Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a GLP compliant, OECD 421, reproduction/developmental toxicity screening test in rats, no adverse effects were observed on the reproductive organs of males and females or on fertility (i.e. reproductive functions up to and including implantation) up to the highest dose (500 mg/kg bw/d). The NOAEL for general toxicity and reproductive toxicity in the parents was 150 mg/kg bw/day (by oral gavage), based on mortality (shortly after parturition; showing signs of complicated parturition), slighly reduced body weight and food consumption, and reduced live birth index at 500 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
According to REACH Annex IX, column 8.7.3. an EOGRTS might be 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.
No further testing on fertility is necessary, because other information is available, and the available data are considered adequate to support a robust risk assessment and classification and labelling.

In December 2017, the Committee for Risk Assessment at ECHA (RAC) decided 2-methylimidazole is to be classified for reproduction toxicity Cat. 1B; H360Df: May damage the unborn child and suspected to impair fertility; based on results from reproduction/developmental screening studies according to OECD421. 2-Methylimidazooe has been included in the 14th ATP to the CLP (EC 1272/2008).
Concerning fertility, RAC decided that there was some evidence for an adverse effect of 2-methylimidazole on female fertility. This is based on the death of two high dose dams (500 mg/kg bw/d) in a reproduction/developmental screening study (OECD421, 2013). The rats died during or shortly after parturition (postnatal days 2 and 3), both showing signs of complicated parturition preceding death. As there were no pathological findings that could explain these deaths and no other effects on fertility were seen, it was concluded, that the deaths appeared to have been a specific adverse effect on female fertility that is not secondary to general toxicity; supporting the classification in Repro Cat. 2;H361f.
Developmental effects were observed in the reproduction/developmental screening study and a follow-up study (both 2013): A decrease in viability index at 500 mg/kg bw/d in the first study, and a dose-related increased incidence of aneurysms in pups exposed to 2-methylimidazole with a developmental NOAEL = 2 mg/kg bw/d in both studies. The observed developmental effects were not considered to be secondary to maternal toxicity because maternal toxicity was limited to BW changes at 500 mg/kg bw/d. Thus, RAC considered the criteria for classification in Category 1B; H360D for developmental toxicity were met.
Concerning repeated dose toxicity studies, no effects on reproductive organ weights were observed in a 28d study in rats up to the highest dose (800 mg/kg bw/d; similar to OECD407, 1975). In a 90d study (NTP TR67, 2004), depressed absolute testes weights and the slight increase in estrous cycle were observed in rats in the high dose group (560 mg), but probably related to the reduced body weight, as discussed in the study. The reduced relative testes weights observed in rats in this study (>= 40 mg/kg bw/d) were not confirmed in the 2-years study (NTP TR516, 2004) and in the more recent OECD421 study.

An EOGRTS, basic test design (cohort 1A and 1B), is not necessary as other information is available as described above. An extension to include the F2 generation (cohort 1B) is not necessary, as there are no consumer or professional uses known. An extension to include developmental neurotoxicity or immunotoxicity (cohort 2A/2B or 3) is not necessary, as no effects on nervous or immune system were observed in available animal studies and 2-methylimidazole is neither classified for STOT SE for transient (e.g. narcotic) or irreversible effects on the CNS, nor it is classified for skin sensitisation or known to be a respiratory sensitiser.

A robust risk assessment for human health is possible based on the developmental NOAEL of 2 mg/kg bw/d, the most sensitive endpoint. The effects which lead to the classification for fertility Cat. 2 were observed at very high doses (500 mg/kg bw/d). Thus, possible effects on fertility are considered to be covered by the risk assessment, which is based on the low NOAEL for developmental effects. It is therefore expected that results from an EOGRTS would not change the outcome of the risk assessment with regard to reproductive toxicity (developmental toxicity and reproductive performance).
As there are only industrial uses for 2-methylimidazole, DNELs are derived for workers. An additional DNEL for the general population is only derived for the assessment man via environment.
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In a GLP compliant reproduction/developmental toxicity screening test according to OECD 421, 2-methylimidazol was given to rats by oral gavage (BASF SE 2013a). Groups of 10 male and 10 female Wistar rats received the test substance, as a suspension in 1% carboxymethylcellulose in (drinking) water, at dose levels of 50, 150 and 500 mg/kg bw/day. Rats of the control group received the vehicle alone (10 mL/kg bw). The duration of treatment covered a 2-week pre-mating period and a mating period (max. of 2 weeks) in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females. Accuracy, homogeneity and stability of test substance formulations were demonstrated by analyses.

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. As this salivation was transient and occurred directly after dosing , it was probably due to the irritation and/or taste of test substance administration, and, therefore, considered not to be adverse. In addition, discoloration was observed in the urine and feces of nearly all mid- and high-dose animals. This discoloration was probably a result of the metabolism and subsequent excretion of the test substance and judged to be not adverse. Two high-dose females died during or shortly after parturition, both showing signs of complicated parturition preceding death (undelivered pups, umbilical cords not cut, newborns 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. These effects on food consumption and growth were considered to be treatment-related and adverse. 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 the test substance 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 (2 male and 4 female) 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. Clinical observations of pups revealed no substance-related changes. 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. Microscopic examination of these macroscopic alterations and of the great vessels from 5 male and 5 female control pups revealed dissecting aneurysms in the aorta, pulmonary trunk and ductus arteriosus, which correlated overall with the macroscopic findings. Several animals showed aneurysms in different vessels simultaneously, but the aorta was the most affected vessel. The number of animals and aneurysms increased with increasing dose levels. Heart and aorta dilations were only observed in single male and female pups of the high-dose group. All of these pathology findings in pups were ascribed to treatment and considered to be adverse.

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, slighly 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. At the mid- and high-dose level dissecting aneurysms were also observed in the pulmonary trunk and ductus arteriosus.  

In conclusion, 150 mg/kg bw/day was a NOAEL for general toxicity and reproductive toxicity in the parents based on mortality (shortly after parturition), slighly reduced body weight and food consumption, and reduced live birth index at 500 mg/kg bw/day. 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).

According to REACH Annex IX, column 8.7.3. an EOGRTS might be 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.

No further testing on fertility is necessary, because other information is available, and the available data are considered adequate to support a robust risk assessment and classification and labelling.

In December 2017, the Committee for Risk Assessment at ECHA (RAC) decided 2-methylimidazole is to be classified for reproduction toxicity Cat. 1B; H360Df: May damage the unborn child and suspected to impair fertility; based on results from reproduction/developmental screening studies according to OECD421. 2-Methylimidazooe has been included in the 14th ATP to the CLP (EC 1272/2008).

Concerning fertility, RAC decided that there was some evidence for an adverse effect of 2-methylimidazole on female fertility. This is based on the death of two high dose dams (500 mg/kg bw/d) in a reproduction/developmental screening study (OECD421, 2013). The rats died during or shortly after parturition (postnatal days 2 and 3), both showing signs of complicated parturition preceding death. As there were no pathological findings that could explain these deaths and no other effects on fertility were seen, it was concluded, that the deaths appeared to have been a specific adverse effect on female fertility that is not secondary to general toxicity; supporting the classification in Repro Cat. 2;H361f.

Developmental effects were observed in the reproduction/developmental screening study and a follow-up study (both 2013): A decrease in viability index at 500 mg/kg bw/d in the first study, and a dose-related increased incidence of aneurysms in pups exposed to 2-methylimidazole with a developmental NOAEL = 2 mg/kg bw/d in both studies. The observed developmental effects were not considered to be secondary to maternal toxicity because maternal toxicity was limited to BW changes at 500 mg/kg bw/d. Thus, RAC considered the criteria for classification in Category 1B; H360D for developmental toxicity were met.  

Concerning repeated dose toxicity studies, no effects on reproductive organ weights were observed in a 28d study in rats up to the highest dose (800 mg/kg bw/d; similar to OECD407, 1975). In a 90d study (NTP TR67, 2004), depressed absolute testes weights and the slight increase in estrous cycle were observed in rats in the high dose group (560 mg), but probably related to the reduced body weight, as discussed in the study. The reduced relative testes weights observed in rats in this study (>= 40 mg/kg bw/d) were not confirmed in the 2-years study (NTP TR516, 2004) and in the more recent OECD421 study.

Effects on developmental toxicity

Description of key information

In a GLP compliant, modified reproduction/developmental toxicity screening test in rats 50 mg/kg bw/day (by oral gavage), the highest dose tested, was a NOAEL for maternal toxicity based on the absence of adverse effects in the adult animals. The NOAEL for developmental toxicity in the F1 offspring is 2 mg/kg bw/d, as dissecting aneurysms in the great vessels of the heart were noted at incidences beyond the historical background range at 10 mg/kg bw/d and above.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
equivalent or similar to
Guideline:
other: Commission Regulation (EC) No 440/2008, Part B: Methods for the determination of toxicity and other health effects: Two-Generation Reproduction Toxicity Study
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 421 (Reproduction/Developmental Toxicity Screening Test)
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)
Principles of method if other than guideline:
Groups of 25 pregnant females received the test substance by oral gavage (control and 3 test groups) from gestation day 6 through day 3 of lactation. The dams and pups were sacrificed on post-natal day 4. Examinations in adult females included clinical signs, parturition and lactation behaviour, body weight, food consumption and gross pathology. Pup examinations included pup status, litter size and external examination at birth, viability, clinical signs, body weight, necropsy and microscopic examination of the great blood vessels of the heart.
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Female rats, time-mated by the breeder (supplied at noon on the day of evidence of mating)
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH.
- Age at arrival: 10-12 weeks.
- Body weight at arrival (gestation day 0): 142.2 – 183.1 g
- Housing: In Makrolon type M III cages, 1 animal per cage (exception: dams were housed together with their litter), with dust-free wooden bedding. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. 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 and storage 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 intervals which guarantee that the test substance in the vehicle remains stable. The formulations were stored in a refrigerator.
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). All determined concentrations in the stability samples were in the range between 90% and 110% of the nominal concentrations.
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: one sample of the control and low-dose formulations, and three samples (withdrawn from the top, middle and bottom of the preparation container) of the low- and high-dose formulations, taken from the container with a magnetic stirrer running. The measured concentrations were in the range between 90% and 110% of the nominal concentration for the mid-dose sample and the high-dose samples. The determined mean values for the low-dose samples were in the range from 116.6 % - 122.4 % (repeat and initial analysis, respectively), which was slightly above the specification limit of 110 %. The low relative standard deviations (maximally 2.4%) indicated that the test substance was homogeneously distributed in the vehicle.
Details on mating procedure:
Time-mated females were obtained from the animal supplier. The animals were supplied at noon on the day of evidence of mating; this day is referred to as GD 0 and the following day as GD 1.
Duration of treatment / exposure:
From implantation to one day prior to sacrifice (gestation day 6 through post-natal day 3).
Frequency of treatment:
Once daily at about the same time in the morning (females in labor were not treated).
Remarks:
Doses / Concentrations:
2, 10 and 50 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
25 females per dose. This group size (22-25 pregnant females) was selected to attain statistical power equivalent to that of a full-scale generation study (e.g. OECD 416).
Control animals:
yes, concurrent vehicle
Details on study design:
- Selection of doses: Based on the results of a previously conducted reproduction/developmental toxicity screening test (OECD421) in which adverse developmental effects (dissecting aneurysms in the great vessels of the heart) were observed up to the lowest dose tested (50 mg/kg bw/day).
- Parturition: The females were allowed to deliver and rear their pups until day 4 after parturition.
- Termination in-life phase: Dams and their pups were sacrificed on PND 4 and subjected to post mortem examinations.
Maternal examinations:
- Mortality: 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 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: Food consumption during pregnancy was determined for GD 0-6, 6-8, 8-10, 10-13, 13-15, 15-17 and 17-20. Food consumption of the females which gave birth to a litter was determined for PND 1-4. Food consumption was not determined in the females without litter during the lactation period.
- Body weight: Body weights during pregnancy were determined on GD 0, 6, 8, 10, 13, 15, 17 and 20. Body weights of the females which gave birth to a litter were determined for PND 0 and PND 4. Body weights were not determined in the females without litter during the lactation period.
- Post mortem examinations: On the day after the last test substance administration the animals were anesthetized with isoflurane (after overnight fasting), sacrificed by cervical dislocation and assessed by gross pathology. Animals without litter were anesthetized with isoflurane, sacrificed by cervical dislocation and assessed by gross pathology shortly after the expected day of delivery. The apparently non-pregnant uteri were stained for about 5 minutes in 1% ammonium sulfide solution according to the method of SALEWSKI (Salewski, E.; 1964). Then the uteri were rinsed carefully with 0.9% NaCl solution.
- Litter observations: see under 'Fetal examinations'.
Fetal examinations:
Litter observations:
- Pup number and status at delivery: All pups delivered 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: All live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.
- Body weight: 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.
- Post mortem examinations of pups sacrificed on schedule: On PND 4, the pups were sacrificed under isoflurane anesthesia with CO2. The pups were examined externally and their organs were assessed macroscopically, paying particular attention to the heart and great vessels of the heart, without removing the organs (except the thymus).
- Post mortem examinations of prematurely died or sacrificed pups: Pups that died or were sacrificed in a moribund state were eviscerated and examined for possible defects and/or the cause of death using appropriate methods, paying particular attention to the heart and great vessels of the heart, without removing the organs (except the thymus).
- Preservation: All pups were preserved in toto (except the thymus) in 4% neutral buffered formaldehyde solution and after 24 - 48 hours they were transferred to 70% alcohol solution for further preservation.
- Histopathology: After fixation, the great blood vessels of the heart were dissected in an appropriate way and processed histotechnically in two paraplast blocks: Block 1 included the base of the heart, aortic arch (aorta ascendens and aorta descendens), pulmonary trunk, and ductus arteriosus. Block 2 included three sections of the abdominal aorta (at the diaphragmatic insertion, region of kidneys [A. renalis] and region of the caudal bifurcatio aortae). Histotechnical processing was followed by light microscopical examination of the aortic arch (block 1; Hart stain combined with Masson-Goldner-Trichrome stain for detection of elastic and collagen fibers of the vessel walls) of all pups/test group, the abdominal aorta (block 2; paraplast embedding) of all pups/test group, and gross lesions (only macroscopic aneurysms/dilations) in all affected pups/test group. Immunohistochemistry investigations (antibody against collagen type I and III) were conducted on the aortic arch of selected control and high-dose pups only. The control pups were selected randomly (4 male and 6 female pups). In the high-dose group, pups with macroscopic findings suspected of aneurysms (2 male and 3 female pups) and additional randomly selected pups wiithout macroscopic or microscopic findings (2 male and 3 female pups) were used. Pups that died or were sacrificed in a moribund state were processed histotechnically and assessed like control pups.
Statistics:
The following statistical methods were used to analyze the data:
- Food consumption, body weight and body weight change (parental females and pups; litter means were used for pup weights), duration of gestation, number of delivered pups per litter, live pups on day x: Simultaneous comparison of all dose groups with the control group using the DUNNETT test (two-sided) for the hypothesis of equal means
- Females delivering, females with liveborn pups, females with stillborn pups, females with all stillborn pups,: Pair-wise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions
- Viability Index: Pair-wise comparison of the dose group with the control group using the WILCOXON test (one-sided) with BONFERRONI-HOLM adjustment for the hypothesis of equal medians
Indices:
- 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

- 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 after birth / number of live pups on the day of birth) x 100

- Sex ratio = (number of live male or female pups on day 0/4 / number of live male and female pups on day 0/4) x 100
Details on maternal toxic effects:
Details on maternal toxic effects:
- Mortality: There were no test substance-related mortalities in any of the parental females in any of the groups.
- Clinical observations: No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the parental females during the whole study. One sperm positive low-dose female (2 mg/kg bw/d) and two control females (Nos. 4 and 21) did not deliver F1 pups.
- Food consumption: Food consumption of the parental females in all test substance-treated groups (test groups 1 - 3; 2, 10 and 50 mg/kg bw/d) was comparable to the concurrent control group during the entire study period. Food consumption was statistically significantly decreased on several occasions during pregnancy in test groups 1 and 2 (2 and 10 mg/kg bw/d). These minor and non-dose related changes were regarded as spontaneous in nature.
- Body weight data: Mean body weights and mean body weight change of the parental females in all test substance-treated groups (test groups 1 - 3) were comparable to the concurrent control group during the entire study period.
- Female reproduction and delivery data: All sperm positive rats delivered pups or had implants in utero with the following exceptions: control female No. 4, control female No. 21, low-dose female No. 33, which were not pregnant, and therefore, did not deliver F1 pups.
The mean duration of gestation was similar in all test groups (i.e. between 22.1 and 22.2 days). The gestation index was 100% in all test groups. The mean number of F1 pups delivered per dam remained unaffected (9.7 / 8.8 / 9.8 and 9.3 pups/dam at 0, 2, 10 and 50 mg/kg bw/d). The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 100% in control and test groups 1 and 3, as well as 99.6% in test group 2. Moreover, the number of stillborn pups was comparable between the groups.
- Maternal necropsy observations: One parental animal showed a spontaneous finding at gross necropsy (dilated renal pelvis). This finding occurred without any relation to dosing.
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
- Pup number and status at delivery: The mean number of delivered F1 pups per dam and the rates of liveborn and stillborn F1 pups were evenly distributed about the groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.
- Pup viability/mortality: The viability index indicating pup mortality during lactation (PND 0 - 4) varied between 100% (control and test groups 2 and 3) and 99.6% (test group 2) without showing any association to the treatment.
- 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.
- Pup clinical observations: There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups. One male pup (dam No. 31, pup No. 1) of dose group 1 (2 mg/kg bw/d) showed a spontaneous finding (short tail).
- Pup body weight data: Mean body weights and mean body weight change of the male and female pups in all test substance-treated groups (test groups 1 - 3) were comparable to the concurrent control group during the entire study period. One male runt was seen in the control, one male and one female runt were seen in test group 1, one male runt was seen in test group 2, two male and four female runts were seen in test group 3.
- Pup necropsy observations: The macroscopic examination of pups at necropsy revealed a number of findings concerning the pericardial vessels in the all-dose groups. More than one of macroscopic findings in the great vessels at the base of the heart (aorta, ductus arteriosus, pulmonary trunk and carotid artery) were simultaneously observed in individual pups. A table showing the numbers of pups with macroscopic findings in the great vessels at the base of the heart is given under 'Any other information on results incl. tables'. A few other pups showed spontaneous findings at gross necropsy, such as dilated ureter, red discolored testis, dilated renal pelvis and white discolored liver lobe. These spontaneous findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences.The two decedent pups (No. 54-11 and 74-5) of test group 2 (10 mg/kg bw/d) showed no abnormalities.

- Histopathology: Dissecting aneurysms result from rupture of the tunica intima occurring in vivo allowing the entry of large amounts of blood through the tunica media that accumulate over time and make the adventitia protrude outside the arterial wall. The criteria for differentiating dissecting aneurysms (intramural blood extravasation occurring in vivo) from artifacts (postmortem blood extravasation occurring during necropsy due to tearing of blood vessels) were taken from Treumann et al., 2011 (Silke Treumann, Steffen Schneider, Sibylle Gröters, Nigel P. Moore and Paul J Boor (2011). Spontaneous occurrence of dissecting aneurysms in the region of the ductus arteriosus in four-day-old Wistar rat pups. Toxicologic Pathology 39: 969-974 ).
In the present study, dissecting aneurysms were characterized by a focal bulging of the arterial wall due to accumulation of densely packed erythrocytes between the outer tunica media and the adventitia (dissecting effect) and additional delicate residues of torn elastic fibers. Hemorrhages were characterized by discrete focal accumulation of erythrocytes within the tunica media that did not cause either bulging of the arterial wall or tearing of elastic fibers. Large aneurysms in some pups extended along different vessels connected with each other (e.g. along ductus arteriosus connecting the pulmonary trunk with the descendent portion of the aorta). Most of the aneurysms occurred in the region of the ductus arteriosus connecting with the descending aorta. Most of the hemorrhages were seen in the descending aorta. Some pups had more than one aneurysm and others had more than one hemorrhage. However, the two findings aneurysm and intramural hemorrhages did never occur simultaneously in the same pup, which means that either aneurysm(s) or intramural hemorrhage(s) occurred in the individual animal. Only single pups in each litter were affected, with one exception each in test group 2 (10 mg/kg bw/d) and test group 3 (50 mg/kg bw/d). In test group 2, two females (Nos. 66-6 and 66-7) of the same litter and in test group 3 (50 mg/kg bw/d), two males (Nos. 91-3 and 91-5) of the same litter were affected. In each of these litters one pup had an aneurysm and the other pup had a hemorrhage. In male pups, histopathology revealed that all aneurysms correlated with the finding “dilation” of the vessels observed at gross pathology. Conversely, in one female pup of test group 2 (10 mg/kg bw/d) and two female pups of test group 3 (50 mg/kg bw/d), macroscopic findings in the vessels had no histopathological correlate and thus the macroscopic findings were regarded as artifacts. The immunohistochemical stain with antibodies against collagen type I and III did not reveal differences between the collagen fibers of control and treated animals with or without dissecting aneurysms. A table showing the numbers of aneurysms and hemorrhages for the various locations is given under 'Any other information on results incl. tables'.
Dose descriptor:
NOAEL
Effect level:
2 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Pup macroscopic findings in the great vessels at the base of the heart

 

Number of findings

Male pups (F1)

Female pups (F1)

Test group

(mg/kg bw/d)

0

(0)

1

(2)

2

(20)

3

(50)

0

(0)

1

(2)

2

(10)

3

(50)

No. of examined pups

99

112

123

114

123

99

119

118

No. of pups with macroscopic findings in the great vessels at the base of the heart

0

1

2

2

0

0

2

3

Aorta

 

 

 

 

 

 

 

 

  - Dilation

 

1

1

1

 

 

2

1

  - Discoloration

 

 

 

 

 

 

 

1

Ductus arteriosus

 

 

 

 

 

 

 

 

  - Dilation

 

1

2

2

 

 

 

1

Pulmonary trunk

 

 

 

 

 

 

 

 

  - Dilation

 

 

1

1

 

 

 

1

Carotid artery

 

 

 

 

 

 

 

 

  - Dilation (right)

 

 

1

 

 

 

 

 

  - Narrow (left)

 

 

1

 

 

 

 

 

 

  

Pup microscopic findings: number of aneurysms and hemorrhages for the various locations

 

 

Male pups (F1)

Female pups (F1)

Test group

(mg/kg bw/d)

0

(0)

1

(2)

2

(10)

3

(50)

0

(0)

1

(2)

2

(10)

3

(50)

No. of examined pups

99

112

123

114

123

99

119

118

Aorta

 

 

 

 

 

 

 

 

  - Aneurysm

 

1

1

1

 

 

1

1

  - Hemorrhage

 

 

 

2

2

3

1

 

Ductus arteriosus

 

 

 

 

 

 

 

 

  - Aneurysm

 

1

2

2

 

 

 

1

  - Hemorrhage

 

 

 

 

2

 

 

 

Pulmonary trunk

 

 

 

 

 

 

 

 

   - Aneurysm

 

 

1

1

 

 

 

 

Carotid artery, right

 

 

 

 

 

 

 

 

  - Aneurysm

 

 

1

 

 

 

 

 

 

Historical control data:

An evaluation of 1016 untreated Wistar rat pups culled at PND 4, from reproductive toxicity studies conducted in the laboratory, revealed low incidences of aneurysms and hemorrhagic lesions, affecting 0.2% of all the offspring that were evaluated (Treumann et al., 2011). Since the analysis was based on pups culled from other studies, the litter-based incidence of these lesions in unexposed animals cannot precisely be determined. Nevertheless, the findings indicate that, although rare, dissecting aneurysms may occur as spontaneous lesions, such that the low incidence in group 1 (1 pup or 0.5% of total offspring affected) cannot be conclusively attributed to treatment.

Reference: Silke Treumann, Steffen Schneider, Sibylle Gröters, Nigel P. Moore and Paul J Boor (2011) Spontaneous occurrence of dissecting aneurysms in the region of the ductus arteriosus in four-day-old Wistar rat pups. Toxicologic Pathology 39: 969-974.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
2 mg/kg bw/day
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

For examination of developmental effects two studies are available, namely a reproduction/developmental toxicity screening test in Wistar rats according to OECD 421 and GLP (BASF SE 2013a) and a follow-up study according to a modified reproduction/developmental toxicity screening protocol (BASF SE 2013b).The follow-up study was conducted because developmental toxicity was observed at all dose levels (50, 150 and 500 mg/kg bw/day) in the original OECD 421 study (pups showed dissecting aneurysms of the great vessels of the heart). The original study is described in this endpoint summary under ‘Effects on fertility’. The follow-up study is presented in the present section ‘Effects on developmental toxicity’.

 

The GLP compliant modified reproduction/developmental toxicity screening study was conducted to define a NOAEL for the developmental toxic effects of the test substance on the great blood vessels of Wistar rats when exposed to 2-methylimidazol during prenatal and early postnatal development (BASF SE 2013b). In this study groups of 25 pregnant females received the test substance by oral gavage, as a suspension in 1% carboxymethylcellulose in (drinking) water, at dose levels of 2, 10 and 50 mg/kg bw/day, once daily from gestation day 6 through lactation day 3 (females in labor were not dosed). Animals of the control group received the vehicle alone (10 mL/kg bw). The dams and pups were sacrificed on post-natal day 4. Accuracy, homogeneity and stability of test substance formulations were demonstrated by analyses (actual concentrations were in the range between 90-110% of the nominal concentrations, except for the low-dose samples which were found to be between 16.6% and 24.0% above the nominal concentrations). Examinations in adult females included clinical signs, parturition and lactation behaviour, body weight, food consumption and gross pathology. Pup examinations included pup status, litter size and external examination at birth, viability, clinical signs, body weight, necropsy and microscopic examination of the great blood vessels of the heart. In addition to routine histochemistry, immunohistochemistry was conducted on the aortic arch of selected pups of the control group (4 male and 6 female, randomly selected) and the high-dose group (all pups with macroscopic findings suspected of aneurysms [2 male, 3 female] and additional randomly selected pups without macroscopic or microscopic findings [2 male, 3 female]).

There was no apparent effect of treatment upon either food consumption or body weight in parental females throughout the entire treatment period, and all 2-Methylimidazol -treatment groups were not toxicologically significantly different from control. Reproduction and delivery of the F0 females were unaffected. The numbers of live litters born were 23, 24, 25, and 25 in groups 1 through 4, respectively. There were no treatment-related effects on litter size, the number of stillborn pups, the weight of the offspring at birth and PND 4, and their postnatal survival.

Regarding pathology of the pups, macroscopic dilations observed in the great vessels at the base of the heart (aorta, ductus arteriosus, pulmonary trunk and carotid artery) mostly correlated microscopically with dissecting aneurysms, which occurred in 1 out of 211 (0.5%) pups of test group 1 (2 mg/kg bw/d), 3 out of 242 (1.2%) pups of test group 2 (10 mg/kg bw/d) and 3 out of 232 pups (1.3%) of test group 3 (50 mg/kg bw/d). Predominant locations were the ductus arteriosus and aorta. No aneurysms were observed in male and female control pups.

An evaluation of 1016 untreated Wistar rat pups culled at PND 4, from reproductive toxicity studies conducted in the laboratory, revealed low incidences of aneurysms and hemorrhagic lesions, affecting 0.2% of all the offspring that were evaluated (Treumann et al., 2011). Since the analysis was based on pups culled from other studies, the litter-based incidence of these lesions in unexposed animals cannot precisely be determined. Nevertheless, the findings indicate that, although rare, dissecting aneurysms may occur as spontaneous lesions, such that the low incidence in group 1 (1 pup or 0.5% of total offspring affected) cannot be conclusively attributed to treatment.

Histopathology also revealed the presence of intramural hemorrhages that were not detected macroscopically. Hemorrhages occurred in 2 out of 222 (0.9%) control pups, 3 out of 211 (1.4%) pups of test group 1, 1 out of 242 (0.4%) pups of test group 2 and 2 out of 232 (0.9%) pups of test group 3. Individual pups had either aneurysms or hemorrhages, but never both lesions together. Although hemorrhages might be considered to be precursor lesions of aneurysms, their location and incidence in this and in earlier studies (Schneider et al., 2012) suggests otherwise, as it was not consistent with the aneurysms. Hemorrhages were mostly located in the descending aorta while aneurysms were predominantly seen in the ductus arteriosus. Hemorrhages were also present in the concurrent control group while no aneurysm was seen in the control offspring. The lack of concordance between these two findings across the dose groups may reflect unrelated pathologies. Alternatively, because dissecting aneurysms are longitudinal lesions, in contrast to focal hemorrhages, they may be more readily captured during transverse sectioning of the tissues. Therefore, the disparity between the dose-response relationships for these two lesions may partly reflect their acquisition during post-mortem processing. The latter presumption and, in particular, the lack of a dose response suggest that a treatment-relationship of the hemorrhages is rather unlikely.

In addition to the above histopathological examinations, an attempt was made to get intial insight into a potential mechanism behind the vessel lesions, like disorganization of the elastic lamellae. For this purpose, an immunhistochemistry stain of vessel walls was conducted in selected animals, using antibodies against collagen type I and III. These investigations did not reveal differences between the collagen fibers of control and treated animals with or without dissecting aneurysms.

In conclusion, under the conditions of the present Modified Reproduction/Developmental Toxicity Screening Test in Wistar Rats, the NOAEL for general parental and reproductive toxicity is 50 mg/kg bw/d, the highest tested dose. The NOAEL for developmental toxicity in the F1 offspring is 2 mg/kg bw/d, as dissecting aneurysms in the great vessels of the heart were noted at incidences beyond the historical background range at 10 mg/kg bw/d and above.

 

Reference:

- Silke Treumann, Steffen Schneider, Sibylle Gröters, Nigel P. Moore and Paul J Boor (2011) Spontaneous occurrence of dissecting aneurysms in the region of the ductus arteriosus in four-day-old Wistar rat pups. Toxicologic Pathology 39: 969-974.

- Steffen Schneider, Silke Treumann and Nigel P. Moore (2012) Malformations of the great vessels in the neonatal rat induced by N-(2-Aminoethyl)ethanolamine. Birth Defects Research (Part B) 95:95-106.

Justification for classification or non-classification

Based on the death of two high dose dams in the reproduction/developmental screening test in rats (OECD421), both showing signs of complicated parturition preceding death, there was some evidence for an adverse effect of 2-methylimidazole on female fertility. As there were no pathological findings that could explain these deaths and no other effects on fertility were seen, it was concluded, that the deaths appeared to have been a specific adverse effect on female fertility that is not secondary to general toxicity; supporting the classification in Repro Cat. 2;H361f (Suspected of damaging fertiliy), according to the CLP Regulation (EC 12727/2008).

Based on the developmental effects which occurred in the absence of maternal toxicity in the reproduction/developmental toxicity screening test in rats (OECD421) and the subsequent modified developmental screening test, the test substance needs to be classified as reproductive toxicant, Cat. 1B;H360D (May damage the unborn child) according to the CLP Regulation (EC 1272/2008).