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 Wo rk Assignment Manager

March 5, 2010HPV1 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 22202Draft HPV1 Test Rule – Data Adequacy Assessment– Chromosomal Aberrations 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 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 (DMS O) 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 a nd up to 500 mg/mL in DMSO and ethanol.

A toxicity assay and a chromosomal aberrati on assay were performed using the following procedures: The preliminary toxicity assay was conducted wi th 9 treatment groups (0.204-2040

μ g/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 μ g/ml with S9 (4-hour exposure) and 1.56, 3.13, 6.25, 12.5, 25, 35 and 50 μ g/ml without S9 (4- and 20-hour exposure).

2Draft 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 10 5 cells/25 cm 2 flask

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

hours. Treatment was carried out by refeeding duplicate flasks w ith 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 μ L 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 cu ltures per treatment condition were exposed for 4 hours or continuously for 20 hours up to cell harves t at 37 ± 1°C in a humi dified atmosphere of

5 ± 1% CO 2 in air. In the four hour exposure gro up, after the exposure period, the treatment medium was removed, the cells were washed w ith calcium and magnesium free buffered saline (CMF PBS), refed with complete medium and re turned 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 cond ition at a final concentration of 0.1 μ g/mL and the flasks

returned to the incuba tor until cell collection.

For exposure with S-9 activation, duplicate cultures pe r 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 μ g/mL and the flasks returned to the incubator until cell collection.

A concurrent toxicity test was conducted in both th e activated and non activated test systems. After cell harvest, an aliquot of the cell susp ension was removed from each culture and counted. If present, test substance precipitate was reco rded. Cell viability was determined by trypan blue dye exclusion. The cell counts a nd percent viability were used to determine cell growth

inhibition relative to the solvent control.

Two hours after the addition of Colcemid, metaph ase cells were harvested for all exposures by trypsinization. Cells were collect ed approximately 20 hours after initiation of treatment, fixed with Carnoy’s fixative and stor ed at least overnight at 2-8° C. Fixed cell suspensions were applied to slides, allowed to air dry, stained with 5% Giem sa, 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 i ndex) 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) we re examined for chromatid and chromosometype aberrations. The following aberrations were scored: chromatid and isochromatid breaks and exchange figures (such as triradials, qudriradia ls, complex rearrangements); chromosome breaks and exchange figures (such as dicentrics and ri ngs); chromatid or acentric fragments; pulverized chromosomes and cells, and severely damaged cells ( ≥ 10 aberrations). Chromatid and isochromatid gaps were recorded but not include d in the analysis of the data. Polyploid and endoreduplicated cells were evaluated from each treatment flask per 100 metaphase cells scored.

3Draft 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 μ g/mL. Cyclophosphamide was used as the positive control in the activated exposures at final concentrations of 10 and 20 μ g/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 w ith lack of a dose leve l with close to 50% reduction in cell growth, selecti on of doses for microscopic anal ysis 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 ce lls was performed using th e 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 contro l. In the event of a positive Fisher’s test at any test substance dose level, the Cochran-Armitage test wa s used to measure dose responsiveness.

The test substance is considered to induce a posit ive response when the percentage of cells with aberrations was increased in a dose-responsive ma nner with one or more concentrations being statistically significant (p ≤ 0.05).

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

solvent control must be within the range of the hist orical solvent contro l (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 choi ce 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 leas t 50% cell growth inhibition, relati ve to the solvent control) was

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

4Draft HPV1 Test Rule – Data Adequacy Assessment– Chromosomal Aberrations CAS No. 112-52-7

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

100 μ g/ml with S9 (4-hour exposure) and 1.56, 3.13, 6.25, 12.5, 25, 35, and 50 μ g/ml without S9 (4- and 20-hour exposure).

Chromosome Aberration Assay: In the chromosome aberration assay, the test arti cle 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 μ g/mL, the highest concentration examine d, was 30% in the 4-hour exposure and 17% in the 20-hour exposure without S9. In th e 4- hour exposure with S9 activation, 57% cell growth inhibition was observed at 35 μ g/mL, the highest concentration tested, as compared to the

solvent control.

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

and 35 μ g/mL for all three exposures.

When treated for four hours wit hout S9, the mitotic index at 35 μ g/mL was 56% reduced relative to the solvent control. The percentage of cells with structural and numer ical aberrations in the test article-treated group was not significantly increased above that of the solvent control at any dose level. Treatment with the pos itive control (Mitomycin C) resulted in an 18% increase in the percentage of cells with structurally damaged ch romosomes as compared to the solvent control, which was statistically significant.

When treated for four hours in the pr esence of S9, the mitotic index at 35 μ g/mL was 53% reduced relative to the solvent control. The percenta ge of cells with structural aberrations at the

35 μ g/mL dose level was statistically increased (12.0%) above that of th e solvent control; the Cochran-Armitage test was also positive for a do se response. The percentage of cells with

numerical aberrations in the test article-treated group was not si gnificantly increased above that of the solvent control at any dose level. Treatm ent with the positive control (Cyclophosphamide)

resulted in a 16% increase in th e percentage of cells with stru cturally 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 μ g/mL was 55% reduced relative to the solvent control. The perc entage of cells with structural and numerical

aberrations in the test articl e-treated group was not significantly increased above that of the solvent control at any dose level. Treatment with the positive control (Mit omycin 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-chlorodode cane was concluded to be negative for the

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

5Draft 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 induc tion of numerical chromo some aberrations in CHO cells in the S9-activated test system.

STUDY RELIABILITY

[1] – Reliable without restrictions

REFERENCE

EPA-HQ-OPPT-2005-0033-0314.3; G udi, 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).

6Review 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 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).