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 Work Assignment Manager 

March 5, 2010 
HPV1 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 22202 
Draft HPV1 Test Rule – Data Adequacy – Chromosomal Aberrations CAS No. 118-82-1

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 – Chromosomal Aberrations CAS No. 118-82-1

GENOTOXICITY – CHROMOSOMAL 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-tert-butylphenol) (AN-2; Phenol, 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; second 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 between 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 medium for culturing the CHO cells was Ham's F-12 

medium (with Glutamax-I) supplemented with heat-inactivated 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 final 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 dose level for the first test. 

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

Exponentially growing cells were seeded in sterile tissue culture flasks containing culture 

medium, and incubated at 37 °C in humidified air containing 5% CO2. 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 the presence of S-9, the culture medium with fetal 

calf serum was replaced by culture medium with penicillin and streptomycin, but without calf 

serum. Two tests were conducted. In the first test (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 cultures 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% CO2. Two hours before the 

end of the culture period, the cells and culture media were checked visually and selected cultures 

were arrested in metaphase by the addition of colcemid. 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 with 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 cells were exposed continuously for 18 hours and harvested 

immediately after the exposure period. 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: cyclophosphamide (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 observed, three concentrations 

of the test substance were selected for the scoring of chromosomal aberrations. If possible, the 

highest concentration chosen reduced the mitotic index by at least 50% (but not more than 70%) 

when compared to the negative control, or exhibited some other clear indication 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-type 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 response are: a concentration-related increase in the percentage of 

cells with chromosomal aberrations over the concurrent control frequencies 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 significant (0.05 < p < 0.1), then the response is 

considered equivocal. 

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Draft 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 exposures with or without 

activation; ≥ 10 μg/mL in continuous exposure without activation 

Results remarks:

In the first test, in the pulse treatment group with metabolic activation, the three concentrations 

of test material chosen for chromosomal aberration analysis were 50, 100, and 200 μg/mL. The 

mitotic index of the highest concentration analyzed (200 μg/mL) was reduced to 71% of the 

concurrent vehicle control. The test substance did not induce a statistically 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 activation, the three concentrations of test 

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

index of the lowest concentration analyzed (50 μg/mL) was not reduced; the mitotic indices of 

the middle concentration (100 μg/ml) and the highest concentration (200 μg/mL) were reduced 

to 59% and 54% respectively of that of the concurrent vehicle control. The test substance did not 

induce a statistically significant increase in the number of aberrant cells at any of the dose levels 

analyzed when compared to the number of aberrant cells in the vehicle control group. 

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

could be demonstrated. The test concentrations chosen for chromosomal aberration analysis 

were 100, 200 and 300 μg/mL. The mitotic index of the lowest concentration 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 concentration analyzed was reduced to 

40% of that of the concurrent vehicle control. The test substance did not induce a statistically 

significant increase in the number of aberrant cells 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 treatment 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 index of the lowest concentration analyzed (2.5 

μg/ml) was 91%, the mitotic index of the mid-concentration 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 vehicle control. At the next higher concentration selected for cell 

sampling (30 μg/ml), the mitotic index was reduced to 11%. This strong reduction was above 

the acceptable toxicity level and thus this dose was not selected for chromosomal aberration 

analysis. The test substance did not induce a statistically significant 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 substance was observed at 100 and 200 μg/mL in the first test and at 300 

μg/mL in the second test. 

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Draft 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 control results falling within the historical range. 

CONCLUSIONS

Under the conditions of the study, 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 Laboratories, Zeist, Netherlands, Project No. 

031.11113/01.42, Study No. 6802/24, May 9, 2007. 

5 
Review 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) 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: 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 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).