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Toxicological information

Developmental toxicity / teratogenicity

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Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
weight of evidence
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)
Qualifier:
according to
Guideline:
other: EPA, Health Effects Test Guidelines; OPPTS 870.3650: Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test (Jul 2000)
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 11-12 weeks
- Weight at study initiation: male animals 293.1 g - 328.9 g and female animals 196.1 g - 236.7 g
- Housing: individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²), with the following exception: during overnight matings, male and female mating partners were housed together in Makrolon type M III cages. Pregnant animals and their litters were housed together until PND 4. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. For enrichment wooden gnawing blocks (Typ NGM E-022; supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria) were added. Dust-free wooden bedding was used in this study.
- Diet (e.g. ad libitum): ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, which was available to the animals ad libitum.
- Water (e.g. ad libitum): from water bottles (ad libitum)
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h.
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
TEST ITEM PREPARATIONS
The test substance solutions in drinking water were prepared at the beginning of the administration period and thereafter in intervals, which took into account the analytical results of the stability verification.
For the preparation of the administration solutions the test substance was weighed in a graduated flask depending on the dose group, topped up with drinking water and subsequently thoroughly mixed by shaking until it was completely dissolved.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.
Analytical verifications of the stability of the test substance in drinking water for a period of 7 days at room temperature were carried out in a comparable batch prior to the start of the study.
Samples of the test substance solutions were sent to the analytical laboratory once at the beginning of the study for verification of the concentrations.Of each sample, one additional reserve sample was retained.
Due to the fact that the test substance preparations were true solutions, it was not considered necessary to prove homogeneity through analytical procedures. All measured values for 1-Vinylimidazol were in the expected range of the target concentrations (90 - 110%), demonstrating the correctness of the diet preparations.
Duration of treatment / exposure:
The duration of treatment covered a 2-week pre-mating and a mating period in both sexes. In males treatment lasted 30 days after beginning of administration. Females were treated the entire gestation period as well as approximately 2 weeks of the lactation period. Females were sacrificed 50 days after beginning of administration.
Frequency of treatment:
Once daily at approximately the same time in the morning.
Remarks:
Doses / Concentrations:
5; 15; 35 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The oral route was selected since administration by gavage has been proven to be appropriate for the detection of a toxicological hazard.
Prior to the OECD422 study, a 14-d dose range finding stuy in Wistar rats was performed, in order to find the apropriate dose regimen for the main study. With 3 animals/sex and group, 0, 35, 75, 125 and 250 mg/kg bw/d 1-vinylimidazole were administered to male and female rats per gavage (01R0335/00R18; BASF, 2012). Besides the clinical observations, food consumption and body weights were examined at day 0, 3, 7, 10 and 14 and the animals were assessed post-mortem by gross pathology. In the highest dose group 3/3 males and 2/3 females were sacrificed moribund, the third female was found dead. In the 125 mg/kg bw/d dose group all males and females were sacrificed moribund. In the 75 mg/kg bw/d dose group one female was sacrificed moribund, all other animals were sacrificed scheduled. In the lowest dose group all animals were sacrificed scheduled. Statistically significant reduced food consumption compared to the control was observed in all males and female groups exept for the low dose females receiving 35 mg/kg bw/d. Statistically significant decreased body weights were observed in the low dose females.
Due to the deaths occuring in the first range finder at doses >= 75 mg/kg bd/d, another 14-d range finder with male and female rats (4/sex and group) was considered to be neccessary with doses of 35 and 70 mg/kg bw/d per gavage (01R0492/11R136; BASF, 2012). This study additionally examined water consumption, clinical pathology (hematology, chlinical chemistry) and some organ weights (adrenal glands, kidneys, liver, spleen). All males and females receiving 1-vinylimidazole showed semiclosed eyelids and piloerection of the fur during the study. Compared to the controls, reduced food consumption was observed in both groups in males and females mainly within the first week of administration. A statistically significant increase in water consumption was observed only once in females of the lower dose group from day 3 to 7. Body weights of the males receiving 70 mg/kg bw/d were significantly reduced at the end of the exposure period (d13) compared to controls: 326.1 +/- 20.1 g vs 371.7 +/- 18.8 g (-12%). These males had a body weight loss of 4.1 g at study day 13 compared to day 0, where as the control animals showed a body weight gain of 43.0 g from day 0 to 13. Bodyweights of the mid and high dose females also showed a statistically significant reduction after the exposure period compared to controls: 229.0 +/- 3.3 g; 211.4 +/- 5.6 g (-8%); 201.1 +/- 10.6 g (-12%).
In conclusion, a dose of 70 mg/kg bw /d 1-vinylimidazole was considered to exceed the maximal tolerated dose, based on the significant body weight loss of the high dose males and the significant reduced body weight of > 10 % in both sexes after 14 days of exposure. Thus, 35 mg/kg bw/d was selected as the high dose for the main study according to OECD test guideline 422.

- Method of formulation: For the preparation of the administration solutions the test substance was weighed in a graduated flask depending on the dose group, topped up with drinking water and subsequently thoroughly mixed by shaking until it was completely dissolved.

- Mating procedures: In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group. The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 07.00 - 09.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted "GD 0" and the following day "GD 1".

- Parturition: The females were allowed to deliver and rear their pups until day 4 after parturition. The female animals were sacrificed 50 days after the beginning of the administration, and examined.

Observations and examinations performed and frequency:
PARENTAL ANIMALS
- Mortality / Viability: At twice daily on working days and once daily on other days.
- Clinical signs: A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each animal. Individual data of daily observations can be found in the raw data. The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis. On weekdays (except Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.
- Food consumption: Generally, food consumption was determined once a week for male and female parental animals, 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 F0 animals). Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0, 7, 14 and 20. Food consumption of F0 females which gave birth to a litter was determined on PND 1 and 4. Food consumption was not determined in females without positive evidence of sperm during the gestation period and in females without litter during the lactation period.
- Body weights: In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice. The body weight change of the animals was calculated from these results. The following exceptions are notable for the female animals: During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. Females with litter were weighed on the day of parturition (PND 0) and on PND 4. Females without positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume.
- Detailed clinical observations (DCO): Detailed clinical observations were performed in all animals once prior to the first administration and at weekly intervals during the administration period. The examinations started in the morning. The findings were ranked according to the degree of severity, if applicable. For observation, the animals were removed from their cages by the investigator and placed in a standard arena (50 x 37.5 cm wide, with side borders which are 25 cm high). The following parameters listed were assessed: 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.
- Functional Observations: A functional observational battery was performed in five male and 5 female animals (with litter) per group at the end of the administration period starting at about 10.00 h. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
Home cage observations: The animals were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to: Posture, Tremors, Convulsions, Abnormal movements, Impairment of gait, Other findings.
Open field observations: The animals were transferred to a standard arena (50 x 50 cm wide, with side borders which are 25 cm high) and observed for at least 2 minutes. The following parameters were examined: Behavior when removed from cage, Fur, Skin, Salivation, Nose discharge, Lacrimation, Eyes/pupil size, Posture, Palpebral closure, Respiration, Tremors, Convulsions, Abnormal movements/stereotypy, Impairment of gait, Activity/arousal level, Feces excreted within 2 minutes (number of scybala discharged/appearance/consistency), Urine excreted within 2 minutes (amount/color), Number of rearings within 2 minutes
Sensory motor tests/Reflexes: The animals were removed from the open field and subjected to following sensory motor or reflex tests: Approach response, Touch response, Vision (“visual placing response”), Pupillary reflex, Pinna reflex, Audition (“startle response”), Coordination of movements (“righting response”), Behavior during “handling”, Vocalization, Pain perception (“tail pinch”), Other findings, Grip strength of forelimbs and hindlimbs, Landing foot-splay test.
Motor activity measurement (MA): The MA was measured on the same day as FOB was performed in 5 parental males and females (with litter) per group. The examinations were performed using the Multi-Varimex system supplied by Columbus Instruments Int. Corp., Ohio, U.S.A. For this purpose, the animals were placed in clean polycarbonate cages for the time of measurement. The number of beam interrupts was counted over 12 intervals for 5 minutes in each case. The sequence at which the animals were placed in the polycarbonate cages was selected at random. The measurement was started at about 14.00 h. On account of the measuring variant "staggered", the starting time was varied by the time needed to place the animals in the cages. For each animal, measurement was started individually when the 1st beam was interrupted and ended exactly 1 hour later. The animals received no food or water during the measurements. After the transfer of the last animal in each case, the room where the measurements were carried out was darkened.
- General 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. The pairing partners, the number of mating days until vaginal sperm was detected and gestational status was recorded for F0 females.
- Clinical laboratory investigations: In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane (Isoba®, Essex GmbH Munich, Germany). The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. Urine samples were evaluated in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results of clinical pathology examinations were expressed in Inter¬national System (SI) units.
- Haematology: The following parameters were determined in blood with EDTA K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany). Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RET). Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany): Prothrombin time (Hepato Quick’s test) (HQT).
- Clinical biochemistry: The following clinical biochemistry parameters were determined using An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany): Alanine aminotransferase, Aspartate aminotransferase, Alkaline Phosphatase,γ-Glutamyltransferase, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Total protein, Albumin, Globulins,Triglycerides, Total Bilirubin, Bile acids, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate.
- Urinalysis: The dry chemical reactions on test strips (Combur 10 test M, Roche, Mannheim, Germany) used to determine urine constituents semiquantitatively were evaluated with a reflection photometer (Miditron M; Roche, Mannheim, Germany): pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color (turbidity), Volume.
Sacrifice and pathology:
- Necropsy: All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology; special attention was given to the reproductive organs. Female animal No. 102 was sacrificed moribund 36 days after start of exposure and was necropsied as soon as possible after its death and assessed by gross pathology. The following organs or tissues of all parental animals were fixed in 4% buffered formaldehyde solution or modified Davidson’s solution: All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, cortex, Cecum, Cervix, Colon, Coagulation gland, Duodenum, Epididymides, Eyes (with optic nerve), Esophagus, Extraorbital lacrimal glands, Epididymides (modified Davidson’s solution), Femur including with kneejoint, Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Larynx, Liver, Lung, Lymph nodes (axillary and mesenteric), Mammary gland (male and female), Nose (nasal cavity), Ovaries (modified Davidson’s solution), Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate gland, Rectum, Salivary glands (mandibular, sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with bone marrow, Stomach (forestomach and glandular stomach), Testes (modified Davidson’s solution), Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina. The ovaries of female animal No. 102 that was sacrificed moribund 36 days after start of exposure were fixed in 4% buffered formaldehyde solution.
- Organ weights: The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): Adrenal glands, Brain, Heart, Kidneys, Liver, Ovaries, Spleen, Thymus. The following weights were determined in all animals sacrificed on schedule: Epididymides, Testes.
- Histopathology: Fixation was followed by histotechnical processing, examination by light microscopy: Adrenal glands, All gross lesions, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum,,Epididymides, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Lymph nodes (axillary and mesenteric), Ovaries, Oviducts, Prostate gland, Peyer’s patches, Rectum, Sciatic nerve, Seminal vesicles,Spinal cord (cervical, thoracic, lumbar), Spleen, Stomach (forestomach and glandular stomach), Testes,,Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus. Special attention was given to stages of spermatogenesis in the male gonads. Female animal No. 102 that was sacrificed moribund 36 days after start of exposure was processed histotechnically and assessed with all organs listed above, which increases the number of female control animals to 6. A correlation between gross lesions and histopathological findings was attempted.
Other examinations:
All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.

PUP VIABILITY/MORTALITY
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. Dead pups were evaluated by the methods, which are described in detail in section “Necropsy observations”. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4. The viability index was calculated.

SEX RATIO
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups was finally confirmed at necropsy. The sex ratio was calculated at day 0 and day 4 after birth.

Additionally, a few pups with macroscopically dilated pericardial vessels from test groups 2 and 3 were processed histotechnically, stained with Hart/ Masson-Goldner Trichrome and examined histopathologically for the presence of aneurysms. After completion of the histopathological assessment by the study pathologist an internal peer review was performed by Dr. Maria Cecilia Rey Moreno (BASF SE, Ludwigshafen) including the liver of all male and female animals. Results presented in this report reflect the consensus opinion of the study pathologist and the peer review pathologist.
Statistics:
Means, medians and standard deviations of each test group were calculated for several parameters.
Blood parameters: For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians. For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
Urinalysis parameters: Urine pH, volume, specific gravity, color and turbidity: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p- value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians. Urine color and turbidity are not evaluated statistically. Other urinary parameters: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
Pathology weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.
Details on results:
See also table 1 and 2

- Mortality: There were no test substance-related mortalities in any of the male and female parental animals in any of the groups. During study week 1, one control female (0 mg/kg bw/d) was sacrificed moribund, after showing labored respiration, piloerection and poor general state.
- Clinical observations: Four of ten high-dose (35 mg/kg bw/d) male and six of ten high-dose female F0 generation parental animals as well as one of ten mid-dose (15 mg/kg bw/d) male and three of ten mid-dose female F0 generation parental animals showed semiclosed eyelid and piloerection during the premating period. One high-dose male and one mid-dose female showed piloerection during premation days 1 – 3 and one mid-dose female showed piloerection during PND 1 - 2. These clinical observations were attributed to the treatment. No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in the low-dose (5 mg/kg bw/d) male or female F0 generation parental animals during the whole study including gestation and lactation periods.
- Food consumption: Food consumption of the high-dose F0 males (35 mg/kg bw/d) was statistically significantly below control during the whole premating period (up to 21%). The mid-dose F0 males (15 mg/kg bw/d) showed statistically significantly reduced food consumption during premating days 0 - 13 (about 9% below control) and during premating days 0 - 7 about 13% below control. The low-dose F0 males (5 mg/kg bw/d) did not show any test substance-related changes in food consumption during the whole treatment period. Food consumption of the high-dose F0 females was statistically significantly below control during premating days 0 - 13 (about 15%), during premating days 0 - 7 (about 23%), during GD 0 - 20 (about 9%), during GD 14 - 20 (about 12%) and during the whole lactation period (about 34%). The mid-dose F0 females showed statistically significantly reduced food consumption during the whole premating period (up to 16% below control). The low-dose F0 females did not show any test substance-related changes in food consumption during the whole treatment period. Food consumption of the mid-dose females was comparable to the concurrent control throughout the gestation and lactation period.
- Body weight data: The high-dose parental males had statistically significantly lower body weights on premating day 7 and 13 (about 4% below control, respectively) and on postmating day 0 (about 5% below control) as well as statistically significantly lower body weight change during premating days 0 - 13 (about 38% below control) and during premating days 0 - 7 (about 62% below control). The body weight gain of the mid-dose parental males was statistically significantly below control during premating days 0 - 13 (about 30%) and during premating days 0 - 7 (about 52%). The high-dose parental females had statistically significantly lower body weights during the whole lactation period (up to 13% below control). The body weights of the mid-dose parental females were statistically significantly below control on PND 0 (about 6%). Neither mean body weights nor mean body weight change of the F0 parental animals in the low-dose group were influenced by the test substance during the whole treatment period.
- Detailed clinical observations (DCO): Male and female animals of all dose groups (35, 15 and 5 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. Any deviations from "zero values" were equally distributed between test substance-treated groups and controls or occurred in single animals only. Therefore, these observations were considered as being incidental. Quantitative Parameters: No test substance-related impaired parameters were observed in male and 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 dose groups in comparison to the concurrent control group.
- Clinical biochemistry (see table 3): No treatment-related changes among hematological parameters were observed. No treatment-related changes among clinical chemistry parameters were observed. In females of test groups 2 and 3 (15 and 35 mg/kg bw/d) alkaline phosphatase (ALP) activities were higher compared to controls, but the values were within the historical controls range (ALP: 0.39-0.87 µkat/L). In males of test group 2 only (15 mg/kg bw/d), total bilirubin and albumin levels were increased. Both parameters were not anymore increased in test group 3 (35 mg/kg bw/d). Some parameters were changed in males of all test groups (test group 1, 2 and 3: 5, 15 and 35 mg/kg bw/d), but the means were either within historical control ranges (for triglycerides and chloride; historical control range: triglycerides 0.41-1.32 mmol/L, chloride 99.9-107.4 mmol/L) or some means were marginally out of the historical control ranges, but the values were not changed dose-dependently (for potassium and inorganic phosphate; historical control range: potassium 4.35-4.95 mmol/L, inorganic phosphate 1.36-1.96 mmol/L). Therefore, all mentioned changes were regarded as incidental and not treatment-related. Urea levels in males of test groups 2 and 3 (15 and 35 mg/kg bw/d) were higher compared to controls, but the values were not dose-dependently changed. The values were slightly above the historical control range (urea 4.91-7.42 mmol/L). However, this was the only relevantly altered parameter in these animals and therefore the change was regarded as maybe treatment-related, but not adverse (ECETOC, 2002).

No treatment-related changes among urinalysis parameters were observed. In rats of both sexes (in females not statistically significantly) of all test groups (test group 1, 2 and 3; 5, 15 and 35 mg/kg bw/d), urine pH values were higher compared to controls. Probably due to this in rats of both sexes of test group 3 (35 mg/kg bw/d) and additionally in females of test group 2 (15 mg/kg bw/d), more crystals (in males phosphate crystals, in females mainly unknown crystals) were found in the urine sediment, because of precipitation of these type of crystals in more alkaline urine. At least phosphate crystals were normal in urine sediments of controls, and higher levels per se without any other alteration of urine parameters were regarded as treatment-related, but not adverse.

- Macroscopic examination: All findings were considered to be incidental or spontaneous in origin and without any relation to treatment. Animal 102 which was sacrificed moribund showed a deposition on its lung.
- Organ weights: The terminal body weight was significantly decreased in males and females of test group 3 (35 mg/kg bw/day), which was considered to be treatment-related. Liver weight increases were regarded to be treatment-related and were correlated with centrilobular hypertrophy histopathologically.
The increased kidney weights in males and females of test groups 2 and 3 were outwith the historical control range and weights showed a dose response. Therefore the increase in kidney weights was regarded to be treatment-related. The increased relative testes weights in males of test group 3 (35 mg/kg bw/day) was related to the decreased terminal body weights in these animals. All other mean absolute and relative weight parameters did not show significant differences when compared to the control group 0.
- Microscopic examination: In the liver, treatment related centrilobular hypertrophy was observed in male and female animals with incidence and grading.
All other findings (including liver cell foci and few foci of necrosis) 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.

- Reproduction data: Animal 102 (sacrificed moribund) showed pleuritis correlating to deposit on the lung and atrophy and inflammation of the thymus with intralesional plant material. The female animals (Nos. 103, 111, 130) which were not pregnant as well as the male mating partners (Nos. 2, 3, 11, 30) were not examined histopathologically. Animals 33/ 133 did not show relevant histopathological lesions.
- Developmental data: The mean number of delivered F1 pups per dam and the rates of liveborn and stillborn F1 pups were comparable between test groups 0, 1 and 2. The statistically significantly increased number of stillborn pups (and therefore significantly reduced number of liveborn pups) in test group 3 reflects a treatment-related effect in this dose group. Statistically significant more pups died in the high-dose litters than control. That is, 19 died or were cannibalized in the high-dose group (35 mg/kg bw/d), compared to none dead/cannibalized in the control group. Consequently, the viability index as indicator for pup mortality during lactation (PND 0-4) was significantly lower in test group 3 (59.6%** [p<= 0.01]) than in the control and test groups 1-2 (i.e. 100% / 99.3% / 92.3% at 0, 5 and 15 mg/kg bw/d). The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature. There were no test substance related adverse clinical signs observed in any of the F1 generation pups of the different test groups. Mean body weights of the high-dose (35 mg/kg bw/d) male pups were statistically significantly below control during the whole lactation period (ranging between 17% on PND 1 and between 26% on PND 4). Mean body weights of the mid-dose (15 mg/kg bw/d) male pups were statistically significantly below control on PND 1 (about 16%). Mean body weights of the high-dose female pups were statistically significantly below control on PND 4 (22%). Both sexes combined showed statistically significantly decreased mean body weights in high-dose pups on PND 1 and 4 (about 17% and 27 % below control, respectively) and in mid-dose pups on PND 1 (about 14% below control). No test compound-related influence on F1 pup body weights were noted in all pups of test group 1 (5 mg/kg bw/d) during the whole lactation period, in female pups of test group 2 during the whole lactation period and in female pups of test group 3 on PND 1. Mean body weight change of the high-dose male and female pups were statistically significantly below control during the whole lactation period (ranging between 37% [females], 45% [males] and 46% [both sexes combined]. No test compound-related influence on F1 pup body weight change were noted in test group 2 and 1 during the whole lactation period. A few other pups showed spontaneous findings at gross necropsy, such as post mortem autolysis, reddish discolored kidney(s) and empty stomach. These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Pups of test group 2 (1 male, 3 females) and 3 (4 males, 4 females) showed aneurysms in the vessels macroscopically described as dilated (aorta, arteries or ductus arteriosus).
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on adverse clincal symptoms and decreased body weights/body weight gain.
Critical effects observed:
not specified

Table 1:     Summary table of statistically significant, substance related findings in the F0 males of the OECD 422 study

 

Control

5 mg/kg bw/d

15 mg/kg bw/d

35 mg/kg bw/d

Clinical observation during premating1)

0/10

0/10

1/10 (piloerection and semi closed eyelid)

5/10 (piloerection and 4/10 semi closed eyelid)

Food consumption/d during premating (d0-13)2)

22.9 ± 1.6

22.1 ± 0.8

20.8 ± 1.3**
(-9%)

18.9 ± 1.3**
(-17%)

Body weight [g] at pre-mating day 132)

343.3 ± 13.4

339.0 ± 8.4

332.1 ± 9.9

328.9 ± 15.5*
(-4%)

Body weight [g] at post-mating day 0 (28 days of exposure)2)

376.9 ± 22.3

371.0 ± 13.2

358.6 ± 13.3

357.1 ± 19.3*
(-5%)

Body weight changes [g] during pre-mating, day 0-132)

33.0 ± 10.1

28.4 ± 6.7

23.2 ± 7.4*
(-30%)

20.3 ± 8.4**
(-38%)

Absolute terminal body weight [g]2)

348.0 ± 23.4

342.4 ± 11.3

329.9 ± 14.1

327.6 ± 17.8*
(-6%)

Absolute kidney weight [g]2)

2.008 ± 0.235

2.234 ± 0.099

2.242 ± 0.156

2.476 ± 0.176*
(+23%)

Absolute liver weight [g]2)

7.120 ± 0.571

7.504 ± 0.335

7.732 ± 0.654

8.756 ± 0.814**
(+23%)

Absolute testes weight [g]2)

3.373 ± 0.336

3.368 ± 0.215

3.344 ± 0.263

3.603 ± 0.252

Relative kidneys weight [%]2)

0.600 ± 0.062

0.651 ± 0.047

0.696 ± 0.303**
(+16%)

0.761 ± 0.032**
(+27%)

Relative liver weight [%]2)

2.126 ± 0.117

2.184 ± 0.061

2.399 ± 0.112*
(+13%)

2.688 ± 0.096**
(+26%)

Relative testes weight [%]2)

0.972 ± 0.113

0.984 ± 0.063

1.014 ± 0.067

1.102 ± 0.084**
(+13%)

Hepatic centrilobular hypertrophy1)

0/10

0/10

9/10 (Grade 1)

10/10 (Grade 3)

1)Number of affected animals / total number per group.

2)Mean ± SD with * p ≤ 0.05, ** p ≤ 0.01; statistically significant differences compared to control group

Table 2:    Summary table of statistically significant, substance related findings in the F0 females of the OECD 422 study

 

Control

5 mg/kg bw/d

15 mg/kg bw/d

35 mg/kg bw/d

Clinical observation during premating1)

1/10 (general poor condition, piloerection, labored respiration, sacrificed moribund

0/10

4/10 (piloerection), 3/10 (semi closed eyelid)

6/10 (piloerection and semi closed eyelid)

Clinical observation during lactation1)

0/8

0/9

1/9 (piloerection)

2/9 (complete litter loss)

Food consumption/d during premating2)

16.4 ± 1.1

15.5 ± 1.0

14.4 ± 0.8**
(-12%)

13.9 ± 1.8**
(-15%)

Food consumption/d during gestation (d0-20)2)

22.2 ± 1.6

22.6 ± 1.3

20.7 ± 1.9

20.2 ± 1.4*
(-9%)

Food consumption/d during lactation (d1-4)2)

34.4 ± 4.3

31.9 ± 4.5

31.2 ± 5.9

22.8 ± 4.7**
(-34%)

Body weight [g] at PND 02)

264.4 ± 16.5

258.2 ± 11.6

248.3 ± 13.2*
(-6%)

235.1 ± 9.2**
(-11%)

Body weight [g] at lactation day 42)

281.8 ± 17.0

270.8 ± 14.9

267.7 ± 14.3

224.2 ± 9.0**
(-20%)

Absolute terminal body weight [g]

247.4 ± 14.8

235.2 ± 8.3

233.43 ± 10.8

222.7 ± 10.0**
(-10%)

Relative kidneys weight [%]

0.656 ± 0 030

0.698 ± 0 038

0.722 ± 0.035*
(+10%)

0.754 ± 0.049**
(+15%)

Relative liver weight [%]

2.431 ± 0.126

2.597 ± 0.191

2.565 ± 0.145

2.869 ± 0.064**
(+18%)

Hepatic centrilobular hypertrophy1)

0/10

0/10

0/10

9/10 (Grade 1)

1)Number of affected animals / total number per group.

2)Mean ± SD with * p ≤ 0.05, ** p ≤ 0.01; statistically significant differences compared to control group

Table 3:       Summary table of statistically significant findings in clinical chemistry in the F0 males and females of the OECD 422 study

 

 

Control

5 mg/kg bw/d

15 mg/kg bw/d

35 mg/kg bw/d

Males

 

Urea (mmol/L)
day 31 [mean
± SD]

6.02± 0.46

6.27± 0.50

8.00± 1.09**

7.54± 0.67*

Total bilirubin (µmol/L) day 31
mean
± SD]

1.92± 0.20

2.18± 0.28

2.67± 0.34**

2.18± 0.45

Albumin (g/L)
day 31 [mean
± SD]

38.62± 0.60

39.27± 0.51

40.38± 0.76*

38.88± 1.09

Triglycerides (mmol/L) day 31
[mean
± SD]

0.35± 0.04

0.53± 0.07**

0.73± 0.23*

0.49± 0.10

K (mmol/L) day 31 [mean± SD]

4.49± 0.19

5.03± 0.22*

4.83± 0.09*

4.95± 0.25*

Cl (mmol/L) day 31
[mean
± SD]

106± 0.8

104± 2.4*

103± 1.1**

101± 2.9**

Inorganic phosphate (mmol/L) day 31
[mean
± SD]

1.70± 0.10

2.01± 0.11**

1.94± 0.14*

2.08± 0.13**

Females

 

ALP (µkat/L) day 50

[mean± SD]

0.54± 0.05

0.66± 0.21

0.80± 0.12**

0.67± 0.05*

 Mean ± SD with * p ≤ 0.05, ** p ≤ 0.01; statistically significant differences compared to control
(Kruskal-Wallis + Wilcoxon test, two sided)

Reason / purpose:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to
Guideline:
other: EPA, Health Effects Test Guidelines; OPPTS 870.3650: Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test (Jul 2000)
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 11-12 weeks
- Weight at study initiation: male animals 293.1 g - 328.9 g and female animals 196.1 g - 236.7 g
- Housing: individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²), with the following exception: during overnight matings, male and female mating partners were housed together in Makrolon type M III cages. Pregnant animals and their litters were housed together until PND 4. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. For enrichment wooden gnawing blocks (Typ NGM E-022; supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria) were added. Dust-free wooden bedding was used in this study.
- Diet (e.g. ad libitum): ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, which was available to the animals ad libitum.
- Water (e.g. ad libitum): from water bottles (ad libitum)
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h.
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
TEST ITEM PREPARATIONS
The test substance solutions in drinking water were prepared at the beginning of the administration period and thereafter in intervals, which took into account the analytical results of the stability verification.
For the preparation of the administration solutions the test substance was weighed in a graduated flask depending on the dose group, topped up with drinking water and subsequently thoroughly mixed by shaking until it was completely dissolved.
Details on mating procedure:
In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group. The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 07.00 - 09.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted "GD 0" and the following day "GD 1".

The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 07.00 - 09.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted "GD 0" and the following day "GD 1".
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.
Analytical verifications of the stability of the test substance in drinking water for a period of 7 days at room temperature were carried out in a comparable batch prior to the start of the study.
Samples of the test substance solutions were sent to the analytical laboratory once at the beginning of the study for verification of the concentrations.Of each sample, one additional reserve sample was retained.
Due to the fact that the test substance preparations were true solutions, it was not considered necessary to prove homogeneity through analytical procedures. All measured values for 1-Vinylimidazol were in the expected range of the target concentrations (90 - 110%), demonstrating the correctness of the diet preparations.
Duration of treatment / exposure:
The duration of treatment covered a 2-week pre-mating and a mating period in both sexes. In males treatment lasted 30 days after beginning of administration. Females were treated the entire gestation period as well as approximately 2 weeks of the lactation period. Females were sacrificed 50 days after beginning of administration.
Frequency of treatment:
Once daily at approximately the same time in the morning.
Dose / conc.:
5 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
35 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The oral route was selected since administration by gavage has been proven to be appropriate for the detection of a toxicological hazard.
Prior to the OECD422 study, a 14-d dose range finding stuy in Wistar rats was performed, in order to find the apropriate dose regimen for the main study. With 3 animals/sex and group, 0, 35, 75, 125 and 250 mg/kg bw/d 1-vinylimidazole were administered to male and female rats per gavage (01R0335/00R18; BASF, 2012). Besides the clinical observations, food consumption and body weights were examined at day 0, 3, 7, 10 and 14 and the animals were assessed post-mortem by gross pathology. In the highest dose group 3/3 males and 2/3 females were sacrificed moribund, the third female was found dead. In the 125 mg/kg bw/d dose group all males and females were sacrificed moribund. In the 75 mg/kg bw/d dose group one female was sacrificed moribund, all other animals were sacrificed scheduled. In the lowest dose group all animals were sacrificed scheduled. Statistically significant reduced food consumption compared to the control was observed in all males and female groups exept for the low dose females receiving 35 mg/kg bw/d. Statistically significant decreased body weights were observed in the low dose females.
Due to the deaths occuring in the first range finder at doses >= 75 mg/kg bd/d, another 14-d range finder with male and female rats (4/sex and group) was considered to be neccessary with doses of 35 and 70 mg/kg bw/d per gavage (01R0492/11R136; BASF, 2012). This study additionally examined water consumption, clinical pathology (hematology, chlinical chemistry) and some organ weights (adrenal glands, kidneys, liver, spleen). All males and females receiving 1-vinylimidazole showed semiclosed eyelids and piloerection of the fur during the study. Compared to the controls, reduced food consumption was observed in both groups in males and females mainly within the first week of administration. A statistically significant increase in water consumption was observed only once in females of the lower dose group from day 3 to 7. Body weights of the males receiving 70 mg/kg bw/d were significantly reduced at the end of the exposure period (d13) compared to controls: 326.1 +/- 20.1 g vs 371.7 +/- 18.8 g (-12%). These males had a body weight loss of 4.1 g at study day 13 compared to day 0, where as the control animals showed a body weight gain of 43.0 g from day 0 to 13. Bodyweights of the mid and high dose females also showed a statistically significant reduction after the exposure period compared to controls: 229.0 +/- 3.3 g; 211.4 +/- 5.6 g (-8%); 201.1 +/- 10.6 g (-12%).
In conclusion, a dose of 70 mg/kg bw /d 1-vinylimidazole was considered to exceed the maximal tolerated dose, based on the significant body weight loss of the high dose males and the significant reduced body weight of > 10 % in both sexes after 14 days of exposure. Thus, 35 mg/kg bw/d was selected as the high dose for the main study according to OECD test guideline 422.

- Method of formulation: For the preparation of the administration solutions the test substance was weighed in a graduated flask depending on the dose group, topped up with drinking water and subsequently thoroughly mixed by shaking until it was completely dissolved.

- Parturition: The females were allowed to deliver and rear their pups until day 4 after parturition. The female animals were sacrificed 50 days after the beginning of the administration, and examined.
Parental animals: Observations and examinations:
- Mortality / Viability: At twice daily on working days and once daily on other days.
- Clinical signs: A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each animal. Individual data of daily observations can be found in the raw data. The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis. On weekdays (except Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.
- Food consumption: Generally, food consumption was determined once a week for male and female parental animals, 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 F0 animals). Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0, 7, 14 and 20. Food consumption of F0 females which gave birth to a litter was determined on PND 1 and 4. Food consumption was not determined in females without positive evidence of sperm during the gestation period and in females without litter during the lactation period.
- Body weights: In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice. The body weight change of the animals was calculated from these results. The following exceptions are notable for the female animals: During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. Females with litter were weighed on the day of parturition (PND 0) and on PND 4. Females without positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume.
- Detailed clinical observations (DCO): Detailed clinical observations were performed in all animals once prior to the first administration and at weekly intervals during the administration period. The examinations started in the morning. The findings were ranked according to the degree of severity, if applicable. For observation, the animals were removed from their cages by the investigator and placed in a standard arena (50 x 37.5 cm wide, with side borders which are 25 cm high). The following parameters listed were assessed: 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.
- Functional Observations: A functional observational battery was performed in five male and 5 female animals (with litter) per group at the end of the administration period starting at about 10.00 h. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
Home cage observations: The animals were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to: Posture, Tremors, Convulsions, Abnormal movements, Impairment of gait, Other findings.
Open field observations: The animals were transferred to a standard arena (50 x 50 cm wide, with side borders which are 25 cm high) and observed for at least 2 minutes. The following parameters were examined: Behavior when removed from cage, Fur, Skin, Salivation, Nose discharge, Lacrimation, Eyes/pupil size, Posture, Palpebral closure, Respiration, Tremors, Convulsions, Abnormal movements/stereotypy, Impairment of gait, Activity/arousal level, Feces excreted within 2 minutes (number of scybala discharged/appearance/consistency), Urine excreted within 2 minutes (amount/color), Number of rearings within 2 minutes
Sensory motor tests/Reflexes: The animals were removed from the open field and subjected to following sensory motor or reflex tests: Approach response, Touch response, Vision (“visual placing response”), Pupillary reflex, Pinna reflex, Audition (“startle response”), Coordination of movements (“righting response”), Behavior during “handling”, Vocalization, Pain perception (“tail pinch”), Other findings, Grip strength of forelimbs and hindlimbs, Landing foot-splay test.
Motor activity measurement (MA): The MA was measured on the same day as FOB was performed in 5 parental males and females (with litter) per group. The examinations were performed using the Multi-Varimex system supplied by Columbus Instruments Int. Corp., Ohio, U.S.A. For this purpose, the animals were placed in clean polycarbonate cages for the time of measurement. The number of beam interrupts was counted over 12 intervals for 5 minutes in each case. The sequence at which the animals were placed in the polycarbonate cages was selected at random. The measurement was started at about 14.00 h. On account of the measuring variant "staggered", the starting time was varied by the time needed to place the animals in the cages. For each animal, measurement was started individually when the 1st beam was interrupted and ended exactly 1 hour later. The animals received no food or water during the measurements. After the transfer of the last animal in each case, the room where the measurements were carried out was darkened.
- General 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. The pairing partners, the number of mating days until vaginal sperm was detected and gestational status was recorded for F0 females.
- Clinical laboratory investigations: In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane (Isoba®, Essex GmbH Munich, Germany). The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. Urine samples were evaluated in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results of clinical pathology examinations were expressed in Inter¬national System (SI) units.
- Haematology: The following parameters were determined in blood with EDTA K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany). Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RET). Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany): Prothrombin time (Hepato Quick’s test) (HQT).
- Clinical biochemistry: The following clinical biochemistry parameters were determined using An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany): Alanine aminotransferase, Aspartate aminotransferase, Alkaline Phosphatase,γ-Glutamyltransferase, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Total protein, Albumin, Globulins,Triglycerides, Total Bilirubin, Bile acids, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate.
- Urinalysis: The dry chemical reactions on test strips (Combur 10 test M, Roche, Mannheim, Germany) used to determine urine constituents semiquantitatively were evaluated with a reflection photometer (Miditron M; Roche, Mannheim, Germany): pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color (turbidity), Volume.
Litter observations:
All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.

PUP VIABILITY/MORTALITY
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. Dead pups were evaluated by the methods, which are described in detail in section “Necropsy observations”.
The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4. The viability index was calculated.

SEX RATIO
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups was finally confirmed at necropsy. The sex ratio was calculated at day 0 and day 4 after birth.
Postmortem examinations (parental animals):
- Necropsy: All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology; special attention was given to the reproductive organs. Female animal No. 102 was sacrificed moribund 36 days after start of exposure and was necropsied as soon as possible after its death and assessed by gross pathology. The following organs or tissues of all parental animals were fixed in 4% buffered formaldehyde solution or modified Davidson’s solution: All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, cortex, Cecum, Cervix, Colon, Coagulation gland, Duodenum, Epididymides, Eyes (with optic nerve), Esophagus, Extraorbital lacrimal glands, Epididymides (modified Davidson’s solution), Femur including with kneejoint, Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Larynx, Liver, Lung, Lymph nodes (axillary and mesenteric), Mammary gland (male and female), Nose (nasal cavity), Ovaries (modified Davidson’s solution), Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate gland, Rectum, Salivary glands (mandibular, sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with bone marrow, Stomach (forestomach and glandular stomach), Testes (modified Davidson’s solution), Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina. The ovaries of female animal No. 102 that was sacrificed moribund 36 days after start of exposure were fixed in 4% buffered formaldehyde solution.
- Organ weights: The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): Adrenal glands, Brain, Heart, Kidneys, Liver, Ovaries, Spleen, Thymus. The following weights were determined in all animals sacrificed on schedule: Epididymides, Testes.
- Histopathology: Fixation was followed by histotechnical processing, examination by light microscopy: Adrenal glands, All gross lesions, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum,,Epididymides, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Lymph nodes (axillary and mesenteric), Ovaries, Oviducts, Prostate gland, Peyer’s patches, Rectum, Sciatic nerve, Seminal vesicles,Spinal cord (cervical, thoracic, lumbar), Spleen, Stomach (forestomach and glandular stomach), Testes,,Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus. Special attention was given to stages of spermatogenesis in the male gonads. Female animal No. 102 that was sacrificed moribund 36 days after start of exposure was processed histotechnically and assessed with all organs listed above, which increases the number of female control animals to 6. A correlation between gross lesions and histopathological findings was attempted.
Postmortem examinations (offspring):
At necropsy on PND 4, all pups were sacrificed with CO2, under isoflurane anesthesia, and examined macroscopically for external and visceral findings.
Statistics:
Means, medians and standard deviations of each test group were calculated for several parameters.
Blood parameters: For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians. For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
Urinalysis parameters: Urine pH, volume, specific gravity, color and turbidity: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p- value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians. Urine color and turbidity are not evaluated statistically. Other urinary parameters: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
Pathology weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.
Reproductive indices:
For each group, the following calculations were performed:
- Male mating index (%) = (number of males with confirmed mating / number of males placed with females) x 100
- Male fertility index (%) = (number of males proving their fertility / number of males placed with females) x 100
- Female mating index (%) = (number of females mated / number of females placed with males) x 100
- Female fertility index (%) = (number of pregnant females / number of females mated) x 100
- Gestation index (%) = (number of females with live pups on the day of birth / number of pregnant females) x 100


Offspring viability indices:
For each group, the following calculations were performed:
- Live birth index (%) = (number of liveborn pups at birth / total number of pups born) x 100
- Postimplantation loss (%) = (number of implantations – number of pups delivered / number of implantations) x 100
- Viability index = (Number of live pups on Day 4 post partum / Number of pups born alive) x 100
- Sex ratio = (number of live male or female pups on day 0/4 / number of live male and female pups on day 0/4) x 100
- Mortality: There were no test substance-related mortalities in any of the male and female parental animals in any of the groups. During study week 1, one control female (0 mg/kg bw/d) was sacrificed moribund, after showing labored respiration, piloerection and poor general state.
- Clinical observations: Four of ten high-dose (35 mg/kg bw/d) male and six of ten high-dose female F0 generation parental animals as well as one of ten mid-dose (15 mg/kg bw/d) male and three of ten mid-dose female F0 generation parental animals showed semiclosed eyelid and piloerection during the premating period. One high-dose male and one mid-dose female showed piloerection during premation days 1 – 3 and one mid-dose female showed piloerection during PND 1 - 2. These clinical observations were attributed to the treatment. No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in the low-dose (5 mg/kg bw/d) male or female F0 generation parental animals during the whole study including gestation and lactation periods.
- Food consumption: Food consumption of the high-dose F0 males (35 mg/kg bw/d) was statistically significantly below control during the whole premating period (up to 21%). The mid-dose F0 males (15 mg/kg bw/d) showed statistically significantly reduced food consumption during premating days 0 - 13 (about 9% below control) and during premating days 0 - 7 about 13% below control. The low-dose F0 males (5 mg/kg bw/d) did not show any test substance-related changes in food consumption during the whole treatment period. Food consumption of the high-dose F0 females was statistically significantly below control during premating days 0 - 13 (about 15%), during premating days 0 - 7 (about 23%), during GD 0 - 20 (about 9%), during GD 14 - 20 (about 12%) and during the whole lactation period (about 34%). The mid-dose F0 females showed statistically significantly reduced food consumption during the whole premating period (up to 16% below control). The low-dose F0 females did not show any test substance-related changes in food consumption during the whole treatment period. Food consumption of the mid-dose females was comparable to the concurrent control throughout the gestation and lactation period.
- Body weight data: The high-dose parental males had statistically significantly lower body weights on premating day 7 and 13 (about 4% below control, respectively) and on postmating day 0 (about 5% below control) as well as statistically significantly lower body weight change during premating days 0 - 13 (about 38% below control) and during premating days 0 - 7 (about 62% below control). The body weight gain of the mid-dose parental males was statistically significantly below control during premating days 0 - 13 (about 30%) and during premating days 0 - 7 (about 52%). The high-dose parental females had statistically significantly lower body weights during the whole lactation period (up to 13% below control). The body weights of the mid-dose parental females were statistically significantly below control on PND 0 (about 6%). Neither mean body weights nor mean body weight change of the F0 parental animals in the low-dose group were influenced by the test substance during the whole treatment period.
- Detailed clinical observations (DCO): Male and female animals of all dose groups (35, 15 and 5 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. Any deviations from "zero values" were equally distributed between test substance-treated groups and controls or occurred in single animals only. Therefore, these observations were considered as being incidental. Quantitative Parameters: No test substance-related impaired parameters were observed in male and 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 dose groups in comparison to the concurrent control group.
- Clinical biochemistry: No treatment-related changes among hematological parameters were observed. No treatment-related changes among clinical chemistry parameters were observed. In females of test groups 2 and 3 (15 and 35 mg/kg bw/d) alkaline phosphatase (ALP) activities were higher compared to controls, but the values were within the historical controls range (ALP: 0.39-0.87 µkat/L). In males of test group 2 only (15 mg/kg bw/d), total bilirubin and albumin levels were increased. Both parameters were not anymore increased in test group 3 (35 mg/kg bw/d). Some parameters were changed in males of all test groups (test group 1, 2 and 3: 5, 15 and 35 mg/kg bw/d), but the means were either within historical control ranges (for triglycerides and chloride; historical control range: triglycerides 0.41-1.32 mmol/L, chloride 99.9-107.4 mmol/L) or some means were marginally out of the historical control ranges, but the values were not changed dose-dependently (for potassium and inorganic phosphate; historical control range: potassium 4.35-4.95 mmol/L, inorganic phosphate 1.36-1.96 mmol/L). Therefore, all mentioned changes were regarded as incidental and not treatment-related. Urea levels in males of test groups 2 and 3 (15 and 35 mg/kg bw/d) were higher compared to controls, but the values were not dose-dependently changed. The values were slightly above the historical control range (urea 4.91-7.42 mmol/L). However, this was the only relevantly altered parameter in these animals and therefore the change was regarded as maybe treatment-related, but not adverse (ECETOC, 2002).
No treatment-related changes among urinalysis parameters were observed. In rats of both sexes (in females not statistically significantly) of all test groups (test group 1, 2 and 3; 5, 15 and 35 mg/kg bw/d), urine pH values were higher compared to controls. Probably due to this in rats of both sexes of test group 3 (35 mg/kg bw/d) and additionally in females of test group 2 (15 mg/kg bw/d), more crystals (in males phosphate crystals, in females mainly unknown crystals) were found in the urine sediment, because of precipitation of these type of crystals in more alkaline urine. At least phosphate crystals were normal in urine sediments of controls, and higher levels per se without any other alteration of urine parameters were regarded as treatment-related, but not adverse.
- Macroscopic examination: All findings were considered to be incidental or spontaneous in origin and without any relation to treatment. Animal 102 which was sacrificed moribund showed a deposition on its lung.
- Organ weights: The terminal body weight was significantly decreased in males and females of test group 3 (35 mg/kg bw/day), which was considered to be treatment-related. Liver weight increases were regarded to be treatment-related and were correlated with centrilobular hypertrophy histopathologically.
The increased kidney weights in males and females of test groups 2 and 3 were outwith the historical control range and weights showed a dose response. Therefore the increase in kidney weights was regarded to be treatment-related. The increased relative testes weights in males of test group 3 (35 mg/kg bw/day) was related to the decreased terminal body weights in these animals. All other mean absolute and relative weight parameters did not show significant differences when compared to the control group 0.
- Microscopic examination: In the liver, treatment related centrilobular hypertrophy was observed in male and female animals with incidence and grading.
All other findings (including liver cell foci and few foci of necrosis) 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.
- Reproduction data: Animal 102 (sacrificed moribund) showed pleuritis correlating to deposit on the lung and atrophy and inflammation of the thymus with intralesional plant material. The female animals (Nos. 103, 111, 130) which were not pregnant as well as the male mating partners (Nos. 2, 3, 11, 30) were not examined histopathologically. Animals 33/ 133 did not show relevant histopathological lesions.
Dose descriptor:
NOAEL
Remarks:
parental
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
35 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects up to the highest doses tested.
The mean number of delivered F1 pups per dam and the rates of liveborn and stillborn F1 pups were comparable between test groups 0, 1 and 2. The statistically significantly increased number of stillborn pups (and therefore significantly reduced number of liveborn pups) in test group 3 reflects a treatment-related effect in this dose group. Statistically significant more pups died in the high-dose litters than control. That is, 19 died or were cannibalized in the high-dose group (35 mg/kg bw/d), compared to none dead/cannibalized in the control group. Consequently, the viability index as indicator for pup mortality during lactation (PND 0-4) was significantly lower in test group 3 (59.6%** [p<= 0.01]) than in the control and test groups 1-2 (i.e. 100% / 99.3% / 92.3% at 0, 5 and 15 mg/kg bw/d). The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature. There were no test substance related adverse clinical signs observed in any of the F1 generation pups of the different test groups. Mean body weights of the high-dose (35 mg/kg bw/d) male pups were statistically significantly below control during the whole lactation period (ranging between 17% on PND 1 and between 26% on PND 4). Mean body weights of the mid-dose (15 mg/kg bw/d) male pups were statistically significantly below control on PND 1 (about 16%). Mean body weights of the high-dose female pups were statistically significantly below control on PND 4 (22%). Both sexes combined showed statistically significantly decreased mean body weights in high-dose pups on PND 1 and 4 (about 17% and 27 % below control, respectively) and in mid-dose pups on PND 1 (about 14% below control). No test compound-related influence on F1 pup body weights were noted in all pups of test group 1 (5 mg/kg bw/d) during the whole lactation period, in female pups of test group 2 during the whole lactation period and in female pups of test group 3 on PND 1. Mean body weight change of the high-dose male and female pups were statistically significantly below control during the whole lactation period (ranging between 37% [females], 45% [males] and 46% [both sexes combined]. No test compound-related influence on F1 pup body weight change were noted in test group 2 and 1 during the whole lactation period. A few other pups showed spontaneous findings at gross necropsy, such as post mortem autolysis, reddish discolored kidney(s) and empty stomach. These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Pups of test group 2 (1 male, 3 females) and 3 (4 males, 4 females) showed aneurysms in the vessels macroscopically described as dilated (aorta, arteries or ductus arteriosus).
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
histopathology: non-neoplastic
Reproductive effects observed:
yes
Lowest effective dose / conc.:
15 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
not specified

Table 1:      Summary table of statistically significant, substance related findings in the F0 males of the OECD TG 422 study

 

Control

5 mg/kg bw/d

15 mg/kg bw/d

35 mg/kg bw/d

Clinical observation during premating1)

0/10

0/10

1/10 (piloerection and semi closed eyelid)

5/10 (piloerection and 4/10 semi closed eyelid)

Food consumption/d during premating (d0-13)2)

22.9 ± 1.6

22.1 ± 0.8

20.8 ± 1.3**
(-9%)

18.9 ± 1.3**
(-17%)

Body weight [g] at pre-mating day 132)

343.3 ± 13.4

339.0 ± 8.4

332.1 ± 9.9

328.9 ± 15.5*
(-4%)

Body weight [g] at post-mating day 0 (28 days of exposure)2)

376.9 ± 22.3

371.0 ± 13.2

358.6 ± 13.3

357.1 ± 19.3*
(-5%)

Body weight changes [g] during pre-mating, day 0-132)

33.0 ± 10.1

28.4 ± 6.7

23.2 ± 7.4*
(-30%)

20.3 ± 8.4**
(-38%)

Absolute terminal body weight [g]2)

348.0 ± 23.4

342.4 ± 11.3

329.9 ± 14.1

327.6 ± 17.8*
(-6%)

Absolute kidney weight [g]2)

2.008 ± 0.235

2.234 ± 0.099

2.242 ± 0.156

2.476 ± 0.176*
(+23%)

Absolute liver weight [g]2)

7.120 ± 0.571

7.504 ± 0.335

7.732 ± 0.654

8.756 ± 0.814**
(+23%)

Absolute testes weight [g]2)

3.373 ± 0.336

3.368 ± 0.215

3.344 ± 0.263

3.603 ± 0.252

Relative kidneys weight [%]2)

0.600 ± 0.062

0.651 ± 0.047

0.696 ± 0.303**
(+16%)

0.761 ± 0.032**
(+27%)

Relative liver weight [%]2)

2.126 ± 0.117

2.184 ± 0.061

2.399 ± 0.112*
(+13%)

2.688 ± 0.096**
(+26%)

Relative testes weight [%]2)

0.972 ± 0.113

0.984 ± 0.063

1.014 ± 0.067

1.102 ± 0.084**
(+13%)

Hepatic centrilobular hypertrophy1)

0/10

0/10

9/10 (Grade 1)

10/10 (Grade 3)


1)
Number of affected animals / total number per group.

2)Mean ± SD with * p ≤ 0.05, ** p ≤ 0.01; statistically significant differences compared to control group.


Table 2:      Summary table of statistically significant, substance related findings in the F0 females of the OECD TG 422 study

 

Control

5 mg/kg bw/d

15 mg/kg bw/d

35 mg/kg bw/d

Clinical observation during premating1)

1/10 (general poor condition, piloerection, labored respiration, sacrificed moribund

0/10

4/10 (piloerection), 3/10 (semi closed eyelid)

6/10 (piloerection and semi closed eyelid)

Clinical observation during lactation1)

0/8

0/9

1/9 (piloerection)

2/9 (complete litter loss)

Food consumption/d during premating2)

16.4 ± 1.1

15.5 ± 1.0

14.4 ± 0.8**
(-12%)

13.9 ± 1.8**
(-15%)

Food consumption/d during gestation (d0-20)2)

22.2 ± 1.6

22.6 ± 1.3

20.7 ± 1.9

20.2 ± 1.4*
(-9%)

Food consumption/d during lactation (d1-4)2)

34.4 ± 4.3

31.9 ± 4.5

31.2 ± 5.9

22.8 ± 4.7**
(-34%)

Body weight [g] at PND 02)

264.4 ± 16.5

258.2 ± 11.6

248.3 ± 13.2*
(-6%)

235.1 ± 9.2**
(-11%)

Body weight [g] at lactation day 42)

281.8 ± 17.0

270.8 ± 14.9

267.7 ± 14.3

224.2 ± 9.0**
(-20%)

Absolute terminal body weight [g]

247.4 ± 14.8

235.2 ± 8.3

233.43 ± 10.8

222.7 ± 10.0**
(-10%)

Relative kidneys weight [%]

0.656 ± 0 030

0.698 ± 0 038

0.722 ± 0.035*
(+10%)

0.754 ± 0.049**
(+15%)

Relative liver weight [%]

2.431 ± 0.126

2.597 ± 0.191

2.565 ± 0.145

2.869 ± 0.064**
(+18%)

Hepatic centrilobular hypertrophy1)

0/10

0/10

0/10

9/10 (Grade 1)


1)
Number of affected animals / total number per group.

2)Mean ± SD with * p ≤ 0.05, ** p ≤ 0.01; statistically significant differences compared to control group.

Table 3:      Mating, reproduction and delivery data

 

Control

5 mg/kg bw/d

15 mg/kg bw/d

35 mg/kg bw/d

No. of females mated

9

10

10

10

No. of females pregnant

8

9

9

9

Female/male mating index [%]

100

100

100

100

Female/male fertility index [%]

88.9

90.0

90.0

90.0

Mating days until day 0 pc [mean]

1.9

2.4

2.4

3.0

No. dams with liveborn pups

[%]

8 (100%)

9 (100%)

9 (100%)

9 (100%)

No. dams with total litter loss

[%]

0 (0%)

0 (0%)

0 (0%)

0 (0%)

No. dams with stillborn pups

1 (12.5%)

0 (0%)

1 (11.1%)

6 (66.7%)

Total no. of liveborn pups

(Live birth index)

88
(98.9%)

91
(100%)

100
(94.3%)

70 **
(74.5%)

Total no. of still born pups

1 (1.1%)

0 (0%)

6 (5.7%)

24 (25.5%) **

Gestation days
[mean ± SD]

22.2 ± 0.5

22.3 ± 0.5

22.6 ± 0.5

22.9 ± 0.8

Implantations/dam
[mean ± SD]

11.5±2.3

11.0 ± 3.6

12.2 ± 2.4

11.9 ± 1.3

Post implantation loss per group (ratio dead implants/total implants)

[mean %]

3.53

6.66

3.34

11.57

Pups delivered/dam
(viable and stillborn)
[mean ± SD]

11.1 ± 2.4

10.1 ± 3.1

11.8 ± 2.1

10.4 ± 2.0

Sex ratio [% live males day 0]

56.8

48.4

47.0

57.1

 

*p ≤ 0.05, ** p ≤ 0.01 (Dunnett test, two-sided), statistically significant differences compared to control group

Table 4: Summary litter report and the pup status

 

Control

5 mg/kg bw/d

15 mg/kg bw/d

35 mg/kg bw/d

Total No. of litters

8

9

9

9

With liveborn pups

8 (100%)

9 (100%)

9 (100%)

9 (100%)

With stillborn pups

1 (12.5%)

0 (0%)

1 (11.1%)

6 (66.7%)

Implantation sites/dam
[mean ± SD]

11.5±2.3

11.0 ± 3.6

12.2 ± 2.4

11.9 ± 1.3

Post implantation loss per test group (ratio dead implants/total implants)
[mean %]

3.53

6.66

3.34

11.57

Pups delivered/dam
(viable and stillborn)
[mean ± SD]

11.1 ± 2.4

10.1 ± 3.1

11.8 ± 2.1

10.4 ± 2.0

Viable litter size day 0

[mean ± SD]

11.0 ± 2.2

10.1 ± 3.1

11.1 ± 2.6

7.8 ± 3.3

Viable litter size day 4

[mean ± SD]

11.0 ± 2.2

10.0 ± 2.9

10.3 ± 2.9

5.7 ± 4.3**

Total no. of liveborn pups

(Live birth index)

88
(98.9%)

91
(100%)

100
(94.3%)

70**
(74.5%)

Total no. of stillborn pups

1 (1.1%)

0 (0%)

6 (5.7%)

24 (25.5%)**

Perinatal loss per group
(= %stillborn/delivered x 100)

[mean %]

0.89

0

5.13

27.29

No. of pups surviving
days 0 to 4

88

90

93

51

Viability index
[mean% ± SD]
(no. live pups on day 4 /
no live pups/day of birth)

100±0

99.3± 2.2

92.3± 15.9

59.6± 43.1**

Sex ratio [% live males day 0]

56.8

48.4

47.0

57.1

* p <= 0.05, ** p <= 0.01 (Dunnett test, two-sided), statistically significant differences compared to control group

Table 5:      Summary of pathology examination pups

 

Control

5 mg/kg bw/d

15 mg/kg bw/d

35 mg/kg bw/d

No. of litters evaluated

8

9

9

9

No. of pups evaluated

89

91

106

94

No. of pups with signs per group (all necropsy observations incl. post mortem autolysis, empty stomach, cannibalized pups, any organ findings)

1

2

10

39

No. pups with dilated aorta

(thereof in stillborn pups)

0

0

1
(1)

7
(4)

Affected litters with pups with dilated aorta (%)
[mean incidence per litter]


0%


0%


11%
(0.11)


22%
(0.77)

No. pups with dilated aortic arch (thereof in stillborn pups)


0


0


3
(1)


3
(2)

Affected litters with pups with dilated aortic arch (%)

[mean incidence per litter]


0


0


2 (22%)
(0.33)


2 (22%)
(0.33)

No. pups with dilated ductus arteriosus

0

0

1

0

No. of pups with dilated subclavian artery

0

0

0

1

No. of pups with empty stomachs

0

0

0

11

Pup weight day 1 (g)

(all viable pups)

[mean% ± SD]

7.1±0.9

7.2 ± 0.8

6.1 ± 0.6*

5.9 ± 0.7*

Pup weight gain (g)

(PND1 to PND4)

[mean% ± SD]

3.9±0.8

3.8 ± 0.9

3.4 ± 0.8

2.1 ± 1.6**

 

* p <= 0.05, ** p <= 0.01 (Dunnett test, two-sided), statistically significant significant differences compared to control

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test substance: 1-Vinylimidazol
- Purity: 99.8%
- Physical state/Appearance: Liquid / yellowish, clear
- Storage conditions: Ambient (RT)
- Test substance No.: 00/0335-3
- Batch identification: 87876088Q0
- Storage stability: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 11-12 weeks
- Weight at study initiation: male animals 293.1 g - 328.9 g and female animals 196.1 g - 236.7 g
- Housing: individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²), with the following exception: during overnight matings, male and female mating partners were housed together in Makrolon type M III cages. Pregnant animals and their litters were housed together until PND 4. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. For enrichment wooden gnawing blocks (Typ NGM E-022; supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria) were added. Dust-free wooden bedding was used in this study.
- Diet (e.g. ad libitum): ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, which was available to the animals ad libitum.
- Water (e.g. ad libitum): from water bottles (ad libitum)
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST ITEM PREPARATIONS
The test substance solutions in drinking water were prepared at the beginning of the administration period and thereafter in intervals, which took into account the analytical results of the stability verification.
For the preparation of the administration solutions the test substance was weighed in a graduated flask depending on the dose group, topped up with drinking water and subsequently thoroughly mixed by shaking until it was completely dissolved.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.
Analytical verifications of the stability of the test substance in drinking water for a period of 7 days at room temperature were carried out in a comparable batch prior to the start of the study.
Samples of the test substance solutions were sent to the analytical laboratory once at the beginning of the study for verification of the concentrations.Of each sample, one additional reserve sample was retained.
Due to the fact that the test substance preparations were true solutions, it was not considered necessary to prove homogeneity through analytical procedures. All measured values for 1-Vinylimidazol were in the expected range of the target concentrations (90 - 110%), demonstrating the correctness of the diet preparations.
Details on mating procedure:
In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group. The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 07.00 - 09.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted "GD 0" and the following day "GD 1".

The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 07.00 - 09.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted "GD 0" and the following day "GD 1".
Duration of treatment / exposure:
The duration of treatment covered a 2-week pre-mating and a mating period in both sexes. In males treatment lasted 30 days after beginning of administration. Females were treated the entire gestation period as well as approximately 2 weeks of the lactation period. Females were sacrificed 50 days after beginning of administration.
Frequency of treatment:
Once daily at approximately the same time in the morning.
Duration of test:
50 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
5 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
35 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The oral route was selected since administration by gavage has been proven to be appropriate for the detection of a toxicological hazard.
Prior to the OECD422 study, a 14-d dose range finding stuy in Wistar rats was performed, in order to find the apropriate dose regimen for the main study. With 3 animals/sex and group, 0, 35, 75, 125 and 250 mg/kg bw/d 1-vinylimidazole were administered to male and female rats per gavage (01R0335/00R18; BASF, 2012). Besides the clinical observations, food consumption and body weights were examined at day 0, 3, 7, 10 and 14 and the animals were assessed post-mortem by gross pathology. In the highest dose group 3/3 males and 2/3 females were sacrificed moribund, the third female was found dead. In the 125 mg/kg bw/d dose group all males and females were sacrificed moribund. In the 75 mg/kg bw/d dose group one female was sacrificed moribund, all other animals were sacrificed scheduled. In the lowest dose group all animals were sacrificed scheduled. Statistically significant reduced food consumption compared to the control was observed in all males and female groups exept for the low dose females receiving 35 mg/kg bw/d. Statistically significant decreased body weights were observed in the low dose females.
Due to the deaths occuring in the first range finder at doses >= 75 mg/kg bd/d, another 14-d range finder with male and female rats (4/sex and group) was considered to be neccessary with doses of 35 and 70 mg/kg bw/d per gavage (01R0492/11R136; BASF, 2012). This study additionally examined water consumption, clinical pathology (hematology, chlinical chemistry) and some organ weights (adrenal glands, kidneys, liver, spleen). All males and females receiving 1-vinylimidazole showed semiclosed eyelids and piloerection of the fur during the study. Compared to the controls, reduced food consumption was observed in both groups in males and females mainly within the first week of administration. A statistically significant increase in water consumption was observed only once in females of the lower dose group from day 3 to 7. Body weights of the males receiving 70 mg/kg bw/d were significantly reduced at the end of the exposure period (d13) compared to controls: 326.1 +/- 20.1 g vs 371.7 +/- 18.8 g (-12%). These males had a body weight loss of 4.1 g at study day 13 compared to day 0, where as the control animals showed a body weight gain of 43.0 g from day 0 to 13. Bodyweights of the mid and high dose females also showed a statistically significant reduction after the exposure period compared to controls: 229.0 +/- 3.3 g; 211.4 +/- 5.6 g (-8%); 201.1 +/- 10.6 g (-12%).
In conclusion, a dose of 70 mg/kg bw /d 1-vinylimidazole was considered to exceed the maximal tolerated dose, based on the significant body weight loss of the high dose males and the significant reduced body weight of > 10 % in both sexes after 14 days of exposure. Thus, 35 mg/kg bw/d was selected as the high dose for the main study according to OECD test guideline 422.

- Method of formulation: For the preparation of the administration solutions the test substance was weighed in a graduated flask depending on the dose group, topped up with drinking water and subsequently thoroughly mixed by shaking until it was completely dissolved.

- Parturition: The females were allowed to deliver and rear their pups until day 4 after parturition. The female animals were sacrificed 50 days after the beginning of the administration, and examined.

Examinations

Maternal examinations:
- Mortality / Viability: At twice daily on working days and once daily on other days.
- Clinical signs: A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each animal. Individual data of daily observations can be found in the raw data. The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis. On weekdays (except Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.
- Food consumption: Generally, food consumption was determined once a week for male and female parental animals, 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 F0 animals). Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0, 7, 14 and 20. Food consumption of F0 females which gave birth to a litter was determined on PND 1 and 4. Food consumption was not determined in females without positive evidence of sperm during the gestation period and in females without litter during the lactation period.
- Body weights: In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice. The body weight change of the animals was calculated from these results. The following exceptions are notable for the female animals: During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. Females with litter were weighed on the day of parturition (PND 0) and on PND 4. Females without positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume.
- Detailed clinical observations (DCO): Detailed clinical observations were performed in all animals once prior to the first administration and at weekly intervals during the administration period. The examinations started in the morning. The findings were ranked according to the degree of severity, if applicable. For observation, the animals were removed from their cages by the investigator and placed in a standard arena (50 x 37.5 cm wide, with side borders which are 25 cm high). The following parameters listed were assessed: 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.
- Functional Observations: for details see IUCLID section 7.5.1
- General 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. The pairing partners, the number of mating days until vaginal sperm was detected and gestational status was recorded for F0 females.
- Clinical laboratory investigations: In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane (Isoba®, Essex GmbH Munich, Germany). The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. Urine samples were evaluated in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results of clinical pathology examinations were expressed in Inter¬national System (SI) units.
- Haematology: The following parameters were determined in blood with EDTA K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany). Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RET). Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany): Prothrombin time (Hepato Quick’s test) (HQT).
- Clinical biochemistry: The following clinical biochemistry parameters were determined using An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany): Alanine aminotransferase, Aspartate aminotransferase, Alkaline Phosphatase,γ-Glutamyltransferase, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Total protein, Albumin, Globulins,Triglycerides, Total Bilirubin, Bile acids, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate.
- Urinalysis: The dry chemical reactions on test strips (Combur 10 test M, Roche, Mannheim, Germany) used to determine urine constituents semiquantitatively were evaluated with a reflection photometer (Miditron M; Roche, Mannheim, Germany): pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color (turbidity), Volume.

POSTMORTEM EXAMINATIONS:
- Necropsy: All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology; special attention was given to the reproductive organs. Female animal No. 102 was sacrificed moribund 36 days after start of exposure and was necropsied as soon as possible after its death and assessed by gross pathology. The following organs or tissues of all parental animals were fixed in 4% buffered formaldehyde solution or modified Davidson’s solution: All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, cortex, Cecum, Cervix, Colon, Coagulation gland, Duodenum, Epididymides, Eyes (with optic nerve), Esophagus, Extraorbital lacrimal glands, Epididymides (modified Davidson’s solution), Femur including with kneejoint, Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Larynx, Liver, Lung, Lymph nodes (axillary and mesenteric), Mammary gland (male and female), Nose (nasal cavity), Ovaries (modified Davidson’s solution), Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate gland, Rectum, Salivary glands (mandibular, sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with bone marrow, Stomach (forestomach and glandular stomach), Testes (modified Davidson’s solution), Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina. The ovaries of female animal No. 102 that was sacrificed moribund 36 days after start of exposure were fixed in 4% buffered formaldehyde solution.
- Organ weights: The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): Adrenal glands, Brain, Heart, Kidneys, Liver, Ovaries, Spleen, Thymus. The following weights were determined in all animals sacrificed on schedule: Epididymides, Testes.
- Histopathology: Fixation was followed by histotechnical processing, examination by light microscopy: Adrenal glands, All gross lesions, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum,,Epididymides, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Lymph nodes (axillary and mesenteric), Ovaries, Oviducts, Prostate gland, Peyer’s patches, Rectum, Sciatic nerve, Seminal vesicles,Spinal cord (cervical, thoracic, lumbar), Spleen, Stomach (forestomach and glandular stomach), Testes,,Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus. Special attention was given to stages of spermatogenesis in the male gonads. Female animal No. 102 that was sacrificed moribund 36 days after start of exposure was processed histotechnically and assessed with all organs listed above, which increases the number of female control animals to 6. A correlation between gross lesions and histopathological findings was attempted.
Fetal examinations:
LITTER EXAMINATIONS:
All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups. At necropsy on PND 4, all pups were sacrificed with CO2, under isoflurane anesthesia, and examined macroscopically for external and visceral findings.
All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically. All pups without notable findings or abnormalities were discarded after their macroscopic
evaluation. Animals with notable findings or abnormalities with regard to dissecting aneurysms in the great vessel of the heart were also evaluated microscopically.
Statistics:
Means, medians and standard deviations of each test group were calculated for several parameters.
Blood parameters: For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians. For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
Urinalysis parameters: Urine pH, volume, specific gravity, color and turbidity: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p- value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians. Urine color and turbidity are not evaluated statistically. Other urinary parameters: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
Pathology weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.
Indices:
REPRODUCTION INDICES
- Male mating index (%) = (number of males with confirmed mating / number of males placed with females) x 100
- Male fertility index (%) = (number of males proving their fertility / number of males placed with females) x 100
- Female mating index (%) = (number of females mated / number of females placed with males) x 100
- Female fertility index (%) = (number of pregnant females / number of females mated) x 100
- Gestation index (%) = (number of females with live pups on the day of birth / number of pregnant females) x 100

OFFSPRING INDIECES
For each group, the following calculations were performed:
- Live birth index (%) = (number of liveborn pups at birth / total number of pups born) x 100
- Postimplantation loss (%) = (number of implantations – number of pups delivered / number of implantations) x 100
- Viability index = (Number of live pups on Day 4 post partum / Number of pups born alive) x 100
- Sex ratio = (number of live male or female pups on day 0/4 / number of live male and female pups on day 0/4) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Details on results:
- Mortality: There were no test substance-related mortalities in any of the male and female parental animals in any of the groups. During study week 1, one control female (0 mg/kg bw/d) was sacrificed moribund, after showing labored respiration, piloerection and poor general state.
- Clinical observations: Four of ten high-dose (35 mg/kg bw/d) male and six of ten high-dose female F0 generation parental animals as well as one of ten mid-dose (15 mg/kg bw/d) male and three of ten mid-dose female F0 generation parental animals showed semiclosed eyelid and piloerection during the premating period. One high-dose male and one mid-dose female showed piloerection during premation days 1 – 3 and one mid-dose female showed piloerection during PND 1 - 2. These clinical observations were attributed to the treatment. No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in the low-dose (5 mg/kg bw/d) male or female F0 generation parental animals during the whole study including gestation and lactation periods.
- Food consumption: Food consumption of the high-dose F0 males (35 mg/kg bw/d) was statistically significantly below control during the whole premating period (up to 21%). The mid-dose F0 males (15 mg/kg bw/d) showed statistically significantly reduced food consumption during premating days 0 - 13 (about 9% below control) and during premating days 0 - 7 about 13% below control. The low-dose F0 males (5 mg/kg bw/d) did not show any test substance-related changes in food consumption during the whole treatment period. Food consumption of the high-dose F0 females was statistically significantly below control during premating days 0 - 13 (about 15%), during premating days 0 - 7 (about 23%), during GD 0 - 20 (about 9%), during GD 14 - 20 (about 12%) and during the whole lactation period (about 34%). The mid-dose F0 females showed statistically significantly reduced food consumption during the whole premating period (up to 16% below control). The low-dose F0 females did not show any test substance-related changes in food consumption during the whole treatment period. Food consumption of the mid-dose females was comparable to the concurrent control throughout the gestation and lactation period.
- Body weight data: The high-dose parental males had statistically significantly lower body weights on premating day 7 and 13 (about 4% below control, respectively) and on postmating day 0 (about 5% below control) as well as statistically significantly lower body weight change during premating days 0 - 13 (about 38% below control) and during premating days 0 - 7 (about 62% below control). The body weight gain of the mid-dose parental males was statistically significantly below control during premating days 0 - 13 (about 30%) and during premating days 0 - 7 (about 52%). The high-dose parental females had statistically significantly lower body weights during the whole lactation period (up to 13% below control). The body weights of the mid-dose parental females were statistically significantly below control on PND 0 (about 6%). Neither mean body weights nor mean body weight change of the F0 parental animals in the low-dose group were influenced by the test substance during the whole treatment period.
- Detailed clinical observations (DCO): Male and female animals of all dose groups (35, 15 and 5 mg/kg bw/d) did not show any abnormalities.
- Functional observational battery (FOB): for details see IUCLID section 7.5.1
- Clinical biochemistry: No treatment-related changes among hematological parameters were observed. No treatment-related changes among clinical chemistry parameters were observed. In females of test groups 2 and 3 (15 and 35 mg/kg bw/d) alkaline phosphatase (ALP) activities were higher compared to controls, but the values were within the historical controls range (ALP: 0.39-0.87 µkat/L). In males of test group 2 only (15 mg/kg bw/d), total bilirubin and albumin levels were increased. Both parameters were not anymore increased in test group 3 (35 mg/kg bw/d). Some parameters were changed in males of all test groups (test group 1, 2 and 3: 5, 15 and 35 mg/kg bw/d), but the means were either within historical control ranges (for triglycerides and chloride; historical control range: triglycerides 0.41-1.32 mmol/L, chloride 99.9-107.4 mmol/L) or some means were marginally out of the historical control ranges, but the values were not changed dose-dependently (for potassium and inorganic phosphate; historical control range: potassium 4.35-4.95 mmol/L, inorganic phosphate 1.36-1.96 mmol/L). Therefore, all mentioned changes were regarded as incidental and not treatment-related. Urea levels in males of test groups 2 and 3 (15 and 35 mg/kg bw/d) were higher compared to controls, but the values were not dose-dependently changed. The values were slightly above the historical control range (urea 4.91-7.42 mmol/L). However, this was the only relevantly altered parameter in these animals and therefore the change was regarded as maybe treatment-related, but not adverse (ECETOC, 2002).
No treatment-related changes among urinalysis parameters were observed. In rats of both sexes (in females not statistically significantly) of all test groups (test group 1, 2 and 3; 5, 15 and 35 mg/kg bw/d), urine pH values were higher compared to controls. Probably due to this in rats of both sexes of test group 3 (35 mg/kg bw/d) and additionally in females of test group 2 (15 mg/kg bw/d), more crystals (in males phosphate crystals, in females mainly unknown crystals) were found in the urine sediment, because of precipitation of these type of crystals in more alkaline urine. At least phosphate crystals were normal in urine sediments of controls, and higher levels per se without any other alteration of urine parameters were regarded as treatment-related, but not adverse.
- Macroscopic examination: All findings were considered to be incidental or spontaneous in origin and without any relation to treatment. Animal 102 which was sacrificed moribund showed a deposition on its lung.
- Organ weights: The terminal body weight was significantly decreased in males and females of test group 3 (35 mg/kg bw/day), which was considered to be treatment-related. Liver weight increases were regarded to be treatment-related and were correlated with centrilobular hypertrophy histopathologically.
The increased kidney weights in males and females of test groups 2 and 3 were outwith the historical control range and weights showed a dose response. Therefore the increase in kidney weights was regarded to be treatment-related. The increased relative testes weights in males of test group 3 (35 mg/kg bw/day) was related to the decreased terminal body weights in these animals. All other mean absolute and relative weight parameters did not show significant differences when compared to the control group 0.
- Microscopic examination: In the liver, treatment related centrilobular hypertrophy was observed in male and female animals with incidence and grading.
All other findings (including liver cell foci and few foci of necrosis) 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.
- Reproduction data: Animal 102 (sacrificed moribund) showed pleuritis correlating to deposit on the lung and atrophy and inflammation of the thymus with intralesional plant material. The female animals (Nos. 103, 111, 130) which were not pregnant as well as the male mating partners (Nos. 2, 3, 11, 30) were not examined histopathologically. Animals 33/ 133 did not show relevant histopathological lesions.

Maternal developmental toxicity

Details on maternal toxic effects:
The mean number of delivered F1 pups per dam and the rates of liveborn and stillborn F1 pups were comparable between test groups 0, 1 and 2. The statistically significantly increased number of stillborn pups (and therefore significantly reduced number of liveborn pups) in test group 3 reflects a treatment-related effect in this dose group. Statistically significant more pups died in the high-dose litters than control. That is, 19 died or were cannibalized in the high-dose group (35 mg/kg bw/d), compared to none dead/cannibalized in the control group. Consequently, the viability index as indicator for pup mortality during lactation (PND 0-4) was significantly lower in test group 3 (59.6%** [p<= 0.01]) than in the control and test groups 1-2 (i.e. 100% / 99.3% / 92.3% at 0, 5 and 15 mg/kg bw/d). The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature. There were no test substance related adverse clinical signs observed in any of the F1 generation pups of the different test groups. Mean body weights of the high-dose (35 mg/kg bw/d) male pups were statistically significantly below control during the whole lactation period (ranging between 17% on PND 1 and between 26% on PND 4). Mean body weights of the mid-dose (15 mg/kg bw/d) male pups were statistically significantly below control on PND 1 (about 16%). Mean body weights of the high-dose female pups were statistically significantly below control on PND 4 (22%). Both sexes combined showed statistically significantly decreased mean body weights in high-dose pups on PND 1 and 4 (about 17% and 27 % below control, respectively) and in mid-dose pups on PND 1 (about 14% below control). No test compound-related influence on F1 pup body weights were noted in all pups of test group 1 (5 mg/kg bw/d) during the whole lactation period, in female pups of test group 2 during the whole lactation period and in female pups of test group 3 on PND 1. Mean body weight change of the high-dose male and female pups were statistically significantly below control during the whole lactation period (ranging between 37% [females], 45% [males] and 46% [both sexes combined]. No test compound-related influence on F1 pup body weight change were noted in test group 2 and 1 during the whole lactation period. A few other pups showed spontaneous findings at gross necropsy, such as post mortem autolysis, reddish discolored kidney(s) and empty stomach. These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Pups of test group 2 (1 male, 3 females) and 3 (4 males, 4 females) showed aneurysms in the vessels macroscopically described as dilated (aorta, arteries or ductus arteriosus).

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:
- MORTALITY: The mean number of delivered F1 pups per dam and the rates of liveborn and stillborn F1 pups were comparable between test groups 0, 1 and 2. The statistically significantly increased number of stillborn pups (and therefore significantly reduced number of liveborn pups) in test group 3 reflects a treatment-related effect in this dose group. Statistically significant more pups died in the high-dose litters than control. That is, 19 died or were cannibalized in the high-dose group (35 mg/kg bw/d), compared to none dead/cannibalized in the control group. Consequently, the viability index as indicator for pup mortality during lactation (PND 0-4) was significantly lower in test group 3 (59.6%** [p<= 0.01]) than in the control and test groups 1-2 (i.e. 100% / 99.3% / 92.3% at 0, 5 and 15 mg/kg bw/d). - SEX DISTRIBUTION: The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.
- CLINICLA SIGNS: There were no test substance related adverse clinical signs observed in any of the F1 generation pups of the different test groups.
- BODY WEIGHTS: Mean body weights of the high-dose (35 mg/kg bw/d) male pups were statistically significantly below control during the whole lactation period (ranging between 17% on PND 1 and between 26% on PND 4). Mean body weights of the mid-dose (15 mg/kg bw/d) male pups were statistically significantly below control on PND 1 (about 16%). Mean body weights of the high-dose female pups were statistically significantly below control on PND 4 (22%). Both sexes combined showed statistically significantly decreased mean body weights in high-dose pups on PND 1 and 4 (about 17% and 27 % below control, respectively) and in mid-dose pups on PND 1 (about 14% below control). No test compound-related influence on F1 pup body weights were noted in all pups of test group 1 (5 mg/kg bw/d) during the whole lactation period, in female pups of test group 2 during the whole lactation period and in female pups of test group 3 on PND 1. Mean body weight change of the high-dose male and female pups were statistically significantly below control during the whole lactation period (ranging between 37% [females], 45% [males] and 46% [both sexes combined]. No test compound-related influence on F1 pup body weight change were noted in test group 2 and 1 during the whole lactation period.
- MACROSCOPIC EXAMINATION: A few other pups showed spontaneous findings at gross necropsy, such as post mortem autolysis, reddish discolored kidney(s) and empty stomach. These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Pups of test group 2 (1 male, 3 females) and 3 (4 males, 4 females) showed aneurysms in the vessels macroscopically described as dilated (aorta, arteries or ductus arteriosus).
- MICRSOCOPIC EXAMINATION: Micrsoscopic findings concerning dissecting aneurysms of the big vessels of the heart were confirmed by microscopic examination.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
other: dissecting aneurysms in the great vessels of the heart in MD and HD pups

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion