SRC TR-12-036

HPV3 TOXICITY STUDY PLAN REVIEW (DRAFT)

Benzenesulfonyl chloride, CASRN 98-09-9

Prepared by:

Peter McClure, Teresa Manyin, William L. Richards, and Gary Diamond

Chemical, Biological and Environmental Center

SRC, Inc.

7502 Round Pond Road

North Syracuse, 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, 2012
Final HPV3 Study Plan Review CASRN 98-09-9

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HPV3 Toxicity Study Plan Review: Benzenesulfonyl 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 toxicity testing. For Group 1 chemicals with log Kow < 4.2, 

such as benzenesulfonyl chloride, the C2 requirement includes tests of Acute Toxicity in 

Daphnia (ASTM E729-96) and Toxicity to Plants (Algae) (ASTM E1218-04 ε1). Also required 

by the test rule for benzenesulfonyl 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) F1 – tests 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 characterize 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 

identities and concentrations of major ingredients and major impurities; this requirement could 

be satisfied by the submission of a certificate of analysis. The test rule specifies that the “Class 

1” chemical substances such as benzenesulfonyl chloride must be tested 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 submitted plan indicated that the study will be performed based on procedures outlined in 

OECD Guideline 202 (2004), Method C.2 of Commission Regulation (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 website2. The submitted study plan is considered adequate to satisfy the acute daphnid 

toxicity testing requirement for chemicals with log Kow < 4.2 (Group 1) with the following 

stipulations, in accordance with ASTM E729-96:

1. As specified by OPPTS 850.1010, a loading rate of ≤ 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/AGGR97b8bf45-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

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indicate the corresponding number of daphnids/L.) Based on the description of the test 

system 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 10-3sec-1for the hydrolysis of 

benzenesulfonyl chloride in a 1% aqueous dioxane solution at 21 °C (which corresponds 

to a half-life of 5.1 min) was reported by Haughton et al. (1975) in a peer-reviewed 

journal. Because of this relatively fast hydrolysis rate, a flow-through test is

recommended, rather than a static or static-renewal test. In addition, the test organisms 

should be placed in test media within 30 minutes of addition of the test substance.

4. The submitted plan indicates that samples will be taken at the beginning and termination 

of exposure for determination of concentration of the test material (benzenesulfonyl 

chloride). The chemical analysis of the samples should also include determination of the 

concentration of the hydrolysis product, benzenesulfonic acid. Final results (e.g., EC50) 

should be expressed separately in terms of the concentration of the parent compound 

(benzenesulfonyl chloride) and in terms of the concentration of the hydrolysis product 

(benzenesulfonic acid).

5. The 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 not 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, 

physical 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. In contrast, ASTM E729-96 (Table 4 and section 

11.3.2) recommends 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 oxygen 

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 concentration at the end of the test should 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

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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 E1218-04ε1, the guideline specified in the test rule, and identify this guideline in the final 

report. The submitted study plan is considered adequate to satisfy the algae toxicity testing 

requirement with the following stipulations, in accordance with ASTM E1218-04ε1:

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 104

cells/mL.

3. The light intensity for illumination of the algae cultures during the test should be 4300 

lm/m2or 60 μEinsteins·m

-2

·s

-1

.

4. The temperature during the test should be 24 ± 2 °C.

5. Because of the expected relatively fast hydrolysis of benzenesulfonyl chloride in aqueous 

media (half-life of 5.1 min at 21 °C; Haughton et al. 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 determination of concentration of the test material (benzenesulfonyl 

chloride). The chemical analysis of the samples should also include determination of the 

concentration of the hydrolysis product, benzenesulfonic acid. Final results (e.g., EC50) 

should be expressed separately in terms of the concentration of the parent compound 

(benzenesulfonyl chloride) and in terms of the concentration of the hydrolysis 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 B13/14 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 bacteria will be used. The 

test guideline specifies that at least five bacterial strains must be tested. All of the strains 
Final HPV3 Study Plan Review CASRN 98-09-9

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listed in the study plan (TA1537, TA98, TA1535, TA100, and WP2 uvrA) must be tested 

in order for the study to be considered acceptable.

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 hydrolysis of 

benzenesulfonyl chloride in aqueous media is expected to be relatively 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 799.9510, 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 

analyses, and concurrent and historical control data.

4. Chromosome Aberration Test in Human Lymphocytes In Vitro 

The submitted study plan is considered adequate to satisfy the chromosomal damage testing 

requirement 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 solvent and positive controls both with and without activation must be included 

in each experiment.

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 hydrolysis of 

benzenesulfonyl chloride in aqueous media is expected to be relatively 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 toxicity and precipitation during the test, definition for 

aberrations (including gaps), number of cells with chromosome aberrations and type of 

chromosome aberrations given separately for each treated and control culture, statistical 

analyses, 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 repeated 

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

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1. The submitted study plan indicated that the test methods will be compatible with OECD 

Guideline 422 and Commission Regulation (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 substance, 

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 hydrolysis of 

benzenesulfonyl chloride in aqueous media is expected to be relatively 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, live births, and runts (pups that are significantly smaller than 

corresponding control pups) and the presence of gross abnormalities.

5. Food consumption should be measured at least weekly during pre-mating, pregnancy and 

lactation.

6. Overnight fasting of animals is recommended before sampling of blood at the planned 

sampling dates (day 42 for males; day 4 for post-partum females).

7. The epididymis should be evaluated for leukocyte infiltration, change in prevalence of 

cell types, aberrant cell types, and phagocytosis 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 signs of toxicity (including the specific types and severity 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 survival, pre- and post-implantation losses, etc.) should be statistically analyzed 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: justification for choice of solvent; rationale for dose level 

selection; sex/strain and condition (e.g., body weight and age) of test animals before the 

test; details of test material formulation, stability, homogeneity, and administration; and 

details of results obtained (toxic response data by sex, dose, and generation) including 

statistical analyses 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) rangefinding toxicity study in Wistar rats. As specified in 40 CFR 799.9365, 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 dose 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 considered 

adequate for a range-finding study.
Final HPV3 Study Plan Review CASRN 98-09-9

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Conclusion: The submitted study plans are adequate to satisfy the toxicity testing requirements 

for benzenesulfonyl chloride with the stipulations listed above.

References

Haughton, AR, Laird, RM, Spence, MJ. 1975. Reactions of organic sulphur compounds. I. The 

hydrolysis of arenesulfonyl chlorides. J Chem Soc 6:637-643.