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Data Evaluation Record - Salmonella/escherichia/ mammalian Activation Gene Mutation Assay - MRID 44487801

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 Cytotoxicity Assay
    2. Mutagenicity Assay
  3. Reviewer's Discussion / Conclusions
    1. Discussion
    2. Study Deficiencies

Study Type: Salmonella/escherichia/mammalian Activation Gene Mutation Assay; OPPTS 870.5265 ['84-2]

Prepared for

Biopesticides and Pollution Prevention Division
Office of Pesticide Programs
U.S. Environmental Protection Agency
Crystal Station I
2800 Jefferson Davis Highway
Arlington, VA 22202

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.

Secondary Reviewers:

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

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

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, Ph.D.
Biopesticides & Pollution Prevention Division

DATA EVALUATION RECORD

Study Type: Salmonella/Escherichia/mammalian activation gene mutation assay; OPPTS 870.5265 ['84-2]

DP Barcode: D243976 Submission Code: S538748 CASE: 061954 Tox. Chem. NO.: not provided

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

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

Citation: Wagner, V.O. and E.W. Walton (1997) Bacterial reverse mutation assay with Granola 97 (SCJ NB # 14735R108). MA BioServices, Inc., 9630 Medical Center Drive, Rockville, MD 20850. Laboratory study number G97BF49.502, November 18, 1997. MRID 44487801. Unpublished

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

Executive Summary:

In a reverse gene mutation assay in bacteria (MRID 44487801), strains TA98, TA100, TA1535 and TA1537 of S. typhimurium and strain WP2(uvrA) of E. coli were exposed to Granola 97 (Batch No. 703001, 98.3% a.i.) in DMSO at concentrations of 25 (WP2(uvrA) only), 75, 200, 600, 1800, and 5000 g/plate in the presence and absence of mammalian metabolic activation (S9-mix). The S9-fraction was obtained from Aroclor 1254 induced male Sprague-Dawley rat liver.

Granola 97 was tested up to a limit concentration of 5000 g/plate. In the preliminary cytotoxicity assay, no thinning of the background lawn was seen at any concentration up to and including 5000 g/plate in any of the five tester strains, with or without S9-mix. Some reduction in the number of revertants per plate was seen both with and without S9-mix at 1000 g/plate and higher concentrations in WP2(uvrA). The mean number of revertants per plate at 1000, 3333 and 5000 g/plate in this strain was reduced, compared to the solvent control value of 21 both with and without S9-mix, to 8, 5, and 3, respectively, with S9-mix and to 7, 8 and 4, respectively, without S9-mix. Granola 97 was not mutagenic to any of the five tester strains at any concentration tested, with or without S9-mix. Solvent and positive control values were appropriate for the respective strains. 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 (bacterial reverse gene mutation) 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:
      2. Granola 97

        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:

        Solvent/final concentration: DMSO/50 L/plate

        Positive:

        Nonactivation

        Sodium azide 1.0 g/plate TA100, TA1535
        2-Nitrofluorene 1.0 g/plate TA98
        9-Aminoacridine 75 g/plate TA1537
        Methyl methanesulfonate 1000 g/plate WP2(uvrA)

        Activation:

        2-Aminoanthracene 1.0 g/plate (TA strains)
        2-Aminoanthracene 10.0 g/plate WP29uvrA

      5. Activation: S9 derived from male Sprague-Dawley rats
      6. x Aroclor 1254    x induced     x rat     x liver

        phenobarbital non-induced mouse lung none other other

        S9 mix composition: in 100 mM phosphate buffer, pH 7.4

        5 mM glucose-6-phosphate

        4 mM NADP

        8 mM MgCl2

        33 mM KCl

        10% S9-fraction

      7. Test organisms: S. typhimurium strains
      8. x TA98 x TA100 TA102 TA104 x TA1535 x TA1537 TA1538

        E. coli strain WP2 (uvrA)

        Properly maintained? Y

        Checked for appropriate genetic markers (rfa mutation, R factor)? Y (The testing laboratory's criteria for a valid study require the bacteria to have the appropriate genetic markers. The authors stated that all criteria for a valid study were met. They did not state directly that the bacteria were checked for the appropriate genetic markers.)

      9. Test compound concentrations used
      10. Preliminary cytotoxicity test: (single plating)

        Nonactivated and activated conditions: 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333, 5000 μg/plate (all strains)

        Main mutagenicity test: (triplicate platings)

        Nonactivated and activated conditions: 25 μg/plate (WP2(uvrA) only); 75, 200, 600, 1800, 5000 μg/plate (all strains)

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    2. Test Performance
      1. Type of Salmonella assay
      2. x standard plate test

        pre-incubation ( minutes)

        "Prival" modification (i.e. azo-reduction method)

        spot test

        other [describe]

      3. Protocol
      4. For each plate, 500 L of S9-mix (or 500 L of 100 mM phosphate buffer), 100 L of an overnight culture of the desired bacteria tester strain and 50 L of solvent or test material were added to 2 mL of molten selective top agar at 45"2°C (media composition is given in the Appendix). The mixture was vortexed and poured onto the surface of 25 mL of minimal bottom agar. After the agar had solidified, the plates were inverted and incubated for 48 to 72 hours at 37"2°C. All platings were in triplicate. Revertant colonies on the plates were either counted immediately after incubation or the plates were stored at 4"2°C until they could be counted. Revertant colonies were usually counted by an automated colony counter; however, manual counting was used in the preliminary cytotoxicity assay, when toxicity was apparent or if a test article precipitate interfered with automatic counting. All plates for a given tester strain and activation condition were counted by the same method.

        To be considered positive, the test material must cause a dose-related increase in the mean number of revertants per plate over at least two increasing concentrations of test material in at least one tester strain. The increase in mean number of revertants must be at lease three times the solvent control value for strains TA1535 and TA1537 and at least two times the solvent control value for strains TA98, TA100 and WP2(uvrA).

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  2. Reported Results
    1. Preliminary Cytotoxicity Assay
    2. Ten concentrations of Granola 97 ranging from 6.7 to 5000 g/plate were tested, with and without S9-mix, in all five tester strains in a preliminary cytotoxicity assay. One plate per test point was used. A reduction in the number of revertants per plate and/or a thinning or absence of the background lawn of bacteria was the measure of cytotoxicity. The background lawn of all five tester strains was normal at all concentrations of Granola 97 studied, with or without S9-mix. Some reduced revertant counts, compared to the solvent controls, were seen but no systematic pattern was evident except for WP2(uvrA). The mean number of revertants per plate at 1000, 3333 and 5000 g/plate in this strain was reduced, compared to the solvent control value of 21 both with and without S9-mix, to 8, 5, and 3, respectively, with S9-mix and to 7, 8 and 4, respectively, without S9-mix. No test material precipitation was observed. The limit concentration of 5000 g/plate was selected as the highest concentration used in the mutagenicity assay.

    3. Mutagenicity Assay
    4. Five concentrations of Granola 97 ranging from 75 to 5000 g/plate were tested, with and without S9-mix in all five bacterial strains. An additional concentration of 25 g/plate was used with strain WP2(uvrA). All plating was in triplicate. Granola 97 was not mutagenic to any of the five tester strains at any concentration tested, with or without S9-mix. Solvent and positive control values were appropriate for the respective strains. Results of the mutagenicity study are summarized in Appendix Table 1 (MRID 44487801, p. 30).

  3. Reviewer's Discussion / Conclusions
    1. This is an acceptable study. Granola 97 was tested to a limit concentration of 5000 g/plate, the experimental protocol was acceptable and the solvent and positive control values were appropriate for the respective strains.


    2. Study Deficiencies No study deficiencies were identified.

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