N-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)-phenyl-aminocarbonyl]-2,6-difluorobenzamide

Neurotoxicity

Description of key information

- Oral: no neurotoxic effects observed, male/female, rats, subchronic, OECD TG 424, Classen 1992.

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records

Reference

Endpoint:

neurotoxicity: sub-chronic oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 May 1991 to 25 Nov 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: CFR 40 §158.82-7

Qualifier:

according to guideline

Guideline:

other: CFR 40 §798.6400

Qualifier:

according to guideline

Guideline:

EPA OTS 798.6050 (Neurotoxicity Screening Battery)

Qualifier:

according to guideline

Guideline:

OECD Guideline 424 (Neurotoxicity Study in Rodents)

Version / remarks:

Draft Guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: FIFRA § 82-5

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.6200 (Neurotoxicity Screening Battery)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Tif:RAIf (SPF)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: about 8 weeks.
- Weight at study initiation: 233 to 322 g.
- Housing: The animals were initially housed in groups of 5 in Macrolon cages type 4 with wire mesh tops and soft wood bedding. Upon weighing about 500 g animals were separated and housed in groups of 2 or 3.
- Diet: Pelleted, certified standard diet, ad libitum. All batches of the diet were assayed for nutritive ingredients and contaminant level by the manufacturer. Analytical results are available at the Animal Supply Office.
- Water: Tap water, ad libitum. Drinking water quality according to the specifications of the "Schweizerisches Lebensmittelbuch" (Ed. 1972 / Appendix B). Results of the routine chemical examination at source as conducted periodically by the water authority (Baudepartement des Kantons Basel, Abteilung Gewasserschutz) are available.
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 10
- Airchanges (per hour): 16- 20
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 20 May 1991 To: 25 Nov 1991

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Remarks:

Stability and homogeneity of the test article in the food has been assessed for previously conducted toxicity studies.

Duration of treatment / exposure:

4 months

Frequency of treatment:

Continuously

Dose / conc.:

5 ppm

Remarks:

Group 2. Dietary concentration equivalent to 0.255 mg/kg bw/day

Dose / conc.:

25 ppm

Remarks:

Group 3. Dietary concentration equivalent to 1.22 mg/kg bw/day

Dose / conc.:

100 ppm

Remarks:

Group 4. Dietary concentration equivalent to 5.43 mg/kg bw/day

Dose / conc.:

500 ppm

Remarks:

Group 5. Dietary concentration equivalent to 27 mg/kg bw/day

No. of animals per sex per dose:

10 males per dose group, and 10 males in Group 1 and 5 as recovery animals.

Control animals:

yes, plain diet

Details on study design:

Dose selection rationale
- Males I: 5 animals per group for evaluation of neurotoxicity, including clinical signs, neurology, motor activity, startle habituation, maze learning, and potentiation of pentylenetetrazol- induced seizures, and for neuropathology
- Males II: 5 animals per group for evaluation of neurotoxicity, including clinical signs, neurology, motor activity, startle habituation, maze learning, and potentiation of pentylenetetrazol- induced seizures, neuropathology, and for determination of tissue concentrations
- Males III: 5 animals per group for evaluation of reversibility after 2 months of recovery including neurotoxicity and neuropathology
- Males IV: 5 animals per group for evaluation of reversibility after 2 months of recovery including neurotoxicity, neuropathology, and determination of tissue concentrations

Observations and clinical examinations performed and frequency:

- Mortality: twice daily, once on weekends and public holidays
- Clinical signs: observed daily except for weekends, reported once weekly
- Body weight: weekly during pre-test, treatment, and recovery periods
- Food consumption: weekly during pre-test, treatment, and recovery periods
- Concentration of test article: after termination of treatment and recovery in tissue periods

Neurobehavioural examinations performed and frequency:

- Neurological investigations: pre-test and monthly during treatment and recovery periods
- Motor activity: pre-test and monthly during treatment and recovery periods
- Startle habituation: pre-test and monthly during treatment and recovery periods
- Maze test: at end of treatment and recovery periods
- Pentylenetetrazol potentiation: at end of treatment and recovery periods

Sacrifice and (histo)pathology:

- Necropsy and neuropathology periods: after termination of treatment and recovery

Statistics:

- For each time point and parameter an univariate statistical analysis was performed.
- In general nonparametric methods were applied, to allow for non normal as well as normal data distribution. Bodyweight and food consumption data from each treated group was compared to the control group by Lepage's two-sample test. This test is a combination of Wilcoxon and Ansari- Bradley statistics, i.e. a combined test for location and dispersion. The Lepage test has a good power against the more general alternative that the distributions differ not only in location but also in dispersion. Increasing or decreasing trends in location from control to the highest dose group were tested by Jonckheere's test for ordered alternatives. This test is sensitive to monotone dose-related treatment effects. Motor activity data were analysed by parametric procedures» using log transformed data. The treated groups were compared to the control group by repeated T-tests, based on the pooled variance estimate. Group means were tested for a trend (linear by group number) from control to the highest dose group. Two-sided p-values are given in the statistics table.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

In the 500 ppm group, a single episode of fasciculations and clonic-tonic convulsions was observed each in one animal between weeks 13-18. There were no other compound-related clinical signs in the treated animals.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no treatment-related mortalities during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weight was comparable for all groups over the whole study period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was comparable for all groups over the whole study period.

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):

no effects observed

Description (incidence and severity):

The maze learning error scores did not differ between controls and treated groups neither at the end of the treatment period nor at the end of the recovery period. Mean error score is a measure for complex memory and primarily depends on intact function of the hippocampus. Percent blind alley visits is a simple left-right discrimination involving long-term memory functions. The brain structures supporting this function have not yet been identified. These two measures cover cognitive functions that are also involved in complex learning tasks in humans.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related macroscopic or microscopic changes were seen in the central and peripheral nervous system and in muscles.

Neuropathological findings:

no effects observed

Description (incidence and severity):

- Motor activity parameters were not affected in any dose group throughout the whole study period.
- Mean peak amplitude (mean of first 10 startle reactions) and mean startle habituation (mean of first 10 – mean of last 10 startle reactions) were comparable for all dose groups throughout the whole study period.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

- At the end of treatment period as well as following the treatment-free recovery period perfusion fixation was performed on all animals on study.
- Macroscopic examination did not show any treatment-related changes in the tissues collected for processing.
- Light microscopic examination was carried out on 10 control males and 10 males treated with 500 ppm and necropsied at the end of treatment period. Brain tissue was embedded in paraplast and stained with haematoxylin and eosin and with cresyl violet; samples of spinal cord, peripheral nerves, ganglia and muscle were embedded in epoxy resin and stained with toluidine blue.
There were no treatment-related microscopic changes in the multiple examined areas of the central and peripheral nervous system, indicating that test substance was devoid of neuropathic effects under conditions of this special experiment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

CONCENTRATION OF TEST ARTICLE IN TISSUE
The compound accumulated in fatty tissue reaching a mean concentration of 2600 mg/kg in the high dose group at the end of the treatment period. At the end of the 2-month recovery period without exposure to test article, mean concentration in fat of high-dose animals was still at 1600 mg/kg. These data indicate that test substance, is preferentially accumulated in fat and has a very slow rate of elimination.

PENTYLENETETRAZOL POTENTIATION
Following intraperitoneal injection with 25 mg/kg of pentylenetetrazol at the end of the treatment period, generalised clonic-tonic convulsions involving the hind limbs were induced in top dose animals only. Comparable motor seizures limited to the head, neck and forelimb areas were induced in all other groups. After a generalised clonic-tonic convulsion subsequent seizure activity was inhibited whereas motor seizures were shown frequently throughout the 30 minute observation period. At the end of the recovery period, incidence rate for generalised convulsions in top dose animals was comparable to that seen at the end of the treatment period. Median convulsion scores in the top dose animals were lower than at the end of the treatment period but still higher than in control animals, thus indicating partial recovery from the proconvulsive effect induced by the test article.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

neuropathology

Remarks on result:

other:

Remarks:

Dietary equivalent to 5.43 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Remarks:

neurotoxicity

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

neuropathology

Remarks on result:

other:

Remarks:

Dietary equivalent to 5.43 mg/kg bw/day

Key result

Critical effects observed:

no

Table 1: Incidence rate for pentylenetetrazol induced convulsions (number of animals)

	Dietary concentration of test substance (ppm)
	0	5	25	100	500
Week 19 Maximum convulsion scores 0-4 Maximum convulsion scores 5	10 0	10 0	10 0	10 0	3 7*
Week 27 Maximum convulsion scores 0-4 Maximum convulsion scores 5	9 1				4 6

* Statistically significant difference from control group mean, p<0.01 (Fisher’s Exact Test, 2-sided)

 Table 2 Concentration in blood (mg/L) and in fat (mg/kg)

	Dietary concentration of test substance (ppm)
	0	5	25	100	500
Concentration in blood (mg/L) Week 19 Week 27	<0.1 <0.1	0.1	0.6	2.6	17 4.3
Concentration in fat (mg/kg) Week 20 Week 28	<2 <2	16	150	660	2600 1600

* Statistically significant difference from control group mean, p<0.01 (Fisher’s Exact Test, 2-sided)

Conclusions:

No effects of treatment with testsubstance was observed 100 ppm or below, hence 100 ppm (equivalent to 5.43 mg/kg/day) was considered to be the no observable effect level (NOEL) in this study.

Executive summary:

The aim of the present study, performed according the OECD Draft Guideline 424≈FIFRA § 82-5 and GLP, was to characterise the neurotoxic potential of test substance by assessing changes in neurological functions, motor activity, and neuro-histopathology. In addition, maze learning, startle habituation, and potentiation of pentylenetetrazol-induced convulsions were assessed. The test substance was administered to 70 male Tif:RAIf (SPF), albino rats at dietary levels of 0, 5, 25, 100 or 500 ppm, corresponding to a dietary concentrations of 0, 0.255, 1.22, 5.43 and 27.0 mg/kg bw/day, respectively. In the study 10 males per dose group were used and an additional 10 males rats were used as recovery group in the control and high dose group.

Male rats given 500 ppm test substance for 4 months induced single episodes of spontaneous clonic-tonic convulsion or fasciculations and facilitated pentylenetetrazol-induced generalised convulsions. Neurological parameters, motor activity, startle response and startle habituation, complex spatial learning, as well as histology of the peripheral and central nervous system were not affected by treatment. The proconvulsive effect partially recovered over a 2-month period without exposure to the test substance. There were no indications for impaired motor or cognitive functions or for permanent lesions in the peripheral or central nervous system. No effects of treatment with test substance were seen at 100 ppm or below. A concentration of 100 ppm (equivalent to 5.43 mg/kg/day) can thus be considered as a no observable effect level (NOEL) in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

5.43 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP compliant OECD TG 424 study.

Effect on neurotoxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Effect on neurotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Neurotoxicity: oral

4-month dietary study in rats

The aim of the present study, performed according the OECD Draft Guideline 424≈FIFRA § 82-5 and GLP, was to characterise the neurotoxic potential of test substance by assessing changes in neurological functions, motor activity, and neuro-histopathology. In addition, maze learning, startle habituation, and potentiation of pentylenetetrazol-induced convulsions were assessed. The test substance was administered to 70 male Tif:RAIf (SPF), albino rats at dietary levels of 0, 5, 25, 100 or 500 ppm, corresponding to a dietary concentrations of 0, 0.255, 1.22, 5.43 and 27.0 mg/kg bw/day, respectively. In the study 10 males per dose group were used and an additional 10 males rats were used as recovery group in the control and high dose group.

Male rats given 500 ppm test substance for 4 months induced single episodes of spontaneous clonic-tonic convulsion or fasciculations and facilitated pentylenetetrazol-induced generalised convulsions. Neurological parameters, motor activity, startle response and startle habituation, complex spatial learning, as well as histology of the peripheral and central nervous system were not affected by treatment. The proconvulsive effect partially recovered over a 2-month period without exposure to the test substance. There were no indications for impaired motor or cognitive functions or for permanent lesions in the peripheral or central nervous system. No effects of treatment with test substance were seen at 100 ppm or below. A concentration of 100 ppm (equivalent to 5.43 mg/kg/day) can thus be considered as a no observable effect level (NOEL) in this study.