SRC TR-10-054

HPV1 TEST RULE STUDY – DATA ADEQUACY ASSESSMENT

Phenol, 4,4’-methylenebis[2,6- bis(1, 1-dimethylethyl)-, CAS No. 118-82-1

Genotoxicity - Chromosomal Aberrations

Prepared by:

Teresa Manyin, Chris Janson, 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 Wo rk Assignment Manager

March 5, 2010HPV1 Test Rule – Data Adequacy Assessment

Phenol, 4,4’-methylenebis[2,6- bis(1,1- dimethylethyl)-, CAS No. 118-82-1

Genotoxicity - 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 – Chromosomal Aberrations CAS No. 118-82-1

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 – Chromosomal Aberrations CAS No. 118-82-1

GENOTOXICITY – CHRO MOSOMAL ABERRATIONS

Title: Chromosomal Aberration test with AN-2 in Cultured Chinese Hamster Ovary (CHO) Cells

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: 4,4'-methylenebis(2,6-di-te rt-butylphenol) (AN-2; Ph enol, 4,4’-methylenebis[2,6- bis(1,1-dimethylethyl) -), CAS No. 118-82-1

Remarks: 99.45% purity; white powder ; production lot: 5127; MW: 424.7; supplier: Albemarle Corporation; TNO Dispense No.: 060123; vehicle: DMSO

METHOD

Method/guideline followed: OECD 473, 40 CFR 799.9537

Test type: Chromosomal Aberration Test in Cultured Chinese Hamster Ovary Cells

GLP compliant?: Yes

Year study performed: 2006

Test species: Chinese hamster ovary cells

Metabolic activation: With and without

Concentrations tested: First test (pulse exposure; with and without activation): 0, 0.1, 0.2, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, and 200 μg/mL; seco nd test (pulse exposure; with activation): 0, 5, 10, 30, 50, 75, 100, 150, 200, and 300 μg/mL; second test (continuous exposure; without activation): 0, 1.25, 2.5, 5, 10, 30, 50, 75, 100, 150, 200, 300 μg/mL

Statistical methods used: Fisher’s exact probability test (two-sided) was conducted to determine significant differences be tween treated and control cultures.

Test method/conditions remarks: Chinese Hamster Ovary cells (CHO K-1 line) were stored as frozen stock cultures, and

subcultures were made from these stocks. Each passage was tested for mycoplasma contamination and karyotype stability. The medi um for culturing the CHO cells was Ham's F-12 medium (with Glutamax-I) supplemented with heat-i nactivated fetal calf serum (10%), penicillin and streptomycin. The S-9 mix consisted of a liver homogenate fraction and cofactors, derived

from Arocolor 1254-induced Wistar rats. The fina l concentration of S-9 mix in culture medium used in the tests requiring activation was 4%. Dimethyl sulfoxide (DMSO) was chosen for the vehicle. Based on the solubility pre-test, 200 μg /ml final concentration in the culture medium was chosen as the highest dos e level for the first test.

2Draft HPV1 Test Rule – Data Adequacy – Chromosomal Aberrations CAS No. 118-82-1

Exponentially growing cells were seeded in st erile tissue culture flasks containing culture medium, and incubated at 37 °C in humidified air containing 5% CO 2 . Approximately 24 hours after seeding, the cells were exposed to the test substance or control substance, with or without S-9 mix. Duplicate cultures were used. In th e presence of S-9, the cult ure medium with fetal calf serum was replaced by culture medium with penicillin and streptom ycin, but without calf

serum. Two tests were conducted. In the first te st (pulse exposure), the cells were exposed to

the test substance for 4 hours at the following concentrations, either with or without activation: 0 (vehicle control), 0.1, 0.2, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, and 200 μg/mL. At the end of the exposure, the culture s were checked, the medium with test substance was removed, and the cells were washed twice and supplied with fresh culture medium. The cells were

incubated for another 14 hours at 37 °C in humidified air with 5% CO 2 . Two hours before the end of the culture period, the ce lls and culture media were checked visually and selected cultures were arrested in metaphase by the addition of co lcemid. The cells were harvested at 18 hours by trypsinization. The following concentrations were selected for cell sampling: 0, 6.25, 12.5, 25, 50, 100, and 200 μg/mL.

In the second test, the 4-hour pulse exposure wi th activation was repeated at the following concentrations: 0, 5, 10, 30, 50, 75, 100, 150, 200, and 300 μg/mL. The following

concentrations were selected for cell sampling: 0, 50, 75, 100, 150, 200, and 300 μg/mL. In the absence of activation, the cel ls were exposed continuously for 18 hours and harvested immediately after the exposure pe riod. The concentrations used for continuous exposure were: 0, 1.25, 2.5, 5, 10, 30, 50, 75, 100, 150, 200, 300 μg/mL. The concentrations selected for cell sampling were: 0, 1.25, 2.5, 5, 10, and 30 μg/mL.

The positive control substances were: cycl ophosphamide (5 μg/mL) with activation and mitomycin C (0.1 μg/mL) without activation.

Two slides were prepared from each culture that had been selected for cell sampling. For each culture, at least 1000 nuclei were examined (500 per slide) to determine the mitotic index (percentage of cells in mitosis). On the basis of the mitotic index obser ved, three concentrations

of the test substance were selected for the scor ing of chromosomal aberra tions. If possible, the highest concentration chosen redu ced the mitotic index by at leas t 50% (but not more than 70%) when compared to the negative control, or exhibi ted some other clear indi cation of cytotoxicity. For each chosen concentration, 200 well-spread metaphases (100 metaphases/culture, each containing 20-22 centromeres) were examined for chromatid-type aberrations (gaps, breaks, fragments, and interchanges) and chromosome-t ype aberrations (gaps, breaks, minutes, rings, and dicentrics). Heavily damaged cells were not included in the 200 analyzed cells.

Evaluation of test results The criteria for a clastogenic res ponse are: a concentratio n-related increase in the percentage of

cells with chromosomal aberrations over the concu rrent control frequencie s or a single positive test point in both tests at approximately the same dose level. Cells with only gaps (achromatic lesions) were not included in the final assessment of clastogenic activity. If the increase in cells with chromosomal aberrations is marginally si gnificant (0.05 < p < 0.1), then the response is considered equivocal.

3Draft HPV1 Test Rule – Data Adequacy – Chromosomal Aberrations CAS No. 118-82-1

RESULTS

Genotoxicity result: Negative

Cytotoxic concentration (if applicable): ≥ 100 μg/mL in pulse expos ures with or without activation; ≥ 10 μg/mL in continuous e xposure without activation

Results remarks: In the first test, in the pulse treatment group with metabolic activ ation, the three concentrations

of test material chosen for chromosomal abe rration analysis were 50, 100, and 200 μg/mL. The mitotic index of the highest c oncentration analyzed (200 μg/mL ) was reduced to 71% of the

concurrent vehicle control. The test substance did not induce a statistical ly significant increase in the number of aberrant cells at any of the dose levels analyzed when compared to the number

of aberrant cells observed in the concurrent control.

In the first test, in the pulse treatment group without act ivation, the three con centrations of test

material chosen for chromosomal aberration anal ysis were 50, 100, and 200 μg/mL. The mitotic

index of the lowest concentrati on analyzed (50 μg/mL) was not reduced; the mitotic indices of the middle concentration (100 μg/ml) and the hi ghest concentration (200 μg/mL) were reduced to 59% and 54% respectively of th at of the concurrent vehicle control. The test substance did not induce a statistically significant in crease in the number of aberrant cells at any of the dose levels analyzed when compared to the number of aber rant cells in the vehi cle control group.

In the second test, in the pulse treatment group with metabolic activatio n, dose-related toxicity

could be demonstrated. The te st concentrations chosen for chromosomal aberration analysis were 100, 200 and 300 μg/mL. The mitotic index of the lowest concen tration analyzed (100 μg/ml) was reduced to 80%; the mitotic index of the mid-concentration analyzed (200 μg/mL)

was reduced to 49% and the mitotic index of the highest concentr ation analyzed was reduced to

40% of that of the concurrent ve hicle control. The test substa nce did not induce a statistically significant increase in the number of aberrant ce lls at any of the dose levels analyzed, when compared to the aberrant cells found in the concurrent control cultures.

In the second test, in the continuous treatmen t group of 18 hours without metabolic activation, dose-related toxicity could be demonstrated. Dose levels chosen for chromosomal aberration analysis were 2.5, 5 and 10 μg/mL. The mitotic i ndex of the lowest conc entration analyzed (2.5 μg/ml) was 91%, the mitotic index of the mid-con centration analyzed (5 μg/mL) was reduced to 87%, and at the highest concentration analyzed (10 μg/mL) the mitotic index was reduced to

47% of that of the concurrent ve hicle control. At th e next higher concentration selected for cell sampling (30 μg/ml), the mitotic index was redu ced to 11%. This strong reduction was above the acceptable toxicity level and thus this dos e was not selected for chromosomal aberration analysis. The test substance di d not induce a statistically signifi cant increase in the number of aberrant cells at any of the dose levels analyzed, when compared to the number of aberrant cells

found in the concurrent control cultures.

Precipitation of the test substan ce was observed at 100 and 200 μg/m L in the first test and at 300 μg/mL in the second test.

4Draft HPV1 Test Rule – Data Adequacy – Chromosomal Aberrations CAS No. 118-82-1

Criteria for validity of the study were met by the positive controls (mitomycin C without S-9, and cyclophosphamide with S-9) giving the expected statistically increased numbers of aberrant cells and the negative cont rol results falling within the historical range.

CONCLUSIONS

Under the conditions of the st udy, the test substance was not clastogenic to CHO cells.

STUDY RELIABILITY

[1] = Reliable without restriction.

REFERENCE

de Vogel, N. and Usta, B. 2007. Chromosomal Aberration test with AN-2 in Cultured Chinese Hamster Ovary (CHO) Cells. TNO Laborator ies, Zeist, Netherlands, Project No. 031.11113/01.42, Study No. 6802/24, May 9, 2007.

5Review of TSCA Section 4 Final Study Report

CITB Project Manager: Catherine Roman

Chemical Name: Phenol, 4,4’-methylenebis[2,6- bis(1,1-dimethylethyl)-

CAS Registry Number: 118-82-1

Report Title: Chromosomal Aberration test with AN -2 in Cultured Chinese Hamster Ovary (CHO) Cells

Test Sponsor: Albemarle Corporation, Baton Rouge, LA

Testing Lab: TNO Laboratories, Zeist, Netherlands

Test Standard: 40 CFR 799.9537

Date Study Received by EPA: July 2007 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.

[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) inspect ions, their dates, and findings were missing or not signed. There is evidence that QA pr ocedures 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: Teresa Manyin/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).