Document ID: EPA-HQ-OPPT-2009-0112-0167
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2013-11-27T05:00Z

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

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).  

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 website.  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:

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 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.

The concentrations of both total organic carbon (TOC) and particulate
matter of the dilution water should be < 5 mg/L.

 A measured overall rate constant of 2.25 x 10-3 sec-1 for 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.

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).

The final study report should include the composition of the test
substance, including identities and concentrations of major ingredients
and major impurities.

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.”

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.

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. 

2.  Algal Growth Inhibition Test

ε1, 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:

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.

At the initiation of the test, each test chamber should be inoculated
with 1-2 x 104 cells/mL.

The light intensity for illumination of the algae cultures during the
test should be 4300 lm/m2 or 60 µEinsteins·m-2·s-1.

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

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.

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).

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:

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.

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 listed in the
study plan (TA1537, TA98, TA1535, TA100, and WP2 uvrA) must be tested in
order for the study to be considered acceptable.

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).

The final study report should include the composition of the test
substance, including identities and concentrations of major ingredients
and major impurities.

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:

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.

Tests of solvent and positive controls both with and without activation
must be included in each experiment.

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).

The final study report should include the composition of the test
substance, including identities and concentrations of major ingredients
and major impurities.

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:

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.

The final study report should include the source and composition of the
test substance, including identities and concentrations of major
ingredients and major impurities.

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).

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.

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

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).

The epididymis should be evaluated for leukocyte infiltration, change in
prevalence of cell types, aberrant cell types, and phagocytosis of
sperm.

Histopathological examination of the ovary should detect qualitative
depletion of the primordial follicle population.

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.

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.

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ification 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) range-finding 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.

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.

 Study Plans were submitted to EPA on April 3, 2012.

   HYPERLINK
"http://apps.echa.europa.eu/registered/data/dossiers/DISS-9d8ca91a-d8a5-
0cd4-e044-00144f67d249/AGGR-97b8bf45-f6da-4702-970e-25793baaab7d_DISS-9d
8ca91a-d8a5-0cd4-e044-00144f67d249.html#AGGR-97b8bf45-f6da-4702-970e-257
93baaab7d" 
http://apps.echa.europa.eu/registered/data/dossiers/DISS-9d8ca91a-d8a5-0
cd4-e044-00144f67d249/AGGR-97b8bf45-f6da-4702-970e-25793baaab7d_DISS-9d8
ca91a-d8a5-0cd4-e044-00144f67d249.html#AGGR-97b8bf45-f6da-4702-970e-2579
3baaab7d   Accessed April, 2012.

Final HPV3 Study Plan Review		CASRN 98-09-9

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