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 bw/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.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 11 weeks
- Weight at study initiation: Males: 281-315g, Females: 189-215g
- Housing: Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm). Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm). Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes. General: Sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment or bedding material.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Results of analyses for nutrients and contaminants were examined and archived.
- Water: Free access to tap-water. Certificates of analysis were examined and archived.
- Acclimation period: At least 5 days prior to start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
- Amount of vehicle: 10 mL/kg body weight
- Preparation of dosing solution: Formulations (w/w) were prepared daily within 6 hours and 11 minutes prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the test substance (0.99 g/cm3 at 40°C). No correction was made for the purity/composition of the test substance. To liquefy the test substance before weighing, where considered necessary the test substance was heated to a maximum temperature of 60.3°C for a maximum of 2 hours and 42 minutes. Information provided by the sponsor indicated that the test substance could be heated up to 60-70°C.
Details on mating procedure:
- Mating procedures: Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from males. Detection of mating was not confirmed for animal nos. 45 (Group 1) and 80 (Group 4) which did deliver live offspring. The mating date of these animals was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Chemical analysis of dose preparations: Analyses were conducted by ABL BV, Assen, the Netherlands; test site study identification ABL12288. Samples (0.5 mL) were taken using a pipette (a clean pipette tip was used for every group), and were weighed on an analytical balance at 4 decimals precision. During sampling, formulations were placed on a magnetic stirrer. Immediately after sampling (accuracy and homogeneity samples) or after 6 hours at room temperature under normal laboratory light conditions (stability samples), samples were stored on dry ice. Samples remained on dry ice until receipt at ABL, The Netherlands. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
- Analysis of dose preparations: No test substance was detected in the Group 1 formulations. The concentrations analysed in the formulations of Group 2 showed a mean accuracy of 89.4%. This mean accuracy was only minimally exceeding the acceptable range of 90-110% of nominal concentration. Given the NOAEL derived in this study (set at the highest dose, i.e. Group 4), this deviating accuracy value for Group 2 formulations did not affect interpretation of the study results. The concentrations analysed in the formulations of Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours (i.e. relative difference ≤ 10%).
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 43-50 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Female nos. 41, 42, 45 (Group 1), 53, 56 (Group 2) and 61 (Group 3) were not dosed during littering.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.
Remarks:
Doses / Concentrations:
15, 50, 150 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a previous 14-day dose range finding study (Project 500561; BASF Project 01R0074/12X163), daily dosing of 300 mg/kg bw resulted in body weight loss in both sexes. Additionally, lethargy, hunched posture and/or piloerection was observed during the last 3-4 days of treatment. Therefore, the dose of 300 mg/kg bw/day was considered to be too high for the present study.
- Treatment: By oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
- Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.
Parental animals: Observations and examinations:
- Mortality / Viability: At least twice daily.
- Clinical signs: Daily from treatment onwards, detailed clinical observations were made for all animals, immediately after dosing (based on the absence of a peak effect of occurrence of clinical signs in the dose range finding study, Project 500561; BASF Project 01R0074/12X163). Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed sign were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.
- Functional Observations: The following tests were performed on the selected 5 animals/sex/group: hearing ability, pupillary reflex, static righting reflex, grip strength, locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations are reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head. During motor activity monitoring, all animals were caged individually (pups were housed in their home cages and kept warm using bottles filled with warm water). These tests were conducted after dosing at no specific time point, but within a similar time period after dosing for the respective animals. These tests were performed after observation for clinical signs (incl. arena observation, if applicable) and before blood sampling. The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period (all before blood sampling).
- Body weights: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.
- Food consumption: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.
- Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.
- General reproduction data: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Palpation was used to aid in confirmation of pregnancy. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.
- Clinical laboratory investigations: Blood samples were collected on the day of necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) between 7.00 and 10.30 a.m. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and Li-heparin treated tubes for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into untreated tubes for determination of bile acids. Furthermore, from the selected 5 animals/sex/group an additional blood sample (1 mL) was collected into serum tubes for possible future measurement of thyriod-stimulating hormone (TSH), and the thyroid hormones triiodothyronine (T3) and thyroxine (T4). After clotting and centrifugation, serum samples were stored at <-75°C. Since histopathological examination of the thyriod glands did not reveal any treatment-related changes, all samples were discarded at finalization of the study report without further investigation.
- Haematology: The following haematology parameters were determined in blood prepared with EDTA as an anticoagulant, using the ADVIA® 120 Hematology System (Siemens Healthcare Diagnostics B.V., Breda, The Netherlands): White blood cells, Differential leucocyte count: Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils, Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets. The following clotting parameters were determined in plasma prepared with citrate as anti-coagulant, using the STA Compact® (Diagnostica Stago S.A.S., Asnières, France): Prothrombin Time, Activated Partial Thromboplastin Time.
- Clinical biochemistry: The following clinical biochemistry parameters were determined using the AU400® Chemistry System (Beckman Coulter Nederland B.V., Woerden, The Netherlands). All parameters were determined in plasma, except for bile acids which were determined in serum: Alanine aminotransferase, Aspartate aminotransferase, Alkaline Phosphatase, Total protein, Albumin, Total Bilirubin, Bile acids, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate.
Litter observations:
- Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made for all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation.
Postmortem examinations (parental animals):
- Necropsy: All males, the selected 5 females/group were deprived of food overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. Non-selected females were not deprived of food. Animals surviving to scheduled necropsy were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated. Necropsy was conducted on the following days: Females which delivered: Lactation Days 5-7, Females which failed to deliver (nos. 63 and 74): Post-coitum Day 27 (female no. 63 with evidence of mating) or 22 days after the last day of the mating period (female no. 74 without evidence of mating), Males: Following completion of the mating period (a minimum of 28 days of dose administration). All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The numbers of former implantation sites and corpora lutea were recorded for all paired females. Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). Selected 5 animals/sex/group (tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination): Adrenal glands, (Aorta), Brain - cerebellum, mid-brain, cortex, Caecum, Cervix, Clitoral gland, Colon, Coagulation gland, Duodenum, Epididymides, Eyes (with optic nerve (if detectable) and Harderian gland), Female mammary gland area, Femur including joint, Heart, Ileum, Jejunum, Kidneys, (Lacrimal gland, exorbital), (Larynx), Liver, Lung - infused with formalin, Lymph nodes - mandibular, mesenteric, (Nasopharynx), (Esophagus), Ovaries, (Pancreas), Peyer's patches [jejunum, ileum] if detectable, Pituitary gland, Preputial gland, Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, (Skin), Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid including parathyroid if detectable, (Tongue), Trachea, Urinary bladder, Uterus, Vagina, All gross lesions. All remaining animals, females which failed to deliver: Cervix, Clitoral gland, Coagulation gland, Epididymides, Mammary gland area, Ovaries, Preputial gland, Prostate gland, Seminal vesicles, Testes, Uterus, Vagina, All gross lesions.
- Organ weights: Terminal body weights were recorded from all males and the selected 5 females/sex/group. The following organ weights were recorded from the following animals on the scheduled day of necropsy. Selected 5 animals/sex/group: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus, Uterus (including cervix), Prostate (weighed when fixed for at least 24 hours), Seminal vesicles including coagulating glands (weighed when fixed for at least 24 hours), Thyroid including parathyroid (weighed when fixed for at least 24 hours). All remaining males: Epididymides, Testes.
- Histotechnology: All organ and tissue samples, as defined under Histopathology (following section), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands). From the selected 5 males of the control and high dose group and male no. 23 (Group 3) suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis. The teses were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).
- Histopathology: The following slides were examined: (1) The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4. (2) The additional slides of the testes of the selected 5 males of Groups 1 and 4 and male no. 23 (Group 3) suspected to be infertile to examine staging of spermatogenesis. (3) All gross lesions of all animals (all dose groups). (4) The reproductive organs (cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina) of male no. 23 (Group 3) that failed to sire and female nos. 63 (Group 3; no offspring) and 74 (Group 4; not mated; male no. 34 was already selected for assessment of reproductive organs) that failed to deliver healthy pups.
Postmortem examinations (offspring):
Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation. All pups were sexed and descriptions of all external abnormalities were recorded. The stomach of pups not surviving to scheduled necropsy was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated. Pups were checked for macroscopically visible anomalies of the greater vessels (aortic arch, thoracic aorta). Pups with findings were fixated in toto in 10% buffered formalin.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences, followed by the Wilcoxon test to compare the treated groups to the control group in case intergroup differences were seen.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Reproductive indices:
For each group, the following calculations were performed:
- Mating index (%) = (Number of females mated / Number of females paired) x 100
- Fertility index (%) = (Number of pregnant females / Number of females paired) x 100
- Conception index (%) = (Number of pregnant females / Number of females mated) x 100
- Gestation index (%) = (Number of females bearing live pups / Number of pregnant females) x 100
- Duration of gestation = Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
For each group, the following calculations were performed:
- Percentage live males at First Litter Check = (Number of live male pups at First Litter Check / Number of live pups at First Litter Check) x 100
- Percentage live females at First Litter Check = (Number of live female pups at First Litter Check / Number of live pups at First Litter Check) x 100
- Percentage of postnatal loss = (Number of dead pups before planned necropsy / Number of live pups at First Litter Check) x 100
- Viability index = (Number of live pups before planned necropsy / Number of pups born alive) x 100
- Mortality: No mortality occurred during the study period that was considered to be related to treatment with the test substance. One control male died during the blood sampling procedure, which was considered incidental in nature.
- Clinical signs: No clinical signs of toxicity were noted during the observation period. Salivation seen after dosing among animals of the 50 (individual males) and 150 mg/kg bw/day dose group (all males and most of the females) was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to taste of the test substance. Pale faeces was noted for most females at 150 mg/kg bw/day, primarily towards the end of the study period. Given that body weight development and food intake was unaffected by treatment, and since blood analysis and morphological assessment did not reveal any toxicologically relevant changes, this finding was considered not to be of an adverse nature. The incidence of scabs, hunched posture and alopecia occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.
- Functional observations: Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. The variation in motor activity did not indicate a relation with treatment. The statistically significant lower mean counts for total movements of males at 50 mg/kg occurred in the absence of a dose-related trend; the control mean was considered to be slightly high. Therefore, no toxicological relevance was ascribed to this change. All groups showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test period.
- Body weights: No toxicologically relevant changes in body weights and body weight gain were noted. Any statistically significant changes in body weight and body weight gain across the dose groups were considered not to be of toxicological relevance since the changes were slight in nature and/or occurred in the absence of a dose-related trend. These changes consisted of lower body weight gain of males at 150 mg/kg bw/day during the premating and mating period (also statistically significant for absolute body weight on Day 15 of the mating period), lower absolute body weight and body weight gain for females at 150 mg/kg bw/day on Day 8 of the premating period and on Day 4 of the lactation period, and lower body weight gain on Day 1 and 15 of the mating period for males at 15 and 50 mg/kg bw/day, respectively.
- Food consumption: No toxicologically relevant changes in food consumption before or after correction for body weight were noted. The statistically significant lower food consumption (before or after correction for body weight) of females at 150 mg/kg bw/day over Days 0-4 of the post-coitum period was slight in nature and did not prevail with continuing treatment. No toxicological relevance was therefore ascribed to these changes.
- Haematology: No toxicologically relevant changes occurred in haematological parameters of treated rats. The higher individual red blood cell distribution width (RDW) and reticulocyte counts in individual males and/or females at 150 mg/kg bw/day (exceeding the range considered normal for rats of this age and strain) were not part of a group response and occurred in the absence of any supportive changes in red blood cell parameters. These changes were therefore considered to be of no toxicological relevance.
- Clinical biochemistry: No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats. Any (statistically significant) changes in clinical biochemistry parameters at 150 mg/kg bw/day were slight in nature and occurred in the absence of morphological changes indicative of organ dysfunction. These changes consisted of higher cholesterol level in males and females (not statistically significant, but means just within the range considered normal for rats of this age and strain), lower sodium and chloride level in males, and higher inorganic phosphate level in males (not statistically significant, but mean outside the range considered normal for rats of this age and strain). The statistically significantly higher bile acid level for females at 150 mg/kg bw/day was due to higher values for two females (nos. 76 and 77). Other selected females showed normal values for this parameter. The statistically significantly higher total bilirubin level of males at 15 mg/kg bw/day occurred in the absence of a dose-related trend and the mean remained within the range considered normal for rats of this age and strain. These changes were therefore considered to be of no toxicological relevance.
- Macroscopic examination: Necropsy did not reveal any toxicologically relevant alterations. The incidence of macroscopic findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, did not show a dose-related incidence trend and occurred in the absence of any treatment-related histopathological changes.
- Organ weights: No toxicologically relevant changes were noted in organ weights and organ to body weight ratios. The statistically significantly higher kidney to body weight ratio and/or kidney weight of males at 50 and 150 mg/kg bw/day occurred in the absence of clear dose-related trend. The statistically significantly higher liver, kidney, spleen and uterus to body weight ratio in females at 150 mg/kg bw/day remained within the range considered normal for rats of this age and strain. Since absolute weights were similar to control levels these changes were ascribed to the lower terminal body weights. Also, since no histopathological correlates were found for these organ weight changes, these were considered to be of no toxicological relevance. Seminal vesicle weight and seminal vesicle to body weight ratio of males at 150 mg/kg bw/day appeared lower (approximately 15% for relative weights) than controls without achieving a level of statistical significance. Since these differences in seminal vesicle weight had no histopathological correlates, and there were no reproductive changes, these differences were considered to be of no toxicological relevance.
- Microscopic examination: There were no treatment-related microscopic findings. All microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar-Han rats of this age. No histopathological changes were noted in the reproductive organs of two Group 3 animals (male no. 23 and female no. 63) and two Group 4 animals (male no. 34 and female no. 74) that could account for failing to deliver healthy offspring. The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item.
- Reproduction data: No toxicologically relevant effects on reproductive parameters were noted. Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.
- Developmental data: Gestation index and duration of gestation were unaffected by treatment. No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
Dose descriptor:
NOAEL
Remarks:
parental
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no findings that were considered to be adverse in nature following treatment up to 150 mg/kg bw/day.
Dose descriptor:
NOAEL
Remarks:
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. A total of five pups of the control group, eight pups at 50 mg/kg bw/day and three pups at 150 mg/kg bw/day were found dead or missing during lactation. Pups missing were most likely cannibalised. The higher number of dead/missing pups at 50 mg/kg bw/day resulted in a statistically significantly lower viability index at this dose level. No toxicological relevance was attributed to these dead/missing pups across the dose groups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age. Incidental clinical symptoms of pups consisted of a dented head, a red spot on the head, absence of milk in the stomach, abnormal posture of the left hindleg, and a scab on the nose and right hindleg. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance. Body weights of pups were unaffected by treatment. Incidental macroscopic findings of pups that were found dead included cannibalism of the head region, absence of milk in the stomach, beginning/slight/moderate autolysis and a wound on the upper jaw. Incidental macroscopic findings among surviving pups included a dented head, and abnormal posture of the left hindleg. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance. No treatment-related anomalies of the greater vessels (aortic arch, thoracic aorta) were noted among the pups. Incidental findings consisted of a supernumary artery originating from the aortic arch after the left subclavian (pup no. 6 of litter 76 at 150 mg/kg bw/day), and a right subclavian originating from the aortic arch (pup no. 6 of litter no. 46, control group). The distribution and nature of these findings showed no dose-related trend and were incidental in nature. No toxicological relevance was therefore ascribed to these findings.
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no findings that were considered to be adverse in nature following treatment up to 150 mg/kg bw/day.
Reproductive effects observed:
not specified
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, 1,2 -dimethylimidazole was given to rats by oral gavage (2013, RL1). Four groups of ten male and ten female Wistar Han rats were exposed to the test substance 7 days/week at 15, 50, or 150 mg/kg bw/day. Rats of the control group received the vehicle, water, alone. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 43-50 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Accuracy, homogeneity and stability of formulations were demonstrated by analyses. Salivation occurred post-dosing in most animals at 150 mg/kg bw/day and in individual males at 50 mg/kg bw/day. This finding was considered to be a local reaction to the taste of the compound and therefore not as adverse. Pale faeces were noted for most females at 150 mg/kg bw/day, primarily towards the end of the study period. Given that body weight development and food intake was unaffected by treatment, and since blood analysis and morphological assessment did not reveal any toxicologically relevant changes, this finding was considered not to be of an adverse nature. No toxicologically significant changes were noted in any of the parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination). In addition, no toxicologically significant changes were noted in any of the reproductive parameters (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites) or in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy). Based on these results, a parental, reproduction and developmental NOAEL of at least 150 mg/kg bw/day was derived.

Effects on developmental toxicity

Description of key information

Testing proposal for an OECD414 study.

Mode of Action Analysis / Human Relevance Framework

No information available.

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.

Additional information