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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:
2013-03-28 until 2013-07-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
, adopted 1995
Deviations:
no
Qualifier:
according to
Guideline:
other: OPPTS 870.3550 Reproduction/Developmental Toxicity Screening Test, EPA Health Effects Test Guidelines, July 2000.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid

Test animals

Species:
rat
Strain:
other: Hsd.Brl.Han:Wist
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Toxi-Coop Zrt. Cserkesz u. 90., H-1103 Budapest, Hungary
- Age at study initiation:
Male animals: 84- 89 days old
Female animals: 84 - 89 days old
- Weight at study initiation:
Male animals: 331 - 392 g
Female animals: 190 - 216 g
- Housing: Type II polypropylene/polycarbonate
Size: 22 x 32 x 19 cm (width x length x height)
Supplier: Charles River Europe
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female / cage
Pregnant females were housed individually
Males after mating: individually
- Diet:
Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany ad libitum.
- Water:
Tap water from municipal supply, as for human consumption from a 500 mL bottle ad libitum.
- Acclimation period:
19 days

ENVIRONMENTAL CONDITIONS
- Temperature:
22 ± 3 °C
- Humidity:
30-70 %
- Air changes (per hr):
8-12
- Photoperiod:
12 hours daily, from 6.00 a.m. to 6.00 p.m.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5 % aqueous methylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in aqueous methylcellulose at concentrations of 20 mg/mL, 60 mg/mL and 200 mg/mL. Formulations were prepared in the laboratory of Test Facility 2-3 days before the use. Analytical control of concentration of dosing solutions was performed in the laboratory of the Test Facility. The measured concentrations varied in the acceptance range in comparison to the nominal values at all analytical occasions.

VEHICLE
- Justification for use and choice of vehicle:
The test item is not soluble in water; therefore aqueous methylcellulose was used for preparing formulations appropriate for oral administration. 0.5 % aqueous methylcellulose was a suitable vehicle to facilitate formulation analysis for the test item.

- Amount of vehicle:
A constant treatment volume of 5 mL dose preparation/kg body weight was administered in all groups. The individual volume of the treatment based on the most recent individual body weight of the animals. In the first week of the pre-mating period, animals received volumes based on the actual body weight on day 0.
Details on mating procedure:
Mating was started 2 weeks after the initiation of treatment. One female and one male of the same dose group (1:1 mating) were placed in a single cage. Females remained with the same male during 14 days. Pairs were changed within a dose group thereafter; females were paired with not mated males then with proven males, if it was necessary.
Each morning a vaginal smear was prepared and stained with 1 % aqueous methylene blue solution. The smear was examined with a light microscope. The presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 421). Sperm positive females were caged individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. Concentration of the test item in the dosing formulations was checked twice during the study. CCPIB concentrations in the dosing formulations varied in the range of 93 and 115 % of the nominal values at both analytical occasions, thereby confirming proper dosing.
Duration of treatment / exposure:
The test item was administered in a single dose by oral gavage (stomach tube) on a 7 days/week basis, every day at a similar time. Control animals were treated concurrently with the vehicle only. Animals were not treated on the day of gross pathology.
Dosing of both sexes began after acclimatization and two weeks before mating and was continued up to and including the day before the necropsy. Rats of this strain have already reached full sexual maturity at the age of 12 weeks.
Male animals were dosed for 43 days and then they were subjected to necropsy one day after the last treatment.
Female animals were dosed for 14 days pre-mating, during mating period, through gestation and up to lactation days 3 - 4 [for 41 - 45 days, depending on day of mating (mating days 1 - 6)]. The day of delivery (viz. when parturition was complete) was defined as day 0 post-partum.
Non-pregnant female animals were treated up to and including the day before necropsy (for 41 or 43 days).
Control animals were handled in an identical manner to the test groups receiving 5 mL vehicle/kg bw. O
Frequency of treatment:
Animals were treated once per day.
Details on study schedule:
Male animals were dosed for 43 days and then they were subjected to necropsy one day after the last treatment.
Female animals were dosed for 14 days pre-mating, during mating period, through gestation and up to lactation days 3, 4 or 5 (for 41 – 45 days, depending on date of mating). The day of delivery (viz. when parturition was complete) was defined as day 0 post-partum. Non-pregnant and not mated female animals were treated up to and including the day before necropsy (for 41 or 43days).
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12 animals/sex in the control and dose groups
Control animals:
yes, concurrent vehicle
Details on study design:
The dose setting with 1000, 300 and 100 mg/kg bw/day based on findings obtained in a previous 28-day oral gavage dose range finding study with CCPIB in the Rat
Positive control:
Not applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).
General clinical observations were made once a day, after treatment at approximately the same time.

DETAILED CLINICAL OBSERVATIONS: Yes
More detailed examinations were made at the times of weekly weighing, prior to and during the mating until necropsy. Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behaviour pattern, changes in gait, posture and response to handling.

BODY WEIGHT: Yes
All parent animals were weighed with an accuracy of 1 g. Parent male animals were weighed on the first day of dosing (day 0), weekly thereafter and on the day of necropsy.
Parent females were weighed on the first day of dosing (day 0) then weekly, on gestation days 0, 7, 14 and 20 and on postpartal days 0 (within 24 hours after parturition) and 4, as well as on the day of necropsy. Body weight of the female animals was weighed on gestational days 10 and 17 in order to give accurate treatment volumes, but these data were not evaluated statistically.
Oestrous cyclicity (parental animals):
Mating was started 2 weeks after the initiation of treatment. One female and one male of the same dose group (1:1 mating) were placed in a single cage. Females remained with the same male during 14 days. Pairs were changed within a dose group thereafter; females were paired with not mated males then with proven males, if it was necessary.
Each morning a vaginal smear was prepared and stained with 1 % aqueous methylene blue solution. The smear was examined with a light microscope. The presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 421). Sperm positive females were caged individually.
Sperm parameters (parental animals):
Parameters examined in male parental generations:
Detailed histological examination was performed on the testes and epididymides of the animals in the control and high dose groups. For testes and epididymides, examinations were performed with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Litter observations:
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), and the presence of gross abnormalities.
Live pups were counted, sexed and weighed within 24 hours of parturition (on the day when parturition was complete) and day 4 post-partum with an accuracy of 0.01 g.
In addition to the observations on parent animals, any abnormal behaviour of the offspring was observed.
All the litters were checked and recorded daily for the number of viable and dead pups. The dead pups found were subjected to necropsy by a macroscopic examination. On day 0 of lactation, a lung flotation test was performed on all pups found dead to separate stillborns from those that died after delivery. The lung flotation test is negative for stillborns (pups that died intrauterine) but positive for pups that died after delivery.
Postmortem examinations (parental animals):
Gross necropsy was performed on each animal one day after the last treatment. Animals were anesthetized by Isoflurane and then were exsanguinated.
After examination of the external appearance the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed, and any abnormality was recorded with details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded.
The uterus with cervix, vagina, testes, epididymides, prostate, and seminal vesicles with coagulating glands, ovaries, pituitary of all adult animals were preserved. Testes and epididymides were preserved in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution.

Postmortem examinations (offspring):
GROSS NECROPSY
Parameters listed below were evaluated.
Litter weight on postnatal days 0 and 4
Mean body weight gain per litter between postnatal days 0-4
Number of live births per litter, and number of viable pups per litter on postnatal days 0 and 4
Survival Index of pups on postnatal day 4
Sex ratio % (on postnatal days 0 and 4)


Statistics:
The statistical evaluation of appropriate data were performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible. The frequency of clinical signs, pathology and histopathology findings were calculated.
Results were evaluated in comparison with values of control group (i.e. control value).
Reproductive indices:
The following reproductive indices were calculated: Male mating index, female mating index, male fertility index, female fertility index, gestation index. The formulas for calculation can be found below in "Any other information on materials and methods incl. tables"

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

Mortality
There was no mortality in any group of this study during the course of study.

Clinical Observations
Daily Observations
There were no toxic signs related to the test item effect at the daily clinical observations. The behavior and physical condition of animals were considered to be normal at each dose level (1000, 300 and 100 mg/kg bw/day) during the entire observation period.
Alopecia was observed on some male and female animals as follows:
Male: 1/12 at 1000 mg/kg bw/day, 2/12 at 300 mg/kg bw/day, 1/12 in the control group between Days 21 and 42, between Days 24-42 and 20-26, as well as between Days 34-42, respectively.
Dams: 1/10 at 1000 mg/kg bw/day and 1/10 in the control group, from gestation day 10 to lactation day 3 and from gestation day 7 to lactation day 4, respectively.
Alopecia is a common finding in this strain of experimental rats and is seen also in untreated rats. Therefore, this isolated finding was considered to be independent from treatment.

Detailed Weekly Observations
The behavior and physical condition of animals was not affected by the test item at any dose level (1000, 300 or 100 mg/kg bw/day) based on the weekly detailed clinical observations during the entire treatment period.
Alopecia as described above was detected in male and female animal at the detailed weekly observations.

Body Weight
The body weight development was unaffected at any treatment group (male and female, 1000, 300 and 100 mg/kg bw/day).
The mean body weight gain of male animals in 300 mg/kg bw/day group was slightly but statistically significantly less with respect to the control during week 2. This finding is considered to be indicative of biological variation and not related to the test item treatment as similar findings were not detected for the animals of the high dose group.
The mean body weight and body weight gain were similar to that of the control group in male and female animals of 1000 and 100 mg/kg bw/day groups during the course of entire treatment period (pre-mating, mating and post mating period in male animals and pre-mating, mating, gestation and lactation periods in female animals.


Food Consumption
There were no test item or treatment related changes in the food consumption of male and female animals at any dose level (1000, 300 and 100 mg/kg bw/day).
Compared to the control group, statistical significances were detected at the slightly higher mean daily food consumption of male animals of the 1000 and 300 mg/kg bw/day groups during the first week of the post-mating period (between days 20 and 27).
The mean daily food consumption was similar to that of the control group in male animals of 100 mg/kg bw/day group and in female animals of 1000, 300 and 100 mg/kg bw/day groups during the course of entire treatment period.

Delivery Data of Dams
The delivery data of dams were not affected by the treatment with the test item at 1000, 300 or 100 mg/kg bw/day doses.
The mean number of corpora lutea, implantation sites, pre-implantation loss, post-implantation loss and total intrauterine mortality were similar in the control and all test item treated groups.
There were no significant differences between the control and test item treated groups in the mean duration of pregnancy, in the mean number of total births, live-borns, stillborns or viable pups.

Reproductive Performance
The examined parameters of reproductive performance were not affected by the treatment with the test item at 1000, 300 or 100 mg/kg bw/day doses in male or female animals.
Compared to the concurrent control groups, statistical significance was noted for the slightly reduced fertility index of male and female animals of 300 and 100 mg/kg bw/day groups, which included also a less percentage of fertile animals and a higher percentage of infertile animals.
Statistical significances were noted for the slightly less percentage of pregnant females and for the slightly higher percentage of non-pregnant females in the 300 and 100 mg/kg bw/day groups comparing to the control group. The percentage of dams delivered and the gestation index of 300 and 100 mg/kg bw/day groups exceeded these values of the control group.
However these slight, but statistically significant differences in the fertility indices, in the percentage of dams delivered, pregnant and non-pregnant females, and gestation index with respect to the control were considered to be indicative of biological variation and not related to the test item treatment. These differences were not found to be dose-dependent and similar findings were not detected for the animals of the high dose group.

Necropsy
No test item related alterations were found at the macroscopic observations of organs or tissues in male or female animals at any dose level (1000, 300 and 100 mg/kg bw/day) at the necropsy.
Alopecia was noted for single male animal in the 1000, 300 mg/kg bw/day and control groups (1/12, each) and in single dam in the 1000 mg/kg bw/day and control groups (1/10, both) at the necropsy observations.
Early death was observed in the uterine horns in some dams of 1000 mg/kg bw/day (1/10), 100 mg/kg bw/day (4/8) and in the control (1/10) groups. In non-pregnant female animals, hydrometra was detected in 300 and 100 mg/kg bw/day groups (1/4 and 2/4 respectively).
These isolated findings were considered to be individual alterations without any toxicological relevance.

Organ Weight
The absolute and relative organ weights of brain, testes and epididymides did not demonstrate any test item related alterations.
There were no significant differences between the control and test item treated groups in the examined organ weights (brain, testes and epididymides).

Histopathology

Histological examination of male and female genital organs (testes, epididymides and ovaries) did not reveal any toxic or other test item related alterations at the highest dose of 1000 mg/kg/bw/day.
The investigated organs of reproductive system (testes, epididymides) were histologically normal in all animals of the high dose and in all male animals which did not fertilize females in 300 and 100 mg/kg bw/day groups. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa), representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically. Compared to the concurrent control, epididymides were histologically normal in all animals.
The ovaries had a normal structure characteristic for the species, age and phase of sexual cycle in all examined female animals of 1000 mg/kg bw/day group and non-pregnant animals of 300 and 100 mg/kg bw/day groups. The cortex contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes and ovulation. The epithelial capsule and ovarian stroma were normal in all cases as well. In one male animal of 1000 mg/kg bw/day group and in one control female animal lack of hair in the intact hair follicles was noted. In the skin tissues, there were no inflammation, necrosis, fungal or parasitic bodies and it was considered to be an individual disorder.









Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Overall effects

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

Mortality
There was no test item related effect on pup’s mortality.
The mean number of live births per litter, the mean number of viable pups per litter on postnatal days 0 and 4 were similar in all groups. There were no differences between the control and test item treated (1000, 300 or 100 mg/kg bw/day) groups in the survival indices.

Sex Distribution
There were no test item related differences between the control and test item treated groups in the ration or in the litter means of genders on postnatal days 0 or 4.

Clinical Observations
Test item related clinical signs were not detected in the offspring.
The number and percentage of pups with findings (cold, no milk in the stomach) were the highest in the control group.

Body Weight
Test item effect on the body weight development of offspring was not found.
The mean litter weights and mean pup weights were similar in the control and in all test item treated groups on postnatal days 0 and 4.

Necropsy
No test item related macroscopic alterations were found in offspring subjected to gross pathological examination.






Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Overall effects

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The reprotoxic properties of the test item were assessed in a study performed according to OECD Guideline 421 in rats. Based on the results obtained from testing the parental NOAEL , the NOAEL for reproductive performance and the NOAEL for the F1 generation were determined to be 1000 mg/kg bw/day.
Executive summary:

The purpose of this reproduction/developmental toxicity screening test was to provide initial information concerning the effect of the test item CCPIB on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 4 post-partum associated with oral administration to rats at repeated doses. Four groups of Hsd.Brl.Han:Wist rats (n=12/sex/group) were administered orally (by gavage) once a day at 0 (vehicle only), 1000, 300 and 100 mg/kg bw/day at concentrations of 200 mg/mL, 60 and 20 mg/mL corresponding to 5 mL/kg bw dose volume. Control animals were administered with 0.5 % aqueous methylcellulose. The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. Concentration of the test item in the dosing formulations was checked twice during the study. CCPIB concentrations in the dosing formulations varied in the range of 93 and 115 % of the nominal values at both analytical occasions, thereby confirming proper dosing. All animals of the parent (P) generation received test item or vehicle prior to mating (14 days) and throughout mating. Test item or vehicle was administered to male animals post mating up to the day before the necropsy (altogether for 43 days). For females, test item was administered through the gestation period and up to lactation days 3-4 i.e. up to the day before the necropsy (altogether for 41 - 45 days).

Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. The dams were allowed to litter, and rear their young up to day 4 postpartum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on postnatal day 4. All parental animals were subjected to gross pathology one day after the last treatment. Histopathology examination was performed on reproductive organs (testes, epididymides and ovaries) in the control and high dose groups. The reproductive organs of non-pregnant females and males cohabited with in the low and mid dose groups were also processed and evaluated histologically. The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with the vehicle (0.5% methylcellulose) only.

Results

Mortality

There was no test item related mortality at any dose level (1000, 300 and 100 mg/kg bw/day).

Clinical observation

The behavior and physical condition of animals were normal during the entire observation period (pre-mating and post mating for male animals, during pre-mating for dams and non-pregnant females, during gestation and lactation periods for dams).

Body weight and body weight gain

The body weight development was undisturbed in the test item treated animals at each dose level (1000, 300 and 100 mg/kg bw/day) during the entire treatment period (pre-mating, mating, post-mating, gestation and lactation periods).

Food consumption

The mean daily food consumption was not affected by the test item in male or female animals at 1000, 300 and 100 mg/kg bw/day during the entire study (pre-mating, post-mating, gestation and lactation periods).

Reproduction

There were no test item related differences between the control and test item treated groups in delivery data of dams and in the reproductive performance of male and female animals.

Necropsy

Specific macroscopic alterations related to the test item were not found during the necropsy.

Organ weight

There were no test item related changes in brain, testes and epididymides weights.

Histopathology

Histopathological examinations of male and female genital organs (ovaries, testes and epididymides) did not reveal any toxic or other test item related changes at any dose level.

Offspring

Negative effects of the test item on offspring development (mortality, clinical signs, and body weight and necropsy findings) were not detected between postnatal days 0 and 4.

Conclusion

Under the conditions of the present study CCPIB did not cause toxic changes and did not influence the reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) in parental male and female Hsd.Brl.Han: Wistar rats or development of the F1 offspring from conception to day 4 post-partum after repeated dose oral administration at 1000, 300 or 100 mg/kg bw/day.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for male rats: 1000 mg/kg bw/day

NOAEL for female rats: 1000 mg/kg bw/day

NOAEL for reproductive performance of the male rats: 1000 mg/kg bw/day

NOAEL for reproductive performance of the female rats: 1000 mg/kg bw/day

NOAEL for F1 Offspring: 1000 mg/kg bw/day