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Data Evaluation Record - Mammalian Cells in Culture Gene Mutation Assay in Mouse Lymphoma L5178Y cells - MRID 44438706

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

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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

STUDY TYPE: Mammalian Cells in Culture Gene Mutation Assay in Mouse Lymphoma L5178Y cells; OPPTS 870.5300 [§84-2]

Prepared for

Biopesticides and Pollution Prevention Division
Office of Pesticide Programs
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.
Signature:
Date:

Secondary Reviewers:

Cheryl B. Bast, Ph.D., D.B.A.T.
Signature:
Date:

Robert H. Ross, M.S., Group Leader
Signature:
Date:

Quality Assurance:

Susan Chang, M.S.
Signature:
Date:

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
Date:
Biopesticides & Pollution Prevention Division

DATA EVALUATION RECORD

STUDY TYPE: Mammalian cells in culture gene mutation assay in mouse lymphoma L5178Y cells; OPPTS 870.5300 [§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:

San, R.H. and J.J. Clarke (1997) In vitro mammalian cell gene mutation test with Granola 97 (SCJ NB # 14735R108). MA BioServices, Inc., 9630 Medical Center Drive, Rockville, MD 20850. Laboratory study number G97BF49.702. November 18, 1997. MRID 44438706. Unpublished

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

EXECUTIVE SUMMARY:

In a mammalian cell gene mutation assay at the thymidine kinase locus (MRID 44438706), L5178Y/TK" cells cultured in vitro were exposed to Granola 97 (98.3% a.i., batch No. 703001) in DMSO at concentrations of 600, 800, 1000, 1250, 1500 and 2000 µg/mL in the absence of mammalian metabolic activation (S9-mix) and to concentrations of 500, 600, 800, 1000, 1250 and 1500 µg/mL in the presence of S9-mix. The S9-fraction was obtained from Aroclor 1254 induced male Sprague-Dawley rat liver.

Granola 97 was tested up to cytotoxic concentrations. In the preliminary cytotoxicity assay, little or no cell growth occurred at 1500 or 5000 µg/mL, with or without S9-mix. Cell growth was unaffected at 500 µg/mL and lower concentrations in the presence of S9-mix or at concentrations of 150 µg/mL and lower in the absence of S9-mix. Cell growth was 70% of the solvent control at 500 µg/mL without S9-mix. In the main mutagenicity assay, the 2000 µg/mL and 1500 µg/mL doses were too toxic to clone in the absence and presence of S9-mix, respectively. Cultures were cloned for five Granola 97 concentrations ranging from 600 to 1500 µg/mL without S9-mix and five concentrations ranging from 500 to 1250 µg/mL with S9-mix. All plating was in triplicate with duplicate cultures at each concentration. No visible precipitate was seen in the treatment medium at any dose level. There was no evidence of a mutagenic effect at any concentration of Granola 97 tested, with or without S9-mix. One of the duplicate cultures at 500 µg/mL with S9-mix did have a mutant frequency of 56 per 106 clonable cells over the solvent control value (a mutant frequency of 55 to 99 mutants per 106 clonable cells was the criterium for an equivocal response). The other duplicate culture at this concentration had a mutant frequency of 8 mutants per 106 clonable cells over the solvent control. The solvent and positive controls induced the appropriate responses, within the laboratory=s historical control ranges. There was no evidence of induced mutant colonies over background.

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

COMPLIANCE:

Signed and dated GLP, Quality Assurance and Data Confidentiality statements were provided. A Flagging statement was not 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

      2. Control materials
        3.

        Solvent/final concentration: DMSO / 100 µL

        Positive:

        Nonactivation (concentrations/solvent): methyl methanesulfonate / 10 and 20 Fg /mL / treatment medium

        Activation (concentrations/solvent): 7,12-dimethyl-benz(a)anthracene / 2.5 and 4.0 µg/mL / treatment medium

      3. Activation: S9 derived from male Sprague-Dawley rats
        4. Chemical Induction Test Animal Organ
        x Aroclor 1254 x induced x rat x liver
        phenobarbital   hamster other
        other     other

        Note: "x" means yes

        S9 mix composition:

        11.25 mg DL-isocitric acid, 6 mg NADP and 0.25 mL S9 homogenate per mL in F0P (Fischer=s Medium for Leukemic Cells of Mice with 0.1% Pluronics). The cofactor mixture was adjusted to pH 7.0 prior to the addition of S9 homogenate.

      4. Test cells: mammalian cells in culture


        • x mouse lymphoma L5178Y cells


        • Chinese hamster ovary (CHO) cells


        • __V79 cells (Chinese hamster lung fibroblasts)


        • __other (list):


        • Properly maintained? Y


        • Periodically checked for Mycoplasma contamination? Y


        • Periodically checked for karyotype stability? Y


        • Periodically "cleansed" against high spontaneous background? Y

        Media: Treatment medium was Fischer's Medium for Leukemic Cells of Mice with 0.1% Pluronics (F0P). Expression medium was F0P supplemented with 10% horse serum and 2 mM L-glutamine (F10P). Selection medium was "cloning medium" (undefined) containing 0.23% granulated agar and 3 µg/mL trifluorothymidine.

      5. Locus examined


        • x thymidine kinase (TK)


        • Selection agent: 3 µg/mL trifluorothymidine (TFT)


      6. Test compound concentrations used:

        7. Preliminary Cytotoxicity assay:

        Nonactivated and activated conditions: 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 Fg/mL

        Mutagenicity assay:

        Nonactivated conditions: 600, 800, 1000, 1250, 1500, 2000 µg/mL

        Activated conditions: 500, 600, 800, 1000, 1250, 1500 µg/mL

    2. Test Performance



      1. Cell treatment:
        2.

        1. Cells exposed to test compound, negative/solvent or positive controls for:

          2. 4 hours (nonactivated) 4 hours (activated)

        2. After washing, cells cultured for 2 days (expression period) before cell selection:


        3. After expression, 1 x 106 cells/dish ( 3 dishes/ group) were cultured for 10-14 days in selection medium to determine numbers of mutants and 200 cells/dish ( 3 dishes/group) were cultured for 10-14 days without selective agent to determine cloning efficiency.


      2. Statistical methods: none used


      3. Evaluation criteria: Criteria for a valid test were:


        1. The mutants frequency of the solvent control must be within 20 to 100 TFT-resistant mutants per 106 surviving cells with a cloning efficiency greater than 50%.


        2. At least one concentration of each positive control must exhibit mutant frequencies of ≥100 mutants per 106 clonable cells over the background level. The colony size distribution for the MMS positive control must show an increase in both small and large colonies.


        3. At least four test material concentrations with analyzable mutant frequency data are required.

        The following criteria were considered when interpreting the data:

        1. The results were considered positive if a dose related increase in mutant frequency was seen and one or more dose levels (with 10% or greater total growth) exhibited mutant frequencies of ≥100 mutants per 106 clonable cells over the background level.


        2. The results were considered equivocal if the mutant frequency in treated cultures was between 55 and 99 mutants per 106 clonable cells over background level.


        3. Results were considered negative if fewer than 55 mutants per 106 clonable cells over the background level were seen in test material treated cultures.

          4. Mutations occurring only at highly toxic concentration of test material (less than 10% total growth) were not considered biologically relevant. Colony size was determined for positive and solvent controls and in test material treated cultures showing a positive response.

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  2. Reported Results

    1. Preliminary Cytotoxicty Assay

      2. Nine concentrations of Granola 97 ranging from 0.5 to 5000 µg/mL were tested with and without S9-mix in the preliminary cytotoxicity assay. In the absence of S9-mix, Granola 97 did not reduce cell growth relative to the solvent control at concentrations up to and including 150 µg/mL. The relative grow was 70%, 5% and 0% at 500, 1500 and 5000 Fg/mL, respectively. In the presence of S9-mix, Granola 97 did not reduce the relative growth at concentrations up to and including 500 µg/mL but completely eliminated growth at 1500 and 5000 µg/mL. Results of the preliminary cytotoxicity assay are presented in Appendix Table 1 (MRID 44438706, p.15).

    2. Mutagenicity Assay

      3. All plating was in triplicate with duplicate cultures at each concentration. The authors stated that concentrations of Granola 97 ranging from 100 to 2000 μg/mL were used for the mutagenesis assay; however, cultures were cloned only for five Granola 97 concentrations ranging from 600 to 1500 μg/mL without S9-mix and five concentrations ranging from 500 to 1250 μg/mL with S9-mix. In the absence of S9-mix, the 5000 μg/mL concentration was too toxic to clone while in the presence of S9-mix, 1500 μg/mL was too toxic to clone. No visible precipitate was seen in the treatment medium at any dose level. There was no evidence of a mutagenic effect at any concentration of Granola 97 tested, with or without S9-mix. One of the duplicate cultures at 500 μg/mL with S9-mix did have a mutant frequency of 56 per 106 clonable cell over the solvent control value (a mutant frequency of 55 to 99 mutants per 106 clonable cells was the criterium for an equivocal response). The other duplicate culture at this concentration had a mutant frequency of 8 mutants per 106 clonable cells over the solvent control. The two positive controls and the solvent control gave appropriate responses within the testing laboratory's historical ranges. Results of the mutagenesis assay are presented in Appendix Tables 2-5 (MRID 44438706, pp. 16-19).

  3. Reviewer's Discussion Conclusions

    1. This is an acceptable study.

      2. Granola 97 was tested to cytotoxic concentrations, the experimental protocol was acceptable and the solvent and positive control values were appropriate. There was no evidence of a mutagenic effect at any tested concentration, with or without S9-mix.

    2. Study Deficiencies

      3. No study deficiencies were identified.

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