SRC TR-13-003

HPV3 TEST RULE DATA ADEQUACY REVIEW (FINAL)

Benzenesulfonyl chloride, CASRN 98-09-9

Prepared by:

Julie Ramage, Fernando Llados, William L. Richards, and Teresa Manyin

Defense and Environmental Solutions Division

SRC, Inc.

7502 Round Pond Road

North Syracuse, NY 13212

Contract No. EP-W-09-027

Task FG007.3.009.006

Submitted to:

OCSPP/OPPT/RAD (7403M)

U.S. Environmental Protection Agency

1200 Pennsylvania Avenue

Washington, D.C. 20460

Bryan Lobar, Project Officer

David Brooks, Alternate Work Assignment Manager

January 31, 2013
Final HPV3 Chemical Data Adequacy Review CASRN 98-09-9

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TABLE OF CONTENTS

A. SOURCE MATERIAL USED FOR ASSESSMENT .............................................................. 1

B. EVALUATING DATA ADEQUACY ..................................................................................... 1

C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE ........ 1

D. DATA FOR SIDS ENDPOINTS.............................................................................................. 3

D-1. Genetic Toxicity – Gene Mutation..................................................................................... 3

D-2. Genetic Toxicity – Chromosomal Aberrations.................................................................. 7
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A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates two studies submitted by Lanxess Corporation to 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-OPPT-2009-0112-0086 as of 

January 31, 2013) for benzenesulfonyl chloride (CASRN 98-09-9). The HPV3 test rule required 

the following toxicological tests for CASRN 98-09-9: C2 (consisting of acute toxicity to 

Daphnia and toxicity to algae tests if log Kow < 4.2, or chronic toxicity to Daphnia and toxicity 

to algae tests if log Kow ≥ 4.2), E1 (bacterial reverse mutation test), E2 (chromosomal aberration 

or micronucleus test), and F1 (combined repeated-dose toxicity study with the 

reproduction/developmental toxicity screening test). The two newly submitted studies consist of 

a bacterial reverse mutation test (submitted via email from D. Vercek to J. Schaeffer on 

December 3, 2012; also submitted to regulations.gov on December 3, 2012 and available online 

at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0128 as of 

January 31, 2013) and an in vitro mammalian chromosome aberration test (submitted via email 

from D. Vercek to J. Schaeffer on December 17, 2012). These studies are evaluated for 

adequacy herein. 

B. EVALUATING DATA ADEQUACY

Data available for SIDS toxicity endpoints are evaluated for adequacy in Section D. Each 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. Studies for a 

common endpoint are grouped together in the document and an endpoint adequacy assessment is 

provided near the top of the first page of the summaries for that endpoint. 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 supplied on December 3, 2012 and December 17, 2012 by Lanxess Corporation are 

considered adequate to satisfy the gene mutation (E1) and chromosomal aberrations (E2) test 

requirements promulgated in the final HPV3 test rule. 

Data have not yet been submitted for the following test rule requirements: C2 (consisting of 

acute toxicity to Daphnia and toxicity to algae tests if log Kow < 4.2, or chronic toxicity to 

Daphnia and toxicity to algae tests if log Kow ≥ 4.2) and F1 (combined repeated-dose toxicity 

study with the reproduction/developmental toxicity screening test). Lanxess Corporation 

submitted a letter of intent for testing of CASRN 98-09-9 to EPA on January 23, 2012 (available 

online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0103 as 

of January 31, 2013) and a study plan for testing of these endpoints (via email from Lanxess 

Corporation to EPA on April 3, 2012). In a letter to EPA dated October 23, 2012 (available 

online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0122 as 
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of January 31, 2013), Lanxess Corporation indicated that study reports for C2 and F1 testing 

would be submitted to EPA by March 31, 2013 and July 31, 2013, respectively.
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D. DATA FOR SIDS ENDPOINTS

D-1. Genetic Toxicity – Gene Mutation

Summary of endpoint: Adequate 

Study 1: Reverse Mutation Assay in Salmonella typhimurium and Escherichia coli

(Thompson, 2012)

Title: Benzenesulfonyl chloride: Reverse mutation assay “Ames test” using Salmonella 

typhimurium and Escherichia coli

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Benzenesulfonyl chloride (CASRN 98-09-9)

Remarks: Clear colorless liquid; purity 99.53%; Batch number KX120302.A; impurities: 

0.043% benzene sulfonic acid, 0.04% diphenylsulfone, 0.03% chlorobenzene

METHOD

Method/guideline followed: OECD TG 471; EU Method B13/14 of Commission Regulation 

(EC) number 440/2008; TSCA 40 CFR 799.9510.

Test type: Bacterial Reverse Mutation Test

GLP compliant?: Yes, with exception: “No analysis was carried out to determine the 

homogeneity, concentration or stability of the test item formulation. The test item was 

formulated within four hours of it being applied to the test system. It is assumed that the 

formulation was stable for this duration. This exception is considered not to affect the purpose 

or integrity of the study.” [Thompson, 2012, p. 3].

Year study performed: 2012

Test species: Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100;

Escherichia coli strain WP2uvrA

Metabolic activation: With and without (S9 fraction of rat liver homogenate)

Concentrations tested: 

Preliminary toxicity screen with and without activation: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 

1500, and 5000 μg/plate

Experiment 1 (plate incorporation test with and without activation): 0, 1.5, 5, 15, 50, 150, 500, 

and 1500 μg/plate
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Experiment 2 (pre-incubation test): 0, 1.5, 5, 15, 50, 150, 500, and 1500 μg/plate (S. 

typhimurium strains with activation); 0, 0.5, 1.5, 5, 15, 50, 150, and 500 μg/plate (S. typhimurium

strains without activation and E. coli with and without activation).

Statistical methods used: According to Mahon, GAT; et al. 1989. Analysis of data from 

microbial colony assays. In: Statistical Evaluation of Mutagenicity Test Data, UKEMS subcommittee on guidelines for mutagenicity testing, (Kirkland, D. J., Ed.), Cambridge University 

Press Report, 26–65.

Test method/conditions remarks: S9 fractions were derived from homogenates of the livers of 

rats induced orally with phenobarbitone/β-naphthoflavone orally for three days. Acetone was 

used as a vehicle for the test substance. 

Test substance concentrations used in Experiment 1 were determined by a preliminary toxicity 

assay (using a single plate for each bacterial strain and concentration) in which concentrations of 

0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, or 5000 μg/plate were tested in strains TA100 and 

WP2uvrA using the plate incorporation method with and without metabolic activation. For 

Experiment 2, the dose range was amended slightly based on the results of Experiment 1. In 

Experiments 1 and 2, testing was conducted in triplicate for each bacterial strain and 

concentration, both with and without metabolic activation. 

In Experiment 1, concentrations of 1.5, 5, 15, 50, 150, 500, or 1500 μg/plate of the test item were 

assayed in each test strain using the direct plate incorporation method. Experiment 2, which 

used the pre-incubation method, tested concentrations of 1.5, 5, 15, 50, 150, 500, or

1500 μg/plate in all Salmonella strains in the presence of S9-mix and tested concentrations of 

0.5, 1.5, 5, 15, 50, 150, or 500 μg/plate in all Salmonella strains in the absence of S9-mix and in 

E. coli strain WP2uvrA in the presence and absence of S9-mix. 

A solvent (acetone) treatment group was used as the vehicle control; positive controls without 

S9-mix included N-ethyl-N’-nitro-N-nitrosoguanidine (2 μg/plate for WP2uvrA, 3 μg/plate for 

TA100, and 5 μg/plate for TA1535), 9-aminoacridine (80 μg/plate for TA1537), and 

4-nitroquinoline-1-oxide (0.2 μg/plate for TA98). Additionally, 2-aminoanthracene (1 μg/plate 

for TA100, 2 μg/plate for TA1535 and TA1537, and 10 μg/plate for WP2uvrA) and 

benzo(a)pyrene (5 μg/plate for TA98), which are non-mutagenic in the absence of metabolizing 

enzymes, were used as positive controls with S9-mix. 

Plates were incubated at 37°C for ~48 hours and revertant colonies were counted using an 

automated colony counter.

The test article was evaluated as positive if a dose-related increase in mutant frequency occurred 

over the dose range tested, a reproducible increase occurred at one or more concentrations, 

biological relevance against in-house historical ranges was seen, statistical analysis of data as 

determined by UKEMS proved significant, and/or an increase of greater than two-fold occurred

as compared to the concurrent solvent control for any tester strain. The test item response was

evaluated as negative if the above criteria were not met. An equivocal result was reported when 

an experiment did not give a clear positive or negative result.
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RESULTS 

Genotoxicity result: Negative in the presence and absence of metabolic activation

Cytotoxic concentration (if applicable): 

Preliminary toxicity test: ≥ 500 μg/plate with and without activation in TA100 and WP2uvrA

Experiment 1: ≥ 150 μg/plate without activation in TA100, TA1535, and TA1537; ≥ 500 

μg/plate with activation in TA100, TA1535, and TA1537; ≥ 500 μg/plate with and without

activation in WP2uvrA and TA98

Experiment 2: ≥ 150 μg/plate without activation in TA1535; ≥ 500 μg/plate with and without 

activation in TA100, WP2uvrA, and TA1537; ≥ 500 μg/plate in TA1535 with activation and in 

TA98 without activation; 1500 μg/plate in TA98 with activation

Results remarks: 

“The test item caused a visible reduction in the growth of the bacterial background lawns of all 

the tester strains, initially from 150 and 500 μg/plate in the absence and presence of S9-mix 

respectively. The test item was, therefore, tested up to the toxic limit. No test item precipitate 

was observed on the plates at any of the doses tested in either the presence or absence of 

S9-mix.” [Thompson, 2012, p. 16]

“No toxicologically significant increases in the frequency of revertant colonies were recorded for 

any of the bacterial strains, with any dose of the test item, either with or without metabolic 

activation or [by either] exposure method. Small, statistically significant increases in revertant 

colony frequency were observed in several tester strains in both the absence and presence of S9-

mix at sub toxic dose levels in Experiment 1. These increases were considered to be of no 

biological relevance because there was no evidence of a dose-response relationship or 

reproducibility in any case. Furthermore, the individual revertant counts at the statistically 

significant dose levels were within the in-house historical untreated/vehicle control range for 

each tester strain and the maximum fold increase was only 1.8 times the concurrent vehicle 

control.” [Thompson, 2012, p. 16].

Negative and solvent controls tested concurrently responded appropriately. Positive controls 

induced marked increases in the frequency of revertant colonies, confirming the activity of the 

S9-mix and the sensitivity of the bacterial strains.

CONCLUSIONS

“The test item, benzenesulfonyl chloride, was considered to be non-mutagenic under the 

conditions of this test.” [Thompson, 2012, p. 16].

STUDY RELIABILITY

[1] Reliable without restrictions. 
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REFERENCE

Thompson, PW. (2012) Benzenesulfonyl chloride: Reverse mutation assay “Ames test” using 

Salmonella typhimurium and Escherichia coli. Harlan Laboratories Ltd., Shardlow, Derbyshire, 

UK. Project number: 41201581, 25 pgs. Sponsor: Lanxess Corporation, Pittsburgh, PA. 

Available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-

0112-0128 as of January 31, 2013.

Text in quotes is taken directly from Thompson (2012). 
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D-2. Genetic Toxicity – Chromosomal Aberrations

Summary of endpoint: Adequate 

Study 1: Chromosomal aberrations study in human lymphocytes (Morris, 2012)

Title: Benzenesulfonyl chloride: Chromosome aberration test in human lymphocytes in vitro

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Benzenesulfonyl chloride (CASRN 98-09-9)

Remarks: 99.53% purity; clear, colorless liquid; Batch # KX120302.A; impurities: 0.043% 

benzene sulfonic acid, 0.04% diphenylsulfone, 0.03% chlorobenzene

METHOD

Method/guideline followed: OECD TG 473; Method B10 of Commission Regulation (EC) No. 

440/2008 (30 May 2008); TSCA 40 CFR 799.9537

Test type: In Vitro Mammalian Chromosome Aberration Test

GLP compliant?: Yes, with exception: “No analysis was carried out to determine the 

homogeneity, concentration or stability of the test item formulation. The test item was 

formulated within two hours of it being applied to the test system; it is assumed that the 

formulation was stable for this duration. This exception is considered not to affect the purpose 

or integrity of the study.” [Morris, 2012, p. 3].

Year study performed: 2012

Test species: Human lymphocytes

Route of administration: In vitro

Vehicle: Acetone

Exposure duration: 4 or 24 hours

Metabolic activation: With and without

Concentrations tested: 

Preliminary toxicity test [4(20)a

-hour with and without S9; 24-hour without S9]: 0, 6.90, 13.80, 

27.59, 55.19, 110.38, 220.75, 441.5, 883, or 1766 μg/mL
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Main tests: 

Experiment 1:

4(20)a

-hour without S9: 0, 13.75, 27.5, 55, 110, 220, or 330 μg/mL

4(20)a-hour with S9 (2%): 0, 13.75, 27.5, 55, 110, 220, or 330 μg/mL

Experiment 2:

24-hour without S9: 0, 3.44, 6.88, 13.75, 27.5, 55, or 110 μg/mL

4(20)a

-hour with S9 (1%): 0, 27.5, 55, 110, 165, 220, or 330 μg/mL

a

Cells were incubated in the presence of vehicle control, test item, or positive control for 4 hours, 

centrifuged, resuspended in treatment-free culture medium, washed, centrifuged, resuspended in 

treatment-free culture medium, and incubated for an additional 20 hours.

Statistical methods used: “The frequency of cells with aberrations excluding gaps and the 

frequency of polyploid cells were compared, where necessary, with the concurrent vehicle 

control value using Fisher's Exact test.” [Morris, 2012, p. 15].

Test method/conditions remarks: “A preliminary toxicity test was performed on cell cultures 

using a 4-hour exposure time with and without metabolic activation followed by a 20-hour 

recovery period, and a continuous exposure of 24 hours without metabolic activation. The dose 

range of test item used was 6.90, 13.80, 27.59, 55.19, 110.38, 220.75, 441.5, 883, and 

1766 μg/mL. Parallel flasks, containing culture medium without whole blood, were established 

for the three exposure conditions so that test material precipitate observations could be made. 

Precipitate observations were recorded at the beginning and end of the exposure periods.”

[Morris, 2012, p. 12].

“Using a qualitative microscopic evaluation of the microscope slide preparations from each 

treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic 

index data was used to estimate test item toxicity and for selection of the dose levels for the main 

study.” [Morris, 2012, p. 12].

In the main study, “duplicate cultures of human lymphocytes, treated with the test item, were 

evaluated for chromosome aberrations at up to four dose levels, together with vehicle and 

positive controls. Four treatment conditions were used for the study; i.e. In Experiment 1, 

4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% 

final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in 

the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 

4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in 

the absence of metabolic activation the exposure time was increased to 24 hours.” [Morris, 2012, 

p. 6].

“Vehicle and positive controls were used in parallel with the test item. The positive control 

items were as follows: In the absence of S9, mitomycin C (MMC) (Sigma, Batch No. 

089K0731) was used at 0.4 and 0.2 μg/mL for cultures in Experiment 1 and 2 respectively. It 

was dissolved in Minimal Essential Medium.” [Morris, 2012, p. 10].
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“In the presence of S9, cyclophosphamide (CP) (Acros, Batch No. A0302605) was used at 

5 μg/mL in both experiments. It was dissolved in dimethyl sulphoxide.” [Morris, 2012, p. 10].

For cell harvest, “[m]itosis was arrested by addition of demecolcine (Colcemid 0.1 μg/mL) two 

hours before the required harvest time. After incubation with demecolcine, the cells were 

centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in 

0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most 

of the hypotonic solution was drawn off and discarded. The cells were resuspended and then 

fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The 

fixative was changed at least three times and the cells stored at approximately 4°C to ensure 

complete fixation... The lymphocytes were resuspended in several mL of fresh fixative before 

centrifugation and resuspension in a small amount of fixative. Several drops of this suspension 

were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently 

labelled with the appropriate identification data... When the slides were dry they were stained in 

5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium...

A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase 

recorded and expressed as the mitotic index and as a percentage of the vehicle control value.”

[Morris, 2012, pp. 13–14].

RESULTS

Genotoxicity result: Positive

Cytotoxic concentration (if applicable): Mitotic index data, along with a subjective evaluation 

of whether or not suitable metaphase spreads were available for scoring, were used to estimate 

test item toxicity. The following cytotoxic concentrations are those that produced >40% 

inhibition of the mitotic index: 

Preliminary toxicity test: 220.75 μg/mL, 4(20)-hour without S9; 55.19 μg/mL, 4(20)-hour with 

S9; 27.59 μg/mL, 24-hour without S9 

Experiment 1: 110 μg/mL, 4(20)-hour in the absence of S9; 220 μg/mL, 4(20)-hour in the 

presence of S9

Experiment 2: 55 μg/mL, 4(20)-hour in the presence of S9 and 24-hour in the absence of S9

Results remarks: 

In the preliminary toxicity study, precipitate was seen in parallel, blood-free cultures at the end 

of the culture period at ≥ 883 μg/mL [4(20)-hour in the absence of S9 and 24-hour in the absence 

of S9] and 1766 μg/mL [4(20)-hour in the presence of S9]. 

In the main tests, “All vehicle (solvent) controls had frequencies of cells with aberrations within 

the range expected for normal human lymphocytes.” [Morris, 2012, p. 7].

“All the positive control items induced statistically significant increases in frequency of cells 

with aberrations indicating the satisfactory performance of the test and of the activity of the 

metabolizing system.” [Morris, 2012, p. 7].

“The test item did not induce any statistically significant increases in the frequency of cells with 

aberrations, in the 4(20)-hour exposure groups in the absence or presence of S9 in Experiment 1 
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or in the 4(20)-hour exposure group in the presence of S9 in Experiment 2 using a dose range 

which exceeded optimum toxicity. [The concentration range analyzed for chromosomal 

aberrations in Experiment 1 was 27.5 – 110 μg/mL without activation and 27.5 – 220 μg/mL 

with activation. The concentration range analyzed for chromosomal aberrations in Experiment 2

was 13.75 – 55 μg/mL without activation and 27.5 – 110 μg/mL with activation.] However in 

the 24-hour exposure group of Experiment 2 [at 27.5 and 55 μg/mL without activation] the test 

item induced statistically significant increases in the frequency of cells with aberrations at a dose 

level which achieved less than optimum toxicity.” [Morris, 2012, p. 7].

CONCLUSIONS

“The test item did not induce any statistically significant increases in the frequency of cells with 

aberrations, in the 4(20)-hour exposure groups in the absence or presence of S9 in Experiment 1 

or in the 4(20)-hour exposure group in the presence of S9 in Experiment 2 using a dose range 

which exceeded optimum toxicity. In the 24-hour exposure group of Experiment 2 the test item 

induced statistically significant increases in the frequency of cells with aberrations at a dose level 

[27.5 μg/mL] which achieved less than optimum toxicity [23% inhibition of the mitotic index].” 

[Morris, 2012, p. 19].

“The test item was therefore considered to be clastogenic to human lymphocytes in vitro.” 

[Morris, 2012, p. 19].

STUDY RELIABILITY

[1] Reliable without restrictions.

REFERENCE

Morris, A. (2012) Benzenesulfonyl chloride: Chromosome aberration test in human lymphocytes 

in vitro. Harlan Laboratories, Ltd. Shardlow, Derbyshire, UK. Project number: 41201582, 32 

pgs. Sponsor: Lanxess Corporation, Pittsburgh, PA.

Text in quotes is taken directly from Morris (2012).