UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D. C. 20460 OFFICE OF PREVENTION PESTICIDES AND TOXIC SUBSTANCES DATE: December 15, 2003 MEMORANDUM SUBJECT: PCNB ( PC Code 056502) Toxicology Disciplinary Chapter for the Reregistration Eligibility Decision ( RED)

From: Laurence D. Chitlik , D. A. B. T., Senior Toxicologist Toxicology Branch Health Effects Division ( 7509C)

THROUGH: Alberto Protzel, Ph. D. , Senior Scientist Toxicology Branch Health Effects Division ( 7509C)

TO: Diana Locke Reregistration Branch 2 Health Effects Division ( 7509C)

PC Code: 056502 TXR No.: 056502 DP Barcode: 296860 ACTION REQUESTED: Prepare a toxicology chapter for the RED for PCNB RESPONSE: The toxicology database for PCNB has been reviewed by Toxicology Branch, the Health Effects Division Hazard Identification Assessment Review Committee ( HIARC), Cancer Peer Review Committee ( CPRC), the HED FQPA Safety Factor Committee, and the Metabolism Assessment Review Committee ( MARC). Although a complete data base is not currently available to support a risk assessment for PCNB ( since key kinetic, metabolism and target organ toxicity are unavailable and need to be submitted by the Registrants), available data for PCNB are presented in this document in support of a Reregistration Eligibility Decision. Page 2 PCNB ( Pentachloronitrobenzene)

PC Code: 056502 Toxicology Disciplinary Chapter for the Reregistration Eligibility Decision August 13, 2003 Prepared by: Laurence D. Chitlik, DABT Senior Toxicologist Toxicology Branch Health Effects Division Mail Code 7509C Peer Reviewed by : Alberto Protzel Senior Scientist Toxicology Branch Health Effects Division Mail Code 7509C _______________________________________ Laurence D. Chitlik, DABT, Senior Toxicologist _______________________________________ Alberto Protzel,, Senior Scientist Page 3 EPA Reviewer: Laurence D. Chitlik____________________________ Date___________ Toxicology Branch ( 7509C) EPA Secondary Reviewer: Alberto Protzel_______________________ Date___________ Toxicology Branch ( 7509C)

TABLE OF CONTENTS 1.0 HAZARD CHARACTERIZATION 4 2.0 REQUIREMENTS 6 3.0 DATA GAPS( S) 8 4.0 HAZARD ASSESSMENT 9 4.1 Acute Toxicity 9 4.2 Subchronic Toxicity 10 4.3 Prenatal Developmental Toxicity 13 4.4 Reproductive Toxicity 16 4.5 Chronic Toxicity 18 4.6 Carcinogenicity 21 4.7 Mutagenicity 24 4.8 Neurotoxicity 25 4.9 Metabolism 26 4.10 Special Studies 29 5.0 TOXICITY ENDPOINT SELECTION ( HIARC) 30 5.1 Dietary Exposure 5.1.1 Acute Reference Dose ( aRfD) 30 5.1.2 Chronic Reference Dose ( cRfD) 31 5.2 Incidental Oral Exposure 32 5.3 Dermal Absorption 33 5.4 Short Term Dermal (( 1 30 days) Exposure 34 5.5 Intermediate Term Dermal ( 6 months) Exposure 34 5.6 Long Term Dermal (> 6 months) Exposure 35 5.7 Inhalation Exposure: Short Term ( 1 30 days) 35 5.8 Inhalation Exposure: Intermediate Term ( 1 6 Months) 35 5.9 Inhalation Exposure: Long Term (> 6 Months) 36 5.10 Margins of Exposure 36 5.11 Recommendation for Aggregate Exposure Risk Assessments 37 6.0 CLASSIFICATION OF CARCINOGENIC POTENTIAL 37 6.1 Combined Chronic Toxicity/ Carcinogenicity Study in Rats 37 6.2 Carcinogenicity Study in Mice 39 6.3 Classification of Carcinogenic Potential 40 7.0 MUTAGENICITY 40 8.0 FQPA CONSIDERATIONS 8.1 Developmental Neurotoxicity Recommendation 42 8.2 Other Issues 43 9.0 REFERENCES 45 10.0 APPENDICES 48 10.1.2 Subchronic , Chronic and Other Toxicity Tables 49 10.1.3 Summary of Toxicology Endpoint Selection Tables 51 Page 4 1.0 HAZARD CHARACTERIZATION Pentachloronitrobenzene is an older organochlorine pesticide and very few of these are still marketed today due to cancer, environmental and bioaccumulation issues which plagued them. Until this reregistration review of the toxicology data base, PCNB data was separated into two distinct data bases since it was concluded that the toxicity of the two technicals of both Amvac and Uniroyal were uniquely distinct and not comparable. For example, recent cancer studies had found one registrant's technical to produce cancer, while findings were negative for the other registrant's technical.

These companies were directed to develop separate data bases in support of their registrations and tolerances. In the 1970' s HCB concentrations were in excess of 10% of the technical. However, by 1992, the level of HCB was much lower than this in both registrant formulations and some level of concern was apparently raised relative to other impurities in PCNB ( although they were not defined). In toxicology reviews conducted during the mid 1990' s it still appeared that the primary concerns were focused on the HCB levels which had by this time been dramatically reduced often less than 0.1%. By 1992, levels of impurities including, but not limited to hexachlorobenzene ( HCB) concentration, were still thought to be so different as to necessitate clear separation of the data bases for Uniroyal and Amvac PCNB. As a result, the Agency required separate toxicology studies supporting tolerances and registrations for each company.

This approach to the data bases changed on October 14, 2001, when the Metabolism Assessment Review Committee convened to consider the Uniroyal and Amvac PCNB data bases. Upon examination of the database, in particular considering the more recent studies, the MARC concluded no evidence could be found that the chemical composition ( i. e. impurity levels) differences among the formulations tested in the toxicity studies affected the NOAELS/ LOAELS and endpoints found. Other unspecified factors likely were considered greater contributors to the differences in the side by side toxicity studies submitted by the two registrants. As a consequence, it was recommended that the two data bases be combined. However, in contradiction to this, since PCNB was first registered over 40 years ago, PCNB impurities have changed dramatically as the manifestations of toxicity have changed. This is of key importance in the hazard characterization of PCNB since the toxicity, tumorigenicity and even basic toxicological effects like sensitization still differ and persist in the two data bases. As well, and complicating the public safety issues further, it seems that as the source of technical material has changed ( including Mexican, Japanese and US sources) there has been a significant impact on the levels of impurities in the technical material as compared to what had originally been tested in available toxicology studies.

Many chronic, oncogenicity and other toxicity studies had been submitted to the Agency in previous years in support of PNCB use and many of these are now excluded from the toxicity data base. A number of these studies are excluded since the technical material tested years ago is so dissimilar to what is currently marketed. The Cancer Peer Review of 1992, determined that many of these studies were inappropriate and found to be Page 5 unacceptable due to study conduct issues and also due to the dramatic reductions in HCB which was expected to reduce or eliminate the carcinogenic risks previously observed. High HCB contamination had been associated with increases in papillomas in a skin painting study and in hepaocellular carcinomas in mouse carcinogenicity studies.

The current review of these data demonstrate that this conclusion may no longer be supportable. The differences in results observed in the Uniroyal chronic/ oncogenicity study ( MRID# 41987301, ( utilizing a more purified technical with less HCB than the Amvac technical ) resulted in an increase in thyroid follicular cell adenomas and carcinomas while the Amvac chronic/ oncogenicity toxicity ( gavage) ( MRID# 43015801, was negative for neoplasia. These data might raise a flag suggesting significant differences in toxicity risk potential of the two technical materials even though in both studies the HCB concentrations and other impurities have been reduced to very low levels. Conversely, this might suggest that it is PCNB itself ( or possibly a metabolite) is responsible for the carcinogenicity findings and not the HCB as thought prevously.

Acute toxicity categories are low with most study results in Toxicity Categories III or IV. A Uniroyal study found PCNB to be a sensitizer while the Amvac study did not.

Data from several 1970' s monkey studies suggest that PCNB bioaccumulates, to some degree, in mammals. In these studies, only a few animals are utilized but data suggest that the half live might be as long as 4 or more days. These investigators also noted that after 20 days only 59% of a given dose was eliminated. It was also suggested that from day 30 to 40, the excretion curve paralleled the dose which means that an equilibrium between uptake and excretion was reached, resulting in a plateau of the storage curve. These data clearly raise the uncertainty as to the biological half life for PCNB and therefore as to its potential for bioaccumulation. Additional kinetic and other data have been required by the HED HIARC and should eventually be available to facilitate the risk assessment process.

Subchronic and chronic studies indicate that the thyroid and the liver are target organs for PCNB. Limited evidence suggest that the thyroid effects might be due, at least in part, to disturbance of thyroid homeosthasis. In addition, aminotransferase activities ( AST/ ALT, particularly ALT) consistently decrease in a dose dependent manner by 30 80% in rats and dogs. The toxicological significance of these findings needs to be further assessed and the current data base is inadequate to define this pattern of PCNB associated toxicity.

Results from NTP genetic toxicology testing indicate that PCNB ( lot and % a. i. not specified) is not mutagenic in bacteria or in cultured mammalian cells. There is, however, evidence showing that PCNB is clastogenic in the absence of S9 activation and equivocal in the presence of S9 activation. PCNB was also negative for the induction of SCEs in vitro. The HIARC recommended that the in vitro test results of clastogenicity be examined in an in vivo cytogenetic assay. Since this HIARC review, an in vivo mammalian cytogenetics study ( Erythrocyte Micronucleus assay in the Mouse, MRID # 45539601) has been submitted and reviewed. Review of the study indicated that there was no reproducible significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time. Page 6 The carcinogenicity of PCNB was assessed in reviews of 1977 ( special review) and in 1986 and 1992. In 1992, the CPRC classified PCNB as a Group C possible human carcinogen and recommended that for the purpose of risk characterization, the Reference Dose approach should be used for quantification of human risk.

Acceptable developmental toxicity studies in rats ( MRID# 40588601 and41361201) and rabbits ( MRID # 41361301 and 40717102) and reproduction studies ( MRID# 43469301, 43469302, 43469303 and 41918701) are available and adequate for FQPA considerations. No findings of significant toxicological concern have been identified in developmental toxicity or reproduction study data. Data gaps were identified and special studies are required for thyroid assessment ( including hormone levels and histopathology), assessment of the significance of ALT/ AST enzyme level reductions, metabolism and pharmacokinetics. The data base is not complete for adequate FQPA assessment.

No neurotoxicity studies have been identified in the data base but no neurobehavioral alterations nor evidence of neuropathological effects have been observed in available data.

2.0 REQUIREMENTS The testing requirements ( CFR 158.340) for Food and Non Food Use for PCNB are listed in Table 1. Use of the new guideline numbers does not imply that the revised guidelines of 1998 were used. Special studies as required by the HIARC are also included in this table. Page 7 Table 1. Toxicology Data Requirements Test Technical Required Satisfied 870.1100 Acute Oral Toxicity 870.1200 Acute Dermal Toxicity 870.1300 Acute Inhalation Toxicity 870.2400 Primary Eye Irritation 870.2500 Primary Dermal Irritation 870.2600 Dermal Sensitization yes yes yes yes yes yes yes yes yes yes yes yes 870.3100 Oral Subchronic ( rodent) 870.3150 Oral Subchronic ( nonrodent) 870.3200 21 Day Dermal 870.3250 90 Day Dermal 870.3465 90 Day Inhalation yes yes yes yes yes yes yes yes yes no 870.3700a Developmental Toxicity ( rodent) 870.3700b Developmental Toxicity ( nonrodent) 870.3800 Reproduction yes yes yes yes yes yes 870.4100a Chronic Toxicity ( rodent) 870.4100b Chronic Toxicity ( nonrodent) 870.4200a Oncogenicity ( rat) 870.4200b Oncogenicity ( mouse) 870.4300 Chronic/ Oncogenicity ( satisfies requirements of 870.4100a and 870.4200a) yes yes yes yes yes yes yes yes yes yes 870.5100 Mutagenicity   Gene Mutation  bacterial 870.5300 Mutagenicity   Gene Mutation  mammalian 870.5375 Mutagenicity   Strucural Chromosomal Aberrations 870.5395 Mutagenicity   in vivo Mammalian Cytogenetics 870.5550 Mutagenicity   Other Genotoxic Effects yes yes yes yes yes yes yes yes 870.6100a Acute Delayed Neurotoxicity ( hen) 870.6100b 90 Day Neurotoxicity ( Hen) 870.6200a Acute Neurotoxicity Screening Battery ( Rat) 870.6300 Developmental Neurotoxicity no no no no n/ a n/ a n/ a n/ a 870.7485 General Metabolism 870.7600 Dermal Penetration yes yes no no Special Studies: A special study to assess the potential for bioaccumulation a A special study to assess thyroid toxicity b The Registrant must satisfactorily address the significance of dose related decreases in AST/ ALT or submit a special study to assess the toxicological significance of these findings c yes yes yes no no no a The HIARC has required that a special study be performed in order to adequately assess the potential of PCNB to bioaccumulate. If bioaccumulation is observed in this study, data must be provided to assist in the risk assessment process ( eg circulating blood levels at various dose levels over time). The registrant will need to submit a protocol for review by the Agency.

b The HIARC has required that the potential for thyroid toxicity be thoroughly assessed in a special study. The registrant will need to submit a protocol for review by the Agency. Page 8 C The HIARC has required that the registrant adequately address the toxicological significance of decreases in AST/ ALT. This might necessitate a special study be performed to assess the toxicological significance of consistent dose related decreases in AST/ ALT observed in a multiple test species in a dose related manner at statistically significant levels. The registrant will need to submit a protocol for review by the Agency.

3.0 DATA GAPS A. Although the thyroid is a significant target organ for PCNB toxicity, effects on the thyroid have been inadequately assessed in nearly all available toxicity studies. Thyroid weights were increased in a number of chronic and subchronic studies in rats. In addition, TSH, T3, and T4 levels were affected in the only non guideline oral study and only measured in male rats using inappropriate dosing ( 0, 1, & 333 mg/ kg/ day) which precludes a meaningful assessment of dose response and time course features of the effect. The HIARC requested that the Registrant conduct a study to assess thyroid toxicity in adults vs. offspring development. The study should include: ( a) Assays of appropriate hormones ( b) Organ Weights ( c) Histopathology. The study should be conducted utilizing an adequate dosing regimen allowing for meaningful toxicological assessment of dose response and comparison to other submitted toxicological studies. In addition, it should be integrated with appropriate kinetic data. The registrant is requested to submit a protocol for review by the Agency. In addition, several special thyroid metabolism studies ( MRIDs 44096601 and 44096602) have deficiencies that need to be addressed by the registrants.

The absence of the comparative thyroid study resulted in a database uncertainty factor of 10x ( UF DB of 10x) which was applied to the dietary ( acute and chronic) as well as all residential exposure ( incidental oral, dermal and inhalation) scenarios. The HIARC determined that the 10X UF DB is required since the available data provide no basis to support reduction or removal of the default 10X factor.

B. Aminotransferase activities ( AST/ ALT, particularly ALT) decrease in a dosedependent manner by as much as 30 80% in rats and dogs. Although the literature indicates that aminotransferase inhibition, may result in increases in GABA, hepatocellular tyrosine, and other aminoacid related compounds, there are no data to evaluate the toxicological significance of the inhibition data for PCNB. The Registrant is requested to address the toxicological significance of these alterations in AST/ ALT activities and determine the toxicological significance of these findings.

C. Limited kinetic data have been identified in the literature which suggest that PCNB could accumulate within mammalian tissues potentially impacting upon the risk assessment process. Because of concerns about the uncertainties of the half life and thus for the accumulation potential of the chemical, the HIARC recommended that a study to determine the biological half life on PCNB be requested. It is therefore requested that the Registrant perform a guideline metabolism study of PCNB ( including a mass balance of radioactivity in excreta, tissues, and carcass) including a Tier II study of blood kinetics, to determine a half life of PCNB. If bioaccumulation is observed, additional kinetic data will also be necessary to relate to endpoints observed in other toxicity studies which should assist in meaningful risk assessment. The Registrant should consult the Agency as Page 9 to the details of the protocol.

D. There are no subchronic inhalation toxicity data for PCNB. Due to the greenhouse use of PCNB, the HIARC requests that a 90 day inhalation toxicity study of PCNB be conducted. In addition to the guideline requirements of ( 870.3465) it is recommended that interim thyroid hormone analyses be made at 7, 14, 30 and 90 days and that histopathology assessment include the thyroid.

E. A dermal penetration study ( 870.7600) is conditionally required since HIARC determined that the available study is not acceptable.

4.0 HAZARD ASSESSMENT 4.1 Acute Toxicity Adequacy of the data base for acute toxicity : The data base for acute toxicity of the technical ` s are considered complete and no additional studies are required at this time. PCNB has a relatively low order of acute toxicity in test animals. Both Uniroyal and Amvac data are presented below. Note that the technical materials of the registrants differ somewhat and that the Uniroyal technical is a sensitizer while the Amvac technical is not. However, for both sets of data, acute toxicity remains relatively low in Categories III and IV.

Table 2. Acute Toxicity of PCNB ( Uniroyal)

Guideline No. Study Type MRIDs # Results Tox Category 81 1 870.1100 Acute Oral 43198201 LD50 = > 5000 mg/ kg IV 81 2 8701200 Acute Dermal 43198202 LD50 = > 5000 mg/ kg IV 81 3 870.1300 Acute Inhalation 43118201 LC50 = > 1.7 mg/ L III 81 4 870.2400 Primary Eye Irritation 43198203 Slight irritant III 81 5 870.2500 Primary Skin Irritation 43198204 Non irritant IV 81 6 870.2600 Dermal Sensitization 4060901 Weak sensitizer n/ a 81 8 Acute Neurotoxicity n/ aa n/ a n/ a a n/ a = not applicable Page 10 Table 2 con't Acute Toxicity of PCNB ( Amvac)

Guideline No. Study Type MRIDs # Results Toxicity Category 81 1 870.1100 Acute Oral 41443101 LD50 = > 5050 mg/ kg IV 81 2 870.1200 Acute Dermal 41443102 LD50 = > 2020 mg/ kg III 81 3 870.1300 Acute Inhalation 41443103 LC50 = > 6.49 mg/ L III 81 4 870.2400 Primary Eye Irritation 41443109 Slight irritant III 81 5 870.2500 Primary Skin Irritation 41443105 PII = 0.0175 IV 81 6 870.2600 Dermal Sensitization 40734001 Non sensitizer n/ a 81 8 Acute Neurotoxicity n/ aa n/ a n/ a a n/ a = not applicable 4.2 Subchronic Toxicity Adequacy of the data base for subchronic toxicity: The data base for subchronic toxicity is partially complete. Despite the fact that 90 day studies have been submitted, reviewed and found to be acceptable, special studies are required to satisfy Agency concerns relative to thyroid toxicity and AST/ ALT findings. In addition, no subchronic inhalation study was available in the data base although exposure would be expected. The HED HIARC has requested that a 90 day inhalation toxicity study be conducted ( HIARC, 4/ 9/ 03). Page 11 870.3100 90 Day Oral Toxicity  Rat A. Subchronic Oral Toxicity rats OPPTS Guideline 870.3100 SPONSOR: Amvac Chemical Corporation In a subchronic oral study ( MRID 42416001), pentachloronitrobenzene ( PCNB; 98% a. i.; Lot/ Batch # 05318 7D, batch II) was administered via gavage to 10 Crl: CDBR rats/ sex/ group at nominal doses of 0, 5, 10, 100, or 1000 ( limit dose) mg/ kg/ day for 13 weeks.

No treatment related deaths occurred. Clinical signs, body weights, body weight gains, food consumption, ophthalmoscopic observations, hematological parameters, and gross pathology were unaffected by the test substance. At 1000 mg/ kg/ day, relative ( to body weight) liver weights were increased in the females (

15%, p  0.05). In addition, centrilobular hepatocellular hypertrophy was observed ( males 6/ 10, females 2/ 10 vs. 0/ 20 controls). In the thyroid, follicular epithelium hypertrophy/ hyperplasia was noted in both sexes ( males 6/ 10, females  5/ 10 vs. 2/ 20 controls). Colloid vacuolation of the thyroid was noted in the females only ( 3/ 10 vs. 0/ 10 controls).

The LOAEL for this study is 1000 mg/ kg/ day ( limit dose) based on increased relative ( to body) liver weight and histopathological changes in the liver and thyroid. The NOAEL for this study is 100 mg/ kg/ day.

The submitted study is classified as acceptable/ guideline ( 82 1a) and satisfies the requirements for a subchronic oral toxicity study in the rat.

Proposed Dose and Endpoint: The NOAEL for this study is 100 mg/ kg/ day. The LOAEL for this study is 1000 mg/ kg/ day ( limit dose) based on increased relative liver weight and histopathological changes in the liver and thyroid.

B. Subchronic Oral Toxicity in male rats ( a non guideline study)

In a special ( nonguideline) subchronic oral toxicity study ( MRID 42630801), 75 male Charles River CD ® rats/ dose were administered pentachloronitrobenzene ( PCNB tech., 99.09% a. i.) in their diet at levels of 0, 20 or 6000 ppm ( equivalent to average daily intakes of 0, 1.0 or 333 mg/ kg/ day). Groups of 15 animals/ dose were sacrificed at 7, 14, 30 or 90 days. The remaining 15 animals/ dose group at day 90 were fed only basal diet for a recovery period of at least 90 days ( sacrificed on day 180 or 183). Levels of circulating thyroid hormones ( TSH, T3 and T4) and thyroid/ liver weights and pathology were evaluated at each sacrifice time.

In the thyroid, follicular epithelial hypertrophy was observed at days 14 and 30 ( trace and Page 12 mild severity, respectively at 6000 ppm) and at day 90 ( mild at 20 ppm and moderate at 6000 ppm). These changes had reversed after the recovery period. In addition, TSH levels were statistically significantly greater than controls starting 14 days after initiation of the study. After 90 days of dosing, no NOAEL was determined in this study. At the high dose level of 6000 ppm, toxicity was observed after only 7 days of dosing. Although this is a non guideline study, since it has sacrifices at 7, 14, 30 or 90 days, it may be especially useful for short term risk assessment.

At 20 ppm, hypertrophy of the liver ( trace) and thyroid ( mild) were observed in 14/ 15 and 15/ 15 animals, respectively ( 0/ 15, controls). At 6000 ppm, statistically significantly decreased mean body weight throughout most of treatment ( at termination,  6.1% less than controls) and decreased body weight gain ( at termination,  20%, due largely to a pronounced decrease during Week 1), decreased food consumption during Week 1 only ( 23% below controls), increased TSH (+ 31% to + 132% above controls; significant at most time points), decreased T3 ( 9 to  26% less than controls; significant at most time points), decreased T4 ( 48 to  54% less than controls, significant at all time points), decreased rT3 ( 28% at day 90; significant), increased relative liver weight (+ 12 to + 18% above controls), decreased absolute thyroid/ parathyroid weights ( 14%, day 30) and increased follicular epithelial hypertrophy of the thyroid ( trace to mild, all animals at all sacrifice times vs. 0/ 15 controls) and hepatocellular hypertrophy ( moderate, all animals at 90 days, vs. 0/ 15, controls). There were no treatment related deaths or clinical signs observed. Animals maintained on basal diet for an additional 90 days showed complete recovery. The following were not evaluated: hematology, urinalysis, organ weights and gross/ microscopic pathology, with the exception of liver, thyroid/ parathyroids. The study LOAEL is 20 ppm ( 1.0 mg/ kg/ day, LDT), based on mild liver and thyroid microscopic hypertrophy. The study NOAEL is < 20 ppm ( 1.0 mg/ kg/ day).

This study is classified Acceptable/ nonguideline ( § 82 1a). It does not satisfy the guideline requirement for a subchronic oral toxicity study in the rodent because it is not a complete Guideline subchronic study. It was intended as a special study to evaluate the effects of PCNB on thyroid hormone levels, rather than a complete Guideline subchronic study, and was adequately conducted to provide this information.

870.3100 90 Day Oral Toxicity  Mouse An NTP mouse 3 month study was conducted as a rangefinding study for establishing dose levels for the mouse oncogenicity study. No MRID number was ever assigned to this study. The study was conducted in 1986 in order to establish dose levels in their oncogenicity study. Males alone received a dose level of 1250 ppm while both males and females received dose levels of 2500, 5000, 10,000 and 20,000 ppm . In addition, females were also tested at 40,000 ppm. A NOAEL was determined to be 1250 in males and 2500 ppm in females. The LOAEL was determined to be 2500 ppm in males and 5000 ppm in females based on increased liver weight and liver to bodyweight ratio. There was no histopathology. This study doses not constitute a subchronic feeding study and is considered as acceptable only as a rangefinding study. Page 13 870.3150 90 Day Oral Toxicity  Dog Not available in the data base. However, this study is not required since there is an acceptable 1 year chronic dog study.

870.3200 21/ 28 Day Dermal Toxicity B Rat SPONSOR: Amvac Chemical Corporation In a 21 day dermal toxicity study ( MRID# 42416002), male and female Crl: CDBR rats received dermal dosages of PCNB ( Lot # 05318 7D, 98 % a. i.) at levels of 0, 100, 300, and 1000 mg/ kg/ day for 5 days per week for a minimum of 3 weeks. The residual material was removed by gently washing the exposure site with water and wiping with a paper towel.

The NOAEL was determined to be 300 mg/ kg/ day and the LOAEL was determined to be 1000 mg/ kg/ day based on the increased incidences of dilatation of the thyroid follicles and hypertrophy of the thyroid follicular epithelium in the high dose males. There were no reported treatment related effects in females.

This study is classified as Acceptable/ Guideline and satisfies the guideline requirement for a repeated dose ( 21 day) dermal toxicity study ( OPPTS 870.3200; § 82.2).

870.3465 90 Day Inhalation B Rat Considered a data gap and required by HIARC ( HIARC , 4/ 9/ 03).

4.3 Prenatal Developmental Toxicity Adequacy of data base for Prenatal Developmental Toxicity: Acceptable developmental toxicity studies in rats ( MRID 40588601 and 41361201) and rabbits ( MRID 41361301 and 40717102) and reproduction studies ( MRID 43469301, 43469302, 43469303 and 41918701) are available and adequate for FQPA considerations. However, special studies are required on the thyroid ( including hormone levels with histopathological assessment), on the toxicological significance of ALT/ AST enzyme level reductions, metabolism and pharmacokinetics. Therefore, the data base is not complete although developmental and reproduction study data are adequate.

870.3700a Prenatal Developmental Toxicity Study  Rat A. Developmental Toxicity Study in the Rat ( gavage) Guideline # 870.3700; 83 3 SPONSOR: Uniroyal Chemical Co., Inc., Bethany, CT In an oral developmental toxicity study ( MRID 40588601), 25 ( presumed) pregnant Page 14 Sprague Dawley COBS CD rats/ dose group were administered pentachloronitrobenzene ( PCNB tech., 96.0% a. i.; 0.025% hexachlorobenzene contaminant) by gavage in 0.2% high viscosity carboxymethylcellulose ( 10 ml/ kg body wt.) at dose levels of 0, 30, 600 or 1200 mg/ kg/ day from gestation days 6 through 15, inclusive.

There were no treatment related maternal or developmental effects observed at any dose level tested. The highest dose tested ( 1200 mg/ kg/ day) exceeded the limit dose requirement. The maternal toxicity LOAEL is > 1200 mg/ kg/ day ( HDT) and the NOAEL is

1200 mg/ kg/ day. The developmental toxicity LOAEL is > 1200 mg/ kg/ day and the NOAEL is

1200 mg/ kg/ day.

This study is classified Acceptable/ Guideline ( § 83 3a; OPPTS 870.3700) and satisfies the guideline requirement for a developmental toxicity study in the rat.

B. Developmental Toxicity Study in the rat ( gavage) Guideline # 870.3700; 83 3 SPONSOR: Amvac Chemical Corporation, Los Angeles, CA In an oral developmental toxicity study ( MRID 41361201), 25 ( presumed) pregnant CRL: CD ® ( SD) BR rats/ dose group were administered pentachloronitrobenzene ( PCNB tech., 98.5% a. i.; hexachlorobenzene contamination 0.09%) at doses of 0, 250, 750 or 1500 mg/ kg/ day from days 6 through 15 of gestation, inclusive. Doses were given by gavage in 1% aqueous carboxymethylcellulose vehicle ( 10 ml/ kg body weight).

Maternal toxicity: No treatment related effects were observed. The highest dose level tested ( 1500 mg/ kg/ day) exceeded the limit dose. The maternal toxicity LOAEL is > 1500 mg/ kg/ day and the NOAEL is

1500 mg/ kg/ day.

Developmental toxicity: At 750 and 1500 mg/ kg/ day, small but statistically significant increases in the average number of thoracic vertebrae/ fetus ( 13.05, 13.16, 13.23 and 13.21, control to high dose; historical control range 13.00 to 13.13), average pairs of thoracic ribs/ fetus ( 13.03, 13.13, 13.17 and 13.15; historical control range 13.00 to 13.09), and decreases in the average number of lumbar vertebrae/ fetus ( 5.94, 5.84, 5.77 and 5.79; historical control range 5.85 to 6.00) were reported ( all historical data from 4852 fetuses in 573 litters, 26 studies). The biological significance of these findings is uncertain due to relatively small magnitude of the changes and lack of a clear doseresponse No treatment related fetal malformations were observed. The developmental toxicity LOAEL is 750 mg/ kg/ day and the NOAEL is 250 mg/ kg/ day.

This study is classified Acceptable/ Guideline ( § 83 3a; OPPTS 870.3700) and satisfies the guideline requirement for a developmental toxicity study in the rat.

A number of older developmental and reproductive studies have been identified in the data base but most of these studies are associated with testing of the older manufactured Page 15 technical ( high in HCB and other contaminants) and therefore these data cannot be used as part of the present assessment on the currently marketed technical.

870.3700b Prenatal Developmental Toxicity Study  Rabbit A. Developmental Toxicity Study in the Rabbit ( Gavage) Guideline # 870.3700; 83 3 SPONSOR: Amvac Chemical Corp., Los Angeles, CA In an oral developmental toxicity study ( MRID 41361301), 20 ( presumed) pregnant New Zealand White rabbits/ dose group were administered pentachloronitrobenzene ( PCNB tech., 98.5% a. i.; 0.09% hexachlorobenzene contaminant) by gavage at 0, 100, 300 or 900 mg/ kg/ day in 10 ml aqueous 1% carboxymethylcellulose vehicle/ kg body weight from gestation days 6 through 18, inclusive.

Maternal toxicity: At 900 mg/ kg/ day, abnormal feces ( soft, liquid or dried; total incidence 45 in 11 does vs. 15 in 4 does, controls), decreased weight gain during dosing ( 75% less than controls; not statistically significant), slightly decreased food consumption ( 16% less than controls; not statistically significant) were observed. Abortions ( days 24 and 25, 2/ 20 vs. 0/ 20, controls) and premature delivery ( day 25, 1/ 20 vs. 0/ 20, controls) were observed and these 3 does also had associated abnormal feces, weight loss and decreased food consumption. The doe with premature delivery also had decreased motor activity and a fluid filled cecum, one abortive female had gastric ulceration and the other had a litter with 4 late resorptions out of 6 conceptuses. No mortality was observed. The maternal toxicity LOAEL is 900 mg/ kg/ day, based on clinical signs of toxicity, decreased body weight gain and food consumption during dosing, abortions and premature delivery. The maternal toxicity NOAEL is 300 mg/ kg/ day.

Developmental toxicity: There were no treatment related developmental effects reported in this study. The developmental toxicity LOAEL is > 900 mg/ kg/ day ( HDT) and the developmental toxicity NOAEL is

900 mg/ kg/ day.

This study is classified Acceptable/ Guideline ( § 83 3b; OPPTS 870.3700) and satisfies the Guideline requirement for a developmental toxicity study in the rabbit. Page 16 B. Developmental Toxicity Study in the Rabbit ( gavage) Guideline # 870.3700; 83 3 SPONSOR: Uniroyal Chemical Co.

In an oral developmental toxicity study ( MRID 40717102) groups of 16 pregnant New Zealand White rabbits were given daily doses of 0 ( two groups) s, 6.25, 12.5, 125 ( two groups) and 250 mg/ kg/ day PCNB ( 96%) by gavage on days 7 through 19 of gestation.

Maternal Toxicity: At the highest dose tested there was mortality, abortions, weight loss during gestation and decreased food consumption. The only effects observed in the 125 mg/ kg/ day dose groups were decreased body weight and weight gain. Group mean maternal body weight was statistically significantly less than control values only during the second of the two trials that were conducted at that dose level. Based on these results, a LOAEL for maternal toxicity was established at 125 mg/ kg/ day and and NOAEL was established at 12.5 mg/ kg/ day.

Developmental Toxicity: Based on statistically significant decreased fetal weights reported in the highest dose group, the LOAEL for developmental toxicity was established at 250 mg/ kg/ day and the NOAEL was determined to be 125 mg/ kg/ day in rabbits.

This study is classified Acceptable ( § 83 3b; OPPTS 870.3700) and satisfies the Guideline requirement for a developmental toxicity study in the rabbit.

4.4 Reproductive Toxicity Adequacy of data base for Reproductive Toxicity: The data base for reproductive toxicity is considered complete. No additional studies are required at this time.

870.3800 Reproduction and Fertility Effects  Rat Two Generation Reproduction Study in Rats ( dietary) OPPTS 870.3800 In a 2 generation reproductive toxicity study ( MRID 41918701, Uniroyal), 26 Sprague Dawley COBS ® CD rats/ sex/ dose group were administered pentachloronitrobenzene ( PCNB tech., > 99% a. i.; < 0.1% hexachlorobenzene contaminant) in their diet at concentrations of 0, 20, 3000 or 6000 ppm ( equivalent to 1.2/ 1.4, 169/ 213 or 344/ 468 mg/ kg/ day, males and 1.5/ 1.7, 218/ 255 or 455/ 640 mg/ kg/ day, females, based on mean F0/ F1 premating compound consumption values), beginning in 55 day old F0 animals at 81 days prior to mating or in weanling F1 animals for at least 90 days prior to mating. Page 17 Each parental generation was mated twice to produce F1/ F2 a and b litters. Dosing of the F0 and F1 parental animals was continued throughout the study period until termination of the animals after day 21 of the second lactation period ( females) or after the second mating period ( males).

Parental toxicity: At 3000 ppm, statistically significantly decreased mean premating body weights were observed in F0 males prior to both matings ( 7% less than controls) and in F1 females prior to the first mating ( 5.7% to  10%; due to decreased initial weights of the F1 weanlings assigned to be parental animals). At 6000 ppm, significantly decreased mean premating body weights ( F0 males  9 to  10% less than controls and females  7%; F1 males, initial weights  35%; thereafter  16%; females initial weights  37%; thereafter  14% to  16%). The small size of almost all F1 animals at 6000 ppm and emaciation in 4 females was related to these decreases. Food consumption was decreased, primarily during the first weeks of each premating period. Increased incidence of pulmonary foci was also observed in F0 females ( 4/ 26) and in F1 males ( 5/ 26) and females ( 8/ 26) ( no controls affected in either sex). The parental systemic toxicity LOAEL is 3000 ppm ( 169 mg/ kg/ day, males and 218 mg/ kg/ day, females), based on decreased body weight/ weight gain. The NOAEL is 20 ppm ( 1.2 mg/ kg/ day, males and 1.5 mg/ kg/ day, females).

Reproductive/ developmental toxicity: At 3000 ppm, decreased mean pup weight ( 6.8% to  8.7% l ess than controls at lactation day 21, both sexes; statistically significant) was observed in F1a, F1b and F2b pups. At 6000 ppm, decreases in male and female pup weights in all 4 litters were pronounced ( 30% to  41% at lactation day 21). The reproductive/ developmental LOAEL is 3000 ppm ( 169 mg/ kg/ day), based on decreased mean pup weight in most generations. The NOAEL is 20 ppm ( 1.2 mg/ kg/ day).

The Offspring NOAEL: Based on decreases in mean pup weights of F1a, F1b, and F2b animals, the offspring NOAEL is 1.2 mg/ kg/ day and the LOAEL is 169 mg/ kg/ day.

This study is classified Acceptable/ Guideline ( § 83 4; OPPTS 870.3800) and satisfies the Guideline requirement for a multigeneration reproductive toxicity study in the rat.

In another 2 generation reproduction study ( MRIDs 43469301, 43469302 and 43469303, Amvac) PCNB,( 98% a. i.) was administered to 35 Crl: CD BR ( Sprague Dawley) VAF/ Plus rats/ sex/ dose by gavage at dose levels of 0, 10, 100 or 1000 mg/ kg/ day. A dose volume of 10 ml/ kg was administered to all groups. P 1 males and females received the test material for 10 weeks prior to mating and the females continued to be dosed through weaning of the F 1 generation. P 2 males and females were dosed with the test material starting at weaning for at least 10 weeks prior to mating and through the mating period for the F 2 litters. The females continued to be dosed until the weaning of the F 2 generation. One litter/ generation was produced and tested.

There were no compound related effects in parental bodyweight, food consumption or Page 18 reproductive performance. In addition, there were no clinical signs of toxicity in the parental animals. In the P 1 generation, hepatocellular hypertrophy was observed at 1000 mg/ kg in both sexes and thyroid follicular cell hypertrophy/ hyperplasia was observed at 100 and 1000 mg/ kg in males and at 1000 mg/ kg in females. There were no changes observed in the reproductive organs. In the P 2 generation, the effects were the same except that the liver effects were only observed in high dose males. No treatment related effects were observed in litter size, pup viability, pup bodyweight or pup macroscopic examinations. No microscopic examinations were conducted on pups.

The LOAEL is 100 mg/ kg/ day in males and 1000 mg/ kg/ day in females based on increases in hepatocellular hypertrophy and thyroid follicular cell hypertrophy/ hyperplasia. The NOAEL is 10 mg/ kg/ day in males and 100 mg/ kg/ day in females.

The reproductive study in the rat is classified as acceptable and satisfies the guideline requirement for a 2 generation reproductive study ( OPPTS 870.3800, § 83 4) in the rat.

4.5 Chronic Toxicity Adequacy of data base for chronic toxicity: The data base for chronic toxicity is considered complete. No additional studies are required at this time.

870.4100a ( 870.4300) Chronic Toxicity B Rat Chronic Oral/ Carcinogenicity Study in rats Guideline #: 83 2( a)

Sponsor: Uniroyal A. In an oral chronic toxicity/ carcinogenicity study ( MRID 41987301), 50 Charles River CD ® rats/ sex/ dose group were administered pentachloronitrobenzene ( PCNB tech., 99.4% a. i.) in the diet at concentrations of 0, 20, 3000 or 6000 ppm ( equivalent to estimated average daily intakes of 0, 1, 150 or 300 mg/ kg/ day; estimated based on a standard conversion factor of 0.05) for 24 months. An additional 10 animals/ sex/ dose group were included for interim sacrifice at 12 months.

At 3000 ppm, statistically significantly increased relative liver weight in males (+ 20% above controls), absolute thyroid/ parathyroid weight in males (+ 26%), relative thyroid/ parathyroid weight (+ 35%, males and + 24%, females), and significantly increased incidence of microscopic lesions including mild hepatocellular hypertrophy ( 27%, males and 38%, females vs. 0%, controls), mild thyroid hyperplasia ( 15%, males vs. 4%, controls and 16%, females vs. 0%, controls) and thyroid hypertrophy ( 42%, males vs. 0%, controls and 36%, females vs. 2%, controls) were observed. Sporadic significantly decreased mean body weights in both sexes were not considered biologically significant. At 6000 ppm, these effects showed a dose response and in addition, statistically significantly decreased mean body weight/ weight gain throughout treatment ( at Page 19 termination,  11%/ 15% less than controls in males and  12%/ 18% in females), decreased food consumption during the first 6 months, increased serum cholesterol ( females), significantly increased relative liver weight in both sexes (+ 25%, males and + 20%, females) and increased incidence of thyroid colloid cysts in males ( 16% vs. 4.1%, controls) were observed. At the 12 month interim sacrifice, both sexes showed slight ( not statistically significant) increases in liver and thyroid weights at mid and high dose and in high dose females, there was a slightly increased incidence of visible tan foci in the lungs. There were no treatment related clinical or ophthalmologic observations and no effects on mortality, hematology or urinalysis parameters. The systemic toxicity LOAEL is 3000 ppm ( 150 mg/ kg/ day), based on hepatocellular hypertrophy, hepatocellular hyperplasia ( females) and thyroid hypertrophy and hyperplasia. The systemic toxicity NOAEL is 20 ppm ( 1 mg/ kg/ day).

PCNB caused an increased incidence of thyroid follicular cell adenomas in males ( 0%, 0%, 12.5% and 10.2%, control to high dose; p< 0.05 at 3000 ppm) and a significantly increasing trend ( p< 0.01). Incidence in females was 2.0%, 0%, 4.0% and 8.7% ( control to high dose; not significant); however, a significantly increasing trend ( p< 0.05) was observed. The incidence of thyroid follicular cell carcinoma was increased at 6000 ppm only in males ( males 0%, 2.0%, 0% and 4.1%; females 2.0%, 0%, 0% and 2.2%). The combined incidence of thyroid follicular cell adenomas and carcinomas was significantly increased in males at 3000 and 6000 ppm ( control to high dose, 0%, 2.0%, 12.5%, 14.3%; p< 0.05) but not females ( 4.0%, 0%, 4.0% and 10.9%), with a significant trend in both sexes ( females p< 0.05 and males p< 0.01). The incidence of follicular cell adenoma in historical control data from this laboratory did not exceed 11.1% in males or 3.2% in females. The incidence of carcinoma did not exceed 9.4% in males or 3.2% in females.

This study is classified Acceptable/ Guideline ( § 83 5; OPPTS 870.4300) and satisfies the Guideline requirement for a chronic toxicity/ carcinogenicity study in the rodent.

COMPLIANCE: Signed and dated GLP, Quality Assurance and Flagging Statements were provided.

Comments about Study/ Endpoint/ Uncertainty Factor( s):. An acceptable Uniroyal One Year Dog feeding study was available in the data base, ( See 870.4100b, Chronic Toxicity  nonrodent ( Dog).

B. In a Chronic Toxicity/ Oncogenicity study ( MRID 43015801) sponsored by Amvac, groups of 60 male and 60 female Crl: CDBR Sprague Dawley rats were given 0, 5, 50, 500, or 1000 mg/ kg bodyweight pentachloronitrobenzene ( Technical 98%) by gavage five days per week for up to two years. When adjusted for continuous exposure, these dose levels were equivalent to 0, 3.6, 36, 357 or 714 mg/ kg/ day. T 3, T 4 and TSH parameters were not examined.

No apparent effects on body weight, food intake, clinical observations, or survival were found. At all dose levels and at all measured time points ( 6, 12, and 18 months and study Page 20 termination), dose dependent decreases in serum AST and ALT activities and the excretion of triple phosphate crystals in the urine were found in both sexes. At 36 mg/ kg/ day and above, increases in minimal to slight hepatocellular hypertrophy were observed in males ( ranging from 9/ 60 to 26/ 60 versus 2/ 60 in controls; p < 0.05 at 36 mg/ kg/ day; p < 0.01 at 357 mg/ kg/ day and above). At 357 mg/ kg/ day and above, increases in minimal to slight hepatocellular hypertrophy were observed in females ( ranging from 10/ 60 to 22/ 60 versus 1/ 60 in controls; p < 0.01). Increases in thyroid follicular cell hypertrophy/ hyperplasia were observed in both sexes ( 6/ 60 versus 2/ 60 in males and 5/ 59 versus 1/ 59 in females) however, the increases were not statistically significant. High dose male rats had an approximate 30% increase of absolute and relative liver weight and an approximate 22% increase in absolute thyroid/ parathyroid weight at necropsy. Microscopically, increases in thyroid follicular cell hypertrophy/ hyperplasia ( mostly minimal to slight; 12/ 60 versus 2/ 60 for males and 10/ 60 versus 1/ 59 for females; p < 0.01) were found. Based on the hepatocellular hypertrophy and the thyroid follicular cell hypertrophy/ hyperplasia, the NOAEL was 3.6 mg/ kg/ day for males and 36 mg/ kg/ day for females. The corresponding LOAELS would be 36 mg/ kg/ day for males and 357 mg/ kg/ day for females. No treatment related increase in neoplasia was found.

The study is classified as core guideline and satisfies the requirements for an § 83 5 Oral Chronic/ oncogenicity Study.

Discussion of Tumor Data: No treatment related increase in neoplasia was noted..

Adequacy of Dosing: Previous concerns for PCNB carcinogenicity were linked to the high HCB concentrations in earlier studies. However, in the Uniroyal feeding study ( MRID 41987301), only 0.04% HCB was reported in the test material and this study demonstrated a positive carcinogenic potential. On the other hand, the Amvac study ( MRID 41987301) was negative for carcinogenicity. This is a gavage study with administration of the test material ( 98% PCNB) only 5 days per week. The interruption in dosing may tend to allow some time for test material body burden to decrease and for for recovery, as compared to a dietary feeding study. This difference in dosing regimen might possibly explain the negative carcinogenicity results ( if the positive oncogenic effects are primarily due to the technical PCNB or metabolites and NOT due to impurities, since apparently the Amvac technical contained a slightly higher level of impurities), although other similar thyroid findings were noted. HIARC concluded that the dose levels tested were judged to be adequate based on thyroid toxicity observed at the mid and high dose levels.

Comments on other Rat Studies: Several rat chronic/ carcinogenicity studies were also conducted and reviewed in the Carcinogenicity Peer Review of 12/ 18/ 92 and not found adequate for risk assessment purposes. One study was conducted in 1974 by the Central Institut voor Voedingsonderzoek TNO ( Netherlands) but the study was performed on a technical containing 2.7% HCB and due to numerous study deficiencies, the study was not Page 21 considered useful for risk assessment purposes ( MRID 00114223). A second study was conducted in 1978 by Hazleton Laboratories and Tracor Jitco ( MRID 00114226) utilizing a technical contaminated with approximately 3% HCB. This study resulted in increases in pituitary chromophobe adenomas in males but numerous study deficiencies were identified and the study was not considered acceptable/ useful to characterize the carcinogenic potential of PCNB.

870.4100b Chronic Toxicity  Dog In a chronic oral toxicity study ( MRIDs 41718600, 41718601) 6 beagle dogs/ sex/ dose were administered pentachloronitrobenzene ( PCNB tech., 99.4% a. i.; 0.04% hexachlorobenzene contaminant) in their diet for 1 year at concentrations of 0, 15, 150 or 1500 ppm ( equivalent to estimated average daily intakes of 0, 0.375, 3.75 or 37.5 mg/ kg/ day; estimated using standard conversion factor for dogs of 0.025).

At 1500 ppm, increased serum alkaline phosphatase (+ 34% to + 56% above controls; both sexes at 6 and 12 months), decreased ALT ( 84% to  93% less than controls, both sexes at 6 and 12 months) increased cholesterol (+ 37% to + 41%, males only at 6 and 12 months), increased liver weight ( relative + 26% above controls, males; abs./ rel + 39%/+ 30%, females) and increased incidence of hepatocellular hypertrophy ( trace, 6/ 6 males; trace to mild, 6/ 6 females vs. 0/ 6 controls) were observed. Increased relative kidney weight was also reported in females (+ 17%). There were no treatment related clinical, ophthalmological or gross anatomical findings nor changes in body weight, food consumption, hematology or urinalysis parameters. The LOAEL is 1500 ppm ( 37.5 mg/ kg/ day), based on increased serum ALP and cholesterol, increased liver weight and hepatocellular hypertrophy. The NOAEL is 150 ppm ( 3.75 mg/ kg/ day).

This study is classified Acceptable/ Guideline ( § 83 1b) and satisfies the Guideline requirement for a chronic oral toxicity study in the dog.

4.6 Carcinogenicity Adequacy of data base for Carcinogenicity: The data base for carcinogenicity is considered complete by the HIARC despite numerous study deficiencies noted in the Mouse Oncogenicity Study conducted by NTP ( NTP, 1987). No additional studies are required at this time.

870.4200a Carcinogenicity Study  rat Note that the study addressing this test requirement is a combined oncogenicity/ chronic toxicity study. Page 22 Combined Chronic Toxicity/ Carcinogenicity Study in Rats OPPTS 870.4300 A. In an oral chronic toxicity/ carcinogenicity study ( MRID 41987301, Uniroyal), 50 Charles River CD ® rats/ sex/ dose group were administered pentachloronitrobenzene ( PCNB tech., 99.4% a. i.) in the diet at concentrations of 0, 20, 3000 or 6000 ppm ( equivalent to estimated average daily intakes of 0, 1, 150 or 300 mg/ kg/ day; estimated based on a standard conversion factor of 0.05) for 24 months. An additional 10 animals/ sex/ dose group were included for interim sacrifice at 12 months.

At 3000 ppm, statistically significantly increased relative liver weight in males (+ 20% above controls), absolute thyroid/ parathyroid weight in males (+ 26%), relative thyroid/ parathyroid weight (+ 35%, males and + 24%, females), and significantly increased incidence of microscopic lesions including mild hepatocellular hypertrophy ( 27%, males and 38%, females vs. 0%, controls), mild thyroid hyperplasia ( 15%, males vs. 4%, controls and 16%, females vs. 0%, controls) and thyroid hypertrophy ( 42%, males vs. 0%, controls and 36%, females vs. 2%, controls) were observed. Sporadic significantly decreased mean body weights in both sexes were not considered biologically significant. At 6000 ppm, these effects showed a dose response and in addition, statistically significantly decreased mean body weight/ weight gain throughout treatment ( at termination,  11%/ 15% less than controls in males and  12%/ 18% in females), decreased food consumption during the first 6 months, increased serum cholesterol ( females), significantly increased relative liver weight in both sexes (+ 25%, males and + 20%, females) and increased incidence of thyroid colloid cysts in males ( 16% vs. 4.1%, controls) were observed. At the 12 month interim sacrifice, both sexes showed slight ( not statistically significant) increases in liver and thyroid weights at mid and high dose and in high dose females, there was a slightly increased incidence of visible tan foci in the lungs. There were no treatment related clinical or ophthalmologic observations and no effects on mortality, hematology or urinalysis parameters. The systemic toxicity LOAEL is 3000 ppm ( 150 mg/ kg/ day), based on hepatocellular hypertrophy, hepatocellular hyperplasia ( females) and thyroid hypertrophy and hyperplasia. The systemic toxicity NOAEL is 20 ppm ( 1 mg/ kg/ day).

This study is classified Acceptable/ Guideline ( § 83 5; OPPTS 870.4300) and satisfies the Guideline requirement for a chronic toxicity/ carcinogenicity study in the rodent.

Discussion of Tumor Data: PCNB caused an increased incidence of thyroid follicular cell adenomas in males ( 0%, 0%, 12.5% and 10.2%, control to high dose; p< 0.05 at 3000 ppm) and a significantly increasing trend ( p< 0.01). Incidence in females was 2.0%, 0%, 4.0% and 8.7% ( control to high dose; not significant); however, a significantly increasing trend ( p< 0.05) was observed. The incidence of thyroid follicular cell carcinoma was increased at 6000 ppm only in males ( males 0%, 2.0%, 0% and 4.1%; females 2.0%, 0%, 0% and 2.2%). The combined incidence of thyroid follicular cell adenomas and carcinomas was significantly increased in males at 3000 and 6000 ppm ( control to high dose, 0%, 2.0%, 12.5%, 14.3%; p< 0.05) but not females ( 4.0%, 0%, 4.0% and 10.9%), with a significant trend in both sexes ( females p< 0.05 and males p< 0.01). The incidence of Page 23 follicular cell adenoma in historical control data from this laboratory did not exceed 11.1% in males or 3.2% in females. The incidence of carcinoma did not exceed 9.4% in males or 3.2% in females ( MRID 41987301).

Adequacy of the Dose Levels Tested: The dose levels are considered adequate. Systemic toxicity was seen at the mid and high dose levels tested.

B. In a second Chronic Toxicity/ Oncogenicity study ( MRID 43015801) sponsored by Amvac, groups of 60 male and 60 female Crl: CDBR Sprague Dawley rats were given 0, 5, 50, 500, or 1000 mg/ kg bodyweight pentachloronitrobenzene ( Technical 98%) by gavage five days per week for up to two years. When adjusted for continuous exposure, these dose levels were equivalent to 0, 3.6, 36, 357 or 714 mg/ kg/ day. T 3, T 4 and TSH parameters were not examined.

No apparent effects on body weight, food intake, clinical observations, or survival were found. At all dose levels and at all measured time points ( 6, 12, and 18 months and study termination), dose dependent decreases in serum AST and ALT activities and the excretion of triple phosphate crystals in the urine were found in both sexes. At 36 mg/ kg/ day and above, increases in minimal to slight hepatocellular hypertrophy were observed in males ( ranging from 9/ 60 to 26/ 60 versus 2/ 60 in controls; p < 0.05 at 36 mg/ kg/ day; p < 0.01 at 357 mg/ kg/ day and above). At 357 mg/ kg/ day and above, increases in minimal to slight hepatocellular hypertrophy were observed in females ( ranging from 10/ 60 to 22/ 60 versus 1/ 60 in controls; p < 0.01). Increases in thyroid follicular cell hypertrophy/ hyperplasia were observed in both sexes ( 6/ 60 versus 2/ 60 in males and 5/ 59 versus 1/ 59 in females) however, the increases were not statistically significant. High dose male rats had an approximate 30% increase of absolute and relative liver weight and an approximate 22% increase in absolute thyroid/ parathyroid weight at necropsy. Microscopically, increases in thyroid follicular cell hypertrophy/ hyperplasia ( mostly minimal to slight; 12/ 60 versus 2/ 60 for males and 10/ 60 versus 1/ 59 for females; p < 0.01) were found. Based on the hepatocellular hypertrophy and the thyroid follicular cell hypertrophy/ hyperplasia, the NOAEL was 3.6 mg/ kg/ day for males and 36 mg/ kg/ day for females. The corresponding LOAELS would be 36 mg/ kg/ day for males and 357 mg/ kg/ day for females. No treatment related increase in neoplasia was found.

The study is classified as core guideline and satisfies the requirements for an § 83 5 Oral Chronic/ oncogenicity Study.

Discussion of Tumor Data: No treatment related increase in neoplasia was noted..

Adequacy of Dosing: Previous concerns for PCNB carcinogenicity were linked to the high HCB concentrations in earlier studies. However, in the Uniroyal feeding study ( MRID No. 41987301), only 0.04% HCB was reported in the test material and this study demonstrated a positive carcinogenic potential. On the other hand, the Amvac study ( MRID No. 41987301) was negative for carcinogenicity. This is a gavage study Page 24 with administration of the test material ( 98% PCNB) only 5 days per week. The interruption in dosing may tend to allow some time for test material body burden to decrease and for for recovery, as compared to a dietary feeding study. This difference in dosing regimen might tend to explain the negative carcinogenicity results, although other similar thyroid findings were noted. HIARC concluded that the dose levels tested were judged to be adequate based on thyroid toxicity observed at the mid and high dose levels.

870.4200b Carcinogenicity ( feeding)  Mouse In an oral carcinogenicity study conducted by NTP ( 1987), ( No assigned MRID number) 50 B6C3F1 mice per sex per dose group were administered PCNB ( purity not reported) in the diet at concentrations of 0, 2500, or 5000 ppm for 103 weeks. At the end of the feeding period, test diets were withdrawn and the animals were fed control diets during a one week observation period before termination of the study.

No increase in the incidence of neoplastic lesions was observed in the treated mice, but the test substance may have predisposed the female mice of the high dose group to a bacterial infection which significantly decreased their survival after 86 weeks. PCNB did not exhibit carcinogenic potential under the conditions of the experiment.

The HIARC recommended that this study be considered as Acceptable. This NTP mouse oncogenicity was completed in 1987 and a number of significant deficiencies have been identified in this study. Previous reviews of the study indicated that nonneoplastic lesions observed in female mice were secondary to bacterial infection. The investigators attempted to explain the high levels of infection observed in this study by suggesting that the test material might have predisposed the female mice ( but curiously not the males) to the infection. The infection was identified as Klebsiella. The mouse onco study may be compromised due to infection confounding interpretation of the study results as well as affecting the longevity of the animals on test. In addition, the lot number of the test material is not identified and there is no indication in the report that the test diets were analyzed. Lack of diet analysis by itself is often considered adequate for invalidation of a carcinogenicity study. Also, a review by R. Gardner, 2/ 2/ 89, states that it is " prudent to use the mouse study in the absence of better data" but at this time I do not find this a reason to continue to use this study as was done in the 1993 Cancer review...." There is also a review by the California Department of Food and Agriculture which states that the study is unacceptable because among other things, only two doses were used, many missing tissues in histopathology examination, a questionable MTD was utilized, and effects of an infectious agent compromised the study.

4.7 Mutagenicity Adequacy of data base for Mutagenicity: The data base for Mutagenicity is considered adequate based on the HIARC determination that there is no concern for mutagenicity Page 25 resulting from exposure to PCNB. Pentachloronitrobenzene was selected for genetic toxicology screening by the National Toxicology Program ( NTP, 1987). Testing included: the Salmonella/ mammalian microsome test, induction of forward gene mutations in L5178Y mouse lymphoma cells and induction of chromosome aberrations and sister chromatid exchanges ( SCEs) in Chinese hamster ovary ( CHO) cells. Although formal DERs do not exist for these assays, they are considered acceptable by HED.

Gene Mutation Guideline # 870.5100, Gene Mutaton Bacterial MRID: n/ a Acceptable NTP ( 1987)

Guideline# 870.5300, Gene Mutation Mammalian MRID: n/ a Acceptable ( NTP( 1987) Salmonella typhimurium/ mammalian microsome reverse gene mutation assay: Using the preincubation procedure, PCNB ( 95%) was negative in S. typhimurium strains TA1535, TA1537, TA198 and TA 100 up to high doses of 6666.7 to 10,000 ug/ plate without S( activation and with S9 activation derived from rat and hamster livers induced with Aroclor 1254. Compound precipitation was seen at > 1000ug/ plate. The study is acceptable and satisfies the guideline requirement for a bacterial gene mutation assay.

L5178Y mouse lymphoma cell forward gene mutation assay: Independent trials were negative up to the highest doses tested ( 15 or 30

g/ mL  S9; 15

/ mL + S9). The study is acceptable and satisfies the guideline requirement for a mammalian cell gene mutation assay.

Cytogenetics Guideline #, 870.5375, Structural Chromosomal Aberrations ( CHO) MRID: n/ a Acceptable NTP ( 1987)

Guideline # 870.5395, in vivo cytogenetics ( Mouse Micronucleus Test) MRID# 45539601 Acceptable In vitro cytogenetics assay in Chinese hamster ovary ( CHO) cells: the test was positive in the absence of S( activation with a significant ( p< 0.05) increase in chromosome aberrations over a concentration range of 7.5 75

g/ mL. With S9 activation, results were equivocal at the highest dose tested ( 75

g/ mL). The study is acceptable and satisfies the guideline requirement for an in vitro cytogenetic assay.

In independently conducted ICR mouse bone marrow micronucleus assays, groups of five male and five female mice were administered Pentachloronitrobenzene as Terraclor Technical ( 99%, lot # 88101) once via single intrapertioneal ( ip) injections of 0, 230, 460 or 920 mg/ kg bw. Comparable doses were administered in the repeat trial. Bone marrow cells were harvested at 24 and 48 hours post treatment. The vehicle was corn oil. Based on preliminary toxicity testing, the highest dose tested ( HDT) was 80% of the LD 50/ 3.

Clinical signs included lethargy and piloerection at 460 and 920 mg/ kg; a sight to moderate ( up to 25%) reduction in the ratio of polychromatic erythrocytes to total erythrocytes ( PCE: NCE) was recorded following exposure to the HDT. Terraclor technical was, therefore, tested up to an adequate dose ( based on overt toxicity in the animals and cytotoxicity in the target cells). The positive control induced the appropriate response. There was no reproducible significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time. Page 26 Other Genotoxicity Guideline # 870.5xxx, Other Genotoxic Effects ( SCE) MRID: n/ a acceptable NTP( 1987) In vitro SCE assay in CHO cells: The test was negative up to the highest concentration tested ( 7.5

g/ mL  S9; 75

g/ mL+ S9). The study is acceptable and satisfies the guideline requirement for an in vitro SCE assay.

4.8 Neurotoxicity Adequacy of data base for Neurotoxicity: No data suggesting that PCNB is a neurotoxicant were apparent in the data base.

870.6100 Delayed Neurotoxicity Study  Hen No data are available.

870.6200 Acute Neurotoxicity Screening Battery No data are available.

870.6200 Subchronic Neurotoxicity Screening Battery No data are available.

870.6300 Developmental Neurotoxicity Study Not required as per HIARC's FQPA assessment.

4.9 Metabolism Adequacy of data base for metabolism: The data base for metabolism is considered to be inadeqate. Guideline and special bioaccumulation studies are required and are considered data gaps.

870.7485 Metabolism  Rat Although the HIARC has identified the rat metabolism study as a significant data gap, available data in the literature suggest that PCNB bioaccumlates to some degree and requires a more complete assessment and a new special study has been required to resolve outstanding issues. Several special non guideline and unacceptable ( but potentially Page 27 upgradable) rat metabolism studies have been submitted to the Agency and data from these are presented below under " Special Studies". The HIARC has required that a new rat metabolism study be submitted.

Data from a number of literature studies have been reviewed and data from these are presented below. Data from several rhesus monkey studies are presented and these involve the use of only a few animals. In addition, it should be noted that available literature data are very old using older methodologies with all identified and referenced studies conducted more than 24 years ago and in one case about 35 years ago. Hence, the determination of residues of greatest toxicological concern prior to having an acceptable and complete assessment of the PCNB metabolism and a full understanding of the potential for bioaccumulation of PCNB and/ or its metabolites is seriously handicapped at this time. However, we do know that PCNB does bioaccumulate within animal tissues as do most other organochlorine pesticides, but the extent of this accumulation within tissues is not adequately quantitated at this time. On the other hand, it is unlikely that PCNB bioaccumulates at the same levels of some of the other organochlorine pestidices including DDT.

PCNB Metabolism Data Available in the Literature In a study by Kogel et al ( 1979a) the metabolism of PCNB in the Rhesus monkey was evaluated after a single low dose of 2mg/ kg, after a single high dose of 91 mg/ kg and after feeding of 2 ppm for 71 days. The major metabolites were determined to be pentachloroaniline ( 55.4% of urinary extract and 70.6% of fecal extract after 71 days), pentachlorobenzene ( 11.8% of urinary extract and 0.5% of fecal extract after 71 days), pentachlorophenol 12.2% of urinary extract after 71 days), pentachlorothioanisole ( 9.8% of urinary extract and 6% of fecal extract after 71 days), and bis methylmercaptotetrachlorobenzene ( 9.7% of urinary extract and 9.3% of fecal extract after 71 days). After the single high dose, the metabolites 2,3,5,6 tetrachlorophenol methylated, tetrachlorothioanisole, tetrachloroaminothioanisole, tetrachloroaminophenylmethylsulfoxide tetrachlorophenylmethyl sulfoxide, and bis methylmercaptoaminotrichlorobenzene and five other unidentified metabolites were also noted which had not been detected after the single low dose exposure.

After the single low dose, 16.3% of the fecal extract was parent PCNB, after the single high dose 12.9% of the fecal extract was parent and after 71 days, 13.4 % of the fecal extract was parent while no parent was reported in the urine during any of the dosing periods. The authors concluded that the two main pathways of metabolism of PCNB in the Rhesus monkey were ( 1) the reduction of the nitro moiety to the corresponding aniline, and ( 2) the cleavage of the C N bond, presumably via conjugation with sulfurcontaining amino acids, e. g. glutathione, with subsequent breakdown of these conjugates. They also noted that the pathways of biotransformation were similar to those observed in the rat. It should be noted that no attempt was made in this study to assess accumulation within tissues and that the percent of extract was assessed rather than the % of dosed PCNB. Page 28 In another study by Kogel et al ( 1979b) purified PCNB was administered to Rhesus monkeys as single oral doses of 0.5, 2, or 91 mg/ kg and in a 70 day feeding study at a level of 2 ppm in the diet. The authors concluded that PCNB was readily absorbed from the gastrointestinal tract primarily by the portal venous route and is fairly rapidly converted to pentachloroaniline and numerous other metabolites. The authors noted that the half life of the PCNB was 1.5 to 1.7 days only after very low doses of 2ppm but they failed to characterize the half life after the higher dose level. However, the investigators noted that " pentachlorophenol, which is comparable to pentachloroaniline in its polarity, has a much longer half life of 15 20 days in rhesus monkeys after only a single dose ( Ballhorn, 1978). The investigators noted that only a small portion of the parent PCNB ( 4.3%) was excreted unmetabolized after a single 91 mg/ kg dose. In addition, they noted that after a single dose of 91 mg/ kg that after 24 hours only 15.1% of the dose was excreted ( 11.8% in urine and 3.3% in feces) and after 20 days, the total excretion was 59.5% ( 25.8% in urine and 33.7% in feces). These findings might suggest that the possibility of greater accumulation of metabolites of PCNB dependent upon the level of exposure/ dose levels.

These investigators also noted that after a low dose of 2ppm for 70 days, that a plateau level of storage of the labeled PCNB was reached after 30 40 days and that by day 71, males had retained 7.7% and females 10.3% of the total dose administered with highest amounts ( 7.73 ppm in bile of males and 3.72 ppm reported in bile of females). PCNB and/ or metabolites were also noted in numerous other tissues including liver, thymus and fat at 0.19 ppm.

It is important to note that these investigators also performed a very limited examination of hematology, clinical chemistry and several hormones. Hematology of a single rhesus monkey given 91 mg/ kg of PCNB was reported as normal with the exception of an increase in methemogolobin which was elevated on the day after dosing and returned to normal the following day. During the 70 day feeding at a dose level of 2ppm, hematology and clinical chemistry data were reported to be within normal limits but data were based on only two animals. The authors also reported that radioimmunoassays for luteinizing hormone, follicle stimulating hormone and progesterone did not show any gross effects in circulating blood. However, they pointed out that their experiment was not designed as an endocrinology study and that more subtle interferences of PCNB with hormone patterns may have remained undetected.

In a still older study by Kuchar et al( 1969), PCNB manufactured by Olin Corporation was incorporated in feed and fed daily to beagle dogs and rats in a two year chronic study. Note that the test material differs from that currently marketed and contained impurities including HCB at 1.8%, PCB at < 0.1% and and 2,3,4,5 tetrachloronitrobenzene at 0.4%. Data obtained from the fat tissues of dogs and rats are indicative of fat storage of the chlorinated impurities such as PCB and HCB as well as other chlorinated metabolites containing groups reported to be  NH2 and  SCH2 but the parent PCNB was not identified. Other tissues were analyzed in this study but only after animals were placed on a two month recovery period ( control diet) and these analyses do not appear especially Page 29 appropriate. The authors concluded that the metabolic products of PCNB are PCA and methyl pentachloro phenyl sulfide. The authors also concluded that extracts of rat tissues from rats fed PCNB and from plants grown in PCNB soil are indicative of an identical metabolism.

In another study by O'Grodnick et al ( 1981) it was reported that S( pentachloro phenyl) N acetylcysteine as the predominant urinary metabolite from rats after a single oral dose of 5 mg/ kg labeled PCNB. PCA was the predominant metabolite in feces and also a major urinary metabolite with small amounts of Pentachloro thioanisole also recovered from urine and feces. PCNB itself was only found in feces.

The investigators concluded that pentachloronitrobenzene is metabolized ( 1) to sulfurcontaining metabolites produced by reaction with glutathione, catalyzed by glutathione Stransferase ( 2) to non sulfur containing metabolites by denitration to pentachlorpphenol, and ( 3) by reduction to pentachloroaniline.

The limited data that are available suggest that generally it is the metabolites that are accumulated within tissues to a greater extent than parent PCNB itself. In addition, it must be recognized that the determination of metabolites of concern is generally carried out utilizing sound and complete metabolism data which is not the current situation since an acceptable rat metabolism study has not been provided by the registrant. Hence, it is possible that the toxicological significance of a particular metabolite( s) might be overlooked at this time. Hence, for the above listed reasons, it is not possible to consider / confirm inclusion of all metabolites of toxicological concern at this time.

870.7600 Dermal Absorption  Rat A dermal penetration study ( MRID no. 250698 and 255226) was available; two formulations ( 20% dust and 75% wettable powder) were tested in rats for four hours or 5 days. After 4 hours, recovery/ dermal penetration was 1.3% for the wettable powder and 3.1% for the dust. After 5 days, the recovery was 32.3% for the wettable powder and 33.8% for the dust.

The HIARC did not use this study since this study: ( 1) did not follow current guidelines; ( 2) test materials are significantly different from the technical product currently marketed: ( 3) the lot number of the test materials was not reported; and ( 4) the report did not account for all radioactivity.

The HIARC extrapolated a dermal absorption factor of 33% based on the ratio of 333 mg/ kg/ day ( based on the presence of thyroid hormone changes observed at 7, 14, and 30 days at the 333 mg/ kg/ day dose level observed in the male 90 day rat feeding study) and the LOAEL of 1000 mg/ kg/ day in the 21 day dermal toxicity study. In rats, it was noted that thyroid toxicity was the common toxicity seen via both routes in the same species. Page 30 4.10 Special/ Other Studies Special Non Guideline Rat Metabolism Data Submitted to the Agency In a special ( non guideline) mechanistic metabolism study ( MRID 44096602), thyroid uptake of 125Iodine and biliary excretion of [ 125I] thyroxine were evaluated in 10 male CD rats/ dose treated with pentachloronitrobenzene for 10 days in the diet at 0, 20, or 6000 ppm. An additional 10 animals/ group were administered Arochlor 1254 as a positive control. Half of the animals per group ( 5) received [ 125I] by iv injection 8.5 hours prior to sacrifice at which time thyroid glands were weighed and [ 125I ] measured. The other half were given [ 125I] thyroxine by iv injection and bile was collected at 15 minute intervals for 45 minutes, then at 30 minute intervals until 135 minutes post dosing. Blood was collected at the midpoints of these intervals and body temperature was measured at 15 minute intervals during bile collection.

At 6000 ppm, statistically significant increases in cumulative biliary excretion and mean clearance of [ 125I] thyroxine (+ 28% and + 63% above basal diet controls for 0 135 minutes post dosing, respectively), significantly decreased mean serum thyroxine concentration ( 47%, 0 135 minutes), slightly increased mean bile flow rate throughout the sampling and significantly decreased cumulative uptake of [ 125I] ( 53%, 0 135 minutes) were observed. There were no treatment related deaths, clinical signs, body weight changes or thyroid weight changes. At 20 ppm of PCNB, no effects were observed. However, the reviewer determined that a NOAELand LOAEL for effects of thyroid uptake of [ 125I] and of biliary excretion of [ 125I] thyroxine could not be determined at that time pending submission of additional required data. This study was classified as unacceptable since raw data were reported to be missing by the testing laboratory itself and information on the purity and lots of labeled thyroxine were not provided.

In another special ( non guideline) mechanistic metabolism study ( MRID 44096601), the effect of pentachloronitrobenzene on thyroid uptake of [ 125 ] Iodine and biliary excretion of [ 125I] thyroxine was evaluated. PCNB was administered in the diet to a total of 18 male SD rats/ dose at concentrations of 0, 20, or 6000 ppm for one week prior to administration of the radiolabel. Positive controls were administered either Aroclor 1254 or benzo( a) pyrene. From the test groups, 6 animals/ dose were injected ip with 10  g [ 125I] kg body weight in 1 ml/ kg 0.9% saline ( thyroid uptake studies) and 6/ dose were injected iv with 1  g [ 125I] thyroxine/ kg body weight in 2.0 ml/ kg 50% aqueous ethanol ( biliary studies). In the negative and positive groups for the biliary excretion studies, only 4 and 5 animals were evaluated, respectively, due to mortality of animals associated with the bile cannulation procedure. For the thyroid uptake studies, animals were sacrificed at 3 hours following administration of [ 125I], retroorbital blood samples were withdrawn at 1.5 hours and thyroids were weighed and evaluated for [ 125I] activity. For the biliary excretion studies, bile was collected at 15 and 30 minutes after administration of [ 125I] thyroxine, then at 30 minute intervals up to 4 hours post dosing. Blood was collected at the Page 31 midpoint between these intervals.

At 6000 ppm, biliary excretion of [ 125I] thyroxine equivalents was increased (+ 23% above negative controls, not statistically significant), bile: blood ratio and biliary clearance of [ 125I] thyroxine equivalents were increased and thyroid uptake of [ 125I] was decreased ( 43% less than negative controls; p< 0.05). Rats treated with benzo( a) pyrene showed a marked increase in biliary excretion of [ 125I] thyroxine (+ 266%, p< 0.01). No changes in the uptake of [ 125I] by the thyroid were observed in the rats treated with Aroclor 1254 and thyroid weights were comparable for all groups. There were not treatment related differences in rats treated with 20 ppm PCNB. A NOAEL/ LOAEL for effects on thyroid uptake of [ 125I] and biliary excretion of [ 125I] thyroxine were not determined at this time, pending receipt of additional information to potentially upgrade this study. This study was classified as unacceptable and can only be upgraded if acceptable data on ( 1) the fate of individual animals and verification of the number of treated animals that died prior to assignment to the experimental groups and ( 20 the different compound consumption values given in the study report and ( 3) verification that the test diets were prepared and used within the time of demonstrated stability.

5.0 TOXICITY ENDPOINT SELECTION ( HIARC)

5.1 See Appendix for Endpoint Selection Table 5.11 Acute Reference Dose ( aRfD) General Population Acute Reference Dose ( aRfD)  An endpoint attributable to a single dose ( exposure) was not available in the database.

5.12 Chronic Reference Dose ( cRfD)

Study Selected: Combined chronic toxicity/ carcinogenicity  rat § OPPTS 870.4300 In an oral chronic toxicity/ carcinogenicity study ( MRID 41987301), 50 Charles River CD ® rats/ sex/ dose group were administered pentachloronitrobenzene ( PCNB tech., 99.4% a. i.) in the diet at concentrations of 0, 20, 3000 or 6000 ppm ( equivalent to estimated average daily intakes of 0, 1, 150 or 300 mg/ kg/ day; estimated based on a standard conversion factor of 0.05) for 24 months. An additional 10 animals/ sex/ dose group were included for interim sacrifice at 12 months.

At 3000 ppm, statistically significantly increased relative liver weight in males (+ 20% above controls), absolute thyroid/ parathyroid weight in males (+ 26%), relative thyroid/ parathyroid weight (+ 35%, males and + 24%, females), and Page 32 significantly increased incidence of microscopic lesions including mild hepatocellular hypertrophy ( 27%, males and 38%, females vs. 0%, controls), mild thyroid hyperplasia ( 15%, males vs. 4%, controls and 16%, females vs. 0%, controls) and thyroid hypertrophy ( 42%, males vs. 0%, controls and 36%, females vs. 2%, controls) were observed. Sporadic significantly decreased mean body weights in both sexes were not considered biologically significant. At 6000 ppm, these effects showed a dose response and in addition, statistically significantly decreased mean body weight/ weight gain throughout treatment ( at termination,  11%/ 15% less than controls in males and  12%/ 18% in females), decreased food consumption during the first 6 months, increased serum cholesterol ( females), significantly increased relative liver weight in both sexes (+ 25%, males and + 20%, females) and increased incidence of thyroid colloid cysts in males ( 16% vs. 4.1%, controls) were observed. At the 12 month interim sacrifice, both sexes showed slight ( not statistically significant) increases in liver and thyroid weights at mid and high dose and in high dose females, there was a slightly increased incidence of visible tan foci in the lungs. There were no treatment related clinical or ophthalmologic observations and no effects on mortality, hematology or urinalysis parameters.

The systemic toxicity LOAEL is 3000 ppm ( 150 mg/ kg/ day), based on hepatocellular hypertrophy, hepatocellular hyperplasia ( females) and thyroid hypertrophy and hyperplasia. The systemic toxicity NOAEL is 20 ppm ( 1 mg/ kg/ day).

PCNB caused an increased incidence of thyroid follicular cell adenomas in males ( 0%, 0%, 12.5% and 10.2%, control to high dose; p< 0.05 at 3000 ppm) and a significantly increasing trend ( p< 0.01). Incidence in females was 2.0%, 0%, 4.0% and 8.7% ( control to high dose; not significant); however, a significantly increasing trend ( p< 0.05) was observed. The incidence of thyroid follicular cell carcinoma was increased at 6000 ppm only in males ( males 0%, 2.0%, 0% and 4.1%; females 2.0%, 0%, 0% and 2.2%). The combined incidence of thyroid follicular cell adenomas and carcinomas was significantly increased in males at 3000 and 6000 ppm ( control to high dose, 0%, 2.0%, 12.5%, 14.3%; p< 0.05) but not females ( 4.0%, 0%, 4.0% and 10.9%), with a significant trend in both sexes ( females p< 0.05 and males p< 0.01). The incidence of follicular cell adenoma in historical control data from this laboratory did not exceed 11.1% in males or 3.2% in females. The incidence of carcinoma did not exceed 9.4% in males or 3.2% in females.

This study is classified Acceptable/ Guideline ( § 83 5; OPPTS 870.4300) and satisfies the Guideline requirement for a chronic toxicity/ carcinogenicity study in the rodent.

Dose and Endpoint for Establishing cRfD: NOAEL = 1 mg/ kg/ day based on hepatocellular hypertrophy, hepatocellular hyperplasia ( females) and thyroid hypertrophy and hyperplasia at The LOAEL = 150 mg/ kg/ day. Page 33 Uncertainty Factor( s): 1000 ( 10x for interspecies extrapolation, 10x for interspecies differences, and UF DB for lack of comparative thyroid assay).

Comments about Study/ Endpoint/ Uncertainty Factor: This study is appropriate for the population and duration considered in a chronic risk assessment.

Chronic RfD = ( NOAEL) 1 mg/ kg/ day = 0.001 mg/ kg/ day ( UF) 1000 5.2 Incidental Oral Exposure: Short Term ( 1 30 days)

Study Selected: Subchronic Oral Toxicity in male rats § Nonguideline In a special ( nonguideline) subchronic oral toxicity study ( MRID 42630801), 75 male Charles River CD ® rats/ dose were administered pentachloronitrobenzene ( PCNB tech., 99.09% a. i.) in their diet at levels of 0, 20 or 6000 ppm ( equivalent to average daily intakes of 0, 1.0 or 333 mg/ kg/ day). Groups of 15 animals/ dose were sacrificed at 7, 14, 30 or 90 days. The remaining 15 animals/ dose group at day 90 were fed only basal diet for a recovery period of at least 90 days ( sacrificed on day 180 or 183). Levels of circulating thyroid hormones ( TSH, T3 and T4) and thyroid/ liver weights and pathology were evaluated at each sacrifice time.

At 20 ppm, hypertrophy of the liver ( trace) and thyroid ( mild) were observed in 14/ 15 and 15/ 15 animals, respectively ( 0/ 15, controls). At 6000 ppm, statistically significantly decreased mean body weight throughout most of treatment ( at termination,  6.1% less than controls) and decreased body weight gain ( at termination,  20%, due largely to a pronounced decrease during Week 1), decreased food consumption during Week 1 only ( 23% below controls), increased TSH (+ 31% to + 132% above controls; significant at most time points), decreased T3 ( 9 to  26% less than controls; significant at most time points), decreased T4 ( 48 to  54% less than controls, significant at all time points), decreased rT3 ( 28% at day 90; significant), increased relative liver weight (+ 12 to + 18% above controls), decreased absolute thyroid/ parathyroid weights ( 14%, day 30) and increased follicular epithelial hypertrophy of the thyroid ( trace to mild, all animals at all sacrifice times vs. 0/ 15 controls) and hepatocellular hypertrophy ( moderate, all animals at 90 days, vs. 0/ 15, controls). There were no treatment related deaths or clinical signs observed. Animals maintained on basal diet for an additional 90 days showed complete recovery. The following were not evaluated: hematology, urinalysis, organ weights and gross/ microscopic pathology, with the exception of liver, thyroid/ parathyroids. The study LOAEL is 20 ppm ( 1.0 mg/ kg/ day), based on liver and thyroid histopathology. The study NOAEL was not determined.

This study is classified Acceptable/ nonguideline ( § 82 1a). It does not satisfy the guideline requirement for a subchronic oral toxicity study in the rodent because it is not a complete Guideline subchronic study. This was a special non guideline Page 34 study designed only to assess effects of PCNB on the thyroid hormone levels, rather than a complete Guideline subchronic study, and was adequately conducted to provide this information. However, only males were tested, only two doses were utilized and hematology, urinalysis, organ weights, and gross and microscopic pathology ( with the exception of thyroid/ parathyroids and liver) were not assessed.

Dose and Endpoint for Risk Assessment: 1 mg/ kg/ day. Although a NOAEL was not identified in the above non guideline male study at 90 days, no effects were observed at the 1 mg/ kg/ day dose level at interim sacrifice/ hormone assessment intervals conducted at 7, 14, and 30 days. This finding was considered an appropriate endpoint for risk assessment by HIARC for periods up to 30 days.

Comments about Study/ Endpoint: Although no effects were observed in this special study at the 1 mg/ kg/ day dose level during interim assessments up to 30 days, it must be recognized that this is not a guideline study and was intended only for a limited assessment of the thyroid and liver function. In addition, it included no assessment for potential effects on females ( since none were utilized in the study), it only utilized two dose levels, and did not include routine assessments in males for hematology, urinalysis, organ weights and gross and microscopic pathology ( with the exception of liver and thyroid/ parathyroids) and other endpoints. HIARC concluded that the effects observed in this study are appropriate for the population ( infants and children) and duration of concern ( 1 30 days).

5.3 Dermal Absorption Dermal Absorption Factor: 33%

A dermal penetration study ( MRID no. 250698 and 255226) was available; two formulations ( 20% dust and 75% wettable powder) were tested in rats for four hours or 5 days. After 4 hours, recovery/ dermal penetration was 1.3% for the wettable powder and 3.1% for the dust. After 5 days, the recovery was 32.3% for the wettable powder and 33.8% for the dust.

The HIARC did not use this study since this study: ( 1) did not follow current guidelines; ( 2) test materials are significantly different from the technical product currently marketed: ( 3) the lot number of the test materials was not reported; and ( 4) the report did not account for all radioactivity.

The HIARC extrapolated a dermal absorption factor of 33% based on the ratio of 333 mg/ kg/ day ( based on the presence of thyroid hormone changes observed at 7, 14, and 30 days at the 333 mg/ kg/ day dose level observed in the male 90 day rat feeding study) and the LOAEL of 1000 mg/ kg/ day in the 21 day dermal toxicity study. In rats, it was noted that thyroid toxicity was the common toxicity seen via both routes in the same species Page 35 5.4 Short term Dermal : ( 1 30days) Exposure Study Selected: 21 Day Dermal Toxicity Study § OPPTS 870.3200 In a 21 day dermal toxicity study ( MRID# 42416002), Charles River rats were exposed dermally to PCNB ( 98% a. i) by application of the moistened solid ( distilled water vehicle) to the shaved dorsal skin at doses of 0, 100, 300 and 1000 mg/ kg for 6 hours per day, five days per week.

In the high dose males, dilatation of the thyroid follicles was observed in 3/ 5 animals and hypertrophy of the thyroid follicular epithelium was observed in 4/ 5 animals. These changes were not observed in any of the other dose groups, including controls or in any females. No other treatment related effects were observed in either males or females. Based on these findings, the NOAEL is 300 mg/ kg/ day and the LOAEL is 1000 mg/ kg/ day based on thyroid effects.

Dose and Endpoint for Risk Assessment: NOAEL = 300 mg/ kg/ day based on hypertrophy of the thyroid follicular epithelium and dilation of the thyroid follicles in males only at the LOAEL = 1000 mg/ kg.

Comments about Study/ Endpoint: This study is appropriate for the route and duration of exposure concerns.

5.5 Intermediate term Dermal: ( 1 6 months) Exposure Study Selected: 21 Day Dermal Toxicity Study § OPPTS 870.3200 MRID No.: 42416002 Executive Summary: See Short Term Dermal ( 1 30 Days) Exposure Dose and Endpoint for Risk Assessment: NOAEL = 300 mg/ kg/ day based on hypertrophy of the thyroid follicular epithelium and dilation of the thyroid follicles in males at the LOAEL = 1000 mg/ kg/ day.

Comments about Study/ Endpoint: This study is appropriate for the route and duration of exposure concerns. Page 36 5.6 Long Term Dermal : (> 6 months) Exposure Study Selected: Combined Chronic Toxicity/ Carcinogenicity StudyRat § OPPTS 870.4300 See Chronic Reference Dose ( RfD)

Dose and Endpoint for Risk Assessment: 1 mg/ kg/ day based on hepatocellular hypertrophy, hepatocellular hyperplasia ( females) and thyroid hypertrophy and hyperplasia at 150 mg/ kg/ day.

Comments about Study/ Endpoint: This dose/ end point study was also used to establish the chronic RFD. Since an oral dose was identified, 33% dermal absorption should be used in route to route extrapolation.

5.7 Inhalation Exposure: Short Term ( 1 30 days)

Study Selected: Subchronic Oral Toxicity Study in Male Rats § Nonguideline See Short Term ( 1 30 Days) Incidental Oral Exposure Dose/ Endpoint for Risk Assessment: 1 mg/ kg/ day. Although a NOAEL was not identified in the non guideline male study at 90 days, no effects were observed at the 1 mg/ kg/ day dose level at interim sacrifice/ hormone assessment intervals conducted at 7, 14, and 30 days. This finding was considered an appropriate endpoint for risk assessment by HIARC for periods up to 30 days.

Comments about Study/ Endpoint: In the absence of an inhalation study, an oral study was selected. Absorption by the inhalation route should be considered to be equivalent to absorption by the oral route.

5.8 Inhalation Exposure: Intermediate Term ( 1 6 months)

Study Selected: Subchronic Oral Toxicity Study in Male Rats, MRID# 42630801 § Nonguideline See Short Term ( 1 30 Days Incidental Oral Exposure Dose/ Endpoint for Risk Assessment: 1 mg/ kg/ day. Although a NOAEL was not identified in the non guideline male study at 90 days, no effects were observed at the 1 mg/ kg/ day dose level at interim sacrifice/ hormone assessment intervals conducted at 7, 14, and 30 days. This finding was considered an appropriate endpoint for risk assessment by HIARC for periods up to 30 days. Page 37 Comments about Study/ Endpoint: In the absence of an inhalation study, an oral study was selected. Absorption by the inhalation route should be considered to be equivalent to absorption by the oral route.

5.9 Inhalation Exposure: Long Term (> 6 months)

Study Selected: Combined Chronic Toxicity/ Carcinogenicity Study  Rat § OPPTS 870.4300 ( MRID# 41987301)

See Chronic Reference Dose ( RfD)

Dose/ Endpoint for Risk Assessment: 1 mg/ kg/ day based on hepatocellular hypertrophy, hepatocellular hyperplasia ( females) and thyroid hypertrophy and hyperplasia at 150 mg/ kg/ day.

Comments about Study/ Endpoint: In the absence of an inhalation study, an oral study was selected. Absorption by the inhalation route should be considered to be equivalent to absorption by the oral route.

5.10 Margins of Exposure Summary of target Margins of Exposure ( MOEs) for risk assessment.

Route Duration Short Term ( 1 30 Days) Intermediate Term ( 1  6 Months) Long Term (> 6 Months)

Occupational ( Worker) Exposure Dermal 100 100 100 Inhalation 100 100 100 Residential ( Non Dietary) Exposure Oral 1000 1000 N/ A Dermal 1000 1000 1000 Inhalation 1000 1000 1000 For Occupational exposure: This is based on the conventional uncertainty factor of 100X ( 10X for intraspecies extrapolation and 10X for interspecies variation)

For Residential exposure: This is based on the conventional uncertainty factor of 100X ( 10X for intraspecies extrapolation and 10X for interspecies variation) in addition to a 10X UF DB due to the lack of a comparative thyroid study. Page 38 5.11 Recommendationfor Aggregate Exposure Risk Assessments As per FQPA, 1996, when there are potential residential exposures to the pesticide, aggregate risk assessment must consider exposures from three major sources: oral, dermal and inhalation exposures. The toxicity endpoints selected for these routes of exposure may be aggregated as follows:

A common toxicological endpoint of concern ( thyroid toxicity) was identified for the oral, dermal, and inhalation ( oral equivalent) routes of exposure. Therefore, for short, intermediate and long term exposure aggregate risk assessments, these routes/ durations can be aggregated for the appropriate populations.

6.0 CLASSIFICATION OF CARCINOGENIC POTENTIAL 6.1 Combined Chronic Toxicity/ Carcinogenicity Study in Rats OPPTS 870.4300 A. In an oral chronic toxicity/ carcinogenicity study ( MRID 41987301), 50 Charles River CD ® rats/ sex/ dose group were administered pentachloronitrobenzene ( PCNB tech., 99.4% a. i.) in the diet at concentrations of 0, 20, 3000 or 6000 ppm ( equivalent to estimated average daily intakes of 0, 1, 150 or 300 mg/ kg/ day; estimated based on a standard conversion factor of 0.05) for 24 months. An additional 10 animals/ sex/ dose group were included for interim sacrifice at 12 months.

At 3000 ppm, statistically significantly increased relative liver weight in males (+ 20% above controls), absolute thyroid/ parathyroid weight in males (+ 26%), relative thyroid/ parathyroid weight (+ 35%, males and + 24%, females), and significantly increased incidence of microscopic lesions including mild hepatocellular hypertrophy ( 27%, males and 38%, females vs. 0%, controls), mild thyroid hyperplasia ( 15%, males vs. 4%, controls and 16%, females vs. 0%, controls) and thyroid hypertrophy ( 42%, males vs. 0%, controls and 36%, females vs. 2%, controls) were observed. Sporadic significantly decreased mean body weights in both sexes were not considered biologically significant. At 6000 ppm, these effects showed a dose response and in addition, statistically significantly decreased mean body weight/ weight gain throughout treatment ( at termination,  11%/ 15% less than controls in males and  12%/ 18% in females), decreased food consumption during the first 6 months, increased serum cholesterol ( females), significantly increased relative liver weight in both sexes (+ 25%, males and + 20%, females) and increased incidence of thyroid colloid cysts in males ( 16% vs. 4.1%, controls) were observed. At the 12 month interim sacrifice, both sexes showed slight ( not statistically significant) increases in liver and thyroid weights at mid and Page 39 high dose and in high dose females, there was a slightly increased incidence of visible tan foci in the lungs. There were no treatment related clinical or ophthalmologic observations and no effects on mortality, hematology or urinalysis parameters. The systemic toxicity LOAEL is 3000 ppm ( 150 mg/ kg/ day), based on hepatocellular hypertrophy, hepatocellular hyperplasia ( females) and thyroid hypertrophy and hyperplasia. The systemic toxicity NOAEL is 20 ppm ( 1 mg/ kg/ day).

This study is classified Acceptable/ Guideline ( § 83 5; OPPTS 870.4300) and satisfies the Guideline requirement for a chronic toxicity/ carcinogenicity study in the rodent.

Discussion of Tumor Data: PCNB caused an increased incidence of thyroid follicular cell adenomas in males ( 0%, 0%, 12.5% and 10.2%, control to high dose; p< 0.05 at 3000 ppm) and a significantly increasing trend ( p< 0.01). Incidence in females was 2.0%, 0%, 4.0% and 8.7% ( control to high dose; not significant); however, a significantly increasing trend ( p< 0.05) was observed. The incidence of thyroid follicular cell carcinoma was increased at 6000 ppm only in males ( males 0%, 2.0%, 0% and 4.1%; females 2.0%, 0%, 0% and 2.2%). The combined incidence of thyroid follicular cell adenomas and carcinomas was significantly increased in males at 3000 and 6000 ppm ( control to high dose, 0%, 2.0%, 12.5%, 14.3%; p< 0.05) but not females ( 4.0%, 0%, 4.0% and 10.9%), with a significant trend in both sexes ( females p< 0.05 and males p< 0.01). The incidence of follicular cell adenoma in historical control data from this laboratory did not exceed 11.1% in males or 3.2% in females. The incidence of carcinoma did not exceed 9.4% in males or 3.2% in females.

Adequacy of the Dose Levels Tested: The dose levels are considered adequate. Systemic toxicity was seen at the mid and high dose levels tested.

B. In a Chronic Toxicity/ Oncogenicity study ( MRID 43015801), groups of 60 male and 60 female Crl: CDBR Sprague Dawley rats were given 0, 5, 50, 500, or 1000 mg/ kg bodyweight pentachloronitrobenzene ( Technical 98%) by gavage five days per week for up to two years. When adjusted for continuous exposure, these dose levels were equivalent to 0, 3.6, 36, 357 or 714 mg/ kg/ day. T 3, T 4 and TSH parameters were not examined.

No apparent effects on body weight, food intake, clinical observations, or survival were found. At all dose levels and at all measured time points ( 6, 12, and 18 months and study termination), dose dependent decreases in serum AST and ALT activities and the excretion of triple phosphate crystals in the urine were found in both sexes. At 36 mg/ kg/ day and above, increases in minimal to slight hepatocellular hypertrophy were observed in males ( ranging from 9/ 60 to 26/ 60 versus 2/ 60 in controls; p < 0.05 at 36 mg/ kg/ day; p < 0.01 at 357 mg/ kg/ day and above). At 357 mg/ kg/ day and above, increases in minimal to slight hepatocellular hypertrophy were observed in females ( ranging from 10/ 60 to 22/ 60 versus 1/ 60 in Page 40 controls; p < 0.01). Increases in thyroid follicular cell hypertrophy/ hyperplasia were observed in both sexes ( 6/ 60 versus 2/ 60 in males and 5/ 59 versus 1/ 59 in females) however, the increases were not statistically significant. High dose male rats had an approximate 30% increase of absolute and relative liver weight and an approximate 22% increase in absolute thyroid/ parathyroid weight at necropsy. Microscopically, increases in thyroid follicular cell hypertrophy/ hyperplasia ( mostly minimal to slight; 12/ 60 versus 2/ 60 for males and 10/ 60 versus 1/ 59 for females; p < 0.01) were found. Based on the hepatocellular hypertrophy and the thyroid follicular cell hypertrophy/ hyperplasia, the NOAEL was 3.6 mg/ kg/ day for males and 36 mg/ kg/ day for females. The corresponding LOAELS would be 36 mg/ kg/ day for males and 357 mg/ kg/ day for females. No treatment related increase in neoplasia was found.

The study is classified as core guideline and satisfies the requirements for an § 83 5 Oral Chronic/ oncogenicity Study.

Discussion of Tumor Data: No treatment related increase in neoplasia was noted..

Adequacy of Dosing: Previous concerns for PCNB carcinogenicity were linked to the high HCB concentrations in earlier studies. However, in the Uniroyal feeding study ( MRID 41987301), only 0.04% HCB was reported in the test material and this study demonstrated a positive carcinogenic potential. On the other hand, the Amvac study ( MRID 41987301) was negative for carcinogenicity. This is a gavage study with administration of the test material ( 98% PCNB) only 5 days per week. The interruption in dosing may tend to allow some time for test material body burden to decrease and for for recovery, as compared to a dietary feeding study. This difference in dosing regimen might possibly explain the negative carcinogenicity results, although other similar thyroid effects were noted. HIARC concluded that the dose levels tested were judged to be adequate based on thyroid toxicity observed at the mid and high dose levels.

6.2 Carcinogenicity Study in Mice In an oral carcinogenicity study, ( MRID 45609101) 50 B6C3F1 mice per sex per dose group were administered PCNB ( purity not reported) in the diet at concentrations of 0, 2500, or 5000 ppm for 103 weeks. At the end of the feeding period, test diets were withdrawn and the animals were fed control diets during a one week observation period before termination of the study.

No increase in the incidence of neoplastic lesions was observed in the treated mice, but the test substance may have predisposed the female mice of the high dose group to a bacterial infection which significantly decreased their survival after 86 weeks. PCNB did not exhibit carcinogenic potential under the conditions of the experiment. Page 41 Due to the decreased survival rate, since the lot and concentration of the test material used in the study are not indicated, and the suggestion that study interpretation may have been confounded due to infection, this study is classified as unacceptable and does not satisfy the requirement for a mouse carcinogenicity study.

Discussion of Tumor Data: No carcinogenic effects were noted.

Adequacy of Dosing: Dosing may have been adequate, but survival was reduced associated with a bacterial infection.

6.3 Classification of Carcinogenic Potential The carcinogenicity of PCNB was assessed in reviews of 1977 ( special review) and in 1986 and 1992. In the most recent carcinogenicity peer review of PCNB ( held on 8/ 26/ 92, memo of 12/ 18/ 92) the peer review committee agreed that PCNB should be classified as a Group C possible human carcinogen and recommended that for the purpose of risk characterization, the Reference Dose approach should be used for quantification of human risk. This review considered the new chronic/ oncogenicity study in the rat ( MRID 41987301) by Uniroyal to be the only appropriate study to use. The report also indicated that a second rat study was being conducted by Amvac and that it was expected at the Agency by December1993. This Amvac study ( MRID 43015801) was later reviewed by P. Hurley, 6/ 27/ 95, and found to be negative for carcinogenicity..

7.0 MUTAGENICITY The HIARC concluded that there is not a concern for mutagenicity resulting from exposure to PCNB.

Pentachloronitrobenzene was selected for genetic toxicology screening by the National Toxicology Program ( NTP). Testing included: the Salmonella/ mammalian microsome test, induction of forward gene mutations in L5178Y mouse lymphoma cells and induction of chromosome aberrations and sister chromatid exchanges ( SCEs) in Chinese hamster ovary ( CHO) cells. Although formal DERs do not exist for these assays, they are considered acceptable by HED.

In independently conducted ICR mouse bone marrow micronucleus assays ( MRID No. 45539601), groups of five male and five female mice were administered Pentachloronitrobenzene as Terraclor Technical ( 99%, lot # 88101) once via single intrapertioneal ( ip) injections of 0, 230, 460 or 920 mg/ kg bw. Comparable doses were administered in the repeat trial. Bone marrow cells were harvested at 24 and 48 hours Page 42 post treatment. The vehicle was corn oil. Based on preliminary toxicity testing, the highest dose tested ( HDT) was 80% of the LD 50/ 3.

Clinical signs included lethargy and piloerection at 460 and 920 mg/ kg; a sight to moderate ( up to 25%) reduction in the ratio of polychromatic erythrocytes to total erythrocytes ( PCE: NCE) was recorded following exposure to the HDT. Terraclor technical was, therefore, tested up to an adequate dose ( based on overt toxicity in the animals and cytotoxicity in the target cells). The positive control induced the appropriate response. There was no reproducible significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

This study is classified as acceptable ( guideline) and satisfies the guideline requirement for Test Guideline OPPTS 870.5395; OECD 474 for in vivo cytogenetic mutagenicity data.

8.0 FQPA CONSIDERATIONS There was no quantitative or qualitative evidence of increased susceptibility of rat or rabbit fetuses after in utero exposure, or after pre or postnatal exposure to rats in multigeneration reproduction studies.

The HIARC, however, did require a comparative thyroid assay in young and adult rats which included hormonal measurements for thyroid function, since thyroid weights were increased in a number of chronic and subchronic studies in rats, and TSH, T3, and T4 levels were affected in a 90 day special oral study in male rats.

The absence of the comparative thyroid study resulted in a database uncertainty factor of 10x ( UF DB of 10x) which was applied to the dietary ( acute and chronic) as well as all residential exposure ( incidental oral, dermal and inhalation) scenarios. The HIARC determined that the 10X UF DB is required since the available data provide no basis to support reduction or removal of the default 10X factor.

8.1 Recommendation for a Developmental Neurotoxicity Study Acute delayed neurotoxicity, acute and subchronic neurotoxicity studies and a developmental neurotoxicity studies are not required at this time since there is no evidence that the compound is a neurotoxicant. Page 43 8.2 OTHER ISSUES Generally, decreases in serum enzyme activities are considered unrelated to treatment with the test material and not adverse toxicological effects. However, PCNB studies consistently show these effects in both subchronic and chronic studies. AST/ ALT activities decrease in a dose dependent manner by as much ast 30 80 %. This shows much more than a general association between decreased AST/ ALT activities and test material treatment.

It is known that penicillamine, cycloserine, hydralazine, and in particular the antituberculin drug isoniazid can decrease the activities of AST and ALT at therapeutic doses. The mechanism by which this occurs is through a biochemical interaction between the drug and Vitamin B6. With isoniazid this is thought to occur through combination with pyridoxal or pyridoxal phosphate to form hydrazone, a potent inhibitor of pridoxal kinase.

Vitamin B6 compromises a group of closely related compounds, pyridoxine, pyridoxal, and pyridoxamine, that are phosphorylated in vivo by pyridoxal kinase to form pyridoxal phosphate. Pyridoxal phosphate serves as a cofactor in many reactions, including decarboxylation and transamination of animo acids, deamination of hydroxy amino acids and cysteine, conversion of tryptophan to niacin, metabolism of fatty acids, protein metabolism, and the transport of certain amino acids across cell membranes. The inhibition of pyridoxal kinase by the isoniazid pyridoxal complex occurs at concentrations 1000 times less than those required to inhibit the enzyme containing the cofactor.

The dose dependent decreases of AST/ ALT activities found consistently in subchronic and chronic studies suggest that PCNB disrupts transferase and other enzyme activities that require pyridoxal phosphate, perhaps by affecting Vitamin B6 homeostasis. Whether the mechanism is similar to that of the isoniazid is not known and cannot be determined from the subchronic or chronic studies. However, the clinical data suggests a disturbance of protein and amino acid metabolism. This is supported not only by the decreased activities of AST/ ALT but also by the dose dependent excretion of triple phosphate crystals in the urine. The excretion of triple phosphate and calcium phosphate, which are urine buffering systems, suggests a disturbance of systemic acid base balance. It is also of interest to note at terminal sacrifice the presence of tyrosine phosphate crystals in urine. Typically these crystals suggest severe hepatic toxicity. However, the clinical pathology of the liver did not suggest toxicity sufficient to disrupt hepatic function. All that was typically found was hepatocellular hypertrophy. Although tyrosine phosphate crystals may have originated from impaired hepatic function, they could have also arisen from tyrosine overload. Excess tyrosine could have resulted from the chronic and cumulative inhibition of various transaminases, particularly tyrosine transaminase, that are essential for protein and amino acid metabolism.

Therefore, in the PCNB studies submitted, it might be reasonable ( in the absence of other relevant kinetic data) to consider the decreases of AST/ ALT as adverse effects. PCNB Page 44 induces a chronic and progressive inhibition of transferase activity. The exact mechanism for the inhibition is speculative, but likely involves the cofactor pyridoxal phosphate. The requirement by HIARC that the Registrant submit new studies to assess AST/ ALT findings as adverse effects of PCNB will eventually resolve these issues.

It seems likely that PCNB induces primary hypothyroidism in both male and female rats. However, the study investigators did not include the assessment of thyroid hormones in any of the submitted studies except one non guideline study ( a 90 day study in male rats). In this study, thyroid hormones were affected after only a 7 day exposure at the high dose level ( 6000 ppm). However, the dose selection in this study did not allow for meaningful assessment of these effects since the next lowest dose level was 20 ppm. At this level the hormones were apparently not affected although, both thyroid and liver hypertrophy were observed in nearly all animals of the 20 ppm dose group after 90 days ( with increased severity observed at the high dose level in all animals). Since this study was only performed in males and since the spread of dose levels is unreasonable ( and there are only two levels at that) it appears that only a new study ( as required by HIARC) will more accurately define PCNB toxicity for the risk assessment process.

Hepatocellular hypertrophy has been well documented to occur following the administration of chlorinated benzenes, polyhalogenated biphenyls, TCDD, and other polyhalogenated hydrocarbons. These compound induced hepatic microsomal enzymes that ultrastructurally result in cellular changes consistent with those reported in the chronic and subchronic PCNB studies. Likewise, the induction of thyroid follicular hyperplasia and hypertrophy in animals is a well documented effect of many polyhalogenated aromatic hydrocarbons. Current evidence suggests that the induction of certain hepatocellular microsomes increases the metabolism of throxine, thereby inducing a hypothyroid state. However, without analysis of TSH, this cannot be firmly established nor can the severity of the hypothyroidism be determined. Further, what effects might be superimposed, or whether the effects would be potentiated, additive, synergistic, or antagonistic is unclear at this time. For this and other reasons, adequate kinetic data should be available for use in the risk assessment process.

Note that in cases where bioaccumulation takes place, MOE and safety factor approaches to risk assessment do not usually apply especially for short duration exposure scenarios. In such cases, blood levels at steady state and other time points for short duration exposure assessment might need to be determined and compared to those of guideline required studies. Such data would then allow for supportable risk assessments for pesticides with longer half lives ( resulting in accumulation within tissues) which likely will have higher risk scenarios. Few of the newer classes of pesticides bioaccumulate. However, PCNB was developed nearly 40 years ago ( an organochlorine pesticide) and it appears to be one of those that does. Clearly, MOE and safety factor approaches to risk assessment by themselves do not take this type of situation into account. Page 45 9.0 REFERENCES in MRID order ( when available)

114223. Sinkeldam, E., Van der Heijden, C., de Groot, A. et al ( 1974) Carcinogenicity Study with Pentachloronitrobenzen ( PCNB) in Rats. Report No. R4442: 3569. Prepared by Central Institut voor Voedingsonderzoek TNO. The Netherlands. Submitted by Olin Cor; poration., Stamford , CT: CDL 248285 H. Unpublished 114226. Weisburger, J., Weisburger, E., Powers, M. et al ( 1978) Bioassay of Pentachloronitrobenzene for Possible Carcinogenicity. DHEW Publication No. ( NIH) 78 1311. Hazelton Laboratories and Tracor Jitco. U. S. National Institutes of Health, NCI, Submitted by Olin Corp., Stamford, CT: CDL: 24825 L 250698 and 255226. Mitoma, C. T., Steeger, S. E., Jackson, and Wheeler K., ( 1983) Skin Penetration Study of Olin's terrachlor Formulations in Rats. Report No. LSC 4675 4 prepared by Stanford Research Institute. January 6, 1983. Unpublished 40588601. Keller, K. A. ( 1988) Developmental Toxicity Study in Rats. International Research and Development Corporation, Mattawan, MI. Laboratory Project ID 399 068. April 11, 1988. Unpublished.

40717102. Keller, K. A. ( 1988) Developmental Toxicity Study in New Zealand White Rabbits, International Research and Development Corporation, Study Number 399 070, Unpublished 41361201. Hoberman, Alan M. ( 1989) Developmental Toxicity ( Embryo Fetal Toxicity and Teratogenic Potential) Study of Pentachloronitrobenzene ( PCNB) Administered Orally Via Gavage to Crl: CD ® ( SD) BR Presumed Pregnant Rats. Argus Research Laboratories, Inc., Horsham, PA, Argus Research Laboratories Protocol 310 005. Unpublished.

41361301, Hoberman, Alan M. ( 1989) Developmental Toxicity ( Embryo Fetal Toxicity and Teratogenic Potential) Study of Pentachloronitrobenzene ( PCNB) Administered Orally ( Stomach Tube) to New Zealand White Rabbits. Argus Research Laboratories, Inc., Horsham, PA. Argus Research Laboratories Protocol 310 006. December 21, 1989. Unpublished.

41718600 and 01. Goldenthal, Edwin I. ( 1993) One Year Chronic Dietary Study in Dogs. International Research and Development Corp., Mattawan, MI. Study No. 399 087. November 16, 1990. Unpublished 41918701. Schardein, J. ( 1991) Two Generation Reproduction Study in Rats. International Research and Development Corporation ( IRDC), Mattawan, MI. Study No. IRDC 399 086. February 1, 1991. Unpublished.

41987301. Goldenthal, E. I. ( 1993) Two Year Dietary Toxicity and Oncogenicity Study in Rats. Internaional research and Development corp., Mattawan, MI. Study Report No. 399 072. August 1, 1991. Unpublished Page 46 42413901, 42713401, 42713402. ( 1992) Twenty one Day Dermal Toxicity Study in the Rat 42416001. Keefe, R. T. ( 1992) 90 Day Subchronic Oral Toxicity Study in Rats with Pentachloronitrobenzene( 89 505). Exxon Biomedical Sciences, Study Number MRD 89 505 Lab Project ID 250570A, Unpublished 42416002. Trimmer, G. W. ( 1992) 21 Day Repeated Dose Dermal toxicity Study in Rats with Pentachloronitrobenzene . Exxon Biomedical Sciences, Inc., Study # 150509, June 22, 1992.

42630801. Goldenthal, Edwin I. ( 1993) 90 Day Dietary Toxicity Study in ( Male) Rats. International research and Development Corp., Mattawan, MI. Study Lab Project No. 399 122. January 5, 1993. Unpublished 43469301, 43469302, 43469303. Phillips, R. ( 1994) Two Generation Reproduction Toxicity Study in Rats with Pentachloronitrobenzene ( PCNB) ( MRD 89 505). Exxon Biomedical Sciencies, Inc. Toxicology Laboratory, east Millstone, NJ, Lab Project ID No. 150535, June 1, 1994. Unpublished 43015801. Plutnick, R. T. ( 1993) Two Year Chronic toxicity/ Oncogenicity Study in Rats with Pentachloronitrobenzene ( PCNB , MRD 890505 batch II) Exxon Biomedical Sciencies, Inc., Toxicology Lab, Mettlers road, CN 2350, east Millstone, NJ 08875 2350. November 8, 1993. Unpublished 43469301, 43469302, 43469303. Phillips, R. ( 1994) Two Generation Reproduction Toxicity Study in Rats with Pentachloronitrobenzene ( MRD 89 505). Exxon Biomedical Sciences, Inc. Toxicology Laboratory, east Millstone, NJ. Laboratory Project ID No. 150535, June 1, 1994. Unpublished.

44096601. Shea, Paul ( 1996) Biliary Excretion of Labeled Thyroxine and Thyroid Uptake of Labeled Iodine in PCNB Treated Rats. Biodevelopment Laboratories, Cambridge, MA. Laboratory Project ID 10601. June 1996. Unpublished 44096602. Kosser, David C. ( 1996) Biliary Excretion of [ 125I] thyroxine and Thyroid Uptake of [ I] in Pentachloronitrobenzene ( PCNB) Treated Rats. MPI research, Mattawan, MI ( formerly IRDC). Laboratory Study Identification No. 399 179. April 17, 1996. Unppublished LITERATURE REFERENCES CPRC ( 1992) Second Carcinogenicity Peer Review of PCNB. Memorandum from Pam Hurley and Esther Rinde dated December 18, 1992 FQPA Consideratons for PCNB ( 2002) Memorandum from Laurence D. Chitlik dated November 21, 2002 Galloway, S. M., Armstrong, M. A., Reuben, C., Colman, S., Brown, B., Cannon, C. et al ( 1987). Chromosome aberration and sister chromatid exchange in Chinese ovary cells: Evaluation of 108 Page 47 chemicals. Environ Molec Mutagen 10( Suppl 10): 1 176.

Haworth, S., Lawlor, T., Mortelmans, K., Speck, W., and Zeiger, E. ( 1983). Salmonella mutagenicity test results for 250 chemicals. Environ Mutagen 5( Suppl 1): 3 142 Helsley, D ( 2000). Personal communication between d. Helsley, cellular and Genetic toxicology Branch, NTP and N. E. McCarroll, OPP/ HED/ Tox 1   mouse lymphoma data.

HIARC ( 2001a) Report of the Hazard Indentification Assessment Committee. Memorandum from Laurence Chitlik, dated January 9, 2001 HIARC ( 2001b) Report of the Hazard Indentification Assessment Committee. Memorandum from Laurence D. Chitlik dated March 4, 2002 HIARC ( 2003) Hazard Identification Assessment Committee Meeting of December 10, 2002 Concerning FQPA Assessment for PCNB.. Memorandum from Elizabeth A. Doyle Kogel, W., Muller, W. F., Coulston, F., Korte, F. 1979a. Fate and Effects of Pentachloronitrobenzene in Rhesus Monkeys. J. Agric. Food Chem., Vol 27, No. 6, 1979 Kogel, W., Muller, W. F., Coulston, F., Korte, F. 1979b. Biotransformation of Pentachloronitrobenzene 14 in Rhesus Monkeys after Single and Chronic Oral Administration, Pergamon Press Ltd. Chemosphere No. 2, pp 97 105 Kuchar, E. J. Geenty, F. O., Griffith, W. P. and Thomas R. J., 1969. Analytical Studies of Metabolism of terrachlor in Beagle Dogs, Rats, and Plants. J. Agr. Food Chem. Vol. 17, No. 6, Nov Dec 1969 MARC ( 2001) Report of the Metabolism Assessment Review Committee ( combining of AMVAC and Uniroyal data bases). Memorandum of October 14, 2001 National Toxicology Program. 1987. Toxicology and Carcinogenesis Studies of Pentachloronitrobenzene in B6C3 F1 Mice. ( CAS No. 82 68 8) NTP Technical Report Series Nol. 325. NIH Publication No. 87 2581. No MRID or accession number assigned.

O'Grodnick, J. S., Adamovics, J. A., Blake, S. M., and Wedig, J. 1981. The Metabolic Fate of 14 CLabeled Pentachloronitrobenzene in Osborne Mendell Rats. Pergamon Press Ltd., Chemosphere Vol. 10, pp 67 72. 1981 Toxicology Branch Peer Review Committee ( 1986) Peer Review on Pentachloronitrobenzene. Memorandum from Judith Hauswirth dated August 27, 1986 Page 48 10.0 APPENDICES Tables for Use in Risk Assessment Page 49 10.1 Toxicity Profile Summary Tables 10.1.1 Acute Toxicity Table  See Section 4.1 10.1.2 Subchronic, Chronic and Other Toxicity Tables Guideline #/ Study Type MRIDNo.( year)/ Classification/ Doses/ SPONSOR Results 1870.4300 2 YR CHRONIC/ CARCINOGENIC RAT FEEDING 41987301 ( 1991) 0, 1, 150, 300 mg/ kg/ day (

/  ) / Acceptable/ UNIROYAL Increase in thyroid follicular cell adenomas and carcinomas in

at 150mg/ kg/ day & 300 mg/ kg/ day ( p< 0.05) & a trend in  Systemic NOAEL= 1 mg/ kg/ day Systemic LOAEL= 150 mg/ kg/ day based on an increase in hepatocellular hypertrophy, hepatocellular hyperplasia ( females) and thyroid hypertrophy and hyperplasia 870.4300 2 YR CHRONIC TOXICITY/ ONCOGENICITY GAVAGE RAT 43015801 ( 1993) 0, 3.6, 36, 357, 714 mg/ kg/ day (

/  ) / Acceptable/ AMVAC NOAEL= 3.6 mg/ kg/ day

and 36 mg/ kg/ day  Negative for neoplasia LOAEL= 36 mg/ kg/ day

and 357 mg/ kg/ day  870.4200b 2 YR CARCINOGENICITY IN MICE NTP TR325 ( 1987) 0, 400, AND 1000 mg/ kg/ day

0, 600 AND 1400 mg/ kg/ day  / Acceptable NO INCREASED NEOPLASIA BUT REDUCED SURVIVAL DECREASED SENSITIVITY OF STUDY 870.4100b 1 YR CHRONIC FEEDING  DOG 41718600 & 41718601 ( 1993) 0, 0.375, 3.75, AND 37.5 mg/ kg/ day ( used dog conversion factor) (

/  ) UNIROYAL NOAEL= 3.75 mg/ kg/ day LOAEL= 37.5 mg/ kg/ day based on increased serum ALP and cholesterol, increased liver weight and hepatocellular hypertrophy.

870.3800 2 GENERATION REPRODUCTION ( RAT) 43469301, 43469302, 43469303/ ( 1994) 0, 10, 100, 1000 mg/ kg/ day/ Acceptable AMVAC NOAEL= 10 mg/ kg/ day

100 mg/ kg/ day  LOAEL= 100 mg/ kg/ day

and 1000 mg/ kg/ day  based on increases in hepatocellular hypertrophy and thyroid follicular cell hypertrophy/ hyperplasia.

870.3800 2 GENERATION REPRODUCTION ( RAT) 41918701 ( 1991) 0, 1.2, 169, 344 mg/ kg/ day (

) & 0, 1.5, 218, 455 mg/ kg/ day (  ) / Acceptable/ UNIROYAL Parental NOAEL = 1.2 mg/ kg/ day

, 1.5 mg/ kg/ day  Reproductive/ Developmental NOAEL= 1.2 mg/ kg/ day Parental LOAEL= 169 mg/ kg/ day

, 218 mg/ kg/ day  based on decreased body weight and weight gain. Reproductive/ developmental LOAEL= 169 mg/ kg/ day based on decreased mean pup weights. Page 50 870.3700a DEVELOPMENTAL TOX RAT ( GAVAGE) 40588601 ( 1988) 0. 30, 600, 1200 mg/ kg/ day / Acceptable/ UNIROYAL Maternal NOAEL= Maternal > 1200 mg/ kg/ day Developmental NOAEL > 1200 mg/ kg/ day Maternal LOAEL > 1200 ( HDT) Developmental NOAEL > 1200 ( HDT)

870.3700b DEVELOPMENTAL TOX RABBIT ( GAVAGE) 41361301 ( 1989) 0, 100, 300, AND 900 mg/ kg/ day / Acceptable/ AMVAC Maternal NOAEL = 300 mg/ kg/ day Developmental NOAEL > 900 mg/ kg/ day Maternal LOAEL= 900 mg/ kg/ day based on clinical signs of toxicity, decreased body weight gain and food consumption, abortions and premature delivery Developmental LOAEL > 900 ( HDT)

870.3700b DEVELOPMENTAL TOX RABBIT ( GAVAGE) 40717102 ( 1988) 0, 6.25, 12.5, 125 and 250 mg/ kg/ day/ Acceptable ( combination of two studies)/ UNIROYAL Maternal NOAEL= 12.5 mg/ kg/ day Developmental NOAEL= 125 mg/ kg/ day Maternal LOAEL 125 mg/ kg/ day based on decreased maternal body weight and body weight gain at 125 mg/ kg/ day and mortality, abortions and weight loss at 250 mg/ kg/ day Developmental LOAEL= 250 mg/ kg/ day based on decreased fetal weights 870.3700a DEVELOPMENTAL TOX RAT ( GAVAGE) 41361201 ( 1989) 0, 250, 750, 1500 mg/ kg/ day AMVAC Maternal NOAEL > 1500 mg/ kg/ day Developmental NOAEL= 250 mg/ kg/ day Maternal LOAEL > 1500 mg/ kg/ day ( HDT) Developmental LOAEL= 750 mg/ kg/ day based on increases in the incidences of thoracic vertebrae/ fetus, average pairs of thoracic ribs/ fetus and other variations non guideline 7, 14, 30 OR 90 DAY SUBCHRONIC ( MALE RAT) 42630801 ( 1993) 0, 1.0, & 333 mg/ kg/ day ACCEPTABLE NONGUIDELINE UNIROYAL NOAEL ( 90 Day) < 1.0 mg/ kg/ day LOAEL ( 90 Day) 1.0 mg/ kg/ day based on liver and thyroid hypertrophy. After 14 & 30 DAYS: thyroid follicular epithelial hypertrophy observed at 333 mg/ kg/ day level 870.3100 13 Week SUBCHRONIC ORAL ( GAVAGE) RAT 42416001 ( 1992) 0, 5, 10, 100, OR 1000 mg/ kg/ day

/  / Acceptable/ AMVAC NOAEL= 100 mg/ kg/ day (

/  ) LOAEL= 1000 mg/ kg/ day (

/  ) ( HDT)

870.3200 21 DAY DERMAL ( RAT) 42413901 42713401& 42713402 / ( 1992) 0, 30, 300, or 1000 mg/ kg/ day / Acceptable/ UNIROYAL Systemic NOAEL > 1000 mg/ kg/ day Systemic LOAEL > 1000 mg/ kg/ day ( HDT)

870.3200 21 DAY DERMAL ( RAT) 42416002 ( 1992) 0, 100, 300 & 1000 mg/ kg/ day (