Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
a sub-chronic toxicity study (90 days) does not need to be conducted because the substance is unreactive, insoluble and not inhalable and there is no evidence of absorption and no evidence of toxicity in a 28-day 'limit test' and human exposure is limited
Cross-reference
Reason / purpose:
read-across: supporting information
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
from 1994-08-09 to 1994-09-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
For justification of read across please refer to the attachment in IUCLID section 13.
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
directive 92/69/EEC
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
12 May 1981
Deviations:
no
Principles of method if other than guideline:
NA
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL Ltd., Basel, Switzerland
- Age at study initiation: approximately 6 weeks.
- Weight at study initiation: males: mean abt. 150 gr; females: mean abt. 120 g
- Fasting period before study: no
- Housing: Group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors.
- Diet: free access to standard pelleted laboratory animal diet (Kliba 343 from Klingentalmuhle AG, Kaiseraugst, Switzerland)
- Water: free access to tap-water.
- Acclimation period: Acclimatisation period was at least 5 days before start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature: 21°C
- Humidity: 50 % rel. humidity
- Air changes: approximately 15 air changes per hour
- Photoperiod: 12 hours artificial fluorescent light and 12 hours dark per day

IN-LIFE DATES: From: 1994 08-09 To: 1994-09-06
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Stability in vehicle: Stable for at least 96 hours in water
Method: Formulations (w/w) were prepared daily immediately prior to dosing. Adjustment was made for specific gravity of the test substance.
Storage conditions: at ambient temperature
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of representative formulations prepared after study termination were analysed to check accuracy of the preparations (all concentrations).
Duration of treatment / exposure:
Oral gavage, using a stainless steel stomach tube.
Frequency of treatment:
Once daily for 28 days, approximately the same time each day, 7 days per week.
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
other: actual ingested
Remarks:
Doses / Concentrations:
50 mg/kg bw/day
Basis:
other: actual ingested
Remarks:
Doses / Concentrations:
200 mg/kg bw/day
Basis:
other: actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
other: actual ingested
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
yes
Details on study design:
Doses volume: 5 mL/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight

Positive control:
none
Observations and examinations performed and frequency:
Observations:
-Mortality/Viability: Twice daily.
-Clinical signs: At least once daily from day 1 onwards. The time of onset, degree and duration were recorded.
-body weights: Weekly and on the day preceding termination, prior to overnight fasting.
-Food consumption: weekly.
-Water consumption: subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
-Ophthalmoscopic examinations: both eyes were examined following instillation of tropicamide solution (5 mg/mL) during the last week of treatment (week 4).
Sacrifice and pathology:
-necropsy: All animals surviving to the end of the observation period (day 29) were deeply anaesthetised using ether vapour and subsequently exsanguinated, except for one male which accidentally died during blood sampling.
All animals assigned to study were necropsied and descriptions of all macroscopic abnormalities recorded.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution: adrenal glands, heart, kidneys, liver, spleen, stomach and testes.
- organ weights: The following organ weights (and terminal body weight) were recorded on the scheduled day of necropsy: Adrenal glands, heart, kidneys, liver, spleen and testes.
-Histotechnology: All organ and tissue samples, as defined under Histopathology, were processed embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.
-Histopathology: Slides of adrenals, heart, kidneys, liver, spleen, stomach and testes, collected at the scheduled sacrifice from all animals of the control and the highest dose group were examined by a pathologist. All abnormalities were described and included in the report.
Other examinations:
NA
Statistics:
The following statistical methods were used to analyse the data:
Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
The extract Fisher-test was applied to the ophthalmoscopic examination data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores).
Test statistics were calculated on the basis of extract values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
Mortality: No mortality occurred during the study period in any of the treated animals. One control male accidentally died during blood sampling.
Clinical signs: There were no clinical signs of toxicity or behavioural changes over the 28 day observation period that were considered to be related to treatment. Salivation was observed in all animals receiving 1000 mg/kg/day. This finding is commonly noted after oral gavage administration and, moreover, a bad taste or irritant effect of the test substance may contribute to this effect. Alopecia noted in one female receiving 1000 mg/kg/day was considered of no toxicological significance.
Body weight: Body weights and body weight gain of treated animals remained in the same range as controls over the 4-week study period.
Food consumption: There were no differences in food consumption before or after allowance for body weight between treated and control animals.
Ophthalmoscopic examination: There were no ophthalmology findings at week 4 that were attributed to treatment with the test item. Opacity of the cornea or lens was noted in treated and control animals. Incidences and severity were considered to be the same for all dose groups and were within the range of normal biological variation.
Haematology: Haematological parameters of treated rats were considered not to have been affected by treatment. In females receiving 200 and/or 1000 mg/kg/day minor statistically significant differences compared to the control animals were apparent, but these were considered to have arisen by chance and not to represent a change of biological significance.
Clinical biochemistry: There were no differences noted between control and treated rats that were considered to be related to treatment with the test item. The value for alanine aminotransferase in treated females receiving 1000 mg/kg/day achieved a level of statistical significance when compared to controls. This was considered to have arisen as a result of a slightly high control value and therefore of no toxicological significance.
Macroscopic examination: Macroscopic observations at necropsy did not reveal any alterations in any of the control and treated animals.
Organ weights: Organ weights and relative organ weights of treated animals were considered to be similar to those of control animals. A statistically significant decrease was observed for the liver weight and liver: body weight ratio in the males receiving 50 mg/kg/day when compared to control weights. As no corroborative findings were noted in the liver of these males or animals of other treatment groups, this finding was considered to have occurred by chance and not to represent a toxic effect.
Microscopic examination: There were no microscopic findings noted that were considered to be treatment related. The small numbers of changes recorded in treated animals were within the range commonly seen for rats of this age and strain.
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
not specified

no remarks

Conclusions:
From the results presented in this report a definitive No Observed Effect Level (NOEL) of 1000 mg/kg bw/day was established.
Executive summary:

The repeated dose toxicity of the test item was investigated in a subacute 28-day GLP and guideline oral toxicity study by daily gavage in the rat. Based on the results of a 5-day range finding study, the dose levels for the 28-day toxicity study were selected to be 0, 50, 200 and 1000 mg/kg bw/day. The test substance was administered daily for 28 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 5 males and 5 females. The following parameters were evaluated: clinical signs daily, body weight and food consumption weekly; ophthalmoscopy at week 4; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

RESULTS:

Formulations: Homogeneity was assumed, based on visually clear solutions obtained after preparation. Accuracy of test substance formulations were demonstrated by analyses.

50 mg/kg bw/day: No treatment-related findings noted.

200 mg/kg bw/day: No treatment-related findings noted.

1000 mg/kg bw/day: No treatment-related findings noted.

CONCLUSION:

From the results presented in this report a definitive No Observed Effect Level (NOEL) of 1000 mg/kg bw/day was established.

Data source

Materials and methods

Results and discussion

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion