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 Wo rk Assignment Manager

March 5, 2010HPV1 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 22202Draft HPV1 Test Rule – Data Adequacy – Gene Mutations CAS No. 112-52-7

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 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 Guidelin e, 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 uvr A

Metabolic activation: With and without (Arocolo r 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 uvr A with and without an exogenous metabolic activating system, Aroclor-indu ced rat liver (S9 mixture), to 50, 150, 500,

1500 and 5000 μ g 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 μ g per plate) were plated (two plates per dose) with ove rnight cultures of TA98, TA100, TA1535 and TA 1537 and WP2uvrA to evaluate toxicity. In the confirmatory mutageni city assay, five dose levels of test article (50, 150, 500, 1500 and 5000 μ g/plate) along with appr opriate vehicle (ethanol) and pos itive controls were plated, in triplicate, with overnight cultures of all five bacterial strains. Both assays were conducted by the plate incor poration method on selective minimal agar with

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

2Draft 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 ve hicle or test article dilution added to 2.0 mL of molten selective top ag ar. When plating the positive controls, the test article aliquot was replaced by a 0.05 mL aliquot of the a ppropriate positive control.

The following positive controls were used without S9: 2-nitrofluorene (1.0 μ g/plate) with TA98; sodium azide (1.0 μ g/plate) with TA100 and TA1535; 9-aminoacridine (75 μ g/plate) with TA1537 and methyl methanesulfonate (1000 μ g/plate) with WP2 uvr A. With S9, 2- aminoanthracene (1.0 or 10 μ g/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 automa ted colony counter or by hand. Individual counts from the plates for each concentration along w ith 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 inc ubation period by visual examination without magnification. Toxicity and degree of precipita tion were scored relative to the vehicle control plate.

The criteria for evaluation were: At least a 2-fold (TA98, TA100 and WP2 uvr A) or 3-fold (TA1535 and TA1537) increase in m ean revertants at the peak of the dose response over the mean vehicle control value was considered positiv e. 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 uvr B gene. Cultures of tester

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

WP2 uvr A cultures must demonstrate the deletion in the uvr A gene. All cultures must demonstrate the characteristic mean number of spont aneous revertants in th e vehicle controls as follows (inclusive): TA98, 10 - 50; TA100, 80 - 240; TA1535, 5 - 45; TA1537, 3 - 21; WP2

uvr A, 10 - 60. To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3x10 9 cells/mL. The mean of each positive control must

exhibit at least a 3.0-fold increase in the num ber 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 consider ed toxic if one or both of the following criteria are met: (1) A

>50 % reduction in the mean numbe r 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 reducti on in the background lawn (background code 3, 4 or 5).

RESULTS

Genotoxicity result: Negative

Cytotoxic concentration (if applicable) : None

3Draft HPV1 Test Rule – Data Adequacy – Gene Mutations CAS No. 112-52-7

Results remarks: 

### No appreciable toxicity was observed at any do se level in the preliminary toxicity-mutation assay. Based on these results, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μ g per plate.

No appreciable toxicity and no increase in re vertants 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 mutageni c 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).

4Review 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 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).