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Toxicological information

Neurotoxicity

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Description of key information

Acute neurotoxicity in the rat (OECD 424), NOAEL = 500 mg/kg bw/day

Subchronic neurotoxicity in the rat (OECD 424), 90-days, NOAEL = 5000 ppm (highest dose tested), equivalent to doses of 345 and 416 mg/kg bw/day in males and females, respectively

Developmental neurotoxicity in the rat (OPPTS 870.6300), NOAEL for maternal animals = 450 ppm, equivalent to a dose of 37.1 mg/kg bw/day; NOAEL for offspring = 45 ppm, equivalent to a dose of 3.8 mg/kg bw/day

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
3.8 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 1) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

Additional information

Reliable oral studies on the potential for the test substance to cause acute, subchronic and developmental neurotoxicity are available.

 

Acute oral neurotoxicity

In this GLP-compliant study that was performed according to OECD 424, the test substance administered by oral gavage once to four groups of 12 male and 12 female Wistar rats at doses of 0, 200, 500, and 2000 mg/kg bw (M-263029-01-1). In a preliminary ADME study (not reported in this dossier), the time after oral gavage dosing of the test substance to the peak blood concentration of the test substance was found to be 30 minutes in both males and females. Thus, conduct of the FOB and motor activity studies began approximately but no less than 30 minutes after dosing, and was concluded at approximately 2.5 hours after treatment. In total, the FOB and motor activity assessments were conducted one week prior to dosing, approximately 30 minutes after dosing, and on days 7 and 14 after dosing. The FOB included standard parameters including home cage and open field observations, reflex testing, and determination of fore-limb and hind-limb grip strength, and landing foot splay. Motor and locomotor activities were measured according to standard methods over a 60-minute period for each animal. On day 14 after dosing, all animals were sacrificed. Half of the animals at each dose level were anesthetized and perfused via the left ventricle. The brain was weighed, and the brain, spinal cord, eyes with optic nerve, sciatic, tibial, and sural nerves, gasserian ganglion, gastrocnemius muscle, both forelimbs, and any gross lesions were removed and preserved for examination. The other half of the animals at each dose level were sacrificed by CO2 asphyxiation and were not perfused. All animals were subjected to a gross necropsy. Histopathology was conducted on the nervous tissues from the 6 perfused animals in the control and high dose groups only.

There were no mortalities at any dose level in this study. Treatment-related clinical signs were limited to urine stains, nasal and oral stains, and stained forepaws. These stains were seen in all treatment groups, with a general dose-related incidence, and were attributed to the excretion of the test substance in the urine leading to intense yellow/orange coloration of the urine. The staining had reversed in almost all animals by the time of sacrifice. As the staining was due to compound excretion, it was considered not to be an adverse or toxicologically significant finding. There were no effects on body weight in either males or females at any dose level. There were no effects on any parameters of the functional observation battery, including grip strength and landing foot splay, in any dose group in either males or females. Motor and locomotor activity was biologically and / or statistically significantly decreased in both males and females at 2000 mg/kg bw/day on day 0 only. There were no other treatment-related effects on either parameter at any other dose level, or at any dose level on days 7 and 14 after dosing. There was no effect on brain weight (the only organ weight measured) at any dose in either males or females. There were no treatment-related macroscopic findings observed at any dose in either males or females. Microscopic findings: There were no treatment-related effects observed at 2000 mg/kg bw when compared to controls, thus histopathological sections were not made at either 200 or 500 mg/kg bw.

Conclusions: The oral gavage administration of the test substance produced no clinical signs indicative of neurotoxicity. Motor activity and locomotor activity were decreased in both males and females at 2000 mg/kg bw, but there was no other effect on any neurotoxicity-related parameter at any time point. There was therefore no indication of neurotoxicity at any dose level. The NOAEL of the acute neurotoxicity study was therefore established at 504 mg/kg bw.

Subchronic neurotoxicity

In this GLP-conform study that was performed according to OECD 424 (M262680-01-1), the test item was incorporated into rodent meal at concentrations of 0, 500, 2500, and 5000 ppm and provided to 12 male and 12 female Wistar rats per group for 90 days. The dietary concentrations prepared provided doses of 0, 32.3, 166, and 345 mg/kg bw/day for males and 0, 41.9, 206, and 416 mg/kg bw/day for females. Clinical signs were assessed twice daily on weekdays and once daily on weekends and holidays. Body weight and food consumption were measured on a weekly basis. The FOB and motor activity were assessed on five occasions, once in the week prior to the start of the feeding period, and once each during weeks 2, 4, 8, and 13. The FOB included standard parameters including home cage and open field observations, reflex testing, and determination of fore-limb and hind-limb grip strength, and landing foot splay. Motor and locomotor activity was measured according to standard methods over a 60-minute period for each animal. Ophthalmological examinations were conducted in all animals prior to the start of the study and during week 12. After at least 90 days dietary administration, all animals were sacrificed. Half of the animals at each dose level were anesthetized and perfused via the left ventricle. The brain was weighed, and the brain, spinal cord, eyes with optic nerve, sciatic, tibial, and sural nerves, gasserian ganglion, gastrocnemius muscle, both forelimbs, and any gross lesions were removed and preserved for examination. The other half of the animals at each dose level were sacrificed by CO2 asphyxiation and were not perfused. All animals were subjected to a gross necropsy. Histopathology was conducted on the nervous tissues from the 6 perfused animals in the control and high dose groups only.

There were no mortalities in either males or females during the study. Treatment-related clinical signs observed in this study were limited to urine staining in both males and females, largely occurring in a dose-related manner. The yellow to brownish-orange stains observed were considered to be due to urinary excretion of the test substance and were considered not to be indicative of toxicity. There was no treatment-related effect on food consumption in either males or females at any dose level. Food consumption was statistically significantly increased in males at 5000 ppm on days 21-28 through days 28-35, and on days 70-77 through days 77-84. However, this was considered not to be indicative of a toxic effect as there was no corresponding increase in food consumption in females nor was body weight affected in males at 5000 ppm. There was no treatment-related effect on body weight in either males or females at any dose level. Neither males nor females revealed any effects at ophthalmological examination which were attributed to treatment. There was no effect on any parameter of the FOB, including grip strength and landing foot splay, in either males or females at any point during the study. There were no treatment-related alterations in either motor or locomotor activity in either males or females at any dose. There were occasional statistically nonsignificant increases in motor or locomotor activity at various time points, but they followed no dose-related pattern, were generally limited to one sex, and were opposite to the treatment-related change observed in the acute neurotoxicity study. Therefore, these increases were considered not to be treatment-related. There was no effect on brain weight in either males or females at any dose. No gross lesions or other macroscopic observations were present at necropsy in either male or female rats which could be attributed to treatment. There were no histopathological findings in male or female rats at 5000 ppm, relative to control, which were indicative of a treatment-related effect. In the absence of any treatment-related effect at 5000 ppm, tissues were not examined at lower doses.

Conclusions: There were no indications of a neurotoxic effect of the test substance after 90 days dietary administration, nor were there effects on body weight or food consumption. The NOAEL for this study was therefore 5000 ppm, or 345 mg/kg bw/day in males and 416 mg/kg bw/day in females.

 

Developmental neurotoxicity

In this GLP-compliant study that was performed according to EPA OPPTS 870.6300 (M-266434-02-1), the test substance was incorporated into rodent diet at concentrations of 0, 45, 450, and 4500 ppm and administered to groups of 30 sperm-positive female Wistar rats from gestation day 6 through lactation day 21. Dietary concentrations were adjusted during lactation to provide for a constant dosage throughout the treatment period. These concentrations provided an average daily intake of 0, 3.8, 37.1, and 354 mg/kg bw/day. Dams were observed for clinical signs at least once daily. Body weight and food consumption were measured on a weekly basis, on gestation days 6, 13, and 20, and lactation days 0, 7, 14, and 21. Maternal body weight was also measured on lactation day 4. Litters were culled on lactation day 4 to eight pups, with four male and four female pups wherever possible. Dams were sacrificed on lactation day 21 following weaning of their litters, without post- mortem examinations.

As soon as possible after pup birth, anogenital distance was measured and pups were tattooed. Surviving pups were counted, sexed, and weighed individually on lactation days 0, 4, 11, 17, and 21. Offspring were monitored daily throughout lactation for clinical signs or morbidity. After weaning on day 21, the pups were monitored twice daily for morbidity and mortality, and were weighed on a weekly basis as well as on the day that vaginal patency or balanopreputial separation were achieved. Food consumption was not measured. Pups were examined daily starting from postnatal day 29 (females) or postnatal day 38 (males) for developmental landmarks, and pupil constriction was tested in all pups on postnatal day 21. All tests used at least 10 offspring per sex per dose, and with the exception of learning and memory, no animal was tested more than once in the same test.

At least 10 male and 10 female offspring per dose group were selected for ophthalmological examination at approximately 50-60 days of age. On postnatal day 21, 10 male and 10 female offspring were sacrificed and the brains were collected whole for micropathological examination and morphometric analysis. The animals were anesthetized (50 mg/kg bw pentobarbital, intraperitoneal injection) and perfused via the left ventricle with sodium nitrite in phosphate buffer followed by in situ fixation with universal fixative in phosphate buffer. On postnatal day 75, a further 10 male and 10 female offspring were sacrificed and perfused, and brain, spinal cord, both eyes with optic nerves, bilateral sciatic, tibial, and sural nerves, gasserian ganglion, gastrocnemius muscle, and both forelimbs were collected and fixed. Brains were weighed. In both cases, prior to sectioning, the anterior-to-posterior lengths of the cerebrum and of the cerebellum were measured with Vernier calipers. Other measurements, made after histologic sectioning, were the thickness of the frontal cortex, parietal cortex, and hippocampal gyrus, horizontal width of the caudate putamen, and height of the cerebellum. The offspring not selected for neuropathological examination were sacrificed without examination.

 

Findings for parental animals: There were no mortalities in parental females during either gestation or lactation. There were no treatment-related clinical signs observed during gestation. During lactation, ocular opacities were observed in 5 females at 450 ppm and in 14 females at 4500 ppm. Ocular opacities, evaluated as related to treatment, were observed during the FOB in three females at 450 ppm and seven females at 4500 ppm. This increase at 4500 ppm was statistically significant compared to controls. Ocular opacities are considered a rat-specific phenomenon of HPPDase inhibitors without relevance for humans. Accordingly, this finding was not used for setting a NOAEL (for a detailed justification see endpoint summary for repeated dose toxicity). There were no other treatment-related findings at FOB evaluation. Body weight and body weight gain were not affected during gestation or lactation at any dose. Food consumption was statistically significantly increased at 4500 ppm during gestation, although this was considered to be due to wastage from palatability issues. Installation of grates to reduce wastage in week 2 of gestational treatment markedly reduced food consumption at 4500 ppm. During lactation, food consumption was statistically significantly reduced at both 450 ppm (lactation days 0-7 and 7-14) and 4500 ppm (all periods). These reductions were attributed to unpalatability, as there was no effect on body weight at either dose. The fertility index was statistically non-significantly decreased at 4500 ppm compared to controls, and was evaluated as treatment related. There were no other effects on reproductive parameters.

 

Findings for offspring: There were no effects of treatment on litter size, viability, or other litter parameters. There were no treatment-related signs observed during lactation in either males or females. In the post-weaning phase, treatment-related findings were restricted to ocular opacities in 6 males and 1 female at 4500 ppm, and 2 females at 450 ppm. There was no effect on the incidence of moribund or found-dead pups. Body weight at birth was similar at all doses. On postnatal day 4 (post-culling), body weight was statistically significantly decreased in males at 4500 ppm, with statistically non-significant decreases in body weight in females at 4500 ppm and both sexes at 450 ppm. Body weight and body weight gain were statistically significantly reduced for males and females combined at 450 and 4500 ppm through weaning at postnatal day 21. After weaning, statistically significantly decreased body weight continued in males at 450 and 4500 ppm through the end of the study, in females at 450 ppm for the first three weeks after weaning, and in females at 4500 ppm for the first two weeks after weaning.

Difference from control body weight during lactation reached a maximum of 7% at 450 ppm and 11% at 4500 ppm. Body weight gain was also reduced, by 8% relative to controls at 450 ppm and by 12% at 4500 ppm. After weaning, body weight reduction in males showed a maximum of 9% at 450 ppm and 13% at 4500 ppm. In females, body weight was reduced by a maximum of 8% at 450 ppm or 11% at 4500 ppm. These effects on body weight were considered to be treatment-related. Preputial separation was statistically significantly delayed at 4500 ppm (46.7 days, p < 0.01) and statistically non-significantly delayed at 450 ppm (46.0 days), compared to controls (44.1 days). This increase in time to preputial separation was considered to be related to treatment as a secondary effect of reduced body weight. There was no effect on time to vaginal patency. Pupillary constriction on day 21 in both males and females was not affected by treatment. The only treatment-related changes at FOB assessment were ocular opacities in one female at 450 ppm and one female at 4500 ppm. These changes were first noted on postnatal days 45 and 35, respectively, and persisted in both cases through postnatal day 60. There were no treatment-related effects observed on either motor or locomotor activity in either males or females at any dose level during either lactation or post-weaning phases. There were no treatment-related effects on auditory startle at any time during the study or at any dose level. There was no effect of treatment during the acquisition phase of the passive avoidance testing. However, during trial 1 of the retention phase, males at 4500 ppm showed a statistically significantly decreased latency to crossing than controls. Females at 4500 ppm showed a similar, although statistically non-significantly, decreased latency to crossing. This effect is considered to be related to treatment. Although there were other statistically significant differences observed during the passive avoidance testing, these were considered not related to treatment as they were generally within historical control data, not related to treatment, and / or seen in one sex only. There were no treatment-related effects on acquisition and retention in either males or females during the water maze testing. Retinal atrophy was statistically significantly increased at 4500 ppm, with four males and four females affected at this dose. This was considered to be a treatment-related finding. Absolute brain weight was statistically significantly decreased for males and females at 4500 ppm and females at 450 ppm on day 21, and for females at 4500 ppm on day 75. Relative brain weight in these groups was no different from controls, indicating that the decreased absolute brain weight was related to the reduced body weight at these doses. At day 21, cerebellum length was statistically significantly decreased in males and females at 4500 ppm. Cerebrum length was also statistically significantly decreased in females at 450 and 4500 ppm. At day 75, cerebellum length was statistically significantly decreased in males at 4500 ppm, and statistically non-significantly decreased in females at 4500 ppm. There was no effect on cerebrum length in either males or females at day 75. These decreases observed on day 21 and 75 were considered to be related to treatment.

On day 21, males and females at 450 and 4500 ppm showed a treatment-related, statistically significant decrease in cerebellum height. On day 75, cerebellar height was statistically significantly decreased at 4500 ppm in males and females. On histopathological examination of the brains and other nervous tissues, the only treatment-related finding was retinal degeneration in one male at 4500 ppm.

 

Conclusions: The relevant maternal findings observed in this study were limited to decreased fertility index at 4500 ppm. The NOAEL for maternal animals administered the test substance by dietary incorporation was therefore 450 ppm (37.1 mg/kg bw/day).

The treatment-related findings in the offspring at 450 and 4500 ppm included decreased body weight both prior to and after weaning, delay in preputial separation, ocular opacities, and retinal degeneration, decreased terminal body weights, and slight decreases in absolute but not relative fixed brain weight at postnatal day 21. There were also slight decreases in postnatal day 21 animals in cerebrum and / or cerebellum length, and cerebellum thickness was decreased in both postnatal day 21 and postnatal day 75 animals at 4500 ppm. The offspring NOAEL was 45 ppm (3.8 mg/kg bw/day).

Justification for classification or non-classification

The available data on neurotoxicity with the test substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008, and is therefore conclusive but not sufficient for classification.