Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
21 September 1998
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
21.08.2001
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Considered suitable for type of study and usually required by regulatory agencies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6 weeks old
- Weight at study initiation: females: 153 - 177g; males 117 - 150g
- Housing: Macrolon cages with wood shavings as bedding material and shredded paper. Animals housed in groups of five, separated by sex
- Diet (e.g. ad libitum): ad libitum, commercial rodent diet (RM3)
- Water (e.g. ad libitum): ad libitum tap water for human comsumption
- Acclimation period: approx. 12 days

DETAILS OF FOOD AND WATER QUALITY:
Each batch of RM3 diet is analysed by the supplier for nutrients and contaminants
Routine microbiological, physical and chemical analysis of water made by supplier

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-3°C
- Humidity (%): 30-70%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hours dark; 12 hours light
Route of administration:
oral: feed
Details on route of administration:
Test item extracted from diet using acetonitrile. Extracts examined using HPLC and Fluorescence detection. Quantitation of test item was obtained by comparing peak areas of the samples with reference solutions containing a known amount of the test substance.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

- DIET PREPARATION
- Rate of preparation of diet (frequency): Once at start of study
- Mixing appropriate amounts with : Homogeneity was obtained by mixing in a mechanical blender. Feed in feeder was replaced by fresh portions once a week and topped up once weekly in between
- Storage temperature of food: Directly after preparation the feed was stored in a freezer (<-10C) until use.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Continuous - dietary
Dose / conc.:
0.12 other: % (w/w)
Remarks:
low-dose
Dose / conc.:
0.4 other: % (w/w)
Remarks:
mid-dose
Dose / conc.:
1.2 other: % (w/w)
Remarks:
high-dose
No. of animals per sex per dose:
10 males per dose
10 females per dose
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale:
- Rationale for animal assignment (if not random): 7 days before start of the treatment, the rats were weighed. On starting day of the treatment (Day 0), they were weighed , checked for normal growth and correct weight variation. The rats were then allocated to groups proportionately by weight class by a computer randomisation program
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: weekdays - twice per day; weekends - once per day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: all rats prior to exposure, once weekly during treatment up to and including Week 12

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0 and once per week thereafter. Final (fasted) body weights were determined on the day of the scheduled necropsy (Day 91 for males and Day 92 for females) in order to calculate the correct organ to body weight ratios

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: Yes
- Time schedule for examinations: Weighing bottles daily over a four day period in Week 1, 6 and 12 of the study.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to start of study (Day -3)
- Dose groups that were examined: All groups
- Time schedule for examinations: Last week of treatment (Day 89)
- Dose groups that were examined: Control and high dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes (CO2/O2)
- Animals fasted: Yes
- How many animals: All groups
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necrospy
- Animals fasted: Yes
- How many animals: All groups
- Parameters checked in table 2 were examined.

URINALYSIS: Yes (renal concentration test)
- Time schedule for collection of urine: Day 88-89 of study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (deprived of water for 24 hours, food in last 16 hours)
- Parameters checked in table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Day 82-25 of the study
- Dose groups that were examined: All groups
- Battery of functions tested: see table 4
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 5)
All rats from each treatment group were subjected to a complete macroscopic examination. Organs in table 5 were weighed (Paried organs together) as soon as possible after dissection to avoid drying

HISTOPATHOLOGY: Yes (see table 6)
Histopathological examination was performed on all tissues and organs in table 6 on all animal of the control and high dose groups
Liver, kidneys and all gross lesions were examined microscopically in all rats of all groups.
Statistics:
-Body weight: one-way analysis of covariance using pre-exposure (Day 0) weights as the covariate. When group means were significantly different (p<0.005), individual pair wise comparisons were made using Dunnett's multiple comparison method.
-Food intake, food conversion efficiency, water intake, red blood cell and clotting potential variable, total white blood cell counts, absolute differential white blood cell counts, clinical chemistry values, volume and density of the urine and organ weights and terminal body weights: ANOVA followed by Dunnett's multiple comparison tests or the least significant difference (LSD) test, Independent from the results of anova, the homogeneity of variances was tested by means of Bartlett's test. Parameters for which the variances differed significantly (p<0.001), were re-evaluated with Kruskal-Wallis non-parametric Anova followed by Mann-Whitney U-tests.
Reticulocytes and relative differential white blood cell counts, semi-quantitative urinary determinations and microscopy of the urinary sediment: Kruskal-Wallis non-parametric anova. When this analysis yielded a signifcant difference, pair wise comparisons between the control- and treatment groups were made by means of Mann-Whitney U-tests.
- Functional observational battery and motor activity results: anova followed followed multiple comparison tests (continuous data), Kruskal-Wallis non-parametric anova followed by multiple comparisaon tests (rank order data) or Paerson chi-square test (categorical data)
- Histopathological changes: Fisher's exact probability test
All analyses were two-sided. Group mean differences with an assciated probability of less thatn 0.05 were considered to be statistically significant.
Clinical signs:
effects observed, non-treatment-related
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:
effects observed, non-treatment-related
Description (incidence and severity):
Food conversion efficiency was comparable between the groups; a statistically significant decrease in high-dose males in Week 9 was considered an incidental finding.
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Increased haemoglobin concentration was noted in females of the low dose group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Cholesterol concentration in the plasma was statistically significantly increased in females of the high-dose group.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Presence of ketones in the urine in almost all males of the mid- and high-dose groups. Ketones were not detected in females in any group.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The relative weight of the liver was increased in males and females of the high-dose group
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Findings observed were common for rats of this strain and age, and occurred only incidental.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Findings observed were common for rats of this strain and age, and occurred only incidentally.
Incidence of focal mineralisation in the overies was statistically significantly lower in the high-dose females that in the control. This is considered fortuitous because mineralization in the ovaries is frequently observed in Wistar rats at variable incidences
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Details on results:
Decrease in feed intake per kg bw due to increasing age of the rats, intake of the test substance per kg bw gradually decreased. Overall mean intake of test substance in the low- mid- and high-dose groups was 74, 244 and 750 mg/kg bw/day for males and 74, 251 and 767 mg/kg bw/day for females
Dose descriptor:
NOAEL
Effect level:
ca. 74 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
urinalysis
Dose descriptor:
NOAEL
Effect level:
ca. 250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
Critical effects observed:
no
Conclusions:
It was concluded that the test substance NOAEL in this 13 week dietary study is placed at 0.12% in the diet (overall intake ca. 74 mg/kg bw/day) as the worst case value in males.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
74 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification