Imidazole

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

One- or two-generation studies are not available. However, a 90-day repeated dose toxicity study performed with imidazole and reproductive toxicity studies performed with five imidazole derivatives are available (1-methylimidazole (CAS no. 616-47-7), 2-methylimidazole (CAS no. 693-98-1), 2-ethyl-4-methylimidazole (CAS no. 931-36-2), 1,2-dimethylimidazole (CAS no. 1739-84-0) and 1-(3-Aminopropyl) imidazole (CAS no. 5036 -48 -6). By modelling the potential metabolism of these analogues in the liver, it is suggested that 1-methylimidazole would toxicologically be closest to imidazole as the target compound is a predicted metabolite of the source substance 1-methylimidazole. It is therefore suggested that a qualitative read-across can be performed for the endpoint fertility impairing effects based on the close structural and the metabolic similarities between imidazole and its analogue 1-methylimidazole. Furthermore, a general concern for fertility effects due to the imidazole substructure is assessed taking also into account data on relevant reproductive toxicity endpoints for all five source substances.

Target chemical:

Imidazole (CAS no. 288-32-4)

Parameters relevant to assess effects of imidazole on fertility were included in a 90 -day repeated dose toxicity study performed according to OECD 408, in which Wistar rats (10 per sex and dose group) were dosed with 0, 20, 60, 180 mg imidazole/kg bw/day via gavage. In this study no changes in weight and histopathology of reproductive organs (uterus, ovaries, oviducts, vagina, female mammary gland, left testes, left epididymis, prostate gland, seminal vesicles) were found at all dose levels. Moreover, the test substance did not cause any effects on sperm parameters (motility, morphology, head count in cauda epididymis and testis) and estrus cyclicity. In addition, no indications of adult neurotoxicity were observed.

Main source substance:

1 -methylimidazole (CAS no. 616-47 -7)

Additional supportive source substances:

2-methylimidazole (CAS no. 693-98-1), 2-ethyl-4-methylimidazole (CAS no. 931-36-2), 1,2-dimethylimidazole (CAS no. 1739-84-0) and 1H-imidazole-1-propylamine (CAS no. 5036-48-6).

These source substances were all tested according to OECD guideline 421 (reproduction/developmental toxicity screening test) or 422 (combined 28 -days repeated dose toxicity study with reproduction/developmental toxicity screening test) and GLP.

 

1-methylimidazole (CAS no. 616-47-7)

In a GLP compliant combined oral repeated dose toxicity study and reproduction/developmental toxicity screening test performed according to OECD 422, 1 -methylimidazole was administered to Wistar rats by oral gavage at doses of 10, 30 and 90 mg/kg bw/day. Regarding clinical pathology, slight dysregulations in the liver cell metabolism of male and female rats of test group 3 (90 mg/kg bw/day) can be assumed because of higher urea levels in both sexes, indicating an increased protein metabolism, as well as higher cholesterol levels in males. In addition, in males of the same test group chloride levels were low whereas inorganic phosphate levels were high. In these animals, a slight functional effect on the kidneys cannot be excluded; this was confirmed by higher incidences of transitional epithelial cells as well as phosphate crystals in the urine. No 1 -methylimidazole related findings were noted in any of the other parameters investigated in this study (mortality / viability, clinical signs, functional observations other than for locomotor activity, body weight, food consumption, haematology, macroscopy, organ weights and histopathology). All other measures of reproductive performance or fertility were normal in both genders and at all doses. Therefore, no alterations to reproductive performance or fertility were identified in the parental rats. No effects on pup body weights or pathology at necropsy were observed; thus no substance-dependent developmental toxicity was identified at any dose. All other findings recorded were considered to be incidental in nature and unrelated to treatment. Thus, under the conditions of this reproduction/developmental toxicity screening test the NOAEL for general, systemic toxicity of 1-methylimidazole is 30 mg/kg bw/day for the F0 parental animals based on the increased urea levels in both sexes at 90 mg/kg bw/day. Additionally, males exhibited signs of increased serum inorganic phosphate and cholesterol levels, as well as decreased serum chloride concentration and a higher incidence of both transitional epithelial cells and phosphate crystals in the urine at this dose. The NOAEL for fertility in the F0 parental rats is 90 mg/kg body weight/day, the highest dose tested. The NOAEL for developmental toxicity was 90 mg/kg body weight/day, the highest dose tested.

In addition, 1-methylimidazole has recently been tested according to OECD guideline 408 in rats (BASF SE, 2016b). 1-Methylimidazol was administered by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0, vehicle control), 10 (test group 1), 30 (test group 2) and 90 mg/kg body weight/day (mg/kg bw/d; test group 3) over a period of 3 months. Clinical pathology indicated a slight effect on liver cell metabolism and a slight functional effect on the kidneys male and female rats at the highest dose. Therefore, the no observed adverse effect level (NOAEL) was 90 mg/kg bw/d for male and female Wistar rats.Regarding pathology, the liver was the target organ. Males of all test groups and females at 30 and 90 mg/kg bw/day revealed a minimal to slight centrilobular hypertrophy which however was not accompanied by any adverse histopathological findings. This was therefore regarded to be treatment-related but not to be adverse.No effects on the reproductive organs were observed.

2-methylimidazole (CAS no. 693-98-1)

2-methylimidazole was tested in a GLP-compliant reproduction/developmental toxicity screening test performed according to OECD 421. 2-methylimidazol was given to Wistar rats by oral gavage at dose levels of 50, 150 and 500 mg/kg bw/day.

Systemic parental toxicity: The daily clinical observations revealed no adverse treatment-related changes. Salivation occurred in male and female mid- and high-dose parental animals throughout major parts of the treatment period. Discoloration was observed in the urine and feces of nearly all mid- and high-dose animals. Two high-dose females died during or shortly after parturition, both showing signs of complicated parturition preceding death (undelivered pups, umbilical cords not cut, new-borns not nursed). As a consequence their pups either died or had to be killed for humane reasons. Slightly reduced food consumption was observed in high-dose females during premating (13%) and lactation (20%). This resulted in statistically significantly decreased body weights (7%) and body weight gains (18%) compared to the controls.

The weights of the testes and epididymides, necropsy findings at scheduled termination and histopathological examination of the testes, epididymides and ovaries revealed no treatment-related changes in the parental animals. Male and female mating and fertility indices, pre-coital time, gestation index, post-implantation loss, litter size and sex ratio were not affected by treatment. Slightly longer gestation was observed in high-dose females compared to concurrent controls. Gestation length in the high-dose group was similar to historical control data from OECD screening studies and within the historical control range from multi-generation studies. However, considering the two high-dose dams which died having parturition difficulties, an influence of 2-methylimidazole on normal term delivery cannot be excluded. The pups in the high-dose group 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 index and viability index (PND 0-4) were strongly reduced (to 90% and 59%, respectively). In addition, 6 runts were born into the high-dose group. Together, these effects were judged to be treatment-related and adverse. Mean body weight and body weight change of the high-dose pups were slightly reduced during lactation, however, without attaining statistical significance. Upon gross pathological examination of the pups, aneurysms of the great vessels of the heart were observed in the low-, mid- and high-dose group but not in the control group. The number of animals and aneurysms increased with increasing dose levels. Under the conditions of this Reproduction/Developmental Toxicity Screening Test the oral administration by gavage of 2-methylimidazol to male and female Wistar rats resulted in signs of systemic toxicity (mortality shortly after parturition, slightly reduced body weight and food consumption in parental females), increased gestation length, reduced live birth index and reduced viability index PND 0-4, and an increased number of runts at 500 mg/kg bw/day. Dissecting aneurysms in the aorta were observed from the lowest dose level and their incidence increased with dose. In conclusion, 150 mg/kg bw/day was a NOAEL for general toxicity in the parents based on mortality (shortly after parturition), slightly reduced body weight and food consumption. In summary, a NOAEL for developmental toxicity could not be established because dissecting aneurysms occurred in the great vessels of the heart up to the lowest dose tested (50 mg/kg bw/day). Regarding reproductive performance, no adverse effects were observed on the reproductive organs or on fertility (i.e. reproductive functions up to and including implantation) up to the highest dose (500 mg/kg bw/d).

2-ethyl-4 -methylimidazole (CAS no. 931-36 -2)

In a GLP compliant combined 28 -days repeated dose toxicity study with reproduction/developmental toxicity screening test, performed in accordance with OECD 422, four groups of ten male and ten female Wistar Han rats were exposed to the test substance at 15, 50, or 150 mg/kg/day by oral gavage. Rats of the control group received the vehicle, water, alone. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 43-56 days, i.e. during 2 weeks prior to mating, during mating, during postcoitum, and during at least 4 days of lactation. Accuracy, homogeneity and stability of formulations were demonstrated by analyses. Locomotor activity as determined by total movements and ambulations were comparable in all groups (both sexes). Mean values remained within the normal range of biological variation. All groups showed a comparable habituation profile with high activity in the first interval that decreased over the duration of the test period. Lower levels of total protein and albumin at 150 mg/kg bw/day were noted for females, and higher liver weights (absolute and relative to body weight) were recorded at 15, 50 and 150 mg/kg bw/day for males. These changes were minor or occurred in the absence of a dose-related trend, means were within the range considered normal and/or histopathological support was absent. Therefore, these changes were not considered to be toxicologically relevant. No test substance related findings were noted in any of the other parameters investigated in this study (mortality / viability, clinical signs, functional observations other than for locomotor activity, body weight, food consumption, haematology, macroscopy, organ weights and histopathology). There were 9, 10, 7 and 8 litters available for evaluation in the control, 15, 50 or 150 mg/kg bw/day groups, respectively. No morphological findings were noted in the reproductive organs of the animals that failed to sire or deliver healthy pups. Moreover, there was no dose-dependency, and the sizes of the litters were within normal ranges up to 150 mg/kg bw/day. Therefore, the lower numbers of pregnant females in Groups 3 and 4 were considered to be a chance finding. The seven litters available for evaluation in the 50 mg/kg bw/day group was slightly lower than the advised minimum of eight litters as mentioned in the guideline. Nevertheless, sufficient data could be obtained from the remaining litters of this group and the other groups for an accurate evaluation of possible developmental effects. In conclusion, no effects on fertility and developmental toxicity was observed up to the highest dose level tested. Based on these results, a parental, fertility and developmental NOAEL of at least 150 mg/kg bw/day was derived.

1,2-dimethylimidazole (CAS No. 1739-84-0)

In a GLP compliant combined 28-days repeated dose toxicity study with reproduction/developmental toxicity screening test, 1,2 -dimethylimidazole was given to rats by oral gavage. Four groups of ten male and ten female Wistar Han rats were exposed to the test substance 7 days/week at 15, 50, or 150 mg/kg bw/day. Rats of the control group received the vehicle, water, alone. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 43-50 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Accuracy, homogeneity and stability of formulations were demonstrated by analysis. Salivation occurred post-dosing in most animals at 150 mg/kg bw/day and in individual males at 50 mg/kg bw/day. This finding was considered to be a local reaction to the taste of the compound and therefore not adverse. Pale faeces were noted for most females at 150 mg/kg bw/day, primarily towards the end of the study period. Given that body weight development and food intake was unaffected by treatment, and since blood analysis and morphological assessment did not reveal any toxicologically relevant changes, this finding was considered not to be of an adverse nature. No toxicologically significant changes were noted in any of the parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination). In addition, no toxicologically significant changes were noted in any of the reproductive parameters (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites) or in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy). Based on these results, a parental, fertility and developmental NOAEL of at least 150 mg/kg bw/day was derived.

N-(3-aminopropyl)-imidazole (CAS no. 5036-48-6)

In a OECD 421 study performed according to GLP, 1H-imidazole-1 -propylamine was given daily to 10 Wistar rats per sex and group per gavage at doses of 0, 40, 200 and 1000 mg/kg bw/day to screen for potential effects on fertility and developmental toxicity. The exposure lasted for at least 28 days in males and for at least 38 days in females (2 weeks prior to mating, during the mating period of max. 2 weeks, about 1 week post-mating for males and for the entire gestation period up to PND 4 in females). The examination of clinical signs and mortality, of food consumption, body weight and organ weights, the gross pathology, histopathology and the reproductive performance revealed no toxicologically relevant adverse effects in parental animals up to 1000 mg/kg bw/day. Furthermore, no toxicologically relevant developmental differences in the F1 pups were detected during this study considering viability, clinical signs, body weight and gross pathology of the F1 pups. Therefore, under the conditions of this study, the NOAEL for fertility as well as for developmental toxicity in the F1 offspring is 1000 mg/kg bw/day.

Conclusion

An Extended One-Generation-Reproductive Toxicity Study is not available for imidazol. According to Council Regulation 1907/2006/EC, Annex IX, an Extended One-Generation-Reproductive Toxicity Study (B.56 of Commission Regulation on test methods or OECD 443), is required if the available repeated dose toxicity studies (e.g. 28-day or 90-day studies, OECD 421 or 422 screening studies) indicate adverse effects on reproductive organs or tissues or reveal other concerns in relation with reproductive toxicity.

However, from the results obtained in the 90-day oral gavage study in rats it can be concluded that imidazole has no adverse effect on fertility. In this study no changes in weight and histopathology of reproductive organs (uterus, ovaries, oviducts, vagina, female mammary gland, left testes, left epididymis, prostate gland, seminal vesicles) were found at all dose levels. Moreover, the test substance did not cause any effects on sperm parameters (motility, morphology, head count in cauda epididymis and testis) and oestrus cyclicity. In addition, no indications of adult neurotoxicity were observed. Additionally there is sufficient data available on analogue substances that make a weight of evidence assessment for fertility effects possible. These data show no adverse effects on fertility.

In addition, read-across from the key analgoue 1-methylimidazole using experimentally derived data from a repeated-dose study (OECD TG 408) and a combined oral repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD TG 422) does not indicate a specific alert of a fertility impairing effect. In addition, this approach is supported by available screening studies according to OECD TG 421 or 422 for additional four structurally closely related imidazole analogues which uniformly did not show a fertility impairing effect.One substance, 2-methylimidazole, showed developmental toxicity (classification as Repro 1B, H360D), but there was no evidence of a fertility impairing effect or damage of the reproductive organs in the same study.

Imidazole is already classified with Repr 1B (H360D) as in a standard developmental toxicity study in rats oral administration resulted in developmental toxicity and teratogenicity (NOAEL developmental toxicity and teratogenicity 60 mg/kg bw/day, LOAEL developmental toxicity and teratogenicity 180 mg/kg bw/day). The classification was confirmed by RAC in 2013 and the classification and labelling was included in the 7^thATP to the CLP. This classification and labelling has to be applied from January 1^st2017 onwards at the latest. For this reason, the exposure to the substance should be minimized and the strict workplace safety precautions and regulatory measures are already in place due to its known developmental toxic property.Based on the toxicological results for imidazole and analogue derivatives and the fact that strict risk reduction measures are already in place because imidazole is a developmental toxicant classified with Repr 1B (H360D), a further animal study addressing the fertility effects of imidazole is not required. Accordingly, the information from a new extended-one-generation reproductive toxicity study (OECD TG 443) relevant for the risk assessment of the substance can be regarded as minimal.Also, taking into account animal welfare reasons an additional study should not be conducted.

Short description of key information:
No changes of the male and female reproductive organs including sperm quality and estrus cycle were noted in any of the dose groups up to and including 180 mg/kg bw/d in a rat 90-d oral gavage study with imidazole. In addition, the key analogue 1-methylimidazole and four additional supportive imidazole derivatives tested for reproductive toxicity under GLP and according to OECD 421 and 422 did not exert effects on fertility which indicates that the imidazol substructure does not possess and inherent hazard for fertility effects. Please see further details in attached Weight of Evidence Assessment in chapter 13 of the IUCLID.

Link to relevant study records

Reference

Reference 1

Endpoint:

toxicity to reproduction

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reproductive effects observed:

not specified

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test, July 2000)

GLP compliance:

yes

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 start of treatment: 11-13 weeks
- Weight at start of treatment: Males: 297.8-324.2g, Females: 195.0-238.7g
- Housing: During the study period, the rats were housed individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²). 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. Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data). Wooden gnawing blocks (Type NGM E-022) supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria, were added for environmental enrichment.
- Diet: Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, available ad libitum.
- Water: drinking water (from water bottles), available ad libitum.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 1% carboxymethylcellulose in drinking water

Details on exposure:

- Amount of vehicle: 10 mL/kg body weight.
- Preparation of test substance formulations: The test substance was weighed in a calibrated beaker, topped up with the vehicle and intensely mixed with a homogenizer. During administration, the formulations were kept homogeneous with a magnetic stirrrer. Fresh formulations were prepared at the start of the administration period and therafter at maximum intervals of 7 days, which took into account the analytical results of the stability verification.

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 test 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 was detected was denoted gestation day (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 for stability, homogeneity and concentration control of the test-substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany, in compliance with the Principles of Good Laboratory Practice. The samples were analysed using a validated HPLC method.
The stability of the test substance in the vehicle upon storage in a refrigerator for a period of 7 days was demonstrated before the start of the toxicity study in a similar batch (BASF Project No. 01Y0098/02Y003).
Samples of the test substance preparations, taken at the beginning of the administration period, were sent to the analytical laboratory once during the study period for verification of the concentration and homogeneity. Three samples (one from the top, middle and bottom) were taken from the beaker with a magnetic stirrer running. The measured concentrations were in the range between 90% and 110% of the nominal concentration for all samples. The low relative standard deviations (4.5% and 1.4% for the low- and high-concentration, respectively) indicated that the test substance was homogeneously distributed in the vehicle.

Duration of treatment / exposure:

Males were exposed for 28 days, namely during 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
Females were exposed during 2 weeks prior to mating, during mating (max. of 2 weeks), during gestation and during 4 days of lactation (last dose on the day prior to scheduled necropsy).

Frequency of treatment:

Once daily at about the same time in the morning (females in labor were not treated).

Remarks:

Doses / Concentrations:
50, 150 and 500 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Selection of doses: By request of the sponsor.
- Parturition: The females were allowed to litter and rear their pups until day 4 after parturition. Pups were sacrificed on PND 4 and gross necropsied.

Parental animals: Observations and examinations:

- Mortality / Viability: A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.
- 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 affected animal. 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-hours 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 (F0 animals) during the mating period (male and female F0 animals); Food consumption of the F0 females with evidence of sperm was determined on GD 0-7, 7-14, 14-20; Food consumption of F0 females which gave birth to a litter was determined for PND 1-4. Food consumption was not determined in females without positive evidence of sperm during mating and gestation periods and in females without litter during the lactation period.
- Body weight data: Body weight was determined three days before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning). The body weight change of the animals was calculated from these results. The following exceptions are notable for the female animals: During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20; Females with litter were weighed on the day of parturition (PND 0) and on PND 4; Females without a litter and females without positive evidence of sperm in the vaginal smear were weighed weekly (these body weight data were solely used for the calculations of the dose volume).
- General reproduction and delivery 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. Male and female mating and fertility indices, gestation index, gestation length and post-implantation loss were calculated. The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters.

Litter observations:

- Pup number and status at delivery: 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 examined for macroscopically evident changes. Pups that 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. 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 PND 0-4 was calculated.
- Sex and 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.
- Clinical observations: The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.
- Body weights: The pups were weighed on the day after birth (PND 1) and on PND 4. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning). “Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.

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. Two females (No. 136 and 140 of the high-dose group) which died intercurrrently were necropsied as soon as possible after their death and assessed by gross pathology.
- Terminal body weight and organ weights: The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, Epididymides, Testes.
- Tissue preservation: The following organs or tissues were fixed in 4% buffered formaldehyde solution or in modified Davidson’s solution: All gross lesions, Cervix, Coagulating glands, Epididymides (modified Davidson’s solution), Ovaries (modified Davidson’s solution), Oviducts, Prostate gland, Seminal vesicles, Testes (modified Davidson’s solution), Vagina, Uterus. The ovaries of the females that died intercurrently were fixed in 4% buffered formaldehyde solution.
- Histopathology: Fixation was followed by histotechnical processing (HE staining) and examination by light microscopy of the Testes, Epididymides and Ovaries of all animals of the control group and the high-dose group.

Postmortem examinations (offspring):

- Necropsy: All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia with CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically, paying particular attention to the heart and aortic vessels. All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and examined for possible defects and/or the cause of death, paying particular attention to the heart and aortic vessels. Moribund pups that were sacrificed before PND 4 were also eviscerated and assessed macroscopically, with special attention given to the basis of the heart and the great vessels.
- Preservation: All pups with findings and 10 control pups (5/sex) were preserved in toto in 4% neutral buffered formaldehyde after gross examination for further histopathological examination. All remaining pups without notable findings or abnormalities were discarded after their macroscopic evaluation.
- Histopathology: Fixation was followed by histotechnical processing and examination by light microscopy of the basis of the heart and great vessels of 5 male and 5 female pups of the control group and all affected pups of the low-, mid- and high-dose groups. The pups were stained with Hart stain combined with Masson-Goldner-Trichrome stain for detection of elastic and collagen fibers of the vessel walls.

Statistics:

The following statistical methods were used to analyze the data:
- Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), number of mating days, duration of gestation, number of implantation sites, postimplantation loss and percentage postimplantation loss, number of pups delivered per litter: Simultaneous comparison of all dose groups with the control group using the DUNNETT test (two-sided) for the hypothesis of equal means.
- Male and female mating indices, male and female fertility indices, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups canniballized, pups sarificed moribund, viability index, number of litters with affected pups at necropsy: Pairwise comparison of each dose group with the control group using FISHER'S EXACT test for the hypothesis of equal proportions.
- Proportions of affected pups per litter with necropsy observations: Pairwise comparison of each dose group with the control group using WILCOXON-test (one-sided) for the hypothesis of equal medians.
- Anesthetized animals and organ weights: 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
- Post-implantation loss (%) =( (Number of implantations - number of pups delivered) / number of implantations) 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
- Viability index (%) = (Number of live pups on Day 4 post partum / Number of pups born alive) x 100
- Sex ratio day 0/4 = ( 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
Two female animals of test group 3 (500 mg/kg bw/d) were found dead during the lactation period. High-dose female No. 136 was found dead on PND 2. For this animal some preceding abnormal clinical findings were recorded, such as yellowish discolored urine, light brown discolored feces, insufficient maternal care of pups and not consumed placentas. High-dose female No. 140 was found dead on PND 3 after showing chromodacryorrhea, piloerection, yellowish discolored urine, light brown discolored feces and a poor general state. Furthermore, this animal had undelivered pup(s) palpable in its abdomen on GD 22 and on PND 0, it showed salivation after treatment, its pups were not properly nursed and the umbilical cords were not cut. As a consequence, no more pups were alive on PND 1. There were no pathological findings which could explain these premature deaths.
There were no test substance-related or spontaneous mortalities in the F0 parental animals of test groups 1 and 2 (50 and 150 mg/kg bw/d).
CLINICAL SIGNS
All 10 male and 10 female F0 parental animals of the high-dose group (500 mg/kg bw/d) showed transient salivation during major parts of the treatment period, inclusive gestation and lactation. In the mid-dose group (150 mg/kg bw/d) nearly all female animals (7 out of 10 at a maximum) showed salivation after treatment during the premating, gestation and lactation periods. Salivation persisted in the respective animals only for some minutes after daily gavage dosing (i.e. up to 10 minutes) and was initially observed during study week 1. Since this salivation was transient and occurred directly after dosing, it was probably due to the irritation and/or taste of substance administration, and therefore was not assessed as adverse.
All high dose male and female F0 parental animals (500 mg/kg bw/d) had yellowish discolored urine during the entire study period, covering premating, gestation and lactation periods. In the mid-dose group (150 mg/kg bw/d) for all male and female F0 parental animals the discolored urine was recorded from study week 2 onwards until scheduled sacrifice. Furthermore, out of 10, respectively, light brown discolored feces were noticed in 3 high-dose females during GD 21-22 and 8 high-dose females during PND 0-4. This discoloration of urine and feces was probably a result of the metabolism and subsequent excretion of the test compound. While this effect was substance-dependent, it was judged not to be adverse.
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any male or female F0 generation parental animals at 50 mg/kg bw/d during the different study periods.
BODY WEIGHT
Mean body weights and mean body weight gain of the F0 males in test groups 1-3 (50, 150 or 500 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study.
On the whole, mean body weights and mean body weight gain of the high-dose F0 females (500 mg/kg bw/d) were comparable to the control throughout the entire premating, gestation and lactation periods. However, if calculated for GD 0-20, mean body weight gain was statistically significantly reduced in this test group (-18%) and, on PND 0, mean body weights were slightly, but statistically significantly, decreased in these animals (-7%).
Mean body weight and mean body weight gain of the F0 females in test groups 1 and 2 (50 and 150 mg/kg bw/d) were comparable to the concurrent control throughout the entire premating, gestation and lactation periods.
FOOD CONSUMPTION
Food consumption of the substance-treated F0 parental males (test groups 1-3; 50, 150 or 500 mg/kg bw/d) was comparable to the concurrent control group throughout the entire study period.
Food consumption of the high-dose F0 parental females (500 mg/kg bw/d) was slightly reduced during premating, gestation and lactation periods, attaining statistical significance in premating week 0-1 (13% less in comparison to the control) and PND 1-4 (20% less).
Low- and mid-dose F0 parental females (50 and 150 mg/kg bw/d) did not show any test substance-related changes in food consumption during the whole treatment period.
REPRODUCTIVE PERFORMANCE
- Copulation was confirmed for all F0 parental males and females which were placed with females to generate F1 pups. Thus, the male and female mating index was 100% in all groups including the controls. The mean duration until sperm was detected (GD 0) varied between 2.3 and 3.3 days without any relation to dosing.
- Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter. Two control males (Nos. 3 and 9) did not generate F1 pups. These two apparently infertile male rats did not show relevant gross lesions, which could explain the infertility.
One female rat (No. 111 - 50 mg/kg bw/d) was not detected as sperm positive, but had pups. All sperm positive rats delivered pups or had implants in utero except for two control females (Nos. 103 and 109, mated with males Nos. 3 and 9). The two non-pregnant control females had no relevant gross lesions. The male and female fertility index varied between 80% (control) and 100% (test groups 1-3). These values reflect the normal range of biological variation inherent in the strain of rats used for this study.
- The gestation index was 100% in all test groups. Although the high-dose value (22.5 days) was slightly, but statistically significantly increased, the mean duration of gestation was comparable between the test substance-treated groups and the control group (i.e. between 21.9 and 22.5 days). The longer gestation time in the high-dose group was similar, when compared to the historical control data for this rat strain from OECD screening studies (21.6 - 22.4 days) and still within the historical control range collected from multi-generation studies with this rat strain (21.5 - 22.5 days). However, considering the two high-dose dams which died having parturition difficulties, and influence of the test substance on normal term delivery cannot be excluded.
- Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the controls, taking normal biological variation into account (12.9 / 12.8 / 13.0 / 11.8 implants/dam in test groups 0-3 (0, 50, 150 and 500 mg/kg bw/d)). There were no statistically significant differences in post-implantation loss between the groups (8.2% / 7.5% / 12.0% / 9.2%), and the mean number of F1 pups delivered per dam remained unaffected (11.8 / 11.9 / 11.5 and 11.1 pups/dam at 0, 50, 150 and 500 mg/kg bw/d).
- Delivery data are presented under ‘Details on results (offspring)’.
ORGAN WEIGHTS
The mean absolute and relative organ weights (testes and epididymides) did not show significant differences when compared to the control group 0.
GROSS PATHOLOGY AND HISTOPATHOLOGY
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Dose descriptor:

NOAEL

Remarks:

parental

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on mortality (shortly after parturition) and slighly reduced body weight and food consumption in females at 500 mg/kg bw/day. Males showed no adverse effect up to 500 mg/kg bw/day.

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects on fertility were observed.

PUP NUMBER AND STATUS AT DELIVERY
The rate of liveborn pups was statistically significantly reduced in the high-dose group (500 mg/kg bw/d), as indicated by a reduced live birth index (100% in test groups 0 and 1, 97% in test group 2 and 90%** [p<=0.01] in test group 3) in this group. Moreover, the number of stillborn pups was statistically significantly increased in the high-dose group (0 / 0 / 4 / 11** [p<=0.01] pups/dam at test groups 0-3). This fact is mainly caused by high-dose dam No. 140, which had 7 stillborn pups (3 male / 4 female) in its litter (12 pups in total). However, the respective values were clearly outside the historical control range (HCD range: 0.0 - 7.3 stillborn pups/dam; 93 - 100% live birth index) and thus reflects a treatment-related effect in this dose group. The mean number of delivered pups per dam in the high-dose group (11.1) did not differ significantly from that in the control group (11.8).
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.
VIABILITY/MORTALITY
Statistically significantly more pups died in the high-dose litters than control. That is, 28 died in the high-dose group (500 mg/kg bw/d), compared to none dead in the control group. Furthermore, 3 pups were cannibalized in the high dose group in comparison to one cannibalized pup in the control and test group 1, respectively, and 2 cannibalized pups in test group 2. Consequently, the viability index as indicator for pup mortality during lactation (PND 0-4) was significantly lower in test group 3 (59%** [p<= 0.01]) than in the control and test groups 1-2 (i.e. 99% / 98% / 97% at 0, 50 and 150 mg/kg bw/d).
SEX RATIO
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.
CLINICAL SIGNS
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.
BODY WEIGHT
Mean pup body weight and pup body weight change of the high-dose F1 pups were slightly reduced during lactation period, however, without attaining statistical significance. Mean pup body weight and pup body weight change of the low- and mid-dose F1 pups were statistically comparable to the concurrent control group.
There were no runts in the control group. Respectively, two female runts were seen in each of test groups 1 and 2 (50 and 150 mg/kg bw/d), despite the lack of alterations in mean pup body weight parameters. Therefore these findings are considered spontaneous. However, the two male and four female runts seen in test group 3 (500 mg/kg bw/d) correspond to marginal reductions in pup body weight and pub body weight change, indicating a possible substance-related effect.
GROSS PATHOLOGY (OFFSPRING)
Aneurysms were present at different levels of the aorta (ascending, descending or aortic arch), in the region of ductus arteriosus and the pulmonary trunk. Frequently, aneurysms were observed simultaneously at different sites in the same pup. The number of affected pups increased from test group 1 (50 mg/kg bw/d) to test group 3 (500 mg/kg bw/d) in both male and female pups. Dilation of the aorta and heart was seen in 2 males and 2 females of test group 3 (500 mg/kg bw/d). A table showing the numbers of pups with macroscopic findings and aneurysms is given under ‘Any other information on results incl. tables’.
HISTOPATHOLOGY
Dissecting aneurysms were found in the aorta (ascending, descending and aortic arch), in the ductus arteriosus and the pulmonary trunk. Several animals showed simultaneously aneurysms at these different sites. In most cases, histopathology correlated with the aneurysms detected at gross pathology. The aneurysms were characterized by bulging of the affected vessel due to a cavity within the artery wall filled with blood and dissecting the elastic fibers of the medial layers. Mostly they were observed in the descending aorta. Occasionally, the aneurysms were compressing the lumen of the affected vessel. Dilation of the heart or of the aorta was noted only in single male and female pups of test group 3 (500 mg/kg bw/d). A table showing the numbers of examined control pups as well as treated pups with gross lesions (mostly aneurysms) is given under ‘Any other information on results incl. tables’.

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the presence of dissecting aneurysms in the great vessels of the heart at the lowest dose tested (50 mg/kg bw/day).

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Reproductive effects observed:

not specified

Gross lesions in pups:

Test group (mg/kg bw/d)	Male pups				Female pups
	0	1	2	3	0	1	2	3
	(0)	(50)	(150)	(500)	(0)	(50)	(150)	(500)
No. of pups with findings	0	1	8	20	0	1	7	24
No. of pups with aneurysms	0	1	8	18	0	1	6	24

Microscopic findings in pups:

Test group (mg/kg bw/d)	Male pups				Female pups
	0	1	2	3	0	1	2	3
	(0)	(50)	(150)	(500)	(0)	(50)	(150)	(500)
No. of examined pups	5	1	8	20	5	1	7	24
No. of pups with aneurysm	0	1	8	16	0	1	6	21
•       No of pups with one location		1	5	10		1	3	16
•       No of pups with multiple locations			3	6			3	5
Aorta		1	7	11		1	6	19
•       descending		1	7	10		1	6	19
•       ascending				1				1
•       arch				1
Ductus arteriosus			4	9			3	3
Pulmonary trunc			3	5			3	5

Reference 3

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

December 2011 - October 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Age at study initiation: (P) 11-13 wks
- Weight at study initiation: (P) Males: 293-321 g; Females: 201-226 g
- Housing: individually in Makrolon type M III cages; exceptions: male and female partners were housed together during overnight matings, pregnant animals and their litters were housed together until PND 4
- Diet (ad libitum): groudn Kliba maintenance diet mouse/rat "GLP meal, Provimi Klibi SA, Kaiseraugust, CH
- Water (ad libitum): in water bottles
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test substance was weighed in a graduated measuring flask depending on the dose group, topped up with highly deionized water and intensely mixed by shaking until completely dissolved.
- Volume administered each day: 10 ml/kg bw

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: overnight (about 04:00 p.m. until 07:00 - 09:00 a.m.)
- Proof of pregnancy: [sperm in vaginal smear] referred to as GD0 = day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

once during the study period

Duration of treatment / exposure:

- females: 14 days before mating, during pregnancy, until PND4
- males: 28 days

Frequency of treatment:

once daily

Remarks:

Doses / Concentrations:
0, 40, 200, 1000 mg/kg bw/d
Basis:
nominal in water

No. of animals per sex per dose:

10 per sex and group

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes, at least daily

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data

BODY WEIGHT: Yes, weekly

FOOD CONSUMPTION: Yes, weekly, but not determined during mating period of F0 animals

WATER CONSUMPTION: No

Oestrous cyclicity (parental animals):

-

Sperm parameters (parental animals):

-

Litter observations:

PARAMETERS EXAMINED in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality up to PND4, clinical observation, presence of gross anomalies, weight gain PND1-4

GROSS EXAMINATION OF DEAD PUPS:
[yes, for external and internal abnormalities]

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals [the next day after 28 days of exposure]
- Maternal animals: All surviving animals [at PND4]

GROSS NECROPSY
- Gross necropsy, special attention was given to the reproductive organs

HISTOPATHOLOGY / ORGAN WEIGHTS
- Weights in all animals: anesthetized animals, epididymes, testes
- Fixation in 4% formaldehyde or in modified Davidson´s solution: all gross lesions, cervix, coagulating gland, epididymes, ovaries, oviducts, prostate gland, seminal vesicles, testes, vagina, uterus
- Histopathology: Testes, epididymes, ovaries of control and high dose group

Postmortem examinations (offspring):

SACRIFICE: on PND4
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
GROSS NECROPSY: external examination and eviceration, macroscopic assessment of organs
Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding.

Statistics:

DUNNETT-test, FISHER´s EXACT test, WILCOXON-test, KRUSKAL-WALLIS test

Reproductive indices:

- Male mating index (%), male fertility index
- Female mating index (%), female fertility index (%), gestation index (%), live birth index (%), postimplantation loss (%)


Females: determination of the number of implantations and calculation of the postimplantation loss

Offspring viability indices:

- Viability index (%), sex ratio

There were no test substance-related adverse findings in any of the groups.

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One male animal (#32) of the high-dose group was found dead in study week 3 without any abnormal clinical or pathological findings. Therefore, this was considered to be incidential and not treatment-related.
Some animals of the high-dose group showed transient salivation, perstisting only for some minutes after daily dosing.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weight of the high-dose males (up to -6% during study week 1 and 2) and food consumption of the high-dose males and females (-21% or -19% during study week 1) were statistically significantly reduced. Later on, both parameters were comparable to the control.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- Male/female mating index = 100% (control, low-, mid-, high-dose). One low- and one mid-dose female (#117, 121) were not detected as being sperm positive. However, both had pups, giving evidence for successful copulation.
- Male/female fertility index = 100 % (control), 90% (low-, mid-dose), 80 % (high-dose). This is in the normal range of biological variation, inherent in the used rat strain. The infertile males (#17, 25, 34, 37) showed no relevant gross lesions. Minimal changes in testicles and epididymes were observed in male #34, but not severe enough to explain infertility. The non-pregnant femalse (#115, 125, 134, 137) had no relevant gross lesions.
- Mean duration of gestation: similar in all groups (22.0 - 22.5 d)
- Gestation index = 100 % in all groups
- Mean numer of implantation sites: comparable between control, low-, mid-, high-dose groups (13.5, 12.4, 13.0, 12.1)
- Post-implantation loss: comparable between control, low-, mid-, high-dose groups (10.7%, 7.7%, 6.7%, 7.4%)
- Mean number of F1 pups, delivered per dam: comparable between control, low-, mid-, high-dose groups (12.0, 11.4, 12.0, 11.3)
- Live birth index: 100% (control, low-dose), 99% (mid-dose), 94%* (p<= 0.05, high-dose); mainly caused by dam #138 with 4/10 stillborn pups.
- Number of stillborn pups: statistically increased in high-dose group (0, 0, 1, 5*); mainly caused by dam #138 with 4/10 stillborn pups. However, all respective values were in the historical control range and thus relect the normal range of biological variation inherent in the used rat strain.

ORGAN WEIGHTS (PARENTAL ANIMALS)
All mean absolute and relative weight parameters did not show significant differences compared to the control.

GROSS PATHOLOGY (PARENTAL ANIMALS)
All findings occured either indivdually or were biologically equally distributed over all groups and were not considered treatment-related.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No findings were observed in the high-dose group, that could explain the lack of offspring in males (#34, 37) or lack of pregnancy in females (#134, 137). Minimal changes observed in the testicles (multifocal tubular degeneration) and epididymes (cell debris) of male #34 frequently occur as background lesions in male rats without impairing fertility. These changes were not considered treatment-related. All other findings occured either indivdually or were biologically equally distributed over all groups and were not considered treatment-related.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no test substance-related adverse findings at any dose

There were no test substance-related adverse findings in any of the groups.

VIABILITY (OFFSPRING)
- Mean number of F1 pups, delivered per dam: comparable between control, low-, mid-, high-dose groups (12.0, 11.4, 12.0, 11.3)
- Live birth index: 100% (control, low-dose), 99% (mid-dose), 94%* (p<= 0.05, high-dose); mainly caused by dam #138 with 4/10 stillborn pups.
- Number of stillborn pups: statistically increased in high-dose group (0, 0, 1, 5*); mainly caused by dam #138 with 4/10 stillborn pups. However, all respective values were in the historical control range and thus relect the normal range of biological variation inherent in the used rat strain.
- Viablilty index during lactation (PND0-4): no test substance related changes between control, low-, mid-, high-dose (99, 93%*, 99, 96%). The slightly reduced index in the low-dose group resulted of the accidental death of 6 pups, which were placed with the wrong dam after weighing and immediatly killed).
- Sex distribution and ratios did not show substational differences between the groups.

CLINICAL SIGNS (OFFSPRING)
No test substance-related adverse were observed.

BODY WEIGHT (OFFSPRING)
Mean pup bw and pup bw change of all groups were statistically comparable.
One female runt each was seen in the control and the high-dose group, one male runt in the mid-dose group.

GROSS PATHOLOGY (OFFSPRING)
- One F1 pup showed a spontaneous finding (dilated renal pelvis). There were no further findings.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no test substance-related findings at any dose

Reproductive effects observed:

not specified

In conclusion, under the conditions of this study the NOAEL for general systemix toxicity, reproductive toxicity performance and fertility is 1000 mg/kg bw/d for the F0 parental rats. Furthermore, the NOAEL for developmental toxicity was also 1000 mg/kg bw/d.

Conclusions:

In conclusion, under the conditions of this OECD421 study the NOAEL for general systemix toxicity, reproductive toxicity performance and fertility is 1000 mg/kg bw/d for the F0 parental rats. Furthermore, the NOAEL for developmental toxicity was also 1000 mg/kg bw/d. Therefore, Aminopropyl-imidazol is considered to be non-toxic to reproduction (neither fertility, nor developmental toxicity) in this study.

Executive summary:

In a GLP OECD421 study, N-(3-Aminopropyl)-imidazol was given daily to 10 Wistar rats per sex and group per gavage at doses of 0, 40, 200 and 1000 mg/kg bw/d (BASF SE, 2012) to screen fo potential reproductive and developmental toxicity. The exposure lasted for at least 28 days in males and for at least 38 days in females (2 weeks prior to mating, during the mating period of max. 2 weeks, about 1 week post-mating for males and for the entire gestation period up to PND 4 in females). The examination of clinical signs and mortality, of food consumption, body weight and organ weights, the gross pathology, histopathology and the reproductive performance relvealed no toxicologically relevant adveres effects in parental animals up to 1000 mg/kg bw/d. Furthermore, no toxicologically relevant developmental differences in the F1 pups were detected during this study considering viability, clincial signs, body weight and gross pathology of the F1 pups. Therefore, under the conditions of this study, the NOAEL for reproductive toxicity as well as for developmental toxicity in the F1 offspring is 1000 mg/kg bw/d.

Reference 4

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

GLP compliance:

yes

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 Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 11 weeks
- Weight at study initiation: Males: 281-315g, Females: 189-215g
- Housing: Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm). Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm). Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes. General: Sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment or bedding material.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Results of analyses for nutrients and contaminants were examined and archived.
- Water: Free access to tap-water. Certificates of analysis were examined and archived.
- Acclimation period: At least 5 days prior to start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

- Amount of vehicle: 10 mL/kg body weight
- Preparation of dosing solution: Formulations (w/w) were prepared daily within 6 hours and 11 minutes prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the test substance (0.99 g/cm3 at 40°C). No correction was made for the purity/composition of the test substance. To liquefy the test substance before weighing, where considered necessary the test substance was heated to a maximum temperature of 60.3°C for a maximum of 2 hours and 42 minutes. Information provided by the sponsor indicated that the test substance could be heated up to 60-70°C.

Details on mating procedure:

- Mating procedures: Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from males. Detection of mating was not confirmed for animal nos. 45 (Group 1) and 80 (Group 4) which did deliver live offspring. The mating date of these animals was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Chemical analysis of dose preparations: Analyses were conducted by ABL BV, Assen, the Netherlands; test site study identification ABL12288. Samples (0.5 mL) were taken using a pipette (a clean pipette tip was used for every group), and were weighed on an analytical balance at 4 decimals precision. During sampling, formulations were placed on a magnetic stirrer. Immediately after sampling (accuracy and homogeneity samples) or after 6 hours at room temperature under normal laboratory light conditions (stability samples), samples were stored on dry ice. Samples remained on dry ice until receipt at ABL, The Netherlands. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
- Analysis of dose preparations: No test substance was detected in the Group 1 formulations. The concentrations analysed in the formulations of Group 2 showed a mean accuracy of 89.4%. This mean accuracy was only minimally exceeding the acceptable range of 90-110% of nominal concentration. Given the NOAEL derived in this study (set at the highest dose, i.e. Group 4), this deviating accuracy value for Group 2 formulations did not affect interpretation of the study results. The concentrations analysed in the formulations of Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours (i.e. relative difference ≤ 10%).

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 43-50 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Female nos. 41, 42, 45 (Group 1), 53, 56 (Group 2) and 61 (Group 3) were not dosed during littering.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.

Remarks:

Doses / Concentrations:
15, 50, 150 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a previous 14-day dose range finding study (Project 500561; BASF Project 01R0074/12X163), daily dosing of 300 mg/kg bw resulted in body weight loss in both sexes. Additionally, lethargy, hunched posture and/or piloerection was observed during the last 3-4 days of treatment. Therefore, the dose of 300 mg/kg bw/day was considered to be too high for the present study.
- Treatment: By oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
- Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.

Parental animals: Observations and examinations:

- Mortality / Viability: At least twice daily.
- Clinical signs: Daily from treatment onwards, detailed clinical observations were made for all animals, immediately after dosing (based on the absence of a peak effect of occurrence of clinical signs in the dose range finding study, Project 500561; BASF Project 01R0074/12X163). Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed sign were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.
- Functional Observations: The following tests were performed on the selected 5 animals/sex/group: hearing ability, pupillary reflex, static righting reflex, grip strength, locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations are reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head. During motor activity monitoring, all animals were caged individually (pups were housed in their home cages and kept warm using bottles filled with warm water). These tests were conducted after dosing at no specific time point, but within a similar time period after dosing for the respective animals. These tests were performed after observation for clinical signs (incl. arena observation, if applicable) and before blood sampling. The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period (all before blood sampling).
- Body weights: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.
- Food consumption: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.
- Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.
- General reproduction data: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Palpation was used to aid in confirmation of pregnancy. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.
- Clinical laboratory investigations: Blood samples were collected on the day of necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) between 7.00 and 10.30 a.m. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and Li-heparin treated tubes for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into untreated tubes for determination of bile acids. Furthermore, from the selected 5 animals/sex/group an additional blood sample (1 mL) was collected into serum tubes for possible future measurement of thyriod-stimulating hormone (TSH), and the thyroid hormones triiodothyronine (T3) and thyroxine (T4). After clotting and centrifugation, serum samples were stored at <-75°C. Since histopathological examination of the thyriod glands did not reveal any treatment-related changes, all samples were discarded at finalization of the study report without further investigation.
- Haematology: The following haematology parameters were determined in blood prepared with EDTA as an anticoagulant, using the ADVIA® 120 Hematology System (Siemens Healthcare Diagnostics B.V., Breda, The Netherlands): White blood cells, Differential leucocyte count: Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils, Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets. The following clotting parameters were determined in plasma prepared with citrate as anti-coagulant, using the STA Compact® (Diagnostica Stago S.A.S., Asnières, France): Prothrombin Time, Activated Partial Thromboplastin Time.
- Clinical biochemistry: The following clinical biochemistry parameters were determined using the AU400® Chemistry System (Beckman Coulter Nederland B.V., Woerden, The Netherlands). All parameters were determined in plasma, except for bile acids which were determined in serum: Alanine aminotransferase, Aspartate aminotransferase, Alkaline Phosphatase, Total protein, Albumin, Total Bilirubin, Bile acids, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate.

Litter observations:

- Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made for all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation.

Postmortem examinations (parental animals):

- Necropsy: All males, the selected 5 females/group were deprived of food overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. Non-selected females were not deprived of food. Animals surviving to scheduled necropsy were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated. Necropsy was conducted on the following days: Females which delivered: Lactation Days 5-7, Females which failed to deliver (nos. 63 and 74): Post-coitum Day 27 (female no. 63 with evidence of mating) or 22 days after the last day of the mating period (female no. 74 without evidence of mating), Males: Following completion of the mating period (a minimum of 28 days of dose administration). All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The numbers of former implantation sites and corpora lutea were recorded for all paired females. Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). Selected 5 animals/sex/group (tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination): Adrenal glands, (Aorta), Brain - cerebellum, mid-brain, cortex, Caecum, Cervix, Clitoral gland, Colon, Coagulation gland, Duodenum, Epididymides, Eyes (with optic nerve (if detectable) and Harderian gland), Female mammary gland area, Femur including joint, Heart, Ileum, Jejunum, Kidneys, (Lacrimal gland, exorbital), (Larynx), Liver, Lung - infused with formalin, Lymph nodes - mandibular, mesenteric, (Nasopharynx), (Esophagus), Ovaries, (Pancreas), Peyer's patches [jejunum, ileum] if detectable, Pituitary gland, Preputial gland, Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, (Skin), Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid including parathyroid if detectable, (Tongue), Trachea, Urinary bladder, Uterus, Vagina, All gross lesions. All remaining animals, females which failed to deliver: Cervix, Clitoral gland, Coagulation gland, Epididymides, Mammary gland area, Ovaries, Preputial gland, Prostate gland, Seminal vesicles, Testes, Uterus, Vagina, All gross lesions.
- Organ weights: Terminal body weights were recorded from all males and the selected 5 females/sex/group. The following organ weights were recorded from the following animals on the scheduled day of necropsy. Selected 5 animals/sex/group: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus, Uterus (including cervix), Prostate (weighed when fixed for at least 24 hours), Seminal vesicles including coagulating glands (weighed when fixed for at least 24 hours), Thyroid including parathyroid (weighed when fixed for at least 24 hours). All remaining males: Epididymides, Testes.
- Histotechnology: All organ and tissue samples, as defined under Histopathology (following section), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands). From the selected 5 males of the control and high dose group and male no. 23 (Group 3) suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis. The teses were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).
- Histopathology: The following slides were examined: (1) The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4. (2) The additional slides of the testes of the selected 5 males of Groups 1 and 4 and male no. 23 (Group 3) suspected to be infertile to examine staging of spermatogenesis. (3) All gross lesions of all animals (all dose groups). (4) The reproductive organs (cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina) of male no. 23 (Group 3) that failed to sire and female nos. 63 (Group 3; no offspring) and 74 (Group 4; not mated; male no. 34 was already selected for assessment of reproductive organs) that failed to deliver healthy pups.

Postmortem examinations (offspring):

Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation. All pups were sexed and descriptions of all external abnormalities were recorded. The stomach of pups not surviving to scheduled necropsy was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated. Pups were checked for macroscopically visible anomalies of the greater vessels (aortic arch, thoracic aorta). Pups with findings were fixated in toto in 10% buffered formalin.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences, followed by the Wilcoxon test to compare the treated groups to the control group in case intergroup differences were seen.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Reproductive indices:

For each group, the following calculations were performed:
- Mating index (%) = (Number of females mated / Number of females paired) x 100
- Fertility index (%) = (Number of pregnant females / Number of females paired) x 100
- Conception index (%) = (Number of pregnant females / Number of females mated) x 100
- Gestation index (%) = (Number of females bearing live pups / Number of pregnant females) x 100
- Duration of gestation = Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

For each group, the following calculations were performed:
- Percentage live males at First Litter Check = (Number of live male pups at First Litter Check / Number of live pups at First Litter Check) x 100
- Percentage live females at First Litter Check = (Number of live female pups at First Litter Check / Number of live pups at First Litter Check) x 100
- Percentage of postnatal loss = (Number of dead pups before planned necropsy / Number of live pups at First Litter Check) x 100
- Viability index = (Number of live pups before planned necropsy / Number of pups born alive) x 100

- Mortality: No mortality occurred during the study period that was considered to be related to treatment with the test substance. One control male died during the blood sampling procedure, which was considered incidental in nature.
- Clinical signs: No clinical signs of toxicity were noted during the observation period. Salivation seen after dosing among animals of the 50 (individual males) and 150 mg/kg bw/day dose group (all males and most of the females) was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to taste of the test substance. Pale faeces was noted for most females at 150 mg/kg bw/day, primarily towards the end of the study period. Given that body weight development and food intake was unaffected by treatment, and since blood analysis and morphological assessment did not reveal any toxicologically relevant changes, this finding was considered not to be of an adverse nature. The incidence of scabs, hunched posture and alopecia occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.
- Functional observations: Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. The variation in motor activity did not indicate a relation with treatment. The statistically significant lower mean counts for total movements of males at 50 mg/kg occurred in the absence of a dose-related trend; the control mean was considered to be slightly high. Therefore, no toxicological relevance was ascribed to this change. All groups showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test period.
- Body weights: No toxicologically relevant changes in body weights and body weight gain were noted. Any statistically significant changes in body weight and body weight gain across the dose groups were considered not to be of toxicological relevance since the changes were slight in nature and/or occurred in the absence of a dose-related trend. These changes consisted of lower body weight gain of males at 150 mg/kg bw/day during the premating and mating period (also statistically significant for absolute body weight on Day 15 of the mating period), lower absolute body weight and body weight gain for females at 150 mg/kg bw/day on Day 8 of the premating period and on Day 4 of the lactation period, and lower body weight gain on Day 1 and 15 of the mating period for males at 15 and 50 mg/kg bw/day, respectively.
- Food consumption: No toxicologically relevant changes in food consumption before or after correction for body weight were noted. The statistically significant lower food consumption (before or after correction for body weight) of females at 150 mg/kg bw/day over Days 0-4 of the post-coitum period was slight in nature and did not prevail with continuing treatment. No toxicological relevance was therefore ascribed to these changes.
- Haematology: No toxicologically relevant changes occurred in haematological parameters of treated rats. The higher individual red blood cell distribution width (RDW) and reticulocyte counts in individual males and/or females at 150 mg/kg bw/day (exceeding the range considered normal for rats of this age and strain) were not part of a group response and occurred in the absence of any supportive changes in red blood cell parameters. These changes were therefore considered to be of no toxicological relevance.
- Clinical biochemistry: No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats. Any (statistically significant) changes in clinical biochemistry parameters at 150 mg/kg bw/day were slight in nature and occurred in the absence of morphological changes indicative of organ dysfunction. These changes consisted of higher cholesterol level in males and females (not statistically significant, but means just within the range considered normal for rats of this age and strain), lower sodium and chloride level in males, and higher inorganic phosphate level in males (not statistically significant, but mean outside the range considered normal for rats of this age and strain). The statistically significantly higher bile acid level for females at 150 mg/kg bw/day was due to higher values for two females (nos. 76 and 77). Other selected females showed normal values for this parameter. The statistically significantly higher total bilirubin level of males at 15 mg/kg bw/day occurred in the absence of a dose-related trend and the mean remained within the range considered normal for rats of this age and strain. These changes were therefore considered to be of no toxicological relevance.
- Macroscopic examination: Necropsy did not reveal any toxicologically relevant alterations. The incidence of macroscopic findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, did not show a dose-related incidence trend and occurred in the absence of any treatment-related histopathological changes.
- Organ weights: No toxicologically relevant changes were noted in organ weights and organ to body weight ratios. The statistically significantly higher kidney to body weight ratio and/or kidney weight of males at 50 and 150 mg/kg bw/day occurred in the absence of clear dose-related trend. The statistically significantly higher liver, kidney, spleen and uterus to body weight ratio in females at 150 mg/kg bw/day remained within the range considered normal for rats of this age and strain. Since absolute weights were similar to control levels these changes were ascribed to the lower terminal body weights. Also, since no histopathological correlates were found for these organ weight changes, these were considered to be of no toxicological relevance. Seminal vesicle weight and seminal vesicle to body weight ratio of males at 150 mg/kg bw/day appeared lower (approximately 15% for relative weights) than controls without achieving a level of statistical significance. Since these differences in seminal vesicle weight had no histopathological correlates, and there were no reproductive changes, these differences were considered to be of no toxicological relevance.
- Microscopic examination: There were no treatment-related microscopic findings. All microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar-Han rats of this age. No histopathological changes were noted in the reproductive organs of two Group 3 animals (male no. 23 and female no. 63) and two Group 4 animals (male no. 34 and female no. 74) that could account for failing to deliver healthy offspring. The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item.
- Reproduction data: No toxicologically relevant effects on reproductive parameters were noted. Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.
- Developmental data: Gestation index and duration of gestation were unaffected by treatment. No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Dose descriptor:

NOAEL

Remarks:

parental

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no findings that were considered to be adverse in nature following treatment up to 150 mg/kg bw/day.

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no findings that were considered to be adverse in nature following treatment up to 150 mg/kg bw/day.

Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings. A total of five pups of the control group, eight pups at 50 mg/kg bw/day and three pups at 150 mg/kg bw/day were found dead or missing during lactation. Pups missing were most likely cannibalised. The higher number of dead/missing pups at 50 mg/kg bw/day resulted in a statistically significantly lower viability index at this dose level. No toxicological relevance was attributed to these dead/missing pups across the dose groups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age. Incidental clinical symptoms of pups consisted of a dented head, a red spot on the head, absence of milk in the stomach, abnormal posture of the left hindleg, and a scab on the nose and right hindleg. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance. Body weights of pups were unaffected by treatment. Incidental macroscopic findings of pups that were found dead included cannibalism of the head region, absence of milk in the stomach, beginning/slight/moderate autolysis and a wound on the upper jaw. Incidental macroscopic findings among surviving pups included a dented head, and abnormal posture of the left hindleg. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance. No treatment-related anomalies of the greater vessels (aortic arch, thoracic aorta) were noted among the pups. Incidental findings consisted of a supernumary artery originating from the aortic arch after the left subclavian (pup no. 6 of litter 76 at 150 mg/kg bw/day), and a right subclavian originating from the aortic arch (pup no. 6 of litter no. 46, control group). The distribution and nature of these findings showed no dose-related trend and were incidental in nature. No toxicological relevance was therefore ascribed to these findings.

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no findings that were considered to be adverse in nature following treatment up to 150 mg/kg bw/day.

Reproductive effects observed:

not specified

Reference 5

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

GLP compliance:

yes

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 Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 11 weeks
- Weight at study initiation: Males: 287-315g, Females: 184-217g
- Housing: Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm). Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm). Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes. General: Sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. Certificates of analysis were examined and then retained in the WIL Research B.V. archives. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment or bedding material.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Results of analyses for nutrients and contaminants were examined and archived.
- Water: Free access to tap-water. Certificates of analysis (performed quarterly)
were examined and archived.
- Acclimation period: At least 5 days prior to start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

- Amount of vehicle: 5 mL/kg body weight

Details on mating procedure:

- Mating procedures: Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated. After 14 days of mating, female nos. 42, 43 and 48 from the control group (Group 1) did not show evidence of mating. Therefore, they were separated from their males and cohabited with proven males (nos. 7, 9 and 10) from the same group on 11 May 2012. Until 14 May 2012, nine Group 1 females in total showed evidence of mating. As this was the required minimum number of mated females, re-mating was suspended.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Chemical analysis of dose preparations: Analyses were conducted on a single occasion during the treatment phase (01 May 2012), according to a validated method (Project 499238; BASF Project 05Y0379/05X022). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
- Analysis of dose preparations: The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test substance was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 43-56 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Female nos. 43, 45 (Group 1), 51, 53, 56, 58 (Group 2) were not dosed during littering.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.

Remarks:

Doses / Concentrations:
15, 50, 150 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a previous dose range finding study (Project 499236; BASF Project 01R0379/05X016), groups of 4 male and 4 female Crl:WI(Han) rats received 2-Ethyl-4-methylimidazole by oral gavage for a period of 14 days at the dose levels of 0, 50, or 150 mg/kg bw/day. Test substance related adverse effects were noted at 150 mg/kg bw/day only. Changes were transient and/or slight and consisted of transient clinical signs (rales, hunched posture, piloerection, chromodacryorrhoea, and/or ptosis; one male and one female), and initially slightly lower body weight gains (males) and food consumption (absolute and relative to body weight; both sexes). Clinical laboratory investigations revealed a higher count of white blood cells and platelets (males), and higher cholesterol levels (both sexes) at 150 mg/kg bw/day. There were no adverse effects on either macroscopy or organ weights. No adverse findings were noted at 50 mg/kg bw/day. Based on these results, dose levels of 0, 15, 50, or 150 mg/kg bw/day were selected for the present study, taking into account the longer administration period and inclusion of pregnant animals.
- Method of formulation: The test substance was melted at approximately 60-65°C, divided in suitable portions and stored at room temperature in the dark until preparation of the formulation. Formulations (w/w) were prepared daily within 6 hours prior to dosing. The vehicle was added to the pre-weighed test substance and the formulations were heated (at approximately 60-65°C) and stirred continuously (magnetic stirrer) for approximately 15 minutes to achieve homogeneity at a visually acceptable level. Subsequently, dose preparations were allowed to cool down to a maximum of 40°C before dosing. No adjustment was made for specific gravity/density of the test substance, vehicle, and/or formulation. No adjustment was made for the purity of the test substance.
- Treatment: By oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing. Due to the relatively high pH-values of the dose preparations for Groups 2-4, preventative measures were taken to avoid possible local esophageal and/or gastrointestinal effects: Wiping of the flexible catheter prior to dosing of each animal and additional rinsing of the flexible catheter with approximately 0.5-0.6 mL water (Elix, Millipore S.A.S., Molsheim, France) after dosing each animal with the test formulations.
- Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.

Parental animals: Observations and examinations:

- Mortality / Viability: At least twice daily.
- Clinical signs: At least once daily from start of treatment onwards, detailed clinical observations were made for all animals. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. In the period from treatment Days 1-12 (i.e. 16-27 April 2012), clinical observations were conducted at least between 1 and 2 hours after dosing (based on results of the dose range finding study, Project 499236; BASF Project 01R0379/05X016). However, since from treatment Day 12 onwards animals from Groups 3 and 4 showed salivation immediately after dosing, the time point for detailed observations for clinical signs (incl. arena observations) was changed to immediately after dosing from treatment Day 13 (28 April 2012) onwards. The time of onset, grade and duration of any observed sign were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.
- Functional Observations: The following tests were performed on the selected 5 animals/sex/group: hearing ability, pupillary reflex, static righting reflex, grip strength, locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations are reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head. During motor activity monitoring, all animals were caged individually (pups were housed in their home cages and kept warm using bottles filled with warm water). The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period (all before blood sampling). These tests were performed as soon as possible after observation for clinical signs (incl. arena observation, if applicable), and before blood sampling.
- Body weights: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.
- Food consumption: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.
- Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.
- General reproduction data: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.
- Clinical laboratory investigations: Blood samples were collected from the selected 5 animals/sex/group under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) between 7.00 and 10.30 a.m. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and Li-heparin treated tubes for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into untreated tubes for determination of bile acids. Furthermore, from the selected 5 animals/sex/group an additional blood sample (0.5 mL) was collected into serum tubes for possible future measurement of the thyroid hormones triiodothyronine (T3) and thyroxine (T4). Under these storage conditions, T3 and T4 was stable for 2 months. Since histopathological examination revealed no treatment related changes in the thyroid glands, no thyroid hormone measurements were required. The blood samples were discarded at finalization of the study report.
- Haematology: The following haematology parameters were determined in blood prepared with EDTA as an anticoagulant, using the ADVIA® 120 Hematology System (Siemens Healthcare Diagnostics B.V., Breda, The Netherlands): White blood cells, Differential leucocyte count: Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils, Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets. The following clotting parameters were determined in plasma prepared with citrate as anti-coagulant, using the STA Compact® (Diagnostica Stago S.A.S., Asnières, France): Prothrombin Time, Activated Partial Thromboplastin Time.
- Clinical biochemistry: The following clinical biochemistry parameters were determined using the AU400® Chemistry System (Beckman Coulter Nederland B.V., Woerden, The Netherlands). All parameters were determined in plasma, except for bile acids which were determined in serum: Alanine aminotransferase, Aspartate aminotransferase, Alkaline Phosphatase, Total protein, Albumin, Total Bilirubin, Bile acids, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate.

Litter observations:

- Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made for all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation.

Postmortem examinations (parental animals):

- Necropsy: All males, the selected females/group were deprived of food overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. Non-selected females were not deprived of food. Animals surviving to scheduled necropsy were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated. Necropsy was conducted on the following days: Females which delivered: Lactation Days 5-7, Females which failed to deliver: Post-coitum Day 28 (females with evidence of mating) or 21 days after the last day of the mating period (female without evidence of mating), Males: Following completion of the mating period (a minimum of 28 days of dose administration). All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The numbers of former implantation sites and corpora lutea were recorded for all paired females (for females which failed to deliver: In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites). Samples of the following tissues and organs were collected from all animals and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands): Adrenal glands, (Aorta), Brain - cerebellum, mid-brain, cortex, Caecum, Cervix, Clitoral gland, Colon, Coagulation gland, Duodenum, Epididymides, Eyes (with optic nerve (if detectable) and Harderian gland), (Male and Female mammary gland area), Femur including joint, Heart, Ileum, Jejunum, Kidneys, (Lacrimal gland, exorbital), (Larynx), Liver, Lung, infused with formalin, Lymph nodes - mandibular, mesenteric, (Nasopharynx), (Esophagus), Ovaries, (Pancreas), Peyer's patches [jejunum, ileum] if detectable, Pituitary gland, Preputial gland, Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, (Skin), Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach (forestomach and glandular stomach), Testes, Thymus, Thyroid including parathyroid if detectable, (Tongue), Trachea, Urinary bladder, Uterus, Vagina, All gross lesions. The epididymides, eyes and testes were fixed in modified Davidson's solution and Milli-Ro water and transferred to formalin after fixation for at least 24 hours. Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.
- Organ weights: The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus, Uterus (including cervix), Prostate (weighed when fixed for at least 24 hours), Seminal vesicles including coagulating glands (weighed when fixed for at least 24 hours), Thyroid including parathyroid (weighed when fixed for at least 24 hours). All remaining males: Epididymides, Testes.
- Histotechnology: All organ and tissue samples, as defined under Histopathology (following section), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands). Of all males of the control and high dose group and all males suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).
- Histopathology: The following slides were examined by a pathologist: (1) The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4. (2) The additional slides of the testes of all males of Groups 1 and 4 and all males suspected to be infertile or which died before mating to examine staging of spermatogenesis. (3) All gross lesions of all animals (all dose groups). (4) The spleen and kidneys of all selected 5 females of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4. (5) The reproductive organs (cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina) from all animals of Groups 1 and 4 and from all males that failed to sire and all females that failed to deliver healthy pups.

Postmortem examinations (offspring):

Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation. All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Reproductive indices:

For each group, the following calculations were performed:
- Mating index (%) = (Number of females mated / Number of females paired) x 100
- Fertility index (%) = (Number of pregnant females / Number of females paired) x 100
- Conception index (%) = (Number of pregnant females / Number of females mated) x 100
- Gestation index (%) = (Number of females bearing live pups / Number of pregnant females) x 100
- Duration of gestation = Number of days between confirmation of mating and the beginning
of parturition

Offspring viability indices:

For each group, the following calculations were performed:
- Percentage live males at First Litter Check = (Number of live male pups at First Litter Check / Number of live pups at First Litter Check) x 100
- Percentage live females at First Litter Check = (Number of live female pups at First Litter Check / Number of live pups at First Litter Check) x 100
- Percentage of postnatal loss Days 0-4 of lactation = (Number of dead pups on Day 4 of lactation / Number of live pups at First Litter Check) x 100
- Viability index = (Number of live pups on Day 4 post partum / Number of pups born alive) x 100

- Mortality: No mortality occurred during the study period that was considered to be related to treatment with the test substance. One female at 50 mg/kg bw/day died after blood sampling. In the absence of any adverse findings for this female, her accidental death was considered to have been caused by complications during the blood sampling procedure rather than to be related to treatment with the test substance.
- Clinical signs: No clinical signs of toxicity were noted during the observation period. Salivation was seen after dosing among animals of the 50 and 150 mg/kg bw/day dose groups (both sexes) from the end of treatment Week 2 onwards. This finding was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). It may be related to irritancy/taste of the test substance. Incidental findings that were noted included alopecia and scabbing. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.
- Functional observations: Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. Locomotor activity as determined by total movements and ambulations were comparable in all groups (both sexes). Mean values remained within the normal range of biological variation. All groups showed a comparable habituation profile with high activity in the first interval that decreased over the duration of the test period. For one control female (no. 45), mean values for total movements (2114) and ambulations (401) were relatively low, with mean values at the lower end of the normal range (total movements: P5 = 2006, ambulations: P5 = 408). No explanation could be given for this finding. All other functional observation parameters were normal and there were no supportive clinical signs (i.e. diminished general activity or abnormal gait).
- Body weights: No toxicologically relevant changes in body weights and body weight gain were noted. The statistically significant changes noted for body weight gain (on Day 1 of mating for females at 15 and 150 mg/kg bw/day and on Days 4 and/or 7 post-coitum for females at 50 and 150 mg/kg bw/day) were considered to be of no toxicological relevance as values remained within the range considered normal for rats of this age and strain and/or changes occurred in the absence of a treatment-related distribution.
- Food consumption: No toxicologically relevant changes in food consumption before or after allowance for body weight were noted. The statistically significant increase in absolute food consumption for females at 150 mg/kg bw/day on Days 4-7 post-coitum was considered to be of no toxicological relevance as it was only a very slight increase and remained within the range considered normal for rats of this age and strain.
- Haematology: No toxicologically relevant changes occurred in haematological parameters of treated rats. Minor statistically significant differences arising between controls and treated animals were considered not to represent a change of biological relevance. These findings included a higher haematrocrit value for males at 50 mg/kg bw/day, lower mean corpuscular haemoglobin concentration for males at 15, 50 and 150 mg/kg bw/day and higher white blood cell count for females at 150 mg/kg bw/day. Since changes were very slight, occurred in the absence of a dose-related trend, and/or values remained within the range considered normal for rats of this age and strain, they were considered of no toxicological relevance.
- Clinical biochemistry: Statistically significant differences arising between controls and treated animals were noted for females only. These changes included decreased levels of total protein at 150 mg/kg bw/day and decreased albumin at 150 mg/kg bw/day Other statistically significant changes in clinical biochemistry parameters of females were considered to be without toxicological relevance. Slightly lower albumin levels at 15 mg/kg bw/day occurred without dose dependency. The slightly higher level of aspartate aminotransferase (ASAT) at 15 mg/kg bw/day occurred in the absence of a dose-related trend. The lower glucose at 150 mg/kg bw/day was not considered to be treatment related because the difference was in part due to slightly high values obtained for controls and remained in the range considered normal for animals of this age and strain. For males, clinical biochemistry parameters were unaffected by treatment up to 150 mg/kg bw/day.
- Macroscopic examination: Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment. The incidence of findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance. They included reddish discolouration or enlargement of the mandibular lymph node(s), alopecia, isolated, several or many tan, reddish or dark-red foci on the thymus, lungs, clitoral gland or glandular mucosa of the stomach, pelvic dilation of the kidneys (unilateral or bilateral), enlarged spleen, liver with the left lateral lobe reduced in size, uterus containing fluid or uterus with an enlarged right horn with black contents. The latter observation corresponds to the microscopy finding of cystic dilation and eosinophilic contents.
- Organ weights: Statistically significant higher liver weights (absolute and relative to body weight) were recorded for males, but not females, at 15, 50 and 150 mg/kg bw/day. There were no other effects on organ weights or organ to body weight ratios at any dose level tested.
- Microscopic examination: No toxicologically relevant changes were noted at the microscopic level. There appeared to be an increased severity of hemopoietic foci in the spleen of female rats treated at 150 mg/kg bw/day. After examination of the spleens of the selected 15 (Group 2) and 50 (Group 3) mg/kg bw/day treated female rats, no dose response relation was present and the severity grades scored in the 150 mg/kg bw/day treated rats were considered to be within normal limits for this study. There appeared to be a small increase in incidence of tubular basophilia in the kidneys of 2/5 female rats treated at 150 mg/kg bw/day. After examination of the kidneys of the selected 15 (Group 2) and 50 (Group 3) mg/kg bw/day treated female rats, no dose response relation was present. The incidences and severity grades scored (up to slight) in the 150 mg/kg bw/day treated rats were considered to be within normal limits and there were no concomitant kidney findings indicating a test item related effect. The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item. All remaining microscopic findings recorded were considered to be within the normal range of background pathology encountered in rats of this age and strain and in this type of study.
- Reproduction data: No toxicologically relevant effects on reproductive parameters were noted. Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment. In the control group, three females did not mate in the first pairing period of 15 days. After cohabiting these females with a proven male, two females showed evidence of mating, though the third female (no. 48) did not. For this female implantation sites were noted in the uterus at necropsy indicating that she had been pregnant, but probably lost the conceptus during pregnancy. At 50 or 150 mg/kg bw/day, there were three and two non-pregnant females, respectively. Consequently, the number of litters available for evaluation of developmental data was 9, 10, 7 and 8 in the control, 15, 50 or 150 mg/kg bw/day groups, respectively.
- Developmental data: Gestation index and duration of gestation were not affected by treatment. No signs of difficult or prolonged parturition were noted among the pregnant females. For one female at 15 mg/kg bw/day (no. 53) bleeding from the vagina was noted on lactation Day 1. No toxicological relevance was attached to this isolated finding. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Dose descriptor:

NOAEL

Remarks:

parental

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no findings that were considered to be adverse in nature following treatment up to 150 mg/kg bw/day.

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no findings that were considered to be adverse in nature following treatment up to 150 mg/kg bw/day.

Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings. The number of pups found dead or went missing during the first days of lactation were 2, 1, 1 and 3 in the control, 15, 50 and 150 mg/kg bw/day groups. Pups that went missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age. There were no clinical signs that were considered a sign of toxicity. Incidental findings included a blue spot on the snout, scabbing on the abdomen, and pale or cold appearance. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance. Body weights of pups were comparable in all groups. Incidental macroscopic findings of pups that were found dead included no milk in the stomach, beginning or moderate autolysis, cannibalism (left inguinal region or all abdominal organs missing). For one surviving pup black discolouration of the tail apex was recorded. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no findings that were considered to be adverse in nature following treatment up to 150 mg/kg bw/day.

Reproductive effects observed:

not specified

Reference 6

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

see WoE assessment reproductive toxicity in chapter 13

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 OPPTS 870.3650 (Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

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: 11-13 weeks
- Weight at study initiation: 329.9-361.9 g (males); 186.2-215.6 g (females)
- Fasting period before study: No
- Housing: During the study period, the rats were housed individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²), with the following exceptions:
* 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.
- Diet: Ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, which was available to the animals ad libitum throughout the study
- Water: Drinking water was supplied from water bottles (ad libitum)
- Acclimation period: About 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
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.
From visual inspection, it seemed that the test substance was not completely miscible with drinking water. Therefore, for the preparation of the administration solution the test substance was weighed in a calibrated beaker depending on the dose group, topped up with drinking water and subsequently intensely mixed with a homogenizer. During administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0, 100, 300 and 900 mg/100mL
- Amount of vehicle (if gavage): 10 mL/kg bw/day

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: Maximum of 2 weeks (the female was placed in the cage of the male mating partner from about 16.00 h until 07.00 - 09.00 h of the following morning)
- Proof of pregnancy: Sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: Individually

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 during the study. From visual inspection, it seemed that the test substance was not completely miscible with drinking water. Therefore, in order to prove that the test substance preparations were true solutions, a homogeneity analysis was carried out. Samples of the test substance solutions were sent to the analytical laboratory once at the beginning of the study for verification of the concentrations.
Concentration control analysis: The mean values of 1-Metylimidazol in drinking water in the low-dose group (samples 3R-5R and 12-14 ) were found in the range of 122.1% to 129.0% of the nominal concentrations. The mean found concentrations at 100 mg/100 mL are above the specification limit of 110%. This slight departure from the nominal concentration range presumably brought about an insignificantly higher dose for the low-dose group than intended. However, since this dose did not produce any effect in the respective animals, the validity of the study is not regarded to be compromised. All other values of 1-Metylimidazol in drinking water were found to be in the range of 90% - 110% of the nominal concentration.

Duration of treatment / exposure:

The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 2 days post-mating in males, and the entire gestation period as well as approximately 2 weeks of the lactation period.

Frequency of treatment:

Once daily at approximately the same time in the morning. Females in labor were not treated.

Remarks:

Doses / Concentrations:
10, 30, 90 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: By request of the sponsor

Parental animals: Observations and examinations:

MORTALITY:
A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). If animals were in a moribund state, they were sacrificed and necropsied.

CAGE SIDE OBSERVATIONS:
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. 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

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: 1. Abnormal behavior in “handling”, 2. Fur, 3. Skin, 4. Posture, 5. Salivation, 6. Respiration, 7. Activity/arousal level, 8. Tremors, 9. Convulsions, 10. Abnormal movements, 11. Gait abnormalities, 12. Lacrimation, 13. Palpebral closure, 14. Exophthalmos, 15. Assessment of the feces discharged during the examination (appearance/consistency), 16. Assessment of the urine discharged during the examination, 17. Pupil size

BODY WEIGHT:
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.

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.

HAEMATOLOGY:
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): 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 and Reticulocytes (RET).
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. (reference: Hematology: Principles and Procedures, 6th Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101). Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany). Parameter and method: Prothrombin time (Hepato Quick’s test) (HQT).

CLINICAL CHEMISTRY:
An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters. Parameters:
- Enzymes: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT).
- Blood chemistry parameters: Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Bile acids (TBA).

URINALYSIS:
The dry chemical reactions on test strips (Combur-10-test M, Roche, Mannheim, Germany) used to semi quantitatively determine urine constituents were evaluated with a reflection photometer (Miditron M; Roche, Mannheim, Germany). Parameters: pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color, turbidity, Volume.

NEUROBEHAVIOURAL EXAMINATION:
A functional observational battery was performed in 5 parental male and 5 parental female animals (with litter) per group at the end of the administration period starting at about 10.00h. 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: 1. Posture, 2. Tremors, 3. Convulsions, 4. Abnormal movements, 5. Impairment of gait, 6. 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: 1. Behavior when removed from cage, 2. Fur, 3. Skin, 4. Salivation, 5. Nose discharge, 6. Lacrimation, 7. Eyes/pupil size, 8. Posture, 9. Palpebral closure, 10. Respiration, 11. Tremors, 12. Convulsions, 13. Abnormal movements/stereotypy, 14. Impairment of gait, 15. Activity/arousal level, 16. Feces excreted within 2 minutes (number of scybala discharged/appearance/consistency), 17. Urine excreted within 2 minutes (amount/color), 18. 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: 1. Approach response, 2. Touch response, 3. Vision (“visual placing response”), 4. Pupillary reflex, 5. Pinna reflex, 6. Audition (“startle response”), 7. Coordination of movements (“righting response”), 8. Behavior during “handling”, 9. Vocalization, 10. Pain perception (“tail pinch”), 11. Other findings, 12. Grip strength of forelimbs and hindlimbs, 13. 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.

Sperm parameters (parental animals):

Parameters examined in male parental generation: testis weight, epididymis weight

Litter observations:

- Pup number and status at delivery: 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.
- 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.
- Pup clinical observations: The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams and documented for each pup.
- Pup body weight data: The pups were weighed on the day after birth (PND 1) and on PND 4. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning). “Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, epididymides, testes
The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): Adrenal glands, brain, heart, kidneys, liver, spleen, thymus
The following organs or tissues of all parental animals were fixed in 4% buffered formaldehyde solution or modified Davidson’s solution: All gross lesions, adrenal glands, aorta, bone marrow (femur), brain, cecum, cervix, coagulating glands, colon, duodenum, eyes with optic nerve, esophagus, extraorbital lacrimal glands, epididymides (modified Davidson’s solution), femur with knee joint, heart, ileum, jejunum (with Peyer’s patches), kidneys, larynx, liver, lungs, lymph nodes (axillary and mesenteric), mammary gland (male and female), nose (nasal cavity), ovaries (modified Davidson’s solution), oviducts, pancreas, parathyroid glands, pharynx, pituitary gland, prostate gland, rectum, salivary glands (mandibular and sublingual), sciatic nerve, seminal vesicles, skeletal muscle, spinal cord (cervical, thoracic and lumbar cord), spleen, sternum with marrow, stomach (fore stomach and glandular stomach), target organs, testes (modified Davidson’s solution), thymus, thyroid glands, trachea, urinary bladder, uterus, vagina
At necropsy additional liver samples (Processus papillaris and caudatus) were taken from all F0 animals per sex and group. The weight of these liver samples was recorded (per animal) for the females only. All samples were then frozen in liquid nitrogen and stored at -80°C.
The uteri of all cohabited female F0 parental animals were examined for the presence and number of implantation sites. The uteri of apparently non-pregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations (SALEWSKI's method (1)). Then the uteri were rinsed carefully with 0.9% NaCl-solution. When the examinations were completed, the uteri were transferred to the Pathology Laboratory for further processing.
(1)SALEWSKI, E.: Färbemethode zum makroskopischen Nachweis von Implantationsstellen am Uterus der Ratte; Naunyn-Schmiedeberg’s Arch. Exp. Pathol. Pharmakol. 247, 367 (1964)

HISTOPATHOLOGY: Yes
For the following tissues fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings: 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, peyers 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, vagina.
Special attention was given on stages of spermatogenesis in the male gonads.

Postmortem examinations (offspring):

- Necropsy observations: All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia with CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically. 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 were evaluated on a case-by-case basis, depending on the type of finding.

Statistics:

- 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 (apart from pH, urine volume, specific gravity, color and turbidity): Pairwise comparison of each dose group with the control
group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
- 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.
Statistics of 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.
Statistics of the clinical examinations:
- Food consumption, body weight and body weight change (parental animals); Simultaneous comparison of all dose groups with the control group using the DUNNETT test (two-sided) for the hypothesis of equal means

Reproductive indices:

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.
For the males, mating and fertility indices were calculated for F1 litters according to the following formulas:
Male mating index (%) = (number of males with confirmed mating* / number of males placed with females) x 100
* defined by a female with vaginal sperm or with implants in utero
Male fertility index (%) = (number of males proving their fertility* / number of males placed with females) x 100
* defined by a female with implants in utero

For the females, mating, fertility and gestation indices were calculated for F1 litters according to the following formulas:
Female mating index (%) = (number of females mated* / number of females placed with males) x 100
* defined as the number of females with vaginal sperm or with implants in utero
Female fertility index (%) = (number of females pregnant* / number of females mated**) x 100
* defined as the number of females with implants in utero
** defined as the number of females with vaginal sperm or with implants in utero
Gestation index (%) = (number of females with live pups on the day of birth / number of females pregnant*) x 100
* defined as the number of females with implants in utero
The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters according to the following formula:
Live birth index (%) = (number of liveborn pups at birth / total number of pups born) x 100
The implantations were counted and the postimplantation loss (in %) was calculated according the following formula:
Postimplantation loss (%) = ((number of implantations – number of pups delivered) / number of implantations) x 100

Offspring viability indices:

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 according to the following formula:
Viability index (%) = (number of live pups on day 4 after birth / number of live pups on the day of birth) x 100

The sex ratio was calculated at day 0 and day 4 after birth according to the following formula:
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

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no test substance-related mortalities in any of the male and female parental animals in any of the groups.
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any male or female F0 generation parental animals during the whole study including gestation and lactation periods.
Several male and female animals of the high-dose group (90 mg/kg bw/day) showed salivation after treatment during mating (males), post-mating, gestation and lactation. This transient salivation for a few minutes immediately after treatment was likely to be induced by the unpleasant taste of the test substance or by local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Mean body weights of the male and female F0 generation parental animals in all test substance-treated groups were comparable to the concurrent control group during the entire study period.
The high-dose parental males had a statistically significantly lower body weight change during premating days 0 - 7 (about 37% below control).
The body weight change of the mid and low-dose parental males was comparable to the concurrent control group during the whole study period.
The high-dose parental females had statistically significantly lower body weight change during PND 0 - 4 (about 65% below control).
The mean body weight change of the high-dose parental females was comparable to the concurrent control group during premating and gestation. The body weight change of the mid and low-dose parental females was comparable to the concurrent control group during the whole study period.
Food consumption of the high-dose F0 males (90 mg/kg bw/day) was statistically significantly below control during premating days 0 - 7 (about 11%).
The mid and low-dose F0 males (30 and 10 mg/kg bw/day) did not show any test substancerelated changes in food consumption during the whole treatment period.
Food consumption of the female F0 generation parental animals in all test substance-treated groups (10, 30 and 90 mg/kg bw/day) was comparable to the concurrent control group during the entire study period.

DETAILED CLINICAL OBSERVATIONS (DCO) (PARENTAL ANIMALS)
Male and female animals of all dose groups (90, 30 and 10 mg/kg bw/d) did not show any abnormalities.

FUNCTIONAL OBSERVATIONAL BATTERY (FOB) (PARENTAL ANIMALS)
- 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.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all groups including the controls. Fertility was proven for all F0 parental males within the scheduled mating interval for F1 litter. Thus, the male fertility index was 100% in all groups including the controls.
The female mating index calculated after the mating period for F1 litter was 100% in all test groups. The mean duration until sperm was detected (GD 0) varied between 3.0 and 4.3 days without any relation to dosing. All sperm positive rats delivered pups. The fertility index was 100% in all test groups.
The mean duration of gestation was similar in all test groups (i.e. between 22.4 and 22.5 days). The gestation index was 100% in all test groups.
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the controls, taking normal biological variation into account (14.0 / 12.0 / 11.9 and 13.5 implants/dam in all test groups (0, 10, 30 and 90 mg/kg bw/day)). There were no statistically significant differences in post-implantation loss between the groups (4.1% / 7.7% / 8.8% / 5.1%). The mean number of F1 pups delivered per dam remained unaffected and was comparable between the test substance-treated groups and the control, taking normal biological variation into account (13.4 / 11.1 / 10.9* [* = p-level of significance ≤0.05] and 12.8 pups/dam at 0, 10, 30 and 90 mg/kg bw/day). The slight, statistically significant reduction in the mean number of F1 pups delivered per dam in the mid-dose females was a result of a small litter size in only two of the females of this group. If these litters are excluded, the remaining average litter size, 12.0, is not different than that of the other dose groups. Since the other litters were similar in size to those found in the control group, there did not appear to be a dose-response relationship. Furthermore, 10.9 pups/litter is within the range of the historical control data at this test facility. All together, these results suggest that the litter size reduction in dose group 2 is spontaneous in nature.
The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 100% control and low-dose group), 99.1% (mid-dose group) and 97.7% (high-dose group). Moreover, the number of stillborn pups was comparable between the groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Absolute weights
When compared to the control group, the mean absolute weight of the liver was significantly increased in the high-dose male and female animals. All other mean absolute weight parameters of treated male and female animals did not show relevant differences when compared to the control group and are therefore considered to be within the normal range.
Relative weights
When compared to the control group, the mean relative weight of the liver was significantly increased in the high-dose male and female animals. All other mean relative organ weights of treated male and female parental animals did not show relevant differences when compared to the control group and are therefore considered to be within the normal range.
The significant absolute and relative weight increase in the liver of males and females in the high-dose group (90 mg/kg bw/day) was consistent with histopathological findings and was considered to be treatment-related.

CLINCAL PATHOLOGY (PARENTAL ANIMALS)
- Hematology: No treatment-related, adverse changes among hematological parameters were observed. In males of test group 3 (90 mg/kg bw/d), hematocrit and prothrombin (Hepatoquick’s test = HQT) values were decreased, whereas relative reticulocyte counts were increased. All mentioned parameter means were within historical control ranges (hematocrit 0.396-0.448 L/L; prothrombin time 30.2-37.9 sec; relative reticulocyte counts 0.9-2.5 %). Therefore, the changes were regarded as incidental and not treatment-related. In males of test group 1 (10 mg/kg bw/d) total white blood cell counts, as well as absolute lymphocyte counts, were decreased, but the alterations were not dose-dependent and therefore, the changes were regarded as incidental and not treatment-related. In males of test group 3 (90 mg/kg bw/d) absolute large unstained cell (LUC) counts were increased and
the mean was slightly above the historical control range (LUC 0.01-0.04 Giga/L). However, this was the only changed differential blood cell parameter in these rats. No change occurred in the females of the corresponding dose group. Therefore, the change was regarded as maybe treatment-related, but not adverse.
- Clinical chemistry: In males of test group 3 (90 mg/kg bw/d), urea, cholesterol and inorganic phosphate levels were increased, but chloride concentrations were decreased. Additionally, in females of the same test group urea levels were higher compared to controls.
- Urinalyses: In males of test group 3 (90 mg/kg bw/d), more phosphate crystals and transitional epithelial cells were observed in the urine sediment.
Additionally, in females of the same test group urine volume was higher. This alteration is per se not regarded as an adverse effect.

GROSS PATHOLOGY (PARENTAL ANIMALS)
All gross findings noted at necropsy in parental animals are regarded as incidental and spontaneous and are not related to treatment.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Treatment-related findings were observed in the liver of parental male and female animals; in 5 out of 5 high-dose male and 5 out of 5 high-dose female animals centrilobular hypertrophy was observed. This finding correlated with a slight absolute and relative liver weight increase. However, this finding is regarded as adaptive and not adverse.
Two out of ten parental female animals of the high-dose group (90 mg/kg bw/day) showed a minimal follicular hypertrophy/hyperplasia in the thyroid gland when compared with the ten females of the control group. Due to the minimal magnitude and the low incidence of this change it was not regarded as treatment related and was considered to be an incidental finding.
All other findings noted in parental animals were either single observations, or were biologically equally distributed between controls and treated rats. All of them are considered to be incidental and/or spontaneous in origin.

Dose descriptor:

NOAEL

Remarks:

parental

Effect level:

30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

90 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at the highest dose tested.

VIABILITY (OFFSPRING)
The viability index indicating pup mortality during lactation (PND 0 - 4) varied between 94.4% (high-dose group), 90.0 (mid-dose group), 100.0 (low-dose group) and 99.3% (control), well within historical control values.
The reduction in pup numbers in the mid-dose group was amplified by PND 4. Eleven pups died/were cannibalized before PND 4, resulting in a small, but statistically significantly decreased, average live litter size of 9.7 (compared to 13.3 in the concurrent control group). A further 5 pups in the high dose group also died/were cannibalized intercurrently. All 16 pups originated from only two litters, nos. 123 and 133 (1 each in the 30 and 90 mg/kg bw/day dose groups, respectively), which showed signs of poor maternal care, such as findings of pups not fed/no milk in stomach and reduced nutritional condition in the pups. As the incidence of pup deaths is restricted to a small number of litters, lies within the normal range of biological variation inherent in the rat strain used and is not dose-dependent, the death of these pups was probably incidental.

CLINICAL SIGNS (OFFSPRING)
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.

BODY WEIGHT (OFFSPRING)
Mean body weights and mean body weight change of the male and female pups in all test substance-treated groups were comparable to the concurrent control group during the entire study period.
One female runt was seen in the high-dose group.
Two male runts were seen in the mid-dose group.
One female runt was seen in the low-dose group.
Five male and two female runts were seen in the control.

GROSS PATHOLOGY (OFFSPRING)
A few pups showed spontaneous findings at gross necropsy, such as post mortem autolysis, empty stomach and discolored liver lobe. These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. None of these findings were considered to be associated with the test substance.

HISTOPATHOLOGY (OFFSPRING)
In one pup of the control group and one pup of mid-dose group (30 mg/kg bw/day), dissecting aneurysms of the ductus arteriosus and aorta correlated with macroscopic changes observed at necropsy. Since these findings occurred without a dose-dependency they were considered to be incidental and not related to treatment.

OTHER FINDINGS (OFFSPRING)
The mean number of delivered F1 pups per dam and the rates of liveborn and stillborn F1 pups were comparable between the dose groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study. The slight, statistically significant reduction in the mean number of F1 pups delivered per dam in the mid-dose females was considered to be spontaneous in nature.

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

90 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at the highest dose tested.

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Imidazole, when tested in the rat according to OECD Guideline 414 at dose levels of 20, 60, and 180 mg/kg bw/d, was developmentally toxic and teratogenic at 180 mg/kg bw/d. The NOAEL for maternal toxicity, developmental toxicity and teratogenicity was 60 mg/kg bw/d.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Oct 2001 to Sep 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was performed according to OECD Guideline 414 and GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, 67056 Ludwigshafen, Germany

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

SPECIES AND STRAIN
Time-mated Wistar rats (CrIGIxBrIHan:WI ) supplied by Charles River Laboratories, Germany
Animals free from clinical signs of disease, were used for the investigations.
The female animals were supplied at an age of about 70 - 84 days on October 09, 10 and 11, 2001.
Based on the pregnant animals the body weight on day 0 varied between 139.4 -10 186.4 g.

ANIMAL IDENTIFICATION
The animals were mated by the breeder and supplied on day 0 post coitum (= detection of vaginal plug/sperm).
They were assigned to the test groups by taken random selection from the transport box.
After randomization the rats were identified uniquely by ear tattoo.

REASON FOR SPECIES SELECTION
This strain was selected since extensive experience is available on Wistar rats and this strain has been proved to be
sensitive to substances with ateratogenic potential.

HOUSING AND DIET
The rats were housed singly from day 0- 20 p.c. in type DK III stainless steel wire mesh cages.

The animals were accommodated in fully air- conditioned rooms in which central air conditioning guaranteed a range of
temperature of 20 - 24°C and a range of relative humidity of 30 - 70%. There were no deviations from these limits.

The day/ night rhythm was 12 hours (12 hours light from 6.00 a.m. to 6.00 p.m. and 12 hours
darkness from 6 .00 p.m. to 6.00 a.m.).
Before the study started, the animal room was completely disinfected using a disinfector ("AUTEX", fully automatic, formalin-ammonia - based terminal disinfection). In general, each week the walls and the floor were cleaned with water containing about 0 .5% Mikro-Quat (supplied by ECOSAN GmbH, FRG).

The food used was ground Kliba maintenance diet rat/mouse/hamster meal, supplied by PROVIMI KLIBA SA, Kaiseraugst, Switzerland.
Foodwas available to the animals ad libitum throughout the study (from the day of supply to the day of necropsy), as was
drinking water of tap water quality from water bottles.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

TS dissolved in doubly distilled water and the oral route was selected, since this has proven to be suitable for the detection of a toxicologicalhazard.
The test substance solutions were administered to the animals orally (by gavage) once a day from implantation to one day prior to the expected day of parturition (day 6 to day 19 p.c.) always at approx. the same time of day (in the morning). The animals of the control group were treated in the same way with the vehicle (doubly distilled water). The volume administered each day was 10 ml/kg body weight. The calculation of the volume administered was based on the last individual body weight. The fetuses were removed from the uterus and further investigated with different methods. Due to technical reasons, the study was carried out in 3 sections. Each dose group was represented in each section. A treatment interval of one day elapsed before the next section.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verifications of the stability of the test substance in doubly distilled water at room temperature were carried out with GC method before andafter the study had been initiated. Samples of the test substance solutions were sent to the analytical laboratory twice during the
study period (at the beginning and towards the end) for verification of the stability and/or the concentrations. Since the test substance preparations were true solutions, investigations concerning homogeneity were not necessary.

Details on mating procedure:

The animals were mated by the breeder ("time-mated") and supplied on day 0 post coiturn (= detection of vaginal plug / sperm).
The animals arrived on the same day (i .e. day 0 p.c.) at the experimental laboratory.
The following day was designed "day 1" post coitum (p.c.). Between start of the study (beginning of the experimental phase) and
first administration (day 6 p.c.) the animals were acclimated to the laboratory conditions.

Duration of treatment / exposure:

TS was administered by oral gavage once a day from implantation to one day prior to expected parturition, i.e. d 6-19 p.c.

Frequency of treatment:

7 d/wk

Duration of test:

Start of the experimental phase (day 0 p.c.) was on Oct 09, 2001; last sacrifice (day 20 p.c.) was on Oct 31, 2001.

Remarks:

Doses / Concentrations:
0, 20, 60, 180 mg/kg bw/d
Basis:
nominal conc.

No. of animals per sex per dose:

25 time-mated female rats per dose, age ca. 70 d at beginning of the study (day 0, detection of sperm), were used.
Body weights ranged between 143 to 186 g on day 0 post coitum (p.c.).

Control animals:

yes, concurrent vehicle

Details on study design:

Only pregnant dams were used for the calculations of mean maternal food consumption, body weight and body weight change.
Only pregnant dams with scheduled sacrifice (day 20 p .c.) were taken for the calculation of mean gravid uterine weights,
mean net maternal body weight change (corrected body weight gain) and summary of reproduction data.

In this study the following females were partially or totally excluded from the above mentioned calculations:
Test group 0 (0 mg/kg bw/d): females Nos. 1, 9 and 15 - not pregnant
Test group 1(20 mg/kg bw/d): female No . 33 - not pregnant
Test group 2 (60 mg/kg bw/d): females Nos . 57 and 62 - not pregnant
Test group 3 (180 mg/kg bw/d): female No. 79 - not pregnant

Thus, according to the requirements of the most relevant OECD test guidelines which are listed in chapter 2 .3., each test group including
the controls contained a sufficient number of females with implantation sites at necropsy (" . . . .approximately 20, but not fewer than 16 females with implantation sites").

Maternal examinations:

During the conduct of the study the animals were examined at least daily for clinical symptoms. Mortality was checked twice a day or once
on Saturday/ Sunday. Food and water consumption, body weight on days 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20 p.c..
Corrected body weight gain was calculated after terminal sacrifice as [bw d 20- (uterus weight + bw d 6 p.c.)].
Dams were sacrificed on day 20 p.c.. Necropsy included gross pathology assessment weight determinations of the unopened
uterus and the placentae.

Ovaries and uterine content:

Uterus and ovaries were removed with subsequent examination and recording of:
unopened uterus weight; no. of corpora lutea; no. and classification of implantation sites as live fetuses or dead implantations
(early, late resorptions, dead fetuses). Calculation of conception rate and pre- and postimplantation losses.

Fetal examinations:

Fetuses were weighed and sexed by anogenital distance observation (which was later confirmed by internal examination of those fetuses
which were preserved in BOUIN's solution), and macroscopically examined (viability, condition of placenta, fetal membranes, umbilical cords,placental weight). Approx. 50% of all fetuses were subjected to soft tissue examinations after fixation in BOUIN's solution, the other
50% of fetuses was examined for skeletal changes.

Statistics:

Statistical evaluation of data included FISHER's Exact Test for conception rate, mortality of the dams, and number of litters with fetal findings; WILCOXON Test for proportions of fetuses with malformations and/or variations in each litter; and DUNNETT's Test for all other data
including water consumption and body weight gain.

Indices:

Calculations of conception rate and pre- and postimplantation losses were carried out:

- The conception rate ( in %) was calculated according to the following formula :
(number of pregnant animals/number of fertilized animals)x100

- The preimplantation loss (in %) was calculated according to the following formula :
(number of corpora lutea - number of implantations/number of corpora lutea)x100

- The postimplantation loss (in %) was calculated from the following formula :
(number of implantations - number of live fetuses/ number of implantations)x100

Historical control data:

Detailed historical control data were included in the study report.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Only pregnant dams or pregnant dams sacrificed at schedule were used. Animals which were totally or partially excluded were: 3 non-pregnant control animals; one non-pregnant animal from each dose group. No mortality was seen in any of the groups. The group mean reproductive and fetal data can be found in table 1 below.

Low dose, 20 mg/kg bw/d: No substance related effects seen.
Intermediate dose, 60 mg/kg bw/d: No substance related effects seen, except slight but significant decrease of body weight gain on d 6-8 p.c.
High dose, 180 mg/kg bw/d:
- transient salivation in 6 females between days 15 - 19 p.c. and vaginal hemorrhage in one dam on day 20 p.c.
- statistically significantly decreased food consumption on days 6 - 8 p.c. (about 13% less food intake as in the concurrent control group)
- statistically significantly impaired body weight gain on days 6 - 8 p.c. (about 45% below controls) and on days 17 - 20 p.c. (33% - 34% below controls)
- statistically significantly lowered mean uterus weight (about 26% below controls)

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
No substance related effects were observed at 20 and 60 mg/kg bw/d. Sex distribution was comparable to control animals in all treatment
groups.

Embryotoxic and teratogenic effects were present at the high dose level of 180 mg/kg bw/d (see also table 2 below):
- statistically significantly increased number of resorptions (especially late ones) and consequent a statistically-significantly increased
postimplantation loss value (43 .4% versus 7.9% in the controls); 3 of the 24 pregnant dams resorbed all implants and had no live fetuses
at necropsy
- statistically significantly increased mean placental weights (about 22% above controls)
- markedly reduced mean fetal body weights (about 14% below controls) due to a higher number of stunted fetuses (so-called runts)
- statistically significantly increased rate of external malformations (anasarca and/or cleft palate); these findings occurred in 13 of 132
fetuses [= 9.8%] in 7 out of 21 litters [= 33%], i.e. 9.0% versus 0.0% affected fetuses/ litter in the control group
- statistically significantly increased rate of soft tissue variations (dilated renal pelvis, dilated ureter) with a total of 27.1 % versus 6.4%
affected fetuses/litter in the control group
- statistically significantly increased rate of skeletal malformations (e.g. shortened scapula, bent radius/ulna, malpositioned and bipartite
sternebra); these findings occurred in 7 of 73 fetuses [= 9.6%] in 5 out of 21 litters [= 24%], i.e. 7.8% versus 1.1% affected fetuses/litter in
the control group
- statistically significantly increased occurrence of several skeletal variations (mainly delays in the ossification process) with a total of 98.4%
versus 91.1 % affected fetuses/litter in the control group
- statistically significantly increased rates of total malformations (10.8% versus 0.6% affected fetuses/ litter in the control group), variations
(7 0.4% versus 52.0% affected fetuses/ litter in the control group) and unclassified skeletal cartilage observations (69.8% versus 50.8%
affected fetuses/ litter in the control group)

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day

Basis for effect level:

other: fetotoxicity

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Clinical data:

Transient salivation was noted in 6/25 animals during days 15-19 p.c. starting few minutes after dosing and lasting 15 to 20 min. Vaginal hemorrhage on d 20 was noted in one animal. Salivation was regarded to be treatment related due to bad taste or local affection of the upper digestive tract, but was not assessed as an adverse or toxic effect.

Food intake, body weights:

Food intake was significantly reduced (-13% compared to controls) during a period at the beginning of treatment on d 6-8 p.c. Body weight gain was also statistically reduced compared to controls on d 6-8 p.c. (-45%) and d 17-20 p.c. (-34%). However, at the end of the treatment period body weight was comparable in all groups. Findings were regarded as being substance-induced and reflecting direct adverse effects on dams (d 6-8) and on fetuses (d 17-20; resorptions, lowered fetal body weight). Corrected body weight gain was comparable in all dose groups.

Table 1 Group mean reproductive and fetal data

 

Dose levels (mg/kg bw/d)	0	20	60	180
Pregnancies on day 20	22	24	23	24
Conception rate	88	96	92	96
Dams with viable fetuses	22	22	23	21
Gravid uterus weight (g)	51.3 (9.52)	45.6 (16.65)	50.0 (11.57)	38.2 (16.81)**
Implantations/dam	9.7 (1.67)	9.0 (2.1)	9.7 (1.69)	9.3 (1.88)
Pre-implantation loss (%)	10.7 (14.07)	18.5 (20.84)	14.5 (13.33)	15.7 (12.46)
Post-implantation loss (%)	7.9 (10.13)	14.8 (28.31)	9.6 (11.77)	43.4 (34.09) **
Resorptions (total)	0.8 (1.05)	0.9 (1.23)	0.9 (1.12)	3.8 (3.06) **
Live fetuses/dam	8.9 (1.61)	8.8 (1.68)	8.8 (1.93)	6.3 (3.15) **
Fetal weight (g) (all viable)	3.7 (0.27)	3.6 (0.2)	3.6 (0.32)	3.2 (0.27) **
Placental weights (mg) (all viable fetuses)	460 (62)	450 (46)	490 (57)	560 (173) **
Sex ratio (% males)	53	41	52	46
SD in brackets ** p < = 0.01 (Dunnett test) (two-sided)

 

Table 2 Summary of all classified fetal external, soft tissue, and skeletal observations

 

Parameter	No. and (%) fetuses at (mg/kg bw/d)
	0	20	60	180
No. litters evaluated	22	22	23	21
No. fetuses evaluated	195	194	202	132
Total malformations, mean (%) (affected fetuses/litter)	0.6 (3.05)	1.1 (3.68)	0.5 (2.32)	10.8 (14.67) **
Total variations, mean (%) (affected fetuses/litter)	52 (11.51)	50 (13.24)	61 (15.8)	70.4 (20.64) **
Unclassified skeletal cartilage observations, mean %, (affected fetuses/litter)	50.8 (29.2)	42.9 (38.11)	51.2 (28.95)	69.8 (28.79) **
External malformations, mean - litter incidence (%) - affected fetuses/litter (%)	0 0	0 0	0 0	33 ## 9 (15.08) **
Skeletal malformations, mean - litter incidence (%) - affected fetuses/litter (%)	4.5 1.1 (5.33)	9.1 2.3 (7.36)	4.3 0.9 (4.17)	24 7.8 (15.95) *
Soft tissue variations, mean - affected fetuses/litter (%)	6.4 (16.25)	9.2 (17.02)	22.7 (29.69) *	27.1 (35.05) *
Skeletal variations, mean - affected fetuses/litter (%)	91.1 (14.91)	87.2 (16.1)	94.2 (9)	98.4 (7.27) *
SD in brackets * p < = 0.05 (Wilcoxon-test, one-sided), ** p < = 0.01 (Wilcoxon-test, one-sided) ## p < = 0.01 (Fischer’s exact test, one-sided)

 

Executive summary:

The developmental toxicity of imidazole was studied in accordance to OECD TG 414 in rats at dose levels of 20, 60 and 180 mg/kg bw/d.

Maternal toxicity was noted exclusively at 180 mg/kg bw/d as substantiated by significantly reduced food intake at initiation of treatment and impaired body weight gains on days 6-8 p.c.. No signs of maternal toxicity were seen at 60 mg/kg bw/d and below. Fetal development was adversely affected at 180 mg/kg bw/d as substantiated by the high rate of late resorptions (3/24 animals showing complete resorption) which resulted in an elevated postimplantation loss and reduced fetal body weights.

At 180 mg/kg bw/d selective teratogenicity was indicated by increased occurrence of external and skeletal malformations and variations of which anasarca, cleft palate, shortened scapula, and incomplete or delayed ossifications were the most prominent. No increases were noted at 20 or 60 mg/kg bw/d.

Based on these findings NOAEL for maternal toxicity and for prenatal developmental toxicity is 60 mg/kg/d.

Reference 2

Endpoint:

developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

no further testing for developmental toxicity is necessary because the substance is known to cause developmental toxicity, meeting the criteria for classification as toxic for reproduction category 1A or 1B: May damage the unborn child (H360D), and the available data are adequate to support a robust risk assessment and the classification and labelling of the substance

Abnormalities:

not specified

Developmental effects observed:

not specified

Reference 3

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

Oct 2015 - June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

see attached WoE Assessment Reproductive Toxicity in chapter 13

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted on Jan 22, 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 440/2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted on Aug 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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
- Strain: Crl:WI(Han)
- Age at study initiation: about 10-12 weeks
- Housing: Polycarbonate cages type III, one animal per cage
- Enrichment/Bedding: Wooden gnawing blocks (Typ NGM E-022), Abedd, Lab. and Vet. Service GmbH, Vienna, Austria. Dust-free wooden bedding
- Diet: ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water: drinking water supplied from water bottles; ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hrs/ 12 hrs

IN-LIFE DATES: From: 12 Oct 2015 To: 28 Oct 2015

Route of administration:

oral: gavage

Vehicle:

other: drinking water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The aqueous test substance preparations were prepared at the beginning of the administration period and thereafter at maximum intervals of 7 days, which took into account the period of established stability.
- To prepare the dose solutions, the specific amount of test substance were weighed, topped up with drinking water in a calibrated beaker and intensely mixed with a magnetic stirrer until it is completely dissolved. A magnetic stirrer was also used to keep the preparations homogeneous during treatment of the animals.

VEHICLE
- Concentration in vehicle: 0, 0.1, 0.3 and 0.9 mg/100 mL water, respectively in the 0, 10, 30 and 90 mg/kg bw dose groups
- Amount of vehicle (if gavage): 10 mL of the aqueous preparations were applied per kg bw.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in drinking water at room temperature over a period of 7 days had been verified prior to the start of the study in a similar batch (Project No.: 01Y0492/11Y063).

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant
The animals arrived on the same day (gestation day [GD] 0) at the experimental laboratory. The following day was designated as “GD 1”.

Duration of treatment / exposure:

from implantation to one day prior to the expected day of parturition (GD 6 to GD 19), always at approx. the same time in the morning

Frequency of treatment:

once a day

Duration of test:

On GD 20, all surviving dams were sacrificed and examined macroscopically.

Remarks:

Doses / Concentrations:
10, 30, 90 mg/kg bw/day
Basis:
analytical conc.

No. of animals per sex per dose:

25 animals per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on available toxicological information for the substance, especially toxicity findings in the Dose-Range-Finder für repeated dose toxicity (BASF SE, 2013).

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. If such signs occur, the animals were examined several times daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily; a check for mortality was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-20).

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights was recorded on GD 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20. Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6).

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption will be recorded for GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
On GD 20, the dams were sacrificed under isoflurane anesthesia by cervical dislocation (in randomized order according to a randomization plan) necropsied and assessed by gross pathology. The uteri and the ovaries were removed and the following data were recorded: weight of the unopened uterus, number and distribution of implantation sites (classified as: live fetuses, dead implantations [early resorptions, late resorptions, and dead fetuses]). After the weight of the uterus had been determined, all subsequent evaluations of the dams and the gestational parameters were conducted by operators unaware of treatment group in order to minimize bias.

Ovaries and uterine content:

The following examinations will be carried out:
- Number of corpora lutea
- Number of implantations (differentiated according to live and dead fetuses and early or late resorptions)
- Early resorptions according to SALEWSKI in animals that do not appear to be pregnant and animals with single-horn pregnancy
- Site of implantations in the uterus

Fetal examinations:

After the fetuses have been removed from the uterus, the following examinations or weight determinations were carried out:
- Weight of each fetus, Sex, Viability of the fetuses and condition of placentae, umbilical cords, fetal membranes, and fluids, Weight of the placentas.
- Gross-pathological examination of the fetuses after dissection from the uterus (including abnormalities of the fetal membranes, placentas, amniotic fluid and umbilical cord); then all fetuses will be sacrificed by subcutaneous injection of pentobarbital (e.g. Narcoren®, dose: 0.1 ml/fetus).
- About half of the fetuses of each dam was skinned, fixed in ethyl alcohol and, after fixation, stained according to a modified method (KIMMEL and TRAMMELL ) to show the skeleton and the cartilage. After the skeletons/cartilage have been examined, these fetuses were archived individually.
- The other half of the fetuses of each dam was fixed in Harrison’s fluid. After fixation, the soft tissues of these fetuses were examined according to a modified microdissection method (BARROW and TAYLOR ). After the examination, these fetuses were discarded.

Statistics:

Means and standard deviations were calculated. In addition, the following statistical analyses will be carried out:
- Food consumption, body weight, body weight change, corrected body weight gain, weight of the unopened uterus, weight of the placentas and fetuses, corpora lutea, implantations, pre and postimplantation losses, resorptions and live fetuses: DUNNETT’s test
- Number of pregnant animals at the end of the study, mortality rate (of the dams) and number of litters with fetal findings: FISHER's exact test
- Proportion of fetuses with findings per litter: WILCOXON test.

Indices:

Conception rate, preimplantation loss, postimplantation loss

Historical control data:

Historical control data were available

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Transient salivation during the treatment period. No clinical signs or changes of general behavior were detected.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean bw were in general comparable. Consistently to the reduced food consumption on GD 6-8, the bw change of the mid and high dose groups was statistically significantly reduced on D6-8 (approx 28%/81% of control). If calculated for the entire treatment period or the entire study period avarage bw change values were quite similar.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not specified

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

Details on maternal toxic effects:
So far, preliminary results do not show any adverse effects of 1-methylimidazole to the dams.
No. of females mated, pregnant and with viable fetuses were comparable to the control animals. Only salivation in 3 of 25 HD animals, but no other clinical signs were observed. At the beginning of the administration period food consumption (d6-10) and body weight gain (d6-8) were significantly reduced, but this was not observed during the rest of the administration period. Uterus and carcass weight as well as corrected body weight gain were comparable to the control group.

Dose descriptor:

NOAEL

Effect level:

90 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other:

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
No. of postimplantational loss, total resorptions, live fetuses per dam, placental and weights as well as external malformations were comparable to the control group.

Dose descriptor:

NOAEL

Effect level:

>= 90 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Abnormalities:

no effects observed

Developmental effects observed:

no

Reference 4

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: In-vitro study (teratogenicity screen).

Principles of method if other than guideline:

Method: other: whole embryo culture technique

GLP compliance:

not specified

Species:

other: rat and mouse embryos

Strain:

other: Sprague Dawley rats and CD-1 mice

Route of administration:

other: in vitro exposure

Duration of treatment / exposure:

not indicated, presumably 2 days

Frequency of treatment:

continuous

Remarks:

Doses / Concentrations:
30 or 60 ug/ml
Basis:

Control animals:

yes, concurrent vehicle

Details on study design:

Sex: no data
Duration of test: 2 days

Abnormalities:

not specified

Developmental effects observed:

not specified

A concentration-related high mortality up to 83.3 % and
a high rate of abnormalities up to 100 % was found in
treated embryos.
Treated embryos showed also reduced yolk sac diameter and
crown rump length and less somites.
Characteristic abnormalities were decreased brain size and
clear blisters. Mouse embryos were generally more sensitive
to substance related effects.

Executive summary:

Some indication of developmental toxicity was obtained in a whole embryo culture test when rat and mouse embryos were exposed in vitro to imidazole at 30 and 60 μg/ml in vitro. The findings of this teratogenicity screen included reduced yolk sac diameter and crown rump length, and decreased brain size observed in up to 100 % of treated embryos. Mortality was up to 83 % in this exploratory study

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

60 mg/kg bw/day

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

In a prenatal developmenal study conducted in accordance with OECD TG 414, imidazole (99.8%) was administered by oral gavage to Wistar rats from day 6 to 19 of gestation. The dose levels were 0 (vehicle control, water), 20, 60 or 180 mg/kg bw/day. During the study the dams were assessed for clinical observations, body weight and food consumption, and corrected body weight was determined upon necroscopy. At necroscopy, dams were examined for gross pathological changes, the number of corpora lutea in the ovaries, conception rate, the number of live fetuses and pre- and postimplantation losses. The fetuses were weighed, sexed and macroscopically examined for external alterations. One half of all fetuses were fixed and examined for effects on the inner organs, while the other half of fetuses were fixed and stained for skeletal and cartilage evaluation. No signs of maternal toxicity, fetal or developmental toxicity were noted at 20 and 60 mg/kg bw per day. At 180 mg/kg bw/d significantly reduced food intake (-13 %) was noted when the treatment was started. This was reflected by a statistical significantly reduced body weight gain on gestational days 6 - 8 (-45 %) and 17 - 20 (-34 %). However, terminal body weight was comparable in all groups, and corrected terminal body weight gain was also comparable in all groups. The effect on body weight gain on gestational days 17 - 20 is due to a significant decrease of the gravid uterus weight (-26 %), high rate of resorptions (see below) and distinctly lower mean fetal body weight (see below), rather than maternal toxicity. The number of live fetuses per litter was significantly reduced and the postimplantation loss was 43 % compared to only 8 % in the control being statistically significant. Examination of the live fetuses from high dose dams revealed no changes with respect to sex distribution. The mean fetal body weight was reduced by 14 %. Further, the incidence of external malformations (anasarca and/or cleft palate) was significantly increased. About 10 % of the high dose fetuses were affected (13/132 fetuses; in 7/22 litters) while no such changes were observed in the control. Skeletal malformations were also statistically significantly increased: 7.8 % affected fetuses per litter (7/73 fetuses in 5/21 litters) were noted in the high dose group compared to 1.1 % in the control. The incidences of shortened scapula, bent radius, bent ulna, malpositioned and bipartite sternebrae were statistically significantly increased. Soft tissue variations (dilated renal pelvis and ureter) were significantly increased in fetuses from high dose dams compared to controls (27 % vs. 6.4 %). The incidences of skeletal variations, mainly delays of the ossification process, were statistically significantly increased from 91 % in the control group to 98.4 % in the high dose group. In historical control animals the mean occurrence of skeletal variations is 92.6 % (range 87.0 -98.1 %). The NOAEL for maternal toxicity, developmental toxicity and teratogenicity was 60 mg/kg bw/day (BASF SE, 2002).

Some indication of developmental toxicity was obtained in a whole embryo culture test when rat and mouse embryos were exposed to imidazole at 30 and 60 μg/mL in vitro. The findings of this teratogenicity screen included reduced yolk sac diameter and crown rump length, and decreased brain size observed in up to 100 % of treated embryos. Mortality was up to 83 % in this exploratory study (Daston et al., 1989).

Toxicity to reproduction: other studies

Additional information

In a paper by Adams et al (1998), increasing single doses of imidazole (0-300 mg/kg bw) were administered to rats (10 male rats/group) via subcutaneous injection and samples of serum and TIF (testicular interstitial fluid) investigated at a single time point only (after 2 hours). Imidazole decreased serum testosterone in a dose-dependent manner. Imidazole dose-dependently also decreased TIF testosterone concentrations, TIF volume, and LH secretion. This study suggests imidazole may have endocrine disrupting properties on sex hormones when given subcutaneously to rats.

However, the findings reported by Adams et al. (1998) are considered to be of limited relevance because the subcutaneous injection does not represent a relevant exposure route. In addition, the precise s.c. injection site is not indicated in the publication, only one time point (2 hours after treatment) was studied and no microscopical examination of the testes was performed. In addition, there were no adverse effects potentially related to endocrine disruption in the available standard oral studies (e.g. no effects on sperm parameters in the 90-day study (OECD 408) and no effects on sex ratio and reproduction organs in the pre-natal developmental toxicity study). It is therefore possible that administration via the subcutaneous route results in larger plasma peaks and slower metabolic clearance than would be seen orally .

Based on the absence of effects related to endocrine disrupting properties in the available studies of imidazole and structural analogues, the potential endocrine disrupting properties of imidazole on sex hormones is unlikely. In addition, imidazole is already classified as Repr 1B; H360D (May damage the unborn child) and further testing is therefore not considered necessary.

Justification for classification or non-classification

Imidazole caused developmental toxicity and teratogenicity in the rat in the developmental toxicity study according to OECD TG 414. Based on this study, imidazole may cause damage to the unborn child and is classified and labelled with H360D Cat. 1B (according to Regulation 1272/2008/EC). This classification was confirmed by RAC in 2013 and the classification and labelling was included in the seventh ATP to the CLP.

Effects on fertility were not observed in a rat 90 -day oral gavage study up to the highest dose level (180 mg/kg bw/day) with thorough histopathological examination of all male and female reproductive organs, sperm and estrus cycle analysis. In addition, five structural analogues of imidazole do not cause fertility impairing effects. Based on a weight of evidence assessment, classification for fertility is not warranted.