Registration Dossier

Diss Factsheets

Administrative data

Description of key information

WoE: Subacute oral toxicity, rat, NOAEL = 100 mg/kg bw/d (reference 7.5.1 -1 & 7.5.1 -2).

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1990-04-27 to 1991-01-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
1981
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: 9 weeks
- Weight at study initiation: males: 316 g (295 - 336); females: 193 g (181 - 208) g
- Fasting period before study: not specified
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature: 22 - 28 °C
- Humidity: 26-45 %
- Air changes: not specified
- Photoperiod: not specified

To provide similar climate and lighting conditions for all rats, the position of the cages was changed once a week according to a rotation schedule.
Route of administration:
oral: gavage
Vehicle:
methylcellulose
Remarks:
0,5 % Methylcellulose in aqua / Tween 80 mixture (5 g Methocel(R) K4M Premium + 5 g Tween 80 ad 1 L)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle: well tolerated
- Concentration in vehicle: according to dose levels: 12.5, 25, 50 g/L
- Amount of vehicle: 20 mL / kg bw
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Test duration: 28 days
Frequency of treatment:
Dosing regime: 7 days/week
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle control
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 per sex per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: 1000 mg/kg are recommended as highest dose for a 4-weektoxicity-study in the OECD-guidelines. According to a dose range finding study the low dose was expected not to produce any adverse effects.
- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical biochemistry: not specified
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: twice daily

FOOD CONSUMPTION AND COMPOUND INTAKE:
- 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 AND COMPOUND INTAKE: Yes
- Time schedule for examinations: twice weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to start of the treatment and in study weeks 1 and 4
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: study week 4
- Anaesthetic used for blood collection: Yes (halothane)
- Animals fasted: Not specified
- How many animals: all animals
- Parameters checked: Erythrocyte count; Leukocyte count; Hemoglobin; Packed cell volume; Platelet count; Reticulocyte count; Differential blood cell count; Absolute number of segmented neutrophilic granulocytes; Absolute number of lymphocytes; mean corpuscular volume; Mean corpuscular hemoglobin content; Mean corpuscular hemoglobin concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: study week 4
- Animals fasted: Not specified
- How many animals: all animals
- Parameters checked: Electrolytes (Sodium, Potassium, Calcium, Chloride, Inorganic phosphorus); Substrates (Glucose, Urea); Creatinine, Bilirubin, Cholesterol, Triglycerides, Enzymes (Alanine aminotransferase; Alkaline Aminotransferase; Aspartate aminotransferase); Proteins (total; Albumin)

URINALYSIS: Yes
- Time schedule for collection of urine: overnight, for 18 h without feed
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: pH value; protein; glucose; Urobilinogen; Bilirubin; Blood; Sediment

NEUROBEHAVIOURAL EXAMINATION: No


IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Statistics:
Body weight gain, food consumption, water consumption, organ weights, the clinico-chemical serum parameters and the hematological parameters hematocrit, hemoglobin and numbers of erythrocytes and leucocytes of the dose groups were compared with those of the control (separately for each sex and time) using the multiple two-sided t-test according to DUNNETT (1955). Different group sizes or variance nonhomogeneity between the dose groups and the control were considered by correcting the critical t-values according to DUNNETT (1964), and in case of variance nonhomogeneity by WELCH'S correction of the degrees of freedom (cf. WINER, 1970, pp. 36 - 39). In cases of very low group sizes or extreme variance nonhomogeneity no statistical comparison was performed. For evaluation of the hematology parameters reticulocytes, eosinophilic, basophilic, juvenile/ bandform neutrophilic granulocytes and the leucocytes (which were not classified
further) the procedure of STOCKY and VOLLMAR (1976) (which is based on a linear rank test acc. to KRAUTH, 1971) with the two sided alternative was used. The parameters segmented neutrophilic granulocytes (SE) and lymphocytes (LY) were evaluated together acc. to UNKELBACH (1980), using the same procedure for the values SE/(SE +LY) .
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Except for dose-dependent burrowing in the litter and salivation, there were no differences to the controls in appearance and behavior of the rats from groups 2 and 3. The same reaction was seen in rats of group 4, some of which showed petechial bleedings at the tail. Burrowing in the litter for a short time and salivation directly after treatment are an indication of bad taste of the test material.
Mortality:
no mortality observed
Description (incidence):
No rat died in the course of the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight gain in group 2 corresponded to that of the controls. In group 3, near the end of the study, moderately reduced body weight gain was seen in both sexes. In group 4, body weight gain was clearly reduced throughout the study.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Decreased food consumption was mainly seen in group 4 in the first two weeks of treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption in group 2 essentially corresponded to that of the controls. At the end of the study, moderately increased water consumption was seen in group 3, while a clear increase occurred in group 4.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
As a finding, which is characteristic of young rats of this strain, rests of the membrana pupillaris persistens were seen in treated and untreated rats with the same frequency.
Haematological findings:
no effects observed
Description (incidence and severity):
The mean values for the hematological parameters corresponded to those of the controls. A slight, but significant decrease of hemoglobin values was indicated in females of the 500 mg/kg group and 1000 mg/kg bw/d group. A slight decrease in erythrocyte numbers was also seen. However, these deviations did not exceed the normal range.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A significant dose-dependent increase in serum creatinine was found in the males and females of groups 3 and 4. Further, a dose-dependent decrease in serum cholesterol values was found, which was significant in all treated groups, except for the males of group 2. Only in the males of group 4 a significant increase in alkaline phosphatase activity was observed. Sporadically mean values occurred which were significantly different from the corresponding control values. However, these deviations did not exceed the normal range and were thus without toxicological relevance.
Urinalysis findings:
not specified
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There was a dose-dependent increase in liver weights in all treated groups. The differences to the controls achieved statistical significance only on the basis of the body weight related data, due to decreased body weight gain of the rats. There was also a dose-dependent effect on the thymus, which was significantly lighter in all groups, except the low dose males. The adrenals were heavier in the dosed as compared to the control rats with statistical significance only in the high dose group. A similar effect was recorded for the prostate of the high dose males.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Adrenal size increased: 500 mg/kg bw/d 1 male and 1 females respectively; 1000 mg/kg bw/d 3 males and 1 female
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The following organ alterations attributable to the test item were found:
Adrenals: Hyperplasia of the adrenal cortex (500 mg/ kg bw/d : 1 m, 2 f; 1000 mg/kg bw/d: 3 m, 1 f)
Thymus: In only one of the 5 females of the 1000 mg/kg group atrophy was diagnosed.
Liver: No alterations attributable to treatment with the test item were found. Thus there was no morphological correlate to the increased weight of this organ.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Remarks on result:
other: Since it was not possible to derive a "no observed adverse effect level" on the basis of this study, a second study was performed.
Key result
Critical effects observed:
no
Conclusions:
Based on this subacute oral toxicity study a no observed adverse effect level could be derived.
Executive summary:

The test item was given via oral gavage to groups of 10 rats in daily doses of 250, 500, and 1000 mg/kg bw/d for a period of 4 weeks. All rats survived the treatment. Treatment and dose-related findings such as reduced food consumption and body weight gain and increased water consumption are frequently found in this kind of rat study. In addition, dose-dependent serum creatinine increase and serum cholesterol decrease, and increased liver weight were found and may be indicative of functional impairment of kidneys and liver. Since, however, these organs were perfectly normal histologically, no irreversible damage was induced in these organs by any of the doses tested. This also applies to all the other organs including thymus and adrenals, which reacted to the treatment by decrease or increase, respectiveley, of their weights.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1991-06-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
In another sub-acute oral toxicity study (reference 7.5.1-1) a No-Observed-Effect-Level (NOEL) could not be determined, even not with the lowest dose of 250 mg/kg bw/d. Therefore, another study was conducted applying lower dose levels of 50 and 100 mg/kg bw/d.
order to get a NOEL.
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
1981
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: 6 weeks
- Weight at study initiation: males: 172 g (159-182 g) ; females: 148 g (142-157 g)
- Fasting period before study: not specified
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature: 21 - 25 °C
- Humidity: 35 - 65 %
- Air changes: not specified
- Photoperiod: not specified
Route of administration:
oral: gavage
Vehicle:
methylcellulose
Remarks:
0,5 % aqueous solution Methylcellulose/Tween 80-mix (5 g Methocel(R) K4M Premium + 5 g Tween 80 ad 1 L)
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Test duration: 28 days
Frequency of treatment:
Dosing regime: 7 days/week
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle control
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In another sub-acute oral toxicity study (reference 7.5.1-1) a No-Observed-Effect-Level (NOEL) could not be determined, even not with the lowest dose of 250 mg/kg bw/d. Therefore, another study was conducted applying lower dose levels of 50 and 100 mg/kg bw/d.
- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical biochemistry: yes
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
daily

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations:
twice daily

FOOD CONSUMPTION AND COMPOUND INTAKE:
- 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 AND COMPOUND INTAKE: Yes
- Time schedule for examinations:
twice weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:
prior to start of the treatment and in study weeks 1 and 4
- Dose groups that were examined:
all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
study week 4
- Anaesthetic used for blood collection: Yes (halothane)
- Animals fasted: Not specified
- How many animals:
all animals
- Parameters checked: Erythrocyte count; Leukocyte count; Hemoglobin; Packed cell volume; Platelet count; Reticulocyte count; Differential blood cell count; Absolute number of segmented neutrophilic granulocytes; Absolute number of lymphocytes; mean corpuscular volume; Mean corpuscular hemoglobin content; Mean corpuscular hemoglobin concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
study week 4
- Animals fasted: Not specified
- How many animals:
all animals
- Parameters checked: Electrolytes (Sodium, Potassium, Calcium, Chloride, Inorganic phosphorus); Substrates (Glucose, Urea); Creatinine, Bilirubin, Cholesterol, Triglycerides, Enzymes (Alanine aminotransferase; Alkaline Aminotransferase; Aspartate aminotransferase); Proteins (total; Albumin)

URINALYSIS: Yes
- Time schedule for collection of urine:
overnight, for 18 h without feed
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: pH value; protein; glucose; Urobilinogen; Bilirubin; Blood; Sediment

NEUROBEHAVIOURAL EXAMINATION: No


IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Statistics:
Body weight gain, food consumption, water consumption, organ weights, the clinico-chemical serum parameters and the hematological parameters hematocrit, hemoglobin and numbers of erythrocytes and leucocytes of the dose groups were compared with those of the control (separately for each sex and time) using the multiple two-sided t-test according to DUNNETT (1955). Different group sizes or variance nonhomogeneity between the dose groups and the control were considered by correcting the critical t-values according to DUNNETT (1964), and in case of variance nonhomogeneity by WELCH'S correction of the degrees of freedom (cf. WINER, 1970, pp. 36 - 39). In cases of very low group sizes or extreme variance nonhomogeneity no statistical comparison was performed. For evaluation of the hematology parameters reticulocytes, eosinophilic, basophilic, juvenile/ bandform neutrophilic granulocytes and the leucocytes (which were not classified
further) the procedure of STOCKY and VOLLMAR (1976) (which is based on a linear rank test acc. to KRAUTH, 1971) with the two sided alternative was used. The parameters segmented neutrophilic granulocytes (SE) and lymphocytes (LY) were evaluated together acc. to UNKELBACH (1980), using the same procedure for the values SE/(SE +LY) .
Clinical signs:
no effects observed
Description (incidence and severity):
No difference in appearance and behavior was seen between the rats of the treatment groups and the controls.
Mortality:
no mortality observed
Description (incidence):
No rat died in the course of the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weight gain of the test item treated rats was equivalent to that of the controls.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The mean food consumption of the test material treated groups corresponded to that of the controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There was no adverse effect on water consumption throughout the study period for all rats of either sex.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
The hematological parameters in the test item treated rats were unremarkable as compared to the controls.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
The clinico-chemical values of the test material treated rats corresponded to those of the controls.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The statistical evaluation in this study revealed diminished ovary weights in females of group 3 and diminished weights of thyroids (group 3) and pituitaries (group 2,3) in males on a low significance level. In a previous study reference 7.5.1-1 much higher doses of the test item (up to 1000 mg/kg bw/d) caused organ weight alterations of thymus, livers, and adrenals. Other endocrine tissues than adrenals were not altered in this previous study. Therefore, in the present study the diminished organ weights are considered incidental. The slight differences of eye weights between treated and control male rats are of no toxicological relevance as well.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The histopathological examination revealed no alterations attributable to treatment with the test item. The lesions observed in this study were spontaneous in nature and occurred in all groups.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at the highest dose tested.
Key result
Critical effects observed:
no
Conclusions:
Under the conditions of this subacute oral toxicity study in the rat a NOAEL of 100 mg/kg bw/d was determined.
Executive summary:

Wistar rats, aged 7 weeks at the start of the study, with a mean weight of 172 g (males) and 148 g (females), were treated orally by gavage with the test item, 7 days a week. The treatment period was 4 weeks. The test material was prepared as a suspension in aqueous 0.5 % Methocel K4M Premium/Tween- 80. The administration volume was 10 mL/kg. The control rats received 10 ml/kg of the carrier medium. Dose levels of 0, 50, and 100 mg/kg bw/d were applied.

Each group consisted of 10 animals (5 m, 5 f). Total number of animals: 30 (15 m, 15 f). Appearance and behavior of all rats were checked daily. Body weight and water consumption were determined twice and food consumption once a week. Hematological examinations and clinico-chemical analyses of serum and urine were carried out in all rats in treatment week 4. At the end of the 4-week treatment period all animals were sacrificed and autopsied. 12 organs were weighed from each animal. 4 organs (liver, kidneys, adrenals, thymus) per animal were subjected to histological examination. No animal died in the course of the study. Appearance and behavior of the rats did not show test material related alterations. Body weight gain, food and water consumption were comparable to the controls. The hematological and clinico-chemical parameters showed no alterations attributable to the test item treatment. Gross pathological and histopathological examinations revealed no toxicologically relevant findings.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP and guideline compliant studies assessed in a WoE approach

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

Reference 7.5.1 -1

The test item was given via oral gavage to groups of 10 rats in daily doses of 250, 500, and 1000 mg/kg bw/d for a period of 4 weeks. All rats survived the treatment. Treatment and dose-related findings such as reduced food consumption and body weight gain and increased water consumption are frequently found in this kind of rat study. In addition, dose-dependent serum creatinine increase and serum cholesterol decrease, and increased liver weight were found and may be indicative of functional impairment of kidneys and liver. Since, however, these organs were perfectly normal histologically, no irreversible damage was induced in these organs by any of the doses tested. This also applies to all the other organs including thymus and adrenals, which reacted to the treatment by decrease or increase, respectiveley, of their weights.

Reference 7.5.1 -2

Wistar rats, aged 7 weeks at the start of the study, with a mean weight of 172 g (males) and 148 g (females), were treated orally by gavage with the test item, 7 days a week. The treatment period was 4 weeks. The test material was prepared as a suspension in aqueous 0.5 % Methocel K4M Premium/Tween- 80. The administration volume was 10 mL/kg. The control rats received 10 mL/kg of the carrier medium. Dose levels of 0, 50, and 100 mg/kg bw/d were applied.

Each group consisted of 10 animals (5 m, 5 f). Total number of animals: 30 (15 m, 15 f). Appearance and behavior of all rats were checked daily. Body weight and water consumption were determined twice and food consumption once a week. Hematological examinations and clinico-chemical analyses of serum and urine were carried out in all rats in treatment week 4. At the end of the 4-week treatment period all animals were sacrificed and autopsied. 12 organs were weighed from each animal. 4 organs (liver, kidneys, adrenals, thymus) per animal were subjected to histological examination. No animal died in the course of the study. Appearance and behavior of the rats did not show test material related alterations. Body weight gain, food and water consumption were comparable to the controls. The hematological and clinico-chemical parameters showed no alterations attributable to the test item treatment. Gross pathological and histopathological examinations revealed no toxicologically relevant findings.

Conclusion

Based on an weight of evidence approach it can be concluded that in the rat the subacute oral toxicity NOAEL is 100 mg/kg bw/d.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on repeated dose toxicity the test item does not require classification for specific target organ toxicity after repeated exposure (STOT RE) according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) 2019/521.