A mixture of: propan-2-one-O,O'(methoxyvinylsilandiyl)dioxime; propan-2-one-O-(dimethoxyvinylsilyl)oxime; propan-2-one-O,O',O''-(vinylsilantriyl)trioxime

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 May 2020 – 18 June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 January 2020 – 21 February 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

This test is used as dose range finding study for a subsequent main test and only 7 females per dose group were used.

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

This test is used as dose range finding study for a subsequent main test and only 7 females per dose group were used.

GLP compliance:

no

Remarks:

As DRFstudy, it was not strictly conducted in accordance to GLP requirements although the test facility is certified under GLP legislation and normally works under GLP principles.

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Experimental Medicine Centre at the Medical University in Białystok.
- Age at study initiation: Approx. 13 to 16 weeks.
- Weight at study initiation: average body weights of all animals introduced to experiment was about 240 g (220 g – 260 g).
- Fasting period before study: No.
- Housing: cages with a plastic bottom and covered with wire-bar lids. Dimensions: 58 × 37 × 21 cm (length × width × height). UV-sterilized wood chips were used as bedding. In each cage wooden blocks, nesting material and tunnels were placed for laboratory animals.
- Diet (e.g. ad libitum): ad libitum access to the "Altromin 1324 P TPF” (Phytoestrogen-poor, Total Pathogen Free)" standard laboratory fodder produced by Altromin Spezialfutter GmbH & Co. KG, Lage, Germany, batch number 2/19.
- Water (e.g. ad libitum): ad libitum access to drinking tap water.
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 – 22.5ºC
- Humidity (%): 46-60 %
- Air changes (per hr): 15-20
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was diluted in corn oil directly before administration, in the appropriate concentration for each dose. The proper concentrations of test item in corn oil was prepared under a nitrogen atmosphere. The proper volume of corn oil for prepared doses was poured to the 15 ml test tubes sealed with a screw cap. Nitrogen was admitted above the oil surface and the proper volume of test item for prepared dose was added to the corn oil simultaneously. After sealing the tubes, the whole content of each tube was thoroughly mixed on a Vortex mixer. For the volumetric preparation of solutions, it was assumed that 1 ml of the test item had a mass of 1000 mg. For maximum accuracy, insulin syringes with 1/100 scale were used to measure the appropriate volumes of corn oil and test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil is universally accepted and routinely used vehicle in oral toxicity studies.
- Concentration in vehicle: 6.125, 35 and 200 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: For 27 females the length of cohabitation was 1 day. For one female the length of cohabitation was 2 days.
- For ten males one male was used to mate with two females. For eight males one male was used to mate with one female.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of gestation.
- Any other deviations from standard protocol: No

Duration of treatment / exposure:

From 5th to 19th day of pregnancy.

Frequency of treatment:

Once a day

Duration of test:

1 month (21 January 2020 – 21 February 2020)

Dose / conc.:

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

Remarks:

G0 - Vehicle control

Dose / conc.:

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

Remarks:

G1 - Low dose

Dose / conc.:

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

Remarks:

G2 - Mid dose

Dose / conc.:

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

Remarks:

G3 - High dose

No. of animals per sex per dose:

7

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: observation for morbidity and mortality was performed twice a day during labour week and once a day on days off.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: on day of beginning of experiment (day 0), and then on 5th, 8th, 11th, 14th, 17th and 20th day of gestation.

BODY WEIGHT: Yes
- Time schedule for examinations: at 0, 5th, 8th, 11th, 14th, 17th and 20th day of gestation.

FOOD CONSUMPTION: yes, food consumption of the females was controlled on days of their weighing

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: females were examined macroscopically for any abnormalities in body structure or pathological changes which could have influenced the gestation.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes.
- Number of implantations: Yes.
- Number of resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No

Statistics:

In case of normal distribution and homogenous variance, data were analyzed by a one-way analysis of variance (ANOVA) and the Dunnett’s test at p ≤ 0.05. In case of absence of normal distribution or homogenous variance, non-parametric statistical test were used i.e. Kruskal-Wallis test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Some clinical changes in locomotor system, respiratory system, urinary system, alopecia and changes in coat and body weight losses were observed in females of group 3.
Alopecia was observed in one female of control group and in one female of group 2.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

In the group 3 one female was euthanized for humane reason on 14th day of experiment. All other females survived the period of the experiment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Average body weight of the females of treated group 1 and group 2 did not differ statistically significantly from average body weight of the females of the control group. Average body weight of the females group 3 was statistically significant lower in comparison with the control group from 8th to 20th gestation day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption of the females of the group 1 was in the same level with average food consumption of the females of the control group and was not statistically significantly different. Average food consumption of the females of the group 2 was statistically lower from 9th to 11th day of experiment and average food consumption of the female of the group 3 was statistically lower from 6th to 14th day in comparison with the control group.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Enlarged spleen was found in seven females of group 2 and in seven females of group 3. The increase of severity in the dose- related manner and the number of cases at the medium and high dose groups states for the test item- related character of the lesion. Some changes in liver were found in one female of group 3. Adipose tissue near the right ovary was found in one female (nonpregnant) of group 1. Emaciation was observed in female of group 3 which was humanely euthanized.
No pathological changes were observed in the remaining females at necropsy.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Description (incidence and severity):

No abortions ocurred in any dose group.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No statistically significant differences in the number of implantations between groups 1, 2, 3 and control group were stated.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There was not any loss of total litter in any dose group.

Early or late resorptions:

no effects observed

Description (incidence and severity):

No statistically significant differences in the number of resorptions between groups 1, 2, 3 and control group were stated.

Dead fetuses:

no effects observed

Description (incidence and severity):

There were not any dead fetuses in any dose group.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

effects observed, treatment-related

Description (incidence and severity):

One female of group 1 and one female of group 2 was not pregnant. This must be considered as accidental. Some percent of mated females with presence of sperms in vaginal swabs are not pregnant.
Two females in group 3 had only implantation sites in uterus. This must be considered as the toxic influence of the test item. In these females the observed clinical changes were very extensive and probably the influence of the test item was the reason of the embryos death.

Other effects:

no effects observed

Description (incidence and severity):

There were no statistically significant differences between groups 1, 2 and 3 and control group in the weight of uterus of pregnant females.

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

food consumption and compound intake

gross pathology

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

food consumption and compound intake

gross pathology

Remarks on result:

other: Effects observed at the medium tested dose of 140 mg/kg bw/day.

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Average weight of placenta in group 1 was statistically lower compared to the control group. Average weight of fetuses with placenta and fetal membranes and fetuses without placenta in group 3 were statistically lower compared to the control group. Average weight of placenta in group 3 was statistically greater compared to the control group.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There were not any dead fetuses in any dose group.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no statistically significant differences observed in the sex ratio of fetuses between the treated groups and the vehicle control group.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

No statistically significant differences regarding the number of fetuses in the litter between groups 1, 2, 3 and control group were stated.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Subcutaneous blood hemorrhages were observed both in the control group and in the treated groups and should not be connected with the test item. Subcutaneous blood hemorrhages had been observed often in fetuses in other prenatal developmental toxicity studies performed in the lab and they should be considered as typical disturbances in fetuses.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Average length of the fetuses with tail and without tail in group 2 was greater compared to the control group. Average length of fetuses with tail and without tail in group 3 was lower compared to the control group. No statistically significant changes in anogenital distance in all treated group were observed compared to the control group.

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

other: placenta weight and length of fetuses changes

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

not specified

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1. WASOX-VMAC2. Results of mating

Parameter	Number of females
	Group0	Group1	Group2	Group3
Number of females per group	7	7	7	7
Numberofpregnant females	7	6	6	7*
Females onlywithimplantations	-	-	-	2 [6,7]
Number of females for evaluation	7	6	6	5

[ ] computer numbers of females

*-includes two females only with implantation sites in uterus

Table 2. WASOX-VMAC2- Clinical signs in females

Parameter	Number of females
	Group 0	Group 1	Group 2	Group 3
Number of females per group	7	7	7	7
Alopecia of forelimbs	1 [7] – (14^th-20^thday)	-	-	-
Alopecia of left side of trunk, abdomen and forelimbs	-	-	1 [6] – (8^th-20^thday)	-
rounded back	-	-	-	1 [7] – (12^th-14^thday)***
slight decrease of locomotor activity and dejection	-	-	-	7 [1] – (9^th-20^thday)  [2] – (11^th-20^thday) [3] – (11^th-20^thday) [4] – (11^th-20^thday) [5] – (9^th-20^thday) [6] – (8^th-11^thday)* [7] – (9^th-20^thday)***
distinct decrease of locomotor activity and dejection	-	-	-	1 [6] – (12^th-14^thday)*
very easy reaction to be caught	-	-	-	1 [6] – (12^th-14^thday)*
passive urination	-	-	-	7 [1] – (10^th-20^thday)  [2] – (16^th-20^thday) [3] – (16^th-20^thday) [4] – (12^th-20^thday) [6] – (10^th-14^thday)* [7] – (16^th-20^thday)***
respiratory murmurs	-	-	-	3 [1] – (14^th-20^thday)  [6] – (7^th-9^thday)* [7] – (13^th-14^thday)***
bristled coat	-	-	-	1 [6] – (11^th-14^thday)* [7] – (12^th-14^thday)***
porphyrin deposition around the nostrils	-	-	-	2 [1] – (14^th-20^thday) [2] – (14^th,17^th-18^thday)
Body weight loss	1 [7] – (8^th-11^thday)	2 [1] – (5^th-8^thday) [7] – (8^th-20^thday)**	2 [1] – (8^th-14^thday)** [5] – (5^th-8^thday)	7 [1] – (5^th-14^thday)  [2] – (5^th-14^thday) [3] – (5^th-14^thday) [4] – (5^th-11^thday) [5] – (5^th-14^thday) [6] – (5^th-14^thday)* [7] – (5^th-20^thday)***

[ ] computer numbers of females; * humanly euthanized female; ** nonpregnant female; *** female only with resorptions

( ) days in which the changes were noticed

Table 3. WASOX-VMAC2. Body weight of females [g]

Day of gestation	Group
	0 n=7	1 n=6	2 n=6	3 n=5
0	240.43 ± 11.30	237.33 ± 10.73	241.83 ± 12.98	237.60 ± 14.84
5	258.29 ± 14.96	248.67 ± 11.78	255.33 ± 19.36	248.80 ± 12.40
8	265.43 ± 16.27	253.83 ± 10.70	259.33 ± 18.74	238.20 ± 15.83 *
11	274.00 ± 12.83	264.67 ± 13.63	268.17 ± 21.81	229.80 ± 8.84 *
14	290.43 ± 15.80	278.00 ± 15.41	282.50 ± 20.84	241.00 ± 9.62 *
17	317.57 ± 17.05	303.00 ± 19.00	308.50 ± 18.71	278.00 ± 9.19 *
20	352.00 ± 18.20	336.17 ± 23.64	345.67 ± 22.59	311.80 ± 12.87 *

 µ ± SD

* statistically significant difference with p ≤ 0.05

Table 4. WASOX-VMAC2. Food consumption [g/100g of b.w./day]

Day of gestation	Group
	0 n=7	1 n=6	2 n=6	3 n=5
0 – 5	6.72 ± 0.59	6.13 ± 0.61	6.75 ± 0.44	6.31 ± 0.38
6 – 8	6.31 ± 0.59	5.69 ± 0.54	6.11 ± 0.73	3.04 ± 1.47 *
9 – 11	6.21 ± 0.88	5.37 ± 0.48	5.04 ± 0.92 *	2.01 ± 0.93 *
12 – 14	6.71 ± 0.78	6.55 ± 0.60	6.25 ± 0.44	4.74 ± 0.88 *
15 – 17	6.18 ± 0.55	6.19 ± 0.43	6.43 ± 0.86	6.19 ± 0.36
18 – 20	6.01 ± 0.47	5.95 ± 0.37	6.01 ± 0.44	5.44 ± 0.54

 µ ± SD

* statistically significant difference with p ≤ 0.05

Table 5. WASOX-VMAC2. Gross lesions in females

Organ	Type of lesion	Group
		0 n=7	1 n=7	2 n=7	3 n=7
Spleen	slightly enlarged	-	-	4 [3,4,5,6]	-
	greatly enlarged	-	-	3 [1,2,7]	7 [1,2,3,4,5,6**,7***]
Liver	slightly enlarged	-	-	-	1 [4]
	lobular structure	-	-	-	1 [4]
	white lesion in caudal lobe 3x4mm	-	-	-	1 [4]
Adipose tissue near the right ovary	yellow pedunculated lesion 2.5x3mm	-	1 [7*]	-	-
Emaciation		-	-	-	1 [6**]
Female non pregnant		-	1 [7*]	1 [1*]
Female only with implantation sites		-	-	-	2 [6**,7***]

* - nonpregnant female

** - female euthanised for humanitarian reasons

***- female only with implantation

Table 6. WASOX-VMAC2. Average weight of uterus of pregnant females

Group	Number of pregnant females	Weight of uterus
0	7	4.706 ± 0.447
1	6	4.679 ± 0.433
2	6	4.686 ± 0.480
3	5	4.904 ± 0.593

Table 7. WASOX-VMAC2. Number of fetuses

Group	Number of pregnant females	Number of fetuses
		Total	in litters min.–max.	average in litter in 1 female	females	males	dead
0	7	95	10-15	13.571 ± 1.813	7.000 ± 2.517	6.571 ± 2.820	0
1	6	73	8-16	12.167 ± 2.563	6.333 ± 2.066	5.833 ± 2.787	0
2	6	79	9-16	13.167 ± 2.714	6.833 ± 2.317	6.333 ± 2.582	0
3	5	64	9-16	12.800 ± 2.588	7.000 ± 1.871	5.800 ± 3.347	0

Table 8. WASOX-VMAC2. Number of corpora lutea, implantations and resorptions

Group	Number of pregnant females	Average number of corpora lutea per one female	Number of implantations in not pregnant females	Number of resorptions
				total	in females min - max	average in one female
0	7	17.714 ± 1.799	0	7	0 – 2	1.000 ± 1.000
1	6	15.000 ± 2.280	0	5	0 – 3	0.833± 1.169
2	6	15.000 ± 2.098	0	3	0 – 1	0.500± 0.548
3	5	15.800 ± 3.114	29*	7	0 – 3	1.400± 1.342

* two females(No 6 and No 7) only with implantation sites in uterus

Table 9. WASOX-VMAC2. Average weight of fetuses and placenta [g]

Group	Number of examined fetuses	Weight of fetuses		Weight of placenta
		with placenta and fetal membranes	without placenta
0	95	4.668 ± 0.412	3.278 ± 0.306	0.573 ± 0.188
1	73	4.586 ± 0.329	3.347 ± 0.328	0.521 ± 0.094*
2	79	4.634 ± 0.352	3.388 ± 0.290	0.539 ± 0.063
3	64	4.508 ± 0.410*	2.963 ± 0.389*	0.676 ± 0.150*

 µ ± SD

* statistically significant difference at p ≤ 0.05, Dunnett's test

Table 10. WASOX-VMAC2. Average length of fetuses [cm] and anogenital distance [mm]

Group	Number of examined fetuses	Length of fetuses		Anogenital distance
		with tail	without tail
0	95	4.882 ± 0.237	3.601 ± 0.198	2.484 ± 0.502
1	73	4.930 ± 0.226	3.629 ± 0.159	2.479 ± 0.503
2	79	4.982 ± 0.159*	3.676 ± 0.161*	2.481 ± 0.503
3	64	4.759 ± 0.210*	3.484 ± 0.190*	2.453 ± 0.502

 µ ± SD

* statistically significant difference at p ≤ 0.05, Dunnett's test

Table 11. WASOX-VMAC2. Lesions in fetuses

Lesion	Number of female [number of fetuses]
	Group 0	Group 1	Group 2	Group 3
Hemorrhage (in different locations of the body)	1 [4] 2 [1] 4 [1] 7 [1]	1 [2] 3 [1] 4 [1]	3 [3] 5 [1]	1 [4] 3 [7] 4 [1]

Conclusions:

In this dose range finding study for a prenatal developmental toxicity study in rats, pathological effects in spleen and reduction in food consumption were observed in females at the mid dose of 140 mg/kg bw/d, and changes in the weight of placenta were observed in fetuses at the low dose of 24.5 mg/kg bw/d. It was therefore recommended to use lower doses in the main study and decrease the low dose to 15 mg/kg bw/d.

Executive summary:

A dose range finding study for a prenatal developmental toxicity study was performed on the test substance WASOX-VMAC2 by oral administration in Wistar rats according to OECD guideline 414. The study was conducted on 28 females divided into 4 groups. Each group (G0, G1, G2 and G3) consisted of 7 females. From day 5 to 19 of gestation, the animals in G0 group were administered with vehicle (corn oil) and the animals in G1, G2 and G3 groups were administered with test item at the dose levels of 24.5, 140 and 800 mg/kg bw/day. Clinical observations for mortality and signs of toxic influence of the test item were performed in females, their body weight and food consumption were controlled. On day 20 of gestation, all females were killed and caesarean section with gross examination was performed. The uterine content was examined. The number of fetuses in the litter, the number of resorptions, implantations and corpora lutea were determined. After removing the fetuses each uterus was weighed. The non-gravid uteri and non-gravid horns were overexposed by electric lamp and were stained using ammonium sulphide in order to confirm the non-pregnant status. Sex and anogenital distance, body weight with and without placenta, weight of placenta, body length with and without tail were determined in all fetuses. Each fetus was subjected to gross examination. 27 females survived the experiment. One pregnant female in group 3 was euthanized by humanitarian reason. Some clinical changes in locomotor system, respiratory system, urinary system, alopecia and changes in coat and body weight losses were observed in females of group 3. Average body weight of pregnant females of group 1 and group 2 was comparable with average body weight of pregnant females of control group. Average body weight of pregnant females of group 3 was statistically significant lower from day 8 to day 20 of the experiment. Average food consumption of pregnant females was statistically significant lower in group 2 from day 9 to 11 and in group 3 from day 6 to 14 of the experiment compared to the control group. One female of group 1 and one female of group 2 were not pregnant. Two females in group 3 had only implantation sites in uterus. Enlarged spleen was found in seven females of group 2 and in seven females of group 3. Some changes in liver were found in one female of group 3. Adipose tissue near the right ovary was found in one female (nonpregnant) of group 1. Emaciation was observed in female of group 3 which was humanely euthanized. No statistically significant differences regarding the number of fetuses in the litter, the number of males and females in the litter, the number of resorptions, implantations and corpora lutea between treated and control groups were stated. Average weight of placenta was statistically lower in group 1 and greater in group 3 compared to the control group. Average weight of fetuses with placenta and fetal membranes and fetuses without placenta in group 3 were statistically lower compared to the control group. Average length of the fetuses with tail and without tail was statistically greater in group 2 and lower in group 3 compared to the control group. Due to these changes found in weight and length of fetuses of group 2 and group 3, the change in placenta weight of fetuses in group 1 was considered to possibly be the first sign of the test item influence in the lowest dose. Based on these results, it was recommended to use lower doses in the main study and the lowest dose should be decreased to 15 mg/kg bw/d.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

A mixture of: propan-2-one-O,O'(methoxyvinylsilandiyl)dioxime; propan-2-one-O-(dimethoxyvinylsilyl)oxime; propan-2-one-O,O',O''-(vinylsilantriyl)trioxime

EC Number:

458-680-3

EC Name:

A mixture of: propan-2-one-O,O'(methoxyvinylsilandiyl)dioxime; propan-2-one-O-(dimethoxyvinylsilyl)oxime; propan-2-one-O,O',O''-(vinylsilantriyl)trioxime

Cas Number:

797751-44-1

Molecular formula:

not applicable, multiconstituent substance

IUPAC Name:

3-ethenyl-3-methoxy-6-methyl-2,4-dioxa-5-aza-3-silahept-5-ene; 5-ethenyl-2,8-dimethyl-5-{[(propan-2-ylidene)amino]oxy}-4,6-dioxa-3,7-diaza-5-silanona-2,7-diene; 5-ethenyl-5-methoxy-2,8-dimethyl-4,6-dioxa-3,7-diaza-5-silanona-2,7-diene

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Experimental Medicine Centre at the Medical University in Białystok.
- Age at study initiation: 14 to 16 weeks.
- Weight at study initiation: average body weights of all animals introduced to experiment was about 249.3 g (208 g – 307g). The body weight of one female in group 1 and one female in group 2 was more than ± 20% of the average value for the group.
- Fasting period before study: No.
- Housing: cages with a plastic bottom and covered with wire-bar lids. Dimensions: 58 × 37 × 21 cm (length × width × height). UV-sterilized wood chips were used as bedding. In each cage wooden blocks, nesting material and tunnels were placed for laboratory animals.
- Diet (e.g. ad libitum): ad libitum access to the "Altromin 1324 P TPF” (Phytoestrogen-poor, Total Pathogen Free)" standard laboratory fodder produced by Altromin Spezialfutter GmbH & Co. KG, Lage, Germany, delivery number 4/20 and 5/20.
- Water (e.g. ad libitum): ad libitum access to drinking tap water.
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 25ºC
- Humidity (%): 38-80 %. The relative air humidity short-term exceeded 70 %: twice during acclimatization and once during the experiment. The changes did not influence on the study course.
- Air changes (per hr): 15-20
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was diluted in corn oil directly before administration, in the appropriate concentration for each dose. The proper concentrations of test item in corn oil was prepared under a nitrogen atmosphere. The proper volume of corn oil for prepared doses was poured to the 50 ml test tubes sealed with a screw cap. Nitrogen was admitted above the oil surface and the proper volume of test item for prepared dose was added to the corn oil simultaneously. After sealing the tubes, the whole content of each tube was thoroughly mixed on a Vortex mixer. For the volumetric preparation of solutions, it was assumed that 1 ml of the test item had a mass of 1.0158 g. For maximum accuracy, insulin syringes with 1/100 scale were used to measure the appropriate volumes of corn oil and test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil is universally accepted and routinely used vehicle in oral toxicity studies.
- Concentration in vehicle: 3.75, 25.125 and 168.35 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
- Lot/batch no.: 19343

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

In order to confirm correct preparation of the test item doses in the study, the solution of the test item in corn oil (i.e. 3.75 mg/mL at dose 15 mg/kg b.w., 25.125 mg/mL at dose 100.5 mg/kg b.w., 168.35 mg/mL at dose 673.4 mg/kg b.w.) was analyzed using a validated GC method with FID detection.
The validation of the method was determined according to SANCO/3029/99 rev. 4 (11/07/00). The mean recoveries at two fortification levels (3.047 mg/mL and 30.474 mg/mL) ranged from 99.8% to 108.2%, RSD values were below 10% and no significant peak occurred at the retention time, so absence of interference was confirmed.
During the study the samples were transferred thrice for chemical analysis: at the beginning, in the middle and at the end of the study. The mean recovery values for the test samples ranged from 87.8% to 107.1%. All results were in the range from 80 to 120%, therefore the acceptance criterion was met.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: For ninety one females the length of cohabitation was 1 day. For five females the length of cohabitation was 2 days. For two females the length of cohabitation was 3 days. For two females the length of cohabitation was 4 days.
- One male was used to mate with two females.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of gestation.
- Any other deviations from standard protocol: No

Duration of treatment / exposure:

From 5th to 19th day of pregnancy.

Frequency of treatment:

Once a day

Duration of test:

07 May 2020 – 18 June 2020

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

G0 - Vehicle control

Doses / concentrations 2

Dose / conc.:

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

Remarks:

G1 - Low dose

Doses / concentrations 3

Dose / conc.:

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

Remarks:

G2 - Mid dose

Doses / concentrations 4

Dose / conc.:

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

Remarks:

G3 - High dose

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were established based on a DRF study where some changes were observed in fetuses at doses of 24.5, 140 and 800 mg/kg bw/d and in females at doses of 140 and 800 mg/kg bw/d.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: observation for morbidity and mortality was performed twice a day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day.

BODY WEIGHT: Yes
- Time schedule for examinations: at 0, 5th, 8th, 11th, 14th, 17th and 20th day of gestation.

FOOD CONSUMPTION: yes, food consumption of the females was controlled on days of their weighing.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: All females were examined macroscopically for any abnormalities in body structure or pathological changes which could have influenced the gestation. The thyroid with parathyroids from all females were collected, fixed in a 10% solution of formalin, embedded in paraffin, stained with hematoxylin and eosin, and evaluated under a light microscope.

OTHER:
Hormones investigation: At the end of experiment (day 20 of gestation) blood samples were taken from hearts of all females. The level of thyroxine (T4), triiodothyronine (T3) and thyroid stimulating hormone (TSH) in serum was determined.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes.
- Number of implantations: Yes.
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: The non-gravid uteri were stained using ammonium sulphide in order to confirm the non-pregnant status.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No

Statistics:

The normality of distribution with the Shapiro-Wilk test was examined and the homogeneity of variance with the Brown-Forsythe test. If the test results were characterized by normal distribution and homogeneous variances, a one-way analysis of variance was used (ANOVA), if necessary confirmed with Dunnett’s test. In the absence of normality of distribution or non-homogeneous variances, the nonparametric Kruskal-Wallis test was used, if necessary confirmed with Dunnett’s test.
Statistical analyses were performed with the use of STATISTICA 10, with p ≤ 0.05. Numerical results were evaluated using the litter as the unit for data analysis. Only females whose pregnancy status was confirmed were taken into consideration for statistical analyses.

Indices:

Preimplantation loss [%] = number of corpora lutea - number of implants/ number of corpora lutea x 100
Postimplantation loss [%] = number of implants - number of viable fetuses/ number of implants x 100
AGD index = AGD /fetal body weight x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs and changes like passive urination, respiratory murmurs, lymphadenophaty or slight decrease of locomotor activity and dejection occurred only in females of group 3. There were no similar cases of changes in control group or in lower doses at group 1 and 2. Changes on coat were observed only in one animal in group 1 and only in two non-pregnant females in group 2. Similar change was observed in two pregnant females in group 0, so they could not be connected with test item.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All females survived the period of the experiment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Average body weight of pregnant females of group 1 and group 2 was comparable with average body weight of pregnant females of control group during the entire experiment. Average body weight of pregnant females of group 3 was statistically significant lower from day 8 to day 20 of the experiment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Average food consumption of pregnant females of group 1 was comparable with average food consumption of pregnant females of control group during the entire experiment.
Average food consumption of pregnant females was statistically significant lower in group 2 from day 9 to 11 and in group 3 from day 6 to 14 of the experiment compared to the control group.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

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):

There were no statistically significant changes in absolute and relative weight of thyroids with parathyroids as well as in absolute weight of uterus with cervix in group 1, 2, and 3 compared to the control group 0. However, statistically significant increase in the relative weight of uterus with cervix was stated in group 2 and 3 compared to the control group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Pathological lesions were stated in spleen as enlargement of the organ in 10 females (9 pregnant and 1 non- pregnant) of group 2, as well as in 2 females of group 3 and significant enlargement of the spleen stated in 23 females (18 pregnant and 5 non- pregnant) of group 3.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The histopathological examination of thyroids with parathyroids revealed the presence of several lesions, among others the hypertrophy of follicular cells, which is common lesion seen in toxicity studies in rats. The highest number of thyroids hypertrophy of follicular cells was observed at the dose 15 mg/kg, however the number of cases did not increase in higher doses and no dose-related response was visible. What is more, this kind of change was observed in some females of control group. Since, there are no statistically significant changes in the level of TSH, TT4 and TT3 concentrations in treated groups compared to the control group and no clinical symptoms of thyroid function disruption, the thyroids hypertrophy of follicular cells was not considered as an adverse effect of the test item action.
So-called background lesions, i.e. lesions often observed and related to species, gender, age or environmental conditions, were seen. These lesions, among others, follicular epithelium hyperplasia and C-cell hyperplasia, should not be associated with the test item administered to animals. The lack of association with the influence of the test item was also evidenced by the presence of the lesions in the control groups, similar number of observed cases and similar degrees of severity of these lesions in animals from all groups.
Other lesions were considered as toxicologically irrelevant.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Hormonal testing, i.e. TSH, TT4, TT3 concentration of pregnant females did not reveal any statistically significant changes in treated groups compared to the control group.

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

No abortions ocurred in any dose group.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

The index for preimplantation loss increased in group 1 (9.51%) compared with control group (5.77%), but the high increase was mainly due to 1 litter (female no. 20), out of 24 litters from pregnant females of group 1, with marked difference between the number of corpora lutea and implantation sites. Since it was singular case, which influenced the whole group result, the increase of preimplantation loss index was regarded as incidental and not test item- related.
The index for postimplantation loss increased in group 3 (9.91%) compared with control group (7.16%). It also corresponds with the highest number of resorptions stated in group 3 (28 early resorptions in group 3 compared with 22 ones in control group). Although the difference is not big, the effect could not be excluded as related to treatment.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There was no loss of total litter in any dose group.

Early or late resorptions:

no effects observed

Description (incidence and severity):

No statistically significant difference was stated in the number of early resorptions in all treated groups compared with the control group.
There was no late resorption found in treated or control groups.

Dead fetuses:

no effects observed

Description (incidence and severity):

Only 1 dead fetus out of 319 was found in control group. No dead fetuses were found in treated groups.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

One female in control group and one female in group 1 were not pregnant, five female in group 2 and five female in group 3 were not pregnant.
All uteri from non-pregnant females were stained using ammonium sulphide in order to confirm the non-pregnant status and in all of them there were no implantation sites. This phenomenon is considered to be completely natural, as for every 25 females who have sperm in a vaginal smear and are considered pregnant, there are 1 to 5 non-pregnant females (no implantation sites identified during necropsy).

Other effects:

no effects observed

Effect levels (maternal animals)

1

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

food consumption and compound intake

gross pathology

organ weights and organ / body weight ratios

2

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

food consumption and compound intake

gross pathology

organ weights and organ / body weight ratios

Remarks on result:

other: Effects observed at the medium tested dose of 100.5 mg/kg bw/day.

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was no statistically significant difference in weight of the fetuses with placenta and fetal membranes in all treated groups in comparison with the control group.
The weight of the fetuses without placenta and fetal membranes, with and without sexes combined, was statistically significantly smaller in group 3 compared with the control group.
The weight of placenta was statistically significantly greater in fetuses of group 3, with and without sexes combined, compared with the control group.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Only 1 dead fetus out of 319 was found in control group. No dead fetuses were found in treated groups.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no statistically significant differences observed in the sex ratio of fetuses between the treated groups and the control group.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

No statistically significant differences regarding the number of fetuses in the litter between groups 1, 2, 3 and control group were stated.

Changes in postnatal survival:

not examined

External malformations:

effects observed, treatment-related

Description (incidence and severity):

Subcutaneous hemorrhages in different parts of the fetal bodies occurred in the treated groups and in the control group and should be considered as typical disturbances in fetuses.
Significantly smaller fetuses were found in control group (1 fetus ), group 1 (3 fetuses) and group 3 (4 fetuses), and although the smaller fetuses were found in almost all groups (except group 2), the biggest number of cases in group 3 may be linked with the decrease of fetal body weight, which was statistically significant in group 3 and interpreted as test item- related.
Disproportion of length of forelimbs was stated in 1 fetus of group 2 and due to presence of 1 case also in control group it was not linked with the test item.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

No statistically significant differences of ossification points in sternum, metacarpus and metatarsus were stated in groups treated with the test item compared with the control group.
The total number of all lesions in skeletal system in group 1 and group 2 is comparable to the control group. However, the total number of all lesions in skeletal system at group 3 is visibly higher compared to the control group and it was considered as a test item-related change.
The summary of percentage of fetuses with a given number of ossification points in sternum showed the lowest value of 63.70% fetuses with 6 ossification points and the highest value of 16.77% fetuses with 4 ossification points in sternum in group 3 compared with the control group (the values for the fetuses with 6 and 4 ossification points in sternum in control group are 79.33% and 3.20% respectively), what was interpreted as test item-related.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

Significantly bigger placenta in fetuses of group 3 found during gross examination in 5 fetuses corresponds with the statistically significant increase of the weight of placenta in group 3, which was interpreted as test item- related.
Polyhydramnios (increased volume of amniotic fluid) was stated only in 1 fetus in group 1 and due to singular occurrence was interpreted as incidental finding, not related with the test item action.
Cases of fused placenta of two fetuses were stated in treated groups 1 ,2 and 3 (1 in each group), but although the lesion was not present in control group, it was observed in other prenatal developmental toxicity studies performed at the lab in control groups, so it was not interpreted as test item- related, but rather as normal incidental finding.
One fetus of group 3 had petechiae in lungs stated. Such lesions are usually euthanasia, agonal or death- related events with no relations to the test item action.
One case of scoliosis in fetus of group 3, due to singular occurrence, was interpreted as incidental finding, not related with the test item.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The length of fetuses was comparable between treated and control groups and slight differences were not statistically significant, so the test item did not influence this parameter.
Although the anogenital distance in fetuses was not statistically different among treated groups and control, anogenital index (with and without sexes combined) was statistically significantly higher in group 3 compared with the control group. However, it may be a secondary effect of the test item influence on the fetuses weight at the highest dose (in group 3), as AGD index rises with the decrease of weight.

Effect levels (fetuses)

1

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

skeletal malformations

other: placenta weight changes

2

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

skeletal malformations

other: placenta weight changes

Remarks on result:

other: Effects observed at the highest tested dose of 673.4 mg/kg bw/day.

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

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

Table 1. WASOX-VMAC2. Results of mating

Parameter	Number of females
	Group 0	Group 1	Group 2	Group 3
Number of females per group	25	25	25	25
Number of pregnant females	24	24	20	20
Number of females for evaluation	24	24	20	20

Table 2. WASOX-VMAC2- Clinical signs in females

Parameter	Group
	0	1	2	3
Number of females per group	25	25	25	25
thinning of coat of forearms	1 [13]–(18^th-20^th day)	-	-	-
thinning of coat on the left side of torso	1 [25]–(17^th-18^th day)	-	-	-
alopecia and scabs on the left side of torso	1 [25]–(19^th-20^th day)	-	-	-
alopecia on forearms and abdomen	-	1 [14]–(17^th-18^th day)	-	-
alopecia on forearms, abdomen and left side of torso	-	1 [14]–(19^th-20^th day)	-	-
thinning of coat of forearms	-	-	2 N [17 N]–(17^th-20^thday) [21 N]–(17^th-20^thday)	-
passive urination	-	-	-	13 and 4 N [1 N] – (14^th-20^thday) [2] – (20^thday) [3] – (14^th-20^thday) [4] – (14^th-15^thday) [6] – (14^th-20^thday) [7] – (9^th-20^thday) [8] – (8^th-20^thday) [10 N] – (17^th-20^thday) [12] – (14^th-20^thday) [13] – (8^th-20^thday) [14] – (11^th-20^thday) [15 N] – (11^th-20^thday)[16 N] – (11^th-20^thday) [17] – (14^th-20^thday) [20] – (11^th-14^thday) [22] – (19^th-20^thday) [23] – (11^th-17^thday)
respiratory murmurs	-	-	-	4 and 1 N [9 N] – (17^th-18^thday) [12] – (19^th-20^thday) [17] – (14^th-16^thday) [19] – (12^th-13^thday) [21] – (9^th-12^thday)
slight decrease of locomotor activity, dejection	-	-	-	1 [21] – (9^th-12^thday)
lymphadenopathy of submandibular lymph nodes	-	-	-	5 [17] – (10^th-20^thday) [18] – (10^th-20^thday) [20] – (10^th-20^thday) [21] – (10^th-20^thday) [22] – (11^th-20^thday)
lymphadenopathy of axillary, submandibular and popliteal lymph nodes	-	-	-	1 [19] – (10^th-20^thday)
Body weight loss – pregnant females	4 [5]–(day 8–6g) [13]–(day8–1g)   [21]–(day8–1g) [24]–(day 14– 6g)	3 [8]–(day 11–1g)   [22]–(day11–2g) [23]–(day14–5g)	3 [20]–(day8–1g) [22]–(day8–1g) [24]–(day8–1g)	20 [2]–(day 8–30g, day11–6g) [3]–(day 8–3g, day11–21g) [4]–(day 8–25g, day11–7g) [5]–(day 8–35g) [6]–(day 8–11g, day11–7g) [7]–(day 8–15g, day11–3g) [8]–(day 8–14g, day11–22g) [11]–(day 8–20g, day11–3g) [12]–(day 8–16g, day11–4g) [13]–(day 8–24g, day11–4g) [14]–(day 8–26g, day11–7g) [17]–(day8–26g) [18]–(day8–15g) [19]–(day 8–25g, day 11–8g) [20]–(day 8–13g, day 11–20g) [21]–(day8–23g) [22]–(day8–20g) [23]–(day 8–13g, day 11–5g) [24]–(day 8–25g) [25]–(day 8–19g, day 11–3g)
Body weight loss – non-pregnant females	1 N [14]–(day 11– 1g, day 14–6g, day 17–2g)	1 N [19]–(day20– 13g)	5 N [8]–(day 14–6g, day 17–3g) [13] –(day 8–2g,day 17 –4g) [14]  –(day 8–2g, day 11–3g, day 14–4g day17-3g, day20-8g) [17]–(day 5–13g, day 8–6g, day 14–5g, day 20-3g) [21]–(day 8–4g, day 14–10g, day 17–4g)	5 N [1]–(day 8–8g, day 11–4g) [9]–(day 8–22g, day 11–9g) [10]–(day 8–26g, day 11–16g) [15]–(day 8–19g, day 20–8g) [16]–(day 8–7g, day 11–10g, day 20–2g)

[ ] computer numbers of females; N - nonpregnant female

( ) days in which the changes were noticed

Table 3. WASOX-VMAC2. Body weight of females [g]

Day of gestation	Group
	0 n=24	1 n=24	2 n=20	3 n=20
0	249.00 ± 18.90	248.84 ± 19.19	249.84 ± 19.76	249.60 ± 13.55
5	262.96 ± 20.14	263.75 ± 17.76	262.40 ± 18.40	262.90 ± 15.46
8	269.71 ± 19.41	270.13 ± 20.17	268.10 ± 20.19	242.30 ± 17.00 *
11	283.29 ± 20.60	281.75 ± 19.73	279.70 ± 20.23	237.50 ± 17.84 *
14	297.38 ± 21.02	295.17 ± 22.34	295.00 ± 22.38	261.25 ± 22.07 *
17	325.38 ± 23.97	321.92 ± 25.78	321.90 ± 25.90	295.25 ± 23.89 *
20	363.88 ± 30.04	357.67 ± 28.69	361.65 ± 30.37	330.55 ± 26.30 *

 µ ± SD

* statistically significant difference with p ≤ 0.05

Table 4. WASOX-VMAC2. Food consumption [g/100g of b.w./day]

Day of gestation	Group
	0 n=24	1 n=24	2 n=20	3 n=20
0 – 5	7.07 ± 0.52	6.93 ± 0.71	6.96 ± 0.44	6.81 ± 0.55
6 – 8	6.43 ± 0.52	6.43 ± 0.63	6.18 ± 0.70	3.24 ± 0.66 *
9 – 11	6.47 ± 0.47	6.39 ± 0.54	5.91 ± 0.47 *	2.39 ± 1.27 *
12 – 14	6.78 ± 0.64	6.62 ± 0.53	6.41 ± 0.67	5.16 ± 1.48 *
15 – 17	6.44 ± 0.57	6.21 ± 0.68	6.21 ± 0.57	6.04 ± 0.95
18 – 20	6.14 ± 0.49	6.03 ± 0.73	6.18 ± 0.65	6.04 ± 0.64

 µ ± SD

* statistically significant difference with p ≤ 0.05

Table 5. Results of hormonal examinations - pregnant females

Group/ dose [mg/kg b.w.]	Number of evaluated pregnant females	TT3 [ng/ml]	TT4 [ng/ml]	TSH [ng/ml]
0/ 0	24	0.83 ± 0.50	19.16 ± 3.40	1.84 ± 0.58
1/ 15	24	0.82 ± 0.47	20.02 ± 5.51	1.92 ± 0.54
2/ 100.5	20	0.88 ± 0.64	18.68 ± 3.93	2.09 ± 0.60
3/ 673.4	20	1.05 ± 0.56	17.56 ± 2.77	2.17 ± 0.50

 µ ± SD

Table 6. Results of hormonal examinations - non pregnant females

Group/ dose [mg/kg b.w.]	Number of evaluated non- pregnant females	TT3 [ng/ml]	TT4 [ng/ml]	TSH [ng/ml]
0/ 0	1	1.00	25.10	1.63
1/ 15	1	1.10	26.43	3.62
2/ 100.5	5	1.47 ± 0.37	30.12 ± 7.96	3.32 ± 0.54
3/ 673.4	5	1.18 ± 0.33	26.66 ± 5.29	2.77 ± 1.02

 µ ± SD

Table 7. Gross lesions in females

Examined organ	Type of change	Group / dose [mg/kg b.w.]
		0/ 0 n=25	1/15 n=25	2/ 100.5 n=25	3/ 673.4 n=25
	enlargement	-	-	10 [4, 5, 6, 7, 10, 19, 20, 21* 24, 25]	2 [11, 12]
Spleen					23 [1*, 2, 3, 4, 5, 6,
	significant enlargement	-	-	-	7, 8, 9*, 10*,13, 14, 15*, 16*,17,
					18, 19, 20, 21, 22,
					23, 24, 25]
Female non-pregnant		1	1	5	5
		[14]	[19]	[8, 13, 14, 17, 21]	[1, 9, 10, 15, 16]

[ ] – computer number of animals with gross lesion

n – number of females

* - female non-pregnant

Table 8. Absolute and relative weight of thyroids with parathyroids and uterus in pregnant females

Organ	Group / dose [mg/kg b.w.]
	0/ 0 n=24	1/15 n=24	2/ 100.5 n=20	3/ 673.4 n=20
Absolute weight of organs [mg]
Thyroids with parathyroids	20.08 ± 3.11	21.63 ± 2.96	21.65 ± 4.03	20.70 ± 4.77
Uterus	4579.38 ± 553.92	4448.50 ± 731.75	5007.45 ± 759.51	4941.75 ± 662.53
Relative weight of organs [%]
Thyroids with parathyroids	0.006 ± 0.001	0.006 ± 0.001	0.006 ± 0.001	0.006 ± 0.001
Uterus	1.26 ± 0.14	1.24 ± 0.16	1.38 ± 0.14*	1.50 ± 0.18*

 µ ± SD

n- number of evaluated pregnant females

* statistically significant difference with p ≤ 0.05, Dunnett's test

Table 9. Average absolute and relative weight of thyroids with parathyroids in non- pregnant females

Group/dose [mg/kgb.w.]	Number of evaluated non- pregnant females	Absolute weight of thyroid with parathyroid [mg]	Relative weight of thyroidwith parathyroid [%]
0/ 0	1	18.00	0.007
1/ 15	1	18.00	0.008
2/ 100.5	5	20.00 ± 3.54	0.008 ± 0.002
3/ 673.4	5	22.20 ± 2.05	0.008 ± 0.001

 µ ± SD

Table 10. Histopathological lesions in pregnant females

Examined organ	Type ofchanges	Degree of severity(numerical score)	Group / dose mg/kg b.w. / sex / number of animals / [computer number of animals with lesions]
			0/0	1/15	2/100.5	3/673.4
			n=24	n=24	n=20	n=20
	Follicle epithelium, hyperplasia	Minimal (1)	6 [7,8,9,15,22,24]	3 [9,10,18]	7 [1,5,10,11,12,19, 24]	5 [17,21,22,23,25]
		Slight (2)	-	-	1 [2]	-
	C-cell, hyperplasia	Minimal (1)	8 [3,6,7,9,11,19, 22,23]	12 [1,3,6,9,10,11,14, 17,20, 21,22,23]	4 [12,18,19,24]	3 [17,20,22]
		Slight (2)	2 [20,25]	-	-	-
			6	9	9	8
		Minimal (1)	[8,12,17,21,24,	[2,3,4,9,14,16,20,	[1,3,5,9,10,11,	[4,5,6,12,17,20,
Left thyroid +	Follicular cell, hypertrophy		25]	22,24]	15,23,24]	22,25]
		Slight (2)	5 [3,6,9,20,22]	10 [6,7,8,10,11,12, 17,18,23,25]	4 [7,18,20,22]	7 [7,11,13,14,18, 21,23]
		Moderate (3)	-	1 [13]	-	-
parathyroid
		Minimal (1)	5	4	5	5
	Follicle epithelium, exfoliation		[6,10,21,24,25]	[4,12,14,16]	[3,7,18,23,24]	[6,8,14,17,20]
		Slight (2)	3 [5,12,20]	3 [2,18,25]	5 [4,9,11,20,22]	2 [12,19]
		Moderate (3)	3	1	2	3
			[15,22,23]	[22]	[10,16]	[3,4,24]
	Parathyroid,	Present (+)	1	1	1	-
	lack of		[8]	[25]	[4]
	Thyroid,	Present (+)	2	1	1	1
	ectopic tissue		[9,25]	[16]	[24]	[24]
	Thyroid, cyst,	Present (+)	-	-	-	1
	congenital					[7]
	Follicle epithelium, hyperplasia	Minimal (1)	3 [12,13,20]	1 [11]	2 [2,18]	8 [4,13,17,19,20, 23,24,25]
	Follicle,	Minimal (1)	1 [19]	-	-	-
	degeneration
	Fibrosis	Present (+)	-	-	1	-
					[9]
	C-cell, hyperplasia	Minimal (1)	10 [2,3,5,8,10,11, 12,18,21,22]	8 [1,2,11,12,13,14, 16,21]	5 [2,9,11,24,25]	5 [8,18,19,23,25]
Right thyroid + parathyroid	Follicular cell, hypertrophy	Minimal (1)	8 [3,4,9,12,13,16, 22,24]	12 [1,4,6,7,11,12,13, 14,15,16,20,23]	8 [1,2,3,5,6,7,22, 25]	4 [19,21,23,25]
		Slight (2)	1 [20]	3 [3,10,24]	6 [4,11,18,19,23, 24]	11 [5,6,11,12,13,14, 17,18,20,22,24]
		Moderate (3)	-	4	1	1
				[8,9,17,18]	[20]	[7]
		Minimal (1)	1 [10]	3 [6,13,20]	4 [2,5,16,24]	2 [13,21]
	Follicle epithelium, exfoliation	Slight (2)	6 [5,9,18,21,22,24]	4 [12,14,22,25]	6 [9,10,11,15,22, 23]	8 [2,5,6,8,12,19, 23,24]
		moderate	3	1	1	2
		(3)	[7,15,23]	[5]	[20]	[3,20]
	Syncytial giant cells, parathyroid	minimal (1)	-	1 [7]	-	-
	Follicle,	present	1	-	-	-
	pigment	(+)	[9]
	Thyroid, ectopic tissue	present (+)	3 [18,19,24]	4 [4,7,22,25]	2 [16,20]	1 [17]
	Parathyroid, lack of	present (+)	4 [3,13,20,22]	1 [13]	-	1 [20]
	Parathyroid, hyperplasia, diffuse	present (+)	1 [10]	-	-	-
	Thyroid, cyst,	present	-	-	1	1
	congenital	(+)			[23]	[20]

 

Table 11. Histopathological lesions in non pregnant females

Examined organ	Type of changes	Degree of severity(numerical score)	Group / dose mg/kg b.w. / sex / number of animals / [computer number of animals with lesions]
			0/0	1/15	2/100.5	3/673.4
			n=1	n=1	n=5	n=5
Leftthyroid + parathyroid	Follicle epithelium, hyperplasia	Minimal (1)	-	-	1 [17]	-
		Slight (2)	-	1 [19]	-	-
	C-cell, hyperplasia	Minimal (1)	-	-	2 [13,14]	-
	Follicular cell, hypertrophy	Minimal (1)	1 [14]	-	2 [14,17]	1 [16]
		Slight (2)	-	-	1 [21]	3 [9,10,15]
		Moderate (3)	-	-	-	1 [1]
	Follicle epithelium, exfoliation	Minimal (1)	-	-	1 [14]	2 [9,16]
		Slight (2)	-	1 [19]	1 [17]	-
Right thyroid + parathyroid	Follicleepithelium, hyperplasia	Minimal (1)	-	-	1 [21]	1 [15]
	C-cell, hyperplasia	Minimal (1)	-	-	2 [8,14]	-
	Follicular cell, hypertrophy	Minimal (1)	-	1 [19]	-	-
		Slight (2)	-	-	2 [14,17]	4 [1,9,10,15]
		Moderate (3)	-	-	1 [13]	-
	Follicle epithelium, exfoliation	Minimal (1)	-	-	1 [14]	2 [9,15]
		Slight (2)	-	1 [19]	1 [17]	-
		Moderate (3)	-	-	1 [21]	1 [16]
	Thyroid, ectopic tissue	Present (+)	1 [14]	-	-	-
	Parathyroid, lack of	present (+)	-	-	1 [21]	1 [16]

Table 12. Number of fetuses

Number of fetuses	Group / dose [mg/kg b.w.]
	0/ 0	1/ 15	2/ 100.5	3/ 673.4
all	319	303	282	266
dead	1	0	0	0
min.–max. in litter	8 - 17	4 - 16	8 - 18	9 - 17
average in litter	13.29 ± 2.61	12.63 ± 2.92	14.10 ± 2.57	13.30 ± 2.70
females	7.21 ± 2.11	6.33 ± 2.48	7.60 ± 2.19	6.95 ± 2.54
males	6.08 ± 2.21	6.29 ± 2.61	6.50 ± 2.48	6.35 ±2.21

 µ ± SD

Table 13. Number of corpora lutea, implantations, resorptions, pre- and postimplantations losses

Number of		Group / dose [mg/kg b.w.]
		0/ 0 n=24	1/ 15 n=24	2/ 100.5 n=20	3/ 673.4 n=20
Corpora lutea	all	363	354	303	303
	average	15.13 ± 1.73	14.75 ± 1.94	15.15 ± 2.03	15.15 ± 1.90
Implantations	all in pregnant females	342	319	296	294
	average in pregnant females	14.25 ± 2.11	13.29 ± 3.04	14.80 ± 2.21	14.70 ± 2.18
	all in non- pregnant females	0	0	0	0
Resorptions (early)	all	22	16	14	28
	min – max in female	0 – 4	0 – 3	0 – 6	0 – 4
	average in pregnant females	0.92 ± 1.18	0.67 ± 0.87	0.70 ± 1.42	1.40 ± 1.19
Preimplantation loss %		5.77	9.51	2.41	2.96
Postimplantation loss %		7.16	4.76	4.89	9.91

 µ ± SD

n- number of evaluated pregnant females

Table 14. Average weight of fetuses and placenta [g]

Group/dose [mg/kg b.w.]	Number of examined fetuses	Weight of fetuses		Weight of placenta
		with placenta and fetal membranes	without placenta and fetal membranes
0/ 0	318	4.67 ± 0.21	3.45 ± 0.15	0.49 ± 0.05
1/ 15	303	4.59 ± 0.19	3.42 ± 0.17	0.48 ± 0.04
2/ 100.5	282	4.67 ± 0.22	3.43 ± 0.18	0.50 ± 0.04
3/ 673.4	266	4.57 ± 0.27	3.18 ± 0.25*	0.63 ± 0.09*

µ ± SD

* statistically significant difference with p ≤ 0.05, Dunnett's test

Table 15. Average weight of fetuses and placenta / sex [g]

Group/dose [mg/kg b.w.]	Weight of fetuses				Weight of placenta
	with placenta and fetal membranes		without placenta and fetal membranes
	♂	♀	♂	♀	♂	♀
0/ 0	4.79 ± 0.22	4.58 ± 0.24	3.53 ±0.18	3.38 ± 0.17	0.50 ± 0.06	0.48 ± 0.05
1/ 15	4.73 ± 0.19	4.49 ± 0.20	3.50 ± 0.20	3.34 ± 0.17	0.49 ± 0.05	0.47 ± 0.04
2/ 100.5	4.76 ± 0.26	4.59 ± 0.22	3.49 ± 0.22	3.37 ± 0.19	0.52 ± 0.06	0.49 ± 0.04
3/ 673.4	4.66 ± 0.32	4.48 ± 0.27	3.26 ± 0.28*	3.10 ± 0.26*	0.64 ± 0.10*	0.63 ± 0.09*

µ ± SD

* statistically significant difference with p ≤ 0.05, Dunnett's test

Table 16. Average length of fetuses [cm]

Group/dose [mg/kg b.w.]	Number of examined fetuses	Length of fetuses
		with tail	without tail
0/ 0	318	4.88 ± 0.15	3.63 ± 0.12
1/ 15	303	4.87 ± 0.18	3.60 ± 0.12
2/ 100.5	282	4.94 ± 0.15	3.60 ± 0.13
3/ 673.4	266	4.79 ± 0.18	3.55 ± 0.15

 µ ± SD

Table 17. Average anogenital distance

Group/dose [mg/kg b.w.]	Number of examined fetuses	Anogenital distance AGD [mm]	anogenital index AGD index
0/ 0	318	2.45 ± 0.15	71.39 ± 5.63
1/ 15	303	2.49 ± 0.17	73.29 ± 6.20
2/ 100.5	282	2.45 ± 0.13	71.72 ± 5.87
3/ 673.4	266	2.49 ± 0.16	79.50 ± 8.97*

µ ± SD

* statistically significant difference with p ≤ 0.05, Dunnett's test

Table 18. Average anogenital distance / sex

Group/dose [mg/kgb.w.]	Anogenital distance AGD [mm]		Anogenital index AGD index
	♂	♀	♂	♀
0/ 0	2.97 ± 0.13	2.02 ± 0.06	84.74 ± 6.26	60.26 ± 3.21
1/ 15	2.97 ±0.09	2.03 ± 0.07	85.26 ± 5.64	61.50 ± 5.04
2/ 100.5	2.99 ± 0.04	2.00 ± 0.00	86.29 ± 5.36	59.66 ± 3.37
3/ 673.4	2.94 ± 0.11	2.06 ± 0.08	91.93 ± 8.66*	67.27 ± 5.33*

µ ± SD

* statistically significant difference with p ≤ 0.05, Dunnett's test

Table 19. Lesions in fetuses

Pathological changes	Number of fetuses
	Group 0 0 mg/kg b.w. n=319	Group 1 15 mg/kg b.w. n=303	Group 2 100.5 mg/kg b.w. n=282	Group 3 673.4 mg/kg b.w. n=266
Hemorrhage on left hindlimb	1 [3]	-	-	-
Hemorrhage on interscapular region	-	1 [17]	-	2 [7,22]
Hemorrhage on the tail	1 [7]	2 [2x25]	1 [3]	1 [8]
Significantly bigger placenta	-	-	-	5 [11,13,20, 2x21]
Fetus significantly smaller	1 [17]*	3 [3,17,18]	-	3 [6,13,18]
Polyhydramnios	-	1 [20]	-	-
Fused placenta*	-	1 [17]	1 [1]	1 [x3]
Disproportion in length of forelimbs	1 [17]	-	1 [4]	-
Right lung- petechiae in caudal lobe	-	-	-	1 [21]
Scoliosis	-	-	-	1 [6]

n- number of evaluated fetuses; [_] computer numbers of females; *-dead fetus 2x- number of fetuses from one female, with the same lesion, for example:

[1] – lesion stated in one fetus from female no. 1; [2x1]- lesion stated in 2 fetuses from female no. 1

* Fused placenta – one common placenta for two fetuses from one female

Table 20. Changes in skeletal system

Lesions		Number of fetuses with lesions
		Group 0 0 mg/kg b.w.	Group 1 15 mg/kg b.w.	Group 2 100.5 mg/kg b.w.	Group 3 673.4 mg/kg b.w.
STERNUM	2^ndossification	-	-	-	3
	point unilateral				[5,13,25]
	3^rdossification	-	1	-	-
	point bipartite		[18]
	4^thossification	-	1	-	1
	point bipartite		[1]		[5]
	4^thossification	2	-	-	-
	point misaligned	[1, 3]
	5^thossification	-	2	-	3
	point bipartite		[8,25]		[6,12,25]
	5^thossification point unilateral	17 [2,3, 5x4,5, 2x6, 9,15, 2x16,17, 18,23]	7 [2,3,6,15,16, 23,25]	18 [2,4, 2x6, 3x10, 12, 4x19,23, 4x24,25]	15 [3x2,3, 2x8,14, 17, 2x18,20,21, 22,24,25]
		33	42	38	42
		[2x1, 4x2,3, 3x4,	[1, 5x2, 3x3, 4x4,	[1, 3x2, 5x5, 2x9,	[6x2, 2x3,4, 3x5,
	6^thossification	5x5, 2x6, 4x8,	2x6,8, 2x9,10,	2x10, 2x11, 4x12,	2x6, 4x8,11,12,
	point bipartite	3x10,13,18,	12, 2x13,15,	15, 2x16, 2x18,	4x14, 2x17, 2x18,
		3x19,21,22,	6x17, 5x22, 6x23,	19, 3x22, 5x23,	2x19, 2x20, 2x21,
		2x24]	24,25]	24, 4x25]	2x22, 3x24, 3x25]
					21
	6^thossification point unilateral	6 [4,6, 2x9,15,20]	7 [3, 2x8,14,15, 17,25]	9 [1,3, 3x6,11,12, 23,25]	[4x3,6, 2x8, 2x11,12,13,17, 2x18, 2x21,
					4x22,25]
	lack of 1^st	-	1	-	-
	ossification point		[3]
	lack of 2^nd	2	3	-	3
	ossification point	[19,21]	[1,3,18]		[6,18,19]
	lack of 3^rd	-	1	-	-
	ossification point		[3]
	lack of 4^th	-	2	-	1
	ossification point		[3,18]		[18]
	lack of 5^thossification point	22 [1, 3x2, 2x3,4, 4x5,8,9,2x13, 2x16, 18,2x19, 21,24]	18 [1,3,4, 2x5,8, 2x11,13,3x16, 3x17,18,20,25]	28 [5x1, 3x2, 3x3,4, 2x5, 3x6,7,10, 4x18,19,23, 3x25]	38 [2, 4x3,5, 5x6, 7, 3x8, 7x13, 2x14, 3x17,18, 3x19, 3x20,21, 2x24,25]
					44
	lack of 6^thossification point	20 [5x3,7,8,11, 4x16, 2x18,19, 20, 2x21,2x23]	25 [1,2,3,5,6, 3x8, 9, 3x11,13,14, 15, 3x16,17,18, 20, 4x25]	18 [2x1, 2x5,3x6,12, 5x18, 2x23, 3x24]	[2x3,5, 6x6,8, 2x11, 8x13,14, 3x17,4x18, 3x19,5x20, 3x21, 2x22,24,
					2x25]
	asymmetrically	-	1	-	-
	located sternum		[1]
OTHER LESIONS	Skull: bipartite ossification of supraoccipital plate	-	1 [1]	-	-
	Vertabral column: thoracic vertebrae - asymmetrically located ossification points in the center of the: 6^th, 8^thand 10^thossification point; bipartite 7^thand dummbell-shaped 9^thossification point	-	1 [1]	-	-
Number of all lesions		102	113	111	171

[ ] computer numbers of females; 2x, 3x etc.- number of fetuses from one female, with the same lesion

for example: [1] – lesion stated in one fetus from female no. 1; [2x1]- lesion stated in 2 fetuses from female no. 1

Table 21. Percentage of fetuses with a given number of ossification points in sternum (%)

Group/dose [mg/kgb.w.]	Sternum ossification points
	0	1	2	3	4	5	6
0/ 0 n=170	-	-	-	1.04	3.20	16.43	79.33
1/15 n=163	0.52	-	0.46	1.04	8.17	12.49	77.32
2/ 100.5 n=152	-	-	-	-	5.14	19.93	74.93
3/ 673.4 n=140	-	-	0.63	0.63	16.77	18.27	63.70

n- number of evaluated fetuses

Table 22. Percentage of fetuses with a given number of ossification points in limbs (%)

Group/dose [mg/kg b.w.]	Metacarpus of forelimbs			Metatarsus of hindlimbs
	3	3.5*	4	3	4
0/ 0 n=170	10.66	2.70	86.64	-	100.00
1/15 n=163	18.90	6.04	75.06	0.98	99.02
2/ 100.5 n=152	14.16	7.25	78.59	-	100.00
3/ 673.4 n=140	14.58	7.56	77.86	-	100.00

n- number of evaluated fetuses

*value 3.5 means 3 ossification points in one limb and 4 in second limb

Table 23. Average number of ossification points of sternum and limbs

Group/dose [mg/kg b.w.]	Sternum	Metacarpus of forelimbs	Metatarsus of hindlimbs
0/ 0 n=170	5.74 ± 0.28	3.88 ± 0.13	4.00 ± 0.00
1/15 n=163	5.63 ± 0.37	3.78 ± 0.23	3.99 ± 0.03
2/ 100.5 n=152	5.70 ± 0.34	3.82 ± 0.24	4.00 ± 0.00
3/ 673.4 n=140	5.44 ± 0.46	3.82 ± 0.22	4.00 ± 0.00

n- number of evaluated fetuses

Applicant's summary and conclusion

Conclusions:

In a prenatal developmental toxicity study performed with WASOX-VMAC2 in Wistar rats, the NOAEL for maternal toxicity was considered to be 15 mg/kg bw/day and the NOAEL for developmental toxicity was considered to be 100.5 mg/kg bw/day.

Executive summary:

A prenatal developmental toxicity study was performed on the test substance WASOX-VMAC2 by oral administration in Wistar rats according to OECD guideline 414, under GLP conditions. The study was conducted on 100 females divided into 4 groups. Each group (G0, G1, G2 and G3) consisted of 25 females. From day 5 to 19 of gestation, the animals in G0 group were administered with vehicle (corn oil) and the animals in G1, G2 and G3 groups were administered with test item at the dose levels of 15, 100.5 and 673.4 mg/kg bw/day, based on results of a preliminary dose range finding study where some changes were observed in females at doses of 140 and 800 mg/kg bw/day and in fetuses at doses of 24.5, 140 and 800 mg/kg bw/day. Clinical observations for mortality and signs of toxic influence of the test item were performed in females, their body weight and food consumption were controlled. At the end of experiment (day 20 of gestation) blood samples were taken from all females in order to determine of thyroxine (T4), triiodothyronine (T3) and thyroid stimulating hormone (TSH). After blood sample collection, all females were euthanized and then subjected to caesarean section with gross examination. Additionally, absolute and relative weights of thyroid with parathyroids were determined and histopathological examinations were conducted. The number of fetuses in the litter, the number of resorptions, implantations and corpora lutea were determined. After removing the fetuses each gravid uteri with the cervix was weighed. The non-gravid uteri were stained using ammonium sulphide in order to confirm the non-pregnant status. Sex and anogenital distance, body weight with and without placenta, weight of placenta, body length with and without tail, fetal motility and reactions to tactile stimuli were determined in all fetuses. Each fetus was subjected to gross examination. Half of fetuses from each litter were subjected to evaluation of skeleton. The rest of them was evaluated for formation of body cavities and internal organs.

All females survived the period of the experiment. Clinical signs and changes like passive urination, respiratory murmurs, lymphadenophaty or slight decrease of locomotor activity and dejection occurred only in females of group 3. Average body weight of pregnant females of group 1 and group 2 was comparable with average body weight of pregnant females of control group during the entire experiment. Average body weight of pregnant females of group 3 was statistically significant lower from day 8 to day 20 of the experiment. Average food consumption of pregnant females was statistically significant lower in group 2 from day 9 to 11 and in group 3 from day 6 to 14 of the experiment compared to the control group. Hormonal testing, i.e. TSH, TT4, TT3 concentration of pregnant females did not reveal any statistically significant changes in treated groups compared to the control group. There were 24 pregnant females in group 0, 24 in group 1, 20 in group 2 and 20 in group 3. Pathological lesions were stated in spleen as enlargement of the organ in 10 females (9 pregnant and 1 non- pregnant) of group 2, as well as in 2 females of group 3 and significant enlargement of the spleen stated in 23 females (18 pregnant and 5 non- pregnant) of group 3. There were no statistically significant changes in absolute and relative weight of thyroids with parathyroids as well as in absolute weight of uterus with cervix in group 1, 2, and 3 compared to the control group. However, statistically significant increase in the relative weight of uterus with cervix was stated in group 2 and 3 compared to the control group. The histopathological examination of thyroids with parathyroids revealed the presence of several lesions, among others the hypertrophy of follicular cells, which is common lesion seen in toxicity studies in rats and was not considered related to treatment. No statistically significant differences regarding the number of fetuses in the litter, the number of males and females in the litter, the number of early resorptions, implantations and corpora lutea between treated and control groups were stated. There was no late resorption found in treated or control groups. The index for postimplantation loss increased in group 3 (9.91%) compared with control group (7.16%) and was considered as related to treatment. The weight of the fetuses without placenta and fetal membranes, with and without sexes combined, was statistically significantly smaller in group 3 compared with the control group. The weight of placenta was statistically significantly greater in fetuses of group 3, with and without sexes combined, compared with the control group. There was no statistically significant difference in average AGD with and without sexes combined in all treated groups in comparison with the control group, but there was statistically significant increase in AGD index stated in fetuses of group 3, with and without sexes combined, in comparison with the control group. No statistically significant differences of ossification points in sternum, metacarpus and metatarsus were stated in groups treated with the test item compared with the control group. The total number of all lesions in skeletal system in group 1 and group 2 is comparable to the control group. However, the total number of all lesions in skeletal system at group 3 is visibly higher compared to the control group and thus it was considered as a test item-related change. Based on these results, the NOAEL for maternal toxicity was considered to be 15 mg/kg bw/day and the NOAEL for developmental toxicity was considered to be 100.5 mg/kg bw/day.