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neurotoxicity: acute oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-05-04 to 2010-10-28
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
EPA OPPTS 870.6200 (Neurotoxicity Screening Battery)
Version / remarks:
August 1998
according to guideline
EU Method B.43 (Neurotoxicity Study in Rodents)
Version / remarks:
30 May 2008
according to guideline
OECD Guideline 424 (Neurotoxicity Study in Rodents)
Version / remarks:
21 July 1997
GLP compliance:
yes (incl. QA statement)
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
Cas Number:
Molecular formula:
C14 H6 Cl2 F4 N2 O2
Test material form:

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles River Laboratories, Research Models and Services, Germany
- Age at study initiation: 48 days
- Weight at study initiation: ca. 198 g, females ca.152 g
- Fasting period before study: no
- Housing: housed singly in Makrolon cages type M III with wire covers
- Diet: ad libitum, ground Kliba maintenance diet, rat-mouse-hamster pellets, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland.
- Water: libitum, tap water
- Acclimation period: at least 12 days

- Temperature (°C): 20-24
- Humidity (%): 30–70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
CMC (carboxymethyl cellulose)
1% CMC in drinking water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was applied as a suspension. To prepare the suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then the vehicle (drinking water containing 1% carboxymethylcellulose) was filled up to the desired volume, subsequently mixed by high speed with an Ultraturrax (maximum 24,000 rpm). During administration of the test substance, preparations were kept homogeneous using a magnetic stirrer. The test substance preparations were made once before the first administration.

- Amount of vehicle: 10 mL/ kg bw
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The stability of the test substance in drinking water containing 1% carboxylmethylcellulose at room temperature over a period of 7 days was demonstrated. The mean values of test substance were found to be in the range of 100.8-107.9% of the nominal concentration.
Duration of treatment / exposure:
single administration followed by 14 days observation
Frequency of treatment:
single treatment
Doses / concentrationsopen allclose all
Dose / conc.:
125 mg/kg bw (total dose)
Dose / conc.:
500 mg/kg bw (total dose)
Dose / conc.:
2 000 mg/kg bw (total dose)
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle


Observations and clinical examinations performed and frequency:
- Time schedule: 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. All animals were checked daily for any clinically abnormal signs. Abnormalities and changes were documented for each animal.


- Time schedule for examinations: The body weight was determined before the first neurofunctional test in order to randomize the animals. During the conduct of the study, the body weight was determined on the days when functional observational batteries were carried out (study days –7, 0, 7 and 14). The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

FOOD CONSUMPTION AND COMPOUND INTAKE: Food consumption was observed daily by visual inspection.

WATER CONSUMPTION AND COMPOUND INTAKE: Water consumption was observed daily by visual inspection of the water bottles for any overt changes in volume.

Neurobehavioural examinations performed and frequency:
FOB was carried out in all animals on study days -7, 0, 7 and 14. In order to guarantee the blind status of the observer, the cages were randomly distributed in the racks at least 30 minutes prior to the examinations, and the cage labels (indicating the test group) were turned. Thus, the observer was only able to identify the animal numbers but not the allocation of the animals to the different test groups. Moreover, the examinations were carried out in randomized order. Another technician knowing the identification of the animals documented the findings and values obtained.

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:
1. posture
2. tremor
3. convulsions
4. abnormal movements
5. impairment of gait
6. other findings

Open field observations:
The animals were transferred to a standard arena (50×50 cm with sides of 25 cm high) and observed for at least 2 minutes. Following parameters were examined:
1. behavior when removed from cage
2. fur
3. skin
4. salivation
5. nose discharge
6. lacrimation
7. eyes/pupil size
8. posture
9. palpebral closure
10. respiration
11. tremors
12. convulsions
13. abnormal movements
14. impairment of gait
15. activity/arousal level
16. feces (number of fecal pellets/appearance/consistency) within two minutes
17. urine (appearance/quantity) within two minutes
18. number of rearings within two minutes

Sensorimotor tests/reflexes:
The animals were removed from the open field and subjected to following sensorimotor and reflex tests:
1. approach response
2. touch response
3. vision ("visual placing response")
4. pupillary reflex
5. pinna reflex
6. audition ("startle response")
7. coordination of movements ("righting response")
8. behavior during "handling"
9. vocalization
10. pain perception ("tail pinch")
11. grip strength of forelimbs
12. grip strength of hindlimbs
13. landing foot-splay test
14. other findings

Motor activity (MA) measurements were carried out in all animals on study days -7, 0, 7 and 14. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed in new clean polycarbonate cages for the time of measurement. Eighteen beams were allocated per cage. During the measurement the animals were kept in polycarbonate cages with absorbent material. The animals were put into the cages in a randomized order. The measurements started at about 14.00 h. The number of beam interrupts was counted over 12 intervals, each lasting 5 minutes. Measurement did not commence at the same instant for all cages; the period of assessment for each animal started when the first beam was interrupted by pushing the cage into the rack (staggered start). Measurements ended exactly 60 minutes later. During the measurements the animals received no food and no water.
Sacrifice and (histo)pathology:
- Time point of sacrifice: The first five surviving animals per sex and test group were subjected to deep anesthesia with isoflurane at the end of the study and sacrificed by perfusion fixation.
- Brain weight: Weight assessment of the brain (without olfactory bulb) was carried out on all perfused animals after removal of the brain but before any further preparation.
- Procedures for perfusion: SOERENSEN's phosphate buffer served as rinsing solution and the fixation solution according to KARNOVSKY served as fixative (Karnovsky, 1965). The animals fixed by perfusion were necropsied with regard to the question of neuropathology, and the visible organs or organ sections were assessed by gross pathology as accurately as it is possible after a perfusion fixation.
- Tissues evaluated: Frontal lobe, Parietal lobe with diencephalon and hippocampus, Midbrain with occipital and temporal lobe, Pons, Cerebellum, Medulla oblongata, Brain-associated tissues (eyes with retina and optic nerve), Spinal cord (cross and longitudinal sections), Peripheral nervous system (Gasserian ganglia with nerve, gastrocnemius muscle)
- Methodology of preparation of sections (see Tables 1 and 2): Paraffin embedding (paraplast), sectioning and staining with hematoxylin-eosin (HE). The hematoxylin-eosin stained sections were examined by light microscopy and assessed. Plastic embedding (epoxy resin), semithin sectioning and staining with Azure IIMethylene blue-basic Fuchsin (AMbF). The AMbF-stained semithin sections were examined by light microscopy and assessed.
- Number of animals evaluated from each sex and treatment group: 5
Positive control:
As required in the test guidelines, positive control studies have been performed by the test facility. These studies demonstrated the ability to detect signs of neurotoxicity and have assigned MRID numbers by the U.S. EPA.
1.Body weight, body weight change: A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means.

2.Feces, rearing, grip strength forelimbs, grip strength hindlimbs, footsplay test, motor activity: Non-parametric one-way analysis using 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) for the equal medians

3. Brain weight parameters: Non-parametric one-way analysis using 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 the WILCOXON test for the hypothesis of equal medians

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No test substance-related, adverse findings were observed.
no mortality observed
Description (incidence):
No animals died prematurely in the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No substance-related, relevant findings concerning body weight data were found.
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 examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional observational battery: Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between test substance-treated groups and controls, were without a dose-response relationship or occurred in single animals, these observations were considered to have been incidental. Beside this, the FOB was performed in different examination segments, which have to be assessed individually.
Home cage observations: No test substance-related findings were observed.
Open field observations: No test substance-related findings were observed.
Quantitative parameters: On study day 0, grip strength of hindlimbs was significantly decreased (-13.42 %) in male animals of test group 2 (500 mg/kg bw). On study day 14, grip strength of hindlimbs in all female test groups were significantly increased, i.e. in test group 1 (125 mg/kg bw) by 25.07 %, in test group 2 (500 mg/kg bw) by 24.53 % and in test group 3 (2000 mg/kg bw) by 19.33 %. All findings were assessed as being incidental as not changes were seen in grip strength of forelimbs or in food splay test. In addition, no dose-response relationship could be observed.
Motor activity measurement: No test substance-related findings were observed. Comparing the single intervals of test groups 1-3 (125, 500 and 2000 mg/kg bw) with the control groups on study day -7, 1 significantly increased value was measured in interval 5 in females of test groups 3 (2000 mg/kg bw). This finding was assessed as being incidental as no test-substance was administered. On study day 14, interval 7 in female animals of test group 3 (2000 mg/kg bw) was significantly increased and interval 8 in female animals of test group 1 (150 mg/kg bw) was significantly decreased. These findings were assessed as spontaneous in nature and, therefore, not test substance-related.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No statistically significant changes in test groups 1-3 (125, 500 and 1000 mg/kg bw/d) were
noted when compared to the control.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross lesions were recorded.
Neuropathological findings:
no effects observed
Description (incidence and severity):
No neuropathological lesions were noted.
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Effect levels

Key result
Dose descriptor:
Effect level:
2 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed up to highest dose tested.

Target system / organ toxicity

Key result
Critical effects observed:

Applicant's summary and conclusion