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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:
01 March 2017 to 26 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: approx. 11-12 weeks old
- Weight at study initiation: males: 369 - 499 g; females: 194 - 264 g
- Fasting period before study: none
- Housing: The animals were kept individually in IVC cages on Altromin saw fibre bedding. During the pre-mating period and after mating, males were housed in groups (2 animals / cage) in IVC cages.
- Diet: Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

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

Administration / exposure

Route of administration:
oral: gavage
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item and control formulations were prepared once in 7-10 days based on the stability information. The prepared formulation was stored at room temperature. The test item was weighed on a suitable precision balance into a tared plastic vial and the vehicle was added to give the appropriate final concentration of the test item. To protect from hydrolysis by humidity, formulation vials were purged with argon after the preparation before closing. Formulates were kept under magnetic stirring during the daily administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle has been selected as suggested by the sponsor based on the test item’s characteristics (hydrolysis in water) and testing guideline. The selected vehicle was dried corn oil.
- Concentration in vehicle: 7.58, 37.88, 189.38, 113.62 mg/mL
- Amount of vehicle (if gavage): not specified
- Lot/batch no. (if required): 22022017; 24022017
- Purity: not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Study pre start stability analysis included samples from high dose and low dose group and the investigation was made for 0 hours, 6 hours (RT), 10 days (RT), 10 days (2-8 °C) and 10 days -15 to -35 °C (10 samples).
Prestart homogeneity investigation included the samples collected from various levels (top, middle and bottom) of high dose and low dose groups (6 samples).
Samples for analysis of concentration of test item in the dosing formulations were taken in the first and last week of the study for all doses (8 samples in total).
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: Mating was performed using a ratio of 1:2 (male to female). Females were paired for cohabitation in batches in order to control the number of animals for terminal sacrifice on a particular day.
- Length of cohabitation: not specified
- Further matings after two unsuccessful attempts: not specified
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: At the subsequent mornings, the vaginal smear of the female was checked to confirm the pregnancy. The day on which sperms were observed in the vaginal smear was considered as gestation day 0.
- Any other deviations from standard protocol: none
Duration of treatment / exposure:
between gestation day 5 until gestation day 19.
Frequency of treatment:
7 days a week
Duration of test:
between gestation day 5 until gestation day 19.
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
Females no. 76- 87 of the HD group were treated with a dose of 750 mg/kg body weight/day for the whole treatment period or until the animals were euthanised for animal welfare reasons.
Dose / conc.:
450 mg/kg bw/day (actual dose received)
Remarks:
Females no. 99, 100-104 of the HD group were treated with a dose of 450 mg/kg body weight/day for the whole treatment period.
No. of animals per sex per dose:
160 animals (52 males and 108 females) were included in the study. 24 Female Wistar rats each in the control and low dose (LD) groups, 25 female Wistar rats in the medium dose (MD) group and 29 in the high dose (HD) group. The HD group comprised 29 females as 4 additional animals were allocated to this group due to high mortality and morbidity.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: According to the results of a previous 28-day study and in consultation with the sponsor the doses were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose) and 1 control group (C).
- Rationale for animal assignment (if not random): random

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily all animals were observed for morbidity and mortality.
- Cage side observations checked included: The health condition of the animals was recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: General clinical observations were made at least once a day. Clinical observations included 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 were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed once before initiation of pairing to ensure that the body weights are within + 20% variation.
The sperm positive females were weighed during gestations 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 gestations days 5, 8, 11, 14, 17 and 20.Food consumption was not measured for males during the entire study or for both male and females during the mating period.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: Macroscopic examination of organs was performed.

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [all per litter]
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. Litter incidence was the primary unit for the statistical analysis and interpretation. The statistics was performed with GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).
Indices:
No data
Historical control data:
Not specified

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.
Low incidences of the clinical signs like alopecia on various body parts were noted in isolated females of the dose groups and the control group. In HD females sacrificed in moribund condition, predominant clinical signs observed were prone position, slight to severe piloerection, reduced spontaneous activity, half eyelid closure, nasal discharge, abnormal breathing and moving the bedding. Piloerection, reduced spontaneous activity, ataxia, hypothermia, prone position, hunched posture, wasp waist, pale skin and dehydration was also noted in few terminally sacrificed females of the HD group.
Mortality:
mortality observed, treatment-related
Description (incidence):
No mortality occurred in the control or any of the dose groups during the treatment period of this study. However, four females (no. 80, 85-86 and 90) of the HD group were euthanized in a moribund condition for animal welfare reasons between gestation day 16-19. This animal morbidity was considered to be related to the treatment with the test item at a dose of 750/450 mg/kg bw/day. With this exception, all animals survived until the end of the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight increased with the progress of the study in the control, the LD, the MD and the HD group. However, after initiation of treatment the mean body weight gain was noted to be moderately but statistically significantly lower (p < 0.05, p < 0.01 and p<0.001) in the HD group when compared to controls during gestation days 11-14 (54.4 % of controls),14-17 (60.1 % of controls), 17-20 (42.3 % of control) and 0-20 (65.2 % of controls). This was based on a loss of body weight of some of the HD females. Lower body weight gain resulted in statistically significantly lower mean body weight of the HD group at the end of the treatment period. Mean body weight of the HD group was slightly lower on gestation day 17 (94 % of controls, p < 0.01) and moderately lower on gestation day 20 (89 % of controls, p < 0.001). Effects on mean body weight and body weight gain in the HD group were considered to be test item related.
The mean body weight of the LD and MD group was unaffected by the test item administration. The mean body weight gain of the LD and the MD group was comparable to the control group throughout the whole study period and was within the normal range of variation for this strain.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In correlation to the body weight and body weight gain, food consumption in the HD group was noted to be moderately and statistically significantly lower compared to the control group on various gestation days like 5-8 (80.3 % of controls), 8-11(80.4 % of controls), 11-14 (79.9 % of controls), 14-17 (69.9 % of controls), 17-20 (70.1 % of controls) and 0-20 (82.3 % of controls) throughout the treatment period. This effect on the food consumption of the HD group was considered to be test item related.
Mean food consumption of the LD and the MD group was comparable to the controls in correlation to the body weight and body weight gain.
Food efficiency:
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, treatment-related
Description (incidence and severity):
No gross pathological changes of toxicological relevance were observed during the macroscopic examination of the females of control LD and MD group.
In the HD group animals that were euthanised for animal welfare reasons (80, 85, 86 and 90), macroscopic findings like small spleen (1/29), discoloured pale /spotted liver (1/29), enlarged adrenal glands (1/29), discoloured dark lung (1/29), gas filled gastrointestinal tract (3/29) were observed. In terminally sacrificed females, enlarged adrenal (1/29) and right atrial auricle enlarged/left atrial auricle rudimentary (1/29) were observed. These gross pathological changes in HD group 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 specified

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
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): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not specified

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: general effects of toxicity

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
The individual mean foetus weight of the HD group was moderately, statistically significantly lower when compared to the control group . The mean male foetus weight of the HD group was also statistically significantly lower when compared to the control group The mean female foetus weight (Individual basis) of the HD group was lower when compared to the control but without achieving statistical significance.
Moderately, statistically significantly lower mean foetus weight (litter basis) of the HD group was observed when compared to the control group.
The lower mean foetus weight (individual and litter basis) and male/female foetus weight (individual basis) of the HD group was considered to be related to the treatment with the test item. There were no test item related effects of toxicological relevance for the total litter weight, male and female litter weight in any of the treatment groups when compared with the controls.

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:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, treatment-related
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 to the control group.
Low incidences of head odema, haematoma on various body parts in few foetuses, red tail, head- subcutaneous edema and anopthalmia, agnathia, anencephaly, absence of maxilla 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 increase for litter incidences of unossified caudal vertebral centrum was observed in HD group when compared with the controls.
A slightly higher litter incidences, but without achieving statistical significance for incomplete ossification of frontal, nasal, premaxilla, 1st sternebra, 2nd sternebra, 4th sternebra -incomplete and misaligned ossification, 5th sternebra, pubis –bilateral, zygomatic arch, zygomatic arch, bilateral 14th rudimentary rib, right 14th full rib, wavy ribs ), rudimentary left 7th cervical rib and right full 7th cervical rib were observed in the HD group when compared to the concurrent control group.
Statistically significant reduced ossification or no statistical significant reduced ossification of several bones that normally exhibit rapid ossification in the last days of gestation was observed in the HD group indicating a generalised skeletal delay in the HD group. This delayed ossification was considered to be associated with the observed maternal toxicity 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 not considered to be adverse. Cervical ribs are skeletal alterations that can occasionally be seen in animals of this strain. Rudimentary/short ribs are considered as transient abnormalities. In contrast, full/long cervical ribs are considered permanent and may cause health effects in humans; however, postnatal consequences in rats are unknown but assumed to be minimal.This finding was not considered to be treatment related but spontaneous in nature.
Wavy ribs are common findings in rodent studies and are considered to be postnatally reversible. Thus, wavy ribs are classified as variations and were not considered as an adverse effect of the treatment with the test item.
There was no statistical significance and no indication of a test item related trend in the type and/or incidences of other skeletal findings and they were therefore considered to be spontaneous in nature.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
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.
All litter incidences were statistically insignificant when compared with the control. Furthermore, slightly higher litter incidence of the discoloured dark liver without achieving statistical significance was observed in HD group but well within the historical control data for discoloured dark liver. There were also one each litter incidences of discolouration of organs like spleen and kidneys observed occasionally in HD group without achieving statistical significance. Discolouration of organs was considered likely to reflect the consequence of a functional disorder and thus not strictly as developmental anomalies. As only one litter was affected in HD group with discolouration of spleen and kidneys, these findings were not considered as toxicologically relevant.
Dilated ureter (bilateral) was observed with a higher litter incidence in the HD group when compared to the control group but without statistical significance. This is a common finding in rodent studies and is classified as a variation as it is transient and likely to be postnatally reversible. Furthermore, values were within the historical control data so that no toxicological relevance was accounted for it.
Other effects:
no effects observed
Description (incidence and severity):
Craniofacial examination by razor blade serial sectioning technique revealed few findings (slightly dilated 3rd ventricle, retinal fold, dilated lateral ventricle, slight subcutaneous edema, small pituitary gland, misshapen pituitary gland, head- subcutaneous haematoma and oedema) at low frequencies generally comparable to or in some cases slightly higher or lower in frequency in the dose groups compared to the controls. These findings were considered to be spontaneous in nature and not related to the treatment with the test item. Statistical analysis of the data revealed no statistical significance of any of the findings. Litter incidence for slightly dilated lateral ventricles in the HD group was higher without achieving statistical significance but well within historical control data when compared with the control.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations

Fetal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: vertebra
other: reduced ossification of some bones
Description (incidence and severity):
Unossified caudal vertebral centrum (65 % compared to 0 % in controls, p< 0.01) was observed in HD group when compared with the controls.

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
450 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects 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 results tables.

Applicant's summary and conclusion

Conclusions:
In the pre-natal developmental toxicity study, conducted according to OECD TG 414 and in compliance with GLP, the reported NOAEL for maternal and foetal toxicity was 150 mg/kg bw/day based on statistically significantly reduced body weight, body weight gain and food consumption during the treatment period in HD group females and a statistically significantly lower mean terminal, adjusted maternal weight and foetal weight in HD group when compared with the control. Furthermore, no treatment related external, visceral and craniofacial findings were observed in HD group. However, reduced ossification of some bones was observed which was indicative of a generalized delayed ossification associated with foetal growth retardation. Observed foetal effects in HD group might be secondary to maternal toxicity.