Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): 2-Ethyl-4-Methyl-Imidazole
- Physical state: liquide, viscous/yellow to amber, clear
- Analytical purity: content: 97.9 g/100 g

Test animals

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

Administration / exposure

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.
Doses / concentrations
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

Examinations

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

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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).

Effect levels (maternal animals)

open allclose all
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

Results (fetuses)

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.









Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
230 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No effects on fetuses.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

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.