SRC TR - 12 - 036

H PV3 TOXICITY STUDY PLAN REVIEW (DRAFT) Benzenesulfon yl chloride , CASRN 98 - 09 - 9

Prepared b y: Peter McClure, Teresa Manyin , William L. Richards , and Gary Diamond Chemical, Biological and Environmental Center

SRC, Inc. 7502 Round Pond Road North Sy racuse, NY 13212 Contract No. EP - W - 09 - 027 Task FG007. 2 .009. 006 Submitted to: OCSPP/OPPT/RAD (7403M) U.S. Environmental Protection Agency 1200 Pennsylvania Avenue Washington, D.C. 20460 Debra Milligan, Project Officer Meena Sonawane , Work Assignment Manager David Brooks, Alternate Work Assignment Manager

April 27 , 2012Final HPV3 Study Plan Review CASRN 98 - 09 - 9

1 **HPV3 Toxicity**  **Study Plan**  **Review: Benzenesu**  **lfonyl chloride, CASRN 98 -**  **09 -**  **9** In response to the test rule (“Testing of Certain High Production Volume Chemicals; Third Group of Chemicals, 40 CFR 65385 , October 21, 2011), Lanxess Corporation submitted study plans for several toxicity

tests to be conducted by

Harlan Laboratories Ltd. with benzenesulfonyl chloride ( CASRN 98 - 09 - 9) . 1 The test rule requires C2 aquatic toxici ty testing. For Group 1 chemicals with log Kow < 4.2 , such as benzenesulfon ylchloride , the C2 requirement includes tests of Acute Toxicity in Daphnia (ASTM E729 - 96) and Toxic ity to Plants (Algae) ( ASTM E1218 - 04 ε1 ) . Also required by the test rule for be nzenesulfonyl chloride are tests of: 1) E1

– Bacterial Reverse Mutation (40 CFR 799. 9510 ); 2) E2

– any one of 3 tests for chromosome damage (40 CFR 799.9537 or 40 CFR 799.9538 or 40 CFR 799.9539 ; the in vitro test [ 799.9537 ] is preferred); and 3) F 1

– test s of mammalian repeated dose/reproductive/developmental toxicity (40 CFR 799.9365 [ preferred ] or 40 CFR 799.9355 and 40 CFR 799.9305). Reviews of submitted study plans for tests required by the test rule follow. The submitted study

plans do not char acterize the properties of the benzenesulfonyl chloride sample that is to be tested . The final report for each study should provide details about the test material including the source, lot number, and composition of the test material, including identitie s and concentrations of major ingredients and major impurities ; this requirement could be satisfied b y the submission of a certificate of analysis. T he test rule specifies that the “Class 1” chemical substances such as benzenesulfon ylchloride must be tes ted at a purity of at least 99%) . If the sponsor believes that a 99% level of purity is unattainable, the sponsor may request a modification under the procedure described in 40 CFR 790.55. **1. Daphnia**  **sp., 48 -**  **Hour Acute Immobilization Test** The submitte d plan indicated that the study will be performed based on procedures outlined in OECD Guideline 202 (2004) , Method C.2 of Commi ssion R e gulation (EC ) No. 440/2008 , and OPPTS 850.1010 . The sponsor should also ensure that the test methods are compatible with ASTM E729 - 96, the guideline specified in the test rule, and identify this guideline in the final report. A measured log Kow of 3.39 has been reported for benzenesulfonyl chloride on the ECHA website 2

. Th e submitted study plan is considered adequate to satisfy the acute daphnid toxicity testing requirement for chemicals with log K ow < 4.2 (Group 1) with the following stipulations, i n accordance with ASTM E729 - 96 : 1. As specified b y OPPTS 850.1010, a loading rate o f ≤ 40 daphnids/L , should be used for a static or static - renewal test , while loading limits in flow - through tests will vary depending upon the flow rate of dilution water . (ASTM E 729 - 96 specifies only that the loading rate should not exceed 0.5 g of organism/L in static tests > 17 °C, but does not

1 Study Plans were submitted to EPA on April 3, 2012. 2 http://apps.echa.europa.eu/registered/data/dossiers/DISS - 9d8ca91a - d8a5 - 0cd4 - e044 - 00144f67d249/AGGR 97b8bf45 - f6da - 4702 - 970e - 25793baaab7d_DISS - 9d8ca91a - d8a5 - 0cd4 - e044 - 00144f67d249.html#AGGR - 97b8bf45 - f6da - 4702 - 970e - 25793baaab7d Accessed April, 2012.Final HPV3 Study Plan Review CASRN 98 - 09 - 9

2 indicate the corresponding number of daphnids/L.) Based on the description of the test sy stem provided in the submitted study plan (i.e., 5 daphnids in test chambers containing ≥ 50 mL), the loading rate would be ≤ 100 daphnids/L . This loading rate should be adjusted, as necessary , to comply with OPPTS 850.1010. 2. The concentrations of both total organic carbon (TOC) and particulate matter of the dilution water should be < 5 mg/L. 3. A measured overall rate constant of 2.25 x 10 - 3 sec - 1 for the h ydrol ys is of benzenesulfon ylchloride in a 1% aqueous dioxane solution at 21 °C (which corresponds to a half - life of 5.1 min) was reported b y Haughton et al. ( 1975) in a peer - reviewed journal . Because of this relativel y fas t hydrol ys is rate , a flow - through test is recommended, rather than a static or static - renewal test. Inaddition, the test organisms should be placed in test media within 30 minutes of addition of the test substance. 4. The submitted plan indicates that sampl es will be taken at the beginning and termination of exposure for determination of concentration of the test material (benzenesulfonyl chloride) . The chemical anal ys is of the samples should also include determination of the concentration of the h ydrolysis product, benzenesulfonic acid. Final results (e.g., EC 50 ) should be expressed separatel y in terms of the concentration of the parent compound (benzenesulfon ylchloride) and in terms of the concentration of the h ydrolysis product (benzenesulfonic acid). 5. T he final study

report should include the composition of the test substance, including identities and concentrations of major ingredients and major impurities. 6. ASTM E729 - 96 indicates that “daphnids from cultures in which ephippia are being produced should n ot be used.” During the proposed test, the former guidance and the following guidance from OPPTS 850.1010 should be observed: “During culturing and acclimation, daphnids should be observed carefully for ephippia and other signs of stress, phy sical damage , and mortality . Dead and abnormal individuals should be discarded. ” 7. The submitted plan states that the water temperature will be maintained at 18

– 22 °C with a maximum deviation of ± 1 °C. Incontrast, ASTM E729 - 96 (Table 4 and section 11.3.2 ) recommen ds an average test temperature of 20 ± 1 °C for studies of daphnids . The study plan should be revised to reflect this guidance . 8. In the submitted test plan (p. 13), the following criterion is not acceptable: “The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/L in the control and test vessels.” According to ASTM E729 - 96 , the concentration of dissolved ox ygen in each test chamber must be at least 60% of saturation during a 48 - hour test. A 60% saturation value at the 20 °C temperature specified for the acute daphnid test by ASTM E729 - 96 and OPPTS 850.1010 corresponds to an oxygen concentration of 5.5 mg/L. Thus, the criterion for oxygen concentration in the submitted test plan should be revised to read “The dissolved oxygen concentr ation at the end of the test sho uld be ≥ 5.5 mg/L in the control and test vessels.” Lanxess Corporation also submitted a plan for a test of chronic toxicity to daphnids , but this was not reviewed because the test rule does not require this test for Group 1 chemicals with log Kow < 4.2.Final HPV3 Study Plan Review CASRN 98 - 09 - 9

3 **2 .**  **Algal Growth Inhibition Test** The submitted plan indicated that the study will be performed based on procedures outlined in OECD Guideline 201 (2006) , Method C.3 of Commission Regulation (EC) No. 761/2009 , and OPPTS 850.5400 . The sponsor should also ensure that the test methods are compatible with ASTM E1218 - 04 ε1 , the guideline specified in the test rule, and identify this guideline in the final report. Th e submitted study plan is considered adequate to satisfy the algae toxicity

testing requirement with the following stipulations, in accordance with ASTM E1218 - 04 ε1 : 1. The test should be conducted with a species of green algae (e.g., Pseudokirchneriella subcapitata ) , as indicated on page 2 of the “Study Specific Supplement” . Testing of cyanobacteria (e.g., Anabaena flos - aquae ) is not appropriate for the Toxicity to Plants (Algae) endpoint because cyanobacteria are not considered to be representative of plants. 2. At the initiation of the test, each test chamber should be inoculated with 1 - 2 x 10 4 cells/mL . 3. The light intensity

for illumination of the algae cultures d uring the test should be 4300 lm/m2 or 60 μE insteins · m - 2 · s - 1

. 4. The temperature during the test should be 24 ± 2 °C. 5. Because of the expected relativel y fast h ydrol ys is of benzenesulfonyl chloride in aqueous media ( half

- life of 5.1 min at 21 °C; Haughton et a l. 1975) , the submitted plan should also specify

that the test organisms be placed in test media within 30 minutes of addition of the test substance. 6. The submitted plan indicates that samples will be taken at the beginning and termination of exposure for d etermination of concentration of the test material (benzenesulfonyl chloride) . The chemical anal ys is of the samples should also include determination of the concentration of the h ydrolysis product, benzenesulfonic acid. Final results (e.g., EC 50 ) should be expressed separatel y in terms of the concentration of the parent compound (benzenesulfon ylchloride) and in terms of the concentration of the h ydrolysis product (benzenesulfonic acid). 7. The final study

report should include the composition of the test substance, including identities and concentrations of major ingredients and major impurities. **3. Reverse Mutation Assay “Ames Test” using Salmonella typhimurium**  **and Escherichia**  **coli** The submitted study

plan is considered adequate to satisfy the bacterial reverse mutation testing requirement with the following stipulations, in accordance with 40 CFR 799.9510: 1. The submitted study

plan indicated that the test methods will be compatible with OECD Test Guideline 471, EPA OPPTS guideline 870.5100, and Method B 13/14 of Commission Regulation (EC) No. 440/2008. The sponsor should also ensure that the test methods are compatible with 40 CFR 799.9510 and identify this guideline in the final report. 2. The submitted study

plan indicated that “up to five” strains of bac teria will be used. The test guideline specifies that **at least** five bacterial strains must be tested. All of the strainsFinal HPV3 Study Plan Review CASRN 98 - 09 - 9

4 listed in the study

plan (TA1537, TA98, TA1535, TA100, and WP2 uvrA ) must be tested in order for the study to be considered acceptabl e. 3. The study

plan states that “all formulations will be used within four hours of preparation and will be assumed to be stable for this period unless specified otherwise by the Sponsor”. The testing laboratory should be made aware that h ydrol ys is of benze nesulfon ylchloride in aqueous media is expected to be relativel y fast ( half - life of 5.1 min at 21 °C; Haughton et al. 1975) . 4. The final study

report should include the composition of the test substance, including identities and concentrations of major ingr edients and major impurities. 5. The test report must contain all study details as specified in 40 CFR 799 .9510 , including, but not limited to, justification for choice of solvent, detailed experimental methods, signs of toxicity

and precipitation during the test, individual plate counts, statistical anal ys es, and concurrent and historical control data. **4 . Chromosome Aberration Test in Human**  **Lymphocytes In V**  **itro** The submitted study

plan is considered adequate to satisfy the chromosomal damage testing requi rement with the following stipulations, in accordance with 40 CFR 799.9537: 1. The submitted study

plan indicated that the test methods will be compatible with OECD Test Guideline 473, EPA OPPTS guideline 870.5375, and Method B10 of Commission Regulation (EC ) No. 440/2008. The sponsor should also ensure that the test methods are compatible with 40 CFR 799.9537 and identify this guideline in the final report. 2. Tests of s olvent and positive controls both with and without activation must be included in each expe riment. 3. The study

plan states that “all formulations will be used within two hours of preparation and will be assumed to be stable for this period unless specified otherwise by the Sponsor”. The testing laboratory should be made aware that h ydrol ys is of b enzenesulfon ylchloride in aqueous media is expected to be relativel y fast (half - life of 5.1 min at 21 °C; Haughton et al. 1975). 4. The final study

report should include the composition of the test substance, including identities and concentrations of major ingredients and major impurities. 5. The test report must contain all study details as specified in 40 CFR 799. 9537, including, but not limited to, justification for choice of solvent, information on cells tested, detailed experimental methods, signs of toxi city and precipitation during the test, definition for aberrations (including gaps), number of cells with chromosome aberrations and ty pe of chromosome aberrations given separatel y for each treated and control culture, statistical anal ys es, and concurrent and historical negative and positive control data. **5. Oral (Gavage)**  **Combined Repeated Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test**  **in the Rat** The submitted study

plan is considered adequate to satisfy the mammalian repeate d dose/reproduction/developmental toxicity testing requirement with the following stipulations, in accordance with 40 CFR 799.9365:Final HPV3 Study Plan Review CASRN 98 - 09 - 9

5 1. The submitted study

plan indicated that the test methods will be compatible with OECD Guideline 422 and Commission Regulati on (EC) No 440/2008 of 30 May 2008. The sponsor should also ensure that the test methods are compatible with 40 CFR 799.9365 and identify

this guideline in the final report. 2. The final study

report should include the source and composition of the test subs tance, including identities and concentrations of major ingredients and major impurities. 3. The study

plan states that “the frequency of test item formulation will be dependent on known stability”. The testing laboratory should be made aware that hydroly sis of benzenesulfon ylchloride in aqueous media is expected to be relativel y fast (half - life of 5.1 min at 21 °C; Haughton et al. 1975). 4. Each litter should be examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, l ive births , and runts (pups that are significantl y smaller than corresponding control pups) and the presence of gross abnormalities . 5. Food consumption should be measured at least weekl y during pre - mating, pregnancy and lactation. 6. Overnight fasting of animal s is recommended before sampling of blood at the planned sampling dates (day

42 for males; day

4 for post - partum females). 7. The epididy

mis should be evaluated for leukocy te infiltration, change in prevalence of cell ty pes, aberrant cell ty

pes , and phagocy

to sis of sperm. 8. Histopathological examination of the ovary should detect qualitative depletion of the primordial follicle population. 9. Individual animal data should be provided, in addition to summary tables showing pertinent data for each dose group (see “(g ) Data and reporting

— (1) Data. ” in 40 CFR 799.9365 for a more complete list) such as the number of animals at the start of the test, the number of animals found dead during the test, the number of pregnant females, the number of animals showing si gns of to xicity (including the specific t ypes and severit y of histopathological changes), individual body weights throughout the entire study , and all relevant litter data. 10. Whenever feasible, reproductive and developmental endpoints (e.g., pup body weights and surv ival, pre - and post - implantation losses, etc.) should be statistically anal yz ed on a per - dam or per - litter basis. 11. The test report must contain all study details as specified in 40 CFR 799. 9365 , including, but not limited to: just ification for choice of solvent; rationale for dose level selection; sex/strain and condition (e.g., bod y weight and age) of test animals before the test; details of test material formulation, stability , homogeneity , and administration; and details of resu lts obtained (toxic response data by sex, dose, and generation) including statistical analy ses of results. The test report must also contain sections discussing the results and describing final conclusions. Lanxess Corporation also submitted a study plan for a 14 - day

repeated dose (gavage) range finding toxicity

study in Wistar rats. As specified in 40 CFR 799.936 5, if no suitable toxicity data for the test material are available, a range - finding study may be performed. The plan calls for 3 rats/sex for each of three d ose groups and a control group and indicates that dose levels may be adjusted during the course of the study so that distinct evidence of toxicity is observed in at least one dose level up to a maximum of 1000 mg/kg - bw - day . The plan is con sidered adequate for a range - finding study .Final HPV3 Study Plan Review CASRN 98 - 09 - 9

6 **Conclusion:** The submitted study plans are adequate to satisfy the toxicity

testing requirements for benzenesulfon ylchloride with the stipulations listed above. **References** Haughton, AR, Laird, RM, Spence, MJ. 1975. Reactions of organic sulphur compounds. I. The hydrol ys is of arenesulfonyl chlorides. J Chem Soc 6:637 - 643.