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 Wo rk Assignment Manager

March 5, 2010HPV1 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 22202Draft HPV1 Test Rule – Data Adequacy – Reproductive/Developmental Toxicity CAS No. 74-95-3

PREPARATION OF THE DAT A ADEQUACY ASSESSMENT

This document contains the review of a toxi city 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 guide s, the study was reviewed for adequacy and evaluated for compliance with the test guideline. (T he applicable test guid eline 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 assi gned a Klimisch reliability code. If the study was determined to be inadequate, a basis for th at conclusion is supplie d. For studies already assigned Klimisch codes, an explanation is prov ided if the reviewer’s assessment of data adequacy differed from the pre-assigned Klimisch code.

A detailed evaluation of the test report a nd study conduct is provided at the end of the summary document in a section entitled “Revie w 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 Procedur es, Good Laboratory Prac tice Standards, and Quality Assurance procedures, as well as the use of appropriate controls and test substances. Any deficiencies in the descri ptions of the study method, test system, animals studied, physical conditions, dosage information, indi vidual 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.

1Draft HPV1 Test Rule – Data Adequacy – Reproductive/Developmental Toxicity CAS No. 74-95-3

REPRODUCTIVE/DEVELOPMENTAL TOXICITY

Title: Oral (Gavage) Reproduction/ Developmental Toxicity Sc reening 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%; indi vidual samples of dibromomethane were supplied by both Co-Sponsors. These two test mate rials were then mixed in equal proportions and the resulting mixture anal yzed 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 Eff ects 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 pha se, 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, im plantation losses and viability indices, offspring bodyweight and bodyweight change, offspring surface righting and adult

2Draft HPV1 Test Rule – Data Adequacy – Reproductive/Developmental Toxicity CAS No. 74-95-3

absolute and bodyweight relative organ weights we re subjected to statistical analysis. Data were assessed for dose response relations hips by linear regression analysis, followed by one way analysis of variance (ANOVA) inco rporating Levene's test for homogeneity

of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett's test. Where Le vene'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 developmenta l markers. In addition, histopathology data were analyzed using the following me thods to determine significant differences between control and treatment groups for the individual sexes:

1. Chi-squared analysis for differences in the in cidence of lesions occu rring with an overall frequency of 1 or greater

2. Kruskal-Wallis one-way non-parametric anal ysis of variance for the comparison of severity grades for the more fre quently observed graded conditions.

Test method/conditions remarks: The test material was administer ed by gavage to three groups each of ten male and ten female Sprague-Daw ley 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 cont rol group of ten males and ten females was similarly dosed with vehi cle alone (Polyethylene glycol 400).

Clinical signs, bodyweight development, food a nd water consumption were monitored during the study.

Pairing of animals within each dose group was unde rtaken on a one male: one female basis on Day 15 of the study, with females subsequently being allowed to litter and re ar their offspring to Day 5 of lactation.

During the lactation phase, daily cl inical observations were perf ormed on all surviving offspring, together with litter size, offspring weights and asse ssment 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 evaluati on of reproductive ti ssues was performed.

RESULTS

Endpoint value(s): NOAEL (systemic/reproductive/developm ental toxicity) = 150 mg/kg/day LOAEL (systemic/reproductive/devel opmental 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 Da y 41 of the study; neither of these deaths were considered to be a consequence of treatment.

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

There were no significant treatme nt-related clinical signs of t oxicity observed at 50, 150 or 500 mg/kg/day.

At 500 mg/kg/day, lower bodyweight gain was appa rent for females during gestation, compared to control, but this was consider ed to be a consequence of the lo wer litter size at this dosage.

At 500 mg/kg/day, lower food conve rsion efficiency was apparent for females during gestation, compared to control, reflecti ng the lower weight gain observe d during this phase of the study. Food intake was lower during lactation and was at tributed 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 no t adversely affected with eight females achieving pregnancy. Gestation length of these females tended to be longer th an 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 represen ting death of the offspring in ut ero. In addition, one female at this dosage was considered to ha ve 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 ma ternal treatment; lowe r litter weights at 500 mg/kg/day reflected the lower l itter size. One control female and one female receiving 500 mg/kg/day showed total litte r loss on Day 1 postpartum.

Necropsy findings among adult animals and their offspring did not indi cate 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 ep ididymal 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 effect s at 500 mg/kg/day, these differences in male reproductive organ weights were considered to be of equi vocal 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.

4Draft 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

5Review 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) inspect ions, their dates, and findings were missing or not signed. There is evidence that QA pr ocedures 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 di fferent 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 summar y 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 ri sk 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/Bran ch/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 me t, 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).