4.17 Phorate (112)

RESIDUE AND ANALYTICAL ASPECTS

Phorate is a systemic organophosphate contact insecticide and acaricide that inhibits acetyl cholinesterase activity. Residue and analytical aspects of phorate were evaluated by the JMPR in 1977, 1984, 1990, 1991, and 1992. The 30^th Session of CCPR (1998) requested priority scheduling of a full review of the compound because of acute intake concerns. Phorate was listed in the Periodic Re-Evaluation Programme at the 36^th Session of the CCPR for periodic review by 2005 JMPR. The JMPR toxicological review was conducted in 2004, which established an ADI of 0-0.0007 mg/kg bw and an ARfD of 0.003 mg/kg bw.

Information on the latest GAP, residue data, metabolism, analytical methods, storage stability and processing studies were provided by the manufacturer to enable the assessment of existing and proposed MRLs on a number of crops or crop groups, including beans, potatoes, sugar beet, sweet corn, maize, sorghum, cotton, and coffee.

In addition, GAP information and/or national MRLs were supplied by Australia and The Netherlands.

The following common names were used for the metabolites discussed below:

Animal metabolism

The Meeting received information on the fate of orally dosed phorate in lactating goats and laying hens. Phorate was ¹⁴C labelled at the methylene position.

Studies on laboratory animal metabolism (rats) were evaluated by the WHO Core Assessment Group of the 2004 JMPR. It was reported that after oral administration of radiolabelled phorate to rats, 77% of the administered dose was recovered in the urine within 24 h after dosing. Faecal excretion accounted for approximately 12% of the administered dose. Over the total duration of the study (192 h), effectively the entire administered dose was eliminated by excretion. The bulk of the administered dose (94%) was biotransformed to nonphosphorylated metabolites. The metabolic pathway responsible for the formation of these metabolites resulted from the cleavage of the phosphorus-sulfur bond, methylation of the liberated thiol group and oxidation of the resulting divalent sulfur moiety to the sulfoxide and sulfone. Thus, these studies demonstrated that in rats phorate is rapidly absorbed and excreted and the accumulation of any toxicologically significant residue is not of concern.

In two consecutive studies, lactating goats were orally treated (balling gun) with ¹⁴C-labelled phorate at dose rates of 1.35 and 5.40 ppm in the feed for either 3 or 7 days. At the highest dose, significant depression in plasma cholinesterase activity was observed. Recovery of total applied radioactivity (in excreta, tissues, milk) was not investigated. After 3 days of treatment, the highest concentration of radioactive residues was found in the liver (0.62 mg/kg eq). Kidney, milk, muscle and fat contained 0.41, 0.26, 0.19, < 0.05 mg/kg eq, respectively. After 7 days of treatment, concentration levels in liver, kidney, milk, muscle and fat were 0.90, 0.76, 0.50, 0.64, and 0.21 mg/kg eq, respectively (all results from highest dose level). Residue levels in milk increased steadily and no plateau was reached during both the dosing periods.

Approximately 95% to 99% total radiolabelled residue (TRR) in extracts of milk, liver, kidney, leg muscle, tenderloin muscle, and omental fat was composed of non-phosphorylated metabolites, which resulted from cleavage of the phosphorus-sulphur bond and the methylation of the resultant mercaptan. The major metabolite in all tissues and milk was, ethylsulfonyl methylsulfonyl methane accounting for 94% to 99% TRR in the tissues and milk. The remaining radioactivity was composed of the parent compound and its various oxidative products (< 0.01% to 2.2% TRR). In the 3 days experiment (highest dose level) the toxicologically significant compounds parent, phorate sulfone and phorate sulfoxide were found at very low levels: milk contained 0.052 mg/kg eq parent and 0.39 mg/kg eq phorate sulfoxide, liver contained 4.34 mg/kg eq parent, kidney 2.50 mg/kg eq phorate sulfoxide, muscle 0.019 mg/kg eq parent, 0.077 mg/kg eq phorate sulfone and 0.14 mg/kg eq phorate sulfoxide, and fat contained none of those at a detectable level.

Groups of laying hens were orally treated (gelatin capsules) for 5 days with ¹⁴C-labelled phorate at dose rates of about 1 and 3 ppm in the feed. Recoveries of the administered doses averaged 64?66%: 62?64% of the total administered radioactivity in excreta, 0.7?1.5% in eggs, 0.5%-0.8% in organs and 1.2% in carcass. Fortification of control excreta with [¹⁴C]phorate resulted in a recovery of 78% after 24 h at room temperature. These results suggest that the low but consistent overall recovery may be associated with the volatility of phorate and/or the low molecular weight of the metabolic products.

Liver and kidney were found to contain the highest level of radioactive material. At the highest dose level, the amounts were 0.31 and 0.24 mg/kg eq respectively. At this dose level total radioactive residues in breast muscle, skin/fat, egg white and egg yolk amounted to 0.031, 0.047, 0.048?0.10, and 0.017?0.20 mg/kg eq respectively. Residue levels in egg yolks and whites increased steadily and no plateau was reached during the dosing period.

Metabolites found in the tissues and eggs include ethylsulfonyl methylsulfonyl methane; (ethylsulfinyl)methyl methyl sulfone; ethyl (methylsulfinyl)methyl sulfone. One additional non-phosphorylated metabolite, ethylsulfinyl methylsulfinyl methane was also found in the egg white. Neither the parent compound phorate, nor any of the oxidative metabolites phorate sulfoxide, phorate sulfone, phoratoxon, phoratoxon sulfoxide or phoratoxon sulfone was found in tissues or in eggs. Significant fractions of the radioactive residues in tissues and eggs (47?59% TRR) were unextractable but were released by enzyme hydrolysis with protease. Since the released activity was highly polar, it was not any of the phorate oxidative metabolites.

In conclusion, the metabolism of phorate in farm animals was similar to that in laboratory animals. Goats and laying hens dosed with phorate quickly detoxify the compound through a set of oxidative, toxic, metabolites. Neither parent nor any of its oxidative metabolites accumulate in edible tissues, milk and eggs.

Plant metabolism

The Meeting received information on the translocation and metabolism of phorate in plants placed in a phorate emulsion, in cotton grown from seeds treated with phorate, and in various plants after soil or foliar application of phorate. Characterization of metabolites was limited to root and foliar parts of young or immature plants. Confined rotational crop studies gave information on the metabolite composition of mature crops (see environmental fate section). Experiments were carried out with ³²P labelled phorate or with phorate ¹⁴C labelled at the methylene position.

The roots of red kidney bean seedlings were placed in an emulsion of ³²P-phorate for 1 day and transplanted afterwards. Leaves were analysed 1, 4 and 12 days after treatment. The bases of young cotton plants, lemon seedlings and alfalfa seedlings were treated with a topical application of ³²P-labelled phorate solution. At various intervals after application (up to 14?17 days) the upper leaves were removed. In all experiments, radioactive residues translocated to the leaves. In red kidney bean leaves analysed 1, 4 and 12 days after application the primary metabolites were phorate sulfoxide and/or phorate sulfone, which could not be separated on the columns used. Small amounts of phoratoxon sulfoxide and/or phoratoxon sulfone and unchanged phorate were also found. No phoratoxon was found. The hydrolysis products formed were phosphoric acid, the diethyl esters of phosphoric acid, phosphorothioic acid and phosphorodithioic acid. Parent and the same four metabolites were also identified in a chloroform extract of cotton, lemon and alfalfa leaves (day 1-17). For cotton leaves, parent was found up to 5 d and never exceeded 5% of the radioactivity. Phorate sulfoxide reached a maximum of 85% of the radioactivity at day 1 after application and thereafter decreased to 35% at day 14. Phorate sulfone, phoratoxon sulfoxide and phoratoxon sulfone increased with time at up to 35%, 15% and 10% radioactivity at day 14, respectively. Phoratoxon was not found at any time point.

A mixture of ³²P-phorate and charcoal was coated on cotton seeds wetted with 2% methylcellulose at a concentration of 160 kg ai/t or 320 kg ai/t. Treated seeds were planted and cotton plants were sampled at 3.9, 7.4, 10.7 or 16 weeks after planting. In addition, foliage from cotton plants grown for 2 weeks from treated seed was sampled for identification of metabolites. Total ³²P residues were less than 0.03 mg/kg eq in leaves and seeds maturing from plants grown from seed treated with 320 kg ai/t at 16 weeks after planting. The residues isolated from the foliage consisted of phorate sulfoxide and phorate sulfone (ratio 61:39) and phoratoxon sulfoxide and phoratoxon sulfone (ratio 70:30). The parent itself was not identified because of interference from plant pigments. Phoratoxon was not found.

Beans, beets, cabbage, carrots, lettuce and peas were treated with radiolabelled ³²P-phorate using both a foliar and a soil application. Applications were made with an EC formulation at a rate of 1.12 kg ai/ha. The vegetable foliage was sampled at 2 hrs and 1, 2, 4, 8, 17 and 32 DAT. In addition pea plants were soil treated and the foliage was sampled at 14 DAT for identification of metabolites. Average values of the distribution of ³²P radioactivity and anti-cholinesterase activity in pooled extracts from beans, beets, cabbage, carrots, lettuce and peas were determined. Anti-cholinesterase activity increased when P=S was replaced by P=O, and increased further upon successive oxidation to sulfoxide and sulfone. Anti-cholinesterase activity increased for about the first four days and then declined but the presence of anti-cholinesterase activity persisted for 20 to 30 days. Residues became more polar in time, showing detoxification of the oxidative metabolites. The residues isolated from the pea foliage consisted of phorate sulfoxide and phorate sulfone (ratio 80:20). Parent itself was not identified because of interference from plant pigments. Phoratoxon was not found.

The translocation and metabolism of ¹⁴C-labelled phorate in maize seedlings, planted in treated test soil (sandy soil), was investigated. After 18 days, 77% of the applied radioactivity was recovered: 71% in soil, 4.0% in maize greens and 1.8% in maize roots. Phorate sulfoxide, phorate sulfone and phoratoxon sulfoxide were the only compounds found in extracts from maize greens; no phorate, phoratoxon or phoratoxon sulfone was found. In nine supervised field trials on maize and sweet corn where phorate was banded at planting and at cultivation, the main metabolites in forage and fodder again were phorate sulfone and phorate sulfoxide. Incidentally parent, phoratoxon, phoratoxon sulfoxide and phoratoxon sulfone were also found.

The translocation and metabolism of [¹⁴C]phorate in oat seedlings, planted in either treated silt loam soil or sandy soil, were investigated. After 13 days, for the sandy soil system 59% of total applied radioactivity was recovered: 26% in soil, 3.5% in roots and 30% in oat greens. For the silt loam soil system 76% of total applied radioactivity was recovered: 68% in soil, 0.5% in roots and 7.4% in oat greens. Although these results do not correspond to those of the maize experiment described above, they seem to indicate that the translocation of [¹⁴C]phorate to the oat seedlings depends on the soil type, where uptake and translocation is more efficient in sandy soils. Phorate sulfoxide and phorate sulfone were the major compounds present in oat greens and roots; the remainder was unknown compounds.

A great difference in the nature of ¹⁴C residues was found between carrots and the other two root crops when extraction efficiency was tested for potatoes, carrots and radishes, which were soil treated with [¹⁴C]phorate phorate. Plants were grown from seeds (or seed potatoes) in pots in a silt loam soil. When plants began to produce edible portions, an aqueous suspension of [¹⁴C]phorate phorate was pipetted onto both the soil and the partly exposed roots/tubers at an application rate equivalent to 2.25-3.35 kg ai/ha. Roots/tubers were harvested 5, 10, and 15 days after treatment (DAT). Total recovered radiolabelled (¹⁴C) residues in the roots/tubers ranged from 0.86 to 12.6 mg/kg eq. With carrots, more than 96% of the radioactivity was extractable with organic solvent, and the ratio of water soluble to organic solvent soluble ¹⁴C residues did not increase with increasing incorporation time. With potatoes and radishes, the ratio of water soluble ¹⁴C residues was much greater than with carrots and this ratio increased with time, while that of carrots remained constant. Phorate sulfoxide (20-75%) and phorate sulfone (15-70%) were the major compounds present in dichloromethane extracts from potatoes and radishes; parent (10-25%), phorate sulfoxide (50-60%) and phorate sulfone (10-25%) were the major compounds present in the dichloromethane extracts from carrots (expressed as % ¹⁴C in dichloromethane extracts). Phorate and phorate sulfoxide decreased with time, while phorate sulfone increased with time. Phoratoxon, phoratoxon sulfoxide and phoratoxon sulfone were found at trace levels (< 3.2% of ¹⁴C in dichloromethane extract). The six phorate residues accounted for 99% of ¹⁴C in dichloromethane extracts of all three crops.

Effects of light intensity and temperature on the translocation and metabolism, in oat, pea, and maize plants, of [¹⁴C]phorate in soil treated were investigated. Higher temperatures caused in most cases an increase in the uptake of ¹⁴C compounds from soil. Higher light intensity also affected the metabolism of translocated ¹⁴C compounds but primarily at 28°C. The relative distribution of benzene-soluble, water-soluble and unextractable radiocarbon was quite similar in all plants under all experimental conditions. Phorate sulfoxide was the major compound present in plant tops and soil. Contrary to soils, parent was not found in plant tops. Further compounds found in plant tops and soils were phorate sulfone, phoratoxon sulfoxide and phoratoxon sulfone.

In conclusion, when phorate is applied to the soil, it and its' degradates are taken up by the plants and translocated. When absorbed by plants, phorate is first oxidized at the thioether sulfur to form the phorate sulfoxide and sulfone and is then oxidized at the thiono sulfur to form the phoratoxon sulfoxide and sulfone. These oxidation products have a similar anticholinesterase activity as the phorate precursor and persist in plants for relatively long periods of time.

Environmental fate in soil

The Meeting received information on laboratory soil degradation and field and confined rotational crop studies. Experiments were carried out with phorate ¹⁴C labelled at the methylene position.

Aerobic soil degradation studies showed that in soil, phorate degrades to phorate sulfoxide which in turn converts to phorate sulfone. Phorate degrades rapidly, while phorate sulfoxide degrades more slowly and phorate sulfone is the most persistent of the three. The half-life of phorate in sandy loam was estimated to be 3 days, while that of phorate sulfoxide was 75 days. The half-life of phorate sulfone could not be determined due to experimental difficulties.

From three confined accumulation studies in which maize, beetroot, lettuce, spring wheat, radish, carrots, peas and barley were grown at several time intervals after treating the soil with phorate (¹⁴C labelled at the methylene position) the following conclusions could be drawn. A decrease of residue levels in soil was seen over time. Most of the radioactivity remained in the top 7.5 cm of soil, indicating that phorate and its metabolites exhibited no appreciable leaching beyond a 15 cm depth of soil. Phorate was rapidly oxidized in the soil and was converted into phorate sulfone. Only 0.2% TRR parent was found in the soil extract one month after treatment. Other metabolites identified in soil were phorate sulfoxide, phoratoxon, phoratoxon sulfoxide, phoratoxon sulfone and ethylsulfonyl methylsulfonyl methane.

In a field rotational crop study, radishes and carrots were planted in either a sand or a muck soil. The soil was treated with phorate at a rate of 3.4 kg ai/ha. The crops were planted either immediately after or one year following treatment. Radishes and carrots were harvested 4 and 14 weeks after planting, respectively. Greater conversion of phorate to phorate sulfoxide and phorate sulfone occurred in the muck soil as compared to the sand. More than 99% and 98% of the applied phorate and its oxidation products disappeared from the sand and muck soil, respectively, within a year of treatment. Low amounts of phorate sulfoxide and phorate sulfone (0.04-0.18 mg/kg eq) were found in radishes grown on both soils in the first year. No residues were present in the second year. No residues were found in carrots grown on either soil in the first year.

Soil residues consisting of phorate sulfone and predominantly non-toxic polar components can be taken up by rotational crops. However phorate applied at a rate of 3.8 kg ai/ha did not lead to the accumulation of phorate and its phosphorylated metabolites in following crops at a plant-back interval of 4 months after treatment (MAT). The assimilated phorate-derived residues are extensively metabolized by plants via non-phosphorylated metabolites to single carbon units, which subsequently are incorporated into endogenous cell components.

Environmental fate in water-sediment systems

The hydrolysis of [¹⁴C]phorate, [¹⁴C]phorate sulfoxide, and [¹⁴C]phorate sulfone in sterile buffer systems was investigated under laboratory conditions. The hydrolysis half-lives of phorate at 25°C were estimated to be 2.36, 2.47, and 2.08 days, for pH 5, 7, and 9 respectively. The major degradate (maximum 31-87% of the total administered radioactivity at termination) observed in all treatments was formaldehyde. Phorate sulfoxide (maximum 5.2-6.6% of the total administered radioactivity at day 1) was formed only at pH 5. The results show that phorate will degrade under abiotic conditions and is not expected to persist in aquatic systems.

Hydrolysis of phorate sulfoxide and phorate sulfone occurs more slowly. At 25°C, hydrolysis half-lives were estimated to be 185, 118, and 7.02 days for phorate sulfoxide and 77.1, 60.2, and 5.25 days for phorate sulfone at pH 5, 7, and 9 respectively. The degradation pathway of phorate sulfoxide and phorate sulfone was pH-dependent at elevated temperatures with de-esterification being the predominant reaction at pH 5-7.

Methods of analysis

The Meeting received descriptions and validation data on methods of residue analysis for enforcement and for residue methods used in the various study reports.

The Pesticide Analytical Manual (PAM) Volume II lists ten methods (1963-1973) for the enforcement of MRLs for phorate residues in/on plants and animal commodities. The description of method I, IA, IB was submitted to the present Meeting. Method I is based on the extraction of the parent and its oxygenated metabolites phorate sulfoxide, phorate sulfone, phoratoxon, phoratoxon sulfoxide and phoratoxon sulfone. The extracts are cleaned-up by liquid-liquid partitioning or alumina column chromatography. Phorate-related residues are oxidized to the common moiety metabolite, phoratoxon sulfone, using 3-chloroperoxybenzoic acid. The oxidized product is then analysed by GC with a phosphorus specific detector.

Method I was validated for animal commodities (milk, meat, fat, offal). Milk, meat and offal are extracted with chloroform, fat is extracted with acetonitrile. The oxidation of phorate to phoratoxon sulfone is about 70% complete. Because of this, parent recoveries are based on oxidized phorate, while phoratoxon sulfone recoveries are based on phoratoxon standards. Recoveries from milk samples resulted in 75-85% for the parent compound at 0.02 mg/kg eq and 95-103% for phoratoxon sulfone at 0.04 mg/kg eq. The reported LOQ was 0.01 mg/kg eq for milk and 0.02 mg/kg eq for meat tissues (cattle, goat, hogs, horses and sheep). Method IA was validated for cottonseed and safflower seed. Validation results are not available. Method IB was validated for sugar beets with a reported LOQ of 0.1 mg/kg eq. This method is considered an identification method in case a confirmatory analysis is required.

Phorate and its five metabolites (phorate, phorate sulfoxide, phorate sulfone, phoratoxon, phoratoxon sulfoxide, and phoratoxon sulfone) were taken through the USA FDA multiresidue method protocols described in PAM Volume I with some success. Protocols C and D gave satisfactory results.

Based on the information available it is unknown what LOQs are achievable for plant products in an enforcement situation. Based on existing CXLs and the available supervised residue trial data, it is assumed that an LOQ of 0.05 mg/kg is a practical value.

Residues in very early residue studies (1961-1963) were analysed by their cholinesterase inhibitive power in an electrometric cholinesterase assay. However, these assays are non-specific. Further, in one of those methods (method A) there is no correlation between total phorate-related residue concentration and cholinesterase activity. Residues with higher cholinesterase activity than oxidized phorate will give an erroneously high residue concentration, whereas compounds with lower cholinesterase activity than oxidized phorate will give an erroneously low residue concentration. This assay is therefore considered inaccurate for the purposes of undertaking residue analyses.

From 1971 on, analytical methods based upon gas chromatography with flame photometric detection (GC-FPD) for determination of total phorate-related residues (oxidizable to phoratoxon sulfone) have been developed for a wide range of substrates. The methods are based on the extraction of the parent and its oxygenated metabolites phorate sulfoxide, phorate sulfone, phoratoxon, phoratoxon sulfoxide and phoratoxon sulfone with either methanol-dichloromethane (10-90; plants) or acetonitrile (animal commodities). The extracts are cleaned-up and phorate-related residues are oxidized to the common moiety metabolite, phoratoxon sulfone, using 3-chloroperoxybenzoic acid in dichloromethane. The reaction mixture is cleaned-up by washing with sodium sulfite and bicarbonate solutions in water, precipitation of oily/fatty residues with aqueous ammonium chloride-phosphoric acid solution and water-dichloromethane partitioning. The dichloromethane is removed and the residue is redissolved in acetone, which is then analysed by GC-FPD in phosphorus mode. GC conditions: packed column 3% OV-210 on Supelcoport 80/100 mesh deactivated with Carbowax 20M at 155-200°C. Calibration is performed by running phorate standards through the oxidation procedure (analysed as phoratoxon sulfone). Oxidation efficiency is verified against a phoratoxon sulfone reference standard and should be at least 50% to start the analysis procedure.

The methods vary in the extraction solvent, in the clean-up procedures used before and after oxidation and in the GC-column conditions. The LOQ for most of the reported trials was 0.05 mg/kg eq. The methods have in general been validated on a wide range of substrates. However, most of the methods were validated with only a limited number of recovery samples per concentration level (n < 5), of compounds used for recovery checks (phorate and 5 oxidized metabolites), and/or of control samples analysed (n < 2). Alternatively calibration data was lacking.

Stability of pesticide residues in stored analytical samples

The Meeting received data on the stability of residues in dry beans, potatoes, sugar beets and maize stored frozen. In addition, the Meeting received data on the stability of residues in milk stored frozen.

Total phorate-related residues (oxidizable to phoratoxon sulfone) are stable for at least two years in dry bean, potato tuber, sugar beet roots and tops, maize grain, green maize plants, and maize straw samples when stored frozen at approximately -10°C to -20°C. In maize meal and maize refined oil stored at £ -23°C total phorate-related residues are stable for at least one year.

No storage data are available on green beans (seeds, seeds with pods). However, storage data for sugar beet tops or green maize forage/fodder (see below) may be extrapolated to green beans (seeds, seeds with pods). Storage data for cotton dry fodder were unavailable as well but the results for dry maize fodder may be extrapolated to cover this.

Storage stability data on cotton seed, coffee beans and on processed potato commodities were not available. The Meeting decided that given the results discussed above, it was highly unlikely that total phorate-related residues were unstable in these commodities.

Total phorate-related residues are stable in cows' milk for at least 4 days when stored in the refrigerator and at least 18 months when stored in the freezer at -20°C or lower. No storage data was submitted on tissues (poultry, ruminants) and eggs. Ruminant tissues from a cow feeding study were stored for less than one month and therefore storage data on these tissues are not needed. However, storage data on poultry tissues and eggs is lacking.

Definition of the residue

In animals, phorate is quickly detoxified and neither parent nor any of its oxidative metabolites accumulate in edible tissues, milk and eggs. The major metabolite in all tissues, milk and eggs was ethylsulfonyl methylsulfonyl methane. Nevertheless, phorate-related residues can be found at low levels.

When absorbed by plants, phorate is first oxidized at the thioether sulfur to form the phorate sulfoxide and sulfone and is then oxidized at the thiono sulfur to form the phoratoxon sulfoxide and sulfone. These oxidation products are all similar anticholinesterase agents to the phorate precursor and persist in plants for relatively long periods of time. In most cases, parent itself is present at low levels, but the ratio of the different metabolites changes from crop to crop.

The analytical methodology available relies on the oxidation of all phorate-related residues to the common moiety metabolite, phoratoxon sulfone. Supervised residue trials show that total phorate-related residues (oxidizable to phoratoxon sulfone) are not to be expected in edible crops, except in potatoes. The composition of the residue in potato tubers after application according to GAP is unknown.

Considering all of the above, the Meeting decided that the residue definition for phorate, both for enforcement and for risk assessment for animal and plant commodities, is:

Sum of parent, its oxygen analogue, and their sulfoxides and sulfones, expressed as phorate.

Although the parent compound has a log K_ow of 3.92, animal metabolism studies indicate that the total residue is not fat-soluble.

Results of supervised trials on crops

Supervised residue trials were available for fruiting vegetables (sweet corn), legume vegetables (green beans, green snap beans), pulses (dry beans, dry soya beans), root and tuber vegetables (potato, sugar beet), cereals (maize, sorghum), oilseeds (cotton), and coffee. Supervised trials on the remaining commodities that currently have a CXL were not provided. Therefore the Meeting decided to withdraw the current recommendations for fodder beet, peanut, peanut oil crude, peanut oil edible, and wheat.

In situations where residues from supervised trials at GAP show nil residues, the MRL was chosen to reflect a sensitivity that is compatible with enforcement activities. Where two different LOQs apply to the residue data, the lowest value was chosen only if the above was true. In this case the lower LOQ will be taken to represent the STMR. The HR value would then be recommended at the highest LOQ used in the studies unless a majority of the observations were derived from the more sensitive LOQ.

In situations where residues from supervised trials at GAP show nil residues even at exaggerated rates, then the MRL will still be chosen to reflect an LOQ that is compatible with enforcement activities. However, both the STMR and HR values will be set at zero.

Sweet corn (corn-on-the-cob)

Nine trials were reported on sweet corn from the USA. No trials were according to GAP of the USA (1.1-1.5 kg ai/ha, PHI 30 days). In the trials Phorate granular formulation was either applied as a single application in a band at planting or as a double application: one in a band at planting followed by a band at cultivation. Rates per application ranged from 1.46 to 7.29 kg ai/ha, with sampling occurring 37-74 days after treatment.

The Meeting decided that there was insufficient data to estimate a maximum residue level for sweet corn (corn-on-the-cob) and decided to withdraw the current recommendation of 0.05 mg/kg.

Legume vegetables

Eighteen trials were reported from the USA. Four trials on green bean seeds (without pods), four trials on green bean pods (with seeds) and ten trials on snap bean pods (with seeds) were reported. At planting, the phorate granular formulation was either drilled to the side of the seed or banded over the row using a granular applicator at application rates between 1.68-4.70 kg ai/ha. Two of the trials on green bean seeds were according to USA GAP (1.1-2.3 kg ai/ha, PHI 60 days) and total phorate-related residues were < 0.05 (2) mg/kg eq. However, these trials could not be used since residues were measured in the beans without pods. The ten snap bean pod trials were all within GAP, albeit with a shorter PHI (48-52 days). All residues were < 0.05 mg/kg eq.

Although the analytical method used in these trials (Method M-1718) was not ideal for green beans, because of low recoveries at 0.05-0.10 mg/kg eq (< 70%) and high relative standard deviation (RSD_r) at 0.01 mg/kg eq (> 20%), the Meeting decided to use the results as no actual residues were measured or expected.

The Meeting agreed to withdraw the previous maximum residue level recommendation for common bean (pods and/or immature seeds) (0.1 mg/kg), to be replaced by a recommendation of 0.05* mg/kg. The Meeting estimated an STMR of 0.05, and a HR of 0.05 mg/kg for phorate on common beans (green pods and/or immature seeds).

Pulses

Dry beans

Twenty-three trials on dry harvested beans were available from the USA. The phorate granular formulation was applied at a rate of 2.0 to 4.7 kg ai/ha in furrow, as a band over the row, or drilled to the side of the seed at planting. In all trials total phorate-related residues were < 0.05 mg/kg eq, except for trials PA-720-010 and PA-720-011, where the actual LOQ was 0.06 mg/kg eq because of matrix interference.

Four of the trials were according to USA GAP (1.1-2.3 kg ai/ha, PHI 60 days) and total phorate-related residues were < 0.05 mg/kg eq. In four other trials where a twofold exaggerated dose was applied (4.7 kg ai/ha) total residues were also < 0.05 mg/kg eq.

The Meeting estimated a maximum residue level of 0.05* mg/kg, and an STMR of 0.05 mg/kg for phorate on dry beans.

Soya bean (dry)

Twenty eight trials on soya beans were available from the USA. At planting, the phorate formulation was applied as a side band in furrow, in a band, or drilled to the side of the seed, at rates of 1.1 to 9.4 kg ai/ha.

None of the trials were according to GAP of the USA (1.7 kg ai/ha, PHI not specified). In four trials rates were below GAP while the remainder were in excess of GAP. However, even at a rate of 9.4 kg ai/ha, total phorate-related residues were < 0.05 mg/kg eq.

The Meeting estimated a maximum residue level of 0.05* mg/kg, and an STMR of 0 mg/kg for phorate on soya beans, dry.

Potatoes

Ware potatoes are normally harvested within 90-120 days after planting. Early maturing varieties can be harvested before 90 days, while late maturing ones (such as Russet Burbank or Maris Piper varieties) are usually harvested after 120 days. The PHI therefore depends on the crop variety. Although on many labels a PHI of 90 days is indicated, the residue measured at maturity was taken for evaluation, as treatment was made before or at planting, and the potatoes are harvested when they are ready. In trials in which the time of maturity of the potatoes was not indicated, the residue level measured at the shortest PHI was used for evaluation.

Trials were reported from the USA and Canada. Twenty-one trials on potatoes were conducted in the USA and twenty-five in Canada. A phorate granulate formulation was applied in-furrow or in a band at planting, at a rate of 2.19 to 266 kg ai/ha. For the post-emergence trials, application was as a band or side dressing at hilling, at the rate of 2.7 to 10 kg ai/ha.

Seven of the USA trials could be evaluated against USA GAP (1.9-4.0 kg ai/ha, PHI 90 days). Total phorate-related residues were < 0.05 (6), 0.08 mg/kg.

Twenty of the Canadian trials could be evaluated against Canadian GAP (2.3-4.3 kg ai/ha, PHI 90 days), yielding total phorate-related residues of < 0.05 (12), 0.07 (2), 0.10, 0.11, 0.12, 0.15, 0.16, 0.27 mg/kg.

The Meeting decided to combine the USA and Canadian trials, yielding the following data set: < 0.05 (18), 0.07 (2), 0.08, 0.10, 0.11, 0.12, 0.15, 0.16, 0.27 mg/kg.

The Meeting agreed to withdraw the previous maximum residue level recommendation for potato (0.2 mg/kg), to be replaced by a recommendation of 0.5 mg/kg. The Meeting estimated an STMR of 0.05, and a HR of 0.27 mg/kg for phorate on potato.

Sugar beet

A total of 16 trials on sugar beets were conducted in the USA in 1985. Ten of these trials, were carried out with a single at planting or post-emergence treatment. In the remaining trials two applications, one at planting and the other post-emergence, were made. Rates ranged from 1.68 to 3.36 kg ai/ha per application.

Two trials were according to GAP of the USA (1.1-1.7 kg ai/ha, PHI 30 days), yielding total phorate-related residues of < 0.05(2) mg/kg eq. In two trials which received double (2 ×) rates total residues found were of < 0.05, 0.06 mg/kg eq. Three trials in which a second application was made yielded total residues of < 0.05 (2) and 0.06 mg/kg eq. In three trials in which a 2 × at planting application rate was combined with a second application, total residues found were < 0.05 (2), 0.06 mg/kg eq.

Based on the above, the Meeting decided to confirm the present recommendation of 0.05* mg/kg, and estimated an STMR and an HR of 0.05 mg/kg.

Cereal grains

Maize

Forty-five trials on maize (field corn) were reported from the USA. A phorate granular formulation was either applied as a single application in a band at planting or as a double application: one in a band at planting followed by either a side dress beside each row or a foliar treatment at cultivation. Rates per application ranged from 1.12 to 8.8 kg ai/ha.

None of the trials were according to the GAP of the USA (1.1-1.5 kg ai/ha, PHI 30 days). In the trials application rates were exaggerated (2 × or 3 ×) and/or the PHI was unacceptably long. However, in 14 maize trials where phorate was applied twice, at an application rate at GAP or two times GAP and the residue was measured at a PHI of 29 or 30 days, the total phorate-related residue was < 0.01(2), < 0.02 (12) mg/kg. From the application of even more exaggerated rates, finite residues were detected.

Based on the above, the Meeting decided to confirm the present recommendation of 0.05* mg/kg, and estimated a STMR of 0.02 mg/kg for maize.

Sorghum

Eighteen trials on sorghum were reported from the USA. Treatments ranged from one application of a granulate phorate formulation at the rate of 1.22-1.46 kg ai/ha at cultivation or at planting, to 2 applications, one at planting and another at cultivation, at rates of 1.12 up to 7.3 kg ai/ha.

In all but four of the trials, total phorate-related residues (oxidizable to phoratoxon sulfone) were determined by GC-FPD, following Method M-1722. This method is considered inaccurate for sorghum grain because of high recoveries at 0.01 mg/kg eq (> 120%) and high RSD_r (> 20%) at concentrations between 0.1-1.0 mg/kg eq. However, as all results were < 0.05 mg/kg eq, the Meeting decided to include them.

None of the trials were according to GAP of the USA (1.1-1.5 kg ai/ha, PHI 30 days). In the trials application rates were exaggerated (2 ×) and/or the PHI was unacceptably long. However, in eight trials where the residue was measured at a PHI of 30 days, the total phorate-related residues found were < 0.05 mg/kg eq.

Based on the above, the Meeting decided to confirm the present recommendation of 0.05* mg/kg, and estimated an STMR of 0.05 mg/kg for sorghum grain.

Cotton seed

Fourteen trials on cotton were reported from the USA. The majority of trials were conducted using two application timings. Rates applied ranged from 0.84 to 7.17 kg ai/ha for the first application to 2.47 to 9.86 kg ai/ha for the second.

None of the trials were according to the GAP of the USA ((0.61-2.4 kg ai/ha, PHI 60 days). In the trials application rates were exaggerated (2 ×) and/or the PHI was unacceptably long. However, in nine trials where the residue was measured at a PHI of 61-65 days, the total phorate-related residue was < 0.05 mg/kg. Two of these trials were at highly exaggerated rates (first application 7.17 kg ai/ha, second application 9.86 kg ai/ha).

Based on the above, the Meeting decided to confirm the present recommendation of 0.05* mg/kg, and estimated an STMR of 0 mg/kg for cotton seed.

Seed for beverages and sweets

Coffee beans

A total of 19 trials on coffee were conducted, two in Colombia, 13 in Brazil, and four in Puerto Rico. Dry unroasted coffee beans were analysed. There was no Colombian or Puerto Rican GAP; Brazilian GAP was 3.0-3.8 g ai/plants for up to 1660 plants/ha, and 5.0-6.2 kg ai/ha for > 1660 plants/ha, with a PHI of 90 days. Trials were considered at GAP when either the application rate in g ai/plant or in kg ai/ha was observed.

Two Colombian trials and two Puerto Rican trials were according to Brazilian GAP, yielding total phorate-related residues of < 0.05 (4) mg/kg eq. All other trials had much higher PHIs and had treatment rates either below or above the GAP rates.

Based on the above and additional information from the remaining trials, the Meeting estimated a maximum residue level of 0.05* mg/kg, and an STMR of 0.05 mg/kg for coffee beans.

Straw, fodder and forage of cereal grains and grasses

Maize forage

For the purpose of estimating the animal dietary burden, the Meeting decided to review data on sweet corn forage coupled with data on maize forage. In all of the nine sweet corn trials and six of the forty-five maize trials residues were measured in green plant material (forage). Rates per application ranged from 1.2 to 8.8 kg ai/ha.

None of the trials were according to GAP of the USA (1.1-1.5 kg ai/ha, waiting period 30 days). In the maize trials residues were measured at PHIs ranging from 83-103 days. In the trials the sweet corn was treated twice at exaggerated rates and/or a PHI that was unacceptably long. Residues were detected at varying levels. The Meeting decided to use data from the trials where two applications were made at 1.46 kg ai/ha, with a PHI of 28-37 days. Total phorate-related residues found were < 0.06, 0.09, and 0.10 mg/kg (wet weight basis).

The Meeting estimated a highest residue of 0.10 mg/kg (wet weight basis) and a median residue of 0.09 mg/kg (wet weight basis) for maize forage.

The Meeting considered that maize forage is not a traded commodity and that the data was insufficient to estimate a maximum residue level. The Meeting decided to withdraw the current recommendation of 0.2 mg/kg.

Maize fodder

In twenty-four of the forty-five trials conducted on maize (field corn) from the USA, residues were measured in dry maize plants (fodder). Rates per application ranged from 1.12 to 8.8 kg ai/ha.

None of the trials were according to the USA GAP (1.1-1.5 kg ai/ha, grazing waiting period 30 days). In the trials the maize was treated twice at rates and above GAP and/or with extended waiting periods. Residues were detected at varying levels. The Meeting decided to use data from trials that were treated twice at rates of 1.12-1.46 kg ai/ha, with a PHI of 29 days. Total phorate-related residues were 0.09 (2), 0.16 and 0.22 mg/kg (wet weight basis).

For the purpose of estimating the animal dietary burden, the Meeting estimated a highest residue of 0.22 mg/kg (wet weight basis) and a median residue of 0.125 mg/kg (wet weight basis) for maize fodder. The Meeting considered that the data was insufficient to estimate a maximum residue level and decided to withdraw the current recommendation of 0.2 mg/kg.

Sorghum forage (green)

In three of the eighteen reported sorghum trials previously reported, residues were measured in the sorghum forage.

None of the trials were according to USA GAP (1.1-1.5 kg ai/ha, PHI 30 days) as the PHI was too long (47-78 days). The Meeting therefore decided that there were insufficient data from which to derive a conclusion on residue levels in sorghum forage.

Sorghum straw and fodder, dry

In 12 of the 18 trials conducted on sorghum, residues were measured in dry sorghum fodder.

None of the trials were according to USA GAP (1.1-1.5 kg ai/ha, PHI 30 days). In the trials the crop was treated twice at rates above GAP and/or with unacceptably long PHIs. However, in eight trials where the residue was measured at a PHI of 30 days, the total phorate-related residue was < 0.05 mg/kg eq. In these trials, total phorate-related residues (oxidizable to phoratoxon sulfone) were determined by GC-FPD, following Method M-1722. This method is considered inaccurate for sorghum dry fodder because of low recoveries below 0.2 mg/kg eq (< 70%).

Based on the above, the Meeting decided not to estimate an MRL, a highest residue or a median residue for sorghum dry fodder.

Miscellaneous fodder and forage crops (group 052)

Cotton fodder, dry

In nine out of the fourteen trials conducted on cotton from the USA, residues were measured in cotton fodder, dry. Most trials consisted of two treatments at rates ranging from 0.84 to 7.17 kg ai/ha for the first application and 2.47 to 9.86 kg ai/ha for the second.

None of the trials were according to USA GAP (0.61-2.4 kg ai/ha, PHI 60 days). In the trials the crop was treated twice at rates above GAP and/or with unacceptably long PHIs. In two trials where the first application was made at a rate of 1.79 kg ai/ha and the second at 2.47 kg ai/ha, with the PHI of 64 or 65 days, residues found were < 0.05 and 0.16 mg/kg.

The Meeting considered that the data was insufficient to estimate a maximum residue level, a highest residue and a median residue for cotton fodder, dry.

Sugar beet tops

In all of the 16 trials on sugar beets conducted in the USA in 1985 residues were measured in sugar beet tops. Ten of these trials were carried out with one treatment either at planting or post-emergence. The rest of the trials consisted of two applications, one at planting and the other post-emergence. Rates ranged from 1.68 to 3.36 kg ai/ha per application.

Four of the trials (study reports PA-724-025 and PA-724-026) were considered not to be acceptable for evaluation because of unacceptably high matrix interferences for sugar beet tops (up to 0.09 mg/kg eq in PA-724-025 and up to 0.41 mg/kg eq in PA-724-026). Only one of the remaining trials was according to USA GAP (1.1-1.7 kg ai/ha, PHI 30 days), yielding total phorate-related residues of < 0.08 mg/kg eq.

The Meeting considered that the data was insufficient to estimate a maximum residue level for sugar beet leaves and tops and decided to withdraw the current recommendation of 1 mg/kg. A highest residue and a median residue also could not be estimated.

Fate of residues in storage and during processing

The Meeting received information on the fate of residues during storage of field treated potatoes at ambient temperatures. Residues declined rapidly, after 23 days in storage it was found that only 33% of the original residue level remained.

The Meeting also received information on the fate of incurred residues of phorate during the processing of potatoes, maize and coffee beans.

Five processing studies were undertaken in which field treated potatoes were either processed into flakes, chips and granules, or were washed, peeled, boiled, baked or fried. In two of those studies, processing factors for potato chips, flakes and granules could not be estimated because residues in the raw agricultural commodity were less than the LOQ. One study was disregarded because residues in the washed potatoes were higher than in the raw agricultural commodity.

Calculated processing factors were < 0.07, < 0.3 for chips, 1.6 for flakes, 1.2, 3.6 for granules, 0.32, 0.49 for washing, 0.25, 0.28 for peeling, 0.13 for boiled with peel, 0.11 for boiled without peel, 0.14 for boiled peel, 0.28 for baked with peel, 0.27 for baked without peel, 2.4 for baked peel, 0.38 for French fries, 0.52, 0.63, 0.73, 0.87 for raw peel, 2.2 for dry peel, 0.36 for microwaved with peel.

In two studies field treated maize was processed into flour and oil. One study was disregarded because residues in the raw agricultural commodity (0.036 mg/kg) were lower than the LOQ (0.05 mg/kg). Calculated processing factors were 12 for hulls, 2.3 for germ, < 0.81 for grits, 2.7 for meal, 2.3 for flour, 4.0 for crude oil, expeller, 1.0 for presscake, expeller, 4.7 for crude oil, solvent extracted, < 0.81 for presscake, solvent extracted, 5.8 for refined oil, < 0.81 for soapstock, and < 0.81 for deodorized oil.

Green coffee beans were sprayed with a phorate sulfone solution in acetone at a final concentration of 0.1 or 4.6 mg/kg. Beans were roasted at 260°C for 5 to 6 minutes in an oven, cooled and ground. Residues in the roasted beans were < 0.05 mg/kg. Because processing was not carried out with incurred residues, no processing factors were calculated.

Field treated green coffee beans were harvested from plots treated at an exaggerated rate. Harvested coffee beans were air dried for a period of 21 days. Green beans were roasted at 260°C for 5 to 6 minutes. The calculated processing factor for roasted beans was 0.067.

In the table below, relevant processing factors for potato, maize and coffee commodities are summarized. Using the HRs for potato, maize and coffee bean (0.27, 0.02 and 0.05mg/kg, respectively) the Meeting estimated HR-Ps for their processed commodities as listed below. Furthermore, using the STMRs for potato, maize and coffee bean (0.05, 0.02, and 0.05 mg/kg) the Meeting estimated STMR-Ps for these commodities.

# taken to be edible oil

Using the highest residue for maize (0.02 mg/kg) and the processing factors as indicated above, the Meeting estimated a maximum residue level of 0.05 mg/kg in maize flour, and 0.1 mg/kg in maize oil, crude, and 0.02 mg/kg in maize oil, edible. For the remaining commodities no maximum residue levels were estimated, either because the commodity is not in the Codex system or because the MRL would be lower than that of the raw agricultural commodity.

The Meeting considered the appropriate HR-P and STMR-P to be used in the dietary intake calculation for potatoes. It was recognized that raw potatoes are not consumed, but that potatoes are not always eaten peeled. The percentage of people who eat unpeeled potatoes however is unknown. Also the ratio of boiled/baked/microwaved/fried for potatoes is unknown. The Meeting therefore decided to use the HR-P and STMR-P on potatoes, microwaved with peel in the dietary intake calculations for potatoes since this represents the worst-case situation.

Farm animal dietary burden

The Meeting estimated the dietary burden of phorate residues in farm animals from the diets listed in Appendix IX of the FAO Manual (FAO, 2002). One feed commodity only from each Codex Commodity Group was used. Calculation from the HR values provides the concentrations in feed suitable for estimating MRLs for animal commodities, while calculation from the STMR values for feed is suitable for estimating STMR values for animal commodities. In the case of processed commodities, the STMR-P value is used for both intake calculations.

Estimated maximum dietary burden of farm animals

# take data raw potato peel; ^§ NU - Not Used

Estimated median dietary burden of farm animals

# take data raw potato peel; § NU - Not Used

Farm animal feeding studies

The Meeting received information on feeding studies for calves, lactating cows and laying hens.

Two groups of three Holstein calves were dosed at levels of 0 and 0.1 mg ai/kg bw for 14 consecutive days by gelatin capsules. The calves weighed 220-234 kg (average 227 kg). Dosage as ppm in the feed was not stated. Assuming a daily feed intake of 4% of the bodyweight, the dose would be 2.5 ppm. Total phorate-related residues (oxidizable to phoratoxon sulfone) in thigh muscle, fat, liver and kidney were all below the LOQ of 0.1 mg/kg. At a dose equivalent to 0.1 mg ai/kg bw, animals showed no significant differences in red blood cell cholinesterase activity as compared to controls. When levels were increased to 0.2 mg ai/kg bw, animals showed significant depression of red blood cell cholinesterase activity.

Three groups of three lactating Holstein cows were dosed twice daily via gelatin capsules at levels of 0, 0.05, and 0.1 mg ai/kg bw per day for 14 consecutive days. The cows weighed 485-505 kg (average 493 kg). Dosage as ppm in the feed was not stated. Assuming a daily feed intake of 4% of the bodyweight, the doses would be 1.25 and 2.5 ppm. Milk was sampled daily and a.m. and p.m. milkings were pooled. Tissues were not collected. Total phorate-related residues (oxidizable to phoratoxon sulfone) were determined by their cholinesterase inhibitive power in an electrometric cholinesterase assay, method B, which is an unspecific method. Total phorate-related residues (oxidizable to phoratoxon sulfone) in milk from the 0.05 mg ai/kg dose rate were all below the LOQ of 0.02 mg/kg eq. In samples from the 0.1 mg ai/kg bw dose group residues were found from day 7 onwards ranging from 0.03-0.06 mg/kg.

Fourteen non-pregnant lactating Holstein cows were divided into three treatment groups. Animals in groups A (4 cows), B (4 cows), and C (6 cows) were dosed orally once a day for 28 consecutive days with gelatin capsules using a balling gun. For two cows of group C, a withdrawal period of up to 14 days was included. Using an average actual daily feed intake of 20 kg dry matter/day, mean actual doses were calculated to be equivalent 0, 1.39 and 3.21 ppm. Milk samples were collected each day and p.m. and a.m. milkings were pooled. Animals were sacrificed within 20 hrs after the last dose and samples of loin muscle, omental fat, both kidneys and whole liver were collected. In cows from all dose groups total phorate-related residues (oxidizable to phoratoxon sulfone) were below the LOQ of < 0.005 mg/kg for whole milk (day 2 to day 28) or < 0.02 mg/kg for tissues.

In a preliminary dose-finding study, animals showed severe signs of organophosphate poisoning (diarrhoea, stiffness, muscular tremors) at doses equivalent to 14 ppm, and mild signs of organophosphate poisoning (depression, salivation, off feed consumption) at doses equivalent to 7 and 5 ppm. At doses equivalent to 1.39 and 3.21 ppm, as used in the final study, animals showed no signs of organophosphate poisoning.

Four groups of six laying hens were dosed at levels of 0, 0.1, 0.3 and 1.0 ppm as total phorate (1:1 phorate: phoratoxon sulfone) for 21 consecutive days. A composite egg sample from each group was collected only on the final day of treatment. The hens were sacrificed 2 to 3 h after final dosing with muscle, fat, liver and kidney samples collected. Total phorate-related residues (oxidizable to phoratoxon sulfone) in muscle, liver and kidney and eggs were all below the LOQ of 0.05 mg/kg and below the LOQ of 0.06 mg/kg in fat.

Residues in animal commodities

In the most recent feeding study where lactating cows were dosed at 1.39 and 3.21 mg ai/kg dry feed, no total phorate-related residues were detected in tissues and milk. Therefore no residues are to be expected at the maximum calculated dietary burden of 1.08 mg/kg feed for beef cattle and 0.67 mg/kg for dairy cattle.

In the feeding study where laying hens were dosed at 0.1, 0.3 and 1.0 mg/kg feed, no total phorate-related residues were detected in tissues and eggs. Therefore no residues are to be expected at the maximum calculated dietary burden of 0.05 mg/kg feed for poultry.

The Meeting estimated a maximum residue level of 0.02* mg/kg in mammalian meat and offal and HRs and STMRs of 0.02 mg/kg. For milk, the Meeting estimated a maximum residue level of 0.01* mg/kg and an STMR of 0.005 mg/kg.

The Meeting estimated a maximum residue level of 0.05* mg/kg in poultry meat and eggs and HRs and STMRs of 0 mg/kg.

DIETARY RISK ASSESSMENT

Long-term intake

The International Estimated Daily Intakes (IEDI) of phorate, based on the STMRs estimated for 18 commodities, for the five GEMS/Food regional diets, were in the range of 9 to 20% of the maximum ADI (0.0007 mg/kg bw), see Annex 3. The Meeting concluded that the long-term intake of residues of phorate resulting from its uses that have been considered by JMPR are unlikely to present a public health concern.

Short-term intake

The International Estimated Short Term Intake (IESTI) for phorate was calculated for 18 food commodities for which maximum residue levels were estimated and for which consumption data was available. The results are shown in Annex 4.

The IESTI represented 0-50% of the ARfD (0.003 mg/kg bw) for the general population and 0-120% of the ARfD for children. The value of 120% represents the IESTI for potato, microwaved with peel. The Meeting concluded that the short-term intake of residues of phorate, resulting from its uses other than on potato that have been considered by the JMPR, is unlikely to present a public health concern. The information provided to the Meeting precludes an estimate that the acute dietary intake from the consumption of potatoes by children aged 6 years and under would be below the ARfD.

4.18 Propamocarb (148)

TOXICOLOGY

Propamocarb (propyl-3-(dimethylamino) propylcarbamate) is a carbamate fungicide that was developed for the control of phycomycetous fungi. A toxicological monograph was prepared by the JMPR in 1984 and a monograph addendum was prepared in 1986. In 1986, an ADI of 0-0.1 mg/kg bw was established based on a NOAEL of 200 ppm, equivalent to 10 mg/kg bw per day, on the basis of minimal non-specific toxicity (i.e. reductions in body weight and food consumption) observed in a 2-year feeding study in rats.

Propamocarb was re-evaluated by the present Meeting within the periodic review programme of the CCPR. The Meeting reviewed a substantial amount of new data on propamocarb that had not been considered previously, as well as relevant data from the previous evaluation.

All pivotal studies with propamocarb were certified as being compliant with GLP.

Biochemical aspects

The kinetics of propamocarb have been studied in rats. After oral administration, propamocarb is rapidly and nearly completely absorbed with peak concentrations being reached within 1 h. Propamocarb is widely distributed, but was predominantly found in organs involved in elimination, i.e. liver and kidney. Elimination from tissues is rapid, with half lives ranging from 11 h to 26 h. Urine is the main route of excretion (about 75-91% of the administered dose within 24 h). Up to 6% of the administered dose is excreted in the faeces. Propamocarb is extensively metabolized. Unchanged propamocarb was found only in small quantities in the urine. Metabolism involves aliphatic oxidation of the propyl chain (to form hydroxypropamocarb) and N-oxidation and N-demethylation of the tertiary amine resulting in propamocarb N-oxide and mono demethyl propamocarb, respectively. No marked sex differences were observed in the absorption, distribution, excretion and metabolism of propamocarb.

Toxicological data

The acute toxicity of propamocarb is low. The oral LD₅₀s in the rat were ³ 2000 mg/kg bw. The dermal LD₅₀s in the rat were > 2000 mg/kg bw. The inhalation LC₅₀ in the rat was > 5.54 mg/L. In studies of acute oral toxicity, clinical signs of toxicity included hypokinesia, lethargy, hunched posture, body tremors, clonic convulsions, nasal haemorrhage, mouth haemorrhage, piloerection, staggering gait and ataxia within 24 h after dosing.

Propamocarb is not irritating to the eye or skin. It induced skin sensitization in a Magnusson & Kligman maximization test, but gave negative results in a Buehler test.

In many studies of short- and long-term toxicity in rats and dogs treated orally, histological examination revealed that propamocarb induces vacuolar alterations in cells. In the rat, propamocarb predominantly induces vacuolization of cells in the choroid plexus of the brain and in the lacrimal glands. In dogs, propamocarb-induced vacuolization was observed in a number of tissues (including the lacrimal glands), but not in the brain.

Short-term studies of oral toxicity were available for mice, rats and dogs. In two 3-month studies in mice, propamocarb did not induce any toxicologically relevant effects when tested at doses of up to 1952 mg/kg bw per day. Propamocarb was tested in two 4-week dose range-finding studies, and at doses of 3-1549 mg/kg bw per day in one 5-week, three 3-month and one 1-year dietary studies in rats. The main toxicological findings were reductions in body weight and vacuolization in the choroid plexus and the lacrimal glands. The lowest NOAEL for these effects, observed in a 1-year dietary study in rats, was 29 mg/kg bw per day, on the basis of vacuolization of the choroid plexus in females receiving a dose of 114 mg/kg bw per day. In a 3-month study with a 28-day recovery period, partial recovery of the choroid plexus lesion was observed after cessation of treatment. The Meeting noted that for one 13-week study in rats the JMPR in 1984 had concluded that, "The study showed the no-effect level to be at least 200 ppm". The JMPR in 1986 had established an ADI of 0-0.1 mg/kg bw per day based, in part, on this study. The present Meeting concluded, however, that the observed effects in the treatment groups in this 13-week study were marginal and not toxicologically significant.

Propamocarb was tested in two 3-month, one 1-year and one 2-year dietary studies in the dog at doses ranging from 2 mg/kg bw per day to 471 mg/kg bw per day. The main toxicological findings were vacuolization in various organs. In a 3-month dietary study in dogs, the NOAEL was 131 mg/kg bw per day on the basis of vacuolar alterations in various organs. In a 1-year dietary study in dogs, the NOAEL was 39 mg/kg bw per day on the basis of vacuolization in various organs. In a 2-year study in dogs, the NOAEL was 71 mg/kg bw per day on the basis of an increase in the severity of glomerulosclerosis and loss of colour and reflectability of the tapetum lucidum of the ocular fundus. Since humans do not have a tapetum lucidum, the Meeting considered that the ocular effects in the dog were not relevant for humans.

The effects of dermal exposure to propamocarb were assessed in rats. In a 3-week study, no treatment-related systemic effects were observed at doses of up to 720 mg/kg bw per day (the highest dose tested). In a 4-week study in rats treated dermally, the NOAEL for systemic effects was 300 mg/kg bw per day on the basis of vacuolization of the choroid plexus of the brain and on reductions in body-weight gain, blood cholesterol and albumin concentrations and liver and thymus weight.

Long-term dietary studies have been performed in mice and rats. No carcinogenic effect of propamocarb was observed in any of these studies. In mice, no toxicologically relevant effects were observed in an 18-month study with doses of up to 883 mg/kg bw per day, and in a 2-year study with doses of up to 54 mg/kg bw per day. In another 18-month study in mice, the NOAEL was 106 mg/kg bw per day on the basis of reductions in body weight and body-weight gain.

In a 2-year study of toxicity and carcinogenicity in rats, minor decreases in food consumption (< 7%) and body weight (< 5%) were observed at a dose of 37 mg/kg bw per day (the highest dose tested). The present Meeting concluded that the small effects on food consumption and body weight were not toxicologically relevant. Since this study had several flaws, it was considered to be of limited value. In a second 2-year study of toxicity and carcinogenicity in rats, the NOAEL was 84 mg/kg bw per day on the basis of a decrease in body weight and body-weight gain and an increased incidence of vacuolization of the ependymal cells of the choroid plexus of the brain. In a third 2-year study of toxicity and carcinogenicity in rats, the LOAEL was 150 mg/kg bw per day (the lowest dose tested) on the basis of an increased incidence of vacuolization of the choroid plexus and the lacrimal gland ducts.

The Meeting concluded that propamocarb is not carcinogenic in rodents.

Propamocarb gave negative results in an adequate range of tests for genotoxicity in vitro and in vivo. The Meeting concluded that propamocarb is unlikely to be genotoxic.

In view of the lack of genotoxicity and the absence of carcinogenicity in mice and rats, the Meeting concluded that propamocarb is unlikely to pose a carcinogenic risk to humans.

In a two-generation dietary study of reproductive toxicity in rats, the NOAEL for parental toxicity was 1250 ppm (equal to 58 mg/kg bw per day for males) on the basis of reductions in body weight and body-weight gain. On the basis of a reduction in body-weight gain in the pups, the NOAEL for offspring toxicity was 1250 ppm (equal to 90 mg/kg bw per day based on the propamocarb intake in females). The NOAEL for reproductive effects was 8000 ppm (the highest dose tested, equal to 336 mg/kg bw per day). In a two-generation study of reproductive toxicity in rats treated by gavage, the NOAEL for parental toxicity was 50 mg/kg bw per day on the basis of clinical signs of toxicity and vacuolar changes in the epithelial cells of the choroid plexus and epididymis. The NOAEL for offspring toxicity was 200 mg/kg bw per day on the basis of decreased pup viability. The NOAEL for reproductive effects was 50 mg/kg bw per day on the basis of a reduced copulation index in females.

The effect of propamocarb on prenatal development was investigated in rats and rabbits. In none of the studies was propamocarb teratogenic. In a study in rats treated by gavage, the NOAEL for maternal toxicity was 680 mg/kg bw per day on the basis of clinical signs of toxicity, reduced body weight and increased mortality. The NOAEL for embryo- and fetotoxicity in this study was 204 mg/kg bw per day on the basis of a slightly increased incidence of number of dead fetuses and a delayed ossification. In a dietary study of developmental toxicity in rats, the NOAEL for maternal toxicity was 123 mg/kg bw per day on the basis of reduced body weight, body-weight gain and food consumption. The NOAEL for embryo- and fetotoxicity was also 123 mg/kg bw per day on the basis of reduced fetal weight and slightly delayed ossification of the cranial bones, cervical and caudal vertebrae, humerus, fore- and hind limb phalanges and metatarsals. The overall NOAEL for developmental toxicity in rats was 204 mg/kg bw per day. In a study in rabbits treated by gavage, the NOAEL for maternal toxicity was 278 mg/kg bw per day, on the basis of reduced body-weight gain. The NOAEL for embryo- and fetotoxicity was 278 mg/kg bw per day on the basis of increased postimplantation loss and increased incidence of a thirteenth rib. In a dietary study of developmental toxicity in rabbits, the NOAEL for maternal toxicity was 76 mg/kg bw per day on the basis of reduced body weight, body-weight gain and food consumption. The NOAEL for embryo- and fetotoxicity in this study was 269 mg/kg bw per day, the highest dose tested).

Studies of acute toxicity and short-term studies of oral toxicity in rats and dogs revealed no effect of propamocarb on cholinesterase activity in blood, plasma or brain, although when tested in vitro an inhibition of cholinesterase activity in rat and dog plasma was observed. In a single-exposure study of neurotoxicity, in which rats received propamocarb by gavage, the NOAEL was 200 mg/kg bw on the basis of reduced motor activity in females and increased incidence of soiled coats in both sexes. In a second single-dose study in rats treated by gavage, the NOAEL was 200 mg/kg bw per day on the basis of decreased activity 1 h after dosing in both sexes. In this study there was no evidence of treatment-related neuropathological effects 14 days after treatment with propamocarb at doses of up to 2000 mg/kg bw. In a 3-month dietary study of neurotoxicity in rats, the NOAEL was 142 mg/kg bw per day on the basis of a reduction in body-weight gain. In a study of neurotoxicity, in which rats received diets containing propamocarb for 101-104 days, the NOAEL was 100 mg/kg bw per day on the basis of intraepithelial vacuolization of the choroid plexus in both sexes and a reduction in body weight and food consumption in females.

The acute toxicity and genotoxicity of four impurities of formulations of propamocarb were tested. In studies of acute toxicity with the impurities N,N'-bis-(3-dimethylaminopropyl)urea dihydrochloride, N,N-bis-3-dimethylaminopropandiamine dihydrochloride, propyl-N-methyl-N-[3-(propoxycarbonylamino)propyl]carbamate and dipropylcarbonate in rats, the LD₅₀s were > 5000, > 3300, > 1045 and > 5000 mg/kg bw respectively. All four impurities gave negative results in tests for reverse mutation in bacteria.

In medical surveillance of manufacturing plant personnel and surveys of data banks of clinical cases and poisoning, no reports on adverse effects on human health were found.

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

Toxicological evaluation

The Meeting established an ADI of 0-0.4 mg/kg bw based on a NOAEL of 39 mg/kg bw per day, on the basis of vacuolization observed in a range of organs in a 52-week study in dogs, and using a safety factor of 100.

The Meeting established an ARfD of 2 mg/kg bw based on a NOAEL of 200 mg/kg bw, on the basis of a decreased in activity in rats 1 h after dosing and using a safety factor of 100. This ARfD is adequately protective for effects observed in studies of developmental toxicity.

A toxicological monograph was prepared.

Levels relevant for risk assessment

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

Estimate of acceptable daily intake for humans

0-0.4 mg/kg bw

Estimate of acute reference dose

2 mg/kg bw

Information that would be useful for the continued evaluation of the compound

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

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