SRC TR-10-069

HPV1 TEST RULE STUDY – DATA ADEQUACY ASSESSMENT

Methyl Heptanone, CAS No. 409-02-9

Genotoxicity – Gene Mutation

Prepared by:

Kendra Moran, Ashlee Aldridge, 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

Methyl Heptanone, CAS No. 409-02-9

Genotoxicity – Gene Mutation

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 – Gene Mutation CAS No. 409-02-9

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 – Gene Mutation CAS No. 409-02-9

GENOTOXICITY – GENE MUTATION

Title: Bacterial Reverse Mutation Assay

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Methyl Heptanone, CAS No. 409-02-9

Remarks: The test substance, Baton Rouge MEK Hea vy Ketone Heart Cut C8 Ketone Fraction, was characterized as a complex mixture.

METHOD

Method/guideline followed: OECD TG 471 (1997); OPPTS 870.5100 (1998).

Test type: Bacterial reverse mutation assay

GLP compliant?: Yes

Year study performed: 2007

Test species: Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100, and Escherichia coli WP2 uvrA(pKM101).

Metabolic activation: With and without

Concentrations tested: Seven concentrations up to 5,000 μg/plate in dimethyl sulphoxide (DMSO).

Statistical methods used: Not stated

Positive controls:

Without metabolic activation: TA100 and TA1535: Sodium azide, 2 μg/plate TA1537: 9-Aminoacridine, 50 μg/plate TA98: 2-Nitrofluorene, 2 μg/plate WP2 uvrA: 4-Nitroquinolin e-1-oxide, 2 μg/plate

With metabolic activation: TA100, TA1535: 2-Aminoanthracene, 5 μg/plate WP2 uvrA: 2-Aminoanthracene, 10 μg/plate TA98, TA1537: Benzo[a]pyrene, 5 μg/plate

Test method/conditions remarks: In the initial test, the plate inco rporation method was used. Briefly, aliquots of the test substance solutions (0.1 ml) were placed in glass vessels , followed by either S9 mix or phosphate buffer

2Draft HPV1 Test Rule – Data Adequacy – Gene Mutation CAS No. 409-02-9

(0.5 ml), bacterial cultures (0.1 ml), and agar (2 ml). The mixtures were mixed thoroughly and overlaid onto Petri dishes pre-coated with mini mal agar. Plates were incubated at 37°C for 72 hours. In a second test, the test substance solutions were pre-incubated with bacteria and S9 mix (or buffer) for 30 minutes at 37°C prior to addition of agar. The remaining steps were identical to the initial test. Each experimental condition (strai n/test concentration/activ ation state) was plated

onto three Petri dishes.

A test was considered valid if the mean of the vehicle control revertant colony numbers for each

strain was within or close to the 99% confiden ce limits of the current historical control range. The historical range is maintained as a rolling record over a maximum of five years. Secondly, positive control compounds must induce an increa se in mean revertant colony number of two times (TA98, TA100, WP2 uvrA) or three times (TA1535, TA1537) the concurrent vehicle controls to be considered valid. Thirdly, mean vi able cell counts in the 10-hour bacterial cultures (to be mixed with test substances) must be at least 109/ml.

A substance was considered to ha ve mutagenic activity if a repr oducible increase in revertant colony numbers of at least tw ice (or three times, in the case of TA1535 and TA1537) the concurrent vehicle controls and some evidence of a positive dose-response relationship was

exhibited. A substance was considered to have no mutagenic activity if a reproducible increase in revertant colony numbers was not exhibited. If cr iteria for a ‘positive’ or ‘negative’ response were not clearly met, statistical methods, in particular Dunnett’s te st, were employed to determine statistical sign ificance. It was considered acceptabl e to conclude that the response was equivocal if no clear re sults were obtained.

RESULTS

Genotoxicity result: Negative

Cytotoxic concentration (if applicable): No evidence of toxicity was observed in the initial test. In the second test, toxicit y, observed as a thin or absent background lawn of non-revertant

colonies and/or reduction in the number of re vertant colonies, was obtained in all strains following exposure to methyl heptenone at one or two concentrations (1500 or 5000 μg/plate),

both with and without metabolic activation.

Results remarks: In the initial test (plate incorporation), no substantial increases in revertant colony numbers over control counts were obtained in any of the tester strains following exposure to methyl heptenone at any concentration up to 5000 μg/plate, with or without metabolic

activation.

In the second test (pre-incubation), no substa ntial increases in reve rtant colony numbers over control counts were obtained in any of the test er strains following exposure to methyl heptenone at any concentration up to 5000 μg/plate, with or without metabolic activation.

CONCLUSIONS Methyl heptenone showed no evidence of mutageni c activity in this bact erial system under the test conditions employed.

3Draft HPV1 Test Rule – Data Adequacy – Gene Mutation CAS No. 409-02-9

STUDY RELIABILITY [1] Reliable without restrictions.

REFERENCE Huntingdon Life Sciences, Ltd. 2008. Methyl he ptenone (CAS #409-02-9) Bacterial Reverse Mutation Test. Study #EXN0095.

4Review of TSCA Section 4 Final Study Report

CITB Project Manager: Catherine Roman

Chemical Name: Methyl Heptanone

CAS Registry Number: 409-02-9

Report Title: Bacterial Reverse Mutation Test

Test Sponsor: Exxon Mobil Chemical Company

Testing Lab: Huntingdon Life Sciences Ltd.

Test Standard: US TSCA TG 40 CFR 799.9510.

Date Study Received by EPA: 6/23/09 (date posted to http://www.regulations.gov )

Date Study Due to EPA: 5/17/07

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.

[D] 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 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/Bran ch/Division/Office: Kendra Moran/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).