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Diss Factsheets

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 Aug - 10 Nov 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 2018
Deviations:
no
Principles of method if other than guideline:
The females of each group were divided in one of 4 subgroups, depending on the Day of coitum, and dosed accordingly. Based on 14/21 early deliveries in Subgroup 2, along with high fetal weights for this subgroup, and low fetal weights for Subgroup 1, it is suspected that Subgroups 1 and 2 may have been switched after mating.
This would indicate that animals of Subgroup 1 (Female Nos. 1-6 (control), 23-27 (100 mg/kg bw/day), 45-49 (300 mg/kg bw/day), 67-72 (1000 mg/kg bw/day)) were dosed on Days 5-19 post-coitum and sacrificed on Day 20 post-coitum, and animals of Subgroup 2 (Female Nos. 7-11 (control), 28-33 (100 mg/kg bw/day), 50-55 (300 mg/kg bw/day), 73-77 (1000 mg/kg bw/day)) were dosed on Days 7-21 post-coitum and sacrificed on Day 22 post-coitum. Subgroups 3 and 4 were dosed according to schedule (i.e., dosing on Days 6-20 post-coitum with euthanasia on Day 21 post-coitum). Full evaluation of the data was still possible, as Subgroups 3 and 4 were dosed according to schedule and sufficient data was available, all dose groups were evenly divided over the different subgroups, data of applicable parameters were evaluated separately for the different subgroups, if deemed necessary and possible test item-related trends were consistent between the different subgroups. Furthermore, for the majority of animals, the critical period of organogenesis was completely included in the dosing period the suspected slight shift in dosing period for Subgroups 1 and 2 (i.e., one day earlier and one day later, respectively) had no impact on the overall conclusion. It was therefore concluded that the overall integrity of the study and the interpretation of the study results and conclusions was not compromised.
Humidity was outside the target range on 4 days (maximum of 80%) without a noticeable effect on the clinical condition of the animals.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Alcohols, C12-14, ethoxylated
EC Number:
500-213-3
EC Name:
Alcohols, C12-14, ethoxylated
Cas Number:
68439-50-9
Molecular formula:
n.a.
IUPAC Name:
Alcohols, C12-14, ethoxylated, < 2.5 EO

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:Wl(Han)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 11 - 15 weeks
- Weight at study initiation: 179 - 267 g
- Fasting period before study: not applicable
- Housing: individually in polycarbonate cages (Makrolon type MIII, height 18 cm) containing sterilized
wooden fibers as bedding material (Lignocel S 8-15, JRS-J.Rettenmaier & Söhne GmbH + CO. KG,
Rosenberg, Germany)
- Diet: SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany, ad libitum
- Water: municipal tap water, ad libitum
- Acclimation period: 5 - 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 57 - 80
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 Aug 2021 To: 10 Sep 2021

Administration / exposure

Route of administration:
oral: feed
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The appropriate amount of the test material was mixed with the vehicle. The dose volume for each animal was based on the most recent body weight me asurement and the dose formulations were stirred continuously during dosing.

VEHICLE
- Concentration in vehicle: 25 mg/mL (for 100 mg/kg bw/day group), 75 mg/mL (for 300 mg/kg bw/day group), 250 mg/mL (for 1000 mg/kg bw/day group),
- Amount of vehicle (if gavage): 4 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analyses of formulations were conducted in Week 1 and 2 to assess accuracy and homogeneity.

The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%).
A small response at the retention time of the ammonium adduct of the C12E6 compound was observed in one of the chromatograms of the Group 1 formulation prepared for use in Week 1. It was considered not to derive from the formulation since the response was not observed in the duplicate study sample. In the other formulation of Group 1, no test item was detected.

The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).
Details on mating procedure:
- Impregnation procedure: not reported
Females were time-mated and arrived at the testing facility as such. Day 0 of gestation is the day of mating and is referred to as Day 0 post-coitum.
Duration of treatment / exposure:
Days 6 - 20 post-coitum
Frequency of treatment:
once daily, 7 days/week
Duration of test:
until necropsy on Day 21 post-coitum
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
Group 4
No. of animals per sex per dose:
22 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels in this study were selected to be 100, 300, 1000 mg/kg bw/day, in consultation with the Sponsor and based on results of a Combined 28-day repeated dose toxicity study with Reproduction/Developmental Toxicity Screening Test with oral exposure of the test substance in rats (Test Facility Study No. 20218715).
- Fasting period before blood sampling for (rat) dam thyroid hormones: no
- Time of day for (rat) dam blood sampling: Sampled between 07.00 and 09.00 from the jugular vein.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily; starting on Day 6 post-coitum up to the day prior to necropsy performed directly post dosing. Mortality and Morbidity were checked twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On Days 2, 6, 15 and 21 post-coitum

BODY WEIGHT: Yes
- Time schedule for examinations: On Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: Over Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post coitum.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: on a regular basis throughout the study (monitored by visual inspection)

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 21 post-coitum
All animals from all groups were subjected to a gross necropsy. All gross lesions were collected.
- Organs weighed: thyroid
- Tissues collected for histopathology: thyroid gland and macroscopic abnormalities.
- Fixative: 10% buffered formalin
- Embedding media: paraffin
- Thickness of sections: 2 - 4 μm
- Staining: hematoxylin and eosin
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
- Number of live and dead fetuses: Yes
Blood sampling:
- Plasma: Yes
- Serum: Yes
- Volume collected: 1.0 mL
- Parameters checked: triiodothyronine (T3), thyroxine (T4) and thyroid-stimulating hormone (TSH).
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
- Anogenital distance of all live rodent pups: Yes (all per litter)
- Body weight: Yes, all per litter
- Sex: Yes, all per litter
Statistics:
Means, standard deviations (or % coefficient of variation or standard error, when deemed appropriate), percentages, numbers, and/or incidences are reported as appropriate by dataset. All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels, unless otherwise noted. All pairwise comparis ons were conducted against the control group (Group 1).

The following statistical tests were used: Levene’s test, ANOVA F-test, Kruskal-Wallis Dunnett’s and Dunn’s test, ANCOVA and Fisher's exact test.
Indices:
Pregnancy rate (%): (No. of pregnant females)/(No. of mated females) * 100
Male fetuses (%): (No. of male fetuses)/(No. of fetuses) * 100
Female fetuses (%): (No. of female fetuses)/(No. of fetuses) * 100
Pre-implantation loss (%): (No. of corpora lutea – No. of implantations)/(No. of corpora lutea) * 100
Post-implantation loss (%): (No. of implantations – No. of live fetuses)/(No. of implantations) * 100
Litter % of fetuses with abnormalities: (No. of fetuses in litter with a given finding)/(No. of fetuses in litter examined) * 100
Historical control data:
Historical control data regarding embryo-fetal development was provided and can be found in Attachment 3 under "Overall Remarks, Attachments".

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In the 100 mg/kg bw/day group, the only clinical sign observed was hunched posture in 1/22 female in the days pre-dosing (Day 2 post coitum). It was therefore not considered treatment-related.

Hunched posture was observed for 2/22 females of the 1000 mg/kg bw/day group on some occasions between Days 7-21 post-coitum and for 1/22 females of the 300 mg/kg bw/day group between Days 9-18 post-coitum. In the high dose group, erected fur was noted for 4/22 females on several occasions and 1/22 female had abnormal breathing sounds on Day 16 post-coitum. These signs were considered treatment-related but not adverse.

Salivation was seen on several occasions in 9/22 and 5/22 females of the 1000 and 300 mg/kg bw/day group, respectively. This was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e., after dosing).

Summarized results can be found in Attachment 2 under "Overall Remarks, Attachments".
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The differences in body weight have been evaluated based on the different subgroups.
At 100 mg/kg bw/day, mean body weights, body weight gain and weight gain corrected for gravid uterus were comparable to control. At 300 mg/kg bw/day, body weight gain and body weight gain corrected for gravid uterus weight was slightly lower in Subgroup 1 only (21 vs 38 g in concurrent controls, the difference in adjusted body weight gain reached statistical significance).
In the 1000 mg/kg bw/day group, all subgroups showed toxicologically relevant differences to the control. Body weight gain for Subgroups 3+4 was lower throughout the dosing period, with a total mean body weight gain of 37% vs 48% in concurrent control. A similar effect on body weight gain was observed in Subgroup 1 (35% vs 47% in the control) and in Subgroup 2 (22% vs 37% in concurrent control). It has to be noted that for Subgroup 2, the body weight of Day 18 post-coitum was used as the last time point for evaluation as several animals of this subgroup delivered early.
Terminal body weight was 11%, 7% and 7% lower compared to the control for Subgroup 1, 3 and 4, respectively. In Subgroup 2, mean body weight of animals that delivered early was 4% lower than control at the end of the dosing period. For the two females in Subgroup 2 that did not deliver early, mean body weight was 9% lower than concurrent control on Day 21 post-coitum.
Slight weight loss was observed in several animals of the 1000 mg/kg bw/day group during the study: 1 female of Subgroup 2 (Days 6 - 9 post-coitum), 3 females of subgroup 1 (Days 6 - 9 post-coitum) and 4 females in Subgroup 3+4 (Days 6 - 9 post-coitum).
At 1000 mg/kg bw/day, body weight gain corrected for gravid uterus weight was lower: 15 vs 38 g and 6 vs 26 g in Subgroups 1 and 3+4, respectively compared to concurrent controls. For Subgroup 2, body weight gain corrected for gravid uterus weight could not be evaluated due to the early delivery of several females of this subgroup.

Summarized results can be found in Attachment 2 under "Overall Remarks, Attachments".
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food intake at 100 mg/kg bw/day was considered comparable to control in all subgroups.
At 300 mg/kg bw/day, food intake for Subgroup 1 animals was reduced during the complete dosing period. The mean overall food intake was -11% compared to the control, but the difference did not reach statistical significance. Food intake in the remaining subgroups at 300 mg/kg bw/day was considered comparable to control. Because of the sporadic finding in only one subgroup and the minor difference, this finding was not considered adverse at 300 mg/kg bw/day.
In the high dose group, food consumption was reduced in all subgroups during the complete dosing period. For most of the intervals in Subgroup 1 and 3+4, these differences reached statistical significance. In Subgroup 2, food consumption was also decreased in all intervals with the exception of Day 18 - 21 post coitum, but none of the differences reached statistical significance. For Subgroups 1, 2 and 3+4, mean overall food intake was 24, 11 and 17% lower than concurrent controls, respectively. For Subgroup 1 and 3+4, this difference was statistically significant.

Summarized results can be found in Attachment 2 under "Overall Remarks, Attachments".
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No difference was observed regarding water consumption between the control and treatment groups up to and including the highest dose level.

Ophthalmological findings:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
not examined
Description (incidence and severity):
not applicable
Clinical biochemistry findings:
not examined
Description (incidence and severity):
not applicble
Endocrine findings:
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant difference was observed regarding thyroid hormones between the control and 100 mg/kg bw/day group.

At 300 and 1000 mg/kg bw/day, total T3 concentration was decreased (0.86x (0.388 ng/mL) and 0.79x (0.356 ng/mL) of control (0.451 ng/mL), respectively) and TSH levels were increased (1.31x (0.3421 mU/L) and 1.66x (0.4347 mU/L) of control (0.2619 mU/L), respectively). The difference in TSH level in the 300 mg/kg bw/day group did not reach statistical significance. However, all mean values were within the available historical control data range, therefore, they were considered non-adverse.

The historical control data ranges were (2020 - 2021):
T3 (ng/mL) mean: 0.439; P5 - P95: 0.280 – 0.616 (n = 263)
TSH (mU/L) mean: 0.353; P5 - P95: 0.127 – 0.699 (n = 373)

Summarized results can be found in Attachment 2 under "Overall Remarks, Attachments".
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No toxicologically relevant difference was observed regarding thyroid weights between the control and treatment groups up to and including the highest dose group.

In the 1000 mg/kg bw/day group, thyroid weights were decreased compared to the control group (-16%), but this change was considered within biological variation as it was small and no associated histopathological findings were noted.

Summarized results can be found in Attachment 2 under "Overall Remarks, Attachments".
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant difference was observed regarding gross pathology between the control and treatment groups up to and including the highest dose group.

A pale discoloration of the liver was noted in 3 females (2/22 of the 300 mg/kg bw/day group, 1/22 of the 1000 mg/kg bw/day group). The liver of the female of the high dose group was also enlarged. 1/22 females of the high-dose group had a a thick, non-glandular stomach and 1/22 females in the 300 mg/kg bw/day group had enlarged (right) and smaller (left) thyroid gland. Given the incidental nature of these findings, no association with histopathology and/or the absence of a dose-related response, these findings were considered unrelated to treatment with the test item.

Summarized results can be found in Attachment 2 under "Overall Remarks, Attachments".
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic observations in the thyroid glands.

Summarized results can be found in Attachment 2 under "Overall Remarks, Attachments".
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Other effects:
not examined
Description (incidence and severity):
not applicable

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
No abortion occurred in this study.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No toxicologically relevant difference was observed regarding pre- and post-implantation loss between the control and treatment groups up to and including the highest dose group.

Pre-implantation loss was slightly higher for females at 1000 mg/kg bw/day group compared to the control group (10.38% vs 7.44% in control). However, as the difference was not statistically significant, mean values remained within the available historical control data (mean: 6.5; P5 - P95: 2.2 – 11.8 (n = 1295) in a period from 2016 to 2020) and individual values were within the concurrent control range, this was considered not related to treatment with the test item.

Summarized results can be found in Attachment 2 under "Overall Remarks, Attachments".
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no total litter losses by resorption in the control or treatment groups up to and including the highest dose group.

Summarized results can be found in Attachment 2 under "Overall Remarks, Attachments".
Early or late resorptions:
no effects observed
Description (incidence and severity):
No difference was observed regarding resorptions between the control and treatment groups up to and including the highest dose group.

Summarized results can be found in Attachment 2 under "Overall Remarks, Attachments".
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead fetuses were observed in any of the groups.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
No toxicologically relevant difference was observed between the control and treatment groups up to and including the highest dose level.

3 control females (Nos. 7, 8, 11), 5 females at 100 mg/kg bw/day (Nos. 29, 30, 31, 32, 33), 4 females at 300 mg/kg bw/day (Nos. 50, 51, 53, 54) and 2 females at 1000 mg/kg bw/day (Nos. 74, 76), all in Subgroup 2, delivered their litter on the day of scheduled necropsy. These females were examined according to scheduled necropsy and included in the evaluation. These early deliveries are not considered treatment-related but caused by the unintentional switch of subgroups, as they occurred in Subgroup 2 only and across all groups, including the control.

Summarized results can be found in Attachment 2 under "Overall Remarks, Attachments".
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
No statistically significant difference was observed regarding the number of pregnant animals between the control and treatment groups up to and including the highest dose group. The numbers of females with viable litters for evaluation were 21, 21, 21 and 22 in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

Summarized results can be found in Attachment 2 under "Overall Remarks, Attachments".
Other effects:
not examined
Description (incidence and severity):
not applicable

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No adverse effects were observed at this dose level.
Key result
Dose descriptor:
LOAEL
Remarks:
systemic toxicity
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 100 mg/kg bw/day mean fetal body weight was within the same range as concurrent controls for all subgroups.
At 300 mg/kg bw/day, mean fetal body weight (male, female and combined) in Subgroup 1 was slightly lower (4% lower than control for combined weights), but the difference did not reached statistical significance and was therefore not considered adverse.
At 1000 mg/kg bw/day, the mean fetal body weights was compared according to subgroup. In Subgroup 1 and 3+4 (male, female and combined) mean fetal body weights were lower compared to the control group (combined weights -8% and -4% in Subgroup 1 and 3+4, respectively), but the difference did not reach statistical significance. Mean fetal body weights at the high dose in Subgroup 4 were below or at the lower end of the historical control range. For Subgroup 2, mean fetal body weights (male, female and combined) was in the same range as the concurrent control.

It has to be noted that fetal body weight for all dose groups of Subgroup 1 (including control) was below the lower range of the available historical control data, while fetal body weight for all
dose groups of Subgroup 2 (including control) was above the upper range of the available historical control data. This was attributed to the suspected switch of Subgroup 1 and 2; fetuses were likely weighed on Day 20 post-coitum in Subgroup 1 and on Day 22 post-coitum in Subgroup 2. Fetal body weight of control Subgroup 3+4 was within the historical control data (5.21 g for males and 5.08 g for females).

Historical control data for body weight of Wistar Han fetuses (period 2016-2020):
Mean male body weight (g) mean: 5.4; P5 - P95: 5.2 – 5.5 (n = 6774).
Mean female body weight (g) mean: 5.1; P5 - P95: 4.9 – 5.3 (n =7024).
Mean fetal body weight (g) mean: 5.3; P5 - P95: 5.0 – 5.4 (n = 13798).

Summarized results can be found in Attachment 2 under "Overall Remarks, Attachments".
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no differences regarding the number of live fetuses between the control and treatment groups.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was unaffected by treatment with the test item up to
1000 mg/kg bw/day. Mean sex ratios (males:females) were 49:51, 50:50, 57:43 and 53:47 for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

Summarized results can be found in Attachment 2 under "Overall Remarks, Attachments".
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no test item-related effects on litter size of any group.
Mean litter sizes were 11.7, 12.2, 11.8 and 11.2 fetuses/litter for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

Summarized results can be found in Attachment 2 under "Overall Remarks, Attachments".
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
The mean anogenital distance (AGD) in male and female fetuses (absolute and normalized for fetal body weight) was considered comparable to control for all treated groups.

The mean anogenital distance for male fetuses of Subgroup 1 of all dose groups (including control) was below the lower range of the available historical control data (1.447 - 1.514), but within the historical control range for the female fetuses. The mean anogenital distance was within the available historical control data for Subgroups 2 and 3+4 for both sexes.


Historical control data for Anogenital distance of Wistar Han fetuses (period 2016-2020):
Male anogenital distance (mm) mean: 2.8; P5 - P95: 2.6 – 3.3 (n = 6774).
Female anogenital distance (mm) mean: 1.3; P5 - P95: 1.1 – 1.7 (n = 7024).

Summarized results can be found in Attachment 2 under "Overall Remarks, Attachments".
Changes in postnatal survival:
not examined
Description (incidence and severity):
not applicable
External malformations:
no effects observed
Description (incidence and severity):
There were no external malformations and variations observed in any groups.

Summarized results can be found in Attachment 2 under "Overall Remarks, Attachments".
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Three skeletal malformations were noted: the thoracic vertebral column and ribs were affected in one fetus of the 300 mg/kg bw/day and in one fetus of the 1000 mg/kg bw/day group, one fetus of the control group had a supernumerary lumbar vertebra. These cases were considered incidental.

Signs of delays of ossification (unossified metacarpals, sternebrae and hyoid body, incompletely ossified sternebrae, skull bones (e.g., supraoccipital, parietal and frontal), vertebra (cervical and sacral) and pelvic girdles (ischium; statistically significant)) were noted at higher incidences at 1000 mg/kg bw/day, although most were not statistically significant. These changes are variations, not malformations and are not considered adverse, but as a pattern was recognized, it will be described here for completeness. To describe the results, the observations for the 4 subgroups have to be viewed separately.

In Subgroup 1, delays of ossification were particularly shown at 1000 mg/kg bw/day (unossified metacarpals and sternebrae and incompletely ossified vertebral centra and arches) and at 300 mg/kg bw/day (unossified sternebrae) and 100 mg/kg bw/day (unossified metacarpals).

In Subgroup 2, fetal skeletal ossification was more advanced than for the fetuses of subgroup 1, which was associated with the higher fetal weights in this subgroup. No ossification delays were noted.

In Subgroup 3+4, a low incidence of incompletely ossified skull bones (e.g., interparietal and parietals) was observed without any test item-relationship between the groups. This implies a more advanced ossification of the fetuses than observed in the fetuses of Subgroup 1, but a poorer ossification when compared to fetuses of Subgroup 2.

In groups treated with the test item, statistically significantly fewer fetuses had wavy ribs when compared to the control group. This is not considered to be treatment-related.

Summarized results can be found in Attachment 2 under "Overall Remarks, Attachments".
Visceral malformations:
no effects observed
Description (incidence and severity):
No toxicologically relevant malformations or variations were observed in any of the control and treatment groups. Only one malformation, which was considered incidental, occurred in one fetus of the 300 mg/kg bw/day group. This fetus had a small eye.

The variations that were noted affected the liver (supernumerary lobes), ureters (convoluted) and urinary bladder (distended) at low incidences or in isolated cases, which did not indicate any test item-relationship.

Summarized results can be found in Attachment 2 under "Overall Remarks, Attachments".

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed up to and including this dose level.
Key result
Dose descriptor:
LOAEL
Remarks:
developmental toxicity
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
The present study was conducted under GLP and according to OECD test guideline 414 (2018). Under the conditions of the study, administration of the test substance once daily by oral gavage for Days 6 - 20 post coitum was well tolerated in pregnant Wistar Han rats at levels up to 300 mg/kg bw/day. The maternal systemic and developmental No Observed Adverse Effect Levels (NOAELs) for the test substance were set at 300 mg/kg bw/day, based on the effects on maternal and fetal body weights. No teratogenicity was observed at any dose level.