1,2-dimethylimidazole

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Remarks:

1st species

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25.06.2018

Deviations:

yes

Remarks:

7 dams per group, 2 dose groups, no examination of visceral and skeletal variations/malformations.

GLP compliance:

not specified

Remarks:

conducted according to good scientific practice and the experimental phases were carried out in accordance with OECD´s and German GLP principles, but not in full compliance with GLP principles (not-QUA checked).

Test material

Test material information

Constituent 1

Reference substance name:

1,2-dimethylimidazole

EC Number:

217-101-2

EC Name:

1,2-dimethylimidazole

Cas Number:

1739-84-0

Molecular formula:

C5H8N2

IUPAC Name:

1,2-dimethyl-1H-imidazole

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: 94180397V0
- Purity, including information on contaminants, isomers, etc.: 98.3 corr. peak area-%

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: about 10-12 weeks
- Weight at study initiation: 180.9 – 226.6 g.
- Housing: individually (Polycarbonate cages type III)
- Diet (e.g. ad libitum): tap water (ad libitum)
- Water (e.g. ad libitum): ad libitum (mouse and rat maintenance diet “GLP”, meal, supplied by Granovit AG, Kaiseraugst, Switzerland)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 45-65%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The 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 deionized water in a graduated flask and intensely mixed with a magnetic stirrer until it was completely dissolved.

Analytical verification of doses or concentrations:

yes

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.

Duration of treatment / exposure:

GD 6 - 19

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

120 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

360 mg/kg bw/day

No. of animals per sex per dose:

7

Control animals:

yes, concurrent vehicle

Details on study design:

On GD 20, blood samples were obtained in a randomized order from all females by retrobulbar venous puncture. After blood sampling all surviving dams were sacrificed and examined.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Mortality: A check was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-20).

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 between 2 and 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. The body weight change of the animals was calculated based on the obtained results.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: 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): Yes
- Time schedule for examinations: The water consumption 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.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Adrenal glands; Kidneys; Liver; Spleen. All paired organs were weighed together (left and right).
- Organ/tissue fixation: The following organs or tissues were fixed in 4% neutral buffered formaldehyde solution from all animals, sacrificed on schedule: All gross lesions; Adrenal glands; Kidneys; Liver; Spleen. No further examinations or procedures were performed in the study.


TERMINAL EXAMINATIONS
Cesarean section
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. The uteri and the ovaries were removed and the following data were recorded:
- Weight of the unopened uterus
- Number and distribution of implantation sites classified as:
• Live fetuses
• 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 dissection from the uterus each fetus, external tissues and all orifices were examined macroscopically.
Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. Thereafter, the fetuses were sacrificed by a subcutaneous injection of pentobarbital (Narcoren®; dose: 0.1 mL/fetus) and discarded.

OTHER:
Corrected (net) body weight gain: 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).

Clinical pathology:
- Blood samples were taken from all females by puncturing the retrobulbar venous plexus following isoflurane anesthesia. Blood sampling and blood and serum examinations were carried out in a randomized sequence. The list of randomization instructions was compiled with a computer.

Parameters Hematology: Leukocyte count (WBC); Erythrocyte count (RBC); Hemoglobin (HGB); Hematocrit (HCT); Mean corpuscular volume (MCV); Mean corpuscular hemoglobin (MCH); Mean corpuscular hemoglobin concentration (MCHC); Platelet count (PLT); Differential blood count; Reticulocytes (RETA)

Parameters Clinical chemistray: Alanine aminotransferase (ALT); Aspartate aminotransferase (AST); Alkaline phosphatase (ALP);gamma-Glutamyltransferase (GGT);
Inorganic phosphate (INP); Calcium (CA); Urea (UREA); Creatinine (CREA); Glucose (GLUC); Total bilirubin (TBIL); Total protein (TPROT); Albumin (ALB); Globulins (GLOB); Triglycerides (TRIG); Cholesterol (CHOL)

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
- Other:

Blood sampling:

- Plasma: Yes
- Serum: Yes

Fetal examinations:

After dissection from the uterus each fetus, external tissues and all orifices were examined macroscopically. Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. Thereafter, the fetuses were sacrificed by a subcutaneous injection of pentobarbital (Narcoren®; dose: 0.1 mL/fetus) and discarded.

Statistics:

Food consumption, Water consumption, Body weight, Body weight change, Carcass weight, Net maternal body weight change: Dunnett test (two-sided)
Uterus weight: Non-parametric one-way analysis using the Kruskal-Wallis test (two-sided). If the resulting p-value was equal to or less than 0.05, a pair-wise com¬parison of each dose group with the control group was performed using the Wilcoxon test (two-sided) for the hypothesis of equal medians.
Pregnant animals at terminal sacrifice: Pair-wise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided; decreased) for the hypothesis of equal proportions.
Female mortality: Pair-wise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided; increased) for the hypothesis of equal proportions.
Implantation sites, Live fetuses, %Live fetuses of implantations: Pair-wise comparison of the dose group with the control group using the WILCOXON test (one-sided; decreased) for the hypothesis of equal medians.
%Postimplantation loss, Resorptions (total, early, late), %Resorptions (total, early, late): WILCOXON test (one-sided; increased)
Blood parameters: For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians.
For parameters with unidirectional changes: WILCOXON-test (one-sided)
Organ weights:Non-parametric one-way analysis using the Kruskal-Wallis test (two-sided). If the resulting p-value was equal to or less than 0.05, a pair-wise com¬parison of each dose group with the control group was performed using the Wilcoxon test (two-sided) for the hypothesis of equal medians.
Carcass weight: Dunnett test (two-sided)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Three females (out of 7) of the high-dose group (360 mg/kg bw/d) showed salivation during the treatment period. It occurred in the respective animals shortly after treatment (within 0-2 h) and was observed on GD 18. This finding is considered to be treatment-related, most likely as a local irritation of the upper digestive tract or as a result of the bad taste of the test substance/vehicle preparation.
All females of the high-dose group (360 mg/kg bw/d) and four females of the low-dose group (120 mg/kg bw/d) showed piloerection during the treatment period. This finding was initially observed on GD 7 (test group 2) or GD 13 (test group 1).
Nearly all (6 out of 7) females of test group 2 had a brown vaginal discharge towards the end of the treatment period (GD 17 - GD 18).
Furthermore, for one female of test group 2 (No. 20) polyuria was recorded on GD 20.
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 120 or 360 mg/kg bw/d during the entire study period.

Mortality:

no mortality observed

Description (incidence):

There were no test substance-related or spontaneous mortalities in any females of all test groups (0, 120 or 360 mg/kg bw/d).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights of the high-dose dams (360 mg/kg bw/d) were statistically significantly impaired from GD 13 onwards (up to 25% below controls). The body weight change of the dams in test group 2 was statistically significantly reduced during the entire treatment period (during GD 6-8 the dams lost weight: -9.67 g; -4% on GD 8 compared to GD 6). If calculated for the entire treatment period (GD 6-19) or the entire study period (0-20), the high-dose dams gained about 88% or 64% less weight than the controls (attaining statistical significance).
The mean body weight of the low-dose dams (120 mg/kg bw/d) was generally comparable to the concurrent control group throughout the entire study period. The average body weight gain was statistically significantly decreased during GD 6-8 (-90% in comparison to the control) but recovered afterwards and was comparable to the concurrent control group again. If calculated for the entire treatment period (GD 6-19) or the entire study period (0-20), the low-dose dams gained a body weight comparable to the controls.
The decreased body weight and body weight change of test group 2 (360 mg/kg bw/d) were evaluated as treatment-related and adverse.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In test group 2 (360 mg/kg bw/d), the mean food consumption was reduced from GD 6 onwards throughout the treatment period attaining statistical significance on GD 6-13 and GD 15-17 (up to 53% below control). If calculated for the entire treatment period (GD 6-19), the high-dose dams consumed about 37% less food than the controls; also attaining statistical significance. The decreased food consumption is assessed as treatment-related and adverse.
Also, in test group 1 (120 mg/kg bw/d), the mean food consumption was statistically significantly reduced on GD 6-13 (up to 20% below control). If calculated for the entire treatment period (GD 6-19), the low-dose dams consumed about 13% less food than the controls. However, the lower food consumption is assessed as treatment-related but not of toxicological relevance.

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

In test group 2 (360 mg/kg bw/d), the mean water consumption was statistically significantly decreased on GD 6 8 (-21% in comparison to the control) but afterwards it recovered and was similar to the control values. If calculated for the entire treatment period (GD 6-19), the mean water consumption in test group 2 was comparable to the concurrent control group and therefore evaluated as treatment-related but not of toxicological relevance.
The mean water consumption of the dams in test group 1 (120 mg/kg bw/d) was generally comparable to the concurrent control group throughout the entire study period.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed.

At the end of the gestation period, in dams of test group 1 (120 mg/kg bw/d) hemoglobin and hematocrit values were significantly decreased. However, both values were within historical control ranges (dams, hemoglobin 6.3-6.9 mmol/L, hematocrit 0.289-0.324 L/L). Mean hemoglobin and hematocrit values of test group 2 (360 mg/kg bw/d) were higher compared to those of test group 1 but cannot be assessed as meaningful data considering the fact that these values consist of 2 dams only. The mentioned changes in dams of test group 1 were regarded as treatment-related indicating an incipient anemia, but not yet adverse.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were observed.

At the end of the gestation period, in dams of test group 1 (120 mg/kg bw/d) albumin and globulin values as well as total protein values as sum of both former parameters were significantly decreased. Total protein and globulin values were below the historical control range whereas albumin values were within its range (dams, total protein 56.50-63.74 g/L, globulins 24.39-30.26 g/L, albumin 31.14-35.55 g/L). Because only globulins were relevantly decreased and total protein levels were low as consequence of the decreased globulin values, and no other clinical chemistry parameter was altered, these changes were regarded as non-adverse if at all treatment-related (ECETOC Technical Report No. 85, 2002). Median values of total protein, albumin and globulin were not dose-dependently changed when comparing to the median values of both dams in test group 2 (360 mg/kg bw/d).

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Uterus: In test group 2 (360 mg/kg bw/d), the mean gravid uterus weight was statistically significantly decreased (Mean 9.5 g** [p ≤ 0.01] vs. 61.2 g of control; 85% below control). The reason for this circumstance is the high rate of post-implantation losses in this group.
The mean gravid uterus weight of the animals of test group 1 (120 mg/kg bw/d) was not influenced by the test substance.

Absolute weights
When compared to control group 0 (= 100%), the mean absolute weights of adrenal glands were significantly increased in test group 2 (statistically significant changes printed in bold). (See table 1).
Relative organ weights
When compared to control group 0 (= 100%), the mean relative weights of following organs were significantly increased in test group 2 (statistically significant changes printed in bold). (See table 2).
All other mean relative weight parameters did not show significant differences when com-pared to the control group 0.
The significantly increased absolute and relative adrenal gland weights in test group 2 were assumed to be treatment related. They lay above historical control values (Mean abs. weight: 73.1 mg [65.5 – 84.3 mg], mean rel. weight: 0.029% [0.027 – 0.034%]. A histopathologic examination of adrenal glands was not performed.

The significantly increased relative kidney weights, as well as the not significantly increased absolute kidney weights (+16.0%) in test group 2 were assumed to be treatment related. Both weight parameters lay above historical control values (Mean rel. weight: 0.617% [0.584 – 0.654%]. A histopathologic examination of adrenal glands was not performed.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In test group 2 (360 mg/kg bw/d), one animal (out of 7) showed an enlarged adrenal gland. Together with the increased adrenal gland weights, the enlargement was regarded as treatment related. A histopathologic examination of the enlarged adrenal gland was not performed.
All other findings occurred individually. They were considered to be incidental or spontane-ous in origin and without any relation to treatment.

Details on results:

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, mean organ weights, corrected (net) body weight gain and summary of reproduction data. No females were excluded from the above-mentioned calculations.

Corrected (net) body weight gain:
The corrected body weight gain (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6) was statistically significantly decreased in test group 2 (360 mg/kg bw/d - about 86% below control). Moreover, mean carcass weight was visibly reduced (about 12% below control, without attaining statistical significance). This is assessed as treatment-related and adverse.
The corrected body weight gain of test group 1 (120 mg/kg bw/d) revealed no difference of any biological relevance to the corresponding control group. Mean carcass weight of test group 1 remained also unaffected by the treatment.

Clinical pathology:
In test group 2 (360 mg/kg bw/d) blood from only two dams was evaluated because the other females in this group were not pregnant anymore on the day of blood sampling. Therefore, statistics could not be applied to this test group. Test group 1 (120 mg/kg bw/d) was compared statistically to the study control with the WILCOXON test.

Maternal developmental toxicity

Details on maternal toxic effects:

The conception rate was 100% in all test groups (0-2; 0, 120 and 360 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of this study.
There were no test substance related and/or biologically relevant differences between the test groups 0-2 in conception rate and the mean number of implantation sites. Furthermore, in test group 1 (120 mg/kg bw/d), there were no test substance related and/or biologically relevant differences in the values calculated for post-implantation loss and the number of resorptions.
In test group 2 (360 mg/kg bw/d), five dams had no viable fetuses (dams with only resorptions). The average number of resorptions per litter (intra-uterine mortality) was higher than in the concurrent control (mean 9.3** [p ≤ 0.01] vs. 1.1 in control). Accordingly, the resorption rate (mean% post-implantation loss) was higher than in the concurrent control (mean% 81.68** [p ≤ 0.01] vs. 9.16 in control). Especially the number of early resorptions (mean 8.9** [p ≤ 0.01] vs. 1.0; mean% 74.73** [p ≤ 0.01] vs. 8.06) were statistically significantly higher in the high-dose group than in the control group. The number of late resorptions was only slightly increased in this test group (mean 0.4 vs. 0.1; mean% 6.96 vs. 1.10) without attaining statistical significance and the value was well within the historical control data. Subsequently, the percentage of live fetuses of implantations (mean% 18.32** [p ≤ 0.01] vs. 90.84) was statistically significantly lower in this test group.
The effects in test group 2 are assessed as treatment-related and adverse.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

120 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

Results (fetuses)

External malformations:

effects observed, treatment-related

Description (incidence and severity):

During external examination of the fetuses the following findings were observed in 3/10 fetuses of the test group 2 (360 mg/kg bw/d):
• multiple external malformations in fetus No. 1 of dam No. 17, such as open eye (right), suspected anophthalmia (left), cleft palate and cleft lip (right upper mandible).
• multiple external malformations in fetus No. 8 of dam No. 17, such as suspected anophthalmia (both), absent nostrils (both), small mouth, pointed lower jaw and paw hyperflexion (right forelimb).
• multiple external malformations in fetus No. 3 of dam No. 21, such as domed head, cleft palate, suspected anophthalmia (right) and small pinna (left).
The above-mentioned malformations were considered to be treatment-related, adverse effects indicating a disturbance of prenatal development.
However, only a low number of fetuses is available due to the high postimplantational lost.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

120 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

reduction in number of live offspring

Fetal abnormalities

Abnormalities:

effects observed, treatment-related

Localisation:

other: external malformations in 3/10 fetuses

Overall developmental toxicity

Developmental effects observed:

yes

Lowest effective dose / conc.:

360 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of this prenatal developmental toxicity study, the oral administration of 1,2-dimethylimidazole to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) provided evidence of maternal toxicity, such as piloerection, reduction in food consumption and body weight at the high-dose level of 360 mg/kg bw/d. Furthermore, increased relative and absolute adrenal gland weights were seen at this dosage. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 120 mg/kg bw/d.

The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 120 mg/kg bw/d due to the high number of post-implantation losses resulting in a decreased number of live fetuses that additionally showed a high incidence of multiple external malformations at the high-dose level of 360 mg/kg bw/d.