SRC TR-10-050 

HPV1 TEST RULE STUDY – DATA ADEQUACY ASSESSMENT 

1-Chlorododecane, CAS No. 112-52-7 

Genetic Toxicity - Gene Mutations 

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 FG007.0.009.002 

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 

1-Chlorododecane, CAS No. 112-52-7 

Genotoxicity – Gene Mutations 

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 Mutations CAS No. 112-52-7

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 Mutations CAS No. 112-52-7

GENOTOXICITY- GENE MUTATIONS 

Title: Bacterial Reverse Mutation Assay of 1-Chlorododecane

The study is: Adequate Not Adequate 

TEST SUBSTANCE 

 

Identity: 1-Chlorododecane, CAS No. 112-52-7 

Remarks: The test substance was a colorless to pale yellow liquid, stored at room temperature 

and was stable for the duration of testing. Batch #N6227945, Purity 97.4% 

METHOD

Method/guideline followed: TSCA Health Effects Guideline, 40CFR Part 799.95 10 (1999), the 

OECD Guideline 471 (1 998) and the ICH (1996 and 1997). 

Test type: Bacterial reverse mutation assay 

GLP compliant?: Yes 

Year study performed: 2008

Test species: Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and 

Escherichia coli tester strain WP2 uvrA 

Metabolic activation: With and without (Arocolor 1254-induced rat liver S9) 

Concentrations tested: 

Initial assay: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate 

Confirmatory assay: 50, 150, 500, 1500 and 5000 μg/plate

Test method/conditions remarks: 

The mutagenic assay was conducted by exposing S. typhimurium tester strains TA98, TA100, 

TA1535 and TA1537, and E. coli tester strain WP2 uvrA with and without an exogenous 

metabolic activating system, Aroclor-induced rat liver (S9 mixture), to 50, 150, 500, 

1500 and 5000 μg 1-Chlorododecane/plate. The concentrations for the main test were determined 

by an initial toxicity-mutation assay in which vehicle controls, positive controls and eight dose 

levels of the test article (1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate) were plated (two 

plates per dose) with overnight cultures of TA98, TA100, TA1535 and TA 1537 and WP2uvrA 

to evaluate toxicity. In the confirmatory mutagenicity assay, five dose levels of test article (50, 

150, 500, 1500 and 5000 μg/plate) along with appropriate vehicle (ethanol) and positive controls 

were plated, in triplicate, with overnight cultures of all five bacterial strains. 

Both assays were conducted by the plate incorporation method on selective minimal agar with 

and without Arochlor-induced rat liver S9. The reaction mixture consisted of 0.5 mL of S9 mix 

2 
Draft HPV1 Test Rule – Data Adequacy – Gene Mutations CAS No. 112-52-7

or Sham (substitution buffer) mix, 0.1 mL of tester strain and 0.05 mL of vehicle or test article 

dilution added to 2.0 mL of molten selective top agar. When plating the positive controls, the test 

article aliquot was replaced by a 0.05 mL aliquot of the appropriate positive control. 

The following positive controls were used without S9: 2-nitrofluorene (1.0 μg/plate) with TA98; 

sodium azide (1.0 μg/plate) with TA100 and TA1535; 9-aminoacridine (75 μg/plate) with 

TA1537 and methyl methanesulfonate (1000 μg/plate) with WP2 uvrA. With S9, 2-

aminoanthracene (1.0 or 10 μg/plate) was used for all strains. After vortexing the mixtures were 

overlaid onto the surface of 25 mL of minimal bottom agar. Once the plates had solidified, they 

were inverted and incubated for approximately 48 to 72 hours at 37 ± 2oC. After incubation, the 

revertant colonies were counted by an automated colony counter or by hand. Individual counts 

from the plates for each concentration along with the means (± standard deviation) were 

determined. 

The condition of the bacterial background lawn was evaluated for evidence of test article toxicity 

by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual 

examination without magnification. Toxicity and degree of precipitation were scored relative to 

the vehicle control plate. 

The criteria for evaluation were: At least a 2-fold (TA98, TA100 and WP2 uvrA) or 3-fold 

(TA1535 and TA1537) increase in mean revertants at the peak of the dose response over the 

mean vehicle control value was considered positive. A response was evaluated as negative if it 

was neither positive, nor equivocal (a biologically relevant increase in a revertant count that 

partially meets the criteria for evaluation as positive). 

The criteria for a valid test were: All Salmonella tester strain cultures must demonstrate the 

presence of the deep rough mutation (rfa) and the deletion in the uvrB gene. Cultures of tester 

strains TA98 and TA100 must demonstrate the presence of the pKM101 plasmid R-factor. All 

WP2 uvrA cultures must demonstrate the deletion in the uvrA gene. All cultures must 

demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls as 

follows (inclusive): TA98, 10 - 50; TA100, 80 - 240; TA1535, 5 - 45; TA1537, 3 - 21; WP2 

uvrA, 10 - 60. To ensure that appropriate numbers of bacteria are plated, tester strain culture 

titers must be greater than or equal to 0.3x109 cells/mL. The mean of each positive control must 

exhibit at least a 3.0-fold increase in the number of revertants over the mean value of the 

respective vehicle control. A minimum of three non-toxic dose levels is required to evaluate 

assay data. A dose level is considered toxic if one or both of the following criteria are met: (1) A 

>50 % reduction in the mean number of revertants per plate as compared to the mean vehicle 

control value. This reduction must be accompanied by an abrupt dose-dependent drop in the 

revertant count. (2) At least a moderate reduction in the background lawn (background code 3, 4 

or 5). 

RESULTS

Genotoxicity result: Negative 

Cytotoxic concentration (if applicable): None

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Draft HPV1 Test Rule – Data Adequacy – Gene Mutations CAS No. 112-52-7

Results remarks: 

No appreciable toxicity was observed at any dose level in the preliminary toxicity-mutation 

assay. Based on these results, the maximum dose plated in the confirmatory mutagenicity 

assay was 5000 μg per plate. 

No appreciable toxicity and no increase in revertants meeting the criteria for a positive 

response were observed at any concentration in any strain with or without metabolic 

activation. All criteria for a valid test were met. 

CONCLUSIONS

Under the conditions of this study, 1-chlorododecane was not mutagenic to bacterial cells with or 

without metabolic activation. 

STUDY RELIABILITY

[1] – Reliable without restrictions 

REFERENCE

EPA-HQ-OPPT-2005-0033-0314.2; Wagner, O.V. and VanDyke, M.R. 2008. Bacterial Reverse 

Mutation Assay of 1-Chlorododecane. BioReliance for Lonza, Inc. Study No. 

AC01UM.503.BTL (unpublished report). 

4 
Review of TSCA Section 4 Final Study Report 

CITB Project Manager: Catherine Roman 

Chemical Name: 1-Chlorododecane 

CAS Registry Number: 112-52-7 

Report Title: Bacterial Reverse Mutation Assay of 1-Chlorododecane

Test Sponsor: Lonza, Inc. 

Testing Lab: BioReliance 

Test Standard: 799.9510 

Date Study Received by EPA: September 19, 2008 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).