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Administrative data

Description of key information

The NOAEL for general, systemic toxicity of the test substance was 20 mg/kg bw/d for male and female based on clinical and pathological findings. At ≥ 60 mg/kg bw/day, local specific effects were observed in the thymus and jejunum.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to guideline
Guideline:
other: US EPA OPPTS 870.3650
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): Amine O
- Analytical purity: 96.6% imidazolines
- Isomers composition: The test substance is a UVCB substance consisting of 96.6% imidazolines
- Lot/batch No.:05774HP8
- Substance type: organic
- Physical state: liquid
- Stability under test conditions: stable
- Storage condition of test material: room temperature
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10-12 weeks
- Weight at study initiation: ca. 350g males, 192 g females
- Housing: individually in Makrolon type M III cages
- Diet: ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: Bottles, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24 °C
- Humidity: 30 - 70 %
- Air changes: 10 per hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was applied as a solution. To prepare the solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then the vehicle (drinking water) was filled up to the desired volume, buffered to pH 7.5 with HCl and subsequently mixed using a magnetic stirrer. Each preparation was prepared once, divided in daily portions and stored deep frozen until the daily use.

VEHICLE
- Amount of vehicle: 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses of the test-substance preparations were carried out at the Analytical Chemistry Laboratory of the Experimental Toxicology and Ecology of BASF SE. The stability of the test substance in drinking water (neutrally buffered) at room temperature for a period of 10 days was proven before the start of the administration period. Homogeneity was given because the test substance was completely miscible with water and solutions were considered to be homogenous without further analysis. Concentration control analyses of the test-substance preparations were performed in samples of all concentrations at the start and at the end of the administration period.
Duration of treatment / exposure:
males: 31 days
females: 51 days
Frequency of treatment:
daily
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Highest dose: First 7 (females) and 8 days (males) of the study
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Remarks:
Highest dose: Reduced dose level from study day 8 (females)/ 9 (males) onwards, toxic effects were seen at 100 mg/kg bw/day
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a previously performed 14-days test study the test article was administered by gavage to groups of 3 male and 3 female Wistar rats at dose levels of 0, 50, 100, 200, and 600 mg/kg body weight/day (mg/kg bw/d). Drinking water containing 1% Carboxylmethylcellulose and Cremophor EL® (5 mg/100 mL) served as vehicle. In this study, the oral administration of the test substance caused severe adverse effects in both sexes, i.e. premature deaths and poor general state, at dose levels of 600 and 200 mg/kg bw/d. Only slight effects on food and water consumption and body weight data were observed at 100 mg/kg bw/d.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.
A cageside examination was conducted before and after treatment for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented for each animal.
The littering and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g., animal could not litter, umbilical cord not cut) were documented on an individual dam basis. On weekdays (except public holidays) the littering behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of littering was considered to be a 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 x 37.5 cm with side borders of 25 cm high). The following parameters were examined: abnormal behavior during “handling”, fur, skin, salivation, exophthalmos, lacrimation, pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements, impairment of gait, activity/arousal level, feces (appearance/consistency), urine, other findings

BODY WEIGHT: Yes
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning).
The body weight change of the animals was calculated from these results. The following exceptions are notable for the female animals:
- During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
- Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
- Females without a litter were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

FOOD CONSUMPTION
Food consumption was determined once a week (in a period of 7 days) for male and female parental animals, with the following exceptions:
- Food consumption was not determined during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on GD 0, 7, 14 and 20.
- Food consumption of F0 females, which gave birth to a litter, was determined on PND 0 and 4.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel).

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: morning
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- How many animals: 5 per sex
- Parameters examined: Leukocyte count, Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, Differential blood count, Reticulocytes, Prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: morning
- Animals fasted: Yes
- How many animals: 5 per sex
- Parameters examined: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, gamma-Glutamyltransferase, Sodium, Potassium, Chloride, Inorganic phosphate, Calcium, Urea, Creatinine, Glucose, Total bilirubin, Total protein, Albumin, Globulins, Triglycerides, Cholesterol, Magnesium

URINALYSIS: Yes
- Time schedule for collection of urine: over night
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color, turbidity, Volume

NEUROBEHAVIOURAL EXAMINATION: Yes
A functional observational battery was performed in the selected five animals per sex and test group at the end of the administration period starting at about 10.00 h. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
- Home cage observations: The animals were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to: posture, tremors, convulsions, abnormal movements, impairment of gait, other findings
- Open field observations: The animals were transferred to a standard arena (50 x 50 cm with side borders of 25 cm high) and observed for at least 2 minutes. The following parameters were examined: behavior when removed from cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements, impairment of gait, activity/arousal level, feces (number of fecal pellets/appearance/consistency within two minutes, urine (appearance/quantity) within two minutes, number of rearings within two minutes
- Sensorimotor Tests/reflexes: The animals were removed from the open field and subjected to following sensorimotor or reflex tests: approach response, touch response, vision (“visual placing response”), pupillary reflex, pinna reflex, audition (“startle response”), coordination of movements (“righting response”), behavior during “handling”, vocalization, pain perception (“tail pinch”), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings.
- Motor activity measurement: The motor activity (MA) was measured on the same day as FOB was performed in 5 parental males and females (with litter) per group. The examinations were performed using the Multi- Varimex system supplied by Columbus Instruments Int. Corp., Ohio, U.S.A. For this purpose, the animals were placed in cages for the time of measurement. Four beams were allocated per cage. The number of beam interrupts was counted over 12 intervals for 5 minutes in each case. The sequence at which the animals were placed in the cages was selected at random. The measurement was started at about 14.00 h. On account of the measuring variant "staggered", the starting time was varied by the time needed to place the animals in the cages. For each animal, measurement was started individually when the 1st beam was interrupted and ended exactly 1 hour later. The animals received no food or water during the measurements. After the transfer of the last animal in each case, the room where the measurements were carried out was darkened.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, Adrenal glands, Brain, Cauda epididymis, Epididymides, Heart, Kidneys, Liver, Ovaries, Pituitary gland, Prostate, Seminal vesicles with coagulation glands, Spleen, Testes, Thymus, Thyroid glands, Uterus with cervix

HISTOPATHOLOGY: Yes
The following organs or tissues were fixed in 4% formaldehyde solution or in modified Davidson’s solution: All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum, Coagulation glands, Colon, Duodenum, Epididymides (modified Davidson’s solution), Esophagus, Eyes with optic nerve, Female mammary gland, Femur with knee joint, Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (mesenteric and axillary lymph nodes), Nose (nasal cavity), Ovaries (modified Davidson’s solution), Oviducts, Pancreas, Pharynx, Pituitary gland, Prostate, Rectum, Salivary glands (mandibular and sublingual glands), Seminal vesicle with coagulation glands, Sciatic nerve, Skeletal muscle, Skin, Spinal cord (cervical, thoracic and lumbar cords), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Testes (modified Davidson’s solution), Thymus, Thyroid glands/parathyroid glands, Trachea, Urinary bladder, Uterus, Vagina

Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings according to the table presented in 'Any other information on materials and methods incl. tables'
Statistics:
Non-parametric one-way analysis using Kruskal-Wallis test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs of toxicity were only seen in test group 3 (100 and 60 mg/kg bw/d). Poor general state was observed in study weeks 1 and 2 in two male animals and two female animals, in one male animal in study weeks 3 and 4 and in one female animal in study weeks 6 and 7. Reduced nutritional state was also observed in study week 2 in one male animal, two female animals in study weeks 1 and 2 and one female animal in week 0. Respiratory sounds was observed in study week 0 in 3 male animals, in study week 1 in two male animals in study week 2 in three male animals, in study week 3 in two animals and in study week 4 in one male animal. In females respiratory sounds were observed in nine animals in study week 0, in eight animals in study week 1, in three animals in study week 2 and also in two animals in study week 6 and in study week 7 one animal.
Piloerection was observed in study week 0 in one female animal. Diarrhea was observed in study week 2 in one female animal. No treatment-related findings were seen in any animal of test groups 1 and 2 (5 and 20 mg/kg bw/d).
Mortality:
mortality observed, treatment-related
Description (incidence):
In test group 3 (100 and 60 mg/kg bw/d) one female animal was found dead within the first week of the study. In addition, one male animal and one female animal of test group 3 (100 and 60 mg/kg bw/d) were sacrificed in a moribund state in study week 2.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In test group 3 (100 and 60 mg/kg bw/d) male animals’ body weight was significantly lower in week 2. In the same test group body weight loss was observed in male and female (not significant) animals between study weeks 0-1 and, in summary, body weight change values of male animals were significantly lower between study weeks 0-4. No effects on body weight data were observed in male and female animals of test groups 1 and 2 (5 and 20 mg/kg bw/d) during the entire study period. The same was true for female animal of test group 3 (100 and 60 mg/kg bw/d) during gestation and lactation periods.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Significantly decreased food consumption in male animals of test group 3 (100 and 60 mg/kg bw/d) was only observed during the first two study weeks. Significantly decreased food consumption in female animals of test group 3 (100 and 60 mg/kg bw/d) was observed in the first study week. Both findings were assessed as being related to treatment. Impaired food consumption was not observed in male and female animals of test groups 1 and 2 (5 and 20 mg/kg bw/d). The same was true for female animals of test group 3 (100 and 60 mg/kg bw/d) during gestation and lactation periods.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related, adverse changes among haematological parameters were observed.
In female rats of test groups 1 (5 mg/kg bw/d) and 3 (100 and 60 mg/kg bw/d) the platelet counts were lower compared to controls. These values were within the historical control ranges (platelet counts: 765-1009 Giga/L). Therefore, the alterations were regarded as incidental rather than treatment-related. In females of test group 2 (20 mg/kg bw/d) the relative lymphocyte counts, and in females of test group 3 (100 and 60 mg/kg bw/d) the relative large unstained cell (LUC) counts were lower compared to controls. These changes in the differential blood cell counts were not accompanied by a change of the total white blood cell counts, and the lymphocyte and LUC cell counts were within the historical control ranges (relative lymphocyte counts: 66.7-83.9%, relative LUC counts: 0.2-0.5%). The alterations were regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related, adverse changes among clinical chemistry parameters were measured. In males of test group 3 (100 and 60 mg/kg bw/d) the creatinine values were decreased. This was the only alteration among the clinical pathology parameters in rats of this sex in this test group. Therefore, it was regarded as a maybe treatment-related but non-adverse effect (ECETOC, Technical Report No. 85, 2002).
Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment-related adverse changes among urinalysis parameters were measured.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No test substance-related or spontaneous findings in male and female animals of all test groups during the home cage observation were observed.
The open field observations did not reveal any test substance-related findings in male and female animals of all test groups. During sensimotor tests, there were no test substance-related findings in male and female animals of all test groups. Any deviations from "zero values" were equally distributed between test substance-treated groups and controls or occurred in single animals only. Therefore, these observations were considered as being incidental. There were no significant deviations concerning single intervals and the overall motor activity (summation of all intervals) in male and female animals of all test groups in comparison to the concurrent control group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Both absolute- and relative organ weight changes did not reveal any relation to treatment for planned sacrificed animals.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Regarding pathology, animals, which died intercurrently or were sacrificed moribund showed some macroscopic and/or microscopic lesions in the gastro-intestinal tract. Dilation of stomach, jejunum, cecum or colon, mucosal/villous atrophy, acute inflammation and erosion(s) and intra-epithelial abscesses in forestomach were observed in one animal. In addition, lesions in some lymphoid organs indicating secondary stress related changes were observed such as severe organ size reduction of thymus (atrophy), lymphoid depletion in spleen and axillary lymph node and lymphocytolysis in axillary and mesenteric lymph nodes. In one animal of test group 3 (100 and 60 mg/kg bw/d) that died on study day 3, also bilateral cortical hypertrophy of the adrenal cortex was observed indicating prolonged stress response. These changes mentioned for animals at moribund sacrifice and spontaneous death were considered to be treatment-related.

No treatment-related macroscopic and/or microscopic changes were observed in animals autopsied at planned sacrifice. For these planned sacrificed animals, all macroscopic and/or microscopic and organ weight changes observed were considered to be normal background changes, bearing no relation to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
clinical signs
gross pathology
mortality
Dose descriptor:
LOAEL
Effect level:
>= 60 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
clinical signs
gross pathology
mortality
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
60 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
jejunum
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
60 mg/kg bw/day (actual dose received)
System:
immune system
Organ:
thymus
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified
Conclusions:
100 mg/kg bw/ day caused mortality, effects in the gastrointestinal tract and signs of stress. The NOAEL for general, systemic toxicity of the test substance was 20 mg/kg bw/d for male and female animals based on clinical and pathological findings.
Executive summary:

The test article was administered orally by gavage to groups of 10 male and 10 female Wistar rats (F0 parental animals) at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; vehicle control; test group 0), 5 mg/kg bw/d (test group 1), 20 mg/kg bw/d (test group 2) and 100 mg/kg bw/d (test group 3). Because of severe clinical findings seen in animals of test group 3 (100 mg/kg bw/d), i.e. respiration sounds, reduced body weights and the death of one female animal on study day 3, the dose level in test group 3 was reduced to 60 mg/kg bw/d for the females on study day 8 as well as for the males on study day 9. Drinking water (neutrally buffered for test substance preparations) served as vehicle. F0 animals were mated 13 days after the beginning of treatment to produce litter (F1 generation pups). Mating pairs were from the same test group. Mating was discontinued as soon as sperm was detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of postimplantation loss for all F0 females. A detailed clinical observation (DCO) was performed in all F0 animals before test substance administration and thereafter at weekly intervals. Food consumption of the F0 parents was determined regularly during premating and after the mating period and during the gestation and lactation periods in dams. In general, body weights of F0 animals were determined once a week. During gestation and lactation period, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, on the parturition day (postnatal day [PND] 0) and on PND 4. Pups were sexed on PND 0 and weighed one day after birth and on PND 4. Their viability was recorded twice daily on each working day or only in the morning on Saturday and Sunday. Clinicochemical and hematological examinations as well as urinalyses were performed in 5 randomly selected F0 parental animals of either sex towards the end of the administration period. At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 randomly selected F0 parental males and females per test group. All surviving F0 parental animals were sacrificed assessed by gross pathology. Organ weights were recorded and a histopathological examination was performed.

 

In test group 3 (100 mg/kg bw/d between study days 0-7 [females] and 0-8 [males], 60 mg/kg bw/d onwards from study day 8 [females] and 9 [males]), one female animal was found dead in study week 1. One male and one female animal were sacrificed moribund state in study week 2. Poor general state was observed in one female animal in study week 0, in two male and two female animals in study weeks 1 and 2, in one male animal in study weeks 3 and 4 and in one female animal in study weeks 6 and 7 as well as in one female animal from GD 0 and 1, and from GD 7 onwards. Reduced nutritional state was recorded in one male animal in study week 2, one female animal in study week 0 and two female animals in study weeks 1 and 2 as well as in one female animal from GD 0 to 5. Respiratory sounds were reported in several male and female F0 animals during the entire study period. Furthermore, piloerection was seen in one female animal in study week 0 and one female animal in study week 2 had diarrhea. Significantly decreased food consumption was reported in males during the first two study weeks and in females in the first study week. In addition, significantly decreased male animals’ body weight in week 2 and significantly decreased body weight change in week 0-1 and in summary between weeks 0-4 were seen. The male animal which was sacrificed moribund showed severe dilation of the gastro-intestinal tract (stomach, jejunum, colon and cecum) with severe organ size reduction of the thymus. The female animal which was sacrificed moribund showed severe dilation of the gastro-intestinal tract (stomach, jejunum and cecum) with severe organ size reduction of the thymus. No test-substance related, relevant findings were observed regarding clinical pathology. Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar Rats the no observed adverse effect level (NOAEL) for general, systemic toxicity of the test substance was 20 mg/kg bw/d for male and female F0 parental animals based on clinical and pathological findings.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
20 mg/kg bw/day
Study duration:
subacute
Species:
rat
System:
other: immune system and gastrointestinal tract
Organ:
jejunum
thymus

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

In a GLP-compliant OECD 422 study, the test article was administered orally by gavage to groups of 10 male and 10 female Wistar rats (F0 parental animals) at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; vehicle control; test group 0), 5 mg/kg bw/d (test group 1), 20 mg/kg bw/d (test group 2) and 100 mg/kg bw/d (test group 3). Because of severe clinical findings seen in animals of test group 3 (100 mg/kg bw/d), i.e. respiration sounds, reduced body weights and the death of one female animal on study day 3, the dose level in test group 3 was reduced to 60 mg/kg bw/d for the females on study day 8 as well as for the males on study day 9. Drinking water (neutrally buffered for test substance preparations) served as vehicle. F0 animals were mated 13 days after the beginning of treatment to produce litter (F1 generation pups). Mating pairs were from the same test group. Mating was discontinued as soon as sperm was detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of postimplantation loss for all F0 females. A detailed clinical observation (DCO) was performed in all F0 animals before test substance administration and thereafter at weekly intervals. Food consumption of the F0 parents was determined regularly during premating and after the mating period and during the gestation and lactation periods in dams. In general, body weights of F0 animals were determined once a week. During gestation and lactation period, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, on the parturition day (postnatal day [PND] 0) and on PND 4. Pups were sexed on PND 0 and weighed one day after birth and on PND 4. Their viability was recorded twice daily on each working day or only in the morning on Saturday and Sunday. Clinicochemical and hematological examinations as well as urinalyses were performed in 5 randomly selected F0 parental animals of either sex towards the end of the administration period. At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 randomly selected F0 parental males and females per test group. All surviving F0 parental animals were sacrificed assessed by gross pathology. Organ weights were recorded and a histopathological examination was performed.

 

In test group 3 (100 mg/kg bw/d between study days 0-7 [females] and 0-8 [males], 60 mg/kg bw/d onwards from study day 8 [females] and 9 [males]), one female animal was found dead in study week 1. One male and one female animal were sacrificed moribund state in study week 2. Poor general state was observed in one female animal in study week 0, in two male and two female animals in study weeks 1 and 2, in one male animal in study weeks 3 and 4 and in one female animal in study weeks 6 and 7 as well as in one female animal from GD 0 and 1, and from GD 7 onwards. Reduced nutritional state was recorded in one male animal in study week 2, one female animal in study week 0 and two female animals in study weeks 1 and 2 as well as in one female animal from GD 0 to 5. Respiratory sounds were reported in several male and female F0 animals during the entire study period. Furthermore, piloerection was seen in one female animal in study week 0 and one female animal in study week 2 had diarrhea. Significantly decreased food consumption was reported in males during the first two study weeks and in females in the first study week. In addition, significantly decreased male animals’ body weight in week 2 and significantly decreased body weight change in week 0-1 and in summary between weeks 0-4 were seen. The male animal which was sacrificed moribund showed severe dilation of the gastro-intestinal tract (stomach, jejunum, colon and cecum) with severe organ size reduction of the thymus. The female animal which was sacrificed moribund showed severe dilation of the gastro-intestinal tract (stomach, jejunum and cecum) with severe organ size reduction of the thymus. No test-substance related, relevant findings were observed regarding clinical pathology. Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar Rats the no observed adverse effect level (NOAEL) for general, systemic toxicity of the test substance was 20 mg/kg bw/d for male and female F0 parental animals based on clinical and pathological findings.



Repeated dose toxicity: via oral route - systemic effects (target organ) cardiovascular / hematological: thymus; digestive: jejunum

Justification for classification or non-classification

There are conclusive and sufficient data available for classification of the test substance with regard to systemic effects following repeated dosing. Based on the data available, the test article is classified as STOT Rep. Exp. 2 H373: 'May cause damage to the intestinal tract and thymus through prolonged or repeated exposure' in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.

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