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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline 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 oral 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.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
males: 35 d, females 49/53 d (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
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: d35 (males)/ d49 (females)
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes
- How many animals: 5/sex
- Parameters: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes, Prothrombin time (Hepato Quick’s test, HQT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: d35 (males)/ d49 (females)
- Animals fasted: Yes
- How many animals: 5/sex
- Parameters: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), gamma-Glutamyltransferase (GGT), Sodium, Potassium, Chloride, Inorganic phosphate, Calcium, Urea, Creatinine, Glucose, Total bilirubin, Total protein, Albumin, Globulins, Triglycerides, Cholesterol, Magnesium


URINALYSIS: Yes
- Time schedule for collection of urine: d30 (males)/ d46 (females)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters: pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color, Turbidity, Volume

NEUROBEHAVIOURAL EXAMINATION: Yes
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.

Functional observation battery (FOB)
A functional observational battery was performed in five parental males and females (with litter) per group at the end of the administration period starting at about 10.00 h. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.

Home cage observations
The animals were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to: Posture, Tremors, Convulsions, Abnormal movements, Impairment of gait, Other findings.
Open field observations
The animals were transferred to a standard arena (50 x 50 cm wide, with side heights of 25 cm) and observed for at least 2 minutes. The following parameters were examined:
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 during the observation period, Urine excreted during the observation period, Number of rearings.

Sensory motor tests/Reflexes:
The animals were removed from the open field and subjected to following sensory motor or reflex tests: 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”), Other findings, Grip strength of forelimbs and hindlimbs, Landing foot-splay test.

Motor activity measurement (MA)
The MA was measured on the same day as FOB was performed in 5 parental males and females (with litter) per group. The examinations were performed using the Multi-Varimex system supplied by Columbus Instruments Int. Corp., Ohio, U.S.A. For this purpose, the animals were placed in cages for the time of measurement. Four beams were allocated per cage. The number of beam interrupts was counted over 12 intervals for 5 minutes in each case. The sequence at which the animals were placed in the cages was selected at random. The measurement was started at about 14.00 h. On account of the measuring variant "staggered", the starting time was varied by the time needed to place the animals in the cages. For each animal, measurement was started individually when the 1st beam was interrupted and ended exactly 1 hour later. The animals received no food or water during the measurements. After the transfer of the last animal in each case, the room where the measurements were carried out was darkened.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see also table below)
HISTOPATHOLOGY: Yes (see also table below)
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.
Statistics:
see tables below

Results and discussion

Results of examinations

Details on results:
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 animal 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.

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, which was considered as effect out of biological relevance due to missing dose-relationsship and the early appearance.

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.

HAEMATOLOGY
No treatment-related changes among hematological parameters were measured.

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

URINALYSIS
No treatment-related changes among urinalysis parameters were measured.

NEUROBEHAVIOUR
Home cage observations
No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.

Open field observations
The open field observations did not reveal any test substance-related findings in male and female animals of all test groups.

Sensorimotor tests/reflexes
There were no test substance-related findings in male and female animals of all test groups. Any deviations from "zero values" were equally distributed between test substance-treated groups and controls or occurred in single animals only. Therefore, these observations were considered as being incidental.

Quantitative Parameters
No test substance-related impaired parameters were observed in male and female animals of all test groups.

Motor activity measurement (MA)
No statistically significant changes in motor activity data were observed in the male and female animals of all dose groups in comparison to the concurrent control group.

Effect levels

Dose descriptor:
NOAEL
Remarks:
systemic and local
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

Target system / organ toxicity

Critical 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 the test item had no adverse effects on the F0 parental animals of both sexes at 100, 300 and 1000 mg/kg bw/d.

No general systemic toxicity was noted in the F0 parental animals at all dose levels tested. The test item 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 biochemical and urine parameters, 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