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Data Evaluation Record - Immunotoxicity - Dermal Exposure - Mice MRID 44438709


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  1. Materials and Methods
    1. Materials
    2. Study Design
    3. Methods
  2. Results
    1. Observations
    2. Body Weight and Weight Gain


Prepared for

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

Prepared by

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

Primary Reviewer:

Melissa D. Halpern, Ph.D.

Secondary Reviewers:

H. T. Borges, Ph.D., MT(ASCP), D.A.B.T

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

Quality Assurance:

LeeAnn Wilson, M.S.


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 Reviewer:

Sheryl K. Reilly, Ph.D.
Biological and Pollution Prevention Division (7511P)

Study Title: Immunotoxicity Screening Study in Mice Exposed Dermally to Granola 97

MRID No.: 44438709
File Jacket Symbol: 4822-UOO
DP Barcode: D243976
Submission: S538748
P.C. Code: 004822
Study No.: 1
Project No.: L08686SN1
Study Completion Date: October 17, 1997

Sponsor: SC Johnson, 1525 Howe Street, Racine, WI, 53403

Testing Facility: IIT Research Institute, Life Sciences Research, 10 West 35th Street, Chicago, IL 60616

Authors: R.V. House, W.D. Johnson, and J.F. Krueger

Quality Assurance (40 CFR 160.12): Included

Executive Summary:

In a dermal immunotoxicity study, female B6C3F1 mice (10/dose) were exposed to undiluted p-Menthane-3,8-diol (a.i. 98.3%) at doses of 0.0, 1000, and 3000 mg/kg once per day for 28 days. Parameters tested were total body weight gains, weekly food consumption, absolute and relative spleen and thymus weights, and antibody plaque forming cell assay

No mortality or clinically related signs of toxicity were observed. Mice exposed to p-Menthane-3,8-diol showed no statistically significant changes in body weight, or relative and absolute spleen and thymus weight compared with controls. Mice from both 1000 and 3000 mg/kg/day dosage groups did show statistically increased food consumption (17% and 16%, respectively) on day 21 but not on days 7, 14, or 28.

There are some problems interpreting the results of the plaque forming cell assay performed in this study. Exposure to 1000 mg/kg p-Menthane-3,8-diol resulted in a statistically significant 43% increase in antibody plaque forming cells/106 viable spleen cells. Total antibody plaque forming cells/spleen was increased 44% in the low dose group, but the enhancement was not statistically significant. Mice exposed to 3000 mg/kg showed no enhancement of either plaque forming cells/106 viable spleen cells or total plaque forming cells. Neither treatment group showed statistically significant changes in total number of viable cells per spleen and there were no differences in absolute and relative spleen and thymus weights in either test group.

The enhancement of the primary antibody response to sheep red blood cells in the low dose but not the high dose group, coupled with only two doses being tested, makes the LOEL appear to be lower than the NOEL. In the immune system, antigens can induce tolerance if injected in sufficiently large and frequent doses over a long enough period of time. Thus, one possible interpretation of the results observed in this study is that immune tolerance to sheep red blood cells was achieved at 3000 mg/kg/day.

However, for the purposes of hazard identification, the NOEL should be considered to be 3000 mg/kg/day. The reason for this is that since the plaque forming cell assay is currently only considered to be sufficiently validated as a test for immune suppression, and no suppression of immune response occurred at a limit dose of 1000 mg/kg/day, the stimulatory effect noted at 1000 mg/kg/day (a limit dose) is not considered to be an endpoint of concern. Thus, repeating the plaque forming cell assay at lower doses is not suggested for the purposes of risk assessment and registration of this technical pesticide product. It is advisable, however, to assess any formulations which include p-Menthane-3,8-diol for effects on the immune system.

This immunotoxicity dermal exposure study is classified as supplementary, as it does not meet the guideline requirements of 152-18. The study only partially fulfills the requirements outlined in the guideline, since only one immunologic parameter, humoral immune function measured by an antibody plaque forming cell assay, was tested. The study cannot be upgraded without the completion of the other studies included in that guideline. However, for the purposes of this risk assessment and the registration of p-Menthane-3,8-diol, further immunotoxicity testing is not required. The reasons for this are as follows: 1) the substance is a technical grade active ingredient, which will ultimately be incorporated into repellents for use on the skin and clothing; 2) no dermal sensitization was observed in a modified Buehler assay in guinea pigs (MRID 444387-05); 3) no effects on absolute and relative spleen and thymus weights, which are valid endpoints for immune suppression, occurred in the 28-day study nor in a 90-day dermal toxicity study (MRID 444387-10); and 4) the results of the 28-day immunotoxicity test indicated that no suppression of the primary antibody response to sheep red blood cells at a limit dose and higher. Thus, there is reasonable certainty that further immunotoxicity testing would not likely change the low level of concern for this endpoint.

  1. Materials and Methods
    1. Materials

      1. Test material: Granola 97 (p-menthane-3,8-diol)
      2. Description: white crystalline solid
        Lot/Batch #: 703001
        Purity: 98.3%
        Stability of compound: stable under ambient conditions

      3. Positive control: cyclophosphamide diluted in sterile saline.

      4. Test animals:
      5. Species: mouse, B6C3F1 (female)
        Age and weight at study initiation: 6 weeks; 16.5 - 18.8 g
        Source: Charles River Laboratories Inc., Portage, MI
        Housing: Singly in stainless steel wire mesh cages
        Diet: Purina Rodent Chow #5002 ad libitum
        Water: tap water ad libitum
        Environmental conditions:

        Temperature: 20 - 24EC
        Humidity: 32 - 74%
        Air changes: unknown

        Photoperiod: 12-hour light/dark cycle
        Acclimation period: 1 week

    2. Study Design

      1. In life dates: Start: 7/24/97; end: 8/21/97

      2. Animal assignment: Animals were assigned to treatment groups using a computerized randomization process constrained by body weight. Mice were individually identified by ear punch and assigned to one of four groups (Table 1).

        TABLE 1. Study designa
        Test Group Dose to animals (mg/kg/day) Treatment duration (days) Number of animals
        Sham controlb
        Positive controlc
        1. Data taken from p. 10 (MRID 44438709.)
        2. Dosed with 3000 mg/kg distilled water.
        3. Received a single dose of 80 mg/kg cyclophosphamide 24 hours prior to euthanasia and assay.
      4. Dose selection rationale: Not stated.

      5. Test substance preparation and analysis: Test substance sufficient for the days dosing was dispensed and warmed slightly to liquefy. These aliquots were kept on a hot plate in the animal room to maintain the liquid state. Tests for homogeneity and stability were not included.

      6. Dose application: The dorsal scapular fur of all mice (including positive controls) was clipped prior to initial dosing. Shaving was repeated weekly during the test period. Correcting for density, the liquefied test substance was administered at 1.036 and 3.109 mL/kg of body weight. Sham control mice received a daily dermal application of 3.109 mL/kg distilled water. Both test substance and water were administered with an automated pipettor and were allowed to spread according to their own physical properties. Positive control mice received no dermal treatments but received a single IP dose of 80 mg/kg cyclophosphamide in sterile saline on day 28. None of the treatment sites were covered after application of the test substance.

      7. Statistics: Terminal body weight, spleen and thymus weight, cell viability, and antibody plaque forming assay data were log- or logit-transformed. Group data were assessed using ANOVA and Dunnett's test.

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    3. Methods
      1. Observations: All mice were observed for mortality and morbidity twice daily on weekdays and once daily on weekends. Mice were observed weekly for adverse clinical signs.

      2. Body weight: Random mice were weighed the day following receipt. All mice were weighed on Day 0 and weekly thereafter for the duration of the study. In addition, all mice were weighed prior to euthanasia and these weights were used to determine organ-to-body weight ratios.

      3. Food consumption: Food consumption was recorded weekly.

      4. Sacrifice and pathology: Mice were sacrificed by CO2 asphyxiation. All animals survived until scheduled termination of the study.

        1. Gross necropsy: The spleen and thymus from each animal were removed and weighed.

        2. Tissue preparation/histopathology: No organs or tissues were preserved.

        3. Cell viability: Spleen cell viability was determined via trypan blue exclusion. Neither thymus nor bone marrow cell viability was assessed.

      5. Primary Antibody Response (Plaque forming cell assay): On day 25, all mice were injected i.v. with 4 x 107 washed sheep red blood cells (SRBC) suspended in Dulbecco's phosphate-buffered saline. Four days post immunization, mice were euthanized and the spleen and thymus were aseptically removed and weighed. Single cell suspensions were prepared by rubbing the spleens through nylon mesh, triturating through a 23-gauge needle and allowing cellular debris to settle. Spleen cells were suspended in RPMI-1640 media supplemented with Hepes buffer, 10 Fg/mL gentamicin, and 2 mM L-glutamine. Spleen cell preparations were diluted 1/30 and 1/120 in RPMI. Aliquots from each dilution were added to tubes containing Bacto-agar/DEAE-dextran, washed SRBC and guinea pig complement. The tubes were mixed and the mixtures poured into petri dishes. A glass cover slip was placed on each mixture to form a homogeneous monolayer. The plates were incubated in 5% CO2 at 37 EC and 95% humidity for 3 hours. The resulting antibody forming cells were enumerated using a plaque counter. Plaques were counted, multiplied by the dilution factor, and the cell suspension volume to determine antibody plaque forming cells/spleen.

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  2. Results
    1. Observations
      1. Clinical signs of toxicity: No clinical signs of toxicity were observed in this study. Alopecia was observed on one sham control mouse during weeks 2-4.

      2. Mortality: No deaths occurred during the study.

    2. Body Weight and Weight Gain:
    3. No effect on body weight (Table 2) or body weight gain (Table 3) was observed for any treatment group.

Day of study Treatment group (mg/kg Granola 97)
Sham control Low dose 1000 High dose 3000 Positive control

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