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Administrative data

developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
In-life phase 13 October 2020 to 30 January 2021; Study completion 09 July 2021
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
GLP compliance:
yes (incl. QA statement)
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:

Test animals

Crl: WI(Han)
Details on test animals or test system and environmental conditions:
- Source: e.g. Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: 11-12 weeks
- Weight at study initiation:
males: 330 - 400 g
(mean: 362.85 g, ± 20% = 290.28 – 435.43 g)
females: 194 - 259 g
(mean: 222.00 g, ± 20% = 177.60 – 266.40 g)
- Fasting period before study:
- Housing: Full barrier in an air-conditioned room, individually in IVCs (type III H, polysulphone cages) on Altromin saw fibre bedding (except during the pre-mating period when females were kept in groups of two animals and during mating period when two females were paired with one male). During the pre-mating period and after mating, males were housed in groups (up to 5 animals / cage) in type IV cages.
- Diet: ad libitum Altromin 1324 maintenance diet for rats and mice
- Water: ad libitum tap water (bottles provided), sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: at least five days

- Temperature (°C): 22 ± 3°C
- Humidity (%): 55 ± 10%
- Air changes (per hr): 10 per hour
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
corn oil
dried and de-acidified corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item formulation was prepared freshly on each day of administration. The test item was diluted in dried and de-acidified corn oil. Dose volumes were adjusted individually based on weekly body weight measurements.

- Justification for use and choice of vehicle (if other than water): in consultation with the sponsor based on the test item’s characteristics.
- Concentration in vehicle: 0, 6.25, 12.5, 25 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
- Lot/batch no. (if required): MKCK6411
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle. The test item formulations were stable for up to 12 days at room temperature. Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups. As the test item was shown to be homogenous after 30 min without stirring samples were not collected during the study for the investigation of homogeneity. Samples were taken for substance concentration in the first and last week of the study for all doses.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1:2 male:female
- Length of cohabitation: no information
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
from gestation day (GD) 5 to gestation day (GD) 19
Frequency of treatment:
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
control group
Dose / conc.:
10 mg/kg bw/day (actual dose received)
low dose (LD)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
medium dose (MD)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
high dose (HD)
No. of animals per sex per dose:
138 animals (46 males and 92 females)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on dose range-finding study
- Rationale for animal assignment (if not random): animals were weighed and assigned to experimental groups to acheive the most homogeneous variation in body weight throughout groups of males and females (randomisation performed with IDBS workbook)


Maternal examinations:
- Time schedule: morbidity and mortality: twice daily (except evenings and weekends when observed once).

- Time schedule: General clinical observations at least once a day
- Clinical observations include spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes or bizarre behaviour were recorded.

- Time schedule for examinations: All animals were weighed once before initiation of pairing to ensure that the body weights were within ±20% variation. The sperm-positive females were weighed on GDs 0, 5, 8, 11, 14, 17 and 20. Males were not weighed in this study except once before initiation of pairing.

- Food consumption of sperm-positive females was determined for the following intervals: GDs 0-5, 5-8, 8-11, 11-14, 14-17 and 17-20. Food consumption was not measured for males during the entire study or for both males and females during the mating period.

- Sacrifice on gestation day #20
- Organs examined: See Table 1 below

OTHER: Thyroid hormones
- Blood samples were collected prior to sacrifice for thyroid hormone (T3, T4, TSH) analysis.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Alive and dead fetuses: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter, at least 20 litters per group
statistical assessment of the results of the body weight and food consumption was performed by comparing values of dosed animals with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, thyroid hormones and foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a posthoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. The statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.3.4 software (p<0.05 is considered as statistically significant).
Historical control data:
See attached historical control data tables.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no relevant test item-related clinical signs observed in any of the treated groups. None of the females showed signs of abortion or premature delivery prior to the scheduled sacrifice.
Clinical signs observed occasionally during the treatment period of the study included skin and fur, hairless area in one control and one LD group rat (nos. 5, 28, respectively) on GDs 18-19 at thorax; one MD group rat (no. 62) on GDs 17-20 at both forelimbs and pale discoloured faeces was observed in one MD group rat (no. 66) on GDs 16-19. All these findings are considered to be incidental findings.
One MD group rat (no. 69) on GD 19 showed moderate salivation and moving the bedding materials. Moving the bedding was observed immediately after dosing in HD groups in animal nos.74, 76, 78, 81 on GD19; animal no. 84 on GDs 16-19, animal no. 85 on GDs 16-18, animal no. 86 on GD 17, animal no. 87 on GDs 17, 19; animal no. 88 on GD 12, animal no. 89 on GDs 12, 14; animal no. 90 on GDs 12-13, 18-19 and animal no. 91 on GDs 11-13 and 18. Slight salivation was observed in HD group animal nos. 75, 78, 87, 88, 90 on GDs 18, 19, 17-19, 12-15 and 18-19 respectively. Moderate salivation was observed in HD group animal nos. 85 and 86 on GDs 16-19 and 17, respectively. Slight to moderate salivation and moving the bedding materials are considered to be a local reaction to the treatment with test item. These clinical signs were observed on a few days of the treatment period immediately after dosing.
no mortality observed
Body weight and weight changes:
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No statistically significant differences in the absolute and relative (to body weight) thyroid/parathyroid weights of the treated groups when compared to the control.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At histopathologic evaluation, degenerative and inflammatory lesions related to the direct irritative effect of the test item were observed in the stomach of HD group animals and consisted of forestomach and glandular stomach ulcerations, forestomach erosions, squamous hyperplasia, forestomach submucosal oedema hyperkeratosis and mixed cell infiltrates (i.e. inflammation). Further, mural necrosis and intramural haemorrhages were also observed in a few animals. These effects are considered to be a result of the direct irritative effect of the test item on the gastric mucosa.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No test item related histopathological findings were observed in thyroid glands and parathyroid of all females.
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormones: no statistically significant or toxicologically relevant effects were observed on group mean T3, T4 and TSH hormone levels and values were comparable to the control.

Maternal developmental toxicity

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
Description (incidence and severity):
No dead fetuses were observed in any treatment group.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no adverse systemic effects were observed.

Maternal 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
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Description (incidence and severity):
No statistically significant or dose-dependent effects were observed.
Visceral malformations:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
The absolute and relative anogenital distance (AGD) in male and female foetuses and testicular descent in males were unaffected by treatment.

Effect levels (fetuses)

Key result
Dose descriptor:
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no effects observed

Fetal abnormalities

no effects observed

Overall developmental toxicity

Developmental effects observed:

Any other information on results incl. tables

See attached tables of results.

Applicant's summary and conclusion

In the prenatal developmental toxicity study, conducted according to OECD Test Guideline 414 and in compliance with GLP, the NOAEL for maternal and developmental toxicity in rats was concluded to be 50 mg/kg bw/day based on the absence of adverse effects in the high dose group.