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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:
27 October 2015 - 19 April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same 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:
OECD Guidelines for Testing Chemicals, No.: 414, Prenatal Developmental Toxicity Study, adopted 22nd January 2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. certificate)
Remarks:
CiToxLAB Hungary Ltd.
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Specific details on test material used for the study:
No correction for purity of test item was applied.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
CRL:WIHan
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, from SPF colony
- Age at study initiation: Young adult female rats, nulliparous and non-pregnant, 11/12 weeks old at mating
- Weight at study initiation: 192-231 g (the variation did not exceed ± 20% of the mean weight)
- Acclimation period: 20 days
- Housing: Standard laboratory conditions; individual housing, deep wood sawdust was use as bedding to allow digging and other normal rodent activities. Nest building material was also added into the cages.
- Diet (e.g. ad libitum): ssniff® SM R/M “Autoclavable Complete Feed for Rats and Mice
- Water (e.g. ad libitum): tap water (in water bottles) as for human consumption ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.2-24.4°C (target: 22 ± 3 °C)
- Humidity (%): 38-56% (target: 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 12 January 2016 To: 4 February 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
distilled water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle (Distilled water) at the appropriate concentrations.
Formulations were prepared freshly or up to four days in advance prior to administration to animals.
The resulted formulations were stored at room temperature until use.
Stability of the test item in the selected vehicle (Distilled water) was assessed under conditions similar to this study during the method validation study
(CiToxLAB Study code: 15/380-901AN). According to the results, the test item was shown to be stable in the selected vehicle in the concentration range of
0.1 mg/mL – 20 mg/mL for 7 days when stored at room temperature and for at least 32 days when stored frozen (-23±8oC).

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test item formulations for concentration and homogeneity was performed using a validated HPLC-MS/MS method at the Test Site. Samples* (top, middle and bottom samples) were taken from the test item formulations
three times during the study (during the first and last two weeks of treatment). Similarly, samples were taken on each occasion from the vehicle control formulation for concentration measurement.
All formulations were found to be in the range of 106.7-109.6% of nominal concentrations (1.5, 4.5 and 12.5 mg/mL) and were homogenous.
Formulations had been shown to have good stability.

REMARK:
Due to a failure of the analytical instrument, at the first sampling (on the first week of treatment) two sets of samples were taken for test item formulations
and control, and were stored refrigerated. Additionally two sets of samples were taken for test item formulations and control and stored frozen for
future analysis as agreed by the Principal Investigator. In case of the second and third sampling (on the last two weeks of the treatment), a total of six
sets were taken for test item formulations and control, two sets were stored at room temperature, two sets were stored refrigerated (5±3oC) and two sets
were stored frozen (-23±8oC). The number of samples analysed chemically was in line with the Study Plan.
Details on mating procedure:
The oestrus cycle of female animals was examined shortly before start of pairing. After acclimation, the females were paired according to their oestrus cycle.
- M/F ratio per cage: 1 male : 1 females
- Length of cohabitation: 2 hours until at least 24 sperm positive females/group are attained
- Proof of pregnancy: After the daily mating period, a vaginal smear was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope; the presence of a vaginal plug or sperm in the
vaginal smear was considered as evidence of copulation (GD0).
Sperm positive females were separated and caged individually.
The number of confirmed pregnant, evaluated dams in the dose groups treated was 22 in the Control group and 23, 23 and 21 in the Low (15 mg/kg bw/day),
Mid (45 mg/kg bw/day) and High (125 mg/kg bw/day) dose groups, respectively.

RANDOMISATION:
The sperm-positive, assumed pregnant females were allocated to each experimental group (on each mating day) in such a way that the group averages of the body weight were as similar as possible.
Duration of treatment / exposure:
Days (GD) 6 - 19 of gestation
Frequency of treatment:
Once daily during exposure period
Duration of test:
Until Day 20 of gestation (GD0 to GD20)
Doses / concentrationsopen allclose all
Dose / conc.:
125 mg/kg bw/day
Remarks:
High dose is expected to produce evident maternal toxicity and most likely some associated foetotoxicity.
Dose / conc.:
45 mg/kg bw/day
Remarks:
Mid dose expected to produce at most, minimal maternal toxicity.
Dose / conc.:
15 mg/kg bw/day
Remarks:
Low dose expected to be without maternal effects.
No. of animals per sex per dose:
96 female animals, 24 mated female animals/group, 4 groups (one control and 3 test item-treated groups); 21-23 pregnant and evaluated female animals/group (with implantation sites at necropsy)
60 male animals for mating; no study-procedures were carried out on the male animals.
Control animals:
yes, concurrent vehicle
Details on study design:
The day of mating (when the sperm-positive vaginal smear, and/or the vaginal plug are identified) is regarded as gestation day 0 (GD0). Treatment will be performed daily by oral administration, between GD6 to GD19. Caesarean section and necropsy with macroscopic examination will be performed on GD20. The dose levels were set based on available data including the results of a dose range finding (DRF) study in the pregnant rat (study code: 15/380-105PE, Ref.4), with the aim of inducing toxic effects but not death or severe suffering at the highest dose and a NOAEL at the lowest dose.
See further information under "Any other information on materials and methods incl. tables".

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS:
Cage-side clinical observations were made at twice daily (at the beginning and end of each working day). Detailed clinical observations were made on all animals at the onset of treatment (GD6) then weekly.

DETAILED CLINICAL OBSERVATIONS:
The animals were monitored for any changes including pertinent behavioural changes and signs of toxicity including mortality, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions,
and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic
movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards), tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
On GD13 and/or 14, the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat, which is considered to confirm implantation).

BODY WEIGHT:
The body weight of each animal was recorded with precision of ±1 g on GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food was measured with precision of ± 1 g on GD0, 3, 6, 8, 10, 12, 14, 16, 18 and 20. Food consumption was calculated for each interval, including GD0-6, GD6-20 and GD0-20.

POST-MORTEM EXAMINATIONS:
Before expected delivery, on GD20, Caesarean section will be performed on each treated dam. Sodium pentobarbital administered by intraperitoneal injection, followed by exsanguination will be used for
euthanasia (pentobarbital sodium for injection; details will be documented and reported). Females showing signs of premature delivery prior to scheduled necropsy will be euthanised and subjected to a thorough
macroscopic examination.
Caesarean sections, necropsy of surviving dams and examination of uterine contents were performed on GD20.
Ovaries and uterine content:
The ovaries and uterus was removed and the pregnancy status ascertained. The uterus including the cervix was weighed and examined for early and late embryonic or foetal deaths and for the number of live foetuses. Gravid uterine weights should not be obtained from animals found dead during the study. The number of corpora lutea in each ovary and implantation sites in each uterine horn, the number of live foetuses, early and late embryonic death and foetal death were counted, the number and percent of pre- and post-implantation losses were calculated. The degree of resorption was described in order to estimate the relative time of death of the conceptus. The placentas were examined macroscopically.

Examinations included:
- GRAVID UTERUS WEIGHT:
Gravid uterine weights should not be obtained from animals found dead during the study.
- NUMBER OF CORPORA LUTEA
- NUMBER OF IMPLANTATIONS, EARLY RESORPTIONS, LATE RESORPTIONS:

Fetal examinations:
Each live foetus was weighed individually and subjected to external examination, plus an additional examination of the great arteries. The gender of foetuses were determined according to the anogenital distance. Thereafter the foetuses were individually identified; approximately half of each litter was subject to visceral examination, and the other half was processed for skeletal examination.
Statistics:
The statistical evaluation of data will be performed with the program package SPSS PC+4.0 or SAS 9.2 in the case of Provantis 9, by an appropriate statistical method (Bartlett, ANOVA/ANCOVA and Dunett’s, Kruskal-Wallis and Mann-Whitney U tests, T-test, Wilcoxon test, Chi2). The homogeneity of variance between groups will be checked by Bartlett`s homogeneity of variance test. Where no significant heterogeneity is detected a one-way analysis of variance (ANOVA/ANCOVA) will be made. If the obtained result is significant Duncan’s Multiple Range test or Kruskal-Wallis test will be used to assess the significance of inter-group differences. Significant results with inter-group comparisons will be further compared using Kruskal-Wallis, and Mann-Whitney U-tests.
Indices:
No information available.
Historical control data:
No information available.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In the High dose group (125 mg/kg bw/day), signs of maternal toxicity were seen including mortality and clinical signs (for example piloerection, noisy respiration, increased salivation, decreased activity).
Slight maternal toxicity was also observed in the Mid dose group of 45 mg/kg bw/day (transient piloerection in more than half of the animals and macroscopic findings in the stomach). The change in the stomach
was considered to reflect a local effect and the transient piloerection was probably related to this local irritation). There was no evidence of any adverse maternal effects in the Low dose group (15 mg/kg bw/day).
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Three animals in the High dose group (125 mg/kg bw/day) were found dead during the treatment period. Dam #186 was found dead on GD16, slight noisy respiration and piloerection was observed in the previous period from GD9;
the animal had hunched back, moderately decreased activity, red discharge, coloured mucoid and it was cold to touch on the previous day (GD15).
Dam #163 was found dead on GD17, slight noisy respiration and piloerection was recorded previously from GD10 for this animal. Dam #165 was found dead on GD19, slight noisy respiration and piloerection was observed in the previous
period from Day 9; the animal had hunched back and slightly decreased activity on the previous day (GD18).
None of these observations such as transient piloerection in the Low and Mid dose group were considered to be related to any systemic effect of the test item, but clear systemic responses were detected in the High dose group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No toxicologically significant changes were observed in the mean body weights or body weight gain values in the Low or Mid dose groups (15 and 45 mg/kg bw/day) when compared to control. Statistically significant decreases in the mean
body weight and body weight gain during the treatment period (GD6-20) or during the overall period of the study (GD0-20) was observed for the High dose group when compared to the vehicle control.
In the High dose group (125 mg/kg bw/day), the corrected terminal body weight and corrected body weight gain throughout the treatment and gestation periods (when the observed values were adjusted for the gravid uterine weight) were significantly lower than the control. As there were no treatment-related changes in the gravid uterine weight recorded in the test item treated pregnant dams when compared to the controls, these effects on body weight (minus gravid uterus)
were considered to be an adverse effect of test substance administration to the dam. The corrected net body weight gain value of the Mid dose group (45 mg/kg bw/day) was also lower than the control, although the difference was statistically not significant, but the individual values of the corrected net body weight gain for six animals in the Mid dose group had lower values than the lower limit of the Historical Control range (mean minus 2SD) while none of the control animals showed such a low value. These facts were considered as a slight test item related effect.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A slight effect of the test item on the food consumption was observed in the study. Statistically significant decrease was observed in the food consumption of the High dose group (125 mg/kg bw/day) only in each period after the start of the treatment (ie. GD6-8, 8-10, 10-12, 12-14, 14-16, 16-18 and 18-20, by approximately 18-42%). Furthermore, the total food consumption during the entire period of the study (GD0-20) or during the treatment period (GD6-20) was also decreased when compared to the vehicle control (by 20.6% and 29.5%, respectively), the difference was statistically significant in both cases at p<0.01 level. No similar effect was observed at the beginning of the study (GD0-6) when no test item was administered to the animals. These facts were considered as a treatment related effect on food intake in the High dose group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Moderately decreased activity was observed in the High dose group for two animals on Day 19, and rooting of bedding was recorded for another animal in the High dose group on Day 19.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no treatment-related changes in the gravid uterine weight recorded in the test item treated pregnant dams when compared to the controls, these effects on body weight (minus gravid uterus) were considered to be a direct effect of test substance administration to the dams.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
During necropsy, findings in the stomach were detected in all animals of the High dose group (125 mg/kg bw/day), similar observations were recorded for four animals in the Mid dose group (45 mg/kg bw/day).
No remarkable internal or external observations were recorded for any Control or Low dose (15 mg/kg bw/day) animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
No abnormalities were observed on the placentas in any examined groups.
Details on results:
EVALUATED ANIMALS:
Fur thin was detected in the control group for three animals (in the period of Days 7-12, 13-20 and 14-19), the same observation was recorded in the Low dose group (15 mg/kg bw/day) for one animal on Days 8-20. Crust on the neck dorsal area
(approx. 1 cm) was observed for one animal in the Control group on Days 7-20. Slight noisy respiration was detected in the Mid dose group (45 mg/kg bw/day) for one to nine animals on Days 10, 12, 15-17 and 19, and in the High dose group for one to thirteen animals on Days 7-20 (in one case on Day 10 the observed effect was moderate). Piloerection was recorded in the Low dose group for three animals on Day 11 or Days 17-18; in the Mid dose group for one to twenty animals on Days 8 and 10-20, and in the High dose group for two to twenty one animals on Days 7-20. Increased salivation was observed for one animal in the High dose group on Day 14. Red discharge was detected in the High dose group for one animal on Day 10 and for another animal on Days 17-18. Moderately decreased activity was observed in the High dose group for two animals on Day 19, and rooting of bedding was recorded for another animal in the High dose group on Day 19.
Findings in the stomach were detected in all animals (21/21) of the High dose group (125 mg/kg bw/day): diffuse, grandular or non-grandular mucosa and/or multifocal, non-grandular mucosa. Similar observations were recorded for 4 out of 23 animals in the Mid dose group (45 mg/kg bw/day). Enlarged (bilateral) adrenal glands were observed in one (1/21) animal of the High dose group. No remarkable internal or external observations were recorded for any Control or Low dose
(15 mg/kg bw/day) animals.
ANIMALS EXCLUDED FROM EVALUATION:
The excluded animals in the Control and Low dose groups were symptom-free. Piloerection was recorded for the Mid dose animal on Days 15-17.
Smaller left kidney and renal pelvis dilatation was observed in one (1/1) Low dose animal, and multifocal non-grandular mucosa in the stomach was observed in one animal (1/1) in the Mid dose group. Diffuse or multifocal, non-grandular mucosa (3/3) was observed in all High dose animals. Diffuse dark red discoloration in all lobes of the lungs were recorded for two (2/3) animals in the High dose group. Enlarged (bilateral) adrenal glands were recorded in all (3/3) High dose animals; while small spleen was observed in one (1/3) High dose animal. Yellowish mucoid material at the urogenital area and red material at the perinasal / perioral area was detected in one (1/3) High dose animal.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
No abortions were observed compared to the contol group. (detailed numbers under "Any other information on results incl. tables" below)
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No adverse effect was observed in pre-implantation loss of the test item treated groups when compared to the control. There was no statistically significant difference in the post-implantation loss between the test item treated and control groups.
(detailed numbers under "Any other information on results incl. tables" below)
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no effects considered related to test item administration on the early and late embryonic loss, post-implantation loss (total resorption, including the early and late embryonic loss) or total intrauterine mortality in the test item-treated dams evaluated.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no effects considered related to test item administration on the early and late embryonic loss, post-implantation loss (total resorption, including the early and late embryonic loss) or total intrauterine mortality in the test item-treated dams evaluated.
Dead fetuses:
no effects observed
Description (incidence and severity):
There was no statistically significant difference in foetal death in any group compared to the control. (see table below).
No dead fetuses were seen.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
No changes on pregnancy duration was noted.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Ninety six females (24 in each group) were mated in the study. The number of confirmed pregnant, evaluated dams was 22, 23, 23 and 21 in the Control, Low (15 mg/kg bw/day), Mid (45 mg/kg bw/day) and High dose groups
(125 mg/kg bw/day), respectively. Three animals were found dead at the highest concentraton (125 mg/kg bw/day).
But no effects on pregnancy duration was noted.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
No changes in number of pregnant. Number of mated females for all concentrations including control: 24; Number of non-pregnant females Control 2, Dose 15 mg/kg bw/day 1, Dose 45 mg/kg bw/day 1, Dose 125 mg/kg bw/day 0
Three animals were found dead at Dose 125 mg/kg bw/day.
Number of evaluated females on GD20 (Caesarean section) control 0: 22; Dose 15 mg/kg bw/day 23; Dose 45 mg/kg bw/day 23; Dose 125 mg/kg bw/day 21
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
In the high dose group (125 mg/kg bw/day) mortality was observed, as well as test item related effects on corrected body weight, corrected body weight gain and food consumption. There was no difference in the intrauterine parameters (number of corpora lutea, number of implantation, early and late embryonic losses etc.) of the test item treated groups when compared to the control.
When administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD 6 to 19, caused maternal toxicity in the High dose group
(125 mg/kg bw/day), and slight maternal toxicity in the Mid dose group (45 mg/kg bw/day). There was no evidence of any adverse maternal effects in the
Low dose group (15 mg/kg bw/day). There was no clear evidence for any systemic toxicity at 45 mg/kg bw/day, but as the findings in this dose group were
treatment-related, thus it was considered that 15 mg/kg bw/day dose represents the NOAEL.


Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day
Based on:
act. ingr.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Abnormalities:
no effects observed
Localisation:
placenta
Description (incidence and severity):
No abnormalities were observed on the placentas of any animals in any examined
groups.

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean weight of foetuses per litter in the Low, Mid dose groups (15 and 45 mg/kg bw/day) did not differ significantly from the control mean, but significantly decreased body weight per litter was observed in the High dose group (125 mg/kg bw/day).
Furthermore, the number of body weight retarded foetuses (runts) and the number of affected litters was also increased in the High dose when compared to the control.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): The mean weight of foetuses per litter in the Low and Mid dose groups (15 and 45 mg/kg bw/day, respectively) did not differ significantly from the control mean value as, but the mean weight of foetuses
per litter in the High dose group (125 mg/kg bw/day) was significantly lower than the control value.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The mean number of viable foetuses was comparable with the control mean.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no toxicologically significant difference in the sex distribution of foetuses between the control and treatment groups as shown in the Table below.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
The total number of retarded foetuses (with a weight of < the concurrent control mean-2SD) was significantly higher in the High dose group (125 mg/kg bw/day) than in the Control group, the number of affected litters was also higher in the High dose group than in the Control group, although the difference in this case was statistically not significant. Additionally, the maximum ratio of foetuses with retarded body weight within a litter also increased from 14% (Control group) to 40% (High dose group). These facts indicated an adverse test item effect causing the retarded body development.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
The mean number of viable foetuses was comparable with the control mean.
External malformations:
no effects observed
Description (incidence and severity):
No external malformations were recorded in the study, and only one external variation was recorded in the control group, but that finding was considered to be incidental and biologically not relevant from
the test item treatment point of view.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No skeletal malformations were recorded. All of the skeletal findings correspond with the current historical control (HC) or the concurrent study control data.
Visceral malformations:
no effects observed
Description (incidence and severity):
Most of the visceral findings are consistent in general nature and incidence with the study concurrent control data or the existing historical control data. Based on the isolated occurrence,
the observation Renal vein, supernumeracy in the Mid dose group was considered incidental, ascribed to individual variability and not related to treatment, however concurrent study control or
existing Historical Control (HC) database did not contain this observation.
In summary, all abnormalities observed at visceral examination in this study were considered as incidental findings; they corresponded with the concurrent study control data, with historical control (HC) data
or were considered to be spontaneous events unrelated to treatment.
Other effects:
no effects observed
Description (incidence and severity):
In conclusion, all of the findings corresponded to the concurrent study control or current historical control data or have isolated occurrence without dose response that
were considered incidental ascribed to individual variability and not related to treatment. Therefore, based on these results, the test item did not affect adversely the
intrauterine development by higher incidence of malformations in the treated groups.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects: no effects

Details on embryotoxic / teratogenic effects:
Four dams were not pregnant. All pregnant dams had one or more live foetuses at scheduled sacrifice. The evaluation of external, visceral and skeletal abnormalities showed no difference between test item treated groups and control group.

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No effects were noted.
Remarks on result:
other:
Remarks:
embryotoxicity
Key result
Dose descriptor:
NOAEL
Effect level:
45 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
changes in litter size and weights
Remarks on result:
other:
Remarks:
foetotoxicity
Key result
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No foetal anomalies, either malformations or variations noted at any dose level.
Remarks on result:
other:
Remarks:
teratogenicity

Fetal abnormalities

Abnormalities:
no effects observed
Description (incidence and severity):
No foetal effects were seen at external, visceral and/or skeletal examination of foetuses
in the study which could be related to the test item administration up to and including
125 mg/kg bw/day.

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
125 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
no
Relevant for humans:
no

Any other information on results incl. tables

Statistically significant lower foetus weight per litter was observed in the High dose group, and the number of foetuses with retarded body weight was also increased in this group.

Summary of the intrauterine evaluation:

Parameters

Dose (mg/kg bw/day)

0

15

45

125

Number of evaluated dams

22

23

23

21

Mean number of corpora lutea

11.50

11.74

11.70

12.14

Mean number of implantations

10.86

11.26

11.09

11.48

Pre-implantation loss, mean

0.64

0.48

0.61

0.67

Pre-implantation loss (%), mean

5.41

4.35

5.30

4.90

Early embryonic loss, mean

0.45

0.35

0.17

0.52

Early embryonic loss (%), mean

4.23

2.83

1.78

4.81

Late embryonic loss, mean

0.18

0.09

0.04

0.05

Late embryonic loss (%), mean

1.91

0.83

0.30

0.33

Dead foetuses, mean

0.00

0.00

0.00

0.00

Post-implantation loss, mean

0.64

0.43

0.22

0.57

Post-implantation loss (%), mean

6.14

3.70

2.09

5.41

Total intrauterine mortality, mean

1.27

0.91

0.83

1.24

Total intrauterine mortality (%), mean

11.05

7.96

7.22

9.95

Viable foetuses, mean

10.23

10.83

10.87

10.90

Viable foetuses (%), mean

93.91

96.30

97.91

94.86

Notes: Mean values were rounded to two decimal places. No statistically significant

differences were observed in these examined parameters of any test item treated groups when

compared to the negative (vehicle) control.

Examination of viable foetuses:

Parameters

Dose (mg/kg bw/day)

0

15

45

125

Number of examined litters

22

23

23

21

Viable foetuses, mean

10.23

10.83

10.87

10.90

Male foetuses, mean                       

5.18

4.78

5.30

5.10

Female foetuses, mean

5.05

6.04

5.57

5.81

Total number of foetuses

225

249

250*CH

229

Total number of male foetuses

114

110

122

107

Total number of female foetuses

111

139

128

122

Mean foetal weight / litter (g)

3.521

3.516

3.531

3.271**DN

Number of fetuses with retarded body weight

5

5

7

20**CH

Number of affected litters

5

5

6

10

Notes: Mean values were rounded to two or three decimal places. CH: Chi square test, DN:

Duncan’s multiple range test; *: p< 0.05; **: p< 0.01

Summary table of the external abnormalities:

 

Dose (mg/kg bw/day)

HC data

Control

15

45

125

Total number of examined litters

22

23

23

21

485

Total number of examined foetuses

225

249

250

229

4955

External malformations

No external malformations were recorded

External variations

Foetus, pale

1 / 1

--

--

--

1 / 1

Notes: Numbers represent the number of abnormalities / number of affected litters.

HC: historical control

Summary table of the visceral abnormalities:

 

Dose (mg/kg bw/day)

HC data

Control

15

45

125

Total number of examined litters

22

23

23

21

485

Total number of examined foetuses

110

124

125

115

2484

Visceral malformations

Situs inversus, total

--

--

--

1 / 1

4 / 4

Visceral variations

Thymic cord

2 / 2

2 / 2

1 / 1

2 / 2

66 / 55

Brachiocephalic trunk, short

2 / 2

--

2 / 2

--

43 / 35

Renal papilla, small

--

1 / 1

3 / 2

1 / 1

18 / 16

Renal vein, supernumeracy

--

--

1 / 1

--

--

Ureter, convoluted

1 / 1

--

1 / 1

1 / 1

3 / 3

Notes: Numbers represent the number of abnormalities / number of affected litters.

HC: historical control

Summary table of the skeletal abnormalities:

 

Dose (mg/kg bw/day)

HC data

Control

15

45

125

Total number of examined litters

22

23

23

21

484

Total number of examined foetuses

115

125

125

114

2467

Skeletal malformations

No skeletal malformations were recorded

Skeletal variations

Skull, 2 or more bones incomplete

ossification

5 / 2

9 / 5

3 / 2

3 / 2

474 / 326

Ossified sternebra (3 or less)

--

--

--

3 / 3

46 / 37

Rib, wavy, marked

1 / 1

--

--

--

243 / 149

Vertebra, dumbbell or asymmetric

ossification (2 or more)

--

1 / 1

1 / 1

2 / 2

79 / 73

Vertebra, bipartite ossification

--

3 / 3

--

--

24 / 24

Vertebra, dumbbell shaped

1 / 1

1 / 1

--

1 / 1

6 / 6

Pubis, unossified

--

1 / 1

--

1 / 1

6 / 6

Tarsal ossified ≤ 3.5

--

1 / 1

--

1 / 1

75 / 53

Notes: Numbers represent the number of abnormalities / number of affected litters.

HC: historical control

 

Applicant's summary and conclusion

Conclusions:
In the High dose group (125 mg/kg bw/day), signs of maternal toxicity were seen including mortality and clinical signs , decreased body weight and body weight gain, decreased food consumption.
Macroscopic findings in the stomach and adrenal glands were also recorded in this dose group at the necropsy.
Slight maternal toxicity was also observed in the Mid dose group of 45 mg/kg bw/day. The change in the stomach was considered to reflect a local effect and the transient piloerection was probably
related to this local irritation. There was no evidence of any adverse maternal effects in the Low dose group (15 mg/kg bw/day).
There was no clear evidence for any systemic toxicity at 45 mg/kg bw/day, but as the findings in this dose group were treatment-related, thus it was considered that 15 mg/kg bw/day dose represents the NOAEL for maternal toxicity.
There were no toxicologically significant differences, or test item related-changes in the evaluated intrauterine parameters examined up to and including 125 mg/kg bw/day.
The mean weight of foetuses per litter in the High dose group (125 mg/kg bw/day) was significantly lower than the control value. The total number of retarded foetuses
was also significantly higher in the High dose group than in the Control group. No similar effects were seen in the Mid and Low dose groups (45 and 15 mg/kg bw/day).
No foetal effects were seen at external, visceral and/or skeletal examination of foetuses in the study which could be related to the test item administration up to and including 125 mg/kg bw/day.

NOAELmaternal toxicity: 15 mg/kg bw/day
NOAELembryotoxicity: 125 mg/kg bw/day
NOAELfoetotoxicity: 45 mg/kg bw/day
NOAELteratogenicity: 125 mg/kg bw/day
No direct developmental toxicity including teratogenicity was observed at any dose level. There are developmental effects, but these are secondary (related) to maternal toxicity.
Executive summary:

The prenatal developmental toxicity study was performed in year 2015/2016 according to OECD Guideline 414 and GLP.

The test substance was administered daily by gavage to pregnant rats on gestation days 6 -19, at concentrations of 0, 15, 45 and 125 mg/kg bw/day. 

The dams were sacrificed at gestation day 20 and the foetuses were examined for visceral and skeletal variations and malformations. Placentas were examined externally.

The test item caused maternal toxicity in the High dose group (125 mg/kg bw/day), and slight maternal toxicity in the Mid dose group (45 mg/kg bw/day). There was no evidence of any adverse maternal effects in the Low dose group (15 mg/kg bw/day). There was no clear evidence for any systemic toxicity at 45 mg/kg bw/day, but as the findings in this dose group were treatment-related, thus it was considered that 15 mg/kg bw/day dose represents the NOAEL. There were no toxicologically significant differences, or test item related-changes in the reproductive parameters examined up to and including 125 mg/kg bw/day under the conditions of this study. The mean weight of foetuses per litter in the High dose group (125 mg/kg bw/day) was significantly lower than the control value; furthermore signs of retarded body development were also recorded in this group. No similar effect were seen in the Mid and Low dose groups (45 and 15 mg/kg bw/day). No foetal effects were seen in any dose groups at external, visceral and/or skeletal examination of foetuses in the study which could be related to the test item administration. In this study, from the observations made in the dams and their foetuses,

the following no-observed-adverse-effect levels were derived:

NOAELmaternal toxicity: 15 mg/kg bw/day

NOAELembryotoxicity: 125 mg/kg bw/day

NOAELfoetotoxicity: 45 mg/kg bw/day

NOAELteratogenecity: 125 mg/kg bw/day

There are developmental effects, but these are secondary (related) to maternal toxicity.