1-vinylimidazole

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A NOAEL for reproductive toxicity/fertility of 35 mg/kg bw/d (highest dose tested) was determined in a GLP compliant, OECD422 study.

Further testing for effects on fertility is considered to be scientifically not necessary for 1-vinylimidazole (VIN), CAS 1072-63-5, as other information is available justifiying the non-classification for effects on fertility and because VIN is a developmental toxicant classified and labelled as Repr. 1B;H360D with a SCL of 0.03% according to Annex VI of the CLP (Regulation (EC) 1272/2008), ensuring an adequate risk assessment with strict risk reduction measures.

Link to relevant study records

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

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Qualifier:

according to guideline

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. QA statement)

Remarks:

BASF SE Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 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 premating^1)	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 13^2)	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 0-13^2)	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 hypertrophy^1)	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 premating^1)	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 lactation^1)	0/8	0/9	1/9 (piloerection)	2/9 (complete litter loss)
Food consumption/d during premating^2)	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 0^2)	264.4 ± 16.5	258.2 ± 11.6	248.3 ± 13.2* (-6%)	235.1 ± 9.2** (-11%)
Body weight [g] at lactation day 4^2)	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 hypertrophy^1)	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

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

35 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a GLP compliant combined repeated dose toxicity study with reproduction/developmental toxicity screening test, 1 -Vinylimidazole was given to rats by oral gavage (2013, RL1). Four groups of ten male and ten female Wistar Han rats were exposed to the test substance at 5, 15, or 35 mg/kg/day. Rats of the control group received the vehicle, water, alone. 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. Accuracy, homogeneity and stability of formulations were demonstrated by analyses. Analyses confirmed the overall accuracy of the prepared concentrations and the homogeneity of the test substance in the vehicle. The stability of these preparations was also demonstrated over a period of 7 days under refrigerated conditions.

In both male and female mid- and high-dose parental animals, piloerection and semi closed eyelids were observed during pre-mating. One mid-dose female showed piloerection during postnatal days 1 and 2. No clinical signs of toxicity or changes of general behavior, which might have been attributed to the treatment were detected in the low-dose male or female generation parental animals during the whole study including gestation and lactation periods. These are considered to be adverse clincial observations. Reduced food consumption was observed in the male and female parental animals at the mid- and high-doses (15 and 35 mg/kg bw/d) during various study phases. This resulted in statistically significantly decreased body weights and body weight gains in comparison to the controls. These effects, while significant, were considered to be treatment-related and adverse. Concerning clinical pathology no treatment-related, adverse effects were observed up to a dose of the compound of 35 mg/kg bw/d. Regarding pathology, target organs were the liver and kidney in parental animals. However, the effects were regarded to be treatment related/adaptive, but not adverse.

The test substance did not influence fertility. Male and female reproduction data (mating index, fertility index, mean drration of gestation, gestation index, mean number of implantation sites, post-implantation loss, number of pups delivered) were not affected by the treatment.

The pups in the high-dose group (35 mg/kg bw/d) were much more likely to be stillborn, die, or be cannibalized in the first four days of life. As a result, both the live birth and viability indices were strongly reduced (74.5 and 59.6 %, respectively). Together, these effects were judged to be both test substance-dependent and adverse. In addition the pup body weights/weight gain were reduced at the 15 and 35 mg/kg bw/d dose. Upon gross pathological examination, a number of mid- and high-dose pups exhibited aneurysms of the great vessels of the heart. When these macroscopic alterations were examined microscopically in selected pups, histopathology revealed dissecting aneurysms in the dilated vessels (aorta, arteries or ductus arteriosus), which correlated overall with the macroscopic findings. The number of affected litters was two both in test groups 2 and 3 (15 and 35 mg/kg bw/d). All of these findings are considered to be treatment-related and adverse.

Under the conditions of the present OECD 422 study with the reproductive/developmental screening study, the NOAEL for general parental toxicity is 5 mg/kg bw/d, based on adverse clinical symptoms and decreased body weights/body weight gain. The NOAEL for reproductive toxicity/fertility is 35 mg/kg bw/d. The NOAEL for developmental toxicity in the F1 offspring is 5 mg/kg bw/d, as decreased pup weights and dissecting aneurysms in the great vessels of the heart were noted at 15 mg/kg bw/d and above.

Waiver for the EOGRTS:

According to the REACH regulation (EC)1907/2006, Annex X, section 8.7.3, column 1 an EOGRTS with basic test design is a standard information requirement for substances manufactured or imported in quantities of 1000 tonnes or more. However, if a substance is known to cause developmental toxicity, is classified as toxic for reproduction Cat. 1A or 1B: May damage the unborn child (H360D), and the available data are adequate to support a robust risk assessment, then no further testing for developmental toxicity will be necessary, but testing for effects on fertility must be considered (REACH, Annex X, section 8.7, column 2).

In December 2016 RAC decided on a harmonised classification and labelling for 1-vinylimidazole (VIN), CAS 1072-63-5, which was included in the 13th ATP of the Regulation (EC) 1272/2008 (CLP) in October 2018. Thus, VIN is classified as Repr. 1B;H360D: May damage the unborn child with a SCL of 0.03%, based on adverse findings in an OECD422 study; e.g. dissecting aneurysms in the great vessels of the heart in pups of the test groups 15 and 35 mg/kg bw/d.

As mentioned above, with this classification for developmental toxicity, an adaptation for further testing for effects on fertility might be possible.

Further testing for effects on fertility is considered to be scientifically not necessary for VIN, as other information is available justifiying the non-classification for effects on fertility: In particular, from the results obtained in a reproduction/developmental toxicity screening test according to OECD test guideline 422 and 90-d oral gavage study in rats did not indicate that VIN has adverse effects on fertility. In the OECD422 study (2013, RL1) no changes in weight and histopathology of reproductive organs (uterus, ovaries, oviducts, vagina, testes, epididymis, prostate gland, seminal vesicles) were found up to the highest dose tested (35 mg/kg bw/d). Male and female mating indices, pre-coital time, gestation index, post-implantation loss, litter size and sex ratio were not affected. In addition, no indications of adult neurotoxicity were observed. Additionally, in the 90-d study in rats (1991, RL2) no adverse findings on reproductive organs were observed during the gross pathology, supporting the findings of the OECD422 study.

This NOAEL for effects on fertility is not based on adverse effects on fertility at higher doses, but based on the general dose limitation for VIN. In a dose range finding study (2013, cited in the IUCLID study entry for the OECD422 study) 70 mg VIN/kg bw/d was proven to exceed the maximal tolerated dose, as a statistically significant body weight loss was observed in males and the body weight was statistically significantly reduced in both sexes (> 10 %) after 14 days of exposure. Thus, even if a further reproduction toxicity study would be performed, higher dosing would not be possible, and detecting any adverse effects on fertility is rather unlikely based on the available data.

With the available data a robust risk assessment is possible: VIN is only used in industrial settings, thus no wide-dispersive uses are expected. Furthermore, the risk assessment for occupational exposure is based on the NOAEL for systemic toxicity (5 mg/kg bw/d) from the OECD422 study as the most sensitive toxicological endpoint. In contrast, the NOAEL for effects on fertility is 7x higher. 

Based on the available data for reproductive toxicity and the fact that strict risk reduction measures are already in place because VIN is a developmental toxicant classified and labelled as Repr. 1B;H360D with a SCL of 0.03% according to Annex VI of the CLP, a further animal study addressing the effects of VIN on fertility is not required.

Effects on developmental toxicity

Description of key information

The NOAEL for developmental toxicity in the F1 offspring of a GLP compliant, OECD422 study is 5 mg/kg bw/d, as decreased pup weights, perinatal mortality, and dissecting aneurysms in the great vessels of the heart were noted at 15 mg/kg bw/d and above.

No further testing for developmental toxicity is necessary, because 1-vinylimidazole, CAS 1072-63-5 is a developmental toxicant classified and labelled as Repr. 1B;H360D with a SCL of 0.03% according to Annex VI of the CLP (Regulation (EC) 1272/2008), based on the results of this OECD422 study.  

Link to relevant study records

Reference

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 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:

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

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.

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

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.

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).

Key result

Dose descriptor:

NOAEL

Effect level:

5 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

Abnormalities:

no effects observed

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.

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

Developmental effects observed:

not specified

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

5 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

1-vinylimidazole caused developmental toxicity and teratogenicity in a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD TG 422 (2013, RL1). Severe effects on embryo-fetal development including increased pup mortality at delivery and during lactation, decreased pup weights and an increased rate of malformations in the pericardial vessels at the mid and high dose (15 and 35 mg/kg bw/d) were observed. According to the CLP regulation, adverse effects on development include (1) death of the developing organism (2) structural abnormality (2) altered growth, and (4) functional deficiency. The observed effects for 1-vinylimidazole fall at least into the categories 1 - 3 of these manifestations and are therefore considered to be clear evidence of an adverse impact on development. The extent of systemic toxicity induced in the F0 generation in the mid and high dose group (moderately decreased food consumption during gestation period, reduced body weights in females during lactation, minimal to moderate centrilobular hepatocellular hypertrophy) is not considered to be attributable to the severe degree of toxicity in the offspring (teratogenicity).

In December 2016 RAC decided on a harmonised classification and labelling for 1-vinylimidazole (VIN), CAS 1072-63-5, which was included in the 13th ATPof the Regulation (EC) 1272/2008 (CLP)in October 2018. Thus, VIN is classified as Repr. 1B;H360D: May damage the unborn child with a SCL of 0.03%, based on adverse findings in the OECD422 study; e.g. dissecting aneurysms in the great vessels of the heart in pups of the test groups 15 and 35 mg/kg bw/d.

It is not possible to assess the effects of the substance on or via lactation due to the experimental design of the study (e.g. pups only investigated until day 4 postnatally, no information on presence of substance or metabolites in milk, measurement of milk yield not studied) although the findings maybe mainly attributed to the teratogenic and fetotoxic potential of 1-vinylimidazole. Based on currently available data, classification for effects on or via lactation is therefore not warranted.

Mode of Action Analysis / Human Relevance Framework

not specified

Justification for classification or non-classification

The substance does not meet the criteria for classification in Category 1 or 2 for adverse effects on sexual function and fertility.

Based on clear evidence for development toxicity in an animal study as indicated by increased pup mortality, decreased pup weights and dissecting aneurysms in the great vessels of the heart, 1-vinylimidazole is a developmental toxicant classified and labelled as Repr. 1B;H360D with a SCL of 0.03% according to Annex VI of the CLP (Regulation (EC) 1272/2008).

Based on currently available data, classification for effects on or via lactation is therefore not warranted.