picoxystrobin (ISO); methyl (2E)-3-methoxy-2-[2-({[6-(trifluoromethyl)pyridin-2-yl]oxy}methyl)phenyl]acrylate

Neurotoxicity

Description of key information

90-Day Rat Diet NOAEL: >3500 ppm; not neurotoxic. OECD 424; Reliability = 1

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:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 424 (Neurotoxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

99.3% purity

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: 219-266 g (males); 141-186 g (females
- Housing: All animals were housed singly in stainless steel, wire-mesh cages suspended above cage boards. Each cage rack contained only animals of one sex.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26°C (64-79°F)
- Humidity (%): 30-70%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 hour light and 12 hour dark

Route of administration:

oral: feed

Vehicle:

other: LLC Certified Rodent LabDiet® 5002

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples (at least duplicate) of all dietary concentrations were collected at the initial diet preparation and analyzed to verify the concentration (average of homogeneity samples) and homogeneity of test substance in the diets. Toward the middle and end of the study, samples were taken to verify concentration. Any remaining samples were discarded following issuance of the report. At the time of analysis, the samples were extracted with acetonitrile and the extracts were analyzed by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily

Dose / conc.:

100 ppm

Dose / conc.:

600 ppm

Dose / conc.:

3 500 ppm

No. of animals per sex per dose:

12

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: In an ongoing 2-year rat chronic/carcinogenicity study (0, 50, 200, 1000, and 3500 ppm), data up to 6 months showed that 3500 ppm caused modest changes in body weight parameters. There were no noteworthy changes in other in-life parameters. Based on these results, the dietary concentrations for the current study were selected to be 0, 100, 600, and 3500 ppm.

Observations and clinical examinations performed and frequency:

Mortality/Moribundity Check

Cage-site examinations to detect moribund or dead animals and abnormal behavior/appearance were conducted twice daily throughout the study. Abnormal behavior/appearance were recorded by exception.

General Clinical Observations

A cage-site examination to detect acute clinical signs of systemic toxicity was conducted once daily at approximately the same time (± 2 hours). Exceptions occurred on the days of neurobehavioral evaluations. Clinical observations were recorded by exception. General Clinical Observations were not done on the days of Careful Clinical Observations except on test day 0.

Careful Clinical Observations
At every weighing, each rat was individually handled and examined for abnormal behavior and appearance.

Neurobehavioural examinations performed and frequency:

A neurobehavioral test battery, consisting of functional observational battery (FOB) assessments and motor activity (MA), was conducted on rats during pretest (baseline) and again on all surviving rats during weeks 4, 8, and 13 of test substance administration. For the baseline evaluation, FOB and MA assessments were also conducted on spare animals in the event that replacements were needed on test day 0.

Animals were counterbalanced by sex and dose and tested over multiple days to minimize the influence of uncontrolled factors. The experimenter conducting the FOB was blind with respect to the group designation of the animal. The testing was performed by the same person each time.
FOB and MA evaluations were conducted in a sound-attenuated room equipped with a whitenoise generation system to minimize environmental variations in the test conditions. Animals were acclimated at least for 10 minutes in the FOB laboratory prior to initiation of evaluation. Animals were weighed on the days of the FOB assessment. Weights are kept in study records and are not reported in the final report.

Sacrifice and (histo)pathology:

On test day 97 and 98, rats selected for neuropathology (6 rats/sex/group) were anesthetized (approximately 1-2 mL of Nembutal® Sodium Solution) and underwent whole-body in situ perfusion. The remaining 6 rats/sex group were sacrificed by carbon dioxide anesthesia and discarded without necropsy.

Tissues were processed as follows: The brain (forebrain, cerebrum (including hippocampus), midbrain, cerebellum, pons, and medulla), spinal cord (cervical and lumbar), eye (with optic nerve), and gastrocnemius muscle were paraffin embedded, sectioned at approximately 5 μm, and stained with hematoxylin and eosin (H&E). Central nervous system tissues (brain and spinal cord) were also stained with Luxol Fast Blue/Periodic Acid Schiff (LFB/PAS) for myelin sheaths and neuronal cell bodies.

Cervical and lumbar dorsal root fibers and ganglia (DRF&G) and ventral root fibers (VRF), as well as sciatic, tibial and sural nerves, and gasserian ganglia were embedded in glycol methacrylate (GMA), sectioned at approximately 3 μm, and stained with H&E.

Only tissues from the right side of animals were processed. All tissues not processed were saved in appropriate fixative as wet tissue.

Tissues from rats in the control and high-dose groups were processed and examined microscopically by a veterinary pathologist. Since there were no test substance-related lesions in the high-dose group, tissues from other dose groups were not processed.

Positive control:

Yes

Statistics:

For body weight, body weight gain, food consumption, and food efficiency, used Levene’s test for homogeneity and the Shapiro-Wilk test for normality; If the preliminary test was not significant, used the One-way analysis of variance test followed by Dunnett’s test; If the preliminary test iwa significant, used the Kruskal-Wallis test followed by Dunn’s test.
For motor activity, grip strength, foot splay, body temperature, and rearing, used Levene’s test for homogeneity and the Shapiro-Wilk test for normality; If the preliminary test was not significant, used the Repeated measures analysis of variance test followed by linear contrasts test; If the preliminary test was significant, used (1) A normalizing, variance stabilizing transformation followed by repeated measures analysis of variance or (2) sequential application of the Jonckheere-Terpstra trend test.

For Incidence of FOB descriptive parameters, used the Cochran-Armitage test for trends

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no early deaths or test substance-related clinical observations in males or females administered up to 3500 ppm of the test substance. All recorded clinical observations were unremarkable and occurred in low incidences without dose response.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males administered 3500 ppm of the test substance exhibited adverse, test substance-related decreases in mean body weight and body weight gain compared to controls. Mean body weights were statistically significantly lower compared to controls in all the weekly intervals (14-23%).
Mean body weight gains were also lower compared to control in all the weekly intervals with most intervals showing statistical significance (28-61%). Final (day 91) mean body weight and overall (day 0-91) mean body weight gain were 77% and 62% (both statistically significant) of
control, respectively. There were no test substance-related effects or statistically significant differences for body weight or body weight gain in males administered up to 600 ppm of the test substance.

Females administered 3500 ppm of the test substance exhibited adverse, test substance-related decreases in body weight and body weight gain compared to control. Mean body weights were statistically significantly lower compared to controls in all the weekly intervals except day 14
(9-15%). While body weight gain was lower compared to control in majority of the weekly intervals, statistical significance (38-56%) was seen only during first month of exposure (days 0-7, 14-21, 21-28). Final (day 91) mean body weight and overall (day 0-91) mean body weight gain were 85% and 69% (both statistically significant) of control, respectively.

Females administered 100 ppm and 600 ppm exhibited non-adverse, test substance-related decreases in body weight and body weight gain compared to control. Body weights in the 100 ppm (3-6%) and 600 ppm (5-9%) groups were lower (not statistically significant except on day 56 at 600 ppm) compared to controls in all the weekly intervals. Final (day 91) body weights were 95% and 92% of controls, respectively. Body weight gains were generally lower, but there were no statistically significant changes in any of the weekly intervals. Overall (day 0-91) mean body weight gain was 91% and 86% of control, respectively, and neither was statistically significant. Decreases seen in overall (day 0-91) body weight gain were due to lower body weight gains during first month (day 0-28) of exposure (91% and 80% of control, respectively, with statistical significance in the 600 ppm group only). Body weight gains during the second and third months of exposure (days 28-56 and 56-91) increased to a level that was comparable to the control, but the increases were insufficient to compensate for the earlier reductions. Due to the low magnitude of the decreases in final body weights, the fact that the decreases in body weight gain were limited to the first 28 days, the lack of any statistical significance in the overall body weight gains, and the lack of significant changes in overall food consumption and food efficiency, the decreased body weights and body weight gains observed in the 100 ppm and 600 ppm groups were considered test substance-related, but not adverse.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males administered 3500 ppm of the test substance exhibited adverse, test substance-related decreases in food consumption compared to control. Mean food consumption values were statistically significantly lower compared to controls in all the weekly intervals (18-35%). Overall (day 0-91) food consumption was 78% of control (statistically significant). There were no test substance-related effects or statistically significant differences for food consumption in males administered up to 600 ppm of the test substance. Females administered 3500 ppm of the test substance exhibited adverse, test substance-related decreases in food consumption compared to control. Mean food consumption values were statistically significantly lower compared to controls in all the weekly intervals (12-22%). Overall (day 0-91) food consumption was 82% (statistically significant) of control. Females administered 100 ppm and 600 ppm exhibited non-adverse, test substance-related decreases in food consumption compared to the control. The decreases in food consumption were in the range of 2-7% and 2-11% in the 100 ppm and 600 ppm groups, respectively, with statistical significance in the food consumption of the 600 ppm group during test days 0-28 and 42-49. Overall (day 0-91) food consumption of the 100 ppm and 600 ppm groups were 96% and 93% of control, respectively. Neither group had a statistically significant decrease in any of the weekly intervals. Due to the low magnitude of the decreases, and the lack of statistical significance in the overall food consumption, the decreases observed in the 100 ppm and 600 ppm groups were considered not adverse. In addition, the changes noted in nutritional parameters in 100 ppm and 600 ppm females were consistent with body weight parameters. Over the course of the study, the mean daily intake of the test substance for males was 6, 36, and 207 mg/kg/day, corresponding to dietary concentrations of 100, 600, and 3500 ppm, respectively. Over the course of the study, the mean daily intake of the test substance for females was 8, 46, and 246 mg/kg/day, corresponding to dietary concentrations of 100, 600, and 3500 ppm, respectively.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Males administered 3500 ppm of the test substance exhibited adverse, test substance-related decreases in food efficiency compared to control. While food efficiency was lower compared to control in the majority of the weekly intervals (20-63%), statistical significance was observed in the weekly intervals of 0-7, 21-28, and 56-63. Overall (day 0-91) food efficiency was 79% of control (statistically significant). There were no test substance-related effects or statistically significant differences for food efficiency in males administered up to 600 ppm of the test substance. Females administered 3500 ppm of the test substance exhibited adverse, test substance-related decreases in food efficiency compared to control. While food efficiency was lower compared to control in the majority of the weekly intervals, statistical significance was observed only during first week of exposure (55% of control). Overall (day 0-91) food efficiency was 86% (not statistically significant) of control. Females administered 100 ppm and 600 ppm exhibited non-adverse, test substance-related decreases in food efficiency compared to control. While food efficiency of 600 ppm was lower than control in the majority of the weekly intervals, it was lower only in a few weekly intervals at 100 ppm. Overall (day 0-91) food efficiency of the 100 ppm and 600 ppm groups were 95% and 92%, respectively. Due to the low magnitude of the decreases, and the lack of statistical significance in the overall food efficiency, the decreases observed in the 100 ppm and 600 ppm groups were considered not adverse.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

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:

effects observed, non-treatment-related

Description (incidence and severity):

Males administered 3500 ppm of the test substance exhibited statistically significant decreases in forelimb and hindlimb grip strength, that were considered test substance-related, but secondary to decreases in body weight. During the week 8 evaluation, forelimb and hindlimb grip strength were statistically significantly lower than control, and during the week 13 evaluation, forelimb grip strength was statistically significantly lower in the 3500 ppm group, compared to control. The decreases were small (6%-9%), they were within the historical control values for rats of a similar age and strain in the test facility, and they were not associated with any noteworthy sign of neurotoxicity. Therefore, the differences were considered secondary to the observed decreases in body weight. There were no test substance-related effects or statistically significant differences for forelimb or hindlimb grip strength in males administered up to 600 ppm of the test substance.

There were no test substance-related effects for forelimb or hindlimb grip strength in females administered up to 3500 ppm of the test substance. During the week 13 evaluation, a statistically significant decrease in forelimb grip strength was noted in females administered 100, 600, and 3500 ppm of the test substance. The differences compared to control were small (9% for all groups), the values were within the historical control values for rats of a similar age and strain in the test facility, they did not occur in a dose-dependent manner, and they were not associated with changes in gait, motor activity, or any microscopic pathological finding. Therefore, the differences were considered spurious.

There were no test substance-related effects or statistically significant differences for footsplay in males administered up to 3500 ppm of the test substance. There were no test substance-related effects for footsplay in females administered up to 3500 ppm of the test substance. During the week 13 evaluation, a statistically significant decrease in footsplay was noted in females administered 3500 ppm of the test substance. However, the increased mean value was within the historical control range for rats of a similar age and strain in the test facility, and it was not associated with any microscopic pathological finding, or any observed abnormality in a behavioural parameter such as arousal, posture, or gait/coordination. Furthermore, the mean control value (5.1 cm) during the week 13 evaluation was at the low end of the historical control data and was lower than the values noted during the weeks 4 (6.2 cm) and 8 (5.8 cm) evaluations. The footsplay in the 3500 ppm group during the week 8 evaluation was slightly higher than the week 13 evaluation (7.4 cm during week 8 and 7.1 cm during week 13), but only the week 13 evaluation was statistically significant due to the slightly lower control mean value. Therefore, the statistical significance noted in the 3500 ppm females during the week 13 evaluation was the function of slightly lower control values and not test substance related.

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

600 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Dose descriptor:

NOAEL

Effect level:

3 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no neurotoxic effects at the highest dose tested

Conclusions:

The no-observed-adverse-effect level (NOAEL) for systemic toxicity in males and females was considered to be 600 ppm (36 and 46 mg/kg/day, respectively), based on decreased body weight and nutritional parameters in males and females administered 3500 ppm. The NOAEL for neurotoxicity in males and females was greater than 3500 ppm (207 and 246 mg/kg/day, respectively), the highest dietary concentration tested, based on the absence of primary test substance-related effects on the nervous system.

Executive summary:

Young adult male and female Crl:CD(SD) rats (12 rats/dose/sex) were administered diet containing 0, 100, 600, and 3500 ppm of the test substance for approximately 90 days (97 or 98 days). Samples of the diets were analyzed to verify the concentration and homogeneity of the test substance in the diet. Body weights and food consumption were determined weekly. Clinical signs of toxicity were assessed daily and weekly. A neurobehavioral test battery, consisting of motor activity and functional observational battery assessments, was conducted on all study rats prior to test substance administration in order to obtain baseline measurements. This neurobehavioral test battery was conducted again during weeks 4, 8, and 13. On test days 97 and 98, 6 rats per sex per group were sacrificed and underwent whole-body in situ perfusion for neuropathology examination.

 The overall mean daily intake of the test substance in the 100, 600, and 3500 ppm groups was 6, 36, and 207 mg/kg/day in males and 8, 46, and 246 mg/kg/day in females, respectively.

 Adverse, test substance-related effects in body weight and nutritional parameters were observed in males and females administered 3500 ppm. Statistically significant decreases in mean body weights were observed in males and females (except day 14 in females) compared to controls. While mean body weight gains were statistically significantly lower compared to controls in most of the weekly intervals in males, statistical significance was seen only during the first month of exposure in females. Final (day 91) mean body weights and overall (day 0-91) mean body weight gains in males and females were 77% and 85% and 62% and 69% of control, respectively. These decreases were statistically significant. Statistically significant decreases in mean food consumption values compared to controls were noted at all the weekly intervals in males and females of 3500 ppm group. While food efficiency was lower compared to controls in the majority of the weekly intervals, statistical significance was observed in only few weekly intervals. Overall (day 0-91) food consumption and food efficiency in male and female rats were 78% and 82% and 79% and 86% of control, respectively. These decreases were statistically significant.

 

There were no test substance-related changes in body weight or nutritional parameters in males administered 100 or 600 ppm. However, body weight and nutritional parameters in female rats in the 100 and 600 ppm groups were generally lower compared to controls throughout the study period. The changes noted in the females were considered test substance-related, but not adverse because these effects were not statistically significant, were lower in magnitude and mainly occurred in the first month of exposure followed by recovery.

 

There were no test substance-related adverse effects on survival, clinical signs of toxicity, motor activity, temperature, behavioral parameters, gross or neuropathological changes in males or females administered any concentration of the test substance. Males administered 3500 ppm of the test substance exhibited statistically significant decreases compared to control in fore- and

hind-limb grip strength during week 8 and fore-limb grip strength during week 13. These changes were considered secondary to decreases in body weight and not a primary effect of the test substance because the decreases were small (6%-9%), they were within the historical control values, and they were not associated with any noteworthy sign of neurotoxicity.

 

The no-observed-adverse-effect level (NOAEL) for systemic toxicity in males and females was considered to be 600 ppm (36 and 46 mg/kg/day, respectively), based on decreased body weight and nutritional parameters in males and females administered 3500 ppm. The NOAEL for neurotoxicity in males and females was greater than 3500 ppm (207 and 246 mg/kg/day, respectively), the highest dietary concentration tested, based on the absence of primary test substance-related effects on the nervous system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

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

In a 90-day neurotoxicity study, young adult male and female rats were administered diet containing 0, 100, 600, and 3500 ppm test substance for approximately 90 days. Adverse, test substance-related effects in body weight and nutritional parameters were observed in males and females administered 3500 ppm. There were no test substance-related adverse effects on survival, clinical signs of toxicity, motor activity, temperature, behavioural parameters, gross or neuropathological changes in males or females administered any concentration of the test substance. The NOAEL for systemic toxicity in males and females was considered to be 600 ppm, or 36 and 46 mg/kg body weight/day, respectively. The NOAEL for neurotoxicity in males and females was greater than 3500 ppm, or 207 and 246 mg/kg body weight/day, respectively.

Justification for classification or non-classification

The test substance was negative for neurotoxicity in a 90-day rat neurotoxicity study. Therefore, the substance does not need to be classified for neurotoxicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.