Iodomethane

Repeated dose toxicity: dermal

Administrative data

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 March 2001 to 29 August 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Iodomethane

EC Number:

200-819-5

EC Name:

Iodomethane

Cas Number:

74-88-4

Molecular formula:

CH3I

IUPAC Name:

iodomethane

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Iodomethane
- Physical state: liquid
- Analytical purity: 99.7%
- Impurities (identity and concentrations): 0.2 % water and <0.1 % methanol
- Composition of test material, percentage of components: not stated
- Isomers composition: not stated

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 9 weeks
- Weight at study initiation: males: 262 to 313 g and females: 182 to 232 g
- Housing: All animals were housed individually in clean, wire-mesh cages suspended above cage-board.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 16 days

DETAILS OF FOOD AND WATER QUALITY: Contaminants were not present in animal feed or water at levels expected to interfere with the objectives of this study.

ENVIRONMENTAL CONDITIONS
- Temperature: 20.6 to 21.7 °C
- Humidity: 36 to 60 %

Administration / exposure

Type of coverage:

occlusive

Vehicle:

corn oil

Details on exposure:

TEST SITE
- On the day prior to dose administration the hair was clipped from the back of each animal, from the scapula (shoulder) to the wing of the ileum (hipbone) and halfway down the flank of each side of the animal.
- The control or appropriate test material was applied to the shaved, intact dorsal skin of each animal for six hours per day for 21 consecutive days. Control group animals were dosed with the vehicle at a volume equal to the dose volume administered to the high dose group. Doses were applied evenly by gentle inunction using a glass rod over the maximum area possible.
- The area of test material application was measured and recorded once per week for an arbitrarily selected animal of each sex in each group [Total body surface area (cm^2) = K x body weight (grams)(^2/3); K = 9 for rats]. The mean area of coverage during the study was approximately 24, 25, 20 and 21 % for males in the control, 30, 300 and 1000 mg/kg/day groups, respectively. The mean area of coverage during the study was approximately 24, 21, 19 and 17 % for the females in these same groups, respectively.
- The application site for each animal was covered with a wrap consisting of a gauze binder around the trunk, which was then covered with an impervious plastic wrap and secured with several overwraps of tape. The corners of the application site were marked with indelible ink to allow proper identification of the treated and untreated skin.

REMOVAL OF TEST SUBSTANCE
- At the end of the six-hour exposure period, the dressings were removed. The test sites were gently washed by disposable paper towels with tepid tap water to remove any residual test material, and then gently dried.

TEST MATERIAL
- Amount(s) applied: 2 mL/kg

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

- On the first day of dosing for the males, 5 mL samples were collected from the top, middle and bottom strata of each test material formulation for stability and homogeneity determinations.
- Subsequent concentration analyses were performed concurrently with administration of newly prepared dosing formulations periodically throughout the study.

- Gas Chromatography:
Instrument: Hewlett Packard 5890A (Series II) gas chromatograph equipped with an FID detector, a HP Headspace analyser
Column: J & W Scientific GS-GasPro, 30 m x 0.316 mm ID (0.25 μm film thickness)
Temperature (Program): 70 °C for 1.0 min, ramp at 40 °C/min to 230 °C, hold 2 min
Carrier gas: Helium set at 12psi (EPC constant flow on)
Injector temperature: 225 °C
Injection volume: 1 mL splitless
Detector: FID at 225 °C
Retention time: Approximately 4.7 min

- Headspace Parameters:
Zone temp: Oven 50 °C, Loop 100 °C and Transfer Line 100 °C
Event Times: GC cycle time 10 min, Vial EQ time 7 min, Pressuriation time 0.20 min, Loop fill time 0.20 min, Loop EQ time 0.05 min and Inject time 0.20 min.

- Preparation of Calibration Stock Solutions: The calibration stock solutions were prepared by transferring under the surface of the oil, the appropriate amount of test material into a 10 mL volumetric flask containing approximately 7 mL of corn oil. The contents were diluted to a final volume of 10 mL with corn oil. These stocks were prepared in the concentration range of 10 to 600 mg/mL.

- Preparation of Quality Control Samples: The quality control (QC) stock solutions were prepared as above. These stocks were prepared at the concentrations of 20, 100 and 400 mg/mL.
- Sample Processing: Calibration, QC’s, and formulation samples were prepared for analysis by transferring 30 μL of the sample, into a headspace vial containing 970 μL of corn oil.
- Concentration Quantitation: A calibration curve was constructed for each set of analyses. The test material peak area (y) and the theoretical concentrations of the calibration standards (x) were fit with a least-squares regression analysis to the ln-quadratic function: ln(y) = a*[ln(x)]^2 + b*ln(x) + c. Concentrations were back-calculated from the results of the regression analysis using a PC spreadsheet program. The concentration data were transferred to another Excel spreadsheet, where appropriate summary statistics, i.e., means, standard deviations (SD), relative standard deviations (RSD), and percent relative error (%RE) were calculated and presented in tabular form.

RESULTS
- Under the described chromatographic conditions, the retention time of the test material was approximately 4.7 minutes. The total analysis time required for each run was approximately 10 minutes. The validity of the assay procedure was established through a careful study of the calibration reproducibility, accuracy, precision, ruggedness, and stability of the test material in quality control stocks and dosing formulations.
- Specificity/Selectivity: The assay specificity/selectivity was confirmed when GC analysis of control formulations revealed that there were no significant peaks near the retention time for the test material.
- Calibration Reproducibility: During each of three validation sets, triplicate calibration samples at five concentration levels were prepared and analysed as described above. Single injections were made of each processed calibration sample. The resulting peak area-concentration data were fit to the ln- quadratic function using the least-squares regression analysis. The results of the regression analyses were used to back-calculate from the peak area data the corresponding concentrations. The reproducibility of the calibration curve data was considered valid when the inter-set variability (RSD) of the back-calculated concentrations at each concentration level was ≤ 15 %, except at the lowest concentration level where ≤ 20 % was acceptable; and the mean back-calculated concentrations at each concentration level were within 15 % of the theoretical values (% RE within ± 15 %), except at the lowest concentration level where % RE ≤ 20 % was acceptable. The inter-set variability (RSD) of the back-calculated concentrations at each level ranged from 2.4 to 6.3 %. The inter-set concentration means had % RE values ranging from -0.89 to 2.5 %. Based on these criteria and the resulting validation data, the reproducibility of the calibration data was acceptable.
- Precision and Accuracy: During each of four validation sets, triplicate quality control (QC) samples at three concentration levels were prepared and analysed as described above. Single injections were made of each processed QC sample. The results of the regression analyses were used to calculate from these QC peak area data the corresponding concentrations. The variability (RSD) of these calculated QC concentration data was used as a measure of assay precision. The precision of the method was considered acceptable when the inter-set RSD of the calculated concentrations at each QC concentration level is ≤ 10 %. The difference from theoretical of the calculated QC concentration means (% RE) was used as a measure of assay accuracy. The accuracy of the method was considered acceptable when the inter-set concentration means of the calculated concentrations at each QC concentration level had % RE values within ± 15 %. The inter-set variability (RSD) of the calculated concentrations at each level (precision) ranged from 2.4 to 9.0 %. The inter-set concentration means had % RE values (accuracy) ranging from –1.1 to 8.2 %. Based on the criteria mentioned above, the precision and accuracy of the test material assay were acceptable.
- Stability of QC Stock Solutions: The QC stock solution prepared on 3/17/01 and initially used in validation set #1 was stored refrigerated. After 4 days, it was used to prepare triplicate samples at each of the three QC concentrations. The analysed concentrations were 79.4, 103, and 101 % of the respective time-zero values. Stability of the refrigerated stock was demonstrated for the mid and high QC stock. The QC stock should be prepared fresh for each analysis.
- Assay Ruggedness: In this assay validation, ruggedness is referred to as the ability of another analyst to successfully perform the procedure as described. Assay ruggedness is demonstrated when two or more different analysts independently and successfully conduct at least one session of the required three validation sets. Assay ruggedness was successfully demonstrated for this procedure.
- Homogeneity of Dosing Formulations: A representative set of formulations was prepared by the pharmacy department on 3/15/01 for the evaluation of homogeneity and stability. Samples from the top, middle and bottom of the formulation batches were collected and analysed. Each group met the requirement for homogeneity, i.e., the RSD for the overall mean concentration was ≤ 10 % at a concentration that is within the acceptable limits (%RE ± 15 %); with the exception of group 2 RSD and the group 4 mean % of target.
- Stability of Dosing Formulations: Samples from the middle of the formulation batches were stored at room temperature for 6-hours. The mean concentrations ranged from 97.1 to 108 % of the time zero concentrations, and, therefore, the formulations were considered stable.
- Summary of Concentration Analysis: The analysed formulations; (3/23/01, 3/29/01, 4/05/01) met the requirements for concentration acceptability for suspension formulations, i.e., the analysed concentrations were within 15 % of the target dose concentrations. Formulations prepared on 3/19/01 (Group 2) and 3/20/01 (all groups) did not meet requirements for concentration acceptability.

Duration of treatment / exposure:

6 hours per day, for 21 consecutive days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

30 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosage levels were selected based on the results of a 5-day range-finding study.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS AND DETAILED CLINICAL OBSERVATIONS
- The animals were observed twice daily, in the morning and afternoon, for mortality and moribundity. All animals received a clinical examination prior to application of the control or test material.

DETAILED CLINICAL OBSERVATIONS: Yes
- Each animal received a detailed physical examination once weekly, beginning approximately one week prior to the initiation of test material administration, and ending on the day of necropsy. During detailed physical examinations, the animals were removed from their home cages and placed in a standard arena for observation.

DERMAL IRRITATION: Yes
- Application sites were examined for erythema, oedema and other dermal findings once per week at the time of the detailed physical examination. Hair was clipped from the backs of the animals as necessary to facilitate dermal scoring. Erythema and oedema were evaluated in accordance with the method of Draize, based on a four-step grading system of very slight, slight, moderate, and severe. Other dermal findings, if present, were noted.

BODY WEIGHT: Yes
- Individual body weights were recorded weekly, beginning approximately one week prior to test material administration. Mean body weight changes were calculated for each corresponding interval. A final (fasted) body weight was collected for each animal on the day of scheduled necropsy.

FOOD CONSUMPTION:
- Individual food consumption was measured weekly, beginning approximately one week prior to test material administration. Food intake was calculated as g/animal/day for the corresponding body weight intervals.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Ocular examinations were conducted on all animals prior to initiation of test material administration (study week -1) and during study week 3. All ocular examinations were conducted using an indirect ophthalmoscope (or other suitable equipment), preceded by mydriasis.

CLINICAL PATHOLOGY: Blood and urine samples for clinical pathology evaluation were collected from all surviving animals at the time of the primary necropsy (study week 3). Blood was collected for hormone level analysis the day prior to necropsy from the lateral tail vein of the non-fasted, non-anesthetiSed animals. For other analyses, the animals were fasted overnight prior to collection of blood samples from the vena cava. Urine was collected overnight using metabolism cages.

- HAEMATOLOGY PARAMETERS: Total Leukocyte Count (White Cell), Erythrocyte Count (Red Cells), Hemoglobin, Hematocrit, Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Platelet Count (Platelet), Prothrombin Time, Activated Partial Thromboplastin Time (APTT), Reticulocyte Count (Percent (Reticulocyte) and Absolute (Retic Absolute)), Differential Leukocyte Count- (Percent and Absolute, -Neutrophil, -Lymphocyte, -Monocyte, -Eosinophil and –Basophil), Platelet Estimate and Red Cell Morphology (RBC Morphology).

- CLINICAL CHEMISTRY PARAMETERS: Albumin, Total Protein, Globulin, Albumin/Globulin Ratio (A/G Ratio), Total Bilirubin (Total Bili), Urea Nitrogen, Creatinine, Alkaline Phosphatase (Alkaline Phos’tse), Alanine Aminotransferase (Alanine Transfer), Aspartate Aminotransferase (Aspartat Transfer), Gamma Glutamyltransferase (Glutamyl Transfer), Glucose, Total Cholesterol (Cholesterol), Calcium, Chloride, Phosphorus, Potassium, Sodium and Triglycerides (Triglyceride).

- URINALYSIS PARAMETERS: Specific gravity (SG), pH, Urobilinogen (URO), Total Volume (TVOL), Colour (CLOR), Appearance (APP), Protein (PRO), Glucose (GLU), Ketones (KET), Bilirubin (BIL), Occult Blood (BLD), Leukocytes (LEU), Nitrites (NIT) and Microscopy of sediment.

- SERUM HORMONE PARAMETERS: T3, TSH, T4 and T3/T4 Ratio

- NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- A complete necropsy was conducted on all animals found dead or euthanised in extremis and at the scheduled necropsies. All animals were euthanised by isoflurane anesthesia followed by exsanguination. The necropsy included examination of the external surface, all orifices, and the cranial, thoracic, abdominal and pelvic cavities including viscera. At the time of necropsy the following tissues and organs were collected and preserved in 10 % neutral buffered formalin: Adrenal glands (2), Aorta, Bone with marrow (Sternum and Femur), Bone marrow smear, Brain (Forebrain, Midbrain and Hindbrain), Epididymides (2, Placed in Bouin’s solution), Exorbital lacrimal glands (2), Eyes with optic nerve (2, placed in Davidson's solution), Gastrointestinal tract (Oesophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon and Rectum), Harderian gland (2), Heart, Kidneys (2), Larynx, Liver (sections of two lobes), Lungs (including bronchi, fixed by inflation with fixative), Lymph node (Mandibular and Mesenteric), Mammary gland (females only), Nose, Ovaries with oviducts (2), Pancreas, Parathyroids (if present), Peripheral nerve (sciatic), Pharynx, Pituitary, Prostate, Salivary glands [mandibular (2)], Seminal vesicles (2), Skeletal muscle (rectus femoris), Skin (treated and untreated), Spinal cord (Cervical, Thoracic and Lumbar), Spleen, Testes (2, Placed in Bouin’s solution), Thymus, Thyroid, Trachea, Urinary bladder, Uterus with cervix, Vagina and Gross lesions (when possible).
- The following organs were weighed from all animals at the scheduled necropsy: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries (with oviducts), Spleen, Testes, Thymus, Thyroid and Uterus. Paired organs were weighed together. Organ to final body weight and organ to brain weight ratios were calculated.

HISTOPATHOLOGY: Yes
- After fixation, protocol-specified tissues were trimmed, sectioned at 5-8 microns, mounted on glass microscope slides and stained with haematoxylin and eosin. All tissues listed in gross pathology were examined microscopically from all animals in the control and 1000 mg/kg/day groups euthanised at the scheduled necropsy and for all animals found dead or euthanised in extremis. The treated and untreated skin, liver, kidneys, adrenal cortex, bone marrow (sternum and femur), lymph node (mandibular and mesenteric), spleen, thymus, glandular stomach, prostate, seminal vesicles and gross lesions (when possible) were examined for all animals in the 30 and 300 mg/kg/day groups.

Statistics:

- All analyses were conducted using two-tailed tests for significance levels of 5 and 1 % comparing the treated groups to the control group by sex. All means are presented with standard deviations (S.D.) and the number of sampling units (N) used to calculate the means. Statistical analyses were not performed if the number of animals was two or less. All statistical tests were performed using appropriate computing devices or programs.
- Body weights, body weight changes, food consumption, clinical pathology values and absolute and relative organ weight data were subjected to a one-way analysis of variance (ANOVA), followed by Dunnett’s test if the ANOVA revealed statistical significance (p<0.05). Clinical laboratory values for cell types that occur at a low incidence (i.e. monocytes, eosinophils and basophils) were not subjected to statistical analysis.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related clinical observations were noted in the 300 and 1000 mg/kg/day group males and females. These findings included wet yellow staining in the urogenital and/or anogential areas. There were no other test material-related clinical findings.
- It should be noted that a 30 mg/kg/day group female was noted with a swollen facial area on study day 19. This animal was observed thrashing itself around the cage in order to get free of the wrapping material applied at dosing. The swollen facial area was most likely the result of the animal hitting its head on the feed jar and cage walls, and not a result of test material treatment.
- Additional clinical findings observed in the test material-treated groups were noted similarly in the control group, were not present in a dose-related manner, were noted at a low incidence, typically in single animals and/or were clinical signs commonly observed in rats of this strain and age.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related dermal findings were noted in the 30, 300 and 1000 mg/kg/day group males and females. These findings included very slight to severe erythema and oedema, fissuring, more pronounced desquamation, eschar, exfoliation, atonia, subcutaneous haemorrhage, ulceration and coriaceousness. These findings were noted as early as the first week of dosing. Very slight erythema was noted for 2/10 and 5/10 animals in the 30 mg/kg/day male and female groups, respectively, and desquamation was noted for 7/10 animals/sex at this dose level. The severity of erythema and oedema tended to increase over the course of the study for the 300 and 1000 mg/kg/day groups. Severe erythema was noted for the 300 and 1000 mg/kg/day group males and females primarily during the last two weeks of the study. The severity of oedema tended to increase with dose, as moderate oedema was generally noted in the 300 mg/kg/day group males and females and severe oedema was primarily noted in the 1000 mg/kg/day group males and females. Desquamation, eschar and exfoliation were noted for all animals in the 300 and 1000 mg/kg/day groups. Atonia was noted for all 1000 mg/kg/day group animals and for 9/10 and 9/10 animals in the 300 mg/kg/day male and female groups, respectively. Fissuring and coriaceousness were common in both the 300 and 1000 mg/kg/day group males and females. Subcutaneous haemorrhage was noted for one 300 mg/kg/day group female after the second week of dosing.
- There were no other definite test material-related dermal findings. Several occurrences of desquamation were also noted in the control group during the study.

Mortality:

mortality observed, treatment-related

Description (incidence):

- Four males were found dead or euthanized in extremis during the study.
- One 300 mg/kg/day group male was found dead on study day 18. Two 1000 mg/kg/day group males were euthanised in extremis on study days 11 and 10, respectively. Another 1000 mg/kg/day group male was found dead on study day 4, this animal was noted with tremors, dried red material around the nose and wet red material on the urogenital area prior to death. The cause of death or moribundity for three animals was urinary tract obstruction. Each of these animals had hydronephrosis, a condition that can be acquired or secondary to an obstructive lesion of the urinary tract distal to the kidney. The cause of death for the other animal could not be determined. The condition that lead to the death of these animals was most likely test material-related.
- All other animals survived to the scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related decreases in mean body weights and body weight gains were noted in the 300 and 1000 mg/kg/day group males. These decreases were statistically significant (p < 0.05 or p < 0.01) throughout the study. A test material-related transient decrease (statistically significant at p < 0.05) in mean body weight gains was also noted in the 1000 mg/kg/day group females during study week 0 to 1.
- There were no other test material-related changes noted during the study. No other remarkable differences were noted when the control and treated groups were compared.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Test material-related decreases in mean food consumption (often statistically significant at p < 0.01) were noted in the 300 and 1000 mg/kg/day group males throughout the study. A test material-related transient decrease in mean food consumption (statistically significant at p<0.01) was also noted in the 1000 mg/kg/day group females during study week 0 to 1.
- There were no other test material-related changes in mean food consumption noted during the study. There were no other remarkable differences noted when the control and treated groups were compared.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

- No oculopathic lesions indicative of a toxic effect were observed.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related changes in haematology parameters were noted in the 300 and 1000 mg/kg/day group males and/or females and consisted of lower red blood counts, haemoglobin, haematocrit, APTT, white blood cell counts and lymphocyte counts and higher neutrophil counts, MCV, MCH and platelet counts.
- Statistically significant decreases (p<0.05 or p<0.01) were noted in mean red blood cell counts, haemoglobin and haematocrit in the 300 and 1000 mg/kg/day group males and females. Mean white blood cell counts were decreased in the 300 and 1000 mg/kg/day group males. Statistically significant (p<0.05 or p<0.01) increases and decreases were also noted in mean absolute and/or relative neutrophil and lymphocyte counts, respectively, for these groups. Necrosis of the treated skin and the subsequent inflammatory response seen microscopically were the direct cause of the white blood cell variations and most of the variations were typical of an acute inflammatory response. Also, the dose-related decrease in white cell counts in treated males and the absence of a definitive leukocytosis in the females suggests that the demand for leukocytes in the skin exceeded marrow production. Additional test material-related changes included decreased APTT and increased platelets in the 300 and 1000 mg/kg/day group males. MCV and MCH were increased in the 1000 mg/kg/day group females due to decreased red blood cell counts.
- There were no other test material-related changes in haematology parameters.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related changes in serum chemistry parameters were noted in the 300 and 1000 mg/kg/day group males and females and consisted of higher globulin, urea nitrogen, alkaline phosphatase, chloride, sodium, aspartate aminotransferase, alanine aminotransferase and gamma glutamyltransferase and lower albumin, A/G ratios, triglycerides and calcium.
- Mean globulin, urea nitrogen, alkaline phosphatase and chloride were increased and statistically significant (p<0.05 or p<0.01) in the 300 and 1000 mg/kg/day group males and females. Mean albumin and A/G ratio values were decreased in these groups. Mean sodium and aspartate aminotransferase were increased in the 300 mg/kg/day group males and females and the 1000 mg/kg/day group females. Mean triglycerides were decreased and alanine aminotransferase was increased in the 300 and 1000 mg/kg/day group females. Mean gamma glutamyltransferase levels were increased in the 1000 mg/kg/day group males and females. Mean calcium was decreased in the 300 and/or 1000 mg/kg/day group females. The decrease in calcium values was considered to be an effect of hypoalbuminemia and not an accurate reflection of true serum calcium concentration.
- There were no other test material-related changes in serum chemistry parameters noted during the study. However, many statistically significant (p < 0.05 or p < 0.01) differences were noted when the control and treated groups were compared. Mean potassium was increased and mean total protein was decreased in the 300 mg/kg/day group males and females, respectively. Mean creatinine was decreased in the 300 and 1000 mg/kg/day group females. Mean cholesterol levels were increased in the 1000 mg/kg/day group males. Mean glucose was increased in the 300 mg/kg/day group females. These changes were not considered test material-related as the alterations were slight, occurred without apparent dose responses and/or were inconsistent between sexes. There were no other remarkable changes when the control and treated groups were compared.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Test material-related changes in urinalysis parameters were noted in the 300 and 1000 mg/kg/day group males and consisted of higher specific gravity values. These changes were statistically significant (p < 0.01) in the 300 and 1000 mg/kg/day group males. This change most likely represented a physiological response to fluid or electrolyte disturbances induced by inflammation and necrosis of the skin or by decreased water consumption that most likely accompanied lower food consumption values. Thus, the differences in specific gravity values were not a direct test material-related effect and were not considered adverse.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related changes in mean absolute and/or relative organ weights (statistically significant at p < 0.05 or p < 0.01) were noted in the brain, spleen, thymus and adrenal glands of the 300 and 1000 mg/kg/day group males and/or females. In the spleen, the inconsistent trends in organ weights were best explained by the effects of lymphoid depletion/necrosis (tendency for lower weights) and extramedullary haematopoiesis (tendency for increased weights) noted microscopically. Both these microscopic changes were considered test material-related; therefore, decreased mean absolute spleen weights in the 300 mg/kg/day group males and increased mean absolute and relative spleen weights in the 1000 mg/kg/day group females were considered test material-related.
- Absolute brain weights in the 300 and 1000 mg/kg/day groups were lower than control group values. These lower values were not related to lower body weights in these groups because brain weights remain relatively unaffected by body weight fluctuations. Furthermore, the gender difference in body weight effects was not present in the brain weight changes. Therefore, the brain weight differences were considered test material-related. However, there were no microscopic changes to explain these lower brain weights.
- In the thymus, decreased absolute and relative weights were noted in the 300 and 1000 mg/kg/day group males and females. These weight changes correlated with lymphoid necrosis/depletion noted microscopically in these groups.
- Increased mean absolute and/or relative adrenal gland weights were noted for males and females in the 300 and 1000 mg/kg/day groups. These weight changes corresponded with vacuolar change and/or extramedullary haematopoiesis noted microscopically in the adrenal cortex of treated animals.
- There were no other test material-related changes in organ weights. However, many statistically significant (p < 0.05 or p < 0.01) differences were noted when the control and treated groups were compared. Mean kidney and heart weights were decreased in the 300 and 1000 mg/kg/day group males. Epididymides and testes weights were decreased in the 1000 mg/kg/day group males and ovary and uterus weights were decreased in the 300 and/or 1000 mg/kg/day group females. Due to the low final body and brain weights in the 300 and 1000 mg/kg/day group males and females, numerous organ weights relative to final body or brain weight were statistically significant when compared to control group values. In the absence of microscopic changes to support these mean absolute weight decreases, these changes were most likely related to lower terminal body weights and not a direct test material effect.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related macroscopic findings were noted in the treated skin, thymus, stomach and seminal vesicles of the 300 and 1000 mg/kg/day group males and/or females. Slough skin was noted for 8/9 males and 10/10 females in the 300 mg/kg/day group and 7/7 males and 9/10 females in the 1000 mg/kg/day group. Scabbing and thickening of the skin were also noted at increased incidences in the 300 and 1000 mg/kg/day group males and females. Microscopic findings of necrosis and exfoliation, both of the epidermis, commonly correlated with the findings noted above.
- Test material-related changes were seen affecting the skin at treated and untreated sites in the skin. Reddened, thickened, slough and/or scabbing of treated skin were seen in all animals in the 300 and 1000 mg/kg/day groups. Single instances of scabbing were noted in both sexes at 30 mg/kg/day and in one control group male. Similar changes were noted at lower incidences in untreated skin primarily in the 1000 mg/kg/day groups. These findings often correlated with microscopic findings of necrosis and exfoliation noted in both treated and untreated skin. Macroscopic changes in the untreated skin were not noted in the 30 mg/kg/day or control groups.
- A small thymus was noted for 2/10 males and 3/10 females in the 300 mg/kg/day group and for 8/10 females in the 1000 mg/kg/day group. This change correlated with the microscopic finding of lymphoid necrosis or depletion noted in these groups.
- In the stomach, dark red areas were noted in 3/10 females in the 1000 mg/kg/day group. Dark red gastric contents were noted for a few males and females at dose levels of 300 and 1000 mg/kg/day. Dark red areas correlated with microscopic findings of erosion in the glandular stomach. Gastric erosion was considered the source of dark red contents throughout the gastrointestinal tract.
- Small and/or soft seminal vesicles were noted for 1/10 and 4/10 males in the 300 and 1000 mg/kg/day groups, respectively. This change correlated with decreased glandular secretion noted microscopically.
- There were no other remarkable macroscopic findings noted at the scheduled necropsy. Findings noted for treated animals were observed similarly in the control group and/or were noted at low incidence, typically single animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- For the unscheduled deaths, urinary tract obstruction, often accompanied by decreased glandular secretion, inflammation and/or oedema of the seminal vesicles or prostate, was a common cause of death or moribundity in the 300 and 1000 mg/kg/day groups. This conclusion was supported by the condition of hydronephrosis in three of the four unscheduled deaths.
- Test material-related microscopic changes were noted at the scheduled necropsy in the skin, adrenal cortex, bone marrow, lymph nodes, spleen, thymus, glandular stomach, prostate and seminal vesicles in the 300 and 1000 mg/kg/day groups.
- The most extensive changes, and the only findings directly related to the test material, were noted in the skin and included exfoliation, necrosis, epithelial hyperplasia, subacute inflammation, oedema and intraepithelial vesicle. Exfoliation was the term used to describe the full-thickness loss of a necrotic epidermis, often including hair follicles, and a small portion of the superficial dermis. This finding correlated with slough noted macroscopically. Histologically, there was an underlying regenerative epithelium that was often hyperplastic. In contrast, necrosis was used to describe a necrotic but otherwise intact epidermis and commonly correlated with the macroscopic findings of scabbing and/or thickened skin. Epidermal necrosis/ulceration commonly has dermal inflammation, oedema and epithelial hyperplasia as associated changes. In this study, subacute inflammation, oedema and epithelial hyperplasia were diagnosed only when they were not associated with epidermal necrosis. However, these three changes were often associated and commonly were subjacent to regions of exfoliation. Epithelial hyperplasia and hyperkeratosis, generally of minimal to mild severity, were commonly seen in the control and low-dose groups and were attributed to mechanical irritation of test material administration. The incidence and severity of hyperplasia increased with dose. Intra-epithelial vesicles were considered to represent oedema within the epidermis. These findings were seen in a small number of animals in the 30 mg/kg/day group, but all males and females in the 300 and 1000 mg/kg/day groups had varying combinations of these test material-related changes.
- In the skin, generally from the ventral abdominal region, necropsy findings similar to those seen in the treated skin were observed. One female from the 300 mg/kg/day group and 3/10 males and 9/10 females in the 1000 mg/kg/day group were noted with test material-related exfoliation and/or necrosis in regions other than the application site. The incidence of hyperkeratosis also tended to increase in a dose-related manner in treated females. Finding treatment-related skin lesions in areas other than the application site probably represents mechanical transfer of the test material between sites on the animal rather than systemic involvement of the skin. In the lymphoid tissues (spleen, gut associated lymphoid tissue, lymph nodes and thymus), lymphoid necrosis and/or depletion was seen in one or more tissues from 8/10 males and all females in the 300 mg/kg/day group and all animals in the 1000 mg/kg/day group. The thymus was the lymphoid tissue most commonly affected, but none of these findings were seen in the control or 30 mg/kg/day groups. Lymphoid depletion and necrosis were considered to be secondary to the stress associated with the treatment-related skin changes.
- In the bone marrow, hypercellularity was seen in 8/10 males and 6/10 females in the 300 mg/kg/day group and 9/10 males and 6/10 females in the 1000 mg/kg/day groups. Increased production of hematopoietic cells was also seen at lower incidences at sites of extramedullary haematopoiesis (spleen, liver and adrenal cortex). These changes were in response to the treatment-related inflammation and necrosis seen in the skin that resulted in the decreased red cell count and increased demand for neutrophils and were not a direct effect of test material treatment.
- In the adrenal cortex, cytoplasmic vacuolation was seen in 1/9 males from the 300 mg/kg/day group and 8/10 males and 5/10 females in the 1000 mg/kg/day group. The vacuoles were clear, round, well-defined and morphologically consistent with lipid. This change was considered to be secondary to stress and not a direct effect of treatment. Extramedullary haematopoiesis (EMH) was seen only in one male and seven females in the 1000 mg/kg/day group. This change was a manifestation of increased EMH in other tissues as well as bone marrow hypercellularity in response to the treatment-related skin changes and not a direct effect of the test material.
- In the glandular stomach, erosion was seen in 2/10 males and 1/10 females in the 300 mg/kg/day group and 3/10 females in the 1000 mg/kg/day group. This change was considered to be secondary to stress and not a direct effect of test material treatment.
- In the males, urinary tract obstruction was a common cause of early death or debilitation, accounting for 3 of 4 cases in the 300 and 1000 mg/kg/day groups. The animals commonly had decreased glandular secretion, inflammation, and/or oedema of the seminal vesicles or prostate that may have contributed to the urinary obstruction. When all animals were considered, 2/10 from the 300 mg/kg/day group and 3/10 from the 1000 mg/kg/day group had acute or chronic active inflammation of the prostate with inflammation extending to the seminal vesicles in one of the 1000 mg/kg/day animals. Since a control group male also had minimal chronic active inflammation of the prostate, the significance of the slightly increased inflammation in treated animals was uncertain. Similarly it was unclear if decreased secretion within the seminal vesicles, noted in 1/10 and 4/10 males in the 300 and 1000 mg/kg/day groups, respectively, was related to inflammation, general debilitation, or a direct effect of test material treatment.
- There were no other test material-related microscopic findings. Other findings noted in test material-treated animals occurred similarly in the control group, were considered spontaneous and/or were common changes observed in laboratory animals.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

SERUM HORMONES:
- Test material-related changes in serum hormones were noted in the 300 mg/kg/day group males and the 30 and 1000 mg/kg/day group females and consisted of decreased mean T4 values and increased mean T3 values during the study. These changes were statistically significant (p < 0.01) when the control and treated groups were compared. Mean T4 was decreased in the 300 mg/kg/day group males and the 1000 mg/kg/day group females. It is unclear if these decreases represented a reduction in thyroid function or were secondary to inflammation or hypoalbuminemia. Mean T3 was increased in the 30 mg/kg/day group females. The absence of a dose response and the inconsistency between sexes suggests that this change in T3 may be incidental and not test material-related. There were no other remarkable differences noted when the control and treated groups were compared.

Details on results:

CONCLUSIONS

- No oculopathic changes indicative of a test material-related effect were noted. One 300 mg/kg/day group male and three 1000 mg/kg/day group males were found dead or euthanized in extremis during the study. The cause of death or moribundity for three of these animals was attributed to possible urinary tract obstruction. The cause of death for the fourth animal (1000 mg/kg/day group male) could not be determined. The condition that lead to the death of these animals was most likely test material-related.
- Test material-related clinical findings were noted in the 300 and 1000 mg/kg/day group males and females and included wet yellow staining in the urogenital and/or anogenital areas.
- Test material-related decreases in mean body weights, body weight gains and food consumption were noted in the 300 and 1000 mg/kg/day group males throughout the study.
- Test material-related dermal findings were noted in the 30, 300 and 1000 mg/kg/day group males and females and included very slight to severe erythema and oedema, fissuring, desquamation, eschar, exfoliation, atonia, subcutaneous haemorrhage, ulceration and/or coriaceousness. These findings were noted as early as the first week of dosing. The severity of erythema and oedema tended to increase with dose and over the course of the study for the 300 and 1000 mg/kg/day groups.
- Test material-related changes in haematology parameters were noted in the 300 and 1000 mg/kg/day group males and females. Decreases were noted in mean red blood cell counts, haemoglobin, haematocrit, APTT and white blood cell counts in the 300 and 1000 mg/kg/day group males and/or females. Increases and decreases were also noted in mean absolute and/or relative neutrophil and lymphocyte counts, respectively, for these groups. Necrosis of the treated skin and the subsequent inflammatory response seen microscopically were the direct cause of the white blood cell variations and most of the variations were typical of an acute inflammatory response. Also, the dose-related decrease in white cell counts in treated males and the absence of a definitive leukocytosis in the females suggests that the demand for leukocytes in the skin exceeded marrow production. Increased platelets were noted in the 300 and 1000 mg/kg/day group males. MCV and MCH were also increased in the 1000 mg/kg/day group females due to decreased red blood cell counts.
- Mean globulin, urea nitrogen, alkaline phosphatase, sodium, aspartate aminotransferase, alanine aminotransferase, gamma glutamyltransferase and chloride were increased in the 300 and/or 1000 mg/kg/day group males and/or females. Mean albumin, triglycerides, calcium and A/G ratio values were decreased in these groups. The decrease in calcium values was considered to be an effect of hypoalbuminemia and not an accurate reflection of true serum calcium concentration.
- Test material-related changes in serum hormones were noted in the 300 mg/kg/day group males and the 30 and 1000 mg/kg/day group females and consisted of decreased mean T4 and increased T3 values.
- Test material-related changes in urinalysis parameters were noted in the 300 and 1000 mg/kg/day group males and consisted of higher specific gravity values.
- The most extensive microscopic changes were noted in the skin and included exfoliation, necrosis, epithelial hyperplasia, subacute inflammation, oedema and intraepithelial vesicle. Exfoliation correlated with slough noted macroscopically. Necrosis was commonly correlated with the macroscopic findings of scabbing and/or thickened skin. Epithelial hyperplasia and hyperkeratosis, generally of minimal to mild severity, were commonly seen in the control and low-dose groups and were attributed to mechanical irritation of test material administration. The incidence and severity of hyperplasia increased with dose as there was a regenerative and often hyperplastic epithelium underlying the exfoliated (sloughed) epidermis. Intra-epithelial vesicles were considered to represent oedema within the epidermis. These findings were seen in a small number of animals in the 30 mg/kg/day group, but all males and females in the 300 and 1000 mg/kg/day groups had varying combinations of these test material-related changes.
- In the lymphoid tissues, test material-related changes were noted for organ weights, macroscopic and/or microscopic examinations in the spleen and thymus of the 300 and 1000 mg/kg/day group males and females. Test material-related changes in organ weights were noted in the spleen and thymus of the 300 and 1000 mg/kg/day group males and females. A small thymus was noted for males and females at necropsy, correlating with decreased absolute and relative thymus weights and the histologic finding of lymphoid necrosis or depletion noted in these groups. The thymus was the lymphoid tissue most commonly affected with necrosis and depletion. In the spleen, the inconsistent trends in organ weights were best explained by the effects of lymphoid depletion/necrosis (tendency for lower weights) and extramedullary haematopoiesis (tendency for increased weights) noted microscopically. Since both these microscopic changes were related to treatment, decreased mean absolute spleen weights in the 300 mg/kg/day group males and increased mean absolute and relative spleen weights in the 1000 mg/kg/day group females were considered test material-related.
- Lymphoid necrosis and/or depletion was also noted in the gut associated lymphoid tissue and/or lymph nodes in the 300 and 1000 mg/kg/day groups. Lymphoid depletion and necrosis in all affected tissues were considered to be secondary to the stress associated with the treatment-related skin changes. Splenic extramedullary haematopoiesis (EMH) was considered to be secondary to the treatment-related inflammation and necrosis seen in the skin.
- Increased mean absolute and/or relative adrenal gland weights were noted for males and females in the 300 and 1000 mg/kg/day groups. These weight changes corresponded with cytoplasmic vacuolation of the adrenal cortex in males and/or females from the 300 and 1000 mg/kg/day groups. The vacuoles were clear, round, well-defined and morphologically consistent with lipid. This change was considered to be secondary to stress and not a direct effect of treatment. Extramedullary haematopoiesis (EMH) was seen only in one male and seven females in the 1000 mg/kg/day group. This change was a manifestation of increased EMH in other tissues as well as bone marrow hypercellularity in response to the treatment-related skin changes and not a direct effect of the test material.
- Test material-related decreases in mean absolute brain weights were noted in the 300 and 1000 mg/kg/day groups.
- In the stomach, dark red areas were noted in the 1000 mg/kg/day group females, correlating with microscopic findings of erosion in the glandular stomach of the males and/or females in the 300 and 1000 mg/kg/day groups. Dark red gastric contents were noted for a few males and females at dose levels higher than 300 mg/kg/day. Gastric erosion was considered the source of dark red contents throughout the gastrointestinal tract. Erosion of the glandular stomach was considered to be secondary to stress and not a direct effect of test material treatment.
- Other test material-related microscopic changes were noted in the bone marrow, prostate and seminal vesicles in the 300 and 1000 mg/kg/day groups. In the bone marrow, hypercellularity was seen in males and females in the 300 and 1000 mg/kg/day groups. Increased production of hematopoietic cells was also seen at lower incidences at sites of extramedullary hematopoiesis (spleen, liver and adrenal cortex). These changes were in response to the treatment-related inflammation and necrosis seen in the skin that resulted in the decreased red cell count and increased demand for neutrophils and were not a direct effect of test material treatment. In the males, urinary tract obstruction was a common cause of early death or debilitation, accounting for 3 of 4 cases in the 300 and 1000 mg/kg/day groups. The animals commonly had decreased glandular secretion (correlating with small and/or soft seminal vesicles noted macroscopically), inflammation, and/or oedema of the seminal vesicles or prostate that may have contributed to the urinary obstruction. Since a control group male also had minimal chronic active inflammation of the prostate, the significance of the slightly increased inflammation in treated animals was uncertain. Similarly it was unclear if decreased secretion within the seminal vesicles, noted in the 300 and 1000 mg/kg/day groups, was related to inflammation, general debilitation, or a direct effect of test material treatment.
- Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for systemic toxicity of the test material when administered dermally to rats for 21 consecutive days was 30 mg/kg/day.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

haematology

histopathology: neoplastic

mortality

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Summary of selected clinical chemistry and pathology findings (at termination)

Dose (mg/kg/day)		0	30	300	1000	0	30	300	1000
Sex		Male				Female
No. in group		10	10	10	10	10	10	10	10
Haematology and clinical chemistry (mean values)
Red cells (mil/µL)		7.19	7.18	6.16**	5.70**	6.11	5.66	5.08**	4.60**
White cells (thous/µL)		10.2	8.8	7.0*	6.4*	8.0	7.9	7.5	9.0
Haemoglobin (g/dL)		14.2	14.2	12.3**	11.4**	12.2	11.4	10.4**	9.9**
Haemocrit (%)		38.8	39.0	33.6**	31.7**	33.2	31.1	28.4	27.0
Globulin (g/dL)		2.1	2.21	3.1*	3.3**	2.3	2.3	3.0*	3.4**
Albumin/ Globulin ratio		1.84	1.77	0.94**	0.86**	1.79	1.77	1.01**	0.87**
Urea nitrogen (mg/dL)		14.0	15.1	32.4**	31.1**	19.4	19.5	34.2**	35.9**
Chloride (mEq/L)		101	100	107**	105**	100	101	108**	106**
Histopathology (incidence), Skin:
Hyperplasia (epithelial)		-	-	1 (1)	2 (3)	-	-	1 (10)	5 (10)
Necrosis		1 (10)	1 (10)	9 (9)	7 (7)	-	-	1 (10)	9 (10)
Exfoliation		-	-	1 (1)	2 (3)	-	-	1 (10)	4 (10)
Sub-acute inflammation		-	-	1 (1)	3 (3)	0 (10)	1 (10)	10 (10)	10 (10)

*Significant (p < 0.05) vs control;**Significant (p < 0.01) vs control

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for systemic toxicity of the test material when administered dermally to rats for 21 consecutive days was 30 mg/kg/day.

Executive summary:

The repeated dose dermal toxicity of the test material was investigated in accordance with the standardised guideline OPPTS 870.3200, under GLP conditions.

The possible toxic effects of the test material were evaluated in this 21-day dermal study in Crl:CD®(SD)IGS BR rats. The test material in the vehicle, corn oil, was applied seven days per week for three weeks to the shaved intact dorsal skin of each rat. The application sites were covered with gauze, occluded with plastic wrap and secured with non-irritating tape for a period of six hours per exposure. Following the exposure period, the application sites were gently washed with water and disposable paper towels to remove residual test material. Each of the three test material groups consisted of 10 males and 10 females. Dosage levels were 30, 300 and 1000 mg/kg/day. A concurrent control group received the vehicle on a comparable regimen. The animals were examined twice daily for mortality and moribundity. Clinical observations were performed once daily prior to exposure. Detailed physical examinations were performed and dermal observations were recorded weekly. Ophthalmological examinations were performed prior to dosing and during study week 3. Clinical pathology evaluations (haematology, serum chemistry, serum hormones and urinalysis) were performed prior to the scheduled necropsy. Complete necropsies were performed for all animals and selected organs weighed. Selected tissues were examined microscopically.

No oculopathic changes indicative of a test material-related effect were noted. One 300 mg/kg/day group male and three 1000 mg/kg/day group males were found dead or euthanized in extremis during the study. These deaths were attributed to test material administration.

Test material-related clinical findings were noted in the 300 and 1000 mg/kg/day group males and females and included wet yellow staining in the urogenital and/or anogenital areas.

Test material-related decreases in mean body weights, body weight gains and food consumption were noted in the 300 and 1000 mg/kg/day group males throughout the study.

Test material-related dermal findings were noted in the 30, 300 and 1000 mg/kg/day group males and females and included very slight to severe erythema and oedema, fissuring, desquamation, eschar, exfoliation, atonia, subcutaneous haemorrhage, ulceration and/or coriaceousness. The severity of the dermal reactions tended to increase with dose and/or over the course of the study. These findings were often accompanied by microscopic findings of exfoliation, necrosis, epithelial hyperplasia, subacute inflammation, oedema and intraepithelial vesicles.

Several haematology, organ weight and histological changes were noted in the 300 and 1000 mg/kg/day groups as secondary effects of treatment with the test material. Necrosis of the treated skin and the subsequent inflammatory response seen microscopically were the direct cause of the white blood cell variations. Most other variations in haematology parameters (red blood cell parameters and APTT) were typical of an acute inflammatory response. Increased urine specific gravity was also considered secondary to test material-related changes in the skin due to possible fluid or electrolyte imbalances. In the lymphoid tissues (spleen, gut associated lymphoid tissue, lymph nodes and thymus), necrosis and/or depletion of these tissues and/or changes in organ weights were considered secondary to stress associated with treatment-related skin changes. Additionally, changes in adrenal gland weights, cytoplasmic vacuolation of the adrenal cortex, erosion of the glandular stomach and hypercellularity of the bone marrow were also attributed to stress and considered secondary to treatment.

Test material-related changes were noted in serum chemistry and serum hormone parameters. Mean globulin, urea nitrogen, alkaline phosphatase, sodium, aspartate aminotransferase, alanine aminotransferase, gamma glutamyltransferase and chloride were increased in the 300 and/or 1000 mg/kg/day group males and/or females. Mean albumin, triglycerides, calcium and A/G ratio values were decreased in these groups. Changes in serum hormones noted in the 300 mg/kg/day group males and the 30 and 1000 mg/kg/day group females consisted of decreased mean T4 and increased mean T3 values.

Additional test material-related changes included decreased mean absolute brain weights in the 300 and 1000 mg/kg/day group males and females and microscopic changes in the prostate and seminal vesicles in the 300 and 1000 mg/kg/day group males. In the males, urinary tract obstruction was a common cause of early death or debilitation. The animals often had decreased glandular secretion (correlating with small and/or soft seminal vesicles noted macroscopically), inflammation, and/or oedema of the seminal vesicles or prostate that may have contributed to the urinary tract obstruction. The significance of the slightly increased inflammation of the prostate in treated animals was uncertain. Similarly, it was unclear if decreased secretion within the seminal vesicles, noted in the 300 and 1000 mg/kg/day groups, was related to inflammation, general debilitation, or a direct effect of test material treatment.

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for systemic toxicity of the test material when administered dermally to rats for 21 consecutive days was 30 mg/kg/day.