SRC TR-10-066 

HPV1 TEST RULE STUDY – DATA ADEQUACY ASSESSMENT 

Dibromomethane, CAS No. 74-95-3 

Genotoxicity – Chromosomal Aberrations 

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 

Genotoxicity – Chromosomal Aberrations 

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 – Chromosomal Aberrations 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 – Chromosomal Aberrations CAS No. 74-95-3

GENOTOXICITY – CHROMOSOMAL ABERRATIONS 

Title: Chromosome Aberration Test in Human Lymphocytes In Vitro 

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: OECD Guidelines for Testing of Chemicals (1997) No. 473 

"Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Directive 

2000/32/EC. The study design also meets the requirements of the UK Department of Health 

Guidelines for Testing of Chemicals for Mutagenicity and US OPPTS 870.5375 and 40 CFR 

799.9537 guidelines. 

Test type: In vitro mammalian chromosomal aberration test 

GLP compliant?: Yes 

Year study performed: 2006 

Test species: Human lymphocytes 

Metabolic activation: With and without S9 (from the livers of male Sprague-Dawley 

rats weighing ~ 250 grams). 

Concentrations tested: 

Preliminary Toxicity Test: 6.80, 13.59, 27.19, 54.38, 108.75, 217.5, 435, 870 and 1740 

μg/mL with and without S9 

Experiment 1: 27.19, 54.38, 108.75, 217.5, 435 and 870 μg/mL with and without S9 

Metaphase Analysis: 54.38, 108.75 and 217.5 μg/mL with and without S9 

Statistical methods used: The frequency of cells with aberrations excluding gaps and the 

frequency of polyploid cells were compared, where necessary, with the concurrent vehicle 

control value using Fisher's Exact test. 

Test method/conditions remarks: A preliminary toxicity test was performed on cell cultures 

using a 4-hour exposure time with and without metabolic activation followed by a 20-hour 

recovery period, and a continuous exposure of 24 hours without metabolic activation. The dose 

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Draft HPV1 Test Rule – Data Adequacy – Chromosomal Aberrations CAS No. 74-95-3

range of test material used was 6.80, 13.59, 27.19, 54.38, 108.75, 217.5, 435, 870 and 1740 

μg/mL. Parallel flasks, containing culture medium without whole blood, were established for the 

three exposure conditions so that test material precipitate observations could be made. 

Precipitate observations were recorded at the beginning and end of the exposure periods. 

Using a qualitative microscopic evaluation of the microscope slide preparations from Each 

treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic 

index data was used to estimate test material toxicity and for selection of the dose levels for the 

main study. 

In the main study, duplicate cultures of human lymphocytes, treated with the test material, were 

evaluated for chromosome aberrations at three dose levels, together with vehicle and positive 

controls. Two treatment conditions were used for the study, i.e. In experiment 1, 4 hours in the 

presence for an induced rat liver homogenate metabolizing system (S9), at a 2% final 

concentration with cell harvest after a 20-hour expression period and 4 hours exposure in the 

absence of metabolic activation (S9) with a 20-hour expression. Experiment 2 was performed 

but not scored because a clear positive result was obtained in Experiment 1. 

The dose levels used in Experiment 1 were selected on the basis of the results of a Preliminary 

toxicity test and were 27.19, 54.38, 108.75, 217.5, 435 and 870 μg/mL for the 4(20)-hour 

exposures both with and without S9. The dose levels selected for metaphase analysis were 

54.38, 108.75 and 217.5 μg/mL in both the absence and presence of S9. The dose levels used in 

Experiment 2 were 54.38, 108.75, 217.5, 435, 652 .5 and 870 μg/mL in the absence of S9, and 

13.60, 27.19, 54.38, 108.75, 217.5 and 435 μg/mL in the presence of S9. 

Vehicle (dimethyl sulphoxide, DMSO) and positive controls were used in parallel with the test 

material. The positive control materials were Mitomycin C (MMC) dissolved in Minimal 

Essential Medium (0.4 μg/mL) in the absence of activation and Cyclophosphamide (CP) 

dissolved in DMSO (5 μg/mL) in the presence of activation. 

A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase 

recorded and expressed as the mitotic index and as a percentage of the vehicle control value. 

RESULTS

Genotoxicity result: Positive 

Cytotoxic concentration (if applicable): ≥ 870 μg/mL 

Results remarks: All vehicle (solvent) controls gave frequencies of cells with aberrations within 

the range expected for normal human lymphocytes. All the positive control materials induced 

statistically significant increases in the frequency of cells with aberrations indicating the 

satisfactory performance of the test and of the activity of the metabolizing system. 

The test material induced a statistically significant increase in the frequency of cells with 

aberrations at all dose levels analyzed, in both exposure groups, using a dose range that included 

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Draft HPV1 Test Rule – Data Adequacy – Chromosomal Aberrations CAS No. 74-95-3

a dose level that induced greater than 50% mitotic inhibition. No increase in the incidence of 

polyploid cells was observed in either the absence or presence of S9. 

The study was terminated after Experiment 1 because a statistically significant increase in 

structural aberrations were observed in the 4(20)-hour exposures for Experiment 1. The response 

was considered to be clear evidence of clastogenic activity and in such cases a second 

experiment is not necessary and not required by the test guidelines. 

CONCLUSIONS

The test material induced a statistically significant dose-related increase in the frequency of cells 

with chromosome aberrations in both the absence and the presence of metabolic activation. The 

material was considered to be clastogenic to human lymphocytes in vitro 

STUDY RELIABILITY

[1] Reliable without restrictions 

REFERENCE

EPA-HQ-OPPT-2005-0033-0254.5, Durward et al. (2007) “Dibromomethane: 

Chromosome Aberration Test in Human Lymphocytes In Vitro”. SPL Project Number 

0466/0262, 1-27 pgs. Sponsor: Bromine Compounds Ltd. and Albemarle Corporation Health, 

Safety and Environment. 

4 
Review of TSCA Section 4 Final Study Report

CITB Project Manager: Catherine Roman 

Chemical Name: Dibromomethane 

CAS Registry Number: 74-95-3 

Report Title: Chromosome Aberration Test in Human Lymphocytes In Vitro 

Test Sponsor: Bromine Compounds Ltd. and Albemarle Corporation

Testing Lab: Safepharm Laboratories Limited

 

Test Standard: 799.9537

Date Study Received by EPA: June 07, 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).