Registration Dossier

Administrative data

Description of key information

The administration of the test item in the diet to male and female rats was tolerated without adverse effects up to 900 ppm, according to about 56 mg/kg body weight/day in males and 78 mg/kg body weight/day in females; these doses can be regarded as no-observed-adverse-effect levels (NOAEL), based on poor general condition, body weight retardation and irritating effects in the stomach in the highest dose group.

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
Study period:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
1998
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
1998
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- stability was proven with 50 ppm for 8 days, 100 and 10000 ppm for 15 days; at -20 °C stability was proven with 2 months;
- Homogeneity was established for a 12 kg mixture: 50, 100 and 10000 ppm
- the content of active ingredient was taken into account
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann, Borchen, Germany
- Weight at study initiation: 142-196 g for males and 141-165 g for females
- Diet: ad libitum
- Water: ad libitum

IN-LIFE DATES: From: 2002-03-22 To: 2002-07-26
Route of administration:
oral: feed
Details on route of administration:
ad libitum in the diet up to necropsy
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): one a week
- Mixing appropriate amounts with (Type of food): the test substance was blended with Provimi Kliba 3883.9.25 (using a mixing granulator). To all diet mixtures (including 0 ppm) peanut oil (DAB 10; content per kg food: 10 g) was added to minimize dust formation. The amounts of test item were calculated on the basis of a 92 % content and applied in the diet (NAFAG 9441 G4 with 1 % peanut oil, DAB 10)

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
four content checks (all doses) including investigation on homogeneity (low and high dose) were done
Duration of treatment / exposure:
93 days
Frequency of treatment:
continuously via diet followed by a recovery period without treatment over 4 weeks
Dose / conc.:
100 ppm
Remarks:
corresponding to about 6.4 mg/kg bw/day in males and 9.6 mg/kg bw/day in females
Dose / conc.:
900 ppm
Remarks:
corresponding to about 55.9 mg/kg bw/day in males and 78.1 mg/kg bw/day in females
Dose / conc.:
8 100 ppm
Remarks:
main groups: corresponding to about 624.6 mg/kg bw/day in males and 852.2 mg/kg bw/day in females
Dose / conc.:
8 100 ppm
Remarks:
pair-fed recovery groups: corresponding to about 702.8 mg/kg bw/day in males and 1040.3 mg/kg bw/day in females
No. of animals per sex per dose:
10 in main study and in pair-fed recovery groups (control and high dose)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale:
Based on the results of the dose-range finding study for the 3-months study a clear toxic effect on body weight (possibly due to the reduced intake of food) was to be expected at a dose of 5000 ppm in males and 10000 ppm in females. Therefore, the high dose was chosen in the range of 5000 to 10000 ppm.

The dose-range finding study showed markedly lower food intake in males at 10000 ppm that may have been due to bad palatability of the test substance. Thus, pair-fed controls were introduced into this study to distinguish between possible effects due to palatability and toxicological alterations. These groups (2 and 7) were started one week (actual dates see Table 3-1) after the start of the other groups. Mean daily food intake of main group males and females treated with 8100 ppm was determined in week n and the same amount was offered daily to pair-fed animals in week n + 1 (relative to main group animals).
During the first weeks of the treatment period it was observed, as expected, that males and females at 8100 ppm spilled some food. Therefore, on day 15 the first 5 cages of control animals (groups 1 and 6) and of animals at 8100 ppm (from groups 5 and 1 0) were put aside so that bedding material and feces could be separated from spilled food by sifting. The procedure was repeated during days 15-21,43-49, and 71- 77. The resulting "correction factors" were used to calculate the amount of food offered daily to pair-fed groups for the following week(s) or until scheduled necropsy of these animals.
Positive control:
no positive control required
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before start of treatment (all groups), at the end of treatment and at the end of the recovery period (control and high-dose groups)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 2 times in main groups, once in recovery groups

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 2 times in main groups, once in recovery groups

URINALYSIS: Yes
- Time schedule for collection of urine: 2 times in main groups, once in recovery groups

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once at the end of the treatment periods
- Battery of functions tested: sensory activity / grip strength / motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (all dose groups and controls)

ORGAN Weights: Yes

HISTOPATHOLOGY: Yes
Statistics:
yes
Description (incidence and severity):
Clinical observations revealed in both sexes at 8100 ppm piloerection and decreased reactivity, which were reversible, as well as spilling of food and increased feces excretion during the treatment period and also within the first weeks of the recovery period. The spilling of food is most likely reflecting a palatability problern and the search of the animals for better tasting food.
Mortality:
no mortality observed
Description (incidence and severity):
Body weight development was retarded in all animals at 8100 ppm, which resulted in lower body weights at the end of the treatment period (reduced by 20/23 % in males and 9/19 % in females; main group/recovery group ). This effect was not reversible during the recovery period in females and only slightly reversible in males. Due to the food·restriction (see below) pair-fed animals showed a similar body weight development. This suggests that the lower body weight gain in main and recovery groups at 81 00 ppm is at least in part due to a reduced food intake (concealed by the spilled food).
Description (incidence and severity):
The determination of food intake suggests a higher intake in main and recovery groups at 8100 ppm; however, these determinations were distorted by the spillage of food observed and, therefore, unsuitable for a definitive assessment at this dose level.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
The average water intake was higher in both sexes at 8100 ppm at the end of treatment, which was not reversible; increased water intake was interpreted as a consequence of the local irritative effect of the test substance. Urinalyses and histopathology revealed no indication of test substance-induced effects on kidney function or morphology.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Hematology revealed a prolonged thromboplastin time (HQUICK) in 8100 ppm main group males at both dates of examination, which was reversible. In the spleen of this group the average grade of hematopoiesis and pigment deposition was slightly reduced.
Description (incidence and severity):
Clinical chemistry showed a lower glucose concentration in main group males and females at 8100 ppm at both dates of examination, which was reversible. At the end of the recovery period, cholesterol and·protein concentrations were lower in both sexes, triglyceride concentration was lower in males and urea concentration was higher in females compared to controls. These changes reflect most likely functional liver changes; no adverse effects are assumed.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Description (incidence and severity):
At necropsy of main groups several significant differences from controls were observed. However, mostly all of the changes seen in treatment groups can be explained by the retardation in body weight development because either there were not significant differences of relative organ weight (taking into account the final body weights) or pair-fed controls showed comparable changes (i.e. liver weights). Beyond that main group females at 8100 ppm showed a tendency to lower relative uteri weights (p>0.05).
Description (incidence and severity):
Necropsy revealed a dilated stomach in main group males at 8100 ppm.
Neuropathological findings:
no effects observed
Description (incidence and severity):
Functional observational battery as well as motor and locomotor activity did not indicate a neurotoxic effect.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathology showed microerosions in the glandular stomach mucosa in this group and also in one recovery male, which is indicative of a local irritative effect.
Individual rats of the pair-fed group or at 8100 ppm showed atrophy of the salivary glands (parotid or sublingual gland), which is caused by body weight retardation and no direct test substance-effect is considered.
In the mesenteric lymph nodes, 8100 ppm main and recovery animals had an increased number of histiocytes with a foamy cytoplasm in their sinuses.
Dose descriptor:
NOAEL
Effect level:
900 ppm
Based on:
other: mean daily intake of test substance via food: 56 mg/kg bw/day for males
Sex:
male
Basis for effect level:
other: body weight retardation and irritating effects in the stomach, general condition
Dose descriptor:
NOAEL
Effect level:
900 ppm
Based on:
other: mean daily intake of test substance via food: 78 mg/kg bw/day for females
Sex:
female
Basis for effect level:
other: body weight retardation and irritating effects in the stomach, general condition
Critical effects observed:
not specified
Conclusions:
The NOAEL for the given test substance was 900 ppm according to 56 mg/kg bw/day in males and 78 mg/kg bw/ day in females.
Executive summary:

The test item was administered in the diet to 10 male and 10 female Wistar rats per dose group, in doses of 0 (control), 0 (as pair-feeding control; food supply adapted to food intake at 8100 ppm), 100, 900 and 8100 ppm for a period of 93 days. In addition, 10 males and 10 females per dose group were treated likewise in doses of 0 and 8100 ppm over a period of 91 days and observed for a subsequent treatment free period of 29 days for reversibility, continuation or delayed occurrence of toxic effects.

Mortality was unaffected by the treatment. Clinical observations revealed in both sexes at 8100 ppm piloerection and decreased reactivity, which were reversible, as well as spilling of food and increased feces excretion during the treatment period and also within the first weeks of the recovery period. The spilling of food is most likely reflecting a palatability problem and the search of the animals for better tasting food. Functional observational battery as well as motor and locomotor activity did not indicate a neurotoxic effect. Ophthalmological and histopathological investigation gave no indication of an oculotoxic effect of the test substance. Body weight development was retarded in all animals at 8100 ppm, which resulted in lower body weights at the end of the treatment period (reduced by 20/23 % in males and 9/19 % in females; main group/recovery group). This effect was not reversible during the recovery period in females and only slightly reversible in males. Due to the food·restriction (see below) pair-fed animals showed a similar body weight development. This suggests that the lower body weight gain in main and recovery groups at 8100 ppm is at least in part due to a reduced food intake (concealed by the spilled food). The food intake determined at 100 and 900 ppm resulted in a test substance intake during the treatment period of about 6.4 and 56 mg/kg body weight/day in males and of about 9.6 and 78 mg/ kg body weight/day in females. The determination of food intake suggests a higher intake in main and recovery groups at 8100 ppm; however, these determinations were distorted by the spillage of food observed and, therefore, unsuitable for a definitive assessment at this dose level.

The average water intake was higher in both sexes at 8100 ppm at the end of treatment, which was not reversible; increased water intake was interpreted as a consequence of the local irritative effect of the test substance (see below). Blood electrolyte concentrations were inconspicuous. Urinalyses and histopathology revealed no indication of test substance-induced effects on kidney function or morphology. Hematology revealed a prolonged thromboplastin time (HQUICK) in 8100 ppm main group males at both dates of examination, which was reversible. In the spleen of this group the average grade of hematopoiesis and pigment deposition was slightly reduced. Clinical chemistry showed a lower glucose concentration in main group males and females at 8100 ppm at both dates of examination, which was reversible. At the end of the recovery period, cholesterol and protein concentrations were lower in both sexes, triglyceride concentration was lower in males and urea concentration was higher in females compared to controls. These changes reflect most likely functional liver changes; no adverse effects are assumed.

Necropsy revealed a dilated stomach in main group males at 8100 ppm. Histopathology showed microerosions in the glandular stomach mucosa in this group and also in one recovery male, which is indicative of a local irritative effect. Individual rats of the pair-fed group or at 8100 ppm showed atrophy of the salivary glands (parotid or sublingual gland), which is caused by body weight retardation and no direct test substance-effect is considered. In the mesenteric lymph nodes, 8100 ppm main and recovery animals had an increased number of histiocytes with a foamy cytoplasm in their sinuses. Gross and histopathological investigation of other organs and tissues did not indicate test substance-related effects.

Under the conditions described the administration of the test item in the diet to male and female rats was tolerated without adverse effects up to 900 ppm, according to about 56 mg/kg body weight/day in males and 78 mg/kg body weight/day in females; these doses can be regarded as no-observed-adverse-effect levels (NOAEL), based on the effects in the highest dose group.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
56 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Additional information

The test item was administered in the diet to 10 male and 10 female Wistar rats per dose group, in doses of 0 (control), 0 (as pair-feeding control; food supply adapted to food intake at 8100 ppm), 100, 900 and 8100 ppm for a period of 93 days. In addition, 10 males and 10 females per dose group were treated likewise in doses of 0 and 8100 ppm over a period of 91 days and observed for a subsequent treatment free period of 29 days for reversibility, continuation or delayed occurrence of toxic effects.

Under the conditions described the administration of the test item in the diet to male and female rats was tolerated without adverse effects up to 900 ppm, according to about 56 mg/kg body weight/day in males and 78 mg/kg body weight/day in females; these doses can be regarded as no-observed-adverse-effect levels (NOAEL), based on poor general condition, body weight retardation and irritating effects in the stomach in the highest dose group.

In a 28-day oral toxicity study in Wistar rats intended as a pilot study for a one-generation reproduction and subchronic toxicity study the test item was given in the diet in concentrations of 100, 1000, 5000, and 100000 ppm in the diet. The highest dose clearly exceeded MTD. The NOAEL was considered with 1000 ppm, i.e. 115 mg/kg bw/day in males and 157 mg/kg bw/ day in females.

Justification for classification or non-classification

Based on the available data no classification is warranted.