1-methylimidazole

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No effects on reproduction and the respective organs was observed in 2 GLP-compliant repeated dose toxicity studies, performed according to OECD guidelines 422 and 408 (IUCLID section 7.5.1.) The NOAEL for reproductive performance and fertility in rats is 90 mg/kg bw/day, the highest dose tested.

Link to relevant study records

Reference

Reference 1

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity

Reproductive effects observed:

not specified

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

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 (incl. QA statement)

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 Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 11-13 weeks
- Weight at study initiation: 329.9-361.9 g (males); 186.2-215.6 g (females)
- Fasting period before study: No
- Housing: During the study period, the rats were housed individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²), with the following exceptions:
* During overnight matings, male and female mating partners were housed together in Makrolon type M III cages.
* Pregnant animals and their litters were housed together until PND 4.
Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
For enrichment wooden gnawing blocks (Typ NGM E-022; supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria) were added.
- Diet: Ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, which was available to the animals ad libitum throughout the study
- Water: Drinking water was supplied from water bottles (ad libitum)
- Acclimation period: About 6 days

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

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance solutions in drinking water were prepared at the beginning of the administration period and thereafter in intervals, which took into account the analytical results of the stability verification.
From visual inspection, it seemed that the test substance was not completely miscible with drinking water. Therefore, for the preparation of the administration solution the test substance was weighed in a calibrated beaker depending on the dose group, topped up with drinking water and subsequently intensely mixed with a homogenizer. During administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0, 100, 300 and 900 mg/100mL
- Amount of vehicle (if gavage): 10 mL/kg bw/day

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: Maximum of 2 weeks (the female was placed in the cage of the male mating partner from about 16.00 h until 07.00 - 09.00 h of the following morning)
- Proof of pregnancy: Sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: Individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.
Analytical verifications of the stability of the test substance in drinking water for a period of 7 days at room temperature were carried out during the study. From visual inspection, it seemed that the test substance was not completely miscible with drinking water. Therefore, in order to prove that the test substance preparations were true solutions, a homogeneity analysis was carried out. Samples of the test substance solutions were sent to the analytical laboratory once at the beginning of the study for verification of the concentrations.
Concentration control analysis: The mean values of 1-Metylimidazol in drinking water in the low-dose group (samples 3R-5R and 12-14 ) were found in the range of 122.1% to 129.0% of the nominal concentrations. The mean found concentrations at 100 mg/100 mL are above the specification limit of 110%. This slight departure from the nominal concentration range presumably brought about an insignificantly higher dose for the low-dose group than intended. However, since this dose did not produce any effect in the respective animals, the validity of the study is not regarded to be compromised. All other values of 1-Metylimidazol in drinking water were found to be in the range of 90% - 110% of the nominal concentration.

Duration of treatment / exposure:

The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 2 days post-mating in males, and the entire gestation period as well as approximately 2 weeks of the lactation period.

Frequency of treatment:

Once daily at approximately the same time in the morning. Females in labor were not treated.

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:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The doses for the OECD422 study were selected based on two sequential 14-d range finding studies [01R0492/11R088 and 01R0492/11R136].
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.

Parental animals: Observations and examinations:

MORTALITY:
A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). If animals were in a moribund state, they were sacrificed and necropsied.

CAGE SIDE OBSERVATIONS:
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each animal. The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g.inability to deliver) were documented on an individual dam basis. On weekdays (except Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day

DETAILED CLINICAL OBSERVATIONS (DCO):
Detailed clinical observations were performed in all animals once prior to the first administration and at weekly intervals during the administration period. The examinations started in the morning. The findings were ranked according to the degree of severity, if applicable. For observation, the animals were removed from their cages by the investigator and placed in a standard arena (50 x 37.5 cm wide, with side borders which are 25 cm high). The following parameters listed were assessed: 1. Abnormal behavior in “handling”, 2. Fur, 3. Skin, 4. Posture, 5. Salivation, 6. Respiration, 7. Activity/arousal level, 8. Tremors, 9. Convulsions, 10. Abnormal movements, 11. Gait abnormalities, 12. Lacrimation, 13. Palpebral closure, 14. Exophthalmos, 15. Assessment of the feces discharged during the examination (appearance/consistency), 16. Assessment of the urine discharged during the examination, 17. Pupil size

BODY WEIGHT:
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice. The body weight change of the animals was calculated from these results. The following exceptions are notable for the female animals:
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
Females without positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

FOOD CONSUMPTION :
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0, 7, 14 and 20.
• Food consumption of F0 females which gave birth to a litter was determined on PND 1 and 4.
Food consumption was not determined in females without positive evidence of sperm during the gestation period and in females without litter during the lactation period.

HAEMATOLOGY:
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): 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 and Reticulocytes (RET).
Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. (reference: Hematology: Principles and Procedures, 6th Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101). Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany). Parameter and method: Prothrombin time (Hepato Quick’s test) (HQT).

CLINICAL CHEMISTRY:
An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters. Parameters:
- Enzymes: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT).
- Blood chemistry parameters: Sodium (NA), Potassium (K), Chloride (CL), 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), Bile acids (TBA).

URINALYSIS:
The dry chemical reactions on test strips (Combur-10-test M, Roche, Mannheim, Germany) used to semi quantitatively determine urine constituents were evaluated with a reflection photometer (Miditron M; Roche, Mannheim, Germany). Parameters: pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color, turbidity, Volume.

NEUROBEHAVIOURAL EXAMINATION:
A functional observational battery was performed in 5 parental male and 5 parental female animals (with litter) per group at the end of the administration period starting at about 10.00h. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
- Home cage observations: The animals were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to: 1. Posture, 2. Tremors, 3. Convulsions, 4. Abnormal movements, 5. Impairment of gait, 6. Other findings
- Open field observations: The animals were transferred to a standard arena (50 x 50 cm wide, with side borders which are 25 cm high) and observed for at least 2 minutes. The following parameters were examined: 1. Behavior when removed from cage, 2. Fur, 3. Skin, 4. Salivation, 5. Nose discharge, 6. Lacrimation, 7. Eyes/pupil size, 8. Posture, 9. Palpebral closure, 10. Respiration, 11. Tremors, 12. Convulsions, 13. Abnormal movements/stereotypy, 14. Impairment of gait, 15. Activity/arousal level, 16. Feces excreted within 2 minutes (number of scybala discharged/appearance/consistency), 17. Urine excreted within 2 minutes (amount/color), 18. Number of rearings within 2 minutes
- Sensory motor tests/Reflexes: The animals were removed from the open field and subjected to following sensory motor or reflex tests: 1. Approach response, 2. Touch response, 3. Vision (“visual placing response”), 4. Pupillary reflex, 5. Pinna reflex, 6. Audition (“startle response”), 7. Coordination of movements (“righting response”), 8. Behavior during “handling”, 9. Vocalization, 10. Pain perception (“tail pinch”), 11. Other findings, 12. Grip strength of forelimbs and hindlimbs, 13. Landing foot-splay test
- Motor activity measurement (MA): The MA was measured on the same day as FOB was performed in 5 parental males and females (with litter) per group. The examinations were performed using the Multi-Varimex system supplied by Columbus Instruments Int. Corp., Ohio, U.S.A. For this purpose, the animals were placed in clean polycarbonate cages for the time of measurement. The number of beam interrupts was counted over 12 intervals for 5 minutes in each case. The sequence at which the animals were placed in the polycarbonate cages was selected at random. The measurement was started at about 14.00 h. On account of the measuring variant "staggered", the starting time was varied by the time needed to place the animals in the cages. For each animal, measurement was started individually when the 1st beam was interrupted and ended exactly 1 hour later. The animals received no food or water during the measurements. After the transfer of the last animal in each case, the room where the measurements were carried out was darkened.

Sperm parameters (parental animals):

Parameters examined in male parental generation: testis weight, epididymis weight

Litter observations:

- Pup number and status at delivery: All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.
- Pup viability/mortality: In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays.
- Sex ratio: On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups was finally confirmed at necropsy.
- Pup clinical observations: The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams and documented for each pup.
- Pup body weight data: The pups were weighed on the day after birth (PND 1) and on PND 4. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning). “Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, epididymides, testes
The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): Adrenal glands, brain, heart, kidneys, liver, spleen, thymus
The following organs or tissues of all parental animals were fixed in 4% buffered formaldehyde solution or modified Davidson’s solution: All gross lesions, adrenal glands, aorta, bone marrow (femur), brain, cecum, cervix, coagulating glands, colon, duodenum, eyes with optic nerve, esophagus, extraorbital lacrimal glands, epididymides (modified Davidson’s solution), femur with knee joint, heart, ileum, jejunum (with Peyer’s patches), kidneys, larynx, liver, lungs, lymph nodes (axillary and mesenteric), mammary gland (male and female), nose (nasal cavity), ovaries (modified Davidson’s solution), oviducts, pancreas, parathyroid glands, pharynx, pituitary gland, prostate gland, rectum, salivary glands (mandibular and sublingual), sciatic nerve, seminal vesicles, skeletal muscle, spinal cord (cervical, thoracic and lumbar cord), spleen, sternum with marrow, stomach (fore stomach and glandular stomach), target organs, testes (modified Davidson’s solution), thymus, thyroid glands, trachea, urinary bladder, uterus, vagina
At necropsy additional liver samples (Processus papillaris and caudatus) were taken from all F0 animals per sex and group. The weight of these liver samples was recorded (per animal) for the females only. All samples were then frozen in liquid nitrogen and stored at -80°C.
The uteri of all cohabited female F0 parental animals were examined for the presence and number of implantation sites. The uteri of apparently non-pregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations (SALEWSKI's method (1)). Then the uteri were rinsed carefully with 0.9% NaCl-solution. When the examinations were completed, the uteri were transferred to the Pathology Laboratory for further processing.
(1)SALEWSKI, E.: Färbemethode zum makroskopischen Nachweis von Implantationsstellen am Uterus der Ratte; Naunyn-Schmiedeberg’s Arch. Exp. Pathol. Pharmakol. 247, 367 (1964)

HISTOPATHOLOGY: Yes
For the following tissues fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings: Adrenal glands, all gross lesions, bone marrow (femur), brain, cecum, cervix, coagulating glands, colon, duodenum, epididymides, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (axillary and mesenteric), ovaries, oviducts, prostate gland, peyers patches, rectum, sciatic nerve, seminal vesicles, spinal cord (cervical, thoracic, lumbar), spleen, stomach (forestomach and glandular stomach), testes, thymus, thyroid glands, trachea, urinary bladder, uterus, vagina.
Special attention was given on stages of spermatogenesis in the male gonads.

Postmortem examinations (offspring):

- Necropsy observations: All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia with CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically. All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated
and their organs were assessed macroscopically. All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding.

Statistics:

- 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: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
- Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity): Pairwise comparison of each dose group with the control
group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
- Urine pH, volume, specific gravity, color and turbidity: 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. Urine color and turbidity are not evaluated statistically.
Statistics of pathology:
- Weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). 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 equal medians.
Statistics of the clinical examinations:
- Food consumption, body weight and body weight change (parental animals); Simultaneous comparison of all dose groups with the control group using the DUNNETT test (two-sided) for the hypothesis of equal means

Reproductive indices:

The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.
For the males, mating and fertility indices were calculated for F1 litters according to the following formulas:
Male mating index (%) = (number of males with confirmed mating* / number of males placed with females) x 100
* defined by a female with vaginal sperm or with implants in utero
Male fertility index (%) = (number of males proving their fertility* / number of males placed with females) x 100
* defined by a female with implants in utero

For the females, mating, fertility and gestation indices were calculated for F1 litters according to the following formulas:
Female mating index (%) = (number of females mated* / number of females placed with males) x 100
* defined as the number of females with vaginal sperm or with implants in utero
Female fertility index (%) = (number of females pregnant* / number of females mated**) x 100
* defined as the number of females with implants in utero
** defined as the number of females with vaginal sperm or with implants in utero
Gestation index (%) = (number of females with live pups on the day of birth / number of females pregnant*) x 100
* defined as the number of females with implants in utero
The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters according to the following formula:
Live birth index (%) = (number of liveborn pups at birth / total number of pups born) x 100
The implantations were counted and the postimplantation loss (in %) was calculated according the following formula:
Postimplantation loss (%) = ((number of implantations – number of pups delivered) / number of implantations) x 100

Offspring viability indices:

The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4. The viability index was calculated according to the following formula:
Viability index (%) = (number of live pups on day 4 after birth / number of live pups on the day of birth) x 100

The sex ratio was calculated at day 0 and day 4 after birth according to the following formula:
Sex ratio = (number of live male or female pups on day 0/4 / number of live male and female pups on day 0/4) x 100

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no test substance-related mortalities in any of the male and female parental animals in any of the groups.
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any male or female F0 generation parental animals during the whole study including gestation and lactation periods.
Several male and female animals of the high-dose group (90 mg/kg bw/day) showed salivation after treatment during mating (males), post-mating, gestation and lactation. This transient salivation for a few minutes immediately after treatment was likely to be induced by the unpleasant taste of the test substance or by local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Mean body weights of the male and female F0 generation parental animals in all test substance-treated groups were comparable to the concurrent control group during the entire study period.
The high-dose parental males had a statistically significantly lower body weight change during premating days 0 - 7 (about 37% below control).
The body weight change of the mid and low-dose parental males was comparable to the concurrent control group during the whole study period.
The high-dose parental females had statistically significantly lower body weight change during PND 0 - 4 (about 65% below control).
The mean body weight change of the high-dose parental females was comparable to the concurrent control group during premating and gestation. The body weight change of the mid and low-dose parental females was comparable to the concurrent control group during the whole study period.
Food consumption of the high-dose F0 males (90 mg/kg bw/day) was statistically significantly below control during premating days 0 - 7 (about 11%).
The mid and low-dose F0 males (30 and 10 mg/kg bw/day) did not show any test substancerelated changes in food consumption during the whole treatment period.
Food consumption of the female F0 generation parental animals in all test substance-treated groups (10, 30 and 90 mg/kg bw/day) was comparable to the concurrent control group during the entire study period.

DETAILED CLINICAL OBSERVATIONS (DCO) (PARENTAL ANIMALS)
Male and female animals of all dose groups (90, 30 and 10 mg/kg bw/d) did not show any abnormalities.

FUNCTIONAL OBSERVATIONAL BATTERY (FOB) (PARENTAL ANIMALS)
- Home cage observations: No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.
- Open field observations: The open field observations did not reveal any test substance-related findings in male and female animals of all test groups.
- Sensorimotor tests/reflexes: There were no test substance-related findings in male and female animals of all test groups.Any deviations from "zero values" were equally distributed between test substance-treated groups and controls or occurred in single animals only. Therefore, these observations were considered as being incidental.
- Quantitative Parameters: No test substance-related impaired parameters were observed in male and female animals of all test groups.
- Motor activity measurement (MA): No statistically significant changes on motor activity data (summation of all intervals) was observed in the male and female animals of all dose groups in comparison to the concurrent control group.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all groups including the controls. Fertility was proven for all F0 parental males within the scheduled mating interval for F1 litter. Thus, the male fertility index was 100% in all groups including the controls.
The female mating index calculated after the mating period for F1 litter was 100% in all test groups. The mean duration until sperm was detected (GD 0) varied between 3.0 and 4.3 days without any relation to dosing. All sperm positive rats delivered pups. The fertility index was 100% in all test groups.
The mean duration of gestation was similar in all test groups (i.e. between 22.4 and 22.5 days). The gestation index was 100% in all test groups.
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the controls, taking normal biological variation into account (14.0 / 12.0 / 11.9 and 13.5 implants/dam in all test groups (0, 10, 30 and 90 mg/kg bw/day)). There were no statistically significant differences in post-implantation loss between the groups (4.1% / 7.7% / 8.8% / 5.1%). The mean number of F1 pups delivered per dam remained unaffected and was comparable between the test substance-treated groups and the control, taking normal biological variation into account (13.4 / 11.1 / 10.9* [* = p-level of significance ≤0.05] and 12.8 pups/dam at 0, 10, 30 and 90 mg/kg bw/day). The slight, statistically significant reduction in the mean number of F1 pups delivered per dam in the mid-dose females was a result of a small litter size in only two of the females of this group. If these litters are excluded, the remaining average litter size, 12.0, is not different than that of the other dose groups. Since the other litters were similar in size to those found in the control group, there did not appear to be a dose-response relationship. Furthermore, 10.9 pups/litter is within the range of the historical control data at this test facility. All together, these results suggest that the litter size reduction in dose group 2 is spontaneous in nature.
The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 100% control and low-dose group), 99.1% (mid-dose group) and 97.7% (high-dose group). Moreover, the number of stillborn pups was comparable between the groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Absolute weights
When compared to the control group, the mean absolute weight of the liver was significantly increased in the high-dose male and female animals. All other mean absolute weight parameters of treated male and female animals did not show relevant differences when compared to the control group and are therefore considered to be within the normal range.
Relative weights
When compared to the control group, the mean relative weight of the liver was significantly increased in the high-dose male and female animals. All other mean relative organ weights of treated male and female parental animals did not show relevant differences when compared to the control group and are therefore considered to be within the normal range.
The significant absolute and relative weight increase in the liver of males and females in the high-dose group (90 mg/kg bw/day) was consistent with histopathological findings and was considered to be treatment-related.

CLINCAL PATHOLOGY (PARENTAL ANIMALS)
- Hematology: No treatment-related, adverse changes among hematological parameters were observed. In males of test group 3 (90 mg/kg bw/d), hematocrit and prothrombin (Hepatoquick’s test = HQT) values were decreased, whereas relative reticulocyte counts were increased. All mentioned parameter means were within historical control ranges (hematocrit 0.396-0.448 L/L; prothrombin time 30.2-37.9 sec; relative reticulocyte counts 0.9-2.5 %). Therefore, the changes were regarded as incidental and not treatment-related. In males of test group 1 (10 mg/kg bw/d) total white blood cell counts, as well as absolute lymphocyte counts, were decreased, but the alterations were not dose-dependent and therefore, the changes were regarded as incidental and not treatment-related. In males of test group 3 (90 mg/kg bw/d) absolute large unstained cell (LUC) counts were increased and
the mean was slightly above the historical control range (LUC 0.01-0.04 Giga/L). However, this was the only changed differential blood cell parameter in these rats. No change occurred in the females of the corresponding dose group. Therefore, the change was regarded as maybe treatment-related, but not adverse.
- Clinical chemistry: In males of test group 3 (90 mg/kg bw/d), urea, cholesterol and inorganic phosphate levels were increased, but chloride concentrations were decreased. Additionally, in females of the same test group urea levels were higher compared to controls.
- Urinalyses: In males of test group 3 (90 mg/kg bw/d), more phosphate crystals and transitional epithelial cells were observed in the urine sediment.
Additionally, in females of the same test group urine volume was higher. This alteration is per se not regarded as an adverse effect.

GROSS PATHOLOGY (PARENTAL ANIMALS)
All gross findings noted at necropsy in parental animals are regarded as incidental and spontaneous and are not related to treatment.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Treatment-related findings were observed in the liver of parental male and female animals; in 5 out of 5 high-dose male and 5 out of 5 high-dose female animals centrilobular hypertrophy was observed. This finding correlated with a slight absolute and relative liver weight increase. However, this finding is regarded as adaptive and not adverse.
Two out of ten parental female animals of the high-dose group (90 mg/kg bw/day) showed a minimal follicular hypertrophy/hyperplasia in the thyroid gland when compared with the ten females of the control group. Due to the minimal magnitude and the low incidence of this change it was not regarded as treatment related and was considered to be an incidental finding.
All other findings noted in parental animals were either single observations, or were biologically equally distributed between controls and treated rats. All of them are considered to be incidental and/or spontaneous in origin.

Dose descriptor:

NOAEL

Remarks:

parental

Effect level:

30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

90 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at the highest dose tested.

VIABILITY (OFFSPRING)
The viability index indicating pup mortality during lactation (PND 0 - 4) varied between 94.4% (high-dose group), 90.0 (mid-dose group), 100.0 (low-dose group) and 99.3% (control), well within historical control values.
The reduction in pup numbers in the mid-dose group was amplified by PND 4. Eleven pups died/were cannibalized before PND 4, resulting in a small, but statistically significantly decreased, average live litter size of 9.7 (compared to 13.3 in the concurrent control group). A further 5 pups in the high dose group also died/were cannibalized intercurrently. All 16 pups originated from only two litters, nos. 123 and 133 (1 each in the 30 and 90 mg/kg bw/day dose groups, respectively), which showed signs of poor maternal care, such as findings of pups not fed/no milk in stomach and reduced nutritional condition in the pups. As the incidence of pup deaths is restricted to a small number of litters, lies within the normal range of biological variation inherent in the rat strain used and is not dose-dependent, the death of these pups was probably incidental.

CLINICAL SIGNS (OFFSPRING)
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.

BODY WEIGHT (OFFSPRING)
Mean body weights and mean body weight change of the male and female pups in all test substance-treated groups were comparable to the concurrent control group during the entire study period.
One female runt was seen in the high-dose group.
Two male runts were seen in the mid-dose group.
One female runt was seen in the low-dose group.
Five male and two female runts were seen in the control.

GROSS PATHOLOGY (OFFSPRING)
A few pups showed spontaneous findings at gross necropsy, such as post mortem autolysis, empty stomach and discolored liver lobe. These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. None of these findings were considered to be associated with the test substance.

HISTOPATHOLOGY (OFFSPRING)
In one pup of the control group and one pup of mid-dose group (30 mg/kg bw/day), dissecting aneurysms of the ductus arteriosus and aorta correlated with macroscopic changes observed at necropsy. Since these findings occurred without a dose-dependency they were considered to be incidental and not related to treatment.

OTHER FINDINGS (OFFSPRING)
The mean number of delivered F1 pups per dam and the rates of liveborn and stillborn F1 pups were comparable between the dose groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study. The slight, statistically significant reduction in the mean number of F1 pups delivered per dam in the mid-dose females was considered to be spontaneous in nature.

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

90 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at the highest dose tested.

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

studies according to the respective OECD test guidelines

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 accordance with Column 1 of REACH Annex IX, an enhanced one-generation toxicity study does not need to be conducted when a 28-day or 90-day study does not indicate adverse effects on reproductive organs or tissues. For this substance a combined repeated dose and reproduction / developmental screening study in rats (2013) and a repeated dose 90 -day oral toxicity study in rats (2016) are available and no adverse effects were observed on the reproductive organs.

In a GLP OECD408 study, 1-Methylimidazol was administered by gavage to 10 male and female Wistar rats per dose levels of 0, 10, 30 and 90 mg/kg bw/d over a period of 3 months (2016). Food consumption and body weight were determined weekly. The animals were examined at least daily for signs of toxicity or mortality and for any clinically abnormal signs before and within 2 h as well as within 5 h after treatment. Detailed clinical examinations were conducted prior to the start of the administration period and weekly thereafter. Further examinations were ophthalmology (before staring and at the end of the administration period), functional observational battery (FOB), measurement of motor activity (MA), clinicochemical and hematological examinations as well as urinalyses at the and of administration period. After the administration period all animals were sacrificed and assessed by gross pathology. Organ weights were determined followed by histopathological examinations. The stability of the test-substance preparations and their correct concentrations were confirmed. No test-substance-related, adverse findings were noted up to the highest dose group of 90 mg/kg bw/d. There were some treatment-related findings, considered to be non-adverse, but rather adaptive: In HD males relative reticulocyte counts and urea values were higher compared to controls and slightly above the historical control range. Furthermore, the urine volume was decreased, which was regarded as maybe treatment-related. In HD females chloride levels were decreased and the values were marginally below the historical control range. A statistically significant increase of relative liver weights was observed in males and females of the mid and/or high dose group. Histopathology revealed a dose-dependent, minimal to slight centrilobular liver hypertrophy in males and females of all test groups. As there were no correlating findings in clinical chemistry, these findings were regarded as non-adverse. The administration of 1-Methylimidazol by gavage to male and female Wistar rats for 3 months caused no signs of systemic toxicity up to the highest dose level tested (90 mg/kg bw/d). Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 90 mg/kg bw/d for male and female Wistar rats.

Further details concerning this study can be found in the IUCLID section 7.5.1 Repeated dose toxicity: oral.

In a GLP compliant combined oral repeated dose toxicity study and reproduction/developmental toxicity screening test according to OECD 422, the substance was administered daily as an aqueous preparation to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 10, 30 and 90 mg/kg bw/day (2013). Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only (drinking water). The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 2 days post-mating in males, and the entire gestation period as well as approximately 2 weeks of the lactation period. Accuracy, homogeneity and stability of formulations were demonstrated by analyses. Regarding clinical pathology, slight dysregulations in the liver cell metabolism of male and female rats of test group 3 (90 mg/kg bw/d) can be assumed because of higher urea levels in both sexes, indicating an increased protein metabolism, as well as higher cholesterol levels in males. In addition, in males of the mentioned test group chloride levels were low whereas inorganic phosphate levels were high. In these animals, a slight functional effect on the kidneys cannot be excluded; this was confirmed by higher incidences of transitional epithelial cells as well as phosphate crystals in the urine. No test substance related findings were noted in any of the other parameters investigated in this study (mortality / viability, clinical signs, functional observations other than for locomotor activity, body weight, food consumption, haematology, macroscopy, organ weights and histopathology). All other measures of reproductive performance or fertility were normal in both genders and at all doses. Therefore, no alterations to reproductive performance or fertility were identified in the parental rats. No effects on pup body weights or pathology at necropsy were observed; thus no substance-dependent developmental toxicity was identified at any dose. All other findings recorded were considered to be incidental in nature and unrelated to treatment. Thus, under the conditions of this reproduction/developmental toxicity screening test the NOAEL for general, systemic toxicity of the test substance is 30 mg/kg body weight/day for the F0 parental animals based on the increased urea levels in both sexes at 90 mg/kg bw/day. Additionally, males exhibited signs of increased serum inorganic phosphate and cholesterol levels, as well as decreased serum chloride concentration and a higher incidence of both transitional epithelial cells and phosphate crystals in the urine at this dose. The NOAEL (no observed adverse effect level) for reproductive performance and fertility in the F0 parental rats is 90 mg/kg body weight/day, the highest dose tested. The NOAEL for developmental toxicity was 90 mg/kg body weight/day, the highest dose tested.

Effects on developmental toxicity

Description of key information

The NOAEL for maternal and prenatal developmental toxicity is 90 mg/kg bw/d in an OECD414 study, based on maternal and fetal adverse findings at 230 mg/kg bw/d.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 2020 - August 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

additionally to available study, with one higher dose

Justification for type of information:

RATIONALE FOR STUDY INITIATION:
Based on ECHA´s final decision in 2018, the registrant was requested by its local enforcement authority in 2020 to conduct a supplementary study according to the OECD test guideline 414.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 440/2008

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 June 2018

Deviations:

yes

Remarks:

one dose, additionally to available OECD414 study

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Name of test substance: 1-Methylimidazole
Test substance No.: 11/0492-4
Batch No.: 51249316K0
CAS No.: 616-47-7
Content: 99.8 ± 0.0 g/100 g (Final Report, Study code: 20L00014)
Identity: confirmed (Final Report, Study code: 20L00014)
Homogeneity: Given (visually)
Storage stability: Expiry date: 11 Dec 2020

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany
- Age at study initiation: About 10-12 weeks
- Weight at study initiation: 174-215 g
- Housing: 1 animals per cage (Polycarbonate cages type III)
- Enrichment: Wooden gnawing blocks (Typ SAFE® block large), J. Rettenmaier & Sohne GmbH + Co KG, Rosenberg, Germany. Play tunnel large (Art. 14153), PLEXX B.V., Elst, Netherlands.
- Nesting material: Cellulose wadding toward the end of gestation (pregnant females)
- Bedding: Dust-free wooden bedding
- Diet (e.g. ad libitum): Mouse and rat maintenance diet "GLP", Granovit AG, Kaiseraugst, Switzerland; ad libitum
- Water (e.g. ad libitum): potable tap water in water bottles, ad libitum
- Acclimation period: From GD0 (day of supply) to the beginning of administration (GD6), the animals will be accustomed to the environmental conditions and to the diet.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 45-65%
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours darkness (18:00 h-06:00 h) / 12 hours light (06:00 h - 18:00 h)

IN-LIFE DATES: From: 05 Aug 2020 To: 26 Aug 2020

Route of administration:

oral: gavage

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the test substance preparation, the specific amount of test substance will be weighed, topped up with drinking water in a graduated flask and intensely mixed with a magnetic stirrer until it is completely dissolved.

VEHICLE
Concentration in vehicle: 2.3 g/100 ml --> 10 ml/kg bw/d, based on the most recent individual body weight.

ADMINISTRATION:
Once a day orally by gavage with disposable syringes (e.g. Becton Dickinson GmbH, Heidelberg, Germany) of suitable size and suitable gavage tubes (e.g. MTL Instruments GmbH, Kaarst, Germany).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical investigations of the test substance preparations at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, 67056 Ludwigshafen, Germany.

Details on mating procedure:

The animals were paired by the breeder and supplied on GD0 (= detection of vaginal plug/sperm).

Duration of treatment / exposure:

GD6-19

Frequency of treatment:

once daily, GD6 - GD19 (14 administrations)

Duration of test:

GD0: day of evidence of mating.
GD6 - GD19: administration period.
GD20: blood sampling, sacrifice and examination.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

230 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In the available pre-natal developmental toxicity study (2016) the NOAEL for maternal and pre-natal developmental toxicity was 90 mg/kg bw/d, the highest dose tested. Thus, in a subacute Dose Range Finder Study (2019) higher doses of 1-methylimidazole were administered dissolved in drinking water for 2 weeks to groups of 5 female non-pregnant Wistar rats, via daily gavage. The dose levels were 0, 90, 180, 270, 360 and 540 mg/kg body weight/day. Two animals at the 540 mg/kg bw/d dose level died during the night after the second administration. The remaining 3 animals of this group were euthanized moribund, after showing distinct signs of toxicity (piloerection, twitching, tremors, hyperexitability, abdominal position). They have been assessed by gross pathology as far as possible, revealing no macroscopic findings. At 360 mg/kg salivation was observed in 3 of 5 animals and one glandular stomach focus was found in 1 of 5 animals. Water consumption was nearly 2x as high as compared to control and thus statistically significantly increased (d0-14: +93.8%). Food consumption was severely decreased from d0-3 (-58.6%) and remained - although not statistically significantly - below the control values (d0-14: -17.3%). Initially, the body weight change was significantly increased, but overall no significant difference of the body weight was observed; probably due to the increased water consumption.
Increased water consumption was also observed in all three lower dose groups (90 mg/kg, 180 mg/kg, 270 mg/kg) and initially decreased food consumption in the 180 and 270 mg/kg group. But there were no differences in BW oder BWC compared to the control. Additionally, significant changes in clinical pathology were observed in the test groups 180 mg/kg and above: decreased total protein and albumin, increased inorganic phosphate levels. In the 360 mg/kg bw group number of absolute monocytes, alanine aminotransferase and triglycerides were statistically significantly increased. Furthermore, absolute and relative liver weights were increased in the 360 mg/kg group (127% and 130% compared to control = 100%), as well as relative kidney weights (111%). Absolute and relative spleen weights were decreased in this group (75% and 77%).
In conclusion, oral administration of 540 mg 1-methylimidazole/kg bw/d exceeded the maximal tolerable dose for female non-pregnant rats, whereas lower doses (180, 270 and 360 mg/kg bw/d) induced adverse effects after 14 days of administration: water and food consumption, clinical pathology, organ weights.

Thus, for the maternal Dose Range Finder Study (2020) the dose levels of 0, 270 and 360 mg/kg body weight/day were chosen and 1-methylimidazole was administered dissolved in drinking water from GD6-19 to groups of seven female pregnant Wistar rats, via daily gavage. The animals of the 360 mg/kg group showed distinct clinical symptoms (unsteady gait, semiclosed eyelid, piloerection, hyperthermie, reduced nutrial condition, twitching abdominal position), markedly decreased food consumption and body weight loss. One animal died and the other dams were sacrificed prematurely for animal welfare reasons at d13. At 270 mg/kg clinical symptoms, e.g. salivation, blood in bedding and piloerection, were observed. Food consumption and several body weight parameters were statistically significantly reduced from d0-20 compared to the control: Food consumption: -21%, BW: -6%, BWC: -14%, carcass weight (= final BW - uterine weight): -9.3%, corrected body weight gain (carcass weight - BW from GD6): -51.9%. Additionally, statistically significant changes in clinical pathology were observed in the lower test group 270 mg/kg: Decrease of RBC, HGB, HCT as well as increase of RETA. Furthermore, TPROT, ALB and GLOB values were decreased, CHOL increased.
In conclusion, both doses showed too severe signs of maternal toxicity and a dose of 230 mg/kg bw/d was selected for the main study, in order to induce sufficient maternal toxicity in line with the OECD TG 414 without exceeding the maximal tolerable dose.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Check for moribund and dead animals: twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays (GD0-20)
- Cage side examination: at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity
- During the administration period (GD6-19) all animals will be checked daily for any abnormal clinically signs before the administration as well as within 2 hours and between 2 and 5 hours after the administration. Abnormalities and changes will be documented for each animal.

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 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: GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17,
17-19 and 19-20

WATER CONSUMPTION: Yes
- Time schedule for examinations: 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
- Clinical pathology after blood sampling: Hematology, clinical chemistry, thyroid hormones
- Gross pathology with special attention to reproductive organs.
- Pathology: necropsy, organ weights (adrenal glands, kidneys, thyroid glands with parathyroid glands, liver, spleen), carcass weight. Organ/tissue fixation (all gross lesions, adrenal glands, kidneys, stomach, thyroid glands with parathyroid glands, liver, spleen).

HISTOPATHOLOGY: Yes (Adrenal glands, kidneys, liver, spleen, thyroid glands)
Fixation, histotechnical processing, examination by light microscopy and assessment of findings.

Ovaries and uterine content:

Uterus: weight unopened, no. of corpora lutea/implantations/early resoprtions, site of implantations

Blood sampling:

Yes, by retrobulbar venous puncture at GD20. 1 ml of blood was sampled and prepared in original Eppendorf tubes with EDTA as anticoagulant (10 µl of a 10% solution), samples were centrifuged, plasma separated. The preparation of the samples was done under cooling. All samples were covered with a N2 atmosphere and then stored at -80°C for research.
- Hematology: Leukocytes, Erythrocytes, Hemoglobin, Hematocrit, Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelets, Differential blood count, Reticulocytes
- Clinical chemistry: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Serum y-glutamyl transferase, Inorg. phosphate,
Calcium, Urea, Creatinine, Glucose, Total bilirubin, Total protein, Albumin, Globulins, Triglycerides, Cholesterol
- Thyroid hormones: T3, T4, TSH

Fetal examinations:

- External examinations: Yes
weight of each fetus, sex (external by gross examination and internal (gonadal) sex), weight of placenta, gross-pathological examination
- Soft tissue examinations: Yes (half of the fetuses of each dam)
- Skeletal examinations: Yes (half of the fetuses of each dam)
- Anogenital distance of all live rodent pups: yes

Statistics:

- DUNNETT´s test for water consumption, food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight, anogenital distance, anogenital index.
- FISHER´s exact test for female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings.
- WILCOXON test for proportions of fetuses with malformations, variations and/or unclassified observations in each litter; for clinical pathology parameters, carcass weights and absolute and relative organ weights of the dams.

Historical control data:

Yes, data attached as background material

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Some females of the treatment group (7/25) showed polyuria on GD18-20. Most likely, this finding was caused by increased water consumption in this test group.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean BW of the dams in the treatment group were lower in comparison to the concurrent control group from GD8 to GD20, attaining statistical significance on GD8-15: up to a maximum of 7% lower BW (table 1 in "Any other information on results incl. tables").
The corrected BWG (terminal BW on GD20 minus weight of the unopened uterus minus BW on GD6) was distinctly and statistically significantly lower in the treatment group compared to the control: about 60% below control.
Furthermore, the carcass weight of these dams was reduced in comparison to the concurrent control group (about 6% below, statistically significant). (table 3a)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the treatment group , the mean FC was statistically significantly reduced during GD6-13 (up to 62% below control) but recovered afterwards and was comparable to the control values throughout the remaining study period. Nevertheless, during the treatment period (GD6-19), these dams consumed about 20% less food than the controls. (table 2)

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

effects observed, treatment-related

Description (incidence and severity):

The mean WC of the dams in the treatment group was statistically significantly increased on GD10-20 (up to a maximum of 67% above the concurrent control). During the entire treatment period (GD6-19), these dams consumed 32% more water than the controls. (table 2)

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At GD20, in dams of the treatment red blood cell (RBC) counts, hemoglobin and hematocrit values as well as mean corpuscular hemoglobin concentration (MCHC) were significantly decreased compared to control values (-10.1%, -9.6%, -8.7%, -1.42%, respectively), whereas absolute reticulocyte counts were significantly increased compared to the control (+95%: 288.2 vs. 147.7 giga/L). These alterations were regarded as treatment related and adverse. (table 3b)

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At GD20, in dams of the treatment group total protein as well as albumin and globulin values were significantly decreased (-12.3%, -12.4%, -12.2%, respectively). These alterations were regarded as treatment related and adverse.
Other value changes (e.g. AST, cholesterol, total bilirubin, calcium) were regarded as incidental and not treatment related.

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

In dams of treatment group TSH levels were significantly increased compared to the control (+51.2%; 12.01 vs. 7.94 µg/L) and were above historical controls (dams TSH 4.92-7.55 µg/L). However, the control value was also slightly above the HCR. T3 and T4 values were not changed compared to the control (230 mg/kg vs. cntrl: 1.14 vs. 1.15 nmol/L T3; 24.4 vs. 26.0 nmol/L T4).

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

When compared to control group 0 (= 100%), the mean absolute weights of the final body weight, adrenal glands, kidneys, liver and spleen were significantly changed: 94%, 128%, 110%, 113%, 93%, respectively; but the thyroid glands weight was not. When compared to control group 0 (= 100%), the mean relative weights of the adrenal glands, kidneys and liver were significantly increased: 137%, 118%, 121%, respectively; but the spleen and thyroid glands weights were not. (table 3c)
The significant decrease of the terminal BW was within the historical control range. The significant absolute and relative weight increases of the adrenal glands (non-adverse) and the liver were both above the historical control ranges. Both were consistent with histopathological findings and were therefore considered treatment-related.
The significant absolute and relative weight increases of the kidneys were within the historical control range and had no histopathological correlate. Although a possible treatment-related effect cannot be excluded, this change was regarded as non-adverse. The significant absolute weight decrease of the spleen was within the historical control range and was regarded as incidental.

Description (incidence and severity):

In 3/25 treated dams thickening of the duodenum wall and in 1/25 a focus in the glandular stomach were observed.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in the adrenal cortex (6/25 dams: minimal hypertrophy of zona fasciculata; non-adverse), liver (increased grading of vacuolation pattern in hepatocytes, increased extramedullary hematopoiesis in 19 vs. 2 control dams), spleen (increased grading in extramedullary hematopoiesis) and thyroid glands (minimal hypertrophy/-plasia of follicular cells in 9 vs. 1 control dams).

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Details on results:

see table 1 - 3 in "Any other information on results incl. tables" and and the historical control data attached as background material.

There is evidence of maternal toxicity, such as increased water consumption, reduction in food consumption, a decrease in (corrected) body weight gain,
changes in clinical pathology parameters (e.g. a hypochromic, normocytic anemia, changed liver cell protein synthesis and accelerated thyroid hormone excretion) and corresponding histopathological findings in liver, spleen and thyroid at the tested dose level of 230 mg/kg bw/d.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

There were no test substance related and/or biologically relevant differences between the treatment and the control group in conception rate, in the mean number of corpora lutea and in the values calculated for the pre-implantation loss (table 4).
The mean number of implantation sites of the treatment group and the post-implantation loss (litter-based mean% of resorptions per group) were outside the historical control range, though non-significantly compared to the control group.
- implantation sites (mean): 12.7 vs. 11.5 in controls, HCR: 10.0 - 11.8;
- post-implantation loss (mean%): 15.9 vs. 9.4 in controls, HCR: 4.1 – 11.9.
Besides the distinct maternal toxicity, the high number of implantation sites in the treatment group, which is not related to treatment, may play a role in the higher post-implantation loss. However, a treatment-related effect - potentially secondary to the maternal toxicity - cannot be excluded.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

The total number of resorptions was statistically significantly higher than in the concurrent control (mean 2.0 vs. 1.0 in control) and outside the historical control range (0 5 - 1 5; mean 0.8 +/- 1.2). But neither the early nor the late resorptions were significantly increased compared to the control.
Besides the distinct maternal toxicity, it must be noted the number of implantation sites of the treatment group was above the historical control range, which could have contributed to the increased resorption rate (table 4).

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

(cesarean section)

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

(time-mated females from the breeder)

Details on maternal toxic effects:

see table 4 in "Any other information on results incl. tables" and and the historical control data attached as background material.

The significant increased number of resorptions and the corresponding post-implantation loss was mainly driven by high resorption rates in dams No. 39 and 41, which showed even more severe signs of toxicity than the whole treatment group (glandular stomach focus in no. 39, net weight change day 6 of -26.1 g in no. 41). The mean value of the net weight change day 6 for the treatment group was 13.4 g, thus 40 % of the control value of 33.3 g (table 3a). Additionally. the red blood cell parameters that were significantly altered in the treatment group (mean values), indicating a maternal anemia, were clearly more influenced in these two dams (table 3b). Furthermore, low resorption rates of a higher number of affected females compared to the control group were observed and included also a considerable number of late resorptions. This had no consequences for the average number of viable fetuses compared to the control.

Dose descriptor:

LOAEL

Effect level:

230 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical biochemistry

early or late resorptions

food consumption and compound intake

haematology

organ weights and organ / body weight ratios

pre and post implantation loss

water consumption and compound intake

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The mean fetal weights were comparable to the control group.
The mean placental weight of the treatment group was statistically significantly decreased in comparison to the concurrent control group. Nevertheless, the mean value (i.e. 0.61 g, both sexes combined) was within the historical control range (HCD both sexes: mean 0.48 g; range of 0.35 - 0.97 g).

Anogenital distance of all rodent fetuses:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Fetal external malformations, variations and unclassified observations were comparable between treatment and control group and within the historical control range: one control fetus with multiple external (and skeletal) malformations, one fetus with one external variation in each test group and two or five fetuses in the control or treatment group, respectively, with unclassified observations. (table 5)

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

MALFORMATIONS: The total incidences did not differ significantly from control (mean% affected fetuses/litter: 3.1 vs. 1.1 in control) and were comparable to the historical control data. Two fetuses of the control group with multiple skeletal malformations and five fetuses of the treatment group with single observations related to cervical and thoracic vertebrae, forelimbs, ribs and sternum.
VARIATIONS: Skeletal variations of different bone structures were observed, with and without effects on corresponding cartilages. They were related to several parts of fetal skeleton and appeared in the majority of cases without relation to treatment. The overall affected fetuses/litter incidence was statistically significantly increased and above HCD , whereas the incidence of the control was below the HCD (mean%: 100.0* vs. 90.8 in the control; HCD: 97.2, range: 94.4 - 98.5). The variations with statistically significant differences were related to skull, cervical and thoracic vertebrae, ribs and sternum (table 7). The significantly increased mean value of ‘supernumerary rib (14th) with cartilage not present’ was clearly inside the historical control range, whereas the mean value of the control was even below. Therefore, this finding maybe incidental and not treatment-related. For all other findings the statistically significantly increased mean values were outside the respective historical control range. For further discussion see "Details on embryotoxic / teratogenic effects".
UNCLASSIFIED CARTILAGE OBSERVATIONS: Some isolated cartilage findings without impact on the respective bone structure occurred in both groups and did not show any relation to dosing (mean% affected fetuses/litter: 59.1 vs. 61.7 in control).

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

MALFORMATIONS: The total incidence of soft tissue malformations in treated animals (3.2 mean% affected fetuses/litter) did not differ significantly from the concurrent control group but was outside the historical control range (HCD: mean% 0.3 [0.0-1.5]: one fetus with anophthalmia, which is within the HCD; two fetuses with misshapen heart. The latter was found in two fetuses of one litter. The respective dam showed even more severe maternal toxicity than the whole treatment group. For further discussion see "Details on embryotoxic / teratogenic effects".
VARIATIONS: The incidences of these variations were not significantly increased in the treated group (12.7 vs. 9.3 mean% in the control). The three variations (malpositioned subclavian origin, dilated renal pelvis, dilated ureter) were either found in the control group or in the historical control data. However, the incidences for dilated renal pelvis were outside the historical control range in both groups (mean%: 12.7 vs. 8.7 in control; HCR: 0.8 - 7.7)
UNCLASSIFIED OBSERVATIONS: No visceral unclassified observations were recorded.

Details on embryotoxic / teratogenic effects:

see tables 4 - 7 in "Any other information on results incl. tables". Further details about all fetal malformations and variations (external, visceral, skeletal) and the historical control data are attached as background material.

In the treated group significantly higher total malformation (external, soft tissue and skeletal combined; 3.2%* [p ≤ 0.05] vs. 0.6% in control) and total variation (external, soft tissue and skeletal combined; 58.8%** [p ≤ 0.01] vs. 54.5% in control) rates were noted which were mainly driven by high incidences of skeletal findings. The majority of these malformations and variations was related to skull, cervical and thoracic vertebrae, forelimbs, ribs and sternum. Over-all incidences and several incidences of individual findings exceeded the historical control range (HCR) of the test facility. Many individual findings represented minor changes, indicating delays of development or disturbances of ossification (table 7). These are accepted to depend on the maternal status; e.g. decreased food consumption and body weight and are considered not to persist, which is supported by the presence of normal cartilage. “Wavy ribs” are a common finding in rodent studies, are not classified as true malformations and rather often occur along with maternal toxicity (Carney & Kimmel, 2007; Daston & Seed, 2007). Furthermore, the significantly increased number of other fetal skeletal variations, e.g. “supraoccipital hole(s)”, “supernumerary thoracic vertebra”, was only slightly above the HCR. The number of “supernumerary rib (14th), cartilage not present” is increased compared to the control, but within the HCR, whereas the control value is below the HCR. There was no significant increase of external observations. The visceral malformation (misshapen heart) which was not found in the HCD, was found in two fetuses of one litter. The respective dam showed even more severe maternal toxicity than the whole treatment group. A net body weight change from d6 of -26.1 g indicates a severe net body weight loss. The mean value for the treatment group was 13.4 g, thus 40 % of the control value of 33.3 g (table 3a). Furthermore, red blood cell parameters that were significantly altered in the treatment group (mean values), indicating a maternal anemia, were clearly more influenced in this dam (table 3b). Maternal anemia resulting in adverse developmental effects are described for several species (Kalisch-Smith et al, 2021, Clark et al., 1984, Shepard et al, 1980; see also ECETOC TR No. 138, 2021).

Thus, the data indicate some but no clear evidence of an adverse effect on developmental toxicity in experimental animals, as the increased malformation and variation incidences, which are maybe indicative for a potential of the test item to impair embryofetal development, occurred only at a high, maternally toxic dose (230 mg/kg bw/d).


References:
- Carney, E.W. & Kimmel, C.A. (2007): Review Article. Interpretation of Skeletal Variations for Human Risk Assessment: Delayed Ossification and Wavy Ribs. Birth Defects Research (Part B) 80:473–496.
- Daston, G.P. & Seed, J. (2007): Introduction. Skeletal Malformations and Variations in Developmental Toxicity Studies: Interpretation Issues for Human Risk Assessment. Birth Defects Research (Part B) 80:421–424.
- Kalisch-Smith, J.I., Ved, N., Szumska, D., Munro, J., Troup, M., Harris, S.E., Rodriguez-Caro, H., Jacquemot, A., Miller, J.J., Stuart, E.M., Wolna, M., Hardman, E., Prin, F., Lana-Elola, E., Aoidi, R., Fisher, E.M.C., Tybulewicz, V.L.J., Mohun, T.J., Lakhal-Littleton, S., De Val, S., Giannoulatou, E. & Sparrow D.B. (2021): Maternal iron deficiency perturbs embryonic cardiovascular development in mice. Nature Communications 12:3447.
- Clark R.L., Roberston, R.T., Minsker, D.H., Cohen, S.M., Tocco, D.J., Allen, H.L., James, M.L., Bokelman, D.L. (1984): Diflunisal-induced maternal anemia as a cause of teratogenicity in rabbits. Teratology 30(3): 319-32.
- Shepard T.H., Mackler B., Finch, C.A. (1980): Reproductive studies in the iron-deficient rat. Teratology 22(3): 329-34.
- ECETOC (2021): ECETOC Guidance on Dose Selection. Technical Report No. 138.

Dose descriptor:

LOAEL

Effect level:

230 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: total fetal malformations and variations

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: skull

skeletal: forelimb

skeletal: sternum

skeletal: rib

skeletal: vertebra

Developmental effects observed:

yes

Lowest effective dose / conc.:

230 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Statistical significance: *: p <= 0.05, **: p <= 0.01

HCD: Historical control data

 

Table 1: Mean maternal body weights during gestation [g]

Study day	0 mg/kg	230 mg/kg	in % to cntrl
0	194.1	195.3	100.6%
1	208.2	209.9	100.8%
3	217.2	219.3	101.0%
6	228.4	231.5	101.4%
8	233.1	216.0**	92.7%
10	242.4	225.9**	93.2%
13	255.6	241.1**	94.3%
15	265.6	254.7*	95.9%
17	282.8	275.8	97.5%
19	306.3	297.5	97.1%
20	319.9	308.7	96.5%

 

Table 2: Mean maternal water and food consumption

	Water consumption [g/animal/day]			Food consumption [g/animal/day]
Study day	0 mg/kg	230 mg/kg	in % to cntrl	0 mg/kg	230 mg/kg	in % to cntrl
0-1	21.8	23.2	106.4%	13.6	14.6	107.4%
1-3	23.5	24.2	103.0%	19.9	20.3	102.0%
3-6	24.1	23.5	97.5%	20.4	21.1	103.4%
6-8	19.5	11.0**	56.4%	20.0	7.7**	38.5%
8-10	21.2	20.7	97.6%	21.0	12.8**	61.0%
10-13	23.8	33.1**	139.1%	22.4	18.5**	82.6%
13-15	27.1	41.1**	151.7%	21.5	20.3	94.4%
15-17	29.6	49.5**	167.2%	23.4	23.3	99.6%
17-19	32.9	48.8**	148.3%	23.6	23.1	97.9%
19-20	31.1	42.5**	136.7%	24.3	22.9	94.2%

 

Table 3a: Gravid uterine weights and net maternal BW change (mean +/- SD) [g]

	0 mg/kg	230 mg/kg	in % to cntrl
gravid uterus	58.3 +/- 18.5	63.8 +/- 14.0	109.4%
carcass (terminal BW minus uterine weight)	261.6 +/- 16.11	245.0** +/- 17.4	93.7%
net weight change from GD6 (carcass weight minus BW at GD6)	33.3 +/- 6.77	13.4** +/- 12.27	40.2%

 

Table 3b: Red blood cell parameters of the dams, incl. individual data of dam no. 39 and 41 (mean +/- SD)

	0 mg/kg bw/d	230 mg/kg bw/d	dam no. 39	dam no. 41
RBC (tera/L), day 20	6.04 +/- 0.40	5.44** +/- 0.43	4.94	4.00
HGB (mmol/L), day 20	6.6 +/- 0.4	6.0** +/- 0.4	5.4	4.5
HCT (L/L), day 20	0.318 +/- 0.021	0.290** +/- 0.019	0.268	0.230
MCHC (mmol/L), day 20	20.94 +/- 0.31	20.64* +/- 0.44	20.23	19.41
RETA (giga/L) day 20	147.7 +/- 29.3	288.2** +/- 98.1	518.2	628.40

 

 

Table 3c: Absolute and relative organ weights of the dams

	absolute weights (mean +/- SD)			relative weights (mean)
	0 mg/kg	230 mg/kg	in % to cntrl	0 mg/kg	230 mg/kg	in % to cntrl
Final body weight [g]	261.6 +/- 16.1	245.0** +/- 17.4	94%	100%	100%	100%
Adrenal glands [mg]	70.0 +/- 7.7	89.4** +/- 15.0	128%	0.027%	0.037%**	137%
Kidneys [g]	1.59 +/- 0.14	1.75** +/- 0.16	110%	0.609%	0.716%**	118%
Liver [g]	11.37 +/- 1.21	12.09** +/- 1.31	113%	4.338%	5.263%**	121%
Spleen [g]	0.53 +/- 0.06	0.49* +/- 0.10	93%	0.202%	0.201%	100%
Thyroid glands [g]	17.48 +/- 2.74	18.4 +/- 4.0	105%	0.007%	0.008%	112%

 

Table 4: Summary of reproduction data

	0 mg/kg	230 mg/kg	HCD	HCD: min - max
Females mated [n]	25	25	499
- Pregnant [n]	25	25	479
- conception rate [%]	100	100	96
- Aborted	0	0	0
- Premature births	0	0	0
- Dams with viable fetuses	25	25	479
- Dams with all resoprtions	0	0	0
Female mortality [n]	0	0	0
Pregnant at terminla sacrifice [n]	25	25	479
Corpora lutea [mean +/- SD]	12.5 +/- 2.5	13.1 +/- 1.6		10.8 - 13.8
- Total [n]	312	327
Implanation sites [mean +/- SD]	11.5 +/- 3.7	12.7 +/- 1.8		10.0 - 11.8
- Total [n]	288	318
Preimplantation loss [mean% +/- SD]	9.5 +/- 19.1	2.9 +/- 5.9		2.2 - 14.1
Postimplantation loss [mean% +/- SD]	9.4 +/- 14.0	15.9 +/- 17.6		4.1 - 11.9
Resorptions, total [mean/litter]	1.0 +/- 1.0	2.0* +/- 2.2		0.5 - 1.5
- Total [n]	24	49
- [mean% +/- SD]	9.4 +/- 14.0	15.9 +/- 17.6		4.1 - 11.9
Resorptions, early [mean/litter]	0.8 +/- 0.9	1.3 +/- 1.2
- Total [n]	19	32
- [mean% +/- SD]	8.0 +/- 13.7	10.2 +/- 9.2
Resorptions, late [mean/litter]	0.2 +/- 0.5	0.7 +/- 1.7
- Total [n]	5	17
- [mean% +/- SD]	1.5 +/- 3.7	5.7 +/- 13.8
Dead fetuses [n]	0	0		0
Dams with viable fetuses [n]	25	25
Live fetuses [mean +/- SD]	10.6 +/- 3.6	10.8 +/- 3.0		9.4 - 11.2
- Females [mean +/- SD]	5.4 +/- 2.0	5.3 +/- 2.0
- Males [mean +/- SD]	5.2 +/- 2.7	5.5 +/- 2.1
- Per cent live females	50.8	49.1
- Per cent live males	49.2	50.9

 

Table 5: Placental and fetal data (mean +/- SD)

	0 mg/kg	230 mg/kg	HCD: mean%	HCD: min - max
Placental weight [g]	0.49 +/- 0.08	0.61** +/- 0.08	0.48	0.35 - 0.97
Fetal weight [g]	3.6 +/- 0.16	3.5 +/- 0.35	3.6	2.6 - 4.5
AGD males [mm]	3.0 +/- 0.12	3.0 +/- 0.12
AGD females [mm]	1.5 +/- 0.07	1.5 +/- 0 06

 

Table 6: Incidences of fetal external, skeletal and visceral findings (malformations, variations and other observations) [mean% affected fetuses/litter  +/- SD]

	0 mg/kg	230 mg/kg	HCD: mean%	HCD: min - max	description
total fetal external malformations	0.3 +/- 1.67	0	0.1	0 - 0.8	no findings in treatment group
total fetal external variations	0.3 +/- 1.54	0.3 +/- 1.54	0.1	0 - 07	not treatment related
total fetal external unclassified findings	0.6 +/- 2.09	1.6 +/- 4.7	0	0	not treatment related
total fetal soft tissue malformations	0	3.2 +/- 13.52	0.3	0 - 1.5	all in HCD except for "missshapen heart" in two fetuses of one dam (no. 41)
total fetal soft tissue variations	9.3 +/- 10.99	12.7 +/- 22.59	4.1	0.8 - 7.7	not significantly increased and/or found in the HCD
total fetal soft tissue unclassified findings	0	0	0	0	no findings
total fetal skeletal malformations	1.1 +/- 3.72	3.1 +/- 6.44	0.7	0 - 3.1	incidences not statistically significantly different from control, comparable to the HCD
total fetal skeletal variations	90.8 +/- 12.28	100.0* +/- 0.0	97.2	94.0 - 98.5	statistically significant findings in table 7 below
total fetal skeletal cartilage	61.7 +/- 24.08	59.1 +/- 28.11	67.4	44.7 - 79.4	no relation to dosing
total fetal malformations	0.6 +/- 2.09	3.2* +/- 6.42	0.59	0 - 2.03	statistically significantly increased and outside HCR; however, except ‘misshapen heart’, all of them can be found in the HCD
total fetal variations	54.5 +/- 11.86	58.8* +/- 10.72	53.24	49.05 - 55 85	statistically significantly increased and outside the HCR; mainly driven by the skeletal variations

 

Table 7: Statistically significant fetal skeletal variations

	0 mg/kg bw/d	230 mg/kg bw/d	HCD: Mean %	HCD: min - max
Supraoccipital hole(s)	0	17.1**	1.3	0.0 - 11.4
Incomplete ossification of cervical arch; cartilage present	1	4.5*	0.7	0.0 - 2.3
Incomplete ossification of thoracic centrum; unchanged cartilage	0	5.1*	0.7	0.0 - 2.8
Supernumerary thoracic vertebra	5.2	9.6*	4.9	2.1 - 9.4
Incomplete ossification of sternebra; unchanged cartilage	73.7	92.4**	78.5	69.3 - 89.1
Misshapen sternebra; unchanged cartilage	29.6	52.8**	25.4	11.4 - 41.8
Supernumerary rib (14^th); cartilage not present	37.5	54.6*	54.4	43.7 - 64.9
Wavy rib	3.1	20.7**	3.6	0.0 - 10.5

Reference 2

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

22 January 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 440/2008

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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
- 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

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.

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 and by the reproduction/ developmental toxicity screening test (OECD 422) in 2013.
In the first 14-d range finding study, 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 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]. Thus, 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.

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:

Yes, data attached as background material

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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. This was considered treatment-related, but not adverse. 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.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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 mid and 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. Thus, theses effects were considered treatment-related, but not adverse. 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.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

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. This was considered treatment-related, but not adverse.
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.
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.

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Description (incidence and severity):

(Not part of the test guideline version) No thyroid glands findings during necropsy: gross pathology.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

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.

Gross pathological findings:

no effects observed

Description (incidence and severity):

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.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

see tables 1 - 3 in "Any other information on results incl. tables".
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.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

(cesarean section)

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

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 .

Details on maternal toxic effects:

see tables 4 - 6 in "Any other information on results incl. tables".
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.

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.

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Furthermore, 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.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Description (incidence and severity):

(not part of the test guideline version)

Changes in postnatal survival:

not examined

Description (incidence and severity):

(cesarean section)

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One fetus, each, in test groups 0, 1 and 3 (0, 10 and 90 mg/kg bw/d) had external malformations. No external variations were recorded. 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). The findings was not considered as biologically relevant, did not differ significantly from the concurrent control group and was also seen in the historical control data.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

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. 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. 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.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No soft tissue malformations were recorded. 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. No unclassified soft tissue observations were recorded.

Details on embryotoxic / teratogenic effects:

Tables with all fetal malformations / variations are attached as background material.

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.

Abnormalities:

no effects observed

Developmental effects observed:

no

*: p <= 0.05, **: p <= 0.01

HCD: Historical control data

 

Table 1: Mean maternal body weights during gestation [g]

					in % to control
Study day	0 mg/kg	10 mg/kg	30 mg/kg	90 mg/kg	10 mg/kg	30 mg/kg	90 mg/kg
0	165.5	163.1	165.4	165.9	98.5%	99.9%	100.2%
1	178.2	175.2	177.6	177.8	98.3%	99.7%	99.8%
3	186.2	183.8	186.2	187.8	98.7%	100.0%	100.9%
6	196.2	194.2	196.0	197.0	99.0%	99.9%	100.4%
8	203.2	201.3	200.9	198.3	99.1%	98.9%	97.6%
10	213.6	211.8	211.8	209.6	99.2%	99.2%	98.1%
13	229.9	227.6	228.5	228.2	99.0%	99.4%	99.3%
15	240.9	238.9	239.7	239.6	99.2%	99.5%	99.5%
17	257.1	254.6	255.9	256.4	99.0%	99.5%	99.7%
19	280.0	277.4	279.8	281.0	99.1%	99.9%	100.4%
20	293.2	290.9	294.1	294.3	99.2%	100.3%	100.4%

 

Table 2: Mean maternal food consumption [g]

					in % to control
Study day	0 mg/kg	10 mg/kg	30 mg/kg	90 mg/kg	10 mg/kg	30 mg/kg	90 mg/kg
0-1	15.3	14.8	14.6	15.1	96.7%	98.6%	103.4%
1-3	17.0	16.9	16.7	17.4	99.4%	98.2%	102.4%
3-6	18.9	19.5	19.3	19.7	103.2%	102.1%	104.2%
6-8	18.7	18.6	16.9**	13.8**	99.5%	90.4%	73.8%
8-10	19.3	19.1	17.8*	16.7**	99.0%	92.2%	86.5%
10-13	20.2	20.5	20.0	19.7	101.5%	99.0%	97.5%
13-15	21.2	20.9	20.6	20.9	98.6%	97.2%	98.6%
15-17	21.7	21.9	22.5	22.8	100.9%	103.7%	105.1%
17-19	22.4	22.7	23.2	23.2	101.3%	103.6%	103.6%
19-20	20.9	21.6	21.7	22.0	103.3%	103.8%	105.3%

 

Table 3: Mean gravid uterine weights and net maternal BW change [g]

					in % to control
	0 mg/kg	10 mg/kg	30 mg/kg	90 mg/kg	10 mg/kg	30 mg/kg	90 mg/kg
gravid uterus	57.1 +/- 14.0	56.8 +/- 11.0	58.1 +/- 8.4	58.7 +/- 11.8	99.5%	101.8%	102.8%
carcass (terminal BW minus uterine weight)	236.1 +/- 12.6	234.0 +/- 10.5	236.0 +/- 10.6	235.6 +/- 13.6	99.1%	100.0%	99.8%
net weight change from GD6 (carcass weight minus BW at GD6)	39.9 +/- 6.9	39.8 +/-5.9	40.1 +/- 6.6	38.6 +/- 7.3	99.7%	100.6%	96.7%

 

Table 4: Summary of reproduction data

	0 mg/kg	10 mg/kg	30 mg/kg	90 mg/kg	HCD	HCD: min - max
Females mated [n]	25	25	25	25	850
- Pregnant [n]	25	25	25	23	835
- conception rate [%]	100	100	100	92	98
- Aborted	0	0	0	0	0
- Premature births	0	0	0	0	0
- Dams with viable fetuses	25	25	25	23	831
- Dams with all resorptions	0	0	0	0	4
Female mortality [n]	0	0	0	0	0
Pregnant at terminla sacrifice [n]	25	25	25	23	835
Corpora lutea [mean +/- SD]	12.0 +/- 1.9	11.4 +/- 1.4	11.5 +/- 1.8	11.7 +/- 1.6		10.5 - 12.3
- Total [n]	300	284	287	269
Implanation sites [mean +/- SD]	11.1 +/- 2.6	10.9 +/- 1.6	10.9 +/- 1.5	11.0 +/- 2.2		9.9 - 11.8
- Total [n]	277	273	273	252
Preimplantation loss [mean% +/- SD]	8.1 +/- 16.9	3.9 +/- 5.6	4.5 +/- 6.1	6.5 +/- 14.9		1.4 - 13.3
Postimplantation loss [mean% +/- SD]	8.7 +/- 11.8	9.5 +/- 14.7	5.2 +/- 6.9	4.5 +/- 5.7		3.2 - 14.0
Resorptions, total [mean/litter]	0.8 +/- 0.9	1.0 +/- 1.5	0.6 +/- 0.7	0.5 +/- 0.7		0.3 - 1.5
- Total [n]	20	26	14	12
- [mean% +/- SD]	5.7 +/- 11.8	9.5 +/- 14.7	5.2 +/- 6.9	4.6 +/- 5.7		3.2 - 14.0
Resorptions, early [mean/litter]	0.8 +/- 0.9	1.0 +/- 1.5	0.5 +/- 0.7	0.5 +/- 0.7
- Total [n]	20	26	13	12
- [mean% +/- SD]	8.7 +/- 11.8	9.5 +/- 14.7	4.9 +/- 6.9	4.6 +/- 5.7
Resorptions, late [mean/litter]	0	0	0 +/- 0.2	0
- Total [n]	0	0	1	0
- [mean% +/- SD]	0	0	0.3 +/- 1.7	0
Dead fetuses [n]	0	0	0	0		0
Dams with viable fetuses [n]	25	25	25	23
Live fetuses [mean +/- SD]	10.3 +/- 2.7	9.9 +/- 2.1	10.4 +/- 1.6	10.4 +/- 2.1		9.3 - 11.2
- Females [mean +/- SD]	4.9 +/- 2.0	5.3 +/- 1.9	5.1 +/- 1.6	5.0 +/- 2.0
- Males [mean +/- SD]	5.4 +/- 1.8	4.6 +/- 1.9	5.2 +/- 1.5	5.4 +/- 2.4
- Per cent live females	47.9	53.8	49.4	47.9
- Per cent live males	52.1	46.2	50.6	52.1

 

Table 5: Placental and fetal body weights (mean +/- SD) [g]

	0 mg/kg	10 mg/kg	30 mg/kg	90 mg/kg	HCD: mean%	HCD: min - max
Placental weight	0.47 +/- 0.11	0.46 +/- 0.04	0.45 +/- 0.03	0.5 +/- 0.09	0.47	0.35 - 1.13
Fetal weight	3.7 +/- 0.33	3.9 +/- 0.24	3.8 +/- 0.19	3.7 +/- 0.20	3.5	2.5 - 5.1

 

Table 6: Incidences of fetal external, skeletal and visceral findings (malformations, variations and other observations) [mean% affected fetuses/litter +/- SD]

	0 mg/kg	10 mg/kg	30 mg/kg	90 mg/kg	HCD: mean%	HCD: min - max
total fetal external malformations	0.3 +/- 1.5	0.3 +/- 1.5	0	0.4 +/- 1.9	0.2	0 - 0.9
total fetal external variations	0	0	0	0	0.1	0 - 0.7
total fetal external unclassified findings	0	0	0	0.9 +/- 2.9	0.1	0 - 0.6
total fetal soft tissue malformations	0	0	0	0	0.3	0 - 2.1
total fetal soft tissue variations	0.8 +/- 4.1	4.7* +/- 10.5	3.9 +/- 8.2	5.5* +/- 9.9	3.1	0 - 6.9
total fetal soft tissue unclassified findings	0	0	0	0	0.1	0 - 1.3
total fetal skeletal malformations	0.5 +/- 2.5	2.7 +/- 10.4	2.1 +/- 5.9	0.7 +/- 3.5	0.8	0 - 3.0
total fetal skeletal variations	96.7 +/- 8.0	98.6 +/- 4.8	96.4 +/- 8.8	98.7 +/- 4.5	98.7	94.3 - 100.0
total fetal skeletal cartilage	70.7 +/- 34.1	78.4 +/- 25.4	74.0 +/- 27.3	66.7 +/- 23.1	74.4	55.3 - 90.7
total fetal malformations	0.3 +/- 1.5	1.7 +/- 6.8	1.1 +/- 3.0	0.4 +/- 1.9	0.62	0 - 1.66
total fetal variations	53.6 +/- 10.4	54.7 +/- 6.8	53.0 +/- 6.8	54.7 +/- 5.5	53.58	51.16 - 57.01

 

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

90 mg/kg bw/day

Species:

rat

Quality of whole database:

studies according to the respective OECD test guidelines

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

1-Methylimidazole, CAS 616-47-7 (1METI) was tested for its prenatal developmental toxicity (OECD 414) in Wistar rats (2016). The test substance was administered as an aqueous preparation to groups of 25 time-mated female Wistar rats by gavage at doses of 0, 10, 30 and 90 mg/kg bw/d on GD6-19.

The state of health, food consumption and body weights of the animals were recorded regularly throughout the study period. On GD20, all females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology (including weight determinations of the unopened uterus and the placentae). For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings.

No test substance-related adverse effects on dams, gestational parameters or fetuses in all test groups were observed in this prenatal developmental toxicity study, 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.

 

In a supplementary OECD414 study (2021) requested by the local enforcement authority the additional high dose was selected to be 230 mg/kg bw/d, based on the results of two range finding studies and 25 time-mated female Wistar rats were gavaged on GD6-19. At terminal sacrifice on GD20, all dams had implantation sites. Analyses confirmed the correctness of the prepared concentration and the stability of the test substance in the vehicle.
The state of health, food consumption and body weights of the animals were recorded regularly throughout the study period. Additionally, water consumption was recorded throughout the study and on GD20, blood samples were obtained from all females. After sacrifice all females were assessed by gross pathology; including sampling of adrenal glands, kidneys, stomach (forestomach and glandular stomach) and thyroid glands (with parathyroid glands) and weight determinations of the adrenal glands, kidneys and thyroid glands (with parathyroid glands), unopened uterus and placentas. For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Anogenital distance measurements were conducted on all liveborn fetuses. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings.
RESULTS: Regarding clinical examinations, females of the treatment group showed signs of maternal toxicity: polyuria towards the end of the treatment period in some treated dams, which correlated to a distinctly increased water consumption of the affected females. During treatment, these dams showed a reduction in mean food consumption (average 20% below control) and a decrease in body weight change (average 15% below control). These effects were most distinct at the beginning of treatment with up to 62% reduction of food consumption and a body weight loss of 15.5 g on GD6-8. Corrected body weight gain and carcass weight of treated dams was distinctly and statistically significantly decreased (60% and 6% below control, respectively).
Regarding clinical pathology, decreased RBC counts, hemoglobin and hematocrit values in dams of the treatment group indicated an anemia. Lower MCHC and normal MCV characterized the anemia as hypochromic but normocytic. Increased absolute reticulocyte counts were a sign of a regenerative anemia. Lower protein levels which includes albumin as well as globulins may be a sign of a changed protein synthesis by liver cells. TSH values were significantly increased, however, even the control was slightly above the HCR. T3 and T4 levels in the serum were not changed. No changes in the absolute or relative weights of the thyroid glands were observed, and histopathology of the thyroid glands revealed only a minimal hypertrophy/hyperplasia of the follicular cells in some treated dams. This finding was considered treatment-related and in combination with increased TSH values assessed as adverse. However, the rather minor effect is considered secondary as it indicates a compensation of the accelerated thyroid hormone excretion via the bile by an increased hormone production in the thyroid gland.

Regarding pathology, a significant decrease of the terminal body weight (-6%) was observed in treated dams, however, within the HCR. Furthermore, statistically significant absolute and relative weight increases above historical control values were noted in the adrenal glands (+28% and +37%, respectively), correlating with minimal hypertrophy/hyperplasia in the zona fasciculata in some dams. These changes were regarded as treatment-related but not as adverse. Statistically significant absolute and relative weight increases in the liver (+13% and +21%, respectively above historical control values) correlated with glycogen storage, which was increased in incidence and grading, when compared with the controls. Additionally, minimal to slight extramedullary hematopoiesis was observed in the liver of 19 out of 25 treated dams. All these findings in the liver occurred in combination with altered liver parameters in the clinical chemistry and with parameters of anemia in the hematology. Therefore, all these changes in the liver were regarded as treatment-related and adverse. In the spleen, treated dams showed an increased extramedullary hematopoiesis when compared to the control dams. In combination with the mentioned signs of anemia these changes were considered as treatment-related and adverse. The significant absolute and relative weight increases of the kidneys (+10% and +18%) were within the historical control range and had no histopathological correlate. Since a possible treatment-related effect cannot be excluded, this change was regarded as possibly treatment-related but not adverse.

No statistically significant differences between the control and the treatment group were determined for most reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre-implantation loss and the number of viable fetuses. In the treatment group, the total number of resorptions was statistically significantly higher than in the concurrent control (mean 2.0* [p ≤ 0.05] vs. 1.0 in control) and outside the historical control range (mean 0.8 [0.5 - 1.5]). Correspondingly, the post-implantation loss (litter-based mean% of resorptions per group) was higher in the treated animals (15.9 vs. 9.4 mean% in controls), though non-significantly but outside the historical control range (4.1 – 11.9 mean%). However, the high number of implantation sites above the HCR, also seen in the rather low number of pre-implantation losses, might also have attributed to the higher post-implantation loss. This effect was mainly driven by high resorption rates in dams No. 39 and 41, which showed even more severe maternal toxicity than the whole treatment group (e.g. glandular stomach focus, net body weight change from d6, red blood cell parameters), as well as by low resorption rates of a higher number of affected females compared to the control group and included also a considerable number of late resorptions. A relationship of the higher resorption rate to the treatment cannot be fully excluded, but this had no consequences for the average number of viable fetuses compared to the control.
No toxicologically relevant influence of the test substance on sex distribution, body weight and anogenital distance/index of the fetuses was noted at the tested dose. In the treated group significantly higher total malformation rates (external, soft tissue and skeletal combined; 3.2%* [p ≤ 0.05] vs. 0.6% in control) and total variation rates (external, soft tissue and skeletal combined; 58.8%** [p ≤ 0.01] vs. 54.5% in control) were noted which were mainly driven by high incidences of skeletal findings, related to skull, cervical and thoracic vertebrae, forelimbs, ribs and sternum. Total incidences of skeletal malformations were not significantly increased and comparable to the HCD. The overall incidence of skeletal variations and several incidences of individual findings were significantly increased compared to the control and/or exceeded the HCR. Many individual findings represented minor changes, indicating delays of development or disturbances of ossification. These are accepted to depend on the maternal status; e.g. decreased food consumption and body weight and are considered not to persist, which is supported by the presence of normal cartilage. “Wavy ribs” are a common finding in rodent studies, are not classified as true malformations and rather often occur along with maternal toxicity (Carney & Kimmel, 2007; Daston & Seed, 2007). Furthermore, the significantly increased number of other fetal skeletal variations, e.g. “supraoccipital hole(s)”, “supernumerary thoracic vertebra”, was only slightly above the HCR. The number of “supernumerary rib (14th), cartilage not present” is increased compared to the control, but within the HCR, whereas the control value is below the HCR. There was no significant increase of external observations. The visceral malformation (misshapen heart) which was not found in the HCD, was found in two fetuses of one litter. The respective dam no. 41 showed even more severe maternal toxicity than the whole treatment group (net body weight change from d6, red blood cell parameters).

Maternal anemia resulting in adverse developmental effects are described for several species (Kalisch-Smith et al, 2021, Clark et al., 1984, Shepard et al, 1980; see also ECETOC TR No. 138, 2021).

Thus, the increased malformation and variation incidences are maybe indicative for a potential of the test item to impair embryofetal development, when tested at a high, maternally toxic dose (230 mg/kg bw/d).

 

Supportingly, in a GLP compliant combined oral repeated dose toxicity study and reproduction/developmental toxicity screening test according to OECD 422 (2013), no effects on pup body weights or pathology at necropsy were observed up to 90 mg/kg bw/d, the highest dose tested.  Further details concerning this study can be found in the IUCLID section 7.8.1 Toxicity to reproduction.

 

In conclusion, the NOAEL for maternal toxicity is 90 mg/kg bw/d, based on the evidence of maternal toxicity, such as a reduction in food consumption, a decrease in (corrected) body weight gain, changes in clinical pathology parameters (e.g. a hypochromic, normocytic anemia, changed liver cell protein synthesis and accelerated thyroid hormone excretion) and corresponding histopathological findings in liver, spleen and thyroid at the higher dose level of 230 mg/kg bw/d.
The NOAEL for prenatal developmental toxicity is also 90 mg/kg bw/d, based on increased intrauterine mortality as well as increased malformation and variation rates, indicative of an impaired embryofetal development, which
were noted at the distinctly maternally toxic dose of 230 mg/kg bw/d. The pattern of fetal findings was not indicative of a potential specific teratogenic action of 1METI.

Mode of Action Analysis / Human Relevance Framework

No further details.

Justification for classification or non-classification

1METI showed no adverse effects on sexual function and fertility in the respective studies (OECD422 in 2013; OECD408 in 2016). However, in the most recent OECD414 study oral administration of 1METI to pregnant Wistar rats from GD6-19 provided evidence of maternal toxicity at 230 mg/kg bw/d as well as an impaired embryofetal development, which was noted at this distinctly maternally toxic dose. Thus, 1METI is classified as a suspected human reproductive toxicant (Repr. Cat. 2; H361d)

According to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures the classification criteria for reproductive toxicity category 2 is the following (CLP, Annex I: 3.7.2.1.): “Substances are classified in Category 2 for reproductive toxicity when there is some evidence from humans or experimental animals, possibly supplemented with other information, of an adverse effect on sexual function and fertility, or on development, and where the evidence is not sufficiently convincing to place the substance in Category 1. If deficiencies in the study make the quality of evidence less convincing, Category 2 could be the more appropriate classification. Such effects shall have been observed in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of the other toxic effects.”
Thus, data providing a clear evidence of an adverse effect in animal studies might justify a classification in Category 1B. Adverse effects, that are considered secondary to a non-specific other effect, might not warrant a classification for reproductive toxicity.
According to CLP Annex I: 3.7.2.4.2. "...maternal toxicity may, depending on severity, influence development via non-specific secondary mechanisms, producing effects such as depressed foetal weight, retarded ossification, and possibly resorptions and certain malformations in some strains of certain species. However, the limited number of studies which have investigated the relationship between developmental effects and general maternal toxicity have failed to demonstrate a consistent, reproducible relationship across species. ..."

The increased intrauterine mortality as well as increased malformation and variation rates, observed in the prenatal developmental toxicity study (2021), are indicative of an impaired embryofetal development, which is observed only at this distinctly maternally toxic dose of 230 mg/kg bw/d. The NOAEL for prenatal developmental and maternal toxicity is 90 mg/kg bw/d, derived from the former prenatal developmental study (2016).
Besides the distinct maternal toxicity, it must be noted the number of implantation sites of the treatment group was above the historical control range, which could have contributed to the increased intrauterine mortality (resorption rate and post-implantation loss). Furthermore, the increased malformation and variation rates were mainly driven by high incidences of skeletal findings. Many individual findings represented minor changes, indicating delays of development or disturbances of ossification, which can be linked to maternal toxicity. Furthermore, the significantly increased number of fetal skeletal variations was only slightly above the historical control range, which is supported by a comparison to the historical control range of the former prenatal developmental toxicity study in 2016. There was no significant increase of skeletal malformations and the findings were comparable to the HCD. The visceral malformation (misshapen heart) was found in two fetuses of one litter. The respective dam showed even more signs of maternal toxicity compared to the whole treatment group. Overall, the pattern of fetal findings was not indicative of a potential specific teratogenic action of the test compound.
In conclusion, the data indicate some but no clear evidence of an adverse effect on developmental toxicity at a high, maternally toxic dose and 1-methylimidazole is classified as a suspected human reproductive toxicant according to the CLP: Repr. 2; H361d: Suspected of damaging the unborn child.