Pesticide residues in food - 2002 - Joint FAO/WHO Meeting on Pesticide Residues

First draft prepared by
C. Vleminckx
Scientific Institute of Public Health, Division Toxicology,
Brussels, Belgium.

Metalaxyl is a 1:1 mixture of (R)-2-[(2,6-dimethylphenyl)methoxyacetylamino]propionic acid methyl ester (R-enantiomer) and (S)-2-[(2,6-dimethylphenyl)methoxyacetylamino]propionic acid methyl ester (S-enantiomer). Technical-grade metalaxyl-M consists of a minimum of 97% of the R-enantiomer and 3% of the S-enantiomer. The two compounds are fungicides used in agriculture, horticulture and forestry, which act by inhibiting mycelial growth and spore formation. Metalaxyl-M has not been evaluated previously; however, the toxicity of metalaxyl was evaluated by the 1982 Joint Meeting (Annex 1, reference 38), which established an ADI of 0–0.03 mg/kg bw on the basis of a NOAEL of 2.5 mg/kg bw per day in a 2-year study in rats.

All the studies with metalaxyl-M were conducted according to current guidelines of the OECD, Commission of the European Communities and the FIFRA of the USA and also in accordance with the principles of good laboratory practice. Several of the studies performed with metalaxyl were finalized before the OECD guidelines and regulations for good laboratory practice were enacted. Nevertheless, all the relevant studies were subjected to quality assurance and, with few exceptions, their protocols complied with today’s guideline requirements.

Absorption, distribution and excretion are generally passive processes that are similar for enantiomers, but enzymatic metabolism and protein binding to plasma or tissue proteins can show a high degree of stereoselectivity. Hence, enantiomers can be metabolized at different rates and even along different routes, although this is less common (Caldwell, 1995). A detailed comparative investigation of the metabolism of both metalaxyl-M and metalaxyl is therefore indicated. All the studies of metabolism were conducted with metalaxyl or metalaxyl-M uniformly labelled with ¹⁴C on the phenyl ring.

1.1 Absorption, distribution and excretion

Rats

The metabolic fate of [¹⁴C]metalaxyl (radiochemical purity, > 99%) was followed in four male and four female RAI rats given a single oral dose of 0.5 or 25 mg/kg bw. The animals were kept in individual metabolism cages, and urine, faeces and expired CO₂ were collected separately for analysis at 24-h intervals. When the rats were killed 144 h after dosing, the liver, fat, kidney, muscle, blood, heart, brain, lungs, spleen, ovary, testis and remaining carcass were examined for residual radioactivity.

In both sexes, irrespective of dose, more than 60% of the administered radioactivity was excreted within 24 h, and the compound was almost completely eliminated within 144 h (Table 1). While renal elimination was the preferred route in female rats, males excreted greater amounts in faeces. Males and females at 0.5 mg/kg bw excreted 37% and 55% of the administered dose in urine and 66% and 45% in faeces, respectively; while males and females at 25 mg/kg bw excreted 38% and 63% in urine and 63% and 35% in faeces, respectively. Less than 0.02% of the dose appeared in expired air. In animals at 0.5 mg/kg bw, residues were found at levels above the limit of quantitative determination only in liver, blood and carcass, still accounting for < 0.005 ppm of metalaxyl equivalents (Table 2). Animals at 25 mg/kg bw showed higher concentrations, with 0.1–0.23 ppm in liver, carcass, fat and blood and < 0.1 ppm of metalaxyl equivalents in all other tissues. The concentration of residual radioactivity in tissues was generally higher in females than in males. Two-dimensional thin-layer chromatography (TLC) of the urine in various solvent systems demonstrated the presence of four to six major metabolite fractions and about 10 minor ones, most of the metabolites being relatively polar. The pattern of metabolites was not significantly influenced by dose or by the sex of the animals. No unchanged metalaxyl was detected in urine. The results of this study show that orally administered metalaxyl is readily absorbed from the gut into the general circulation and rapidly excreted in rats. The preferred route of excretion is via the urine for females and the faeces for males. Because of the rapid elimination of the compound, the residual radioactivity in tissues was generally low (Hamboeck, 1977, 1981a).

Table 1. Kinetics of excretion of [¹⁴C]metalaxyl (% of administered dose) by rats treated by gavage

From Hamboeck (1977)

Table 2. Residual radioactivity (ppm of metalaxyl equivalents) in rat tissues 6 days after a single oral doses by gavage

From Hamboeck (1977); LOQ, limit of quantification; LOD, limit of detection

The absorption, distribution and excretion of [¹⁴C]metalaxyl (radiochemical purity, > 98%) were studied in groups of five male and five female Sprague-Dawley rats given metalaxyl at a single oral dose of 2 or 80 mg/kg bw or 2 mg/kg bw by intravenous injection. The animals were kept in individual metabolism cages, and urine, faeces and expired CO₂ were collected separately for analysis at 24-h intervals for 3 days after dosing. Biliary excretion was investigated, and enterohepatic circulation was monitored for 24 h by injecting 0.4 ml of bile collected for 6 h from male rats given metalaxyl at 80 mg/kg bw intravenously. Blood samples were taken from the caudal vein (after oral administration) or jugular vein (after intravenous injection) at various times, and portions of the samples were radioassayed. To prevent enterohepatic circulation during determination of the rate of disappearance of radioactivity from blood in animals given 2 mg/kg bw intravenously, their bile ducts were cannulated under anaesthesia, and the concentration of metalaxyl in whole blood and plasma was measured. The distribution of radioactivity in plasma, blood, brain, thyroid, lung, heart, thymus, liver, kidney, adrenal, spleen, pancreas, duodenum, testis, uterus, ovary, abdominal fat and hypogastrium, abdominal and dorsal skin, femoral muscle and bone marrow was measured 1, 24 and 72 h after administration.

After gavage, the compound was taken up readily into the general circulation. At 2 mg/kg bw, the maximum concentration (C_max) of radioactivity in blood was reached after 20 min in males (0.48 µg/ml) and 40 min in females (0.93 µg/ml) (Table 3). The decline in radioactivity showed a biphasic relationship, with half-times of 1.1 and 72 h in males and 2 and 22 h in females for the first and second phase, respectively. In the group given [¹⁴C]metalaxyl at 80 mg/kg bw, the concentration in blood reached a maximum more slowly than with 2 mg/kg bw, by 40 min after administration in males and by 100 min in females, the C_max in females (38 µg/ml) again being higher than that in males (19 µg/ml). The half-times were 1.5 h and 125 h in males and 3 h and 96 h in females for the first and second phases, respectively. The decreasing blood concentrations after 6 h suggested enterohepatic circulation of metalaxyl or its metabolites.

Table 3. Concentrations of radioactivity in blood (µg equivalents/ml) after a single oral or intravenous dose of [¹⁴C]metalaxyl

From Uesugi (1988)

The rate of disappearance of radioactivity from blood of animals treated intravenously with metalaxyl at 2 mg/kg bw fitted a two-compartment model. The half-time in whole blood was 0.42 h in males and 0.64 h in females, and the half-time in plasma was 0.41 h in males and 0.56 h in females.

The distribution of radioactivity in tissues after oral administration of metalaxyl is shown in Table 4. The concentration of radioactivity in all organs except brain and in tissues of all treated animals reached a maximum 1 h after administration and was higher than that in plasma. At 2 mg/kg bw, high concentrations of radioactivity were observed after 1 h in liver, kidney and duodenum in males and in thyroid, liver, kidney, duodenum and abdominal fat in females. The concentrations in these organs and in plasma in females was higher than that in males. Thereafter, the concentrations in most organs declined gradually, and by 72 h after administration the concentrations in liver and kidney had decreased to one-sixth to one-tenth of the values at 1 h. At 80 mg/kg bw, high concentrations of radioactivity were observed in thyroid, liver, kidney, duodenum and abdominal fat in males and in thyroid, liver, kidney, adrenal, spleen, duodenum and fat in females. After 1 h, the concentrations in most organs declined gradually, and the values at 72 h were one-half to one-tenth of that at 1 h.

Table 4. Distribution of radioactivity (µg equivalent/g) in rat tissues after a single oral administration of [¹⁴C]metalaxyl at 2 / 80 mg/kg bw

From Uesugi (1988)

The amounts of radioactivity excreted in urine and faeces and in expired air are shown in Table 5. Urinary and faecal excretion was rapid, both males and females excreting 67–84% of the administered dose within 24 h and 92–100% within 72 h. The amounts excreted in expired air by rats at 2 mg/kg bw was below the level of detection at all times, while those at 80 mg/kg bw excreted 0.001–0.006% of the administered dose.

Table 5. Cumulative excretion of radioactivity (% of dose) in rat urine, faeces and expired air after a single oral administration of [¹⁴C]metalaxyl

ND, not detected

Biliary excretion of radioactivity is shown in Table 6. When cannulated rats were given [¹⁴C]metalaxyl at 2 mg/kg bw orally, males excreted 31% in bile within 1 h, 49% within 2 h and 71% within 24 h, while females excreted 11% within 1 h, 33% within 2 h and 66% within 24 h. A clear difference between males and females was seen in the amount excreted in bile after the high dose of metalaxyl, males excreting 15% within 1 h, 29% within 2 h and 69% within 24 h and females excreting 1.2% within 1 h, 4.4% within 2 h and 54% within 24 h. After intravenous injection of [¹⁴C]metalaxyl, male rats excreted 30% in bile within 10 min, 67% within 30 min and 91% within 5 h, and females excreted 9.1% within 10 min, 36% within 30 min and 91% within 5 h, indicating differences in the transport and metabolism of metalaxyl by the liver and bile duct.

Table 6. Cumulative biliary excretion of radioactivity (% of administered dose) after a single oral dose of [¹⁴C]metalaxyl to bile-cannulated rats

From Uesugi (1988)

In rats that received bile from male rats given [¹⁴C]metalaxyl intravenously at 80 mg/kg bw, males again excreted 0.9% of the administered dose in bile within 1 h, 8.9% within 10 h and 46% within 24 h, and females excreted 0.8% within 1 h, 11% within 10 h and 19% within 24 h. Males excreted 9.1% in urine within 24 h and females excreted 6.3%. These results strongly support the existence of enterohepatic circulation of metalaxyl or its metabolites.

The results of this study show that orally administered metalaxyl is rapidly absorbed in rats through the digestive tract. The twofold higher C_max in females than in males may have been due to different excretory rates into bile, and the subsequent biphasic pattern of disappearance showed enterohepatic circulation. Metalaxyl and its metabolites were excreted rapidly in urine and faeces, the differences between the sexes being due to the differences in biliary excretion. The higher excretion rate in urine by females suggests qualitative and quantitative differences in the metabolites in bile, a difference in the reabsorption rate from the digestive tract and a different flow to enterohepatic circulation. The rates of excretion in bile were high in both sexes. No difference was found in the total amounts excreted by males and females over 24 h, but males showed a higher excretion rate at earlier times, as confirmed in experiments with intravenous administration. The average bioavailability of metalaxyl was thus about 90%. Metalaxyl translocated readily to all tissues except brain, with maximal amounts 1 h after oral administration, decreasing to relatively low concentrations in all tissues by 72 h (Uesugi, 1988).

The absorption, distribution and excretion of [¹⁴C]metalaxyl (radiochemical purity, 97.2–97.3%) were studied after intravenous and oral administrations to groups of five male and five female rats (Taconic Farms, Germantown, New York, USA). Group 1 received a single intravenous injection of 1.1 mg/kg bw, groups 2 and 4 received single oral doses of 1.1 and 200 mg/kg bw, respectively, and Group 3 received 14 daily doses of 1.1 mg/kg bw unradiolabelled compound (purity, 96.5%) orally, followed by a single oral dose of [¹⁴C]metalaxyl. For each group, one additional male and one female were designated as controls and received the vehicle only (isotonic saline for intravenous administration, ethanol and PEG-200 for oral administration). Faeces and urine were collected at various times for 7 days. Rats were killed at the completion of the study, and specified tissues collected. The study was performed in compliance with the principles of good laboratory practice (GLP) with quality assurance (QA) certification, and the protocol was in accordance with guideline 85-1 of the FIFRA Subdivision F and mostly in compliance with OECD TG 417 (1984) and TM B36 from Annex V of Directive 87/302 of the European Commission.

The total average recoveries were 102.8% for group 1, 99.7% for group 2, 103.0% for group 3 and 101.6% for group 4 (Table 7). Excretion of radioactivity was rapid and complete in all groups (Table 8), the amount eliminated in excreta ranging from 95.5% to 109.4%. More than 89% was eliminated within the first 48 h after dosing. The pattern of elimination was different in males and females, elimination via faeces predominating in males (60%) and elimination in urine predominating in females (70%). The similar patterns of excretion after oral and intravenous administration indicate that the compound was well absorbed. The high recovery of radioactivity in the faeces of intravenously dosed rats (59% in males, 36% n females) suggests the involvement of biliary secretion, which was more extensive in males.

Table 7. Recovery of radioactivity (per cent of total dose) in rats after oral or intravenous administration of [¹⁴C]metalaxyl

From Jameson (1990)

Table 8. Elimination of [¹⁴C]metalaxyl (per cent of administered dose) by rats after oral or intravenous treatment

From Jameson (1990); NS, no sample collected

At the low dose, the concentrations of [¹⁴C]metalaxyl equivalents were low in all tissues (Table 9). After 7 days, the highest concentrations of residue (average for males and females) were found in intestine (0.03 ppm) and liver (0.007 ppm). The tissue levels were not affected by the route of administration. At the high dose, residues were measurable in all tissues, the highest concentrations at 7 days being observed in the intestine (3.1 ppm) and liver (0.81 ppm). No significant difference between the sexes was seen at either dose. In all groups, < 1% of the dose was recovered in tissues after 7 days. Low concentrations were found in blood fractions; the highest average values at the high dose (males and females) were 0.62 ppm for erythrocytes and 0.057 ppm for plasma.

Table 9. Residual radioactivity (ppm metalaxyl equivalents) in rats after oral or intravenous administration of [¹⁴C]metalaxyl

From Jameson (1990); LOQ, limit of quantification

This study confirmed the previous finding that metalaxyl is rapidly and well absorbed and eliminated, with differences in excretion between males and females. Pre-treatment with 14 daily doses of 1 mg/kg bw of unlabelled metalaxyl or intravenous administration did not affect the rate or route of excretion. Reflecting the rapid elimination of the compound, the residual radioactivity in tissues was generally low; even after an oral dose of 200 mg/kg bw, the concentrations was < 1 ppm in all tissues (Jameson, 1990).

The absorption, distribution, metabolism and excretion of metalaxyl-M and metalaxyl were compared at two doses of [phenyl-U-¹⁴C]-labelled test substance (radiochemical purity, 98.5% and 97.3%, respectively) in groups of four male and four female Tif:RAIf (SPF) rats. Groups received metalaxyl or metalaxyl-M at a single oral dose of 1 or 100 mg/kg bw. Urine and faeces were collected at 24-h intervals up to 168 h after treatment, and urine was collected after 8 h (12 h from the group given the higher dose of metalaxyl-M). Blood samples were taken 0.25, 0.5, 1, 2, 4, 8, 24 and 48 h after dosing from all animals and from three additional rats of each sex per dose in the group given metalaxyl-M at the higher dose. Seven days after dosing, bone, brain, fat, gonads, heart, kidneys, liver, lungs, plasma, skeletal muscle, spleen, uterus, whole blood and residual carcass were taken for analysis. The study was performed in compliance with the principles of GLP (with QA certification).

The concentrations of radioactivity from both compounds reached a maximum in blood within 0.5–1 h after administration, irrespective of the dose, except that the maximum in females at the higher dose group of metalaxyl occurred at 4 h (Table 10). The short half-times of 9–14 h, indicating rapid depletion from blood, were also independent of test substance, dose or sex of the animals. The areas under the blood concentration–time curve (AUC) of metalaxyl-M and metalaxyl were similar for the lower dose but increased proportionally at the higher dose, except in females at the higher dose of metalaxyl, for which the AUC was 179-fold higher than at the lower dose. Generally, the bioavailability of both compounds was higher in females than in males.

Table 10. Blood kinetics of metalaxyl-M and metalaxyl after oral administration to rats

From Müller (1997)

Actual doses: metalaxyl-M, 1 and 110 mg/kg bw for males and females; metalaxyl, 1.2 and 100 mg/kg bw for males and 1.1 and 120 mg/kg bw for females

The urinary excretion and tissue residues indicated the extent of absorption was similar for metalaxyl-M (37–62%) and metalaxyl (48–61%) (Table 11). As shown previously, most of an oral dose of metalaxyl is eliminated with the bile. As the absorption process is generally not influenced by chirality, it can be assumed that both test compounds were completely absorbed. Distribution occurred rapidly: 7 days after the low dose of either substance, the concentrations of residues in all tissues were very low, not exceeding 0.010 ppm of parent equivalents (Table 12). The pattern of distribution was similar at the higher dose but approximately 100-fold greater. Depletion of the racemate metalaxyl from adipose tissue was markedly slower than that of the R-enantiomer metalaxyl-M, owing to a slightly greater tendency of the racemate to form lipophilic metabolites than the R-enantiomer and subsequent deposition in adipose tissue. However, this metabolic difference applied only to 0.01% and 0.03% of the dose of metalaxyl-M and metalaxyl, respectively.

Table 11. Absorption (per cent of dose) of metalaxyl-M and metalaxyl after oral administration to rats

From Müller (1997)

Table 12. Tissue residues (ppm of parent equivalents) 7 days after oral administration to rats

From Müller (1997); LOQ, limit of quantification: LOD, limit of detection

LOD, limit of detection; LOQ, limit of quantification

The excretion pattern was essentially the same for metalaxyl-M and metalaxyl (Table 13). In all groups, females showed slightly greater renal elimination than males. With both compounds, the administered dose was rapidly and almost completely eliminated, independently of dose or the sex of the animals. The blood kinetics, absorption, distribution and rate and route of excretion were not influenced by chirality (Müller, 1997).

Table 13. Excretion of metalaxyl-M and metalaxyl (per cent of dose) after oral administration

From Müller (1997)

1.2 Biotransformation

Rats

A generalized metabolic pathway for metalaxyl and metalaxyl-M in rats is shown in Figure 1.

Figure 1. Proposed metabolic pathway for metalaxyl and metalaxyl-M in rats, goats and hens

Metabolite 1, N-(2,6-dimethylphenyl)-N-(methoxyacetyl)alanine; metabolite 2,
N-[(2-hydroxymethyl)-6-methylphenyl]-N-(methoxyacetyl)alanine; metabolite 3,
N-(2,6-dimethyphenyl)-N-(hydroxyacetyl)alanine methyl ester; metabolite 4,
N-(carboxycarbonyl)-N-(2,6-dimethylphenyl)alanine methyl ester; metabolite 5,
N-hydroxyacetyl-2,6-dimethylaniline; metabolite 6, N-(2,6-dimethylphenyl)-N-(hydroxyacetyl)alanine;
metabolite 7, N-(2,6-dimethyl-5-hydroxyphenyl)-N-(methoxyacetyl)alanine methyl ester;
metabolite 8, N-(2-hydroxymethyl-6-methylphenyl)-N-(methoxyacetyl)alanine methyl ester;
metabolite 9, N-(2-carboxy-6-methylphenyl)-N-(methoxyacetyl)alanine methyl ester; metabolite 10,
N-(2,6-dimethylphenyl)methoxy-acetamide; metabolite 11, N-[(2,6-dimethylphenyl)alanine; metabolite 12,
N-(2-carboxy-6-methylphenyl)-N-methoxyacetyl)alanine; metabolite 13,
N-(carboxycarbonyl)-N-(2,6-dimethylphenyl)alanine; metabolite 14, [(2,6-dimethylphenyl)amino]oxoacetic acid

Conjugates not shown

* Chiral centre

The degradation of [¹⁴C]metalaxyl (purity, > 99%) was investigated in a preliminary study in 16 female Tif:RAI (SPF) rats after a single oral dose of 28 mg/kg bw. Urine and faeces were collected for 48 h. Within that time, 64% of the radioactivity was excreted in urine and 33% in faeces. The identified metabolites accounted for about 30% of the radioactivity in urine, equivalent to 19% of the dose. The following urinary metabolites were identified chromato-graphically and spectroscopically: N-(2-hydroxymethyl-6-methylphenyl)-N-(methoxyacetyl)-alanine methyl ester (14% of the dose, metabolite 8 ‘B’ isomer), N-(2,6-dimethylphenyl)hydroxy-acetamide or N-hydroxyacetyl-2,6-dimethylaniline (3%, metabolite 5), N-(2,6-dimethylphenyl)-N-(methoxyacetyl)alanine (2%, metabolite 1) and N-(2,6-dimethylphenyl)methoxyacetamide (only in free form, 0.3%, metabolite 10).

The structures identified so far show that the metabolism of metalaxyl proceeds primarily via oxidative and hydrolytic processes: (i) methyl ester hydrolysis, (ii) N-dealkylation, (iii) methyl ether cleavage and (iv) benzylic methyl oxidation. Most of the metabolites formed were subsequently conjugated by glucuronic acid and excreted via the kidney. They were therefore found in urine in both free and conjugated forms. Products formed by ring hydroxylation in the aniline moiety, as reported for lidocaine, mepivacaine and bupivacaine, which contain the same aniline moiety, were not found in this study (Hamboeck, 1978).

In a follow-up to the previous studies, the metabolic fate of [¹⁴C]metalaxyl (radiochemical purity, > 98%) was investigated in 24 female Tif:RAI (SPF) rats after administration of a single oral dose of 28 mg/kg bw. Urine and faeces were collected for 48 h. Within this time, 58% of the radioactivity was excreted in urine and 32% in faeces. The metabolites present in urine and faeces that were identified chromatographically and/or spectroscopically were N-(2,6-dimethylphenyl)-N-(hydroxyacetyl)alanine (39% of the dose, metabolite 6), N-(2-hydroxymethyl-6-methylphenyl)-N-(methoxyacetyl)alanine methyl ester (14%, metabolite 8 ‘A’ isomer), N-(2-6-dimethylphenyl)-N-(methoxyacetyl)alanine (4.1%, metabolite 1), N-hydroxyacetyl-2,6-dimethylaniline (2.9%, metabolite 5), N-(2-carboxy-6-methylphenyl)-N-(methoxyacetyl)alanine methyl ester (1.2%, metabolite 9), N-(2,6-dimethyphenyl)-N-(hydroxyacetyl)alanine methyl ester (0.9%, metabolite 3), 4-(2,6-dimethylphenyl)-3-methylmorpholine-2,5-dione (0.6%, isomeric lactone form of metabolite 6), metalaxyl (0.4%) and N-(2,6-dimethyl-5-hydroxyphenyl)-N-(methoxyacetyl)alanine methyl ester (0.3%, metabolite 7). The urinary metabolites were partially conjugated with glucuronic acid.

At least four independent pathways of biotransformation degrade metalaxyl in rats: (i) hydrolytic cleavage of the carboxyl methyl ester group, (ii) hydrolytic (or oxidative) cleavage of the methyl ether moiety, (iii) oxidation of the toluene methyl side-chain to the benzylic alcohol derivative and (iv) oxidation of the phenyl moiety to form phenols. Secondary biotransformation pathways are N-dealkylation at the 2-aniline propionic acid bond, oxidation of benzylic alcohol to the benzoic acid derivative and conjugation of metabolites with glucuronic acid.

Metalaxyl was effectively metabolized by rats, preferably by hydrolytic and oxidative reactions. The products formed were readily excreted in urine and faeces, and also as conjugates with glucuronic acid in urine. Urine and faeces generally contained the same metabolite structures, indicating their common origin from the general circulation (Hamboeck, 1981b).

The distribution, excretion and metabolism of [¹⁴C]metalaxyl (radiochemical purity, 96.9%) were studied after intravenous and oral administrations to groups of five male and five female Sprague-Dawley rats. Groups 1 and 2 received a single dose of 1.1 mg/kg bw intravenously or orally. Group 3 received metalaxyl (purity, 96.5%) in 14 daily oral doses of 1.4 mg/kg bw and on day 15 received [¹⁴C]metalaxyl at a single oral dose of 1.1 mg/kg bw. Group 4 received [¹⁴C]metalaxyl at a single oral dose of 200 mg/kg bw. Urine and faeces were collected for 7 days, and the radioactive residues were quantified. Rats were killed 168 h after dosing, and tissues were taken for determination of residues. Urine was characterized by TLC and sequential enzymatic hydrolysis. Faeces were extracted with methanol:water (80:20, v:v) to solubilize a minimum of 91% of the radioactive residue. The study was performed in accordance with the FIFRA guidelines (40 CFR part 158.135) for general metabolism studies and in compliance with the principles of GLP (with QA certification).

[¹⁴C]Metalaxyl was almost completely eliminated (93–98%) in the urine and faeces of rats within 72 h after intravenous or oral administration. Excretion was rapid and complete. Excretion occurred primarily via the kidneys in females (70%) and in faeces in males (60%). The similarity of the elimination profiles in rats treated intravenously and orally indicated that metalaxyl was well absorbed. Biliary excretion was suggested by the high faecal recoveries in male (59%) and female (36%) rats after intravenous administration. The concentrations in tissues were low in all groups at the lower dose, the highest values being found in intestine (0.045 ppm) and liver (0.008 ppm). A 200-fold increase was found with the higher dose, the highest values again being found in intestine (3.5 ppm) and liver (0.98 ppm). These findings would result from localized dynamic enterohepatic circulation, with metalaxyl conjugates and intestinal beta-glucuronidase moving through a conjugation, deconjugation, reabsorption and reconjugation cycle to eliminate the xenobiotic.

In urine, the patterns of metabolites were qualitatively similar, regardless of sex and dose. Metabolism was extensive, with a possible 33 metabolites found. Ten metabolites were identified by co-chromatography with standards. Nine metabolites (including metalaxyl) were purified and identified from mass and nuclear magnetic resonance spectra. Metabolites 6, 1, 9 and 8 ‘B’ isomer were the major metabolites in rat urine (Table 14), while metabolites 5 and 4 (N-(carboxycarbonyl)-N-(2,6-dimethylphenyl)alanine methyl ester) were present in moderate amounts. Metabolite 8 ‘A’ isomer, N-(2,6-dimethylphenyl)alanine (metabolite 11) and metabolite 7 were minor metabolites. Metalaxyl co-chromatographed with metabolite 3 as minor components, and a new metabolite, N-[(2-hydroxymethyl)-6-methylphenyl]-N-(methoxyacetyl)alanine (metabolite 2), was identified. Free metabolites represented 7.9–30% of the dose, accounting for 20–51% of the total ¹⁴C-labelled residue in urine. Conjugates represented 50–97% of urinary radioactivity. After enzyme hydrolysis, most of the metabolites could be partitioned into ethyl acetate and co-chromatographed with free metabolites and standards. Urinary conjugation included glucuronide, sulfate and possibly peptide adducts.

Table 14. Major metabolites of metalaxyl in rats

From Itterly (1990)

In faeces, metabolite 6 was the main metabolite in females. Metabolites 6 and 9 co-chromatographed as a major zone for males. Metabolite 9 was present in moderate amounts in females, and metabolites 1 and 8 ‘B’ isomer were found in both sexes. Metalaxyl and metabolite 3 co-chromatographed as minor components. Metabolites 3 ‘A’ isomer and 5, 4, 11, 7 and 2 were all minor faecal metabolites. Unconjugated metabolites identified in faecal extracts represented 11–19% of the dose and accounted for 46–76% of the total ¹⁴C-labelled residue. Metabolite conjugation accounted for 29–51% of the faecal radioactivity, which was hydrolysed predominantly to aglycones with beta-glucuronidase (18–42%). Sulfate conjugates accounted for 3.8–12% of the radioactivity.

The extensive biotransformation of metalaxyl in rats is accounted for by demethylation, N-dealkylation and hydroxylation, followed by glucuronide and sulfate conjugation. Metalaxyl was extensively metabolized in all groups, little or no unchanged metalaxyl being eliminated in urine or faeces. TLC showed that the biotransformation products were similar in all groups, and approximately the same relative proportions were observed in urine and faeces. A sizeable proportion of metabolites was conjugated in both urine and faeces, especially at the lower dose. The metabolic route appeared to involve three major and one minor pathways (see Figure 1). Demethylation of the ether gave the alcohol, metabolite 3, with stepwise demethylation of the ester forming the alcohol acid, metabolite 6, which was the major metabolite in urine and faeces. Further oxidation of the alcohol, metabolite 3, formed the acid, metabolite 4. N-Dealkylation of metabolite 3 gave metabolite 5, the hydroxyacetamide. Oxidation of the aromatic methyl of metalaxyl formed the benzylic alcohol isomers, A and B, of metabolite 8. The methyl ester of isomer A was demethylated, forming an acid, metabolite 2, the benzylic alcohol of metabolite 1. The B isomer was oxidized to the benzoic acid, metabolite 9. Demethylation of the ester of metalaxyl formed the acid ether, metabolite 1, which was a major urinary metabolite in females at the higher dose and a major faecal metabolite in males. In the minor pathway, metalaxyl underwent hydroxylation at the meta position on the phenyl ring, forming a mixture of isomers of metabolite 7. All the metabolites that were isolated undergo phase II conjugation reactions and are present as glucuronide and sulfate conjugates (Itterly, 1990).

The pattern of metabolites of the R-enantiomer, metalaxyl-M, and the racemate, metalaxyl, in rats was also investigated in the study of Müller (1997), the protocol of which is described above. Faeces were extracted with acetonitrile and acetonitrile:water, such that 91–95% of the radioactivity present was extracted, and the composite urines from each group and the faecal extracts were analysed quantitatively by two-dimentional TLC.

Apart from stereochemistry, the metabolic patterns of metalaxyl-M and metalaxl were similar (Tables 15 and 16). Both were extensively metabolized, yielding 17 and 13 metabolite fractions in urine and faeces, respectively, independently of dose and the sex of the animals. The dose- and sex-related differences in the concentrations of metabolites metalaxyl found by Itterly (1990) were also seen for metalaxyl-M. Only the concentration of metabolite fraction U4 in male rats at the higher dose depended markedly on stereochemistry. Therefore, the metabolic pathways deduced for metalaxyl are also valid for metalaxyl-M (see Figure 1).

Table 15. Quantitative pattern of metabolites of metalaxyl-M and metalaxyl in urine of rats (per cent of dose)

From Müller (1997)

U, urinary metabolite

Table 16. Quantitative pattern of metabolites of metalaxyl-M and metalaxyl in faeces of rats (per cent of dose)

From Müller (1997)

F, faecal metabolite

The study of Itterly (1990) showed that metalaxyl undergoes extensive phase II reactions, namely conjugation with sulfuric acid and glucuronic acid. Sulfonation and glucuronidation are competing pathways for hydroxylated metalaxyl metabolites, and sulfonation can be superseded by glucuronidation at increasing concentrations of substrate, resulting in a quantitative shift of metabolite distribution. These reactions are catalysed by sulfontransferases and UDP-glucuronyltransferases, which are known to discriminate between enantiomers. In the case of metabolite U4, the preferred substrate for a sex- and dose-dependent shift of conjugation appeared to be the S-enantiomer, as the concentration of the R-enantiomer was not affected by the dose. The pattern of metabolites in urine and faeces thus indicated that, aside from stereochemistry, the metabolic pathways of metalaxyl-M (the R-enantiomer) and metalaxyl (the racemate) are similar (Müller, 1997).

Goats

One lactating goat was given 7 ppm of [¹⁴C]metalaxyl in gelatine capsules orally for 10 consecutive days, and urine, faeces, milk, volatiles and CO₂ were collected daily. Blood was collected every other day and on the day of sacrifice, day 10. At sacrifice, 24 h after the last dose, brain, omental fat, skeletal fat, tenderloin muscle, leg muscle, heart, kidney, liver and intestinal contents were sampled. The radioactivity in urine was characterized by partitioning, TLC and electrophoresis.

At sacrifice, 94% of the administered radioactivity was found in urine and 12% in faeces; the rumen and intestinal contents contained only 0.77% of the total. Less than 0.01% of the administered dose was excreted in milk, which contained concentrations of 0.003–0.008 ppm, and little was found in the blood (0.06%) or tissues (0.87%). The residual radioactivity was highest in liver (0.057 ppm), skeletal fat (0.023 ppm) and kidneys (0.019 ppm). The total recovery of radioactivity was 107.22%. The goat metabolized metalaxyl to polar, organic acidic products. Enzyme analysis indicated that the metabolites were natural conjugates rather than simple metabolites. Two-dimensional TLC comparisons of the metabolites in goat and rat urine showed that the metabolites were the same, goat urine containing more polar metabolites than rat urine.

Thus, [¹⁴C]metalaxyl fed to one goat was rapidly absorbed, metabolized and excreted. Radioactivity did not accumulate in tissues and was not secreted in milk. The results of TLC suggested that metalaxyl is metabolized by the same pathways in goats and rats (Marco, 1978).

In a study conducted according to Guideline 171-4 of FIFRA Subdivision O and in compliance with the principles of GLP (with QA certification), two lactating goats were given [¹⁴C]metalaxyl (radiochemical purity, > 97%) in gelatine capsules at a dose of 150 mg/kg bw per day for 4 days. The dose was equivalent to a dietary concentration of 77 ppm. During the dosing period, urine and faeces were collected daily and milk twice a day. The goats were killed 6 and 7 h after the last dose, and blood and samples of leg muscle, omental fat, perirenal fat, kidney, tenderloin, gall-bladder, liver, heart and rumen were collected. Urine, tissue and milk extracts were treated with glucuronidase and profiled on TLC.

During the observation period, a total of 76% of the administered radioactivity was eliminated, with 67% in urine, 9.3% in faeces and 0.1% in milk (Table 17). The concentrations of residues in milk were highest on day 4, amounting to 0.12 and 0.415 ppm metalaxyl equivalents for the two goats. At sacrifice, 3.8% of the administered radioactivity was found in the intestinal tract. The tissues contained 1% of the total dose, individual residual concentrations ranging from 0.065 ppm metalaxyl equivalents in tenderloin to 2.3 ppm in kidney (Table 18).

Table 17. Mean excretion of [¹⁴C]metalaxyl in two goats given a concentration of 77 ppm

From Emrani (1990, 1991)

Table 18. Mean residues of [¹⁴C]metalaxyl equivalents in two goats 6–7 h after administration of a dose equivalent to 77 ppm

From Emrani (1990, 1991)

Co-chromatography allowed identification of 28–73% of the radiactive residues present in tissues, 90% of those in milk and 78% of the radioactive materials in urine. The main metabolites of metalaxyl in urine were metabolites 6 (43%), 3 (3.9%), 1 (1.6%), 7 (< 1.7%) and both isomers of metabolite 8 (8.7% A; 19% B) (see Figure 1). In tissues, the main metabolites identified were metabolite 6 and both isomers of metabolite 8. The main metabolite in milk was metabolite 3, mostly conjugated to fatty acids (66%).

Thus, in goats, metalaxyl was hydrolysed to the ester alcohol and the acid alcohol, which may be N-dealkylated. Alternatively, oxidation can lead to either benzylic alcohol or phenolic derivatives. Most of the urinary metabolites were present as glucuronic acid conjugates. The main metabolites in milk appeared to be lipophilic conjugates of the acid alcohol or the ester alcohol (Emrani, 1990, 1991).

Chickens

Five laying hens were given a diet containing [¹⁴C]metalaxyl (radiochemical purity, > 97%) at a concentration of 100 ppm, equivalent to 10 mg/kg bw per day, for 4 days. Eggs and excreta were collected daily. The birds were killed 6 h after the last dose, and selected tissues were taken to determine the amount and nature of the metabolites. The study was conducted according to Guideline 171-4 of the FIFRA Subdivision O and in compliance with the principles of GLP (with QA certification). Metalaxyl was almost completely eliminated in the excreta (91%) (Table 19). Only marginal amounts of radioactivity were detected in eggs (0.04% in whites and < 0.04% in yolks) and 0.9% in edible tissues. The concentrations of residue levels were about 0.5 ppm in most organs, only gizzard, liver and kidney containing larger amounts (Table 20). In eggs, the concentrations were 0.13–0.18 ppm in whites and 0.014–0.21 ppm in yolks. The excreta contained mostly (28%) metabolite 6, 2-[(1-carboxyethyl) (methoxyacetyl)amino]-3-methyl benzoic acid (metabolite 12), metabolites 3 and 8 and unchanged metalaxyl (see Figure 1). Minor amounts of metabolite 9, [(2,6-dimethylphenyl)amino]-oxoacetic acid (metabolite 14), N-(carboxycarbonyl)-N-(2,6-dimethylphenyl) alanine (metabolite 13) and metabolite 4 were also found. Additional, unidentified metabolites were detected. Chromatographic examination showed that most of the residual radioactivity in tissues was in the form of the A and B isomers of N-[2-(hydroxymethyl)-6-methylphenyl]-N-(hydroxyacetyl)alanine (metabolites P1 and P2) and metabolites 6 and 8. The main residues in egg yolk were metabolites 6, P1 and P2. The whites contained mainly metabolites P1, 8, P2 and P3 (the structure of which is only partially known) and unchanged metalaxyl. Most of the metabolites were glucuronic, sulfuric or fatty acid conjugates.

Table 19. Excretion of [¹⁴C]metalaxyl by hens given a concentration of 100 ppm

From Kennedy (1990, 1991)

Table 20. Mean residues of [¹⁴C]metalaxyl equivalents in hens 6 h after administration of a diet containing 100 ppm

From Kennedy (1990, 1991)

The results show that the metabolism of metalaxyl in laying hens initially involves oxidation and demethylation of the parent compound. Sequential demethylation of the ether and the ester groups gives first the alcohol, metabolite 3, and then the hydroxy acid, metabolite 6. The ester alcohol, metabolite 3, undergoes conjugation with both fatty acids and glucuronic acid, the latter known as metabolite P3a. Oxidation of the benzylic carbon of metalaxyl produces the benzylic alcohol, metabolite 8, which undergoes sequential demethylation of the ether moiety to give metabolite P0 (N-[(2-hydroxymethyl)-6-methylphenyl]-N-(methoxyacetyl)alanine), the free acid of metabolite 8, and the ester moieties to give metabolites P1 and P2. These metabolites can undergo conjugation with fatty acids. The benzylic alcohol of metabolite 8 also forms the sulfuric acid conjugate P4 and to a minor extent the benzoic acid, metabolite 9.

Comparison of the metabolites found in hen excreta with those found in goat and rat urine indicates that the major metabolic pathways in hens are substantially the same as those in goats and rats (Figure 1). The differences between species are due primarily to the faster metabolic rate and greater tendency for oxidative transformation in hens (Kennedy, 1990, 1991).

All the data suggest similar pathways in all three species (Emrani, 1990, 1991).

1.3 Dermal absorption

The dermal absorption of [phenyl-U-¹⁴C]-metalaxyl-M (purity, 97.3%), formulated as an emulsifiable concentrate containing active substance at 480 g/l, was tested in groups of 12 male Tif:RAIf rats at a dose of 0.094 mg/cm² (2% dilution) or 4.7 mg/cm² (undiluted formulation) in a volume of 100 µl. The substance was applied for 8 h inside a 10-cm² ring, which was glued to the clipped skin of the animals and covered with non-occlusive tape. Subgroups of four animals per dose were killed at 8 (directly after washing of the skin), 24 and 48 h, and urine and faeces were collected. The washing solution, including cotton swabs, ring and covering tape, and the cage washing solution were also collected. Whole blood, plasma, treated and untreated skin and carcasses were retained after sacrifice. Blood samples were taken 0.5, 1, 2, 4, 6, 8, 12, 24 and 48 h after application. The study was performed in compliance with the principles of GLP (with QA certification).

The lower dose of metalaxyl-M was rapidly absorbed; a first maximum in blood (0.06 ppm) was reached 1 h after application (Table 21), and a second maximum (0.045 ppm) was reached after 12 h. The concentration of radioactivity in blood decreased considerably between 12 and 48 h after treatment. After administration of the higher dose, the first maximum in blood (0.44 ppm) was reached 8 h after application. The wash-off effect—an increase in blood concentration after removal of the compound from the skin—was more pronounced than at the lower dose, and the second maximum (1.5 ppm) was reached at 24 h. The concentration of radioactivity in blood decreased between 24 and 48 h.

Table 21. Kinetics of radioactivity (ppm of equivalents) from [phenyl-U-¹⁴C]metalaxyl-M in four rats 48 h after receiving a dermal application

From Mewes (1998a); LOQ, limit of quantification

Within 8 h, 26% of the lower dose and 3% of the higher dose had been absorbed systemically (Table 22); 35% of the lower and 16% of the higher dose were absorbed within 48 h. At both doses, systemic absorption increased even after the substance had been removed from skin, but the absorbed metalaxyl-M was rapidly excreted in the urine and faeces. Some substance remained on the skin after washing at 8 h, but the amount decreased rapidly up to 48 h, indicating that metalaxyl-M in the epidermis and dermis during exposure was systemically absorbed after washing. The actual amounts absorbed suggest that the absorption process was saturated at the higher dose. The rate of penetration was only six times higher at the higher dose (18 µg cm²/h) than at the lower dose (3 µg cm²/h) although the dose increased by a factor of 50. The absorbed radioactivity was excreted in similar amounts in urine and feces. The terminal concentrations in blood and plasma after the lower dose were similar 8 and 24 h after application but had decreased considerably by 48 h (Table 23). With the higher dose, the blood and plasma concentrations increased between 8 and 24 h but decreased thereafter.

Table 22. Recovery (% applied dose) of radioactivity after dermal exposure of rats to [phenyl-U-¹⁴C]metalaxyl-M

From Mewes (1998a)

^a Systemic absorption plus treated skin

Table 23. Terminal concentrations in blood and plasma (ppm equivalents) of radioactivity after dermal exposure of rats to [phenyl-U-¹⁴C]metalaxyl-M

From Mewes (1998a)

The Meeting considered that at least some of the substantial amount of metalaxyl-M remaining on treated skin after washing was available for systemic absorption. On the basis of the average values for the sum of systemic absorption and skin deposition at 8, 24 and 48 h, the absorption was 44% of the lower dose and 22% of the higher dose (Mewes, 1998a).

Penetration of [phenyl-U-¹⁴C]metalaxyl-M (purity, 97.3%), formulated as an emulsifiable concentrate containing 480 g/l of active substance, through rat and human epidermis was compared in vitro. Epidermal membranes were set up in flow-through diffusion cells, and the perfusates were collected at defined intervals. Metalaxyl-M was applied at 0.083, 0.76 or 40 mg/cm² to rat epidermis and 0.083, 0.77 or 40 mg/cm² to human epidermis for 48 h. The two lower doses reflected the concentrations used for foliar (0.2% dilution) and soil application (2% dilution), respectively, and the highest dose represented undiluted formulation. The study was performed in compliance with the principles of GLP (with QA certification).

As seen in Table 24, metalaxyl-M at concentrations used in the field and as undiluted formulation penetrated human skin more slowly and to a significantly smaller extent than through rat skin. The ratio for rat:human was 6:1 at the lowest dose and 3:1 at the highest dose. The 50:50 mixture of ethanol:water used as receptor fluid in the study may have resulted in an artificially high value for absorption (Mewes, 1998b).

Table 24. Penetration of metalaxyl-M through rat and human epidermis in vitro

From Mewes (1998b)

The results of this study were used to compare dermal absorption between species semi-quantitatively. Thus, human dermal absorption in vivo was estimated as the systemic absorption in rats in vivo divided by the factors for species differences determined in vitro. A figure of 10% for dermal absorption was considered an appropriate compromise, in view of the uncertainties in the individual studies. This figure was based on a dermal absorption of 40% for rats in vivo and a fourfold correction for human skin in vitro.

2.1 Acute toxicity

(a) Lethal doses

The acute toxicity of metalaxyl-M after administration by the oral, dermal and inhalation routes is summarized in Table 25.

Table 25. Studies of the acute toxicity of metalaxyl-M

All studies were performed in male and female animals according to good laboratory practice, with quality assurance certfication

CMC, carboxymethylcellulose

Groups of five male and five female fasted mice were given metalaxyl-M (purity, 97.1%) orally at a dose of 500 or 1000 mg/kg bw for males and 200, 500 or 1000 mg/kg bw for females, and were observed for 14 days before sacrifice. The experimental protocol was not fully in compliance with OECD TG 401 (1987) or TM B1 from Annex V of Directive 92/69/EEC, as only two doses were given to males. Two of five males at 1000 mg/kg bw and one of five females at doses >500 mg/kg bw died, and and three of five females were found dead and two were killed for humane reasons. At 1000 mg/kg bw, male and female animals showed the following treatment-related symptoms: ventral or lateral recumbency, severe dyspnoea, reduced locomotor activity, convulsions, tremor and tonic spasms. Ataxia, hunched posture and piloerection were found in surviving males only, all of which recovered fully by day 7. At 500 mg/kg bw, ventral or lateral recumbency were seen in two males and two females, and dyspnoea, reduced locomotor activity, hunched posture and piloerection were seen in all animals, whereas tonic spasms, convulsions and ataxia were seen in individual animals. All males had recovered by day 4 and all surviving females by day 2. At 200 mg/kg bw, all females presented with hunched posture and piloerection but fully recovered within 1 day. At autopsy, no deviations from normal morphology were found. The body weights of surviving animals were not affected by the treatment (Winkler, 1996c).

Metalaxyl-M (purity, 97.9%) was administered orally to groups of five male and five female fasted rats at a dose of 500, 1000 or 2000 mg/kg bw for males and 200 or 500 mg/kg bw for females, and the animals were observed for 14 days before sacrifice. The experimental protocol was not fully in compliance with OECD TG 401 (1987) or TM B1 from Annex V of Directive 92/69/EEC, as only two doses were given to females. Four of five females at 500 mg/kg bw, three of five males at 1000 mg/kg bw and all males at 2000 mg/kg bw died on the day of administration after observation of non-specific symptoms such as piloerection, abnormal body position, dyspnoea and reduced locomotor activity. Convulsions and/or tonic spasms occurred in two males and four females given 500 mg/kg bw and all males at 1000 and 2000 mg/kg bw. Ataxia was seen in two females at 200 mg/kg bw and tremor in one male at 500 mg/kg bw. Four females at 500 mg/kg showed increased irritability. Males at 2000 mg/kg bw showed vocalization, respiratory sounds and cyanosis. The surviving animals recovered within 3–6 days. At autopsy, one male at 2000 mg/kg bw (which died shortly after treatment) had a spotted thymus. No deviations from normal morphology were found in the other animals (Schoch, 1994a).

Five male and five female rats were exposed to metalaxyl-M (purity, 97.1%) for 4 h in a nose-only system to a mean aerial concentration of 2.3 mg/l, the highest attainable concentration, measured in the breathing zone. The test atmosphere was generated by nebulizing the material into small droplets with a compressed air-driven machine. The mass median aerodynamic diameter of the particles was 2.1 µm with a geometric standard deviation of 1.4 µm, which ensured exposure of the bronchioles and alveoli of the animals. The animals were observed for 14 days before sacrifice. The experimental protocol was in compliance with OECD TG 403 (1981) and TM B2 from Annex V of Directive 92/69/EEC. No deaths occurred. Slight shallow breathing was observed in all rats during the first 3 h of exposure, and clear restlessness was seen from the second hour of exposure and onwards. Two female rats also showed slightly decreased breathing frequency during the last hour of exposure. Shortly after exposure, four females had a hunched appearance, and one female had slightly decreased breathing frequency and incoordination. The only abnormalities seen during the 14-day observation period were fatty, yellow, discoloured fur and a small alopecic area in one female. No abnormalities in body-weight gain were observed. At autopsy, no abnormalities were found (Arts, 1995).

Five male and five female rats received dermal applications of metalaxyl-M (purity, 97.3%) at a dose of 2000 mg/kg bw under a semi-occlusive dressing. The dressing was removed after 24 h, and the animals were observed for 14 days before sacrifice. The experimental protocol was in compliance with OECD TG 402 (1987) and TM B3 from Annex V of Directive 92/69/EEC. No deaths occurred, and no clinical symptoms or signs of local irritation were found. At autopsy, no deviations from normal morphology were noted (Schoch, 1994b).

Thus, in rats, the oral LD₅₀ was 380 mg/kg bw for females and 950 mg/kg bw for males; the dermal LD₅₀ was > 2000 mg/kg bw, and the LC₅₀ (4-h exposure) was > 2.3 mg/l of air. In addition to nonspecific clinical signs, reduced locomotor activity, ataxia, convulsions, tremor and spasms were seen after treatment with metalaxyl-M; however, these effects occurred only at or near lethal doses and were considered not to be indicative of neurotoxic potential (see below). Surviving animals recovered within 3–6 days.

The acute toxicity of metalaxyl is summarized in Table 26. Metalaxyl had qualitatively and quantitatively similar effects to the R-enantiomer, metalaxyl-M. All of the studies except that reported by Hartmann (1992) were considered only as providing additional information in view of the limited data presented. In the study of Naidu & Radhakrishnamurty (1978), rats developed symptoms of central nervous system poisoning including tremors, twiches, tonic extension, loss of rightening reflex, ataxia and hypnosis within 5–10 min of intraperitoneal injection of metalaxyl. Death occurred within 10–15 min at 300–400 mg/kg bw and within 1–2 h at lower doses.

Table 26. Studies of the acute toxicity of metalaxyl

CMC, carboxymethylcellulose

(b) Dermal and ocular irritation and dermal sensitization

Dermal irritation: The dermal irritation potential of metalaxyl-M (purity, 97.3%) was tested in three male New Zealand white rabbits to which 0.5 ml of test material was applied under an occlusive dressing for 4 h. The skin reactions were evaluated 1, 24, 48 and 72 h after removal of the patch. Slight erythema (score 1 in two rabbits) was observed only 1 h after removal of the bandages, but no skin reactions were seen subsequently in any animals, and the study was terminated. Metalaxyl-M was not irritating to the skin. The study was performed according to GLP, and the protocol was in compliance with TM B4 from Annex V of Directive 92/69/EEC (Marty, 1994a).

Ocular irritation: The ocular irritation potential of metalaxyl-M (purity, 97.3%) was tested in one male and two female New Zealand white rabbits which received 0.1 ml of the substance in the conjunctival sac of the left eye; the other eye was left untreated to serve as a control. The eyes were examined for irritation with a slip-lamp, and any ocular reactions were recorded 1, 24, 48, 72 h and 7, 10, 14, 17 and 21 days after instillation. Clear signs of ocular irritation were seen in all animals, comprising corneal opacity grades 1–2, grade 1 iridal lesions and conjunctival redness and chemosis (grades 2 and 1, respectively). Vascularization of the cornea was observed in two animals on days 7 and 10, but all symptoms were reversed within 14 days. In the third animal, vascularization was observed on day 14, and corneal opacity was still present at the end of the observation period. As the changes were not fully reversible in all animals, the substance is considered a severe ocular irritant. The study was performed according to GLP, and the protocol was in compliance with TM B5 from Annex V of Directive 92/69/EEC (Marty, 1994b).

Dermal sensitization: The skin sensitization potential of metalaxyl-M (purity, 97.3%) was assessed in 10 male and 10 female Pirbright white guinea-pigs in the Magnusson-Kligman maximization test. The animals received one intradermal injection of 0.1 ml of a 5% solution of metalaxyl-M in peanut oil with Freund adjuvant, followed after 1 week by one epidermal application of 0.4 g of undiluted metalaxyl-M. The animals were challenged on day 21 with a 30% emulsion of metalaxyl-M in vaseline. After the dressing had been removed, on day 10, irritation of the application site was observed in all treated animals. One animal showed grade 1 skin reactions 24 and 48 h after the challenge application, but all the others remained unaffected. The test substance was considered not to be sensitizing. The study was performed according to GLP, and the protocol was in compliance with TM B6 from Annex V of Directive 92/69/EEC (Marty, 1994c).

The potential of metalaxyl-M (purity, 96.6%) to induce delayed contact hypersensitivity was evaluated in a Buehler test. On the basis of the results of a preliminary test, 10 albino Crl:(HA)BR guinea-pigs were given undiluted material for both the induction phase and the challenge. In the induction phase, each animal received metalaxyl-M once a week for 3 weeks, at a dose of 0.4 ml on an adhesive patch, which was semi-occluded and left in place for 6 h. Two weeks after application of the third induction dose, a challenge dose of 0.4 ml was administered in the same manner. No dermal reactions were observed in the animals in either the induction or the challenge phase of the study. The study was performed according to GLP and to Guideline 81-6 of the Environmental Protection Agency (USA) (EPA). The protocol was not fully in compliance with TM B6 from Annex V of Directive 92/69/EE, as 10 animals were used instead of 20 (Glaza, 1995).

Studies on dermal and ocular irritation and dermal sensitization with metalaxyl are summarized in Table 27. The studies of Sachsse & Ullmann (1976b,c) were considered only to provide additional information, since limited data were presented. In the study of Sachsse & Ullmann (1976c), approximately 100 mg of metalaxyl were placed in the conjunctival sacs of three male and three female rabbits. The eyes of three of the six rabbits were rinsed 30 s after instillation of the test material. No irritation was found in any of the rinsed eyes, but the unwashed eyes showed irritation of the conjunctiva and cornea for 3 days. They were clear by day 4. While the degree of corneal involvement appeared to be minimal, it could not be evaluated subjectively as only total scores according to the Draize (1977) scale were presented. As rinsing the eyes prevented irritation, the irritation in the unwashed eyes may have been related to the physical nature of the particles rather than to the chemical itself.

Table 27. Irritation and sensitization potential of metalaxyl

In the study of Sachsse & Ullmann (1976d), groups of 10 male and 10 female guinea-pigs were given a series of intracutaneous injections of metalaxyl as a 0.1% suspension in polyethylene glycol and saline, according to the optimization test of Mauer et al. (1975). Dinitrochlorobenzene served as the positive control. Seven of 20 treated animals and one of 20 vehicle control animals showed positive reactions after challenge, while all 20 animals in the positive control group gave positive reactions. The authors considered that metalaxyl did not have skin sensitizing potential in guinea-pigs. In the metalaxyl-treated group, the average increase in skin reactions after challenge over that seen after induction was only marginal, by a factor of 1.2. In contrast, in the positive control group, the average increase in skin reactions at challenge was 18-fold. The reactions in the vehicle control group during induction were more marked than in the metalaxyl-treated group at induction and challenge by factors of 1.7 and 1.3, respectively. Use of an irritating vehicle and irritating concentrations of metalaxyl at challenge made it difficult to differentiate sensitization reactions from irritation. The results were below the threshold for significance set by the laboratory (p = 0.02). The Meeting considered that the results were equivocal but do not represent a positive effect.

Metalaxyl was not irritating to the skin of rabbits and did not sensitize guinea-pigs skin. It was slightly irritating to the eyes of rabbits, in contrast to metalaxyl-M, which was a severe ocular irritant.

2.2 Short-term studies of toxicity

Several short-term studies of toxicity have been conducted with metalaxyl-M, by oral administration in rats and dogs and by dermal application in rats. In earlier studies, racemic metalaxyl was tested by oral administration in rats and dogs, dermal application in rabbits and inhalation in rats.

Rats

A study was conducted to detect possible differences in the toxicological properties of metalaxyl-M and the racemate metalaxyl. The study was in compliance with the principles of GLP (with QA certification), and the protocol was fully in compliance with OECD TG 407 (1981) and TM B7 from Annex V of Directive 92/69/EEC. Metalaxyl-M (purity, 96.1%) and metalaxyl (purity, 97.3%) suspended in water containing 0.5% carboxymethylcellulose (CMC) and 0.1% Tween 80 were administered to groups of five male and five female Sprague-Dawley-derived rats (Tif:RAIf (SPF) hybrids of RII/1  RII/2) at a dose of 0, 10, 50, 150 or 300 mg/kg bw per day, 7 days/week for 4 weeks by gavage. The study was terminated after 28 days of treatment, and all animals were examined clinically and histopathologically.

No deaths occurred. Animals given doses > 150 mg/kg bw per day showed transient hypoactivity after the first treatment with metalaxyl-M, and two females at this dose were prostrate. No further clinical signs were observed. Body-weight gain and food consumption were similar in treated and untreated rats. The mean water consumption of males at the highest dose was reduced by 10% throughout treatment with metalaxyl-M, whereas treatment with metalaxyl increased the mean water consumption of animals at this dose by 10% in males and 22% in females.

The haematological examinations revealed no changes. A minimally lower plasma sodium concentration, a minimally higher plasma chloride concentration and a tendency to decreased urea concentration were recorded among males at the highest dose of both metalaxyl-M and metalaxyl. In addition, males treated with metalaxyl-M at this dose had a minimally lower plasma bilirubin concentration, and females showed increased plasma albumin and globulin concentrations and reduced plasma bilirubin. The differences from untreated controls were statistically significant for animals receiving metalaxyl-M at 300 mg/kg bw per day and for those given metalaxyl at 150 and 300 mg/kg bw per day.

No treatment-related gross changes in organs were seen at necropsy. The absolute and relative weights of the liver were increased by 9–12% in males and females at the highest dose of metalaxyl and females at the highest dose of metalaxyl-M.

Histopathological examination revealed minimal-to-moderate hypertrophy of centrilobular hepatocytes in females given metalaxyl-M at 150 mg/kg bw per day and in both sexes at 300 mg/kg bw per day. Minimal hepatocellular hypertrophy was also seen in the females given metalaxyl at 300 mg/kg bw per day. A minimally increased incidence of extramedullary haematopoiesis in the spleen was found in females given the two higher doses of metalaxyl.

In summary, treatment of rats with metalaxyl and metalaxyl-M resulted in similar effects. The main target organ of both substances was the liver, which reacted to treatment with hypertrophy. Metalaxyl-M had no unexpected toxicological properties that would differentiate the R-enantiomer from the racemic form of metalaxyl qualitatively. Quantitative differences in the effects of the two substances were generally minimal and remained within the range of biological variation. It is therefore justified to conclude that the two compounds are toxicologically equivalent. The NOAEL for both substances was 300 mg/kg bw per day, the highest dose tested, as the effects on the liver observed at doses > 150 mg/kg bw per day were considered not to be adverse (Gerspach, 1994).

Metalaxyl (purity, 94.6%) was administered by gastric intubation to groups of 10 male and 10 female Tif:RAIf (SPF) rats at an initial daily dose of 0, 10, 30 or 100 mg/kg bw. As the treatment provoked no overt toxic reaction, the doses were raised to 0, 30, 100 and 300 mg/kg bw per day from day 15 to 21 and finally to 0, 60, 200 and 600 mg/kg bw per day from day 22 to 28. The study was performed before GLP guidelines and EEC or OECD test guidelines were enacted. The study was not considered in the final evaluation, as the doses were increased during treatment, no neurological examination beyond normal clinical inspections were conducted and no histopathological examination was done.

No deaths occurred. After the dose had been increased to 600 mg/kg bw per day, i.e. to near the acute oral LD₅₀ for rats, tremors were observed. On subsequent days, the animals adapted to the treatment, and no clinical symptoms were seen on days 26, 27 and 28.

No treatment-related effects were noted on body-weight gain or food consumption. Ophthalmic examination, haematology, blood biochemistry and urine analysis revealed no treatment-related changes. The mean absolute and relative weights of the liver were increased in all treated groups, with a positive trend from control to highest dose. This effect was more pronounced in females than in males. In males at the highest dose, the absolute weight of the testes was significantly increased. The relative weight of the adrenals was significantly increased in females at the highest dose. The only gross pathological change observed was atrophy of the left testis in one male at the highest dose. This study indicated that the liver responded to treatment with hypertrophy, as shown by the dose-related increases in absolute and relative liver weights (Sachsse, 1979).

In a study conducted in compliance with the principles of GLP (with QA certification), groups of 10 male and 10 female Sprague-Dawley-derived rats (Tif:RAIf (SPF), hybrids of RII/1 × RII/2) were given diets containing technical-grade metalaxyl-M (purity, 97.1%) at a concentration of 25, 50, 250, 625 or 1250 ppm for 3 months. A group of 10 rats of each sex receiving the vehicle (acetone) served as controls. An additional group of 10 rats of each sex from the control and highest-dose groups were kept for a 4-week recovery period before sacrifice. The doses were selected to allow direct comparison with the previous short-term study conducted with metalaxyl. The doses were equal to mean intakes of 1.7, 3.5, 17, 45 and 91 mg/kg bw per day for males and 1.9, 3.7, 18, 49 and 95 mg/kg bw per day for females. Overt signs of toxicity were recorded daily, and body weight, food consumption and water consumption were recorded weekly throughout the study. Ophthalmoscopy was performed on animals from the control and highest-dose groups before treatment, towards the end (day 87) of treatment and towards the end of the recovery period (day 115). Haematological, blood chemical and urine analyses were carried out on all surviving treated animals at the end of treatment and at the end of the recovery period. All animals killed after treatment underwent a detailed necropsy and comprehensive microscopic evaluation of tissues; the livers of animals allowed to recover for 4 weeks were examined. The protocol complied generally with OECD TG 408 (1981) and TM B26 from Annex V of Directive 87/302/EEC except that the maximum tolerated dose was not reached.

No treatment-related differences were found between treated animals and controls during treatment, with no deaths or clinical signs and similar body-weight gain and food and water consumption in treated and control animals. No ophthalmological changes were noted, and haematology, clinical chemistry and urine analysis revealed no changes that could be attributed to treatment.

At termination, no treatment-related gross changes were found in organs. The absolute and relative weights of the organs were unaffected by treatment, although a slight (maximum, 7%) increase in mean liver:body weight ratio was seen in females at concentrations > 625 ppm. Histopathological examination revealed a dose-related occurrence of hepatocellular inclusion bodies (consisting of ring- or whorl-shaped eosinophilic particles located within the cytoplasm of perilobular hepatocytes) in males at 625 and 1250 ppm. The occurrence of these inclusions was sometimes associated with enlargement of perilobular hepatocytes. Females at these concentrations showed increased incidences of minimal hepatocellular hypertrophy, located centrilobularly. Both findings were completely reversed within the 4-week recovery period.

Treatment with metalaxyl-M at these concentrations was thus well tolerated. The NOAEL was 1250 ppm, equal to 91 mg/kg bw per day, the highest dose tested, as the effects on the liver observed at 625 ppm were considered not to be adverse (Gerspach, 1995).

Groups of 20 male and 20 female Sprague-Dawley rats received diets containing metalaxyl (purity, 99%) at a concentration of 0, 50, 250 or 1250 ppm for 3 months. Five males and five females from the control and highest-dose groups were kept for a 4-week recovery period before they were killed. The doses were equal to mean intakes of 3.2, 16 and 79 mg/kg bw per day for males and 3.5, 18 and 86 mg/kg bw per day for females. Overt signs of toxicity were recorded daily, and body weight and food consumption were recorded weekly throughout the study. Ophthalmoscopy was performed on all animals before treatment and on 10 males and females from the control and highest-dose groups during weeks 5, 9 and 13. Animals allowed to recover were examined during week 17. Haematological, blood chemical and urine analyses were carried out on all surviving treated animals at weeks 5, 9 and 13 and at week 17 for rats allowed to recover. Animals that died or were killed during the the study and all animals that survived until scheduled sacrifice were autopsied. A comprehensive microscopic evaluation of tissues from all treated animals was carried out, and the livers and ovaries from the five female rats killed after the 4-week recovery period were examined. The study was performed before GLP guidelines and EEC or OECD test guidelines were enacted; however, the protocol complied with the major requirements of OECD TG 408 (1981).

No deaths and no overt signs of toxicity were reported. Ophthalmic examination revealed no treatment-related changes. Males at the highest dose had minimally decreased body-weight gain (terminal weight, 97% of control) and food consumption (week 13, 94% of control). Haematology, blood biochemistry and urine analysis revealed no treatment-related changes. At autopsy, no macroscopic changes were seen, and there were no relevant changes in organ weights. Histopathological examination revealed minimal hypertrophy in a few hepatocytes in the females at the two higher concentrations, with incidences of 0/20, 0/20, 2/20 and 7/20. Large ovarian cysts were reported in 5/20 females at 1250 ppm, but these were regarded as of doubtful significance. In the group allowed to recover, no changes were noted in the liver, and the ovaries were similar to those of controls.

The maximum tolerated dose was not reached in this study. As the liver-cell hypertrophy at 1250 ppm was considered not to be adverse, this concentration, 79 mg/kg bw per day, was the NOAEL (Drake, 1977).

In a study performed in compliance with the principles of GLP (with QA certification), groups of 20 male and 20 female Sprague-Dawley-derived rats (Tif:RAIf (SPF)) received diets containing metalaxyl (purity, 93.5%) at a concentration of 0, 10, 50, 250 or 1250 ppm for 3 months. The doses were equal to mean intakes of 0.66, 3.5, 15 and 72 mg/kg bw per day for males and 0.67, 3.6, 16 and 74 mg/kg bw per day for females. Overt signs of toxicity were recorded daily, and body weight and food consumption were recorded weekly throughout the study. Ocular examinations and an auditory test were performed before and after treatment. Animals allowed to recover were examined during week 17. Haematological, blood chemical and urine analyses were carried out on 10 rats of each sex per group in weeks 4, 8 and 12. At the end of treatment, all animals were subjected to a detailed necropsy and a comprehensive microscopic evaluation of tissues. The study was performed before EEC or OECD test guidelines were enacted; however, the protocol complied with the major requirements of OECD TG 408 (1981).

No deaths or clinical signs were seen. Body-weight gain and food consumption remained unaffected by treatment. The laboratory investigations revealed a slight decrease in leukocyte count for males at the highest concentration at week 12. At necropsy, slightly increased adrenal weights were seen in males at concentrations > 50 ppm (160%, 180% and 170% of control values). Histopathological examination of the organs and tissues revealed no changes related to administration of metalaxyl.

The maximum tolerated dose was not reached in this study. The concentration of 10 ppm, equal to 0.66 mg/kg bw per day, represents a conservative NOAEL on the basis of the changes in adrenal weights in males. However, the effect was not observed in females and was not confirmed by blood biochemistry or histopathology. Moreover, this effect has not been observed in other studies. This study was not taken into account in the final evaluation (Gfeller, 1980).

In a study of the toxicological effects of repeated inhalation of the pyrolysis products of cigarette tobacco treated with metalaxyl, groups of 10 male and 10 female Fischer 344 rats were exposed for 5 days per week, for 13 weeks, to the smoke from 18 cigarettes that had been spiked with 0, 130, 3900 or 13 000 ppm of metalaxyl (technical-grade, purity not specified). In addition, five groups of two rats of each sex were exposed once to the smoke of each concentration of spiked tobacco and to ambient air, before initiation of the 90-day study, for analyses of plasma nicotine. The criteria used to evaluate treatment-related effects included death, moribundity, appearance, behaviour, body weight, clinical pathology, absolute and relative organ weights, gross pathology and histopathology. The study was conducted in compliance with the principles of GLP (with QA).

No distinct clinical changes were noted in any of the treated animals during the study. No treatment-related effects or trends were observed in body-weight gain, food consumption, haematological or clinical chemical parameters or gross or histological appearance. This study was not used in the final evaluation (Coate, 1982).

In a study conducted in compliance with the principles of GLP (with QA certification), metalaxyl-M (purity, 97.1%) suspended in 1% (w/v) carboxymethylcellulose in 0.1% (w/v) aqueous polysorbate 80 was applied to the clipped skin of groups of five Sprague-Dawley-derived (Tif:RAIf (SPF) hybrids of RII/1 × RII/2) rats of each sex at a dose of 0, 50, 250 or 1000 mg/kg bw per day under an occlusive dressing for 6 h/day, 5 days/week, for 4 weeks. No application was made on the weekend days of weeks 1–3. Clinical signs, body weight, food consumption and deaths were monitored throughout the study. Haematological and blood chemical analyses were performed at the end of treatment. At sacrifice, the animals were examined macroscopically and organ weights were recorded. Organs and tissues were collected, prepared for histopathological evaluation and examined microscopically. The protocol of the study complied with the major requirements of OECD TG 410 (1981) and TM B9 from Annex V of Directive 92/69/EEC.

Treatment with metalaxyl-M produced no clinical signs or behavioural changes and no signs of local irritation. Although the food intake of treated and control animals was similar, males at the highest dose gained less weight than controls. At the end of treatment, the mean body weight of males at the highest dose was 6% lower and the mean body-weight gain 21% lower than that of controls. Haematological and blood chemical analyses gave no indication of a treatment-related effect. Males at the highest dose had a 16% decreased mean spleen weight, and the liver:body weight ratios were increased by 8% in males and 16% in females. Macroscopic and histopathological examination showed no treatment-related findings. Dermal treatment with metalaxyl-M was thus well tolerated, with no irritating effect. The NOAEL for systemic effects was 1000 mg/kg bw per day, the highest dose tested, as the modifications in liver and spleen weight were not confirmed at necropsy or by histopathological findings (Gerspach, 1998).

Rabbits

In a study conducted in compliance with the principles of GLP (with QA certification), powdered metalaxyl (purity, 92%) was applied to the clipped skin of groups of 10 male and 10 female New Zealand white rabbits at a dose of 0, 10, 100 or 1000 mg/kg bw per day under a semi-occlusive dressing for 6 h/day, 5 days/week, for 3 weeks. The skin of half of the animals was abraded once a week in order to enhance dermal absorption. Clinical signs, deaths and signs of dermal irritation were recorded daily throughout the study. Body weight and food consumption were monitored at the beginning of treatment and then twice a week until sacrifice or death. Haematological and blood chemical analyses were performed before and at the end of treatment. After death or at sacrifice, the animals were examined macroscopically, and organ weights were recorded. Organs and tissues were collected, prepared for histopathological evaluation and examined microscopically. The study was performed before EEC or OECD test guidelines were enacted; however, the protocol of the study complied with the major requirements of OECD TG 410 (1981).

The application was tolerated at all doses with no signs of systemic toxicity. Body weight and food consumption were similar in all groups. One male at 100 mg/kg bw per day was found dead on day 20, but the cause of death was not reported. Abnormal skin reactions were limited to lesions from the tape used to secure the dressing, and pimple-like eruptions were seen in all groups at a frequency that was not dose-related; they were considered not to be related to treatment. Haematological and clinical chemical analyses and post-mortem examinations for organ weight, gross and histopathological lesions in brain, pituitary, heart, thyroid, adrenals, genital organs, liver, kidney and skin revealed no treatment-related changes. The experimental procedure induced multifocal dermatitis in treated and untreated areas of the skin in both treated and control rabbits. Dermal treatment with metalaxyl was thus well tolerated. The NOAEL was 1000 mg/kg bw per day, the highest dose tested (Calkins, 1980).

Dogs

The toxicity of metalaxyl-M in dogs was investigated in a 13-week study of dietary administration. Metalaxyl was previously tested in a 6-month study with administration in the diet and in a 2-year study with administration in capsules.

In a study conducted in compliance with the principles of GLP (with QA certification), groups of four male and four female beagle dogs were given diets containing technical-grade metalaxyl-M (purity, 97.1%) in acetone at a concentration of 0, 50, 125, 250 or 1250 ppm for 13 weeks. The doses were selected to allow direct comparison with the previous short-term study conducted with metalaxyl and were equal to a mean daily intake of 1.6, 4.1, 7.3 and 39 mg/kg bw per day for males and 1.6, 4.3, 7.9 and 40 mg/kg bw per day for females. Overt signs of toxicity, body weight and food consumption were recorded daily throughout the study. Ophthalmoscopy was performed before and at the end of treatment. Haematological, blood chemical and urine analyses were conducted before treatment and at weeks 7 and 13. At 13 weeks, all animals were killed and necropsied, and a comprehensive histological examination was carried out. The protocol was generally in compliance with OECD TG 409 (1981) and TM B27 from Annex V of Directive 87/302/EEC, except that the maximum tolerated dose was not reached.

Treatment caused no deaths or any clinical signs of toxicological significance, and the ophthalmic examination revealed no changes. All dogs ate similar quantities of food, and the body-weight development was similar in treated and control groups. No treatment-related changes in haematological parameters were observed. Increased alkaline phosphatase activity was seen in males and females at the highest concentration after 7 weeks (190% increase over controls for males and 220% increase for females) and 13 weeks (210% for males and 260% for females) of treatment. Treatment had no effect on the urine parameters investigated. At sacrifice, the mean absolute (25% in males, 28% in females) and relative (25% in males, 33% in females) weights of the liver were increased in animals at the highest concentration. Macro- and histopathological examination revealed no changes of toxicological significance. Metalaxyl-M was thus well tolerated at concentrations < 1250 ppm. The liver was the target organ, as indicated by increases in relative and absolute weights and in alkaline phosphatase activity. The NOAEL was 250 ppm, equal to 7.3 mg/kg bw per day (Altmann, 1995).

In a study conducted in compliance with the principles of GLP (with QA certification), groups of six male and six female beagle dogs were given diets containing technical-grade metalaxyl (purity, 92%) in acetone at a concentration of 0, 50, 250 or 1000 ppm for 6 months. Two additional dogs of each sex from the control group and that at 1000 ppm were kept for a 4-week recovery period before they were killed. The doses were equal to mean daily intakes of 1.6, 7.8 and 31 mg/kg bw per day for males and 1.7, 7.4 and 32 mg/kg bw per day for females. Overt signs of toxicity, body weight and food consumption were recorded daily throughout the study. Ophthalmoscopy was performed before and at the end of treatment. Haematological and blood chemical analyses were conducted before treatment and at 30-day intervals until termination of dosing. Urine was analysed before treatment and at 60-day intervals until termination of dosing. Blood samples were collected from dogs allowed to recover 4 weeks after termination of dosing, and urine was collected before necropsy. At 6 months, all animals were killed and subjected to a detailed necropsy and a comprehensive histological examination. The protocol generally complied with OECD TG 409 (1981) and TM B27 from Annex V of Directive 87/302/EEC, although the duration of treatment exceeded the basic guideline requirements. Nevertheless, it did not exceed 10% of the normal life span of the dogs, and the investigation can be considered a short-term study.

No deaths occurred and no treatment-related changes were found in clinical signs, body-weight gain, food consumption or ophthalmic parameters. In comparison with the control animals, the erythrocyte count, erythrocyte volume fraction and haemoglobin concentration were significantly lower in males at the highest concentration from day 60 of treatment onwards, and these haematological changes were still present in males allowed to recover, though they were not statistically significant. Blood biochemical analyses revealed a slight increase in plasma alkaline phosphatase activity in animals of each sex at the highest concentration from day 30 onwards, which became statistically significant during the second half of the treatment period. After 180 days of treatment, the activity was 170% of the control value in males and 190% in females. In the group allowed to recover, the alkaline phosphatase activity was only minimally above the control values (120% of control value in males and 110% in females). Urinary parameters were all within normal limits, and no trends by dietary concentration were detected.

Although not statistically significant, there appeared to be a dose-related increase in absolute liver weight, with increases of 8% at 50 ppm, 14% at 250 ppm and 16% at 1000 ppm, and in the liver:body weights ratios in males and especially in females, with increases of 2% at 50 ppm, 13% at 250 ppm and 20% at 1000 ppm. These changes were reversible in males and less pronounced in females when allowed to recover. The difference in liver:brain weight ratio attained statistical significance (120% of control values) in females at 1000 ppm after 180 days of treatment; the value was 110% of control in the group allowed to recover. Gross and histopathological examination showed no treatment-related changes.

The NOAEL was 250 ppm, equal to 7.8 mg/kg bw per day, on the basis of increases in alkaline phosphatase activity and liver weight at 1000 ppm (Beck & deWard, 1981).

Metalaxyl (purity, 92.7–94.1%) was administered in gelatine capsules to groups of six male and six female beagle dogs at a dose of 0, 0.8, 8 or 80 mg/kg bw per day once a day, 7 days a week, for 2 years. All surviving animals were killed at termination of the study at week 103. The study complied with the principles of GLP (with QA certification), and the protocol was generally in compliance with OECD TG 409 (1981) although it was begun in 1980, before adoption of OECD TG.

Animals at the highest dose frequently showed spasms and/or salivation, especially during the first 52 weeks of treatment. The symptoms usually occurred 10–30 min after dosing and disappeared within 0.5–2 min of their onset. Two males and two females died during weeks 20–52. There were no treatment-related effects on body weight, although male dogs at 0.8 mg/kg bw per day weighed slightly more than controls during the study. There were no treatment-related effects on food consumption, water consumption or ophthalmic end-points.

A slight decrease or a decreasing trend in the specific gravity of the urine was observed in males at 80 mg/kg bw per day from 13 weeks of treatment. Mild anaemia was observed in animals at the highest dose. The reductions (10–20%) in erythrocyte count, haemoglobin concentration and erythrocyte volume fraction became evident only at 26 weeks and were slightly more pronounced in males than in females. The values for all three haematological end-points in males were within the range of other controls in the laboratory in weeks 26 and 52. For females, the values for all three end-points in week 26 and for erythrocyte volume fraction in week 52 were within the range of other controls; the other two parameters were marginally lower than those of other controls in week 52.

Animals at 80 mg/kg bw per day had significantly increased serum activities of alkaline phosphatase and alanine aminotransferase as compared with controls (Table 28). The values for alkaline phosphatase during the study were 1.5–2.5 times higher than those determined before initiation of treatment and 2–5.6 times those in the corresponding control groups. The activity in males at 8 mg/kg bw per day and in controls decreased gradually during the study from that determined before initiation of treatment, and only a slight (maximum, twofold) but statistically significant increase in alkaline phosphatase activity was found. The increase in alanine aminotransferase activity in males at the intermediate dose was slight (maximum, 50%) and often not dose-related. Animals at the highest dose also had elevated albumin, total protein and calcium concentrations. Males showed decreased aspartate aminotransferase activity and globulin and creatinine concentrations.

Table 28. Activity of alkaline phosphatase and alanine aminotransferase in dogs given metalaxyl in gelatine capsules for 2 years (U/l; group mean ± standard deviation)

From Harada (1984)

* Significantly different from controls at p < 0.05; ** significantly different from controls at p < 0.001

At necropsy, animals at the highest dose frequently showed enlargement of the liver and increased liver weights, with increases in absolute weight of 58% in males and 16% in females and an increase in relative weight of 16% in both sexes. Males also had significantly increased kidney weights (by 34%) in comparison with controls. Histopathology revealed no specific changes attributable to treatment. The incidences of hepatic lesions such as focal inflammation, fibrosis and brown pigment deposition tended to be higher in animals at 80 mg/kg bw per day than in controls; however, the lesions could not definitively be related to treatment as they were relatively mild.

The changes in alkaline phosphatase and alanine aminotransferase activity observed in animals at the highest dose indicated hepatic effects of treatment and were considered to correspond to the increased liver weights. However, even though the difference in alkaline phosphatase activity between males at 8 mg/kg bw per day and concurrent controls was statistically significant, it was slight, and, in the absence of other biologically significant changes in clinical chemistry and histopathology, was considered not to be adverse

The liver was the target organ of metalaxyl, as indicated by the changes in blood chemistry and increased liver weight. Although a decrease in urine specific gravity and increased kidney weights were observed in males, there were no histopathological abnormalities in the kidney, and it is not clear whether this effect was related to treatment. The haematological end-points indicating anaemia occurred only after long-term treatment and were not relevant to acute exposure. The NOAEL was 8 mg/kg bw per day (Harada, 1984).

2.3 Long-term studies of toxicity and carcinogenicity

Mice

Groups of 60 male and 60 female ICI-derived Swiss mice were given diets containing metalaxyl (purity, 93–94.6%) at a concentration of 0, 50, 250 or 1250 ppm for 104 weeks, equal to mean daily intakes of 4, 19 and 100 mg/kg bw per day for males and 4.6, 23 and 120 mg/kg bw per day for females. These values were re-calculated from individual values for food consumption and body weight, taking into account the content of the test compound in the food batches used. In earlier evaluations of this study, standard conversion factors were used to calculate the intake in mg/kg bw per day. The study was performed in compliance with the principles of GLP (with QA certification), and the design complied with the requirements of OECD TG 451 (1981), although the treatment period was longer than that usually recommended for mice (18 months).

The animals showed no clinical signs of toxicity, and the mortality rate was similar in all groups. At 78 weeks, the survival rates were 53%, 55%, 57% and 50% for males and 67%, 53%, 53% and 52% for females in the control group and at the low, intermdiate and high dietary concentrations, respectively. At 104 weeks, the mortality rates were increased, with survival rates of 8%, 10%, 10% and 17% for males and 13%, 13%, 8% and 10% for females, respectively. During weeks 11–30 of treatment, males at 1250 ppm gained less weight (12 g) than control males (17 g), corresponding to a reduction of 31% and leading to a difference of 9% at week 30. The animals recovered slowly thereafter, but the cumulative body-weight gain was still reduced at week 30 (by 12%) and week 56 (by 10%) for males at 1250 ppm. A slight, 8% reduction in body-weight gain in comparison with controls was observed in weeks 0–10 among females at 1250 ppm. The terminal body weights of treated and untreated groups were similar. The food consumption and water intake of treated and control animals were essentially identical throughout the treatment period. Food conversion efficiency was reduced in males at 1250 ppm during weeks 11–30.

Macroscopic and histopathological examination of a wide range of tissues, including blood and bone marrow smears, revealed no significant treatment-related changes. The histopathological results for the main target organ, the liver, are outlined in Table 29.

Table 29. Incidences of histopathological liver changes in mice given diets containing metalaxyl for 2 years

From McSheehy et al. (1980a,b)

There was no evidence of a carcinogenic response to treatment. A re-evaluation of the slides of the liver did not alter this conclusion, and the liver tumours in male mice were considered not to be treatment-related. This conclusion is supported by the following findings: (i) The liver tumours were age-related, occurring predominantly after 18 months of the study. (ii) The survival of the animals was not affected by administration of metalaxyl. (iii) Neither general health nor body weight or food consumption was compromised by treatment. (iv) The presence of Tyzzeria did not obscure the presence of liver tumours, and the non-neoplastic lesions in the liver (hepatocytic fatty vacuolation and bile-duct proliferation) were no different in control and treated groups and would therefore not have affected the distribution of liver tumours. (v) The liver tumour incidence was not statistically significantly different (chi², 2 × 4) between control and treated groups (p = 0.12). (vi) The liver tumour incidence did not show a dose-related trend (Mantel trend test; p = 0.15). (vii) The liver tumour incidence in the control group (23%) was within the range of that in six other control groups in the same laboratory (12–37%).

Treatment with metalaxyl did not increase the incidence of benign or malignant tumours in mice of either sex. The NOAEL was 250 ppm, equal to 19 mg/kg bw per day, on the basis of slight body-weight loss in male mice at 1250 ppm (McSheehy et al., 1980a,b).

Rats

Groups of 80 male and 80 female CD Sprague-Dawley-derived rats were fed diets containing technical-grade metalaxyl (purity, 93–96.4%) at a concentration of 0, 50, 250 or 1250 ppm for 2 years, equal to mean daily intakes of 1.7, 8.7 and 43 mg/kg bw per day for males and 2, 10 and 55 mg/kg bw per day for females. The doses were selected on the basis of a 3-month study of toxicity, which had shown decreased body-weight gain and effects on the liver at 1250 ppm. Ten animals of each sex per dose were killed after 55 weeks of treatment, while the remaining animals were maintained on the treated diet until terminal sacrifice after 105 weeks. The study was performed in compliance with the principles of GLP (with QA certification), and the design complied generally with the requirements of OECD TG 453 (1981), although the highest concentration was high enough for a carcinogenicity study but not for a toxicity study, gamma-glutamyl transpeptidase activity was not measured and the ovaries were not weighed.

No deaths and no clinical signs of toxicity resulted from treatment. While no differences between treated and control animals were seen in food consumption or absolute body weight, the body-weight gain of females at 1250 ppm was reduced transiently in weeks 26–52 (by 10% in comparison with controls). Haematological, clinical chemical and urine parameters remained within normal limits, and no remarkable intergroup differences were found.

At interim sacrifice, the liver:body weight ratio was significantly increased (120% of control) in females at 1250 ppm. At terminal sacrifice, increased absolute and relative liver weights were found in animals of each sex at this concentration. The increase in relative liver weight in males at 250 ppm was due at least partly to a decreased carcass weight in this group. The increased relative weights of the liver observed in females at 1250 ppm in week 55 and in males at 250 and 1250 ppm in weeks 105 were not accompanied by underlying hepatic damage, indicating that the changes in weight indicated a mild change in the liver that was not adverse.

An increased severity of eosinophilic vacuolation was seen in males at the two higher concentrations that were killed at week 55. This finding was considered to represent a transitory response that was not adverse because it was not observed in animals dying during the study or at terminal sacrifice. Centriacinar, periacinar and panacinar hepatocytic vacuolation in the liver of various degrees of severity was observed, indicating fatty changes. The centriacinar and panacinar vacuolation was not related to treatment. In males, no significant, dose-dependent increase in the incidence of hepatocytic vacuolation was observed in any treated group. In animals that died up to week 52, no relationship with treatment was found for hepatocyte vacuolation in the few animals affected. Increased incidences of periacinar hepatocyte vacuolation were observed in all treated females at 55 and 105 weeks, but the distribution among groups was uneven and there was no clear relationship with dose. The increase in severity was not dose-related at any time, except in females at 1250 ppm at terminal sacrifice, in which the severity was increased in comparison with concurrent controls and with females at this concentration at earlier times. Therefore, only a usually slight-to-moderate increase in the incidence of periacinar hepatocytic vacuolation, which was considered to be a fatty change and occurred spontaneously in control animals, was observed in metalaxyl-treated females, with an increase in severity only for females at the highest concentration at week 105. Except in the these animals, there was a slight decrease in the overall average severity of hepatic vacuolation between sacrifice at week 55 and at week 105. A second histopathological evaluation (Faccini, 1985) confirmed the overall result of marginal changes in the liver. It also showed an increased incidence of centrilobular hepatocytomegaly in male and female rats at 1250 ppm and possibly a marginally higher incidence in males at 250 ppm. A higher incidence of foci of cellular alteration was seen in males at 250 ppm, with a possible trend towards the same effect in females at this concentration.

A statistically significant increase (p < 0.05) was found in the incidence of C-cell (parafollicular cell) adenomas in females at 250 ppm (Table 30). However, no tumours were detected in animals that died before 78 weeks of treatment, no dose–response relationship was established, and the incidence was within that of controls from 15 contemporary studies in the same laboratory (total adenomas, 0–15%; adenomas in females killed at termination of study: 0–30%). Therefore, the distribution was considered to be fortuitous and not related to treatment with metalaxyl.

Table 30. Incidences of C-cell adenomas of the thyroid in rats given diets containing metalaxyl for 2 years

From Ashby & Whitney (1980a,b)

* Statistically significant at p < 0.005

Thus, metalaxyl administered to rats at concentrations < 1250 ppm in the diet for 2 years was well tolerated. The finding of hepatocytic vacuolation is an age-related finding and was considered not to be adverse. The hepatocellular changes were not accompanied by degenerative, irreversible or cytotoxic lesions such as necroses, which can be observed in the cases of severe fatty change of the liver, and no accompanying inflammatory changes were found. Blood chemistry showed no altered liver parameters pointing to hepatocellular damage. In general, most evidence suggests that moderate hepatic fatty change alone does not impair hepatic function (Haschek & Rousseaux, 1991). Centrilobular hepatocytomegaly was considered to be the main effect, occurring in rats of each sex at the highest concentration. This change might have been a consequence of a mild enzyme-inducing effect of the compound (Uesugi, 1988). The treatment had no effect on the incidence or distribution of neoplastic lesions. A re-evaluation of C-cell (parafollicular cell) adenomas in the thyroid gland did not alter this position. The NOAEL was 1250 ppm, equal to 43 mg/kg bw per day, the highest concentration tested, as the mild liver changes seen at this concentration were considered not to be adverse (Ashby & Whitney, 1980a,b).

2.4 Genotoxicity

The mutagenic or genotoxic potential of technical-grade metalaxyl and metalaxyl-M was investigated in a battery of tests in vitro and in vivo (Tables 31 and 32).

Table 31. Results of the studies of genotoxicity conducted with metalaxyl