SRC TR

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HPV 3 TEST RULE DATA ADEQUACY REVIEW ( FINAL )

Benzenesulfonyl chloride , CASRN 98

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- 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 .009.00 6

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

, 201 3Final HPV3 Chemical Data Adequacy Review CASRN 98

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i 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

................................ ................................ .. 7Draft HPV3 Chemical Data Adequacy Review CASRN 98

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1 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 : C 2 (consisting of acute toxicity to Daphnia and toxicity to algae tests if log K ow < 4.2

, or chronic toxicity to Daphnia and toxicity to algae tests if log K ow ≥ 4.2), E1 (bacterial reverse mutation test), E2 (chromosomal aberration or micronucleus test), an d 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 Januar y 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 d ocument 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 K ow < 4.2, or chronic toxicity to Daphnia and toxicity to algae tests if log K ow ≥ 4.2 ) and F1 (combined repeated

- dose toxic ity 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 Lanxe ss 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

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- 0122 asDraft HPV3 Chemical Data Adequacy Review CASRN 98

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2 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.Draft HPV3 Chemical Data Adequacy Review CASRN 98

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3 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 a ssay “Ames t est” using Salmonella typhimurium and Escherichia coli

This study is : Adequate Not Adequate

TEST SUBSTANCE

Identity : Benzenesulfonyl chloride ( CAS R N 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% chlorobenz e ne

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 : “N o analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test i tem 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, TA153 7 , TA98

, and TA100 ; Escherichia coli strain WP2 uvrA

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

Concentrations tested : P reliminary 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/plateDraft HPV3 Chemical Data Adequacy Review CASRN 98

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4 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 with out activation and E. coli with and without activation )

.

Statistical methods used : According to Mahon

, GAT ; et a l. 1989

. Analysis of data from microbial colony assays. In : Statistical Evaluation of Mutagenicity Test Data, UKEMS sub

committee 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.

T est substance c oncentrations used in E xperiment 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 μ g/plate were tested in strains TA100 and WP2 uvrA 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 E xperiment 1, concentrations of 1.5, 5, 15, 50, 150, 500, or 1500 μ g/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 μ g/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 μ g/plate in all Salmonella strains i n the absence of S9

- mix and in E. coli strain WP2 uvrA 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 μ g/plate for WP2 uvrA, 3 μ g/plate for TA100, and 5 μ g/plate for TA1535), 9

- aminoacridine (80 μ g/plate for TA1537), and 4

- nitroquinoline

- 1

- oxide (0.2 μ g/plate for TA98). Additionally, 2

- aminoanthracene (1 μ g/plate for TA100, 2 μ g/plate for TA1535 and TA1537, and 10 μ g/plate for WP2 uvrA ) and benzo(a)pyrene (5 μ g/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 occur red over the dose range tested, a reproducible increase occur red at one or more concentrations, biological relevance against in

- house historical ranges wa s seen, statistical analysis of data as determined by UKEMS prove d 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 w as evaluated as negative if the above criteria we re not met . An equivocal result was reported when an experiment d id not give a clear positive or negative result.Draft HPV3 Chemical Data Adequacy Review CASRN 98

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5 RESULTS

Genotoxicity result : Nega tive in the presence and absence of metabolic activation

Cytotoxic concentration (if applicable) : Preliminary toxicity test : ≥ 500 μ g/plate with and without activation in TA100 and WP2 uvrA Experiment 1 : ≥ 1 50 μ g/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 WP2 uvrA and TA98 Experiment 2 : ≥ 150 μ g/plate without activation in TA1535; ≥ 500 μ g/plate with and without activation in TA100, WP2 uvrA

, and TA1537; ≥ 500 μ g/plate in TA1535 with activation and in TA98 without activation; 1500 μ g/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 5 00 μ g/plate in the absence and presence of S9

- mix respectively. The test item was, therefore, tested up to the tox ic 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. Furthermor e, 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.” [T hompson, 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 bacteria l 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.Draft HPV3 Chemical Data Adequacy Review CASRN 98

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6 REFERENCE

Thompson, PW. ( 2012 ) Benzenesulfonyl chloride : Reverse mutation a ssay “Ames t est” using Salmonella typhimurium and Escherichia coli

. Harlan Laboratories Ltd., Shardlow, Derbyshire, UK. Project n umber : 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 ( 20 12 )

.Draft HPV3 Chemical Data Adequacy Review CASRN 98

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7 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% chlorobenz e ne

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/mLDraft HPV3 Chemical Data Adequacy Review CASRN 98

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8 Main test s : Experiment 1: 4(20) a

- hour without S9 : 0, 13.75, 27.5, 55, 110, 220, or 330 μ g/mL 4(20) a

- hour with S9 (2%) : 0, 13.75, 27.5, 55, 110, 220, or 330 μ g/mL Experiment 2: 24

- hour without S9 : 0, 3.44, 6.88, 13.75, 27.5, 55, or 110 μ g/mL 4(20) a

- hour with S9 (1%) : 0, 27.5, 55, 110, 165, 220, or 330 μ g/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, 11 0.38, 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 evalua tion. 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, “ d uplicate 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 metaboli s ing 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 (usin g 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 μ g/mL for cultures in Experiment 1 and 2 respectively. It was dissolved in Minimal Essential Medium.” [Morris, 2012, p. 10].Draft HPV3 Chemical Data Adequacy Review CASRN 98

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9 “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 μ g/ 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 KC l

. After approximately fourteen minutes (including cent rifugation), most of the hypotonic solution was drawn off and discarded. The cells were resuspended and then fixed by dropping the KC l cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and th e 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 cle an, 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, p p

. 13

– 14].

RESULTS

Genotoxicity result : Positive

Cytotoxic concentration (if app licable) : 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/m L , 4(20)

- hour in the absence of S9 ; 220 μg/m L

, 4(20)

- hour i n the presence of S9 Experiment 2 : 55 μg/m L

, 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 1Draft HPV3 Chemical Data Adequacy Review CASRN 98

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10 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 ana lyzed for chromosomal aberrations in Experiment 1 was 27.5

– 110 μg/mL without activation and 27.5

– 22 0 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

– 11 0 μg/mL with activation. ] However in the 24

- hour exposure group of Experiment 2 [at 27.5 and 55 μg/mL with out 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 i n 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 fre quency 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 v itro

. Harlan Laboratories, Ltd. Shardlow, Derbyshire , UK

. Project n umber : 41201582 , 32
pgs . Sponsor : Lanxess Corporation, Pittsburgh, PA.

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