Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 May 2017 to 27 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: 12 weeks old females and from 19 to 26 weeks old males
- Weight at study initiation: 357 - 485 g males and 227 - 283 g females
- Fasting period before study: not specified
- Housing: Female animals were kept individually in IVC cages during most of the study period except during pre-mating period when females were kept in groups of two and the mating period when two females were paired with one male. Male rats were housed in groups of two during the pre-mating period and after mating period.
- Diet (e.g. ad libitum): Free access to Altromin 1324 maintenance diet for rats and mice, ad libitum.
- Water (e.g. ad libitum): Free access to tap water, sulphur acidified to a pH of approximately 2.8
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 55 ± 10%
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: sesame oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item and control formulations were prepared freshly once every 7-10 days based on the stability information. The prepared formulation was stored at room temperature. For the dose formulation the test substance was weighed and the vehicle was added to give the appropriate final concentration of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle, sesame oil, was selected as suggested by the sponsor based on test item's characteristics, hydrolysis in water and testing guideline.
- Lot/batch no. (if required): 16312506
- Concentration in vehicle: LD: 1.25; MD: 2.5; HD: 5
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle at Eurofins Munich as part of a separate GLP study. Samples for analysis of concentration of the test item in the dosing formulations were taken in the first and last week of the study for all doses.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 male : 2 females
- Length of cohabitation: until pregnancy was confirmed by sperm in vaginal smear
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. Not applicable
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: none
Duration of treatment / exposure:
between gestation day 5 and until gestation day 19.
Frequency of treatment:
7 days per week
Duration of test:
from pre-mating until necropsy of females
Doses / concentrationsopen allclose all
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Remarks:
low dose (LD)
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Remarks:
middle dose (MD)
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Remarks:
high dose (HD)
No. of animals per sex per dose:
25 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The test concentrations were selected based on a 90-day study, one generation reproductive toxicity study and in consultation with the sponsor. The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels was selected with the intention to demonstrate any dose-related response and a NOAEL.
- Rationale for animal assignment (if not random): random

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked included: health condition, morbidity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily checked for spontaneous activity, lethargy, recumbent position,convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin
and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed once before pairing, then sperm positive females were weighed on gestation days 0, 5, 8, 11, 14, 17 and 20. Males were not weighed in this study except once before initiation of pairing.

FOOD CONSUMPTION: Yes. Food consumption of sperm positive females was measured on gestation days 5, 8, 11, 14, 17 and 20. Food consumption was not measured for males.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: macroscopic examination of for structural abnormalities or pathological changes; any macroscopic findings were preserved in 4% neutral-buffered formaldehyde; uteri were removed and pregnancy status was confirmed; non-gravid uteri were further examined; each gravid uterus with the cervix was weighed; number of corpora lutea was counted; the uterine content was examined for embryonic or foetal deaths as well as the number of viable foetuses; degree of resorption was confirmed; position and number of foetuses in each uterine horn was recorded; males were sacrificed without any observations at any time after completion of mating procedure.
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: [the first 20 processed by Alizarin red staining from second half litter per group]
- Head examinations: Yes: [the first 20 processed by Alizarin red staining from second half litter per group]
Statistics:
A statistical assessment of the results of the body weight, food consumption was performed by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were analysed using Fisher’s exact test. The litter were used as the unit for data analysis.The statistics were performed with GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no clinical signs of toxicological relevance observed in the control, LD and MD dose groups. Specific clinical signs observed in dead/ moribund females were alopecia on right shoulder, moderate piloerection, reduced spontaneous activity, diarrhoea, apathy, spontaneous activity severely reduced, ataxia, , and hunched posture, piloerection (moderate), slow movements, moderately reduced spontaneous activity, half eyelid closure. Moreover, sunken flanks in female no. 93., exophthalmos in female no. 84 and slow movements in female no. 87 were observed. In terminally sacrificed females low incidences of clinical signs like alopecia were noted in the dose groups as well as in the control group. There were also occasional clinical signs like abnormal breathing in one HD female, red to dark red nasal discharge in one each female of the LD and HD groups, piloerection in 3 HD females and exophthalmos in one control female. Moving the bedding was also noted mainly immediately after administration and just for a short period in one control, 2 LD and 1 HD group females. This effect was considered to be mainly a local reaction to the test item rather than a systemic effect. All clinical signs observed in terminally sacrificed females were incidental or non adverse in nature. However, clinical signs observed in dead/moribund females could be attributed to the treatment with test item.
Mortality:
mortality observed, treatment-related
Description (incidence):
One mortality occurred at the HD group during the treatment period of the study, female no. 87 on gestation day 17. Two females (no. 84 and 93) of the HD group were euthanised in a moribund condition for animals welfare reasons on gestation days 9 and 19. These instances of mortality and morbidity were considered to be treatment-related. No other deaths were observed during the treatment period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight remained unaffected and increased with the progress of the study in the control, LD, MD and HD groups. However, after initiation of treatment the
mean body weight gain was noted to be marginally but statistically significantly lower (p < 0.05) in the HD group during gestation days 8-11 (65.05 % of controls) when
compared to the controls. There was also marginally lower overall body weight gain (gestation day 0-20) observed in the HD group (89.24 % of controls) without achieving
statistical significance when compared to the controls. Although this effect on body weight gain in the HD group was marginal, it could be attributed to treatment with test
item.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was unaffected by the treatment in any of the dosed animals and food consumption of LD, MD and HD was comparable to the control group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No effects were noted on gravid uterine weight.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
No gross pathological changes of toxicological relevance were observed during the macroscopic examination of the females of the control, LD and MD groups.
In the HD group found dead female (87), enlarged adrenal glands were observed during the gross pathological examination. This finding in the HD group dead female could be
considered as test item-related.
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:
no effects observed
Description (incidence and severity):
No test item-related effects of toxicological relevance.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No test item-related effects of toxicological relevance were noted for implantation sites.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No test item-related effects of toxicological relevance were noted for early and late resorptions.
Early or late resorptions:
no effects observed
Description (incidence and severity):
No test item-related effects of toxicological relevance were noted for early and late resorptions.
Dead fetuses:
no effects observed
Description (incidence and severity):
No test item-related effects of toxicological relevance were noted for number of live fetuses. No dead foetuses were noted in any of the groups.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Successful mating resulted in 24/25 pregnancies in the LD group, 24/25 in the MD group and 19/22 in the HD group compared to 23/25 pregnancies in the control group. The marginally low pregnancy rates (no. of pregnancies / no. of females mated or sperm positive x 100) of 86.36 % in the HD group compared to 92% in the control group was considered to be a biological variation and well above standard pregnancy rate of minimum 80% in rat.
Other effects:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain

Maternal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
other: reduced body weight gain at high dose level

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean foetus weight (individual basis) of the HD group (3.26 g) was moderately, statistically significantly lower when compared to the control group (3.53 g) (92.35 % of controls, p< 0.001). The mean male and female foetus weight (Individual basis) of the HD group (male- 3.33 g and female -3.19 g) was also statistically significantly lower when compared to the control group (male- 3.60 g and female- 3.45 g) (92.5 % of controls in males and 92.46 % of controls in females, p< 0.001). The mean foetus weight (litter basis) of the HD group (3.30 g) was lower when compared to the control (3.54 g) but without achieving statistical significance (63.22 % of controls).
Moderately, statistically significantly lower male litter weight (litter basis) of the HD group (18.03 g) (73.35 % of controls, p< 0.05) was observed when compared to the control group (24.58 g). This effect on male litter weight could be attributed to relatively lower group mean male number in the HD group (5.42) when compared with the controls (6.52). There was also statistically significantly higher group mean foetus weight (3.64 g) and male foetus weight (3.73 g) (Individual basis) observed in the LD group when compared to the control which was not considered as test item-related.
The lower mean foetus weight (individual and litter basis) and male/female foetus weight (individual basis) of the HD group were considered to be related to the treatment with the test item.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No test item-related effects of toxicological relevance were noted for number of live foetuses.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No test item-related effects of toxicological relevance were noted for sex ratio.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no test item-related effects of toxicological relevance for the total litter weight and female litter weight in any of the treatment groups when compared with the controls.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There were no external abnormalities considered to be of toxicological relevance in any of the dose groups. Statistical analysis of data revealed no significant differences compared to the control group. Low incidences of astomia (1 in MD), haematoma on various body parts in a few foetuses (1 in control, 1 in LD, 2 in MD and 1 in HD), lower jaw small (1 in control), small mandible (1 in MD), small foetus (1 in control) and small snout (1 in MD) were noted in isolated females of the control group and/or the dose groups without dose dependency. As these findings were observed mostly in single foetuses, they were considered to be incidental in nature and unrelated to the treatment.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decrease in litter incidence for incomplete ossification of left and right parietal in the LD group and fused sacral vertebral centrum and arch in the MD group compared to the control group were considered to be incidental as frequencies were even less in numbers compared to the controls. Therefore, these findings are not to be considered as treatment-related and solely spontaneous in nature. A statistically significant increase for litter incidences of incomplete ossification of right squamosal (31.58 % compared to 5 % in controls, p< 0.05) was observed in the HD group when compared with the controls. Slightly higher litter incidences, but without achieving statistical significance for incomplete ossification were observed in the HD group when compared to the concurrent control group of basioccipital,frontal, hyoid body, parietal, squamosal, zygomatic arch, scapula, 2nd sternebra, 5th sternebra, cervical vertebral arch, pubis, humerus, misshapen humerus, irregularly ossified thoracic vertebral centrum, unossified 6th sternebra, unossified thoracic vertebral centrum, unossified forelimb metacarpals,full 14th rib, wavy ribs.
The observed statistically significantly reduced ossification without achieving statistical significance of several bones in the HD group that normally exhibit rapid ossification in the last days of gestation indicates a generalised skeletal delay in the HD group. This delayed ossification was considered to be associated with the observed slight maternal toxicity (lower body weight) and reduced foetal body weight of the HD group. Generally delayed ossification is not regarded to persist postnatally and not associated with long-term consequences on survival, general growth and development and therefore is not considered to be adverse.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There was higher litter incidence of umbilical artery transposed and bilateral dilatation of ureter observed in all treatment groups including control group. However, values were well within historical control data range (85.71 % - umbilical artery transposed and 63.64 % - dilated ureter). There was discolouration of a few organs like liver, spleen and thymus observed without dose dependency. Discolouration of organs was considered likely to reflect the consequence of a functional disorder and thus not strictly as developmental anomalies.
Visceral findings observed in the dose groups were at frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to controls. As observed findings were either minor variations and/or due to a lack of dose dependency and consistency, no toxicological significance can be attributed to these findings and they were considered to be spontaneous in nature.
Other effects:
not examined

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
changes in litter size and weights

Fetal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
other: reduced body weight in high dose group

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
20 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

See attachment for result tables.

Applicant's summary and conclusion

Conclusions:
In the prenatal developmental toxicity study, conducted according to OECD TG 414 and GLP, the concluded NOAEL for maternal and developmental toxicity was 10 mg/kg bw/day based on reduced body weight gain in maternal animals and reduced foetal body weight at high dose group females and litters. The reduced foetal weights are likely to be secondary to reduced maternal body weights.