Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
developmental toxicity
Remarks:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2018 - 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, D-55116 Mainz.
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 16315004.
- Expiration date of the lot/batch: 01 May 2019.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature.
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Solubility and stability of the test substance in the solvent/vehicle: The stability of test substance in 0.5% CMC suspension in deionized water (with 10 mg/100mL Cremophor) was demonstrated for a period of 7 days at room temperature.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: For the preparation of the administration suspensions the test substance was weighed in a calibrated beaker depending on the dose group, topped up with 0.5% CMC suspension in
deionized water with 10 mg/100 mL Cremophor and intensely mixed with a homogenizer. During administration, the preparations were kept homogeneous with a magnetic stirrer.
- Final preparation of a solid: suspension in 0.5% CMC suspension in deionized water (with 10 mg/100mL Cremophor)

FORM AS APPLIED IN THE TEST (if different from that of starting material) : suspension.
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH/ Charles River Laboratories, France.
- Females (if applicable) nulliparous and non-pregnant: yes.
- Age at study initiation: 10 - 12 weeks old when arrived, 13 - 15 weeks old at the beginning of treatment.
- Weight at study initiation: 376.3 - 380.3 g (males), 210.0-212.7 g (females). Weight variation did not exceed 20 percent of the mean weight of each sex.
- Fasting period before study: no.
- Housing: During the study period, the rats were housed individually in Polycarbonate cages type III supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany, with the following exceptions: During overnight matings, male and female mating partners were housed together in Polycarbonate cages type III and pregnant animals and their litters were housed together until PND 13 in Polycarbonate
cages type III. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. For enrichment wooden gnawing blocks (Typ Lignocel® block large, J. Rettenmaier & Söhne
GmbH + Co KG, Rosenberg, Germany) were added. In addition in Polysulfonate cages large play tunnels (Art. 14153; supplied by PLEXX B.V., Elst, Netherlands) were added. The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured. The animals were housed in fully air-conditioned rooms in which central air conditioning guaranteed a range of temperature and a range of relative humidity.
- Diet: ad libitum.
- Water: ad libitum.
- Acclimation period:

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C.
- Humidity (%): 45-65%.
- Air changes (per hr): 15 times per hour.
- Photoperiod (hrs dark / hrs light): The day/night cycle was 12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h.

IN-LIFE DATES: From: 03 Jul 2018 To: 25 Sep 2018.
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% CMC suspension in deionized water with 10 mg/100mL Cremophor
Details on exposure:
After the acclimatization period, the test substance was administered to the parental animals orally by gavage, once daily at approximately the same time in the morning. Females in labor were not treated. The treatment lasted up to one day prior to sacrifice. The animals of the control group were treated with the vehicle (0.5% CMC suspension in deionized water with 10 mg/100mL Cremophor), in the same way. The volume administered each day was 10 mL/kg body weight. The calculation of the administration volume was based on the most recent individual body weight.

PREPARATION OF DOSING SOLUTIONS: The test substance suspensions in 0.5% CMC suspension in deionized water with 10 mg/100 mL Cremophor were prepared in intervals, which took into account the analytical results of the stability verification. For the preparation of the administration suspensions the test substance was weighed in a calibrated beaker depending on the dose group, topped up with 0.5% CMC suspension in deionized water with 10 mg/100 mL Cremophor and intensely mixed with a homogenizer. During administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0, 0.15, 0.50 and 1.50 g/100 mL.
- Amount of vehicle (if gavage): 10 mL/kg bw/d.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the test substance in 0.5% CMC suspension in deionized water with 10 mg/100 mL Cremophor for a period of 7 days at room temperature had been verified prior to the start of the study.
At the beginning (during premating), twice during gestation and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. The 3 samples were withdrawn from the top, middle and bottom of the preparation vessel. 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.
Details on mating procedure:
- M/F ratio per cage: 1:1.
- Length of cohabitation: from about 16.00 h until 6.30 - 9.00 h of the following morning.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy (gestation day (GD) 0).
- After successful mating each pregnant female was caged (how): Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
Duration of treatment / exposure:
males: 30 days; females: 58/63 days
The duration of treatment covered a 2-weeks premating and mating period in both sexes (mating pairs were from the same test group), 3 days postmating in males, and the entire gestation and approximately 3 weeks of lactation period in females up to the day of scheduled sacrifice of the animals.
Frequency of treatment:
daily at the same time in the morning (exception: no administration to animals being in labor).
Duration of test:
63 days
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: By request of the sponsor.
- Fasting period before blood sampling for clinical biochemistry: 16 hours fasting before blood sampling.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes (for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity; parturition and lactation behavior of the dams).
- Time schedule: at least once daily.

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: prior to the first administration and thereafter at weekly intervals
- Examined parameters: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size.

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period (to randomize the animals), study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning) with the following exceptions for the females:
• 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), PNDs 4, 7, 10 and 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 administration volume).

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
- Time schedule for examinations: once a week with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined for GD 0-7, 7-14 and 14-20.
• Food consumption of the F0 females which gave birth to a litter was determined on PND 1-4, 4-7, 7-10 and 10-13.
- Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period in females without litter during the lactation period.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Generally, water consumption was determined once a week as representative value over a period of 3 days for the male and female parental animals, with the following exceptions:
• Water consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals).
• Water consumption of the females with evidence of sperm was determined on gestation days (GD) 0-1, 6-7, 13-14 and 19-20.
• Water consumption of the F0 females, which gave birth to a litter was determined for PND 1-2, 3-4, 6-7 and 12-13.
- Water consumption was not determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.

HAEMATOLOGY: Yes.
- Time schedule for collection of blood: at termination (males); at PND 14 (females).
- Anaesthetic used for blood collection: Yes (isoflurane).
- Animals fasted: Yes (about 16 hours).
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes.
- Time schedule for collection of blood: at termination (males); at PND 14 (females).
- Anaesthetic used for blood collection: Yes (isoflurane).
- Animals fasted: Yes (about 16 hours).
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group.
- Parameters checked in table 2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes; functional observational battery and motor activity assessment (see "OTHER")

POST-MORTEM EXAMINATIONS: Yes.
GROSS PATHOLOGY: Yes (see table 4, Organ weights)
HISTOPATHOLOGY: Yes (see table 4)


OTHER:
Functional observational battery (FOB):
- A functional observational battery was performed in the first five parental male animals per test group and the first five surviving females with litter (in order of delivery) of all test groups at the end of the administration period starting at about 10.00 h on study day 27 (males) and 52 (females).
- Examined parameters:
• Home cage observations: posture, tremors, convulsions, abnormal movements, gait, other findings
• Open field observations (at least for 2 minutes): 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), rearing within 2 minutes, other findings
• Sensory motor tests/ reflexes: reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (auditory 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 assessment:
- measured from 14:00 h onwards on the same day as the FOB was performed in the first five parental males and the first five surviving females with litter (in order of delivery) per group

Estrous cycle:
- In all parental females in the premating phase, estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating, mating and throughout cohabitation until there is evidence of sperm in the vaginal smear. Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all female F0 rats.

Female reproduction and delivery data
- The pairing partners, the number of mating days until vaginal sperm were detected and gestational status were recorded for F0 females.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes.
Examinations included:
- Gravid uterus weight: Yes.
- Number of corpora lutea: Yes.
- Number of implantations: Yes.
Fetal examinations:
- External examinations: No.
- Soft tissue examinations: No.
- Skeletal examinations: No.
- Head examinations: No.

Fetal examinations are not applicable for OECD TG 422. Litter were investigated:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and status of delivery (number of live born and stillborn), pub viability/mortality at least once daily, sex ratio (by anogenital distance, mammary line, necropsy), pub clinical observations (clinical symptoms including gross-morphological findings), pub body weight at PND 1, 4, 7 and 13, runts, anogenital distance, anogenital index, nipple/aereola anlagen (male pubs at PND13), pub necropsy observations, thyroid hormone concentrations.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.
Statistics:
See table 5.
Indices:
Viability index of pups, Survival index of pups (for detailed formulas see "Any other information on materials and methods")
Historical control data:
Historical control data to allow comparison with concurrent controls were provided by the test facility for the following parameters: maternal body weights, mating data, estrous cycle, delivery data, motor activity, functional observational battery, clinical pathology, organ weights, litter data, pup weights, sex ratios, pup necropsy observations, anogenital distance, presence of areolas/nipples.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Several male and female animals of the mid-dose group and almost all male and female animals of the high-dose group (50 and 150 mg/kg bw/d) showed salivation immediately after dosing (up to 2 hours post dosing) during the treatment period. It is likely, that this temporary finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse toxicologically relevant finding.
No other clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F0 parental animals in any of the test groups (1 – 3; 15, 50 and 150 mg/kg bw/d) during the study.
One high-dose male (No. 39) showed a distended, hard abdomen and piloerection during postmating days 1 – 2 which was assessed to be incidental.
Two sperm positive control females (Nos. 102 and 107), one sperm positive low-dose female (No. 118) and one sperm positive high-dose female (No. 133) did not deliver F1 pups.
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In high-dose males, mean body weights were statistically significantly below the concurrent control values on mating day 14 (about 5%). Furthermore, mean body weight change was statistically significantly below the concurrent control during premating days 0 - 7 (about 80%). In high-dose females, mean body weights were statistically significantly below the concurrent control values during lactation (PND 4, PND 10 – 13, up to 7% below control) showing a downward trend towards the end of the study (see graphs below). This finding in high-dose males and females was assessed as treatment-related and adverse.
Mean body weight (change) of the mid- and low-dose males and females during the entire study period were comparable to the concurrent control values.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of the high-dose group was statistically significantly reduced compared to the concurrent control values in males during premating days 0 - 7 and in females during premating days 0 - 13 (both up to 9% below control). Food consumption in females showed a downward trend towards the end of the study since food consumption of high-dose females was further reduced (up to 17 % below control, without statistical significance) during lactation. The reduction in food consumption in male and females of the high-dose was assessed as treatment-related and adverse.
Food consumption of the high-dose females during gestation and of the low- and mid-dose males and females during the entire study was comparable to the concurrent control values.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
In males of the high-dose (150 mg/kg bw/d), water consumption was statistically significantly increased compared to the concurrent control values during premating days 7 - 10 (about 36%). In the high-dose F0 females, water consumption was statistically significantly increased compared to the concurrent control values during the entire gestation period (up to 43%). Together with the finding salivation, it is most likely that the increase in water consumption occurred due to the bad taste of the test substance or local affection of the upper digestive tract. It was assessed as treatment related but not adverse.
Water consumption of the high-dose females during premating and lactation and of the lowand mid-dose males and females during the entire study was comparable to the concurrent control values. The statistically significantly higher water consumption in the low-dose females during premating days 0 - 3 was considered to be spontaneous in nature and not treatment related since it was not related to dose.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period in male and female rats of test group 3 (150 mg/kg bw/d), absolute eosinophil cell counts were decreased (in males not statistically significantly). Additionally, in females of this test group absolute (not statistically significant) and relative monocyte counts were increased, and relative eosinophil cell counts were decreased. These changes were regarded as treatment-related and adverse.

In males of test group 3 (150 mg/kg bw/d) relative basophil cell counts were significantly decreased. However, no pathophysiological correlate exists regarding low basophil counts and, therefore, this change was regarded as incidental and not treatment-related. In addition, in males of this test group mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) were significantly decreased. MCV was within, MCH marginally below the historical control range (males, MCV 48.1-51.7 fL, MCH 1.03-1.11 fmol). However, these were calculated red blood cell indices and the measured red blood cell parameters (i.e. hemoglobin, hematocrit and red blood cell (RBC) counts) were not changed. Therefore, the MCV and MCH alterations in males of test group 3 were regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, in females of test group 3 (150 mg/kg bw/d) glucose levels were significantly decreased. This change combined with the hematological alterations is regarded as treatment-related and adverse.

In males of test group 3 (150 mg/kg bw/d) total protein and globulin values were significantly decreased and inorganic phosphate levels were significantly increased. However, all values were within historical control ranges (males, total protein 59.06-65.10 g/L, globulins 21.95- 28.93 g/L, inorganic phosphate 1.45-2.06 mmol/L). Therefore, these alterations were regarded as incidental and not treatment-related.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See table 6 and 7 for details.
Absolute organ weights: When compared to control group 0 (=100%), the mean absolute weights of following organs were significantly increased: Adrenal glands, liver, kidneys, ovaries.
Relative organ weights: When compared to control group 0 (=100%), the mean relative weights of following organs were significantly increased: Adrenal glands, liver, kidneys, spleen, thyroid glands.

The significant decrease of the final body weight in males and females, as well as the significant absolute and relative weight increase of the liver in both sexes, and of the adrenal glands and kidneys in females of test group 3 were considered treatment-related and had a histopathological correlate. The significant relative weight increase of the kidneys in males was considered secondary to the final body weight decrease, since it was marginally above the maximal historical control value, had an absolute weight within the historical control range and showed no histopathological correlate. The significant relative weight increase of the thyroid glands in females of the high dose group was within the historical control range and had no histopathological correlate. Therefore, it was regarded as incidental and not treatment-related.
The significant absolute weight increase of the ovaries and the significant relative weight increase of the spleen in females of test group 2 were regarded as incidental and not related to treatment.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered incidental or spontaneous in origin and without any relation to treatment.

Fertility: The female animals (Nos. 102, 107, 118 and 133), which were not pregnant as well as the male mating partners (Nos. 2, 7, 18 and 33) did not show relevant gross lesions.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were observed in following organs: brain, eyes with optic nerves, liver, lungs, skeletal muscle, seminal vesicles, epididymides, adrenal cortex, axillary and mesenteric lymph nodes, glandular stomach, kidneys, spleen and uterus. The main finding in each org n was a cytoplasmic vacuolation, characterized by the presence of multiple fine to small vacuoles, that were no larger than the nuclei of the cell. This type of vacuolation is referred here as “microvesicular” and most frequently increased the size of the affected cells. On the other hand, large cytoplasmic vacuoles, that displaced the nuclei or deformed the cells are mentioned here as “macrovesicular type” and were seen only in the kidneys and glands of the uteri. Incidences and grading of findings in the different target organs are given in the tables 8-21.

Fertility: The female animals (Nos. 102, 107, 118 and 133), which were not pregnant as well as thmale mating partners (Nos. 2, 7, 18 and 33) did not show relevant histopathological findings that could have impaired fertility.
Other effects:
no effects observed
Description (incidence and severity):
THYROID HORMONES
- In parental males (test groups 1, 2 and 3; 15, 50 and 150 mg/kg bw/d) and in male and female pups at PND13 (test groups 11, 12 and 13; 15, 0 and 150 mg/kg bw/d), no treatment-related alterations of T4 and TSH levels were observed.

DETAILED CLINICAL OBSERVATIONS (DCO):
- All male and all female animals of all dose groups (15, 50 and 150 mg/kg bw/d) did not show any abnormalities.

FUNCTIONAL OBSERVATIONAL BATTERY (FOB)
- Home cage observations: No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.
- Open field observations: The open field observations did not reveal any test substance-related findings in male and female animals of all test groups.
- Sensorimotor tests/reflexes: There were no test substance-related findings in male and female animals of all test groups.
- Quantitative Parameters: The landing foot-splay test (FST) in the mid- and high-dose male animals was statistically significantly below the concurrent control values (9.5 and 9.7 cm, respectively, vs. 11.8 cm in the control). However, there was no relation to dose and both values were within the range of the historical control data (Supplement, HCD, FST males: range of 9.3 – 13.8 cm). Therefore, it was not assessed as treatment-related and adverse. No test substance-related impaired parameters were observed in the low-dose males and all female animals of all test groups.

MOTOR ACTIVITY MEASUREMENT (MA)
- No statistically significant changes on motor activity data (summation of all intervals) was observed in the male and female animals of all test substance-treated groups in comparison to the concurrent control values.
Details on results:
Regarding clinical examination, several male and female animals of the mid-dose group and almost all male and female animals of the high-dose group (50 and 150 mg/kg bw/d) showed salivation immediately after dosing (up to 2 hours post dosing) during the treatment period. It is likely, that this temporary finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse toxicologically relevant finding.
In F0 males and females of the high-dose, water consumption was statistically significantly increased compared to control during premating (males: about 36% above control) or gestation (up to 43% above control). Together with the clinical finding salivation, it is most likely that the increase in water consumption occurred due to the bad taste of the test substance or local affection of the upper digestive tract. It was assessed as treatment related but not adverse.

Adverse signs of toxicity were observed at the highest dose (150 mg/kg bw/d).
Food consumption of the high-dose was statistically significantly reduced compared to control in males and females during premating (both up to 9% below control) and in females during lactation (without statistical significance, up to 17 % below control).
Mean body weight (change) was statistically significantly below the control values in males during premating (BWC) and during mating (BW: about 5% below control) and in females during lactation (up to 7% below control). Food consumption and body weight in high-dose animals showed a downward trend towards the end of the study and were assessed as treatment-related and adverse.
No test substance related, adverse findings were observable in the mid- and low-dose (50 and 15 mg/kg bw/d).

Regarding clinical pathology, in females of test group 3 (150 mg/kg bw/d) decreased absolute and relative eosinophils and increased absolute and relative monocyte counts as well as decreased glucose levels indicated some subacute stress event which is regarded as treatment-related. Lower absolute eosinophil counts were also observed in males of the same test group.

Regarding pathology, a significant decrease of the final body weight in males and females of test group 3 (150 mg/mg bw/d) was accompanied by a significant absolute and relative weight increase of the liver in both sexes, and of the adrenal glands and kidneys in females. These organ weight changes were considered treatment-related and were consistent with histopathological findings, characterized mainly by cytoplasmic vacuolation. Vacuolation was also observed in several other organs of males and females in test group 3 (brain, eyes, lungs, skeletal muscle, seminal vesicles, epididymides, axillary and mesenteric lymph nodes, glandular stomach, spleen and uterus) and in test group 2 (epididymides). The cytoplasmic vacuolation was of microvesicular type (multiple fine to small vacuoles, that were no larger than the nuclei of the respective cell types and did not displace it). A macrovesicular type (large and single cytoplasmic vacuoles, that displaced the nuclei or altered the cellular form) was distinctively and additionally seen in the kidneys (all females) and uteri (two females), and only as background in the remaining organs.
The vacuolation was regarded as treatment-related and assessed as adverse, if it was accompanied by cytotoxicity (cell death, degeneration and/or inflammation). In test group 3 (150 mg/kg bw/d) animals, these criteria were accomplished for the liver and skeletal muscle of males and females; axillary and mesenteric lymph nodes and kidneys of females. Finally, vacuolation alone without sings of cytotoxicity, occurring in special organs with central vital functions or special senses, was also assessed as adverse, assuming a potential impairment of vital functions. This was true for the spleen in females and brain and eyes in males and females of test group 3 (150 mg/kg bw/d). A summary of histopathological findings assessed as treatment-related and adverse is given as follows:
In test group 3 (150 mg/kg bw/d), histopathology revealed in the liver a centrilobular microvesicular vacuolation in all males and females accompanied by single cell necrosis. It cannot be excluded if other hepatic findings like multifocal necrosis, multinuclear hepatocytes and karyomegaly, affecting very few animals were treatment-related and were, therefore, assumed to be possibly treatment-related and adverse. In the skeletal muscle of males and females, the fiber vacuolation was accompanied by necrosis and regeneration of fibers. In the axillary lymph node of females, the vacuolation was present in the high endothelial venules (HEV) and was accompanied by apoptosis. The mesenteric lymph nodes of females showed vacuolation in the HEV, lymphocytes of medullary cords and macrophages of medullary sinuses, apoptosis and macrophage aggregates in the periphery of paracortical areas, and decreased cellularity in paracortical regions (T-lymphocytes). In the kidneys of females, a tubular cytoplasmic macro- and microvesicular vacuolation affecting all animals was accompanied by tubular degeneration/regeneration and granulomatous inflammation. Both types of vacuolation affected predominantly the cortical tubular epithelium but the microvesicular vacuolation extended also into the outer stripe of the outer medulla. The spleen of females displayed a vacuolation of the B-lymphocytes (follicular and marginal zone) and a decreased cellularity of T-lymphocytes in the periarteriolar lymphocyte sheath (PALS). Finally, a microvesicular vacuolation without signs of cytotoxicity was detected in the plexus choroideus of the brain, and in the stromal iris and choroid of the eyes of males and females.

Treatment-related vacuolation not accompanied by signs of cytotoxicity and assessed as not adverse, was seen in following organs in test group 3:
Adrenal cortex of females (zona fasciculata and reticularis) correlating with significantly increased weights; lungs of all male and females (bronchial/bronchiolar epithelium) and glandular stomach of females (base of the glands in the pyloric region). In the epithelium of epididymides (corpus), seminal vesicles and uterus (surface and/or glandular epithelium, and smooth muscle of the wall). Fertility in these animals was not relevantly altered when compared with the control animals.

In test group 2 (50 mg/kg bw/d), histopathology revealed vacuolation in the epididymal epithelium. This change was assessed as treatment-related but not adverse, since no alteration was seen in the sperm density and morphology and no relevant alteration of the fertility was noted in these male animals.

Special stains performed exemplarily in selected organs (liver, kidneys, lungs and skeletal muscle) of animals in test group 3 revealed that the vacuolation was negative for the presence of neutral fat (Oil-red-O stain negative) and was positive for the presence of phosphorylated lipids (Sudan black positive). An increased accumulation of glycogen in the skeletal muscle was discarded (PAS with diastase negative).

Transmission electron microscopy examination performed exemplarily in the liver and kidneys of selected male and female animals of test group 3 revealed the presence of intracytoplasmic myelin figures, characterized by concentric, multilaminated membrane whorls in phagolysosomes (Ghadially, 1988,). This morphological picture is indicative of phospholipidosis and represents lysosomal accumulation of phospholipids. It is inferred that the vacuolation observed in all other organs was most likely of the same nature (Nonoyama and Fukada, 2008; Rudmann et al, 2004).

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. Furthermore, neither the synchrony of the morphology of the estrous cycle in ovaries, uterus, cervix, and vagina nor the stages of seminiferous tubules were altered.
Number of abortions:
not examined
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
postimplantation loss did not show any significant differences between the groups
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
not examined
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
The mean duration of gestation values varied between 22.1 (test group 1), 22.2 (control and test group 2) and 22.5 (test group 3)
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
All female rats delivered pups with the following exception:
- Control female No. 102 (mated with male No. 2) did not become pregnant.
- Control female No. 107 (mated with male No. 7) did not become pregnant.
- Low-dose female No. 118 (mated with male No. 18) did not become pregnant.
- High-dose female No. 133 (mated with male No. 33) did not become pregnant.
The fertility index ranged between 80% and 100% without showing any relation to dosing.
Other effects:
no effects observed
Details on maternal toxic effects:
Regarding fertility and reproductive performance, no signs of toxicity were observed in male or female parental animals of all test groups during the entire study. F0 parental animals proved to be fertile. Mating behavior, conception, implantation and parturition were not affected.
The female mating index calculated after the mating period for F1 litter was 100% in all test groups.
The mean duration until sperm was detected (GD 0) varied between 1.9 and 3.9 days. The mean value of 3.9 days in the high-dose group was well within the range of the historical control data (HCD, mating days: 1.6 – 5.8) and can be explained by female No. 135 which had 14 mating days. The female was possibly pseudo-pregnant indicated by 12 days of diestrus. Afterwards, the female mated and was pregnant.
All female rats delivered pups with the following exception:
- Control female No. 102 (mated with male No. 2) did not become pregnant.
- Control female No. 107 (mated with male No. 7) did not become pregnant.
- Low-dose female No. 118 (mated with male No. 18) did not become pregnant.
- High-dose female No. 133 (mated with male No. 33) did not become pregnant.
The fertility index ranged between 80% and 100% without showing any relation to dosing. The non-pregnant females had no relevant gross lesions or microscopic findings. The mean duration of gestation values varied between 22.1 (test group 1), 22.2 (control and test group 2) and 22.5 (test group 3)
The gestation index was 100% in all test groups 0 - 3.
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (12.0 / 13.7 / 12.1 and 10.3 implants/dam in test groups 0 - 3,
respectively). The value of test group 3 was marginally outside the range of the historical control data (HCD, implantation sites: 10.4 - 14.9), without statistical significance. It was assessed as spontaneous and not treatment-related since the mean value of the related parameter pups delivered of test group 3 (9.6) was within the historical control range (HCD, pups delivered: 9.3 -13.9).
Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any significant differences between the groups, and the mean number of F1 pups delivered per dam remained unaffected (11.1 / 13.2 / 10.6 and 9.6 pups/dam in test groups 0 - 3, respectively)
The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 100% / 98.3% / 100% and 100% in test groups 0 - 3, respectively. Moreover, the number of stillborn pups was not significantly different between the test groups.
Thus, the test substance did not adversely affect reproduction and delivery of the F0 generation parental females.
Dose descriptor:
LOAEL
Remarks:
systemic toxicity
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
haematology
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
other: no adverse effect observed
Dose descriptor:
NOAEL
Remarks:
reproductive performance & fertility
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
other: no adverse effect observed
Abnormalities:
no effects observed
Fetal body weight changes:
not examined
Reduction in number of live offspring:
not examined
Changes in sex ratio:
not examined
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
not examined
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Pup number and status at delivery:
- The mean number of delivered F1 pups per dam and the rates of liveborn, stillborn, found dead and cannibalized F1 pups were evenly distributed among the test groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.

Pup viability:
- The viability index indicating pup survival during lactation (PND 0 - 4) varied between 98.1%/ 100% / 99.2% and 100% in test groups 0 - 3, respectively. The pups surviving index indicating pup survival during lactation (PND 4 - 13) varied between 100%/ 98.6% / 100% and 100% in test groups 0 - 3, respectively.

Pup mortality:
- The pup mortality shown in Figure 4.2.2.1.3.1 based on total number of stillborn pups, dead pups, pups sacrificed moribund and cannibalized pups.

Pub sex ratio:
- In general, the sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature. The statistically significant change in sex ratio of the high-dose group on PND 0 (males 40.9* versus [vs.] 56.0 in control and females 59.1* vs. 44.0 in control) was within the historical control range (HCD: 36.6% - 60.5% (males) 39.5% - 63.4% (females)). It was assessed as not treatment-related. Further endocrine relevant parameters (AGD, nipple development) were not influenced by the test substance in any of the dose levels (see below).

Pup clinical observations:
- There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups. One low-dose female pup (No. 11 of dam No. 113) showed and injury of the left hindlimb on PND 11 without relation to treatment and was therefore sacrificed moribund on PND 11.

Pup body weight data:
- Two male and three female runts were seen in test group 1, one male and one female runt was seen in test group 2 and one female runt was seen in test group 3. In test group 3, mean body weight development was comparable to control on PND 1 and 4. On PND 13, mean body weights of the high-dose F1 male pups and both sexes combined were statistically significantly below the concurrent control (about 11% and 10%, respectively). The pup body weight change in the high-dose male pups was statistically significantly below the concurrent control values during the intervals PND 4 - 7, 7 - 13 and 1 - 13 (up to 15%) and in the high-dose female pups and both sexes combined during PND 7 - 13 and 1 - 13 (up to13%). The decrease in body weights of males and both sexes combined was at the very lower end of the range of the historical control data (Supplement, HCD, pup weights, males: 28.5-33.4 g, both: 28.1-33.0 g). A relation to treatment cannot be excluded. In the highest test group, maternal / parental toxicity was observed. The decrease in body weight of the high dose pups was considered to be a secondary, minor effect in presence of maternal toxicity. No test substance-related influence on body weight (change) of the low- and mid-dose male and female F1 pups were noted.

Anogenital distance/anogenital index:
- Neither on anogenital distance nor anogenital index test substance-related effects were noted in all treated F1 offspring (test groups 1 - 3 [15, 50 and 150 mg/kg bw/d]).

Nipple/ areola anlagen:
- The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.

Pup necropsy observations:
- A few pups showed spontaneous findings at gross necropsy, such as discolored thymus (red), discolored testis (red), small testis, supernumerary liver lobe (cystic), post mortem autolysis, partly cannibalized, limb lesion, diaphragmatic hernia and dilated renal pelvis. These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, all these findings were not considered to be associated to the test substance.
Details on embryotoxic / teratogenic effects:
Regarding developmental toxicity, a statistically significant change in sex ratio of the high-dose group on PND 0 (males 40.9* versus [vs.] 56.0 in control and females 59.1* vs. 44.0 in control) was within the historical control range (Supplement, HCD: 36.6% - 60.5% (males) 39.5% - 63.4% (females)). Therefore, it was assessed as not treatment-related. Furthermore, neither determination of anogenital distance/index nor the count of nipple/areola anlagen revealed any treatment-related, adverse changes up to and including a dose of 150 mg/kg bw/d. All parameters concerning mating and delivery were not adversely affected by the test substance. Regarding F1 pups and litter data, pup status and viability showed no treatmentrelated, adverse findings.
Mean body weights of the high-dose F1 male pups and both sexes combined were statistically significantly below the concurrent control (about 11% and 10%, respectively) on PND 13. The body weight change in high-dose male and female pups was statistically significantly below the control during PND 1-13. The decrease in body weights of males and both sexes combined was at the very lower end of the range of the historical control data (Supplement, HCD, pup weights, males: 28.5 - 33.4 g, both: 28.1-33.0 g). A relation to treatment cannot be excluded. In the highest test group, maternal / parental toxicity was observed (see above). The decrease in body weight of the high dose pups was considered to be a secondary, minor effect in presence of maternal toxicity.
No test substance-related influence on body weight (change) of the low- and mid-dose male and female F1 pups were noted.
Dose descriptor:
LOAEL
Remarks:
developmental toxicity
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Remarks on result:
other: fetus analysis is not applicable for OECD TG 422
Remarks:
maternal toxic dose
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at this dose
Remarks on result:
other: fetus analysis is not applicable for OECD TG 422
Abnormalities:
not examined
Developmental effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
not specified
Relevant for humans:
yes

The various analyses:

  • Demonstrated the stability of the test substance preparations over a period of 7 days at room temperature
  • Confirmed the homogeneous distribution of the test substance in 0.5% CMC suspension in deionized water (with 10 mg/100mL Cremophor)
  • Confirmed the overall accuracy of the prepared test substance concentrations

Concentration control analysis:

Measured values for the test substance were in the expected range of the target concentrations (90 - 110%) demonstrating the correctness of the preparations. For the low dose (15 mg/kg bw/d), one single value (sample No. 3) was marginally above the tolerance range of the test facility (111%). However, the test substance preparation was homogeneously distributed for this dose level and all other values of the low dose were within the tolerance range. Therefore, the measured values of the low dose were assessed to be in an acceptable range of the nominal concentrations in this study.

Table 6: Absolute organ weights

 

Male animals

Female animals

Test group (mg/kg bw/d)

1

(15)

2

(50)

3

(150)

1

(15)

2

(50)

3

(150)

Final body

weight

-1.7%

-0.5%

-6.1%**

+0.2%

+2.5%

-6.2%**

Adrenal glands

 

 

 

+10.649%

+7.273%

+29.610%**

Liver

-0.55%

+2.45%

+8.90%*

+0.31%

+8.12%

+26.47%**

Kidneys

%

%

%

-3.50%

+6.99%

+49.06%**

Ovaries

%

%

%

+11.907%

+27.043%**

+9.183%

*p <= 0.05; **p <= 0.01

Table 7: Relative organ weights

 

Male animals

Female animals

Test group (mg/kg bw/d)

1

(15)

2

(50)

3

(150)

1

(15)

2

(50)

3

(150)

Adrenal glands

 

 

 

+10.246%

+3.863%

+39.463%**

Liver

+1.37%

+2.27%

+15.01%**

+0.25%

+4.48%

+36.04%**

Kidneys

-3.94%

-5.39%

+9.79%*

-3.65%

+3.52%

+60.60%**

Spleen

%

%

%

-6.65%

-13.51%*

+7.87%

Thyroid glands

 

 

 

+15.735%

+13.353%

+19.488%**

 *p <= 0.05; **p <= 0.01

Table 8: Histopathology/ Brain

Brain

Male animals

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

10

10

10

10

Vacuolation, choroid plexus, diffuse

0

0

0

10

0

0

0

10

·        Grade1

 

 

 

6

 

 

 

3

·        Grade2

 

 

 

4

 

 

 

2

·        Grade3

 

 

 

 

 

 

 

5

The cytoplasmic vacuolation was observed mainly in the epithelium of the choroid plexus of the lateral ventricle and was of microvesicular type.

Table 9: Histopathology/ Eyes with optic nerve

Eyes with optic nerve

Male animals

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

10

10

10

10

Vacuolation, diffuse

0

0

0

5

0

0

0

10

·        Grade1

 

 

 

4

 

 

 

 

·        Grade2

 

 

 

1

 

 

 

3

·        Grade3

 

 

 

 

 

 

 

7

The vacuolation was of microvesicular type, most likely in macrophages of the iris stroma. In several animals the vacuolation extended from here into the peripheral choroidal layer.

Table 10: Histopathology/ Liver

Liver

Male animals

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

10

10

10

10

Vacuolation, centrilobular

0

0

0

10

0

0

0

10

·        Grade1

 

 

 

 

 

 

 

1

·        Grade2

 

 

 

4

 

 

 

4

·        Grade3

 

 

 

6

 

 

 

3

·        Grade4

 

 

 

 

 

 

 

2

Necrosis, single cell

0

0

0

2

0

0

0

9

·        Grade1

 

 

 

1

 

 

 

4

·        Grade2

 

 

 

1

 

 

 

5

Necrosis, multifocal

0

0

0

2

0

0

0

1

·        Grade1

 

 

 

1

 

 

 

1

·        Grade2

 

 

 

1

 

 

 

 

Multinuclear hepatocytes

0

0

0

1

0

0

0

2

·        Grade1

 

 

 

 

 

 

 

1

·        Grade2

 

 

 

1

 

 

 

1

Karyomegaly

0

0

0

0

0

0

0

1

·        Grade1

 

 

 

 

 

 

 

1

The vacuolation was of microvesicular type and was accompanied by single cell necrosis of vacuolated cells. The centrilobular vacuoles were negative for Oil-Red-O and positive for Sudan black. Findings like multifocal necrosis, multinuclear hepatocytes and karyomegaly, did affect only very few animals and treatment-relationship remains unclear.

Table 11: Histopathology/Lungs

Lungs

Male animals

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

10

10

10

10

Vacuolation, epith., bronchial/bronchiolar

0

0

0

10

 

 

 

10

·        Grade1

 

 

 

10

 

 

 

3

·        Grade2

 

 

 

 

 

 

 

5

·        Grade3

 

 

 

 

 

 

 

2

Histiocytosis, alveolar, multifocal

2

2

2

3

3

1

2

9

·        Grade1

2

2

2

1

3

1

2

4

·        Grade2

 

 

 

1

 

 

 

4

·        Grade3

 

 

 

1

 

 

 

1

The vacuolation in the lungs was of microvesicular type and affected most frequently the bronchiolar epithelium including terminal bronchioles, and in extensive cases also the bronchial epithelium. It was accompanied by an increase in alveolar histiocytosis, which was stronger in females than in males. The vacuoles as well as the alveolar histiocytes were negative for Oil- Red-O and positive for Sudan black.

Table 12: Histopathology/Skeletal muscle

Skeletal muscle

Male animals

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

10

10

10

10

Vacuolation, fiber

0

0

0

7

0

0

0

9

·        Grade1

 

 

 

6

 

 

 

7

·        Grade2

 

 

 

 

 

 

 

1

·        Grade3

 

 

 

 

 

 

 

1

·        Grade4

 

 

 

1

 

 

 

 

Necrosis, multifocal

0

0

0

0

0

0

0

2

·        Grade1

 

 

 

2

 

 

 

2

Regeneration, multifocal

0

0

0

1

0

0

0

1

·        Grade1

 

 

 

 

 

 

 

1

·        Grade3

 

 

 

1

 

 

 

 

The vacuolation in the skeletal muscle was characterized by the presence of very tiny microvacuoles with a multifocal distribution pattern within the myofibers. In addition, some of the affected fibers were retracted and strongly eosinophilic, indicating a hyaline necrosis in 2 males and 2 females. One male animal and one female with severe and moderate vacuolation, respectively, showed also an regeneration of fibers. The tiny vacuoles in the affected fibers were negative for Oil-Red-O, positive for Sudan-black and were negative in the PAS-with diastase stain.

Table 13: Histopathology/ Epididymides

Epididymides

Male animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

Vacuolation, epithelial

0

0

10

10

·        Grade1

 

 

2

 

·        Grade2

 

 

8

 

·        Grade3

 

 

 

10

The vacuolation was of microvesicular type. In test group 3, it was diffusely distributed along the tubules of the corpus, starting slightly distally of the head and extended to the junction with the cauda. In test group 2, the vacuolation was mostly present in the caudal part of the corpus, reaching the junction with the cauda.

Table 14: Histopathology/ Seminal vesicles

Seminal vesicles

Male animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

Vacuolation, epithelial

0

0

0

2

·        Grade1

 

 

 

1

·        Grade2

 

 

 

1

The diffuse epithelial vacuolation was of microvesicular type, with a distinctive basal localization.

Table 15: Histopathology/ Adrenal cortex

Adrenal cortex

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

Vacuolation, increased

0

0

0

10

·        Grade1

 

 

 

4

·        Grade2

 

 

 

4

·        Grade3

 

 

 

2

The cytoplasmic vacuolation was of microvesicular type and included the zona fasciculata and reticularis.

Table 16: Histopathology/ Axillary lymph node

Axillary lymph node

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

Vacuolation, multifocal

0

0

0

4

·        Grade1

 

 

 

3

·        Grade2

 

 

 

1

Apoptosis

0

0

0

1

·        Grade1

 

 

 

1

The vacuolation was of microvesicular type and was noted mainly in the high endothelial venules (HEV). The apoptosis was seen in the periphery of the paracortex.

Table 17: Histopathology/ Mesenteric lymph node

Mesenteric lymph node

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

Vacuolation, multifocal

0

0

0

8

·        Grade1

 

 

 

6

·        Grade2

 

 

 

2

Apoptosis

0

0

0

7

·        Grade1

 

 

 

6

·        Grade2

 

 

 

1

Cellularity, decreased

0

0

0

6

·        Grade1

 

 

 

4

·        Grade2

 

 

 

2

Aggregates, macrophage, increased

0

1

3

9

·        Grade1

 

1

2

7

·        Grade2

 

 

1

2

The microvesicular vacuolation was seen in the HEV (high endothelial venules) of all animals, and lymphocytes of medullary cords and macrophages of medullary sinuses. The apoptosis and macrophage aggregates were noted in the periphery of paracortical areas. The decreased cellularity occurred in paracortical regions (T-lymphocytes).

Table 18: Histopathology/ Glandular stomach

Glandular stomach

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

Vacuolation, epithelial

0

0

0

7

·        Grade1

 

 

 

5

·        Grade2

 

 

 

2

The vacuolation affected the cytoplasm of the cells at the base of the glands in the pyloric region of the stomach.

Table 19: Histopathology/ Kidneys

Kidneys

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

Vacuolation, macrovesicular

0

0

0

10

·        Grade1

 

 

 

2

·        Grade2

 

 

 

3

·        Grade3

 

 

 

5

Vacuolation, microvesicular

0

0

0

10

·        Grade1

 

 

 

5

·        Grade2

 

 

 

3

·        Grade3

 

 

 

2

Degeneration/ regeneration, tubular

0

0

0

8

·        Grade1

 

 

 

1

·        Grade2

 

 

 

2

·        Grade3

 

 

 

5

Inflammation, granulomatous

0

0

0

2

·        Grade2

 

 

 

2

A tubular cytoplasmic macro- and microvesicular vacuolation accompanied by tubular degeneration/regeneration was distinguished in the kidneys of females. Both types of vacuolation affected predominantly the cortical tubular epithelium, but the microvesicular vacuolation extended also into the outer stripe of the outer medulla. Macrovesicular vacuoles seemed to be empty (by light microscopy) and were most likely noted in collecting ducts, whereas the microvesicular vacuoles seemed to affect more the convoluted proximal and distal tubules and the pars recta. However, a clear distinction was not always possible. Figures of tubular vacuoles might indicate that the macrovesicular vacuolation could originate by exaggerated distention and coalescence of less sized vacuoles. The microvesicular vacuoles were negative for Oil-Red-O and positive for Sudan black stain. The macrovesicular vacuoles did not stain.

Table 20: Histopathology/ Spleen

Spleen

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

Vacuolation, lymphoid cell

0

0

0

10

·        Grade1

 

 

 

4

·        Grade2

 

 

 

4

·        Grade3

 

 

 

2

Cellularity decreased

0

0

0

6

·        Grade1

 

 

 

1

·        Grade2

 

 

 

3

·        Grade3

 

 

 

2

The vacuolation in the spleen of females was of microvesicular type and affected the cytoplasm of lymphoid cell of the follicles and marginal zone (B-lymphocytes). A decreased cellularity of T-lymphocytes was observed in the periarteriolar lymphocyte sheath (PALS).

Table 21: Histopathology/ Uterus

Uterus

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

Vacuolation, epithelial

0

0

0

8

·        Grade1

 

 

 

4

·        Grade2

 

 

 

1

·        Grade3

 

 

 

3

Vacuolation, smooth muscle

0

0

0

5

·        Grade1

 

 

 

4

·        Grade2

 

 

 

1

A diffuse microvesicular vacuolation occurred in the cytoplasm of the surface epithelium. However, in two females, a macrovesicular vacuolation was visualized in the glandular epithelium. The smooth muscle of the uterus wall showed a microvesicular type of vacuolation.

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.

Conclusions:
Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats the oral administration of the test substance by gavage to male and female Wistar rats resulted in signs of systemic toxicity at the highest dose of 150 mg/kg bw/d (LOAEL), such as a reduction in food consumption and a decrease in body weight (change), adverse changes in clinical pathology parameters and histopathology. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 50 mg/kg bw/d for male and female Wistar rats.
The NOAEL for reproductive performance and fertility was set to 150 mg/kg bw/d for male and female Wistar rats.
The NOAEL for developmental toxicity was 50 mg/kg bw/d. This was based on the decrease in pup body weight at 150 mg/kg bw/d. This decrease, however, was considered to be secondary to the significant maternal toxicity and was not considered to be a selective adverse effect.
Executive summary:

In an OECD 422 study, the test substance was administered daily to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 15, 50 and 150 mg/kg body weight/day (mg/kg bw/d, test groups 1-3, respectively) to screen for potential systemic, reproductive and developmental toxicity.Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only (0.5% Sodium carboxymethyl cellulose (CMC) suspension in deionized water (with 10 mg/100mL Cremophor). The duration of treatment covered a 2-weeks premating and mating period in both sexes (mating pairs were from the same test group), 3 days postmating in males, and the entire gestation and approximately 3 weeks of lactation period in females up to the day of scheduled sacrifice of the animals.

 

After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm were 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 animals before the start of the administration period and, as a rule, thereafter at weekly intervals.

Water consumption of the F0 parents was determined for premating days 0 - 3 and 7 - 10. In dams water consumption was determined for gestation days (GD) 0 - 1, 6 - 7, 13 - 14 and 19 - 20 and lactation days (PND) 1 - 2, 3 - 4, 6 - 7 and 12 - 13.

Food consumption of the F0 parents was determined once weekly during premating. In dams food consumption was determined for GD 0 - 7, 7 - 14, 14 - 20 and PND 1 - 4, 4 - 7, 7 - 10 and 10 - 13.

Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating. During gestation and lactation period, F0 females were weighed on GD 0, 7, 14 and 20, on the day of parturition (PND 0) and on PND 4, 7, 10 and 13.

Estrous cycle data were evaluated for F0 generation females over a two weeks period prior to mating until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice.

The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1, 4, 7 and 13. Their viability was recorded. At necropsy on PND 13, all pups were sacrificed with CO2, under isoflurane anesthesia, and examined macroscopically for external and visceral findings

Anogenital distance (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements were conducted in a blind randomized fashion, using a measuring ocular on all live male and female pups on PND 1. All surviving pups were examined for the presence or absence of nipple/areola anlagen on PND 13. The number of nipple/areola anlagen were counted.

Blood samples 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 for hormone measurement.

Clinico-chemical and hematological examinations were performed in 5 animals per sex and group towards the end of the administration period. Blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia for hormone measurement.

At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 parental males and females per group. 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:

·        Demonstrated the stability of the test substance preparations over a period of 7 days at room temperature

·        Confirmed the homogeneous distribution of the test substance in 0.5% CMC suspension in deionized water (with 10 mg/100mL Cremophor)

·        Confirmed the overall accuracy of the prepared test substance concentrations

 

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

150 mg/kg bw/d

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

·        Decreased food consumption in males and females during premating (up to 9 % below control) and lactation (females: up to 17% below control, without statistical significance)

·        Decreased body weights in males on mating day 14 (up to 5% below control) and in females during lactation (PND 4, 10 – 13; up to 7% below control)

·        Decreased body weight change in males during premating days 0 - 7 (about 80% below control)

·        Decreased absolute eosinophil cell counts in both sexes and decreased relative eosinophil counts in females

·        Increased absolute and relative monocyte counts in females

·        Decreased glucose levels in females

·        Brain: vacuolation, plexus choroideus: all males (minimal to slight) and females (minimal to moderate)

·        Eyes with optic nerve: vacuolation, diffuse, iris and choroidal layer: 5 out of 10 males (minimal to slight) and all females (slight to moderate)

·        Liver:

o  Significant absolute and relative weight increase in males (+8.90% and +15.01%) and females (+29.61% and 36.04%)

o  Vacuolation: all males (slight to moderate) and females (minimal to severe)

o  Necrosis, single cell: 8 out of 10 males (minimal), 9 out of 10 females (minimal to slight)

o  Necrosis, multifocal: 2 out of 10 males (minimal to slight), 1 out of 10 females (minimal)

o  Multinuclear hepatocytes: 1 out of 10 males (slight) and 2 out of 10 females (minimal to slight)

o  Karyomegaly: 1 out of 10 females (minimal)

·        Skeletal muscle:

o  Vacuolation, fiber: 6 out of 10 males (minimal and severe), 9 out of 10 females (minimal to moderate)

o  Necrosis: 2 out of 10 males and 2 out of 10 females (minimal)

o  Regeneration: 1 out of 10 males (moderate) and 1 out of 10 females (minimal)

·        Axillary lymph node:

o  Vacuolation, multifocal: 4 out of 10 females (minimal to slight)

o  Apoptosis: 1 out of 10 females (minimal)

·        Mesenteric lymph node:

o  Vacuolation, multifocal: 8 out of 10 females (minimal to slight)

o  Apoptosis: 7 out of 10 females (minimal to slight)

o  Cellularity decreased (paracortex): 6 out of 10 females (minimal to slight)

o  Macrophage aggregates, increased: 9 out of 10 females (minimal to slight)

·        Kidneys:

o  Significant increase of the absolute (+49.06%) and relative (+60.60%) weight in females

o  Vacuolation, macrovesicular: all females (minimal to moderate)

o  Vacuolation, microvesicular: all females (minimal to moderate)

o  Degeneration/regeneration, tubular: 8 out of 10 females (minimal to moderate)

o  Inflammation, granulomatous: 2 out of 10 females (slight)

·        Spleen:

o  Vacuolation, lymphoid cell (B-lymphocytes): all females (minimal to moderate)

o  Cellularity decreased (T-lymphocytes): 6 out of 10 females (minimal to moderate)

 

F1 PUPS

CLINICAL EXAMINATIONS/ GROSS FINDINGS

·        Decreased mean body weights of male pups and both sexes combined (PND 13: about 11% and 10%, respectively, below control) and decreased body weight change in male and female pups during PND 1-13.

 

 

50 mg/kg bw/d

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

·        No test substance-related, adverse findings were noted

F1 PUPS

CLINICAL EXAMINATIONS/ GROSS FINDINGS

·        No test substance-related, adverse findings were noted

 

 

15 mg/kg bw/d

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

·        No test substance-related, adverse findings were noted

F1 PUPS

CLINICAL EXAMINATIONS/ GROSS FINDINGS

·        No test substance-related, adverse findings were noted

 

Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats the oral administration of the test substance by gavage to male and female Wistar rats resulted in signs of systemic toxicity at the highest dose of 150 mg/kg bw/d (LOAEL), such as a reduction in food consumption and a decrease in body weight (change), adverse changes in clinical pathology parameters and histopathology. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 50 mg/kg bw/d for male and female Wistar rats.

The NOAEL for reproductive performance and fertility was set to 150 mg/kg bw/d for male and female Wistar rats.

The NOAEL for developmental toxicity was 50 mg/kg bw/d. This was based on the decrease in pup body weight at 150 mg/kg bw/d. This decrease, however, was considered to be secondary to the significant maternal toxicity and was not considered to be a selective adverse effect.

 

Reason / purpose:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018 - 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 Jul 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, D-55116 Mainz
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 16315004.
- Expiration date of the lot/batch: 01 May 2019.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature.
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Solubility and stability of the test substance in the solvent/vehicle: The stability of test substance in 0.5% CMC suspension in deionized water (with 10 mg/100mL Cremophor) was demonstrated for a period of 7 days at room temperature.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: For the preparation of the administration suspensions the test substance was weighed in a calibrated beaker depending on the dose group, topped up with 0.5% CMC suspension in
deionized water with 10 mg/100 mL Cremophor and intensely mixed with a homogenizer. During administration, the preparations were kept homogeneous with a magnetic stirrer.
- Final preparation of a solid: suspension in 0.5% CMC suspension in deionized water (with 10 mg/100mL Cremophor)

FORM AS APPLIED IN THE TEST (if different from that of starting material) : suspension.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH/ Charles River Laboratories, France.
- Females (if applicable) nulliparous and non-pregnant: yes.
- Age at study initiation: 10 - 12 weeks old when arrived, 13 - 15 weeks old at the beginning of treatment.
- Weight at study initiation: 376.3 - 380.3 g (males), 210.0-212.7 g (females). Weight variation did not exceed 20 percent of the mean weight of each sex.
- Fasting period before study: no.
- Housing: During the study period, the rats were housed individually in Polycarbonate cages type III supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany, with the following exceptions: During overnight matings, male and female mating partners were housed together in Polycarbonate cages type III and pregnant animals and their litters were housed together until PND 13 in Polycarbonate
cages type III. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. For enrichment wooden gnawing blocks (Typ Lignocel® block large, J. Rettenmaier & Söhne
GmbH + Co KG, Rosenberg, Germany) were added. In addition in Polysulfonate cages large play tunnels (Art. 14153; supplied by PLEXX B.V., Elst, Netherlands) were added. The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured. The animals were housed in fully air-conditioned rooms in which central air conditioning guaranteed a range of temperature and a range of relative humidity.
- Diet: ad libitum.
- Water: ad libitum.
- Acclimation period:

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C.
- Humidity (%): 45-65%.
- Air changes (per hr): 15 times per hour.
- Photoperiod (hrs dark / hrs light): The day/night cycle was 12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h.

IN-LIFE DATES: From: 03 Jul 2018 To: 25 Sep 2018.
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% CMC suspension in deionized water with 10 mg/100mL Cremophor
Details on exposure:
After the acclimatization period, the test substance was administered to the parental animals orally by gavage, once daily at approximately the same time in the morning. Females in labor were not treated. The treatment lasted up to one day prior to sacrifice. The animals of the control group were treated with the vehicle (0.5% CMC suspension in deionized water with 10 mg/100mL Cremophor), in the same way. The volume administered each day was 10 mL/kg body weight. The calculation of the administration volume was based on the most recent individual body weight.

PREPARATION OF DOSING SOLUTIONS: The test substance suspensions in 0.5% CMC suspension in deionized water with 10 mg/100 mL Cremophor were prepared in intervals, which took into account the analytical results of the stability verification. For the preparation of the administration suspensions the test substance was weighed in a calibrated beaker depending on the dose group, topped up with 0.5% CMC suspension in deionized water with 10 mg/100 mL Cremophor and intensely mixed with a homogenizer. During administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0, 0.15, 0.50 and 1.50 g/100 mL.
- Amount of vehicle (if gavage): 10 mL/kg bw/d.
Details on mating procedure:
- M/F ratio per cage: 1:1.
- Length of cohabitation: from about 16.00 h until 6.30 - 9.00 h of the following morning.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy (gestation day (GD) 0).
- After successful mating each pregnant female was caged (how): Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the test substance in 0.5% CMC suspension in deionized water with 10 mg/100 mL Cremophor for a period of 7 days at room temperature had been verified prior to the start of the study.
At the beginning (during premating), twice during gestation and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. The 3 samples were withdrawn from the top, middle and bottom of the preparation vessel. 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.
Duration of treatment / exposure:
males: 30 days; females: 58/63 days
The duration of treatment covered a 2-weeks premating and mating period in both sexes (mating pairs were from the same test group), 3 days postmating in males, and the entire gestation and approximately 3 weeks of lactation period in females up to the day of scheduled sacrifice of the animals.
Frequency of treatment:
daily at the same time in the morning (exception: no administration to animals being in labor).
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Fasting period before blood sampling for clinical biochemistry: 16 hours fasting before blood sampling.
Positive control:
no.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes (for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity; parturition and lactation behavior of the dams).
- Time schedule: at least once daily.

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: prior to the first administration and thereafter at weekly intervals
- Examined parameters: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size.

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period (to randomize the animals), study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning) with the following exceptions for the females:
• 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), PNDs 4, 7, 10 and 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 administration volume).

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
- Time schedule for examinations: once a week with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined for GD 0-7, 7-14 and 14-20.
• Food consumption of the F0 females which gave birth to a litter was determined on PND 1-4, 4-7, 7-10 and 10-13.
- Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period in females without litter during the lactation period.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Generally, water consumption was determined once a week as representative value over a period of 3 days for the male and female parental animals, with the following exceptions:
• Water consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals).
• Water consumption of the females with evidence of sperm was determined on gestation days (GD) 0-1, 6-7, 13-14 and 19-20.
• Water consumption of the F0 females, which gave birth to a litter was determined for PND 1-2, 3-4, 6-7 and 12-13.
- Water consumption was not determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.

HAEMATOLOGY: Yes.
- Time schedule for collection of blood: at termination (males); at PND 14 (females).
- Anaesthetic used for blood collection: Yes (isoflurane).
- Animals fasted: Yes (about 16 hours).
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes.
- Time schedule for collection of blood: at termination (males); at PND 14 (females).
- Anaesthetic used for blood collection: Yes (isoflurane).
- Animals fasted: Yes (about 16 hours).
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group.
- Parameters checked in table 2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes; functional observational battery and motor activity assessment (see "OTHER")

OTHER:
Functional observational battery (FOB):
- A functional observational battery was performed in the first five parental male animals per test group and the first five surviving females with litter (in order of delivery) of all test groups at the end of the administration period starting at about 10.00 h on study day 27 (males) and 52 (females).
- Examined parameters:
• Home cage observations: posture, tremors, convulsions, abnormal movements, gait, other findings
• Open field observations (at least for 2 minutes): 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), rearing within 2 minutes, other findings
• Sensory motor tests/ reflexes: reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (auditory 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 assessment:
- measured from 14:00 h onwards on the same day as the FOB was performed in the first five parental males and the first five surviving females with litter (in order of delivery) per group

Estrous cycle:
- In all parental females in the premating phase, estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating, mating and throughout cohabitation until there is evidence of sperm in the vaginal smear. Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all female F0 rats.

Male reproduction data:
- The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.

Female reproduction and delivery data
- The pairing partners, the number of mating days until vaginal sperm were detected and gestational status were recorded for F0 females.

Thyroid hormones (males only)
- Time schedule for collection of blood: at termination.
- Anaesthetic used for blood collection: Yes (isoflurane).
- Animals fasted: Yes (about 16 hours).
- How many animals: all surviving males at termination.
- Parameters checked in table 3 were examined.
Oestrous cyclicity (parental animals):
Estrous cycle:
- In all parental females in the premating phase, estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating, mating and throughout cohabitation until there is evidence of sperm in the vaginal smear. Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all female F0 rats.
Sperm parameters (parental animals):
Parameters examined in parental males:
Testis weight, epididymides weight, prostate weight, weight of seminal vesicles with coagulating glands; histopathological examination of epididymides, prostate gland, seminal vesicles, testes; spermatogenic staging profiles.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes.
- If yes, maximum of 8 pups/litter (4 pups/sex/litter as nearly as possible); surplus pups were sacrificed under isoflurane anesthesia by decapitation, blood was sampled for determination of thyroid hormone concentrations and pups were examined externally, eviscerated and their organs were assessed macroscopically.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and status of delivery (number of live born and stillborn), pub viability/mortality at least once daily, sex ratio (by anogenital distance, mammary line, necropsy), pub clinical observations (clinical symptoms including gross-morphological findings), pub body weight at PND 1, 4, 7 and 13, runts, anogenital distance, anogenital index, nipple/aereola anlagen (male pubs at PND13), pub necropsy observations, thyroid hormone concentrations.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, on study day 30.
- Maternal animals: All surviving animals, on study day 58/63.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera (see table 4, Organ weights).

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 4 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 (surplus pups after litter standardization) and 13 (remaining pups after litter standardization) days of age.
- These animals were subjected to postmortem examinations (macroscopic and microscopic (if required) as follows: the pups were examined externally and eviscerated, and the organs were assessed macroscopically. All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY
Thyroid glands/parathyroid glands of one male and one female pup per litter at 13 days of age were fixed in neutral buffered 4% formaldehyde solution for possible further processing.
Statistics:
See table 5.
Reproductive indices:
Male reproduction data:
- The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.
- mating and fertility indices were calculated for F1 litters (for formulas see "Any other information on materials and methods")

Female reproduction and delivery data
- The pairing partners, the number of mating days until vaginal sperm were detected and gestational status were recorded for F0 females.
- mating, fertility and gestation indices, live birth index, postimplantation loss were calculated for F1 litters (for formulas see "Any other information on materials and methods")
Offspring viability indices:
- viability index, survival index (for formulas see "Any other information on materials and methods")
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Several male and female animals of the mid-dose group and almost all male and female animals of the high-dose group (50 and 150 mg/kg bw/d) showed salivation immediately after dosing (up to 2 hours post dosing) during the treatment period. It is likely, that this temporary finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse toxicologically relevant finding.
No other clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F0 parental animals in any of the test groups (1 – 3; 15, 50 and 150 mg/kg bw/d) during the study.
One high-dose male (No. 39) showed a distended, hard abdomen and piloerection during postmating days 1 – 2 which was assessed to be incidental.
Two sperm positive control females (Nos. 102 and 107), one sperm positive low-dose female (No. 118) and one sperm positive high-dose female (No. 133) did not deliver F1 pups.
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In high-dose males, mean body weights were statistically significantly below the concurrent control values on mating day 14 (about 5%). Furthermore, mean body weight change was statistically significantly below the concurrent control during premating days 0 - 7 (about 80%). In high-dose females, mean body weights were statistically significantly below the concurrent control values during lactation (PND 4, PND 10 – 13, up to 7% below control) showing a downward trend towards the end of the study (see graphs below). This finding in high-dose males and females was assessed as treatment-related and adverse.
Mean body weight (change) of the mid- and low-dose males and females during the entire study period were comparable to the concurrent control values.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of the high-dose group was statistically significantly reduced compared to the concurrent control values in males during premating days 0 - 7 and in females during premating days 0 - 13 (both up to 9% below control). Food consumption in females showed a downward trend towards the end of the study since food consumption of high-dose females was further reduced (up to 17 % below control, without statistical significance) during lactation. The reduction in food consumption in male and females of the high-dose was assessed as treatment-related and adverse.
Food consumption of the high-dose females during gestation and of the low- and mid-dose males and females during the entire study was comparable to the concurrent control values.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
In males of the high-dose (150 mg/kg bw/d), water consumption was statistically significantly increased compared to the concurrent control values during premating days 7 - 10 (about 36%). In the high-dose F0 females, water consumption was statistically significantly increased compared to the concurrent control values during the entire gestation period (up to 43%). Together with the finding salivation, it is most likely that the increase in water consumption occurred due to the bad taste of the test substance or local affection of the upper digestive tract. It was assessed as treatment related but not adverse.
Water consumption of the high-dose females during premating and lactation and of the lowand mid-dose males and females during the entire study was comparable to the concurrent control values. The statistically significantly higher water consumption in the low-dose females during premating days 0 - 3 was considered to be spontaneous in nature and not treatment related since it was not related to dose.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period in male and female rats of test group 3 (150 mg/kg bw/d), absolute eosinophil cell counts were decreased (in males not statistically significantly). Additionally, in females of this test group absolute (not statistically significant) and relative monocyte counts were increased, and relative eosinophil cell counts were decreased. These changes were regarded as treatment-related and adverse.

In males of test group 3 (150 mg/kg bw/d) relative basophil cell counts were significantly decreased. However, no pathophysiological correlate exists regarding low basophil counts and, therefore, this change was regarded as incidental and not treatment-related. In addition, in males of this test group mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) were significantly decreased. MCV was within, MCH marginally below the historical control range (males, MCV 48.1-51.7 fL, MCH 1.03-1.11 fmol). However, these were calculated red blood cell indices and the measured red blood cell parameters (i.e. hemoglobin, hematocrit and red blood cell (RBC) counts) were not changed. Therefore, the MCV and MCH alterations in males of test group 3 were regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, in females of test group 3 (150 mg/kg bw/d) glucose levels were significantly decreased. This change combined with the hematological alterations is regarded as treatment-related and adverse.

In males of test group 3 (150 mg/kg bw/d) total protein and globulin values were significantly decreased and inorganic phosphate levels were significantly increased. However, all values were within historical control ranges (males, total protein 59.06-65.10 g/L, globulins 21.95- 28.93 g/L, inorganic phosphate 1.45-2.06 mmol/L). Therefore, these alterations were regarded as incidental and not treatment-related.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were observed in following organs: brain, eyes with optic nerves, liver, lungs, skeletal muscle, seminal vesicles, epididymides, adrenal cortex, axillary and mesenteric lymph nodes, glandular stomach, kidneys, spleen and uterus. The main finding in each org n was a cytoplasmic vacuolation, characterized by the presence of multiple fine to small vacuoles, that were no larger than the nuclei of the cell. This type of vacuolation is referred here as “microvesicular” and most frequently increased the size of the affected cells. On the other hand, large cytoplasmic vacuoles, that displaced the nuclei or deformed the cells are mentioned here as “macrovesicular type” and were seen only in the kidneys and glands of the uteri. Incidences and grading of findings in the different target organs are given in the tables 8-21.

Fertility: The female animals (Nos. 102, 107, 118 and 133), which were not pregnant as well as thmale mating partners (Nos. 2, 7, 18 and 33) did not show relevant histopathological findings that could have impaired fertility.
Other effects:
no effects observed
Description (incidence and severity):
THYROID HORMONES
- In parental males (test groups 1, 2 and 3; 15, 50 and 150 mg/kg bw/d) and in male and female pups at PND13 (test groups 11, 12 and 13; 15, 0 and 150 mg/kg bw/d), no treatment-related alterations of T4 and TSH levels were observed.

DETAILED CLINICAL OBSERVATIONS (DCO):
- All male and all female animals of all dose groups (15, 50 and 150 mg/kg bw/d) did not show any abnormalities.

FUNCTIONAL OBSERVATIONAL BATTERY (FOB)
- Home cage observations: No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.
- Open field observations: The open field observations did not reveal any test substance-related findings in male and female animals of all test groups.
- Sensorimotor tests/reflexes: There were no test substance-related findings in male and female animals of all test groups.
- Quantitative Parameters: The landing foot-splay test (FST) in the mid- and high-dose male animals was statistically significantly below the concurrent control values (9.5 and 9.7 cm, respectively, vs. 11.8 cm in the control). However, there was no relation to dose and both values were within the range of the historical control data (Supplement, HCD, FST males: range of 9.3 – 13.8 cm). Therefore, it was not assessed as treatment-related and adverse. No test substance-related impaired parameters were observed in the low-dose males and all female animals of all test groups.

MOTOR ACTIVITY MEASUREMENT (MA)
- No statistically significant changes on motor activity data (summation of all intervals) was observed in the male and female animals of all test substance-treated groups in comparison to the concurrent control values.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
- Estrous cycle data, generated during the last 2 weeks prior to mating for the F1 litter, revealed regular cycles in the females of all test groups 0 - 3. The mean estrous cycle duration ranged between 4.0 and 4.1 days.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The stages of spermatogenesis in the testes of males of the high dose test group were comparable to those of the controls.
Reproductive performance:
no effects observed
Description (incidence and severity):
MALE REPRODUCTION DATA
- For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all test groups (0 - 3).
- Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter.
- Two control males (No. 2 and 7), one low-dose male (No. 18) and one high-dose male (No. 33) did not generate pregnancy.
- Thus, the male fertility index ranged between 80% and 100% without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study.
- The apparently infertile male rat did not show relevant gross lesions.

FEMALE REPRODUCTION AND DELIVERY DATA
- The female mating index calculated after the mating period for F1 litter was 100% in all test groups.
- The mean duration until sperm was detected (GD 0) varied between 1.9 and 3.9 days. The mean value of 3.9 days in the high-dose group was well within the range of the historical control data (Supplement, HCD, mating days: 1.6 – 5.8) and can be explained by female No. 135
which had 14 mating days. The female was possibly pseudo-pregnant indicated by 12 days of diestrus. Afterwards, the female mated and was pregnant.
- All female rats delivered pups with the following exception: Control female No. 102 (mated with male No. 2) did not become pregnant. Control female No. 107 (mated with male No. 7) did not become pregnant. Low-dose female No. 118 (mated with male No. 18) did not become pregnant. High-dose female No. 133 (mated with male No. 33) did not become pregnant.
- The fertility index ranged between 80% and 100% without showing any relation to dosing.
- The non-pregnant females had no relevant gross lesions or microscopic findings.
- The mean duration of gestation values varied between 22.1 (test group 1), 22.2 (control and test group 2) and 22.5 (test group 3)
- The gestation index was 100% in all test groups 0 - 3.
- Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (12.0 / 13.7 / 12.1 and 10.3 implants/dam in test groups 0 - 3, respectively). The value of test group 3 was marginally outside the range of the historical control data (Supplement, HCD, implantation sites: 10.4 - 14.9), without statistical significance. It was assessed as spontaneous and not treatment-related since the mean value of the related parameter pups delivered of test group 3 (9.6) was within the historical control range (HCD, pups delivered: 9.3 -13.9). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any significant differences between the groups, and the mean number of F1 pups delivered per dam remained unaffected (11.1 / 13.2 / 10.6 and 9.6 pups/dam in test groups 0 - 3, respectively)
- The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 100% / 98.3% / 100% and 100% in test groups 0 - 3, respectively. Moreover, the number of stillborn pups was not significantly different between the test groups.
- Thus, the test substance did not adversely affect reproduction and delivery of the F0 generation parental females.
Regarding clinical examination, several male and female animals of the mid-dose group and almost all male and female animals of the high-dose group (50 and 150 mg/kg bw/d) showed salivation immediately after dosing (up to 2 hours post dosing) during the treatment period. It is likely, that this temporary finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse toxicologically relevant finding.
In F0 males and females of the high-dose, water consumption was statistically significantly increased compared to control during premating (males: about 36% above control) or gestation (up to 43% above control). Together with the clinical finding salivation, it is most likely that the increase in water consumption occurred due to the bad taste of the test substance or local affection of the upper digestive tract. It was assessed as treatment related but not adverse.

Adverse signs of toxicity were observed at the highest dose (150 mg/kg bw/d).
Food consumption of the high-dose was statistically significantly reduced compared to control in males and females during premating (both up to 9% below control) and in females during lactation (without statistical significance, up to 17 % below control).
Mean body weight (change) was statistically significantly below the control values in males during premating (BWC) and during mating (BW: about 5% below control) and in females during lactation (up to 7% below control). Food consumption and body weight in high-dose animals showed a downward trend towards the end of the study and were assessed as treatment-related and adverse.
No test substance related, adverse findings were observable in the mid- and low-dose (50 and 15 mg/kg bw/d).

Regarding clinical pathology, in females of test group 3 (150 mg/kg bw/d) decreased absolute and relative eosinophils and increased absolute and relative monocyte counts as well as decreased glucose levels indicated some subacute stress event which is regarded as treatment-related. Lower absolute eosinophil counts were also observed in males of the same test group.

Regarding pathology, a significant decrease of the final body weight in males and females of test group 3 (150 mg/mg bw/d) was accompanied by a significant absolute and relative weight increase of the liver in both sexes, and of the adrenal glands and kidneys in females. These organ weight changes were considered treatment-related and were consistent with histopathological findings, characterized mainly by cytoplasmic vacuolation. Vacuolation was also observed in several other organs of males and females in test group 3 (brain, eyes, lungs, skeletal muscle, seminal vesicles, epididymides, axillary and mesenteric lymph nodes, glandular stomach, spleen and uterus) and in test group 2 (epididymides). The cytoplasmic vacuolation was of microvesicular type (multiple fine to small vacuoles, that were no larger than the nuclei of the respective cell types and did not displace it). A macrovesicular type (large and single cytoplasmic vacuoles, that displaced the nuclei or altered the cellular form) was distinctively and additionally seen in the kidneys (all females) and uteri (two females), and only as background in the remaining organs.
The vacuolation was regarded as treatment-related and assessed as adverse, if it was accompanied by cytotoxicity (cell death, degeneration and/or inflammation). In test group 3 (150 mg/kg bw/d) animals, these criteria were accomplished for the liver and skeletal muscle of males and females; axillary and mesenteric lymph nodes and kidneys of females. Finally, vacuolation alone without sings of cytotoxicity, occurring in special organs with central vital functions or special senses, was also assessed as adverse, assuming a potential impairment of vital functions. This was true for the spleen in females and brain and eyes in males and females of test group 3 (150 mg/kg bw/d). A summary of histopathological findings assessed as treatment-related and adverse is given as follows:
In test group 3 (150 mg/kg bw/d), histopathology revealed in the liver a centrilobular microvesicular vacuolation in all males and females accompanied by single cell necrosis. It cannot be excluded if other hepatic findings like multifocal necrosis, multinuclear hepatocytes and karyomegaly, affecting very few animals were treatment-related and were, therefore, assumed to be possibly treatment-related and adverse. In the skeletal muscle of males and females, the fiber vacuolation was accompanied by necrosis and regeneration of fibers. In the axillary lymph node of females, the vacuolation was present in the high endothelial venules (HEV) and was accompanied by apoptosis. The mesenteric lymph nodes of females showed vacuolation in the HEV, lymphocytes of medullary cords and macrophages of medullary sinuses, apoptosis and macrophage aggregates in the periphery of paracortical areas, and decreased cellularity in paracortical regions (T-lymphocytes). In the kidneys of females, a tubular cytoplasmic macro- and microvesicular vacuolation affecting all animals was accompanied by tubular degeneration/regeneration and granulomatous inflammation. Both types of vacuolation affected predominantly the cortical tubular epithelium but the microvesicular vacuolation extended also into the outer stripe of the outer medulla. The spleen of females displayed a vacuolation of the B-lymphocytes (follicular and marginal zone) and a decreased cellularity of T-lymphocytes in the periarteriolar lymphocyte sheath (PALS). Finally, a microvesicular vacuolation without signs of cytotoxicity was detected in the plexus choroideus of the brain, and in the stromal iris and choroid of the eyes of males and females.

Treatment-related vacuolation not accompanied by signs of cytotoxicity and assessed as not adverse, was seen in following organs in test group 3:
Adrenal cortex of females (zona fasciculata and reticularis) correlating with significantly increased weights; lungs of all male and females (bronchial/bronchiolar epithelium) and glandular stomach of females (base of the glands in the pyloric region). In the epithelium of epididymides (corpus), seminal vesicles and uterus (surface and/or glandular epithelium, and smooth muscle of the wall). Fertility in these animals was not relevantly altered when compared with the control animals.

In test group 2 (50 mg/kg bw/d), histopathology revealed vacuolation in the epididymal epithelium. This change was assessed as treatment-related but not adverse, since no alteration was seen in the sperm density and morphology and no relevant alteration of the fertility was noted in these male animals.

Special stains performed exemplarily in selected organs (liver, kidneys, lungs and skeletal muscle) of animals in test group 3 revealed that the vacuolation was negative for the presence of neutral fat (Oil-red-O stain negative) and was positive for the presence of phosphorylated lipids (Sudan black positive). An increased accumulation of glycogen in the skeletal muscle was discarded (PAS with diastase negative).

Transmission electron microscopy examination performed exemplarily in the liver and kidneys of selected male and female animals of test group 3 revealed the presence of intracytoplasmic myelin figures, characterized by concentric, multilaminated membrane whorls in phagolysosomes (Ghadially, 1988,). This morphological picture is indicative of phospholipidosis and represents lysosomal accumulation of phospholipids. It is inferred that the vacuolation observed in all other organs was most likely of the same nature (Nonoyama and Fukada, 2008; Rudmann et al, 2004).

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. Furthermore, neither the synchrony of the morphology of the estrous cycle in ovaries, uterus, cervix, and vagina nor the stages of seminiferous tubules were altered.

Regarding fertility and reproductive performance, no signs of toxicity were observed in male or female parental animals of all test groups during the entire study. F0 parental animals proved to be fertile. Mating behavior, conception, implantation and parturition were not affected.
Dose descriptor:
LOAEL
Remarks:
systemic toxicity
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
haematology
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no adverse effect observed
Dose descriptor:
NOAEL
Remarks:
reproductive performance & fertility
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no adverse effect observed
Critical effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (actual dose received)
System:
other: divers
Organ:
other: brain, eyes with optic nerve, liver and skeletal muscle (male/female); lymph nodes, kidneys, spleen (females)
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
yes
Remarks on result:
other: not applicable for OECD TG 422
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups. One low-dose female pup (No. 11 of dam No. 113) showed and injury of the left hindlimb on PND 11 without relation to treatment.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
One low-dose female pup (No. 11 of dam No. 113) showed and injury of the left hindlimb on PND 11 without relation to treatment and was therefore sacrificed moribund on PND 11.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Two male and three female runts were seen in test group 1, one male and one female runt was seen in test group 2 and one female runt was seen in test group 3. In test group 3, mean body weight development was comparable to control on PND 1 and 4. On PND 13, mean body weights of the high-dose F1 male pups and both sexes combined were statistically significantly below the concurrent control (about 11% and 10%, respectively). The pup body weight change in the high-dose male pups was statistically significantly below the concurrent control values during the intervals PND 4 - 7, 7 - 13 and 1 - 13 (up to 15%) and in the high-dose female pups and both sexes combined during PND 7 - 13 and 1 - 13 (up to 13%). The decrease in body weights of males and both sexes combined was at the very lower end of the range of the historical control data (Supplement, HCD, pup weights, males: 28.5-33.4 g, both: 28.1-33.0 g). A relation to treatment cannot be excluded. In the highest test group, maternal / parental toxicity was observed. The decrease in body weight of the high dose pups was considered to be a secondary, minor effect in presence of maternal toxicity. No test substance-related influence on body weight (change) of the low- and mid-dose male and female F1 pups were noted.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Neither on anogenital distance nor anogenital index test substance-related effects were noted in all treated F1 offspring (test groups 1 - 3 [15, 50 and 150 mg/kg bw/d]).
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A few pups showed spontaneous findings at gross necropsy, such as discolored thymus (red), discolored testis (red), small testis, supernumerary liver lobe (cystic), post mortem autolysis, partly cannibalized, limb lesion, diaphragmatic hernia and dilated renal pelvis. These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, all these findings were not considered to be associated to the test substance.
Dose descriptor:
LOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: maternal toxic dose
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no adverse effect observed
Critical effects observed:
no
Reproductive effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
not specified
Relevant for humans:
yes

The various analyses:

  • Demonstrated the stability of the test substance preparations over a period of 7 days at room temperature
  • Confirmed the homogeneous distribution of the test substance in 0.5% CMC suspension in deionized water (with 10 mg/100mL Cremophor)
  • Confirmed the overall accuracy of the prepared test substance concentrations

Concentration control analysis:

Measured values for the test substance were in the expected range of the target concentrations (90 - 110%) demonstrating the correctness of the preparations. For the low dose (15 mg/kg bw/d), one single value (sample No. 3) was marginally above the tolerance range of the test facility (111%). However, the test substance preparation was homogeneously distributed for this dose level and all other values of the low dose were within the tolerance range. Therefore, the measured values of the low dose were assessed to be in an acceptable range of the nominal concentrations in this study.

Table 6: Absolute organ weights

 

Male animals

Female animals

Test group (mg/kg bw/d)

1

(15)

2

(50)

3

(150)

1

(15)

2

(50)

3

(150)

Final body

weight

-1.7%

-0.5%

-6.1%**

+0.2%

+2.5%

-6.2%**

Adrenal glands

 

 

 

+10.649%

+7.273%

+29.610%**

Liver

-0.55%

+2.45%

+8.90%*

+0.31%

+8.12%

+26.47%**

Kidneys

%

%

%

-3.50%

+6.99%

+49.06%**

Ovaries

%

%

%

+11.907%

+27.043%**

+9.183%

*p <= 0.05; **p <= 0.01

Table 7: Relative organ weights

 

Male animals

Female animals

Test group (mg/kg bw/d)

1

(15)

2

(50)

3

(150)

1

(15)

2

(50)

3

(150)

Adrenal glands

 

 

 

+10.246%

+3.863%

+39.463%**

Liver

+1.37%

+2.27%

+15.01%**

+0.25%

+4.48%

+36.04%**

Kidneys

-3.94%

-5.39%

+9.79%*

-3.65%

+3.52%

+60.60%**

Spleen

%

%

%

-6.65%

-13.51%*

+7.87%

Thyroid glands

 

 

 

+15.735%

+13.353%

+19.488%**

 *p <= 0.05; **p <= 0.01

Table 8: Histopathology/ Brain

Brain

Male animals

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

10

10

10

10

Vacuolation, choroid plexus, diffuse

0

0

0

10

0

0

0

10

·        Grade1

 

 

 

6

 

 

 

3

·        Grade2

 

 

 

4

 

 

 

2

·        Grade3

 

 

 

 

 

 

 

5

The cytoplasmic vacuolation was observed mainly in the epithelium of the choroid plexus of the lateral ventricle and was of microvesicular type.

Table 9: Histopathology/ Eyes with optic nerve

Eyes with optic nerve

Male animals

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

10

10

10

10

Vacuolation, diffuse

0

0

0

5

0

0

0

10

·        Grade1

 

 

 

4

 

 

 

 

·        Grade2

 

 

 

1

 

 

 

3

·        Grade3

 

 

 

 

 

 

 

7

The vacuolation was of microvesicular type, most likely in macrophages of the iris stroma. In several animals the vacuolation extended from here into the peripheral choroidal layer.

Table 10: Histopathology/ Liver

Liver

Male animals

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

10

10

10

10

Vacuolation, centrilobular

0

0

0

10

0

0

0

10

·        Grade1

 

 

 

 

 

 

 

1

·        Grade2

 

 

 

4

 

 

 

4

·        Grade3

 

 

 

6

 

 

 

3

·        Grade4

 

 

 

 

 

 

 

2

Necrosis, single cell

0

0

0

2

0

0

0

9

·        Grade1

 

 

 

1

 

 

 

4

·        Grade2

 

 

 

1

 

 

 

5

Necrosis, multifocal

0

0

0

2

0

0

0

1

·        Grade1

 

 

 

1

 

 

 

1

·        Grade2

 

 

 

1

 

 

 

 

Multinuclear hepatocytes

0

0

0

1

0

0

0

2

·        Grade1

 

 

 

 

 

 

 

1

·        Grade2

 

 

 

1

 

 

 

1

Karyomegaly

0

0

0

0

0

0

0

1

·        Grade1

 

 

 

 

 

 

 

1

The vacuolation was of microvesicular type and was accompanied by single cell necrosis of vacuolated cells. The centrilobular vacuoles were negative for Oil-Red-O and positive for Sudan black. Findings like multifocal necrosis, multinuclear hepatocytes and karyomegaly, did affect only very few animals and treatment-relationship remains unclear.

Table 11: Histopathology/Lungs

Lungs

Male animals

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

10

10

10

10

Vacuolation, epith., bronchial/bronchiolar

0

0

0

10

 

 

 

10

·        Grade1

 

 

 

10

 

 

 

3

·        Grade2

 

 

 

 

 

 

 

5

·        Grade3

 

 

 

 

 

 

 

2

Histiocytosis, alveolar, multifocal

2

2

2

3

3

1

2

9

·        Grade1

2

2

2

1

3

1

2

4

·        Grade2

 

 

 

1

 

 

 

4

·        Grade3

 

 

 

1

 

 

 

1

The vacuolation in the lungs was of microvesicular type and affected most frequently the bronchiolar epithelium including terminal bronchioles, and in extensive cases also the bronchial epithelium. It was accompanied by an increase in alveolar histiocytosis, which was stronger in females than in males. The vacuoles as well as the alveolar histiocytes were negative for Oil- Red-O and positive for Sudan black.

Table 12: Histopathology/Skeletal muscle

Skeletal muscle

Male animals

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

10

10

10

10

Vacuolation, fiber

0

0

0

7

0

0

0

9

·        Grade1

 

 

 

6

 

 

 

7

·        Grade2

 

 

 

 

 

 

 

1

·        Grade3

 

 

 

 

 

 

 

1

·        Grade4

 

 

 

1

 

 

 

 

Necrosis, multifocal

0

0

0

0

0

0

0

2

·        Grade1

 

 

 

2

 

 

 

2

Regeneration, multifocal

0

0

0

1

0

0

0

1

·        Grade1

 

 

 

 

 

 

 

1

·        Grade3

 

 

 

1

 

 

 

 

The vacuolation in the skeletal muscle was characterized by the presence of very tiny microvacuoles with a multifocal distribution pattern within the myofibers. In addition, some of the affected fibers were retracted and strongly eosinophilic, indicating a hyaline necrosis in 2 males and 2 females. One male animal and one female with severe and moderate vacuolation, respectively, showed also an regeneration of fibers. The tiny vacuoles in the affected fibers were negative for Oil-Red-O, positive for Sudan-black and were negative in the PAS-with diastase stain.

Table 13: Histopathology/ Epididymides

Epididymides

Male animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

Vacuolation, epithelial

0

0

10

10

·        Grade1

 

 

2

 

·        Grade2

 

 

8

 

·        Grade3

 

 

 

10

The vacuolation was of microvesicular type. In test group 3, it was diffusely distributed along the tubules of the corpus, starting slightly distally of the head and extended to the junction with the cauda. In test group 2, the vacuolation was mostly present in the caudal part of the corpus, reaching the junction with the cauda.

Table 14: Histopathology/ Seminal vesicles

Seminal vesicles

Male animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

Vacuolation, epithelial

0

0

0

2

·        Grade1

 

 

 

1

·        Grade2

 

 

 

1

The diffuse epithelial vacuolation was of microvesicular type, with a distinctive basal localization.

Table 15: Histopathology/ Adrenal cortex

Adrenal cortex

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

Vacuolation, increased

0

0

0

10

·        Grade1

 

 

 

4

·        Grade2

 

 

 

4

·        Grade3

 

 

 

2

The cytoplasmic vacuolation was of microvesicular type and included the zona fasciculata and reticularis.

Table 16: Histopathology/ Axillary lymph node

Axillary lymph node

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

Vacuolation, multifocal

0

0

0

4

·        Grade1

 

 

 

3

·        Grade2

 

 

 

1

Apoptosis

0

0

0

1

·        Grade1

 

 

 

1

The vacuolation was of microvesicular type and was noted mainly in the high endothelial venules (HEV). The apoptosis was seen in the periphery of the paracortex.

Table 17: Histopathology/ Mesenteric lymph node

Mesenteric lymph node

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

Vacuolation, multifocal

0

0

0

8

·        Grade1

 

 

 

6

·        Grade2

 

 

 

2

Apoptosis

0

0

0

7

·        Grade1

 

 

 

6

·        Grade2

 

 

 

1

Cellularity, decreased

0

0

0

6

·        Grade1

 

 

 

4

·        Grade2

 

 

 

2

Aggregates, macrophage, increased

0

1

3

9

·        Grade1

 

1

2

7

·        Grade2

 

 

1

2

The microvesicular vacuolation was seen in the HEV (high endothelial venules) of all animals, and lymphocytes of medullary cords and macrophages of medullary sinuses. The apoptosis and macrophage aggregates were noted in the periphery of paracortical areas. The decreased cellularity occurred in paracortical regions (T-lymphocytes).

Table 18: Histopathology/ Glandular stomach

Glandular stomach

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

Vacuolation, epithelial

0

0

0

7

·        Grade1

 

 

 

5

·        Grade2

 

 

 

2

The vacuolation affected the cytoplasm of the cells at the base of the glands in the pyloric region of the stomach.

Table 19: Histopathology/ Kidneys

Kidneys

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

Vacuolation, macrovesicular

0

0

0

10

·        Grade1

 

 

 

2

·        Grade2

 

 

 

3

·        Grade3

 

 

 

5

Vacuolation, microvesicular

0

0

0

10

·        Grade1

 

 

 

5

·        Grade2

 

 

 

3

·        Grade3

 

 

 

2

Degeneration/ regeneration, tubular

0

0

0

8

·        Grade1

 

 

 

1

·        Grade2

 

 

 

2

·        Grade3

 

 

 

5

Inflammation, granulomatous

0

0

0

2

·        Grade2

 

 

 

2

A tubular cytoplasmic macro- and microvesicular vacuolation accompanied by tubular degeneration/regeneration was distinguished in the kidneys of females. Both types of vacuolation affected predominantly the cortical tubular epithelium, but the microvesicular vacuolation extended also into the outer stripe of the outer medulla. Macrovesicular vacuoles seemed to be empty (by light microscopy) and were most likely noted in collecting ducts, whereas the microvesicular vacuoles seemed to affect more the convoluted proximal and distal tubules and the pars recta. However, a clear distinction was not always possible. Figures of tubular vacuoles might indicate that the macrovesicular vacuolation could originate by exaggerated distention and coalescence of less sized vacuoles. The microvesicular vacuoles were negative for Oil-Red-O and positive for Sudan black stain. The macrovesicular vacuoles did not stain.

Table 20: Histopathology/ Spleen

Spleen

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

Vacuolation, lymphoid cell

0

0

0

10

·        Grade1

 

 

 

4

·        Grade2

 

 

 

4

·        Grade3

 

 

 

2

Cellularity decreased

0

0

0

6

·        Grade1

 

 

 

1

·        Grade2

 

 

 

3

·        Grade3

 

 

 

2

The vacuolation in the spleen of females was of microvesicular type and affected the cytoplasm of lymphoid cell of the follicles and marginal zone (B-lymphocytes). A decreased cellularity of T-lymphocytes was observed in the periarteriolar lymphocyte sheath (PALS).

Table 21: Histopathology/ Uterus

Uterus

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

Vacuolation, epithelial

0

0

0

8

·        Grade1

 

 

 

4

·        Grade2

 

 

 

1

·        Grade3

 

 

 

3

Vacuolation, smooth muscle

0

0

0

5

·        Grade1

 

 

 

4

·        Grade2

 

 

 

1

A diffuse microvesicular vacuolation occurred in the cytoplasm of the surface epithelium. However, in two females, a macrovesicular vacuolation was visualized in the glandular epithelium. The smooth muscle of the uterus wall showed a microvesicular type of vacuolation.

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.

Conclusions:
Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats the oral administration of the test substance by gavage to male and female Wistar rats resulted in signs of systemic toxicity at the highest dose of 150 mg/kg bw/d (LOAEL), such as a reduction in food consumption and a decrease in body weight (change), adverse changes in clinical pathology parameters and histopathology. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 50 mg/kg bw/d for male and female Wistar rats.
The NOAEL for reproductive performance and fertility was set to 150 mg/kg bw/d for male and female Wistar rats.
The NOAEL for developmental toxicity was 50 mg/kg bw/d. This was based on the decrease in pup body weight at 150 mg/kg bw/d. This decrease, however, was considered to be secondary to the significant maternal toxicity and was not considered to be a selective adverse effect.
Executive summary:

In an OECD 422 study, the test substance was administered daily to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 15, 50 and 150 mg/kg body weight/day (mg/kg bw/d, test groups 1-3, respectively) to screen for potential systemic, reproductive and developmental toxicity.Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only (0.5% Sodium carboxymethyl cellulose (CMC) suspension in deionized water (with 10 mg/100mL Cremophor). The duration of treatment covered a 2-weeks premating and mating period in both sexes (mating pairs were from the same test group), 3 days postmating in males, and the entire gestation and approximately 3 weeks of lactation period in females up to the day of scheduled sacrifice of the animals.

 

After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm were 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 animals before the start of the administration period and, as a rule, thereafter at weekly intervals.

Water consumption of the F0 parents was determined for premating days 0 - 3 and 7 - 10. In dams water consumption was determined for gestation days (GD) 0 - 1, 6 - 7, 13 - 14 and 19 - 20 and lactation days (PND) 1 - 2, 3 - 4, 6 - 7 and 12 - 13.

Food consumption of the F0 parents was determined once weekly during premating. In dams food consumption was determined for GD 0 - 7, 7 - 14, 14 - 20 and PND 1 - 4, 4 - 7, 7 - 10 and 10 - 13.

Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating. During gestation and lactation period, F0 females were weighed on GD 0, 7, 14 and 20, on the day of parturition (PND 0) and on PND 4, 7, 10 and 13.

Estrous cycle data were evaluated for F0 generation females over a two weeks period prior to mating until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice.

The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1, 4, 7 and 13. Their viability was recorded. At necropsy on PND 13, all pups were sacrificed with CO2, under isoflurane anesthesia, and examined macroscopically for external and visceral findings

Anogenital distance (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements were conducted in a blind randomized fashion, using a measuring ocular on all live male and female pups on PND 1. All surviving pups were examined for the presence or absence of nipple/areola anlagen on PND 13. The number of nipple/areola anlagen were counted.

Blood samples 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 for hormone measurement.

Clinico-chemical and hematological examinations were performed in 5 animals per sex and group towards the end of the administration period. Blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia for hormone measurement.

At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 parental males and females per group. 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:

·        Demonstrated the stability of the test substance preparations over a period of 7 days at room temperature

·        Confirmed the homogeneous distribution of the test substance in 0.5% CMC suspension in deionized water (with 10 mg/100mL Cremophor)

·        Confirmed the overall accuracy of the prepared test substance concentrations

 

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

150 mg/kg bw/d

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

·        Decreased food consumption in males and females during premating (up to 9 % below control) and lactation (females: up to 17% below control, without statistical significance)

·        Decreased body weights in males on mating day 14 (up to 5% below control) and in females during lactation (PND 4, 10 – 13; up to 7% below control)

·        Decreased body weight change in males during premating days 0 - 7 (about 80% below control)

·        Decreased absolute eosinophil cell counts in both sexes and decreased relative eosinophil counts in females

·        Increased absolute and relative monocyte counts in females

·        Decreased glucose levels in females

·        Brain: vacuolation, plexus choroideus: all males (minimal to slight) and females (minimal to moderate)

·        Eyes with optic nerve: vacuolation, diffuse, iris and choroidal layer: 5 out of 10 males (minimal to slight) and all females (slight to moderate)

·        Liver:

o  Significant absolute and relative weight increase in males (+8.90% and +15.01%) and females (+29.61% and 36.04%)

o  Vacuolation: all males (slight to moderate) and females (minimal to severe)

o  Necrosis, single cell: 8 out of 10 males (minimal), 9 out of 10 females (minimal to slight)

o  Necrosis, multifocal: 2 out of 10 males (minimal to slight), 1 out of 10 females (minimal)

o  Multinuclear hepatocytes: 1 out of 10 males (slight) and 2 out of 10 females (minimal to slight)

o  Karyomegaly: 1 out of 10 females (minimal)

·        Skeletal muscle:

o  Vacuolation, fiber: 6 out of 10 males (minimal and severe), 9 out of 10 females (minimal to moderate)

o  Necrosis: 2 out of 10 males and 2 out of 10 females (minimal)

o  Regeneration: 1 out of 10 males (moderate) and 1 out of 10 females (minimal)

·        Axillary lymph node:

o  Vacuolation, multifocal: 4 out of 10 females (minimal to slight)

o  Apoptosis: 1 out of 10 females (minimal)

·        Mesenteric lymph node:

o  Vacuolation, multifocal: 8 out of 10 females (minimal to slight)

o  Apoptosis: 7 out of 10 females (minimal to slight)

o  Cellularity decreased (paracortex): 6 out of 10 females (minimal to slight)

o  Macrophage aggregates, increased: 9 out of 10 females (minimal to slight)

·        Kidneys:

o  Significant increase of the absolute (+49.06%) and relative (+60.60%) weight in females

o  Vacuolation, macrovesicular: all females (minimal to moderate)

o  Vacuolation, microvesicular: all females (minimal to moderate)

o  Degeneration/regeneration, tubular: 8 out of 10 females (minimal to moderate)

o  Inflammation, granulomatous: 2 out of 10 females (slight)

·        Spleen:

o  Vacuolation, lymphoid cell (B-lymphocytes): all females (minimal to moderate)

o  Cellularity decreased (T-lymphocytes): 6 out of 10 females (minimal to moderate)

 

F1 PUPS

CLINICAL EXAMINATIONS/ GROSS FINDINGS

·        Decreased mean body weights of male pups and both sexes combined (PND 13: about 11% and 10%, respectively, below control) and decreased body weight change in male and female pups during PND 1-13.

 

 

50 mg/kg bw/d

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

·        No test substance-related, adverse findings were noted

F1 PUPS

CLINICAL EXAMINATIONS/ GROSS FINDINGS

·        No test substance-related, adverse findings were noted

 

 

15 mg/kg bw/d

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

·        No test substance-related, adverse findings were noted

F1 PUPS

CLINICAL EXAMINATIONS/ GROSS FINDINGS

·        No test substance-related, adverse findings were noted

 

Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats the oral administration of the test substance by gavage to male and female Wistar rats resulted in signs of systemic toxicity at the highest dose of 150 mg/kg bw/d (LOAEL), such as a reduction in food consumption and a decrease in body weight (change), adverse changes in clinical pathology parameters and histopathology. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 50 mg/kg bw/d for male and female Wistar rats.

The NOAEL for reproductive performance and fertility was set to 150 mg/kg bw/d for male and female Wistar rats.

The NOAEL for developmental toxicity was 50 mg/kg bw/d. This was based on the decrease in pup body weight at 150 mg/kg bw/d. This decrease, however, was considered to be secondary to the significant maternal toxicity and was not considered to be a selective adverse effect.

 

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 Jul 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, D-55116 Mainz
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 16315004.
- Expiration date of the lot/batch: 01 May 2019.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature.
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Solubility and stability of the test substance in the solvent/vehicle: The stability of test substance in 0.5% CMC suspension in deionized water (with 10 mg/100mL Cremophor) was demonstrated for a period of 7 days at room temperature.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: For the preparation of the administration suspensions the test substance was weighed in a calibrated beaker depending on the dose group, topped up with 0.5% CMC suspension in
deionized water with 10 mg/100 mL Cremophor and intensely mixed with a homogenizer. During administration, the preparations were kept homogeneous with a magnetic stirrer.
- Final preparation of a solid: suspension in 0.5% CMC suspension in deionized water (with 10 mg/100mL Cremophor)

FORM AS APPLIED IN THE TEST (if different from that of starting material) : suspension.

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH/ Charles River Laboratories, France.
- Females (if applicable) nulliparous and non-pregnant: yes.
- Age at study initiation: 10 - 12 weeks old when arrived, 13 - 15 weeks old at the beginning of treatment.
- Weight at study initiation: 376.3 - 380.3 g (males), 210.0-212.7 g (females). Weight variation did not exceed 20 percent of the mean weight of each sex.
- Fasting period before study: no.
- Housing: During the study period, the rats were housed individually in Polycarbonate cages type III supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany, with the following exceptions: During overnight matings, male and female mating partners were housed together in Polycarbonate cages type III and pregnant animals and their litters were housed together until PND 13 in Polycarbonate
cages type III. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. For enrichment wooden gnawing blocks (Typ Lignocel® block large, J. Rettenmaier & Söhne
GmbH + Co KG, Rosenberg, Germany) were added. In addition in Polysulfonate cages large play tunnels (Art. 14153; supplied by PLEXX B.V., Elst, Netherlands) were added. The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured. The animals were housed in fully air-conditioned rooms in which central air conditioning guaranteed a range of temperature and a range of relative humidity.
- Diet: ad libitum.
- Water: ad libitum.
- Acclimation period:

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C.
- Humidity (%): 45-65%.
- Air changes (per hr): 15 times per hour.
- Photoperiod (hrs dark / hrs light): The day/night cycle was 12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h.

IN-LIFE DATES: From: 03 Jul 2018 To: 25 Sep 2018.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard.
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% CMC suspension in deionized water with 10 mg/100mL Cremophor
Details on oral exposure:
After the acclimatization period, the test substance was administered to the parental animals orally by gavage, once daily at approximately the same time in the morning. Females in labor were not treated. The treatment lasted up to one day prior to sacrifice. The animals of the control group were treated with the vehicle (0.5% CMC suspension in deionized water with 10 mg/100mL Cremophor), in the same way. The volume administered each day was 10 mL/kg body weight. The calculation of the administration volume was based on the most recent individual body weight.

PREPARATION OF DOSING SOLUTIONS: The test substance suspensions in 0.5% CMC suspension in deionized water with 10 mg/100 mL Cremophor were prepared in intervals, which took into account the analytical results of the stability verification. For the preparation of the administration suspensions the test substance was weighed in a calibrated beaker depending on the dose group, topped up with 0.5% CMC suspension in deionized water with 10 mg/100 mL Cremophor and intensely mixed with a homogenizer. During administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0, 0.15, 0.50 and 1.50 g/100 mL.
- Amount of vehicle (if gavage): 10 mL/kg bw/d.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the test substance in 0.5% CMC suspension in deionized water with 10 mg/100 mL Cremophor for a period of 7 days at room temperature had been verified prior to the start of the study.
At the beginning (during premating), twice during gestation and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. The 3 samples were withdrawn from the top, middle and bottom of the preparation vessel. 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.
Duration of treatment / exposure:
males: 30 days; females: 58/63 days
The duration of treatment covered a 2-weeks premating and mating period in both sexes (mating pairs were from the same test group), 3 days postmating in males, and the entire gestation and approximately 3 weeks of lactation period in females up to the day of scheduled sacrifice of the animals.
Frequency of treatment:
daily at the same time in the morning (exception: no administration to animals being in labor)
Doses / concentrationsopen allclose all
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Fasting period before blood sampling for clinical biochemistry: 16 hours fasting before blood sampling.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes (for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity; parturition and lactation behavior of the dams).
- Time schedule: at least once daily.

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: prior to the first administration and thereafter at weekly intervals
- Examined parameters: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size.

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period (to randomize the animals), study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning) with the following exceptions for the females:
• 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), PNDs 4, 7, 10 and 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 administration volume).

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
- Time schedule for examinations: once a week with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined for GD 0-7, 7-14 and 14-20.
• Food consumption of the F0 females which gave birth to a litter was determined on PND 1-4, 4-7, 7-10 and 10-13.
- Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period in females without litter during the lactation period.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Generally, water consumption was determined once a week as representative value over a period of 3 days for the male and female parental animals, with the following exceptions:
• Water consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals).
• Water consumption of the females with evidence of sperm was determined on gestation days (GD) 0-1, 6-7, 13-14 and 19-20.
• Water consumption of the F0 females, which gave birth to a litter was determined for PND 1-2, 3-4, 6-7 and 12-13.
- Water consumption was not determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.

HAEMATOLOGY: Yes.
- Time schedule for collection of blood: at termination (males); at PND 14 (females).
- Anaesthetic used for blood collection: Yes (isoflurane).
- Animals fasted: Yes (about 16 hours).
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes.
- Time schedule for collection of blood: at termination (males); at PND 14 (females).
- Anaesthetic used for blood collection: Yes (isoflurane).
- Animals fasted: Yes (about 16 hours).
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group.
- Parameters checked in table 2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes; functional observational battery and motor activity assessment (see "OTHER")

OTHER:

FUNCTIONAL OBSERVATIONAL BATTERY (FOB):
- A functional observational battery was performed in the first five parental male animals per test group and the first five surviving females with litter (in order of delivery) of all test groups at the end of the administration period starting at about 10.00 h on study day 27 (males) and 52 (females).
- Examined parameters:
• Home cage observations: posture, tremors, convulsions, abnormal movements, gait, other findings
• Open field observations (at least for 2 minutes): 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), rearing within 2 minutes, other findings
• Sensory motor tests/ reflexes: reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (auditory 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 ASSESSMENT:
- measured from 14:00 h onwards on the same day as the FOB was performed in the first five parental males and the first five surviving females with litter (in order of delivery) per group

ESTROUS CYCLE:
- In all parental females in the premating phase, estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating, mating and throughout cohabitation until there is evidence of sperm in the vaginal smear. Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all female F0 rats.

MALE REPRODUCTION DATA:
- The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.

FEMALE REPRODUCTION AND DELIVERY DATA:
- The pairing partners, the number of mating days until vaginal sperm were detected and gestational status were recorded for F0 females.

THYROID HORMONES: (males only)
- Time schedule for collection of blood: at termination.
- Anaesthetic used for blood collection: Yes (isoflurane).
- Animals fasted: Yes (about 16 hours).
- How many animals: all surviving males at termination.
- Parameters checked in table 3 were examined.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 4, Organ weights)

HISTOPATHOLOGY: Yes (see table 4)
Statistics:
see table 5

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Several male and female animals of the mid-dose group and almost all male and female animals of the high-dose group (50 and 150 mg/kg bw/d) showed salivation immediately after dosing (up to 2 hours post dosing) during the treatment period. It is likely, that this temporary finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse toxicologically relevant finding.
No other clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F0 parental animals in any of the test groups (1 – 3; 15, 50 and 150 mg/kg bw/d) during the study.
One high-dose male (No. 39) showed a distended, hard abdomen and piloerection during postmating days 1 – 2 which was assessed to be incidental.
Two sperm positive control females (Nos. 102 and 107), one sperm positive low-dose female (No. 118) and one sperm positive high-dose female (No. 133) did not deliver F1 pups.

Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In high-dose males, mean body weights were statistically significantly below the concurrent control values on mating day 14 (about 5%). Furthermore, mean body weight change was statistically significantly below the concurrent control during premating days 0 - 7 (about 80%). In high-dose females, mean body weights were statistically significantly below the concurrent control values during lactation (PND 4, PND 10 – 13, up to 7% below control) showing a downward trend towards the end of the study (see graphs below). This finding in high-dose males and females was assessed as treatment-related and adverse.
Mean body weight (change) of the mid- and low-dose males and females during the entire study period were comparable to the concurrent control values.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of the high-dose group was statistically significantly reduced compared to the concurrent control values in males during premating days 0 - 7 and in females during premating days 0 - 13 (both up to 9% below control). Food consumption in females showed a downward trend towards the end of the study since food consumption of high-dose females was further reduced (up to 17 % below control, without statistical significance) during lactation. The reduction in food consumption in male and females of the high-dose was assessed as treatment-related and adverse.
Food consumption of the high-dose females during gestation and of the low- and mid-dose males and females during the entire study was comparable to the concurrent control values.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
In males of the high-dose (150 mg/kg bw/d), water consumption was statistically significantly increased compared to the concurrent control values during premating days 7 - 10 (about 36%). In the high-dose F0 females, water consumption was statistically significantly increased compared to the concurrent control values during the entire gestation period (up to 43%). Together with the finding salivation, it is most likely that the increase in water consumption occurred due to the bad taste of the test substance or local affection of the upper digestive tract. It was assessed as treatment related but not adverse.
Water consumption of the high-dose females during premating and lactation and of the lowand mid-dose males and females during the entire study was comparable to the concurrent control values. The statistically significantly higher water consumption in the low-dose females during premating days 0 - 3 was considered to be spontaneous in nature and not treatment related since it was not related to dose.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period in male and female rats of test group 3 (150 mg/kg bw/d), absolute eosinophil cell counts were decreased (in males not statistically significantly). Additionally, in females of this test group absolute (not statistically significant) and relative monocyte counts were increased, and relative eosinophil cell counts were decreased. These changes were regarded as treatment-related and adverse.

In males of test group 3 (150 mg/kg bw/d) relative basophil cell counts were significantly decreased. However, no pathophysiological correlate exists regarding low basophil counts and, therefore, this change was regarded as incidental and not treatment-related. In addition, in males of this test group mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) were significantly decreased. MCV was within, MCH marginally below the historical control range (males, MCV 48.1-51.7 fL, MCH 1.03-1.11 fmol). However, these were calculated red blood cell indices and the measured red blood cell parameters (i.e. hemoglobin, hematocrit and red blood cell (RBC) counts) were not changed. Therefore, the MCV and MCH alterations in males of test group 3 were regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, in females of test group 3 (150 mg/kg bw/d) glucose levels were significantly decreased. This change combined with the hematological alterations is regarded as treatment-related and adverse.

In males of test group 3 (150 mg/kg bw/d) total protein and globulin values were significantly decreased and inorganic phosphate levels were significantly increased. However, all values were within historical control ranges (males, total protein 59.06-65.10 g/L, globulins 21.95- 28.93 g/L, inorganic phosphate 1.45-2.06 mmol/L). Therefore, these alterations were regarded as incidental and not treatment-related.
Urinalysis findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See table 6 and 7 for details.
Absolute organ weights: When compared to control group 0 (=100%), the mean absolute weights of following organs were significantly increased: Adrenal glands, liver, kidneys, ovaries.
Relative organ weights: When compared to control group 0 (=100%), the mean relative weights of following organs were significantly increased: Adrenal glands, liver, kidneys, spleen, thyroid glands.

The significant decrease of the final body weight in males and females, as well as the significant absolute and relative weight increase of the liver in both sexes, and of the adrenal glands and kidneys in females of test group 3 were considered treatment-related and had a histopathological correlate. The significant relative weight increase of the kidneys in males was considered secondary to the final body weight decrease, since it was marginally above themaximal historical control value, had an absolute weight within the historical control range and showed no histopathological correlate. The significant relative weight increase of the thyroid glands in females of the high dose group was within the historical control range and had no histopathological correlate. Therefore, it was regarded as incidental and not treatment-related.
The ignificant absolute weight increase of the ovaries and the significant relative weight increase of the spleen in females of test group 2 were regarded as incidental and not related to treatment.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered incidental or spontaneous in origin and without any relation to treatment.

Fertility: The female animals (Nos. 102, 107, 118 and 133), which were not pregnant as well as the male mating partners (Nos. 2, 7, 18 and 33) did not show relevant gross lesions.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were observed in following organs: brain, eyes with optic nerves, liver, lungs, skeletal muscle, seminal vesicles, epididymides, adrenal cortex, axillary and mesenteric lymph nodes, glandular stomach, kidneys, spleen and uterus. The main finding in each org n was a cytoplasmic vacuolation, characterized by the presence of multiple fine to small vacuoles, that were no larger than the nuclei of the cell. This type of vacuolation is referred here as “microvesicular” and most frequently increased the size of the affected cells. On the other hand, large cytoplasmic vacuoles, that displaced the nuclei or deformed the cells are mentioned here as “macrovesicular type” and were seen only in the kidneys and glands of the uteri. Incidences and grading of findings in the different target organs are given in the tables 8-21.

Fertility: The female animals (Nos. 102, 107, 118 and 133), which were not pregnant as well as thmale mating partners (Nos. 2, 7, 18 and 33) did not show relevant histopathological findings that could have impaired fertility.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
THYROID HORMONES
- In parental males (test groups 1, 2 and 3; 15, 50 and 150 mg/kg bw/d) and in male and female pups at PND13 (test groups 11, 12 and 13; 15, 0 and 150 mg/kg bw/d), no treatment-related alterations of T4 and TSH levels were observed.

DETAILED CLINICAL OBSERVATIONS (DCO):
- All male and all female animals of all dose groups (15, 50 and 150 mg/kg bw/d) did not show any abnormalities.

FUNCTIONAL OBSERVATIONAL BATTERY (FOB)
- Home cage observations: No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.
- Open field observations: The open field observations did not reveal any test substance-related findings in male and female animals of all test groups.
- Sensorimotor tests/reflexes: There were no test substance-related findings in male and female animals of all test groups.
- Quantitative Parameters: The landing foot-splay test (FST) in the mid- and high-dose male animals was statistically significantly below the concurrent control values (9.5 and 9.7 cm, respectively, vs. 11.8 cm in the control). However, there was no relation to dose and both values were within the range of the historical control data (Supplement, HCD, FST males: range of 9.3 – 13.8 cm). Therefore, it was not assessed as treatment-related and adverse. No test substance-related impaired parameters were observed in the low-dose males and all female animals of all test groups.

MOTOR ACTIVITY MEASUREMENT (MA)
- No statistically significant changes on motor activity data (summation of all intervals) was observed in the male and female animals of all test substance-treated groups in comparison to the concurrent control values.

ESTROUS CYCLE DATA
- Estrous cycle data, generated during the last 2 weeks prior to mating for the F1 litter, revealed regular cycles in the females of all test groups 0 - 3. The mean estrous cycle duration ranged between 4.0 and 4.1 days.

MALE REPRODUCTION DATA
- For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all test groups (0 - 3).
- Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter.
- Two control males (No. 2 and 7), one low-dose male (No. 18) and one high-dose male (No. 33) did not generate pregnancy.
- Thus, the male fertility index ranged between 80% and 100% without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study.
- The apparently infertile male rat did not show relevant gross lesions.

FEMALE REPRODUCTION AND DELIVERY DATA
- The female mating index calculated after the mating period for F1 litter was 100% in all test
groups.
- The mean duration until sperm was detected (GD 0) varied between 1.9 and 3.9 days. The mean value of 3.9 days in the high-dose group was well within the range of the historical control data (Supplement, HCD, mating days: 1.6 – 5.8) and can be explained by female No. 135
which had 14 mating days. The female was possibly pseudo-pregnant indicated by 12 days of diestrus. Afterwards, the female mated and was pregnant.
- All female rats delivered pups with the following exception: Control female No. 102 (mated with male No. 2) did not become pregnant. Control female No. 107 (mated with male No. 7) did not become pregnant. Low-dose female No. 118 (mated with male No. 18) did not become pregnant. High-dose female No. 133 (mated with male No. 33) did not become pregnant.
- The fertility index ranged between 80% and 100% without showing any relation to dosing.
- The non-pregnant females had no relevant gross lesions or microscopic findings.
- The mean duration of gestation values varied between 22.1 (test group 1), 22.2 (control and test group 2) and 22.5 (test group 3)
- The gestation index was 100% in all test groups 0 - 3.
- Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (12.0 / 13.7 / 12.1 and 10.3 implants/dam in test groups 0 - 3,
respectively). The value of test group 3 was marginally outside the range of the historical control data (Supplement, HCD, implantation sites: 10.4 - 14.9), without statistical significance. It was assessed as spontaneous and not treatment-related since the mean value of the related parameter pups delivered of test group 3 (9.6) was within the historical control range (HCD, pups delivered: 9.3 -13.9). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any significant differences between the groups, and the mean number of F1 pups delivered per dam remained unaffected (11.1 / 13.2 / 10.6 and 9.6 pups/dam in test groups 0 - 3, respectively)
- The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 100% / 98.3% / 100% and 100% in test groups 0 - 3, respectively. Moreover, the number of stillborn pups was not significantly different between the test groups.
- Thus, the test substance did not adversely affect reproduction and delivery of the F0 generation parental females.

F1 GENERATION PUBS/LITTERS
- Pup number and status at delivery: The mean number of delivered F1 pups per dam and the rates of liveborn, stillborn, found dead and cannibalized F1 pups were evenly distributed among the test groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.
- Pup viability: The viability index indicating pup survival during lactation (PND 0 - 4) varied between 98.1%/100% / 99.2% and 100% in test groups 0 - 3, respectively. The pups surviving index indicating pup survival during lactation (PND 4 - 13) varied between 100%/ 98.6% / 100% and 100% in test groups 0 - 3, respectively.
- Pup mortality: No adverse effect observed.
- Sex ratio: In general, the sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature. The statistically significant change in sex ratio of the high-dose group on PND 0 (males 40.9* versus [vs.] 56.0 in control and females 59.1* vs. 44.0 in control) was within the historical control range (Supplement, HCD: 36.6% - 60.5% (males) 39.5% - 63.4% (females)). It was assessed as not treatment-related. Further endocrine relevant parameters (AGD, nipple development) were not influenced by the test substance in any of the dose levels (see below).
- Pup clinical observations: There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups. One low-dose female pup (No. 11 of dam No. 113) showed and injury of the left hindlimb on PND 11 without relation to treatment and was therefore sacrificed moribund on PND 11.
- Pup body weight data: Two male and three female runts were seen in test group 1, one male and one female runt was seen in test group 2 and one female runt was seen in test group 3. In test group 3, mean body weight development was comparable to control on PND 1 and 4. On PND 13, mean body weights of the high-dose F1 male pups and both sexes combined were statistically significantly below the concurrent control (about 11% and 10%, respectively). The pup body weight change in the high-dose male pups was statistically significantly below the concurrent control values during the intervals PND 4 - 7, 7 - 13 and 1 - 13 (up to 15%) and in the high-dose female pups and both sexes combined during PND 7 - 13 and 1 - 13 (up to 13%). The decrease in body weights of males and both sexes combined was at the very lower end of the range of the historical control data (Supplement, HCD, pup weights, males: 28.5-33.4 g, both: 28.1-33.0 g). A relation to treatment cannot be excluded. In the highest test group, maternal / parental toxicity was observed. The decrease in body weight of the high dose pups was considered to be a secondary, minor effect in presence of maternal toxicity. No test substance-related influence on body weight (change) of the low- and mid-dose male and female F1 pups were noted.
- Anogenital distance/anogenital index: Neither on anogenital distance nor anogenital index test substance-related effects were noted in all treated F1 offspring (test groups 1 - 3 [15, 50 and 150 mg/kg bw/d]).
- Nipple/ areola anlagen: The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.
- Pup necropsy observations: A few pups showed spontaneous findings at gross necropsy, such as discolored thymus (red), discolored testis (red), small testis, supernumerary liver lobe (cystic), post mortem autolysis, partly cannibalized, limb lesion, diaphragmatic hernia and dilated renal pelvis. These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, all these findings were not considered to be associated to the test substance.
Details on results:
Regarding clinical examination, several male and female animals of the mid-dose group and almost all male and female animals of the high-dose group (50 and 150 mg/kg bw/d) showed salivation immediately after dosing (up to 2 hours post dosing) during the treatment period. It is likely, that this temporary finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse
toxicologically relevant finding.
In F0 males and females of the high-dose, water consumption was statistically significantly increased compared to control during premating (males: about 36% above control) or gestation (up to 43% above control). Together with the clinical finding salivation, it is most likely that the increase in water consumption occurred due to the bad taste of the test substance or local affection of the upper digestive tract. It was assessed as treatment related but not adverse.

Adverse signs of toxicity were observed at the highest dose (150 mg/kg bw/d).
Food consumption of the high-dose was statistically significantly reduced compared to control in males and females during premating (both up to 9% below control) and in females during lactation (without statistical significance, up to 17 % below control).
Mean body weight (change) was statistically significantly below the control values in males during premating (BWC) and during mating (BW: about 5% below control) and in females during lactation (up to 7% below control). Food consumption and body weight in high-dose animals showed a downward trend towards the end of the study and were assessed as treatment-related and adverse.
No test substance related, adverse findings were observable in the mid- and low-dose (50 and 15 mg/kg bw/d).

Regarding clinical pathology, in females of test group 3 (150 mg/kg bw/d) decreased absolute and relative eosinophils and increased absolute and relative monocyte counts as well as decreased glucose levels indicated some subacute stress event which is regarded as treatment-related. Lower absolute eosinophil counts were also observed in males of the same test group.

Regarding pathology, a significant decrease of the final body weight in males and females of test group 3 (150 mg/mg bw/d) was accompanied by a significant absolute and relative weight increase of the liver in both sexes, and of the adrenal glands and kidneys in females. These organ weight changes were considered treatment-related and were consistent with histopathological findings, characterized mainly by cytoplasmic vacuolation. Vacuolation was also observed in several other organs of males and females in test group 3 (brain, eyes, lungs, skeletal muscle, seminal vesicles, epididymides, axillary and mesenteric lymph nodes, glandular stomach, spleen and uterus) and in test group 2 (epididymides). The cytoplasmic vacuolation was of microvesicular type (multiple fine to small vacuoles, that were no larger than the nuclei of the respective cell types and did not displace it). A macrovesicular type (large and single cytoplasmic vacuoles, that displaced the nuclei or altered the cellular form) was distinctively and additionally seen in the kidneys (all females) and uteri (two females), and only as background in the remaining organs.
The vacuolation was regarded as treatment-related and assessed as adverse, if it was accompanied by cytotoxicity (cell death, degeneration and/or inflammation). In test group 3 (150 mg/kg bw/d) animals, these criteria were accomplished for the liver and skeletal muscle of males and females; axillary and mesenteric lymph nodes and kidneys of females. Finally, vacuolation alone without sings of cytotoxicity, occurring in special organs with central vital functions or special senses, was also assessed as adverse, assuming a potential impairment of vital functions. This was true for the spleen in females and brain and eyes in males and females of test group 3 (150 mg/kg bw/d). A summary of histopathological findings assessed as treatment-related and adverse is given as follows:
In test group 3 (150 mg/kg bw/d), histopathology revealed in the liver a centrilobular microvesicular vacuolation in all males and females accompanied by single cell necrosis. It cannot be excluded if other hepatic findings like multifocal necrosis, multinuclear hepatocytes and karyomegaly, affecting very few animals were treatment-related and were, therefore, assumed to be possibly treatment-related and adverse. In the skeletal muscle of males and females, the fiber vacuolation was accompanied by necrosis and regeneration of fibers. In the axillary lymph node of females, the vacuolation was present in the high endothelial venules (HEV) and was accompanied by apoptosis. The mesenteric lymph nodes of females showed vacuolation in the HEV, lymphocytes of medullary cords and macrophages of medullary sinuses, apoptosis and macrophage aggregates in the periphery of paracortical areas, and decreased cellularity in paracortical regions (T-lymphocytes). In the kidneys of females, a tubular cytoplasmic macro- and microvesicular vacuolation affecting all animals was accompanied by tubular degeneration/regeneration and granulomatous inflammation. Both types of vacuolation affected predominantly the cortical tubular epithelium but the microvesicular vacuolation extended also into the outer stripe of the outer medulla. The spleen of females displayed a vacuolation of the B-lymphocytes (follicular and marginal zone) and a decreased cellularity of T-lymphocytes in the periarteriolar lymphocyte sheath (PALS). Finally, a microvesicular vacuolation without signs of cytotoxicity was detected in the plexus choroideus of the brain, and in the stromal iris and choroid of the eyes of males and females.

Treatment-related vacuolation not accompanied by signs of cytotoxicity and assessed as not adverse, was seen in following organs in test group 3:
Adrenal cortex of females (zona fasciculata and reticularis) correlating with significantly increased weights; lungs of all male and females (bronchial/bronchiolar epithelium) and glandular stomach of females (base of the glands in the pyloric region). In the epithelium of epididymides (corpus), seminal vesicles and uterus (surface and/or glandular epithelium, and smooth muscle of the wall). Fertility in these animals was not relevantly altered when compared with the control animals.

In test group 2 (50 mg/kg bw/d), histopathology revealed vacuolation in the epididymal epithelium. This change was assessed as treatment-related but not adverse, since no alteration was seen in the sperm density and morphology and no relevant alteration of the fertility was noted in these male animals.

Special stains performed exemplarily in selected organs (liver, kidneys, lungs and skeletal muscle) of animals in test group 3 revealed that the vacuolation was negative for the presence of neutral fat (Oil-red-O stain negative) and was positive for the presence of phosphorylated lipids (Sudan black positive). An increased accumulation of glycogen in the skeletal muscle was discarded (PAS with diastase negative).

Transmission electron microscopy examination performed exemplarily in the liver and kidneys of selected male and female animals of test group 3 revealed the presence of intracytoplasmic myelin figures, characterized by concentric, multilaminated membrane whorls in phagolysosomes (Ghadially, 1988,). This morphological picture is indicative of phospholipidosis and represents lysosomal accumulation of phospholipids. It is inferred that the vacuolation observed in all other organs was most likely of the same nature (Nonoyama and Fukada, 2008; Rudmann et al, 2004).

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. Furthermore, neither the synchrony of the morphology of the estrous cycle in ovaries, uterus, cervix, and vagina nor the stages of seminiferous tubules were altered.

Regarding fertility and reproductive performance, no signs of toxicity were observed in male or female parental animals of all test groups during the entire study. F0 parental animals proved to be fertile. Mating behavior, conception, implantation and parturition were not affected.

Regarding developmental toxicity, a statistically significant change in sex ratio of the high-dose group on PND 0 (males 40.9* versus [vs.] 56.0 in control and females 59.1* vs. 44.0 in control) was within the historical control range (Supplement, HCD: 36.6% - 60.5% (males) 39.5% - 63.4% (females)). Therefore, it was assessed as not treatment-related. Furthermore, neither determination of anogenital distance/index nor the count of nipple/areola anlagen revealed any treatment-related, adverse changes up to and including a dose of 150 mg/kg bw/d. All parameters concerning mating and delivery were not adversely affected by the test substance. Regarding F1 pups and litter data, pup status and viability showed no treatment related, adverse findings.
Mean body weights of the high-dose F1 male pups and both sexes combined were statistically significantly below the concurrent control (about 11% and 10%, respectively) on PND 13. The body weight change in high-dose male and female pups was statistically significantly below the control during PND 1-13. The decrease in body weights of males and both sexes combined was at the very lower end of the range of the historical control data (Supplement, HCD, pup weights, males: 28.5 - 33.4 g, both: 28.1-33.0 g). A relation to treatment cannot be excluded. In the highest test group, maternal / parental toxicity was observed (see above). The decrease in body weight of the high dose pups was considered to be a secondary, minor effect in presence of maternal toxicity. No test substance-related influence on body weight (change) of the low- and mid-dose male and female F1 pups were noted.

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.

Effect levels

open allclose all
Dose descriptor:
LOAEL
Remarks:
systemic toxicity
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
haematology
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no adverse effect observed
Dose descriptor:
NOAEL
Remarks:
reproductive performance & fertility
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no adverse effect observed
Dose descriptor:
LOAEL
Remarks:
developmental toxicity
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Remarks on result:
other:
Remarks:
maternal toxic dose
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no adverse effects observed

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (actual dose received)
System:
other: divers
Organ:
other: brain, eyes with optic nerve, liver and skeletal muscle (male/female); lymph nodes, kidneys, spleen (females)
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
yes

Any other information on results incl. tables

The various analyses:

  • Demonstrated the stability of the test substance preparations over a period of 7 days at room temperature
  • Confirmed the homogeneous distribution of the test substance in 0.5% CMC suspension in deionized water (with 10 mg/100mL Cremophor)
  • Confirmed the overall accuracy of the prepared test substance concentrations

Concentration control analysis:

Measured values for the test substance were in the expected range of the target concentrations (90 - 110%) demonstrating the correctness of the preparations. For the low dose (15 mg/kg bw/d), one single value (sample No. 3) was marginally above the tolerance range of the test facility (111%). However, the test substance preparation was homogeneously distributed for this dose level and all other values of the low dose were within the tolerance range. Therefore, the measured values of the low dose were assessed to be in an acceptable range of the nominal concentrations in this study.

Table 6: Absolute organ weights

 

Male animals

Female animals

Test group (mg/kg bw/d)

1

(15)

2

(50)

3

(150)

1

(15)

2

(50)

3

(150)

Final body

weight

-1.7%

-0.5%

-6.1%**

+0.2%

+2.5%

-6.2%**

Adrenal glands

 

 

 

+10.649%

+7.273%

+29.610%**

Liver

-0.55%

+2.45%

+8.90%*

+0.31%

+8.12%

+26.47%**

Kidneys

%

%

%

-3.50%

+6.99%

+49.06%**

Ovaries

%

%

%

+11.907%

+27.043%**

+9.183%

*p <= 0.05; **p <= 0.01

Table 7: Relative organ weights

 

Male animals

Female animals

Test group (mg/kg bw/d)

1

(15)

2

(50)

3

(150)

1

(15)

2

(50)

3

(150)

Adrenal glands

 

 

 

+10.246%

+3.863%

+39.463%**

Liver

+1.37%

+2.27%

+15.01%**

+0.25%

+4.48%

+36.04%**

Kidneys

-3.94%

-5.39%

+9.79%*

-3.65%

+3.52%

+60.60%**

Spleen

%

%

%

-6.65%

-13.51%*

+7.87%

Thyroid glands

 

 

 

+15.735%

+13.353%

+19.488%**

 *p <= 0.05; **p <= 0.01

Table 8: Histopathology/ Brain

Brain

Male animals

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

10

10

10

10

Vacuolation, choroid plexus, diffuse

0

0

0

10

0

0

0

10

·        Grade1

 

 

 

6

 

 

 

3

·        Grade2

 

 

 

4

 

 

 

2

·        Grade3

 

 

 

 

 

 

 

5

The cytoplasmic vacuolation was observed mainly in the epithelium of the choroid plexus of the lateral ventricle and was of microvesicular type.

Table 9: Histopathology/ Eyes with optic nerve

Eyes with optic nerve

Male animals

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

10

10

10

10

Vacuolation, diffuse

0

0

0

5

0

0

0

10

·        Grade1

 

 

 

4

 

 

 

 

·        Grade2

 

 

 

1

 

 

 

3

·        Grade3

 

 

 

 

 

 

 

7

The vacuolation was of microvesicular type, most likely in macrophages of the iris stroma. In several animals the vacuolation extended from here into the peripheral choroidal layer.

Table 10: Histopathology/ Liver

Liver

Male animals

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

10

10

10

10

Vacuolation, centrilobular

0

0

0

10

0

0

0

10

·        Grade1

 

 

 

 

 

 

 

1

·        Grade2

 

 

 

4

 

 

 

4

·        Grade3

 

 

 

6

 

 

 

3

·        Grade4

 

 

 

 

 

 

 

2

Necrosis, single cell

0

0

0

2

0

0

0

9

·        Grade1

 

 

 

1

 

 

 

4

·        Grade2

 

 

 

1

 

 

 

5

Necrosis, multifocal

0

0

0

2

0

0

0

1

·        Grade1

 

 

 

1

 

 

 

1

·        Grade2

 

 

 

1

 

 

 

 

Multinuclear hepatocytes

0

0

0

1

0

0

0

2

·        Grade1

 

 

 

 

 

 

 

1

·        Grade2

 

 

 

1

 

 

 

1

Karyomegaly

0

0

0

0

0

0

0

1

·        Grade1

 

 

 

 

 

 

 

1

The vacuolation was of microvesicular type and was accompanied by single cell necrosis of vacuolated cells. The centrilobular vacuoles were negative for Oil-Red-O and positive for Sudan black. Findings like multifocal necrosis, multinuclear hepatocytes and karyomegaly, did affect only very few animals and treatment-relationship remains unclear.

Table 11: Histopathology/Lungs

Lungs

Male animals

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

10

10

10

10

Vacuolation, epith., bronchial/bronchiolar

0

0

0

10

 

 

 

10

·        Grade1

 

 

 

10

 

 

 

3

·        Grade2

 

 

 

 

 

 

 

5

·        Grade3

 

 

 

 

 

 

 

2

Histiocytosis, alveolar, multifocal

2

2

2

3

3

1

2

9

·        Grade1

2

2

2

1

3

1

2

4

·        Grade2

 

 

 

1

 

 

 

4

·        Grade3

 

 

 

1

 

 

 

1

The vacuolation in the lungs was of microvesicular type and affected most frequently the bronchiolar epithelium including terminal bronchioles, and in extensive cases also the bronchial epithelium. It was accompanied by an increase in alveolar histiocytosis, which was stronger in females than in males. The vacuoles as well as the alveolar histiocytes were negative for Oil- Red-O and positive for Sudan black.

Table 12: Histopathology/Skeletal muscle

Skeletal muscle

Male animals

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

10

10

10

10

Vacuolation, fiber

0

0

0

7

0

0

0

9

·        Grade1

 

 

 

6

 

 

 

7

·        Grade2

 

 

 

 

 

 

 

1

·        Grade3

 

 

 

 

 

 

 

1

·        Grade4

 

 

 

1

 

 

 

 

Necrosis, multifocal

0

0

0

0

0

0

0

2

·        Grade1

 

 

 

2

 

 

 

2

Regeneration, multifocal

0

0

0

1

0

0

0

1

·        Grade1

 

 

 

 

 

 

 

1

·        Grade3

 

 

 

1

 

 

 

 

The vacuolation in the skeletal muscle was characterized by the presence of very tiny microvacuoles with a multifocal distribution pattern within the myofibers. In addition, some of the affected fibers were retracted and strongly eosinophilic, indicating a hyaline necrosis in 2 males and 2 females. One male animal and one female with severe and moderate vacuolation, respectively, showed also an regeneration of fibers. The tiny vacuoles in the affected fibers were negative for Oil-Red-O, positive for Sudan-black and were negative in the PAS-with diastase stain.

Table 13: Histopathology/ Epididymides

Epididymides

Male animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

Vacuolation, epithelial

0

0

10

10

·        Grade1

 

 

2

 

·        Grade2

 

 

8

 

·        Grade3

 

 

 

10

The vacuolation was of microvesicular type. In test group 3, it was diffusely distributed along the tubules of the corpus, starting slightly distally of the head and extended to the junction with the cauda. In test group 2, the vacuolation was mostly present in the caudal part of the corpus, reaching the junction with the cauda.

Table 14: Histopathology/ Seminal vesicles

Seminal vesicles

Male animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

Vacuolation, epithelial

0

0

0

2

·        Grade1

 

 

 

1

·        Grade2

 

 

 

1

The diffuse epithelial vacuolation was of microvesicular type, with a distinctive basal localization.

Table 15: Histopathology/ Adrenal cortex

Adrenal cortex

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

Vacuolation, increased

0

0

0

10

·        Grade1

 

 

 

4

·        Grade2

 

 

 

4

·        Grade3

 

 

 

2

The cytoplasmic vacuolation was of microvesicular type and included the zona fasciculata and reticularis.

Table 16: Histopathology/ Axillary lymph node

Axillary lymph node

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

Vacuolation, multifocal

0

0

0

4

·        Grade1

 

 

 

3

·        Grade2

 

 

 

1

Apoptosis

0

0

0

1

·        Grade1

 

 

 

1

The vacuolation was of microvesicular type and was noted mainly in the high endothelial venules (HEV). The apoptosis was seen in the periphery of the paracortex.

Table 17: Histopathology/ Mesenteric lymph node

Mesenteric lymph node

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

Vacuolation, multifocal

0

0

0

8

·        Grade1

 

 

 

6

·        Grade2

 

 

 

2

Apoptosis

0

0

0

7

·        Grade1

 

 

 

6

·        Grade2

 

 

 

1

Cellularity, decreased

0

0

0

6

·        Grade1

 

 

 

4

·        Grade2

 

 

 

2

Aggregates, macrophage, increased

0

1

3

9

·        Grade1

 

1

2

7

·        Grade2

 

 

1

2

The microvesicular vacuolation was seen in the HEV (high endothelial venules) of all animals, and lymphocytes of medullary cords and macrophages of medullary sinuses. The apoptosis and macrophage aggregates were noted in the periphery of paracortical areas. The decreased cellularity occurred in paracortical regions (T-lymphocytes).

Table 18: Histopathology/ Glandular stomach

Glandular stomach

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

Vacuolation, epithelial

0

0

0

7

·        Grade1

 

 

 

5

·        Grade2

 

 

 

2

The vacuolation affected the cytoplasm of the cells at the base of the glands in the pyloric region of the stomach.

Table 19: Histopathology/ Kidneys

Kidneys

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

Vacuolation, macrovesicular

0

0

0

10

·        Grade1

 

 

 

2

·        Grade2

 

 

 

3

·        Grade3

 

 

 

5

Vacuolation, microvesicular

0

0

0

10

·        Grade1

 

 

 

5

·        Grade2

 

 

 

3

·        Grade3

 

 

 

2

Degeneration/ regeneration, tubular

0

0

0

8

·        Grade1

 

 

 

1

·        Grade2

 

 

 

2

·        Grade3

 

 

 

5

Inflammation, granulomatous

0

0

0

2

·        Grade2

 

 

 

2

A tubular cytoplasmic macro- and microvesicular vacuolation accompanied by tubular degeneration/regeneration was distinguished in the kidneys of females. Both types of vacuolation affected predominantly the cortical tubular epithelium, but the microvesicular vacuolation extended also into the outer stripe of the outer medulla. Macrovesicular vacuoles seemed to be empty (by light microscopy) and were most likely noted in collecting ducts, whereas the microvesicular vacuoles seemed to affect more the convoluted proximal and distal tubules and the pars recta. However, a clear distinction was not always possible. Figures of tubular vacuoles might indicate that the macrovesicular vacuolation could originate by exaggerated distention and coalescence of less sized vacuoles. The microvesicular vacuoles were negative for Oil-Red-O and positive for Sudan black stain. The macrovesicular vacuoles did not stain.

Table 20: Histopathology/ Spleen

Spleen

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

Vacuolation, lymphoid cell

0

0

0

10

·        Grade1

 

 

 

4

·        Grade2

 

 

 

4

·        Grade3

 

 

 

2

Cellularity decreased

0

0

0

6

·        Grade1

 

 

 

1

·        Grade2

 

 

 

3

·        Grade3

 

 

 

2

The vacuolation in the spleen of females was of microvesicular type and affected the cytoplasm of lymphoid cell of the follicles and marginal zone (B-lymphocytes). A decreased cellularity of T-lymphocytes was observed in the periarteriolar lymphocyte sheath (PALS).

Table 21: Histopathology/ Uterus

Uterus

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(15)

2

(50)

3

(150)

No. of animals

10

10

10

10

Vacuolation, epithelial

0

0

0

8

·        Grade1

 

 

 

4

·        Grade2

 

 

 

1

·        Grade3

 

 

 

3

Vacuolation, smooth muscle

0

0

0

5

·        Grade1

 

 

 

4

·        Grade2

 

 

 

1

A diffuse microvesicular vacuolation occurred in the cytoplasm of the surface epithelium. However, in two females, a macrovesicular vacuolation was visualized in the glandular epithelium. The smooth muscle of the uterus wall showed a microvesicular type of vacuolation.

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.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats the oral administration of the test substance by gavage to male and female Wistar rats resulted in signs of systemic toxicity at the highest dose of 150 mg/kg bw/d (LOAEL), such as a reduction in food consumption and a decrease in body weight (change), adverse changes in clinical pathology parameters and histopathology. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 50 mg/kg bw/d for male and female Wistar rats.
The NOAEL for reproductive performance and fertility was set to 150 mg/kg bw/d for male and female Wistar rats.
The NOAEL for developmental toxicity was 50 mg/kg bw/d. This was based on the decrease in pup body weight at 150 mg/kg bw/d. This decrease, however, was considered to be secondary to the significant maternal toxicity and was not considered to be a selective adverse effect.
Executive summary:

In an OECD 422 study, the test substance was administered daily to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 15, 50 and 150 mg/kg body weight/day (mg/kg bw/d, test groups 1-3, respectively) to screen for potential systemic, reproductive and developmental toxicity. Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only (0.5% Sodium carboxymethyl cellulose (CMC) suspension in deionized water (with 10 mg/100mL Cremophor). The duration of treatment covered a 2-weeks premating and mating period in both sexes (mating pairs were from the same test group), 3 days postmating in males, and the entire gestation and approximately 3 weeks of lactation period in females up to the day of scheduled sacrifice of the animals.

 

After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm were 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 animals before the start of the administration period and, as a rule, thereafter at weekly intervals.

Water consumption of the F0 parents was determined for premating days 0 - 3 and 7 - 10. In dams water consumption was determined for gestation days (GD) 0 - 1, 6 - 7, 13 - 14 and 19 - 20 and lactation days (PND) 1 - 2, 3 - 4, 6 - 7 and 12 - 13.

Food consumption of the F0 parents was determined once weekly during premating. In dams food consumption was determined for GD 0 - 7, 7 - 14, 14 - 20 and PND 1 - 4, 4 - 7, 7 - 10 and 10 - 13.

Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating. During gestation and lactation period, F0 females were weighed on GD 0, 7, 14 and 20, on the day of parturition (PND 0) and on PND 4, 7, 10 and 13.

Estrous cycle data were evaluated for F0 generation females over a two weeks period prior to mating until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice.

The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1, 4, 7 and 13. Their viability was recorded. At necropsy on PND 13, all pups were sacrificed with CO2, under isoflurane anesthesia, and examined macroscopically for external and visceral findings

Anogenital distance (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements were conducted in a blind randomized fashion, using a measuring ocular on all live male and female pups on PND 1. All surviving pups were examined for the presence or absence of nipple/areola anlagen on PND 13. The number of nipple/areola anlagen were counted.

Blood samples 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 for hormone measurement.

Clinico-chemical and hematological examinations were performed in 5 animals per sex and group towards the end of the administration period. Blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia for hormone measurement.

At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 parental males and females per group. 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:

·        Demonstrated the stability of the test substance preparations over a period of 7 days at room temperature

·        Confirmed the homogeneous distribution of the test substance in 0.5% CMC suspension in deionized water (with 10 mg/100mL Cremophor)

·        Confirmed the overall accuracy of the prepared test substance concentrations

 

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

150 mg/kg bw/d

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

·        Decreased food consumption in males and females during premating (up to 9 % below control) and lactation (females: up to 17% below control, without statistical significance)

·        Decreased body weights in males on mating day 14 (up to 5% below control) and in females during lactation (PND 4, 10 – 13; up to 7% below control)

·        Decreased body weight change in males during premating days 0 - 7 (about 80% below control)

·        Decreased absolute eosinophil cell counts in both sexes and decreased relative eosinophil counts in females

·        Increased absolute and relative monocyte counts in females

·        Decreased glucose levels in females

·        Brain: vacuolation, plexus choroideus: all males (minimal to slight) and females (minimal to moderate)

·        Eyes with optic nerve: vacuolation, diffuse, iris and choroidal layer: 5 out of 10 males (minimal to slight) and all females (slight to moderate)

·        Liver:

o  Significant absolute and relative weight increase in males (+8.90% and +15.01%) and females (+29.61% and 36.04%)

o  Vacuolation: all males (slight to moderate) and females (minimal to severe)

o  Necrosis, single cell: 8 out of 10 males (minimal), 9 out of 10 females (minimal to slight)

o  Necrosis, multifocal: 2 out of 10 males (minimal to slight), 1 out of 10 females (minimal)

o  Multinuclear hepatocytes: 1 out of 10 males (slight) and 2 out of 10 females (minimal to slight)

o  Karyomegaly: 1 out of 10 females (minimal)

·        Skeletal muscle:

o  Vacuolation, fiber: 6 out of 10 males (minimal and severe), 9 out of 10 females (minimal to moderate)

o  Necrosis: 2 out of 10 males and 2 out of 10 females (minimal)

o  Regeneration: 1 out of 10 males (moderate) and 1 out of 10 females (minimal)

·        Axillary lymph node:

o  Vacuolation, multifocal: 4 out of 10 females (minimal to slight)

o  Apoptosis: 1 out of 10 females (minimal)

·        Mesenteric lymph node:

o  Vacuolation, multifocal: 8 out of 10 females (minimal to slight)

o  Apoptosis: 7 out of 10 females (minimal to slight)

o  Cellularity decreased (paracortex): 6 out of 10 females (minimal to slight)

o  Macrophage aggregates, increased: 9 out of 10 females (minimal to slight)

·        Kidneys:

o  Significant increase of the absolute (+49.06%) and relative (+60.60%) weight in females

o  Vacuolation, macrovesicular: all females (minimal to moderate)

o  Vacuolation, microvesicular: all females (minimal to moderate)

o  Degeneration/regeneration, tubular: 8 out of 10 females (minimal to moderate)

o  Inflammation, granulomatous: 2 out of 10 females (slight)

·        Spleen:

o  Vacuolation, lymphoid cell (B-lymphocytes): all females (minimal to moderate)

o  Cellularity decreased (T-lymphocytes): 6 out of 10 females (minimal to moderate)

 

F1 PUPS

CLINICAL EXAMINATIONS/ GROSS FINDINGS

·        Decreased mean body weights of male pups and both sexes combined (PND 13: about 11% and 10%, respectively, below control) and decreased body weight change in male and female pups during PND 1-13.

 

 

50 mg/kg bw/d

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

·        No test substance-related, adverse findings were noted

F1 PUPS

CLINICAL EXAMINATIONS/ GROSS FINDINGS

·        No test substance-related, adverse findings were noted

 

 

15 mg/kg bw/d

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

·        No test substance-related, adverse findings were noted

F1 PUPS

CLINICAL EXAMINATIONS/ GROSS FINDINGS

·        No test substance-related, adverse findings were noted

 

Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats the oral administration of the test substance by gavage to male and female Wistar rats resulted in signs of systemic toxicity at the highest dose of 150 mg/kg bw/d (LOAEL), such as a reduction in food consumption and a decrease in body weight (change), adverse changes in clinical pathology parameters and histopathology. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 50 mg/kg bw/d for male and female Wistar rats.

The NOAEL for reproductive performance and fertility was set to 150 mg/kg bw/d for male and female Wistar rats.

The NOAEL for developmental toxicity was 50 mg/kg bw/d. This was based on the decrease in pup body weight at 150 mg/kg bw/d. This decrease, however, was considered to be secondary to the significant maternal toxicity and was not considered to be a selective adverse effect.