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 Work Assignment Manager 

March 5, 2010 
HPV1 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 22202 
Draft HPV1 Test Rule – Data Adequacy – Gene Mutation CAS No. 409-02-9

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 – 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 Heavy 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-Nitroquinoline-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 incorporation 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 

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Draft 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 minimal 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 (strain/test concentration/activation 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% confidence 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 increase 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 viable 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 have mutagenic activity if a reproducible increase in revertant 

colony numbers of at least twice (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 criteria for a ‘positive’ or ‘negative’ response 

were not clearly met, statistical methods, in particular Dunnett’s test, were employed to 

determine statistical significance. It was considered acceptable to conclude that the response was 

equivocal if no clear results were obtained. 

RESULTS

Genotoxicity result: Negative 

Cytotoxic concentration (if applicable): No evidence of toxicity was observed in the initial 

test. In the second test, toxicity, observed as a thin or absent background lawn of non-revertant 

colonies and/or reduction in the number of revertant 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 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. 

CONCLUSIONS

Methyl heptenone showed no evidence of mutagenic activity in this bacterial system under the 

test conditions employed. 

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Draft 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 heptenone (CAS #409-02-9) Bacterial Reverse 

Mutation Test. Study #EXN0095. 

4 
Review 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) 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: 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 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).