Registration Dossier

Administrative data

Endpoint:
neurotoxicity: acute oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jan - March 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 418 (Delayed Neurotoxicity of Organophosphorus Substances Following Acute Exposure)
Version / remarks:
4 April 1984
Deviations:
yes
Remarks:
deviation from the current TG version: no biochemical analysis (NTE) performed
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, tightly sealed

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: dissolution in vehicle

FORM AS APPLIED IN THE TEST (if different from that of starting material): dissolved in vehicle

Test animals

Species:
hen
Strain:
other: Hisex white
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Werner Maerki, Gefluegelzucht, CH-5443 Niederrohrdorf
- Age at study initiation: approximately one year (after one laying period)
- Weight at study initiation: 1490 to 2100 g
- Housing: 4 hens per cage
- Diet: certified standard diet NAFAG No. 261; ad libitum
- Water: ad libitum
- Acclimation period: at least one week under test conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: Jan 27 To: March 16, 1992

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% in 0.1% aqueous polysorbate 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: not specified

VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified
- Amount of vehicle (if gavage): 10 ml/kg bw
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
41 days observation period
Frequency of treatment:
once;
A second dose was administered on day 21 after the first administration because no neurotoxic signs have been observed.
Doses / concentrations
Dose / conc.:
2 000 mg/kg bw (total dose)
Remarks:
limit dose
No. of animals per sex per dose:
4 animals as vehicle group; 7 animals as treatment group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The selection of the doses was based on an acute oral toxicity study in rats, where the LD50 was determined to be higher than 5000 mg/kg body weight. According to the requirements of the OECD guideline 418 the acute oral LD50 or the limit dose has to be administered in acute delayed neurotoxicity studies.

Examinations

Observations and clinical examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: at the day of the first administration of the test substance and weekly thereafter
Sacrifice and (histo)pathology:
- Time point of sacrifice: on day 41
- Number of animals sacrificed: all surviving and moribund animals
- Parameters measured: macroscopical examination of brain, spinal cord and peripheral nerve
- Procedures for perfusion: The animals were injected intraperitoneally with an overdose of barbiturate with addition of 1000 IU heparin (per animal). Subsequently they were perfused in situ under pressure of about 140 mm Hg with 4% neutral buffered formalin, for at least 15 minutes, preceded for 60 seconds by phosphate buffer alone (0.1M, pH 7.4).
- Number of animals perfused: all sacrificed animals
- Tissues evaluated: cerebellum, medulla oblongata, cervical spinal cord, thoracic spinal cord, lumbar spinal cord, sciatic nerve, tibial nerve
- Type of staining: Bodian' s silver stain for demonstration of axons combined with luxol fast blue counter stain to reveal the myelin sheaths
- Methodology of preparation of sections: After the fixation, organ samples were embedded in paraplast and sectioned.
- Thickness: 3-5 microns
- Number of animals evaluated from each sex and treatment group: all animals (sacrificed scheduled and prematurely)
Positive control:
As positive control tri-orthocresyl phosphate (TOCP) was administered to four hens (in a separate study).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
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 examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Effect levels

Dose descriptor:
NOAEL
Effect level:
2 000 mg/kg bw (total dose)
Based on:
test mat.
Sex:
female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Effects observed in the positive control:

- ruffled feathers and hunched poture; later on ataxia and progressive paralysis developed

- one hen was found dead on day 8, the remaining 3 animals had to be euthanasized on day 17 due to progressive paralysis

- body weight loss was recorded in the second week after administration until spontaneous death or sacrifice

- 3/4 positive control animals had a minimal to marked degeneration of peripheral nerve fibres associated with a minimal to moderate degeneration of terminal spinocerebellar fibres in the cerebellum. This pattern of lesion is compatible with central/peripheral neuropathy known to be produced by triorthocresylphosphate (TOCP) that was administered to these animals.

Applicant's summary and conclusion

Conclusions:
The test substance administered orally in two doses of 2000 mg/kg did not produce clinical, macropathological, or micropathological signs of neurotoxicity.
Executive summary:

In the present study the test substance has been administered orally in a single dose of 2000 mg/kg to eight domestic hens. Four hens served as vehicle control. As positive control, tri-orthocresyl phosphate (TOCP) was administered to four hens.

Due to the absence of neurotoxic signs in the test article dosed animals during the first three weeks of the observation period, a second dose of 2000 mg/kg was administered. On day 42 after first administration the test was terminated. The results can be summarized as follows:

Mortality

No spontaneous mortalities occurred in the animals treated with the test article. Of the TOCP-treated positive control group, one hen was found dead on day 8, the remaining three animals had to be euthanasized on day 17 due to progressive paralysis.

In-life observations

No clinical signs or symptoms were observed in all animals dosed with the test substance and in the controls. In the TOCP-treated animals ruffled feathers and hunched posture were observed; later on ataxia and progressive paralysis developed.

Body weight

Compared to the vehicle control group, no significant influence of the test substance treatment could be observed. In the TOCP-treated animals body weight loss was recorded in the second week after administration until spontaneous death or sacrifice.

Histopathology

Macroscopical and microscopical examination did not reveal any treatment-related neuropathic chanes in the treated or vehicle-control animals, while 3 out of 4 positive control animals showed distally accentuated central/peripheral neuropathy.

The test substance did not produce toxic neuropathy under the conditions of thís study