4.18 Pirimiphos-methyl (086)(R)**

R-residue and analytical aspects

** Evaluated within the Periodic Review Programme of the Codex Committee on Pesticide Residues

RESIDUE AND ANALYTICAL ASPECTS

Pirimiphos-methyl, a broad-spectrum insecticide, was first evaluated in 1974 for toxicology and residues. Subsequently, it was reviewed for toxicology in 1976 and 1992 and for residues in 1976, 1977, 1979, 1983, 1985 and 1994. The current ADI of 0-0.03 mg/kg body weight was established by the 1992 JMPR. Currently there are 44 Codex MRLs for residues resulting from pre- and post-harvest uses of pirimiphos-methyl.

The 30th Session of the CCPR identified pirimiphos-methyl as a priority compound for periodic re-evaluation by the present Meeting.

The Meeting received data on metabolism, analytical methods, storage stability, supervised field trials, processing and farm animal feeding trials. The manufacturer and the governments of Australia, France, Germany and The Netherlands provided information on use patterns.

Animal metabolism

When a single dose of 50 mg/kg [2-¹⁴C]pirimiphos-methyl was administered by gavage to rats fitted with a bile duct cannula, 33-38% of the administered radioactivity was excreted in urine, 17-21% in the bile, and 16-30% in the faeces within 48 h. Uncannulated rats receiving the same dose excreted 61-76% of the administered radioactivity in urine and 15-29% in faeces in 48 h.

After a single dose of 1 mg/kg given to normal rats, the main urinary metabolite was 2-ethylamino-6-methylpyrimidin-4-ol (R35510). At a single dose of 250 mg/kg, the main metabolites were O-2-ethylamino-6-methylpyrimidin-4-yl O-methyl O-hydrogen phosphorothioate (desethyl-R402186) and R35510 in male rats and O-2-diethylamino-6-methylpyrimidin-4-yl O-methyl O-hydrogen phosphorothioate (R402186) and desethyl R402186 in female rats. No parent compound was present in urine or bile. Faeces of bile-cannulated rats contained only pirimiphos-methyl while those of normal rats also contained several metabolites.

These results indicate that pirimiphos-methyl was incorporated, metabolized, and eventually excreted in urine. Re-absorption of pirimiphos-methyl metabolites from bile appeared to occur.

A lactating goat dosed with 50 mg/kg [2-¹⁴C]pirimiphos-methyl in gelatin capsules twice daily for 7 days at a rate equivalent to 45 ppm in the diet excreted 89% of the administered dose in urine and faeces and 0.2% in milk. In fat samples (TRR 0.067 mg/kg pirimiphos-methyl equivalents) the major residues were pirimiphos-methyl (55% of the TRR) and O-2-ethylamino-6-methylpyrimidin-4-yl O,O-dimethyl phosphorothioate (R36341) (17% of the TRR). In other tissues (TRR 0.042 mg/kg in meat, 0.32 mg/kg in liver and 0.50 mg/kg in kidney as pirimiphos-methyl) and milk (TRR 0.18 mg/kg pirimiphos-methyl equivalents) they were R35510 (12-35% of the TRR), 2-amino-6-methylpyrimidin-4-ol (R4039) (7-20% of the TRR) and 2-diethylamino-6-methylpyrimidin-4-ol (R46382) (3-5% of the TRR). Conjugates of R46382 and R35510 were found in liver and kidney. Up to 32% of the total radioactive residues were unextracted from liver. Refluxing the unextracted material in 4M HCl released 27% of the TRR originally in the liver.

Radioactivity in the milk increased sharply after the first dose and reached a peak in the afternoon of day 2. After some decrease, it stabilized on day 4.

Laying hens were dosed with [2-¹⁴C]pirimiphos-methyl in gelatin capsules twice daily for 14 days at a rate equivalent to 50 ppm in the diet, and 97.5% of the administered radioactivity was recovered from excreta collected over 14 days. Pirimiphos-methyl was the predominant residue in the fat (73% of TRR; 0.056 mg/kg) and was also present in egg yolk (9.5% of TRR; 0.022 mg/kg) but not found in muscle, liver or egg albumen.

R35510 and R4039 were the major residues in liver (12 and 6% of TRR), egg yolk (34 and 11% of TRR) and egg albumen (43 and 22% of TRR). Conjugates of these compounds were present in liver while a conjugate of R4039 was the major component of the leg and breast muscle. Thirty-nine per cent of the TRR in liver was unextracted. After refluxing the unextracted material in acid TLC of the extract showed that the major components were R35510 and R4039.

Radioactivity in eggs reached a plateau after about 6 days.

Pirimiphos-methyl was absorbed and extensively metabolized. Five transformation processes seem to occur: hydrolysis of a methyl ester group, de-ethylation of the N-diethyl group, conjugation with glucuronic acid or other biological compounds, hydrolysis of the pyrimidinyl group, and oxidation of phosphorothioate to phosphate.

Plant metabolism

Seventy g of wheat grains, rice grains (with husk) and husked rice were treated with a 2% dust formulation containing [2-¹⁴C]pirimiphos-methyl at 4 or 8 mg/kg (g/t). The treated grains were stored at 25°C for 8 months at low (12-15%) or high (17-20%) moisture content. On wheat grains treated at 4 mg/kg, pirimiphos-methyl decreased from the maximum of 2.7 mg/kg to 2.1 mg/kg at the lower moisture content and to 0.4 mg/kg at the higher moisture content in 32 weeks. The percentage of unextracted radioactivity increased from 0.02 to 0.11 mg/kg (lower moisture content) and to 1.60 mg/kg (higher moisture content) expressed as pirimiphos-methyl in 32 weeks after treatment. The main products were pyrimidinols, R46382, R35510 and R4039, with R46382 representing at least 90% of these. In all samples, the main product was R46382 which increased gradually over 8 months to a maximum of 0.17 mg/kg (lower moisture content) or 0.62 mg/kg (higher moisture content).

The degradation pattern of pirimiphos-methyl and the quantities of degradation products in rice and wheat were similar. The main product was R46382.

Radioautograms showed that radioactivity was concentrated in the pericarp of treated grain, indicating that residues in white flour and bread would be lower than in bran and wholemeal products.

Wheat, rice with husk, and husked rice grains were treated with aqueous formulations containing [2-¹⁴C]pirimiphos-methyl at rates equivalent to 15 mg/kg (g/t) (wheat) or 22.5 mg/kg (rice) and stored in the dark in desiccators to keep them at low (10-14%) or high (19-24%) moisture content for 24 weeks at 20°C. The degradation pattern and the quantities of degradation products were similar in wheat and rice. Pirimiphos-methyl accounted for 50-95% of the radioactive residues and R46382 and an unknown compound which was hydrolysed to it by acid reflux accounted for 70-85% of the remaining radioactivity. Other minor products were R36341, R35510 and R4039.

Duplicate samples of maize grain with 14% of moisture were sprayed three times with an EC formulation containing ¹⁴C-pirimiphos-methyl, each at a rate of approximately 47 mg ai/kg grain. This resulted in a total application rate of 96 mg ai/kg, an exaggerated rate. The treated grain was stored under conditions that maintained the moisture content at about 14%. In the first 12 weeks, a decrease of radioactivity corresponding to 44-63% of that applied occurred for unknown reasons. Most of the radioactivity in the grain was extractable with methanol, with 6% of the total radioactivity remaining unextracted at 0, 12 and 24 weeks after the last application. The predominant residue (no less than 60% of the TRR in week 24) was the parent compound with up to 18% of R46382, R46382 and R35510.

The studies on stored grains showed similar profiles. The predominant residue was the unchanged parent compound which accounted for no less than 60% of the TRR at the end of each experiment. The major components of the remaining residues were the pyrimidinols R46382, R35510 and R4039. These were derived from the parent compound by the same transformation processes as in animals. The main pyrimidinol was R46382 which was present at up to 10% of the TRR under conditions reflecting current GAP. Unknown compound(s) were also present in wheat grain, which were converted to R46382 by hydrolysis. R35510 and R4039 were present only at <5% of the TRR. R36341, resulting from the loss of one N-ethyl group from the parent compound, was also detected in small amounts.

Environmental fate in soil and water-sediment systems

No studies on environmental fate in soil or water-sediment systems or on rotational crops were reported. The supervised trial data were only on stored cereal grains, where use is only indoors, with little or no impact on the environment or succeeding crops.

However, pre-harvest uses are registered in many countries. Since pirimiphos-methyl is susceptible to photolysis (the half-life in sterile aqueous buffer solution is 0.46 h at pH 5 and 0.47 h at pH 7) with the major photolytic degradation product being R46382, it was estimated that the impact on the environment might not be significant.

Methods of analysis

The Meeting received information on gas chromatographic methods for determining pirimiphos-methyl in a variety of fruits and vegetables and wheat, and both pirimiphos-methyl and its metabolites in animal tissues, milk and eggs.

All methods for the determination of pirimiphos-methyl involve extraction with acetone/hexane (2:8), maceration, addition of water, shaking and centrifugation. The resulting hexane layer derived from plant samples was analysed directly by gas chromatography, and that from animal tissues, milk or eggs underwent clean-up in a silica solid-phase extraction column. The hexane layer from fat samples was subjected to an additional hexane/acetonitrile partition procedure before clean-up.

A gas chromatographic method using specific thermionic detection showed linearity between 0.0125 and 2.0 mg/ml in the final extract (3.75-600 pg injected) for all plant samples tested including wheat. The limit of quantification was 0.05 mg/kg and the average recovery within an acceptable range (70-110%) although individual recovery values ranged from 60% to 117%. Gas chromatographic methods using mass-selective detection showed linearity between 0.0125 and 2.0 mg/ml (12.5-2000 pg injected) for all plant samples except cotton seed and olives, and between 0.001 and 1.0 mg/kg (2-2000 pg injected) for animal samples. The limit of quantification was 0.05 mg/kg for plant samples and 0.01 mg/kg for animal samples, and the average recovery within an acceptable range although individual recovery values ranged from 65% to 118% for plant samples. The methods were therefore suitable for analysing both plant and animal samples.

A method for the determination of pyrimidinols in animal samples involves extraction of animal tissues with methanol/2N HCl (1:1), centrifugation, extraction with hexane, evaporation of methanol, hydrolysis of the aqueous extract in acid, butanol partition and clean-up by adsorption chromatography. Milk samples were extracted with concentrated HCl, methanol and hexane, and egg samples with methanol/2N HCl (9:1) to remove protein. No hydrolysis was used for egg or milk samples. The final extract was analysed by gas chromatography with mass spectrometric detection after trimethylsilylation. The method showed a limit of quantification of 0.01 mg/kg and an average recovery within the acceptable range for R46382 and R35510. The recovery of R4039 from animal tissues was lower (65 ± 13%) than from other samples. Since this was attributed to the inhibition of trimethylsilylation, R31680 was added as an internal standard. The validity of using R31680 was confirmed by the linear calibration for 0.1-1.0 mg/kg and 0.01-0.10 mg/kg of R4039 with the addition of 5 mg/kg and 0.5 mg/kg of R31680 respectively. The modified method was shown to be suitable for determining pyrimidinols in animal tissues, milk and eggs.

Stability of residues in stored analytical samples

The stability of pirimiphos-methyl in barley, carrot, lettuce, olive and tomato stored at <-16°C for 24 months was investigated. No significant loss of pirimiphos-methyl residues occurred during the 24-month storage.

Analytical extracts of plant samples (the final extracts for GC with thermionic or MS detection) retained in vials and stored at a temperature of 5-7°C were stable for 7 days, the maximum tested period.

No data on the storage stability of pirimiphos-methyl or its main metabolites in animal tissues or eggs were provided. A storage stability study for 2 months showed that pirimiphos-methyl and R35311 added to milk and milk fat at 0.01 or 0.1 mg/kg were stable for 2 months at -14°C. When R36341 was added to milk at the same fortification level it was degraded to below the LOQ of 0.005 mg/kg after 2 months but it was shown to be stable for 2 months when added to milk fat.

Definition of the residue

Pirimiphos-methyl is metabolized in plants and animals through two major biotransformation routes: hydrolysis of the phosphorothioate group to produce the pyrimidinol R46382, and successive loss of the two N-ethyl groups. In muscle, liver, kidney, milk and eggs, and in plants, the main metabolites were the pyrimidinols R46382, R35510 and R4039.

The present Meeting received supervised data only on stored grains in which the predominant residue was pirimiphos-methyl.

In animal fat, the predominant residue was pirimiphos-methyl. Little or no pirimiphos-methyl was found in animal tissues other than fat, or in milk and eggs, although no storage stability studies were conducted on pirimiphos-methyl or its metabolites in animal commodities except milk. A feeding study on cows indicated that pirimiphos-methyl residues were below the limit of quantification in all tissues analysed including fat. In another study pyrimidinols were also below the limit of quantification or very low. A feeding study on hens showed 0.03-0.96 mg/kg R4039 in the muscle of hens dosed with 3.3-38 mg/kg pirimiphos-methyl. The other two pyrimidinols (R46483 and R35510) were in most cases below the limit of quantification or less than 0.06 mg/kg (R35510 in liver). These pyrimidinols were thought to be of much lower toxicity than the parent and their analysis requires a different method from that for pirimiphos-methyl.

The definition of the residue in the all countries whose national MRLs were reported to the Meeting is pirimiphos-methyl.

Pirimiphos-methyl has a log P_ow of 3.90 at 20°C and in animals was found only in fat and egg yolk,, indicating that pirimiphos-methyl should be categorized as fat-soluble.

The Meeting agreed that the definition of the residue for plant and animal commodities should be pirimiphos-methyl for compliance with MRLs and for the estimation of dietary intake.

The residue is fat-soluble.

Results of supervised trials on crops

Supervised post-harvest trials on stored cereal grains were conducted in Germany and the UK. Approved application rates for stored cereal grains are in general 4-8 g ai/t. Only three of 20 countries whose information was available approved rates outside this range.

In wheat trials in Germany and the UK, pirimiphos-methyl residues resulting from 12 trials using rates within the range mentioned above were 1.8, 1.9, 2.2, 2.3 (2), 2.6 (2), 3.2 (2), 3.7, 3.8 and 4.5 mg/kg, and those from 16 barley trials within the same range were 0.74, 0.80, 1.0 (2), 1.3 (2), 1.4, 1.5, 1.6, 2.0, 2.4, 2.6, 2.7, 2.8, 3.1 and 3.7 mg/kg.

Trials on oats, rye and maize were conducted in accordance with the GAP of many countries. The residues were 2.9 mg/kg in oats, 1.9 mg/kg in rye and 2.4 mg/kg in maize. Only a single trial on each crop was reported, but in the studies on the fate of pirimiphos-methyl in stored grain it was estimated that the degradation profiles after the application of pirimiphos-methyl were similar qualitatively and quantitatively among the grains analysed, namely wheat, rice and maize. The Meeting therefore agreed to combine the results of these trials to recommend a group MRL for cereal grains.

The combined values are 0.74, 0.80, 1.0 (2), 1.3 (2), 1.4, 1.5, 1.6, 1.8, 1.9 (2), 2.0, 2.2, 2.3 (2), 2.4 (2), 2.6 (3), 2.7, 2.8, 2.9, 3.1, 3.2 (2), 3.7 (2), 3.8 and 4.5 mg/kg.

The Meeting recommended an MRL of 7 mg/kg Po for cereal grains to replace the existing CXL of 10 mg/kg Po. The STMR and HR were 2.3 and 4.5 mg/kg respectively.

No data on supervised trials were provided on the following commodities: apples, Brussels sprouts, head cabbages, carrots, cauliflower, cherries, citrus fruits, common beans, cucumbers, blackcurrants, dates, dried fish, gooseberries, kiwifruit, lettuce, mushrooms, olives, peanuts, peanut oil, pears, peas, peppers, plums, potatoes, raspberries, spinach, spring onions, strawberries, and tomatoes. The Meeting therefore decided to recommend withdrawal of the MRLs for these commodities.

Fate of residues during processing

In a laboratory scale baking of flour treated with radiolabelled pirimiphos-methyl to bread and biscuits there was little degradation of pirimiphos-methyl, with up to 10% of the TRR attributed to R46382 and R4039. R46382 was present at 25% of the TRR in bread crusts and R4039 at 12% in breadcrumbs.

Processing wheat grain treated at 4 g ai/t on a commercial scale resulted in a concentration of pirimiphos-methyl in bran and offal and a reduction in white and wholemeal flours and breads.

The calculated processing factors and STMR-Ps are shown in the Table below. A maximum residue level was calculated for bran, in which the highest concentration of pirimiphos-methyl was found, from the HR for wheat grain, 4.5 mg/kg.

The Meeting recommended an MRL of 15 mg/kg (PoP) for unprocessed wheat bran to replace the existing XL of 20 mg/kg and recommended withdrawal of the existing CXls for wheat wholemeal, wheat flour, white bread and wholemeal bread as STMR-Ps were calculated for intake estimation.

Processing factors for wheat products

Residues in the grain used for processing were 1.9 mg/kg for preparing bran, offal, white flour and white bread, and 2.9 mg/kg for preparing wholemeal flour and wholemeal bread.

Processing wheat grain to processed fractions and to bran breakfast cereals on a commercial scale showed a concentration of pirimiphos-methyl in fine bran (PF 1.7) and light bran (PF 1.6) but a reduction in heavy bran (PF 0.70). The processing factor from grain to bran breakfast cereals was calculated to be 2.3-4.

Residues of pirimiphos-methyl were extremely low, close to or below the limit of quantification of 0.01 mg/kg, in beer produced from barley grain treated with pirimiphos-methyl at a normal rate. Only two of 22 samples brewed separately in single brews in two experiments contained pirimiphos-methyl above the LOQ, with one showing 0.08 mg/kg. In malt, malt germ, wort and spent malt, low-level residues were detected showing significant degradation (more than 90%) of pirimiphos-methyl during malting. In 16 beer samples obtained from sequential brews pirimiphos-methyl residues were <0.01-0.04 mg/kg. The Meeting calculated a processing factor from these results of <0.002 and an STMR-P for beer of 0.01 mg/kg.

Less than 4% of the residue in treated oat grain was found in rolled oats, but there were insufficient data to calculate a processing factor.

Studies with barley and oats indicated that no more than 10% (2-9%) of the pirimiphos-methyl residue in grain was found in the kernels of treated grains when the application rate was 4 g ai/t. Most of the residues were associated with the husks. There were too few trials to estimate the ratio of pirimiphos-methyl between the husks and kernels.

Since no processing studies were available for rice or rye, the Meeting decided to recommend withdrawal of the existing CXLs for rice bran, unprocessed; rice, husked; rice, polished and rye wholemeal.

Residues in animal commodities

Farm animal dietary burdens

The Meeting estimated farm animal burdens of pirimiphos-methyl residues with the diets in Appendix IX of the FAO Manual. A plateau was reached rapidly in milk (4 days). In eggs, a metabolism study indicated that a plateau was reached in 6 days and feeding studies indicated 15 days and 6 days. The Meeting agreed that calculation from MRLs would provide the feed levels suitable for recommending animal commodity MRLs, and calculation from feed STMRs would be suitable for the estimation of animal commodity STMRs.

¹ HR

The pirimiphos-methyl dietary burdens for animal commodity MRL and STMR estimation are beef cattle, 6.4 and 2.1 mg/kg, dairy cattle 5.6 and 2.9 mg/kg, and poultry 6.3 and 2.1 mg/kg.

Farm animal feeding studies

Milk obtained from lactating cows fed diets containing 0, 5, 15 or 50 ppm (dry weight basis) of pirimiphos-methyl for 30 days contained only very low concentrations of pirimiphos-methyl throughout the trial; residue concentrations higher than those found in controls were seen only in milk from cows given 15 ppm (<0.005 to 0.02 mg/kg) and 50 ppm in the diet (<0.005 to 0.03 mg/kg), and were below 0.01 mg/kg (<0.005-0.008 mg/kg) from 5 ppm. No trend of accumulation in milk was observed. Pirimiphos-methyl residues were below the limit of quantification in all tissues analysed (heart, liver, kidney, fat, cardiac muscle, adductor and pectoral muscle) from all the cows.

Lactating cows were fed twice a day for 30 days with feed containing 0, 8.3, 31 and 94 ppm of pirimiphos-methyl. Residues of the pyrimidinols R46382, R35510 and R4039 were extremely low in milk, even from animals that received the highest dose; residues above the LOQ of 0.01 mg/kg were found only in isolated cases. At 94 ppm up to 0.03 mg/kg of these hydroxypirimidines were found in the liver, and slightly higher concentrations in the kidneys, especially of R46382 (up to 0.16 mg/kg) and R35510 (up to 0.14 mg/kg).

Laying hens given single oral doses of [2-¹⁴C]pirimiphos-methyl excreted only 0.16-0.33% of the administered radioactivity in eggs. With unlabelled doses of 1-8 mg/kg 12% of the collected eggs contained pirimiphos-methyl above 0.001 mg/kg, the highest residue being 0.008 mg/kg. Radioactivity in egg albumen and yolk from hens given daily doses of [2-¹⁴C]pirimiphos-methyl for 28 days at 4 mg/kg reached a maximum (0.04 mg/kg) 15 days after the start of dosing, and remained fairly constant thereafter. In egg yolks pirimiphos-methyl did not exceed 0.001 mg/kg. Egg white from hens receiving 32 ppm pirimiphos-methyl for 7 days contained pirimiphos-methyl at 0.001-0.007 mg/kg, which remained constant, while in the yolk the pirimiphos-methyl concentration increased to a maximum of 0.012 mg/kg at day 6. No pirimiphos-methyl was found in any of the muscle samples taken at the end of the study. There was no evidence of accumulation of residues in eggs.

Muscles from laying hens given 3.3-38 ppm pirimiphos-methyl in the diet for 28 days were found to contain 0.03-0.96 mg/kg of R4039. R35510 and R46382 were in most cases below the limit of quantification in muscle, liver and eggs. The highest residue was 0.06 mg/kg R35510 in liver from 38 ppm.

Maximum residue levels for animal commodities

Since no data was reported on the storage stability of pirimiphos-methyl or its metabolites in animal tissues, milk or eggs, the Meeting concluded that it could not exclude the possibility that the low or negligible concentrations of pirimiphos-methyl and metabolites reported were due to the unstable nature of these compounds in the samples.

According to one feeding study pirimiphos-methyl was present at <0.005-0.008 mg/kg (mean 0.0053 mg/kg calculated from 0.005 mg/kg instead of <0.005 mg/kg) in whole milk from cows fed at 5 ppm pirimiphos-methyl, and at <0.005-0.02 mg/kg (mean 0.0064 mg/kg) in whole milk from cows fed at 15 ppm. Pirimiphos-methyl was found to be stable in milk for two months when stored at -14°C. Concentrations of pirimiphos-methyl in butter prepared from the milk of cows fed at 50 ppm were on average twice those in whole milk, indicating that most of the pirimiphos-methyl residue was in the non-fat fraction of the milk.

At the calculated maximum burden of 5.6 mg/kg, it is unlikely that whole milk would contain pirimiphos-methyl above 0.01 mg/kg, and at the STMR burden of 2.9 mg/kg STMR its level was calculated to be less than 0.003 mg/kg.

The Meeting estimated a maximum residue level of 0.01 mg/kg for milks, recommended to replace the existing CXL of 0.05 mg/kg, and an STMR of 0.003 mg/kg. The Meeting concluded that any preferential solubility of pirimiphos-methyl in milk fat was insufficient to attach the suffix "F" to the maximum residue level.

The Meeting decided not to estimate maximum residues levels for animal commodities except milk pending a storage stability study with animal commodities. It therefore recommended withdrawal of the CXLs for eggs at 0.05 mg/kg and meat (from mammals other than marine mammals) at 0.05 mg/kg.

FURTHER WORK OR INFORMATION

Desirable

1. A study on the storage stability of pirimiphos-methyl and metabolites in animal tissues and eggs.

2. Pirimiphos-methyl concentrations in fat in animal feeding studies.

DIETARY RISK ASSESSMENT

Long-term intake

The International Estimated Dietary Intakes (IEDIs) were calculated for the five GEMS/Food regional diets using the STMR for cereal grains and STMR-Ps for milks, beer and processed wheat products estimated by the current Meeting (Annex 3). The current ADI is 0-0.03 mg/kg bw and the calculated IEDIs were 10-50% of the maximum ADI. The Meeting concluded that the intake of residues of pirimiphos-methyl resulting from the uses considered by the current JMPR was unlikely to present a public health concern.

Short-term intake

The International Estimated Short-Term Intakes (IESTI) of pirimiphos-methyl by the general population and by children were calculated for commodities for which STMRs or STMR-Ps were estimated by the current Meeting (Annex 4). The Meeting considered that it might be necessary to establish an acute reference dose for pirimiphos-methyl, but as one has not been established the short-term risk assessment for pirimiphos-methyl could not finalized.