Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 22 Mar 1996
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Golpanol ALS wasserfrei
- Physical state: Solid / white
- Analytical purity: 71.3%
- Impurities (identity and concentrations): 26% NaCl
- Lot/batch No.: 04749356P0
- Stability under test conditions: guaranteed by the sponsor
- Storage condition of test material: Room temperature; avoidance of temperatures >60°C

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Crl:WI(Han), supplied by Charles River Laboratories, Research Models and Services, Germany GmbH
- Age: ca. 12 weeks at study initiation
- Mean weight: males 279-345 g, females 180-205 g at study initiation
- Housing: individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (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).
• For motor activity (MA) measurements the animals were housed individually in polycarbonate cages with wire covers from Ehret, Emmendingen (floor area of about 800 cm2) and small amounts of bedding material.
- Diet: ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: Drinking water was supplied from water bottles, ad libitum
- Acclimation period: ca. 5 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
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. The maximum period for which each preparation was used was 7 days.
For the preparation of the administration solutions the test substance was weighed in a graduated measuring flask depending on the dose group, topped up with drinking water and subsequently thoroughly shaken until completely dissolved.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight
- Proof of pregnancy: sperm in vaginal smear referred to as gestation day 0 (GD0)
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
The duration of treatment covered a 2-week pre-mating and a mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation and approximately 1 week thereafter in females.
Frequency of treatment:
daily (females in labor were not treated)
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first administration and thereafter at weekly intervals
For observation, the animals were removed from their cages by the investigator and placed in a standard arena for at least 20 seconds/animal (50 x 37.5 cm wide, with side heights of 25 cm). The following parameters were assessed:
Abnormal behavior during “handling”, Fur, Skin, Posture, Salivation, Respiration, Activity/arousal level, Tremors, Convulsions, Abnormal movements, Gait abnormalities, Lacrimation, Palpebral closure, Exophthalmos, Assessment of the feces discharged during the examination (appearance/consistency), Assessment of the urine discharged during the examination, Pupil size.

BODY WEIGHT: Yes
- Time schedule for examinations: once a week at the same time of the day

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, generally once a week

OTHER
On study day 31, a functional observational battery and motor activity measurement were carried out in five male animals per group.
On study day 45, a functional observational battery and motor activity measurement was carried out in five female animals (with litter) per group.
Urinalysis were carried out from 5 male and 5 female animals (with litter) per group on study day 30 (males) and 46 (females). Clinicochemical and hematological examinations were carried out on study days 35 (males) and 49 (females).
Litter observations:
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams. If pups showed particular findings, these were documented with the dam concerned. The pups were weighed on the day after birth (PND 1) and on PND 4.
Postmortem examinations (parental animals):
All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

Organ weights
The following weights were determined in all animals sacrificed on schedule:
Anesthetized animals, Adrenal glands, Brain, Cauda epididymis, Epididymides, Heart, Kidneys, Liver, Ovaries, Pituitary gland, Prostate, Seminal vesicles with coagulation glands, Spleen, Testes, Thymus, Thyroid glands, Uterus with cervix.

Organ /tissue fixation
The following organs or tissues were fixed in 4% formaldehyde solution or in modified Davidson’s solution:
All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulation glands, Colon, Duodenum, Epididymides (modified Davidson’s solution), Esophagus, Eyes with optic nerve, Female mammary gland, Femur with knee joint, Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (mesenteric and axillary lymph nodes), Nose (nasal cavity), Ovaries (modified Davidson’s solution), Oviducts, Pancreas, Parathyroid glands, Pharynx, Prostate, Rectum, Salivary glands (mandibular and sublingual glands), Sciatic nerve, Seminal vesicle, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cords), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Testes (modified Davidson’s solution), Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina.

Histopathology
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings according to the table below.
Postmortem examinations (offspring):
All pups with scheduled sacrifice on PND 4 were sacrificed by means of CO2 under Isofluran anesthesia. All pups were examined externally and eviscerated; their organs were assessed macroscopically.
All pups without notable findings or abnormalities were discarded after their macroscopic evaluation.
Statistics:
see tables below
Reproductive indices:
Male mating index (%)
Male fertility index (%)
Female mating index (%)
Female fertility index (%)
Gestation index (%)
Live birth index (%)
Postimplantation loss (%)
Offspring viability indices:
Viability index (%)
Sex ratio

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed

Details on results (P0)

Mortality
There were no test substance-related or spontaneous mortalities in any of the groups.

Clinical observations
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any male or female F0 generation parental animals during the whole study including gestation and lactation periods.
Six high-dose males showed salivation after treatment (weeks 1 - 4). This transient salivation for a few minutes immediately after each treatment was likely to be induced by the unpleasant taste of the test substance or by local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity.
Two sperm positive control females did not deliver F1 pups.

Food consumption
Low-, mid- and high dose males and females did not show any test substance-related changes of food consumption during the whole study.

Body weight data
Body weights/ body weight gain of all treated males and females were not influenced by the treatment during the whole study.
This statement includes the statistically significantly decreased body weight change of the mid-dose males between weeks 0-1.

Detailed clinical observations (DCO)
Male and female animals of all dose groups (100, 300 and 1000 mg/kg bw/d) did not show any abnormalities.

Male reproduction data
For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all groups including the controls.
Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter.
Two control males did not generate F1 pups. No histomorphological correlate was found to explain these apparent infertilities.
Thus, the male fertility index ranged between 80% and 100% without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study.


Female reproduction and delivery data
The female mating index for F1 litter was 100% in all test groups.
The mean duration until sperm was detected (GD 0) varied between 2.1 and 3.6 days without any relation to dosing.
All sperm positive rats delivered pups or had implants in utero with the following exceptions:
2 control females did not become pregnant
The fertility index varied between 80% in control and 100% in test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d). These values reflect the normal range of biological variation inherent in the strain of rats used for this study.
One non-pregnant female control rat did not show macroscopically visible lesions to explain the apparent infertility. However, the fixed tissue of the uterus was damaged or incomplete and precluded further diagnosis. In the second non-pregnant female control rat a severe dilation of both uterine horns containing suppurative cloudy fluids indicated inflammatory processes in the uterus which most likely explained the infertility.
The mean duration of gestation was similar in all test groups (i.e. between 21.8 and 22.0 days). The gestation index was 100% in the control and all dose groups.
Implantation, prenatal development and delivery were not affected by the treatment since neither the mean number of implantation sites nor the postimplantation loss or the average litter size showed any statistically significant differences between the groups.
The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 99% (control and low-dose groups) or 100% (mid- and high-dose groups). Moreover, the number of stillborn pups was comparable between the groups.

Pathology
Concerning gross pathology as well as histopathology all findings occurred either singly or were biologically equally distributed over the control group and the treatment groups. They are considered to be incidental or spontaneous in origin and without any relation to treatment up to 1000 mg/kg bw/d.
---
No treatment-related changes among hematological parameters were measured.

No treatment-related changes among clinical chemistry parameters were measured.

No treatment-related changes among urinalysis parameters were measured.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not specified

Details on results (F1)

Pup number and status at delivery
The mean number of delivered F1 pups per dam and the rates of liveborn and stillborn F1 pups were evenly distributed about the groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.

Pup viability/mortality
The viability index indicating pup mortality during lactation (PND 0 - 4) varied between 99% (control and test group 2) and 100% (test groups 1 and 3) without showing any association to the treatment.

Sex ratio
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; slight differences were regarded to be spontaneous in nature.

Pup clinical observations
There were no test substance related adverse clinical signs observed in any of the F1 generation pups of the different test groups.

Pup body weight data
Pup body weights/body weight gain of the low-, mid- and high-dose pups were not influenced by the treatment. The number of "runts" was highest in the low-dose group.

Pup necropsy observations
One control and one low-dose F1 pup showed post mortem autolysis, a spontaneous finding, at gross necropsy. No malformations were observed, indications for embryotoxic/teratogenic effects were not noted.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
other: parental
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
other: see remarks on result
Sex:
male/female
Basis for effect level:
other: P0 data
Remarks on result:
other:
Remarks:
(i) the substance is of low toxicological activity (no evidence of toxicity seen in any of the tests available), (ii) it can be proven from toxicokinetic data that no systemic absorption occurs via relevant routes of exposure (e.g. plasma/blood concentrations below detection limit using a sensitive method and absence of the substance and of metabolites of the substance in urine, bile or exhaled air) and (iii) there is no or no significant human exposure (In accordance with column 2 of REACH Annex IX , studies for reproductive toxicity do not need to be conducted as a very low toxicological activity was documented in a combined study for repeated dose and reproductive/ developmental toxicity in doses up to the limit dose of 1000 mg/kg bw/day.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar Rats Golpanol ALS wasserfrei had no adverse effects on fertility of the F0 parental animals of both sexes at 100, 300 and 1000 mg/kg bw/d.

The test substance caused also no impairments of the reproductive performance. Mating behaviour, conception, gestation, parturition, as well as sexual organ weights and gross and histopathological findings of these organs were not influenced up to 1000 mg/kg bw/d.

No general systemic toxicity was noted in the F0 parental animals at all dose levels tested. Golpanol ALS wasserfrei caused no indications of test substance-induced effects in low-, mid- and high-dose rats regarding food consumption and body weights/ body weight gain as well as detailed clinical examinations in an open field, in a functional observational battery (FOB) and measurements of motor activity.

Concerning clinical and gross pathology as well as histopathology all findings occurred either singly or were biologically equally distributed over the control group and the treatment groups. They are considered to be incidental or spontaneous in origin and without any relation to treatment up to 1000 mg/kg bw/d.

Applicant's summary and conclusion