Registration Dossier

Administrative data

Description of key information

Under the conditions of a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422), the oral administration by gavage of the test item to Wistar rats revealed signs of systemic toxicity at a dose level of 250 mg/kg bw/d in animals of both sexes. Thus, the NOAEL for general systemic toxicity was 80 mg/kg bw/d for male and female Wistar rats.

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:
2017-03-13 to 2018-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Version / remarks:
July 2000
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and batch No.of test material: 002-150504
- Expiration date of the batch: 29 Apr 2025
- Purity: 99.22 area-% (sum of all peaks, HPLC 231 nm), 99.05 area-% (sum of all peaks, HPLC 323 nm)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under storage conditions: guaranteed by the sponsor
- Storage conditions: ambient
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 14-15 weeks (males animals); about 13 weeks (female animals)
- Weight at study initiation: (P) Males: 374.6 +/- 13.5 g; Females: 221.4 +/- 7.8 g
- Housing during pre-mating, mating, gestation, lactation, males after mating and females after weaning: Polycarbonate cages type III
- Housing during pre-treatment: Polysulfonate cages Typ 2000P (H-Temp), floor area about 2065 cm2 (610 x 435 x 215 mm); supplied by TECHNIPLAST, Hohenpeiflenberg, Germany
- No of animals per cage: 1; exceptions: during pre-treatment: 5 animals per sex and cage; during mating: 1 male/1 female per cage; during rearing up to PND 13: 1 dam with her litter.
- Bedding: Dust-free wooden bedding
- Diet and water: ad libitum
- Type of diet: Ground Kliba maintenance diet mouse/rat "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland
- Acclimation period: 28 days prior to the beginning of administration period

REASON FOR SELECTION:
The rat is a frequently used laboratory animal, and there is comprehensive experience with this animal species. Moreover, the rat has been proposed as a suitable animal species by the OECD and the EPA. This Wistar rat strain (Crl:WI(Han)) is selected because extensive historical control data is available for these rats.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 /12
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% Carboxymethylcellulose suspension in drinking water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- Route of administration: Orally by gavage using 3 or 5 mL syringes
- Preparation frequency: The test substance preparations were prepared at intervals which guarantee that the test substance concentrations in the vehicle will remain stable.
- Preparation: Test substance preparations in 0.5% Carboxymethylcellulose suspension in drinking water.
- Form of preparation: suspension
- Carrier: 0.5% Carboxymethylcellulose suspension in drinking water
- Reason for the selection of the route of administration: The oral administration of a test substance has been proved useful worldwide in numerous studies for discovering a potential toxicological profile.

DIET PREPARATION
- Rate of preparation of diet (frequency): at least weekly
- Mixing appropriate amounts with (Type of food): Ground Kliba maintenance diet mouse/rat "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland
- Storage conditions of the preparations: Ambient

VEHICLE
- Concentration in vehicle: 0.5% Carboxymethylcellulose suspension in drinking water
- Amount of vehicle: 10 mL/kg bw/d; the body weight determined most recently was used to calculate the administration volume.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- At the beginning (during pre-mating), twice during gestation and once during lactation of the study each 3 samples (from the top, middle and bottom of the preparation vessel) were taken from the lowest and highest concentration for potential homogeneity analyses. These samples were used as a concentration control at the same time. At the above mentioned time points additionally one sample from the mid concentration was taken for concentration control analysis.
- All test samples, plus a duplicate set of reserve samples, were withdrawn by staff of the Sub-chronic/Chronic Toxicology Laboratory Rodents.
- The samples collected at the beginning of the administration period and during lactation were analysed in the Analytical Laboratory.
- The samples of the gestation were analysed only if any imprecision occurs during the analysis of the samples from the beginning and lactation of the study.
- The last sample date was identified on the accompanying documentation to inform the Analytical Laboratory that the report can be prepared.
- Retain samples were stored frozen at the Laboratory Subchronic/Chronic Toxicity (at -20°C).
Duration of treatment / exposure:
All animals, with the exception of the controls, received the test substance daily by gavage according to the time schedule (exception: no administration to animals being in labor). All animals were daily observed for any clinical signs during the study period. The duration of exposure was at least 28 days, including 14 days of pre-mating.
Frequency of treatment:
Once daily for 7 days/week
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
low-dose level
Dose / conc.:
80 mg/kg bw/day (nominal)
Remarks:
mid-dose level
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
high-dose level
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
yes, historical
Details on study design:
- Dose selection rationale: Doses were determined by performing a 14-day dose range finding study in which doses of 300 and 1000 mg/kg bw/day were tested described in the supporting study record with report number "01R0051/16N02" in section 'Repeated dose toxicity: oral" (BASF, 2017).

Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Parameters observed: Posture, Tremors, Convulsions, Abnormal movements, Gait, Other findings.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily from Mondays to Fridays and once daily on weekends and public holidays
- Parameters observed: Abnormal behavior in handling; Fur; Skin; Posture; Salivation; Respiration; Activity/arousal level; Tremors; Convulsions; Abnormal movements; Gait abnormalities; Lacrimation; Palpebral closure; Exophthalmus (Protruding eyeball); Assessment of the feces excreted during the examination (appearance, consistency); Assessment of the urine excreted during the examination; Pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: once a week at the same time of the day (in the morning)
- During the mating period, the 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), PND 4. PND 7 PND 10 and PND 13.
- Females showing no positive evidence of sperm in the vaginal smear were weighed once a week during this mating interval as were the males (for the calculation of the administration volume, body weight data of these individuals were only reported in the individual tables).
- Females without litter and after weaning (PND 13) were weighed once a week (for the calculation of the administration volume, body weight data of these individuals were only reported in the individual tables).

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Food consumption was determined once a week for the male and female parental animals.
- Food consumption was determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals).
- Food consumption of the females with evidence of sperm was determined for GD 0-7, 7-14 and 14-20.
- Food consumption of the females which gave birth to a litter was determined for PND 1-4, 4-7, 7-10 and 10-13.

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily by visual inspection of the water bottles for any changes in volume

HAEMATOLOGY: Yes
- Time schedule: Prenatal day14
- Anaesthetic used for blood collection: Yes, under isoflurane anesthesia
- Animals fasted: Yes
- How many animals: the first 5 surviving parental males per group at termination and the first 5 females with litters (in order of delivery) per group at PND 14.
- The following parameters were observed: leukocytes; erythrocytes; haemoglobin; haematocrit; mean corpuscular volume (MCV); mean corpuscular haemoglobin (MCH); mean corpuscular haemoglobin concentration (MCHC); platelets; differential blood count; reticulocytes; preparation of blood smears (only evaluated blood smears were archived); prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule: Prenatal day 14
- Anaesthetic used for blood collection: Yes, under isoflurane anesthesia
- Animals fasted: Yes
- How many animals: the first 5 surviving parental males per group at termination and the first 5 females with litters (in order of delivery) per group at PND 14.
- The following parameters were observed: alanine aminotransferase; aspartate aminotransferase; alkaline phosphatase; serum y-glutamyl transferase; sodium; potassium ; chloride; inorg. phosphate; calcium; urea; creatinine; glucose; total bilirubin; total protein; albumin; globulins; triglycerides; cholesterol; bile acids.

THYROID HORMONES: Yes
- Blood samples for T3, T4 and TSH measurement were taken from all surplus pups at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia.
- If not sufficient serum could be sampled from PND 4 pups, samples were pooled per sex and litter. If not at least 8 pools per sex were sufficient for the hormone measurements, samples were pooled regardless of sex per litter.
- Additionally, blood samples for the above mentioned hormones were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia from all dams at PND 14 and all adult males at termination. The adults were fastened before the blood sampling.
- Blood samples from the PND 13 pups and the adult males were assessed for serum levels for T4 and TSH.
All generated serum samples were frozen at -80° at least until finalization of the report.

BEHAVIOUR (FUNCTIONAL FINDINGS): Yes
FUNCTIONAL OBSERVATIONAL BATTERY
- The functional observational battery (FOB) was carried out once, towards the end of the administration period, in the first 5 surviving parental males and the first 5 surviving parental females with litter per group (in order of delivery).
- The examinations were generally started in the morning at about 10:00 h. The FOB was carried out in a randomized sequence. The animals were not h transferred to new cages before the test, nor were food or drinking water withdrawn. The FOB was started with passive observations without disturbing the rats, followed by removal from the home cage, open field observations in a standard arena and sensory motor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable.
- Home cage observation: besides other abnormalities, posture; tremors ; convulsions; abnormal movements; gait were observed.
- Open field observation: besides other abnormalities, behavior on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/stereotypes, gait, activity/arousal level, feces excreted within 2 minutes (appearance/consistency), urine excreted within 2 minutes (amount/color), rearings within 2 minutes and other findings were observed.
- Sensory motor tests/reflex tests: the animals were removed from the open field and were subjected to the sensory motor and reflex tests; reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (startte response), coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), other findings, grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test were performed.

MEASUREMENT OF MOTOR ACTIVITY
- The measurement of motor activity (MA) was carried out once, towards the end of the administration period in the first 5 surviving parental males per group and the first 5 surviving parental females with litter per group (in order of delivery).
- For this purpose, the animals were placed in clean polycarbonate cages with a small amount of bedding for the duration of the measurement. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. Eighteen beams will be allocated per cage.
- The number of beam interrupts were counted over 12 intervals for 5 minutes in each case. The sequence at which the animals were placed in the polycarbonate cages was selected at random. Motor activity measurements were carried out from 14.00 h onwards.
- On account of the measuring variant "staggered", the starting time varied by the time which was needed to place the animals in the cages. For each animal, measurement started individually when the 1st beam was interrupted and ended exactly 1 hour later.
- The animals were given no food or water during the measurements. During measurement the pups were placed in a different room, separated from the dams. After the transfer of the last animal in each case, the room of measurement was darkened. The program required a file name for the measured data to be stored. This name consists of the reference number and a serial number
Sacrifice and pathology:
SACRIFICE
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention was given to the reproductive organs. Animals which died intercurrently or were sacrificed in a moribund state were necropsied as soon as possible after their death and assessed by gross pathology.

GROSS NECROPSY
Gross necropsy consisted of external and internal examinations including the cervical and thoracic

HISTOPATHOLOGY / ORGAN WEIGHTS
- The following weights were determined in all animals sacrificed on schedule: Anesthetized animals; Epididymides; Ovaries; Prostate; Seminal vesicles with coagulating glands; Testes; Thyroid glands; Uterus (with cervix)
- The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): Adrenal glands; Brain; Heart; Kidneys; Liver; Spleen; Thymus
- The following organs or tissues of all parental animals were fixed in 4% buffered formaldehyde solution or modified Davidson's solution: All gross lesions; Adrenal glands; Aorta; Bone marrow (femur); Brain; Cecum; Cervix; Coagulating glands; Colon; Duodenum; Eyes with optic nerve; Esophagus; Extraorbital lacrimal glands; Epididymides (modified Davidson's solution); Femur with knee joint; Heart; Ileum; Jejunum (with Peyer's patches); Kidneys; Larynx; Liver; Lungs; Lymph nodes (axillary and mesenteric); Mammary gland (male and female); Nose (nasal cavity); Ovaries (modified Davidson's solution); Oviducts; Pancreas; Parathyroid glands; Pharynx; Pituitary gland; Prostate gland; Rectum; Salivary glands (mandibular and sublingual); Sciatic nerve; Seminal vesicles; Skeletal muscle; Spinal cord (cervical, thoracic and lumbar cord); Spleen; Sternum with marrow; Stomach (forestomach and glandular stomach); Testes (modified Davidson's solution); Thymus; Thyroid glands;Trachea ;Urinary bladder; Uterus; Vagina.
Statistics:
Statistics of clinical examinations:
- Means and standard deviations were calculated. In addition, the following statistical analyses were carried out:
- Food consumption (parental animals), body weight and body weight change (parental animals): DUNNETT test (two-sided)

Statistics of clinical pathology
- Means, medians and standard deviations were calculated
- Clinical pathology parameters: KRUSKAL-WALLIS and WILCOXON
Clinical signs:
no effects observed
Description (incidence and severity):
In test group 3 (250 mg/kg bw/d) black discoloration of feces was observed in male and female animals from pre-mating day 3 onwards until sacrifice. Grey discoloration of skin started on pre-mating day 4, grey discoloration of eyes was observed first on pre-mating day 5 in both sexes. These findings lasted until sacrifice for each animal. In test group 2 (80 mg/kg bw/d) black discoloration of feces, grey discoloration of skin and grey discoloration of eyes were observed in male animals from post-mating day 1 onwards until sacrifice. All findings which appeared in test groups 2 and 3 were assessed to be related to treatment and demonstrated the bioavailability of the test item. None of the mentioned findings occurred in male animals of test group 1 (25 mg/kg bw/d) during pre- and post-mating periods.

During Gestation
In test group 3 (250 mg/kg bw/d) black discoloration of feces, grey discoloration of skin and grey discoloration of eyes were observed in all female animals from gestational day (GD) 0 onwards until end of gestation. In test group 2 (80 mg/kg bw/d) the same kind of findings, i.e. black discoloration of feces, grey discoloration of skin and grey discoloration of eyes, occurred in all female animals starting in individual animals on GD 12. From GD 15 onwards, all animals were affected until end of gestation. All findings which appeared in test groups 2 and 3 were assessed to be related to treatment and demonstrated the bioavailability of the test item. No findings were observed in test group 1 (25 mg/kg bw/d). In test group 0 (control), female animal No. 107 was unable to deliver. A mass in the abdominal region was palpable between GD 25 to GD 39. The mass disappeared on GD 40. A relation to treatment could be excluded.

During Lactation
In test groups 2 and 3 (80 and 250 mg/kg bw/d) black discoloration of feces, grey discoloration of skin and grey discoloration of eyes were still observable in all female animals from postnatal day (PND) 0 onwards until end of the lactation period. Again, no findings were observed in test group 1 (25 mg/kg bw/d).

Detailed clinical observations
In test group 3 (250 mg/kg bw/d) black discoloration of feces grey discoloration of skin and grey discoloration of eyes were observed on study days 7, 14, 21 and 28 in both sexes and, in addition, on study days 35, 42, 49 and 56 in females. In test group 2 (80 mg/kg bw/d) black discoloration of feces, grey discoloration of skin and grey discoloration of eyes were also observed in female animals on study days 35, 42, 49 and 56. These effects were related to the test substance but assessed as being non-adverse. In test group 0 (control), a palpable mass was observed in female animal No. 107 on study days 42 and 49. The mass was not palpable any more on study day 56. A relation to treatment could be excluded.
Mortality:
no mortality observed
Description (incidence):
No animal died prematurely in the present study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During the pre-mating period, body weight loss was observed for male and female animals (250 mg/kg bw/d). At later stages, the animals recovered but mean body weights were significantly lower in males and females of test group 3 during the entire application period. These changes were considered to be treatment-related and adverse. No test substance-related changes in mean body weights and mean body weight change values were observed for male and female animals of test groups 1 (25 mg/kg bw/d) and 2 (80 mg/kg bw/d) when compared to the control group. The significantly lower mean body weight of female animals in test group 2 (80 mg/kg bw/d) at the beginning of lactation as well as the significantly lower mean body weight change values of male animals in this test group in the pre-mating phase were regarded to be incidental and not related to treatment as these changes occurred only sporadically.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption values in male animals of test group 3 (250 mg/kg bw/d) were significantly lower during pre-mating (maximum of -15% between study days 0-7). Food consumption values in females of test group 3 (250 mg/kg bw/d) were also significantly lower during premating (except between days 7 -13), gestation and lactation. These changes were assessed to be related to treatment and adverse. During the entire application period, no impairment of food consumption was observed in male and female animals of test groups 1 and 2 (25 and 80 mg/kg bw/d).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test substance-related changes in water consumption were observed.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment-related, adverse changes among hematological parameters were observed. In females of test group 3 (250 mg/kg bw/d) absolute reticulocyte counts were significantly increased. This was the only altered red blood cell parameter. No relevant histopathological findings in the spleen were found. Therefore, higher reticulocyte counts were regarded as treatment-related, but not adverse (ECETOC Technical Report No. 85, 2002).
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment-related, adverse changes among clinical chemistry parameters were observed. In rats of both sexes of test groups 2 and 3 (80 and 250 mg/kg bw/d) total bilirubin levels were significantly increased. This was the only relevantly changed clinical chemistry parameter. No relevant histopathologic findings were observed in the liver. Because of the bluish-black serum color in these individuals, total bilirubin photometric measurement is especially prone to an interference. Therefore, this change was regarded as incidental and not treatment-related. In females of test group 3 (250 mg/kg bw/d) aspartate aminotransferase (AST) activities and in males of test groups 2 and 3 (80 and 250 mg/kg bw/d) creatinine levels were significantly decreased. The AST activity decrease in females of test group 3 was slight (decrease of mean versus control -27%). The mean value for creatinine in males of test group 2 was within, that one of test group 3 marginally below the historical control range (males: creatinine 22.2-29.8 μmol/L). Both alterations were isolated in the individuals of the respective groups. Therefore, these alterations were regarded as maybe treatment-related, but not adverse (ECETOC Technical Report No. 85, 2002).
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observational battery:
Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between test-substance treated groups and controls, without a doseresponse relationship or occurred in single animals only, these observations were considered as incidental.
The following examinations were performed during FOB and are assessed individually:
- Home cage observations: No test substance-related, adverse effects were observed.
- Open field observations: No test substance-related, adverse effects were observed.
- Male and female animals of test group 3 (250 mg/kg bw/d) as well as female animals of test group 2 (80 mg/kg bw/d) showed grey discolored skin. One female animal of test group 3 showed black discolored feces.
- Sensorimotor tests/reflexes: No test substance-related, adverse effects were observed.
- Quantitative Parameters: No test substance-related effects were observed.

Motor activity measurements:
Regarding the overall motor activity, no test substance-related deviations were noted for male and female animals. Comparing the single intervals with the control groups, significantly decreased value was measured for female animals of test group 3 (250 mg/kg bw/d) at interval 4. The difference was regarded to be incidental and not related to treatment as no other interval as well as the overall motor activity was not affected. No significant changes were observed for male animals of test groups 1, 2 and 3 (25, 80 and 250 mg/kg bw/d) as well as for female animals of test groups 1 and 2 when compared to the control group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Absolute organ weights:
The significant decrease of the terminal body weight in males of test group 3 (354.37 g) was within the historical control range (338.67 – 395.56 g). The same occurred in females of test groups 1, 2 and 3 (234.24 g, 231.52 g and 223.10 g), which were all within the historical control values (216.65 – 255.02 g). However, the decreases in males and females of test group 3 (250 mg/kg bw/d) were consistent with clinical observations, and were therefore assumed to be treatment-related. All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights:
The significant relative weight increase of the kidneys (0.688%) was marginally above the historical control range (0.577 – 0.671%), whereas the significant liver weight increase (2.39%) was within the historical control range (2.126 – 2.45%). Since no treatment-related histopathological changes were noted in both organs, these changes were considered to be secondary to the terminal body weight decreased. All other mean relative weight parameters did not show significant differences when compared to the control group 0.
Gross pathological findings:
no effects observed
Description (incidence and severity):
In test group 3, dark grey discolorations were noted in several organs of males and females including liver, lungs, thymus, kidneys, brain, pancreas, skin, axillary and mesenteric lymph nodes, mandibular glands, mesentery, uterus, ovaries, testes, epididymides, seminal vesicles, prostate and forestomach. In test group 2, the same type of discoloration was noted only in the liver, uterus, testes, seminal vesicles, pancreas and forestomach. Gastrointestinal contents were also dark grey discolored. In addition, several black foci were observed in the margins of the lungs of 2 females in test group 3. These findings were considered treatment-related and reflected the presence of the test substance characterized by a black color. The dark grey discolorations in the organs were no longer observable after the histotechnical processing of the tissue samples and no histopathologic correlate was found. The black color of the foci in the lungs was also no longer
observable, however the foci correlated with multifocal histiocytosis. All other findings occurred either individually or were biologically equally distributed over
control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Fertility:
The non-pregnant female animals Nos. 132 and 139 as well as one male mating partner (Nos. 32) did not show relevant gross lesions. However, the male mating partner No. 39 showed a reduced size of the testes and epididymides.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The incidence and grading of multifocal histiocytosis was increased in males and females of test group 3. The black foci in the margins of the lungs of 2 females of test group 3 correlated with moderate multifocal subpleural histiocytosis. However, neither the moderate nor the slight or minimal histiocytosis showed black contents. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. The stages of spermatogenesis in the testes of males of the high-dose test group were comparable to those of the controls. In high-dose females, the different stages of functional bodies in the ovaries were present and comparable to the control animals.

Fertility
The non-pregnant female animals Nos. 132 and 139 as well as one male mating partner No. 32) did not show relevant histopathological findings. The male mating partner No. 39 presented a hypoplasia of the testes and epididymides with aspermia, which correlated with the reduced size of both organs and explained the infertility of this animal. This finding was considered to be incidental and not treatment-related.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Estrous cycle:
Estrous cycle data revealed regular cycles in the rearing F1 females of all test groups including the control. The mean estrous cycle duration in the different test groups was 4.0 days in test group 0 (control) and 3.9 days in test groups 1 (25 mg/kg bw/d), 2 (80 mg/kg bw/d) and 3 (250 mg/kg bw/d).

Male mating index:
The male mating index calculated after the mating period for F1 litter was 100% in test groups 0 (control), 1 (25 mg/kg bw/d) and 2 (80 mg/kg bw/d) and 90% in test group 3 (250 mg/kg bw/d) as male animal No. 39, which was placed overnight with female animal No. 139, did not mate.

Male fertility index:
Fertility was proven for most of the F0 parental males within the scheduled mating interval to produce F1 litter. Male animal Nos. 32 and 39 of test group 3 (250 mg/kg bw/d), which were placed with female animal Nos. 132 and 139, did not generate F1 pups although sperm was detected at least in female animal No 132. Thus, the male fertility index was 100.0% in test group 0 (control), test group 1 (25 mg/kg bw/d) and test groups 2 (80 mg/kg bw/d) but only 80% in test group 3 (250 mg/kg bw/d; see table 4.2.1.12.2.1.). However, these values reflected the normal range of biological variation inherent in the strain of rats used for this study. A relation to treatment was not considered for male animals of test group 3.

Female mating index:
The female mating index calculated after the mating period for F1 litter was 100% in test groups 0, 1 and 2 (control, 25 and 80 mg/kg bw/d) and 90% in test group 3 (250 mg/kg bw/d). The mean duration until sperm was detected (GD 0) was 3.2 days for test group 0, 2.8 days for test group 1, 2.9 days for test group 2 and 2.1 days for test group 3. No sperm was detected in female animal No. 139 of test group 3. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

Female fertility index:
Most sperm positive rats delivered pups with the exception of female animal No. 132, which was mated with male animal No. 32 of test group 3 (250 mg/kg bw/d). The animal had spermin vaginal smear but delivered no pups and showed no implants. Thus, the female fertility index was 100% in test group 0 (control), 1 (25 mg/kg bw/d), and 2 (80 mg/kg bw/d) and 88.9% in test group 3 (250 mg/kg bw/d). These values reflected the normal range of biological variation inherent in the strain of rats. The mean duration of gestation was similar in all test groups, i.e. 22.0 days in test groups 0 (0 mg/kg bw/d; control), 1 (25 mg/kg bw/d) and 3 (250 mg/kg bw/d) and 22.2 days in test group 2 (80 mg/kg bw/d). These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

Gestation index:
The gestation index was 100% in test groups 1 (25 mg/kg bw/d), 2 (80 mg/kg bw/d) and 3 (250 mg/kg bw/d) and 90% in test group 0 (control).

Live Birth Indices:
The rate live birth indices were 100% in all test groups including the control.

Postimplantation loss:
The postimplantation loss was 16.7% in test group 0 (control), 4.4% in test group 1 (25 mg/kg bw/d), 5.9% in test group 2 (80 mg/kg bw/d) and 25.9% in test group 3 (250 mg/kg bw/d). The values in test groups 0, 1 and 2 reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data. The postimplantation loss in test group 3 was clearly outside the range. This change was assessed to be related to treatment and adverse.

Thyroid hormones:
In parental males of test groups 2 and 3 (80 and 250 mg/kg bw/d) and in female pups of test group 13 (250 mg/kg bw/d) at PND 13 T4 values were significantly increased. However, the values were within historical ranges (adult males T4 44.87-88.29 nmol/L; female pups at PND13 50.04-75.97 nmol/L). TSH values of the respective individuals were not significantly altered. At PND 13 no treatment-related alterations of T4 and TSH levels were observed in male pups
of test groups 1, 2 and 3 (25, 80 and 250 mg/kg bw/d).
Key result
Dose descriptor:
NOAEL
Effect level:
80 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
Key result
Critical effects observed:
no

Analyses

Stability

The stability of the test substance in 0.5% sodium carboxymethyl cellulose in drinking water was demonstrated over a period of 7 days at room temperature. As the test substance preparations were not stored longer than this time period, the stability was guaranteed.

Homogeneity

Considering the low relative standard deviation in the homogeneity analysis, it can be concluded that the test item was distributed homogeneously in 0.5% sodium carboxymethyl cellulose in drinking water.

Concentration

The concentrations of Orasol Black X45 in 0.5% sodium carboxymethyl cellulose in drinking water were found to be in the range of 90-110% of the nominal concentration. These results demonstrated the correctness of the concentrations of the test item in 0.5% sodium carboxymethyl cellulose in drinking water.

Food

On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants, the diet was found to be suitable. Fed. Reg. Vol. 44, No. 91 of 09 May 1979, p. 27354 (EPA), served as a guideline for maximum tolerable chemical contaminants. The number of microorganisms did not exceed 1*105/g food.

Drinking water

On the basis of the analytical findings the drinking water was found to be suitable. German “Trinkwasserverordnung” (Drinking Water Regulation) served as a guideline for maximum

Bedding and enrichment

On the basis of the analytical findings, the bedding and the enrichment are found to be suitable. Levels given in Lab. Animal, Nov-Dec 1979, pp. 24-34, served as a guideline for maximum tolerable contaminants.

 

Table 1 Absolute and relative organ weights

 

Males

Females

Test group

(mg/kg bw/d)

0 (0)

01 (25)

02 (80)

03 (250)

0 (0)

01 (25)

02 (80)

03 (250)

Terminal body weight

-

97 %

96 %

92 %**

-

96 %*

95 %*

91 %**

Relative organ weight

Kidneys

-

105 %

109 %

116 %**

-

-

-

-

Liver

-

103 %

104 %

111 %**

-

-

-

-

Gross lesions

Several organs,

discoloration

0

0

8

10

0

0

10

10

Cecum,

discoloration of

contents

0

0

10

10

0

0

5

10

Colon,

discoloration of

contents

0

0

9

10

0

0

5

10

Forestomach,

discoloration

0

0

0

0

0

0

1

0

Glandular

stomach,

discoloration of

contents

0

0

0

10

0

0

0

10

Jejunum,

discoloration of

contents

0

0

0

10

0

0

5

10

Lungs,

several foci,

black

0

0

0

0

0

0

0

2

Histopathology

Histiocytosis, multifocal

1

3

1

6

0

0

0

5

Grade 1

1

3

1

1

 

 

 

3

Grade 2

 

 

 

5

 

 

 

2

*p ≤ 0.05; **p ≤ 0.01

 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
80 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP and guideline study.

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

OECD 422 Study

The test item was administered by gavage to groups of 10 male and 10 female Wistar rats (F0 generation) at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; test group 0), 25 mg/kg bw/d (test group 1), 80 mg/kg bw/d (test group 2) and 250 mg/kg bw/d (test group 3). Drinking water containing 0.5% sodium carboxymethyl cellulose was served as vehicle, control animals were dosed daily with the vehicle only. The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same dose group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals. The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances. F0 animals were mated for a maximum of two weeks after the beginning of treatment to produce a litter (F1 generation pups). As soon as sperm was detected in the vaginal smear, mating was discontinued. F0 animals were examined for their reproductive performance including determinations of the number of implantations and the calculation of the postimplantation loss in all F0 females. A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals. Food consumption of the F0 parents was determined regularly once weekly before and after the mating period, as well as in dams during gestation days 7,14 and 20 and lactation days 4, 7, 10 and 13. In general, the body weights of F0 animals were determined once a week. However, during gestation and lactation, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, and on postnatal days (PND) 0, 4, 7, 10 and 13. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PNDs 1, 4, 7 and on PND 13 and their viability was recorded. On day 1 after birth the anogenital distance (AGD) was determined on all live male, female and uncertain pups. On PND 4, the individual litters were standardized in such a way that, whenever possible, each litter contains 4 male and 4 female pups (as a rule, the first 4 surviving pups/sex in each litter were taken for further rearing). On PND 13, all male F1 pups were examined for retention of nipples/areolae. The number of nipples/areolae anlagen were counted. At necropsy on PNDs 4 and 13, all pups were sacrificed with CO2 under isoflurane anesthesia and examined macroscopically for external and visceral findings. Blood samples were taken from all surplus pups or 2 preferably female pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Additionally, blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. Thyroid glands/parathyroidglands were fixed in neutral buffered 4% formaldehyde solution and transferred to the Laboratory Pathology for possible further processing. Towards the end of the administration period a functional observational battery was performed and motor activity was measured in 5 parental animals per sex and test group. Clinico-chemical and hematological examinations were performed in 5 parental animals per sex and group towards the end of the administration period. All F0 parental animals were sacrificed by decapitation under isoflurane anesthesia and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

The various analyses confirmed:

- the stability of the test-substance preparations for a period of 7 days at room temperature,

- the homogeneous distribution of the test substance in the vehicle,

- the correctness of the prepared concentrations.

The following test substance-related effects/findings were noted:

 

Test group 3: 250 mg/kg bw/d

 

F0 PARENTAL ANIMALS

Clinical Examinations

- Discolored feces, grey skin and grey eyes were observed from the first application week onwards.

- Reduced food consumption in male and female over the entire administration period (mostly significantly altered).

- Reduce mean body weight and body weight change values over the over the entire administration period.

 

Reproductive Performance

- Postimplantation loss was clearly increased (25.9%).

- The mean number of delivered pups per litter was significantly lower.

- Decreased viability index (-19.2%; not statistically significant)

 

Clinical Pathology

- No treatment-related, adverse effects were observed.

Pathology
- Dark grey discolorations were noted in several organs of males and females including liver, lungs, thymus, kidneys, brain, pancreas, skin, axillary and mesenteric lymph nodes, mandibular glands, mesentery, uterus, ovaries, testes, epididymides, seminal vesicles, prostate and forestomach. Gastrointestinal contents were also dark grey discolored.
- Several black foci were observed in the margins of the lungs of 2 females in test group 3.

F1 PUPS

Clinical Examinations/ Gross Findings

- Grey discoloration of skin was observed in all male and female pups.

- Tail abnormalities were observed in 16 male and 15 female pups during the lactation period. The findings included kink tails, short tails and curled tails (pigtails). Skeletal evaluations revealed misshapen, fused, bipartite or absent caudal vertebrae.

- Abnormal limb flexibility was observed in one male pup.

- Impaired pup weight development during the lactation period.

- At necropsy, several pups showed grey discoloration of liver, thymus and kidneys as well as black discoloration of stomach and intestinal contents.

 

Test group 2: 80 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical Examinations and Reproductive Performance

-Discolored feces, grey skin and grey eyes were observed from during the post-mating phase in males and from mid-gestation onwards in females.

 

Clinical Pathology

-No treatment-related, adverse effects were observed.

 

Pathology

-Dark grey discolorations were noted in several organs of males and females including liver, uterus, testes, seminal vesicles, pancreas and forestomach. Gastrointestinal contents were also dark grey discolored.

 

F1 PUPS

Clinical Examinations/ Gross Findings

-Grey discoloration was observed in all male and female pups.

-Tail abnormalities were observed in 2 male and 5 female pups during the lactation period. The findings included kink tails and curled tails (pigtails). The skeletal evaluations revealed misshapen, fused, bipartite or absent caudal vertebrae.

-At necropsy, several pups showed grey discoloration of liver, thymus and kidneys as well as black discoloration of stomach and intestinal contents.

 

Test group 1: 25 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical Examinations and Reproductive Performance

-No treatment-related, adverse effects were observed.

 

Clinical Pathology

-No treatment-related, adverse effects were observed.

 

Pathology

-No treatment-related, adverse effects were observed.

F1 PUPS

Clinical Examinations/ Gross Findings

-Grey discoloration of skin was observed in all male and female pups.

-At necropsy, several pups showed grey discoloration of liver and thymus as well as black discoloration of intestinal contents.

 

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage of the test item to Wistar rats revealed signs of systemic toxicity at a dose level of 250 mg/kg bw/d in animals of both sexes. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 80 mg/kg bw/d for male and female Wistar rats. The NOAEL for reproductive performance and fertility was also set to 80 mg/kg bw/d for male and female Wistar rats. The NOAEL for developmental toxicity was 25 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. As a result the substance is not considered to be classified for specific target organ toxicity following repeated exposure (STOT RE) under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.