Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25th November 2004 to 30th December 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was designed to comply with the following guideline: ⋅ EEC Directive No. 96/54, B7, 30th September 1996, and was based on the following guidelines for neurotoxicological investigations: ⋅ EEC Directive No. 2004/73, B43, 29 April 2004, ⋅ EPA, Guideline 799, 9620-62-158, 15 August 1997.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
yes
Remarks:
The samples for analysis of homogeneity immediately after preparation of the dosage forms were not taken with administration device used in the study, . parathyroid glands of two animals were not present on the histological slide of thyroids: - G23731 (1
Qualifier:
according to
Guideline:
EU Method B.43 (Neurotoxicity Study in Rodents)
Deviations:
yes
Remarks:
the samples for analysis of homogeneity immediately after preparation of the dosage forms were not taken with administration device used in the study, . parathyroid glands of two animals were not present on the histological slide of thyroids: - G23731 (1F
Qualifier:
according to
Guideline:
other: EPA, Guideline 799, 9620-62-158, 15 August 1997.
Deviations:
yes
Remarks:
The samples for analysis of homogeneity immediately after preparation of the dosage forms were not taken with administration device used in the study, . parathyroid glands of two animals were not present on the histological slide of thyroids: - G23731 (1
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Beige powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Number: 90 rats (45 males and 45 females) were received at CIT on 25 November 2004.
Strain and Sanitary status: Wistar rat, Rj: WI (IOPS Han).
Breeder: Janvier, Le Genest-Saint-Isle, France.
Age/Weight: on the first day of treatment, the animals were approximately 6 weeks old and had a mean body weight of 205 g (range: 192 g to 221 g) for the males and 178 g (range: 167 g to 191 g) for the females.
Receipt: on arrival, the animals were given a clinical examination to ensure that they were in good condition.
Acclimation: the animals were acclimated to the study conditions for a period of 6 days before the beginning of the treatment period. A larger
number of animals than necessary were acclimated to permit the selection and/or replacement of individuals.
Allocation to groups: during the acclimation period, the required number of animals (40 males and 40 females) were selected according to body
weight and clinical condition. They were then randomly allocated to groups (by sex), so that the average body weight of each group was
similar.
Identification: each animal was identified by an individual ear tattoo. At the beginning of the study, each animal received a unique CIT identity number.

From arrival at CIT, the animals were housed in a barriered rodent unit, under specific pathogen free (SPF) standard laboratory conditions.
The animal room conditions are set as follows:
. temperature : 22 ± 2°C
. relative humidity : 50 ± 20%
. light/dark cycle : 12h/12h (7:00 - 19:00)
. ventilation : approximately 12 cycles/hour of filtered, non-recycled air.
The corresponding instrumentation and equipment are checked and calibrated at regular intervals. The temperature and relative humidity are
recorded continuously and the records checked daily and filed.
The animal room was disinfected before the arrival of the animals and cleaned regularly thereafter.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
The vehicle was 0.5% carboxymethylcellulose aqueous solution in purified water.
The test item was administered as a suspension in the vehicle.
The test item was ground to a fine powder using a mortar and pestle, suspended in the vehicle in order to achieve the concentrations of 30, 90 and 200 mg/mL and then homogenized using a magnetic stirrer.
The test item dosage forms were prepared for use for up to 8 days according to satisfactory results of the stability assay performed in the present
study, and were stored at +4°C prior to use.

The dosage forms were administered by gavage using a plastic syringe fitted with a metal gavage tube, once a day, at approximately the same time.
The quantity of dosage form administered to each animal was adjusted according to the most recently recorded body weight.
A constant dosage-volume of 5 mL/kg/day was used.
Control animals (group 1) received the vehicle alone.
The dosage forms were stirred continuously throughout the dosing procedure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity:
The results of the analyses demonstrated the homogeneity of each dosage form analyzed (30 and 200 mg/mL) just after preparation and after 9 days storage at +4°C.
Furthermore, there was a satisfactory correspondence between the nominal and the measured concentrations of the test item in the vehicle.

Stability:
The results of the analyses demonstrated the satisfactory stability of the two dosage forms investigated (30 and 200 mg/mL) over a 9-day period at +4°C.

Concentration:
A satisfactory agreement was observed between the nominal and actual concentrations of the test item in the administered dosage forms since the
deviations from nominal concentration were in an acceptable range of ± 10%.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:

Basis:
other: The dosage forms were administered by gavage.
No. of animals per sex per dose:
Three treated groups of five male and five female Wistar rats received the test item, SR28043, by
gavage at the dose-levels of 150, 450 or 1000 mg/kg/day for 29 days. An additional group of
five males and five females received the vehicle alone (0.5% carboxymethylcellulose) under the
same experimental conditions and acted as a control group.
Control animals:
yes
Details on study design:
The dose-levels (150, 450 and 1000 mg/kg/day) were selected in agreement with the Sponsor on
the basis of the results of a 7-day range-finding toxicity study by oral route performed in the
same species (CIT/Study No. 28694 TSR).

Examinations

Observations and examinations performed and frequency:
Morbidity and mortality:
Each animal was checked for mortality or signs of morbidity at least twice a day during the treatment period, including weekends and public holidays.

General clinical observations:
Each animal was observed at least once a day, at approximately the same time, for the recording of clinical signs.

Detailed clinical examination (principal animals):
Detailed clinical observations were made on all animals outside the home cage, in a standard arena, once before the beginning of the treatment period and then once a week until the end of the study.
Observations included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and
autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to
handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self-mutilation, walking backwards) were also recorded.

Functional Observation Battery (FOB) (principal animals):
All animals were evaluated once at the end of the treatment period.
This included a detailed clinical examination, measurement of reactivity to manipulation or to different stimuli and motor activity.
The animals were randomized in order to ensure "blind" evaluation.
All animals were observed in the cage, in the hand and in the standard arena.
The following parameters were assessed and graded:
⋅ "touch escape" or ease of removal from the cage,
⋅ in the hand: fur appearance, salivation, lachrymation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or
mydriasis),
⋅ in the standard arena (2-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions,
gait, arousal (hypo- and hyper-activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia.
Then, the following parameter measurements, reflexes and responses were recorded:
⋅ touch response,
⋅ forelimb grip strength (qualitative approach),
⋅ pupillary reflex,
⋅ visual stimulus response,
⋅ auditory startle reflex,
⋅ tail pinch response,
⋅ righting reflex,
⋅ landing foot splay,
⋅ at the end of observation: rectal temperature.
Finally, motor activity of all animals was measured once by automated infra-red sensor equipment over a 60-minute period.

Body weight:
The body weight of each animal was recorded at least once before group allocation, on the first day of treatment, and then once a week until the end of the study.

Food consumption (principal animals):
The quantity of food consumed by the animals in each cage was recorded once a week until the end of the study.

Monitoring of estrus cycle (principal animals):
The estrous cycle stage of all principal females was determined from a fresh vaginal lavage on each morning for 5 consecutive days (from days 25 to 29 inclusive) before the day of blood collection (day 30).
The slides were stained using the Harris-Schorr staining technique and preserved for possible future evaluation.
Sacrifice and pathology:
Principal:
On completion of the treatment period, after at least 14 hours fasting, all animals were asphyxiated by carbon dioxide and sacrificed by
exsanguination.

Satellite:
After the last blood sampling, the satellite animals were asphyxiated by isoflurane inhalation excess and subsequently discarded without necropsy.
Other examinations:
Organ weights (principal animals):
The body weight of all animals sacrificed at the end of the treatment period was recorded before sacrifice, and the organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection.
The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

Epididymal sperm analysis (principal animals):
Before sacrifice, each male was slightly anesthetized and kept under isoflurane anesthesia. The left epididymis was removed, weighed and sperm from the cauda was sampled for motility and morphology investigations.
Animals were then asphyxiated by carbon dioxide and exsanguinated.
The cauda of the left epididymis was separated from the corpus using a scalpel and subsequently frozen at -20°C for further investigation.
These investigations were performed for all principal male animals.

Epididymal sperm motility:
The sperm were evaluated on a slide, after appropriate dilution. The number of motile and immotile spermatozoa from a sample of 200 was evaluated under microscope using a 40-fold magnification.
Results are expressed as a proportion of motile and non-motile spermatozoa.

Epididymal sperm count (cauda sperm reserve):
After thawing, the left cauda epididymis was weighed, minced and homogenized using a Polytron in a saline-triton solution.
An aliquot of the suspension was sampled and the number of spermatozoa was counted in a Malassez cell.
Results are expressed as a number of spermatozoa per cauda and per gram of cauda.

Epididymal sperm morphology:
The morphology was determined from a sperm smear, after eosin staining, and counting 100 spermatozoa per slide.
Results are expressed as a proportion of spermatozoa in each of the following categories:
. normal,
. normally shaped head separated from flagellum,
. mis-shapen head separated from flagellum,
. mis-shapen head with normal flagellum,
. mis-shapen head with abnormal flagellum,
. degenerative flagellar defect(s) with normal head,
. other flagellar defect(s) with normal head.

Macroscopic post-mortem examination (principal animals):
A complete macroscopic post-mortem examination was performed on all principal study animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated
organs and tissues and the neck with its associated organs and tissues.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Some animals had soiled urogenital regions
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Administration of the test item by oral route (gavage) to Wistar rats for 4 weeks did not elicit any effects on body weights, body weight changes or
food consumption when given at 150, 450 or 1000 mg/kg/day.
The only relevant clinical sign observed was soiled urogenital region in some principal and satellite females given 1000 mg/kg/day or 450 mg/kg/day. The estrous cycle stage was not affected at any dose-level and there were no treatment-related changes in neurotoxicological
parameters. Hematological, blood biochemical parameters or organ weights were not affected by the treatment. No relevant necropsy and
histopathology findings were noted in any treated group.
Consequently, under the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) was 1000 mg/kg/day.
Executive summary:

Administration of the test item by oral route (gavage) to Wistar rats for 4 weeks did not elicit any effects on body weights, body weight changes or food consumption when given at 150, 450 or 1000 mg/kg/day. The only relevant clinical sign observed was soiled urogenital region in some principal and satellite females given 1000 mg/kg/day or 450 mg/kg/day. The estrous cycle stage was not affected at any dose-level and there were no treatment-related changes in neurotoxicological parameters. Hematological, blood biochemical parameters or organ weights were not affected by the treatment. No relevant necropsy and histopathology findings were noted in any treated group. Consequently, under the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) was 1000 mg/kg/day.