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Data Evaluation Record - Developmental Toxicity B Rat MRID 44438711

DATA EVALUATION REPORT
GRANOLA 97

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  1. Materials and Methods
    1. Materials
    2. Procedures and Study Design
    3. Observations
    4. Data Analysis
  2. Results
    1. Maternal Toxicity
    2. Developmental Toxicity
  3. Discussion
    1. Investigator's Conclusions
    2. Reviewer's Discussion
    3. Study Deficiencies
    4. Core Classification

STUDY TYPE:

DEVELOPMENTAL TOXICITY B RAT (83-3a)

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:

Carol S. Forsyth, Ph.D., D.A.B.T.

Secondary Reviewers:

Claudia M. Troxel, Ph.D.
Robert H. Ross, M.S., Group Leader

Quality Assurance:

Susan Chang, M.S.

Disclaimer

This review may have been altered subsequent to the contractor's signatures above.

Managed by Lockheed Martin Energy Research, Corp., for the U.S. Department of Energy under Contract No. DE-AC05-96OR22464.

EPA Reviewer:

Sheryl Reilly, Ph.D.
Biological and Pollution Prevention Division

DATA EVALUATION RECORD

Study Type: Developmental Toxicity - Rat OPPTS 870.3700 ['83-3a]

DP Barcode: D243976 Submissission Code: S538748 P.C. Code: 061954 Tox. Chem. NO.:

Test Material (PURITY): Granola 97 (SCJ NB# 14735R108) (98.5% a.i.)

Synonyms: p-Menthane-3,8-diol

Citation: Wakefield, A.E. (1997) Rat prenatal developmental toxicity study with Granola 97 (SCJ NB# 14735R108). Covance Laboratories, Inc., 9200 Leesburg Pike, Vienna, VA 22182-1699. Laboratory Study Identification, Covance 6106-116. October 30, 1997. MRID 44438711. Unpublished.

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

Executive Summary:

In a developmental toxicity study (MRID 44438711), 25 pregnant Sprague-Dawley Crl:CD7BR rats per group were administered Granola 97 (SCJ NB# 14735R108) (98.5% a.i.) by dermal application at doses of 0, 1, and 3 g/kg/day on gestation days (GD) 6-19, inclusive. The doses were adjusted based on a specific gravity of 0.98 and the control group received tap water. On GD 20, all dams were sacrificed and all fetuses were examined for external malformation/variations. Approximately one-half of each litter was processed for visceral examination and the remainder stained for skeletal and cartilage evaluation.

All animals survived to scheduled sacrifice. No treatment-related clinical signs of toxicity were observed in any animal during the study. The skin at the application site of animals in both treated groups did not show signs of irritation. No statistically significant differences in absolute body weights occurred between the treated and control groups during the study. Body weight gains were significantly (91% of controls; p # 0.05) lower in the high-dose group during GD 6-20 as compared to the controls. The reduced body weight gain by the high-dose group was a result of significantly lower (90%of controls; p # 0.01) food consumption than the controls during the GD 6-9 interval. At all other times during the study, food consumption and body weight gains by the treated groups were comparable to the controls. No abnormalities were noted at maternal necropsy.

Therefore, the maternal toxicity NOEL is >3 g/kg/day and the maternal toxicity LOEL was not identified.

No dose- or treatment-related statistically significant effects on pregnancy rate, number of corpora lutea, pre- or postimplantation losses, resorptions/dam, fetuses/litter, fetal body weights, or fetal sex ratios were observed in the treated groups as compared to the controls. Two low-dose dams had complete litter resorption.

No treatment-related external, visceral, or skeletal malformations/variations were observed in any litter. The number of litters in the 0, 1, and 3 g/kg/day groups containing fetuses with major malformations was 1/23, 2/21, and 1/22, respectively. All treated and control litters contained fetuses with minor variations in skeletal ossification.

Therefore, the developmental toxicity NOEL is >3 g/kg/day and the developmental toxicity LOEL was not identified.

This study is classified as Unacceptable (guideline; upgradable) and does not satisfy the guideline requirements for a developmental toxicity study (83-3a) in rats. Justification for the dermal route was not provided nor were data available indicating that the test article is absorbed dermally.

Compliance: Signed and dated Quality Assurance, Good Laboratory Practice, and Data confidentially statements were included.

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  1. Materials and Methods

    1. Materials

      1. Test material:

        2. Granola 97 (SCJ NB# 14735R108)

        Description: clear, viscous liquid
        Lot/Batch No.: 703002
        Purity: 98.5% a.i.
        Stability of compound: not stated, responsibility of sponsor
        CAS No.: 42822-86-6
        Structure: not provided

      2. Vehicle and/or positive control

        3. Tap water served as the control material. The test material was applied neat without the use of a vehicle.

      3. Test animals

        4. Species: Rat
        Strain: Sprague-Dawley Crl:CD7BR
        Age and weight at study initiation: 11 weeks; 205-280 g on GD 0
        Source: Charles River Laboratories, Inc., Portage, MI (females); Kingston, NY (males)
        Housing: Animals were housed individually in a stainless-steel, hanging, wire-mesh cage.
        Diet:         PMI7 Certified Rodent Diet7 #5002 and tap water were available ad libitum.

      4. Environmental conditions:

        5. Temperature: 19.5-25.8EC
        Humidity: 40.1-62.4%
        Air changes: ±10/hour
        Photoperiod: 12-hour light/12-hour dark
        Acclimation period: 1 week

    2. Procedures and Study Design

      3. This study was designed to assess the developmental toxicity potential of Granola 97 when administered dermally to rats on gestation days 6 through 19, inclusive.

      1. In life dates

        2. Start: June 11, 1997; end: July 5, 1997

      2. Mating

        3. One female was mated with one male of the same strain until mating was confirmed by the presence of sperm in a vaginal lavage or a retained copulatory plug. The day evidence of mating was observed was designated GD 0.

      3. Animal assignment and dose selection are presented in Table 1. Confirmed-mated females were assigned to groups using a table of random numbers.

        4.  

        TABLE 1. Animal assignment
        Group
        Dose
        (g/kg/day)
        		 Number
        of animals
        Control
        0
        25
        Low
        1
        25
        High
        3
        25
        Data taken from text table p. 14, MRID 44438711.
      4. Dose selection rationale

        5. Doses were based on data from pharmacology, other toxicology, and kinetics studies. The study author stated that the high-dose level was close to the maximum feasible dermal dose. No other details on dose selection were included in the study.

      5. Dosing

        6. The test material was applied neat, as supplied. Prior to the first dose, and as needed thereafter (at least weekly), animals were shaved on the dorsal surface of the back. Test material was administered to the site once daily on GD 6-19. The application site was covered with 4-ply gauze and a dermal harness was placed on the animal. At least 6 hours but not more than 6 hours and 55 minutes after dose application, the site was wiped with gauze to remove any residue. Doses were based on the most recently recorded body weight.

      6. Dose solution preparation and analysis

        7. Doses were adjusted based on the specific gravity of 0.98.

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    3. Observations

      1. Maternal observations and evaluations

        2. The animals were checked twice daily for mortality and moribundity. Observation for clinical signs of toxicity was performed once daily at the time of dermal harness removal. A thorough physical examination was conducted at each weighing interval and included an examination of the treatment site for irritation. Maternal body weights and food consumption were measured on GD 0, 3, 6, 9, 12, 15, 18, and 20. Dams were sacrificed on GD 20 by carbon dioxide inhalation and exsanguination and examined grossly. The uterus from each gravid female was excised, weighed, and examined for the number and placement of implantation sites, live and dead fetuses, early and late resorptions, and any abnormalities of the placenta or amniotic sac. The ovaries were examined for the number of corpora lutea.

      2. Fetal evaluations

        3. Each fetus was sexed, weighed, examined for external abnormalities, and sacrificed via intraperitoneal injection of sodium pentobarbital. Approximately one-half of all fetuses from each litter were randomly selected and processed for visceral examination. The remaining fetuses were eviscerated and processed for skeletal and cartilage evaluation.

    4. Data Analysis

      1. Statistical analysis

        2. Mean maternal body weight, body weight change, food consumption, uterine weights, and cesarean section data of the treated groups were compared to the control group using a One-Way analysis of Variance. Levene's test was used to determine homogeniety of variances. If the variances of the untransformed data were heterogeneous, analyses were performed on rank-transformed data. Dunnett's t-test served as the post-hoc group comparison test. Mean live fetal weights were analyzed by One-Way Analysis of Covariance using the number of fetuses in each litter as the covariate.

      2. Historical control data were not provided to allow comparison with concurrent controls.

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  2. Results

    1. Maternal Toxicity

      1. Mortality and clinical signs

        2. All animals survived to scheduled sacrifice. No treatment-related clinical signs of toxicity were observed in any animal during the study. The skin at the application site of animals in both treated groups did not show signs of irritation.

      2. Body weight

        3. Selected maternal body weights and body weight gains are given in Table 2. No statistically significant differences in absolute body weights occurred between the treated and control groups during the study. Body weight gains were significantly (91% of controls; p # 0.05) lower in the high-dose group during the treatment period as compared to the controls.

         

        TABLE 2. Maternal body weight and body weight gain (g)
        Day of gestation 0 g/kg/day 1 g/kg/day 3 g/kg/day
        0 250.5 " 14.4 253.3 " 14.5 251.1 " 15.2
        6 289.5 " 14.0 291.8 " 17.4 286.8 " 15.6
        18 384.0 " 26.8 374.9 " 42.6 374.3 " 20.1
        20 417.2 " 33.3 403.7 " 52.6 402.9 " 22.7
        weight gain 6-20 127.74 " 24.07 111.96 " 44.73 116.05 " 15.56*
        weight gain 0-20 166.70 " 25.71 150.48 " 47.32 151.73 " 18.96
        Corrected body weighta 329.77 " 19.71 327.77 " 31.56 316.69 " 20.60

        Data taken from Tables 2A, 2B, and 5, pp. 28, 30, and 37, respectively, MRID 44438711.

        *Significantly different from control, p # 0.05. aCorrected body weight = terminal body weight - gravid uterine weight.

      3. Food consumption

        4. Food consumption data are summarized in Table 3. The high-dose group had significantly lower (90% of controls; p # 0.01) food consumption than the controls during the GD 6-9 interval. At all other times during the study, food consumption by the treated groups was comparable to the controls.

         

        TABLE 3. Maternal food consumption (g/animal/day)
        Gestation interval 0 g/kg/day 1 g/kg/day 3 g/kg/day
        0-6 24.4 " 1.7 24.9 " 2.4 24.5 " 1.7
        6-9 26.8 " 1.9 25.2 " 3.2 24.2 " 1.6**
        6-20 29.8 " 2.2 29.1 " 3.8 29.8 " 2.2
        0-20 28.2 " 1.9 27.8 " 3.1 28.2 " 1.9

        Data taken from Table 3, pp. 32-33, MRID 44438711.

        **Significantly different from control, p # 0.01.

      4. Gross pathology

        5. No treatment-related abnormalities were observed at maternal necropsy.

      5. Cesarean section data

        6. Data obtained at cesarean section are given in Table 4. No dose- or treatment-related statistically significant effects on pregnancy rate, number of corpora lutea, pre- or postimplantation losses, resorptions/dam, fetuses/litter, fetal body weights, or fetal sex ratio were observed in the treated groups as compared to the controls. One control dam had only two implantation sites with a single viable fetus. The slightly higher percentage of resorptions/dam and fewer live fetuses/litter in the low-dose group are a result of two animals with total litter resorption. If these two dams are excluded from the group means, the values are similar to the control group levels. Because the high-dose group was not similarly affected, the differences in the low-dose group are not considered to be treatment-related.

         

        TABLE 4. Cesarean section observations
        Observation 0 g/kg/day 1 g/kg/day 3 g/kg/day
        No. Animals Assigned 25 25 25
        No. Animals Mated/Inseminated 23 23 22
        Pregnancy Rate (%) 92 92 88
        Maternal Mortality 0 0 0
        Delivered early/aborted 0 0 0
        Total Corpora Lutea 393 365 378
        Corpora Lutea/Dam 17.1 15.9 17.2
        Total Implantation 347 313 341
        Implantation/Dam 15.1 13.6 15.5
        Preimplantation loss (%) 12.6 16.6 9.3
        Postimplantation loss (%) 6.1 15.8 8.2
        Total Live Fetuses 332 284 313
        Live Fetuses/litter 14.4 12.3 14.2
        Mean Fetal Weight (g) 3.76 3.78 3.64
        Sex Ratio (% Male) 53 49 50
        Total Resorptions 15 29 28
        Resorptions/Dam (%)a 0.7 (6.1) 1.3 (15.8) 1.3 (8.2)
        Early resorptions/dama  0.7 0.7 0.8
        Late resorptions/dama 0.0 0.6 0.5
        Total Dead Fetuses 0 0 0
        Dams with all resorptions 0 2 0

        Data taken from Tables 6 and 7, pp. 39-41 and 45, respectively, MRID 44438711.

        aIncludes dams with all resorptions.

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    2. Developmental Toxicity

      3. No treatment-related external, visceral, or skeletal malformations/variations were observed in any litter. The number of litters in the 0, 1, and 3 g/kg/day groups containing fetuses with major malformations was 1/23, 2/21, and 1/22. All litters contained fetuses with minor variations in skeletal ossification.

      1. External examination

        2. No treatment-related external malformations/variations were observed in any fetus from any treated litter. One control fetus had macrophthalmia, one low-dose fetus had anasarca, and another low-dose fetus from a different litter was acaudate with anal atresia.

      2. Visceral examination

        3. No treatment-related visceral malformations/variations were observed in any fetus from any treated litter. The single fetus from a control group dam was examined viscerally. A common finding in control and treated litters was renal pelvic cavitation. One low-dose fetus had multiple malformations.

      3. Skeletal examination

        4. All treated and control litters contained fetuses with skeletal variations. Because one control dam had a single viable fetus which was processed for visceral examination, only 22 control group litters were available for skeletal examination. The most common variations were bipartite vertebral centra, sternebrae unossified or incompletely ossified, and incomplete ossification of the skull. The number of litters in the high-dose group containing fetuses with sternebrae asymmetrically ossified was significantly (p = 0.047) greater than the control group with 1/22, 1/21, and 6/22 litters in the 0, 1, and 3 g/kg/day groups, respectively, containing affected fetuses. One fetus in a low-dose litter had only the first sacral vertebrae present with the second through the fourth sacral vertebrae and all caudal vertebrae absent.

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  3. Discussion

    1. Investigator's Conclusions

      2. The study author concluded that dermal administration of Granola 97 to pregnant rats during organogenesis did not result in overt maternal or fetal toxicity. The NOAEL for maternal and fetal toxicity is 3 g/kg/day.

    2. Reviewer's Discussion

      1. Maternal Toxicity

        2. Maternal toxicity was not observed during the study. Slightly decreased overall body weight gains occurred in the high-dose group and were most pronounced during the GD 6-9 interval; body weight gains during other intervals of the treatment period were comparable to the control group levels. The reduced weight gain was most likely due to the decrease in food consumption during days 6-9 of the treatment period. While body weight gains for GD 6-20 and food consumption for GD 6-9 reached statistical significance for the high-dose group, the changes are of questionable biological significance. The reductions were transient, were not dose-related, and were within 10% of the control group value. Therefore, the reduced body weight gain in the high-dose group resulting from decreased food consumption is not considered to be a treatment-related toxicity.

        The study author did not justify the dermal route of treatment except to state that this is the expected route of human exposure. No information was included that showed dermal absorption of Granola 97 occurred to an appreciable amount. In a 90-day dermal study in rats (MRID 44438710), the main finding was inflammation and acanthosis at the application site on males and females administered 3 g/kg/day for 6 hr/day. There was also a trend towards increasing severity of kidney lesions in male rats indicating some dermal absorption may occur, but absorption is probably minimal. Although the doses used in both the current and subchronic studies exceed the limit dose, experimental evidence of dermal absorption is necessary before the current study can be considered valid. Therefore, further justification of the dermal route and pharmacokinetic data showing dermal absorption are required before this study is acceptable.

        Although three doses are generally recommended to adequately assess dose-response relationships, only two doses were used in the current study. Lack of a third dose is not considered a deficiency because no toxicity occurred at the high dose and because the high dose was above the limit dose.

      2. Developmental Toxicity


        1. Deaths/resorptions

          2. Maternal treatment with Granola 97 during gestation did not result in fetal death or an increase in resorptions.

        2. Altered growth

          3. Fetal growth was not affected by maternal treatment with Granola 97.

        3. Developmental variations

          4. No treatment-related variations were observed in any fetuses. Although the number of high-dose litters containing fetuses with asymmetrically ossified sternebrae was significantly greater than the controls, this is not considered a compound-related effect. The variation was not dose-related and there were only seven fetuses in a total of six affected litters. Historical control data would have been helpful to determine the background incidence range of this anomaly.

        4. Malformations

          5. Major malformations did not result from maternal treatment with Granola 97 during gestation. Two fetuses from different low-dose litters had multiple malformations, but these are considered incidental to treatment.

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    3. Study Deficiencies

      4. Justification for the dermal route was not provided nor were data available indicating that the test article is absorbed dermally. Without evidence that Granola 97 is absorbed dermally, it is unknown whether the chemical is available to the fetus. Historical control data were not provided.

    4. Core Classification

      5. This study is classified as Unacceptable (guideline; upgradable) and does not satisfy the guideline requirements for a developmental toxicity study (83-3a) in rats. Justification for the dermal route was not provided nor were data available indicating that the test article is absorbed dermally.

      1. Maternal NOEL = >3 g/kg/day


      2. Maternal LOEL = not identified


      3. Developmental toxicity NOEL = >3 g/kg/day


      4. Developmental toxicity LOEL = not identified

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