SRC TR-10-064 

HPV1 TEST RULE STUDY – DATA ADEQUACY ASSESSMENT 

Dibromomethane, CAS No. 74-95-3 

Reproductive/Developmental Toxicity 

Prepared by: 

Ashlee Aldridge, Justine Von Runnen, and Joan D. Garey 

Environmental Science Center 

SRC, Inc. 

7502 Round Pond Road 

North Syracuse, NY 13212 

Contract No. EP-W-09-027 

Task FG007.0.009.004 

Submitted to: 

OPPT/RAD/HPVCB (7403M) 

U.S. Environmental Protection Agency 

1200 Pennsylvania Avenue 

Washington, D.C. 20460 

Elizabeth Margosches, Project Officer 

Meena Sonawane, Work Assignment Manager 

David Brooks, Alternate Work Assignment Manager 

March 5, 2010 
HPV1 Test Rule – Data Adequacy Assessment 

Dibromomethane, CAS No. 74-95-3 

Reproductive/Developmental Toxicity 

March 2010 

Submitted to: 

High Production Volume Chemicals Branch 

Risk Assessment Division 

Office of Pollution Prevention and Toxics 

Environmental Protection Agency 

WAM: Meena Sonawane 

Alternate WAM: David Brooks 

1200 Pennsylvania Avenue, NW 

Washington, DC 20460-0001 

Prepared By: 

SRC, Inc. 

Environmental Science Center 

2451 Crystal Drive, Suite 804 

Arlington, VA 22202 
Draft HPV1 Test Rule – Data Adequacy – Reproductive/Developmental Toxicity CAS No. 74-95-3

PREPARATION OF THE DATA ADEQUACY ASSESSMENT

This document contains the review of a toxicity study conducted in response to the first 

HPV TSCA Section 4 Test Rule (71 FR 13708, March 16, 2006). Using the applicable test 

guideline and professional judgment as guides, the study was reviewed for adequacy and 

evaluated for compliance with the test guideline. (The applicable test guideline is not always the 

test guideline required by the test rule, because some test sponsors requested and received 

approval from EPA to use a different guideline.) The study was summarized according to a 

standard format, assessed for adequacy, and assigned a Klimisch reliability code. If the study 

was determined to be inadequate, a basis for that conclusion is supplied. For studies already 

assigned Klimisch codes, an explanation is provided if the reviewer’s assessment of data 

adequacy differed from the pre-assigned Klimisch code. 

A detailed evaluation of the test report and study conduct is provided at the end of the 

summary document in a section entitled “Review of TSCA Section 4 Final Study Report”. Any 

deviations from the applicable test guideline are described. The study is also evaluated for 

compliance with Standard Operating Procedures, Good Laboratory Practice Standards, and 

Quality Assurance procedures, as well as the use of appropriate controls and test substances. 

Any deficiencies in the descriptions of the study method, test system, animals studied, physical 

conditions, dosage information, individual animal data, calculations, and statistical analyses are 

described, and internal inconsistencies within the study report are identified. An overall 

assessment is provided for each of the following: study conduct, data reliability, and data utility. 

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Draft HPV1 Test Rule – Data Adequacy – Reproductive/Developmental Toxicity CAS No. 74-95-3

REPRODUCTIVE/DEVELOPMENTAL TOXICITY

Title: Oral (Gavage) Reproduction/Developmental Toxicity Screening Test in the Rat 

The study is: Adequate Not Adequate 

TEST SUBSTANCE 

 

Identity: Dibromomethane, CAS No. 74-95-3 

Remarks: Clear colorless liquid; Purity: 99.4%; individual samples of dibromomethane were 

supplied by both Co-Sponsors. These two test materials were then mixed in equal proportions 

and the resulting mixture analyzed to determine its purity. 

METHOD

Method/guideline followed: TSCA test guideline 40 CFR 799.9355, March 16, 2006 

(comparable to the OECD Guidelines for Testing of Chemicals No. 421 

"Reproduction/Developmental Toxicity Screening Test" (adopted 27 July 1996 and are in 

accordance with the EPA Health Effects Test Guidelines: OPPTS 870.3550 

Reproduction/Developmental Toxicity Screening Test, July 2000). 

Test type: Reproduction/Developmental Toxicity Screening Test

GLP compliant?: Yes 

Year study performed: 2006

Test species: Sprague-Dawley Crl:CD (SD) IGS BR rats obtained from Charles River (UK) 

Limited, Margate, Kent; 268 – 336g (males); 176 – 226g (females) and approximately 8 weeks 

old at the start of treatment.

Number of animals/sex/dose: 10/sex/group 

Route of administration: Gavage 

Exposure duration: ~ 40 days (two week maturation phase, pairing, gestation and early 

lactation) 

Doses/concentrations tested: 0 (vehicle control), 50, 150 and 500 mg/kg-bw/day 

Vehicle: Polyethylene glycol 400 (4 mL/kg/day) 

Statistical methods used: Bodyweight and bodyweight change, food consumption 

during gestation and lactation, litter data, implantation losses and viability indices, 

offspring bodyweight and bodyweight change, offspring surface righting and adult 

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Draft HPV1 Test Rule – Data Adequacy – Reproductive/Developmental Toxicity CAS No. 74-95-3

absolute and bodyweight relative organ weights were subjected to statistical analysis. 

Data were assessed for dose response relationships by linear regression analysis, followed 

by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity 

of variance. Where variances were shown to be homogenous, pairwise comparisons 

were conducted using Dunnett's test. Where Levene's test showed unequal variances the 

data were analyzed using nonparametric methods: Kruskal-Wallis ANOVA and MannWhitney `U' test . Non-parametric methods were used to analyze implantation loss, 

offspring sex ratio and landmark developmental markers. In addition, histopathology 

data were analyzed using the following methods to determine significant differences 

between control and treatment groups for the individual sexes: 

1. Chi-squared analysis for differences in the incidence of lesions occurring with an overall 

frequency of 1 or greater 

2. Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of 

severity grades for the more frequently observed graded conditions. 

Test method/conditions remarks: The test material was administered by gavage to three groups 

each of ten male and ten female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, for 

approximately forty days (to include a two week maturation phase, pairing, gestation and early 

lactation) at dose levels of 500, 150 and 50 mg/kg/day . A control group of ten males and ten 

females was similarly dosed with vehicle alone (Polyethylene glycol 400). 

Clinical signs, bodyweight development, food and water consumption were monitored during the 

study. 

Pairing of animals within each dose group was undertaken on a one male: one female basis on 

Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to 

Day 5 of lactation. 

During the lactation phase, daily clinical observations were performed on all surviving offspring, 

together with litter size, offspring weights and assessment of righting reflex. 

Surviving males were terminated on Day 43 of the study, with all the surviving females and 

offspring being killed on Day 5 post partum . All animals were subjected to a gross necropsy 

examination and histopathological evaluation of reproductive tissues was performed. 

RESULTS

Endpoint value(s): 

NOAEL (systemic/reproductive/developmental toxicity) = 150 mg/kg/day 

LOAEL (systemic/reproductive/developmental toxicity) = 500 mg/kg/day 

Results remarks: One male, receiving 500 mg/kg/day, was killed in extremis on Day 2 of the 

study and one control female was found dead on Day 41 of the study; neither of these deaths 

were considered to be a consequence of treatment. 

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Draft HPV1 Test Rule – Data Adequacy – Reproductive/Developmental Toxicity CAS No. 74-95-3

There were no significant treatment-related clinical signs of toxicity observed at 50, 150 or 500 

mg/kg/day. 

At 500 mg/kg/day, lower bodyweight gain was apparent for females during gestation, compared 

to control, but this was considered to be a consequence of the lower litter size at this dosage. 

At 500 mg/kg/day, lower food conversion efficiency was apparent for females during gestation, 

compared to control, reflecting the lower weight gain observed during this phase of the study. 

Food intake was lower during lactation and was attributed to the lower physiological demand on 

parent females due to the smaller litter size. 

At 500 mg/kg/day, there was a clear increase in pre-coital interval, compared with control, with 

only four females mating during the first four days of pairing. Copulation plug count was 

generally lower than control, but fertility was not adversely affected with eight females achieving 

pregnancy. Gestation length of these females tended to be longer than control, possibly 

influenced by lower litter size. 

At 500 mg/kg/day, litter size was lower than control on Day 1 due to increased post-implantation 

loss; this loss probably representing death of the offspring in utero. In addition, one female at 

this dosage was considered to have lost her litter in-utero. 

At 50, 150 or 500 mg/kg/day, offspring bodyweight on Day 1 and subsequent survival, growth 

and development to Day 4 were unaffected by maternal treatment; lower litter weights at 500 

mg/kg/day reflected the lower litter size. One control female and one female receiving 500 

mg/kg/day showed total litter loss on Day 1 postpartum. 

Necropsy findings among adult animals and their offspring did not indicate any effect of 

maternal treatment at dosages of 50, 150 or 500 mg/kg/day. 

At 500 mg/kg/day, males showed lower absolute and bodyweight-relative epididymal and testes 

weights compared to control. At 150 mg/kg/day, absolute and bodyweight relative 

testes weights were also lower than control. In the absences of any treatment-related 

histopathological effects at 500 mg/kg/day, these differences in male reproductive organ weights 

were considered to be of equivocal toxicological significance. 

Microscopic examination of adult reproductive tissues did not reveal any treatment-related 

effects at dosages of 50, 150 or 500 mg/kg/day. 

CONCLUSIONS

Treatment with Dibromomethane at 500 mg/kg/day was associated with a clear effect on mating 

performance and a reduction in litter size at birth, most probably due to increased in-utero 

mortality. The `No Observed Adverse Effect Level' (NOAEL) for reproduction observed in this 

study was considered to be 150 mg/kg/day. 

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Draft HPV1 Test Rule – Data Adequacy – Reproductive/Developmental Toxicity CAS No. 74-95-3

STUDY RELIABILITY

[1] Reliable without restrictions 

REFERENCE

EPA-HQ-OPPT-2005-0259.3 and 0259.4, Fulcher et al. (2007) Dibromomethane: Oral (Gavage) 

Reproduction/Development Toxicity Screening Test in the Rat. SafePharm Laboratories, SPL 

Project Number 0466/0261, 1-180 pgs. Sponsor: Bromine Compounds Ltd and Albemarle 

Coroporation Health Safety and Environment 

5 
Review of TSCA Section 4 Final Study Report

CITB Project Manager: Catherine Roman 

Chemical Name: Dibromomethane 

CAS Registry Number: 74-95-3 

Report Title: Oral (Gavage) Reproduction/Development Toxicity Screening Test in the Rat 

Test Sponsor: Bromine Compounds Ltd. and Albemarle Corporation 

Testing Lab: Safepharm Laboratories Limited 

 

Test Standard: 799.9355 

Date Study Received by EPA: July 12, 2007 Date Study Due to EPA: May 17, 2007 

Special Instructions (e.g., regarding approved protocol modifications, etc.): 

 

Part I. Test Report and Study Conduct

Enter one of the following codes into each of the checkboxes 1 through 13: Agree [A], Disagree [D], Not 

Applicable [NA], or Cannot Determine [C], and add comment, below, as needed

 [D] 1. The final study report indicates evidence of gross, unauthorized or undocumented deviation from 

 the EPA test standard requirements, including “shall,” “must,” or “will” violations. List 

 significant test standard deviations and “shall,” “must,” or “will” violations in comments below. 

 [D] 2. The final study report indicates evidence of deviations from the “should” statements in the study protocol 

that may have a bearing on the study conclusions. List these in comments below. 

 [NA] 3. Approved modifications of test standard or testing protocol (CITB PM describe or attach) were not 

followed or are inadequately documented. 

 [D] 4. There is evidence that laboratory Standard Operating Procedures (SOPs) were inadequately followed. 

 [D] 5. The Statement of Quality Assurance (QA) inspections, their dates, and findings were missing or not signed. 

There is evidence that QA procedures may be inadequate. 

[D] 6. There is evidence that Good Laboratory Practice Standards (GLPS) were inadequately followed. 

[D] 7. The test, control, or reference substances are different from that specified in the section 4 test rule or 

Enforceable Consent Agreement (ECA). 

[D] 8. Animal identification is inadequate or unclear; use of unhealthy animals or other factors (e.g., deviations 

from temperature, lighting, dosing) are apparent. 

[D] 9. The final study report inadequately describes the study method. 

[D] 10. The final study report inadequately describes the test system. 

[D] 11. The final study report inadequately describes the dosage information. 
[D] 12. The final study report does not accurately reflect the individual animal data (or that of other discrete 

observations pertinent to the type of study conducted), or individual data were not provided. 

[D] 13. The final study report inadequately describes the calculations, interpolations, data transformations, 

statistical analyses, or modeling parameters used. 

Part II. Conclusions

Place a check mark beside the summary statement(s) that apply to this review and add comments below as 

appropriate

Part A. To be filled out by Reviewer

 

Study Conduct 1. The study was well-conducted. [X] 

 2. The study was generally well-conducted, with some minor [ ] 

 deficiencies (list in notes below). 

 3. The study conduct was significantly deficient or questionable, (give [ ] 

 reasons in notes below). 

Data Reliability 4. The study results are precise and accurate. [X] 

 5. Appropriate methods were used to determine [X] 

 statistical significance. 

 6. If repeated, the test results would be expected to be the [X] 

 same. 

Data Utility 7. The data are useful for risk assessment for the endpoint tested. [X] 

 8. The data are useful for some other purpose (e.g., dose-setting). [ ] 

 9. The data are not useful for any purpose (describe in notes below). [ ] 

Reviewer Comments (continue on separate page, if needed): 

Reviewer Name/Title/Branch/Division/Office: Ashlee Aldridge/SRC, Inc.

Reviewer Signature: David Brooks/HPVCB/RAD/OPPTS 

Part B. To be filled out by CITB PM

Tiered Testing Decision 1. The tiered testing trigger was met, additional testing is indicated. [ ] 

(when applicable) 2. The trigger for additional (tiered) testing was not met. [ ] 

 

Study Acceptability 3. The study is acceptable. [ ] 

 4. The study may not be acceptable [ ] 

 (provide rationale in memo to CITB chief).