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Toxicological information

Repeated dose toxicity: dermal

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Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1995-09-26 until 1995-10-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance to GLP and according to current guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Test animals

Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan UK Ltd, Bicester, Oxon, England
- Age at study initiation: approximately 14 weeks
- Weight at study initiation: 2.2 to 2.7 kg
- Housing: individually, in Macrolon size 3 plastic cages
- Diet: standard laboratory diet SDS Rabbit Diet SQC, ad libitum
- Water: ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 21°C
- Humidity: 41 to 72%
- Photoperiod: 12/12 (hours dark / hours light)
- Air changes (per hr): approximately 19

Administration / exposure

Type of coverage:
occlusive
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: dorsal region
- % coverage: 10%
- Type of wrap if used: Impervious bandage "Elastoplast"

REMOVAL OF TEST SUBSTANCE
The test substance remained on the back of each rabbit for approximately 6 hours each day after which time the dressings were removed and the treated skin washed with warm (30 to 40 °C) water and gently blotted dry.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2 mL/kg bw
- Constant volume or concentration used for each dose group: yes
- For solids, paste formed: yes

VEHICLE
distilled water to for moistening the powder and to ensure good contact with the skin.
USE OF RESTRAINERS FOR PREVENTING INGESTION: no
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
not performed
Duration of treatment / exposure:
6 h per day for 21 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
5 animlas per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The appropriate weight of test substance was spread evenly over the prepared skin of Group 2 to 4 rabbits inclusive. As the test substance is a powder, it was moistened using distilled water to ensure good contact with the skin. The control group was treated in a similar manner receiving water alone.
The treatment site was occluded by covering with an impervious bandage consisting of "Elastoplast" adhesive dressing backed with impervious "Sleek" plaster.
The test substance remained on the back of each rabbit for approximately 6 hours each day after which time the dressings were removed and the treated skin washed with warm (30 to 40 °C) water and gently blotted dry. Each animal received a constant dosage based on its most recently recorded body weight. Animals were treated once daily for at least twenty-one consecutive days. Treatment of all animals not scheduled for sacrifice on Day 22 was continued up to 24 hours prior to sacrifice.
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
Clinical signs
All animals were observed three times daily for signs of ill health, behavioural changes or toxicosis. Any observed changes were recorded.
All animals were checked early in each working day and again in the late afternoon to look for dead or moribund animals. This allowed a post mortem examination to be undertaken during the working part of that day.

Local dermal irritation
Local irritation was recorded immediately prior to the first daily application of the test substance and subsequently daily. Reactions (erythema and oedema) resulting from treatment were assessed on a numerical basis according to a Draize scoring system as follows:

Erythema and eschar formation:
No erythema 0
Slight erythema 1
Well-defined erythema 2
Moderate erythema 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) 4

Oedema formation:
No oedema 0
Slight oedema 1
Well-defined oedema (area well defined by definite raising) 2
Moderate oedema (raised approximately 1 millimetre) 3 Severe oedema (raised more than 1 millimetre and extending beyond
the area of exposure) 4
Any other lesion not covered by this scoring system, was described.

Bodyweight
All rabbits were weighed prior to dosing on Day 1 (Week 0) and subsequently at weekly intervals throughout the study.
Food consumption
The quantity of food consumed by each rabbit was measured at weekly intervals throughout the study.

Removal of blood samples
Food was withdrawn overnight prior to collection of samples. Blood was withdrawn from the median artery of the ear of all rabbits prior to termination (Week 3). Further removal of blood samples for re-analysis was carried out for individual animals, and the results of these analyses are presented in the appendices.

The collected blood samples were divided as follows:
EDTA anticoagulant tubes for haematological investigations
Citrate anticoagulant tubes for coagulation test
Heparin anticoagulant microtainer tubes for biochemical tests

Haematology
Packed cell volume (PCV)
Haemoglobin (Hb)
Red cell count (RBC)
Mean corpuscular haemoglobin concentration
Mean corpuscular volume
Mean corpuscular haemoglobin

Total white blood cell (WBC) count
Differential WBC count
Neutrophils
Lymphocytes
Eosinophils
Basophils
Monocytes
Large unstained cells
Cell morphology: the most common morphological changes (anisocytosis, micro/macrocytosis, variation in colour, hypo/hyperchromasia, left shift, atypical/blast cells) were recorded.
Platelet count
Thrombotest (TT)

Biochemistry
The following parameters were analysed with a Hitachi 737 Clinical Chemistry Analyser
Glucose
Total protein
Albumin (Alb)
Globulin (Glob)
Urea nitrogen
Creatinine
Alkaline phosphatase
Alanine aminotrnasferase
Aspartate aminotransferase
Bilirubin
Sodium
Potassium
Calcium
Chloride
Inorganic phosphorus
Cholesterol
Sacrifice and pathology:
Termination
All animals surviving treatment were randomly killed on Day 22, Day 23 or Day 24 by means of an intravenous overdose of pentobarbitone sodiu and a complete autopsy undertaken. Specified organs were weighed and relevant tissue samples were fixed for microscopic examination.

Organ weight
The following organs from each animal were dissected free of fat and weighed:
adrenals
ovaries
brain
spleen
kidneys testes (with epididymides)

Macroscopic pathology
The macroscopic appearance of the tissues of all rabbits was recorded and samples of the following tissues were preserved in 10% buffered formalin:
adrenals
aorta
brain (medullary, cerebellar
and cortical sections)
caecum colon duodenum
eyes (Davidson's fluid as fixative)
gall bladder
heart
ileum
jejunum
kidneys
larynx
liver
lungs
lymph nodes (cervical and mesenteric)
mammary glands
oesophagus
ovaries
pancreas
pharynx
pituitary
prostate
salivary
gland
sciatic nerve
skeletal muscle
skin (treated and untreated)
spleen
sternum (for bone and marrow sections)
stomach
testes (including epididymis)
thymus (where present)
thyroid (with parathyroid)
tongue
trachea
urinary bladder
uterus (with cervix)
vagina
any macroscopically abnormal tissue

Microscopic pathology
Fixed tissue samples required for microscopic examination were prepared by embedding in paraffin wax - sections were cut at 4 µm and stained with haematoxylin and eosin.
Microscopic examination of prepared slides (from tissues indicated under "Macroscopic pathology" were carried out for all rabbits of Group 1 (control group) and Group 4 (high dosage group) killed on Day 22/23/24.






Statistics:
All statistical analyses were carried out separately for males and females.
The following sequence of statistical tests were used for bodyweight gains, food consumption, organ weight and clinical pathology data:
If the data consisted predominantly of one particular value (relative frequency of the mode exceeded 75%), the proportion of values different from the mode were analysed by Fisher's exact test followed by Mantel's test for a trend in proportions.
Otherwise:
Bartlett's test was applied to test for heterogeneity of variance between treatments. If significant heterogeneity was found at the 1% level, a logarithmic transformation was tried to see if a more stable variance structure could be obtained.
If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance was carried out followed by Williams' test for a dose related response.
If significant heterogeneity of variance was present and could not be removed by a logarithmic transformation, the Kruskal-Wallis analysis of ranks was used. This analysis was followed by the non-parametric equivalent of Williams' test (Shirley's test).
Covariate analysis of organ weight data (with final bodyweight as covariate) was also performed using adjusted weights for organs where a correlation between organ weight and bodyweight was established at the 10% level of significance.
Significant differences between control animals and those treated with the test substance were expressed at the 5% (* p <0.05) or 1% (** p <0.01) level.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS
There were no mortalities. No treatment-related clinical signs were noted for any of the animals throughout the study.

DERMAL REACTIONS
No dermal reactions were seen during the first five days of the study. Dermal irritation, accompanied by yellow staining and cracking, developed during the latter part of Week 1 and during Weeks 2 and 3 for all treatment groups, and the incidence and severity was dosage related. For some animals treated at the low dosage of 100 mg/kg/day dermal irritation (slight to well defined erythema accompanied by slight to well defined oedema) was recorded. Irritation was accompanied by yellow staining and cracking. Most rabbits of the intermediate dosage of 300 mg/kg/day, dermal irritation (slight to moderate erythema accompanied by slight to well defined oedema) was observed. Dermal irritation was accompanied by yellow staining and cracking and occasionally by sloughing. At the high dosage of 1000 mg/kg/day dermal irritation (slight to moderate erythema accompanied by slight to moderate oedema) was recorded. Dermal irritation was accompanied by yellow staining and cracking and occasionally by sloughing.There were no dermal reactions seen for any animals from the control group.

BODYWEIGHT
There were no statistically significant differences from control noted for bodyweight gains in any of the treatment groups. Bodyweight gains showed considerable variation for all groups of rabbits. This is not uncommon for laboratory rabbits and in the absence of any dosage relationship, differences from control were considered to reflect variation.

FOOD CONSUMPTION
There were no statistically significant differences from control noted for the food consumption in any of the treatment groups. Food consumption for low dosage group female rabbits was slightly higher than control and lower than control for intermediate dosage group male rabbits. These small differences from control were considered to reflect variation and not an effect of treatment.

HAEMATOLOGY
There were no differences from control in the haematological parameters measured that were considered to be related to treatment. Statistically significantly higher than control monocyte levels were recorded for male rabbits of the high dosage group. The magnitude of this change was small and a treatment related effect was considered unlikely. nLymphocyte levels were statistically significantly lower than control for female rabbits treated at 1000 mg/kg/day. However, there was wide variation for individual values and the total white blood cell count was not affected. A treatment-related effect was considered unlikely.


BIOCHEMISTRY
There were no differences from control in the biochemical parameters measured.

ORGAN WEIGHTS
There were no differences from control in the organ weights measured.

MACROSCOPIC PATHOLOGY
The macroscopic examination performed at termination revealed yellow staining of the skin in some rabbits at all dosage levels.

MICROSCOPIC PATHOLOGY
Minimal or trace diffuse acanthosis was seen in the majority of male and female rabbits from all treated groups.

A related minimal or trace diffuse inflammation of the superficial dermis was observed in a proportion of male and female rabbits of the 1000 and 300 mg/kg/day treatment groups and male rabbits of the 100 mg/kg/day treatment group. These changes were considered to be dose-related.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Dose descriptor:
NOEL
Effect level:
< 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion