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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:
17 July 2018 to 9 November 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

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, 97633 Sulzfeld, Germany
- Age at study initiation: 11-12 weeks old females and between 12 weeks and not older than 24 weeks males
- Weight at study initiation (before pairing): males: 329 - 380 g (mean: 354.58 g, ± 20 % = 283.66 – 425.49 g); females: 190 - 251 g (mean: 217.31 g, ± 20 % = 173.85- 260.77 g)
- Fasting period before study: no
- Housing: The animals were kept individually in IVC cages (type III H, polysulphone cages) on Altromin saw fibre bedding (except during the pre-mating period when females were kept in groups of two animals and during mating period when two females were paired with one male). During the pre-mating period and after mating, males were housed in groups (up to 5 animals / cage) in type IV cages.
- Diet (e.g. ad libitum): Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water (e.g. ad libitum): tap water, sulphur acidified to a pH of approximately 2.8, ad libitum
- Acclimation period: at least 5 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item. The formulation was vortexed and/or stirred until visual homogeneity is achieved. Based on the results of stability testing, the test item formulations were prepared at least once every 10 days. The prepared formulation were stored protected from light and at room temperature.

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil (dried and deacidified).The vehicle has been selected based in consultation with the sponsor based on the test item’s characteristics
- Concentration in vehicle: 25, 75, 250 mg/mL
- Amount of vehicle (if gavage): The volume of vehicle was dependent on the dose and body weight of the animal. The total dose volume for all groups was 4 ml/kg bw.
- Lot/batch no. (if required): MKCF8882
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Study pre-start stability analysis were performed on the samples from high dose and low dose group and the investigation was made for 0 h, 6 h (RT), 10 day (RT), 10 day (2-8 °C) and 10 day -15 to -35 °C.
Pre-start homogeneity investigation was performed on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups.
As the test item was shown to be homogenous according to Eurofins Study No. 171154 (after 30 min without stirring), samples were not collected during the study for the investigation of homogeneity and only samples were taken for substance concentration 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: 1:2 (male to female)
- Length of cohabitation: Until sperm in vaginal smear is observed.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: not applicable
- Further matings after two unsuccessful attempts: not required
- 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 until gestation day 19
Frequency of treatment:
daily
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
low dose (LD)
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
medium dose (MD)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
high dose (HD)
No. of animals per sex per dose:
Total: 156 animals (52 males and 104 females)
25 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the data generated from a dose range finding study, dose levels of 100, 300 and 1000 mg/kg body weight/day were proposed. No test item-related toxicological effects in terms of clinical signs, morbidity/mortality, body weight development, food consumption and gross pathological observations of dams were observed in the treatment group pregnant females when compared with the controls and no signs of foetal toxicity were seen in terms of effect on prenatal data, litter data and foetal external findings up to the highest dose level of 1000 mg/kg body weight/day in the dose range-finding study. This dose is also the limit dose for this type of study.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for mortality and morbidity and once daily for general clinical observations
- Cage side observations checked in 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.

DETAILED CLINICAL OBSERVATIONS: No

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 on 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
- Time schedule for examinations: Food consumption of sperm positive females was measured on gestations days 5, 8, 11, 14, 17 and 20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: uterus and uterine content

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: [ one half per litter]
- Skeletal examinations: Yes: [other half per litter]
- Head examinations: Yes: [foetuses used for the soft tissue examination of the first 20 litters per group]
Statistics:
A statistical assessment of the results of the body weight, food consumption were 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 statistics were performed with GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).
Indices:
- number of implantations, number and percent of live and dead fetuses and resorptions;
- number and percent of pre- and post-implantation losses.
Developmental endpoints by dose for litters with live fetuses, including:
- number and percent of live offspring;
- sex ratio
Historical control data:
See 'Overall remarks, attachments' for control data

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
In terminally sacrificed females, predominant clinical signs observed on few days during the treatment period of the study included moving the bedding (1/25 in LD and 21/25 in HD), increased salivation (1/25 in LD, 3/25 in MD and 9/25 in HD), piloerection (1/25 in control and LD, 2/25 in MD and 15/25 in HD), ataxia (5/25 in HD), reduced spontaneous activity (15/25 in HD), hunched posture (5/25 in HD), prone position (4/25 in HD), apathy (5/25 in HD) and half eyelid closure (4/25 in HD) were observed. There were also low incidences of clinical signs including hairless area on various body parts (2/25 in control and LD, 3/25 in MD and 4/25 in HD), scratch, crust, abnormal breathing and wound (1/25 in MD), bloody vagina (1/25 in control) and regurgitation of test item (1/25 in LD and 2/25 in MD) observed in isolated females of all groups including the control group.

As moving the bedding and salivation were noted mainly immediately after test item administration and just for a short period, these transient signs were considered to be due to a local reaction to the test item or unpalatability rather than an adverse systemic effect. Piloerection, ataxia, reduced spontaneous activity, hunched posture, prone position and apathy were observed on very few days without any consistency in a few females of HD group. In the light of no effect observed on body weight development and food consumption in HD group, all clinical signs observed in terminally sacrificed treatment group females were incidental and considered to be of no toxicological relevance or non adverse in nature.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-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 throughout the study period. No statistical significance was achieved in any treatment groups on any day or interval of body weight measurement and all values in the treatment groups were comparable to the controls However, mean body weight gain was noted to be marginally but statistically significantly lower (p < 0.01) in the HD group during GD 11-14 (64.23 % of controls) when compared to the controls. As this effect on group mean body weight gain in the HD group on just one occasion was marginal and attributed to minus values from 2 females (animal nos. 87 and 99), it was not considered related to treatment with test item.
Food consumption and compound intake (if feeding study):
no effects observed
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, non-treatment-related
Description (incidence and severity):
In terminally sacrificed females, predominant clinical signs observed on few days during the treatment period of the study included moving the bedding, increased salivation, piloerection, ataxia, reduced spontaneous activity, hunched posture, prone position, apathy and half eyelid closure.
As moving the bedding and salivation were noted mainly immediately after test item administration and just for a short period, these transient signs were considered to be due to a local reaction to the test item or unpalatability rather than an adverse systemic effect. Piloerection, ataxia, reduced spontaneous activity, hunched posture, prone position and apathy were observed on very few days without any consistency in a few females of HD group. In the light of no effect observed on body weight development and food consumption in HD group, all clinical signs observed in terminally sacrificed treatment group females were incidental and considered to be of no toxicological relevance or non adverse in nature
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross pathological changes were observed during the macroscopic examination of the females of the control, LD and MD groups except a spotted red thymus in 1/25 females from the LD group. In the HD group, discoloured kidneys (1/25), spotted white kidneys (3/25) and 1mm mass on right kidney (1/25) were observed during the gross pathological examination. These finding in the few HD group females are considered be spontaneous in nature and of no toxicological significance.
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
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no difference in percent pre- and post-implantation loss in treatment groups when compared to the controls.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
There was no difference between treated and control animals with regard to early, late and total resorptions.
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead foetuses were noted in any of the groups.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
There were no adverse findings relating to pregnancy duration.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Successful mating resulted in 22/25 pregnancies in the LD group, 21/25 in the MD group and 23/25 in the HD group compared to 22/26 pregnancies in the control group. The variation in pregnancy rates (no. of pregnancies / no. of females mated or sperm positive x 100) of 88 % in the control group compared to treatment groups (88% in LD, 84 % in MD and 92 % in HD) was considered to be a biological variation.
Other effects:
no effects observed
Description (incidence and severity):
There was no difference between the treated and controls animals with regard to uterus weight, number of corpora lutea, implantation sites, number of live foetuses and the number of foetuses in each uterine horn.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed.
Remarks on result:
not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no difference in the number of male and female and the sex ratio of offspring between treated groups and controls.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no test item-related effects of toxicological relevance observed for the mean foetus weight (individual and litter basis), male and female foetus weight (Individual basis), total litter weight (litter basis), male and female litter weight (litter basis) 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 haematoma on various body parts in a few foetuses (3 in MD, 1 in LD and 1 in control), subcutaneous area completely fluid filled (1 in LD), subcutaneous oedema, anasarca (1 in control), umbilical hernia (1 in LD), left eye half open, right eye open (1 in LD), exencephaly (1 in LD) and subcutaneous hemorrhage on right forelimb (1 in control) were noted in isolated foetuses 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:
no effects observed
Description (incidence and severity):
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, non-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 the 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. There was a 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 including liver, adrenal and spleen 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. Due to lack of dose dependency and consistency, these discolouration findings were not considered as toxicologically relevant.
Other effects:
no effects observed
Description (incidence and severity):
Craniofacial examination by razor blade serial sectioning technique revealed a few predominant findings (ear subcutaneous haematoma, subcutaneous edema of head, small pituitary gland, misshapen pituitary gland, retinal fold, dilated 3rd ventricle and dilated lateral ventricle) 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 for any of these findings. Litter incidence for dilated lateral ventricles (B) in the HD group was higher (20 %) without achieving statistical significance, but well within historical control data (69.57 %) when compared with the control (10 %).
Details on embryotoxic / teratogenic effects:
No embryotoxic or teratogenic effects were observed.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse findings.
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
In the prenatal developmental toxicity study, conducted according to OECD 414 Test Guideline and in compliance with GLP, in the rat, the reported NOAEL for both maternal and foetal toxicity was at least 1000 mg/kg bw/day based on no observed mortality; non adverse or incidental clinical signs and no treatment-related effect in the gross pathology up to highest dose tested.