Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2016-11-02 to 2017-05-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Cerium trinitrate
EC Number:
233-297-2
EC Name:
Cerium trinitrate
Cas Number:
10108-73-3
Molecular formula:
Ce.3HNO3
IUPAC Name:
cerium trinitrate
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Hannover Wistar (CRL:WI(Han))
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633, Sulzfeld, Germany) from SPF colony.
- Age at study initiation: approximately 15 weeks old at mating, young adult female rats, nulliparous and non-pregnant
- Weight at study initiation: 215-269 grams ; the variation did not exceed +/- 20% of the mean weight.
- Fasting period before study: No data
- Housing: Standard laboratory conditions; individual housing in Type II polycarbonate cages during mating and gestation period and Type III polycarbonate cages during the acclimatization period. Lignocel Hygenic Animal Bedding were used in the study. Arbocel crinklets natural nesting material was used in the study. Successfully mated animals were housed individually. Deep wood sawdust was used as bedding to allow digging and other normal rodent activities. Nest building material was also added into the cages.
- Diet (e.g. ad libitum): ad libitum, ssniff® SM Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance
- Water (e.g. ad libitum): ad libitum, tap water (in water bottles) for human consumption
- Acclimation period: At least 27 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.4-24.9°C
- Humidity (%): 25-55%
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light

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 at the appropriate concentrations according to the dose level and volume. During the formulation process, the pH of each formulation was adjusted using 0.1M NaOH solution to achieve a pH >= 3.5, to prevent non-specific pH damage to the stomach. The final pH values of the applied dose formulations were measured on each day of administration. Formulations were prepared fresh daily prior to administration to animals (not more than 2 hours before use) to allow their use according to the available stability information. Formulations were kept protected from light.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Distilled water; based on available information of a previous rodent study and the results of a DRF study
- Concentration in vehicle: 0, 10, 30, and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg. A constant volume of 10mL/kg was administered to all dose groups, including the controls. The individual volume of the treatment was based on the most recent individual body weight of the animals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test item formulations for concentraton and/or homogeneity was performed using a validated ICP/AES method to determine the cerium content. Top, middle and bottom samples were taken from the test item formulations two times during the study (during the first and last week of the treatment). Samples were taken in duplicate (2mL/each), one set to analyse and one set as back-up. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.
Formulation samples were kept at room temperature and protected from light (in brown glass containers) until shipment to the Test Site.
Acceptance criteria of the concentration analysis was set according to the analytical method validation, at 100 +/- 15% of the nominal concentration.
Mean measured concentrations of each dose group were analyzed on 08 November 2016 and on 24 November 2016, as follows:
- 08 November analysis- 10.4 mg/mL (104% of nominal); 31.1 mg/mL (103% of nominal); 102 mg/mL (102% of nominal)
- 24 November analysis- 10.0 mg/mL (100% of nominal); 29.8 mg/mL (99% of nominal); 102 mg/mL (102% of nominal)
Details on mating procedure:
- The oaestrus cycle of female animals was examined shortly before start of pairing
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 male, 1 female
- Length of cohabitation: Approximately 2 hours in the morning, daily, until at least 24 sperm-positive females/group are obtained.
- Proof of pregnancy: after the daily mating period, a vaginal smear was prepared and stained with 1% methylene blue solution. The smear was examined with a light microscope; the presence of a vaginal plug or sperm in vaginal smear was considered as evidence of copulation (referred to as day 0 of pregnancy, gestation day 0 or GD0). Sperm positive females were seperated and caged individually.

Duration of treatment / exposure:
from gestation day 6 to gestation day 19
Frequency of treatment:
7 days/week, daily administration, approx at similar times
Duration of test:
20 days from confirmed mating until necropsy
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle control group, group 1, 0mg/mL
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low dose group, group 2, 10 mg/mL
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Mid dose group, group 3, 30 mg/mL
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High dose group, group 4, 100 mg/mL
No. of animals per sex per dose:
24 mated females per dose group, 4 dose groups
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were set based on available information of the chemical nature and characteristics of the test item and available results of an acute toxicity study, a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test and a dose range finding study in pregnant rats.
- In an acute oral toxicity study (OECD no. 401) in Sprague-Dawley rats, LD50 data of 4200 mg/kg was determined.
- In a combined repeated dose toxicity study with reproduction/development screening study (according to OECD guideline 422), a possible local irritation effect on stomach and mortality was observed. The NOAEL was established at 330 mg/kg/day.
- In the pregnant DRF study, concentrations up to 1000 mg/kg/day were examined using pregnant Hannover wistar rats. There were no signs of maternal toxicityother than a possible slight transient body weight difference during the first days of treatment and no embryotoxicity or foetotoxicity were observed in any test item treated dose group.
Based on this information, the doses of 100, 300 and 1000 mg/kg bw/day were deemed suitable for the purpose of the study.
- Rationale for animal assignment (if not random): The sperm-positive, assumed pregnant females were allocated to each experimental group (on each mating day) in such a way that the group average of the body weight was as similar as possible. Females paired with the same male were allocated to different groups on the same mating day.
- Justification of the strain: The rat is regarded as a suitable rodent species for reproduction studies and the test guideline states it is the preferred rodent species. The Hannover Wistar rat was selected due to the experience of the Test Facility with this strain in teratology studies.
- Route of administration: the oral route is a potential route of human exposure to the test item and it was considered suitable to provide the exposure required for this developmental toxicology study.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily (at the beginning and end of each working day); only one general clinical observation in the afternoon on days when detailed clinical observation is made in the morning. When signs of toxicity were noted in a certain animal, this animal was observed more frequently.
- Parameters:
- signs of morbidity and mortality
- pertinent behavioural changes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: on all animals at the onset of treatment (GD6), then weekly. On necropsy days, detailed clinical observation was made only once.
- Parameters:
- signs of toxicity like changes in fur, skin, eyes, mucous membranes, occurence of secretions and excretions, and autonomic activity (eg 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 (eg excessive grooming, repetitive circling), bizarre behaviour (eg self-mutilation, walking backwards), tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
- on GD13 and/or 14, the sperm positive females were examined for 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: Yes
- Time schedule for examinations: 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.
- Corrected body weight was calculated: body weight on GD20 minus eight of the gravid uterus

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food was weighed 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.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Before expected delivery, on gestation day 20, Caesarean section was performed on each treated dam. Sodium pentobarbital administered by intraperitoneal injection, followed by exsanguination was used for euthanasia.
Females showing signs of premature delivery prior to scheduled necropsy will be euthanised and subjected to a thorough macroscopic examination
- Gross pathology: The dams' viscera were examined macroscopically for any structural abnormalities or pathological changes. All the gross findings were retained in 10% buffered formalin solution (or modified davidson fixative, in case of the eyes) for possible future evaluation
- Histopathology: no histopathological evaluation was performed, unless deemed necessary and depending on the effects observed.
Ovaries and uterine content:
The ovaries and uterine were removed and pregnancy status was ascertained
Examinations included:
- Gravid uterus weight: Yes, including cervix; not from animals found dead during the study. The uterus was examined for for early and late embryonic or foetal deaths and for the number of live foetuses.
- Number of corpora lutea: Yes, in each ovary
- Number of implantations: Yes , in each uterine horn
- Number of early resorptions: Yes , degree of resorption was described in order to estimate the relative time of death of the conceptus
- Number of late resorptions: Yes , degree of resorption was described in order to estimate the relative time of death of the conceptus
- Other:
- Placentas were examined macroscopically
- Uteri that appeared non-gravid were further examined to confirm the non-pregnant status
- Classified according to time of death: preimplantation loss, postimplantation loss, early and late embryonic loss as well as foetal death.
Fetal examinations:
- External examinations: Yes; all per litter; plus an additional examination of the great arteries
- Soft tissue examinations: Yes; half of the litter
- the abdominal and thoracic region was opened and the thymus and great arteries were freshly examined by means of a dissecting microscope. The rest of the body was fixed in sanomya mixture then after fixation the body was microdissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method.
- Skeletal examinations: Yes; half of the litter
- the abdominal region was opened, and the viscera and skin of foetuses were removed and the cadaver was fixed in alcian-blue - acetic acid - ethanol mixture. After fixation in isopropanol the skeletons were stained by KOH-Alizarin red-S method and examined by means of a diszsecting microscope.
- Head examinations: Yes; half of the litter (see above)
- Each foetus was weighed individually (accuracy +/-0.01g)
- The gender of foetuses was determined according to appearance of the anogenital distance
- All abnormalities (external, soft tissue and skeletal malformations, and variations) found during the foetal examinations were recorded; photographic records were made additionally.
Statistics:
-SAS v9.2 software package in case of Provantis v9.2:
- normality and heterogeneity of variance between groups: checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified).
- Where both tests showed no significant heterogeneity: Anova/Ancova (one-way analysis of variance) test
- If obtained result was positive, Dunnett's (Multiple Range) test to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis was the better option when the normality and heterogeneity assumptions implicit in the tests were adequate.
- If Shapiro-Wilk or Levene tests showed significance: non-parametric analysis . A Kruskal-Wallis analysis of variance was used after Rank Transformation.
-If there was a positive result: inter-group comparisons using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.
-SPSS PC+4.0 program package (for data tabulated in Excel), the heterogeneity of variance between groups was checked by Bartlett's test.
- no significant heterogeneity detected: one-way analysis of variance (ANOVA)
- If obtained result significant: duncan's Multiple Range test to assess the significance of inter-group differences.
- significant heterogeneity: normal distribution of data examined by Kolmogorow-Smirnow test.
- In case of not normal distribution: non-parametric method of Kruskal-Wallis One-Way analysis of variance
- If positive result : inter-group comparisons using Mann-Whitney U-test.
- The Chi-squared test was used for comparing group incidences against the controls.
Indices:
Necropsy data
- preimplantation loss (%, group mean): (number of corpora lutea - number of implantations)/number of corpora lutea x 100
- postimplantation loss (%, group mean): (number of implantations - number of live foetuses)/number of implantations x 100
Foetal data
- sex distribution (%, group mean): (number of male(female) foetuses)/number of foetuses x 100
- external abnormalities/litter (%, group mean): (number of foetuses with abnormality)/number of foetuses x 100
- visceral abnormalities/litter (%, group mean): (number of foetuses with abnormality)/number of foetuses x 100
- skeletal abnormalities/litter (%, group mean): (number of foetuses with abnormality)/number of foetuses x 100
Historical control data:
No data

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related effects or systemic clinical signs were noted in the treated animals of the Low (100 mg/kg bw/day), Mid (300 mg/kg bw/day) or High (1000 mg/kg bw/day) dose groups.
Slight noisy respiration (animals 3503, 3507, 3511 and 3514 in the Mid dose group; and 4510 and 4516 in the High dose group) and fur thin (animal 1510 in the Control group; and 4502 and 4513 in the High dose group) was recorded on some days, but those findings were not considered as a test item related effect.
One animal (ID 148) of the Low dose group was excluded from the study and euthanized due to scientific reason, as it delivered pups on GD 14, thus could not be used for the study purpose.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality was recorded for any animals in the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No test item related effect on body weight was observed in the Low (100 mg/kg bw/day) and Mid (300 mg/kg bw/day) dose groups, but an effect on body weight was noticed in the High (1000 mg/kg bw/day) dose group when compared to control.
No toxicologically significant changes were observed in the mean body weights of the Low, Mid or High dose groups when compared to control although the High dose weights remained below control until study termination.
Statistically significant differences (p<0.01) were seen in the body weight gain of the High dose group in the beginning of the treatment period (GD 6-8, GD 8-10) when compared to the control value. Although the body weight gain values recovered later, but the difference between High dose group and control (approx. 13-16%) remained statistically significant (p<0.01) when evaluated for the treatment period (GD 6-20) or entire study (GD 0-20).
Furthermore, statistically significant decreases were recorded in the corrected body weight, corrected body weight gain (GD 0-20) and net body weight gain (GD 6-20) values of the High dose group compared to the control values.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No biologically relevant changes were observed in the mean daily food consumption of dams in the Low (100 mg/kg bw/day) and Mid (300 mg/kg bw/day) dose group, but reduced food consumption was recorded in the High dose group (1000 mg/kg bw/day) compared to the control value.
The food consumption of the Low dose group was comparable with the Control group. Decreased food consumption (statistically significant in the Mid and High dose groups at p<0.05 and p<0.01 level, respectively) was recorded in the first half of the treatment period, although they partially recovered later. The mean daily food consumption was significantly decreased in the Mid and High dose group when calculated for the complete treatment period (by 8% and 14%, respectively). However, the difference was statistically not significant in the Mid dose group (6%) when calculated for the entire study, but it still gained statistical significance (p<0.01) in the High dose group (11%). Due to the lack of statistical significance for the study period and there was no effect on body weight or body weight gain, the observed changes in the Mid dose group were not considered as an adverse effect, in contrast to the High dose group where the observed body weight changes were seen as adverse. The major drop in food intake at the High dose group immediately after treatment initiation was a clear effect which could be expected to have the potential for secondary foetal effects.
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):
No treatment related observations were recorded for any evaluated animals in the study.
Multifocal red discoloration of the glandular mucosa was recorded on the stomach of one high dose group animal (4523). This fur on the neck was noticed for another high dose animal (4513). However, these facts were considered as biologically not relevant findings and not related to the test item administration.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
No further data

Maternal developmental toxicity

Number of abortions:
not specified
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
- Number of females with <=5 implantation sites: 0 in all groups
- There was no statistically significant difference in foetal death in any test item treated groups compared to the control
- The mean number of corpora lutea and the mean number of implantation sites were comparable with the controls in all test item treated groups.
- No effect was observed in preimplantation loss of the test item treated groups when compared to the control
- The early and the late embryonic loss did not differ significantly from the control in the test item treated groups.
- There was no statistically significant difference in the postimplantation loss between the test item treated and control groups.
Total litter losses by resorption:
not specified
Description (incidence and severity):
Number of corpora lutea: 11.88 (control), 12.23 (low dose), 12.61 (mid dose), 12.04 (high dose)
Early or late resorptions:
not specified
Dead fetuses:
not specified
Description (incidence and severity):
- The total intrauterine mortality was comparable with the control; no statistically significant differences were observed
- There was no statitically significant difference in the number of dead foetuses in any test item treated group compared to the control
Changes in pregnancy duration:
no effects observed
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 specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
- 96 (24 in each group) were mated in the study.
- the number of confirmed pregnant, evaluated dams was 24 in the control group, 22 in the low dose group, 23 in the mid dose group and 24 in the high dose group
Other effects:
no effects observed
Description (incidence and severity):
Evaluation of placentas: No abnormalities were observed on the placentas of any animals in the control, low, mid or high dose groups.
Details on maternal toxic effects:
No further data

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
300 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

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean weight of foetuses per litter in the low, mid, and high dose groups did not differ significantly from the control mean value. In the case of the parameter 'mean body weight/foetus' significantly higher value (p<0.05) was observed in the low dose group compared to the control, but the slightly higher foetal body weight with a lack of dose response, was not considered as a test item related effect.
The total number of retarded fetuses (runts) as well as the number of affected litters was similar or lower in the test item treated group compared to the control group.
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 specified
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
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The mean weight of foetuses per litter in the low, mid, and high dose groups did not differ significantly from the control mean value
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
The total number of retarded foetuses (runts) as well as the number of affected litters was similar or lower in the test item treated group compared to the Control group, indicating no effect on this parameter
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
All of the external findings correspond with the current historical control or the concurrent study control data, or were considered to be incidental findings
- Number of foetuses with malformation : 0/249 (control), 1/232 (low dose), 0/245 (mid dose), 0/260 (high dose)
- Number of foetuses with variation: 2/249 (control), 0/232 (low dose), 0/245 (mid dose), 2/260 (high dose)
Two malformed foetuses were recorded in the low dose group (with craniorachischisis or with absent tail). Although no similar findigns were detected in the current study control data, but based on the isolated occurrence of both malformations and the lack of dose response, these observations were considered incidental, ascribed to individual variability and not related to treatment.
The occurence of subcutaneous haemorrhage variation was comparable with the control group data, thus these facts were considered as irrelevant findings and not related to the test item treatment.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
All of the skeletal findings correspond with the historical control or the concurrent study control data, or were considered to be incidental findings without dose response
- Number of foetuses with malformation : 1/123 (control), 2/116 (low dose), 2/123 (mid dose), 1/131(high dose)
- Number of foetuses with variation: 7/123 (control), 15/116 (low dose), 17/123 (mid dose), 3/131 (high dose)

- total number of intact foetuses: significantly lower in the low and mid dose groups than in the control group but no similar differences were noted in the high dose group. Similarly, the total number of foetuses with skeletal variation was slightly higher in the low and mid dose groups than in the control group but the incidence in the high dose group was comparable with the control group. due to lack of dose response, these facts were considered as no test item related effect.
- other effects, considered not related to test item treatment:
- malformation of malpositioned pelvic girdle for one foetus in mid dose group, and one foetus in control group
- other malformations as malformed or absent vertebrae (one foetus in each group) and craniorachischisis (one foetus in the low dose group)
- in case of several variations (incomplete ossification of skull, ossified sternebra, dumbbell shaped or assymmetric ossification of vertebras or way ribs, the foetal or litter based incidence in the treated groups was comparable with the current study control or historical control database
- incomplete ossification of hyoid body in 4 mid dose foetuses in two litters; no dose response
- incidence of bipartite ossification of vertebra was higher in low dose group than in current study control value , but no dose response
- incomplete ossification of tarsal bones, increased incidence observed in low and mid dose groups when compared to historical xontrol data, the foetal incidence was statistically significant in case of the mid dose group when compared to the control. However, incidence in the high dose group was similar to the historical control data.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
All of the visceral findings are consistent in general nature and incidence with the study concurrent control data or the existing historical control data, therefore considered as incidental findings.
- Number of foetuses with malformation: 0/126 (control), 0/116 (low dose), 1/122 (mid dose), 0/129 (high dose)
- Number of foetuses with variation: 3/126 (control), 1/116 (low dose), 7/122 (mid dose), 17/129 (high dose)
The number of malformed/variant/intact foetuses were comparable with the control in the low and mid dose groups. In the high dose group the total number of intact foetuses were significantly lower and the number of foetuses with visceral variation was significantly higher than control. However, as these were in connection with only one variation, which was considered not being treatment related by closer special comparison with the historical control database, these facts were considered not showing a treatment related effect. In view of the maternal toxicity observed and the fact that none of the foetal visceral findings were considered to be significantly outside of the normal range, it is concluded that there was no adverse effect of the test item on the visceral observations.
No visceral malformations were recorded in any test item treated dose groups except of one malformed foetus (with total situs inversus) in the mid dose group. Based on the isolated occurrence and the lack of dose response , this observation was considered incidental, ascribed to individual variability and not related to treatment; furthermore, the historical control database contained this finding.
In case of some variations recorded in the test item treated groups (small renal papilla, convoluted ureter or dilated ureter) the calculated incidence was slightly higher than the study control or historical control values. But based on the low absolute values (only one or two litters were affected per dose group) and the lack of statistical significance, they were considered incidental findings, unrelated to test item administration.
Variation of thymic cord: in 1 low dose foetus, 4 mid dose foetuses and in 3 high dose foetuses, while only one foetus was recorded in the control group. However, there was no clear dose response, and the calcualted litter based incidences were comparable with incidences found in the historical control database for this common finding. Therefore these facts were considered as having no toxicological relevance and not being a test item related effect.
In case of short brachiocephalic trunk variation, increased incidences were observed in the mid and high dose groups compared to the control values. The increased number of foetuses with this variation in the high dose group was statistically significant compared to the control group. However, the calculated litter based incidences correlated with the frequencies seen in the historical control database. Taking into account the very slight changes and the borderline subjective judgement during foetal examination for this specific finding, the high dose was considered similar to to the upper end of the historical range, and as such it was not considered to be attributable to an adverse effect caused by the test item.
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
No further data

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: absence of significant toxic effects
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the observations made in the dams and their foetuses, signs of maternal toxicity was noted in the High dose group of 1000 mg/kg bw/day, but there were no toxicologically relevant changes on embryos or foetuses in any test item treated groups. The following no-observed-adverse-effect (NOAEL) levels were derived:
- NOAELmaternal toxicity: 300 mg/kg bw/day
- NOAELembryotoxicity: at least 1000 mg/kg bw/day
- NOAELfoetotoxicity: at least 1000 mg/kg bw/day
- NOAELteratogenecity: at least 1000 mg/kg bw/day