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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:
2016-05-11 to 2016-09-02
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:
2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
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: Females: Young adult and nulliparous females, 11-12 weeks of age at start of the mating period
Males: experienced males, 38-39 weeks of age at start of the mating period
- Fasting period before study: None
- 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: Animals will receive ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany ad libitum.
- Water: Tap water from municipal supply, as for human consumption from 500 mL bottle ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): Above 10 air exchanges/hour by central air-condition system.
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: sunflower oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was formulated in the vehicle in concentrations of 10 mg/mL, 30 mg/mL and 100 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility not longer than 3 days before administration and were stored in the refrigerator (5 +/- 3°C) until application.

VEHICLE
- Justification for use and choice of vehicle: Sunflower oil (Helianthii annui oleum raffinatum), The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. Recovery was 98 and 102 % of the nominal concentrations at 1 and 500 mg/mL in sunflower oil, respectively.
- Lot/batch no.: 1506-4604
- Purity: 98 and 102 %
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 at least twice during the study.
The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. TBPND proved to be stable at room temperature for four hours (recovery was 105 % of starting concentration at 1 mg/mL and 100 % at 500 mg/mL) and at 5 +/- 3°C for 3 days (recovery was 98 % of starting concentration at 1 mg/mL and 101 % at 500 mg/mL). A separate analytical report provided this information.
Details on mating procedure:
- Impregnation procedure: The females will be paired to males in the mornings for two to four hours (one male: one to three females) until the number of sperm positive females per group achieves at least twenty two.
- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male to three females
- Length of cohabitation: two to four hours
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
The sperm positive females were treated from gestational day 5 to 19. The test item was administered in a single dose by oral gavage (stomach tube) on a 7 days/week basis every day at similar time.
Frequency of treatment:
Daily
Duration of test:
The sperm positive females were treated from gestational day 5 to 19. Gross pathology afterwards.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
10 mg/mL
Dose / conc.:
60 mg/kg bw/day (nominal)
Remarks:
30 mg/mL
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
100 mg/mL
No. of animals per sex per dose:
80 males, 130 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 is based on findings obtained in a GLP Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422). 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:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once a day

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of the female rats was measured at least once in the pre-mating period, but will not be statistically evaluated. Body weight of sperm positive females was measured on gestation days 0, 3, 5, 8, 11, 14, 17 and 20 (accuracy of 1 g). The corrected body weight was calculated for the 20th day of pregnancy (body weight on day 20 minus the weight of the gravid uterus).

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: All sperm positive females were sacrificed by decapitation after anesthetizing with Isofluranum on day 20 of gestation.
- Organs examined: The abdomen was opened, the uterus with cervix and the left ovary was removed and weighed. The right ovary was placed into a Petri dish after removal. After removing the uterus gross pathology of dams' viscera was performed.

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
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 statistical evaluation of data was performed with the program package SPSS PC+4.0.
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.
Indices:
Pre-implanation loss:
((number of corpora lutea)-(number of implantations))/(number of corpora lutea)*100

Post-implanation loss:
((number of implantations)-(number of live fetuses))/(number of implantations)*100

Sex distribution:
(number of male (female)fetuses)/(number of fetuses)*100

External abnormalities/ litter:
(number of fetuses with abnormality/(number of fetuses)*100

Visceral abnormalities/litter:
(number of fetuses with abnormality/(number of fetuses examined)*100

Skeletal abnormalities/litter
(number of fetuses with abnormality/(number of fetuses examined)*100
Historical control data:
Findings were compared to historical control data.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related clinical observations. Alopecia and small wound on the skin were observed with a low incidence without a relationship to the test item. Piloerection was recorded in one dam in the high dose group on the day before Caesarean section. Considering that this occurred only in one female, this sign was not attributed to the treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were no significant differences in the body weight values among the dose groups. Statistically significantly, slight to moderate lower body weight gain was indicated in the 60 (-11%) and 200 (-24%) mg/kg bw/day groups for the days between 17 and 20 (p<0.05) and in the 200 mg/kg bw/day group for between days 0 to 20 (-12%). The differences in the body weight parameters in the 200 mg/kg bw/day dose group were considered as treatment-related as there was also a statistically significant (p<0.05), moderate reduction of the corrected body weight gain as most relevant weight parameter in this group. The slightly lower body weight gain in the 60 mg/kg bw/day group was considered as non-adverse as no other weight parameter was affected accordingly.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Slight but statistically significant reduction of the food consumption was observed (p<0.05, -8%) between gestation days 17 and 20 in the 200 mg/kg bw/day dose group. Considering that this reduction correlated with a moderately lower body weight- and corrected body weight gain, a relationship to the treatment was considered as likely. There were no significant differences indicated in the food consumption of the animals in the 20 and 60 mg/kg bw/day groups relative to the control.
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, non-treatment-related
Description (incidence and severity):
There were no treatment related macroscopic alterations recorded. At necropsy the stomach was found to be empty in case of three dams in the 200 mg/kg bw/day group. Also an empty stomach except some bedding was recorded for two dams in the 60 mg/kg bw/day group. These females were considered to have no macroscopic alterations. There was one female in the 200 mg/kg bw/day group where pin-prick sized dark coloured points were to see in the glandular stomach. Clotted bloody stomach content with intact stomach surface was observed in one dam in the 20 mg/kg bw/day dose group. These findings were judged not to be in relationship with the treatment considering the isolated occurrence .
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
not examined
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
There was no effect related to the administration of the test item in the mean percent of pre-implantation loss, early embryonic, late- and fetal death, the mean number of implantations, the sex distribution of the fetuses as well as in the mean number of viable fetuses in the test item treated groups. Moreover the preimplantation loss as non-relevant finding was statistically significantly lower in the 200 mg/kg bw/day group. Statistically significantly (p<0.05) higher mean number of early embryonic death in all test item treated groups as well as postimplantation loss in the 60 mg/kg bw/day group and in the 200 mg/kg bw/day group both without a dose response-relationship considering the mean percentage values was indicated only by the Chi square test and not in case the mean percentage was calculated. Therefore, the observed differences were finally judged to be not related to the treatment.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not 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 25 in the control and high dose as well as 22 both in the low and mid dose groups. There were two non- pregnant females each in the control and 200 mg/kg bw/day and five in the 20 mg/kg bw/day group. In the 60 mg/kg bw/day dose group all females were pregnant. Two females which had less than 3 implantations (one each in the control and 200 mg/kg bw/day group) were excluded from the data evaluation. Data of one litter (with one fetus) were excluded from the data evaluation in the 200 mg/kg bw/day dose group. In total, on gestation day 20 there were 22 evaluated litters each in the control and 60 mg/kg bw/day group, 17 and 21 in the 20, 60 and 200 mg/kg bw/day groups respectively.
Other effects:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight of the fetuses and absolute placental weight (but not the most relevant relative placental weight) was slightly but statistically significantly (p<0.05 to (p<0.01) lower in the 200 mg/kg bw/day group compared to the control. The reduction of the mean fetal weights was judged to be in relationship with the treatment. In contrast, the observation regarding the reduced mean absolute placental weight was considered as not relevant as the most relevant relative placental weight due to its relation to body weight was not affected and moreover, the mean relative placental weights were slightly higher, although without statistically significance, than the respective, control values.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
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, treatment-related
Description (incidence and severity):
The number of evaluated fetuses was 253, 198, 225 and 253 in the control, 20, 60 and 200 mg/kg bw/day groups, respectively.

Malformations
One fetus was found with a malformation (umbilical hernia) at external examination in the 200 mg/kg bw/day group. According to the experience with this species in this laboratory, umbilical hernia occurs sporadically without a relationship to the treatment. This is in line with historical control data of other Wistar rats (Crl:WI(Han), Ginkis and Clifford, 2009) and another strain of rats, i.e. CrL:CD(R) BR rats (Lang, 1993). Both are known to have low incidences of umbilical hernia as spontaneous finding. Consequently, this single malformation in the high dose group in this study was judged to be incidental.

Variations
Body weight retardation (below 3.03 g for males and 2.79 g for females) was evaluated as an external variation. The incidence of body weight retarded fetuses increased statistically significantly (p<0.01) in the 200 mg/kg bw/day dose group which was considered to be in a relationship with the treatment of the dams.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The number of examined fetuses was 126, 99, 113 and 128 in the control, 20, 60 and 200 mg/kg bw/day group respectively. There were no significant differences in the incidence of fetuses with abnormalities among the experimental groups.

Malformations:
Malformations were found only in single cases and distributed among allthe experimental groups. Slightly split or split xiphoid process was recorded for one fetus both in the control and 200 mg/kg bw/day group. Split sternum, bent scapula in two separate fetuses in the 20, dumb-bell shaped cartilage of thoracic vertebral centrum in the 60 mg/kg bw/day dose group and a hemicentric thoracic centrum in one control fetus were considered to be without any relationship to the treatment of the dams with the test item.

Variations:
Incompletely ossified skull bones, bipartite supra occipital, unossified hyoid, incompletely or not ossified, misaligned or bipartite sternebra, wavy, interrupted ribs, shorter 13th rib, neck rib anlage, bipartite (with or without asymmetric ossification), incomplete ossification or unossified vertebral arches, incompletely ossified pubic bones, asymmetric or incomplete ossification of metacarpal and metatarsal were evaluated as variations during the skeletal examination. Slightly but statistically significantly (p<0.05) increased incidence if only 3 or less sternebra were ossified in the high dose group.
Opposed to this the incidence of fetuses with incomplete ossification of the skull bones which was statistically significantly lower in the 200 mg/kg bw/day group as well as the occurrence of wavy ribs was statistically significantly lower in the 60 and 200 mg/kg bw/day groups. In summary the slight increases and decreases in the incidence of skeletal variations were judged to be not related to the treatment.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The number of examined fetuses was 127, 99, 112 and 125 in the control, 20, 60 and 200 mg/kg bw/day group respectively. There were no significant differences in the incidence of variations and malformations among the experimental groups.

Malformations:
Besides the umbilical hernia discussed above at the section external examination, an isolated case of unilateral microphthalmia was found in one fetus in the high dose group. According to the background database of Toxi-Coop Zrt. (Appendix XXIV/A and B) this finding may occur sporadically in control fetuses. Considering the background data and that it was a single fetus with this abnormality in this study, this isolated observation was judged to be not related to the test item. Hydronephrosis was recorded for one fetus in the 20 mg/kg bw/day dose group and none in the higher dose groups hence was judged to be incidental.

Variations:
Hydroureter as variation was found in all groups including control. In addition dilated renal pelvis was found in one single fetus in the high dose group. Considering the low incidence and lack of dose response both variations were found to be without a test item response
Other effects:
not examined

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
Developmental toxicity
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
fetal/pup body weight changes
Key result
Dose descriptor:
NOAEL
Remarks:
Teratogenicity
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
external malformations
skeletal malformations
visceral malformations

Fetal abnormalities

Key result
Abnormalities:
no effects observed
Description (incidence and severity):
Only slightly lower fetal weight was observed at 200 mg/kg bw/day

Overall developmental toxicity

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

Applicant's summary and conclusion

Conclusions:
Oral treatment of pregnant Hsd. Han: WISTAR rats from gestation day 5 up to day 19 (the day before Caesarean section) with the test substance at the dose levels of 60 and 20 mg/kg bw/day did not cause death, clinical signs and necropsy findings. The body weight gains and food consumption were slightly to moderately reduced in the 200 mg/kg bw/day group from gestation day 17 onwards. The test substance did not reveal any adverse effect on the pre- and postimplantation loss, number of implantation and the sex distribution of the fetuses. The slightly lower fetal weight was observed at 200 mg/kg bw/day at a dose level with slight maternal effects. The test substance did not increase the incidence of visceral and skeletal variations and induced no fetal malformations. Based on these observations the No Observed Adverse Effect Level (NOAELs) were determined as follows:
NOAEL (maternal toxicity): 60 mg/kg bw/day
NOAEL (developmental toxicity): 60 mg/kg bw/day
NOAEL (teratogenicity): 200 mg/kg bw/day (high dose)
Executive summary:

The test substance was examined for its possible prenatal developmental toxicity. Groups of 22, 22 and 25 sperm-positive female Hsd. Han: Wistar rats were treated with the test substance by oral (gavage) administration daily at three dose levels of 20, 60 and 200 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of 25 sperm positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 2 mL/kg bw. Sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. The test substance in sunflower oil was stable at room temperature for 4 hours and in a refrigerator (5 ± 3 °C) for 3 days at the concentrations of 1, 10 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 95 and 106 % 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, there were 22 evaluated litters each in the control and 60 mg/kg bw/day group, 17 and 21 in the 60 and 200 mg/kg bw/day groups respectively. None of the females died before scheduled necropsy during the study. There were no treatment related clinical signs and necropsy findings observed. The body weight gains (between 17 and 20 as well as for days 0 to 20 including corrected body weight gain) in the 200 mg/kg bw/day group were judged to be moderately decreased by the treatment. At this dose also a slight reduction of the food consumption between gestation days 17 and 20 in the 200 mg/kg bw/day dose group was noted. The mean number of implantations, pre- and post-implantation loss as well as sex distribution of the fetuses were not influenced by the treatment. Fetal weight was slightly lower in the 200 mg/kg bw/day dose group and the incidence of body weight retardation increased moderately at 200 mg/kg bw/day, both were considered to be related to the treatment of the dams. The test item was judged not to influence the incidences of visceral and skeletal variations. The different type of malformations found at the fetal examinations (umbilical hernia, microphthalmia, split xiphoid cartilage in the 200 mg/kg bw/day group each in one fetus, a dumb-bell shaped cartilage of a thoracic centrum in the 60 mg/kg bw/day group as well as hydronephrosis, split sternum and bent scapula in three different fetuses in the 20 mg/kg bw/day group were judged to be incidental according to the experience with this species in this laboratory and in line with historical control data of other Wistar rats as well as due to the lack of a clear dose response-relationship and/or occurrence in the actual control group. Oral treatment of pregnant Hsd. Han: WISTAR rats from gestation day 5 up to day 19 (the day before Caesarean section) with the test substance at the dose levels of 60 and 20 mg/kg bw/day did not cause death, clinical signs and necropsy findings. The body weight gains and food consumption were slightly to moderately reduced in the 200 mg/kg bw/day group from gestation day 17 onwards. The test substance did not reveal any adverse effect on the pre- and postimplantation loss, number of implantation and the sex distribution of the fetuses. The slightly lower fetal weight was observed at 200 mg/kg bw/day at a dose level with slight maternal effects. The test substance did not increase the incidence of visceral and skeletal variations and induced no fetal malformations. Based on these observations the No Observed Adverse Effect Level (NOAELs) were determined as follows: NOAEL (maternal toxicity): 60mg/kg bw/day, NOAEL (developmental toxicity): 60 mg/kg bw/day, NOAEL (teratogenicity): 200 mg/kg bw/day (high dose).