, spleen, lymph nodes, thymus, heart, salivary gland, liver, bile duct, pancreas, esophagus, stomach, small and large intestines, kidney, urinary bladder, pituitary, adrenal, thyroid, parathyroid, pancreatic islets, testis, prostate, brain, muscle, uterus, mammary gland, and ovary. Time-adjusted analysis was used on animals that survived at least 52 weeks, or earlier, if the first tumor was found before this time (NCI, 1978c). Animals with early mortality and no tumors were excluded. Life-table methods were used when necessary to examine tumor incidence. Using the exact interval on the odds ratio, study authors calculated the 95% confidence interval for the relative risk of each dose group compared to the control. In male and female rats, the study authors reported a dose-related depression of body-weight gain throughout the treatment period (NCI, 1978c). In general, through the first 10 weeks of treatment, animal appearance and behavior were normal; however, hunched appearance, abdominal urine stains, and labored respiration did occur intermittently. From Week 14 onward, the number of animals with a hunched appearance increased with time. At the end of treatment at 78 weeks, 75% of low-dose and 95% of high-dose animals exhibited a hunched appearance. Other clinical observations included urine stains and bloody vaginal discharge in females, which was noted most consistently in the last 3 months of the study. Labored respiration and other respiratory signs (at low-to-moderate frequency) occurred in all groups, particularly during the second year of the study. Decreased survival was seen in both males and females at all doses. In male rats, 45% of controls, 60% of low-dose, and 30% of high-dose animals survived until the end of the study. By Week 45, about 50% of high-dose males were dead. The study authors concluded that this precluded meaningful statistical analysis to evaluate the occurrence of late-developing tumors in this group. In females, 80% of controls, 15 Nitrofen ------- FINAL 11-5-2012 74% of low-dose, and 56% of high-dose animals survived until the end of the study. The study authors noted that adequate survival was seen in all female groups to evaluate tumor development. The incidence of primary neoplasms in female rats treated with nitrofen is summarized in Table B.8 (NCI, 1978c). In females, long-term dietary exposure to nitrofen was associated with highly invasive neoplasms of the pancreas (2/50 rats or 4%, in the low-dose group, and 7/50 or 14%), in the high-dose group). Most of the pancreatic tumors appeared to be ductal carcinomas that were highly anaplastic and poorly differentiated and were also associated with marked desmoplasia, ischemic necrosis, inflammation, and hemorrhage. Invasion of the abdominal cavity was also observed, and all metastasized to the lung (data not shown). Although few animals suffered from neoplasms in the pancreas, study authors noted that these types of neoplasms are rare for this strain of rat, so their occurrence is likely related to nitrofen administration. Various tumors affecting the reproductive system, including the vagina, uterus, and ovary, were also observed in female rats. The study authors reported that vaginal and uterine carcinomas are unusual neoplasms in Osborne-Mendel rats. High-dose female rats also developed an increase in lymphomas (see Table B.8). No other treatment-related neoplasms were observed. In male rats, nitrofen intake was related to a life-shortening effect, especially in high-dose males (NCI, 1978c). The primary effect of treatment in high-dose males was massive hemorrhage of genitalia and the pelvic cavity. In addition, males of the high-dose group experienced massive centrilobular necrosis in the liver. There was also a high incidence of pneumonia in the low-dose males, which may have been increased by stress. Although carcinogenicity was not observed in males, the study authors noted that the high rate of early mortality may have precluded males from displaying carcinogenic responses. Study authors concluded that under the study conditions, dietary administration of nitrofen was carcinogenic to the female rat pancreas and reproductive system and that carcinogenicity in male rats could not be adequately determined. For the chronic-duration study in Fischer F344 rats (6 weeks old), NCI (1979c) administered nitrofen in the diet for 78 weeks. GLP compliance is unknown. Technical-grade nitrofen (purity not specified) was administered in feed to Fischer F344 rats. The rats (4 weeks old) were obtained from A. R. Schmidt (Madison, Wisconsin) and Laboratory Supply Company, Inc. (Indianapolis, IN). The basal laboratory diet for all animals consisted of Wayne Lab-Blox® meal (Allied Mills, Inc.). Food and acidulated water (pH 2.5) were provided ad libitum. Animals were grouped and distributed among cages where the average body weight per cage was approximately equal for the particular species and sex. Animals were housed by sex in groups of four in polycarbonate cages suspended from aluminum racks. Temperature was maintained at 22-26°C, and relative humidity was maintained at 45-55%). Fluorescent light was provided for 8 hours per day. All animals were weighed immediately prior to the start of the experiment. Nitrofen was administered in the feed at a concentration of 0 (control; 20/sex) and low and high concentrations of 3000 ppm and 6000 ppm, respectively, for males and females (50/sex/dose) (NCI, 1979c). Adjusted daily doses (HEDs) are calculated using body-weight data reported by the study authors and default food consumption data (U.S. EPA, 1988) and are 0, 195 (51.75), and 419 (109.23) mg/kg-day for males and 0, 221 (52.03), and 470 (108.85) mg/kg-day for females. All treated animals were dosed for 78 weeks. Following the treatment 16 Nitrofen ------- FINAL 11-5-2012 period, rats were observed (untreated) for an additional 26 weeks. Control animals received the basal laboratory diet for the total 104 weeks. All animals were weighed before the study and at monthly intervals throughout the duration (NCI, 1979c). They were examined two times a day, and food consumption was monitored in 20% of each dose group on a monthly basis. This study was designed to evaluate nitrofen for possible carcinogenicity and did not evaluate organ weights or serum or clinical chemistry endpoints. At the end of the treatment period, animals were euthanized and necropsied. Gross and microscopic examinations were performed on all major tissues, organs, and gross lesions. The following tissues were also microscopically examined: skin, subcutaneous tissue, lungs and bronchi, trachea, bone marrow, spleen, lymph nodes, thymus, heart, salivary gland, liver, pancreas, esophagus, stomach, small and large intestine, kidney, urinary bladder, pituitary, adrenal, thyroid, parathyroid, testis, prostate, brain, uterus, mammary gland, and ovary. Some animals, due to early death or cannibalization, did not have all organs examined. Time-adjusted analysis was used on animals that survived at least 52 weeks, or earlier, if the first tumor was found before this time. Animals with early mortality and no tumors were excluded. Life-table methods were used when necessary to examine tumor incidence. Using the exact interval on the odds ratio, study authors calculated the 95% confidence interval for the relative risk of each dose group compared to the control. For the chronic testing results in Fischer F344 rats, study authors reported dose-related mean body-weight depression in male rats until Week 75 and in female rats throughout the study (NCI, 1979c). However, the data were only reported as line graphs with no data points. Furthermore, the appearance of the female body-weight graphic suggests that values were comparable to controls at the end of the study. No clinical observations were reported. For survival, results of Tarone's test for dose-related mortality were not significant for males or females. There was no significant excess in neoplastic or nonneoplastic lesions in treated rats when compared to control animals. Therefore, the NOAEL for noncancer effects in this study is 6000 ppm (108.85adj,hed mg/kg-day; derivation of a LOAEL is precluded. NCI (1978d) is selected as the principal study for deriving the oral slope factor (p-OSF). NCI (1978d) conducted a chronic-duration study in B6C3Fi mice in which nitrofen was administered in the feed. Compound purity and GLP compliance are as reported in NCI (1978c). Experimental design was identical to that reported for NCI (1978c) with some exceptions. B6C3Fi mice were obtained from Charles River Breeding Laboratories, Inc. (Wilmington, MA). Mice were housed by sex in groups of 10 in polypropylene cages with solid bottoms and filter tops. As with the rat bioassay (NCI, 1978c), this study focused on the ability of nitrofen to form neoplastic lesions in mice. Nitrofen was administered at a concentration of 0 (control; 20/sex) and initial low and high concentrations of 1775 ppm and 3550 ppm, respectively, for males and females (50/sex/dose). Mice were treated for 78 weeks. Following the treatment period, mice were observed (untreated; fed basal diet and corn oil mixture) for an additional 12 weeks. During Week 7, after appearing to tolerate the initial low and high doses, the low dose was increased from 1775 ppm to 2000 ppm, and the high dose was increased from 3550 ppm to 4000 ppm. During Week 22, the low dose was increased from 2000 ppm to 2500 ppm, and the high dose was increased from 4000 ppm to 5000 ppm. These final concentrations were maintained for the remainder of the 78-week treatment period. The resulting time-weighted average concentrations for the low- and high-dose groups over the 78-week treatment period were 2348 ppm and 4696 ppm, respectively. Human equivalent daily 17 Nitrofen ------- FINAL 11-5-2012 doses (HEDs) are calculated using body-weight data provided by the study authors and default data for food consumption (U.S. EPA, 1988) and are 0, 418 (60.70), and 899 (128.26) mg/kg-day for males and 0, 469 (65.38), and 1004 (137.68) mg/kg-day for females. In male mice, no effects on weight gain were seen compared to controls (NCI, 1978d). Female mice displayed a dose-related depression in body-weight gain (data provided in the form of a line graph). In general, throughout the first year of the study, animal appearance and behavior were comparable among treated and control groups; however, beginning in Week 54 and through the end of the study, treated mice, particularly the females, showed pronounced bloating/abdominal distension (data not reported). Necropsy of these animals later revealed liver tumors determined to be hepatocellular carcinomas. In female mice, nitrofen did not have any effects on survival. In male mice, survivability was unusually low in male controls. The study authors noted that no common cause for the decrease in the survival rate was available. Only 10% of the control group males survived until the end of the study, compared to 54% and 34% in the low- and high-dose groups, respectively. In females, 85% of controls, 54% of low-dose, and 62%) of high-dose animals survived. The study authors concluded that survival was considered adequate to evaluate the carcinogenicity of nitrofen in the female mice. The incidence of neoplasms in male and female mice treated with nitrofen is summarized in Table B.9 (NCI, 1978d). In both males and females, long-term dietary exposure to nitrofen was associated with a high occurrence of hepatocellular carcinomas at all treatment levels, and results were statistically significant for low- (36/49, 73%) and high-dose (46/48, 96%) groups. Tumors were generally well differentiated, and most were confined to the liver, but a few metastasized. The occurrence of fibromas or fibrosarcomas in male mice was significant for low-dose males; however, given that these tumors were not observed in the high-dose males, the study authors were uncertain whether the occurrence of these tumors was treatment related. NCI (1979d) further explored the chronic-duration/carcinogenic effects of nitrofen in B6C3Fi mice. GLP compliance is unknown, and compound purity was not reported. Study methods are the same as reported in NCI (1979c) with some exceptions. Mice (4 weeks old) were obtained from Charles River Breeding Laboratories, Inc. (Wilmington, MA). Animals were housed by sex in groups of five in polycarbonate cages suspended from aluminum racks. Similar to the study conducted in rats (NCI, 1979d), this study focused on the possible carcinogenicity of nitrofen and did not report organ weights or serum or clinical chemistry. For the chronic-duration study in B6C3Fi mice (6 weeks old), nitrofen was administered in the feed at a concentration of 0 (control; 20/sex) and low and high concentrations of 3000 ppm and 6000 ppm, respectively, for males and females (50/sex/dose). Adjusted daily doses (HEDs) are calculated using body-weight data provided by the study authors and default data for food consumption (U.S. EPA, 1988) and are 0, 473 (70.66), and 998 (147.03) mg/kg-day for males and 0, 534 (76.47), and 1136 (160.09) mg/kg-day for females. All treated animals were dosed for 78 weeks. Following the treatment period, rats were observed (untreated) for an additional 13 weeks. Control animals received the basal laboratory diet for the total 91 weeks. NCI (1979d) reported dose-related mean body-weight depression in both male and female mice throughout the study (data were provided in graphical form only and did not include data points). No clinical observations were reported. For survival, results of Tarone's test for dose-related mortality were not significant for males or females. In males, 95% (19/20) of controls, 96% (48/50) of low-dose animals, and 80% (40/50) of high-dose animals survived until 18 Nitrofen ------- FINAL 11-5-2012 the end of the study. In females, 60% (12/20) of controls, 86% (43/50) of low-dose animals, and 96%) (48/50) of high-dose animals survived until the end of the study. The study authors reported treatment-related incidences of liver neoplasms and hyperplasias (see Table B.10). There were significant positive dose-related trends in incidences of hepatocellular carcinomas and adenomas in both sexes of mice. These carcinomas had areas of prominent trabecular formations and hepatocytes that formed in cords that were several cells thick. Hepatocellular adenomas were expansile lesions. Livers with hyperplasia consisted of single or multiple foci in which hepatocytes (and their nuclei) were enlarged. The cytoplasm was described as abundant and frequently vacuolated. Bile duct carcinomas were characterized by small, elongated cells consisting of scanty cytoplasm and dark nuclei. Treatment-related neoplasms were not seen in any other tissues. Under the conditions of the study, the study authors concluded that dietary administration of nitrofen is carcinogenic to the liver of B6C3Fi mice, causing hepatocellular carcinomas in males and females. Ambrose et al. (1971d), in a published chronic-duration study, investigated the oral toxicity of nitrofen (95% purity) in 6-month-old purebred beagle dogs. Impurities consisted of 3% /;-chloronitrobenzene, 1% dichlorophenol, and 1% unknown. It is unknown whether the study was conducted in compliance with GLP. The study authors administered 0-, 20-, 200-, or 2000-ppm nitrofen in basal diet to two dogs/sex/dose for 2 years. Adjusted daily doses are calculated using terminal body-weight data provided in the study (Ambrose et al., 197Id) and default food consumption data (U.S. EPA, 1988) and are 0, 0.36, 3.9, and 38 mg/kg-day. The basic diet consisted of 87% ground basal diet, 12% corn oil, and 1% USP cod liver oil. Dogs were immunized against distemper, infectious hepatitis, and leptospirosis, and were treated for intestinal parasites. The study authors measured animal weights weekly and food consumption daily. The study authors conducted hematological examinations and urinary analyses at the beginning of the study and every 3 months thereafter. At study termination, the study authors tested for bromosulfalein (BSP) retention, serum glutamic-oxaloacetic acid transaminase (SGOT), serum alkaline phosphatase (SAP), and blood urea nitrogen (BUN). Additionally, at study termination, the study authors performed histological examinations; weighed the heart, spleen, kidneys, liver, and testes of surviving dogs; and calculated organ-to-body weight ratios. The statistical methods were not reported. No changes in mortality, food consumption, or growth were observed in any of the test animals (Ambrose et al., 1971d). Values from hematological examinations and urinary analyses were not significantly different from the control. BSP, SGOT, SAP, and BUN values indicated no adverse effects. Dogs administered 2000-ppm nitrofen had significantly increased liver-to-body weight ratios (see Table B.l 1). No histopathological findings in any of the dose groups differed from those found in the control groups. A LOAEL of 2000 ppm (38 mg/kg-day) and aNOAEL of 200 ppm (3.9 mg/kg-day) are determined based on increased relative liver weights in combined males and females. Reproductive and Developmental Studies A total of 44 assays covering four species of animals (rats, mice, rabbits, and hamsters) have been performed to evaluate the reproductive and developmental effects of nitrofen. No reproductive effects were seen following exposure to the nitrofen. Administration of nitrofen to pregnant animals resulted in a number of developmental effects, with the most prevalent being neonatal lethality, diaphragmatic hernias, renal defects, and malformation of the Harderian gland. The rat was determined to be most sensitive species. Table 3 provides a summary of the 19 Nitrofen ------- FINAL 11-5-2012 available literature concerning the reproductive and developmental effects of nitrofen. The selected principal study is included in Tables 2 and 3 and summarized below. The study by Ostby et al. (1985) is selected as the principal study for deriving the subchronic and chronic p-RfDs. Ostby et al. (1985) published a study examining the developmental effects of prenatal exposure to nitrofen in the Sprague-Dawley CD rat. Rats were obtained from Charles River Breeding Laboratory in Wilmington, MA, and were provided Purina Rodent Laboratory Chow and water ad libitum. Study authors did not report whether the study was conducted in compliance with GLP guidelines. Nitrofen was recrystallized from technical grade to a purity of 99.59% and administered in 0.2-mL corn oil by gavage to female 90-day-old pregnant Sprague-Dawley CD rats at doses of 0, 0.46, 1.39, 4.17, or 12.5 mg/kg-day on GDs 8-16. Dosing solutions were prepared based on the average weight of all rats on GD 7. Dams were weighed on GDs 7 and 17. Any dams that had not delivered by GD 24 were necropsied. Dead pups found on postnatal day (PND) 0 (the day of birth) were necropsied for cause of death. Numbers of pups per litter and litter weights were recorded on PNDs 0, 1,2, and 6. Percentages of eyes open on PND 16 were calculated. Two blocks of behavioral tests were performed in which the offspring were run through activity mazes for 1 hour, and the number of photocell beam interruptions per individual was recorded (Ostby et al., 1985). In the first block, 86 pups (20 control, -16 per dose group) were tested on PNDs 17 and 24; 64 pups (22 control, -10 per dose group) were tested on PNDs 45 and 49; and 32 pups (8 control, -7 per dose group) were tested on PND 90. Each dose group contained an equal number of males and females for the first two tests; only male pups were tested in the last test. Block 2 differed from the first block in that it only tested 24 controls and 16 rats from the 12.5-mg/kg-day dose group on PNDs 17 and 24. Pups were weaned on PND 29 (Ostby et al., 1985). Females were checked daily from PNDs 32-44 for age at vaginal opening and first estrus. On PND 54, offspring were placed into nonlittermate pairs and mated. Females were allowed to deliver three litters, and number of pups per litter was recorded. Males were sacrificed by asphyxiation and necropsied on PNDs 133-161. Weights of the body, seminal vesicles, testes, liver, right kidney, lungs, and Harderian glands were recorded. Kidneys were examined for hydronephrosis and kidney cortex diameter. Eyes were examined for the presence of porphyrin rings. Data were evaluated using analysis of variance in SAS. Significant effects were then tested using Mests and analyzed using linear regression. The unit of analysis for all preweanling data was the litter mean, and the unit of analysis for all postweanling data was the individual animal. No effects were observed on maternal viability or weight change from GDs 7-17 at any dose level (Ostby et al., 1985). Examination of dams that had not delivered by GD 24 revealed one case of resorbed pups at the 4.17-mg/kg-day dose level. One female delivered only dead pups at the 4.17-mg/kg-day dose level. The number of pups per litter on PND 0 was significantly reduced at the 0.46- and 4.17-mg/kg-day dose levels, and the number of live pups on PNDs 1, 2, and 6 was statistically significantly reduced at the 0.46-, 4.17-, and 12.5 mg/kg day dose levels. Necropsy of pups found dead shortly after birth revealed that most of the dead pups at 1.39 mg/kg-day (3/4, 75%), 4.17 mg/kg-day (2/3, 67%), and 12.5 mg/kg-day (5/5, 100%) had diaphragmatic hernias (see Table B. 12). The percentages of litters containing pups with diaphragmatic hernias were increased at 1.39 (3/11, 27%), 4.17 (2/10, 20%), and 12.5 mg/kg-day (3/7, 43%) (see Table B.12). This increase did not reach statistical significance 20 Nitrofen ------- FINAL 11-5-2012 but is deemed biologically relevant as a number of other studies have reported statistically increased numbers of diaphragmatic hernias in rat and mouse litters at higher dose levels following gestational exposure to nitrofen (see Table 3). Furthermore, given that most of the pups found dead on Day 0 had diaphragmatic hernias, it appears that this malformation may be the primary cause of early neonatal mortality following exposure to low levels of nitrofen. No effects were seen on average pup weight on PND 1 or 2 (see Table B. 12). No delay in eye opening was observed in pups on PND 16. Results of the behavioral locomotor tests are shown in Table B.13. Pups in the 1.39-, 4.17-, and 12.5-mg/kg-day dose groups were hyperactive compared to controls on PNDs 17 and 24. However, these effects were transient because no difference in locomotor activity was observed in tests performed on PNDs 45, 49, or 90. No significant difference in the age at vaginal opening and first estrus between treated females and controls was observed, and no effects were seen in the difference of litter size over three cycles between treated and control groups. Results of the necropsy performed on adult offspring on PNDs 133-161 are presented in Table B.14. Weight of Harderian glands was significantly reduced at the 4.17- and 12.5-mg/kg-day dose levels, and porphyrin rings (indicative of chromodacryorrhea) were observed in 13 individuals in the 12.5-mg/kg-day dose group. Severe cases of hydronephrotic kidneys were observed in animals treated with 4.17 (3 cases) and 12.5 mg/kg-day (6 cases), while no instances were observed in controls or lower dose groups. No effects were observed in body weight, weight of testes, seminal vesicle weight, liver weight, lung weight, right kidney weight, or kidney cortex diameter (data not shown). Based on data for number of litters with pups having diaphragmatic hernias, the NOAEL and LOAEL are 0.46 and 1.39 mg/kg-day, respectively. 21 Nitrofen ------- FINAL 11-5-2012 Table 3. Summary of Oral Reproductive and Developmental Studies for Nitrofen (CASRN 1836-75-5) Study Type, Number of Male/Female, Strain Species, Route of Administration, Study Duration Dosimetry, Purity of Nitrofen" Critical Effects NOAELb LOAELb Reference Rat Developmental, 0/13 (8 at highest dose), Sprague-Dawley CD rat, gavage, dosed on GDs 8-16, offspring observed until PND 133-161, then necropsied 0,0.46, 1.39,4.17, or 12.5 mg/kg-day, purity 99.59% Decreased live pups/litter at birth at PNDs 1, 2, and 6 at >0.46 mg/kg-day; diaphragmatic hernias found in pups that died immediately after birth at 1.39- (3/4), 4.17- (2/3), and 12.5- (5/5) mg/kg-day dose levels; increased percentage of litters containing pups with diaphragmatic hernias at 1.39 (3/11, 27%), 4.17 (2/10, 20%), and 12.5 mg/kg-day (3/7, 43%); decreased Harderian gland weight at >4.17 mg/kg-day; transient effect on locomotor activity (hyperactivity) at >1.39 mg/kg-day on PNDs 17 and 24 as determined by figure-8 maze activity assay; activity returned to normal by PND 45 0.46 mg/kg-day 1.39 mg/kg-day Ostby et al. (1985) Developmental, 0/unreported number, Sprague-Dawley rat, gavage, dosed on GDs 8-16, offspring evaluated for kidney effects on PNDs 3 and 6 0,4.17, 12.5, or 25 mg/kg-day, purity not reported Altered physiological responses in kidneys at >4.17 mg/kg-day; decreased kidney weight and body weight at >12.5 mg/kg-day; renal protein content and glomerular counts decreased at >12.5 mg/kg-day; diaphragmatic hernias in dying pups at 4.17 (2/3), 12.5 (35/35), and 25 mg/kg-day (19/32); decreased pup survival at 12.5 mg/kg-day (69%) and 25 mg/kg-day (37%) None 4.17 mg/kg-day Kavlock and Gray (1983a) Developmental, 0/131 (20 in highest dose group, 37 per group at other doses), Long-Evans rat, gavage, dosed on GDs 6-15, dams were weighed on GDs 6, 8, 10, 13, 16, and 20; half of dams were sacrificed on GD 20, and half were allowed to give birth (generally on GD 21) and were kept with pups until weaning on GD 24 0,6.25, 12.5, or 25 mg/kg-day, technical grade nitrofen, purity 96.6% Decreased pup weight at birth at the 25-mg/kg-day dose level; increase in incidence of chromodacryorrhea indicative of Harderian gland dysfunction at 6.25 mg/kg-day (10.6%) and 12.5 mg/kg-day (35.5%); increase in renal defects (kidney and ureter dilation, absence of renal papilla) at >6.25 mg/kg-day; delayed development of the renal papilla and decreased pup survival (0% survival by PND 2) in 25-mg/kg-day dose group None 6.25 mg/kg-day Kavlock et al. (1988) 22 Nitrofen ------- FINAL 11-5-2012 Table 3. Summary of Oral Reproductive and Developmental Studies for Nitrofen (CASRN 1836-75-5) Study Type, Number of Male/Female, Strain Species, Route of Administration, Study Duration Dosimetry, Purity of Nitrofen" Critical Effects NOAELb LOAELb Reference 2-Generation reproductive study, 10/20 per dose, Sherman rat, diet, Fla generation bred on Day 68, Fib generation bred on Day 200, F2a generation bred at time of weaning of Fib generation, exact duration not reported 0, 1.5, 7.6, or 38 mg/kg-day, technical grade nitrofen, purity 89% Decreased percent survival at >7.6 mg/kg-day in the Fla, Fib, and F2a pups; no effects regarding food consumption or body-weight gain 1.5 mg/kg-day 7.6 mg/kg-day Kimbrough et al. (1974) 3-Generation reproductive study, 25/25 per dose, Wistar albino rat, diet, dosed for 11 weeks, then 20 females (F0) from each dose level were mated to produce Fla and Fib generations; females from Fib were mated to produce F2a and F2b generations; females from F2b were mated to form F3a and F3b generations 0, 0.97, 9.7, or 97 mg/kg-day, purity not reported Decreased pup viability at birth, and 0% pup survival to PND 5 at 97 mg/kg-day; increased number of stillborn pups at 9.7 mg/kg-day (effect most prominent in the Fla and Fib offspring) 0.97 mg/kg-day 9.7 mg/kg-day Ambrose et al. (1971e) Developmental, 0/exact number not reported (5 or 6 per dose group, 24 [control]), CD rat, administration route not specified, dosed on GDs 8-16; treated dams sacrificed and fetuses examined on GD 21; 6 control dams sacrificed and fetuses examined on GDs 19, 20, 21, and 22 0, 12.5, or 25 mg/kg-day, purity not reported Dose-related decrease in fetal body weight and decrease in fetal body-weight index at both dose levels; dose-related decrease in absolute brain, lung, liver, and kidney weight and development; average fetal survival decreased with dose (no statistics reported) None 12.5 mg/kg-day Kavlock et al. (1982) Developmental, 0/unspecified number, 10-16 pups per litter, Sprague-Dawley rat, gavage, dosed on GDs 7-21 0, 12.5 mg/kg-day, purity not reported Significantly reduced absolute and relative kidney weights at birth and significantly reduced absolute kidney weight at PND 10; decreased renal concentrating ability; postnatal survival through PND 10 was significantly decreased by treatment to 22% None 12.5 mg/kg-day Chase- Deesing et al. (1986) 23 Nitrofen ------- FINAL 11-5-2012 Table 3. Summary of Oral Reproductive and Developmental Studies for Nitrofen (CASRN 1836-75-5) Study Type, Number of Male/Female, Strain Species, Route of Administration, Study Duration Dosimetry, Purity of Nitrofen" Critical Effects NOAELb LOAELb Reference Developmental, 0/unspecified number, Sprague-Dawley CD rat, gavage, dosed on GDs 8-16, male offspring sacrificed and necropsied on PND 70 0, 12.5 mg/kg-day, purity not reported Decrease in litter size on PND 3; defects of Harderian gland in 5 males necropsied on PND 70 (1 missing both Harderian glands, 2 missing 1 Harderian gland, 2 with abnormal secretions of Harderian gland) None 12.5 mg/kg-day Gray et al. (1982a) Developmental, 0/unreported number, Fischer-344 rat, gavage, dosed on GDs 10-13 0, 15 mg/kg-day, purity not reported Decreased average pup weight at birth None 15 mg/kg-day Raub et al. (1983) Developmental, 0/number not reported, Sprague-Dawley rat, gavage, dosed on GDs 8-18 0, 20,31.2, or 50 mg/kg-day, purity >98% Decreased birth weights in 31.2- and 50-mg/kg-day groups; labored breathing and cyanosis in all treated pups; decreased pup survival on PNDs 1 and 25 at all doses None 20 mg/kg-day Stone and Manson (1981) Developmental, 0/exact number not reported, Fischer-344 rat, gavage, dosed on GDs 10-13, some dams (numbers not reported) sacrificed and offspring recovered on GD 21, the rest carried to term and were examined until 70 days after birth 0, 20, or 40 mg/kg-day, purity -99% Labored breathing and cyanosis shortly after birth in both dose groups; increased incidence of diaphragmatic hernia in pups at both doses; decreased heart rates in pups (doses not reported); decreased body weights at birth in both dose groups; increased percentage of offspring stillborn at 20 mg/kg-day (11%) and 40 mg/kg-day (25%); 0% survival to PND 2 in 40-mg/kg-day dose group None 20 mg/kg-day Lau et al. (1986) Developmental, 0/exact number not reported, Sprague-Dawley rat, gavage, dosed on GDs 10-13; some dams (numbers not reported) sacrificed and offspring recovered on GD 21, the rest carried to term and were examined until 70 days after birth 0, 20, or 40 mg/kg-day, purity -99% Labored breathing and cyanosis shortly after birth in both dose groups; decreased heart rates in pups (doses not reported); increased incidence of diaphragmatic hernia in pups at >20 mg/kg-day; decreased body weights at birth in both dose groups; increased percentage of offspring stillborn at 20 and 40 mg/kg-day (data not provided); 0% survival to PND 2 in 40-mg/kg-day dose group None 20 mg/kg-day Lau et al. (1986) 24 Nitrofen ------- FINAL 11-5-2012 Table 3. Summary of Oral Reproductive and Developmental Studies for Nitrofen (CASRN 1836-75-5) Study Type, Number of Male/Female, Strain Species, Route of Administration, Study Duration Dosimetry, Purity of Nitrofen" Critical Effects NOAELb LOAELb Reference Developmental, 0/9-11 per dose group, Sherman rat, gavage, dosed on GDs 7-15, pups observed until weaning 0, 10, 20, or 50 mg/kg-day, technical grade nitrofen, purity 89%, or 0, 20, or 50 mg/kg-day, purity 99% Decreased number of live offspring per litter at birth (7.1 for technical and 7.5 for pure nitrofen compared to 12.4 in control) and at weaning (3.6 for technical and 3.9 for pure nitrofen compared to 12.4 in control) at 20 mg/kg-day; low number of live offspring per litter at birth (2.7 for technical and 2.2 for pure nitrofen compared to 12.7 in control) and no live offspring at weaning at 50-mg/kg-day dose level 10 mg/kg-day 20 mg/kg-day Kimbrough et al. (1974) Developmental, 0/number not reported, Sprague-Dawley rat, dosed on GDs 7-21, fetal rats recovered by cesarean section on GD 21 0, 25 mg/kg-day, purity 99% Absolute fetal body, liver, kidney, intestine, heart, and lung weights, as well as organ DNA, RNA, and protein levels decreased in treated pups None 25 mg/kg-day Zeman et al. (1986) Developmental, 0/12 per dose group, Sherman rat, gavage, dosed on GDs 7-18, cesarean sections performed on GD 21 0, 50 mg/kg-day, technical grade nitrofen, purity 89% Fetal cyanosis at birth in pups of treated dams; high fetal mortality in pups of treated dams (data not provided) None 50 mg/kg-day Kimbrough et al. (1974) Developmental, 0/18 (10 treated, 8 control), Sprague-Dawley rat, dosed on GDs 8-18, dams sacrificed and fetuses recovered by cesarean section on GDs 20 or 21 0, 50 mg/kg-day, purity >98% Decreased fetal body and lung weights (absolute and relative) in treated pups apparent at GD 20; histological analysis revealed no effects None 50 mg/kg-day Stone and Manson (1981) Developmental, 0/unspecified number, Sprague-Dawley rat, gavage, dosed on GD 11 0, 50, or 100 mg/kg-bw, purity not reported Hydronephrosis combined with decreased maximal urine osmotic concentration at 50 and 100 mg/kg- bw; decreased fetal percentage survival to PND 2 (63%) and PND 29 (21%) at 100 mg/kg-bw None 50 mg/kg-bw Daston et al. (1988) 25 Nitrofen ------- FINAL 11-5-2012 Table 3. Summary of Oral Reproductive and Developmental Studies for Nitrofen (CASRN 1836-75-5) Study Type, Number of Male/Female, Strain Species, Route of Administration, Study Duration Dosimetry, Purity of Nitrofen" Critical Effects NOAELb LOAELb Reference Developmental, 0/unspecified number, Long-Evans rat, gavage, dosed on GD 11, fetuses recovered on GD 22 0, 70, 115, 265, or 400 mg/kg-bw, trace contaminant identified as reduction product, 2,4-dichloro-4' -amino diphenyl ether present in less than ppm amounts Significant increase in incidence of hydronephrosis and diaphragmatic hernias at all dose levels; dose-related increase in neonatal lethality None 70 mg/kg-day Costlow and Manson (1981) Developmental, 0/unreported number, Long-Evans rat, gavage, dosed on GD 11, offspring observed until sacrificed and necropsied on PND 35 75 mg/kg-bw, purity > 99.9% Decreased number of pups with eyes open on PND 16 (58% compared to 76% in control); decrease in Harderian gland weights compared to controls; no effects on postnatal viability or growth through weaning None 75 mg/kg-day Kavlock and Gray (1983b) Developmental, 0/unspecified number, Long-Evans rat, gavage, dosed on GD 11, pups observed until maturity 0, 75, 115, 150, 200, or 250 mg/kg-bw, trace contaminant identified as reduction product, 2,4-dichloro-4' -amino diphenyl ether present in less than ppm amounts Decrease in body weight at > 115 mg/kg-bw; increased incidence of hydronephrosis at all doses except 150 mg/kg-bw; increased incidence of cardiac malformation at 150 and 250 mg/kg-bw 75 mg/kg-day 115 mg/kg-day Costlow and Manson (1981) Developmental, 0/unspecified number, Long-Evans rat, gavage, dosed on GDs 7-9, 9-11, or 12-14; pups observed until 48 hours after birth 0, 150 mg/kg-day, trace contaminant identified as reduction product, 2,4-dichloro-4' -amino diphenyl ether present in less than ppm amounts Decreased birth weight (86-87% of controls) and survival rate (0-26% of controls) to PND 2 in groups of treated pups for each dosing period None 150 mg/kg-day Costlow and Manson (1981) 26 Nitrofen ------- FINAL 11-5-2012 Table 3. Summary of Oral Reproductive and Developmental Studies for Nitrofen (CASRN 1836-75-5) Study Type, Number of Male/Female, Strain Species, Route of Administration, Study Duration Dosimetry, Purity of Nitrofen" Critical Effects NOAELb LOAELb Reference Developmental, 0/unspecified number, Long-Evans rat, gavage, dosed on GDs 9, 10, 11, or 12; pups observed after birth and sacrificed and autopsied on PND 35 0, 150 mg/kg-bw, trace contaminant identified as reduction product, 2,4-dichloro-4' -amino diphenyl ether present in less than ppm amounts Decreased birth weight (84-92% compared to controls) and decreased survival rate (44-96% compared to controls) at PND 2 in groups of treated pups for each dosing period None 150 mg/kg-day Costlow and Manson (1981) Developmental, 0/unreported number, Sprague-Dawley rat, gavage, dosed on GD 11, single administration, dams sacrificed and fetuses recovered on GD 21 0, 200, 250, 300, 350, or 400 mg/kg-bw, purity not reported Decreased fetal average body weight at all doses; increased number of cardiovascular anomalies (study authors noted ventricular septal defects and anomalous right subclavian arteries, but no further detail was provided) at all dose levels None 200 mg/kg-day Kim et al. (1999) Developmental, 0/17 (10 dosed, 7 control), Sprague-Dawley rat, single administration, administration route not reported, dosed on GD 10 0, 400 mg/kg-bwc, purity not reported Decreased birth weight (87% of control) and lung- to-body-weight ratio (60% of control) in nitrofen-treated pups None 400 mg/kg-bw Ijsselstijn et al. (1997) Developmental, 0/unspecified number, Sprague-Dawley rat, gavage, dosed on GD 9.5, fetuses recovered on GD 13.5 0, 490 mg/kg-bwd, purity not reported Defects in propagation of calcium across airway smooth muscle (ASM) None 490 mg/kg-bw Featherstone et al. (2006) Developmental, 0/unspecified number, Sprague-Dawley rat, gavage, single administration, GD of dosing not reported 0, 735 mg/kg-bwd, purity ~ 98% Abnormal development of the pleuroperitoneal fold in exposed embryos recovered on GDs 13.5-14 None 735 mg/kg-bw Clugston et al. (2010) Developmental, 0/unspecified number, Long-Evans rat, gavage, dosed on GDs 15-20; pups observed until 48 hours after birth 20-100 mg/kg-day, trace contaminant identified as reduction product, 2,4-dichloro- 4'-amino diphenyl ether present in less than ppm amounts No effects observed in newborns (no data provided) 100 mg/kg-day None Costlow and Manson (1981) 27 Nitrofen ------- FINAL 11-5-2012 Table 3. Summary of Oral Reproductive and Developmental Studies for Nitrofen (CASRN 1836-75-5) Study Type, Number of Male/Female, Strain Species, Route of Administration, Study Duration Dosimetry, Purity of Nitrofen" Critical Effects NOAELb LOAELb Reference Developmental, 0/unreported number, rat (strain unreported), administration route not reported, dosed on GDs 7-16 5 dose levels from 0-25 mg/kg-day, purity not reported Diaphragmatic hernias at 1.39 mg/kg-day and above; Harderian gland effects (not specified) at 12.5 mg/kg-day and above None None Gray et al. (1982b)e Developmental, 0/number not reported, rat (strain not reported), route of administration not reported, dosed on GD 10 Not reported Diaphragmatic hernia; decreased growth of lung primordium None None Piersma et al. (1993)e Developmental, 0/unreported number, Fischer F344 rat, administration route not reported, dosed on GDs 10-13; ECGs recorded on GD 21, immediately following birth, and on PNDs 2 and 70 0, 20, 40 mg/kg-day, purity not reported Diaphragmatic hernias (doses unreported); irregular and gasping respiratory movements and cyanosis of all treated litters; 0% pup survival to PND 1, and distension of abdomen in high-dose group; low body weights in low-dose group until PND 70; dose-related decrease in heart rate None None Robinson and Cameron (1984)e Developmental, 0/unreported number, LEH rat, administration route not reported, dosed on GDs 10-12 200 mg/kg-day (no control reported), purity not reported Effects not reported None None Kang and Manson (1987)e Developmental, 0/unreported number, rat (strain unreported), gavage, dosed on GDs7-21 0, 25 mg/kg-day, purity not reported Reduced body weight, intestinal weight, and intestinal length; elevated enzyme activity None None Mahboob et al. (1985)e Mouse Developmental, 0/unspecified number, CD-I mouse, gavage, dosed on GDs 7-17, dams carried to term, and pups were weaned on PND 30, males sacrificed and necropsied on PND 110, females sacrificed and necropsied on PND 130 0,6.25, 12.5,25,50, 100, 150, or 200 mg/kg-day, purity 99.6% Decreased lung and liver weights at all dose levels on PND 110; decreased absolute Harderian gland weight at all dose levels, and absence of glands at 25 (4%), 50 (65%), and 100 (97%) mg/kg-day; retardation of growth rates at >12.5 mg/kg-day; reduced body weights at birth in the 150- and 200-mg/kg-day dose groups, and at PND 3 in the 100-mg/kg-day group; incidence of diaphragmatic hernia (6%), cleft palate (15%), and distended abdomen (22%) at 200 mg/kg-day; 100% pup mortality by PND 3 at 150 and 200 mg/kg-day None 6.25 mg/kg-day Gray et al. (1983a) 28 Nitrofen ------- FINAL 11-5-2012 Table 3. Summary of Oral Reproductive and Developmental Studies for Nitrofen (CASRN 1836-75-5) Study Type, Number of Male/Female, Strain Species, Route of Administration, Study Duration Dosimetry, Purity of Nitrofen" Critical Effects NOAELb LOAELb Reference Developmental, 0/12-14, CD-I mouse, gavage, dosed on GDs 7-17, litters from prenatal untreated control dams (CON) were cross-fostered with prenatal nitrofen treated (N) dams or untreated (CON) dams. Litters from treated (N) dams were cross-fostered with CON dams. Final groups consisted of N/CON, N/N, CON/N and CON/CON. Growth and viability of pups evaluated on PNDs 1,3,10 and 20. Pups were necropsied on PND 110. 0 (corn oil control) or 100 mg/kg-day, purity 99.6% Decreased lung and liver weights on PND 110; decreased absolute Harderian gland weights, and absence of glands (97%); retardation of growth rates; reduced body weights on PND 3 among groups (N/CON and N/N) treated prenatally. Developmental effects not seen in groups (CON/N and CON/CON) without prenatal nitrofen treatment. NA NA Gray et al. (1983b) Developmental, 0/unreported number, Swiss-Webster CD-I mouse, gavage, dosed on GDs 6-15, females carried litters to term, offspring sacrificed at weaning on PNDs 27-33 0, 10, 50, 100, 250, or 500 mg/kg-day, purity >99% Decreased pup body weight in males (80% of control) and females (83% of control), and in Harderian gland size in males (69% of control) and females (76% of control) at 10 mg/kg-day; decrease in the percentage prenatal litter survival at 100 (87%), 250 (63%), and 500 (0%) mg/kg-day (compared to 94% in control); decreased survival per litter to PND 15 at 50 (81% of control), 100 (48% of control), and 250 (2% of control) mg/kg-day Decreased maternal weight gain per female at 10 (71% of control), 50 (67% of control), and 100 (88% of control) mg/kg-day Developmental: None Maternal: None Developmental: 10 mg/kg-day Maternal: 10 mg/kg-day Francis et al. (1999) Developmental, 0/unspecified, CD-I mouse, gavage, dosed on GDs 7-17, pups sacrificed and examined on PNDs 3,8, 13, and 110 0, 100 mg/kg-day, purity not reported Delayed eye opening and decreased weight/absence of Harderian glands in treated pups None 100 mg/kg-day Gray et al. (1982a) 29 Nitrofen ------- FINAL 11-5-2012 Table 3. Summary of Oral Reproductive and Developmental Studies for Nitrofen (CASRN 1836-75-5) Study Type, Number of Male/Female, Strain Species, Route of Administration, Study Duration Dosimetry, Purity of Nitrofen" Critical Effects NOAELb LOAELb Reference Developmental, 0/169 (76 treated, 93 control), Swiss Webster mouse, gavage, dosed on GD 8, embryos recovered by cesarean section and examined on GDs 12, 14, 16, or 17 0, 1111 mg/kg-bwd, purity not reported Severe fetal craniofacial defects (no further detail provided) in groups recovered on GDs 12, 14, 16, and 17 Developmental: None Developmental: 1111 mg/kg-day Acosta et al. (2001) Developmental, 0/unreported number, mouse (strain unreported), administration route not reported, dosed on GDs 8-12 0, 200 mg/kg-day, purity not reported 0% pup viability in treatment group; eyeless offspring; reduction in size of Harderian gland None None Gray et al. (1982b)e Developmental, 0/unreported number, mouse (strain unreported), administration route not reported, dosed on GDs 7-17 0-200 mg/kg-day (exact dose levels not reported), purity not reported Reduced size of Harderian gland at 6.25 mg/kg-day and above, and destruction of Harderian gland in 4% of pups at 50 mg/kg-day and 35% at 100 mg/kg-day; 50% pup viability at 100 mg/kg-day and 0% viability at 150 mg/kg-day and above; eyeless offspring at 200 mg/kg-day None None Gray et al. (1982b)e Developmental, 0/unreported number, mouse (strain unreported), administration route not reported, dosed on GDs 7-17 0, 50-200 mg/kg-day (exact dose levels not reported, control not specified), purity not reported Reproductive problems at 50 and 100 mg/kg-day; stunted growth in all treated groups None None Gray et al. (1982b)e Rabbit Developmental, 0/15 per dose group, New Zealand White Rabbit, capsule, dosed on GDs 6-18, 10 rabbits per group sacrificed on GD 28 and examined for developmental effects, remaining 5 rabbits per group were sacrificed along with offspring on PND 2 0, 5, 20, or 80 mg/kg-day, purity not reported Decreased live fetuses per litter at 80 mg/kg-day 20 mg/kg-day 80 mg/kg-day Siou (1979) as reported in Burke Hurt et al. (1983) Hamster Developmental, 0/unreported number, hamster (strain unreported), administration route not reported, dosed on GDs 7-11 0, 25, 50, 100, 200, or 400 mg/kg-day, purity not reported Harderian glands, lungs, adrenals, seminal vesicles, epididymides, testes, sperm counts, and flank gland development were reduced (doses not reported) None None Gray (1984)e 30 Nitrofen ------- FINAL 11-5-2012 Table 3. Summary of Oral Reproductive and Developmental Studies for Nitrofen (CASRN 1836-75-5) Study Type, Number of Male/Female, Strain Species, Route of Administration, Study Duration Dosimetry, Purity of Nitrofen" Critical Effects NOAELb LOAELb Reference Developmental, 0/unreported number, hamster (strain unreported), administration route not reported, dosed on GDs 8-9, 11-12, or 14-15 0, 400 mg/kg-day, purity unreported Uterus unicornis and ipsilateral renal agenesis in females and unilateral agenesis of vas deferens and/or epididymis and seminal vesicle in male pups following treatment on GDs 8-9; spermatic granulomas in male pups treated later in gestation; serum thyroxin levels reduced in males at PND 25 None None Gray (1984)e Developmental, 0/unreported number, hamster (strain unreported), administration route not reported, dosed on GDs 7-11 0-400 mg/kg-day (exact dose levels not reported), purity not reported Reduced size of Harderian gland at 100 mg/kg-day and above; severe reproductive effects (further details not provided) None None Gray et al. (1982b)e aUnits are reported in mg/kg-day or mg/kg-bw (for single-administration studies). bNot reported by the study author(s) but determined from data for this review. °Dose reported in study as mg; converted using the following equation: Dose (mg/kg-day) = provided dose (mg) average body weight (kg), where average body weight is the body weight provided by the study authors. dDose reported in study as mg; converted using the following equation: Dose (mg/kg-day) = provided dose (mg) average body weight (kg), where average body weight is the average subchronic body weight for females of the species and strain provided by the study authors. eOnly abstract available; no NOAELs or LOAELs were derived due to lack of information. NA = Not applicable because the study (Gray et al., 1983b) was not designed to identify a LOAEL. 31 Nitrofen ------- FINAL 11-5-2012 Inhalation Exposures Subchronic-duration Studies No studies were identified. Chronic-duration Studies No studies were identified. Developmental and Reproductive Studies No studies were identified. OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS) Studies investigating the kinetics of nitrofen in rats, sheep, and cows (Brown and Mason, 1986; Costlow and Manson, 1983; Hunt et al., 1977; Gutenmann and Lisk, 1967) and the potential mode of action for nitrofen (Noble et al., 2007; Brandsma et al., 1994; Manson et al., 1984, as cited by Manson, 1986) are summarized in Table 4. The data evaluating the genotoxicity/mutagenicity of nitrofen are also included in Table 4. Ames assays testing the genotoxicity of nitrofen found equivocal evidence of genotoxic effects in various strains of Salmonella typhimurium (Dunkel et al., 1985; Moriya et al., 1983; Tanaka et al., 1996). Data reported in other Ames assays were also available (Byeon et al., 1976; Jeang and Li, 1980; Shirasu et al., 1982, as cited in Burke Hurt et al., 1983). The TA98 and TA100 S. typhimurium strains exhibited positive and negative results, both with or without rat liver S9 activation. The YG1026 and YG1029 strains exhibited a positive result with S9 activation in one study (Tanaka et al., 1996). A possible explanation of these positive genotoxicity results may be that a low-level impurity in technical grade nitrofen preparations—4,4'-dichlorobenzene—could have caused genotoxic effects to strains TA98 and TA100 (Paik and Lee, 1977; Burke Hurt et al., 1983). Cultured mouse lymphoma cells and human lymphocytes were also used to assess the genotoxicity of nitrofen (Paik and Lee, 1977; McGregor et al., 1996). These studies did not indicate any increase in forward mutation or unscheduled DNA repair synthesis due to nitrofen. Several chromosomal aberration studies were conducted with nitrofen in rats (McLeod and McCarthy., 1981; Reustle and Scribner, 1980; Kiryushin, 1975) and in freshly germinated barley roots (Oku, 1976, as cited by Burke Hurt et al., 1983). No chromosomal aberrations were observed in these studies. Furthermore, mice exposed to nitrofen in doses up to 1.69 mg/kg did not exhibit any increases in the number of micronuclei present in polychromatic erythrocytes (Siou, 1978, as cited by Burke Hurt et al., 1983). These findings suggest that nitrofen is not genotoxic. Noble et al. (2007) conducted in vitro cell assays as well as whole animal rodent studies in order to test hypotheses concerning the mechanisms by which nitrofen induces diaphragmatic hernias in rodents. Study authors investigated the interactions of nitrofen with various stages of the retinoid signaling pathway; with vitamins A, C, and E; and with the thyroid signaling pathway. A luciferase assay conducted with P19 cells revealed that nitrofen application did not affect the mRNA expression of any elements in the retinoid signaling cascade. A yeast-HRE assay revealed significant inhibition of receptor binding at >100|iM nitrofen, but no significant effect at concentrations that induce diaphragmatic hernia without being lethal. A clear dose-dependent decrease in retinoic acid levels was observed with increasing nitrofen concentration in a dual assay system with luciferase and Retinoic Acid Receptor Response 32 Nitrofen ------- FINAL 11-5-2012 Element (RARE)-luciferase, and significantly lower levels of retinoic acid were observed in embryos exposed to nitrofen on GD 9. Study authors observed a rescue effect when vitamin A was administered with nitrofen, but no significant effects when nitrofen was administered with vitamins C or E. Study authors attributed this effect to an increase in the substrate for RALDH in the retinoid signaling cascade, not to the oxidizing properties of the vitamin. Tests conducted to determine the thyromimetic effects of nitrofen returned negative results; nitrofen does not exert teratogenic effects through interactions with the thyroid signaling pathway. Study authors concluded that the perturbation of retinoic acid is the primary effect of nitrofen on the retinoid signaling pathway, and that this may be an underlying cause of nitrofen-induced diaphragmatic hernia in rodent models. 33 Nitrofen ------- FINAL 11-5-2012 Table 4. Other Studies Tests Methods Dosimetry Results References Ames assay Salmonella typhimurium, TA98, TA100, TA1535, TA1537, TA1538, Escherichia coli WP2 urvA, ± S9 mix Unknown (partial PDF missing doses) + without S9 in TA98, TA100, and TA1538; - without S9 at TA1535, TA1537, WP2 urvA + with S9 in TA98, TA100, and TA1538; - with S9 at TA1535, TA1537, WP2 urvA Dunkel et al. (1985) Ames assay S. typhimurium, TA98, TA100, TA1535, TA1537, TA1538, WP2 her, ± S9 mix 0-500 ng/plate + without S9 in TA100; - without S9 at TA1535, TA1537, TA1538, WP2 her + with S9 in TA98; - with S9 at TA1535, TA1537, TA1538, WP2 her Moriya et al. (1983) Ames assay S. typhimurium, TA98, TA100, YG1021, YG1024, YG1026, YG1029, + S9 mix Not reported + with S9 mix in YG1026 and YG1029; - with S9 at TA98, TA100, YG1021 Tanaka et al. (1996) Ames assay Unspecified, Ames system Not reported Positive Byeonetal. (1976), Jeang and Li (1980), Shirasu et al. (1982), as cited by Burke Hurt et al. (1983) Lymphoma assay (mouse and human lymphocytes) L5178Y mouse lymphoma cells, human lymphocytes grown in culture 0, 10, and 20 mM negative for methotrexate-resistant mutants in mouse lymphoma cells; did not induce unscheduled DNA repair synthesis in human lymphocytes Paik and Lee (1977) Mouse lymphoma assay L5178Y mouse lymphoma cells 0, 30, and 60 ng/mL Borderline response at tk locus in L5178Y mouse cells McGregor et al. (1996) Chromosomal aberrations 8 male Charles River CD-I rat 0,0.05, 0.125, and 0.5 g/kg-day for 5 days No increase in chromosomal aberrations from bone marrow McLeod and McCarthy (1981) Chromosomal aberrations 8 male Charles River CD-I rat 0, 0.39, 0.79, and 1.59 g/kg-day for 5 days No increase in chromosomal aberrations from bone marrow Reustle and Scribner (1980) Chromosomal aberrations Rat, strain not specified 1/15th or 1/200*11)50 to rats for 6 days No increase in chromosomal aberrations Kiryushin (1975) Chromosomal aberrations Freshly germinated barley roots 800- and 8000-ppm nitrofen emulsion No increase in chromosomal aberrations Oku (1976), as cited by Burke Hurt et al. (1983) 34 Nitrofen ------- FINAL 11-5-2012 Table 4. Other Studies Tests Methods Dosimetry Results References In vivo micronuclei assay 5-10 male Swiss mouse, gavage, examined for the presence of micronuclei in polychromatic erythrocytes in the bone marrow 0, 0.34, 1.35, and 1.69 mg/kg No increase in micronuclei Siou (1978), as cited by Burke Hurt et al. (1983) Toxicokinetics study Long-Evans Hooded rat; single oral maternal dose of 240 mg/kg 14C-labeled nitrofen on GD 10; maternal and embryonic tissues collected after 1.5-72 hours; HPLC of maternal fat, plasma, liver, heart, and embryo-placental complex 240 mg/kg 14C-labeled nitrofen on GD 10 After 3-12 hours: uptake, peak maternal tissue concentrations (highest in liver); after 3 hours: radioactivity detected in embryo-placental complex; after 12 hours: accumulation in maternal fat underway; after 24 hours: 100-fold higher accumulation in maternal fat compared to blood; after 48 hours: redistribution of nitrofen to maternal heart, liver, and the embryonic compartment; half-life of measured radioactivity in blood estimated to be 42 hours; half- life of detectable nitrofen (parent compound) in blood calculated to be 68 hours; embryonic compartment contained only nitrofen parent compound and was a deep compartment for the compound; 4'-amino, 4'-acetylamine, 5-hydroxy were the dominant metabolites detected in maternal tissues Brown and Manson (1986) Toxicokinetics study 8 Long-Evans rat; single oral maternal dose of 240 mg/kg 14C-labeled nitrofen on GD 10; toxicokinetic analysis of dam tissues; UV profile HPLC analysis of metabolites in embryo-placental complex extracts; analysis of 4'-amino, acetylamine and hydroxyl metabolites 120 mg/kg 14C-labeled nitrofen on GD 11 8-hour absorption phase in dams, maximal maternal blood concentration of 10 (ig/mL; half-life in maternal blood of 8 days; 100-fold higher accumulation in maternal fat compared to blood; volume of distribution (V) of 41.3 liters (0.413 if adjusted to the 1% absorption reported elsewhere based on concentration in excreta); no detectable radiation from 14C-labeled nitrofen in embryo-placental complex extracts; acetylamine metabolite only HPLC-detectable nitrofen compound in embryo-placental complex extracts Costlow and Manson (1983) 35 Nitrofen ------- FINAL 11-5-2012 Table 4. Other Studies Tests Methods Dosimetry Results References Toxicokinetics study Delaine ewe; gelatin capsule of 14C-labeled nitrofen and grain; blood, urine, and feces collected every 0.5 hour (0-4 hours), hourly (4-9 hours), and thereafter every 4 hours (until 100 hours); combustion analysis, TLC analysis, audioradiography, liquid scintillation counting of extracts 40 mg/kg Radioactivity detected at highest levels in blood after 19 hours; aminonitrofen and nitrofen the most recovered radioactive compounds; 11% of applied dose excreted as nitrofen in feces; 39% of the applied dose was recovered in blood, urine, and feces (37.2% of applied dose) after 99 hours; urine contained conjugated nitrofen that was 25% or 70% liberated and extractable by piO-glucoronidase or sulfatase, respectively Hunt et al. (1977) Toxicokinetics study Holsteincow; 5-ppmpure recrystallized nitrofen in feed (based on a daily ration of 26.2 kg); urine feces, milk collected in morning and evening; deconjugation via orthophosphoric acid digestion; samples extracted in acetone; methylation with diazomethane; affinity gas chromatography; also examined stability of nitrofen in rumen fluid 5 ppm in feed for 4 days No detection of nitrofen in milk, urine, or feces; recovery of nitrofen spiked into milk, urine, or feces samples was possible to levels as low as 0.2 ppm; 2,4-dichloro-4'-aminodiphenyl ether detected in rumen fluid Gutenmann and Lisk (1967) Mode-of-action study Bacterial recombinant (ai and (3i forms of thyroid hormone receptor) binding assay; chicken a type rat (3 type thyroxine hormone receptor as recombinant in an it. coli pop 2136 strain vector; measured [125I] labeled triiodothyronine ([125I]T3) specific binding to ai chicken and (3i rat thyroid hormone receptor protein in the presence of excess nonradioactive T3 and nitrofen at various concentrations 10, 100, or 1000 \M nitrofen Decreased maximal binding capacity of T3 to the ai chicken and (3i rat forms of thyroid hormone receptor in a noncompetitive (allosteric) way; these effects of nitrofen were dose dependent; this result indicates that nitrofen inhibits the binding of T3 to the T3 receptor Brandsma et al. (1994) 36 Nitrofen ------- FINAL 11-5-2012 Table 4. Other Studies Tests Methods Dosimetry Results References Mode-of-action study Euthyroid and thyroparathyroidectomized (TPTX) adult female rats, strain not specified; coadministered thyroxine (T4) to test recovery of hypothalamic-pituitary- thyroid function in nonpregnant, pregnant, and fetal rats 15 and 30 mg/kg-day for 2 weeks Euthyroid rats: significant decrease in thyroid stimulating hormone and T4; TPTX rats: coadministration of T4 with nitrofen resulted in a 70% reduction in the frequency of malformed fetuses compared to nitrofen alone Manson et al. (1984), as cited by Manson (1986) Mode-of-action study Retinoid and thyroid hormone signaling pathways examined in vitro in conjunction with rat whole-animal in vivo studies; thyroid hormone, thyroid hormone receptor function assays; yeast hormone response element (HRE) assay: binding to nuclear receptors including thyroid receptor and retinoic acid receptors (transgenic yeast); dual luciferase assay: retinoic acid production measured by retinoic acid receptor RARE-luciferase enzymes lOnM, lOOnM, l\iM, IOjjM, and 100|iM Nitrofen interacts with various elements of the retinoid signaling cascade to ultimately disturb the levels of retinoic acid; no significant effects were observed on the thyroid signaling pathway; no effect was observed when P19 cells were treated with vitamins C or E along with nitrofen, but vitamin A produced a rescue effect when administered with nitrofen Noble et al. (2007) 37 Nitrofen ------- FINAL 11-5-2012 DERIVATION OF PROVISIONAL VALUES Table 5 presents a summary of noncancer reference values. Table 6 presents a summary of cancer values. For cancer, the toxicity value was converted to HED units, and the conversion process is presented in the section on derivation of provisional cancer potency values. Table 5. Summary of Noncancer Reference Values for Nitrofen (CASRN 1836-75-5) Toxicity Type (Units) Species/Sex Critical Effect p-Reference Value POD Method POD UFC Principal Study Subchronic p-RfD (mg/kg-day) Rat/litter data Diaphragmatic hernias 3 x 10"3 BMDL05 0.29 100 Ostby et al. (1985) Chronic p-RfD (mg/kg-day) Rat/litter data Diaphragmatic hernias 3 x 10"3 bmdl05 0.29 100 Ostby et al. (1985) Subchronic p-RfC (mg/m3) None None None None None None None Chronic p-RfC (mg/m3) None None None None None None None Table 6. Summary of Cancer Reference Values for Nitrofen (CASRN 1836-75-5) Toxicity Type Species/ Sex Tumor Type Cancer Value Principal Study p-OSF Rat/F Hepatocellular carcinomas 3.8 x 10"2(mg/kg-day)"1 NCI (1978d) p-IUR None None None None DERIVATION OF ORAL REFERENCE DOSES Derivation of Subchronic Provisional RfD (Subchronic p-RfD) Based on the available literature, there were seven sub chronic-duration, four chronic-duration, several developmental and reproductive studies (see Table 3), and four carcinogenic studies involving oral exposures to nitrofen. When compared to adult animals exposed for any duration, the effects in fetal animals are seen at much lower relative doses, indicating that the critical effect for nitrofen is developmental in nature. The developmental study by Ostby et al. (1985) is selected as the principal study for derivation of the subchronic p-RfD. Details of this study are provided in the "Review of Potentially Relevant Data" section. Table 3 summarizes the available studies reporting developmental effects in animals following oral exposure to nitrofen. The developmental effect of diaphragmatic hernias is the most consistent, with other reported effects (i.e., renal, Harderian gland, heart, and neurobehavioral) not consistently reported. Diaphragmatic hernias showed a strong biological gradient as this effect was related to dose over several independent studies. Diaphragmatic hernias were also reported across species (rat and mouse) with effects in the rat being more sensitive, as indicated by Ostby et al. (1985). Furthermore, at the higher dose levels, most of the pups found dead on PND 0 had diaphragmatic hernias (75% at 1.39 mg/kg-day, 67% at 4.17 mg/kg-day, and 100% at 12.5 mg/kg-day), suggesting that this malformation may be a primary cause of neonatal mortality following maternal exposure to nitrofen (see Table B.12). Thus, diaphragmatic hernias in rat pups is chosen as the critical effect. 38 Nitrofen ------- FINAL 11-5-2012 The Ostby et al. (1985) rat data for the number of litters with pups having diaphragmatic hernias gives a BMDL05 of 0.292 mg/kg-day. Litter-specific data for this study were not available, and the results were reported as mean data. Therefore, use of nested models provided by BMD software was precluded, and regular dichotomous BMD models were used to determine the point of departure (POD) (U.S. EPA, 2012). The dichotomous data models in the EPA BMDS (version 2.1.2) were fit to the data for the number of litters with pups having diaphragmatic hernias following exposure of maternal rats to nitrofen by gavage on GDs 8-16 (see Table B. 12). Table C. 1 lists the BMD output models considered for derivation of the chronic and subchronic p-RfD with curve and BMD output text provided for the selected model in the BMD supplement to this document provided in Appendix C (see Figure C.l and the subsequent text output). All models provided adequate fit to the diaphragmatic hernia litter data. The LogLogistic model is considered the best model to fit the data and was used as the POD as it provided the lowest AIC (37.935) and a BMDL05 of 0.292 mg/kg-day (see Table C.l). The subchronic p-RfD for nitrofen, based on the BMDL05 of 0.292 mg/kg-day in nested rat data (Ostby et al., 1985), is derived as follows: Subchronic p-RfD = BMDL05 UFc = 0.292 mg/kg-day -M00 = 3 x 10 3 mg/kg-day Table 7 summarizes the uncertainty factors for the subchronic p-RfD for nitrofen. Table 7. Uncertainty Factors Used to Derive a Subchronic p-RfD for Nitrofen UF Value Justification ufa 10 A UFa of 10 is applied for interspecies extrapolation to account for potential toxicokinetic and toxicodynamic differences between rats and humans. There are no data to determine whether humans are more or less sensitive than rats to the developmental effects of nitrofen. ufd 1 A UFd of 1 is applied because the database includes 1 acceptable 2-generation reproduction study in rats (Kimbrough et al., 1974), 1 acceptable 3-generation reproduction studies in rats (Ambrose et al., 1971e), and multiple developmental studies across 4 species (rat, mouse, rabbit, hamster; see Table 3). 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 in humans. ufl 1 A UFl of 1 is applied because the POD is a BMDL. UFS 1 A UFS of 1 is applied because a developmental study (Ostby et al., 1985) was utilized as the principal study to derive the subchronic p-RfD. UFC 100 39 Nitrofen ------- FINAL 11-5-2012 The confidence of the subchronic p-RfD for nitrofen is high as explained in Table 8. Table 8. Confidence Descriptors for Subchronic p-RfD for Nitrofen Confidence Categories Designation" Discussion Confidence in study H Confidence in the key study is high. Ostby et al. (1985) examined appropriate developmental toxicity endpoints, although only 8-13 maternal rats per dose group were used, resulting in a small number of litters per dose (7-11). The study was peer reviewed. GLP compliance is unknown. The study included multiple effect levels, and both a NOAEL and LOAEL are identified. The data used as the critical effect were modeled using BMD software and provided a good fit. The key endpoint of diaphragmatic hernias in the litters of pups is seen in multiple independent studies and in two species—rat and mouse. Confidence in database H The database includes subchronic-duration toxicity studies in 2 species (rat and mouse), chronic-duration toxicity studies in 2 species (rat and mouse), developmental toxicity studies in 4 species (rat, mouse, rabbit, and hamster), and one 2-generation reproductive study and one 3-generation reproductive study in rats. Confidence in subchronic p-RfDb H The overall confidence in the subchronic p-RfD is high. aL = Low, M = Medium, H = High. bThe overall confidence cannot be greater than lowest entry in table. Derivation of Chronic Provisional RfD (Chronic p-RfD) Although chronic toxicity testing of nitrofen has been conducted, effects in fetal animals occurred at much lower relative doses indicating that the critical effect is developmental. Therefore, the critical endpoint is diaphragmatic hernias as indicated by Ostby et al. (1985). This is the same critical effect used to derive the subchronic p-RfD. A full description concerning the selection of this endpoint as the critical effect and calculation of the appropriate BMDL0s are provided in the section on the derivation of the subchronic p-RfD. Consistent with the practice of the EPA, the developmental period is recognized as a susceptible lifestage where exposure during certain time windows is more relevant to the induction of developmental effects than lifetime exposure (U.S. EPA, 1991b). Therefore, a UF for extrapolation from less-than-chronic results is not used, and the chronic p-RfD is derived as follows: Chronic p-RfD = BMDL05 UFc = 0.292 mg/kg-day -MOO = 3 x 10 3 mg/kg-day 40 Nitrofen ------- FINAL 11-5-2012 Table 9 summarizes the uncertainty factors for the chronic p-RfD for nitrofen. Table 9. Uncertainty Factors Used to Derive a Chronic p-RfD for Nitrofen UF Value Justification ufa 10 A UFa of 10 is applied for interspecies extrapolation to account for potential toxicokinetic and toxicodynamic differences between rats and humans. There are no data to determine whether humans are more or less sensitive than rats to the developmental effects of nitrofen. ufd 1 A UFd of 1 is applied because the database includes 1 acceptable 2-generation reproduction study in rats (Kimbrough et al., 1974), 1 acceptable 3-generation reproduction study in rats (Ambrose et al., 1971e), and multiple developmental studies across 4 species (rat, mouse, rabbit, hamster; see Table 3). 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 in humans. ufl 1 A UFl of 1 is applied because the POD is a BMDL. UFS 1 A UFS of 1 is applied because a developmental study (Ostby et al., 1985) was utilized as the principal study to derive the chronic p-RfD. Because the developmental period is identified by the EPA as a susceptible lifestage where exposure during times of development may be more relevant than exposure over a lifetime, a UF was not used to account for extrapolation from less than chronic results. UFC 100 The confidence of the chronic p-RfD for nitrofen is high as explained in Table 10. Table 10. Confidence Descriptors for Chronic p-RfD for Nitrofen Confidence Categories Designation" Discussion Confidence in study H Confidence in the key study is high. Ostby et al. (1985) examined appropriate developmental toxicity endpoints, although only 8-13 maternal rats per dose group were used, resulting in a small number of litters per dose (7-11). The study was peer reviewed. GLP compliance is unknown. The study included multiple effect levels, and both a NOAEL and LOAEL are identified. The data used as the critical effect were modeled using BMD software and provided a good fit. The key endpoint of diaphragmatic hernias in the litters of pups is seen in multiple independent studies and in two species—rat and mouse. Confidence in database H The database includes subchronic-duration toxicity studies in 2 species (rat and mouse), chronic-duration toxicity studies in 2 species (rat and mouse), developmental toxicity studies in 4 species (rat, mouse, rabbit, and hamster), and one 2-generation reproductive study and one 3-generation reproductive study in rats. Confidence in chronic p-RfDb H The overall confidence in the chronic p-RfD is high. "L = Low, M = Medium, H = High. bThe overall confidence cannot be greater than lowest entry in table. 41 Nitrofen ------- FINAL 11-5-2012 DERIVATION OF INHALATION REFERENCE CONCENTRATIONS Derivation of a provisional subchronic or chronic RfC for nitrofen is precluded because no quantitative human or animal studies examining the effects of subchronic or chronic inhalation exposure to nitrofen have been identified. Derivation of a screening value is precluded for the same reason. CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR Table 11 identifies the cancer weight-of-evidence descriptor for nitrofen as "Likely to be Carcinogenic to Humans." NCI (1978c) reported treatment-related tumor increases in the hematopoietic system (lymphoma), pancreas (carcinoma), and ovaries (granulosa cell tumor) of female Osborn-Mendel rats at the highest dose (see Table B.8) following 78 weeks of exposure to nitrofen in feed and an additional 32 weeks of untreated observation. No treatment-related tumors were found in the male rat. A study conducted in F344 rats reported no treatment-related tumors in males or females following exposure to nitrofen in feed for 78 weeks, with 26 weeks of untreated observation (NCI, 1979c). NCI (1978d) found an increased incidence of hepatocellular carcinomas in both male and female B6C3Fi mice (see Table B.9) following 78 weeks of exposure to nitrofen in feed and an additional 12 weeks of untreated observation. The liver tumors were increased in a dose-dependent manner. NCI (1979d) conducted an additional study in B6C3Fi mice, which also reported an increased incidence in hepatocellular carcinomas and hepatocellular carcinomas and adenomas (combined) in both males and females (see Table B. 10). These liver tumors were increased at all doses in both sexes except for female hepatocellular carcinomas, which were only increased at the high dose. Table 11. Cancer WOE Descriptor for Nitrofen Possible WOE Descriptor Designation Route of Entry (Oral, Inhalation, or Both) Comments "Carcinogenic to Humans " N/A N/A Convincing epidemiologic evidence of a causal association between human exposure to nitrofen and cancer does not exist. "Likely to Be Carcinogenic to Humans" Selected Oral feed The available evidence of carcinogenicity in Osborn-Mendel rats (lymphoma, pancreatic carcinomas, and ovary granulose cell tumors in females) and B6C3Fi mice (hepatocellular carcinomas and adenomas in males and females) exposed orally (in feed) to nitrofen indicates that nitrofen is likely to be carcinogenic to humans. "Suggestive Evidence of Carcinogenic Potential" N/A N/A The evidence from human and animal data is more than suggestive of carcinogenicity, which raises a concern for carcinogenic effects but is judged sufficient for a stronger conclusion. "Inadequate Information to Assess Carcinogenic Potential" N/A N/A Available adequate information exists to assess carcinogenic potential. "Not Likely to Be Carcinogenic to Humans " N/A N/A No strong evidence of noncarcinogenicity in humans is available. 42 Nitrofen ------- FINAL 11-5-2012 DERIVATION OF PROVISIONAL CANCER POTENCY VALUES Derivation of Provisional Oral Slope Factor (p-OSF) NCI (1978d) is selected as the principal study for derivation of the p-OSF. The cancer endpoint is the incidence of hepatocellular carcinomas in female mice. Details of this study are provided in the "Review of Potentially Relevant Data" section. It should be noted that while pancreatic tumors in the rat reported by NCI (1978c) were observed at a lower DOSEadj,hed (46.97 mg/kg-day) than the lowest DOSEadj,hed for liver tumors in the mouse (65.38 mg/kg-day) (NCI, 1978d), the mouse liver tumors reported by NCI (1978d, 1979d) provided the clearest evidence for carcinogenic potential and the greatest statistical power for analysis. For example, NCI (1978c) reported pancreatic carcinomas in 14% (7/50) of female rats at the adjusted human equivalency dose of 46.97 mg/kg-day. In comparison, hepatocellular carcinomas were seen in 88% (36/41) of female mice at the slightly higher adjusted human equivalency dose of 65.38 mg/kg-day (NCI, 1978d). For further comparison, BMD modeling was conducted using data for liver tumor formation reported by the two NCI mouse studies and pancreatic carcinomas seen in rats (NCI, 1978c). The lowest BMDLioadj,hed of 2.6 for hepatocellular carcinomas in female mice (see Figure C.2.) was over 9 times lower than the BMDLioadj,hed of 24.1 for rat pancreatic carcinomas. Dosimetric adjustments were made for oral dietary administration of nitrofen by adjusting doses for oral cancer analysis (p-OSF). A sample calculation is shown below for the female low-dose group in NCI (1978d). DOSEadj,hed = dose x food consumption per day x (l -h body weight) x (days dosed total days) x (body weight animal ^ body weight human)0'25 = 2348 ppm x 0.0061 kg/day x (1 - 0.026475258 kg) x (546 - 630) x (0.026475258 kg - 70 kg)0'25 = 65.38 mg/kg-day Table B.9 presents BMD input data for incidence of hepatocellular carcinomas in female mice exposed to nitrofen in feed for 78 weeks. Hoover et al. (1980) compared the histological characteristics of the liver tumors induced by nitrofen to the spontaneous neoplasms seen in the control animals. The nitrofen-induced tumors generally consisted of solid sheets or nodules composed of large eosinophilic hepatocytes. A total of three basophilic tumors were seen in the low-dose males. However, all of the tumors examined in treated females were eosinophilic in nature. Spontaneously formed tumors seen in the controls consisted of small basophilic cells arranged in solid or trabecular fashion. The differences in histological characteristics between the liver tumors from mice exposed to nitrofen and those occurring in control mice suggest that nitrofen induces unique liver tumors and does not act as a promoter of neoplasms formed spontaneously. Given the large increase in incidence of the mouse liver tumors compared to controls along with the observed morphological differences, it is concluded that the mouse liver tumors are relevant for deriving the p-OSF. As shown in Table C.2, hepatocellular carcinomas in female mice from the NCI (1978d) study provide the lowest credible POD and are, therefore, used for derivation of the p-OSF. Adequate model fit is obtained for the hepatocellular carcinomas using the multistage-cancer model. The modeling results yield a BMDLioadj,hed of 2.6 mg/kg-day. The curve and BMD output text are provided for the selected model in the BMD supplement to this document found in Appendix C (see Figure C.2 and the text output that follows the figure). 43 Nitrofen ------- FINAL 11-5-2012 p-OSF - 0.1 BMDLioadj,hed = 0.1 2.6 mg/kg-day = 3.8 x 10"2 (mg/kg-day)"1 Derivation of Provisional Inhalation Unit Risk (p-IUR) No human or animal studies examining the carcinogenicity of nitrofen following inhalation exposure have been identified. Therefore, derivation of a p-IUR is precluded. 44 Nitrofen ------- FINAL 11-5-2012 APPENDIX A. PROVISIONAL SCREENING VALUES No screening values are presented. 45 Nitrofen ------- FINAL 11-5-2012 APPENDIX B. DATA TABLES Table B.l. Clinical Chemistry Values of Male Sprague-Dawley Rats Orally Exposed to Nitrofen for 15 Weeks" Parameter Exposure Group, ppm (Adjusted Daily Dose, mg/kg-day) 0 100 (7) 500 (37) 2500 (186) Sample size 25 25 25 24 Glucose (mg/dL) 102.0 ± 11.1 98.6 ± 13.7 (97) 99.3 ± 10.4 (97) 88.0 ± 12.0b (86) Total protein (g/dL) 6.1 ±0.2 6.0 ±0.3 (98) 6.1 ±0.3 (100) 6.9 ± 0.3b (113) Albumin (g/dL) 3.5 ±0.2 3.5 ±0.2 (100) 3.5 ±0.2 (100) 3.9 ± 0.3b (111) Globulin (g/dL) 2.5 ±0.3 2.5 ±0.2 (100) 2.5 ±0.2 (100) 2.9 ± 0.3b (116) Cholesterol (mg/dL) 55.8 ± 12.5 52.9 ± 15.4(95) 64.8 ± 17.4 (116) 119.9 ±32.5b (215) aValues are mean ± SD (% of control) bSignificantly different from control (p < 0.05) as reported by study authors. Source: O'Hara et al. (1983). Table B.2. Body and Organ Weights of Male Sprague-Dawley Rats Orally Exposed to Nitrofen for 15 Weeks" Parameter Exposure Group, ppm (Adjusted Daily Dose, mg/kg-day) 0 100 (7) 500 (37) 2500 (186) Sample size 25 25 25 24 Terminal body weight Absolute (g) 479 ± 43 460 ± 42 (96) 462 ± 39 (96) 420 ± 39b (88) Kidney weight Absolute (g) 3.33 ±0.47 3.31 ±0.41 (99) 3.48 ±0.37 (105) 3.44 ±0.32 (103) Relative 0.70 ±8.1 0.72 ±6.2 (104) 0.75 ±4.7b (108) 0.82 ±5.7b (118) Liver weight Absolute (g) 12.5 ±2.2 12.0 ± 1.6 (96) 13.7 ± 1.7 (110) 19.5 ±2.4b (156) Relative 2.61 ±36 2.61 ± 19(100) 2.96 ± 17b (113) 4.66 ±42b (179) Testes weight Absolute (g) 3.15 ± 0.41 3.14 ±0.32 (100) 3.48 ±0.33b (110) 3.52 ± 0.23b(112) Relative 0.67 ± 10.9 0.69 ±8.9 (103) 0.76 ±8.3b (114) 0.85 ± 10.5b (127) aValues are mean ± SD (% of control); relative organ weights reported as percentage of body weight / 100. bSignificantly different from control (p < 0.05) as reported by study authors. Source: O'Hara et al. (1983). 46 Nitrofen ------- FINAL 11-5-2012 Table B.3. Relative Organ Weights of Male and Female Wistar Rats Orally Exposed to Nitrofen for 13 Weeks3 Parameter Exposure Group, ppm (Adjusted Daily Dose, mg/kg-day) Male 0 100 (9) 500 (46) 2500 (230) 12,500 (1152) Sample size 9 10 9 10 6 Heart (g/kg) 2.7 ±0.3 2.7 ±0.3 (100) 2.8 ±0.2 (104) 3.0 ± 0.6 (111) 3.5 ± 0.3b (130) Spleen (g/kg) 1.6 ±0.2 1.6 ± 0.1 (100) 1.5 ±0.1 (94) 1.9 ± 0.7 (119) 2.5 ±0.6b (156) Kidney (g/kg) 7.2 ±0.7 7.1 ±0.7 (99) 7.6 ±0.6 (106) 8.5 ± 0.6b (118) 10.5 ± 0.7b (146) Liver (g/kg) 33.7 ±5.0 34.9 ±5.1 (104) 40.9 ±6.2b (121) 56.7 ± 5.9b (168) 121.7 ± 18. lb (361) Testes (g/kg) 8.7 ± 1.3 9.1 ±0.6 (105) 9.5 ±0.9 (109) 10.3 ±1.0b (118) 10.9 ± 3.9b (125) Female 0 100 (10) 500 (51) 2500 (256) 12,500 (1282) Sample size 9 10 9 10 6 Heart (g/kg) 3.0 ±0.1 3.3 ±0.4 (111) 3.0 ±0.2 (100) 3.2 ±0.5 (111) 4.3 ±0.7b (143) Spleen (g/kg) 2.5 ± 1.0 2.9 ±1.2 (113) 2.7 ±0.9 (108) 2.6 ±1.0 (107) 2.8 ±1.1 (112) Kidney (g/kg) 7.1 ± 0.5 7.6 ±0.6 (116) 7.8 ± 0.8 (110) 8.2 ±0.5 (104) 11.1 ±1.3b (156) Liver (g/kg) 33.5 ±2.4 37.3 ± 3.9b (107) 39.5 ± 2.9b (118) 52.1 ± 4.5b (115) 101.0 ±5.4b (301) aValues are mean ± SD (% of control). bSignificantly different from control (p < 0.05) as reported by study authors. Source: Ambrose et al. (1971a). Table B.4. Survival, Clinical Effects, and Mean Weight Loss of Male and Female Fischer F344 Rats Orally Exposed to Nitrofen for 4 Weeks Parameter Exposure Group, ppm (Adjusted Daily Dose, mg/kg-day) Male 0 6800 (453) 10,000 (667) 14,670 (978) 21,560 (1437) 31,530 (2102) Sample size 5 5 5 5 5 5 No. survived (%) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 1(20) Mean weight gain3 -- -13 -17 -36 -52 -58 No. with arched backs (%) 0 0 0 0 0 4 (80) Female 0 6800 (512) 10,000 (753) 14,670 (1104) 21,560 (1623) 31,530 (2373) Sample size 5 5 5 5 5 5 No. survived (%) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 1(20) Mean weight gain3 -- -24 -16 -34 -43 -47 No. with arched backs (%) 0 0 0 0 0 4 (80) "Mean body-weight gain less than that of controls indicated by values are percentages as reported by study authors. Source: NCI (1979a). 47 Nitrofen ------- FINAL 11-5-2012 Table B.5. Survival, Clinical Observations, and Mean Weight Loss of Male and Female B6C3Fi Mice Orally Exposed to Nitrofen for 4 Weeks Parameter Exposure Group, ppm (Adjusted Daily Dose, mg/kg-day) Male 0 1180 (142) 2550 (307) 5500 (661) 13,900 (1672) 25,520 (3069) Sample size 5 5 5 5 5 5 No. survived (%) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 2(40) Mean weight gain3 -- 5 -5 -4 -4 1 Clinical signs (%) 0 0 0 0 5b(100) 5b (100) Female 0 1180 (153) 2550 (332) 5500 (715) 13,900 (1808) 25,520 (3320) Sample size 5 5 5 5 5 5 No. survived (%) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 3 (60) Mean weight gain3 -- 7 -1 8 -2 3 Clinical signs (%) 0 0 0 0 5b(100) 5b'° (100) "Mean body-weight gain relative to controls (±%). bRough hair and arched backs. °Mottled kidneys. Source: NCI (1979b). 48 Nitrofen ------- FINAL 11-5-2012 Table B.6. Mean Body Weights and Mortalities of Male and Female Wistar-Derived Albino Rats Orally Exposed to Nitrofen for 97 Weeks" Parameter Exposure Group, ppm (Adjusted Daily Dose, mg/kg-day) Male 0 10 (1.09) 100 (11.5) 1000 (116) Start 58 ±6 58 ±7 [100] 58 ±6 [100] 58 ±6 [100] Week 1 83 ±9 80 ±13 [96] 78 ± 11 [94] 75 ± 12 [90] Week 3 153 ±26 149 ± 29 [97] 146 ± 23 [95] 139 ±22 [91] Week 6 264 ± 33 258 ±32(1) [98] 252 ± 30 [95] 231 ±30b [88] Week 13 375 ±38 369 ± 45 [98] 362 ± 34 [97] 351 ±32 (2) [94] Week 26 458 ±55 450 ± 45 (2) [98] 435 ±42(1) [95] 432 ± 30 [94] Week 52 504 ± 65 (3) 486 ± 46 (7) [98] 465 ± 50b (5) [92] 458 ± 37b (5) [91] Week 78 513 ±69 (7) 506 ± 97 (14) [99] 452 ± 55b (10) [88] 457 ± 45b (10) [89] Week 97 489 ±65 (10) 460 ± 58 (20) [94] 423 ± 58b (14) [87] 447 ±41 (16) [91] Female 0 10 (1.17) 100 (12.2) 1000 (127) Start 54 ±5 54 ±7 [100] 54 ± 5 [100] 53 ± 7 [98] Week 1 73 ±9 73 ±10 [100] 68 ± 12 [93] 70 ± 9 [96] Week 3 129 ± 13 127 ± 13 (2) [98] 118 ± 16b (1) [91] 120 ± 14b [93] Week 6 182 ± 17 185 ± 12(3) [102] 174 ± 12 [96] 172 ± 18b [95] Week 13 232 ± 20 (2) 233 ± 16 [100] 221 ± 14 (2) [95] 215 ± 22b (3) [93] Week 26 281 ±28 275 ± 20 [98] 264 ± 15b (3) [94] 259 ± 32b [92] Week 52 321 ±46 (3) 308 ±31 (5) [96] 311 ±31 (5) [97] 300 ± 46 (7) [93] Week 78 320 ±53 (15) 333 ±53 (11) [104] 325 ±41 (12) [102] 307 ± 46 (13) [96] Week 97 320 ± 12 (20) 340 ±31 (17) 313 ±47 (20) 274 ± 44 (19) [86] aWeights expressed as mean (g) ± SD; (cumulative mortality [% of control]). bSignificantly different from control (p < 0.05) as reported by the study authors. Source: Ambrose et al. (1971c). 49 Nitrofen ------- FINAL 11-5-2012 Table B.7. Organ-to-Body-Weight Ratios of Male and Female Wistar-Derived Rats Orally Exposed to Nitrofen for 97 Weeks" Parameter Exposure Group, ppm (Adjusted Daily Dose, mg/kg-day) Male 0 10 (1.09) 100 (11.5) 1000 (116) Sample sizeb 5 8 5 6 Heart (g/kg) 3.5 ±0.7 3.0 ±0.4 (86) 3.2 ±0.5 (91) 3.4 ±0.4 (97) Spleen (g/kg) 2.1 ±0.6 1.8 ±0.4 (86) 2.0 ±0.4 (95) 2.1 ±0.5 (100) Kidney (g/kg) 8.4 ±0.9 8.8 ±2.2 (105) 8.8 ± 1.6(105) 10.7 ±2.5C (127) Liver (g/kg) 37.1 ±3.9 34.9 ±2.7 (94) 37.0 ±7.9 (100) 50.4 ±5.9C (136) Testes (g/kg) 7.3 ± 1.2 6.8 ±0.8 (93) 8.2 ±2.0 (112) 7.8 ± 1.1 (107) Female 0 10 (1.17) 100 (12.2) 1000 (127) Sample sizeb 15 5 11 9 Heart (g/kg) 4.0 ±0.3 3.6 ±0.4 (90) 4.3 ± 1.1 (108) 4.0 ±0.3 (100) Spleen (g/kg) 3.6 ±0.8 2.8 ± 0.7 (78) 3.2 ±0.9 (89) 2.6 ± 0.5° (72) Kidney (g/kg) 10.9 ± 1.4 9.5 ± 1.4 (87) 10.2 ± 1.3 (94) 10.0 ± 2.4 (92) Liver (g/kg) 42.7 ±4.9 39.8 ±3.4 (93) 43.4 ±6.6 (102) 46.9 ±9.2 (110) aValues are mean ratios of organ to body weight ± SD (% of control). bAnimals that survived to study termination were included. Significantly different from control (p < 0.05), as reported by the study authors. Source: Ambrose et al. (1971c). 50 Nitrofen ------- FINAL 11-5-2012 Table B.8. Incidence of Neoplasms in Female Osborne-Mendel Rats Orally Exposed to Nitrofen for 78 Weeks Exposure Group, ppm (Human Equivalency Dose, mg/kg-day) Parameter 0 1300 (21.88) 2600 (46.97) Hematopoietic System Lymphoma 0/20 (0%) 0/50 (0%) 4/50 (6%)* Pancreas Carcinoma 0/20 (0%) 2/50 (4%) 7/50 (14%)** Uterus Carcinoma 0 0 2/49 (4%) Squamous cell carcinoma 0 1/50 (2%) 0 Adenocarcinoma 0 1/50 (2%) 1/49 (2%) Endometrial stromal polyp 2/20 (10%) 3/50 (6%) 1/49 (2%) Endometrial stromal sarcoma 0 1/50 (2%) 0 Vagina Carcinoma 0 0 1/50 (2%) Squamous cell carcinoma 0 0 1/50 (2%) Ovary Carcinoma 0 1/50 (2%) 2/49 (4%) Squamous cell carcinoma 0 1/50 (2%) 0 Cystadenocarcinoma 0 1/50 (2%) 0 Granulosa-cell tumor 0 0 4/49 (8%)* Granulosa-cell carcinoma 1/20 (5%) 1/50 (2%) 0 * Significantly different from control (p < 0.05), as reported by the study authors. **Significantly different from control (p < 0.01), as reported by the study authors. Source: NCI (1978c). 51 Nitrofen ------- FINAL 11-5-2012 Table B.9. Incidence of Neoplasms in Male and Female B6C3Fi Mice Orally Exposed to Nitrofen for 78 Weeks Parameter Exposure Group, ppma (Human Equivalency Dose, mg/kg-day) Male 0 2348 (60.70) 4696 (128.26) Subcutaneous tissue Fibroma 0 2/44 (5%) 0 Fibrosarcoma 0 8/44 (18%)* 0 Hemangiopericytoma 0 1/44 (2%) 0 Neurofibroma 0 1/44 (2%) 0 Liver Hepatocellular carcinoma 4/20 (20%) 36/49 (73%)** 46/48 (96%)** Hemangiosarcoma 0 1/44 (2%) 4/48 (8%) Urinary bladder Transitional-cell carcinoma 0 0 2/40 (5%) Female 0 2348 (65.38) 4696 (137.68) Liver Hepatocellular carcinoma 0 36/41 (88%)** 43/44 (98%)** Hemangiosarcoma 0 0 4/44 (9%) Urinary Bladder Transitional-cell papilloma 0 0 1/41 (2%) "Represents time-weighted average concentrations over the 78-week treatment period as reported by study authors. * Significantly different from control (p < 0.05), as reported by the study authors. **Significantly different from control (p < 0.01), as reported by the study authors. Source: NCI (1978d). 52 Nitrofen ------- FINAL 11-5-2012 Table B.10. Incidence of Neoplasms in the Livers of Male and Female B6C3Fi Mice Orally Exposed to Nitrofen for 78 Weeks Parameter Exposure Group, ppm (Human Equivalency Dose, mg/kg-day) Male 0 3000 (70.66) 6000 (147.03) Sample size 20 49 48 Hyperplasia3 0 9 (18%) 4 (8%) Hepatocellular adenoma or carcinoma 1 (5%) 31 (63%)** 40 (83%)** Hepatocellular carcinoma 0 13 (27%)** 20 (42%)** Bile duct carcinoma (hepatoblastoma) 0 3 (6%) 4 (8%) Female 0 3000 (76.47) 6000 (160.09) Sample size 18 48 50 Hyperplasia13 0 11 (23%) 22 (22%) Hepatocellular adenoma or carcinoma 0 14 (29%)** 30 (60%)** Hepatocellular carcinoma 0 5 (10%) 13 (26%)* Bile duct carcinoma (hepatoblastoma) 0 1 (2%) 0 aAdjusted daily doses calculated for noncancerous endpoints in males are 0, 515, and 1029 mg/kg-day bAdjusted daily doses calculated for noncancerous endpoints in females are 0,518, and 1037 mg/kg-day. * Significantly different from control (p < 0.05), as reported by the study authors. **Significantly different from control (p < 0.01), as reported by the study authors. Source: NCI (1979d). Table B.ll. Terminal Body Weight and Organ-to-Body-Weight Ratios of Male and Female Beagle Dogs Orally Exposed to Nitrofen for 2 Years3 Parameter Exposure Group, ppm (Adjusted Daily Dose, mg/kg-day) 0 20 (0.36) 200 (3.9) 2000 (38) Sample sizeb 4 4 4 4 Terminal body weight (kg) 10.99 ±0.36 11.30 ± 1.39 (103) 10.50 ± 1.83 (96) 10.65 ± 2.02 (97) Heart (g/kg) 8.17 ± 1.53 7.87 ±0.99 (96) 7.73 ± 1.37 (95) 7.93 ± 1.25 (97) Spleen (g/kg) 8.85 ±2.40 6.42 ± 1.04 (73) 6.06 ± 2.67 (68) 7.55 ± 3.09 (85) Kidney (g/kg) 4.58 ±0.29 5.23 ±0.82 (114) 4.63 ±0.69 (101) 5.67 ±0.64 (124) Liver (g/kg) 26.25 ± 2.8 27.13 ±3.3 (103) 28.63 ± 1.8 (109) 40.45 ±4.3* (154) Testes0 (g/kg) 1.94 2.14(110) 1.85 (95) 1.97 (102) aValues are mean ± SD (% relative to control). bTwo dogs of each sex per diet level. °SD not reported by study authors. * Significantly different from control (p < 0.05), as reported by the study authors. Source: Ambrose et al. (1971d). 53 Nitrofen ------- FINAL 11-5-2012 Table B.12. Neonatal Effects on Sprague-Dawley Rat Pups Exposed to Nitrofen on GDs 8-16 Parameter Exposure Group, mg/kg-day 0 0.46 1.39 4.17 12.5 No. of litters 9 9 11 10 7 No. of pups/litter Day 0 (%)a 12.1 8.2b(68) 10.5 (87) 9.2° (76) 10.1 (83) No. live pups Day 1 (%)a 11.9 8.0b (67) 9.9 (83) 8.9° (75) 8.4° (71) No. live pups Day 2 (%)d 11.8(98) 8.0b (98) 9.8 (93) 8.9° (97) 8.4° (83) No. live pups Day 6 (%)d 11.6 (96) 7.7b (89) 9.3 (88) 8.5° (84) 8.1° (78) No. dead pups recovered Day 0 4 2 4 3 5 No. pups with diaphragmatic hernias (%)e 0 0 3(75) 2(67) 5 (100) No. litters from which dead pups recovered Day 0 (%)f 2(22) 2 (22) 3 (27) 3 (30) 3(43) No. litters with pups having diaphragmatic hernias (%)f 0 0 3 (27) 2(20) 3(43) Average pup weight Day 1 (g) (%)a 6.9 7.2 (104) 7.0 (101) 6.8 (99) 6.7 (97) Average pup weight Day 2 (g) (%)a 8.1 8.2 (101) 7.8 (96) 8.0 (99) 7.7 (95) "Percent of control; calculated for this review. bSignificantly different from control (p < 0.01) by t-test performed by study authors. Significantly different from control (p < 0.05) by /-test performed by study authors. dPercent survival from Day 1; provided by study authors. "Percent of total no. dead pups Day 0; calculated for this review. fPercent of total no. litters; calculated for this review. Source: Ostby et al. (1985). 54 Nitrofen ------- FINAL 11-5-2012 Table B.13. Effects on Locomotor Activity of Sprague-Dawley Rats Exposed to Nitrofen on GDs 8-16a Parameter Exposure Group, mg/kg-day 0 0.46 1.39 4.17 12.5 Block 1 Day 17 251 ± 31 305 ±38 (122) 323 ±38 (129) 273 ± 34 (109) 345 ± 34b (137) Day 24 266 ± 38 307 ±38 (115) 363 ± 38 (136) 421± 38° (158) 359 ±44 (135) Days 45 & 49 682 ± 66 632 ± 63 (93) 638 ±61 (94) 660 ± 69 (97) 689 ±59 (101) Day 90 749 ± 53 734 ± 50 (97) 648 ± 63 (87) 719 ±95 (96) 748 ± 65 (100) Block 2 Day 17 234 ± 22 - - " 306 ± 23b (131) Day 24 294 ± 25 - - " 377 ± 32b (128) Blocks 1 & 2 Days 17 & 24 264 ± 16 304 ±29 (115) 341 ±29b (129) 345 ± 28b (131) 341 ± 20° (129) aValues are mean ± SD (% of control, calculated for this review); values represent number of photocell beam interruptions in a figure-8 maze with 8 photocells over a 1-hour period. Blocks refer to sequential experiments. bSignificantly different from control (p < 0.05) as reported by study authors. Significantly different from control (p < 0.01) as reported by study authors. Source: Ostby et al. (1985). 55 Nitrofen ------- FINAL 11-5-2012 Table B.14. Necropsy Results of 133-161-Day-Old Male Sprague-Dawley Rats Exposed to Nitrofen on GDs 8-16a Parameter Exposure Group, mg/kg-day 0 0.46 1.39 4.17 12.5 Sample size 25 25 25 22 17 Body weight (g) 640.9 ± 76b 652.9 ±70 (102) 621.2 ±53 (97) 644.9 ±66 (101) 655.3 ± 59 (102) Seminal vesicle weight (g) 1.96 ±0.29 1.98 ±0.33 (101) 1.97 ±0.38 (101) 1.90 ±0.31 (97) 1.90 ±0.35 (97) Harderian gland weight (g) 0.392 ±0.06 0.385 ±0.05 (98) 0.391 ±0.04 (100) 0.358 ±0.07c (91) 0.303 ± 0.04d (77) No. animals with hydronephrotic kidneys (%) 0 0 0 3(14) 6(35) No. animals with porphyrin rings around one or both eyes (%) 0 0 0 0 13 (76) Sample size 6 6 6 6 6 Testes weight (g) 3.85 ±0.29 3.39 ±0.61 (88) 3.71 ±0.22 (96) 3.86 ±0.42 (100) 3.90 ±0.26 (101) Liver weight (g) 27.01 ±4.46 27.78 ±3.96 (103) 24.83 ±2.76 (92) 30.79 ±5.12 (114) 25.72 ±3.90 (95) Lung weight (g) 1.80 ±0.19 1.85 ± 0.11 (103) 1.98 ±0.42 (110) 2.13 ±0.23 (118) 1.88 ±0.13 (104) Right kidney weight (g) 2.40 ±0.17 2.47 ± 0.22 (103) 2.31 ±0.28 (96) 2.70 ±0.21 (113) 2.39 ±0.24 (100) "Absolute weights expressed as mean ± SD (% of control). bSample size = 24. Significantly different from control (p < 0.05) as reported by study authors. Significantly different from control (p < 0.01) as reported by study authors. Source: Ostby et al. (1985). 56 Nitrofen ------- FINAL 11-5-2012 APPENDIX C. BMD OUTPUTS Subchronic and Chronic Endpoints: Table C.l. Model Predictions for Rat Litters with Pups Having Diaphragmatic Hernias Model Goodness-of-Fit p-Valuea AIC for Fitted Model BMD0S mg/kg-day BMDL0S mg/kg-day Conclusions Gamma 0.16 40.761 0.828 0.453 Weibull 0.16 40.761 0.828 0.453 LogProbit 0.14 42.643 2.866 1.191 LogLogistic 0.43 37.935 0.587 0.292 Lowest AIC Lowest BMDL Multistage 0.16 40.761 0.828 0.453 Logistic 0.20 42.235 2.624 1.547 Probit 0.20 42.125 2.386 1.427 Quantal Linear 0.16 40.761 0.828 0.453 "Values < 0.10 fail to meet conventional goodness-of-fit criteria. AIC = Akaike's Information Criteria; BMD = benchmark dose; BMDL = BMD lower limit 57 Nitrofen ------- FINAL 11-5-2012 Log-Logistic Model with 0.95 Confidence Level Log-Logistic 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 I3MDL BMD 0 2 4 6 8 10 12 dose 12:26 07/26 2010 Figure C.l. LogLogistic BMD Model for Rat Litters with Pups Having Diaphragmatic Hernia Data (Ostby et al., 1985) Text Output for LogLogistic BMD Model for Rat Litters with Pups Having Diaphragmatic Hernia Data (Ostby et al., 1985) Logistic Model. (Version: 2.13; Date: 10/28/2009) Input Data File: C:/l/Ostby_1985_LittersWithPupsDiaHern05_LogLogistic_l.(d) Gnuplot Plotting File: C:/l/Ostby_1985_LittersWithPupsDiaHern05_LogLogistic_l.plt Mon Jul 26 12:26:26 2010 [add_notes_here] The form of the probability function is: Pfresponse] = background+(l-background)/[l+EXP(-intercept-slope*Log(dose))] Dependent variable = DichEff Independent variable = Dose Slope parameter is restricted as slope >= 1 58 Nitrofen ------- FINAL 11-5-2012 Total number of observations = 5 Total number of records with missing values = 0 Maximum number of iterations = 250 Relative Function Convergence has been set to: le-008 Parameter Convergence has been set to: le-008 User has chosen the log transformed model Default Initial Parameter Values background = 0 intercept = -2.57481 slope = 1 Asymptotic Correlation Matrix of Parameter Estimates (*** The model parameters) -background -slope have been estimated at a boundary point, or have been specified by the user, and do not appear in the correlation matrix ) intercept intercept 1 Parameter Estimates 95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit background 0 * * * intercept -2.41098 * * * slope 1 * * * * - Indicates that this value is not calculated. Analysis of Deviance Table Model Log(likelihood) # Param's Deviance Test d.f. P-value Full model -16.2299 5 Fitted model -17.9674 1 3.47512 4 0.4817 Reduced model -21.2537 1 10.0477 4 0.03963 AIC: 37.9348 Goodness of Fit Scaled Dose Est._Prob. Expected Observed Size Residual 0.0000 0.0000 0.000 0.000 9 0.000 0.4600 0.0396 0.357 0.000 9 -0.609 1.3900 0.1109 1.220 3.000 11 1.709 4.1700 0.2723 2.723 2.000 10 -0.514 12.5000 0.5287 3.701 3.000 7 -0.530 ChiA2 = 3.84 d.f. = 4 P-value = 0.4283 59 Nitrofen ------- FINAL 11-5-2012 Benchmark Dose Specified effect = Risk Type Confidence level = BMD = BMDL = Computation 0.05 Extra risk 0.95 0.58657 0.292378 60 Nitrofen ------- FINAL 11-5-2012 Cancer Endpoints: Table C.2. Summary of the PODs for the p-OSF for Nitrofen Study and Year Endpoint Gender/ Species File Name BMD BMDL BMD/ BMDL /7-Value Test AIC Scaled Residual of Interest Model Notes NCI, 1978d Hepatocellular Carcinoma Female Mouse NCI_1978_new_Liver_HepaCarc _mouse_F_MultiCanc2_l.out 3.5 2.6 1.3 0.8849 42.18 0.000 Lowest BMDL NCI, 1978d Hepatocellular Carcinoma Male Mouse NCI_1978_new_Liver_HepaCarc_ mouse_M_MultiCanc2_l .out 7.3 4.1 1.8 NA 99.34 0.000 Failed p-valuc test NCI, 1979d Hepatocellular Adenoma or Carcinoma Male Mouse NCI_1979_new_Liver_HepaAdem Carc_mouse_M_MultiCanc2_ 1. out 8.4 6.7 1.3 0.6397 119.85 -0.052 NCI, 1979d Hepatocellular Adenoma or Carcinoma Female Mouse NCI_1979_new_Liver_HepaAdem Carc_mouse_F_MultiCanc2_ 1. out 27.7 16.1 1.7 1.0000 129.25 0.000 NCI, 1979d Hepatocellular Adenoma Male Mouse NCI_1979_new_Liver_HepaAdeno ma_mouse_M_MultiCanc2_ 1. out 27.3 19.2 1.4 0.1514 143.61 -0.397 NCI, 1979d Hepatocellular Carcinoma Male Mouse NCI_1979_new_Liver_HepaCarc_ mouse_M_MultiCanc2_l .out 26.9 20.4 1.3 0.8891 124.13 0.000 NCI, 1978c Pancreatic Carcinoma Female Rat NCI_1978_ne wPancCarcN 0 S_r at_F_MultiCanc2_ 1. out 38.3 24.1 1.6 1.0000 61.29 0.000 NCI, 1979d Hepatocellular Adenoma Female Mouse NCI_1979_new_Liver_HepaAdem _mouse_F_MultiCanc2_ 1. out 40.0 29.4 1.4 0.9941 112.44 0.088 NCI, 1979d Hepatocellular Carcinoma Female Mouse NCI_1979_new_Liver_HepaCarc_ mouse_F_MultiCanc2_l .out 73.9 43.0 1.7 1.0000 93.38 0.000 61 Nitrofen ------- FINAL 11-5-2012 Multistage Cancer Model with 0.95 Confidence Level ESMDL 60 80 dose 11:43 11/04 2010 Figure C.2. Multistage-Cancer BMD Model for Hepatocellular Carcinoma in Female Mouse Data (NCI, 1978d) Multistage Cancer Linear extrapolation Text Output for Multistage-Cancer BMD Model for Hepatocellular Carcinoma in Female Mouse Data (NCI, 1978d) Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010) Input Data File: C:/36/NCI_1978_new_Liver_HepaCarc_mouse_F_MultiCanc2_l.(d) Gnuplot Plotting File: C:/36/NCI_1978_new_Liver_HepaCarc_mouse_F_MultiCanc2_l.pit Thu Nov 04 12:43:27 2010 Incidence_of_Hepatocellular_Carcinoma_in_Female_B6C3Fl_Mice The form of the probability function is: P[response] = background + (1-background)*[1-EXP( -betal*dose/sl-beta2*dose/s2) ] The parameter betas are restricted to be positive 62 Nitrofen ------- FINAL 11-5-2012 Dependent variable = DichEff Independent variable = Dose Total number of observations = 3 Total number of records with missing values = 0 Total number of parameters in model = 3 Total number of specified parameters = 0 Degree of polynomial = 2 Maximum number of iterations = 250 Relative Function Convergence has been set to: le-008 Parameter Convergence has been set to: le-008 Default Initial Parameter Values Background = 0.101802 Beta(1) = 0.0274117 Beta(2) = 0 Asymptotic Correlation Matrix of Parameter Estimates ( *** The model parameter(s) -Background -Beta(2) have been estimated at a boundary point, or have been specified by the user, and do not appear in the correlation matrix ) Beta(1) Beta (1) 1 Parameter Estimates Interval Variable Limit Background Beta(1) Beta(2) Estimate 0. 0304142 0 Std. Err. 95.0% Wald Confidence Lower Conf. Limit Upper Conf. Indicates that this value is not calculated. Analysis of Deviance Table Model Full model Fitted model Reduced model Log(likelihood) -19.9753 -20.0879 -57.3584 Param's 3 1 1 Deviance Test d.f. 0.225146 74.7661 P-value 0.8935 <.0001 AIC: 42.1758 Goodness of Fit Scaled Dose Est._Prob. Expected Observed Size Residual 63 Nitrofen ------- FINAL 11-5-2012 0.0000 0.0000 0.000 0.000 19 0.000 65.3846 0.8631 35.388 36.000 41 0.278 137.6762 0.9848 43.332 43.000 44 -0.409 Chi^2 = 0.24 d.f. = 2 P-value = 0.8849 Benchmark Dose Computation Specified effect = 0.1 Risk Type = Extra risk Confidence level = 0.95 BMD = 3.4 6419 BMDL = 2.64773 BMDU = 7.22885 Taken together, (2.64773, 7.22885) is a 90 % two-sided confidence interval for the BMD Multistage Cancer Slope Factor = 0.0377682 64 Nitrofen ------- FINAL 11-5-2012 APPENDIX D. REFERENCES ACGIH (American Conference of Governmental Industrial Hygienists). (2011) Threshold limit values for chemical substances and physical agents and biological exposure indices. Cincinnati, OH. As cited in HSDB (Hazardous Substances Data Bank). Available online at http://toxnet.nlm.nih.eov/cei-bin/sis/htmleen7HSDB. Accessed on 12/07/2011. Acosta, JM; Chai, Y; Meara, JG; et al. (2001) Prenatal exposure to nitrofen induces Fryns phenotype in mice. Ann Plast Surg 46: 635-640. 625662 Ambrose, AM; Larson, PS; Borzelleca, JF; et al. (1971) (a,b,c,d,e) Toxicologic studies on 2,4-dichlorophenyl-p-nitrophenyl ether. ToxicolApplPharmacol 19(2):263-275. doi: 10.1016/0041 -008X(71)90111-6. 625664 ATSDR (Agency for Toxic Substances and Disease Registry). (2009) Toxicological profile information sheet. Atlanta, GA: U.S. Department of Health and Human Services, Public Health Service. Available online at http://www.atsdr.cdc.eov/toxprofites/index.asp. Accessed on 12/07/2011. Brandsma, AE; Tibboel, D; Vulto, IM; et al. (1994) Inhibition of T3-receptor binding by Nitrofen. Biochim Biophys Acta 1201(2):266-270. 627515 Brown, TJ; Manson, JM. (1986) Further characterization of the distribution and metabolism of nitrofen in the pregnant rat. Teratology 34(2): 129-139. doi: 10.1002/tera. 1420340202. 625670 Burke Hurt, SS; Smith, JM; Hayes, AW. (1983) Nitrofen: A review and perspective. Toxicology 29(1-2): 1-37. doi:10.1016/0300-483X(83)90036-7. 625661 Byeon, WH; Hyun, HH; Lee, SY. (1976) Mutagenicity of pesticides in the Salmonella/microsome system. J Microbiol 14:128-134. 628017. As cited in Burke Hurt (1983). CalEPA (California Environmental Protection Agency). (2007) Office of Environmental Health Hazard Assessment. Proposition 65. Available online at http://oehha.ca.eov/prop65/prop65 list/files/P65sinele040210.pdf and http://oehha.ca.eov/prop65/prop65 list/files/sinelelist040210.xls. Accessed on 12/07/2011. CalEPA (California Environmental Protection Agency). (2008) All OEHHA acute, 8-hour and chronic reference exposure levels (chRELs) as on December 18, 2008. Office of Environmental Health Hazard Assessment, Sacramento, CA. Available online at http://www.oehha.ca.eov/air/allrels.html. Accessed on 12/07/2011. Chase-Deesing, C; Kavlock, RJ; Zeman, FJ. (1986) Kidney morphology and function in the young of rats malnourished and exposed to nitrofen during pregnancy. J Toxicol Environ Health A Curr Iss 19: 1-21. doi: 10.1080/15287398609530902. 625657 65 Nitrofen ------- FINAL 11-5-2012 Clugston, RD, Zhang, W, Greer, JJ. (2010) Early development of the primordial mammalian diaphragm and cellular mechanisms of nitrofen-induced congenital diaphragmatic hernia. Birth Defects Res A ClinMol Teratol 88:15-24. doi:10.1002/bdra.20613. 625663 Costlow, RD; Manson, JM. (1981) The heart and diaphragm: target organs in the neonatal death induced by nitrofen (2,4-dichlorophenyl-p-nitrophenyl ether). Toxicology 20:209-227. doi: 10.1016/0300-483X(81)90052-4. 625653 Costlow, RD; Manson, JM. (1983) Distribution and metabolism of the teratogen nitrofen (2,4- dichloro-4'-nitro diphenyl ether) in pregnant rats. Toxicology 26:11-23. doi:10.1016/0300- 483X(83)90052-5. 627463 Daston, GP; Rehnberg, BF; Carver, B; et al. (1988) Functional teratogens of the rat kidney. II. Nitrofen and ethylenethiourea. Fundam Appl Toxicol 11 (3):401 —415. doi: 10.1016/0272- 0590(88)90105-4. 627500 Dunkel, VC; Zeiger, E; Brusick, D; et al. (1985) Reproducibility of microbial mutagenicity assays: II Testing of carcinogens and noncarcinogens in Salmonella typhimurium and Escherichia coli. Environ Mutagen 5:1-248. doi: 10.1002/em.2860070902. 017367 Featherstone, NC; Connell, MG; Fernig DG; et al. (2006) Airway smooth muscle dysfunction precedes teratogenic congenital diaphragmatic hernia and may contribute to hypoplastic lung morphogenesis. Am JRespir CellMolBiol 35(5):571—578. doi:10.1165/rcmb.2006-00790C. 627232 Francis, BM; Metcalf, RL; Lewis, PA; et al. (1999) Maternal and developmental toxicity of halogenated 4'-nitrodiphenyl ethers in mice. Teratology 59(2):69-80. 625680 Gray, LE Jr. (1984) An assessment of reproductive and thyroid function following prenatal nitrofen exposure in hamsters. Teratology 29: 32A-33A. doi: 10.1002/tera. 1420290219. 627257 Gray, LE Jr; Kavlock, RJ; Chernoff, N; et al. (1982a) Prenatal exposure to the herbicide 2,4-dichlorophenyl-p-nitrophenyl ether destroys the rodent Harderian gland. Science 215(4530):293-294. doi:10.1126/science.7053576. 627608 Gray, LE; Kavlock, RJ; Chernoff, N; et al. (1982b) The effects of prenatal exposure to the herbicide TOK on the postnatal-development of the harderian-gland of the mouse, rat, and hamster. Teratology 25:45A. doi: 10.1002/tera. 1420250218. 629656 Gray, LE Jr; Kavlock, RJ; Chernoff, N; et al. (1983a,b) Postnatal developmental alterations following prenatal exposure to the herbicide 2,4-dichlorophenyl-p-nitrophenyl ether: a dose response evaluation in the mouse. Toxicol Appl Pharmacol 67:1-14. doi: 10.1016/0041- 008X(83)90239-9. 625669 Gutenmann, WH; Lisk, DJ. (1967) Metabolism of TOK herbicide in the dairy cow. J Dairy Sci 50(9): 1516-1518. 629679 66 Nitrofen ------- FINAL 11-5-2012 Hoover, KL; Ward, JM; Stinson, SF. (1980) Histopathologic differences between liver tumors in untreated (C57BL/6 X C3H)F1 (B6C3F1) mice and nitrofen-fed mice. J Natl Cancer Inst 65:937-948. 625671 Hunt, LM; Chamberlain, WF; Gilbert, BN; et al. (1977) Absorption, excretion, and metabolism of nitrofenby a sheep. JAgric Food Chem 25(5): 1062-1065. doi:10.1021/jf60213a008. 627226 HSDB (Hazardous Substances Data Bank). (2011) Chemical database. National Library of Medicine, Toxicology Data Network. Available online at http://toxnet.nlm.nih.gov/cgi- bin/sis/htmleen?HSDB. IARC (International Agency for Research on Cancer). (2011) Monographs on the evaluation of carcinogenic risks to humans. Lyon, France: IARC. Available online at http://monographs.iarc.fr/ENG/Monographs/PDFs/index.php. Accessed on 12/07/2011. Ijsselstijn, H; Pacheco, BA; Albert, A; et al. (1997) Prenatal hormones alter antioxidant enzymes and lung histology in rats with congenital diaphragmatic hernia. Am J Physiol 272(6 Pt 1):L1059-L1065. 627609 IPCS (International Programme on Chemical Safety). (1999) Nitrofen. IPCS data sheet. Available online at http://www.inchem.org/documents/icsc/icsc/eics0929.htm. Jeang, CL; Li, GC. (1980) Screening of pesticides for mutagenicity in the microbial system: 2, with mammalian activation. Natl Sci CouncMon 8:551-559. 632494. As cited in Burke Hurt (1983). Kang YJ; Manson JM. (1987) Effect of prenatal propranolol-nitrofen exposure on pregnant rats. Teratology 35: 58A. doi:10.1002/tera. 1420350219. 627492 Kavlock, RJ; Gray, JA. (1983a) Morphometric, biochemical, and physiological assessment of perinatally induced renal dysfunction. J Toxicol Environ Health A 11: 1-13. doi: 10.1080/15287398309530316. 629654 Kavlock, RJ; Gray, LE Jr. (1983b) Postnatal evaluation of morphological and functional effects of prenatal exposure to nitrofen in the Long-Evans rat. J Toxicol Environ Health A ll(4-6):679-690. doi: 10.1080/15287398309530376. 625654 Kavlock, RJ; Chernoff, N; Rogers, E; et al. (1982) An analysis of fetotoxicity using biochemical endpoints of organ differentiation. Teratology 26(2): 183-194. doi: 10.1002/tera. 1420260211. 066110 Kavlock, RJ; Hoyle, BR; Rehnberg, BF; et al. (1988) The significance of the dilated renal pelvis in the nitrofen-exposed rat fetus: effects on morphology and function. Toxicol Appl Pharmacol 94(2):287-296. doi:10.1016/0041-008X(88)90270-0. 627668 Kim, WG; Suh, JW; Chi, JG. (1999) Nitrofen-induced congenital malformations of the heart and great vessels in rats: an animal model. JPediatr Surg 34(12): 1782-1786. doi: 10.1016/S0022-3468(99)90312-3. 625679 67 Nitrofen ------- FINAL 11-5-2012 Kimbrough, RD; Gaines, TB; Linder, RE. (1974) 2,4-Dichlorophenyl-p-nitrophenyl ether (TOK): effects on the lung maturation of rat fetus. Arch Environ Health 28(6):316-320. 627686 Kiryushin, VA. (1975) Mutagenic action of various pesticides in case of their successive entry into the body of albino rats. Gig Sanit 9:43. 632486 . As cited in Burke Hurt (1983). Lau, C; Cameron, AM; Irsula, O. (1986) Effects of prenatal nitrofen exposure on cardiac structure and function in the rat. ToxicolApplPharmacol 86:22-32. doi: 10.1016/0041 - 008X(86)90396-0. 031682 Mahboob, S; Kavlock, RJ; Zeman, FJ. (1985) Interaction between protein deprivation and nitrofen exposure on intestinal dipeptidase activities of the rat fetus. FedProc 44: 938. 627554 Manson, JM. (1986) Mechanism of nitrofen teratogenesis. Environ Health Perspect 70:137-147. 625655 Manson, JM; Brown, T; Baldwin, DM. (1984) Teratogenicity of nitrofen (2,4-dichloro- 4'-nitrodiphenyl ether) and its effects on thyroid function in the rat. Toxicol Appl Pharmacol 73(2):323-335. 625668. As Cited in Manson (1986). McGregor, DB; Riach, C; Cattanach, P. (1996) Mutagenic responses of L5178Y mouse cells at the tk and hprt loci. Toxicol In Vitro 10(5):643-647. 626421 McLeod, PL; McCarthy, KL. (1981) Tok dermal cytogenicity study in rats. Rohm and Haas report no. 81R-268 (unpublished). As cited in Burke Hurt (1983). Moriya, M; Ohta, T; Watanabe, K; et al. (1983) Further mutagenicity studies on pesticides in bacterial reversion assay systems. MutatRes 116(3-4): 185-216. 200489 NCI (National Cancer Institute). (1978). (a,b,c,d) Bioassay of nitrofen for possible carcinogenicity. Technical report series no. 26. U.S. Department of Health, Education and Welfare, Public Health Service, National Institutes of Health, Bethesda, MD. Available online at http://ntp.niehs.nih.gov/ntp/htdocs/LT rpts/tr026.pdf. 625659 NCI (National Cancer Institute). (1979). (a,b,c,d) Bioassay of nitrofen for possible carcinogenicity. Technical report series No. 184. U.S. Department of Health, Education and Welfare, Public Health Service, National Institutes of Health, Bethesda, MD. Available online at http://ntp.niehs.nih.gov/ntp/htdocs/LT rptsZtrl84.pdf. 625658 NIOSH (National Institute for Occupational Safety and Health). (2010) NIOSH pocket guide to chemical hazards. Index of chemical abstracts service registry numbers (CAS No.). Center for Disease Control and Prevention, U.S. Department of Health, Education and Welfare, Atlanta, GA. Available online at http://www.cdc.gov/niosh/npg/npgdcas.html. Accessed on 12/07/2011. Noble, BR; Babiuk, RP; Clugston, RD; et al. (2007) Mechanisms of action of the congenital diaphragmatic hernia-inducing teratogen nitrofen. Am J Physiol Lung Cell Mol Physiol 293:L1079-L1087. doi:10.1152/ajplung.00286.2007. 625660 68 Nitrofen ------- FINAL 11-5-2012 NTP (National Toxicology Program). (2011) 12th Report on carcinogens. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC. Available online at http://ntp-server.niehs.nih.gov/index.cfm7obiecticN 32BA9724-F1F6-975E-7FCE50709CB4C932. Accessed on 12/07/2011. O'Hara, GP; Chan, PK; Harris, JC; et al. (1983) The effect of nitrofen [4-(2,4- dichlorophenoxy)nitrobenzene] on the reproductive performance of male rats. Toxicology 28(4):323-333. doi:10.1016/0300-483X(83)90006-9. 625675 Oku, K. (1976) Effects of some herbicides on somatic cell division in barley. La chromosomo II-2:63. As cited in Burke Hurt (1983). OSHA (Occupational Safety and Health Administration). (2006) Air contaminants: occupational safety and health standards for shipyard employment, subpart Z, toxic and hazardous substances. U.S. Department of Labor, Washington, DC. OSHA Standard 1915.1000. Available online at http://www.osha.gov/pls/oshaweb/owadisp.show document?p table ST.WDARDS&p id=102 86. Accessed on 12/07/201 1. Ostby, JS; Gray, Le; Kavlock, RJ; et al. (1985) The postnatal effects of prenatal exposure to low doses of nitrofen (2,4-dichlorophenyl-p-nitrophenyl ether) in Sprague-Dawley rats. Toxicology 34(4):285-297. doi:10.1016/0300-483X(85)90139-8. 627666 Paik, SG; Lee, SY. (1977) Genetic effects of pesticides in the mammalian cells. II. Mutagenesis in L5178Y cells and DNA repair induction. Korean JZool 20:159-168. 627563. As cited in Burke Hurt (1983). Piersma, AH; Hofstee-Hooftman, MW; Verhoef, A. (1993) Early lung development in the rat and its perturbation by nitrofen treatment. Teratology 48:31 A. doi: 10.1002/tera. 1420480215. 627485 Raub, JA; Mercer, RR; Kavlock, RJ; et al. (1983) Effects of prenatal nitrofen exposure on postnatal lung function in the rat. Prog Clin Biol Res 140:119-134. 625672 Reustle, JA; Scribner, HE. (1980) Tok cytogenetic study in mice. Rohm and Haas report no. 79R-173 (unpublished). As cited in Burke Hurt (1983). Robinson, KS; Cameron, AM. (1984) In utero exposure to nitrofen induces postnatal changes in rat electrocardiograms. Teratology 29:54A~55A. doi: 10.1002/tera. 1420290219. 627511 Shirasu, Y; Moriya, M; Tezuka, H; et al. (1982) Mutagenicity screening studies on pesticides. Presented at 3rd International Conference on Environmental Mutagens, 09/21/1981-09/27/1981, New York. 201669. As cited in Burke Hurt (1983). Siou, G. (1978) Study of the potential mutagenic activity of TOK by the Howell-Jolly body technique (Unpublished). Cabinet d'Etudes et de Recherches en Toxicologic Industrielle. Versailles. MBL-2199. 627497 69 Nitrofen ------- FINAL 11-5-2012 Siou, G. (1979) Study of the effect of TOK on the prenatal and postnatal development of the rabbit (Unpublished). Cabinet d'Etudes et de Recherches en Toxicologie Industrielle. Versailles. MBL-2212. 627495 Stone, LC; Manson, JM. (1981) Effects of the herbicide 2, 4-dichlorophenyl-p-nitrophenyl ether (nitrofen) on fetal lung development in rats. Toxicology 20(2-3): 195-207. doi: 10.1016/0300-483X(81)90051-2. 625656 Tanaka, Y; Iwasaki, H; Kitamori, S. (1996) Biodegradation of herbicide chlornitrofen (CNP) and mutagenicity of its degradation products. Water Sci Technol 34(7-8): 15-20. 626418 U.S. EPA (Environmental Protection Agency). (1988) Recommendations for and documentation of biological values for use in risk assessment. Environmental Criteria and Assessment Office, Cincinnati, OH; EPA/600/6-87/008. Available online at http://cfpub.epa. gov/ncea/cfm/recordisplay.cfm?deid=34855#Download. 064560 U.S. (Environmental Protection Agency). (1991a) Chemical assessments and related activities (CARA). Office of Health and Environmental Assessment, Washington, DC. 596442 U.S. EPA (Environmental Protection Agency). (1991b) Guidelines for developmental toxicity risk assessment. U.S. Environmental Protection Agency, Risk Assessment Forum. Washington, DC; EPA/600/FR-91/001. Available online at http://cfpub.epa.gov/ncea/cfm/recordisplav.cfm?deid=23162. 008567 U.S. EPA (Environmental Protection Agency). (1994a) Chemical assessments and related activities (CARA). Office of Health and Environmental Assessment, Washington, DC; EPA/600/R-94/904. Available online at http://nepis.epa.gov/Exe/ZyPURL.cgi?Dockev=6000 lG8L.txt. 596444 U.S. EPA (Environmental Protection Agency). (1994b) Methods for derivation of inhalation reference concentrations (RfCs) and application of inhalation dosimetry. U.S. Environmental Protection Agency, Office of Research and Development, Office of Health and Environmental Assessment, Washington, DC; EPA/600/8-90/066F. Available online at http://cfpub.epa.gov/ncea/cfm/recordisplav.cfm?deid=71993. 006488 U.S. EPA (Environmental Protection Agency). (1997) Health effects assessment summary tables (HEAST). FY-1997 Update. Prepared by the Office of Research and Development, National Center for Environmental Assessment, Cincinnati, OH for the Office of Emergency and Remedial Response, Washington, DC; EPA/540/R-97/036. NTIS PB97-921199. Available online at nepis.epa.gov/Exe/ZvPURL.cgi?Dockev=2000Q0GZ.txt Accessed 12/07/2011. 595422 U.S. EPA (U.S. Environmental Protection Agency). (1998) Status of pesticides in registration, reregi strati on, and special review (aka rainbow report). Office of Pesticide Programs, Washington, DC. Available online at http://www.epa.gov/oppsrrdl/Rainbow/98rainbo.pdf. Accessed on 12/07/2011. 70 Nitrofen ------- FINAL 11-5-2012 U.S. EPA (Environmental Protection Agency). (2006) 2006 edition of the drinking water standards and health advisories. Office of Water, Washington, DC; EPA/822/R-06/013. Available online at http://www.epa.gov/waterscience/drinkine/standards/dwstandards.pdf. Accessed on 12/07/2011. U.S. EPA (Environmental Protection Agency). (2012) Benchmark dose technical guidance. Risk Assessment Forum, Washington, DC; EPA/100/R-12/001. Available online at http://www.epa.gov/raf/publications/pdfs/benchm.ark dose guidance.pdf. U.S. EPA (U.S. Environmental Protection Agency). (2012) Integrated risk information system (IRIS). Office of Research and Development, National Center for Environmental Assessment, Washington, DC. Available online at http://www.epa.gov/iris/. Accessed on 1/10/2012. WHO/FAO (World Health Organization/Food and Agriculture Organization). (1996) Nitrofen. WHO/FAO data sheets on pesticides, No. 84. Geneva: WHO. Available online at http://www.incheni.org/docum.ents/pds/pds/pest84 e.htm. WHO (World Health Organization). (2011) Online catalogs for the Environmental Health Criteria Series. Available online at http://www.who.int/ipcs/publications/ehc/en/. Accessed on 12/07/2011. Zeman, FJ; Heng, H; Hoogenboom, ER; et al. (1986) Cell number and size in selected organs of fetuses of rats malnourished and exposed to nitrofen. Teratog CarcinogMutagen 6(4):339-347. doi: 10.1002/tcm. 1770060409. 627351 71 Nitrofen ------- |