Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11.09.2018 – 23.01.2019
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
O.J.L. 142, 2008
Deviations:
yes
Remarks:
see Any other information ...
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Adopted on January 22nd 2001
Deviations:
yes
Remarks:
see Any other information ...
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): Ethylcentralit
- Physical state: White, solid, powder
- Analytical purity: 99.9% (w/w)
- Lot/batch No.: OP.1000418
- Expiration date of the lot/batch: 06/2023
- Stability under test conditions: stable
- Storage conditions: Store in a closed container. Store in a well-ventilated place. Store in a dry place. Keep cool.
- Storage conditions during the study: The test item was stored in dry room in dark in closed container.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar CRL (SPF quality - guaranteed)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River SPF breeding, supplied via VELAZ s.r.o., Czech Republic, RČH CZ 11760500
- Age at study initiation: 10-11 weeks
- Sex: sexually adult females (males - only for mating)
- Fasting period before study: no
- Housing: plastic cages, sterilised clean shaving of soft wood or sterilized LIGNOCEL (raw material - spruce)
Before mating - 2 rats of the same sex in one cage.
During mating period – one male and two females in one cage were housed. Pregnant females were then placed individually.

- Diet: ad libitum, complete peleted diet for rats Altromin Spezialfutter, diet was sterilised before using.
- Water: ad libitum (quality corresponded to Regulation No. 252/2004 Czech Coll. of Law)

- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3°C
- Humidity (%): 30-70%
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12 hour dark cycle

STUDY TIME SCHEDULE
Test item delivery: 12. 06. 2018
Date of animal arrival: 29. 08. 2018
Acclimatisation: 29. 08. 2018 - 10. 09. 2018
Mating: 11. 09. – 24. 09. 2018
Start of administration: 17. 09. 2018
End of administration: 08. 10. 2018
Clinical observation: 17. 09. – 08. 10. 2018
Necropsies: 02. 10. – 09. 10. 2018
Microscopical examination:30. 10. – 23. 01. 2018
Evaluation of results and final report elaboration:23. 01. – 10. 03. 2019










Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% methylcellulose in aqua pro iniectione
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was weighted into glass beaker and then a smaller volume of 0.5% methylcellulose in aqua pro iniectione was added. This suspension was well mixed and then added with 0.5% methylcellulose in aqua pro iniectione to the required volume. After this the application form was dissolving in ultrasonic bath for 10 minutes and then application form was mixed by magnetic stirrer for 10 minutes at 1000 rpm before application and then during administration.
Prepared daily just before administration, laboratory conditions.

DIET PREPARATION
- Type of food: Complete pelleted diet for rats and mice in SPF breeding was used (Altromin Spezialfutter GmbH & Co. KG, Germany). Diet was sterilized before using.
- Storage temperature of food: laboratory conditions

VEHICLE
0.5% methylcellulose in aqua pro iniectione
Methylcellulose
Batch No.: D180HBK042; Expiration:11/2022
Aqua pro iniectione
Batch No.:1703240253; Expiration: 03/2019
Batch No.:1801290066; Expiration: 01/2020
Manufacturer: Dr. Kulich Pharma, s.r.o., Hradec Králové, Czech Republic

- Amount of vehicle (if gavage):
The test item was administered suspended in 0.5% methylcellulose in aqua pro iniectione by stomach tube and the concentrations of suspensions at all dose levels were adjusted to ensure the administered volume of 1 mL per 100 g of body weight.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
The procedure for application form preparation was taken from the analytical report ‘Determination of homogeneity and stability of Centralit in vehicle’, (Annex 1 of Final report, Study No. 127/15/8, Centralit – Repeated Dose 90-day Oral Toxicity Study/Subchronic Oral Toxicity Test, VUOS-CETA report No. 16-266, 2016.
Details on mating procedure:
MATING
- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male/2 female
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear - referred to as day 0 of pregnancy
Duration of treatment / exposure:
the 5th to the 19th day of pregnancy
Frequency of treatment:
7 days per week at the same time
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
23 pregnant females per dose for 0, 50 and 150 mg/kg bw/day dose level
26 pregnant females for 300 mg/kg b.w./day
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels which have been chosen for this study, 50, 150 and 300 mg/kg b.w./day, were based on doses used in previous long-term toxicity studies performed at Test Facility.

Groups of animals
1. Control (vehicle) 0 mg/kg b.w./day 23 prob. pregnant females
2. Low dose 50 mg/kg b.w./day 23 prob. pregnant females
3. Intermediate dose 150 mg/kg b.w./day 23 prob. pregnant females
4. High dose 300 mg/kg b.w./day 26 prob. pregnant females

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily at the similar time
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
MORTALITY CONTROL: Yes
- Time schedule: daily
BODY WEIGHT: Yes
- Time schedule for examinations: on the 1st, 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy
FOOD CONSUMPTION: Yes
- Time schedule for examinations: on the 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on the 20th day of pregnancy
- Organs examined: uterus
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 viable foteus: Yes
- Number of dead foteus: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations:
yes, all per litter - sex, body weights, symmetry of fore and hind limbs, number of fingers, closing or opening of eye fissures and external auditory canal, symmetry of head, integrity of superior palatum, status of umbilicus and genital papilla

- Soft tissue examinations:
yes, half per litter - detailed gross dissections of foetuses were performed. Placing and morphology of organs and big vessels was reviewed during examination of internal alterations

- Skeletal examinations:
yes, half per litter - skull, clavicle, scapula, sternebra and sternum, ribs, vertebrae, pelvic girdle, forelimb/hindlimb.
Statistics:
For statistical evaluation the software Statgraphic ® Centurion (version XV, USA) was used. The data from control group were compared with data from treated groups. The results statistically significant on probability level 0.05 (p<0.05) are indicated in the summary tables.
Indices:
Preimplantation loss, postimplatation loss were calculated from number of implantations, corpora lutea and resorptions.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Description (incidence and severity):
Four females died in short time after first application of the test item at the highest dose level. The negative effect on neurology system manifested as dyspnoea, body spasm and tremor was observed in these females. The effect on neurology system was observed after first application in others females at the highest dose level. Straub phenomenon accompanied with increased response to stimuli, body spasm and tremor were observed at the highest dose level. Straub reaction consisted of the tail becoming rigid and erected across the back of the animals in a S-shaped curve.
Mortality:
mortality observed, treatment-related
Description (incidence):
Four females died in short time after first application of the test item at the highest dose level.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The test item had a negative effect on the growth of maternal animals at the highest dose level. The body weight increment of females was decreased at the highest dose level compared to the control group, but without statistical significance. Body weight deficits of 5% or greater that are sustained over a period of several days are generally considered to be a signal of an adverse effect on maternal growth (Hood, 2016).
The corrected body weights of females (without weight of uterus) were mildly decreased compared to the control at all dose levels, but without statistical significance.
Description (incidence and severity):
The food consumption was statistically significantly decreased at the middle and the highest dose level from 5th to 8th day of pregnancy. But from 8th up to 20th day of pregnancy the food consumption was comparable to control group at these dose levels.
Description (incidence and severity):
Control of health condition and clinical examinations of mothers at the highest dose level detected clinical symptoms of neurotoxicity related to the test item treatment. Straub phenomenon, increased response to stimuli, dyspnoea, abdominal position, body spasm and tremor was observed after first application in females at the highest dose level. The Straub phenomenon, clonic movements of head and salivation was observed after the first application in one female at the middle dose level. From 6th up to 19th day of pregnancy the symptoms of neurotoxicity were not observed in all treated groups.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The corrected body weights of females (without weight of uterus) were mildly decreased compared to the control at all dose levels, but without statistical significance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
no pathological findings in all dose levels
Evaluation of uterus biometry detected a slightly decrease of absolute weight at the highest dose level, but without statistical significance. Reducing gravid uterine weight was due to decreased fetal weights in this group. This decrease in the absolute weight of the uterus was also related to the lower necropsy body weight of the females at the highest dose level.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
Reproduction parameters – number of live foetuses, early and late intra uterine deaths were evaluated on the basis of examination of uterus content.
The examinations of reproductive parameters revealed that the mean number of implantations, corpora lutea and resorptions in treated females was not statistically significantly changed in comparison with the control females.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
not statistically significantly changed in comparison with the control females
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
not statistically significantly changed in comparison with the control females
Early or late resorptions:
no effects observed
Description (incidence and severity):
not statistically significantly changed in comparison with the control females
Dead fetuses:
no effects observed
Description (incidence and severity):
not statistically significantly changed in comparison with the control females
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
not statistically significantly changed in comparison with the control females
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
not statistically significantly changed in comparison with the control females

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: based on no occurrence of mortality, no serious changes in health condition status, no reproductive-related pathological findings and no changes in reproduction parameters at the dose 150 mg/kg b.w./day

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean body weight of foetuses was slightly decreased at the highest dose level without statistical significance. This reduction in fetal body weight could be associated with a decrease in body weight in females at the highest dose level. Males were heavier than females in all groups (including control). This weight imbalance is common.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Dead foetuses were found sporadically in the middle and highest dose levels (0-0-1-2). The mean number of live foetuses per litter was similar in comparison to the control in treated groups and the sex ratio (mean value) was also comparable in all groups.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The mean number of live foetuses per litter was similar in comparison to the control in treated groups and the sex ratio (mean value) was also comparable in all groups.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No macroscopic changes of soft tissues or external alteration were found, except for dead foetuses. But dead foetuses were observed sporadically (0-0-1-2). Examination of internal alterations of dead foetuses could not be performed due to organ autolysis.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Examination of foetal skeletons revealed incomplete ossification of cranium bones especially in the interparietal, parietal, supraoccipital bone. This delayed development of the foetal skull was found in all groups including control, without dose relation.
Specific anomalies of skull ossification, such as holes in supraoccipital bone was also found in all groups, even with the highest incidence in foetuses in the control group.
Other changes in the foetal skull were found sporadically.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The influence of the test item on the development of the conceptus in uterus was assessed according to the results of weighing, careful necropsy and skeletal examination of foetuses.
Other anomalies of ossification
Examination of sternebrae revealed delayed ossification of ossification sites in foetuses of all treated groups, including the control, generally without dose relation. The ossification sites were either incompletely ossified or unossified. This is normal variability in the schedule of ossification; ossification of the sternum has not been completed on the 20th day of pregnancy (Hood, 2016).
Examination of vertebrae revealed higher occurrence of dumbbell ossification in treated groups in comparison with controls (63.16 % - 75. 00 %- 80.00 % - 94.12 %) with dose dependence. The incidence of asymmetric ossification of vertebrae was slightly increased in treated groups compared to the control group (21.05 % - 35.00 % - 35. 00 % - 35.29 %). The above mentioned findings such the dumbbell ossification an asymmetric ossification of vertebrae are classified as transitional findings. These transitional findings may be upgraded to malformation or downgraded to variation status, depending on severity and/or frequency of occurrence. Transitional findings are considered nonlethal and generally not detrimental to postnatal survival (Solecki, 2001; Hood, 2016). The occurrences of dumbbell ossification and asymmetric ossification are comparatively high in control foetuses in this study, so these findings could be downgraded to variation. But the highest occurrence in foetuses at the high dose level could be associated with a decrease in the weight of the foetuses and maternal toxicity at the highest dose level.

Other changes of foetus skeleton were found very sporadically and without treatment relation.
Serious anomalies of skull or skeleton were not observed in treated groups.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat. (total fraction)
Sex:
male/female
Basis for effect level:
other: based on the no occurrence of anomalies of foetal skull and skeleton in treated groups

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
Treatment related:
yes

Applicant's summary and conclusion

Conclusions:
The oral administration of the test item, Ethylcentralit, at the 50, 150 levels did not cause mortality of pregnant females. The higher sensitivity of pregnant females to the test item treatment was observed in the period of implantation of conceptus at the highest dose level. Four females died after first application and clinical symptoms of neurointoxication in others females after first application were observed at the dose 300 mg/kg b.w./day.

An adverse effect of test the item treatment on the growth of maternal animals was observed at the highest dose 300 mg/kg b.w./day. The body weight increment was decreased in this group. The food consumption was statistically significantly decreased in the first 3 days of application period at the dose levels150 mg/kg b.w./day and 300 mg b.w./day.

Macroscopical structure of organs of pregnant females were unaffected by treatment with the test item.
There were no serious changes in reproduction parameters in treated groups.

The examination of foetuses revealed a slight decrease in the mean body weights at the dose level 300 mg/kg b.w./day.

Examination of foetal skull and skeleton did not revealed serious anomalies in treated groups.
Examination of vertebrae revealed higher occurrence of dumbbell ossification in treated groups in comparison with controls with dose dependence. The incidence of asymmetric ossification of vertebrae was slightly increased in treated groups compared to the control group.
The occurrences of dumbbell ossification and asymmetric ossification of vertebrae are comparatively high in control foetuses in this study, so these findings could not be considered serious anomalies. But the highest occurrence in foetuses at the high dose level could be associated with a decrease in the weight of the foetuses and maternal toxicity at the highest dose level.

Other changes of foetus skeleton were found very sporadically and without treatment relation.

The NOAEL (No Observed Adverse Effect Level) for toxicity in PREGNANT FEMALES was established 150 mg/kg b.w./day. This NOAEL value is based on no occurrence of mortality, no serious changes in health condition status, no reproductive-related pathological findings and no changes in reproduction parameters at the dose 150 mg/kg b.w./day.

The NOAEL (No Observed Adverse Effect Level) for PRENATAL DEVELOPMENT is
300 mg/kg b.w./day. This NOAEL value is based on the no occurrence of anomalies of foetal skull and skeleton in treated groups.


Executive summary:

Introduction

The test item, Ethylcentralit, was tested for prenatal developmental toxicity using the Method B.31, Prenatal Developmental Toxicity Study, Council Regulation (EC) No. 440/2008, Published in O.J. L. 142, 2008 and OECD Test Guideline No. 414, Prenatal Developmental Toxicity Study, Adopted by the Council on January 22nd 2001.

 

Study performance

Wistar rat females (SPF quality) were used for testing. After acclimatization the females were mated with males. The test item was then administered to pregnant females - daily from the 5th to the 19th day of pregnancy. The study included four groups of females – 3 treated groups and 1 control group (vehicle only). The test item was administered suspended in 0.5% methylcellulose in aqua pro iniectione by stomach tube and the concentrations of suspensions at all dose levels were adjusted to ensure the administered volume of 1 mL per 100 g of body weight. 

Dose levels which have been chosen for this study, 50, 150 and 300 mg/kg b.w./day, were based on doses used in previous long-term toxicity studies performed at Test Facility.

 

The health condition, clinical status after application, body weight and food consumption of maternal animals were monitored during the study. On the 20th day of pregnancy, the maternal animals were euthanized, the uterine contents were examined and the foetuses were assessed for soft tissue and skeletal alterations.

 

Results

Maternal animals toxicity and reproduction parameters

The sensitivity of pregnant females to the test item treatment was found out in the conceptus implantation period at the highest dose level.

The unscheduled deaths were recorded at the highest dose level. Four females died in short time after first application of the test item. The effect on neurology system was observed after first application in others females at the highest dose level - Straub phenomenon accompanied with increased response to stimuli, body spasm and tremor were observed.

The test item had a negative effect on the growth of maternal animals at the highest dose level

300 mg/kg b.w./day.

The food consumption was statistically significantly decreased at the 300 mg/kg b.w./day dose level from 5th to 8th day of pregnancy.

Macroscopical structure of organs of pregnant females were unaffected by treatment with the test item. 

 

There were no serious changes in reproduction parameters in treated groups.

 

Foetuses

Dead foetuses were found sporadically at the middle and highest dose levels (0-0-1-2). The mean number of live foetuses per litter was similar in treated groups in comparison to the control group, the sex ratio (mean value) was also comparable in all groups.

 

The mean body weight of foetuses was slightly decreased at the highest dose level without statistical significance.

Detailed necropsy of foetuses did not reveal macroscopic changes in soft tissues or external alterations.

Examination of foetal skull and skeleton did not revealed serious anomalies in treated groups.

Examination of vertebrae revealed higher occurrence of dumbbell ossification in treated groups in comparison with controls with dose dependence. The incidence of asymmetric ossification of vertebrae was slightly increased in treated groups compared to the control group.

The occurrences of dumbbell ossification and asymmetric ossification of vertebrae are comparatively high in control foetuses in this study, so these findings could not be considered serious anomalies. But the highest occurrence in foetuses at the high dose level could be associated with a decrease in the weight of the foetuses and maternal toxicity at the highest dose level.

 

Other changes of foetus skeleton were found very sporadically and without treatment relation.

 

The NOAEL (No Observed Adverse Effect Level) for toxicity in PREGNANT FEMALES was established 150 mg/kg b.w./day. This NOAEL value is based on no occurrence of mortality, no serious changes in health condition status, no reproductive-related pathological findings and no changes in reproduction parameters at the dose 150 mg/kg b.w./day. 

The NOAEL (No Observed Adverse Effect Level) for PRENATAL DEVELOPMENT is 300 mg/kg b.w./day. This NOAEL value is based on the no occurrence of anomalies of foetal skull and skeleton in treated groups.