Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Stoneridge, Kingston, New York, USA
- Age at study initiation: 35 days
- Weight at study initiation: 104-156 g (males); 116-153 g (females)
- Fasting period before study: not reported
- Housing: 1/cage in suspended stainless steel cages
- Diet (e.g. ad libitum): Purina certified rodent chow 5002M
- Water (e.g. ad libitum): ad libitum
- Acclimation period: approximately one week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-23 degrees C
- Humidity (%): 28-79%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark


IN-LIFE DATES: From: September 16, 1992 To: October 15, 1992

Administration / exposure

Type of coverage:
open
Vehicle:
other: Neutral Oil 100 N
Details on exposure:
Route of Administration: dermal
TEST SITE
- Area of exposure: the hair around the entire trunk between the flank and shoulders of each animal was closely clipped
- % coverage: 10% of the bosy surface area
- Type of wrap if used: none
- Time intervals for shavings or clipplings: repeated as required


REMOVAL OF TEST SUBSTANCE
- Washing (if done): washed with paper towels saturated with 1% aqueous soap solution. The exposure sight was then rinsed with paper towels saturated with tap water and gently blotted dry with paper towels.
- Time after start of exposure:6 hours


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2.0 ml/kg/day; 0.0 ml/kg/day- Sham control
- Concentration (if solution): 0.0025, 0.010, 0.030 g/mL
- Constant volume or concentration used: yes
- For solids, paste formed: not applicable


VEHICLE
- Justification for use and choice of vehicle (if other than water): test substance was not soluble in water but in the Neutral Oil 100N
- Amount(s) applied (volume or weight with unit): 2.0 mL/kg/day
- Concentration (if solution): suspension: 0.0025, 0.010, 0.030 g mL
- Lot/batch no. (if required): TD92-145 (number assigned internal to company)
- Purity: 100% of a refined paraffinic distillate


USE OF RESTRAINERS FOR PREVENTING INGESTION: yes; cardboard collars
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples (1 mL) of each dosing solution, 0.0025, 0.010 and 0.030 g/mL, were submitted weekly for analysis in order to verify proximity to target concentration and stability. Gas chromatography was used to measure the amount of test substance in the samples. Before analysis, samples were stored at room temperature and kept at that temperature throughout the study. The first and last week's samples were analyed. Each sample was diluted 2 parts sample to 1 part MDC. Duplicate injections of each sample were run and the levels were calculated using a four-level calibration curve generated before sample analysis.
Duration of treatment / exposure:
6 hrs/day
Frequency of treatment:
5 days/week for 4 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (Sham Control), 0 (Solvent Control), 5, 20, 60 mg/kg/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
6
Control animals:
other: Sham Control and Solvent Control (Neutral Oil 100N)
Details on study design:
- Dose selection rationale: based on a 5-day pilot dermal study
- Rationale for animal assignment (if not random): computer randomiation
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): males follwed by females next day
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
Mortality, signs of ill health or reaction to treatment


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: each treatment day prior to the application and prior to necropsy


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes; weekly


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


OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of necropsy
- Anaesthetic used for blood collection: Yes (identity) pentobarbital
- Animals fasted: Yes
- How many animals: all surviving animals on the day of necropsy
- Hematocrit
Erythrocytes
Hemoglobin
Total white blood cell count and differential
Platelets
Mean corpuscular volume
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of necropsy
- Animals fasted: Yes
- How many animals: all surviving animals on the day of necropsy
- Serum glutamic pyruvic transaminase
Serum glutamic oxaloacetic transaminase
Cholesterol
Triglycerides
Total Bilirubin
Alkaline Phosphatase
Glucose
Gamma glutamyl transpeptidase
Blood urea nitrogen
Creatinine
Total protein
Albumin
Albumin/globulin ratio
Calcium
Sodium
Potassium
Chloride
Inorganic Phosphorus


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: weekly
- Dose groups that were examined: performed on each animal
- Battery of functions tested: sensory activity / grip strength / motor activity / other: appearance, posture, gait,


OTHER: external structures, body orifices, palpation for body masses weekly
Other examinations:
none
Statistics:
One-way analysis of covariance models were used to assess the presence or absence of a treatment effect for body weight, cumulative body weight gain and feed consumption. Separate analyses were conducted at each sampling time for each sex. Pairwise comparisons of least square means between control and each treatment level were evaluated using Dunnett's t-test. The criterion for statistical significance for all comparisons was p-value of 0.05 or less.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Dermal irritation:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: no treatment-related mortalities; - Time of death: 2 rats sacrificed for humane reasons on day 11 - Number of deaths at each dose: 2 females at 60 mg/kg/day;
Red-stained muzzle and red-stained eyes were observed in all treatment groups and were judged to be caused by the collar. Alopecia of the forelegs and scabs on the head or neck were observed and judged unrelated to test substance exposure.


BODY WEIGHT AND WEIGHT GAIN: No treatment-related effect on body weight or cumulative body weight gain was evident in females at any dose level. In males, both body weight and cumulative body weight gain were statistically significantly less than the Sham control in Groups 4 (20 mg/kg/day) and 5 (60 mg/kg/day) throughout the treatment period, and in Group 3 (5 mg/kg/day) at the end of weeks 1 and 4. Body weight was also consistently less in Group 2 (Solvent Control) males throughout the treatment period. Though this difference was statistically significant only at the end of week 4. When compared to Group 2 (Solvent Control), body weight and cumulative body weight gain were significantly lower only in Group 5 males at the end of weeks 1 and 2. These results indicate that body weight effects observed in Groups 3 and 4 (5 and 20 mg/kg/day) males were attributable to Neutral Oil 100N exposure, and only in Group 5 were effects on body weights directly related to test material exposure.


FOOD CONSUMPTION: There was no treatment-related effect on feed consumption in male or female rats at any dose level. A statistically significant reduction in feed consumption was noted in male rats at 60 mg/kg/day during week two of exposure; but this reduction was judged incidental and not test substance-related.


FOOD EFFICIENCY: not conducted


WATER CONSUMPTION: not conducted


OPHTHALMOSCOPIC EXAMINATION: not conducted


HAEMATOLOGY: No treatment-related effect on any hematologic parameter at any dose level. A significant increase in hemoglobin concentration was observed in Group 2 (Solvent Control) females and a significant reduction in lymphocyte count was observed in females at 60 mg/kg/day dose group. These observations were judged incidental due to the minimal change observed and the lack of dose response.


CLINICAL CHEMISTRY: No treatment-related clinical chemistry effects were observed in any group. However, numerous statistically significant changes in clinical chemistry parameters were noted but were judged to be incidental since there was a general lack of dose response or consistent pattern between endpoints and sexes, and absence of correlative histopathologic changes. Alkaline phosphatase levels were significantly increased in males at 60 mg/kg/day. Triglyceride levels were significantly reduced in males at 5 and 60 mg/kg/day groups and chloride was significantly increased in males at 20 mg/kg/day. In females, blood urea nitrogen levels were significantly increased in 60 mg/kg/day dose group, and total protein was significantly increased in Solvent Control and 20 mg/kg/day dose groups.


URINALYSIS: not conducted


NEUROBEHAVIOUR: no effects


ORGAN WEIGHTS: The only treatment-related organ weight changes observed were significantly increased absolute and relative adrenal weights in both sexes at the 60 mg/kg/day dose group.


GROSS PATHOLOGY: Treatment-related gross pathologic observations were confined to the dermal application site (treated skin) of the test material. The severity and distribution as well as the number of lesions at the application site were dose-dependent. Gross observations included scales, erosions, scabs and/or cracking fissures of the epidermis.


HISTOPATHOLOGY: NON-NEOPLASTIC: Treatment-related histopathologic observations were confined to the dermal application site (treated skin) of the test material. The severity and distribution as well as the number of lesions at the application site were dose-dependent. Microscopic observations included multifocal to diffuse, and moderate to severe hyperkeratosis, acanthosis and dermal inflammation. In addition, inflammation of the subcutaneous tissue underlying the skin at the application site was observed with low incidence.


HISTOPATHOLOGY: NEOPLASTIC (if applicable): not applicable


Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
20 other: mg/kg
Sex:
male/female
Basis for effect level:
other: There were no test substance related effects observed at this concentration
Key result
Dose descriptor:
LOAEL
Effect level:
60 other: mg/kg
Sex:
male/female
Basis for effect level:
dermal irritation
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Daily dermal application of the test material to the skin of rats for four weeks (5 days/week) resulted in a No Observed Effect Level (NOEL) for systemic toxicity of 20 mg/kg/day. The minimum effect level was 60 mg/kg/day.
Executive summary:

The dermal toxicity of the test material was assessed after topical application of the test substancec to the shaved intact skin of Crl:CD BR rats. The test material was dispersed in Neutral Oil 100 N for application. Five groups of six male and six female rats were administered the test substancce or Neutral Oil 100 N (solvent control) dermally, to non-occluded sites for 6 hrs/day, 5 days/week for 4 weeks. Group 1 (sham control) animals were treated in a manner identical to animals in other groups with th exception that they did not receive any test material. group 2 (solvent control) received Neutral Oil 100 N at a constant volume of 2.0 mg/kg/day. Groups 3, 4 and 5 received the test material, dispersed in Neutral Oil 100 N at doses of 5, 20 and 60 mg/kg/day, respectively, using a constant dosing volume of 2.0 ml/kg/day.

All animals were observed at least once daily for mortality, signs of ill health or reaction to treatment. Application sites were evaluated for skin irrittation prior to each daily dose and before terminal sacrifice. Body weight and feed consumption were measured weekly; physical examinations were perfomred weekly at the time of weighing. At the end of the 4 - week treatment period, all rats were killed and necropsied. Blood samples for hematology and clinical chemistry were collected and analyzed. Adrenals, brain, kidneys, liver and testes were weighed in all groups.

Histopathologic evaluations were performed on all gross lesions as well as liver, treated and untreated skin, kidney, brain, spinal cord (cervical, thoracic, lumbar), sciatic nerve, skeletal muscle, adrenals and tested collected from all animals in Group 5 ( Test material; 60 mg/kg/day), Groups 1 and 2 (Sham and Solvent controls, respectively).

There were no treatment-related mortalities, clinical signs or effects on feed consumption. Two females of Group 5 (60 mg/kg/day) were sacrificed following the 11th day of test material application due to severe skin irritation and signs of extreme discomfort.

Body weight and cumulative body weight gain were not affected in femlaes of any treatment group. Treatment-related effects on body weight and cumulative body weight gain were observed in Group 5 males at the end of weeks 1 and 2. Other statistically significant changes in body weight and body weight gain were attributed to solvent exposure.

In both males and females, local skin irritation at the application site was observed at all dose levels of the test material, the duration and severity of which were dose-dependent. Signs of slight application site irritation were alos observed in animals of Group 2 (solvent control).

There was no treatment-related effect on any hematologic or clinical chemistry parameter.

Test material exposure resulted in increased absolute and relative adrenal weights in both males and females of Group 5 (60 mg/kg). Other statistically significant changes in absolute and relative organ weights were attributed to solvent exposure or judged incidental.

Treatment-related gross and histopathologic effects were confined to the skin (epidermis and dermis) and underlying subcutaneous tissues at the treatment site. Doses of 60 mg/kg/day proved severely irritating to the skin of rats.

Daily application of the test material to the skin of rats for four weeks at doses up to and including 60 mg/kg/day had a no observed effect level (NOEL) for systemic toxicity of 20 mg/kg/day. The minimum effect level was 60 mg/kg/day.