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Data Evaluation Record - In vitro mammalian cytogenetics (chromosomal aberration) assay in Chinese hamster ovary CHO-K1 cells MRID 44438708

DATA EVALUATION REPORT
p-MENTHANE-3,8-DIO

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  1. Materials and Methods
    1. Materials
    2. Test Performance
  2. Reported Results
    1. Preliminary Cytotoxicty Assay
    2. Mutagenicity Assay
  3. Reviewer's Discussion Conclusions
    1. This is an acceptable study.

STUDY TYPE:

In vitro mammalian cytogenetics (chromosomal aberration) assay in Chinese hamster ovary CHO-K1 cells; OPPTS 870.5375 [§84-2]

Prepared for

Office of Pesticide Programs (7511C)
Environmental Protection Agency
1200 Pennsylvania Avenue, NW
Washington, D.C. 20460

Prepared by

Chemical Hazard Evaluation Group
Toxicology and Risk Analysis Section
Life Sciences Division
Oak Ridge National Laboratory
Oak Ridge, TN 37831
Task Order No. 22

Primary Reviewer:

Bradford L. Whitfield, Ph.D.

Robert H. Ross, M.S., Group Leader

Secondary Reviewers:

Cheryl B. Bast, Ph.D., D.B.A.T.

Quality Assurance:

Susan Chang, M.S.

Disclaimer

This Data Evaluation Report may have been altered by the Biopesticides and Pollution Prevention Division subsequent to signing by Oak Ridge National Laboratory personnel.

Oak Ridge National Laboratory, managed by Lockheed Martin Energy Research Corp. for the U.S. Department of Energy under contract number DE-AC05-96OR22464.

EPA Work Assignment Manager:

Sheryl Reilly
Biopesticides & Pollution Prevention Division

DATA EVALUATION RECORD

STUDY TYPE: In vitro mammalian cytogenetics (chromosomal aberration) assay in Chinese hamster ovary CHO-K1 cells; OPPTS 870.5375 [§84-2]

DP BARCODE: D243976 SUBMISSION CODE: S538748 CASE: 061954 TOX. CHEM. NO.: 011550

TEST MATERIAL (PURITY): Granola 97 (p-methane-3,8-diol)(98.3% a.i.)

SYNONYMS: p-Menthane-3,8-diol; SCJ NB # 14735R108

CITATION: Gudi, R. and E.H. Schadly (1997) In vitro mammalian cytogenetic test using Chinese hamster ovary (CHO) cells with Granola 97 (SCJ NB # 14735R108). MA BioServices, Inc., 9630 Medical Center Drive, Rockville, MD 20850. Laboratory study number G97BF49.335. November 20, 1997. MRID 44438708. Unpublished

SPONSOR: S.C. Johnson & Son, Inc., 1525 Howe Street, Racine, WI 53403-2236

EXECUTIVE SUMMARY:

In a mammalian cell chromosomal aberration cytogenetics assay (MRID 44438708), Chinese hamster ovary CHO-K1 cell cultures were exposed to Granola 97 (98.3% a.i., batch No. 703001) in DMSO in two independent assays. In the initial assay, concentrations of 50, 150, 500 and 1500 g/mL, with and without metabolic activation (S9-mix), were evaluated following a 6 hour treatment and a 14 hour recovery period. In the repeat assay without S9-mix, concentrations of 250, 500, 1000 and 1500 g/mL were evaluated after 20 hours continuous treatment and concentrations of 125, 250, 500 and 1000 g/mL were evaluated after 44 hours continuous treatment. In the repeat assay with S9-mix, concentrations of 250, 500, 1000 and 1500 g/mL were evaluated after 6 hours treatment and either a 14 hour or 38 hour recovery period. The S9-fraction was obtained from Aroclor 1254 induced male Sprague-Dawley rat liver.

Granola 97 was tested up to cytotoxic concentrations. In the initial assay without S9-mix, cell growth was essentially uneffected at Granola 97 concentrations up to and including 500 g/mL, growth was reduced to 72% compared to the solvent control at 1500 g/mL and reduced to 0% at 5000 g/mL. In the presence of S9-mix, cell growth was 55% of the solvent control at 1500 g/mL and 0% at 5000 g/mL. In the repeat assay after 20 hours exposure to Granola 97 without S9-mix, cell growth remained above 50% of the solvent control value at all concentrations except 3000 g/mL where growth was 4% of the solvent control. After 44 hours exposure, cell growth remained above 50% of the solvent control value at concentrations up to and including 500 g/mL and was reduced to 42%, 18% and 0% at 1000, 1500 and 3000 g/mL, respectively. In the presence of S9-mix (6 hour exposure), cell growth remained above 50% of the solvent control value at all concentrations except 3000 g/mL where growth was 3% of the solvent control. The test material precipitated in treatment medium at 5000 g/mL with S9-mix but not in the absence of S9-mix or at any other concentration. In the initial assay without S9-mix, no statistically significant increase (p# 0.05) over the solvent control was seen in the percentage of cells with structural or numerical aberrations at any tested concentrations of Granola 97. In the presence of S9-mix, there was no significant increase in the percentage of cells with numerical aberrations at any concentration of Granola 97 tested; however, there was a statistically significant increase in the percentage of cells with structural aberrations relative to the solvent control at 150 and 1500 g/mL. The percentages of aberrant cells found at these two concentrations (3.5% each) were within the historical solvent control range (0 - 6%) and were not statistically increased compared to untreated controls. Also the Cochran-Armitage trend test was negative for a dose response (p$0.05). Therefore, the percentage increase in cells with structural aberrations is unlikely to be biologically significant. Positive and solvent control values were appropriate in the initial assay. In the repeat assay without S9-mix, there was no statistically significant increase in structural or numerical aberrations at any test material concentration at the 20 hour harvest time. At the 44 hour harvest time, a significant increase was seen in the percentage of cells with numerical aberrations at 1000 g/mL (6.5% vs 2.5% for the solvent control) and in the percentage of cells with structural aberrations at 125 g/mL (10.5% vs 4.0% for the solvent control). No statistically significant increase in structrual aberrations was seen at 250, 500 or 1000 g/mL although a dose-related increase in cytotoxicity was seen. A positive response at the lowest dose tested with no supporting evidence at higher concentrations makes it unlikely that the increase in percentage of cells with structural aberrations at 125 g/mL is biologically relevant. Likewise, the statistically significant increase in numerical aberrations at 1000 g/mL was not considered biologically significant because the percentage of aberrant cells (6.5%) was only 1% above the historical solvent control range of 0 to 5.5% and within the historical untreated control range of 0 to 6.5%. Positive and solvent control values were appropriate. There was no evidence of chromosomal aberrations induced over background.

This study is classified as acceptable (guideline). It satisfies the requirement for FIFRA Test Guideline 84-2 for in vitro cytogenetic mutagenicity data.

COMPLIANCE: Signed and dated GLP, Quality Assurance and Data Confidentiality statements were provided.

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  1. Materials and Methods
    1. Materials
      1. Test material: Granola 97
      2. Description: thick, clear, colorless liquid
        Lot/Batch #: 703001
        Purity: 98.3% a.i.
        Stability of compound: responsibility of sponsor
        CAS #: 42822-86-6
        Structure: not provided
        Solvent used: DMSO
        Other comments: store at 37"2°C, protected from light
      3. Control materials
      4. Negative: untreated
        Solvent/final concentration: DMSO /
        Positive: (concentrations/solvent)

        Nonactivation: mitomycin C / 0.08 and 0.15 μg/mL / H2O
        Activation: cyclophosphamide / 5 and 10 μg/mL / H2O

      5. Activation: S9 derived from male Sprague-Dawley rats
      6. Chemical Induction Test Animal Organ
        x Aroclor 1254 x induced x rat x liver
        phenobarbital non-induced mouse  lung
        none   hamster other
        other   other  

        Note: "x" means yes

        9 mix composition:

        2 mM MgCl2, 6 mM KCl, 1 mM glucose-6-phosphate, 1 mM NADP and 20 μL S9 fraction per mL of medium (McCoy's 5A serum-free medium supplemented with 100 units penicillin and 100 μg streptomycin/mL and 2 mM L-glutamine)

      7. Test compound concentrations used:
      8. Initial assay: (6 hour treatment, 14 hour recovery)

        Toxicity test:

        Nonactivated conditions: 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/mL

        Activated conditions: 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/mL

        Cytogenetic test:

        Nonactivated conditions: 50, 150, 500, 1500 g/mL

        Activated conditions: 50, 150, 500, 1500 g/mL

        Repeat assay:

        Toxicity test:

        Nonactivated conditions: 62.5, 125, 250, 500, 1000, 1500, 3000 μg/mL (20 and 44 hour continuous treatment)

        Activated conditions: 62.5, 125, 250, 500, 1000, 1500, 3000 μg/mL (6 hour treatment, 14 hour recovery and 6 hour treatment, 38 hour recovery)

        Cytogenetic test:

        Nonactivated conditions: 250, 500, 1000, 1500 μg/mL (20 hour continuous treatment); 125, 250, 500, 1000 μg/mL (44 hour continuous treatment)

        Activated conditions: 250, 500, 1000, 1500 μg/mL (6 hour treatment, 14 hour recovery and 6 hour treatment, 38 hour recovery)

      9. Test cells: mammalian cells in culture - Chinese hamster ovary CHO-K1 cells
      10. Properly maintained? Y
        Cell line or strain periodically checked for Mycoplasma contamination? Y
        Cell line or strain periodically checked for karyotype stability? Y (not specifically checked but not used beyond passage 20 to assure karyotypic stability)

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    2. Test Performance
      1. Preliminary cytotoxicity assay
      2. A cytotoxicity evaluation was included as part of the initial and repeat cytogenetic assays with cytotoxicity determinations based upon cell growth inhibition relative to the solvent control.

      3. Cytogenetic assay


        1. Cell treatment:
        2. Cells exposed to test compound, solvent, or positive control for 6, 20 or 44 hours (nonactivated), 6 hours (activated)

        3. Spindle inhibition
        4. Inhibition used/concentration: Colcemid7 / 0.1 g/mL

          Administration time: 2 hours (before cell harvest)

        5. Cell harvest
        6. Cells exposed to test material, solvent or positive control were harvested 0 or 14 hours after termination of treatment (nonactivated), 14 or 38 hours after termination of treatment (activated)

        7. Details of slide preparation
        8. Cells were harvested by trypsinization and collected by centrifugation at approximately 800 rpm for five minutes. The cell pellet was resuspended in two to four mL of 0.075 M KCl and allowed to stand at room temperature for about four to eight minutes. The cells were collected again by centrifugation, the supernatant aspirated and the cells fixed with two washes of approximately two mL fixative (methanol:glacial acetic acid, 3:1, v/v).

          The fixed cells were centrifuged at 800 rpm for five minutes, the supernatant decanted and the cells resuspended to opalescence in fresh fixative. A portion of this suspension was dropped onto the center of a glass slide and allowed to air dry. The slides were then stained with 5% Giemsa, air dried, permanently mounted and coded.

        9. Metaphase analysis
        10. No. of cells examined per dose: 200
          Scored for structural: Y
          Scored for numerical: Y (polyploid and endoreduplicated cells)
          Coded prior to analysis: Y

        11. Evaluation criteria
        12. Whenever possible, 200 metaphase spreads (20"2 centromeres), 100 per duplicate flask, were scored per treatment group. The chromatid aberrations scored were chromatid and isochromatid breaks, exchange figures such as quadriradials and triradials and complex rearrangements. The chromosome aberrations scored were breaks and exchange figures such as dicentrics and rings. Fragments observed in the absence of any exchange figure were scored as breaks while those with an exchange figure were not scored as an aberration but were considered part of the incomplete exchange. Pulverized cells, pulverized chromosomes and cells with ±10 aberrations were recorded. Chromatid and isochromatid gaps were recorded but not included in the analysis. The percentage of polyploid and endoreduplicated cells was recorded per 100 metaphase cells and the mitotic index recorded as the percentage of cells in mitosis per 500 cells counted.

        13. Statistical analysis
        14. Data were evaluated for statistical significance at p #0.05 , using Fisher's exact test

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  2. Reported Results
    1. Preliminary Cytotoxicty Assay
    2. No preliminary cytotoxicity assay was performed; however, cytotoxicity determinations were part of the initial and repeat cytogenetic assays.

    3. Mutagenicity Assay
    4. Initial: Cells were exposed to Granola 97 concentrations ranging from 1.5 to 5000 μg/mL, with and without S9-mix. The test material precipitated in treatment medium at 5000 μg/mL with S9-mix but not in the absence of S9-mix or at any other concentration. In the absence of S9-mix, cell growth was essentially uneffected at Granola 97 concentrations up to and including 500 μg/mL, growth was reduced to 72% compared to the solvent control at 1500 μg/mL and reduced to 0% at 5000 μg/mL. In the presence of S9-mix, cell growth was 55% of the solvent control at 1500 μg/mL and 0% at 5000 μg/mL. Results of the concurrent cytotoxicity test are given in Appendix Table 1 (MRID 44438708, p.19). Four concentrations of Granola 97 were evaluated for cytogenetic effects: 50, 150, 500 and 1500 μg/mL with and without S9-mix. The mitotic index at 1500 μg/mL was not reduced relative to the solvent control in the absence of S9-mix but was reduced by 18% in the presence of S9-mix.

      In the absence of S9-mix, no statistically significant increase (statistical significance defined as p# 0.05 unless otherwise specified) over the solvent control was seen in the percentage of cells with structural or numerical aberrations at any tested concentrations of Granola 97. Positive and solvent control values were appropriate. In the presence of S9-mix, there was no significant increase in the percentage of cells with numerical aberrations at any concentration of Granola 97 tested; however, there was a statistically significant increase in the percentage of cells with structural aberrations relative to the solvent control at 150 and 1500 g/mL. The percentages of aberrant cells found at these two concentrations (3.5% each) were within the historical solvent control range (0 - 6%) and were not statistically increased compared to untreated controls. Historical control values are given as an attachment to the Appendix (from MRID 44438708, pp. 34 and 35). Also the Cochran-Armitage trend test was negative for a dose response (p±0.05). Therefore, the percentage increase in cells with structural aberrations is unlikely to be biologically significant. The solvent and positive control values were appropriate. Results of the initial cytogenetics assay are summarized in Appendix Table 2 (MRID 44438708, p. 23).

      Repeat: Seven concentrations of Granola 97 ranging from 62.5 to 3000 g/mL were tested with and without S9-mix in the repeat cytogenetic assay. Exposure times were 20 and 44 hours without S9-mix and 6 hours with S9-mix. In the absence of S9-mix, after 20 hours exposure to Granola 97 cell growth remained above 50% of the solvent control value at all concentrations except 3000 g/mL where growth was 4% of the solvent control. After 44 hours exposure, cell growth remained above 50% of the solvent control value at concentrations up to and including 500 g/mL and was reduced to 42%, 18% and 0% at 1000, 1500 and 3000 g/mL, respectively. In the presence of S9-mix (6 hour exposure), cell growth remained above 50% of the solvent control value at all concentrations except 3000 g/mL where growth was 3% of the solvent control. Results of the concurrent cytotoxicity test are given in Appendix Table 3 (MRID 44438708, p. 24).

      In the absence of S9-mix, four Granola 97 concentrations ranging from 250 to 1500 g/mL were evaluated for cytogenetic effects at the 20 hour harvest time and four concentrations ranging from 125 to 1000 g/mL were evaluated at the 44 hour harvest time. No dose-related effect on the mitotic index was seen at the 20 hour harvest time; however, at 44 hours, the mitotic index was reduced to 50% of the concurrent solvent control at 1000 g/mL. There was no statistically significant increase in structural or numerical aberrations at any test material concentration at the 20 hour harvest time. At the 44 hour harvest time, a significant increase was seen in the percentage of cells with numerical aberrations at 1000 g/mL (6.5% vs 2.5% for the solvent control) and in the percentage of cells with structural aberrations at 125 g/mL (10.5% vs 4.0% for the solvent control). Because of the apparent positive effect at 125 g/mL, extra slides from this dose level were evaluated and a statistical analysis, performed on the pooled data from all slides at this dose, showed significance at p#0.01. No statistically significant increase in structrual aberrations was seen at 250, 500 or 1000 g/mL although a dose-related increase in cytotoxicity was seen. A positive response at the lowest dose tested with no supporting evidence at higher concentrations led the authors to conclude that the increase at 125 g/mL was not scientifically relevant. Likewise, the statistically significant increase in numerical aberrations at 1000 g/mL was not considered biological significant because the percentage of aberrant cells (6.5%) was only 1% above the historical solvent control range of 0 to 5.5% and within the historical untreated control range of 0 to 6.5%. Positive and solvent control values were appropriate. Results from the 20 hour harvest time are summarized in the top half of Appendix Table 4 (MRID 44438708, p.32).

      In the presence of S9-mix, the mitotic index varied somewhat compared to the solvent control at both the 20 and 44 hours harvest times but not in a dose-related manner. No statistically significant increase in the percentage of cells with numerical or structural aberrations was seen at any tested dose at the 20 hour harvest time. At the 44 hour harvest time, no significant increase in percentage of cells with numerical aberrations was seen at any tested dose; however, a statistically significant, dose-related increase in the percentage of cells with structural aberrations (2.5 to 3.5%) was seen. The percentage of aberrant cells at all doses was within the historical solvent control range of 0 to 6.0% and, therefore, not considered biologically significant by the authors. Positive and solvent control values were appropriate. Results from the 44 hour harvest time are summarized in the bottom half of Appendix Table 4 (MRID 44438708, p.32).

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  3. Reviewer's Discussion Conclusions
    1. This is an acceptable study.
    2. Granola 97 was tested to cytotoxic concentrations, acceptable experimental protocol was followed and the positive and solvent control values were appropriate. As discussed in section II.B., a statistically significant increase in the percentage of cells with structural aberrations compared with the solvent control was seen at a number of experimental points in this study. Although statistically significant, the results at 150 and 1500 g/mL with S9-mix in the initial assay and at 250, 500, 1000 and 1500 g/mL with S9-mix in the repeat assay (44 hour harvest) were not considered biologically significant because the values were within the historical solvent control or untreated control ranges. The increase in percentage of cells with structural aberrations after a 44 hour exposure to 125 g/mL without S9-mix in the repeat assay was outside the historical solvent or untreated control ranges but was considered not relevant because the increase occurred only at this dose (the lowest dose tested) and not at three higher doses. One incidence of a statistically significant increase in percentage of cells with numerical aberrations occurred. This increase, seen after a 44 hour exposure to 1000 g/mL Granola 97 without S9-mix in the repeat assay, was not considered biologically significant because the increase was only 1% above the historical solvent control range and within the untreated control range. The reviewers concur with the authors' conclusions that none of increases represent a test material induced increase in percentage of cells with structural or numerical aberrations.

    3. Study Deficiencies
    4. No study deficiencies were identified.

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