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Toxicological information

Toxicity to reproduction

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Administrative data

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:
other: GLP Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Centralit- Physical state: white crystalline powder- Analytical purity: 99.9% (w/w)- Expiration date of the lot/batch: 16.9.2016- Storage condition of test material: The sample was stored in ventilable room in original packaging, at the ambient temperature.

Test 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- 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 daysENVIRONMENTAL 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 cycleSTUDY TIME SCHEDULEDate of animal arrival: 4. 1. 2012Start of administration: 11. 1. 2012End of administration: males 3.2. 2012, females 4. 3. 2012Clinical examination: 4. 1. – 5. 3. 2012Necropsy and biometry of organs: males 08. 02. 2012, females 20. 2. – 5. 3. 2012

Administration / exposure

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
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:50 mg/kg bwBasis:actual ingested
Remarks:
Doses / Concentrations:150 mg/kg bwBasis:actual ingested
Remarks:
Doses / Concentrations:450 mg/kg bwBasis: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.

Examinations

Parental animals: Observations and examinations:
HEALTH CONDITION CONTROL: Yes- Time schedule: dailyDETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: dailyBODY 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 systemsHISTOPATHOLOGY- 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 vaginaORGAN 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 indexFemale mating indexMales fertility indexFemales fertility indexGestation index
Offspring viability indices:
Pre-implantation lossPost-implantation lossPost-natal lossSurvival index

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
effects observed, treatment-related

Effect levels (P0)

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.

Results: F1 generation

General toxicity (F1)

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

Effect levels (F1)

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.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.