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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-07-02 and 2016-02-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 January 2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest Cserkesz u. 90. Hungary
- Age at study initiation: 8.5-9.5 weeks (females), 34-36 weeks (males)
- Weight at study initiation: 162-206 g (females)
- Housing: Before mating: 1-3 females per cage 1-2 males per cage; Mating: 1 male and 1-3 females / cage; During gestation: 2 sperm positive females per cage, if not possible 1 sperm positive female per cage
- Diet: ad libitum (ssniff® SM R/M-Z+H)
- Water: ad libitum (tap water)
- Acclimation period: 33 days for females, 180 days for males

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 43-62
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Sunflower oil (Helianthii annui oleum raffinatum)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was formulated in the vehicle (sunflower oil) in concentrations of 50 mg/mL, 150 mg/mL and 500 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility not longer than for three days and stored at 5 ± 3 °C until use.

VEHICLE
- Justification for use and choice of vehicle: The test item was not soluble in water therefore Sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 50, 150 and 500 mg/mL
- Amount of vehicle: 2 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of dosing solutions (control of concentration) was performed in the Analytical Laboratory of Test Facility two times during the study. Five samples from different places were taken from each concentration for analysis of concentration and homogeneity on 2 occasions. Similarly, five samples were taken from the vehicle and analyzed by a reverse phase HPLC method UV detection.
The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. Recovery of Sika Hardener LH from sunflower oil was ≥ 97 % at ~25 and ~500 mg/mL concentrations, respectively.
A sufficient stability in the chosen vehicle was also verified over the range of relevant concentrations. The test item proved to be stable in the vehicle at ~25 and ~500 mg/mL concentration levels at least for 3 days in the refrigerator (5 ± 3 °C) and for 4 hours at room temperature. A separate analytical report – Toxi-Coop Study no. 644-100-0844– provided these data.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: one male: one to three females
- Length of cohabitation: two to four hours (until the number of sperm positive females per group achieved at least twenty two)
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:
Sperm positive females were treated from gestational day 5 to 19.
Frequency of treatment:
7 days/week
Duration of test:
Day 0 of gestation - Day 20 of gestation
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
80 males, 100 females to achieve at least 22 sperm positive females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose setting was based on findings obtained in a previous GLP 28-Day Oral Toxicity Study in the Rat with the test item (OECD 407, LAB Research Centre Hungary Ltd. 04/915-100P). The high dose was chosen with the aim of inducing toxic effects but no deaths or severe suffering. The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically.

Examinations

Maternal examinations:
CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day (after treatment at approximately the same time)
- Behaviour and general conditions
- Signs of morbidity and mortality were made twice daily, at the beginning and end of the working period.

BODY WEIGHT: Yes
- Time schedule for examinations: at least once in the pre-mating period (female rats), on gestation days 0, 3, 5, 8, 11, 14, 17 and 20 (sperm positive females)

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule: between gestation days 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20 by re-weighing the non-consumed diet

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus with cervix and ovaries

OTHER:
- Examination of placental signs (All sperm positive animals were examined for vaginal bleeding (placental sign of gestation) on the 13th gestational day. If negative on day 13, the examination was repeated on day 14 of gestation.)

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: number of live and dead fetuses in each uterine horn
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity is detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result is significant Duncan’s Multiple Range test was used to access the significance of inter-group differences. If significance is the result of the Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test.
Dams or litters were excluded from the data evaluation in cases of:
- Non pregnant females or dams (total exclusion)
Although these animals were excluded from the data evaluation the Study Report contains all data of these animals, too. A male/female fetus was considered as retarded in body weight, when its weight was below the average minus twofold standard deviation of the control male/female fetuses.
Historical control data:
Summary of Laboratory's historical control data is available.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related significant differences in the body weight and body weight gain parameters of the dams in the test item treated groups versus control. The body weight gain was slightly but statistically significantly higher (p<0.01) in the 300 mg/kg bw/day group between gestation days 11 and 14 without a dose response and this difference was considered as incidental.
Food consumption and compound intake (if feeding study):
no effects observed
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, non-treatment-related
Description (incidence and severity):
The gravid uterine weight which was measured in order to calculate the corrected body weight was slightly higher (p<0.01) in the 1000 mg/kg bw/day group, this was attributed to the slightly higher mean number of viable fetuses in this group and unrelated from the treatment.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Details on results:
- CLINICAL SYMPTOMS; MORTALITY
None of the females died in the course of the study and there were no clinical signs observed in the females.

- BODY WEIGHT; BODY WEIGHT GAIN AND CORRECTED BODY WEIGHT
There were no treatment-related significant differences in the body weight and body weight gain parameters of the dams in the test item treated groups versus control. The body weight gain was slightly but statistically significantly higher (p<0.01) in the 300 mg/kg bw/day group between gestation days 11 and 14 without a dose response and this difference was considered as incidental. The gravid uterine weight which was measured in order to calculate the corrected body weight was slightly higher (p<0.01) in the 1000 mg/kg bw/day group, this was attributed to the slightly higher mean number of viable fetuses in this group and unrelated from the treatment.

- FOOD CONSUMPTION
There was no statistical difference in the food consumption of the dams among the experimental groups.

- NECROPSY
There were no macroscopic alterations recorded for the dams during necropsy in the other experimental groups.

Maternal developmental toxicity

Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
not examined
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
The number of sperm positive females was 22 in all groups. There were two, five and three females each in the 100, 300 and 1000 mg/kg bw/day dose groups respectively with no implantation and no corpora lutea. These females were excluded from the evaluation. In total, on gestation day 20 there were 22, 20, 17 and 19 evaluated litters each in the control, 100, 300 and 1000 mg/kg bw/day groups respectively.
Other effects:
no effects observed
Details on maternal toxic effects:
INTRAUTERINE MORTALITY AND VIABLE FETUSES
There was no adverse effect indicated related to the administration in the mean percent of pre- and postimplantation loss (early embryonic, late- and fetal death), the mean number of implantations as well as in the mean number of viable fetuses in the test item treated groups. Moreover, the mean percent and whole number of pre- and postimplantation loss, early embryonic death and total intrauterine mortality were statistically significantly lower in the 1000 mg/kg bw/day group as well as the whole number of postimplantation loss in the 300 mg/kg bw/day group. In addition the mean number of viable fetuses was statistically significantly higher in the 300 and 1000 mg/kg bw/day group ((p<0.05 and 0.01 respectively).

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There was one malformed fetus found with agnathy in the control group during external examination.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The agnathy of the fetus in the control group was partially confirmed at skeletal examination (as a markedly hypoplastic mandible). Other malformations in this fetus of the skull (displaced tympanic rings, misshapen palate and sphenoid) as well as ribs (common articulation of two ribs to a vertebra) and vertebrae (Th IV bipartite, cartilage dumb-bell shaped; Th VII rotated, slight; Th VIII asymmetric dumb-bell shaped, cartilage and arches asymmetric; Th IX hemicentric, cartilage hemicentric; Th X displaced, cartilage asymmetric dumb-bell shaped; Th XII asymmetric bipartitewith bipartite cartilage were observed. A split and misshapen (wide) sternum was recorded for one fetus in the 1000 mg/kg bw/day group.
Considering the low incidence (one single fetus) and that split sternum occurs sporadically with low incidence according to the historical control data, this malformation was judged to be unrelated to an effect of the test item.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no malformations found at visceral examination in the 300 and 1000 mg/kg bw/day groups. Internal hydrocephaly (dilated lateral ventricles) in one control fetus and situs inversus totalis in a fetus in the 100 mg/kg bw/day group were recorded.
Considering that situs inversus totalis occurs in fetuses with low incidence unrelated from the treatment according to the historical background data of Toxi-Coop Zrt. this malformation was judged to be unrelated from the treatment.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Please refer to "Details on embryotoxic/teratogenic effects"
Details on embryotoxic / teratogenic effects:
BODY WEIGHT OF FETUSES AND PLACENTAL WEIGHT
There were no treatment related differences in the body weight of the fetuses and placental weight. Statistically significantly lower placental weight was indicated for the fetuses (p<0.01) in the 300 and 1000 mg/kg bw/day dose group and in the relative placental weight in the 1000 mg/kg bw/day group. Considering that the values were within the historical control range, these differences were not attributed to an effect of the test item.

EXTERNAL EXAMINATION
The number of evaluated fetuses was 225, 218, 196 and 225 in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
- Malformations: There was one malformed fetus found with agnathy in the control group during external examination.

- Variations: Body weight retardation (below 2.92 g for males and 2.68 g for females) was evaluated as an external variation. There were no treatment related differences among the experimental groups.

VISCERAL EXAMINATION
The number of evaluated fetuses was 225, 218, 196 and 225 in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
- Malformations: There were no malformations found at visceral examination in the 300 and 1000 mg/kg bw/day groups. Internal hydrocephaly (dilated lateral ventricles) in one control fetus and situs inversus totalis in a fetus in the 100 mg/kg bw/day group were recorded. Considering that situs inversus totalis occurs in fetuses with low incidence unrelated from the treatment according to the historical background data of Toxi-Coop Zrt. this malformation was judged to be unrelated from the treatment.

- Variations: Hydroureter was evaluated as variation and was found only in one fetus each in the control and 300 mg/kg bw/day group, hence there was no test item effect indicated.

SKELETAL EXAMINATION
The number of examined fetuses was 112, 110, 97 and 110 in the control, 100, 300 and 1000 mg/kg bw/day group respectively.
- Malformations: The agnathy of the fetus in the control group was partially confirmed at skeletal examination (as a markedly hypoplastic mandible). Other malformations in this fetus of the skull (displaced tympanic rings, misshapen palate and sphenoid) as well as ribs (common articulation of two ribs to a vertebra) and vertebrae (Th IV bipartite, cartilage dumb-bell shaped; Th VII rotated, slight; Th VIII asymmetric dumb-bell shaped, cartilage and arches asymmetric; Th IX hemicentric, cartilage hemicentric; Th X displaced, cartilage asymmetric dumb-bell shaped; Th XII asymmetric bipartitewith bipartite cartilage were observed. A split and misshapen (wide) sternum was recorded for one fetus in the 1000 mg/kg bw/day group.
Considering the low incidence (one single fetus) and that split sternum occurs sporadically with low incidence according to the historical control data, this malformation was judged to be unrelated to an effect of the test item.

- Variations: Incompletely or not ossified skull bones (including hyoid), incompletely ossified sternebra, sacral II vertebra or metacarpal/metatarsal, wavy ribs, bipartite sternebra, dumb-bell shaped, bipartite, asymmetrically ossified thoracic centra or slightly dumb-bell shaped cartilage as well as asymmetrically ossified metacarpal or metatarsal were evaluated as variations during the skeletal examination. There were no treatment related significant differences in the incidence of the different variations. The incidence of variations due to the markedly incompletely ossified skull bones was statistically significantly (p<0.01) and slightly higher in the 300 mg/kg bw/day group versus control. Considering the lack of dose response, this difference was judged to be unrelated from the treatment.


Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:
NOAEL (maternal toxicity): 1000 mg/kg bw/day
NOAEL (developmental toxicity including teratogenicity): 1000 mg/kg bw/day
Executive summary:

The test item was examined for its possible prenatal developmental toxicity. Groups of 22 sperm-positive female Hsd. Han: Wistar rats were treated with the test item by oral administration daily at three dose levels of 100, 300 and 1000 mg/kg bw/day from day 5 up to and including day 19 post coitum. A control group of 22 sperm positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 2 mL/kg bw.

A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. The item in sunflower oil was stable at room temperature for four hours and for 3 days if stored in the refrigerator at the concentrations of 25 and 500 mg/mL. Analytical control of dosing solutions was performed on the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 92 and 100 % of nominal concentrations at both analytical occasions confirming proper dosing.

During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day, when sperm was detected in the vaginal smear, was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method.

After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.

 

In total, on gestation day 20 there were 22, 20, 17 and 19 evaluated litters each in the control, 100, 300 and 1000 mg/kg bw/day groups respectively. None of the female died before scheduled necropsy and there were no clinical signs recorded. No treatment related necropsy findings were observed. There was no treatment related reduction indicated. Number of implantations, intrauterine mortality and sex distribution of the fetuses were not influenced by the treatment. With regard to fetal examinations, there were no test item related differences in the fetal-and placental weight, body weight retardation and other external, visceral and skeletal variations. There were no malformed fetuses found in the experimental groups besides one control fetus with agnathy during external examination. There were no malformations found at visceral examination in the 300 and 1000 mg/kg bw/day groups. Internal hydrocephaly (dilated lateral ventricles) was found in one control fetus at visceral examination. Situs inversus totalis in a fetus in the 100 mg/kg bw/day group was considered to be unrelated from the treatment taking into accound the low incidence (one single fetus) and that situs inversus totalis occurs in fetuses with low incidence unrelated from the treatment according to the historical background data of Toxi-Coop Zrt. The same control fetus which had agnathy was found with multiple skeletal malformations (skull including mandible, ribs, and vertebrae). In the test item treated groups only a split and misshapen (wide) sternum was recorded for one fetus in the 1000 mg/kg bw/day group. Considering the low incidence and that split sternum occurs sporadically according to the historical control data, this malformation was judged to be unrelated to an effect of the test item.

 

In conclusion, oral treatment of pregnant Hsd. Han: WISTAR rats from gestation day 5 up to day 19 (the day before Caesarean section) with the test item at the dose levels of 100, 300 and 1000 mg/kg bw/day did not cause death, clinical signs and necropsy findings and did not influence the food intake and body weight development of the maternal animals. The test item did not reveal any adverse effect on the pre- and postimplantation loss, number of implantation, sex distribution, body weight, and placental weight, external, visceral and skeletal development of the fetuses.

Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:

NOAEL (maternal toxicity): 1000 mg/kg bw/day

NOAEL (developmental toxicity including teratogenicity): 1000 mg/kg bw/day