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Data Evaluation Record - In Vivo Mammalian Cytogenetics - Micronucleus Assay in Mouse Bone Marrow MRID 44438707
DATA EVALUATION REPORT p-MENTHANE-3,8-DIOL
Related Information
Information related to this page:On This Page
- Materials and Methods
- Materials
- Test Performance
- Reported Results
- Preliminary toxicity assay:
- Micronucleus Assay
- Reviewer's Discussion/Conclusions
- This is an acceptable study
- Study Deficiencies
STUDY TYPE: In Vivo Mammalian Cytogenetics - Micronucleus Assay in Mouse Bone Marrow; OPPTS 870.5395 [§84-2]
Prepared for
Office of Pesticide Programs (7511P)
Environmental Protection Agency
1200 Pennsylvania Avenue, NW
Washington, D.C. 20460Prepared by
Chemical Hazard Evaluation Group
Toxicology and Risk Analysis Section
Life Sciences Division
Oak Ridge National Laboratory
Oak Ridge, TN 37831
Task Order No. 22Primary Reviewer:
Bradford L. Whitfield, Ph.D.
Secondary Reviewers:
Cheryl B. Bast, Ph.D., D.B.A.T.
Robert H. Ross, M.S., Group LeaderQuality 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
STUDY TYPE: In vivo mammalian cytogenetics - micronucleus assay in mouse bone marrow; OPPTS 870.5395 [§'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 P. Ritter (1997) Mammalian erythrocyte micronucleus test with granola 97 (SCJ NB # 14735R108). MA BioServices, Inc., 9630 Medical Center Drive, Rockville, MD 20850. Laboratory study number G97BF49.123001. November 20, 1997. MRID 44438707. Unpublished
SPONSOR: S.C. Johnson & Son, Inc., 1525 Howe Street, Racine, WI 53403-2236
Executive Summary:
In an ICR mouse bone marrow micronucleus assay (MRID 44438707), five mice/sex/dose were treated once i.p. with Granola 97 in corn oil (98.3% a.i., batch No. 703001) at doses of 104, 208, 416 mg/kg or dermally over four days with 3 mL/kg total of neat agent. Bone marrow cells were harvested at 24 hours (all doses) and at 48 hours (416 mg/kg only) post-treatment.
Granola 97 was tested at an adequate dose. All mice in the 208 and 416 mg/kg groups were lethargic following treatment. Convulsions and prostration were also seen in all mice in the 416 mg/kg group. Seven of 15 males and 7/15 females in the 416 mg/kg group displayed piloerection. All mice in the dermal application group showed both hyperactivity and lethargy after treatment. There was no statistically significant increase in the number of micronucleated PCEs at any dose, harvest time or route of exposure tested in this study. The solvent and positive control values were appropriate (The mean number of micronucleated PCEs per 1000 PCEs was, for the corn oil control, 0.7 " 1.10 in males and 0.5 " 0.61 in females at 24 hours and 0.7 " 0.84 in males and 0.5 " 0.35 in females at 48 hours. Positive control values were 19.4 " 3.13 and 17.1 " 4.60 for males and females, respectively, at 24 hours). There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow at any dose, harvest time or route of exposure.
- Materials and Methods
- Materials
- Test material: Granola 97
- Control materials
- Test compound administration
- Test animals
- Species mouse
- No. animals used per dose: 5 males 5 females
- Properly maintained? Y
- Test Performance
- Treatment and sampling times
- Test compound and vehicle control
- Positive control
- Tissues and cells examined:
- Details of slide preparation
- Statistical methods
- Evaluation criteria
- Reported Results
- Preliminary toxicity assay
- Micronucleus Assay
- Reviewer's Discussion/Conclusions
- This is an acceptable study.
- Study Deficiencies
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
Vehicle/Final volume/Route of administrationcorn oil / 20 mL/kg body weight / i.p.;
corn oil / 20 mL/kg body weight / dermal
Positive/Final dose(s)/Route of administration:
cyclophosphamide / 60 mg/kg body weight / i.p.
Volume of test substance administered:
20 mL/kg body weight i.p.; 3 mL/kg body weight dermal
Route of administration: i.p. and dermal
Dose levels used:
Pilot assay:1, 10, 100, 1000 mg/kg (males only); 2000 mg/kg (males and females)
Cytotoxicity assay: 100, 300, 600, 800 mg/kg (males and females)
Micronucleus assay:
i.p. - 104, 208, 416 mg/kg (males and females); dermal -3 mL/kg total of neat agent over four days (male and female)
Strain ICR
Age 6-8 wks
Weight:Pilot study - male 30.3-33.7 g female 25.2-27.5 g
Cytotoxicity study - male 32.0-35.3 g female 24.9-27.8 g
Micronucleus assay - males 30.2-34.7 g female 23.7-30.0 gSource: Harlan Sprague Dawley, Inc., Frederick, MD
i.p. dosing: x once twice (24 hr apart)
other (describe): Dermal dosing: once twice (24 hr apart)
x other (describe): daily for four daysSampling (after last dose): 6 hr 12 hrx 24 hr x 48 hr 72 hr (mark all that are appropriate),
Dosing: x once twice (24 hr apart)
other (describe): Sampling (after last dose): 6 hr 12 hr
x 24 hr 48 hr 72 hr (mark all that are appropriate)
x bone marrow other (list):
No. of polychromatic erythrocytes (PCE) examined per animal: 2000
No. of normochromatic erythrocytes (NCE; more mature RBCs) examined per animal: the number found while screening 2000 PCEs
Mice were killed by CO2 inhalation at the designated sacrifice time, the femurs were exposed and cut just above the knee and the bone marrow aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a centrifuge tube containing 1 mL of fetal bovine serum, the tube capped and the cells pelleted by centrifugation at approximately 100 x g for five minutes. Most of the supernatant was removed and the cells resuspended in the remaining serum. A small drop of the cell suspension was spread onto a clean glass slide (two to four slides per mouse), the slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted. Slides were independently coded using a random number table.
Statistical significance (p#0.05) was determined using the Kastenbaum-Bowman tables. All analyses were performed separately for each sex and sampling time.
Micronuclei were defined as "round, darkly staining nuclear fragments, having a sharp contour with diameters usually from 1/20 to 1/5 of the erythrocyte". Results were considered positive if there was a positive dose-responsive increase in micronucleated PCEs and the increase in micronucleated PCEs at one or more doses was statistically elevated relative to the vehicle control (p#0.05) at any sampling time. A significant increase at one sacrifice time in a single treatment group with no dose-response was considered a suspect or unconfirmed positive. The test article was considered negative if no statistically significant increase in micronucleated PCEs above the concurrent solvent control was seen at any sampling time. To be considered a valid test, the mean incidence of micronucleated PCEs must not exceed 5 per 1000 PCEs in the solvent control and the incidence of micronucleated PCEs in the positive control group must be significantly increased relative to the solvent control group (p#0.05).
In a pilot study, two male mice were treated once i.p. with one of four concentrations of Granola 97 ranging from 1 to 1000 mg/kg. Five males and five female mice were treated once with 2000 mg/kg. All mice treated with 2000 mg/kg Granola 97 and both males treated with 1000 mg/kg had convulsions and died within four hours of treatment. All other mice appeared clinically normal throughout the three day observation period.
A preliminary toxicity assay was also conducted using Granola 97 concentrations of 100, 300, 600 and 800 mg/kg. Groups of five males and five females were treated i.p. at each dose level. One of five females at 600 mg/kg and three of five males and two of five females at 800 mg/kg died within one day of dosing. Clinical signs seen after dosing were lethargy, convulsions and prostration in both males and females at 300, 600 and 800 mg/kg. Ataxia was seen in males and females and piloerection in males at 600 mg/kg. Irregular breathing was seen in males and females at 800 mg/kg. An LD50/3 of 831.6 mg/kg for both male and female mice was calculated by probit analysis and 416 mg/kg (50% of the LD50/3) selected as the upper dose for the micronucleus assay. Details of the preliminary toxicity assay are given in Appendix Table 1 (MRID 44438707, p. 16).
Five males and five females per dose group were treated i.p. once with 104, 208 or 416 mg/kg Granola 97 in a volume of 20 mL/kg body weight. Another group of five mice of each sex was treated dermally with neat Granola 97 for four consecutive days at a total volume of 3 mL/kg. No deaths occurred in any of the dose groups. All mice in the 208 and 416 mg/kg groups were lethargic following treatment. Convulsions and prostration were also seen in all mice in the 416 mg/kg group. Seven of 15 males and 7/15 females in the 416 mg/kg group displayed piloerection. All mice in the dermal application group showed both hyperactivity and lethargy after treatment.
There was no statistically significant increase (p#0.05) in the incidence of micronucleated PCEs over solvent control values in either sex at any Granola 97 concentration, route of exposure or sacrifice time. Slightly reduced PCE/total erythrocyte ratios were reported in males at 104 and 208 mg/kg i.p. at 24 hours(11% decrease compared to solvent control values at both concentrations). No reduction in the PCE/total erythrocyte ratio was seen in either sex at the 24 hour harvest time at 416 mg/kg but at 48 hours the ratios at this dose were reduced by 4% in males and 2% in females. The reductions are unlikely to be biologically significant. The 11% reduction at 104 mg/kg was due to a very low value in one mouse (0.20 compared to the solvent control value of 0.54, also only 980 PCEs were found on the slides from this mouse compared to at least 2000 from other mice). When this mouse is excluded from the calculation, the PCE/total erythrocyte ratio is the same as the solvent control value. Solvent and positive control values were appropriate (the testing laboratory's historical control data are given in an attachment to the Appendix (from MRID 44438707, p. 25)).
Results from the i.p. and dermal routes of exposure are summarized in Appendix Table 2 (MRID 44438707, p. 19) and Appendix Table 3, (MRID 44438707, p. 22), respectively.
Granola 97 was tested to toxic levels and appropriate experimental protocol was followed. Positive and solvent control values were acceptable. Although the authors report slight reductions in the PCE/total erythrocyte ratio, as discussed in section II.B., this is not likely to represent bone marrow toxicity. No evidence was found in this study for a micronuclei inducing potential of Granola 97.
No study deficiencies were identified.