Registration Dossier

Administrative data

screening for reproductive / developmental toxicity
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD guideline compliant study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
according to
other: EPA, Health Effects Test Guidelines; OPPTS 870.3550: Reproduction/Developmental Toxicity Screening Test (Jul 2000)
GLP compliance:
yes (incl. certificate)
Limit test:

Test material

Details on test material:
- Substance type: orange solid
- Physical state: soild

Test animals

Details on test animals and environmental conditions:
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany
- Age at study initiation: 11-12 weeks
- Weight at study initiation: (P) Males: 328.9 g - 357.4 g; Females: 192.4 g - 218.3 g
- Housing: individually in Makrolon type M III cages (floor area of about 800 cm²), with the following 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).
- Diet: ground Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: ad libitum from water bottles
- Acclimation period: 6 days

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

IN-LIFE DATES: From: 07 Oct 2014 To: 09 Dec 2014

Administration / exposure

Route of administration:
oral: gavage
other: drinking water containing 1 % Carboxymethylcellulose
Details on exposure:
The test article was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, the vehicle was filled up to the desired volume, subsequently released with a magnetic stirrer.

- Concentration in vehicle: 1, 3 and 10 g/100 ml
- Amount of vehicle (if gavage): 10 ml /kg bw
Details on mating procedure:
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 test group.
The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 07.00-09.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. 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 was detected was denoted gestation day (GD) 0 and the following day "GD 1".
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the test substance in in drinking water at room temperature for a period of 7 days were carried out prior to the start of the study.Samples of the test substance preparations were sent to the analytical laboratory once during the study period for verification of the concentration. The samples, which were taken for the concentration control analysis at the beginning of the administration period, were also used to verify the homogeneity of the samples of the low- and high-concentration. From these concentrations three samples (one from the top, middle and bottom in each case) were taken from the beaker with a magnetic stirrer running.
Duration of treatment / exposure:
The duration of treatment covered a 2 week premating period and mating in both sexes (mating pairs were from the same dose group) as well as entire gestation and 4 days of lactation period in females up to one day prior to the day of schedule sacrifice of the animals.
Frequency of treatment:
daily (exception: no administration to animals being in labor)
Doses / concentrations
Doses / Concentrations:
100, 300 and 1000 mg/kg bw
actual ingested
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Absence of adverse effects in the existing OECD 407 study
- Rationale for animal assignment: distributed randomly according to weight among the individual test groups, separated by sex


Parental animals: Observations and examinations:
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each affected animal.
The littering and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis.
On weekdays (except public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.
The day of littering was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.

Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).
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 or waiting for necropsy were weighed weekly. These body weight data were solely used for the calculations of the dose volume. Therefore, these values are only documented in the Individual Tables.

Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
• Food consumption was not determined 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 and 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) and in males after the premating period.

Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

The eyes of all male animals were examined prior to the start and at the end of the administration period using an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after administration of a mydriatic agent (Mydrum, Chauvin ankerpharm GmbH, Rudolstadt, Germany).

In a previously performed repeated-dose 28-day toxicity study in Wistar rats, a (multi)focal to extensive inflammation was observed in the lungs of all high-dose animals (1000 mg/kg bw/d). This was regarded to be caused by the test substance and adverse in nature. Therefore, bronchoalveolar lavage fluid examinations were carried out in the second 5 animals of each test group.
The animals designated for lung lavage were killed by exsanguination from aorta abdominalis and vena cava under Narcoren® anesthesia. The lung was lavaged by two instillations of physiologic saline. Cytology was examined in bronchoalveolar lavage fluid (BAL). Total cell counts were determined using a haematology analyzer (Advia 120 Siemens Diagnostics, Fernwald, Germany). Cytocentrifuge preparations were stained according to Wright and evaluated microscopically. The following parameters were examined: Total cell count (BALTCN), Macrophages (BALMPH), Polymorphonuclear neutrophils (BALPMN), Lymphocytes (BALLY), Eosinophils (BALEO), Monocytes (BALMO), Atypical cells (BALATY).
Oestrous cyclicity (parental animals):
no data
Sperm parameters (parental animals):
Epididymides weights, testes weights, staging of spermatogenesis
Litter observations:
Pup number and status at delivery:
All pups delivered from the F0 parents (F1 litter) were examined as soon as possible 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.

In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays.
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 these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4.

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.

Clinical observations:
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.

Pup body weight data:
The pups were weighed on the day after birth (PND 1) and on PND 4. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning). In the summary tables pup body weights and pup body weight change are listed for males, females and males + females.
“Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.
Postmortem examinations (parental animals):
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.

Organ weights:
Weight assessment was carried out on all animals. The following weights were determined:
Anesthetized animals, Epididymides, Testes

The following organs / tissues of all parental animals were preserved in neutral-buffered 4% formaldehyde or in modified Davidson’s solution:
All gross lesions, Cervix, Coagulating gland, Epididymides (modified Davidson’s solution), Ovaries (modified Davidson’s solution), Oviducts, Prostate gland, Seminal vesicles, Testes (modified Davidson’s solution), Vagina, Uterus.
After the organs were fixed, processing, examination by light microscopy and evaluation of findings were performed. The following tissues were examined: testes, epididymides, ovaries from all control and high dose animals.
Postmortem examinations (offspring):
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.
The following statistical methods were used:
• Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), gestation days: DUNNETT test (two-sided).
• Male and female mating indices, male and female fertility indices, females mated, females delivering, gestation index (females with liveborn pups), females with stillborn pups, females with all stillborn pups: FISHER'S EXACT test (one-sided).
• Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal loss, implantation sites, pups delivered, pups liveborn, live pups day x, viability Index: WILCOXON test (one-sided+) with BONFERRONI-HOLM adjustment.
• % live male day x, %live female day x: WILCOXON test (two-sided).
• Broncho-alveolar lavage fluid (BALF) and lung tissue parameters: WILCOXON-test (one-sided).
• Weight parameters: KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided).
Reproductive indices:
male and female mating index, male and female fertility index, gestation index, postimplantation loss
Offspring viability indices:
Live birth index, pup number and status at delivery, viability index

Results and discussion

Results: P0 (first parental generation)

Effect levels (P0)

Dose descriptor:
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: Absence of adverse effects

Results: F1 generation

Effect levels (F1)

Dose descriptor:
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: Absence of adverse effects

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion