1-methylimidazole

Developmental toxicity / teratogenicity

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

1-methylimidazole

EC Number:

210-484-7

EC Name:

1-methylimidazole

Cas Number:

616-47-7

Molecular formula:

C4H6N2

IUPAC Name:

1-methyl-1H-imidazole

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

Test animals

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

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

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.

Examinations

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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.

Maternal developmental toxicity

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.

Effect levels (maternal animals)

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

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

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.

Effect levels (fetuses)

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

Fetal abnormalities

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: skull

skeletal: forelimb

skeletal: sternum

skeletal: rib

skeletal: vertebra

Overall developmental toxicity

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

Any other information on results incl. tables

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