Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

A parental, reproduction, and developmental NOAEL of 150 mg/kg/day (the highest dose tested) was determined in a GLP compliant, OECD 422, combined 28 day repeated dose toxicity study with reproduction/developmental toxicity screening test. Furthermore, no effects on reproductive organs were observed in a 90 -day study (oral, rat) up to the highest dose of 230 mg/kg bw/d tested.

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to
Guideline:
other: EPA 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 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:
reproduction
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
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In a GLP compliant combined 28 -days repeated dose toxicity study with reproduction/developmental toxicity screening test, 2 -ethyl-4 -methylimidazole was given to rats by oral gavage (2012). Four groups of ten male and ten female Wistar Han rats were exposed to the test substance at 15, 50, or 150 mg/kg/d. 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/d were noted for females, and higher liver weights (absolute and relative to body weight) were recorded at 15, 50 and 150 mg/kg bw/d 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/d 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/d. 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/d 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 reproduction and developmental toxicity was observed up to the highest dose level tested (150 mg/kg bw/d). Based on these results, a parental, reproduction and developmental NOAEL of at least 150 mg/kg bw/d was derived.

Furthermore, no effects on reproductive organs were observed in a 90 -day study (oral, rat) up to the highest dose of 230 mg/kg bw/d tested (see IUCLID chapter 7.5.1 for further details).

Effects on developmental toxicity

Description of key information
A prenatal developmental NOAEL of 230 mg/kg/day (the highest dose tested) was determined in a GLP compliant, OECD 414 toxicity test.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation:10-12 weeks
- Weight at study initiation: 148.4 - 184.7 g
- Housing: individually (Polycarbonate cages type III supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany (floor area about 800 cm²))
- Diet (e.g. ad libitum): ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kai-seraugst, Switzerland (ad libitum)
- Water (e.g. ad libitum): drinking water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The aqueous test substance preparations were prepared at the beginning of the administra-tion period and thereafter at intervals, which took into account the period of established sta-bility. The preparations were kept at room temperature.
For the test substance preparations, the specific amount of test substance was weighed, topped up with deionized water in a calibrated beaker and intensely mixed with a magnetic stirrer until it is completely dissolved.
During administration, the preparations were kept homogeneous with a magnetic stirrer.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses of the test substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.
The stability of the test substance in deionized water at room temperature over a period of 7 days had been verified prior to the start of the study.
Samples of the test substance preparations were sent to the analytical laboratory at the be-ginning of administration for verification of the concentrations.
Details on mating procedure:
The animals were paired by the breeder (“time-mated”); the day of evidence of mating (= de-tection of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same day (GD 0) at the experimental laboratory. The following day was designated as “GD 1”.
Duration of treatment / exposure:
GD6-19
Frequency of treatment:
daily
Duration of test:
On GD 20, the females were sacrificed in a randomized order and examined macroscopical-ly. The fetuses were removed from the uterus and investigated.
Remarks:
Doses / Concentrations:
25, 80 and 230 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice on workdays, daily on weekends/public holidays


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A cage-side examination was conducted at least once daily before and after treatment period (GD 0-5 and 20). During treatment period (GD 6-19) all rats were checked daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity before administration as well as within 2 hours and within 5 hours after administration.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20. All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- The consumption of food was recorded for the intervals 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
- Sacrifice on gestation day 20
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No
Statistics:
DUNNETT-test (two-sided), FISHER'S EXACT test (one-sided), WILCOXON-test (one-sided)
Indices:
Conception rate, preimplantation loss, postimplantation loss
Historical control data:
Historical control data were available
Details on maternal toxic effects:
Details on maternal toxic effects:
Clinical examinations of the dams:
Only pregnant dams were used for the calculations of mean maternal water consumption, food consumption, body weight and body weight change. Only pregnant dams with sched-uled sacrifice (GD 20) were used for the calculation of mean gravid uterine weights, correct-ed (net) body weight gain and summary of reproduction data.The following female was excluded from the above-mentioned calculations: Test group 2 (80 mg/kg bw/d)/female No. 64 - not pregnant.
Mortality:
There were no substance-related or spontaneous mortalities in any females of all test groups (0, 25, 80 or 230 mg/kg bw/d).
Clinical symptoms:
Most of the females (22 out of 25) of the high-dose group (230 mg/kg bw/d) and 10 females of the mid-dose group (80 mg/kg bw/d) showed transient salivation during the treatment pe-riod. Salivation occurred in the respective animals only within the 2-hour examination inter-val immediately after treatment and was initially observed on GD 7. During the subsequent 5-hour examination interval (i.e. >2h<5h after treatment), no clinical signs or changes of general behavior were detected in any female of all test groups.No further clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female at dose levels of 25, 80 or 230 mg/kg bw/d during the entire study period.
Food consumption:
The mean food consumption of the dams in test group 3 (230 mg/kg bw/d) was statistically significantly reduced from GD 6-13, but recovered afterwards. Nevertheless, during the treatment period (GD 6-19) the high-dose dams consumed 12% less food in comparison to the concurrent control group. The reduced food consumption was considered as test substance-related. The mean food consumption of the dams in test groups 1 and 2 (25 and 80 mg/kg bw/d) was generally comparable to the concurrent control group throughout the study.
Body weight:
The mean body weight (BW) of the high-dose rats (230 mg/kg bw/d) was statistically signifi-cantly below control from GD 8 onwards until the end of the study (GD 20). The mean body weight gain (BWC) was distinctly and statistically significantly reduced on GD 6-10 (by 34-59%) but recovered afterwards. However, the overall weight gain during the treatment period remained 12% below control (statistically significant.). The lower weights/weight gain were likely to be a subsequent effect of reduced food consumption and considered as test substance-related.The BW and the average BWC of the low- and mid-dose dams (25 and 80 mg/kg bw/d) were generally comparable to the controls throughout the entire study.
Corrected body weight:
The corrected body weight gain (terminal body weight on GD 20 minus weight of the uno-pened uterus minus body weight on GD 6) was statistically significantly lower in the high-dose dams (230 mg/kg bw/d - about 14% below the concurrent control value), as was the carcass weight of of these dams (about 4% below controls). The corrected body weight gain and mean carcass weights of test groups 1 and 2 (25 and 80 mg/kg bw/d) remained unaffected by the treatment.
Uterus weight:
The mean gravid uterus weights of the animals of test groups 1-3 (25, 80 and 230 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the concurrent control groups showed no dose-dependency and were assessed to be without biological relevance.
Necropsy findings:
No necropsy findings which could be attributed to the test substance were seen in any dam. There occurred one spontaneous finding in individual females of test groups 0, 1 and 3 (0, 25 and 230 mg/kg bw/d), i.e. dilated renal pelvis. This finding was detected in three control, one low-dose and two high-dose dams and was therefore assessed as incidental.
Reproduction data:
The conception rate reached 96% in the mid-dose group (80 mg/kg bw/d) and 100% in the control, low- and high-dose groups (0, 25 and 230 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of the study. No test substance related and/or biologically relevant differences between the test groups 0-3 (0, 25, 80 and 230 mg/kg bw/d) were observed with regard to conception rate, mean num-ber of corpora lutea and implantation sites or values calculated for the pre- and the postim-plantation losses, the number of resorptions as well as viable fetuses. All observed differ-ences were considered to reflect the normal range of fluctuations for animals of this strain and age (see also PART III, Supplement, for historical control data). This statement includes the apparently lower mean number of viable male fetuses in test group 1 (25 mg/kg bw/d).
Key result
Dose descriptor:
NOAEL
Effect level:
80 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
230 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Sex distribution of the fetuses:
The sex distribution of the fetuses in test groups 1-3 (25, 80 and 230 mg/kg bw/d) was comparable to the concurrent control fetuses.
Weight of the placentae:
The mean placental weights of the low-, mid- and high-dose groups were comparable to the corresponding control group.
Weight of the fetuses:
The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the concurrent control group.
Fetal external malformations:
One fetus each in test groups 0, 1 and 2 (0, 25 and 80 mg/kg bw/d) had external malfor-mationtions. In two cases, these external malformations were associated with either soft tissue or skeletal malformations. The total incidence of external malformations in treated animals did not differ significantly from the concurrent control group and was covered by the historical control data.
Fetal external variations:
No external variations were recorded.
Fetal external unclassified observations:
One unclassified external observation, i.e. hemorrhage, was recorded in one fetus of the low-dose group (25 mg/kg bw/d). This finding was not considered biologically relevant.
Fetal soft tissue malfromations:
Soft tissue malformations were recorded for one control fetus, which had an associated ex-ternal finding. There were no soft tissue malformations in any of the treated groups.
Fetal soft tissue variations:
Two soft tissue variations were detected in all test groups (0, 25, 80 and 230 mg/kg bw/d), i.e. dilated renal pelvis and dilated ureter. These variations were neither statistically significantly different from control nor dose-dependent and therefore, not considered biologically relevant.
Fetal soft tissue unclassified observations:
No unclassified soft tissue observations were recorded.
Fetal skeletal malformations:
One fetus of test group 1 (No. 31-08) had multiple skeletal malformations in the area of ster-num, skull, thoracic vertebral column and ribs, a specification of each and every detail was impractical. Furthermore, some skeletal malformations were detected in fetuses of test groups 0-2 (0, 25 and 80 mg/kg bw/d) affecting the skull, vertebral column and humerus. The incidences of these malformations were neither statistically significant-ly different from control nor dose-dependent and therefore, not considered biologically relevant.
Fetal skeletal variations:
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeleton and appeared without a relation to dosing. The overall incidences of skeletal variations were covered by the historical control data. Two variations - affecting the skull and ribs - were, at the top dose (230 mg/kg bw/d), present at statistically significant incidences slightly above the historical control range. Other findings were either not dose-related or covered by the historical control range.
Fetal skeletal unclassified cartilage observations:
Additionally, some isolated cartilage findings without impact on the respective bone struc-tures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the sternum and ribs and did not show any relation to dosing.









Dose descriptor:
NOAEL
Effect level:
230 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No effects on fetuses.
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Under the conditions of this prenatal developmental toxicity study, the oral administration of 2-Ethyl-4-Methyl-Imidazole to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at doses as high as 230 mg/kg bw/d caused evidence of maternal but no developmental toxicity.
In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 80 mg/kg bw/d based on reduced food consumption and body weight gain at the LOAEL (230 mg/kg bw/d). The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 230 mg/kg bw/d, the highest dose tested.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
230 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP guideline study
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

2-Ethyl-4-Methyl-Imidazole was tested for its prenatal developmental toxicity in Wistar rats (2016). The test substance was administered as an aqueous preparation to groups of 25 time-mated female Wistar rats by gavage at doses of 25, 80 and 230 mg/kg bw/d on GD 6 through 19. One control group, consisting of 25 females, was dosed with the vehicle (deionized water) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group. At terminal sacrifice on GD 20, 24 - 25 females per group had implantation sites. Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day. On GD 20, all females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology (including weight determinations of the unopened uterus and the placentae). For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings. The stability of the test substance preparations was demonstrated over a period of 7 days at room temperature. The correctness of the prepared concentrations was shown. The following test substance-related, adverse effects/findings were noted at test group 3 (230 mg/kg bw/d):Dams;Statistically significantly reduced food consumption from GD 6-13, overall about 12% less food consumption during treatment period (GD 6-19).Body weight statistically significantly reduced from GD 8-20. Reduced body weight change on GD 6-10, overall about 12% less weight gain during treatment period (GD 6-19). Reduced corrected (net) body weight gain: about 14% below the concurrent control value.No test substance-related adverse effects on fetuses. No test substance-related adverse effects on dams, gestational parameters or fetuses were seen at test group (80 mg/kg bw/d) and test group 1 (25 mg/kg bw/d). Under the conditions of this prenatal developmental toxicity study, the oral administration of 2-Ethyl-4-Methyl-Imidazole to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at doses as high as 230 mg/kg bw/d caused evidence of maternal but no developmental toxicity. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 80 mg/kg bw/d based on reduced food consumption and body weight gain at the LOAEL (230 mg/kg bw/d).The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 230 mg/kg bw/d, the highest dose tested.

Justification for classification or non-classification

Based on the available data, the test substance does not need to be classified for reproduction toxicity according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.