SRC TR-12-043

HPV3 REPRODUCTION/DEVELOPMENTAL TOXICITY STUDY PLAN REVIEW

(FINAL – DOES NOT CONTAIN CBI)

Benzenesulfonic acid, 3-nitro-, sodium salt (1:1), CASRN 127-68-4

Prepared by:

Teresa Manyin, William L. Richards, and Laura Morlacci

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

June 5, 2012
HPV3 Reproduction/Developmental Toxicity Study Plan Review (Final) CASRN 127-68-4

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HPV3 Reproduction/Developmental Toxicity Study Plan Review: 

Benzenesulfonic acid, 3-nitro-, sodium salt (1:1), CASRN 127-68-4

The test rule, “Testing of Certain High Production Volume Chemicals; Third Group of 

Chemicals” (76 FR 65385, October 21, 2011), requires F2 toxicity testing for benzenesulfonic 

acid, 3-nitro-, sodium salt (1:1) (CASRN 127-68-4). The F2 requirement consists of a 

Reproduction/Developmental Toxicity Screening Test following 40 CFR 799.9355. A study

plan for this test, to be conducted by Jai Research Foundation, was submitted to EPA by Tiarco 

Chemical on February 24, 2012. Given that the study plan stated that the purity of the test 

substance would be 96.9%, which does not meet the test rule requirement for a purity of at least 

99%, EPA requested that Tiarco Chemical submit a detailed description of the test substance’s 

composition to determine if the proposed test substance would yield toxicity test data that were 

representative of the test rule chemical. The composition of the proposed test substance was 

submitted to EPA by Tiarco Chemical in a CBI submission (DCN 40120000059) received by 

EPA on April 11, 2012.

The study plan is reviewed herein for compliance with the test rule guideline, 40 CFR 799.9355. 

The composition of the proposed test substance is also evaluated.

Review of Study Plan:

The submitted study plan, “Reproduction/Developmental Toxicity Screening Test of Sodium 3-

Nitrobenzene Sulfonate (CAS # 127-68-4; US EPA HPV3) in Wistar Rats” is considered 

adequate to satisfy the mammalian reproduction/developmental toxicity testing requirement with 

the following stipulations and modifications:

1. In section 5.11: Mating Procedure (p. 10), the study plan states: “If mating has not 

occurred during two weeks mating period, re-mating of females with proven males of the 

same group may be performed.” This appears to indicate that the mating period could be 

longer than two weeks, whereas the 40 CFR 799.9355 indicates that the mating period 

should be no longer than two weeks. This statement should be revised to more clearly 

explain the mating procedure, while adhering to the test rule guideline.

2. The study plan does not indicate when unmated females will be sacrificed. According to 

40 CFR 799.9355, females showing no evidence of copulation are sacrificed 24-26 days 

after the last day of the mating period. These details should be added to the study plan.

3. The test guideline cited in section 2.2: Regulatory Guidelines (p. 5 of the study plan) is 

OPPTS 870.3550. This section should also cite the test rule guideline, 40 CFR 799.9355 

(“TSCA Reproduction/Developmental Toxicity Screening Test”, December 15, 2000); 

the laboratory conducting this test should ensure compliance with 40 CFR 799.9355.

4. In section 5.7: Housing (p. 9), the study plan states: “Throughout experiment period, the 

rats will be housed individually. Except during the mating period, the rats will be housed 

in groups of 2 rats/cage (one male plus one female).” This is unclear. This section 

should be revised to state: “Throughout the experiment period, the rats will be housed 

individually, with the exception that during the mating period, the rats will be housed in 

groups of 2 rats/cage (one male plus one female).” 
HPV3 Reproduction/Developmental Toxicity Study Plan Review (Final) CASRN 127-68-4

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5. In section 7.2: Pups (p. 12), the study plan should be modified to include the 

identification of runts (pups that are significantly smaller than corresponding control 

pups) during the examination of pups at birth.

6. In section 7.2.1: Pup Body Weight (p. 12), the study plan states that individual pup body

weights will be recorded. Additionally, the total weight of pups in each litter should be 

calculated (by summing the individual pup body weights for each litter); litter weights 

should be statistically analyzed to determine treatment effects.

7. In section 7.3: Necropsy (p. 13), the word “scarified” should be replaced with 

“sacrificed”.

8. In section 8.2: Data Evaluation (p. 14), a statement should be added to indicate that 

statistical analyses on pup data (e.g., number of pups born, pup body weights, pup 

survival) will be conducted on a per-litter or per-dam basis.

Review of Test Substance Composition:

The composition of the proposed test substance, which was submitted to EPA by Tiarco 

Chemical in a CBI submission (DCN 40120000059), is considered to be acceptable, with the 

stipulation that the dose levels must be expressed as the concentration of active ingredient (i.e., 

CASRN 127-68-4). A more detailed review of the test substance composition, which includes 

CBI, is contained in DCN 40120000064.

Conclusion:

The submitted study plan and proposed test substance are acceptable with the stipulations 

described above.