4.3 Benalaxyl (155)

TOXICOLOGY

Benalaxyl, the ISO approved name for methyl N-(2,6-dimethylphenyl)-N-(phenylacetyl)-DL-alaninate (a racemic mixture), is a broad-spectrum phenylamide fungicide that inhibits mycelial growth of fungi and germination of zoospores. Benalaxyl was first evaluated by the 1987 JMPR (Annex 1, reference 52), when an ADI of 0-0.05 mg/kg bw was established on the basis of a NOEL of 5.0 mg/kg bw per day for hepatic enlargement in a 13-week dietary study in rats and a safety factor of 100.

Benalaxyl was considered by the present Meeting within the periodic review programme of the CCPR. The Meeting reviewed new data on benalaxyl (studies of toxicokinetics, metabolism, acute toxicity after inhalation, eye irritation, mutagenesis and several studies of toxicity with the two main soil metabolites) that had not been reviewed previously, as well as relevant data from the previous evaluation.

All pivotal studies with benalaxyl were certified as complying with GLP.

Biochemical aspects

Several toxicokinetic studies in rats given ¹⁴C-labelled benalaxyl as single and repeated oral doses showed that the active substance is rapidly and extensively absorbed and distributed by all organs and tissues, with the greatest proportion of radioactivity remaining in the intestine and its contents, and in the liver and kidneys (minor quantities). Seven days after treatment, only approximately 0.3% of the administered radiolabelled dose remained in the rat and was distributed among organs and tissues. The half-life of elimination was about 30 h after administration of single doses and 36 h after administration of repeated doses. The pattern of elimination in the urine and faeces was also similar in all situations (administration of single and repeated oral doses) and was not sex-dependent. At 48 h after dosing, the radioactivity was mainly excreted in the faeces (at least 80%), via the bile and in the urine (approximately 8%).

The metabolites of benalaxyl that appeared in the faeces and urine were similar, irrespective of dose and type of administration (single or repeated doses). Unchanged benalaxyl was not detected in the urine. Eight metabolites were identified and corresponded to approximately 65% of the radioactivity present in the faeces and urine. The identity of three additional very polar metabolites remained unknown, but their proportions were very low compared with those of some other identified compounds. Benalaxyl undergoes extensive metabolism, mainly by oxidation of the methyl group of the aniline ring to a hydroxymethyl group, and finally to the carboxylic acid; minor metabolic pathways were the hydroxylation of the phenyl ring and hydrolysis of the carboxymethyl group.

Toxicological data

Benalaxyl has low acute oral toxicity in rats and mice (LD₅₀ values were 4200 mg/kg bw and 680 mg/kg bw, respectively), low acute dermal toxicity in rats and rabbits (LD₅₀ values were > 5000 mg/kg bw and > 2000 mg/kg bw, respectively) and low acute toxicity in rats exposed by inhalation (the 4-h LC₅₀ value was > 4.2 mg/L, the highest achievable concentration). Although no significant clinical signs were observed in rats treated by oral or dermal administration, signs of intoxication including loss of equilibrium, uncoordinated movements and asthenia occurred in mice treated by oral administration. Benalaxyl is not an irritant to the skin and eyes of rabbits. In a maximization test in guinea-pigs, benalaxyl did not show sensitizing potential.

The toxicity of benalaxyl administered orally was investigated in short-term studies: a 90-day dose range-finding study for a long-term study of toxicity and carcinogenicity in mice, 5-week and 90-day studies in rats, and a 1-year study in dogs. The major target organs were the liver in mice and rats, and the testes in dogs. In the absence of any changes in clinical chemistry or histopathology, the Meeting considered that hepatic enlargement was an adaptive response and not an adverse effect.

In a 90-day study in Swiss mice, a dose-related increase in liver weights occurred at dietary concentrations of 1000 ppm and greater at 96 days and of 2000 ppm and greater at 42 days. There were no histopathological lesions associated with this increase in liver weight. The NOAEL was 5000 ppm, equal to 842 mg/kg bw per day, the highest dose tested.

In a 5-week study in Wistar rats treated by gavage, changes in haematological (coagulation time) and biochemical (increases in cholesterol, albumin and total protein, decreases in aspartate amino transferase and alkaline phosphatase activities) parameters were observed at the highest dose of 800 mg/kg bw per day. The relative weight of the liver was increased in groups treated with benalaxyl at doses of 100 mg/kg bw per day and greater. All these changes had returned to normal relative to values for controls by the end of the 2-week recovery period. The NOAEL was 100 mg/kg bw per day on the basis of changes in haematological and biochemical parameters.

In a study in Sprague-Dawley rats given diets containing benalaxyl at concentrations of up to 10 000 ppm for 13 weeks, or 12 000 ppm for 4 weeks followed by a 9-week recovery period, animals treated at 10 000 and 12 000 ppm had decreased body-weight gain and increased serum cholesterol values relative to those for controls. At 12 000 ppm, there were also some changes in haematological parameters (decreases in erythrocyte count, haemoglobin concentration and erythrocyte volume fraction in both sexes); all changes were reversible after a recovery period. Liver weight was reversibly increased in animals at 1000 ppm (males) and above (both sexes) and lobulation was observed in males in these groups, sometimes associated with rounded edges (this finding was also observed sporadically in other groups). The livers of females at 10 000 ppm were darker than normal, and diffuse steatosis was seen in both sexes at this dietary concentration, although the pattern was more severe in males. The NOAEL was 1000 ppm (equal to 59 mg/kg bw per day).

In a 1-year study in beagle dogs, the only finding that could be attributed to treatment was atrophy of the seminiferous tubules of the testes in two out of six males treated with benalaxyl at the highest dietary concentration of 800 ppm. The NOAEL in males was 200 ppm (equal to 6.5 mg/kg bw per day).

Long-term studies of toxicity and carcinogenicity were carried out in Swiss mice and Sprague-Dawley rats.

In a long-term study of toxicity and carcinogenicity, Swiss mice were given diets containing benalaxyl at concentrations of up to 3000 ppm for 78 consecutive weeks. While there was no effect on survival in female mice, a high incidence of mortality occurred in males at 1000 and 3000 ppm, mainly during the second year of the study. Because 25% of the males at the highest dose survived to termination, this study was considered to be acceptable. In males, body-weight gain was slightly depressed in all treated groups, particularly during the second year of treatment, without a dose-related effect. In females, there was no effect of treatment on body weight. In females at 3000 ppm, absolute and relative weights of the liver were significantly increased (as observed in the 90-day preliminary test). No increase in the incidence of tumours was observed when compared with the control group. The NOAEL was 250 ppm (equal to 43 mg/kg bw per day) on the basis of mortality in males. There was no evidence for carcinogenic potential in Swiss mice treated with benalaxyl for 78 consecutive weeks.

In rats given diets containing benalaxyl at concentrations of up to 1000 ppm for 104 weeks, there was no evidence of neoplastic or non-neoplastic effects related to administration of the test article. Although the incidence of hepatocellular neoplasms was found to be greater in females at the highest dose than in controls, the difference was not statistically significant, no dose-response relationship was observed and the frequency was compatible with that of spontaneous hepatocellular neoplasms. The NOAEL was 1000 ppm (equal to 44 mg/kg bw per day, the highest dose tested), in the absence of any significant findings in either sex.

The Meeting concluded that benalaxyl is not carcinogenic in rodents.

A comprehensive range of studies of genotoxicity in vitro and in vivo with benalaxyl gave consistently negative results. The Meeting concluded that benalaxyl is unlikely to be genotoxic.

In view of the absence of genotoxicity and the lack of carcinogenicity in mice and rats (albeit noting the limitation of the study in rats because the maximum tolerated dose was not attained), the Meeting concluded that benalaxyl is unlikely to pose a carcinogenic risk to humans at dietary doses and anticipated exposures of consumers or workers.

The reproductive toxicity of benalaxyl has been examined in a two-generation study in rats, and in studies of developmental toxicity in rats and rabbits.

In a two-generation (two litters per generation) dietary study of reproductive toxicity in rats, the NOAEL was 1000 ppm (equal to 53 mg/kg bw per day for the F₀ generation) for general toxicity in parent animals (decreased body weight) and adverse effects in pups (decreased pup weight and liver weight) at 5000 ppm, although fertility and reproductive parameters were not affected in the F₀ generation at dietary concentrations of up to 5000 ppm (equal to 289 mg/kg bw per day, the highest dose tested).

In Sprague-Dawley female rats given benalaxyl at doses of up to 200 mg/kg bw per day by gavage from day 6 to day 15 of gestation, no toxicity was apparent in dams. Benalaxyl induced a marginal but statistically significant increase in the delay in ossification of the cranial bones at 50 and 200 mg/kg bw per day (10%, 16%, 18% and 26% of the fetuses in the control group, and at the lowest, intermediate and highest dose, respectively). In addition, in the group receiving the highest dose a statistically significant increase of the percentage of pre-implantation losses was observed. The NOAELs for maternal toxicity, embryotoxicity and developmental toxicity were 200 mg/kg bw per day (the highest dose tested), 50 mg/kg bw per day and 12.5 mg/kg bw per day, respectively.

In female New Zealand White rabbits given benalaxyl by gavage from day 6 to day 27 of gestation, minimal maternal toxicity was manifest as weight loss during late gestation and a low gravid uterus weight at a dose of 250 mg/kg bw per day. There were no treatment-related effects on implantations. No teratogenic potential was seen, but there were statistically significant effects at a dose of 250 mg/kg bw per day on fetal weight and crown-rump lengths and on the incidence of fetuses with delayed skeletal development. The NOAELs for maternal and developmental toxicity were both 50 mg/kg bw per day.

No specific studies of neurotoxicity with benalaxyl were available; however, no evidence of neurotoxicity was apparent from the available studies of toxicity.

No adverse effects were reported in personnel involved in the production and formulation of benalaxyl, or in the use of this product in the field.

The two major soil metabolites, methyl-N-malonyl-N-2,6-xylyl-DL-alaninate (metabolite A) and N-maolonyl-N-2,6-xylyl-DL-alanine (metabolite B) were also investigated. The results of studies of acute toxicity and 90-day studies of oral toxicity with both metabolites in rats, showed that both metabolites have very low toxicity (oral LD₅₀s > 2000 mg/kg bw; NOAEL in 90-day dietary studies in rats, 923/1073 and 819/978 mg/kg bw per day for metabolite A and metabolite B, respectively, the highest doses tested) and are thus less toxic than the parent.

The results of a range of studies of genotoxicity, including tests in vitro with metabolite A and metabolite B, and a test for micronucleus formation in vivo with metabolite A, indicated that neither metabolite was genotoxic.

The Meeting concluded that the existing database on benalaxyl was adequate to characterize the potential hazards to fetuses, infants and children.

Toxicological evaluation

The Meeting established an ADI of 0-0.07 mg/kg bw based on a NOAEL of 6.5 mg/kg bw per day for atrophy of the seminiferous tubules occurring at 25 mg/kg bw per day in a 1-year study in dogs and using a safety factor of 100.

Benalaxyl has little acute toxicity and short-term dosing produced no significant general toxicity; however, a delay in ossification of cranial bones was observed at a dose of 50 mg/kg bw per day in the absence of maternal toxicity and of other markers of developmental delay in a study of developmental toxicity in rats. Although statistically significant, this is a marginal effect, but in the absence of data on historical controls, it was considered to be treatment-related. The Meeting established a conservative ARfD of 0.1 mg/kg bw for benalaxyl for women of childbearing age on the basis of a NOAEL of 12.5 mg/kg bw per day in a study of developmental toxicity in rats, and a safety factor of 100. There is no concern regarding the acute toxicity of this compound for the rest of the population, including children.

A toxicological monograph was prepared.

Levels relevant to risk assessment

^a Dietary administration
^b Gavage administration
^c Highest dose tested

Estimate of acceptable daily intake for humans

0-0.07 mg/kg bw

Estimate of acute reference dose

0.1 mg/kg bw for women of childbearing age

Unnecessary for the rest of the population

Information that would be useful for continued evaluation of the compound

Results from epidemiological, occupational health and other such observational studies of human exposures.

Critical end-points for setting guidance values for exposure to benalaxyl

* For women of childbearing age, unnecessary for the rest of the population

4.4 Carbendazim (072)

TOXICOLOGY

Evaluation for an acute reference dose

Carbendazim is the ISO approved common name for methyl 2-benzimidazole carbamate, a systemically active benzimidazole fungicide that inhibits the synthesis of â-tubulin. Carbendazim was previously evaluated by the Joint Meeting in 1973, 1977, 1983, 1985, and 1995. In 1995, an ADI of 0-0.03 mg/kg bw was established based on the NOAEL of 2.5 mg/kg bw per day in a 2-year study in dogs and a safety factor of 100.

The Meeting had been asked by the CCPR to consider the need for an ARfD for carbendazim. The present Meeting therefore evaluated relevant original studies that had been considered by previous Meetings, and newly submitted data on genotoxicity and reproductive toxicity.

Toxicological data

Carbendazim has low acute toxicity: the oral LD₅₀ is > 10 000 mg/kg bw in rats. The clinical signs of toxicity after single high doses were generally nonspecific. Degenerative changes in the testes and epididymides were observed in rats given single oral doses at ³ 1000 mg/kg bw.

In two short-term studies of toxicity in rats, the overall NOAEL was 2000 ppm (equal to 163 mg/kg bw per day) on the basis of reduced body weight and inhibition of spermatogenesis at 10 000 ppm (equal to 780 mg/kg bw per day) and above. In a 28-day dose range-finding study in dogs, the NOAEL was 500 ppm (equal to 19 mg/kg bw per day) on the basis of liver toxicity at 2500 ppm (equal to 96 mg/kg bw per day).

Carbendazim has been adequately tested in a range of assays for genotoxicity. Carbendazim causes changes in chromosome number (aneuploidy) both in vitro and in vivo (in somatic cells and germ cells) as a result of its interference with mitotic spindle proteins. The effects were seen in tests for the induction of micronuclei or aneuploidy in vivo after single high doses (100 mg/kg bw and above), with a NOAEL of 50 mg/kg bw. The mechanism by which aneuploidy is induced by carbendazim is well understood and consists of inhibition of the polymerization of tubulin, the protein that is essential for the segregation of the chromosomes during cell division. The nature of the mechanism is thus consistent with the identification of a dose that has no toxicological effect. Carbendazim does not cause gene mutations or structural chromosomal aberrations.

The Meeting concluded that the genotoxic effect of carbendazim is a threshold phenomenon.

In a study of developmental toxicity in rats given diets containing carbendazim, the NOAEL for both maternal and developmental toxicity was 10 000 ppm (equal to 747 mg/kg bw per day, the highest dose tested). There was no evidence for embryo- and fetotoxicity or teratogenicity after dietary administration of carbendazim.

Studies of developmental toxicity in rats and rabbits given carbendazim by oral gavage clearly demonstrated that carbendazim is a developmental toxicant and teratogen.

In three studies of developmental toxicity in rats treated by gavage, maternal toxicity (clinical signs, decreased body-weight gain, abortion) was observed at doses of 60 mg/kg bw per day and above. Developmental toxicity consisting of decreased fetal weights and an increased percentage of fetuses with variations per litter was seen at doses of 20 mg/kg bw per day and above. The increased incidence in variations was largely attributable to delayed development and thus correlated with the reduction in fetal weight. The incidence of malformations including hydrocephaly, anophthalmia, microphthalmia, axial skeletal malformations or malformed scapulae was significantly increased at doses of 30 mg/kg bw per day and above in two studies and at 90 mg/kg bw per day in one study, with a slightly higher incidence of skeletal malformations at 20 mg/kg bw per day than in controls. The threshold for embryo/fetotoxicity and teratogenicity was thus considered to be 20 mg/kg bw per day. For the three studies, the overall NOAEL for maternal toxicity was 30 mg/kg bw per day, while the overall NOAEL for developmental toxicity was 10 mg/kg bw per day.

In a study of developmental toxicity in rabbits treated by gavage, maternal toxicity (reduction of feed consumption and body-weight gain, abortion) was observed at 125 mg/kg bw per day, the highest dose tested. Treatment at 20 and 125 mg/kg bw per day resulted in decreased implantation, increased resorption and decreased size of live litters. Additional effects consisting of decreased fetal body weights and increased incidence of malformations of the cervical vertebrae, ribs and thoracic vertebrae were seen at 125 mg/kg bw per day. The NOAEL for maternal toxicity was 20 mg/kg bw per day and the NOAEL for developmental toxicity was 10 mg/kg bw per day.

In a study of toxicity to the male reproductive system in rats, significant testicular and efferent ductal alterations were seen 2 days after the administration of single doses at 100 mg/kg bw and above by gavage. The major cause of testicular atrophy observed at later times (70 days) after dosing was occlusion of the efferent ductules. The NOAEL was 50 mg/kg bw.

Toxicological evaluation

The Meeting established an ARfD of 0.1 mg/kg bw based on an overall NOAEL of 10 mg/kg bw per day for developmental toxicity from three studies in rats and one study in rabbits, and a safety factor of 100. The Meeting concluded that this ARfD applies only to women of childbearing age.

For the general population, including children, the Meeting established an ARfD of 0.5 mg/kg bw based on the NOAEL of 50 mg/kg bw in the study of toxicity to the male reproductive system in rats and supported by the studies on micronucleus or aneuploidy induction in vivo, using a safety factor of 100.

An additional safety factor for the severity of the effects was considered to be unnecessary, since the underlying mechanism is clearly understood and there is a clear threshold for these effects.

An addendum to the toxicological monograph was prepared.

Levels relevant to risk assessment

^a Three studies combined

Estimate of acute reference dose

0.1 mg/kg bw for women of childbearing age

0.5 mg/kg bw for the general population, including children

DIETARY RISK ASSESSMENT

Short-term intake

The International Estimated Short Term Intake (IESTI) for carbendazim, coming from the use of benomyl, carbendazim and thiophanate methyl, was calculated for 31 food commodities for which maximum residue levels were estimated by the JMPR in 1998 and 2003 and for which consumption data was available. These results are shown in Annex 4.

In 2005 the Meeting established for carbendazim an ARfD of 0.5 mg/kg bw for the general population, including children and an ARfD of 0.1 mg/kg bw for women of childbearing age. The IESTI ranged from 0 to 11% ARfD for the general population, from 0 to 30% for children and from 0 to 55% for women of childbearing age. Consumption data generated for the general population was used to assess the intake of women of childbearing age, as no consumption data is available for this group of the population.

The Meeting concluded that the short-term intake of residues of carbendazim from uses of benomyl, carbendazim and thiophanate methyl on commodities that have been considered by the JMPR is unlikely to present a public health concern.