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Repeated dose toxicity: dermal

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Administrative data

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 7, 1989-April 27, 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Well documented study with significant information on study design and methodology to evaluate results according to OECD Guideline 411 Subchronic Dermal Toxicity: 90 Day Study. Deviations from OECD Guideline: females rats are below the suggested weight range, number of animals used was below the suggested 20 for each dose group; details on test substance analysis were lacking.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
females rats are below the suggested weight range, number of animals are below the suggested 20 for each dose group.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: liquid
- Storage condition of test material: Room temperature
Specific details on test material used for the study:
- Name of test material (as cited in study report): 6398-9-20
- Molecular weight: 230
- Substance type: Clear liquid
- Physical state: There was no apparent change in the physical state of the test substance during administration.
- Analytical purity: The purity, identify, strength and stability of the test substance were the responsibility of the sponsor.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, Massachusetts
- Age at study initiation: 47 days old
- Weight at study initiation:
Males (Mean weight in grams)
Control: 204.5
50 mg/kg: 203.9
80 mg/kg: 206.7
250 mg/kg: 204.2
Control recovery: 202.2
250 mg/kg recovery: 203.5

Females (Mean weight in grams)
Control: 147.9
50 mg/kg: 142.3
80 mg/kg: 144.6
250 mg/kg: 146.3
Control recovery: 146.4
250 mg/kg recovery: 141.9

- Housing:Stainless steel 1/2 inch wire mesh cages. Cage sizes were in accordance with the Guide for the /care and Use of Laboratory Animals. of the Institute of Laboratory Resources, National Research Council (NIH 86-23, 1985).
- Diet (e.g. ad libitum): Purina Certified Rodent Lab Meal, ad libitum. Feeders are designed to reduce soiling, bridging and scattering.
- Water (e.g. ad libitum): Fresh tap water, ad libitum
- Acclimation period: 11 days prior to study initiation. During acclimation, animals were weighed and observed for clinical signs of disease and weight gain and were found to be normal at study initiation.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 deg C +/- 3 deg C (63 deg F to 74 deg F)
- Humidity (%): 30 to 70%
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light

IN-LIFE DATES: From: To: August 7, 1089-December 4, 1989

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: Dorsal
- Type of wrap if used: Gauze patch (2 x 2) and wrapped with an elastic bandage. The wrapping was then taped to the animal.
- Time intervals for shavings or clipplings: Clipping was employed weekly or as needed.


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 5 ml/kg
- Concentration (if solution): 0, 50, 80, 250 mg/kg
- Constant volume or concentration used: constant concentration


VEHICLE
- Justification for use and choice of vehicle (if other than water): deionized water
- Amount(s) applied (volume or weight with unit): 5 ml/kg
- Concentration (if solution): 5 ml/kg


USE OF RESTRAINERS FOR PREVENTING INGESTION: no
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
The duration of exposure was approximately 6 hours.
Frequency of treatment:
Once daily, five days per week for a period of 90 days.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Doses / Concentrations:
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
80 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Pretest (Baseline data for hematology and clinical chemistries): 5 males, 5 females
0, 50, 80, 250 mg/kg : 15 males, 15 females
0, 250 mg/kg recovery group: 10 males, 10 females.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected by the sponsor and based upon a 28 Day Dermal Toxicity Study in Rats
- Rationale for animal assignment (if not random): All rats were weighed, ranked according to body weight and assigned to treatment groups using a table for random numbers so that each treatment group had a similar distribution according to body weight.
- Rationale for selecting satellite groups: A negative control recovery group and a high dose recovery groups remained on test, untreated , for an additional 28 days to determine reversibility, persistence, or delayed occurrence of toxic effects.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: Daily observations to document onset, degree and duration of: changes in skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behavior pattern. Mortality checks were made daily and recorded.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Scheduled sacrifice periods of the study ( 30, 90 and 118 days)

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Study did not specify

BODY WEIGHT: Yes
- Time schedule for examinations: Weeky

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No
- Time schedule for examinations: N/A

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pretest, and on Days 25, 88 and 116
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Scheduled sacrifice periods of the study ( 30, 90 and 118 days)
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes, all animals were fasted overnight prior to euthanasia
- How many animals: 10 rats (5/sex)
- Parameters examined: hemoglobin, hematocrit, erythrocyte count, total and differential leukocyte count, platelet count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Scheduled sacrifice periods of the study ( 30, 90 and 118 days)
- Animals fasted: Yes, all animals were fasted overnight prior to euthanasia
- How many animals: 10 rats (5/sex)
- Parameters examined: calcium, phosphorus, chloride, sodium, potassium, fasting glucose, serum alamine aminotransferase, serum aspartate aminotransferase, gamma glutamyl transpeptidase, urea nitrogen, albumin, globulin, blood creatinine, total bilirubin, total serum protein measurements

URINALYSIS: No
- Time schedule for collection of urine: N/A
- Metabolism cages used for collection of urine: N/A
- Animals fasted: N/A
- Parameters checked in table [No.?] were examined. N/A

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations: N/A
- Dose groups that were examined: N/A
- Battery of functions tested: sensory activity / grip strength / motor activity / other: N/A

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Necropsy was performed on all animals and included examination of the external surface of the body, all orifices, and the cranial, thoracic, abdominal and pelvis cavities and their contents.
HISTOPATHOLOGY: Yes
The following organs and tissues were fixed and preserved in 10% neutral buffered formal for possible histopathological examination: gross lesions, brain-including section of medulla/pons, cerebellar cortex and cerebral cortex, pituitary, thyroid/parathyroid, thymus, lungs (intact), trachea, heart, sternum with bone marrow, salivary glands, liver, spleen, kidneys/adrenals, pancreas, gonads, uterus, accessory genital organ (epididymides, prostate, and if present, seminal vesicles), aorta, skin (treated and untreated areas), esophagus, nasal turbinates, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, representative lymph nodes (submandibular), mammary gland, thigh musculature, peripheral nerve, eyes, femur-including articular surface, spinal cord at three levels- cervical, midthoracic and lumbar, exorbital lachrymal glands.
Other examinations:
The following organs were weighed: liver, adrenal glands, kidneys, gonads, brain.
Statistics:
Evaluation of equality of means was made by the one way analysis of variance using the F distribution to assess significance. If significant differences among the means ere indicated, Dunnett¿s or Student¿s t test was used to determine significant differences from control means.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Mortality:
no mortality observed
Description (incidence):
Two rats died on test. One male from the high dose group died on Day 1 and one female from the recovery control group died on Day 11. The deaths were judged coincidental and not related to the administration of the test substance.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No statistically significant differences in body weight were detected between males of the control and low and mid dose groups or females of the control and low and high dose groups. Statistically significant reductions in body weights were observed in mid dose females on Day 14 when compared to the control group. The male high dose group mean body weights were significantly reduced on Days 7, 14 ,and 28.

Daily body weight gain was comparable between control groups and test substance treated groups except on four occasions. The male group mean daily body weight gains were significantly increased in the low dose group on Day 30 (fasted weight) and significantly reduced in the high dose group on Days 7 and 14. The female high dose daily body weight gains were significantly increased on Day 21.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No statistically significant differences in food consumption were detected between the non-recovery control group and non-recovery test substance treated group. The male high dose recovery group mean daily food consumption was significantly reduced on Day 112 when compared to the recovery control group. Female high dose recovery group mean daily food consumption was significantly reduced on Days 42 and 112.
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no ocular findings identified in this study that were attributed to test substance administration.
Haematological findings:
no effects observed
Description (incidence and severity):
A significant decrease was observed in segmented neutrophils on Day 90 for the mid dose females. This decrease was considered not to be biologically significant. There were no additional statistical significant differences in hematological values on Day 30, 90 or 118.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Evaluation of the clinical chemistry data just prior to the Day 30 interim sacrifice revealed significantly higher male values in calcium for the mid dose group, phosphorus for the mid and high dose groups and blood urea nitrogen for the high dose group. Significant lower male total protein values were observed for the low dose group and higher chloride values were noted for each treated male dose group. Significantly higher female phosphorus values were noted for the mid dose group. Significantly lower female values were noted in potassium for the mid and high dose groups and blood urea nitrogen for each treated female dose group. These differences were not dose dependent, are within normal limits and are considered not biologically significant. Evaluation of the clinical chemistry data at the Day 90 sacrifice revealed significantly lower male potassium for the low dose group and creatinine for mid and high dose groups. Significantly higher male chloride values were noted for the mid dose group. Female chloride and phosphorus values were significantly higher for the mid dose group and lower blood urea nitrogen values for the mid and high dose groups. These differences are within normal limits and are considered not biologically significant. Evaluation of the clinical chemistry data for the male high dose recovery group revealed significantly higher creatinine and blood urea nitrogen values when compared to the recovery control group. These values are within normal limits and are considered not biologically significant. There were no significant differences in the clinical chemistry values for the high dose recovery females.
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in absolute organ weights between the control and treated groups. A statistically significant decrease was observed in relative liver to body weight ratios for the male high dose group on Day 30 wen compared to the control group. There were no statistically significant differences in relative organ to brain weight ratios between the control and treated groups. The differences were not dose dependent and not considered biologically significant.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No statistically significant, biologically relevant effects were noted. The majority of gross findings at the Day 30 interim necropsy, Day 90 terminal necropsy and after 28 day recovery (day 118) were non-specific and low in incidence. Based on the histopathology, these lesions were not considered to be related to administration of the test substance.
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
When compared to the male and female rats in the control group, except for mild irritation of the skin at the treatment site of some of the rats, there were no histomorphologic changes present in the tissues evaluated which were related to the dermal exposure to the test substance. The skin irritation was characterized by a minimal to slight hyperkeratosis and/or slight epidermal hyperplasia in five males and seven females. Five of these seven females had multifocal areas of minimal to moderate acute inflammation of the epidermis of the skin (multifocal epidermatitis).
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
not applicable
Description (incidence and severity):
Relative Organ to Body Weights Ratios:
A statistically significant decrease was observed in relative liver to body weight ratios for the ale high dose group on Day 30 when compared to the control group. There were no significant differences noted for the female groups or the high dose recovery group.
Relative Organ to Brain Weights Ratios:
There were no statistically significant differences in relative organ to brain weight ratios between the control groups and treated groups.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: NOAEL for systemic toxicity. Basis for effect level: mortality; body weight; food consumption; clinical chemistry; organ weights; histopathology; haematology;
Dose descriptor:
NOAEL
Effect level:
80 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: NOAEL for local (dermal) effects. Basis for effect level: erythema, edema, necrosis, fissuring/sloughing of skin and alopecia (hair loss).

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

No definitive evidence of test substance related dermal irritation for the low or mid dose males groups. Infrequent dermal irritation was noted for the low dose females during a few day of the study. Several mid dose females displayed test substance related dermal irritation toward the latter part of the treatment interval. In the high dose group, clinical findings indicative of dermal irritation at the application site were noted and included some or all of the following observations: erythema, edema, scattered areas of necrosis, 25-50% necrosis at the application site, scab formation, fissuring and /or sloughing of skin, and alopecia. The incidence and severity of dermal effects varied throughout the study period, but generally increased with duration of treatment. Clinical observations indicative of dermal irritation in the high dose groups on Day 90 were no longer evident for the recovery group following the end of the 28 day reversibility period (Day 118).

Applicant's summary and conclusion

Conclusions:
Based upon the results of the Subchronic 90 Day Dermal Toxicity Study in rats with test substance, there were no clinical signs of systemic toxicity which were attributable to the administration of the vehicle control or test substance. The test substance related mild skin irritation was primarily observed in the high dose group. The skin irritation was reversible after discontinuation of treatment during the recovery period. Two non-treatment related deaths occurred during this study. The deaths were judged coincidental. Significant differences in body weight, daily body weight gain and daily food consumption were detected during the few days of the study. There were no statistically significant differences in the absolute organ weights, or relative organ to brain weight ratios. One significant difference was noted for the male relative to liver to body weight data. Several significant differences were noted in the clinical chemistry data. There was one significant difference noted in the hematological data. These differences were not dose dependent, are within limits and are considered not biologically significant or attributable to the administration of the test substance. Except for the mild skin irritation at the treatment site of the high dose animals, there were not histomorphological alterations attributable to the administration of the test substance. Based upon these findings, dermal application of the test substance to rats did not produce a systemic toxicity when administered five days per week for 30 and 90 days at doses 50, 80 or 250 mg/kg. The no observable effect level (NOEL) for systemic toxicity was the highest dose level tested, 250 mg/kg/day. The NOEL for dermal effects was 80 mg/kd/day.