Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 July 2020 – 02 December 2020
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
A few times the relative air humidity short-term exceeded 70% (four times during acclimatization and eight times during experiment). These changes did not influence the study course.
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
A few times the relative air humidity short-term exceeded 70% (four times during acclimatization and eight times during experiment). These changes did not influence the study course.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
tri{[(2S)-1-ethoxy-1-oxopropan-2-yl]oxy}(vinyl)silane
Molecular formula:
C17H30O9Si
IUPAC Name:
tri{[(2S)-1-ethoxy-1-oxopropan-2-yl]oxy}(vinyl)silane
Test material form:
liquid
Details on test material:
- Stabilisation: in water undergoes hydrolysis
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Analytical purity: 93%
- Expiry date: 22.04.2022

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: 13 to 15 weeks.
- Weight at study initiation: average body weights:
group 0: 241.1 g (207 g – 276 g)
group 1: 241.2 g (206 g – 282 g)
group 2: 241.4 g (213 g – 282 g)
group 3: 241.2 g (210 g – 282 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). Autoclaved and additionally 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.
- Water (e.g. ad libitum): ad libitum access to drinking tap water.
- Acclimation period: All animals were quarantined and observed for at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 23ºC
- Humidity (%): 44-83%. The relative air humidity short-term exceeded 70 %: 4 times during acclimatization and 8 times during the experiment. The changes did not influence the study course.
- Air changes (per hr): 15-20
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: Experimental starting date 29.07.2020 To: Experimental completion date 02.12.2020

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item formulation in the corn oil was prepared freshly every day before administration. It was protected against moisture, therefore all surfaces in the rooms and utensils for preparation and administration of formulations were dry. After merging the appropriate amount of the tested material with the appropriate amount of corn oil, the vessels with the solutions were placed on a magnetic stirrer in order to mix evenly. Room temperature was used for the preparation. During the administration, formulations were kept at room temperature and were thoroughly mixed every few minutes with a dry stirring rod or, if necessary, placed back on the magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): most suitable vehicle, as the test item hydrolyses in water.
- Concentration in vehicle: 7.5, 30 and 120 mg/mL.
- Amount of vehicle (if gavage): the total volume administered was 0.4 mL/100 g b.w. (including test item)
- Lot/batch no.: 20259
- Purity: 100 % corn oil
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
It was conducted by Łukasiewicz Research Network – Institute of Industrial Organic Chemistry, Branch Pszczyna. Department of Toxicological Studies Doświadczalna 27, 43-200 Pszczyna, Poland, which holds the Good Laboratory Practice Certificate GLP.
The test item formulation in the corn oil was prepared freshly every day before administration. Samples of the test item solutions (8.0 mg/g at dose of 30 mg/kg bw, 33.0 mg/g at dose of 120 mg/kg bw and 131.0 mg/g at dose of 480 mg/kg bw) were transferred thrice for chemical analysis: at the beginning, in the middle and at the end of the study.
TECHNIQUE AND TEST METHOD: Gas chromatography coupled with flame ionization detector (FID).
TEST PARAMETERS: Chromatographic column: DB-17 (30m × 0.32 mm) film 0.25 μm. Oven Temperature: 80 ºC (1 minute), gradient 35 ºC/minute, 260 ºC (4 minute), intel temperature: 175 ºC, detector temperature: 250 ºC; flow (splitless): 1.5 mL/min N2; injection volume: 2 µL.
METHOD VALIDATION: The validation of the method was determined according to SANCO/3029/99 rev. 4 (11/07/00). The mean recoveries at two fortification levels (5.0 mg/g and 50 mg/g) ranged from 96.6% to 100.6%, RSD values were below 10% and no significant peak occurred at the retention time, so absence of interference was confirmed.
QUANTITATIVE AND QUALITATIVE DETERMINATION: the mean recovery values for test samples ranged from 90.0 to 100.9%. All results were in the range from 80 to 120%. RSD values were below 10%. Therefore, the acceptance criteria for biological sample 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 four females the length of cohabitation was 2 days. For one female the length of cohabitation was 3 days. For two females the length of cohabitation was 4 days and for two females the length of cohabitation was 5 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:
29.07.2020 – 02.12.2020
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
G0 - Vehicle control
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
G1 - Low dose
Dose / conc.:
120 mg/kg bw/day (nominal)
Remarks:
G2 - Mid dose
Dose / conc.:
480 mg/kg bw/day (nominal)
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 on the basis of existing data from ‘Combined repeated dose toxicity study with the reproduction/ developmental toxicity screening test (according to OECD 422), in which at the doses of 100 and 300 mg/kg b.w. test item- related stillbirths and mortality of pups were stated. Bearing in mind longer time of exposition in the previous study, doses in the current study have been modified by increasing the interval between doses from 3 to 4- fold, (2 to 4- fold intervals are frequently optimal according to the Guideline OECD 414).

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day or once a day (on days off)
- Cage side observations checked: General condition of the animals, i.e. the observation of all animals for morbidity and mortality was conducted.

DETAILED CLINICAL OBSERVATIONS: Yes, evaluation of skin changes, coat changes, changes in eyes and mucous membranes, respiratory system, circulatory system, nervous system, somatic activity and behavior.
- 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 body weight determination.

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 absolute and relative weight determination and histopathological assessment of thyroid with parathyroids was conducted in all females.

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 (TT4), triiodothyronine (TT3) 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: Yes
- 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.
Blood sampling:
- Plasma: No
- Serum: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: yes
- Anogenital distance of all live rodent pups: yes.
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. Only the live fetuses data were statistically evaluated.
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, non-treatment-related
Description (incidence and severity):
Coat changes were observed only in 2 females in group 1 and in 1 female in group 2, and skin changes (scabs) were observed in 1 female in group 1. These changes should not be connected with the test item action. Similar changes were observed in 2 females of control group, and no coat or skin changes were observed in females of group 3 treated with the highest dose of the 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, non-treatment-related
Description (incidence and severity):
Average body weight of pregnant females of group 1, 2 and 3 was comparable with average body weight of pregnant females of control group during the entire experiment. Some decrease of individual body weight was observed in five pregnant females: two in group 2 and three in group 3. Although these cases were observed in treated groups in middle and the highest dose, the decrease concerned single animals and did not exceed 4 g. It was considered to be connected with stress and habituation of animals to administration and manual operation. Additionally, body weight loss was observed in every non-pregnant female of all groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
During the experiment, food consumption of the females of treated group 1, 2 and 3 did not differ statistically significantly from average food consumption of the females of 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
Endocrine findings:
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.
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):
There were no statistically significant changes in absolute and relative weight of thyroids with parathyroids as well as in absolute and relative weight of uterus with cervix in group 1, 2, and 3 compared to the control group 0.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There was one case of bloating cecum with enlarged lymphoid follicles stated in group 3, but due to single occurrence it is not considered as test-item related. In one female of group 2 vaginal purulent plug was observed, and since it was non- pregnant female it cannot be excluded that the failure in mating this female could be associated with the local inflammation, however due to single occurrence, it was not regarded as test-item related. There were also 3 cases of one uteri horn non-gravid, 2 females of group 0 and 1 of group 2.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The histopathological examination of thyroids did not reveal the presence of test-item related lesions.
The histopathological examination revealed the circulatory disorders (i.e. haemorrhage/s), which most likely represent euthanasia-related, agonal and/or death-related events and should not be connected with the test item. This can also be evidenced by varying degrees of severity in all groups (treated and controls).
During the histopathological examination, so-called background lesions were seen. Background lesions are often observed and related to species, gender, age or environmental conditions and should not be associated with the test item administered to animals. Such changes in the rat thyroid with parathyroid may include, among others: fibrosis, hypertrophy of follicular cells, follicular epithelium hyperplasia and C-cell hyperplasia. The lack of association with the influence of the test item is 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.
Lack of parathyroid in several pregnant females (6 from group 0, 2 from group 1, 2 from group 2 and 5 from group 3), total 18 per 100 when considering also non-pregnant females, could be a symptom of its aplasia or could result from technical processing of the specimen due to character of organ. Either way, due to small number of cases, presence also in control group and lack of toxicologically significant lesions in present parathyroids, it can be concluded that it has no influence on interpretation of results and conclusions of the study.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

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:
no effects observed
Description (incidence and severity):
There was no statistically significant difference in the number of implantations in all treated groups compared with the control group.
The indexes for preimplantation loss and postimplantation loss were comparable between treated groups and control group, with no statistically significant differences revealed.
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 in the number of resorptions in all treated groups compared with the control group was stated.
Dead fetuses:
no effects observed
Description (incidence and severity):
Low intrauterine mortality was stated in control group and in group 2, where respectively 1 dead fetus out of 304 and 1 dead fetus out of 282 was found.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
During gross examination lack of pregancy was stated in 2 females in control group, 3 females in group 1 and 2 and 6 females in group 3. 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 usually 1 to 5 non-pregnant females (no implantation sites identified during necropsy). In the current study there were up to 6 non-pregnant females per group. The number of females required by the Guideline OECD 414 was obtained, as according to the guideline each group should contain approximately 20 females with implantation sites at necropsy, but not less than 16.
Other effects:
no effects observed
Description (incidence and severity):
No statistically significant difference in the number of corpora lutea in all treated groups compared with the control group was stated.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 480 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No test item adverse effects observed at the highest dose tested.

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The differences between treated groups and control group in weight of the fetuses with and without placenta and fetal membranes and weight of placenta itself (with and without sexes combined) were statistically irrelevant.

Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Low intrauterine mortality was stated in control group and in group 2, where respectively 1 dead fetus out of 304 and 1 dead fetus out of 282 was found.
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.
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
The anogenital distance in fetuses as well as anogenital index (both with and without sexes combined) were not statistically different between treated groups and control group.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Subcutaneous hemorrhages in different parts of the fetal bodies which occurred in the treated groups and in the control group, were not considered as the effects of the test item, as hemorrhages in fetuses have often been observed in other prenatal developmental toxicity studies performed at the lab and should be considered as typical disturbances in fetuses.
The case of severe oedema of the body in one fetus of group 2 was identified as caused by the accidental injection connected with the anesthesia of the dam and therefore it was excluded from the statistical analysis of parameters that could be affected (weight, length and anogenital distance).
Cyanosis and oedema of the body stated in 1 dead fetus of group 2 was a natural phenomenon related to decay. The case of cranium oedema and dorsalis defection of the body was stated in 1 fetus of group 3, but due to singular occurrence it was not regarded as test-item related.
In one female of group 2 all three fetuses from the litter were significantly bigger than the rest of fetuses in the study, but again it was one litter in the middle dose group out of all 63 litters from all treated groups, so the test item influence is considered doubtful.
There was one significantly smaller fetus in the low dose group, but due to comparable case in control group it was not considered to be connected with the test item action.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
During the evaluation of skeletal system, the skull ossification delay and vertebral lesions were observed. Variations in sternum ossification centers were mainly of unilateral, bipartite and rarely misaligned character and also as lack of one or more ossification points and occurred in all treated groups and in control group. These esions in skeletal system were regarded as variations or retardations which can be defined as changes that occur within the normal population (especially that they occurred also in control group), kind of delay in growth or morphogenesis and are unlikely to affect adversely survival or health. It should be also pointed out that the total number of all lesions in skeletal system in each treated group did not exceed the number in control group, so the changes were not linked with the test item action.
The summary of percentage of fetuses with a given number of ossification points in sternum showed comparable distribution of percentage of fetuses in all treated and control groups. Some delay of sternum ossification in group 0 (0.62% - no ossification points in 1 fetus and 1.16% - 3 ossification points in 2 fetuses) and in group 3 (0.75% - 3 ossification points in 1 fetus) occurred, but due to occurrence in control group and minor scale was regarded as incidental.
The percentage of fetuses with a given number of metacarpal ossification points in the forelimb and metatarsal ossification points in hindlimb are comparable in the treated and control groups, except for the delayed metacarpal ossification in group 3 (decrease of percentage of fetuses with 4 ossification points and the increase in fetuses with 3 ossification points) and the unexpected appearance of fetuses with 5 metatarsal ossification points in group 2. However, in group 3, the shift is not strong enough to affect the average number of ossification points in metacarpus, hence there were no statistically significant differences of this parameter between treated and control groups. Thus, the change in group 3 should be considered rather as some delay of development which could be easily compensated in postnatal period. In group 2 there was 4.55% of fetuses (2 fetuses) with 5 ossification points in metatarsus. Those were the ones from the litter with 3 significantly bigger fetuses. Their size and development were more advanced than in normal rat fetus on gestation day 20. But it was one litter out of all 63 litters from all treated groups, thus phenomenon was rather incidental than test item- related, with no influence on average value of the number of ossification points in metatarsus.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Sagittal sections of fetuses revealed hyperemia of the lung edges of fetuses in all treated and control groups. It should rather be considered as euthanasia-related, agonal and/or death-related events and thus it was not connected with the test item.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
The length of fetuses (with tail and without tail) was comparable between treated and control groups and slight differences were not statistically significant, so the test item did not influence this parameter.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 480 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test item adverse effects observed at the highest dose tested.

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Table 2. 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

23

22

22

19

Number of females for evaluation

23

22

22

19

Table 3. Clinical signs in females

Parameter

Group / dose

0 / 0

1 / 30

2 / 120

3 / 480

Number of females per group

25

25

25

25

thinning of coat of forearms

1

[13 N]–(6th-20th

day)

1

[7]–(18th-20hday)

 

-

 

-

thinning of coat on the right side of torso

1

[13N]–(13th-20th

day)

 

-

 

-

 

-

alopecia on forearms

1

[22]–(5th-20th

day)

 

-

 

-

 

-

scabs on forearms

1

[22]–(17th-20th

day)

 

-

 

-

 

-

thinning of coat of the left forearm

 

-

1

[17]–(6th-20th

day)

1

[20]–(13th-15hday)

 

-

thinning of coat of the right forearm

 

-

1

[17]–(10th-16th

day)

-

 

-

scabs on the left forearm

 

-

1

[17]–(9th-12th

day)

 

-

 

-

scabs on the right forearm

 

-

1

[17]–(9th-12thday)

 

-

 

-

Body weight loss

– pregnant females

 

-

 

-

2

[2]–(day 8–2g),

[23]–(day 11–1g)

3

[7]–(day 8–4g)

[13]–(day 8–3g)

[23]–(day 14–2g)

Body weight loss– non-pregnant females

 

2 N

[13]–(day14–

16g, day20–1g,[20]–(day11–

1g, day 14–2g, day 17–5g.

 

3 N

[6]–(day 14–12g),

[11]–(day8–4g,

day 14–3g), [14]–(day8–3g,

day 14–11g, day 20–3g),

3 N

[10]–(day 14–14g,

day 20–2g), [13]–(day 11–4g,

day 14 – 5g, day17

– 8g, day 20 –3g),

[19]–(day 14–4g,

day 17–2g, day 20– 4g)

6 N

[5]–(day 14–7g, day 17–3g, day 20–2g)

[10]–(day 11–6g, day 14–4g)

[15]–(day 17–13g, day 20–2g)

[20]–(day 17–7g, day 20–1g)

[24]–(day 14–16g, day 17–1g, day 20–5g)

[25]–(day 14–6g, day 17–9g)

[ ] computer numbers of females; N - nonpregnant female

( ) days in which the changes were noticed

Table 4. Body weight of females [g]

Day of gestation

Group / dose / number of evaluated females

0 /0

n=23

1 / 30

n=22

2 / 120

n=22

3 / 480

n=19

0

241.44 ± 17.51

241.86 ± 22.06

240.82 ± 19.54

236.68 ± 15.38

5

258.35 ± 17.27

257.91 ± 23.81

257.50 ± 20.60

249.16 ± 16.81

8

264.52 ± 16.32

264.68 ± 25.81

264.96 ± 21.27

255.05 ± 16.93

11

277.83 ± 17.77

278.55 ± 28.71

276.32 ± 22.54

266.53 ± 18.46

14

291.70 ± 19.19

293.91 ± 28.64

293.32 ± 23.02

281.90 ± 18.25

17

318.30 ± 20.62

320.23 ± 30.78

317.50 ± 23.21

308.68 ± 22.35

20

356.52 ± 26.89

360.82 ± 31.64

353.73 ± 28.23

347.95 ± 22.94

 µ ± SD

Table 5. Food consumption [g/100g of b.w./day]

Day of gestation

Group / dose / number of evaluated females

0 /0

n=23

1 / 30

n=22

2 / 120

n=22

3 / 480

n=19

0 – 5

6.78 ± 0.64

6.85 ± 0.57

6.74 ± 0.68

6.67 ± 0.59

6 – 8

6.79 ± 0.67

6.71 ± 0.61

6.70 ± 0.57

6.56 ± 0.73

9 – 11

6.95 ± 1.14

6.69 ± 0.74

6.69 ± 0.56

6.42 ± 0.76

12 – 14

6.98 ± 0.86

6.99 ± 0.66

7.35 ± 0.65

7.04 ± 0.73

15 – 17

6.88 ± 0.63

6.67 ± 0.45

6.69 ± 0.56

6.81 ± 0.55

18 – 20

6.41 ± 0.58

6.44 ± 0.36

6.26 ± 0.41

6.30 ± 0.58

 µ ± SD

Table 6. 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

21*

0.83 ± 0.26

21.36 ± 4.91

1.15 ± 0.33

1/ 30

22

0.91 ± 0.30

19.31 ± 3.38

0.88 ± 0.42

2/ 120

22

0.95 ± 0.27

19.89 ± 3.99

0.89 ± 0.46

3/ 480

19

0.86 ± 0.25

18.55 ± 4.21

0.87 ± 0.41

 µ ± SD

* 2 pregnant females in group 0 were excluded from statistical evaluation of TSH concentration, due to significantly exceeding values comparing to the rest of the control group.

Table 7. Results of hormonal examinations - non pregnant females

Group/ dose [mg/kgb.w.]

Number of evaluated non- pregnant females

TT3 [ng/ml]

TT4 [ng/ml]

TSH [ng/ml]

0/ 0

2

0.60 ± 0.00

26.09 ± 1.32

0.85 ± 0.41

1/ 30

3

0.55 ± 0.23

35.49 ± 14.48

0.73 ± 0.53

2/ 120

3

0.58 ± 0.14

34.86 ± 10.77

0.73 ± 0.11

3/ 480

6

0.70± 0.13

33.40 ± 3.50

1.10 ± 0.45

 µ ± SD

no statistical analysis was conducted for non- pregnant females

Table 8. Gross lesions in females

Examined organ

Type of change

Group / dose [mg/kg b.w.]

0/ 0

n=25

1/ 30

n=25

2/ 120

n=25

3/ 480

n=25

Cecum

bloating, enlarged lymphoid follicles

-

-

-

1

[21]

Uterus

one horn non-gravid

2

[6,15]

-

1

[22]

-

Vagina

purulent plug

-

-

1 [19*]

-

Female non-pregnant

2

[13,20]

3

[6,11,14]

3

[10,13,19]

6

[5,10,15,20,

24,25]

[ ] – computer number of animals with gross lesion

n – number of females

* - female non-pregnant

Table 9. Absolute and relative weight of thyroids with parathyroids and uterus in pregnant females

 

Organ

Group / dose [mg/kg b.w.]

0/ 0

n=23

1/30

n=22

2/ 120

n=22

3/ 480

n=19

Absolute weight of organs [mg]

Thyroids with parathyroids

19.391 ± 3.500

19.955 ± 3.592

20.500 ± 3.218

19.421 ± 4.260

Uterus

4467.174 ±

736.088

4518.773 ±

501.979

4375.500 ±

968.447

4430.895 ±

689.754

Relative weight of organs [%]

Thyroids with parathyroids

0.005 ± 0.001

0.006 ± 0.001

0.006 ± 0.001

0.006 ± 0.001

Uterus

1.252 ± 0.168

1.256 ± 0.132

1.232 ± 0.242

1.272 ± 0.165

µ ± SD

n- number of evaluated pregnant females

Table 10. 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 thyroid with parathyroid [%]

0/ 0

2

17.000 ± 2.828

0.007 ± 0.001

1/ 30

3

18.000 ± 2.000

0.007 ± 0.0005

2/ 120

3

17.333 ± 1.528

0.007 ± 0.002

3/ 480

6

17.333 ± 4.412

0.006 ± 0.002

 µ ± SD

no statistical analysis was conducted for non- pregnant females

Table 11. Histopathological lesions in 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

n=23

1/30 n=22

2/120 n=22

3/480 n=19

Left thyroid

+

parathyroid

Follicle epithelium, hyperplasia

Minimal (1)

2

[7,18]

 

-

 

-

1

[1]

C-cell, hyperplasia

Minimal (1)

8

[2,3,5,8,14,

15,18,21]

3

[1,21,22]

6

[1,2,3,7,9,14]

4

[1,4,8,11]

Slight (2)

-

1

-

-

[7]

Follicular cell, hypertrophy

Minimal (1)

8

[3,4,5,7,15,16]

6

[1,2,3,22,23,24]

8

[4,6,14,15,16,

17,21,22]

7

[1,2,9,13,16,17,

21]

Slight (2)

6

[1,8,10,19,

21,22,25]

7

[4,7,9,12,13,16,

20]

10

[1,2,3,5,7,8,11,

18,20,23]

7

[3,4,8,12,14,18,

19]

Moderate (3)

5

[2,9,14,23,

24]

4

[15,17,18,21]

3

[12,24,25]

3

[6,7,11]

Follicle epithelium, exfoliation

Minimal (1)

2

[11,19]

7

[4,5,7,8,18,20,

23]

6

[11,15,16,17,20,

23]

5

[9,12,14,19,21]

Slight (2)

2

[12,23]

3

[19,22,24]

1

[21]

5

[6,7,13,22,23]

Moderate (3)

1

1

1

-

[4]

[25]

[22]

Thyroid,

Present (+)

1

4

1

1

ectopic tissue

[14]

[8,10,19,21]

[24]

[4]

 

Minimal (1)

3

1

-

-

Hemorrhage/s

[7,9,23]

[8]

Slight (2)

-

3

1

-

 

[7,10,15]

[25]

Fibrosis,

Present (+)

2

2

3

-

thyroid

[3,23]

[1,8]

[6,20,21]

Fibrosis, parathyroid

 

Present (+)

9

[2,3,6,10,12,14,

17,19,22]

10

[12,13,16,17,20,

21,22,23,24,25]

10

[3,8,11,14,15,

20,21,22,24,25]

6

[2,7,12,13,14,

18]

Cyst,

Present (+)

1

2

2

1

congenital

[17]

[12,18]

[9,11]

[16]

Parathyroid, lack of

Present (+)

4

-

1

4

[7,8,18,25]

[12]

[1,3,4,21]

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

n=23

1/30 n=22

2/120 n=22

3/480 n=19

Right thyroid + parathyroid

Fibrosis,

Present (+)

5

-

3

3

thyroid

[2,3,18,23,24]

[11,22,25]

[1,4,6]

Fibrosis, parathyroid

 

Present (+)

10

[3,8,9,11,14,16,

17,21,22,24]

10

[10,12,15,16,17,

18,19,20,23,25]

7

[9,11,12,17,

22,24,25]

6

[3,4,6,7,23]

C-cell,

Minimal (1)

5

2

3

3

hyperplasia

[1,15,18,19,24]

[3,15]

[8,16,24]

[2,14,17]

 

 

Minimal (1)

8

[2,3,7,10,12,19,

21,24]

5

[3,4,12,13,20]

6

[2,5,9,12,17,21]

7

[3,4,9,12,16,17,

22]

Follicular cell, hypertrophy

 

Slight (2)

9

[5,8,9,14,15,16,

17,22,23]

6

[2,9,16,19,22,23]

9

[1,6,7,8,11,15,

16,18,25]

5

[6,7,8,14,18]

 

Moderate (3)

2

4

4

3

 

[4,11]

[5,15,17,18]

[3,20,22,24]

[11,13,19]

Thyroid,

Present (+)

2

-

3

3

ectopic tissue

[3,14]

[6,7,17]

[7,8,12]

Parathyroid,

Present (+)

-

-

1

-

ectopic tissue

[9]

Cyst/s, congenital

Present (+)

6

[1,2,6,8,10,17]

1

[8]

1

[5]

4

[3,4,11,16]

 

 

Follicle epithelium, exfolation

 

Minimal (1)

2

[9,19]

2

[2,5]

5

[5,15,22,23,25]

6

[12,13,16,17,19,

22]

 

Slight (2)

2

[17,25]

6

[1,8,10,12,13,

19,22,23]

3

[8,20,21]

4

[1,6,21,23]

 

Moderate (3)

1

3

-

-

 

[18]

[4,24,25]

 

Minimal (1)

4

2

1

-

 

[15,19,23,25]

[8,15]

[24]

Hemorrhage/s

Slight (2)

1

[4]

-

-

-

 

Moderate (3)

-

1

-

-

 

[10]

Follicle, dilation

Present (+)

1

-

-

-

[11]

Parathyroid, hyperplasia, diffuse

 

Present (+)

1

[22]

 

-

 

-

 

-

Parathyroid,

Present (+)

2

2

1

1

lack of

[19,23]

[1,7]

[14]

[14]

Table 12. 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

n=2

1/30

n=3

2/120

n=3

3/480

n=6

Left thyroid

+

parathyroid

Follicle epithelium, hyperplasia

 

Minimal (1)

 

-

 

-

 

-

1

[15]

C-cell, hyperplasia

Minimal (1)

1

[13]

1

[14]

-

1

[10]

Follicular cell, hypertrophy

Minimal (1)

 

1

[11]

2

[10,13]

3

[10,15,25]

Slight (2)

1

[20]

2

[6,14]

1

[19]

-

Moderate (3)

1

[13]

-

-

1

[25]

 

Follicle epithelium, exfoliation

Minimal (1)

-

-

-

4

[5,20,24,25]

Slight (2)

1

[20]

-

-

-

Moderate (3)

 

1

[6]

-

-

Hemorrhage/s

Moderate (3)

1

[20]

1

[6]

-

-

Thyroid, ectopic tissue

Present (+)

-

-

1

[13]

-

Fibrosis thyroid

Present (+)

-

-

 

1

[5]

Parathyroid, lack of

Present (+)

-

-

1

[19]

2

[10,15]

Right thyroid + parathyroid

C-cell, hyperplasia

Minimal (1)

-

-

-

1

[25]

 

Follicular cell, hypertrophy

Slight (2)

1

[13,20]

3

[6,11,14]

3

[10,13,19]

2

[10,24]

Moderate (3)

-

-

-

3

[15,20,25]

Follicle epithelium, exfoliation

Minimal (1)

-

-

1

[10]

1

[24]

Slight (2)

-

1

[6]

-

2

[5,20]

Thyroid, ectopic tissue

Present (+)

-

2

[11,14]

1

[10]

-

Hemorrhage/s

Slight (2)

-

1

[6]

-

-

Fibrosis, parathyroid

Minimal (1)

-

-

1

[10]

1

[25]

Fibrosis thyroid

Present (+)

-

-

-

1

[5]

Parathyroid, hyperplasia, diffuse

 

Present (+)

 

-

 

-

 

-

1

[5]

Parathyroid, lack of

Present (+)

-

-

-

2

[15,24]

Table 13. Number of fetuses

Number of fetuses

Group / dose [mg/kg b.w.]

0/ 0

1/ 30

2/ 120

3/ 480

all

304

309

282

259

dead

1

0

1

0

min.max. in litter

7 - 18

8 - 19

2 - 17

9 - 17

average in litter

13.17 ± 2.64

14.05 ± 2.15

12.77 ± 3.79

13.63 ± 1.83

females

6.87 ± 2.49

7.00 ± 1.77

6.18 ± 2.24

6.47 ± 1.78

males

6.30 ± 2.34

7.05 ± 2.28

6.59 ± 2.02

7.16 ± 2.22

 µ ± SD

Table 14. Number of corpora lutea, implantations, resorptions, pre- and postimplantations losses

Number of

Group / dose [mg/kg b.w.]

0/ 0

n=23

1/30

n=22

2/ 120

n=22

3/ 480

n=19

Corpora lutea

all

338

338

330

277

average

14.70 ± 1.82

15.36 ± 1.79

15.00 ± 1.63

14.58 ± 1.64

Implantations

all in pregnant females

319

323

303

271

average in pregnant females

13.87 ± 2.32

14.68 ± 2.03

13.77 ± 3.74

14.26 ± 1.56

all in non- pregnant females

0

0

0

0

Resorptions

all

15

14

21

12

early / late

15 / 0

14 / 0

21 / 0

11 / 1

min – max in female

0 - 6

0 - 3

0 - 2

0 - 4

average in pregnant females

0.65 ± 1.30

0.64 ± 0.90

0.95 ± 0.79

0.63 ± 1.01

Preimplantation loss %

5.64 ± 10.18

4.23 ± 9.77

8.01± 22.60

2.03 ± 4.47

Postimplantation loss %

5.08 ± 9.89

4.37 ± 6.07

8.77 ± 8.94

4.47 ± 7.19

 µ ± SD

n- number of evaluated pregnant females

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

Group/dose

[mg/kgb.w.]

Number of examined fetuses

Weight of fetuses [g]

Weight of placenta [g]

with placenta and fetal membranes

without placenta and fetal membranes

0/ 0

303

4.556 ± 0.220

3.312 ± 0.196

0.511 ± 0.044

1/ 30

309

4.549 ± 0.195

3.390 ± 0.150

0.491 ± 0.052

2/ 120

280

4.762 ± 0.734

3.552 ± 0.706

0.524 ± 0.061

3/ 480

259

4.557 ± 0.234

3.377 ± 0.203

0.515 ± 0.059

µ ± SD

Table 16. Average weight of fetuses and placenta / sex [g]

Group/dose

[mg/kgb.w.]

Weight of fetuses [g]

Weight of placenta [g]

with placenta and fetal membranes

without placenta and fetal membranes

0/ 0

4.649 ± 0.253

4.453 ± 0.245

3.397 ± 0.189

3.239 ± 0.218

0.517 ± 0.043

0.509 ± 0.047

1/ 30

4.649 ± 0.198

4.461 ± 0.208

3.466 ± 0.140

3.326 ± 0.173

0.496 ± 0.054

0.486 ± 0.056

2/ 120

4.859 ± 0.741

4.510 ± 0.289

3.627 ± 0.696

3.324 ± 0.235

0.534 ± 0.072

0.503 ± 0.064

3/ 480

4.657 ± 0.228

4.438 ± 0.271

3.458 ± 0.207

3.279 ± 0.224

0.523 ± 0.064

0.504 ± 0.054

µ ± SD

Table 17. Average length of fetuses [cm]

Group/dose

[mg/kgb.w.]

Number of examined fetuses

Length of fetuses [cm]

with tail

without tail

0/ 0

303

4.87 ± 0.15

3.58 ± 0.13

1/ 30

309

4.87 ± 0.13

3.57 ± 0.11

2/ 120

280

4.94 ± 0.39

3.64 ± 0.34

3/ 480

259

4.84 ± 0.15

3.56 ± 0.13

 µ ± SD

Table 18. Average anogenital distance

Group/dose

[mg/kgb.w.]

Number of examined fetuses

Anogenital distance AGD [mm]

anogenital index AGD index

0/ 0

303

2.42 ± 0.28

73.14 ± 8.36

1/ 30

309

2.37 ± 0.28

69.83 ± 7.81

2/ 120

280

2.46 ± 0.31

69.99 ± 7.39

3/ 480

259

2.42 ± 0.18

71.85 ± 7.84

µ ± SD

Table 19. Average anogenital distance / sex

Group/dose

[mg/kgb.w.]

Anogenital distance AGD [mm]

Anogenital index AGD index

0/ 0

3.08 ± 0.14

1.80 ± 0.25

91.50 ± 7.61

55.88 ± 5.52

1/ 30

3.08 ± 0.10

1.68 ± 0.27

89.45 ± 3.97

50.65 ± 8.19

2/ 120

3.14 ± 0.13

1.66 ± 0.30

88.64 ± 9.31

50.32 ± 10.34

3/ 480

3.06 ± 0.14

1.69 ± 0.25

88.95 ± 6.85

52.09 ± 8.43

µ ± SD

Table 20. Lesions in fetuses

Pathological changes

Group/ dose [mg/kg b.w.] / number of fetuses

0 / 0

1 / 30

2 / 120

3 / 480

Gross examination

 

n=304

n=309

n=282

n=259

Subcutaneous hemorrhages in different locations

Hemorrhage on the head and neck

4 [6,2x17,22]

3

[3,21,24]

1

[18]

1

[23]

Hemorrhage on the thorax

 

8 [1,5,12,5x25]

8       [3,4,5,2x8,10,23,24]

15     [2x2,2x3,4,2x5

,2x6,8,9,2x11,23,24]

11    [2x1,3,4,2x7,

2x8,17,22,23]

Hemorrhage on the forelimbs

2

[2,12]

1

[22]

-

4 [4,6,2x22]

Hemorrhage on the hindlimbs

2

[3,4]

3 [13,2x22]

-

2

[14,16]

Hemorrhage on the tail

-

-

5

[1,6,14,15,17]

1

[17]

total

16

15

21

19

Dead fetus

1

[16]

-

1

[4]

-

Cyanosis and oedema of the body (in dead fetus)

-

-

1

[4]

-

Fetus significantly bigger

-

-

3 [3x22]

-

Fetus significantly smaller

1

[7]

1

[22]

-

-

Severe oedema of the body

-

-

1

[16]

-

Cranium oedema, backward (dorsalis) defection of the body

-

-

-

1

[19]

Sagittal sections

 

n=138

n=144

n=134

n=121

Hyperemia of the lung edges

9       [2x9,10,2x12,18,2x24,25]

11    [8,5x9,10,12, 2x17,22]

4 [9,21,2x24]

11     [1,3,6,7,8,2x9,12,13,2x18]

n- number of evaluated fetuses; [_] computer numbers of females;

2x- number of fetuses from one female, with the same lesion, e.g.: [1] – lesion stated in one fetus from female no. 1; [2x1]- lesion stated in 2 fetuses from female no. 1

Table 21. Changes in skeletal system

Location and type of lesion

Group/ dose [mg/kg b.w.] / number of fetuses

0 / 0 n=166

1 / 30 n=165

2 / 120 n=148

3 / 480 n=138

Sternum ossification points

1st

lack of

1

-

-

-

[7]

 

lack of

3

-

-

2

 

[7,8,25]

[8,23]

2nd

unilateral

-

1

[16]

1

[18]

1

[14]

 

bipartite

-

-

1

1

 

[20]

[1]

 

lack of

1

-

-

-

 

[7]

3rd

bipartite

-

-

-

1

[23]

 

misaligned

-

1

1

1

 

[22]

[16]

[23]

 

lack of

1

-

-

-

 

[7]

 

unilateral

-

-

-

1

4th

[1]

bipartite

1

1

1

-

 

[16]

[22]

[16]

 

misaligned

-

2

2

1

 

[22,24]

[9,16]

[13]

 

lack of

31     [1,2,2x4,3x5,6,

3x7,2x8,2x9,2x10,

11,3x12,4x14,16,

18,4x25]

18        [2x4,9,3x10,12, 15,16,17,19,2x21,

2x22,3x23]

 

11     [3,2x8,9,11,14, 2x18,20,2x21]

18     [4x1,2x6,7,8, 12,2x13,2x14,

3x19,2x21]

 

 

 

22

 

 

5th

unilateral

14       [2,8,3x12,14,15, 2x16,18,19,3x24]

[4,2x7,8,9,2x10, 12,13,2x15,16,

4x20,21,22,2x23,

2x24]

11      [2x3,2x9,11,14, 16,18,21,2x23]

13      [1,3x6,13,3x14, 2x17,18,2x22]

 

bipartite

4

2

3

2

 

[2x16,2x19]

[15,19]

[2,17,23]

[6,13]

 

misaligned

-

-

1

-

 

[2]

 

 

24

25      [1,2x3,2x4,3x8, 2x9,10,4x19,20,

21,22,5x23,2x25]

21 [2x4,5,2x6,8,9, 5x14,15,18,20,

3x21,2x23,24]

32

 

 

[2,3x3,4,2x6,7,

[4x1,2,4,3x6,4x7,

 

lack of

8,9,12,2x14,15,

8,11,12,4x13,

 

 

2x16,2x19,21,

5x14,2x17,2x18,

 

 

2x24,3x25]

2x21,23]

6th

unilateral

6 [5,8,17,2x19,25]

4 [2,2x9,25]

7      [2,4,6,2x12,

21,23]

                   8 [1,3,2x6,14,22,

2x23]

 

 

48

 

 

22      [3x2,3,4,2x6, 2x8,2x17,18,20,

4x21,3x23,2x24]

40      [2x1,3x2,3,3x4,

2x6,4x7,8,9,

4x11,2x12,13,

17,5x18,4x21,

3x22,3x23]

 

 

[1,6x2,3x3,4,

25

 

 

3x5,6,2x8,2x11,

[2,2x3,4x4,8,2x9,

 

bipartite

12,3x14,2x15,16,

13,2x17,18,2x19,

 

 

17,3x18,4x19,

3x20,21,2x23,

 

 

4x21,2x22,2x23,

3x24,3x25]

3x25]

skull

lack of ossification of supraoccipital plate; delay of ossification of interparietal and parietal plates

1

[7]

-

-

-

delayed and uneven ossification of supraoccipital and interparietal plates

-

-

-

1

[13]

bipartite ossification of supraoccipital plate

1

[9]

-

1

[20]

5 [2x6,7,8,14]

Vertebral column

lack of ossification in sacral (3rd and 4th) and caudal vertebrae

1

[7]

-

-

-

TOTAL

137

101

83

127

[ ] 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 22. 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=166

0.62

-

-

1.16

5.08

21.05

72.08

1/30

n=165

-

-

-

-

5.36

15.36

79.28

2/ 120

n=148

-

-

-

-

4.28

12.07

83.65

3/ 480

n=138

-

-

-

0.75

8.50

19.02

71.72

n- number of evaluated fetuses

Table 23. Percentage of fetuses with a given number of ossification points in limbs (%)

Group/ dose [mg/kgb.w.]

Metacarpus of forelimbs [%]

Metatarsus of hindlimbs [%]

3

3.5*

4

3

4

5

0/ 0

n=166

19.74

3.74

76.51

0.62

99.38

-

1/30

n=165

7.93

3.43

88.65

-

100.00

-

2/ 120

n=148

14.05

2.44

83.52

0.65

94.81

4.55

3/ 480

n=138

27.78

5.55

66.67

0.75

99.25

-

n- number of evaluated fetuses

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

Table 24. 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=166

5.62 ± 0.31

3.78 ± 0.20

3.99 ± 0.03

1/30

n=165

5.74 ± 0.25

3.90 ± 0.19

4.00 ± 0.00

2/ 120

n=148

5.79 ± 0.27

3.85 ± 0.19

4.04 ± 0.22

3/ 480

n=138

5.62 ± 0.34

3.69 ± 0.30

3.99 ± 0.03

n- number of evaluated fetuses

Applicant's summary and conclusion

Conclusions:
In a prenatal developmental toxicity study performed with the test item in Wistar rats, the NOAEL for maternal and developmental toxicity was found to be the highest dose tested, i.e. 480 mg/kg bw/day.



Executive summary:

A prenatal developmental toxicity study was performed on the test substance 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 30, 120 and 480 mg/kg bw/day, based on existing experimental data on the substance from ‘Combined repeated dose toxicity study with the reproduction/ developmental toxicity screening test (according to OECD 422), in which at the doses of 100 and 300 mg/kg b.w. test item- related stillbirths and mortality of pups were stated. 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. Coat or skin changes (scabs) were only observed in 1 or 2 females of groups 0, 1 and 2. No clinical effects were observed in group 3. Average body weight and food consumption of pregnant females of group 1, 2 and 3 were comparable with the respective average values in 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 23 pregnant females in group 0, 22 in group 1, 22 in group 2 and 19 in group 3. Lack of pregnancy was stated in 6 females in group 3. 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 usually 1 to 5 non-pregnant females. The number of females required by the Guideline was obtained (not less than 16).

In the pathological examination, only a case of bloating cecum with enlarged lymphoid follicles in 1 female and a single case of vaginal purulent plug in another female were found in the treated groups. They were not regarded as test-item related due to single occurrence. There were no statistically significant changes in absolute and relative weight of thyroids with parathyroids as well as in absolute and relative weight of uterus with cervix in group 1, 2, and 3 compared to the control group. The histopathological examination of thyroids did not reveal the presence of test-item related lesions. No statistically significant difference in the number of fetuses and number of males and females in litters, corpora lutea, implantations, resorptions and preimplantation loss as well as postimplantation loss in all treated groups compared with the control group was stated. There was no statistically significant difference in weight of the fetuses with and without placenta and fetal membranes and weight of placenta, with and without sexes combined, in all treated groups in comparison with the control group. There were no statistically significant difference in length of the fetuses with and without tail in all treated groups in comparison with the control group. There was no statistically significant difference in average anogenital distance and anogenital index with and without sexes combined in all treated groups in comparison with the control group. There were no gross lesions in fetuses connected with the test item action in all treated groups. Sagittal sections of fetuses revealed hyperemia of the lung edges of fetuses in all treated and control groups, which was not connected with the test item. The total number of all lesions in skeletal system in each treated group did not exceed the number in control group, so the changes were not linked with the test item action. No statistically significant differences of average number of ossification points in sternum, metacarpus and metatarsus were stated in groups treated with the test item compared with the control group.

Based on these results, the NOAEL for maternal toxicity and developmental toxicity was considered to be 480 mg/kg bw/day.