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Key value for chemical safety assessment

Effects on fertility

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Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4.1.2012 – 31.5.2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Koleč u Kladna, Czech Republic
- Age at study initiation: (P) 9 wks
- Weight at study initiation: (P) Males: 348-350 g; Females: 227-228 g
- Fasting period before study: no
- Housing: SPF animal room, 2 rats of the same sex in one plastic cage containing sterilised clean shavings of soft wood. During mating period – one male and one female in one cage, pregnant females – individually, offspring – with mother.
- Diet (e.g. ad libitum): Complete peleted diet for rats and mice in SPF breeding - ST 1 BERGMAN, ad libitum
- Water (e.g. ad libitum): Free access to drinking water, ad libitum
- Acclimation period: 5 days

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

STUDY TIME SCHEDULE
Date of animal arrival: 4. 1. 2012
Start of administration: 11. 1. 2012
End of administration: males 3.2. 2012, females 4. 3. 2012
Clinical examination: 4. 1. – 5. 3. 2012
Necropsy and biometry of organs: males 08. 02. 2012, females 20. 2. – 5. 3. 2012
Route of administration:
oral: gavage
Vehicle:
other: 0.5%methylcellulose in aqua pro injections
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The application form was prepared daily just before administration by mixing with 0.5% methylcelulose. The mixture was mixed by magnetic stirrer (ca 1000 rpm) for 3 minutes. The stability and the homogeneity of application form were determined by GC method.

VEHICLE
- Amount of vehicle (if gavage): The concentrations of suspension in all dose level were adjusted to ensure administration of 1 mL per 100 g of body weight.
- Lot/batch no. (if required): DT 265896
Details on mating procedure:
- M/F ratio per cage: 1:1
- Proof of pregnancy: sperm in vaginal smear referred to as day 0
- After successful mating each pregnant female was caged: individually



Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Males and females – 2 weeks prior to the mating period and 2 weeks during the mating period.
Pregnant females - during pregnancy and till 3rd day of lactation.
Males - after mating period – totally for 28 days.
Non-pregnant females (mated females without parturition) - 25 days after the confirmed mating
Frequency of treatment:
7 days per week at the same time
Remarks:
Doses / Concentrations:
50 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
150 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
450 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
12F/12M
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses for the study were chosen with respect to the results of the dose-range finding experiment. Two females at the dose level 600 mg/kg/day died during the dose-range finding experiment. Clinical sings (Straub phenomenon – consisted of the tail becoming rigid and erected across the back of the animal in an S-shaped curve, increased response to stimuli.) were observed at the dose levels 600 mg/kg/day from the 1st day till the 4th day of the experiment. In males at this dose level only mild apathy after application was observed in the 3rd and 4th day and no deaths occurred.
With regard to the high toxicity of the test substance Centralit for females, the application of the dose level higher than 450 mg/kg/day was impossible in main study.

Parental animals: Observations and examinations:
HEALTH CONDITION CONTROL: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations:
males – the first day of administration and than weekly,
females – the first day of administration and than weekly in premating and mating period, during pregnancy: day 0, 7th, 14th, 20th day, during lactation: 0. or 1st and 4th day;

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
-Time schedule:
males - weekly
females - weekly during premating period and after mating period, during pregnancy and lactation – on the same days as body weight (day consumption was counted)

WATER CONSUMPTION: No
Sperm parameters (parental animals):
Parameters examined in [P] male parental generations:
- sperm motility, sperm morphology
Litter observations:
CLINICAL OBSERVATION OF PUPS
- Time schedule: daily
- Changes in behavioural abnormalities were recorded. Detailed examination of each litter was performed as soon as possible after delivery (day 0 or 1 post-partum) and than daily till 4th day of lactation. The number and sex of pups, still births, live births and presence of gross anomalies, changes of physical condition and behavioural abnormalities were recorded.

BODY WEIGHT
- pups (litters) – 0. or 1st and 4th day
Postmortem examinations (parental animals):
SACRIFICE
- Parental males and nonpregnant females - at the end of the administration period – after 28 days of administration.
- Parental females - on the 4th day of lactation.
- Mated females without delivery - 26th day after confirmed mating.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cranial, thoracic, and abdominal cavity, with special attention to the organs of the reproductive systems

HISTOPATHOLOGY
- all gross lesions, pituitary gland, coagulation gland, prostate gland, seminal vesicles, testes and one epididymis (fixed in Davidson´s solutions), cervix of uterus, ovaries, uterus and vagina

ORGAN WEIGHTS
- males - the absolute weights of testes, epididymis, prostate gland and pituitary gland
- females - absolute weight of ovaries, uterus (incl. uterine tube and cervix) and pituitary gland
- the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight.
Postmortem examinations (offspring):
SACRIFICE AND GROSS NECROPSY
- Dead pups or pups killed before the 4th day of lactation were sexed and externally examined; the stomach was examined for the presence of milk. All surviving pups were killed on the 4th day of lactation, they were sexed and subjected to external examination of the cranium, and to macroscopic examination of the thoracic and abdominal tissues and organs.

HISTOPATHOLOGY
- Gross lesions of pups (eventually)


Statistics:
The ANOVA test - Analysis of Variance (QC.Expert 2.5) at significance level 0.05 was used for the statistical analysis. This statistical analysis was used for the results of body weight, biometry of organs and number of pups. Control group with vehicle was compared with three treated groups.

Reproductive indices:
Male mating index
Female mating index
Males fertility index
Females fertility index
Gestation index
Offspring viability indices:
Pre-implantation loss
Post-implantation loss
Post-natal loss
Survival index
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
effects observed, treatment-related
Dose descriptor:
NOAEL
Effect level:
450 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Administration of the test substance Centralit at the dose levels 450 mg/kg/day had no adverse effect to ability of male and female animals to successfully mate and produce viable offspring.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Dose descriptor:
NOEL
Generation:
F1
Effect level:
450 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The development of pups was not changed in treatment groups.
Reproductive effects observed:
not specified

DISCUSSION

The test substance, Centralit, was orally administered by gavage in graduated doses (50, 150 and 450 mg/kg/day) to three groups of males and females. The dose levels were established on the basis of results of Acute Oral Toxicity Study and DRFE (Dose-range finding experiment). Different toxic effect of the test substance for males and females was found. The females were shown more sensitive than males, therefore for this reproductive test, the dose level of 450 mg/kg was chosen as the highest dose levels.

Before mating, the males and females were dosed for two weeks. Females were dosed for at least two complete oestrous cycles in order to elicit any adverse effect on oestrous. All animals were treated during the whole time of mating period. Dosing of females continued during pregnancy and lactation period. Males were administered for 28 days (in total).

 

Effect of the test substance on the health condition of treated females was observed during the 1st week of study. Negative effect on neurology system of animals manifested as Straub phenomenon was recorded. Straub reaction consisted of the tail becoming rigid and erected across the back of the animals in a S-shaped curve. This reaction was accompanied by restlessness, excitability, and extension rigidity of the hindlimbs. This reaction is typical for animals after application of opiate substance, e.g. morphine. During next weeks of study, these changes were not observed any more, which can relate with adaptation of organism of treated animals to the test substance.

 

The first application of the test substance caused mortality in one female at the highest dose level.

The test substance had no negative effect on growth of parental males and females. The slightly increased food consumption and accompanying slightly increased the body weight increment were recorded in treated females since the second week of application.

 

The biometry of organs proved no statistically significant differences in treated parental males and females. Pathological examination of reproductive organs revealed no significant changes in both sexes.

Observation of sperm motility did not detect impaired quality of sperm in treated males versus control. The proportion of morphologically changed sperms was slightly increased in treated males in dose-dependent manner. The detailed histological examination of spermatogenesis stages did not even ascertain any damage. But the animals in the reproduction screening study are treated for less than the duration of the spermatogenic cycle so that an effect on spermatogenesis may not have had adequate time to become evident as reduced sperm counts that affect fertility.

 

Examination of reproductive system of parental females showed insignificant incidence of findings in all groups. No pathological changes were found in reproductive organs of previously pregnant females except for focal accumulation of lipophages and siderophages in the mesometrium as signs of previous gravidity. In nonpregnant females, pathological changes were insignificant and were not considered to be treatment-related.

 

Observation of pups – sex ratio, mean weight of litter and mean body weight of pups were unaffected by the test substance treatment. The average number of pups was slightly increased at highest dose levels. Mortality or presence of macroscopic abnormalities sporadically occurred in pups of all control and treated groups.

Administration of the test substance had no negative effect on reproduction parameters. The numbers of females achieving pregnancy and accompanying fertility indexes and gestation index were found to be comparable to the controls. Mating indexes of males and females at the lowest dose level were slightly decreased than at the control groups.

 

Duration of pregnancy of treated groups was similar to control group. Survival index was well-balanced at all groups. Pre-implantation loss, post-implantation losses and post-natal losses were unaffected by application of the test substance.

Conclusions:
With regard to the high toxicity of the test substance Centralit for females, the application of the dose level higher than 450 mg/kg/day was impossible. Administration of the test substance Centralit at the dose levels 450 mg/kg/day had no adverse effect to ability of male and female animals to successfully mate and produce viable offspring. Also development of pups was not changed in treatment groups.

The NOAEL (No Observed Adverse Effect Level) for the REPRODUCTION was established at 450 mg/kg body weight/day.
The NOEL (No Observed Effect Level) for the DEVELOPMENT of pups was established at 450 mg/kg body weight/day.
Executive summary:

INTRODUCTION

The test substance, Centralit, was tested for reproduction toxicity using the OECD Test Guideline No. 421 Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on July 27th 1995.

 

METHODS

Wistar rats of SPF quality were used for testing. The test substance was administered dissolved in 0.5 % methylcelulose using a stomach tube; oral application of rats was made daily. The concentrations of suspensions at all dose levels were adjusted to ensure the administered volume of 1 mL per 100 g of body weight. Four groups of animals were included in the study - 3 treated groups (doses 50, 150, 450 mg/kg of body weight/day) and one control group (vehicle only). Each group consisted of 12 males and 12 females. The dose levels were established on the basis of results of Acute Oral Toxicity Study and DRFE (Dose-range finding experiment). Different toxic effect of the test substance on sex of treated animals was found. The females were shown as more sensitive than males, therefore the dose level of 450 mg/kg were chosen for this reproductive test as the highest dose levels.

The treated groups were administered daily for the following periods:

males and females – 2 weeks prior to the mating period and during the mating period,

pregnant females – during pregnancy and till the 3rd day of lactation,

males  after mating period – totally for 28 days,

nonpregnant females (mated females without parturition) – for 25 days after the confirmed mating.

During the study clinical observation and health status control were performed daily. The body weight and food consumption were measured weekly or in specified time intervals. Vaginal smears were prepared daily during mating period (until the presence of spermatozoa). Reproduction parameters relevant to pups (number of pups, weight of litters, sex or vitality) were also recorded.

The study was finished by gross necropsy of animals. In all males of all groups the sperm parameters: sperm motility and sperm morphology were examined. The selected organs from parental animals were removed for weighing and histopathological examination.

 

 

RESULTS

The negative clinical effect of the test substance on the health condition of treated females was observed during the 1st week of study. Negative effect on neurology system of animals manifested as Straub phenomenon was recorded. Straub reaction consisted of the tail becoming rigid and erected across the back of the animals in a S-shaped curve. This reaction was accompanied by restlessness, excitability, and extension rigidity of the hindlimbs. This reaction is typical for animals after application of opiate substance, e.g. morphine. During next weeks of study, these changes were not observed any more, which can relate with adaptation of organism of treated animals to the test substance.

The first application of the test substance caused mortality of one female at the highest dose level.

The test substance had no negative effect on growth of parental males and females. The biometry of organs proved no statistically significant differences in treated parental males and females. Pathological examination of reproductive organs revealed no significant changes in both sexes.

 

In treated parental males, the proportion of morphologically changed sperms was slightly dose-dependent increased. The detailed histological examination of spermatogenesis stages did not even ascertain any damage. But the animals in the reproduction screening study are treated for less than the duration of the spermatogenic cycle so that an effect on spermatogenesis may not have had adequate time to become evident as reduced sperm counts that affect fertility.

Examination of reproductive system of parental females showed insignificant incidence of findings in all groups.

Observations of offspring did not reveal significant changes in sex ratio of pups, mean weight of litter and mean body weight of pups. The average number of pups was slightly increased at the dose level 450 mg/kg/day. Mortality of pups or presence of macroscopic abnormalities was recorded only sporadically and it was not related to the test substance administration. 

Administration of the test substance had no negative effect on reproduction parameters. The numbers of females achieving pregnancy and accompanying fertility indexes and gestation index were relative well-balanced with the control groups. Mating indexes of males and females at the lowest dose level were slightly decreased than at the control groups.

Duration of pregnancy of treated groups was similar to control group. Survival index was well-balanced at all groups. Pre-implantation loss, post-implantation looses and post-natal losses were unaffected by application of the test substance.

 

CONCLUSION

With regard to the high toxicity of the test substance Centralit for female, the application of the dose level higher than 450 mg/kg/day was impossible. Administration of the test substance Centralit at the dose levels 450 mg/kg/day had no adverse effect to ability of male and female animals to successfully mate and produce viable offspring. Also development of pups was not changed in treatment groups.

 

The NOAEL (No Observed Adverse Effect Level) for the REPRODUCTION was established at 450 mg/kg body weight/day.     

The NOEL (No Observed Effect Level) for the DEVELOPMENT of pups was established at 450 mg/kg body weight/day.

Endpoint:
extended one-generation reproductive toxicity - with developmental neurotoxicity (Cohorts 1A, 1B without extension, 2A and 2B)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07.11.18 - 25.11.19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
Adopted 25th June 2018
Deviations:
yes
Remarks:
only from Study plan; see section Any other information
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals : 10 weeks to cover the full spermatogenesis and folliculogenesis before the mating

- Basis for dose level selection :
The dose levels for study – 50, 150 and 400 mg/kg/day were chosen on the basis of results of the Study No. 127/15/7 Centralit – Repeated Dose 90-day Oral Toxicity Study (VUOS-CETA Report No. 16-266), and the Study No. 194/11/18 – Centralit – Reproduction/Developmental Toxicity Screening Test (VUOS-CETA Report No. 11-47).

- Inclusion/exclusion of extension of Cohort 1B: without extension
- Termination time for F2 : no F2 generation
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B :
Developmental neurotoxicity cohorts 2A and 2B were conducted for evaluation of potential neurotoxicity effect of the test item.
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: without Cohort 3

- Route of administration : Oral application by stomach tube was performed daily.

- Other considerations, e.g. on choice of species, strain, vehicle and number of animals
Laboratory rat Wistar Han (SPF quality - guaranteed) has been chosen because testing laboratory has long experience with this species and comparability with the studies already carried out. The test item was administered in 0.5% methylcellulose in aqua pro injectione.
27 females and 27 males per dose group + 2 males and 2 females for control of microbiological background (totally 220 animals)
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Koleč u Kladna, Czech Republic, RČH CZ 21760118

- Females (if applicable) nulliparous and non-pregnant:
- Age at study initiation: (P) 5 wks; (F1) from PND 22
- Weight at study initiation:
P Males: 209.5 - 237.6 g; P Females: 162.97 - 170.92 g;
F1–1A males 57.04 - 61.73 g; F1–1A females 54.36 - 59.09 g
F1–2A males 55.64 - 59.67 g; F1–2A females 56.30 - 60.56 g

- Fasting period before study: no
- Housing: SPF conditions according to internal SOP No.12

- Diet (e.g. ad libitum): ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): relative humidity 30-70%
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark

IN-LIFE DATES:
Animal arrival: 7.11.2018
Acclimatisation: 5 days
Test item administration:
P animals – from 13.11. – 16.11.2018 (gradual inclusion of animals in the study)
F1 animals – from PND 22
Termination and necropsy:
P males – after 15 weeks of administration (26.2. - 1.3.2019)
P females – after lactation period (7.3. – 14.3.2019)
Surplus pups after standardisation: at PND 4
Surplus pups not allocated to cohorts: at weaning (PND22)
Animals of cohort 1A: after 13 weeks of age (19.5. – 25.5. 2019)
cohort 1B: after 14 weeks of age (26.5. – 2.6.2019)
cohort 2A: after 11 weeks of age (30.4. – 15.5.2019)
cohort 2B: after weaning, at PND 22
Route of administration:
oral: gavage
Vehicle:
other: 0.5% methylcellulose in aqua pro injections
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): daily
The application form of the test substance (suspension in 0.5% methylcelulose) was prepared daily just before administration. The application form was dissolved in ultrasonic bath for a 10 minutes. Then the application form was stirred by using of laboratory stirrer for 10 minutes.

VEHICLE
- Concentration in vehicle:
The application form was prepared by mixing with 0.5% methylcellulose in aqua pro injectione. Two concentrations of application form were prepared (50 mg/10 mL and 600 mg/10 mL).
Concentration level 50 mg/10mL
Ca. 250 mg of the test substance was weighted into 100 mL glass beaker calibrated to 50 mL. 50 mL of the vehicle were measured out by volumetric cylinder and added to the beaker. Dissolving in ultrasonic bath for a 10 minutes follows. Then the application form (white suspension) was stirred by using of laboratory stirrer for 5 minutes (1000 rpm).
Concentration level 600 mg/10 mL
Ca. 3 g of the test substance was weighted into 100 mL glass beaker calibrated to 50 mL. 50 mL of the vehicle were measured out by volumetric cylinder and added to the beaker. Dissolving in ultrasonic bath for 10 minutes follows. Then the application form (white pasty suspension) was stirred by using of laboratory stirrer for 5 minutes (1000 rpm).

- Amount of vehicle (if gavage):
Individual application volume
The application volume of the test item suspension was based on the recent individual body weight determination and was adjusted weekly for adult P animals.
In pregnant females and in pups allocated into the cohorts after weaning, the application volume was adjusted more often. During pregnancy, the application volume was adjusted on days 0, 7, 14, 16, 18 and 20; during the lactation period on days 1, 4, 7, 14 and 21 and in pups after weaning at interval 2-2-3 days for two weeks and weakly thereafter.

- Lot/batch no. (if required):
The test item was administered in 0.5% methylcellulose in aqua pro injectione.
The preparation of the stock solution of methylcellulose was prepared according to internal SOP M/11. The stock solution was stored in a closed bottle in a refrigerator.
Methylcellulose D180HBK042 (exp. 11/2022)
Details on mating procedure:
- M/F ratio per cage: 2 rats of the same sex in one cage in pre-mating period, during mating period – one male and one female in one cage

- Length of cohabitation:
Parental males and females were mated after 10 weeks of administration: each female of P generation was placed with a single male of P generation from the same dose level (1:1 mating) until copulation occurs or 2 weeks had elapsed.

- Proof of pregnancy: Each morning the females were examined for presence of spermatozoa in vaginal smears. Day 0 of pregnancy was defined as the day the sperms were found.

- After successful mating each pregnant female was caged (how): pregnant females were caged individually, offspring – with mother
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Stability and homogeneity were determined by means of measuring of a peak area of the test substance by a high-performance liquid chromatography based on a method developed at the test facility.
It follows from the results of analyses (homogeneity and stability) that the application form of the test substance at both concentration levels (50 mg/10 mL and 600 mg/10 mL), at defined laboratory conditions (laboratory temperature, preparation of application form by defined manner) is homogenous. It is recommended to sample this application form from the middle of the beaker content.
The application form of the test substance at both concentration levels (50 mg/10 mL and 600 mg/10 mL) is stable for 120 minutes from the finalization of application form preparation.
Duration of treatment / exposure:
P Males and females
The treatment period for the P males and females lasted to the end of lactation period - respectively 15 weeks for males (10 weeks of pre-mating, during the mating and approximately 5 weeks after mating) and at least 16 weeks (10 weeks of pre-mating, during the mating, 3 weeks during the pregnancy and 3 weeks of lactation) for females (mothers). Parental males and females were mated after 10 weeks of administration.
The females were examined for presence of spermatozoa in vaginal smears. Day 0 of pregnancy was defined as the day the sperms will be found.
The standardisation of litter size was performed on the 4th day of lactation period to a number of five males and five females (ideally). Surplus pups were necropsied and examined macroscopically.

F1 Males and females
The treatment period for the males and females of the first filial (F1) generation started after weaning on post-natal day (PND) 22 and persisted to an appropriate week of necropsy in accordance of assignment of animals into the appropriate cohort.



Thus the animals of P generation was administered as follows:
Males: 10 weeks (pre-mating) → during the mating → 5 weeks post-mating (approximately 15 weeks of application)
Females: 10 weeks (pre-mating) → during the mating → pregnancy → lactation (approximately 16 weeks of application)

The animals of F1 generation was administered by the test item suspension to the stomach by gavage post weaning (from PND22).
At weaning (at PND 21) the animals were divided into the cohorts 1A, 1B, 2A, 2B.
Frequency of treatment:
daily
Details on study schedule:
- F1 parental animals not mated until [...] weeks after selected from the F1 litters.
The treatment period for the males and females of the first filial (F1) generation started after weaning on post-natal day (PND) 22 and persisted to an appropriate week of necropsy in accordance of assignment of animals into the appropriate cohort.

- Selection of parents from F1 generation when pups were [...] days of age.
At weaning (at PND 21) the animals were divided into the cohorts.

- Age at mating of the mated animals in the study:
P: premating 10 weeks
F1 - 1A: the vaginal smears for checking of oestrous cyclicity were prepared daily from onset of vaginal patency until first oestrus and daily for two weeks (from PND 75)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
P generation
1. control - vehicle: 0 mg/kg 27 F and 27 M → 20 pregnant F
2. the lowest dose: 50 mg/kg 27 F and 27 M → 20 pregnant F
3. intermediate dose: 150 mg/kg 27 F and 27 M → 20 pregnant F
4. the highest dose: 400 mg/kg 27 F and 27 M → 20 pregnant F
Note: The highest dose level 400 mg/kg was chosen with respect to a several deaths of F at the highest dose levels 450 mg/kg in previous studies.

F1 generation (reproduction cohorts 1A and 1B)
1. control - vehicle: 0 mg/kg 20 M + 20 F
2. the lowest dose: 50 mg/kg 20 M + 20 F
3. intermediate dose: 150 mg/kg 20 M + 20 F
4. the highest dose: 400 mg/kg 20 M + 20 F
F1 generation (neurotoxicity cohorts 2A)
1. control - vehicle: 0 mg/kg 10 M + 10 F
2. the lowest dose: 50 mg/kg 10 M + 10 F
3. intermediate dose: 150 mg/kg 10 M + 10 F
4. the highest dose: 400 mg/kg 10 M + 10 F
Note: F1 animals from 2B cohort (10 animals/sex/dose) are used for brain histopathology assessment at weaning. Test substance administration was not performed.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
According to the results of previous study (Centralit – Repeated Dose 90-day Oral Toxicity Study (VUOS-CETA Report No. 16-266) and Centralit – Reproduction/Developmental Toxicity Screening Test (VUOS-CETA Report No. 11-47) the dose levels 0, 50, 150 and 400 mg/kg/day were determined.
Note: The highest dose level 400 mg/kg was chosen with respect to a several deaths of F at the highest dose levels 450 mg/kg in previous studies.

- Fasting period before blood sampling for clinical biochemistry: The animals were fasted approximately for 18 hours before blood collection but they were supplied drinking water ad libitum.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily after administration in natural conditions.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week.
Changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, and unusual respiratory pattern) were monitored. Changes in gait, posture, response to handling, as well as the presence of clonic or tonic movements, stereotypy or bizarre behaviour were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations:
The body weights of males, females and pups were recorded on automatic balances with group mean computing module. All animals were weighed on the 1st day of administration, then on specified days and also immediately before euthanasia.
Parental animals (P) were weighed on the 1st day of dosing and at least weekly thereafter. In addition, P females were weighed during lactation on the same days as the weighing of the pups.
All F1 animals were weighed individually at weaning (PND 21) and at least weekly thereafter. Body weight was also recorded on the day when they attain puberty (completion of preputial separation or vaginal patency). All animals were weighed at sacrifice.
Weight increment was computed as a mean per group (in grams). Non-pregnant females (females without parturition) were not included in calculation of means in pregnancy and lactation period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as:
The remainder of pellets in each gage was weighed on specified days on automatic balances with group mean computing module. The new food was weighed out and the food consumption for the previous week/time period was computed.
Food consumption for animal/day was calculated from mean values of each group. The same way of calculation of mean food consumption was used for females in pre-mating period. In pregnancy and lactation period mean individual values (grams/animal/day) were calculated for each week of the study. Mean food consumption for each group was calculated from individual values. Nonpregnant females (females without parturition) were not included in calculation of mean food consumption in pregnancy and lactation period.
Food consumption for F1 animals divided into the cohorts was calculated from the time, when two animals were placed in the cage. That means from the first full week to the last full week (due to gradual integration of animals) were recorded.
Oestrous cyclicity (parental animals):
Each morning in the mating period vaginal smears were prepared from all the mated females. These smears were examined for presence of spermatozoa.
Vaginal smears were made also on necropsy day to determine the stage of oestrous cycle.
Vaginal smears were examined daily for all F1 females in cohort 1A, after the onset of vaginal patency, until the first cornified smear was recorded. Oestrous cycles for all F1 females in cohort 1A were monitored for a period of two weeks (beginning PND 75).
Sperm parameters (parental animals):
Parameters examined in P+F1 male parental generations:
In P and F1 males of all groups surviving to scheduled necropsy the sperm parameters (motility, morphology) were examined. Sperm motility was evaluated immediately after sacrifice in all males (P + F1) subjectively, using a microscope. For the evaluation of sperm morphology, an epididymal sperm sample was examined as fixed preparations and 200 spermatozoa per sample were evaluated. The morphology was generated for control and high-dose P and F1 animals only (lower doses were not evaluated because the exposure-related effects were not recorded).

Sperm Motility
Sperm samples were taken from one epididymis and sperm motility was assessed from these samples. The motility of sperm was determined by microscopic examination of the prepared sperm suspension. The result of observation was evaluated subjectively according to following grades: 1 - fast progressive motility, 2 - slow progressive motility, 3 - no progressive motility, 4 - non-motile sperm.

Sperm Morphology
Sperm samples were taken from one epididymis and sperm morphology was assessed from these samples. A smear from the sperm suspension was prepared and stained (Giemsa staining). The morphology of sperm was determined by microscopic examination. All deviations – e.g. broken tail, abnormal form of tail, double head, amorphous head, abnormal form of neck – were recorded.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (ideally 5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Each litter was examined as soon as possible after delivery. The number and sex of pups, still- births, live-births and presence of gross anomalies were recorded. Changes of physical condition and behavioural abnormalities were recorded.
Live pups were counted and weighed as whole litter on PND 1, and then individually on PND 4, 7, 14, and 21.
In addition, the first clinical examination of the pups included recording of body temperature (indicatively), state of activity and reaction to handling.
Standardisation of litter size was carried out by random removal of pups on the 4th day after the birth to a maximum of 10 pups (ideally 5 females + 5 males).

The AGD of each pup of standardized litter was measured on day 4 of lactation. For measuring digital calliper was used. Also individual body weight was recorded.
The presence of nipples in male pups was checked on day 13 of lactation (the presence of nipples on day 13 in male pups is not desirable).
F1 animals were evaluated daily for balano-preputial separation or vaginal patency from PND30 (for male/female). Any abnormalities of genital organs were noted.

GROSS EXAMINATION OF DEAD PUPS:
All pups were examined macroscopically with special attention to the organs of the reproductive system. Dead pups or pups humanely killed in a moribund status were also macroscopically examined.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
Assessment of neurotoxicity (F1 animals – cohort 2A)
– Auditory startle test (ASR), Functional Observation battery (FOB) and Open Field test
An auditory startle test (ASR): on PND 25 using animals in cohort 2A. The startle reflex is a motor response to an intense and unexpected stimulus. The amplitude of a study subject´s response (a “jump”) was quantified.

The cohort 2A animals were subjected to a functional observational battery (FOB) according to internal SOP M/1. Reactions to touch, noise, pain and pupillary reflex, numbers of defecations and emictions, number of upstanding, the values of grip strength were evaluated.

The test of activity of animals by using an Open Field according to internal SOP 262 was also performed. The Open Field test was used to assess locomotor, exploratory and anxiety-like behaviour to novelty of animals. Conditions of experiment were as follows: animals were placed in the centre of arena, 10 sec latency time and 3 minutes test (recording time). Parameters of locomotor activity as distance, speed and resting time were recorded. Parameters of explorative behaviour as entries at zones, time at zones and counter clockwise/clockwise rotation were also recorded.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
For the investigation of pre- and postnatal induced immunotoxic effect, 10 male and 10 female cohort F1-1A animals per group were used. The splenic lymphocyte subpopulation analysis (CD4+ and CD8+ T lymphocytes, B lymphocytes and natural killer cells) was performed.
Postmortem examinations (parental animals):
SACRIFICE
Termination and necropsy:
P males – after 15 weeks of administration
P females – after lactation period
Surplus pups after standardisation: at PND 4
Surplus pups not allocated to cohorts: at weaning (PND22)
Animals of cohort 1A: after 13 weeks of age
cohort 1B: after 14 weeks of age
cohort 2A: after 11 weeks of age
cohort 2B: after weaning, at PND 22

GROSS NECROPSY
At the time of termination or premature death, all P and F1 animals were necropsied and examined macroscopically for any structural abnormalities or pathological changes.
For adult P and F1 females, a vaginal smear was examined on the day of necropsy to determine the stage of the oestrous cycle. The uteri of all P females were examined for the presence and number of implantation sites.
During the necropsy a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities were carried out. Organs for consequent histopathological examination were taken out and stored in containers with fixative (buffered 4% formaldehyde). Testes and epididymides were fixed in modified Davidson’s fixative.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table No.15 (M) and No. 26(F) were prepared for microscopic examination and weighed, respectively.

Biometry of organs (P animals + F1 animals)
Terminal body weight and the weight of the following organs were recorded:
All P animals: weight of uterus (with oviducts and cervix), ovaries, testes, epididymides, prostate, seminal vesicles with coagulating glands and their fluids (as one unit), brain, liver, kidneys, heart, spleen, thymus, pituitary gland, thyroid gland (post-fixation) and adrenal glands.
All F1 adult animals – cohort 1A: weight of uterus (with oviducts and cervix), ovaries, testes, epididymides, prostate, seminal vesicles with coagulating glands and their fluids (as one unit), brain, liver, kidneys, heart, spleen, thymus, pituitary gland, thyroid gland (post-fixation) and adrenal glands.
10 male and 10 female cohort 1A adult animals: additionally weight of the lymph nodes
All F1 animals – cohort 1B: weight of uterus (with oviducts and cervix), ovaries, testes, epididymides, prostate, seminal vesicles with coagulating glands and their fluids (as one unit) and pituitary gland.
All F1 animals – cohort 2A: brain only.
All F1 animals – cohort 2B: brain only.
F1 weanlings not selected for cohorts (on PND 21) - 10 pups per sex per dose group: brain, spleen, and thymus (mammary tissues preserved)

Histopathological examination (P animals + F1 animals)
Full histopathology was performed in the control and high dose group of parental (P) animals and F1 animals of the 1A cohort. The histopathological examination was no extended to animals of low and middle dose group because treatment-related changes were not observed in the high dose group. Only macroscopically changed organs were examined.
No histopathology was performed in animals of cohort 1B. Histopathology in cohort 1B would be conducted if results from cohort 1A would be equivocal.
Neurohistopathology was performed for 10 males and 10 females of the highest dose level and control group of cohort 2A animals, following completion of neurobehavioral testing. The perfusion fixation was required for animals of 2A cohort (internal SOP M/19). The brain, the eyes (retina and optic nerve) and samples of muscle (longitudinal and transverse sections) and spinal cord were also examined. The seven transverse cuts were performed. Multiple sections were examined from the brain to allow examination of olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, mid-brain, brain-stem and cerebellum.
Paraffin sections were prepared and histologic sectioning was made. Haematoxylin-eosin staining was used.
Brain histopathology was performed for 10 males and 10 females of the highest dose level and control group of cohort 2B animals per sex necropsied on PND 22.

Tissues and organs collected from:
P animals and cohort 1 animals
The following tissues and organs were collected from P and F1 adult cohort 1A males and females at necropsy and fixed in buffered 4% formaldehyde solution (v/v) for further histopathology evaluation: all gross lesions and previously identified target organs, uterus (with oviducts and cervix), ovaries, testes, epididymides, prostate, seminal vesicles with coagulating glands and their fluids (as one unit), brain, liver, kidneys, heart, spleen, thymus, pituitary, thyroid, adrenal glands, samples of peripheral nerve, muscle, spinal cord, eye plus optic nerve, gastrointestinal tract, urinary bladder, lung, trachea (with thyroid and parathyroid attached), bone marrow, mammary gland (males and females) and vagina.
Full histopathology of the preserved organs and tissues was performed for all high dose and control. Organs demonstrating treatment-related changes were examined in the lowest and middle dose groups.
For the evaluation of pre- and postnatally induced effects on lymphoid organs also the histopathology on the collected lymph nodes and bone marrow were evaluated of 10 male and 10 female cohort 1A animals.
Detailed histopathological examination was done in testes and epididymides of cohort 1A males (10 control and 10 high dosed animals). A quantitative evaluation of ovaries was performed in cohort 1A females (10 control and 10 high dosed animals).
Cohort 1B: organ preserved were vagina, uterus (with cervix), ovaries, testes, epididymides, seminal vesicles, coagulation glands, prostate, pituitary and/or identified target organs.
Cohort 2A: brain, the eyes (retina and optic nerve), muscle and spinal cord were examined.
Cohort 2B: brain was examined.
Postmortem examinations (offspring):
SACRIFICE
Termination and necropsy
Surplus pups after standardisation: at PND 4
Surplus pups not allocated to cohorts: at weaning (PND22)
Animals of cohort 1A: after 13 weeks of age
cohort 1B: after 14 weeks of age
cohort 2A: after 11 weeks of age
cohort 2B: after weaning, at PND 22

GROSS NECROPSY
All pups were examined macroscopically with special attention to the organs of the reproductive system. Dead pups or pups humanely killed in a moribund status were also macroscopically examined.
During the necropsy a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities were carried out. Organs for consequent histopathological examination were taken out and stored in containers with fixative (buffered 4% formaldehyde). Testes and epididymides were fixed in modified Davidson’s fixative.

HISTOPATHOLOGY / ORGAN WEIGTHS
Weights of organs
The tissues indicated in Table No.54 (F1-1A males) and No. 55(F1-1A females) were weighed.

Histopathological examination
The tissues indicated in Table No.56 (F1-1A males) and No. 57(F1-1A females) were prepared for microscopic examination.
Statistics:
The software Statgraphic®Centurion (version XVII, USA) was used for statistical evaluation.
Males/females from control group were compared with males/females from three treated groups. The results statistically significant on probability level 0.05 were indicated in the summary tables. Some of numerical results were evaluated by an appropriate statistical method. In general:
The parametric tests were used for statistical evaluation of e.g. body weight of males and females, mean pup weight, litter weight, anogenital distance of pups, selected haematology parameters, selected blood biochemistry parameters, data from urinalysis (pH, volume), data from biometry of organs.

As the first step the test for normality (Shapiro-Wilk test) was used. If the data were not normally distributed than the transformation of data was performed (Box-Cox transformation). If still the normal distributed distribution was not achieved than non-parametric tests (Kruskal-Wallis Test, Mann-Whitney test) were used.
For normally distributed data have at first the variance check has been performed (Levene´s test) to verify if standard deviations within each group are equal. One-Way ANOVA (probability level p ≤ 0.05) was used to detect whether there are any significant differences amongst the means. In case of significant differences, the post hoc statistical testing was performed (Fisher's least significant difference - LSD test).

The non-parametric tests were used for statistical evaluation of e.g. selected reproduction parameters with non-continuous distribution (number of live born pups, number of pups, number of implantations), selected haematology parameters with non-continuous distribution.

The Kruskal-Wallis test was used for the comparison of the measured effect in all treatment groups with the vehicle control group (as global test) and the two-groups Mann-Whitney test (probability level p ≤ 0.05)
Reproductive indices:
Ranged in sequence of the dose levels 0-50-150-400 mg/kg/day further in the text

Results of reproductive indices:
Male mating index: 96.3-100.0-100.0-100.0
Female mating index: 96.3-100.0-100.0-100.0
Male Fertility index: 81.5-85.2-80.8-85.2
Female fertility index: 84.6-85.2-80.8-85.2
Gestation index: 100.0-100.0-100.0-100.0
Viability index on PND4: 99.0-99.4-97.3-99.7
Offspring viability indices:
The number of females who mated was 27-27-26-27 (one female from the dose level 150 mg/kg/day died before mating).
The number males who mated was 27-27-26-27.
The 26-27-26-27 females showed evidence of copulation and 22-23-21-23 females achieved pregnancy.
The duration of mating (mean) was 2.00-2.19-1.96-1.85 days.
The duration of pregnancy (mean) was 22.00-21.70-21.86-22.04 days.
The number of females with live pups born was 22-23-21-23.
The number (mean) of implantation was 15.48-15.79-17.57-16.78.
The number (mean) of post-implantation loss was 1.91-2.00-1.86-1.57.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Negative effect manifested as Straub phenomenon was recorded in 7 of 27 females after the first administration of the test item (only) at the dose level 400 mg/kg/day. Straub reaction consisted of the tail becoming rigid and erected across the back of the animals in a S-shaped curve. This reaction was accompanied by restlessness, tremor and rapid breathing.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two females (control group and dose level 150 mg/kg/day) died during the study of an unknown cause.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
M: OK
F: Statistically significantly decreased mean body weight was recorded in females on day 1 and 4 of lactation at the dose level 150 mg/kg/day. In females at the dose level 50 mg/kg/day was detected increased body weight compared to control animals.
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:
effects observed, treatment-related
Description (incidence and severity):
M: statistically significantly increased value of blood clotting time in males at the dose levels 150 and 400 mg/kg/day. Delayed Activated Partial Thromboplastin Time (APTT) was recorded in a dose-dependent manner in all dosed groups in comparison with the control group of males.
F: statistically significantly increased value of blood clotting time in females at all dose levels (without dose dependence). Delayed APTT was recorded.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
M: Biochemical examination showed the changed mean value of hepatic enzymes. The value of ALT was decreased in all groups of dosed males statistically significantly; the value of ALP and AST was decreased insignificantly in males at the dose levels 150 and 400 mg/kg/day.
F: The mean value of total cholesterol was increased (statistically significantly) in females at the dose level 150 and 400 mg/kg/day.
M/F: Concentration of the thyroid hormone T4 and TSH of treated males/females did not show significant difference in comparison with the control M/F.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The behaviour, clinical status and activity of M/F were not different from M/F of the control group.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
M/F: Histopathological examination did not reveal any significant differences in findings between the dosed groups and the M/F control group.
M: Spermatogenesis in the testes of the high dose administered males was without any pathological findings. Epididymides of both control and high dose males were without any pathological findings.
F: The most of findings related to previous gravidity. No treatment related changes were found in female genital tract.
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Ethylcentralit had no effect on P reproductive indices, including mating, fertility, time to mating, gestation length. Litter sizes, pup survival, sperm parameters and follicle counts were also unaffected by Ethylcentralit. Postnatal development of locomotion and sensory development of pups of treated groups were not negatively affected by the test substance item. No differences in development of pups were recorded.
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat. (total fraction)
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat. (total fraction)
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Clinical signs:
no effects observed
Description (incidence and severity):
F1/1A - M/F: In treated males and females of all dose levels the treatment-related effects were not detected during the health condition control and clinical observation in application period.
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F1/1A - M: The statistic evaluation of body weight in 10th week of application and necropsy body weight did not show significant changes.
F1/1A - F: The statistic evaluation of body weight showed significant differences during the study in females at the dose levels 150 and 400 mg/kg/day in comparison with the control females.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
F1/1A - M: the mean food consumption of treated males at the dose level 400 mg/kg/day was from the 5th week of application higher in comparison with the control group. From the same time, the food consumption of males from the dose level 150 mg/kg/day was lower in comparison with the control group of animals.
F1/1A - F: the mean food consumption of treated females was well-balanced with the control females.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
F1/1A - M: no statistically significantly changed values of blood parameters
F1/1A - F: Following statistically significantly changed values were recorded: decreased concentration of haemoglobin and haematocrit in females at the dose level 50 mg/kg/day, decreased value of platelet count in females at the dose level 150 mg/kg/day; delayed activated partial thromboplastin time (APTT) in females at the dose level 50 and 400 mg/kg/day and decreased prothrombin time in all dosed groups of females in comparison with the control females.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
F1/1A - M: Statistically significantly changed value were recorded. Increased value of total protein (T-Pro) and albumin was recorded in males at the dose level 400 mg/kg/day. The value of ALP and ALT were decreased in males at the dose levels 150 and 400 mg/kg/day. Decreased value of creatinine was recorded in males at the dose level 50 and 400 mg/kg/day. Significantly decreased value of urea (BUN) was recorded in all dosed groups of males.
F1/1A - F: Statistically significantly changed value were recorded. Increased value of total protein (T-Pro) and albumin was recorded in females at the dose level 400 mg/kg/day. The value of ALP and ALT were decreased in males at the dose level 400 mg/kg/day. Decreased value of cholesterol and increased value of AST were recorded in females at the dose level 50 mg/kg/day.
F1/1A - M/F:No statistically significant differences in T4 and TSH concentration were noted between the treatment groups and the control group.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
F1/1A - M: a statistically significant decreased volume and pH of urine was detected in males at all dose levels.
F1/1A - F: no statistically significantly changed values were recorded
Sexual maturation:
no effects observed
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
F1/1A - M: The anogenital distance (AGD) of treated F1 males was not affected by the test item treatment though the corrected AGD in males was increased at the dose level 150 and 400 mg/kg/day in comparison with the control males (which does not indicate a negative effect of the test item).
F1/1A - F: The anogenital distance (AGD) of treated F1 female pups was not affected by the test item treatment though the corrected AGD was decreased in females at the dose level 50 and 150 mg/kg/day in comparison with the control animals (which does not indicate a negative effect of the test item).
Nipple retention in male pups:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
F1/1A - M: Statistically significantly changed weights of organs were recorded sporadically. Increase of absolute and relative weights of liver in males at the dose levels 150 and 400 mg/kg/day was recorded. Statistically significantly increased relative weight of spleen was recorded in males at the dose level 400 mg/kg/day. Relative weight of kidneys was increased significantly in males at the dose levels 150 and 400 mg/kg/day.
F1/1A - F: Biometry of organs: the statistical analysis of the data revealed the following significant intergroup differences: Increase of relative weights of liver in females at the dose levels 150 and 400 mg/kg/day. Absolute weight of thyroid gland was increased in females at the dose level 50 and 150 mg/kg/day; relative weight of thyroid gland was increased in females at the dose levels 150 and 400 mg/kg/day.
Gross pathological findings:
no effects observed
Description (incidence and severity):
F1/1A - M/F: macroscopical structure of reproductive organs in treated males were unaffected by treatment of the test item.
Histopathological findings:
no effects observed
Description (incidence and severity):
F1/1A - M:Histopathological examination did not reveal any differences in findings between the dosed groups and the treated groups of males.
F1/1A - F: Histopathological examination did not reveal any differences in findings between the dosed groups and the female control group. Spermatogenesis in males and quantitative evaluation of ovaries in females did not reveal significant differences between control and high dosed animals.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
F1/1A - F: significant changes in weight of axillary lymph nodes were detected in females only. Absolute and relative weight of axillary lymph nodes was detected in females at the dose level 150 mg/kg/day. Significantly decreased relative weight of axillary lymph nodes in females at the dose level 50 mg/kg/day was also detected. No dose-response was observed, so this is unlikely to be related to administration of the test item.
Percentage representation of T-; B-lymphocytes and NK cells in spleen of treated animals was comparable with the control animals. Statistically significant differences were not detected except significantly decreased ratio of CD8+ T lymphocytes in females at the dose level 400 mg/kg/day. Dose dependency was not recorded
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
F1/2A: There were no significant, exposure-related effects on FOB parameters, parameters characterized by an Open Field test and an ASR.
Neurohistopathologic examination of brain after perfusion fixation did not show pathological findings in males and females dosed by dose level 400 mg/kg/day in comparison with the control animals.
F1/2B: Macroscopic brain measurements did not show significant differences in cerebral and cerebellar length. Also absolute and relative weight of brain (after perfusion fixation) of treated animals was comparable with the control animals.
Histopathologic examination of brain did not show pathological findings in males and females dosed by dose level 400 mg/kg/day in comparison with the control animals.
Developmental immunotoxicity:
not examined
Sperm analysis revealed no changes in sperm motility and morphology of dosed males compared to control males.
Key result
Dose descriptor:
NOAEL
Generation:
F1 (cohort 1A)
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat. (total fraction)
Sex:
male/female
Basis for effect level:
other: influence of the test substance on the weight of liver and thyroid gland, some hematological parameters, biochemical parameters especially liver enzymes
Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
400 mg/kg bw/day (actual dose received)

Deviation

The deviations from Study Plan occurred. Because of the necessity and difficulty of terminal examinations due to a big number of animals, the gross necropsy of the P and F1 animals were divided into multiple days (which is a deviation from the study plans, not from the guidelines). The animals were necropsied within the time interval mentioned in guideline (approximate age at necropsy at weeks) not exactly on the day of necropsy mentioned in study plan.

Conclusions:
Test item, Ethylcentralit, was tested in extended one-generation reproductive toxicity study (EOGRTS) in rats, with ten weeks premating exposure duration for the parental (P) generation.
Ten weeks premating exposure duration for the parental (P) generation was ordered by the ECHA (European Chemical Agency) authorities based on previous testing result.
This ten weeks premating exposure period allows to cover the full spermatogenesis and folliculogenesis before the mating.

Effects on reproduction
The administration of the test item did not affected reproductive organs and ability of reproduction. Spermatogenesis in the testes of the high dose administered P and F1 males was without any pathological findings. No treatment-related changes were found in the male genital tract. No treatment-related histological changes were found in the female genital tract. Ovarian follicle count in high dosed F1 females was similar with the control females. Reproductive indexes were unaffected in treated groups of females in comparison with the control group of females.
Across life stages, the EOGRTS has established that test item, Ethylcentralit, showed no evidence of adverse effect on reproduction.

Developmental neurotoxicity cohorts 2A and 2B were included for evaluation of potential neurotoxicity effect of the test item. Cohort 2A was subjected to an auditory startle test, functional observation battery and locomotor activity. There were no significant exposure-related effects on FOB (functional observational battery) parameters, ASR (auditory startle reflex). Also explorative behaviour and locomotor activity examined by the Open Field did not show significant differences between the treated and control animals. Detailed neurohistopathology of brain after perfusion fixation did not reveal a negative effect of the test item on brain. Brain histology was performed for cohort 2B animals and did not reveal a negative effect of the test item.
Across life stages, the EOGRTS has established that Ethylcentralit showed no evidence of developmental neurotoxicity.

The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION of P animals and for DEVELOPMENTAL NEUROTOXICITY in F1 animals was established as 400 mg/kg/day for males and females under the given study conditions.


Systemic toxicity in parental (P) generation
Adverse effect on health status of females only was observed after the first application of the test item only. Negative effect manifested as Straub phenomena – the tail becoming rigid and erected across the back of the animals in a S-shaped curve, tremor, restlessness and rapid breathing were recorded.
In both sexes the adverse influence of the test item on the weight of liver (statistically significant increasing of absolute and relative weight of liver in males and females at the dose levels 150 and 400 mg/kg/day, dose-dependently), some haematological parameters (statistically significant delayed Activated Partial Thromboplastin Time (APTT) was recorded in a dose-dependent manner in males and females at the dose level 150 and 400 mg/kg/day), biochemical parameters especially liver enzymes (decreased value of ALP,AST and ALT in males at the dose level 150 and 400 mg/kg/day, statistically significantly increased value of T-Chol in females at the dose level 150 and 400 mg/kg) were observed in P animals.

Systemic toxicity in first filial (F1) generation
In females decreased body weight at the dose levels 150 and 400 mg/kg/day was recorded during the whole study.
In both sexes the adverse influence of the test item on the weight of liver (statistically significant increasing of absolute and relative weight of liver in males at the dose levels 150 and 400 mg/kg/day, dose-dependently; dose-dependently increased weight of liver in females), some biochemical parameters especially liver enzymes (statistically significantly decreased value of ALP and ALT in males at the dose level 150 and 400 mg/kg/day and the value of ALP in females at the dose level 400 mg/kg/day were observed in F1 animals.

For SYSTEMIC TOXICITY of Ethylcentralit, the value of NOAEL (Not Observed Adverse Effect Level) for MALES and FEMALES was established as 50 mg/kg/day under the given study conditions.
Executive summary:

Introduction

The test item, Ethylcentralit, was tested for repeated toxicity, reproduction toxicity and developmental neurotoxicity using the method OECD Test Guideline No. 443, Extended One-Generation Reproductive Toxicity Study, Adopted 25th June 2018.

Study performance

Wistar rats of SPF quality were used for testing. The test item was administered suspended in methylcellulose (0.5% methylcellulose in aqua pro injection). Oral application by stomach tube was performed daily.

The parental (P) generation, 108 males and 108 females (Wistar rats), approximately 5 weeks of age, were acclimatised for 6 days before initiation of dosing.

The study included four groups of parental animals – 3 treated groups and 1 control group (vehicle only). Each group in the parental (P) generation consisted of 27 males and 27 females at the beginning of the study.

 

The groups of the 1st (F1) generation were formed after weaning into the cohorts as follows:

Cohort 1 (1A and 1B) = Reproductive/developmental toxicity testing

Cohort 2 (2A and 2B) = Developmental neurotoxicity testing.

Cohort 1A: One male and one female/litter/group (20/sex/group): for primary assessment of effects upon reproductive systems and of general toxicity.

Cohort 1B: One male and one female/litter/group (20/sex/group): for obtaining additional histopathology data when results from cohort 1A are equivocal.

Cohort 2A: Total of 20 pups per group (10 males and 10 females per group; one male or one female per litter) assigned for neurobehavioral testing followed by neurohistopathology assessment at adult age.

Cohort 2B: Total of 20 pups per group (10 males and 10 females per group; one male or one female per litter) assigned for neurohistopathology assessment at weaning (PND 22).

Pups not selected into the cohorts (surplus pups) were euthanized and submitted for gross necropsy.

 

Dose levels

The dose levels for study – 50, 150 and 400 mg/kg/day were chosen on the basis of results of the Study No. 127/15/7 Centralit – Repeated Dose 90-day Oral Toxicity Study (VUOS-CETA Report No. 16-266), and the Study No. 194/11/18 – Centralit – Reproduction/Developmental Toxicity Screening Test (VUOS-CETA Report No. 11-47).

 

Treatment

The treatment period for the P males and females lasted to the end of lactation period - respectively 15 weeks for males (10 weeks of pre-mating, during the mating and approximately 5 weeks after mating) and at least 16 weeks (10 weeks of pre-mating, during the mating, 3 weeks during the pregnancy and 3 weeks of lactation) for females (mothers). Parental males and females were mated after 10 weeks of administration. The females were examined for presence of spermatozoa in vaginal smears. Day 0 of pregnancy was defined as the day the sperms will be found.

The standardisation of litter size was performed on the 4th day of lactation period to a number of five males and five females (ideally). Surplus pups were necropsied and examined macroscopically.

The treatment period for the males and females of the first filial (F1) generation started after weaning on post-natal day (PND) 22 and persisted to an appropriate week of necropsy in accordance of assignment of animals into the appropriate cohort.

 

Examinations

The health condition, clinical status after application, body weight and food consumption of males and females of both generations were monitored during the study. Detailed clinical observation was carried out weekly in P males and females during 10-week pre-mating period. Vaginal smears were prepared daily during mating (until presence of spermatozoa) in P generation. Then in females of F1 generation cohort 1A, the vaginal smears for checking of oestrous cyclicity were prepared daily from onset of vaginal patency until first oestrus and daily for two weeks (from PND 75). During the lactation period, the parameters relevant to the pups (number of pups, body weight, sex, vitality and development) were monitored and evaluated. The age and body weight at vaginal opening and preputial separation were determined for F1 offspring.

After the end of administration period P animals and F1 animals, haematological and biochemical examinations in selected animals were performed, animals were sacrificed and gross necropsy was performed. In P and F1-1A males the following sperm parameters were examined: sperm motility and sperm morphology. The selected organs were removed for weighing and histopathological examination.

The assessment of neurotoxicity was performed in animals of cohort F1-2A. The auditory startle test at PND 25 and a functional investigation was evaluated before necropsy. Neurohistopathology of brain after perfusion fixation was performed in animals of cohort 2A. Animals selected for perfusion were anesthetized and cannulated via the left ventricle of the hearth.

Examination of the brain for purposes of neurotoxicity assessment was performed in animals of cohort 2B at weaning.

 

 

Results and Conclusions

The test item, Ethylcentralit, was assessed for induction of systemic toxicity, reproductive toxicity and developmental neurotoxicity in rats.

Negative effect of the test item on the health status of animals was observed in P females after first administration (only) of the test item. Negative effect manifested as Straub phenomenon was recorded. Straub reaction consisted of the tail becoming rigid and erected across the back of the animals in a S-shaped curve. This reaction was accompanied by restlessness, tremor and rapid breathing. This reaction was not observed in males and females of F1 generation.

 

Liver was probably primary target organ – the weight was increased dose dependently in P males, P females and F1 males. Concentration of liver enzymes (ALT, AST and ALP) was changed in treated P and F1 animals in comparison with control animals. No pathological findings were recorded during the histopathological examination of liver in treated parental and first filial generation of animals.

 

Ten weeks premating exposure duration for the parental (P) generation was ordered. The length of the premating exposure period was ten weeks to cover the full spermatogenesis and folliculogenesis before the mating. The administration of the test item did not affect reproductive organs and ability of reproduction. Spermatogenesis in the testes of the high dose administered P and F1 males was without any pathological findings. No treatment-related changes were found in the male genital tract. No treatment-related histological changes were found in the female genital tract. Ovarian follicle count in high dosed F1 females was similar with the control females. Reproductive indexes were unaffected in treated groups of females in comparison with the control group of females.

 

Across life stages, the EOGRTS has established that Ethylcentralit showed no evidence of adverse effect on reproduction.

 

Developmental neurotoxicity cohorts 2A and 2B were conducted for evaluation of potential neurotoxicity effect of the test item. Cohort 2A was subjected to an auditory startle test, functional observation battery and locomotor activity. There were no significant exposure-related effects on FOB (functional observational battery) parameters, ASR (auditory startle reflex). Also explorative behaviour and locomotor activity examined by the Open Field did not show significant differences between the treated and control animals. Detailed neurohistopathology of brain after perfusion fixation did not reveal a negative effect of the test item on brain. Brain histology was performed for cohort 2B animals and did not reveal a negative effect of the test item. 

 

Across life stages, the EOGRTS has established that Ethylcentralit showed no evidence of developmental neurotoxicity.

 

The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION of P animals and DEVELOPMENTAL NEUROTOXICITY was established as 400 mg/kg/day for males and females under the given study conditions.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
400 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The study is a GLP compliant and has Klimish score 1.
Additional information

The test item, Ethylcentralit, was tested for reproduction toxicity using the OECD Test Guideline No. 421 Reproduction/Developmental Toxicity Screening Test. With regard to the high toxicity of the test item for female, the application of the dose level higher than 450 mg/kg/day was impossible. Administration of the test item at the dose levels 450 mg/kg/day had no adverse effect to ability of male and female animals to successfully mate and produce viable offspring. Also development of pups was not changed in treatment groups.

Further the test item was tested using the OECD Test Guideline No. 443 Extended One-Generation Reproductive Toxicity Study and across life stages, the EOGRT study has established that Ethylcentralit showed no evidence of adverse effect on reproduction with NOAEL derived from the highest dose 400 mg/kg/day.

Effects on developmental toxicity

Link to relevant study records
Reference
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
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
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










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
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
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.
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.
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
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
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.
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
Abnormalities:
not specified
Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
Treatment related:
yes
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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The study is a GLP compliant and has Klimish score 1.
Additional information

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.

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.

Further the test item was tested using the OECD Test Guideline No. 443 Extended One-Generation Reproductive Toxicity Study. The NOAEL for REPRODUCTION of P animals and DEVELOPMENTAL NEUROTOXICITY was established as 400 mg/kg/day for males and females under the given study conditions.

Justification for classification or non-classification

Based on the available data, the substance is not classified.