SRC TR-10-052 

HPV1 TEST RULE STUDY – DATA ADEQUACY ASSESSMENT 

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

Genotoxicity - Chromosomal Aberrations 

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 

Genetic Toxicity- Chromosomal Aberrations 

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 Assessment– Chromosomal Aberrations 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 Assessment– Chromosomal Aberrations CAS No. 112-52-7

GENOTOXICITY- CHROMOSOMAL ABERRATIONS 

Title: In Vitro Mammalian Chromosome Aberration Test

The study is: Adequate Not Adequate 

TEST SUBSTANCE 

 

Identity: 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 97.42%. 

METHOD

Method/guideline followed: ICH (1996 and 1997), OECD 473 (1998), and the EPA (TSCA) 40 

CFR 799.9537 

Test type: Chromosomal aberration test (in vitro)

GLP compliant?: Yes 

Year study performed: 2008

Test species: Chinese Hamster ovary (CHO) cells 

Metabolic activation: With and without (Aroclor 1254-induced S9 activation system) 

Concentrations tested: 

Non-activated: 1.56, 3.13, 6.25, 12.5, 25, 35 and 50 μg/mL (4 and 20 hr) 

S9-activated: 6.25, 12.5, 25, 35, 50, 75 and 100 μg/mL (4 hr) 

Test method/conditions remarks: 

Solubility Test: 

A solubility test was conducted to determine the maximum soluble concentration or workable 

suspension using water, dimethyl sulfoxide (DMSO) and ethanol. The vehicle of choice was the 

solvent, selected in order of preference that permitted preparation of the highest soluble stock 

concentration, up to 50 mg/mL in water and up to 500 mg/mL in DMSO and ethanol. 

A toxicity assay and a chromosomal aberration assay were performed using the following 

procedures: The preliminary toxicity assay was conducted with 9 treatment groups (0.204-2040 

μg/mL) and solvent alone (ethanol). Based on the results of the preliminary toxicity assay, the 

test substance concentrations used in the chromosomal aberration assay were 6.25, 12.5, 25, 35, 

50, 75 and 100 μg/ml with S9 (4-hour exposure) and 1.56, 3.13, 6.25, 12.5, 25, 35 and 50 μg/ml 

without S9 (4- and 20-hour exposure). 

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Draft HPV1 Test Rule – Data Adequacy Assessment– Chromosomal Aberrations CAS No. 112-52-7

CHO cells were seeded in duplicate for each treatment condition at 5.0 x 105 cells/25 cm2 flask 

and were incubated at 37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air for 16-24 

hours. Treatment was carried out by refeeding duplicate flasks with 5 mL complete medium for 

the non activated system or 5 mL Aroclor 1254-induced rat liver S9 mix for the activated system, 

to which 50 μL of dosing solution of test or control substance in solvent or solvent alone was 

added. The osmolality of the solvent and of the highest concentration of dosing solution in the 

treatment medium was measured. The pH of the highest concentration of dosing solution in the 

treatment medium was also measured. 

For exposure without S-9 activation, duplicate cultures per treatment condition were exposed for 

4 hours or continuously for 20 hours up to cell harvest at 37 ± 1°C in a humidified atmosphere of 

5 ± 1% CO2 in air. In the four hour exposure group, after the exposure period, the treatment 

medium was removed, the cells were washed with calcium and magnesium free buffered saline 

(CMF PBS), refed with complete medium and returned to the incubator for an additional 16 

hours. For each exposure, two hours prior to the scheduled harvest, Colcemid was added to the 

duplicate flasks for each treatment condition at a final concentration of 0.1 μg/mL and the flasks 

returned to the incubator until cell collection. 

For exposure with S-9 activation, duplicate cultures per treatment condition were exposed for 4 

hours at 37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air. After the exposure period, 

the treatment medium was removed, the cells were washed with CMF PBS, refed with complete 

medium and returned to the incubator for an additional 16 hours. Two hours prior to the 

scheduled harvest, Colcemid was added to the duplicate flasks for each treatment condition at a 

final concentration of 0.1 μg/mL and the flasks returned to the incubator until cell collection. 

A concurrent toxicity test was conducted in both the activated and non activated test systems. 

After cell harvest, an aliquot of the cell suspension was removed from each culture and counted. 

If present, test substance precipitate was recorded. Cell viability was determined by trypan blue 

dye exclusion. The cell counts and percent viability were used to determine cell growth 

inhibition relative to the solvent control. 

Two hours after the addition of Colcemid, metaphase cells were harvested for all exposures by 

trypsinization. Cells were collected approximately 20 hours after initiation of treatment, fixed 

with Carnoy’s fixative and stored at least overnight at 2-8°C. Fixed cell suspensions were 

applied to slides, allowed to air dry, stained with 5% Giemsa, air dried and permanently 

mounted. Slides were coded by random number and scored in a blind fashion. The percentage of 

cells in mitosis per 500 cells scored (mitotic index) was determined for each treatment group. 

Metaphase cells with 20 ± 2 centromeres were examined under oil immersion. A minimum of 

200 metaphase spreads (100 per duplicate flask) were examined for chromatid and chromosometype aberrations. The following aberrations were scored: chromatid and isochromatid breaks and 

exchange figures (such as triradials, qudriradials, complex rearrangements); chromosome breaks 

and exchange figures (such as dicentrics and rings); chromatid or acentric fragments; pulverized 

chromosomes and cells, and severely damaged cells (≥ 10 aberrations). Chromatid and 

isochromatid gaps were recorded but not included in the analysis of the data. Polyploid and 

endoreduplicated cells were evaluated from each treatment flask per 100 metaphase cells scored. 

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Draft HPV1 Test Rule – Data Adequacy Assessment– Chromosomal Aberrations CAS No. 112-52-7

Mitomycin C was used as the positive control in the non-activated exposures at final 

concentrations of 0.1 and 0.2 μg/mL. Cyclophosphamide was used as the positive control in the 

activated exposures at final concentrations of 10 and 20 μg/mL. The solvent vehicle for the test 

substance was used as the solvent control at the same concentration as it was used in the test 

substance treated groups. 

The highest dose level selected for evaluation was the dose which induced at least 50% toxicity, 

as measured by cell growth inhibition, relative to the solvent control, with a sufficient number of 

scoreable metaphase cells. In treatment groups with lack of a dose level with close to 50% 

reduction in cell growth, selection of doses for microscopic analysis was based on mitotic index 

(the lowest dose with at least 50% reduction in mitotic index). Two additional lower dose levels 

were included in the evaluation. 

Statistical analysis of the percent aberrant cells was performed using the Fisher’s exact test. 

Fisher’s test was used for pairwise comparison of the percent aberrant cells of each treatment 

group with that of the solvent control. In the event of a positive Fisher’s test at any test substance 

dose level, the Cochran-Armitage test was used to measure dose responsiveness. 

The test substance is considered to induce a positive response when the percentage of cells with 

aberrations was increased in a dose-responsive manner with one or more concentrations being 

statistically significant (p≤0.05). 

For the test to be valid, the frequency of cells with structural chromosome aberrations in the 

solvent control must be within the range of the historical solvent control (i.e. 0-5%). The 

percentage of cells with chromosomal aberrations in the positive control must be statistically 

increased (p≤0.05) relative to the solvent control. 

RESULTS

Genotoxicity result: Positive 

Cytotoxic concentration (if applicable): ≥ 61.2 μg/mL 

Results remarks: 

Solubility Test: 

Ethanol was the solvent of choice based on the solubility of the test article and compatibility 

with the target cells. The test article was soluble in ethanol at a concentration of 

approximately 500 mg/mL, the maximum concentration tested for solubility. 

Preliminary Toxicity Assay: 

Substantial toxicity (i.e., at least 50% cell growth inhibition, relative to the solvent control) was 

observed at dose levels ≥ 61.2 μg/mL in all three treatment groups. 

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Draft HPV1 Test Rule – Data Adequacy Assessment– Chromosomal Aberrations CAS No. 112-52-7

The dose levels selected for the chromosome aberration assay were 6.25, 12.5, 25, 35, 50, 75 and 

100 μg/ml with S9 (4-hour exposure) and 1.56, 3.13, 6.25, 12.5, 25, 35, and 50 μg/ml without S9 

(4- and 20-hour exposure). 

Chromosome Aberration Assay: 

In the chromosome aberration assay, the test article was soluble in ethanol and in the treatment 

medium at all dose levels tested at the beginning and conclusion of the treatment period. The pH 

of the highest concentration of test article in treatment medium was approximately 7.0. 

Toxicity at 35 μg/mL, the highest concentration examined, was 30% in the 4-hour exposure and 

17% in the 20-hour exposure without S9. In the 4- hour exposure with S9 activation, 57% cell 

growth inhibition was observed at 35 μg/mL, the highest concentration tested, as compared to the 

solvent control. 

The concentrations chosen for microscopic analysis of chromosome aberrations were 6.25, 12.5 

and 35 μg/mL for all three exposures. 

When treated for four hours without S9, the mitotic index at 35 μg/mL was 56% reduced relative 

to the solvent control. The percentage of cells with structural and numerical aberrations in the 

test article-treated group was not significantly increased above that of the solvent control at any 

dose level. Treatment with the positive control (Mitomycin C) resulted in an 18% increase in the 

percentage of cells with structurally damaged chromosomes as compared to the solvent control, 

which was statistically significant. 

When treated for four hours in the presence of S9, the mitotic index at 35 μg/mL was 53% 

reduced relative to the solvent control. The percentage of cells with structural aberrations at the 

35 μg/mL dose level was statistically increased (12.0%) above that of the solvent control; the 

Cochran-Armitage test was also positive for a dose response. The percentage of cells with 

numerical aberrations in the test article-treated group was not significantly increased above that 

of the solvent control at any dose level. Treatment with the positive control (Cyclophosphamide) 

resulted in a 16% increase in the percentage of cells with structurally damaged chromosomes as 

compared to the solvent control, which was statistically significant. 

When treated for 20 hours continuously without S9, the mitotic index at 35 μg/mL was 55% 

reduced relative to the solvent control. The percentage of cells with structural and numerical 

aberrations in the test article-treated group was not significantly increased above that of the 

solvent control at any dose level. Treatment with the positive control (Mitomycin C) resulted in 

an 18% increase in the percentage of cells with structurally damaged chromosomes as compared 

to the solvent control, which was statistically significant. 

CONCLUSIONS

The positive and solvent controls fulfilled the requirements for a valid test. 

Based on the findings of this study, 1-chlorododecane was concluded to be negative for the 

induction of structural and numerical chromosome aberrations in CHO cells in the non-activated 

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Draft HPV1 Test Rule – Data Adequacy Assessment– Chromosomal Aberrations CAS No. 112-52-7

test system. 1–Chlorododecane was concluded to be positive for the induction of structural 

chromosome aberrations and negative for the induction of numerical chromosome aberrations in 

CHO cells in the S9-activated test system. 

STUDY RELIABILITY

[1] – Reliable without restrictions 

REFERENCE

EPA-HQ-OPPT-2005-0033-0314.3; Gudi, R. and Rao, M. 2008. In Vitro Mammalian 

Chromosome Aberration Test with 1-Chlorododecane. BioReliance for Lonza Inc. Study No. 

AC01UM.331.BTL (unpublished report). 

6 
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: In Vitro Mammalian Chromosome Aberration Test with 1-Chlorododecane 

Test Sponsor: Lonza, Inc. 

Testing Lab: BioReliance 

Test Standard: 799.9537 

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).