SRC TR

- 13

- 069

HPV 3 TEST RULE DATA ADEQU ACY REVIEW ( FINAL ): REPEATED

- DOSE, REPRODUCTIVE, AND DEVELOPMENTAL TO XICITY Benzenesulfonyl chloride, CASRN 98

- 09

- 9

Prepared by: Justine von Runnen, Amy Nguyen , Teresa Manyin, and William L. Richards

Defense and Environmental Solutions Division SRC, Inc. 7502 Round Pond Road North Syracuse, NY 13212

Contract No. EP

- W

- 09

- 027 Task FG007. 4 .009.006

Submitted to: OCSPP/OPPT/RAD (7403M) U.S. Environmental Protection Agency 1200 Pennsylvania Avenue

Washington, D.C. 20460 Bryan Lobar, Project Officer David Brooks, Work Assignment Manager Amy Benson, Alternate Work Assignment Manager
October 2 4 , 2 013Final HPV 3 Chemical Data Adequacy Review (October 2013) CASRN 98
- 09
- 9

1 A. SOURCE MATERIAL USED FOR ASSESSMENT
This document evaluates a submission from Lanxess Corporation (dated July 2013) that includes data from toxicity testing of benzenesulfonyl chloride (CASRN 98

- 09

- 9 ). These data were received by EPA in response to the final test rule, “Testing of Certain High Production Volume Chemicals; Third G roup of Chemicals” (76 FR 65385 , October 21, 2011; referred to herein as HPV3 ; available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

2009

- 0112

- 0086 as of October 24 , 2013). The HPV3 test rule requires the following toxicological testing for CASRN 98

- 09

- 9: C2 (consisting of acute toxicity to D aphnia and toxicity to algae testing because the log K ow of CASRN 98

- 09

- 9 is < 4.2 1 ) , E1 (bacterial reverse mutation test), E2 (chromosomal aberration or micronucleus test) , and F1 (combined repeated

dose toxicity study with the reproduction/development al toxicity screening test)

. In th e current submission , Lanxess Corporation supplied data from a combined repeated

- dose toxicity study with the reproduction/developmental toxicity screening test conducted with CASRN 98

- 09

- 9, submitted for the F1 testing r equirement

. These newly submitted data are evaluated for adequacy herein. No other toxicity data are evaluated in this report. B. EVALUATING DATA ADEQUACY The submitted data are evaluated for adequacy in Section D. The study was summarized according to a standard format and assigned a Klimisch reliability code based on its scientific merit and conduct, using professional judgment. To determine whether data were adequate to satisfy SIDS endpoints, study details were compared to current standard test guidelines to establish whether the study met current testing requirements. If the data available for the endpoint were determined to be inadequate, a basis for that conclusion is supplied. C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE Data submitted by Lanxess Corporation and reviewed in this document are adequate to satisfy the F 1 ( combined repeated

- dose toxicity with reproduction/development toxicity screening ) testing requirement for CASRN 98

- 09

- 9 promulgated in the final HPV3 test rule. In a previous submission (dated March 26, 2013 ; available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0140 as of October 24 , 2013) , Lanxess Corporation supplied data for the acute toxicity to Daphnia and toxicity to algae (C2) testing requirements

. These data were reviewed in a separate report (SRC Technical Report #13

- 048) and determined to be adequate to satisfy the C2 testing requirement for CASRN 98

- 09

- 9.

1 Log K ow = 3.39 (experimental); value obtained from the ECHA website (available at http://apps.echa.europa.eu/registered/data/dossiers/DISS

- 9d8ca91a

- d8a5

- 0cd4

- e044

- 00144f67d249/AGGR

e7cad612

- 601f

- 4077

- bf75

- 88ef184348e6_DISS

- 9d8ca91a

- d8a5

- 0cd4

- e044

- 00144f67d249.html#AGGR

- e7cad 612

- 601f

- 4077

- bf75

- 88ef184348e6 as of October 24 , 2013).Final HPV 3 Chemical Data Adequacy Review (October 2013) CASRN 98
- 09
- 9

2 Lanxess Corporation also supplied data for the gene mutation (E1) and chromosomal aberration (E2) testing requirements in previous submissions (dated December 3, 2012 and December 17, 2012; available online at http://www.regulations.gov/#!documentDetail;D=EPA

- H Q

- OPPT

2009

- 0112

- 0128 and http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0134 , respectively, as of October 24 , 2013). These submissions were previously reviewed by EPA (review available online at http://www.regulations.gov/#!documentDetail; D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0136 as of October 24 , 2013) and determined to be adequate to satisfy the E1 and E2 testing requirements for CASRN 98

- 09

- 9. There are no outstanding HPV test rule requirements for toxicity testing of CASRN 98

- 09

- 9

.Final HPV 3 Chemical Data Adequacy Review (October 2013) CASRN 98
- 09
- 9

3 D. DATA FOR SIDS ENDPOINTS
D
- 1
. Repeated
- Dose/Reproductive/Developmental Toxicity Summary of endpoint: Adequate Study 1: Combined repeated
- dose oral toxicity study with reproduction/developmental toxicity screening test in rats ( Fulcher and Watson, 201 3 )
Title: Benzenesulfonyl chloride: O ral (gavage) combined repeat dose toxicity study with reproduction/developmental toxicity screening test in the rat (OECD 422) This study is: Adequate Not Adequate
TEST SUBSTANCE Identity: Benzenesulfonyl chloride, CASRN 98

- 09

- 9 1
Remarks: 99.53% purity; clear, colorless liquid; Lot No. KX120302.A
METHOD Method/guideline followed: OECD TG 422 (1996) “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” ; Commission Regulation (EC) No. 440/2008; 40 CFR 799.9365 “ TSCA Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test”

. Test type: Combined Repeated

- Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test GLP compliant?: Yes Year study performed: 2012 Test species: Wistar HanTM:RccHan TM:WIST strain rats from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK ; at initiation, animals weighed 299

- 345 g (males) and 192

- 226 g (f

emales) and were approximately 12 weeks.
Number of animals/sex/dose: 12
Route of administration: Oral (gavage)
Vehicle: D ried Arachis oil BP

1 The test substance was identified in the study report as “benzenesulfonyl chloride” with a CASRN of 98

- 09

- 8, which is not a valid CAS registry number. Based on the chemical name, the correct CASRN is 98

- 09

- 9.Final HPV 3 Chemical Data Adequacy Review (October 2013) CASRN 98
- 09
- 9

4 Exposure duration : Males: 42 days; Females: 41

- 45 days ( throughout 14 days of pre

- mating, mating [maximum of 14 days] , gestation , and up to lactation day 4)
Doses/concentrations tested: 0, 5, 20 , and 60 mg/kg

- bw/day
Statistical methods used: “Where considered appropriate, quantitative data [were] subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p < 0.05. Statistical analysis was performed on the following parameters: Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Water Consumption during gestation and lactation, Pre

- Coital Interval , Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Haematology, Blood Chemistry, Absolute Or gan Weights, Body Weight

- Relative Organ Weights”

. [ Fulcher and Watson 2013 , p. 33] “Where possible, data were analysed using the decision tree from the Provantis TM Tables and Statistics Module as detailed below: Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analysed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA [Analysis of Variance] , or if required, ANCOVA [Analysis of Covariance] with appropriate covariates. Any transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non

- parametr ic data. If no dose response was found, but the data show [ed] non
- homogeneity of means, the data were analysed by a stepwise Dunnett’s (parametric) or Steel (non

- parametric) test to determine [if there were any] significant difference [ s ] from the control group. Where the data were unsuitable for these analyses, pair

wise tests [were] performed using the Student [’ s] t

- test (parametric) or the Mann

- Whitney U test (nonparametric).” [ Fulcher and Watson 2013 , p. 33] “Data not analysed by the Provantis data ca pture system were assessed separately using the SPSS statistical package. Initially, the homogeneity of the data was assessed using Levene’s test. Where Levene’s test was shown to be non

- significant (p ≥ 0.05), parametric analysis of the data was applied , incorporating analysis of variance (ANOVA). If this data was shown to be significant, this analysis was followed by pair

- wise comparisons using Dunnett’s test. Where Levene’s test was significant, non

- parametric analysis of the data was analysed incorp orating the Kruskal

- Wallis test which if significant, was followed by the Mann

- Whitney U test. Dose response relationship [s] [were] investigated by linear regression. Where the data [were] unsuitable for these analyses, then pair

- wise tests were performed using the Student t

- test (parametric) or the Mann

- Whitney U test (non

- parametric).” [ Fulcher and Watson 2013 , p. 33] “Due to the preponderance of non

- normally distributed data, reproducti ve parameters (implantation losses, offspring sex ratio and offspring surface righting) were generally analysed using non

- parametric analyses.” [ Fulcher and Watson 2013 , p. 34]Final HPV 3 Chemical Data Adequacy Review (October 2013) CASRN 98
- 09
- 9

5 “In addition, suspected treatment

- related histopathology findings were statistically analysed (excluding any decedents, non

- mated females and females not producing a pregnancy/litter).” [ Fulcher and Watson 2013 , p. 34]
Test method/conditions remarks: Range

- Finding Test Procedure: Dose levels in the definitive test were b ased on available data, including a 14

- day range

- finding toxicity test in rats 1 . According to limited information provided in the definitive test study report, adverse effects were seen in the range

- finding test in the stomach of one animal at 100 mg/kg

- bw/day and in most animals at 300 mg/kg

- bw/day

. No adverse effects were seen at 50 mg/kg

- bw/day

. Definitive Test Procedure: Analyses of Test Item Formulations: The stability of prepared test item formulations was determined by high performance liquid chromatography (HPLC) with a UV detector (wavelength : 240 nm)

. Homogeneity of formulations was assessed visually. Analyses of t est item formulations ( nominal concentrations up to 253 mg/mL ) showed that 99

- 102% of initial concentrations remained after storage for 11 days in the dark at 4 oC. Formulations were deemed homogeneous by visual inspection. Based on the demonstrat ed stability of the test item under these conditions, bulk formulations were prepared on a weekly basis and daily aliquots were stored under the same conditions

. During the study, s amples of the test item formulations (nominal concentrations of 0, 1.25, 5 , and 15 mg/mL, correspondin g to doses of 0, 5, 20 , and 60 mg/kg

- bw/day, respectively) were collected within two days of preparation and analyzed by HPLC to verify exposure concentrations. Dosing and Environmental Conditions: Groups of sexually mature rats (12/sex/group) were administered the test item daily via gavage at dose levels of 0 (vehicle only), 5, 20 or 60 mg/kg

- bw/day at a co nstant dos ing volume of 4 mL/kg

- bw . Exposure continued throughout the pre
- mating (14 days) , mating (maximum of 14 days) , and post

- mating periods. After the mating period, exposure continued in males until the day of euthanasia, for a total of 42 consecutive days of exposure. Females continued to receive the test item through lactation day 4, for a total of 41

- 45 consecutive days of expos ure
. For mating, one male was paired with one female for a maximum of 14 days until evidence of mating ( i.e., the presence of sperm or a vaginal plug) . Parental animals were housed in groups of four prior to mating and in pairs (one male: one female) during mating. After mating, males were returned their original cages and females were housed individually (or with their litters after parturition ) throughout gestation and lactation

. F 0 males were euthanized on day 43. F 0 females were allowed to deliver and rear their pups until lactation day 5, when F 0 females and their pups were euthanized. F 0 females that failed to deliver were euthanized on or after day 25 post

- coitum.

1 The 14

- day range
- finding study is described in a separate study report ( Harlan Laboratories Ltd., Project No. 41201578 ) , which was not available for review

.Final HPV 3 Chemical Data Adequacy Review (October 2013) CASRN 98
- 09
- 9

6 Clinical Observations: Rats were observed at least twice daily for mortality , moribundity , and c linical observations

. Body Weight and Food/Water Consumption: B ody weights and food consumption were recorded at least weekly throughout the study . Water consumption was recorded daily throughout the study

. Neuro logical Assessments : Animals were observed for signs of functional/behavioral toxicity prior to the start of treatment and at weekly intervals thereafter. Functional performance tests (motor activity and forelimb/hindlimb grip strength) and sensory reactivity to stimuli assessments were also performed on five F 0 rats/sex/group after mating and prior to termination. Blood and Serum Chemistry Analyses : I nvestigations were performed on blood samples collected from five F 0 rats/sex/group on the day prior to sacrifice

. Animals we re not fasted prior to sampling 1

. Hematological parameters that were evaluated included the following: total leukocyte count, erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, prothrom bin time, activated partial thromboplastin time, reticulocyte count , and differential leukocyte count (including neutrophil s , lymphocyte s , monocyte s , eosinophil s , and basophil s)

. Serum chemistry parameters that were evaluated included the following: urea, glucose, total protein, albumin, albumin/globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphorus, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatinine, total cholesterol, total bilirubin , and bile acids

. Gross Necropsy and Histopathology: All F 0 animals were subjected to a full external and internal examination and selected organs were weighed. Organ weights from five F 0 rats/sex/group were recorded for the adrenal glands, brain, heart, liver, spleen, thymus , and thyroids with parathyroids. Organ weights from all animals were recorded for the epididymides, kidneys, ovaries, pituitary glands, prostate, seminal vesicles, testes , and uterus with cervix.
S amples were collected from the following organs and tissues from five F 0 rats/sex/group and preserved for microscopic examination : adrenal glands, aorta (thoracic), bone and bone marrow (sternum and femur including stifle joint), brain (including cerebrum, cerebellum and pons), caecum, colon, duodenum, eyes, gross lesions, heart, ileum (including Peyer’s patches), jejunum, kidneys, liver, l u ngs (including bronchi), lymph nodes (mandibular and mesenteric), muscle (skeletal), pancreas, esophagus, rectum, salivary gland (submaxillary), sciatic ner ve, skin (hind limb), spinal cord (cervical, mid

- thoracic and lumbar), spleen, stomach, thyroid/parathyroid, trachea, thymus , and urinary bladder . Tissue histopathology examinations were performed on

1 Overnight fasting prior to collect ing blood samples is recommended, but not required, by OECD TG 422 and 40 CFR 799.9365

.Final HPV 3 Chemical Data Adequacy Review (October 2013) CASRN 98
- 09
- 9

7 the samples above from the control and high

- dose (i.e., 60 mg/kg

- bw/day) group s, as well as any from animals dying during the study and animals that failed to achieve pregnancy

. Additionally, s amples were collected from selected organs (coagulating gland, epididymides, mammary gland, ovaries, pituitary gland, prostate, seminal vesicles, testes, uterus/cervix , and vagina) and examined for all animals in the control and high

- dose groups. For the stomach, liver, and kidneys , histological examinations were extended to lower

- dose groups ( five rats/sex/group for stomach and five male rats/group for liver and kidneys) because treatment

- related effects were seen at 60 mg/kg

- bw/day. Reproductive /Developmental Toxicity: At necropsy, selected reproductive organs collected from F 0 animals were weighed and histopathologically examined

. Organ weights from all animals were recorded for the epididymides, ovaries, prostate, seminal vesicles, testes , and uterus with cervix. Reproductive t issues preserved from all animals for histological examination included the coagulating gland, epididymides, mammary gland, ovaries , prostate, seminal vesicles, testes, uterus/cervix , and vagina ; these tissues were examined for all control and high

- dose animals

. The number of implantations was recorded upon examination of the uterus. The following reproductive parameters were calculated: p re
- coital interval, mating index , pregnancy index, gestation length , parturition index, pre
- and post

- implantation loss, live birth index, viability index , and sex ratio. For each litter, the following parameters were recorded: number of live and dead offspring on day 0 postpartum , number of live offspring on days 1 and 4 postpartum, sex of offspring on days 0, 1 and 4 p ostpartum, clinical condition of offspring from birth to day 5 postpartum , and individual offspring weights on days 1 and 4 postpartum. All live offspring were assessed for surface righting reflex on day 1 postpartum. At necropsy (day 5 postpartum), all offspring were subjected to an external and internal examination and all macroscopic abnormalities were recorded.
RESULTS Endpoint value(s): Systemic Toxicity: LOAEL (male s , systemic toxicity) 1 = 5 mg/kg

- bw/day, based on histo patholo gical effects in the kidneys, including minimal to mild foci of basophilic (regenerating) tubules, minimal to mild granular cast formation, mild to moderate intra

- epithelial hyaline droplets , and minimal single cell death

. NOAEL (male s , systemic toxicity) = Not established

1 The presence of nephropathy in association with the hyaline droplet accumulation in male rats suggests that the nephropathy in the males is occurring by an alpha 2u - globulin

- mediated mechanism, which appears to be unique to male rats and the response is probably not releva nt to humans for purposes of risk assessment. EPA’s Risk Assessment Forum has outlined the key events and the data that are necessary to demonstrate this mode of action (Alpha 2u - Globulin: Association with Chemically Induced Renal Toxicity and Neoplasia i n the Rat, EPA/625/3

- 91/019F). One of the key events, alpha 2u - globulin accumulation, has not been demonstrated. Therefore, the nephropathy is assumed to be relevant to human health and it is concluded that a NOAEL for nephropathy in male rats was not est ablished.Final HPV 3 Chemical Data Adequacy Review (October 2013) CASRN 98
- 09
- 9

8 LOAEL (females, systemic toxicity) = 60 mg/kg

- bw/day, based on increased mean cholesterol levels and effects on the stomach, including increased incidence of thickening of the non

glandular region of the stomach and histopathological cha nges in the stomach , consisting of m inimal to moderate spongiosis (epithelial vacuolation) , minimal to moderate focal or diffuse epithelial hyperplasia of the squamous epithelium , and minimal to moderate submucosal inflammation. NOAEL (females, systemic to xicity) = 20 mg/kg

- bw/day Reproductive Toxicity: NOAEL (male and female, reproductive toxicity) = 60 m g/kg

- bw/day, based on no effects at the highest dose tested. Developmental Toxicity: N OAEL (developmental toxicity) = 60 m g/kg

- bw/day, based on no effects at the highest dose tested

. Results remarks: Analyses of Test Item Formulations: Measurement of test item concentrations by HPLC in formulations prepared for dosing (nominal concentrations of 1.25, 5 , and 15 mg/mL, which corresponded to doses of 5, 20 , and 60 mg/kg

bw/day, respectively) showed that formulations contained 95

- 105% of nominal concentrations.
Clinical Observations : No deaths that were considered to be treatment

- related were observed in F 0 animals

. Two males from the high

- dose group were terminated prior to scheduled termination due to suspected intubation errors during the dosing procedure; therefore, both deaths were not cons idered to be treatment

- related. The first male was observed t o have gasping respiration, lethargy, hunched posture , and staining around the snout on d ay 23 and was killed for animal welfare considerations. Necropsy of this animal revealed a mass filled with clear fluid

- like material under the left forelimb and dark lungs. The second male was observed to have a tip

- toed gait, labored and decreased respiration rate, ptosis, pilo erection, lethargy, hunched posture , and pallor of the extremities on d ay 41 . This animal was also killed for animal welfare considerations and n ecropsy revealed fibrous adhesions to the heart and lungs, gaseous distension of the stomach, duodenum, jejunum, ileum, caecum and colon, enlarged adrenals , and dark liver, thymus , and lungs.
A transient increase in salivation was observed post

- dosing on days 13 and 14 in five F 0 males from the high

- dose group . E xophthalmos was observed in one female from the low

- dose group , but not in animals from higher dose groups

. Other signs were unremarkable , and were not considered to be adverse or related to the treatment

. Body Weight and Food/Water Consumption: There were no treatment

- related effects on F 0 animal mean body weights, body weight gain or food consumption. Slightly i ncreased water consumption was noted in males from the mid

- and high

- dose groups, as compared to the controls , but the increase did not appear to be statisticallyFinal HPV 3 Chemical Data Adequacy Review (October 2013) CASRN 98
- 09
- 9

9 significant and did not have a consistent dosage relationship ; therefore, it may have represented normal biological variation. There were no effects on water consumption in F 0 males and females from the low

- dose group or in females from the mid

- and high

- dose groups. Neurological Assessments : There were no treatment

- related effects in F 0 animals on behavior, grip strength, motor activity , or sensory reactivity to different stimuli (auditory, visual , and proprioceptive). Blood and Serum Chemistry Analyses : Statistically significant decrease s in mean blood h emoglobin (p < 0.01) , erythrocy te count (p < 0.05) , and h ematocrit (p < 0.01) were observed in F 0 males from the high

- dose group as compared to the control group
. High

- dose male mean values for hemoglobin, erythrocyte count , and hematocrit , respectively, were 7%, 7%, and 8% less than control means
. No other treatment

related effects on hematological parameters were observed.
Mean cholesterol was statistically significantly increased (by approximately 2 6 % ; p < 0.05 ) in high

- dose F 0 females relative to the control group. No other treatment

- related effects on clinical chemistry parameters were observed. Gross Necropsy and Histopathology: Gross necropsy revealed an increased incidence of thickening of the non

- glandular region of the stomach i n surviving F 0 males and females in the high

- dose group. This effect was seen in 3/10 treated males and 3/12 treated females compared with 0/12 and 0/12 control males and females , respectively. Microscopic changes in the stomach were seen in both F 0 males and females in the 60 mg/kg

bw/day group. “Minimal to moderate spongiosis (epithelial vacuolation) and minimal to moderate focal or diffuse epithelial hyperplasia of the squamous epithelium was observed in 6/8 males and all the females [6/6] given 6 0 mg/kg

- bw/day. These findings were accompanied by minimal to moderate submucosal inflammation in 4/8 males and 4/6 females and minimal hyperkeratosis in 1/8 males given 60 mg/kg

- bw/day. None of these findings were observed in animals from any of the oth er groups on study.” [Fulcher and Watson 2013 , p. 460] Histopathological examinations showed minimal to mild centrilobular hypertrophy of hepatocytes in the liver in F 0 males dosed at 60 mg/kg

- bw/day ( 7/ 7 males in the 60 mg/kg

bw/day group versus 0/ 5 males in the control group)

. This finding was not observed in females dosed with 60 mg/kg

- bw/day. No other treatment

- related effects were seen in the liver of treated F 0 males or females.
Males treated with 20 and 60 mg/kg

- bw/day had statistically sign ificant increases (p < 0.0 1 ) in mean absolute and relative (to body weight) kidney weights. An increased incidence of enlargement and/or pallor of the kidneys was seen in males in the 60 mg/kg

- bw/day group . In this group, 4 /12 males displayed kidney enlargement and 2 /12 displayed kidney pallor

. Kidney enlargement was also observed in 1/12 males in the 20 mg/kg

- bw/day group; pallor was not observed at this dose. Neither kidney enlargement nor pallor was observed in males in the control and 5 mg/kg

- bw /day group s or in females in any dose group. Histological examinationsFinal HPV 3 Chemical Data Adequacy Review (October 2013) CASRN 98
- 09
- 9

10 revealed dose

- related increases in abnormalities in the kidneys of males

. These findings and their respective incidences in males in the 0, 5, 20, and 60 mg/kg

- bw/day dose groups incl uded mi nimal to moderate foci of basophilic (regenerating) tubules in 1/5, 4/5, 4/5, and 8/8 males ; m ild to m arked increases in intra

- epithelial hyaline droplets in 0/5, 5/5, 5/5, and 8/8 males ; m i nimal to moderate granular cast formation in 0/5 , 3/5, 5/5, and 7/8 males ; and m i nimal or mild single cell death in 0/5, 3/5, 4/5, and 8/ 8 males

. The dose

- related increase in intra

- epithelial hyaline droplets in the kidneys of males in all treatment groups (i.e., 5, 20, and 60 mg/kg

- bw/day) is suggestive of alpha 2u - globulin nephropathy , a species

- specific effect that does not appear to be relevant to humans

. However, in the absence of direct measurement of alpha 2u - globulin, there is no conclusive evidence that nephropathy in males was the result of an alpha 2u - glo bulin

- mediated mechanism; therefore, the nephropathy is assumed to be relevant to human health. No treatment

- related kidney effects were seen in females. Absolute and relative (to body weight) mean pituitary weights were statistically significantly (p < 0.05) reduced in females of all dose groups, and in males in the 20 mg/kg

- bw/day dose group, compared to the control group. Because a dose

- related pattern was not evident and no corroborating histopathological changes were seen in the pituitary gland of males or females, these findings may not be toxicologically relevant. Males treated with 20 mg/kg

- bw/day had significantly lower absolute and relative (to body weight) mean thyroid/parathyroid weights (p < 0.05) compared to the control group
. Because ch anges in thyroid/parathyroid weights (absolute or relative) were not observed in males of any other dose group, the changes observed at 20 mg/kg

- bw/day do not appear to be treatment

related. No changes in thyroid/parathyroid weights were seen in treated females. Reproductive /Developmental Toxicity : M ating performance , fertility , and gestation length w ere unaffected by treatment. Reproductive and o ffspring assessments were based on 10, 11, 10 , and 9 litters successfully reared to postpartum day 5 for the control, 5, 20 , and 60 mg/kg

- bw/day groups, respectively. There were no adverse treatment

- related effects on the mean number of corpora lutea or implantations, postnatal litter size, offspring survival , or sex ratio. One female from the high

- dose group showed total litter loss postpartum; however, in the absence of any increased postnatal losses for other females at this dosage, as compared to th e control, this finding was considered incidental and unrelated to treatment.
No treatment

- related effects were seen during histology of reproductive organs collected from F 0 animals. Reproductive organ weights of treated animals were similar to contro ls.
There was no treatment

- related pup mortality. Pups displayed no clinical signs of exposure to the test item during gestation and lactation. I nitial offspring body weight s and offspring body weight gain (through post partum day 4 ) were unaffected in treated groups relative to controls . In the control, 5, 20 , and 60 mg/kg

- bw/day groups , respectively, 1, 2, 17 , and 2 pups were either missing or found dead; however, in the absence of a dose relationship, th e increased incidence of pups missing or dead at 20 mg/kg

- bw/day was not considered to be treatment

- related.Final HPV 3 Chemical Data Adequacy Review (October 2013) CASRN 98
- 09
- 9

11 Surface righting ability of offspring from the high

- dose group was slightly inferior to the control group. However, this finding was not statistically significant and was in the range of hi storical control values for the same rat strain. Surface righting ability of offspring from the low

- and mid

- dose groups was similar to control s
.

“ No treatment

- related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings for offspring were typical for the age observed and were considered to be unrelated to toxicity of the test item. ” [ Fulcher and Watson 2013 , p. 40]
CONCLUSIONS Systemic Toxicity: The LOAEL for systemic toxicity in males was 5 mg/kg

- bw/day, based on histopathological effects in the kidneys, including minimal to mild foci of basophilic (regenerating) tubules, minimal to mild granular cast formation, mild to moderate intra

- epithelial hyaline droplets, and minimal single cell death

. 1 The NOAEL for systemic toxicity in males was not established.
The L OAEL for systemic toxicity in females was 60 mg/kg

- bw/da y, based on increased mean cholesterol levels and effects on the stomach, including increased incidence of thickening of the non
- glandular region of the stomach and histopathological changes in the stomach , consisting of m inimal to moderate spongiosis (epithelial vacuolation) , minimal to moderate focal or diffuse epithelial hyperplasia of the squamous epithelium , and minimal to mod erate submucosal inflammation. The NOAEL for systemic toxicity in females was 20 mg/kg

- bw/day

. Reproductive Toxicity: The NOAEL for reproductive toxicity in males and females was 60 mg/kg

- bw/day, based on no effects at the highest dose tested. Developmental Toxicity: The NOAEL for developmental toxicity was 60 m g/kg

- bw/day, based on no eff ects at the highest dose tested

. STUDY RELIABILITY [ 2] Reliable with restrictions. Animals were not fasted overnight prior to blood sampling, as is recommended, but not required, by OECD TG 422 and 40 CFR 799.9365

. The source or manufacturer of the test item was not reported. Additionally, the study authors did not provide a ju stification for the use of dried Arachis oil BP as the vehicle or addres s the potential toxicity, or

1 The presence of nephropathy in association with the hyaline droplet accumulation in male rats suggests that the nephropathy in the males is occurring by an alpha 2u - globulin

- mediated mechanism, which appears to be unique to male rats and the res ponse is probably not relevant to humans for purposes of risk assessment. EPA’s Risk Assessment Forum has outlined the key events and the data that are necessary to demonstrate this mode of action (Alpha 2u - Globulin: Association with Chemically Induced Re nal Toxicity and Neoplasia in the Rat, EPA/625/3

- 91/019F). One of the key events, alpha 2u - globulin accumulation, has not been demonstrated. Therefore, the nephropathy is assumed to be relevant to human health and it is concluded that a NOAEL for nephropa thy in male rats was not established.Final HPV 3 Chemical Data Adequacy Review (October 2013) CASRN 98
- 09
- 9

12 lack thereof, of the vehicle

. Missing information included details on the methods used for preparation of test item formulations (i.e., bulk formulations prepared on a weekly basis and used for daily dosing ).
REFERENCES Fulcher, S. and Watson, P. 201 3 . Benzenesulfonyl chloride: O ral (gavage) combined repeat dose toxicity study with reproduction/developmental toxicity screening test in the rat (OECD 422). Harlan Laboratories Ltd., Derbyshire, UK , Project No. 41201579. Sponsor: Lanxess Corporation, Pittsburgh, PA. Text in quotes is taken directly from Fulcher and Watson ( 2013 )

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