4.19 Pyraclostrobin (210)(T)*

T- toxicological evaluation
*New compound

TOXICOLOGY

Pyraclostrobin is the provisionally approved ISO name for methyl N-{2-[1-(4-chlorophenyl)-1H-pyrazol-3-yloxymethyl]phenyl}(N-methoxy)carbamate. Pyraclostrobin is a member of the strobilurin group of fungicides. The strobilurin fungicides act through inhibition of mitochondrial respiration by blocking electron transfer within the respiratory chain, which in turn causes important cellular biochemical processes to be severely disrupted, and results in cessation of fungal growth. Pyraclostrobin has not been evaluated previously by the JMPR.

After oral administration of radiolabelled pyraclostrobin to rats, about 50% of the dose was absorbed. Concentrations of radiolabel in the blood peaked initially after 30 minutes, followed by a secondary peak at 8 or 24 h. The majority (74-91%) of the radiolabelled dose was eliminated in the faeces, with the remainder (10-13%) in the urine. The excretion pattern was not affected by repeated administration. In rats, the metabolism of pyraclostrobin proceeds through three main pathways. The methoxy group on the tolyl-methoxycarbamate moiety is readily lost, with few major metabolites retaining this group. Hydroxylation of the benzene and/or pyrazole rings is followed by conjugation with glucuronide and, to a lesser extent, sulphate. Many metabolites are derived from the chlorophenol-pyrazole or tolyl-methoxycarbamate moieties of pyraclostrobin after cleavage of the ether linkage between these two groups, with subsequent ring hydroxylation and glucuronide or sulphate conjugation. Metabolites were similar in both sexes and across all dose groups. No unchanged parent compound was found in the bile or urine and only small amounts were found in the faeces. The majority of the radiolabel isolated from kidney tissues was in the form of the unchanged parent compound and a demethoxylated derivative.

Pyraclostrobin has low acute toxicity when administered orally or dermally, with LD₅₀s of > 5000 and > 2000 mg/kg bw respectively, and no deaths in either case. The compound has moderate toxicity when administered by inhalation, with an LC₅₀ of 0.31-1.07 mg/l when acetone is used as the solvent, and 4.07-7.3 mg/l when Solvesso is used as the solvent. Pyraclostrobin is a mild skin and eye irritant, but is not a skin sensitizer. Clinical signs after oral administration consisted of dyspnoea, staggering, piloerection, and diarrhoea in all animals, which resolved by day 6. There were no pathology findings.

In short-term studies in mice, rats and dogs, the major toxicological findings after repeated doses of pyraclostrobin involved duodenal mucosal hypertrophy and, in some studies in rodents, erosion/ulceration of the stomach mucosa. These findings are suggestive of a local irritant action, a conclusion supported by the occurrence of vomiting in dogs. However, pyraclostrobin is not a severe skin irritant, although in rabbits the irritation was somewhat prolonged.

Reductions in body-weight gain and food consumption were observed in all species, although the pattern of the response and relationship to treatment varied. To some extent, these effects suggest local disturbance to the gastrointestinal tract and taste aversion, particularly in rabbits, although a systemic effect may also be involved, especially in rodents.

In short-term studies using repeated doses of pyraclostrobin, reduced body-weight gains were accompanied by reductions in clinical chemistry parameters (including total protein, globulin, glucose, triglycerides and creatinine) and reduced fat storage in the liver. These observations may be secondary to a disturbance of normal metabolic processes following the disruption of mitochondrial respiration, the primary biochemical mechanism by which pyraclostrobin acts. These effects may also reflect a reduced nutritional status caused by reduced food intake or food conversion. Reduced body-weight gain largely determined the minimally toxic dose in lifetime studies in rats and mice, but was not associated with toxicologically relevant alterations in clinical pathology values where these were measured in rats.

Mild anaemia associated with extramedullary haematopoiesis in the spleen was observed in rodents fed with repeated doses of approximately 400 ppm (equal to 120 mg/kg bw per day in mice and 42 mg/kg bw per day in rats) and above in short-term studies. Hepatocellular hypertrophy, in the absence of significant alterations in relevant clinical chemistry parameters or other histological evidence of liver injury, was also observed in rats receiving 120 mg/kg bw per day.

Pyraclostrobin gave negative results in an adequate battery of in vitro studies of genotoxicity and in an in vivo assay for bone marrow micronuclei in mice.

The Meeting concluded that pyraclostrobin was unlikely to be genotoxic.

The carcinogenic potential of pyraclostrobin was studied in rats and mice. While the incidence of hepatocellular adenomas was slightly increased in one study in rats fed with 200 ppm pyraclostrobin (equal to 9 mg/kg bw per day), no increase was observed in a concurrent lifetime study. Moreover, the incidence of liver adenomas in controls was considerably higher (20% versus 8%), suggesting that a low value for controls contributed to the apparent effect in the first study. There was no evidence of carcinogenic potential in mice and rats. This conclusion is supported by the observation that other strobilurin fungicides have not shown carcinogenic activity of relevance to human risk assessment.

On the basis of the above consideration and the absence of genotoxicity, the Meeting concluded that pyraclostrobin is unlikely to pose a carcinogenic risk to humans.

In a two-generation study of reproductive toxicity in rats, body-weight gains and food consumption were reduced in adults and lower body-weight gains and slightly delayed vaginal patency were observed in pups at a dose of 300 ppm (equal to 33 mg/kg bw per day). The NOAEL for general and pup toxicity was 75 ppm (equal to 8.2 mg/kg bw per day). The NOAEL for effects on reproductive performance was 300 ppm, the highest dose tested.

Two studies of developmental toxicity were conducted in rabbits and one in rats. Maternal toxicity consisting of reduced body-weight gains and food consumption was observed at 25 mg/kg bw per day and above in rats, and at 5 mg/kg bw per day and above in rabbits. In rats, the reduction in food consumption persisted beyond the treatment period and the corrected body-weight gains at termination were also reduced. In rabbits, a transient but marked reduction in food intake (and consequently in body-weight gain) after initiation of dosing was observed which resolved within 3-5 days, despite continued dosing. The pattern of the reduced body-weight gains and food consumption in rabbits indicates that they are likely to be caused by local effects on the gastrointestinal tract related to high concentrations of pyraclostrobin or taste disturbance resulting from regurgitation or leakage of the gavaging solution. Consequently, the Meeting concluded that these effects did not reflect systemic toxicity caused by pyraclostrobin. Nonetheless, the reduced nutritional status of dams, which was caused by lower food intakes at a critical time in gestation at and immediately after implantation, must be taken into account when considering the significance of the observed fetal effects at doses that were not otherwise maternally toxic. The NOAEL for maternal toxicity was 10 mg/kg bw per day in the rat and 3 mg/kg bw per day in the rabbit.

Pyraclostrobin was not teratogenic in rats, but fetal effects consisting primarily of developmental delay (incomplete ossification of sternebra and rudimentary cervical ribs) and an increased incidence of dilated renal pelves, were observed at a dose of 50 mg/kg bw per day. In rabbits, fetal effects consisting of increased post-implantation losses were observed at and above 10 mg/kg bw per day. A slight, non-significant increase in the incidence of skeletal malformations observed at 20 mg/kg bw per day was driven by an increase in the incidence of absent lumbar vertebrae at this dose. Although the incidence was not statistically significant, it exceeded the mean of historical control values and the upper bound of the range, and the Meeting could not exclude the possibility that the effect was potentially treatment related. The effects seen in rabbit fetuses were likely to be secondary to the marked nutritional deficit in the dams at a critical time in gestation. The Meeting concluded, however, that the available data did not provide a sufficient basis on which to confidently exclude other potential mechanisms, and consequently set the NOAEL for developmental toxicity in the study in rabbits at 5 mg/kg bw per day, on the basis of these fetal effects. The developmental NOAEL for rats was 25 mg/kg bw per day.

Pyraclostrobin was investigated for neurotoxicity in rats in a study in which a single dose was administered by gavage and in a 90-day study of pyraclostrobin in the diet. The NOAELs for neurotoxicity were 2000 mg/kg bw and 750 ppm (equal to 50 mg/kg bw per day) respectively, the highest doses tested. Pyraclostrobin was not neurotoxic.

The Meeting concluded that the existing database was adequate to characterize the potential hazard of pyraclostrobin to fetuses, infants and children.

Toxicological evaluation

The Meeting established an ADI of 0-0.03 mg/kg bw based on a NOAEL of 3.4 mg/kg bw per day identified in two 2-year studies in rats, on the basis of reduced body-weight gain and altered liver and stomach histology at 200 ppm and using a 100-fold safety factor.

Pyraclostrobin is not acutely toxic and short-term dosing produced no significant general toxicity, however, fetal resorptions were increased at a dose of 10 mg/kg bw per day in a study of developmental toxicity in rabbits. Although a transient but marked reduction in food intake, and consequently in body-weight gain, was observed at doses of 5 mg/kg bw per day and above after initiation of dosing in studies of developmental toxicity in rabbits, this effect resolved within 3-5 days, despite continued dosing. The pattern of the observations indicates they are likely to be caused by local gastrointestinal tract effects related to high concentrations of pyraclostrobin, or to taste disturbance resulting from regurgitation or leakage of the gavaging solution. Consequently, the Meeting concluded that these observations did not reflect systemic toxicity caused by pyraclostrobin and were not used to establish the acute RfD. The Meeting established an acute RfD of 0.05 mg/kg bw, based on the NOAEL of 5 mg/kg bw per day for fetal toxicity at 10 mg/kg bw per day in the study of developmental toxicity in rabbits and using a 100-fold safety factor. Further information on the relationship between irritation of the gastrointestinal tract and reduced body-weight gains in pregnant rabbits, and the effect of maternal nutritional deficit on fetal resorptions, may allow the acute RfD to be refined.

A toxicological monograph was prepared.

Levels relevant to risk assessment

^a Diet
^b Highest dose tested
^c Gavage
^d A marked but transient reduction in maternal food intake occurred immediately after initiation of dosing at higher concentrations

Estimate of acceptable daily intake for humans

0-0.03 mg/kg bw

Estimate of acute reference dose

0.05 mg/kg bw

Studies that would provide information useful for continued evaluation of the compound

- Observations in humans

- Studies in rabbits to explore the relationship between a marked reduction in food intake at the start of pregnancy and fetal survival and development

Summary of critical end-points for pyraclostrobin