Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Oct 2015 - May 2016
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted on Jan 22, 2001
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No. 440/2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted on Aug 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1-Methylimidazol
- Physical state: liquid/yellowish
- Analytical purity: > 99.9 area %
- Stability under test conditions: the stability of the test substance under storage conditions over the test period was guaranteed.
- Storage condition of test material: room temperature
- Other: Homogeneity analyses was carried out at the beginning and at the end of the study of the low dose (10 mg/kg bw/d) and high dose (90 mg/kg bw/d). Three samples (from the low, mid, and high dose) were taken and analyzed.

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

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

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: drinking water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- The aqueous test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations were kept at room temperature.
For the test substance preparations, the specific amount of test substance was weighed, topped up with drinking water in a graduated flask and intensely mixed with a magnetic stirrer until it is completely dissolved. During administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0, 0.1, 0.3 and 0.9 mg/100 mL water, respectively in the 0, 10, 30 and 90 mg/kg bw dose groups
- Amount of vehicle (if gavage): 10 mL of the aqueous preparations were applied per kg bw.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in drinking water at room temperature over a period of 7 days had been verified prior to the start of the study in a similar batch (Project No.: 01Y0492/11Y063).
Details on mating procedure:
The animals were paired by the breeder (“time-mated”); the day of evidence of mating (= detection 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:
From implantation to one day prior to the expected day of parturition (GD 6 to GD 19), always at approx. the same time in the morning
Frequency of treatment:
Once a day
Duration of test:
On GD 20, all surviving dams were sacrificed and examined macroscopically.
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
90 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25 animals per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The doses for the OECD414 study were selected based on two sequential 14-d range finding studies [01R0492/11R088 and 01R0492/11R136], followed by the reproduction/ developmental toxicity screening test [OECD 422; 85R0492/11R130]
In the first 14-d range finding study [01R0492/11R088] 1-methylimidazole was administered daily to groups of 3 male and 3 female Wistar rats by gavage at doses of 60, 125 and 250 mg/kg bw/d. Severe effects on body weight and food consumption were observed in the mid and high dose groups at study day 3 (= day 4 of administration). Compared to the control the food consumption was statistically significantly decreased in males and females [125 mg/kg bw/d: -52.9 % (m), -35.3% (f); 250 mg/kg bw/d: -73.0% (m), -39.4% (f)]. The body weight at SD3 was reduced compared to the control group at SD 3 [125 mg/kg bw/d: -10.1 % (m), -6.2% (f); 250 mg/kg bw/d: -12.9% (m), -7.9% (f)]. Body weight loss at SD 3 compared to SD0 occurred in both sexes, but was more prominent in males than in females [125 mg/kg bw/d: -20.0 g (m), -8.2g (f); 250 mg/kg bw/d: -36.3 g (m), -4.0 (f)]. Thus, the doses were reduced from 125 to 15 mg/kg bw/d and from 250 to 30 mg/kg bw/d, respectively, as from study day 4. A continuous administration of 125 mg/kg bw/d and above would have caused severe suffering and most likely end in death of the animals and had to be stopped because of animal welfare reasons. Compared to the controls body weights of male and female rats remained marginally reduced until termination of the study, but no further severe findings were observed. In conclusion, a dose of 125 mg/kg bw/d or more exceeded the maximal tolerated dose (MTD), but 60 mg/kg bw/d was too low.
Therefore, a second 14-d range finding study [01R0492/11R136] was performed with daily gavage of 90 mg/kg bw/d to 4 male and 4 female Wistar rats. Females showed piloerection of the fur on several study days and again food consumption was statistically significantly decreased at study day 3, but recovered afterwards [-41.5% (m), -26.8% (f)]. Additionally performed clinical pathology showed increased cholesterol levels [control vs. 90 mg/kg bw/d: 1.45 +/- 0.2 vs. 2.25 +/- 0.25 mmol/L (m); 1.17 +/- 0.42 vs. 2.05 +/- 0.45 mmol/L (f)] indicating systemic toxicity. Again, male rats were more susceptible, as additionally their body weight gain was statistically significantly reduced at study day 3 compared to SD0 [-11.6 g] and their urea levels were increased at study day 14 [control vs. 90 mg/kg bw/d: 6.51 +/- 0.51 vs. 10.43 +/- 0.51 mmol/L]. Based on these observed adverse effects and taking into account the results of the first range-finding study, 90 mg/kg bw/d was selected as the top dose for the OECD422 study.
In the OECD422 study 1-methylimidazole was administered daily to groups of 10 male and 10 female Wistar rats (F0) by gavage at doses of 10, 30 and 90 mg/kg bw/d. The food consumption of the high dose males was statistically significantly compared to the control at study day 7 (-11%). However, no other severe effects on food consumption or body weight/body weight change were observed. In the high dose group, statistically significant increased serum urea levels were observed in both sexes of the parental animals (+32.5% or +14.5%, respectively), indicating an increased protein metabolism. This was considered as a test substance-related, adverse finding and thus, the NOAEL (no observed adverse effect level) for general, systemic toxicity was 30 mg/kg bw/d for the F0 parental animals.
The test substance 1-methylimidazole is classified as Skin Corr. 1B;H314: Causes severe skin burns and eye damage according to EC/1272/2008; with a pH = 11.3 of an aqueous solution (100 g/L). The males were probably more susceptible to its corrosive property, because they received higher amounts of the basic aqueous solution, as these were calculated based on the body weight (10 mL/kg bw). In both range finding studies and the OECD422 study, the males were about 50 % heavier than the females (mean body weight at day 0 (m/f): 327 g/196 g; 330 g/224 g; 344 g/203 g). This effect seems to be crucial especially during the first days of the administration period.
Although the defined criteria for maternal toxicity (clinical signs or decreased body weight) could not fully be reached in the OECD422 study, 90 mg/kg bw/d was selected as the top dose also for the OECD414 study, based on the following reasons: In the first dose range finding study the administration of 125 mg/kg bw/d 1-methylimidazole (not even a factor of 1.5 compared to 90 mg/kg bw/d) showed a considerable and statistically significant decrease in food consumption (-35.3%) and a reduced body weight (-6.2%) in females – caused by body weight loss (-8.2 g) – already within the first three study days. It is generally accepted that pregnant animals are more susceptible to changes in food and water consumption due to their increased energy rate during pregnancy. Therefore, an additional maternal dose range finding study was not performed and 90 mg/kg bw/d was selected as the top dose for the OECD414 study.

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:The animals were paired by the breeder (“time-mated”); the day of evidence of mating (= detection 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”. A check was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-20).

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

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

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

POST-MORTEM EXAMINATIONS: Yes
On GD 20, the dams were sacrificed under isoflurane anesthesia by decapitation, in randomized order. After the dams had been sacrificed, they were necropsied and assessed for gross pathology, in randomized order. The uteri and the ovaries were removed and the following data were recorded: weight of the unopened uterus, number of corpora lutea, number and distribution of implantation sites classified as: live fetuses and dead implantations ( a) Early resorptions (only decidual or placental tissues visible or according to SALEWSKI from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single horn pregnancy); b) Late resorptions (embryonic or fetal tissue in addition to placental tissue visible), c) Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)). After the weight of the uterus had been determined, all subsequent evaluations of the dams and the gestational parameters were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose animal numbers were encoded.
Ovaries and uterine content:
The following examinations will be carried out:
- Number of corpora lutea
- Number of implantations (differentiated according to live and dead fetuses and early or late resorptions)
- Early resorptions according to SALEWSKI in animals that do not appear to be pregnant and animals with single-horn pregnancy
- Site of implantations in the uterus
Fetal examinations:
- External examinations: Yes: [all per litter] At necropsy each fetus was weighed, sexed, and external tissues and all orifices were examined macroscopically. The sex was determined by observing the distance between the anus and the base of the genitalia. Furthermore, the viability of the fetuses and the condition of placentae, umbilical cords, fetal membranes, and fluids were examined. The placentas were weighed and their individual weights were recorded. Thereafter, the fetuses were sacrificed by a subcutaneous injection of pentobarbital (Narcoren®; dose: 0.1 mL/fetus). After these examinations, approximately one half of the fetuses per dam were eviscerated, skinned and fixed in ethanol; the other half were placed in Harrison’s fluid for fixation.
- Soft tissue examinations: Yes: [half per litter] The fetuses fixed in Harrison’s fluid were examined for any visceral findings according to the method of BARROW and TAYLOR. After this examination these fetuses were discarded.
- Skeletal examinations: Yes: [half per litter] The skeletons of the fetuses fixed in ethanol were stained according to a modified method of KIMMEL and TRAMMELL. Thereafter, the skeletons of these fetuses were examined under a stereomicroscope. After this examination the stained fetal skeletons were archived individually.




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

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no 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 scheduled sacrifice (GD 20) were used for the calculation of mean gravid uterine weights, corrected (net) body weight gain and summary of reproduction data. The following females were excluded from the above-mentioned calculations: Test group 3 (90 mg/kg bw/d) - Females Nos. 86, 92 - not pregnant.
Mortality:
There were no substance-related or spontaneous mortalities in any females of all test groups (0, 10, 30 or 90 mg/kg bw/d).
Clinical symptoms:
Three females of the high-dose group (90 mg/kg bw/d) showed transient salivation during the treatment period. Salivation occurred in the respective animals only within the 2-hour examination interval (i.e. <2h after treatment) and was observed on GD 17-19. During the 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 10, 30 or 90 mg/kg bw/d during the entire study period.
Food consumption:
The mean food consumption of the mid- and high-dose dams (30 and 90 mg/kg bw/d) was statistically significantly reduced (up to 10% and 26% below control, respectively) at the beginning of the treatment period (GD 6-10), but recovered afterwards. If calculated for the entire treatment period (GD 6-19) or the entire study period (GD 0-20), the food values of test groups 2 and 3 were comparable to the control value. The mean food consumption of the dams in test group 1 (10 mg/kg bw/d) was generally comparable to the concurrent control throughout the entire study period.
Body weight:
The mean body weights of the dams in test groups 1, 2 and 3 (10, 30 and 90 mg/kg bw/d) were in general comparable to the controls throughout the entire study period. Consistently to the reduced food consumption on GD 6-8, the body weight change of the midand high-dose dams was statistically significantly reduced on GD 6-8 (approx. 29% / 81% of control). However, if calculated for the entire treatment period (GD 6-19) or the entire study period (GD 0-20), average body weight change values of these test groups were quite similar to the concurrent control value. The mean body weight gain of the low-dose dams (10 mg/kg bw/d) were generally comparable to the controls throughout the entire study period.
Corrected body weight:
The corrected body weight gain of test groups 1-3 (10, 30 and 90 mg/kg bw/d) revealed no difference of any biological relevance to the concurrent control group. Moreover, mean carcass weights remained also unaffected by the treatment.
Uterus weight:
The mean gravid uterus weights of the animals of test groups 1-3 (10, 30 and 90 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the concurrent control groups revealed 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 test group 2 (30 mg/kg bw/d), i.e. dilated renal pelvis. This finding was detected in one single animal and was therefore assessed as incidental.
Reproduction data:
The conception rate reached 92% in the high-dose group (90 mg/kg bw/d) and 100% in the control, low- and mid-dose groups (0, 10 and 30 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of the study (according to the test guidelines listed in chapter 2.3.).
There were no test substance-related and/or biologically relevant differences between the test groups 0-3 in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age, see also PART III (SUPPLEMENT) for historical control data.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
90 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No effects observed up to highest dose tested.

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 (10, 30 and 90 mg/kg bw/d) was comparable to the concurrent control fetuses.
Weight of the placentae:
The mean placental weights were comparable between the control and test groups 1-3 (0, 10, 30 and 90 mg/kg bw/d). Biologically relevant differences were not observed. The significantly higher mean placental weight (0.49 g) of female fetuses in test group 3 was marginally above the other dose groups and also within the historical control data (female fetuses - mean 0.46 g; range of 0.32 - 1.10 g). Therefore, the increase was considered to be not biologically relevant.
Weight of the fetuses:
The mean fetal weights of test groups 1, 2 and 3 (10, 30 and 90 mg/kg bw/d) 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 3 (0, 10 and 90 mg/kg bw/d) had external 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. blood coagulum around placenta, was recorded in two fetuses of the high-dose group (90 mg/kg bw/d). This finding was not considered as biologically relevant and was also seen in the historical control data.
Fetal soft tissue malformations:
No soft tissue malformations were recorded.
Fetal soft tissue variations:
Three soft tissue variations were detected, i.e. short innominate, dilated renal pelvis and dilated ureter. The incidences of dilated renal pelvis were statistically significantly increased in the low- and high-dose groups (10 and 90 mg/kg bw/d). As a consequence, the total incidence of fetal soft tissue variations was also increased in these groups. As these incidences were not related to dose and the findings were also seen in the historical control data, they were considered as incidental.
Fetal soft tissue unclassified observations:
No unclassified soft tissue observations were recorded.
Fetal skeletal malformations:
A number of skeletal malformations were detected in fetuses of test groups 0-3 (0, 10, 30 and 90 mg/kg bw/d) affecting the skull, vertebral column, sternum and humerus. Each one fetus of test groups 0, 1 and 3 had associated external findings. All other findings were single cases, most of them can be found in the historical control data. Therefore, the findings were not considered to be treatment-related.
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.
Fetal skeletal unclassified cartilage observations:
Additionally, some isolated cartilage findings without impact on the respective bone structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the vertebral column, the sternum and ribs and did not show any relation to dosing.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
90 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
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:
In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is the highest tested dose of 90 mg/kg bw/d.