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: GLP - guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:
(adopted 12 May 1981)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))
Version / remarks:
(adopted 31 Jul 1992)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)
Version / remarks:
(Nov 1984)
Deviations:
no
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 UK Ltd., Margate, Kent, UK
- Age at study initiation: approx 42 d (males and females)
- Weight at study initiation: 180 - 196 g (males), 188 - 207 g (females)
- Fasting period before study: no
- Housing: individually, in polycarbonate cages with mesh bases suspended over waste trays. The waste tray of each cage was lined with absorbent paper which was replaced 3 times each week. Trays and cages were changed at fortnightly intervals.
- Diet: Modified SQC Expanded Rat and Mouse Maintenance Diet No. 1 supplied by Special Diet Services Ltd., Stepfield, Witham, Essex, UK, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Type of coverage:
occlusive
Vehicle:
other: 1% w/v methyl cellulose in water
Details on exposure:
TEST SITE
- Area of exposure:dorsal area of the trunk
- % coverage: 10%
- Type of wrap if used: the test substance was held in contact with the skin on a 2 x 2 cm pad of porous gauze pad backed by aluminium foil which was held in place over the treatment site by elastic bandage
- Time intervals for shavings or clippings: clipping was repeated as necessary during the study

REMOVAL OF TEST SUBSTANCE
- Washing (if done): residual test material was rinsed of with soap and water
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount applied: 5 mL/kg bw, daily preparation of test suspension
- Concentration: 2, 20 and 200 mg/mL
- Constant concentration used: yes

VEHICLE
- Amount(s) applied (volume or weight with unit): 5 mL/kg bw

USE OF RESTRAINERS FOR PREVENTING INGESTION: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The samples were analysed by extraction of AE F122006 from the suspensions by shaking with acetonitrile + methanol (90 + 10, v/v). The concentration of the test substance in the extract was measured by HPLC using UV detection.The mean results for the test suspension samples analysed and prepared for dosing were within the range 89.5 to 122.3% of nominal at the time of preparation (specified range +20% to -20% of nominal).
Duration of treatment / exposure:
28 days (males)
29 days (females)
Frequency of treatment:
5 days/week, excluding weekends
Doses / concentrations
Remarks:
Doses / Concentrations:
10, 100, 1000 mg/kg bw/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected on the basis of findings in an earlier dose ranging study conducted immediately prior to this study with 4 groups of 3 males dosed at 0, 250, 500 and 1000mg test substance kg/day for 7 days, excluding the weekend. There were no treatment-related effects at 1000 mg/kg/day, equivalent to the accepted international regulatory limit dose, in this range-finding study.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed each morning for abnormal behaviour, including neuro-muscular coordination and physical appearance. Animals were also observed in the afternoon on Mondays to Fridays except on public and Company holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed at randomisation, at the start of treatment, weekly thereafter and at
necropsy.

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

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 29
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No
- How many animals: 5 per sex and dose
- Parameters examined: Haematocrit (HCT), White blood cells (WBC), Haemoglobin (HB), Neutrophils (NEUT), Red blood cells (RBC), Lymphocytes (LYMP), Mean corpuscular volume (MCV), Monocytes (MONO), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Eosinophils (EOS), Basophils (BASO), Large unstained cells (LUC), Platelets (PLT), Reticulocyte count (RET), Prothrombin time (PT), Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 29
- Animals fasted: No
- How many animals: 5 per sex and dose
- Parameters examined: Total protein (PROT), Albumin (ALB), Total globulin (GLOB), A/G ratio (A/G), Calcium (CA), Phosphate (PO4), Sodium (Na), Potassium (K), Urea (UREA), Creatinine (CREAT), Glucose (GLUC), Total cholesterol (CHOL), Total bilirubin (TBIL), Chloride (Cl), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Alkaline phosphatase (AP), G-glutamyl transpeptidase (GGT), Creatine kinase (CPK)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, organ weights of kidneys, adrenals, liver and testes were recorded. Following tissues were fixed in 10% neutral buffered formalin: kidneys, liver, ovaries, pinnae, skin (treated and untreated), testes, adrenals, any other tissue showing macroscopic abnormalities
HISTOPATHOLOGY: Yes, of kidney and liver sections and treated and untreated skin
Statistics:
The significance of differences between control and treated groups was analysed by either parametric or non-parametric statistical tests as appropriate. A maximum 2-tailed probability value of 5% (p <0.05) was considered statistically significant.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: slight dermal irritation in 10/10 rats, slight sloughing at the test site in 3/5 males and 3/5 females; 100 mg/kg bw/day: slight dermal irritation in 2/5 female rats
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
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):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
100 mg/kg bw/day: scabs at the application site in 2/5 female rats
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: mononuclear cell infiltration of the dermis in 3/5 males and 5/5 females; 100 mg/kg bw/day: mononuclear cell infiltration in 2/5 males and 3/5 females; 10 mg/kg bw/day: mononuclear cell infiltration in 1/5 males
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY:
No mortalities occured during the study, test substance related clinical signs other than local dermal irritation were not evident.

BODY WEIGHT AND WEIGHT GAIN
There were no statistically significant treatment-related effects on body weight gain.

FOOD CONSUMPTION
There were no treatment-related effects on food consumption.

HAEMATOLOGY
No treatment-related effects were observed.

CLINICAL CHEMISTRY
No treatment-related effects were observed. At the 1000 mg/kg/day dose level a very mild decrease in cholesterol was observed in female animals, but this decrease was within the normal range of controls (non-adverse).

ORGAN WEIGHTS
At the 1000 mg/kg/day dose level a slight increase in group mean adrenal weight, relative to body weight, was observed in females only which is a reflection of the slight but toxicologically insignificant reduction in body weight gain also observed in females at this dose level (non-adverse).

GROSS PATHOLOGY
There were no treatment-related findings other than scab formation (local dermal effect) at necropsy.

HISTOPATHOLOGY: NON-NEOPLASTIC
At 1000 mg/kg/day,, 3/5 males and 5/5 females had mononuclear cell infiltration of the dermis to a moderate degree.
At 100 mg/kg/day, 2/5 males and 3/5 females had moderate cell infiltration.
At 10 mg/kg/day, 1/5 males had a mild infiltration but because a similar lesion was observed in a control female this is considered not to be induced by application of the test compound.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: gross pathology, histopathology
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
0.5 mg/cm² per day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: calculated based on a bodyweight of 200 g and a patch area of 4 cm²

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion