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Toxicological information

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:
4th November 1997 - 22nd May 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Version / remarks:
1981
Qualifier:
according to
Guideline:
EU Method B.28 (Sub-Chronic Dermal Toxicity Test: 90-Day Repeated Dermal Dose Study Using Rodent Species)
Version / remarks:
1988
Qualifier:
according to
Guideline:
other: TSCA guideline reference CFR 798.2250
Version / remarks:
draft June 1996
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Ex Fluka, 352776-1796
- Purity: 97.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature in the dark

Test animals

Species:
rat
Strain:
other: Alpk:APrSD
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Rodent Breeding Unit, Zeneca Pharmaceuticals, Alderley Park
- Weight at study initiation: 120 - 145 g (Males), 100 - 125 g (Females) upon arrival at CTL
- Fasting period before study: No
- Housing: The rats were individually housed in multiple rat racks suitable for animals of this strain and weight range expected during the course of the study.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): At least 15
- Photoperiod (hrs dark / hrs light): Artificial light 12/12

Administration / exposure

Type of coverage:
occlusive
Vehicle:
ethanol
Details on exposure:
TEST SITE
- Area of exposure: at least 7 cm x 7 cm of the dorso-Iumbar skin.
- % coverage: ca. 10 %
- Type of wrap if used: Each dressing consisted of a 4 ply gauze patch (approximately 7 cm x 7 cm) to cover the treated area. This patch was covered by a patch of plastic film held in position by an adhesive bandage (approximately 25 cm x 5 or 7.5 cm) around which were wrapped 2 pieces of PVC tape.
- Time intervals for shavings or clipplings: Sixteen to thirty-two hours before the first application of the test substance

REMOVAL OF TEST SUBSTANCE
- Washing: warm water
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount applied: 2 mL/kg bodyweight
- Constant volume or concentration used: no
- For solids, paste formed: no

VEHICLE
- Lot/batch No: Y00332/005

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose preparations were analysed to confIrm satisfactory achieved concentration at all dose levels. This analysis was performed twice during the study. The stability of the test substance in the vehicle at room temperature was evaluated for the low and high dose solutions.
Duration of treatment / exposure:
The animals were dosed dermally, for a total of 50 applications (4-6 applications per 7 day period), over a period of 70 days.
Frequency of treatment:
4 - 6 applications per 7 day period
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Group 1
Dose / conc.:
3 mg/kg bw/day
Remarks:
Group 2
Dose / conc.:
30 mg/kg bw/day
Remarks:
Group 3
Dose / conc.:
60 mg/kg bw/day
Remarks:
Group 4
Dose / conc.:
90 mg/kg bw/day
Remarks:
Group 5
No. of animals per sex per dose:
10 males and 10 females

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at the time of dosing and after decontamination on each application day

DERMAL IRRITATION: Yes
- Time schedule for examinations: at the time of dosing and after decontamination on each application day

BODY WEIGHT: Yes
- Time schedule for examinations: each day of dosing throughout the study

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: Yes

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of all animals were examined pre-experimentally and those of the control and high dose group animals during the week of termination.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At study termination
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all animals
- Parameters examined: haemoglobin, red cell count, mean cell volume, mean cell haemoglobin concentration, red cell distribution width, total and differential white blood cell count, platelet count, blood cell morphology, mean cell haemoglobin, haematocrit

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At study termination
- Animals fasted: No
- How many animals: all animals
- Parameters examined: urea, creatinine, glucose, total protein, triglycerides, potassium, calcium, total bilirubin, creatine kinase activity, alkaline phosphatase activity, alanine aminotransferase activity, albumin, albumin/globulin ratio, cholesterol, sodium, chloride, phosphorus (as phosphate), gamma-glutamyl transferase activity, aspartate aminotransferase activity

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were subjected to a full examination post mortem. This involved an external observation and a careful internal examination of all organs and structures.

HISTOPATHOLOGY: Yes
Abnormal tissues, liver, thyroid and skin (treated and untreated) were processed from all animals. The kidneys and lungs were processed from the control and high dose group animals only.
Statistics:
All data were evaluated using the GLM procedure in SAS (1989).

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Dermal irritation:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Urinalysis findings:
not specified
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
It was noted that some animals on some days of dosing exhibited signs of distress whilst bandaged for 6 hours. Consequently, alternative types of dressings, bandages and methods to retain the test substance were evaluated to try and alleviate the distress of the animals. As the alternative methods did not significantly reduce the stress experienced by some of the animals, the study was terminated early on day 71.
There were four early terminations unrelated to treatment with the test item. In addition, one male rat (30 mg test item/kg body weight/day group) was killed for humane reasons on day 11 since necrotic skin was noted on the area of application. With regards to general clinical observations, there were no toxicologically relevant findings at any dose level in either males or females treated with the test item.

BODY WEIGHT AND WEIGHT GAIN
Treatment of male and female rats had no significant effects on bodyweights throughout the study.

FOOD CONSUMPTION
Food consumption was slightly lower in female rats in the 90 mg /kg bodyweight/day group, and the maximum effect was 7 % below control values. This small effect is considered to be of little toxicological relevance.

FOOD EFFICIENCY
There was no effect of treatment on food utilisation.

OPHTHALMOSCOPIC EXAMINATION
The administration of 90 mg /kg bodyweight/day to both male and female rats for 5 days per week for 70 days had no effect on the appearance of the eyes as evaluated by ophthalmoscopy

HAEMATOLOGY
Overall, there were no treatment related findings on any haematological parameters evaluated.

CLINICAL CHEMISTRY
In the high dose group, plasma cholesterol was increased in male rats, plasma albumin to globulin ratio was decreased in males, and plasma levels of glucose, albumin and total protein were significantly decreased in female rats which may reflect the lower food consumption in this group. All of these changes are minimal and are considered to be of no toxicological significance. Certain other parameters were also statistically significantly changed in either male or female rats, but as they only occurred in the 2 lowest groups, it is considered that they are unrelated to treatment.

ORGAN WEIGHTS
Kidney weights for the males in the high dose group were slightly lower than the control values. However, in the absence of pathological findings, this small decrease is considered to be of little toxicological significance. There were no other effects of treatment on organ weights in either male or female rats.

GROSS PATHOLOGY
There were treatment-related macroscopic findings in the treated skin. There was an increased incidence of scabs in males and females treated with 30, 60 or 90 mg/kg bodyweight/day dose groups, and discoloration of the skin in males from all treatment groups and in females treated with 30, 60 or 90 mg/kg bodyweight/day dose groups. In addition, a small number of animals from treated groups had scabs in the untreated skin. There were no other treatment-related macroscopic findings.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were treatment-related microscopic findings in the treated skin of rats killed intercurrently and at termination. There was an increased incidence of lesions which were considered to represent dermatitis in males and females treated with 30, 60 or 90 mg/kg bodyweight/day.
No treatment-related effects were seen in any of the other tissues examined.

Effect levels

open allclose all
Key result
Dose descriptor:
NOEL
Remarks:
systemic toxicity
Effect level:
90 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: highest test dose
Dose descriptor:
NOEL
Remarks:
skin lesions
Effect level:
3 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: scabs and discoloration of the skin

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion