Reaction products of acrylic acid with 2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol]

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 07, 2020 to January 21, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted in 2018

Deviations:

yes

Remarks:

These deviations have no impact of the integrity of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-({2,2-bis[(prop-2-enoyloxy)methyl]butoxy}methyl)-2-[(prop-2-enoyloxy)methyl]butyl prop-2-enoate

EC Number:

830-217-3

Cas Number:

1393932-71-2

Molecular formula:

Not applicable for this UVCB

IUPAC Name:

2-({2,2-bis[(prop-2-enoyloxy)methyl]butoxy}methyl)-2-[(prop-2-enoyloxy)methyl]butyl prop-2-enoate

Test material form:

liquid

Test animals

Species:

rat

Strain:

other: Hannover Wistar rat

Details on test animals or test system and environmental conditions:

- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Sex: females, nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation: 222 - 287 g (the variation did not exceed ± 20% of the mean weight).
- Fasting period before study: no
- Housing: After mating, the sperm-positive females were housed individually

Acclimatization: Animals were housed at least for 5 days
Mating: Females were caged together with stock males for approximately 2 hours on a one-to-one-basis in the morning.

General:
- Housing: SAFE 3/4-S Hygienic Animal Bedding
- Diet: Free access to rodent diet (SM R/M from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: Free access to tap water

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

- Method of formulation: The test substance was formulated fresh at the appropriate concentrations in the selected vehicle (corn oil) in the Pharmacy of the Test Facility on the day of administration. No correction was made for the purity of the test substance.
- Storage conditions: Freshly prepared prior to administration to animals
- Dose volume: A constant volume of 5 mL/kg body weight was administered to all dose groups, including the controls

Administration of Test and Control Items
A constant volume of 5 mL/kg body weight was administered to all dose groups, including the controls. The individual volume of the treatment was based on the most recent individual body weight of the animals. The control or test item dose formulations were administered to mated, sperm-positive assumed pregnant female rats daily by oral gavage on a 7 days/week basis, at approximately similar times with minor variations as practical, from Gestation Day (GD) 6 to Gestation Day (GD) 19.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for test item concentration and homogeneity determination of the dosing formulations were collected two times during the treatment period. The test item was analysed using a validated HPLC-UV method in the Analytical Department of the Test Facility. The analysis showed that the mean concentration of all formulations was found to be in the range of 98-106% of their nominal concentrations (20, 60, and 200 mg/mL) and were found to be homogenous. No test item was detected in the vehicle control formulations.

Details on mating procedure:

The oestrous cycle of female animals was examined a day before start of pairing, and then daily, up to the day of mating. On the first week of the mating, approximately half of the animals was examined daily for their oestrous cycles. After acclimatisation the females, according to their oestrous cycle, were paired with males for approximately 2 hours (1 male: 1 female) in the morning, until at least 24 sperm-positive females/group are obtained. Spermpositive females were separated and caged individually.

Duration of treatment / exposure:

From Days 6 to 19 post-coitum, inclusive.

Frequency of treatment:

Once daily for 7 d/w.

Duration of test:

Administration of test substance was 14 days.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Number of groups: 4 groups; one control and 3 test item-treated groups
Number/sex of animals: 96 female animals, 24 mated female animals/group, 4 groups. A sufficient number of spare animals were ordered. Up to 56 male animals for mating; untreated, proven breeders from Test Facility were used. No study procedures were carried out on
the male animals.

Control animals:

yes, concurrent vehicle

Details on study design:

The test item was administered daily to pregnant animals from implantation (Gestation Day (GD) 6) to one day prior to the day of scheduled kill (Gestation Day (GD) 19), which should be as close as possible to the normal day of delivery without risking loss of data resulting from early delivery. The day of mating (when the sperm-positive vaginal smear and/or the vaginal plug are identified) is regarded as Gestation Day 0 (GD 0). Caesarean section and necropsy with macroscopic examination was performed on Gestation Day (GD) 20. The control group was treated with the vehicle only (corn oil).

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS
- Time schedule: At least twice daily. Only one clinical observation was made in the afternoon on those days when detailed clinical observation was made in the morning. Only one detailed clinical observation was made on necropsy days. When signs of toxicity were noted, animals were observed more frequently.
MORTALITY/MORBIDITY
-Animals were inspected for signs of morbidity and mortality twice daily
CLINICAL OBSERVATIONS
-Time schedule: At least once daily from Day 6 post-coitum onwards up to the day prior to necropsy
BODY WEIGHTS
-Time schedule for examinations: Days 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20 post-coitum. Body weight gain was calculated for each interval, including GD0-6, GD6-20 and GD0-20.
FOOD CONSUMPTION
-Time schedule for examinations: Days 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20 post-coitum. Food consumption was calculated for each interval, including GD0-6, GD6-10, GD6-20.
WATER CONSUMPTION
- No water consumption was measured in the study
OPHTHALMIC EXAMINATIONS
- No ophthalmoscopy was conducted in the study
NEUROLOGICAL ASSESSMENT
-Not performed
EXAMINATION OF VAGINAL SMEARS
- After the daily mating period, a vaginal smear was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope; the presence of a vaginal plug or sperm in the vaginal smear is considered as evidence of copulation (GD0).
ORGAN WEIGHTS
-The weight of the thyroid gland with parathyroid glands for all dams were measured
-The uterus including the cervix was weighed and examined for early and late embryonic or foetal deaths and for the number of live foetuses

Ovaries and uterine content:

Each ovary and uterine horn of animals surviving to planned necropsy were dissected and examined as quickly as possible to determine:
-The number of corpora lutea in each ovary
-Implantation sites in each uterine horn
-The number of live foetuses
-Early and late embryonic death
-Foetal death were counted
-Number and percentage of pre- and post-implantation losses
-The degree of resorption
The placentas were examined macroscopically
If no implantation sites were evident but corpora lutea were present, the uterus was stretched and held in front of a light source to clearly identify the implantation sites. Uteri that appeared non-gravid were further examined to confirm the non-pregnant status (i.e. by ammonium sulphide staining or a suitable alternative method).

Blood sampling:

For thyroid hormone analysis, blood samples were taken from all dams at termination of the study

Fetal examinations:

Histopathology
-No histopathology evaluation was performed
Foetopathology
-abdominal and thoracic region
-Thymus and great arteries
-Testicular descent or cryptorchidism for male foetuses
-The heads were examined by Wilson's free-hand razor blade method
External, visceral and skeletal fetal findings were recorded as developmental variations or malformations.
- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes

Statistics:

Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test.
In the case of non-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test.
The Chi-squared test was used for non-continuous data.
The normality and heterogeneity of variance between groups was checked by Shapiro-Wilk and Levene tests. Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett’s (Multiple Range) test was used to assess the significance of inter-group differences.
If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.
For non-continuous data, the Cochran-Armitage test for trend was applied and the Chi-squared test is used for statistical differences relative to control.
For pathology data (macroscopic and microscopic data) the Cochran-Armitage test for trend was applied, then if appropriate, the Chi-squared test homogeneity test. If significance was plausible based on a user-defined value (0.05), a pairwise test of each treatment group versus the control group was made. If the group size is <5 then Fisher’s Exact Test was used, if the group sizes were bigger then the Chi-squared test was used; identifying differences of <0.05, <0.01 or <0.001 as appropriate.

Indices:

For each animal the following calculations were performed:
-Preimplantation loss: %, group mean = (Number of corpora lutea-Number of implantations) / (Number of corpora lutea) x100
-Postimplantation loss: %, group mean = (Number of implantations-Number of live foetuses) / (Number of implantations) x100
For each fetus following calculations were performed:
-Sex distribution: %, group mean = (Number of male (female) foetuses) / (Number of foetuses) x100
-External abnormalities/litter: %, group mean = (Number of foetuses with abnormality) / Number of foetuses) x100
-Visceral abnormalities/litter: %, group mean = (Number of foetuses with abnormality) / (Number of foetuses) x100
-Skeletal abnormalities/litter: %, group mean = (Number of foetuses with abnormality) / (Number of foetuses) x100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Decreased activity was observed in 1 out of 23 (only pregnant females) and 1 out of 24 animals at 300 and 1000 mg/kg bw/day. The finding appeared on Day 13 in the 300 mg/kg bw/day group and from Day 11 until Day 12, the symptom occurred for a maximum of two days. Hunched back was observed in 1 out of 23 and 1 out of 24 animalsat 300 and 1000 mg/kg bw/day. The finding appeared on Day 12 in the 300 mg/kg bw/day group and on Day 10 in the 1000 mg/kg bw/day dose group. Noisy respiration was observed in 2 out of 24 in the 1000 mg/kg bw/day dose group. The finding appeared from Day 9 until Day 10, the symptom occurred for a maximum of two days. Piloerection was present in 1 out of 23 and 2 out of 24 animals in the 300 and 1000 mg/kg bw/day groups, respectively. The finding appeared on Day 12 in the 1000 mg/kg bw/day dose group and from Day 9 until Day 11 in the 1000 mg/kg bw/day group, the symptom occurred for a maximum of two days. None of the observations were considered to represent adverse systemic effects of the test item.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

One High dose (1000 mg/kg bw/day) female was pre-terminally euthanised and replaced by another female

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item related body weight differences were observed in any of the dose groups. No significantly lower or higher corrected body weight, corrected body weight gain, or net body weight gain was observed in animals in any of the dose groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No changes in the food consumption were observed in any of the dose groups during the treatment period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant increase (p<0.01) in the concentration of the T4 level at 1000 mg/kg bw/day compared to the control, but without dose response it is considered as not test substance related effect.

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):

During the macroscopic examination of dams, the thyroid gland with parathyroid glands were retained from all animals and the organ weights recorded. There were no statistical differences in the organ weights between the control and the Dose groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

For pregnant females, no evidence of Test Item-related macroscopic changes was observed in animals dosed at 100, 300 and 1000 mg/kg bw/day on necropsy Day 20. There were no gross changes observed in non-pregnant female (1 animal (3523) in the Mid dose group) on necropsy Day 20.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

For pregnant females, no evidence of Test Item-related microscopic changes was observed in animals dosed at 100, 300 and 1000 mg/kg bw/day on necropsy Day 20.

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

Number of abortions:

not examined

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:

not specified

Details on maternal toxic effects:

No maternal systemic toxicity and no effect on body weight or growth was observed

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: no maternal systemic effects at 1000 mg/kg bw/day

Remarks on result:

other:

Remarks:

no maternal systemic toxicity

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Description (incidence and severity):

No maternal systemic toxicity and no effect on body weight or growth

Results (fetuses)

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant increase in the body weights were observed for male foetuses in the Low and High dose groups (100 and 1000 mg/kg bw/day). The actual values are within the historical control range (mean ± SD: 3.36 ± 0.23). There was higher number of foetuses with retarded body weight in the Mid dose group (300 mg/kg bw/day), but it was not significant. It was considered that there was no adverse effect on foetal body weights.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

There was no significant difference between the mean anogenital distances of the litters compared to the control

External malformations:

no effects observed

Description (incidence and severity):

There were no external variations in the test substance treated groups. There was no external malformation

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Most of the skeletal findings correspond to the current historical control or the concurrent study control data, or were considered to be incidental findings without dose response. Based on the skeletal findings the number of malformed / intact foetuses were comparable
between the control in the Low, Mid and High dose groups. The number of variations were higher in all test item treated groups. All data was considered to be in the normal range and there was no dose related trend.

In case of Skeletal Variations Rib Wavy the incidence was higher in High treated groups when compared to concurrent control with statistical significance but without dose response, hence the numerical differences were considered to be without toxicological relevance. In case of the variation Skull, Incomplete Ossification (3 or more) an apparent increased incidence of changes in absolute numbers were observed and in the High dose group with statistical significance when compared to control, however the incidence of litters with the finding in the High dose, is slightly lower than the average historic control litter incidence, hence it is probable not considered as a treatment related effect. Based on these results the test item is considered not to have affected adversely the intrauterine development and there was no higher incidence of malformations.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The changes correspond with the current historical control or the study control data or have isolated occurrence that was considered incidental, ascribed to individual variability and not related to treatment

Details on embryotoxic / teratogenic effects:

There were no effects on the embryotoxicity and teratogenic effects observed in the study. NOAEL for embryotoxicity is considered to be 1000 mg/kg bw/day, based on the lack of any test substance related intra-uterine effect in any treatment group.
NOAEL for teratogenecity is considered to be 1000 mg/kg bw/day, based on the lack of treatment related malformations in any dose group.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effect on foetal weight or incidence of runts at 1000 mg/kg bw/day

Remarks on result:

other:

Remarks:

No developmental effects observed up to 1000 mg/kg bw/d (highest dose tested).

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Description (incidence and severity):

no indication of any foetal malformations related to treatment

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Table 1:Summaryof the gravimetricdose formulation analysis

Formulation	07September2020		21September2020
	Mean measured concentration (mg/mL)	Relative to the nominal concentration (%)	Measured concentration (mg/mL)	Relative to the nominal concentration (%)
Control	not detected	-	not detected	-
20 mg/mL	19.8±0.09	99	21.2±0.10	106
60 mg/mL	59.8±0.77	100	63.5±0.73	106
200mg/mL	196.2±3.65	98	209.4±2.06	105

Table 2: Summary of thyroid hormone analysis

	Dose group (mg/kg bw/day)
		Control	100	300	1000
Females
Adult T3 concentration(ng/mL)	Mean	1.2267	1.2546	1.1935	1.2238	NS
	SD	0.2167	0.1837	0.1599	0.2790
Adult T4 concentration(ng/mL)	Mean	30.04	31.13	30.52	33.63**	U
	SD	4.14	4.13	2.92	4.70
Adult TSH concentration (pg/mL)	Mean	236.9	267.9	356.9	311.1	NS
	SD	115.3	194.6	318.5	201.6

NS: Statistically not significant when compared to the vehicle

U:Dunn2sidedtest(**= p<0.01)

Table 3: Summary of pregnancy data

Parameters	Dose(mg/kg bw/day)
	0	100	300	1000
Number of mated females	24	24	24	24
Pre-terminal death or euthanasia	0	0	0	0
Number of non-pregnant females	0	0	1	0
Number of females with ≤5 implantation sites	0	0	0	0
Number of evaluated females on GD20(Caesarean section)	24	24	23	24

Table 4: Summary of intra-uterine evaluation

Parameters	Dose(mg/kg bw/day)
	0	100	300	1000
Number of evaluated dams	24	24	23	24
Mean number of corpora lutea	12.54	12.04	11.83	11.83	NS
Pre-implantation loss,mean	2.04	2.04	1.52	1.79	NS
Pre-implantation loss (%),mean	15.04	16.03	12.02	13.57	NS
Mean number of implantations	10.50	10.00	10.30	10.04	NS
Early embryonic loss,mean	0.33	0.50	0.13	0.21	NS
Early embryonic loss (%),mean	3.04	4.34	1.03	2.01	NS
Late embryonic loss, mean	0.13	0.21	0.13	0.21	NS
Late embryonic loss (%), mean	1.26	2.06	1.36	1.84	NS
Dead foetuses, mean	0.00	0.00	0.00	0.00	NA
Dead foetuses (%), mean	0.00	0.00	0.00	0.00	NA
Post-implantation loss, mean	0.46	0.71	0.26	0.42	NS
Post-implantation loss (%),mean	4.30	6.40	2.39	3.85	NS
Total intra-uterine mortality,mean	2.50	2.75	1.78	2.21	NS
Total intra-uterine mortality(%),mean	18.63	21.69	14.32	16.92	NS
Viable foetuses, mean	10.04	9.29	10.04	9.63	NS

NS: Statistically not significant when compared to the vehicle control

NA:Not applicable.

Table 5: Examination of viable foetuses

Parameters	Dose (mg/kg bw/day)
	0	100	300	1000
Number of examined litters	24	24	23	24
Viable foetuses,mean	10.04	9.29	10.04	9.63	NS
Male foetuses, mean	5.08	4.88	4.83	4.79	NS
Female foetuses, mean	4.96	4.42	5.22	4.83	NS
Total number of foetuses	241	223	231	231	NS
Total number of male foetuses	122	117	111	115	NS
Total number of female foetuses	119	106	120	116	NS
Sex distribution (% of males/females)	51/49	53/47	48/52	50/50	NS
Mean foetal weight/litter(g)	3.414	3.498	3.449	3.507	NS
Number of foetuses with retarded bodyweight	5	5	10	4	NS
Number of affected litters (with runts)	5	4	4	3	NS

NS: Statistically not significant when compared to the vehicle

Table 6: Summary table of the anogenital distances

Dose group(mg/kgbw/day)
	Control	100	300	1000
Anogenital Distance Data Male & Female(mm)
Mean	2.192	2.258	2.119	2.173	NS
SD	0.980	0.981	0.972	0.984
Anogenital Distance Data Male(mm)
Mean	3.132	3.164	3.101	3.137	NS
SD	0.226	0.245	0.262	0.250
Anogenital Distance Data Female(mm)
Mean	1.228	1.257	1.211	1.217	NS
SD	0.226	0.201	0.179	0.156

NS: Statistically not significant when compared to the vehicle

Table7:Summary table of the external abnormalities

Parameter	Dose(mg/kgbw/day)
	0	100	300	1000
Total number of examined litters	24	24	23	24
Total number of examined foetuses	241	223	231	231
Total number of intact (normal)foetuses	241	223	231	231
Total number of foetuses / litters with malformation	0 / 0	0 / 0	0 / 0	0 / 0
Total number of foetuses / litters with variation	0 / 0	0 / 0	0 / 0	0 / 0

Table 8:Summary table of the visceral abnormalities

Parameter	Dose(mg/kg bw/day)
	0	100	300	1000
Total number of examined litters	24	24	23	24
Total number of examined foetuses	120	113	115	117
Total number of intact (normal)foetuses	118	111	112	114
Total number of foetuses / litters with malformation	0 / 0	2 / 2	0 / 0	0 / 0
Total number of foetuses / litters with variation	2 / 2	0 / 0	3 / 3	3 / 3

Table 9:Details of the visceral abnormalities

Parameter			Dose(mg/kg bw/day)				HC data
			0	100	300	1000
Total number of examined litters			24	24	23	24	671
Total number of examined foetuses			120	113	115	117	6853
Visceralmalformations
Situs Inversus(Total)	Litter incidence	n	0	2	0	0	3
		%	0.0	8.3	0.0	0.0	0.5
	Foetal incidence	n	0	2	0	0	3
		%	0.000	1.770	0.000	0.000	0.044
Lung, Abnormal Lobation orAbsent	Litter incidence	n	0	1	0	0	1
		%	0.0	4.2	0.0	0.0	0.2
	Foetal incidence	n	0	1	0	0	1
		%	0.000	0.885	0.000	0.000	0.015
Visceralvariations
Parameter			Dose(mg/kg bw/day)				HC data
			0	100	300	1000
Total number of examined litters			24	24	23	24	670
Total number of examined foetuses			120	113	115	117	3428
Brachiocephalic Trunk,Short	Litter incidence	n	1	0	2	1	1
		%	4.2	0.0	8.7	4.2	0.2
	Foetal incidence	n	1	0	2	1	1
		%	0.833	0.000	1.739	0.855	0.029
Ureterconvoluted	Litter incidence	n	1	0	0	2	10
		%	4.2	0.0	0.0	8.3	1.5
	Foetal incidence	n	1	0	0	2	10
		%	0.833	0.000	0.000	1.709	0.292
Thymiccord	Litter incidence	n	0	0	1	0	66
		%	0.0	0.0	4.3	0.0	9.9
	Foetal incidence	n	0	0	1	0	77
		%	0.000	0.000	0.870	0.000	2.246

Table 10: Summary table of skeletal abnormalities

Parameter	Dose(mg/kg bw/day)
	0	100	300	1000
Total number of examined litters	24	24	23	24
Total number of examined foetuses	121	110	116	114
Total number of intact (normal)foetuses	103	85	98	87
Total number of foetuses / litters with malformation	3 / 3	6 / 5	0 / 0	3 / 3
Total number of foetuses / litters with variation	15 / 9	19/ 12	18/ 10	24/ 13

Table 11:Details of the skeletal abnormalities

Parameter			Dose(mg/kg bw/day)				HC data
			0	100	300	1000
Total number of examined litters			24	24	23	24	671
Total number of examined foetuses			121	110	116	114	3420
Skeletalmalformations
Transverse process fused; Pelvic girdle,malpositioned	Litter incidence	n	3	3	0	2	18
		%	12.5	12.5	0.0	8.3	2.7
	Foetal incidence	n	3	4	0	2	18
		%	2.479	3.636	0.000	1.754	0.526
VertebraeHemicentric	Litter incidence	n	0	0	0	1	2
		%	0.0	0.0	0.0	4.2	0.3
	Foetal incidence	n	0	0	0	1	2
		%	0.000	0.000	0.000	8.772	0.058
Scapula, Humerus, Radius,Short, Bent	Litter incidence	n	0	1	0	0	1
		%	0.000	4.2	0.0	0.0	0.1
	Foetal incidence	n	0	1	0	0	1
		%	0.000	0.909	0.000	0.000	0.029

Parameter			Dose(mg/kg bw/day)				HC data
			0	100	300	1000
Total number of examined litters			24	24	23	24	671
Total number of examined foetuses			121	110	116	114	3420
Skeletalvariations
Skull: Incomplete ossification (3 or More)	Litter incidence	n	2	2	3	4	117
		%	8.3	8.3	13.0	16.7	17.4
	Foetal incidence	n	2	4	5	9*CH	163
		%	1.653	3.636	4.310	7.895	4.766
Skull:Hyoid Body Unossified	Litter incidence	n	3	0	1	0	16
		%	12.5	0.0	4.3	0.0	2.4
	Foetal incidence	n	3	0	1	0	21
		%	2.479	0.000	0.862	0.000	0.614
Sternum: Unossified Sternebra (2 or More)	Litter incidence	n	4	4	6	3	16
		%	16.7	16.7	26.1	12.5	2.4
	Foetal incidence	n	4	5	10	3	17
		%	3.306	4.545	8.620	2.631	0.497
Sternum: Sternebra, Bipartite Ossification	Litter incidence	n	0	0	0	1	2
		%	0.0	0.0	0.0	4.2	0.3
	Foetal incidence	n	0	0	0	1	2
		%	0.000	0.000	0.000	0.877	0.058
Ribs:Wavy	Litter incidence	n	3	9	4	5	179
		%	12.5	37.5	17.4	20.8	26.7
	Foetal incidence	n	4	15**^CH	7	11*^CH	296
		%	3.306	13.636	6.034	9.649	8.655
Ribs: (Costal Cartilage)Interrupted	Litter incidence	n	2	1	2	2	19
		%	8.3	4.2	8.7	8.3	2.8
	Foetal incidence	n	3	2	2	3	27
		%	2.479	1.818	1.724	2.631	0.789
Vertebrae: 2 Dumbbell or Asymmetric Ossification	Litter incidence	n	0	3	0	2	44
		%	0.0	12.5	0.0	8.3	6.6
	Foetalincidence	n	0	3	0	2	50
		%	0.000	2.727	0.000	1.754	1.462

Parameter			Dose(mg/kgbw/day)				HC data
			0	100	300	1000
Total number of examined litters			24	24	23	24	671
Total number of examined foetuses			121	110	116	114	3420
Skeletalvariations
Vertebrae:Dumbbell Shaped	Litter incidence	n	0	0	1	1	8
		%	0.0	0.0	4.3	4.2	1.2
	Foetal incidence	n	0	0	1	1	8
		%	0.000	0.000	0.862	0.877	0.234
Vertebrae: Bipartite Ossification	Litter incidence	n	0	0	1	1	15
		%	0.0	0.0	4.3	4.2	2.2
	Foetal incidence	n	0	0	1	1	15
		%	0.000	0.000	0.862	0.877	0.439
Limbs: Unossified Meta tarsal 3 or less	Litter incidence	n	0	1	1	0	105
		%	0.0	4.2	4.3	0.0	15.6
	Foetal incidence	n	0	1	1	0	52
		%	0.000	0.909	0.862	0.000	1.520

HC:historical control (data provided where considered useful)

CH:Chi square test,*=p<0.05,***=p<0.01

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the maternal toxicity, embryotoxicity, foetotoxicity and teratogenicity NOAELs were established to be 1000 mg/kg bw/day.

Executive summary:

A study was conducted to determine the potential developmental toxicity / teratogenicity of the test substance according to OECD Test Guideline 414, in compliance with GLP. Hannover Wistar rats were exposed to the test substance once daily by oral gavage from Days 6 to 19 post-coitum at doses of 0, 100, 300 and 1000 mg/kg bw/day. Control dams were treated with the vehicle (corn oil) only. The following parameters were evaluated during the study: mortality and clinical observations, body weight, body weight gain and individual food consumption. Maternal reproductive parameters associated with uterine examination were evaluated, and the foetuses were weighed and examined for external, visceral and skeletal abnormalities. Placentas were examined macroscopically. No substance related toxicity was observed in the dams up to 1000 mg/kg bw/day dose levels. No changes in the body weight and food consumption in any of the test substance treated groups were observed during the treatment period. No macroscopic changes were present at necropsy in the animals. There was statistically significant increase in the concentration of the T4 level at 1000 mg/kg bw/day but it was not treatment related increase. No treatment-related changes were noted in organ weights, intra-uterine parameters, male/female foetal weights, sex distribution, anogenital distance, external, visceral or skeletal development of foetuses in the study. There was no indication of any foetal malformations related to treatment. No developmental toxicity was observed at any of the dose levels. Under the study conditions, the maternal toxicity, embryotoxicity, foetotoxicity and teratogenicity NOAELs were established to be 1000 mg/kg bw/day (Szaloki, 2021).