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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:
2012-03-26 - 2012-05-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-conform OECD Guideline study.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA, Health Effects Test Guidelines; OPPTS 870.3650: Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Purity: 99.75 area%

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
- Age at supplied: 10-12 weeks (11 - 13 weeks old at the beginning of treatment)
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Weight at study initiation: Males: 298.3 g - 335.9 g, Females 213.1 g - 236.9 g
- Fasting period before study: no
- Housing: individually in Makrolon type M III cages;
- Exceptions:
- During overnight matings, male and female mating partners were housed together in Makrolon type M III cages
- Pregnant animals and their litters were housed together until PND 4 (end of lactation).
- The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
- Diet, ad libitum: Ground Kliba maintenance diet mouse-rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Water, ad libitum: drinking water
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24°C
- Humidity: 30-70%
- Air changes (per hr): 15
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES: From: 2012-04-02 To: 2012-05-20

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance solutions in drinking water were prepared at the beginning of the administration period and thereafter in intervals, which took into
account the analytical results of the stability verification.
For the preparation of the administration solutions the test substance was weighed in a graduated flask depending on the dose group, topped up
with drinking water and subsequently thoroughly mixed by shaking until it was completely dissolved.

VEHICLE
- Justification for use and choice of vehicle: solubility
- Concentration in vehicle:
50.00 mg/100 mL (5 mg/kg bw/d), 150.00 mg/100 mL (15 mg/kg bw/d), 500.00 mg/100 mL (50 mg/kg bw/d)
- Amount of vehicle: 10 mL/kg body weight
Details on mating procedure:
Pairing of F0 generation parental animals
In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group.
A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted "GD 0" and the following day "GD 1".
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the test substance in drinking water for a period of 7 days at room temperature were carried out in a comparable batch prior to the start of the study. Samples of the test substance solutions were sent to the analytical laboratory once at the beginning of the study for verification of the concentrations. The analytical results confirmed the stability and expected concentrations of the test substance solutions. Due to the fact that the test substance preparations were true solutions, the prove of homogeneity by analytical methods was not necessary.
Duration of treatment / exposure:
Males were exposed for 30 days (prior to mating, during mating, and up to termination) and females 49 days (prior to mating, during mating, during post-coitum, and at least 4 days of lactation).
Frequency of treatment:
Daily
Details on study schedule:
- Dose selection rationale: Dose levels were selected by request of the sponsor
- Route of administration: The oral route was selected since administration by gavage has been proven to be appropriate for the detection of a toxicological hazard.
Doses / concentrations
Remarks:
Doses / Concentrations:
5, 15 and 50 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
10 animals
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
MORTALITY / CAGE SIDE OBSERVATIONS: Yes
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity.
The littering and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams.
On weekdays (except public Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition
to the evaluations in the mornings.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high).
The following parameters were examined: abnormal behavior when handled, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmus, feces (appearance/consistency), urine, pupil size

BODY WEIGHT: Yes
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice.
The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female animals:
- During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
- Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
- Females without a litter and without positive evidence of sperm in the vaginal smear were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

FOOD CONSUMPTION:
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
- Food consumption was not determined after the second premating week (male parental
animals) and during the mating period (male and female F0 animals)
- Food consumption of the F0 females with evidence of sperm was determined on GD 0-7, 7-14, 14-20.
- Food consumption of F0 females, which gave birth to a litter, was determined for PND 1-4.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel).

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: In the morning towards the end of the administrative period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group
- Parameters examined: see Table 1 in 'Any other information on materials and methods incl. tables'.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: In the morning towards the end of the administrative period
- Animals fasted: Yes
- How many animals: first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group
- Parameters examined: see Table 2 in 'Any other information on materials and methods incl. tables'.

URINALYSIS: Yes
- Time schedule for collection of urine: overnight towards the end of the administrative period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: see Table 3 in 'Any other information on materials and methods incl. tables'.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Functional observational battery (FOB) was performed in five male and 5 female animals (with litter) per group at the end of the administration
period
- Dose groups that were examined: all animals
- Battery of functions tested:
Home cage observations:
Attention was paid to: Posture, Tremors, Convulsions, Abnormal movements, Impairment of gait.
Open field observations:
Behavior when removed from cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration,
tremors, convulsions, abnormal movements/stereotypy, impairment of gait, activity/arousal level, feces excreted within two minutes, urine excreted within two minutes, number of rearings within two minutes.
Sensorimotor tests/reflexes:
Approach response, touch response, vision ("visual placing response"), pupillary reflex, pinna reflex, audition ("startle response"), coordination of
movements ("righting response"), behavior during "handling", vocalization, pain perception ("tail pinch"), grip strength of forelimbs and hindlimbs,
landing foot-splay test, other findings.
Motor activity (MA) measured on the same day as the FOB was performed.
Oestrous cyclicity (parental animals):
No.
Sperm parameters (parental animals):
Parameters examined in male parental generation:
- testis weight
- epididymis weight
- stages of spermatogenesis
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- number and sex of pups
- stillbirths
- live births
- postnatal mortality
- presence of gross anomalies
- weight gain, physical or behavioural abnormalities

BODY WEIGHT
- Pups were weighed on the day after birth (PND 1) and on PND 4

CLINICAL OBSERVATIONS
- Live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no; pups were scheduled sacrifice on PND 4
Postmortem examinations (parental animals):
SACRIFICE /GROSS NECROPSY
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weights:
- The following weights were determined in all animals: Anesthetized animals, Epididymides, Testes.
- The following weights were determined in 5 animals/sex and test group (females with litters, same animals as used for clinical pathology
examinations): Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus
- The following organs/ tissues were preserved in 4% buffered formaldehyde or in modified Davidson’s solution: Adrenal glands, All gross lesions, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Eyes with optic nerve, Esophagus, Extraorbital lacrimal gland, Epididymides (modified Davidson’s solution), Femur with knee joint, Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (axillary and mesenteric), Mammary gland (male and female), Nose (nasal cavity), Ovaries (modified Davidson’s solution), Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate gland, Rectum, Salivary glands, (mandibular and sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Target organs,Testes (modified Davidson’s solution), Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina.

For further evaluation proceeding, see Table 4 in 'Any other information on materials and methods incl. tables'.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was scheduled sacrifice on PND 4 under isoflurane anesthesia with CO2.
- These animals were subjected to postmortem examinations as follows: All pups were examined externally and eviscerated; their organs were assessed macroscopically. All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically.
All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.

GROSS EXAMINATION OF DEAD PUPS: yes, Gross necropsy consisted of external examination
All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia with CO2.
All pups were examined externally and eviscerated; their organs were assessed macroscopically.
All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically.
All pups without notable findings or abnormalities were discarded after their macroscopic evaluation.
Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.

HISTOPATHOLOGY / ORGAN WEIGTHS
Not performed
Statistics:
Please refer to 'Any other information on materials and methods incl. tables'.
Reproductive indices:
Male reproduction data
For the males, mating and fertility indices were calculated:

Male mating index (%) = (number of males with confirmed mating* / number of males placed with females) x 100
*defined by a female with vaginal sperm or with implants in utero

Male fertility index (%) = (number of males proving their fertility* / number of males placed with females x 100
*defined by a female with implants in utero


Female reproduction and delivery data
The number of mating days until vaginal sperm were detected, and gestational status were recorded for F0 females.
For the females, mating, fertility and gestation indices were calculated:

Female mating index (%) = (number of females mated* / number of females placed with males) x 100
*defined as the number of females with vaginal sperm or with implants in utero

Female fertility index (%) = (number of females pregnant* / number of females mated**) x 100
*defined as the number of females with implants in utero
**defined as the number of females with vaginal sperm or with implants in utero

Gestation index (%) = (number of females with live pups on the day of birth / number of females pregnant*) x 100
*defined as the number of females with implants in utero
The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for
F1 litters.

Live birth index (%) = (number of liveborn pups at birth / total number of pups born) x 100

The implantations were counted* and the postimplantation loss (in %) was calculated.
Postimplantation loss (%) = ((number of implantations – number of pups delivered) / number of implantations) x 100

*) apparently non-pregnant uteri were stained according to the method of SALEWSKI (1964)
Offspring viability indices:
Pup number and status at delivery
All pups delivered from the F0 parents (F1 litter) were examined on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.

Pup viability/mortality:
The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1 – 4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal death were not included in thesecalculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4.

Viability index (%) = (number of live pups on day 4 after birth / number of live pups on the day of birth) x 100

Sex ratio:
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups was finally confirmed at necropsy. The sex ratio was calculated at day 0 and day 4 after birth.

Sex ratio = (number of live male or female pups on day 0/4 / number of live male and female pups on day 0/4) x 100

Results and discussion

Results: P0 (first parental animals)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (all groups)
Parental animals:
There were no test substance-related or spontaneous mortalities in any of the groups. No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any male or female animals during the whole study.
Detailed clinical observations: Male and female animals of all dose groups (50, 15 and 5 mg/kg bw/d) did not show any abnormalities.

BODY WEIGHT AND WEIGHT GAIN
High-dose females had statistically significantly lower body weights on PND 7 (about 7% below control) and statistically significantly lower body weight change during GD 7 – 14 (about 29% below control). Mean body weights and mean body weight change of the male animals in all test substance-treated groups were comparable to the concurrent control group during the entire study period. Mean body weights of the high-dose
females were comparable to the concurrent control group during premating and gestation period. Additionally, the mean body weight change of the high-dosed females was comparable to the concurrent control group during premating and lactation period. Neither mean body weights nor mean body weight change of the females in the mid and low-dose groups were influenced by the test substance during the whole treatment period.

FOOD CONSUMPTION AND COMPOUND INTAKE
Food consumption of the high-dose F0 females (50 mg/kg bw/d) was statistically significantly below control during GD 14 - 20 (about 13%). Food consumption of the male F0 rats in all dose groups (50, 15 and 5 mg/kg bw/d) was comparable to the concurrent control throughout the entire study. Mid and low-dose females (15 and 5 mg/kg bw/d) did not show any test substance-related changes in food consumption during the whole treatment period.

HAEMATOLOGY
In high dose male rats (50 mg/kg bw/d) total white blood cell (WBC) counts were decreased, which was due to a decrease of the absolute cell counts of lymphocytes, monocytes and eosinophils.

CLINICAL CHEMISTRY
No treatment-related, adverse changes among clinical chemistry parameters were observed.

URINALYSIS
No treatment-related, adverse changes among urinalysis parameters were observed.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS) –

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS) --

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No alterations to reproductive performance or fertility were identified at any dose in the parental rats. However, two sperm positive high-dose females (50 mg/kg bw/d), one sperm positive mid-dose female (15 mg/kg bw/d) and one sperm positive low-dose female (5 mg/kg bw/d) did not deliver F1 pups.

ORGAN WEIGHTS
The liver weight increase and the thymus weight decrease in males of high dose group (50 mg/kg bw/d) were considered to be treatment-related.

GROSS PATHOLOGY
In 5 out of 10 males of the high dose group (50 mg/kg bw/d), the thymus was found to be reduced in size. All other gross findings noted at necropsy are regarded as incidental and spontaneous in nature and are not related to treatment.
Fertility: The five female animals, which were not pregnant as well as the male mating partners did not show relevant gross lesions.

HISTOPATHOLOGY
In 9 out of 10 males of the high dose group (50 mg/kg bw/d), the thymus showed a reduced cellularity in the cortex and medulla. This change ranged from minimal to severe and was characterized by a decrease of the cellular density in the cortex and medulla and loss of the corticomedullary demarcation.
No histopathological correlate was found for the relative liver weight increase in males of the high dose group (50 mg/kg bw/d).
All other findings were either single observations, or were biologically equally distributed between controls and treated rats. All of them were considered to be incidental and/or spontaneous in origin.
Fertility: No histopathological findings were observed in 3 out of 5 non-pregnant females and their corresponding mating males that can explain the lack of offspring. In test groups 1 and 2 (5 and 15 mg/kg bw/d), no histopathology was performed in the other non-pregnant females and their respective mating males.

NEUROBEHAVIOUR
Home cage observations: No test substance-related effects were observed.
Open field observations: No test substance-related effects were observed.
Sensorimotor tests/reflexes: No test substance-related effects were observed.
Quantitative Parameters: No test substance-related effects were observed.
Motor activity measurement: There were no significant deviations concerning the overall motor activity (summation of all intervals) and regarding the single intervals, the statistically significantly decreased number of interrupted beams in mid-dose males (15 mg/kg bw/d) in one interval was considered spontaneous in nature and not treatment related.

OTHER FINDINGS (PARENTAL ANIMALS)
Findings which occurred either individually or were biologically equally distributed over control and treatment groups, were considered to be incidental or spontaneous in origin and without any relation to treatment.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
parental systemic toxicity
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Lymphopenia and/or thymus pathology at 50 mg/kg bw/d
Dose descriptor:
NOAEL
Remarks:
parental systemic toxicity
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Changes to body weight parameters at 50 mg/kg bw/d dose in females.
Dose descriptor:
NOAEL
Remarks:
parental reproductive toxicity - fertility
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No alterations to reproductive performance or fertility were identified at any dose

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
The viability index indicating pup mortality during lactation (PND 0 - 4) varied between 98.2% (50 mg/kg bw/d dose), 99.2 (15 mg/kg bw/d dose), 98.6 (5 mg/kg bw/d dose) and 99.1% (control) without showing any association to the treatment.

CLINICAL SIGNS (OFFSPRING)
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.

BODY WEIGHT (OFFSPRING)
The pup body weights were reduced at the 50 mg/kg bw/d dose. Both male and female pup body weights were approximately 12-13% low on PND 1, a difference which became more evident by PND 4 when the body weights were nearly 22% below controls. Pup body weight gain was similarly decreased by as much as a third over this period. The reduction in these body weight parameters was substantial enough to be considered to be a general sign of developmental toxicity. No differences in pup body weight parameters were noted at either the low- (5 mg/kg bw/d) or mid-doses (15 mg/kg bw/d). Neither were any specific substance-dependent developmental toxic effects identified at any dose.

GROSS PATHOLOGY (OFFSPRING)
A few pups showed spontaneous findings at gross necropsy, such as hydroureter, reddish discolored testis, hydronephrosis and post mortem autolysis. All these findings were not considered to be associated to the test substance.

OTHER FINDINGS (OFFSPRING)
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
15 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reduced postnatal offspring weight and weight gain at 50 mg/kg bw/d

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion