Jump to main content.

Data Evaluation Record - Subchronic Dermal Toxicity - Rat using Granola 97 (p-menthane-3,8-diol) MRID 44438710

DATA EVALUATION REPORT
GRANOLA 97 (p-menthane-3,8-diol)

Related Information
Information related to this page:

On This Page

  1. Materials and Methods
    1. Materials
    2. Study Design
    3. Methods
  2. Results
    1. Observations
    2. Body Weight and Weight Gain
    3. Food Consumption and Compound Intake
    4. Ophthalmoscopic ExaminationOphthalmoscopic Examination
    5. Blood Work
    6. Neurotoxicity
    7. Sacrifice and Pathology
  3. Discussion
    1. Discussion
    2. Study Deficiencies

Study Type: Subchronic Dermal Toxicity - Rat (82-3)

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:

Melissa D. Halpern, Ph.D.

Secondary Reviewers:

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

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

Quality Assurance:

Eric B. Lewis, 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.
Biopesticides and Pollution Prevention Division , Date

DATA EVALUATION RECORD

Study Type: Subchronic Dermal Toxicity-Rat; OPPTS 870.3250 ['82-3)]

DP Barcode: D243976 Submission Code: S538748 P.C. Code: 011550 TOX. CHEM. NO.: unknown

Test Material (Purity): Granola 97 (98.3%)

Synonyms: p-menthane-3,8-diol

Citation: Rush, R.E. (1997) A 90-day dermal toxicity study of Granola 97 in rats. Springborn Laboratories, Inc., Health and Environmental Sciences, 640 North Elizabeth Street, Spencerville, OH, 45887. Study # SCJ 14735R108, October 14, 1997. MRID 44438710. Unpublished.

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

Executive Summary

In a 90 day subchronic dermal toxicity study (MRID 44438710), groups of 15 male and female Sprague-Dawley rats were treated with Granola 97 (98.3%) at doses of 0, 1000 or 3000 mg/kg/day for 6 hours per day.

Decreased body weight (-8% day 36; -9% day 43, p # 0.05) and body weight gain (-30% days 29-36, p # 0.05) were observed in the high dose males. Low dose males displayed decreased (-71% days 64-71, p # 0.05) and increased (+260% days 71-78, p # 0.01) body weight gain. No other effects on body weight were observed.

Barely perceptible erythema and desquamation was reported in all low dose male and female animals. In addition, a number of high dose male and female animals displayed well-defined erythema (23% male, 33% female), slight edema (8% male, 0% female) and pinpoint to moderate eschar (77% male, 40% female). Dermal findings in the control group were limited to one female with desquamation.

Treatment-related microscopic lesions were observed in the kidneys from high dose males and in treated skin from high dose males and females. Hyaline droplets, likely due to alpha-2u-globulin inclusions, were seen in kidneys of control males (20%, minimal to mild) and high dose males (100%, 73% moderate). Minimal acanthosis was observed in 53% of control males' treated skin, while minimal to mild acanthosis was seen in 93% of high dose males. Chronic inflammation was observed in male control (20%, minimal to mild), female control (13%, minimal), and high dose male (100%, 67% mild) and female (100%, 60% mild) animals. In addition, parakeratosis was seen in 7% of high dose males and 27% of high dose females.

Statistically significant increased absolute liver weight (+18%, p#0.001) and relative liver weight (+15%, p#0.001) were observed in high dose females. Relative liver weight (+9%, p#0.05), relative kidney weight (+12%, p#0.001), and relative adrenal weight (+15%, p#0.05) were increased in high dose males. There were no statistical differences noted for low dose male or female animals.

No treatment-related effects were observed with regard to hematology, clinical chemistry, neurotoxicity, or ophthalmology.

Based on the data presented in this study, the NOEL is 1000 mg/kg/day; the LOEL is 3000 mg/kg/day. The LOEL is based on dermal observations in treated skin (increased erythema, edema and eschar) and histological observations in treated skin ( increased acanthosis and inflammation).

This subchronic dermal toxicity study in rats is classified as acceptable (guideline) ('82-3) and satisfies the Subdivision F guideline requirements.

Compliance: Signed and dated GLP, Quality Assurance, Data Confidentiality, and Flagging statements were provided.

Top of Page

  1. Materials and Methods

    1. Materials



      1. Test material: Granola 97

        2. Description: clear, oily liquid/clear, solid material
        Lot/Batch #: 703001
        Purity: a.i. 98.3%
        Stability of compound: not stated

        CAS #: 42822-86-6

        Structure: not provided

      2. Vehicle and/or positive control: no vehicle used


      3. Test animals

        4. Species: Rat
        Strain: Sprague-Dawley Crl: CDBR VAF/Plus
        Age/weight at study initiation: males: 7 weeks, 209-277g ; females: 7 weeks, 150-190g.
        Source: Charles River Laboratories, Inc., Portage, Michigan.
        Housing: individually, in suspended stainless steel, wire mesh cages
        Diet: PMI Certified Rodent Chow #5002, ad libitum
        Water: tap water, ad libitum

      4. Environmental conditions

        5. Temperature: 65-790 F
        Humidity: 30-70%
        Air changes: 10-15 per hour
        Photoperiod: 12 hour light/dark cycle
        Acclimation period: 12 days

    2. Study Design



      1. In life dates
        2. Start: 4/16/1997; End: 7/21/1997
      2. Animal assignment

        3. Animals were assigned to one of three groups based on body weights using a computer randomization program (Table 1). Fifteen rats/sex/dose were utilized.

        Table 1. Study Design
        Dose mg/kg/day No. of animals
        male Positive control
        0   (control)
        15
        15
        1000 (low dose)
        15
        15
        3000
        (high dose)
        15
        15

         

      3. Dose selection rationale

        4. The doses utilized in the study were chosen "to meet and exceed the EPA's Limit Test dose level of 1000 mg/kg/day and to provide an adequate safety factor relative to anticipated human exposure."

      4. Test substance preparation and analysis

        5. Bulk containers of Granola 97 and distilled water used for controls were kept in a 37-450 C oven to maintain a liquid state. On each day of dosing, a container of Granola 97 was removed and dispensed into daily dosing aliquots. These aliquots were placed on a heated stir plate and stirred continuously during dosing.

      5. Dose application

        6. An area of fur equivalent to approximately 15% of the body surface was clipped from the dorsal area of the rats two days before initiation of dosing. The animals were clipped at least weekly thereafter. Granola 97 or distilled water for controls were applied and evenly spread over the clipped area of each animal using a syringe and ball-tipped gavage needle. After application, the treatment area was covered with four-ply gauze secured with elastic wrap. The cut ends of the elastic wrap were secured with athletic tape. Due to wrapping related mortality in 5 animals during the first 17 days of the trial, this wrapping procedure was modified. A less restricting binder consisting of a harness and one piece of tape accompanied by athletic tape applied to the hindpaws was utilized.

        After each 6 hour application, the wrapping material was removed and the treatment site was wiped with gauze soaked in deionized tap water.

        Animals were treated daily except during the functional observation battery intervals (days 43-45 and 85-87). Five animals/sex/group were evaluated on each day.

        At the end of the testing period, animals were sacrificed "when possible" via CO2 asphyxiation. No indication was made of the mode of euthanasia when this was not possible.

      6. Statistics

        7. Data was analyzed using ANOVA. When statistically significant differences were determined, control to treatment group comparisons were performed using the Tukey-Kramer method.

      Top of Page

    3. Methods

      1. Observations

        2. Mortality and moribundity were checked twice daily. Animals were also observed for overt toxic effects prior to dosing and approximately 30-120 minutes after wrap removal. Once a week, detailed clinical observations were performed.

      2. Body weight

        3. Animals were weighed at study initiation and once per week throughout the study.

      3. Food consumption

        4. Individual food consumption was calculated on the same days as body weights, once per week throughout the study.

      4. Ophthalmoscopic examination

        5. Ophthalmological examinations were performed by a board-certified veterinary ophthalmologist prior to study initiation (day -2) and near the study's conclusion (day 94). Eyes were dilated using 0.5% Mydriacyl opthalmic solution prior to biomicroscopic and indirect opthalmoscopic examination.

      5. Blood was collected after overnight fasting from the orbital plexus for hematology and clinical analysis from all surviving animals. The CHECKED (X) parameters were examined.


        1. Hematology


          2. X Hematocrit (HCT)* X Leukocyte differential count*
          X Hemoglobin (HGB)* X Mean corpuscular HGB (MCH)
          X Leukocyte count (WBC)* #  
          X Erythrocyte count (RBC)*   Mean corpusc. HGB conc.(MCHC)
          X Platelet count*   Mean corpusc. volume (MCV)
            Blood clotting measurements*   Reticulocyte count
            x (Thromboplastin time)    
              (Clotting time)    
            x (Prothrombin time)    

          * Required for subchronic studies based on Subdivision F Guidelines

          # Reticulocyte slides were prepared, but not examined.

        2. Clinical chemistry


          3. ELECTROLYTES
          OTHER
          X Calcium* X Albumin*
          X Chloride* X Blood creatinine*
            Magnesium X Blood urea nitrogen*
          X Phosphorus* X Total Cholesterol
          X Potassium* X Globulins
            Sodium* X Glucose*
          Enzymes X Total bilirubin
          x Alkaline phosphatase (ALK) X Total serum protein (TP)*
            Cholinesterase (ChE)   Triglycerides
            Creatine phosphokinase   Serum protein electrophores
            Lactic acid dehydrogenase (LDH)    
              Serum alanine amino-transferase (also SGPT)*    
              Serum aspartate amino-transferase (also SGOT)*    
            Gamma glutamyl transferase (GGT)    
            Glutamate dehydrogenase    

          * Required for subchronic studies based on Subdivision F Guidelines

      6. Urinalysis

        7. Urinalysis was not required and not performed.

      7. Neurotoxicity Screening

        8. A functional observation battery was conducted on animals during days 43-45 and 85-87 and included the following observations:

        1. Home cage observations: body posture, tremors, convulsions, piloerection, respiratory rate/pattern, eyelid closure, unusual urination, abnormal feces and bizarre behavior.


        2. Removal from home cage: ease of removal, reactivity to being handled, vocalization, general appearance, lacrimation, salivation, pupil size, exophthalmus, urine staining and fecal staining.


        3. Open field observations: body posture, tremors, convulsions, gait, alertness of unperturbed animals, excessive/repetitive actions, urination, defecation, rearing and bizarre behavior.


        4. Manipulative tests: tail pinch, startle response, righting ability, pupil response, approach response, touch response, grip strength and landing foot splay.

        Top of Page

      8. Sacrifice and pathology

        9. All animals that died and those sacrificed on schedule were subjected to gross pathological examination. All tissues and organs collected from control and high dose animals, all gross lesions from all mid dose animals and selected tissues from mid dose animals found dead during the study were processed for histopathology. The CHECKED (X) tissues were collected for histological examination. The (XX) organs, in addition, were weighed

        X Digestive System X Cardiovasc./Hemat. X Neurologic
            x Aorta* xx Brain*
        x Tongue xx Heart* x Periph. nerve*
        x Salivary glands* x Bone marrow* x Spinal cord (3 levels) T
        x Esophagus* x Lymph nodes* x Pituitary*
        x Stomach* x Spleen* x Eyes (optic n.)
        x Duodenum* xx Thymus*    
        x Jejunum*        
        x Ileum*   Urogenital   Glandular
        x Cecum* xx Kidneys*+ xx Adrenal gland*
        x Colon* xx Urinary bladder* x Lacrimal gland T
        xx Rectum* xx Testes*+ x Mammary glandT
          Liver*+ x Epididymides xx Parathyroids++
        x Gall bladder* x Prostate xx Thyroids++
          Pancreas* x Seminal vesicle    
            xx Ovaries x Other
        x Respiratory x Uterus* x Bone
        xx Trachea*     x Skeletal muscle
          Lung*     x Skin (treated & untreated)
          Nose       All gross lesions and masses*
          Pharynx        
          Larynx        

        * Required for subchronic studies based on Subdivision F Guidelines
        + Organ weight required in subchronic and chronic studies.
        T = required only when toxicity or target organ

    Top of Page

  2. Results

    1. Observations

      2. No treatment-related mortality was observed. Five deaths (1 female control, 2 low dose females and 2 high dose males) during the first 17 days of the treatment period were attributed to the original wrapping procedure.

      Treatment-related dermal reactions were observed (Table 2) in this study. An increase in barely perceptible erythema, desquamation and focal/pinpoint eschar was observed in low and high dose males. A few incidences of well-defined erythema, very slight edema and mild to moderate eschar were observed in the high dose males. In females, an increase in barely perceptible to well-defined erythema and desquamation was seen in both low and high dose groups. A few incidences of focal/pinpoint to mild eschar were also noted in the high dose females.

      TABLE 2. Incidence of dermal observations after exposure to Granola 97
      Dermal observation Dosage Groups
      Control (0 mg/kg/day) Low (1000 mg/kg/day) High (3000 mg/kg/day)
      malefemale male female male female
      erythema, grade 1
      0
      0
      13
      12
      13
      15
      erthema, grade 2
      0
      0
      0
      1
      1
      4
      maximized, grade 4
      0
      0
      0
      0
      2
      1
      edema, grade 1
      0
      0
      0
      0
      1
      0
      eschar, focal/pinpoint
      0
      0
      2
      0
      8
      5
      eschar, mild
      0
      0
      0
      0
      1
      1
      eschar, moderate
      0
      0
      0
      0
      1
      0
      desquamation
      0
      1
      14
      13
      13
      15
      Data taken from p. 33 and 34, MRID 44438710

      Top of Page

    2. Body Weight and Weight Gain

      3. Small, but statistically significant (p # 0.05) decreases in body weight of high dose males were observed on days 36 (-8%) and 43 (-9%). No decreases were seen at the other time points tested and there were no changes in body weight in low dose males or in any female dosed group.

      Low dose males showed a decrease in body weight gain for days 64-71 (-71%, p#0.05) but an increase for days 71-78 (+260%, p#0.01). High dose males had a decrease in body weight gain for days 29-36 only (-30%, p#0.05). Female mice showed no changes in body weight gain during the study.

    3. Food Consumption and Compound Intake



      1. Food consumption

        2. Mean food consumption was increased in high dose females for days 57-64 (+4%, p # 0.01), 71-78 (+5%, p # 0.05) and 78-85 (+10%, p # 0.05). No changes were observed for low dose females or males of any dose group.

    4. Ophthalmoscopic Examination

      5. No ocular lesions associated with the test substance were observed.

    5. Blood Work



      1. Hematology

        2. No statistically significant differences were observed except for increased segmented neutrophils in low dose males and high dose females (both +100%, p#0.05), and increased activated partial thromboplastin time in high dose females (+6%, p#0.05). Although these values were statistically increased above controls, they were still within normal ranges seen historically at the testing site and are not considered treatment-related.

      2. Clinical chemistry

        3. In males, phosphorus was increased in the low dose group (+13%, p#0.05), while total protein, globulin and urea nitrogen were increased in the high dose group (+5%, p#0.05; +8%, p#0.01; +6%, p#0.01; respectively). Glucose was decreased in both low and high dose males (-17%, p#0.001; and -20%, p#0.001). In females, chloride was decreased in the high dose group (-2%, p#0.05). In both low and high dose females, globulin was increased (+6%, p#0.05; +11%, p#0.001), A/G ratio (-8%, p#0.01; -12%, 0.001) and glucose (-14%, p#0.001; -15%, p#0.001) were decreased. Although these values were statistically significantly different than controls, they remained within normal ranges seen historically at the testing site and are not considered treatment-related.

    6. Neurotoxicity

      7. No evidence of treatment-related neurological effects were observed.

      Top of Page

    7. Sacrifice and Pathology



      1. Organ weight

        2. Selected organ weights are summarized in Table 3. Absolute liver weight was statistically significantly increased in high dose females (+18%, p#0.001) and relative liver weight was increased in both high dose males (+9%, p#0.05) and females (+15%, p#0.001). Relative kidney weight (+12%, p#0.001) and adrenal weight (+15%, p#0.05) was increased only in high dose males.

        No changes in absolute or relative organ weights were observed in low dose male or low dose female groups.

        TABLE 3. Selected absolute and relative organ weights (g) for rats exposed dermally to Granola 97
        Organ Males Females
        Control Low dose High dose Control Low dose High dose
        Liver
        Absolute organ weights
        13.84
        13.28
        14.30
        7.58
        7.96
        8.92**
        Liver/total final body weight Relative organ weights
        3.22
        3.11
        3.53*
        3.28
        3.36
        3.77**
        Kidney/total final body weight
        0.87
        0.90
        0.97**
        0.92
        0.94
        0.94
        Adrenal/final brain weight
        2.81
        3.12
        3.23*
        3.65
        3.68
        3.79

        * p # 0.05
        ** p # 0.001

        Data taken from Tables 10,11 and 12, pgs 92-102 MRID 44438710

      2. Gross pathology

        3. No treatment-related gross lesions were observed in either animals surviving until termination of the study or in the five animals found dead during the study.

      3. Microscopic pathology -


        1. Non-neoplastic

          2. Treatment-related skin lesions were noted in the treated skin of high dose male and female animals (Table 4). These lesions included an increase in the incidence and severity of acanthosis, chronic inflammation and parakeratosis.

          Treatment-related lesions were also observed in the kidneys of high dose male animals. Eosinophilic hyaline droplets were present within the cytoplasm of the tubular lumen in the renal cortex of 3/15 control males and 15/15 high dose males. Because of the morphology of the droplets and the occurrence in only male rats, the authors suggested that the droplets were alpha-2u-globulin inclusions. High dose males also showed an increase in the incidence and severity of chronic progressive nephropathy. The authors suggested that these lesions were a secondary effect of the injury that results from the hyaline droplet formation.

          TABLE 4. Selected microscopic pathology for rats exposed dermally to Granola 97
          Pathological finding Males Females
          Control
          n=15          		 Low dose
          n=2          		 High dose
          n=15          		 Control
          n=15          		 Low dose
          n=2          		 High dose
          n=15
          Treated skin
          		  
          Acanthosis
          8
          0
          14
          0
          1
          12
          minimal
          8
          0
          5
          0
          1
          6
          mild
          0
          0
          9
          0
          0
          6
           
          Inflammation
          3
          0
          15
          2
          1
          15
          chronic-minimal
          1
          0
          4
          2
          1
          6
          chronic-mild
          2
          0
          10
          0
          0
          9
          chronic-active
          0
          0
          1
          0
          0
          0
           
          Parakeratosis
          0
          0
          1
          0
          0
          4
          minimal
          0
          0
          0
          0
          0
          4
          mild
          0
          0
          1
          0
          0
          0
          Kidneys  
          Hyaline droplets
          3
          0
          15
          0
          0
          0
          mild
          2
          0
          1
          0
          0
          0
          minimal
          1
          0
          3
          0
          0
          0
          moderate
          0
          0
          11
          0
          0
          0
           
          CP* nephropathy
          2
          0
          10
          2
          0
          0
          minimal
          2
          0
          4
          2
          0
          0
          mild
          0
          0
          6
          0
          0
          0

          *chronic progressive

          Data taken from Appendix O, pages 340-350, MRID 44438710

        2. Neoplastic

          3. No neoplastic lesion were reported.

    Top of Page

    Discussion

    1. Discussion

      2. The data presented in this 90 day subchronic dermal study show that moderate toxic effects are observed in rats exposed to 3000 mg/kg/day of Granola 97. These effects include dermal observations (increased erythema, edema and eschar) and histological changes in treated skin (increased acanthosis and inflammation) and kidneys (hyaline droplet formation and chronic progressive nephropathy in males). No significant toxic effects were observed in animals exposed to 1000 mg/kg/day.

      No treatment-related mortalities were seen in this study. Exposure to Granola 97 produced a variety of effects in a number of other parameters, but these observations are unlikely to be biologically significant. Decreased body weight and/or body weight gain in low and high dose males was observed only at a few time points, and the final differences in these parameters were not statistically significant. Female animals showed no changes in body weight or body weight gain, but food consumption was increased at certain time points in the high dose females. While any treatment-related weight loss in the female high dose group may have been masked by this increased food consumption, overall food consumption was not significantly different over the course of the study.

      Differences in hematologic and clinical chemistry values were also observed. However, no clear dose-related trends were established and the differences, while statistically valid, were not biologically significant. Changes in relative and absolute organ weights were noted as well, but again no dose-related trends were obvious.

      The author of this study states that the NOEL is 3000 mg/kg/day. The author suggests that the increased incidence and severity of hyaline droplet formation and chronic progressive nephropathy in male rats are associated with alpha-2u -globulin inclusions and these inclusions are specific to the male rat and have no known significance in humans. The reviewers agree with the assumptions concerning the nephropathy. However, 3000 mg/kg clearly produced an increase in the incidence and severity of acanthosis and inflammation in treated skin. In addition, the increased severity of erythema, eschar and edema in the high dose groups suggest toxic effects. Consequently, the reviewer disagrees with the author's conclusions and establishes the NOEL as 1000 mg/kg/day and the LOEL as 3000 mg/kg/day based on dermal effects.

    2. Study Deficiencies

      3. Minor: Only two doses of Granola 97 were tested, but a NOEL and LOEL could be established from the data presented. The lack of an intermediate dose does not compromise the study.

Top of Page

Publications | Glossary | A-Z Index | Jobs

Local Navigation

Jump to main content.