SRC TR-13-069

HPV3 TEST RULE DATA ADEQUACY REVIEW (FINAL):

REPEATED-DOSE, REPRODUCTIVE, AND DEVELOPMENTAL TOXICITY

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 24, 2013
Final HPV3 Chemical Data Adequacy Review (October 2013) CASRN 98-09-9

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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 Group of Chemicals” (76 FR 65385, October 21, 2011; referred to herein as 

HPV3; available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT2009-0112-0086 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 Daphnia and 

toxicity to algae testing because the log Kow of CASRN 98-09-9 is < 4.21), E1 (bacterial reverse 

mutation test), E2 (chromosomal aberration or micronucleus test), and F1 (combined repeateddose toxicity study with the reproduction/developmental toxicity screening test). 

In the 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 requirement. 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 F1 (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 Kow = 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/AGGRe7cad612-601f-4077-bf75-88ef184348e6_DISS-9d8ca91a-d8a5-0cd4-e044-00144f67d249.html#AGGR-e7cad612-

601f-4077-bf75-88ef184348e6 as of October 24, 2013).
Final HPV3 Chemical Data Adequacy Review (October 2013) CASRN 98-09-9

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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-HQ-OPPT2009-0112-0128 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.
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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, 2013)

Title: Benzenesulfonyl chloride: Oral (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 98-09-91

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:RccHanTM: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 

(females) and were approximately 12 weeks.

Number of animals/sex/dose: 12

Route of administration: Oral (gavage)

Vehicle: Dried 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 HPV3 Chemical Data Adequacy Review (October 2013) CASRN 98-09-9

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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 

Organ Weights, Body Weight-Relative Organ Weights”. [Fulcher and Watson 2013, p. 33]

“Where possible, data were analysed using the decision tree from the ProvantisTM 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-parametric 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, pairwise 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 capture 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 incorporating 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, reproductive parameters 

(implantation losses, offspring sex ratio and offspring surface righting) were generally analysed 

using non-parametric analyses.” [Fulcher and Watson 2013, p. 34]
Final HPV3 Chemical Data Adequacy Review (October 2013) CASRN 98-09-9

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“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 based on available data, including a 14-day range-finding 

toxicity test in rats1. 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 test 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 demonstrated 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, samples of the test item formulations (nominal concentrations of 0, 1.25, 5, and 

15 mg/mL, corresponding 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 constant dosing 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 exposure. 

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.

F0 males were euthanized on day 43. F0 females were allowed to deliver and rear their pups until 

lactation day 5, when F0 females and their pups were euthanized. F0 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.
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Clinical Observations:

Rats were observed at least twice daily for mortality, moribundity, and clinical observations.

 

Body Weight and Food/Water Consumption:

Body weights and food consumption were recorded at least weekly throughout the study. Water 

consumption was recorded daily throughout the study. 

Neurological 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 F0 rats/sex/group after mating and prior to termination.

Blood and Serum Chemistry Analyses:

Investigations were performed on blood samples collected from five F0 rats/sex/group on the day 

prior to sacrifice. Animals were not fasted prior to sampling1

. 

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, prothrombin time, 

activated partial thromboplastin time, reticulocyte count, and differential leukocyte count 

(including neutrophils, lymphocytes, monocytes, eosinophils, and basophils).

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 F0 animals were subjected to a full external and internal examination and selected organs 

were weighed. Organ weights from five F0 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. 

Samples were collected from the following organs and tissues from five F0 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, lungs (including bronchi), lymph nodes (mandibular and mesenteric), muscle 

(skeletal), pancreas, esophagus, rectum, salivary gland (submaxillary), sciatic nerve, 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 collecting blood samples is recommended, but not required, by OECD TG 422 and 40 

CFR 799.9365.
Final HPV3 Chemical Data Adequacy Review (October 2013) CASRN 98-09-9

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the samples above from the control and high-dose (i.e., 60 mg/kg-bw/day) groups, as well as any

from animals dying during the study and animals that failed to achieve pregnancy. Additionally, 

samples 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 F0 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 

tissues 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: pre-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 postpartum, 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 (males, systemic toxicity)1= 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.

NOAEL (males, 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 alpha2u-globulin-mediated mechanism, which appears to be unique to 

male rats and the response 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 

(Alpha2u-Globulin: Association with Chemically Induced Renal Toxicity and Neoplasia in the Rat, EPA/625/3-

91/019F). One of the key events, alpha2u-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 established.
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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 nonglandular region of the stomach and histopathological changes in the stomach, consisting of

minimal 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 toxicity) = 20 mg/kg-bw/day

Reproductive Toxicity:

NOAEL (male and female, reproductive toxicity) = 60 mg/kg-bw/day, based on no effects at the 

highest dose tested. 

Developmental Toxicity:

NOAEL (developmental toxicity) = 60 mg/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/kgbw/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 F0 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 considered to be 

treatment-related. The first male was observed to have gasping respiration, lethargy, hunched 

posture, and staining around the snout on day 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, piloerection, lethargy, hunched posture, and pallor 

of the extremities on day 41. This animal was also killed for animal welfare considerations and 

necropsy 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 F0 males 

from the high-dose group. Exophthalmos 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 F0 animal mean body weights, body weight gain or 

food consumption. Slightly increased 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 statistically 
Final HPV3 Chemical Data Adequacy Review (October 2013) CASRN 98-09-9

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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 F0 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 F0 animals on behavior, grip strength, motor activity,

or sensory reactivity to different stimuli (auditory, visual, and proprioceptive). 

Blood and Serum Chemistry Analyses:

Statistically significant decreases in mean blood hemoglobin (p < 0.01), erythrocyte count (p < 

0.05), and hematocrit (p < 0.01) were observed in F0 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 treatmentrelated effects on hematological parameters were observed.

Mean cholesterol was statistically significantly increased (by approximately 26%; p < 0.05) in 

high-dose F0 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 in surviving F0 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 F0 males and females in the 60 mg/kgbw/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 60 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 other groups on study.” [Fulcher and Watson 2013, p. 460] 

Histopathological examinations showed minimal to mild centrilobular hypertrophy of 

hepatocytes in the liver in F0 males dosed at 60 mg/kg-bw/day (7/7 males in the 60 mg/kgbw/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 

F0 males or females.

Males treated with 20 and 60 mg/kg-bw/day had statistically significant increases (p < 0.01) 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 groups or in females in any dose group. Histological examinations 
Final HPV3 Chemical Data Adequacy Review (October 2013) CASRN 98-09-9

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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 included 

minimal to moderate foci of basophilic (regenerating) tubules in 1/5, 4/5, 4/5, and 8/8 males;

mild to marked increases in intra-epithelial hyaline droplets in 0/5, 5/5, 5/5, and 8/8 males;

minimal to moderate granular cast formation in 0/5, 3/5, 5/5, and 7/8 males; and minimal 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 alpha2u-globulin 

nephropathy, a species-specific effect that does not appear to be relevant to humans. However, 

in the absence of direct measurement of alpha2u-globulin, there is no conclusive evidence that 

nephropathy in males was the result of an alpha2u-globulin-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 

changes 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 treatmentrelated. No changes in thyroid/parathyroid weights were seen in treated females. 

Reproductive/Developmental Toxicity:

Mating performance, fertility, and gestation length were unaffected by treatment. Reproductive 

and offspring 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 the control, this finding was considered incidental 

and unrelated to treatment. 

No treatment-related effects were seen during histology of reproductive organs collected from F0

animals. Reproductive organ weights of treated animals were similar to controls. 

There was no treatment-related pup mortality. Pups displayed no clinical signs of exposure to 

the test item during gestation and lactation. Initial offspring body weights and offspring body 

weight gain (through postpartum 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, the increased incidence of 

pups missing or dead at 20 mg/kg-bw/day was not considered to be treatment-related. 
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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 historical 

control values for the same rat strain. Surface righting ability of offspring from the low- and 

mid-dose groups was similar to controls.

“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 LOAEL for systemic toxicity in females was 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 changes in the stomach, consisting of

minimal to moderate spongiosis (epithelial vacuolation), minimal to moderate focal or diffuse 

epithelial hyperplasia of the squamous epithelium, and minimal to moderate 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 mg/kg-bw/day, based on no effects 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 

justification for the use of dried Arachis oil BP as the vehicle or address 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 alpha2u-globulin-mediated mechanism, which appears to be unique to 

male rats and the response 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 

(Alpha2u-Globulin: Association with Chemically Induced Renal Toxicity and Neoplasia in the Rat, EPA/625/3-

91/019F). One of the key events, alpha2u-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 established.
Final HPV3 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. 2013. Benzenesulfonyl chloride: Oral (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).