ALPHA-CYPERMETHRIN First draft prepared by Adriana Fernández Suárez, Buenos Aires, Argentina Richard Ellis, Washington, DC, United States

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1 ALPHA-CYPERMETHRIN First draft prepared by Adriana Fernández Suárez, Buenos Aires, Argentina Richard Ellis, Washington, DC, United States ADDENDUM To the monograph prepared by the 47 th meeting of the Committee and published in the FAO Food and Nutrition Paper 41/9 IDENTITY Chemical name: A racemate of (S)-alpha-cyano-3-phenoxybenzyl (lr,3r)-3-(2,2-dichlorovinyl)-2,2- dimethylcyclopropane carboxylate and (R)-alpha-cyano-3-phenoxybenzy1 (IS,3S)-3-(2,2- dichloroviny1)-2,2-dimethylcyclopropane carboxylate; and a racemate of (S)-alpha-cyano- 3-phenoxybenzyl (1R)-cis-3- (2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylate and (R)-alpha-cyano-3-phenoxybenzyl (1S)-cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylate {International Union Pure and Applied Chemistry (IUPAC) name} Common trade names: FASTAC, CONCORD, FENDONA, RENEGADE. CAS Number: (correct stereochemistry) Structural formula: See next page. Molecular Formula: C 22 H 19 Cl 2 NO 3 Molecular weight: Appearance: Stability: OTHER INFORMATION ON IDENTITY AND PROPERTIES White-to-cream crystalline solid Highly stable to light and elevated temperatures. It is resistant to acidic hydrolysis but undergoes ester cleavage in environmental (basic) aquatic conditions. It has optimum stability at ph = 4. Its low solubility in water indicates a low bioavailability in aquatic situations. Melting point: C Boiling point: 200 C at 9.31 Pa Vapour pressure: 3.4x10-7 Pa at 25 C (pure material) Octanol-Water p = 3.16 x 10s partition coefficient: Refractive index: 0.19 x c (c = concentration in kg/l up to 0.25, only in acetonitrile) Density: g/ml (typical for pure material) Solubility (g/l at n-hexane C): Propanol Methanol 21.3 Ethyl acetate 584 Toluene 596 Fat 78 Water 2.06 µg/l at 20 C Alpha-cypermethrin was determined to be miscible with acetone and dichloromethane at room temperature FAO FNP 41/14

2 Structural formula: AAlpha-cypermethrin Cis 2: (D) and (G) isomers Alpha-cypermethrin Cis 1: (C) and (H) isomer INTRODUCTION Alpha-cypermethrin was first reviewed by the Committee at its 47th meeting in 1996 (JECFA, 1998). In reaching its decision on MRLs for alpha-cypermethrin, the Committee took the following factors into consideration: - The ADI is 0-20 µg/kg of body weight, which is equivalent to a maximum ADI of µg for a 60-kg person. - The parent drug is the marker residue. - Fat is the target tissue, but muscle, liver and kidney should also be considered. Whole milk or milk fat is suitable for monitoring residues in milk, and egg yolk for monitoring residues in eggs.. - The metabolism of the two isomers which form alpha-cypermethrin is similar to that of the other six isomers in cypermethrin. - The metabolism and residue-depletion studies using the radiolabelled drug were not adequate, and therefore very conservative estimates of the proportion of the total residues accounted for by the marker compound in all target species were proposed. Metabolite profiles were not determined in sheep or poultry. Limited studies were presented for cattle. The percentages proposed for the estimation in individual tissues of total residues from the parent drug were 30% for muscle, 10% for liver, 5% for kidney and 60% for fat. In milk and eggs, alphacypermethrin was estimated to account for 80% and 30% of the total residues, respectively. - There was adequate information on residue from the residue depletion studies using the unlabelled drug in the recommended formulations. - Analytical methods were available, but validation was needed. Temporary MRLs for cattle, sheep and poultry were recommended : 500 µg/kg in fat, 100 µg/kg in muscle, liver and kidney, 25 µg/kg for cows whole milk and 50 µg/kg for eggs, expressed as parent drug. The temporary MRLs accommodate the ADI and the recommended use of alpha-cypermethrin as a veterinary drug: The theoretical maximum intake of its residues would be 406 µg per day, compatible with the maximum 1200 µg based upon the ADI of 0-20 µg/kg body weight. The Committee requested at the 47th meeting the following information: FAO FNP 41/

3 1. The results of residue-depletion studies using radiolabelled alpha-cypermethrin in sheep and chickens that extend beyond the recommended withdrawal times for the drug in its topical formulation. The depletion of the total residues and of the parent drug should be determined. 2. The residue-depletion study using radiolabelled alpha-cypermethrin in cattle should be reassessed to determine the depletion of total residues and of the parent drug; 3. Evidence that interconversion of the isomeric forms does not occur during metabolism in the target species. 4. Further information on the validation of the analytical methods, particularly data on the derivation of detection and quantification limits. Since the information required was not provided, at the 54th meeting the Committee did not extend the temporary MRLs for cattle, sheep and chicken.. The Committee requested similar data to be provided for evaluation at the 58 th meeting of JECFA. RESIDUES IN FOOD AND THEIR EVALUATION Conditions of use General Alpha-cypermethrin is a pyrethroid insecticide consisting of two of the four cis isomers present in cypermethrin. These isomers comprise the most biologically-active enantiomeric pair. Alpha-cypermethrin is a highly active broad spectrum insecticide effective against target pests. It is used in veterinary medicine for the control of ectoparasites such as ticks, fleas, lice and blowflies (CVMP, 1998). Dosage Alpha-cypermethrin is applied in pour-on preparation for cattle (15 g/l, g/animal) and sheep (12.5 g/l, g/animal) (sponsors communication; 2002) and also as a dip for sheep and as a spray for poultry (10 mg/animal) (CVMP, 1998). Previous studies of the metabolism of alpha-cypermethrin 1 Cattle Studies summarizing the use of alpha-cypermethrin using an oral or pour-on treatment are reported below. One lactating cow was orally dosed (twice daily for 8 days, 0.25 g/day) with 14 C- alpha-cypermethrin. 58% of administered dose was recovered (34% via faeces, 23% urine, < 1% milk) (Morrison and Richardson,1994). Residue profile were as follows: Table 1 Residues of alpha-cypermethrin (µg equivalents/kg) in an orally dosed lactating cow Tissue µg/kg Alpha-cypermethrin 1 (%) Fat Liver (8 metabolites) Kidney (9 metabolites) Muscle Milk Compound with similar chromatographic properties to alpha-cypermetrhin. 2. Separate values for backfat and peritoneal fat Urine metabolites were characterized as 14 C-3-PBA-glutamic acid conjugate (44%), 3-PBA-glycine (20%) and 3-PBA by co-chromatography (JECFA, 1997, p 62, reference study 1, table 1) Four cows were dosed with 0.15 g pour-on treatment and residues sampled at 7, 14, 28 and 35 days post-dose (Redgrave et al.,1992). Total radiolabelled residues results were mostly below the limit of quantitation (10-30 µg/kg) with the exception of some samples of fat (maximum 30 µg/kg). Radiocounting estimates were not different from levels of alpha-cypermethrin measured by GC-ECD (LOQ = 10 µg/kg). In milk, maximum levels rose to 7 µg/l by day 2-3 (71% determined as alpha-cypermethrin by GC), falling to the LOD for radiocounting (1 µg/l) by day 7. (JECFA, 1997, p 62, reference study 2, table 1) 1 The following abbreviations are used throughout: 3PBA: 3-phenoxybenzoic acid; 3-PBA-glut: N-3-phenoxybenzoylglutamic acid; 3PBA-gly: N-3-phenoxybenzoyl-glycine; 4-OH-3PBA: 3-(4-hydroxyphenoxy) benzoic acid; 3PBA-4-Osulf: 4-hydroxyphenoxybenzoyl-O-sulfate; 4-OH-alpha-cyper: 4-hydroxy-alpha-cypermethrin; α-cyper: alphacypermethrin FAO FNP 41/14

4 Sheep Radiodepletion studies for sheep were not presented. Other residue depletion studies (with unlabelled drug) Cattle 15 calves were dosed with 0.16g per animal as a pour-on treatment (Sherren, 1988b). Sampling time of residues were 3, 7 and 14 days post-dose. Maximum residues in both subcutaneous and perirenal fat occurred at day 7 ( µg/kg). Residues were detected in kidney (< 30 µg/kg), but were no detectable in muscle and liver (LOQ = 10 µg/kg) (JECFA, 1997, p 64, reference study 2, table 2). Two groups of 11 female calves were dosed at 0.15g pour-on treatment per animal (Cameron et al., 1993). Residue sampling times were 3, 7, 14, 21 and 28 days post-dose. Maximum residues in both subcutaneous and perirenal fat occurred at day 14 ( µg/kg), then declining until day 28 (< 40 µg/kg) (JECFA 1997, p 64, reference study 3, table 2). In both studies residues were higher in perirenal fat than in subcutaneous fat 15 cows (three per treatment group) were treated at 0.1, 0.15 and 0.2 g per cow (Sherren, 1988a). Residues were sampled at 1, 2, 3, 4, 7, 14 and 21 days post-dose. Maximum residues (up to 5 µg/kg) were observed between days 2-5 after treatment and were all less than the LOQ (2 µg/l) by day 21 for all treatments. The residue profile follows closely that seen with the radiolabel study using the 0.15 g dose (JECFA, 1997, p 64, reference study 1, table 2). Similar residue profiles to those obtained in the radiolabel studies are found, showing residues principally in fat tissues (perirenal higher than subcutaneous), followed by kidney and minor quantities in muscle. Maximum residues in fat tissues occurred between 7 and 14 days at the same pour-on dose. Results were not corrected for recovery although they were determined. In milk maximum residues were generally observed at short times after treatment (2-5 days) declining thereafter. Analytical evidence suggests that the residues were mainly alpha-cypermethrin. Most measures are near the LOQ of methods employed. Sheep Six sheep, three treated with a pour-on and three dip treated, dosed at 0.2 g pour-on and 60 mg/l dip, were analyzed for residues in fat, skin and wool at 3, 7 and 14 days post-dose (Francis and Gill,1989). High residues were found in skin (up to 1400 µg/kg) for at least two weeks in both treatments. Subcutaneous fat residues were not detectable within 7 days of dosing in the pour-on treated, but in dip treated, residues were 40 µg/kg at 7 and 14 days of dosing (minimum concentration measured was 10 µg/kg) (JECFA, 1997, p 64, reference study 4, table 2). Sheep, pour-on treated (five dosed at 0.01 g/kg bw and five at 0.02 g/kg bw, were sampled at 7 days post-dose (White, 1987). Residues were present after treatment in both perirenal and omental fat with residues at µg/kg and 3-11 µg/kg, respectively, at the 0.1 g/kg bw treatment and 5-18 µg/kg and 2-19 µg/kg, respectively, at the 0.2 g/kg bw treatment (JECFA, 1997, p 64, reference study 5, table 2). In these studies, residues were measured only in fat and were not corrected for recovery. Other tissue residues were not measured. The majority of residues seemed to remain unabsorbed (high concentrations in skin and wool) after external treatments. Bound residues Bound residues for cypermethrin were less than 20% in liver and 10% in other tissues (JECFA, 1997, p. 53). New metabolism and residue studies with 14 C-alpha-cypermethrin Two new radiolabel studies were submitted for evaluation - one in sheep and one in cattle. Studies for poultry were not carried out. One additional report was provided on analytical methods. All the studies were carried out using appropriate and applicable good laboratory practices. Cattle Alpha-cypermethrin was formulated at a nominal concentration of 15 g/l and was topically administered to sixteen animals (8 steers, 3-7 months old, kg and 8 lactating cows, 4-7 years old, kg) along the region of the back between the shoulders and the rump along the mid-dorsal line. Cows were treated following the morning milking. Mean dose level achieved was 3 mg/kg bw ( mg/g of dose solution). The two control animals were dosed with placebo formulations. Two steers and two cows were sacrificed at each 3, 7 (phase I) and 14, 21 (phase II) days post-dose. Each lactating cow was milked twice daily until sacrifice. The study was conducted in two phases. The test substance used was an isotopic mixture of 12 C-, 13 C- and 14 C-alpha-cypermethrin. The 14 C label at the benzyl ring was relatively stable to metabolic transformation. 13 C was labelled at the alpha carbon (chemical purity 96.1%, radiochemical purity 99.4%, specific radioactivity 7.94µCi mg -1 ). Only two samples (one male and one female containing significant residue levels) were used to determine the metabolic profile at each sacrifice interval. The total radioactive residue (TRR) concentration and extractability of different FAO FNP 41/

5 fractions in the liver, kidney, muscle, milk and fat samples from each animal was determined using a radiometric method: fat, and milk by direct liquid scintillation counting (LSC). The limit of quantitation (LOQ) was 4-5 µg/kg. Liver, kidney and muscle were analysed by LSC of the 14 CO 2 absorbed following combustion (LOQ 7 µg/kg). Procedural recoveries were determined. Details are summarized below. Table 2. Depletion of 14 C-alpha-cypermethrin (mg/kg equivalents) in tissues and milk of cattle Sample Sacrifice time 3 day 7 day 14 day 21 day Back fat ± ± ± ±0.069 Omental fat ± ± ± ±0.118 Liver ± ± ± ±.051 Kidney ± ± ± ±.015 Muscle* <0.001(NA) ± ± ±0.004 Milk ± ± ± (NA) NA= not applicable Data expressed as mean (n=4) ±SD in tissue, number of milk samples is variable * Many results were calculated from data less than 30 dpm above background. Mean TRR in milk increased between 12 and 84 hours (3.5 days) after application and reached a peak value of mg/kg between 60 and 108 hours, then declined steadily to a mean concentration of mg/kg at 228 h (9.5 days) post-dose and more slowly thereafter. A half-life of 129 hours was estimated. Mean TRR are highest in fat tissues (back fat > omental fat), followed by kidney, liver, and muscle. Considerable quantity of radioactivity is eliminated by urinary excretion. Mean concentration of total radioactivity reached the maximum values in both fat tissues at 21 days. Back fat was shown to contain the highest TRR at all time points excluding 14-day samples. Residues appear to fluctuate at 7, 14 and 21 days without notable depletion. In omental fat, there seems to be minimal differences between 7, 14 and 21 days (considering SD). Other tissues (liver, muscle) reached maximum values by day 7 with minor mean levels of radioactivity although there are no significant differences in most cases for subsequent time points due to high individual sample variability. Residues in kidney tissue reached a maximum at day 14. Most measures in muscle were below the LOQ. Extractability of residues: In milk, radiolabelled residues were partitioned into an organic solvent mixture following the addition of 5% (w/v) potassium oxalate, ethanol, diethyl ether and hexane. Hexane clean up and concentration steps were included prior to analysis. Radiolabelled residues were extracted from back and omental fat by maceration with acetonitrile. The same technique was used for kidney and muscle, although post-extracted solids (PES) were further extracted with methanol:water (80:20 v/v). Liver was subjected to two enzyme hydrolysis steps (pepsin and protease), followed by precipitation of the combined enzyme extracts and solvent extractions to obtain a enzyme hydrolysis organic extract. Residual tissue material was subsequently hydrolyzed with 6N HCl and extracted with solvent. The combined organic extracts were then analysed. Radioactivity was measured by liquid scintillation counting (LSC) of solvent extracts. PES samples were analysed by LSC following sample combustion. Individual data for all samples were presented. Procedural recoveries were determined. Extraction efficiency values ranged 82-96% TRR in milk, 70-99% TRR for fat, 47-84% TRR for liver and 50-66% TRR for kidney after complete processing, some normal losses were observed through process in these tissues. Residues were extracted in only two muscle samples due to the low residue levels (90% and 77% TRR). Methanol:water (80:20 v/v) extracts recovered 18-32% in kidney and 3.2% in one muscle sample. Analysis of radiolabelled extracted metabolites: Radiolabelled extractable residue components were analyzed using an HPLC radioanalysis procedure (urine was analysed directly). Data of the quantification of radiolabelled metabolites in edible tissues are presented as ranges through the 21 days post-treatment. The limit of quantification was 5 µg/kg equivalents in fat, 7 µg/kg for liver, kidney and muscle and 4 µg/kg in milk. Quantification and identification of radiolabelled residues in tissues clearly showed that unchanged 14C-alphacypermethrin was the major residue component in both omental and back fat and milk (and in the only sample of muscle analyzed). Extensive metabolism of alpha-cypermethrin was observed in liver and kidney, with more polar metabolites than alpha-cypermethrin present in both cases. Minor unknown components were detected in liver, kidney and milk. A small amount of unchanged alpha-cypermethrin was observed in kidney up to 14 days post treatment but not in liver. The main metabolite found in liver and kidney was the 3-PBA-glut. In the case of liver, enzyme and 6N HCl hydrolysates from post extracted solids (PES) were found to contain the same metabolites (3-PBA and 3-PBA-glut) and there were no obvious sex differences. This is also the case for the methanol:water extracts in kidney. In urine, two major components that co-chromatographed with 4-OH-3-PBA and 3-PBA-glut were detected (70-80%) and a number of non-identified very polar metabolites FAO FNP 41/14

6 Table C-alpha-cypermethrin metabolites in cattle tissues and milk Tissue Back Fat (n = 8) Omental Fat (n = 8) Liver (n = 8) Kidney (n = 7) Milk (n = 78) Muscle (n = 1) FAO FNP 41/ %TRR 1 3-PBA 4-O-sulf 3-PBAglut 3-PBA 4-OH α- cyper ND ND ND 1-6 [4] (3-5) ND ND ND 1-8 [5] (2-5) [6] 4-6 [3] (5-24) (29-90) (4-20) (5-11) 5-7 [3] (2-4) (12-39) 3-19 [7] (1-10) 4-22 [3] (2-12) ND ND ND 1-10 [6] (1-2) α -cyper (35-647) (20-421) Unknowns [number of] ND ND ND [5] (17-44) 2-37 [6] (1-22) (10-83) 7-24 [4] (4-13) + ND ND ND ND 90 ND (35) 1. The second entries in parenthesis in each cell are concentrations expressed in µg/kg by calculation. Numbers in square brackets are the number of positive samples, except in column labelled unknowns. + means two samples indicated the presence of one or two radiolabelled metabolites. ND is non detected. Muscle sample is from day 7. Values of % TRR and µg/kg may seem different because the TRR in µg/kg is different for each sample. 14 C-Alpha-cypermethrin at different time points measured by HPLC-radioanalysis. In milk, the mean values of 14 C alpha-cypermethrin equivalents, calculated from the 60h (earliest time point) was 51 µg/kg; at 84h the value was 66 µg/kg, then decaying steadily to 36µg/kg at 120h, 29 µg/kg at 168h, and 20 µg/kg at the last measured time of 216h. These measures are determined from only two samples analyzed at each milking time. 14 C alpha-cypermethrin was detected at all times post-treatment in all tissues except liver. On average (n=2), calculated µg/kg of this compound shows no differences between 7, 14, and 21 days for omental fat (237, 263, 287 µg/kg). Day 3 samples showed an average value of 41 µg/kg. Substantial fluctuation was observed for back fat with time. Alphacypermethrin residues were 62, 406, 143, 567 µg/kg at 3, 7, 14, 21 days, respectively. Values for back fat are considerably higher than omental fat. These are the same tendencies found in analysing TRR values. No analysis can be made in muscle because only one sample at 7 days post-treatment contained parent drug (35 µg/kg). No parent drug was detected in any liver sample and only one kidney sample at 14 days (22 µg/kg) had residues of more than 8 µg/kg. Confirmation of alpha-cypermethrin as main residue. An APCI LC-MS method was used to confirm alphacypermethrin as the main residue in a single fat and milk sample. Based on the retention time, the presence of the ions at m/z 207 and 209 and the ratio of these ions, the peaks in the radiochromatograms for both tissues provided confirmation of alpha-cypermethrin as the main residue. This conclusion can be extrapolated to all samples where alpha-cypermethrin was identified by co-chromatography. Analysis of alpha-cypermethrin by a GC-ECD method. Same tissue samples were analyzed for alpha-cypermethrin content using a GC-ECD method (LOQ 50µg/kg). The results of the GC analysis showed that alpha-cypermethrin, on an average (n=8), accounted for 76 ±44% of the back fat TRR with high variability and 70 ±16% of the omental fat (n=7); the ratios are higher if measured by radioanalysis (84 ±17% and 91 ±10%, respectively). For both types of fat, alpha-cypermethrin on an average (n=16) accounted for 71 ±33% of the fat TRR by GC-ECD analysis which is lower than the 87 ±11% obtained from radioanalysis of the fat samples. Residues of alpha-cypermethrin (GC-method) in fat reached levels up to 713 µg/kg in back fat and up to 337 µg/kg in omental fat at 21 days post treatment. Residues (µg/kg) calculated from radioanalysis shows some differences with those found by the GC method in both fats, but follows the same tendencies with time as the radiolabel measures. Muscle, kidney and liver samples from 3 to 21 days post-dose contained levels of alpha-cypermethrin below the limit of quantification (50 µg/kg) of the GC-ECD method. In milk, alpha-cypermethrin residues (µg/kg) calculated by radioanalysis are similar to those obtained by GC analysis at different milking times (60-126h, n=13). Residues of alpha-cypermethrin (GC-ECD) were higher in milk (maximum value of 89 µg/kg in 60 hours) and in fat (maximum value of 713 µg/kg in back fat and maximum value of 337 µg/kg in omental fat at 21 days) than other tissues and was present as the main component in the extracts analyzed. HPLC radioanalysis of selected milk samples near the maximum values were analyzed for alpha-cypermethrin content. On an average (n=17), 14 C- alpha-cypermethrin accounted for 86 ±6% in milk TRR (range 74-95%). Some milk samples analyzed by GC-ECD (LOQ=10µg/kg) showed that alpha-cypermethrin accounted for 76 ±13% (n=81) of milk

7 TRR. This value which included several extreme data points was slightly lower than that obtained by radioanalysis. Mean values of residues in tissues and milk are summarized in Table 4 and Table 5. Table 4. Alpha-cypermethrin residues in cattle tissues Tissue Post treatment (days) Alpha-cypermethrin GC-ECD method HPLC radioanalysis µg/kg Ratio (α-cyp/trr) µg/kg Ratio(α-cyp/TRR) Back fat 3 173, , , , , , , , , , , , , , , , 0.93 Mean ±SD 0.76 ± ±0.11 Omental fat 3 70, <LOQ , , , , , , , , , , , , , , 0.97 Mean ±SD 0.70 ± ±0.10 Kidney 3 <LOQ NA 6, , <LOQ NA 8, , <LOQ NA 22, NA 0.37 Mean ±SD NA 0.16 ±0.13 Muscle 7 <LOQ NA Mean ±SD NA 0.90 ND= Not detected. NA= concentration measured by GC residue method is below LOQ. Ratio of alphacypermethrin to TRR could not be calculated. Table 5. Residues of alpha-cypermethrin in cattle milk following topical dosing at 3 mg/kg body weight Hours posttreatment GC-ECD analysis µg/kg (number of samples) Alpha-cypermethrin 1 HPLC radioanalysis Total µg/kg equivalents (number of samples) ±11 (n = 3) 16 ±14 (n = 8) ±34 (n = 3) 36 ±31 (n = 8) ±19 (n = 5) 47 ±26 (n = 6) ±18 (n = 5) 48 ±16 (n =6) ±7 (n = 6) 27 ±6 (n = 6) ±3 (n = 3) 21 ±6 (n = 4) ±1 (n = 2) 13 ±3 (n = 4) ± Results <LOQ or not sampled are not included in the table. Maximum number of values is 8. LOQ for HPLCradioanalysis = 3 µg/kg; for the GC-ECD = 10 µg/kg. Insufficient data points for >240h. Bound residues. Radioactivity remaining after extraction in the solid samples were <3% in omental fat (one value of 7.5%), <10% in back fat, 13-47% in liver, <17% in kidney (one value of 59%) and 7% in muscle. In the case of liver, the enzyme and 6N HCl hydrolysates from PES were found to contain the same metabolites. Analysis of cis- and trans-isomers. Normal phase HPLC-LSC analysis was also used to analyse the cis-trans isometric ratio of the parent compound in a single fat, milk and muscle sample. Cis-2 cypermethrin was the major residue component (>92%). A minor component that did not co-chromatograph with trans-3- or trans-4-cypermethrin was detected in muscle and back fat. An additional minor component was detected with a retention time similar to the cis-1 isomer was detected in back fat and milk, but also in the treatment solution. There was no evidence, therefore, of interconversion of the cis to the trans isomers during metabolism following topical treatment. Sheep The sheep study included data points extending beyond the recommended withdrawal time of 7 days for the pour-on treatment on sheep. Ratio FAO FNP 41/14

8 The test substance used was an isotopic mixture of 12 C-, 13 C- and 14 C-alpha-cypermethrin with the same characteristics of that used in the cattle study (chemical purity 96.1%, radiochemical purity 99.8%, specific radioactivity 10µCi.mg -1 ). The radiochemical purity of the test compound in dose formulation (pre and post dose) ranged from 99.5% to 99.7% throughout the period of the study. Fourteen 12 month-old sheep (7 male, 7 female, kg) were acclimatised in the experimental unit before being randomly assigned to the study groups. The test animals were housed in individual wooden boxes in two rooms at C, relative humidity: 34-79% and a 12 h/12 h light/dark cycle. The animals were offered pre-weight diet of up to 400 g twice daily and water and good quality meadow hay ad libitum. Animal appearance and behaviour was monitored at least daily. Alpha-cypermethrin was formulated at a nominal concentration of 12.5 g/l and was topically administered on either side of the spine and around the rump in a zigzag line to 12 animals (6 male and 6 female) at a mean dose level of 0.5 g per animal (15.02 mg/kg). The two control animals were dosed with placebo formulations. Three treated sheep were sacrificed at each 2, 4, 7 and 14 days post-dose. Only two samples (one male and one female containing significant residue concentrations) were used to determine the metabolic profile and analyzed for residues at each sacrifice interval. The total radioactive residue (TRR) concentration and extractability of different fractions in the liver, kidney, muscle, and fat samples from each animal was determined using a radiometric method: fat by direct LSC (LOQ = 5-6 µg/kg) and liver, kidney and muscle by LSC of the 14 CO 2 absorbed following combustion (LOQ = 4 µg/kg). Procedural recoveries were determined. Data on TRR are presented in Table 6. Table 6. Depletion of 14 C-alpha-cypermethrin (mg/kg equivalents) in sheep tissues Sample Sacrifice time 2 day 4 day 7 day 14 day Back fat ± ± ± ±0.025 Omental fat ± ± ± ±0.057 Liver ± ± ± ±0.003 Kidney ± ± ± ±0.007 Muscle ± ± ± ±0.003 Urine ± ± ± ±0.319 Data expressed as the mean (n=3 ±SD) Mean TRR values are highest in fat tissues (back fat > omental fat), followed by kidney, liver, and muscle. A considerable quantity of radioactivity is eliminated by urine. The mean concentration of total radioactivity reached maximum values at 4 days in liver kidney and fat, with the exception of muscle that presents maximum mean levels at 2 days. In general, the amounts are highly variable (high SD). In back fat tissues the high variability made almost no difference on residue levels between days 2 and 4, while decaying slowly after; in omental fat, small differences between 4, 7 and 14 days were observed. There are minimal differences in most cases for TRR decay with the posttreatment time for liver, kidney, and muscle due to high SD. Extractability of residues. Residues from back and omental fat were extracted from macerated fat with acetonitrile. Extraction efficiency values were 88-95% TRR for fat. The same general technique was applied for kidney and muscle % of the TRR for kidney and 85-99% TRR for muscle were accomplished after complete processing. Some normal losses were observed through further processing in liver and kidney. Methanol:water (80:20 v/v) extracts recovered 11-20% of the TRR in kidney. In muscle tissue, extracts were not processed further due to the low residue levels. For liver, 43-90% TRR were extracted, however, two enzymatic digestions with pepsin and protease and subsequently hydrolysis of precipitated tissue material with 6N HCl were necessary. Analysis of extracted metabolites. Radiolabelled extractable residue components were analyzed by reverse-phase HPLC-LSC (LOD = 3-4 µg/kg). Only samples containing a significant residue level from one male and one female animal at each sacrifice interval were used to determine the metabolite profile by HPLC. Urine was not analyzed. Retention times of components were compared to those of metabolite reference standards (UV detection in the same HPLC system). Data of the quantification of radiolabelled metabolites in edible tissues are presented in Table 7 as the range of values through the 14 days post-treatment. FAO FNP 41/

9 Table C-Alpha-cypermethrin metabolites in sheep tissues Tissue % TRR 1 3-PBA-4- O-sulf 3-PBAglut 3-PBA 4-OH-3PBA 4-OHα-cyper α-cyper Unknowns [number of] Back fat ND + <1-2 [5] (7-16) ND <1-2 [7] (1-7) [8] ( ) 1-12 [6] (3-44) Omental Fat ND + + ND 1-4 [8] (1-9) [8] (28-314) [10] (1-25) Liver [5] (1-3) 1-7 [5] (1-4) < 3 [2] (< 1) 6-29 [5] (4-13) 2-16 [5] (1-10) [10] (7-10) Kidney <1-3 [4] (1-3) 12-51[8] (3-55) 7-42 [8] (2-67) 1-6 [5] (< 13) <1-4 [6] (<1-5) 2-23 [6] (1-22) 2-21 [10] (<1-30) Muscle ND [5] (< 1-1) ND 3-40 [6] (<1-6) [6] (4-18) < [5] (<1-9) 1. The second entries in parenthesis in each cell are concentrations expressed in µg/kg by calculation. Numbers in square brackets are the number of positive samples, except in column labelled unknowns. + : two or more samples indicated the presence of one or two radiolabelled metabolites. ND: non detected. 2. Next lowest percent value was 82%. 3. Next highest value was 6%. 4. Next lowest value was 52%. 5. Next highest value was 6%. Quantitation and identity of radiolabelled residues in tissues showed that unchanged alpha-cypermethrin was the major residue component in fat and muscle, with only one exception of the samples of muscle analyzed (4-OH-alphacypermethrin). Extensive metabolism of alpha-cypermethrin was shown in liver and kidney where a small amount of unchanged alpha-cypermethrin was observed. The main metabolite found in liver was the 4-hydroxy parent and in kidney, the 3-PBA-glut. More polar metabolites than alpha-cypermethrin are present in both cases. Minor unknown components were detected in all tissues. In the case of liver, enzyme and 6N HCl hydrolysates from PES were found to contain the same metabolites. Same case for the methanol:water extracts in kidney. 14 C-alpha-cypermethrin at different time points measured by HPLC-radioanalysis: Alpha-cypermethrin was detected at all times post-treatment in all tissues. In omental fat, total residues declined from a maximum concentration at day 4. On average (n=2), calculated concentrations of alpha-cypermethrin (in µg/kg) show no differences between 4, 7, and 14 days for omental fat (87, 189, 212 and 135 µg/kg at 2, 4, 7 and 14 days after treatment). For back fat, radiolabelled alpha-cypermethrin declined rapidly after four days p-t. (796, 768, 118, 63 µg/kg at 2, 4, 7 and 14 days, respectively). Values for back fat are considerably higher than omental fat. For liver, the highest amount of 14 C-alphacypermethrin occurred at day 4 and residues declined with time (7 to 3 µg/kg, from day 4 to 7 after treatment); similarly, for kidney, maximum concentrations of radiolabelled alpha-cypermethrin occurred at day 4 and residues declined after (22 to 1 µg/kg at day 2 to 14 after treatment). For muscle, maximum radiolabelled residues occurred at day 2 and alpha-cypermethrin residues declined (15 to 7 µg/kg from day 2 to 7 after treatment). Most radioactive measures were made near the LOD of the method (about 3-4 µg/kg) in muscle, liver and kidney. This was not the case for fat. Confirmation of alpha-cypermethrin as main residue: The same APCI LC-MS method applied to cattle tissues was used to confirm alpha-cypermethrin as main residue in a single fat and muscle sample. One sheep fat sample was selected for analysis by negative ion chemical ionization-gas chromatography-mass spectrometry (NCI-GC-MS). The spectrum contained the ions at m/z 207 and 209 observed by negative APCI LC-MS. These diagnostic ions were consistent with the spectrum of authentic alpha-cypermethrin. Analysis of alpha-cypermethrin by a GC-ECD method. The results of the GC analysis showed that alphacypermethrin on an average (n=8) accounted for 85 ±20% of the back fat TRR with high variability, equal ratio with less variability was obtained by radioanalysis (85 ±5%). Alpha-cypermethrin on average (n=8) accounted for 59 ±18% of the omental fat TRR, this ratio was higher if measured by radioanalysis (83 ±17%). Ratios present no differences with times post-treatment. For both types of fat, alpha-cypermethrin on an average (n=16) accounted for 72 ±23% of the fat TRR which is lower than the 84 ±12% obtained from radioanalysis of the fat samples. Muscle, kidney and liver samples from 2 to 14 days post-dose generally contained levels of alpha-cypermethrin below the limit of quantification (20 µg/kg), except one muscle sample at 22 µg/kg). Analysis of the ratio of alpha-cypermethrin to TRR is only practical in fat tissues. Residues of alpha-cypermethrin decay with time post-treatment more markedly in back fat than in omental fat. Values of residues (µg/kg) calculated from the radioanalysis were similar to those found by the GC method in both fat tissues. Results are summarized in Table FAO FNP 41/14

10 Table 8. Alpha-cypermethrin residues in sheep tissues Tissue Back fat Post treatment (days) FAO FNP 41/ Alpha-cypermethrin GC-ECD method HPLC radioanalysis µg/kg Ratio (α-cyp/trr) µg/kg Ratio (α-cyp/trr) , , , , , , , , , , , , , , , , 0.88 Mean ±S.D ± ±0.05 Omental fat 2 31, , , , , , , , , , , , , , , , 0.92 Mean ±S.D. 0.59± ±0.17 Liver 2 <LOQ NA 1, , <LOQ NA NA, 7 NA, <LOQ NA 5, , 0.04 Mean ±S.D. NA 0.09±0.06 Kidney 2 <LOQ NA NA, 22 NA, <LOQ NA 7, , <LOQ NA 10, , <LOQ NA NA, 1 NA, 0.03 Mean ±S.D. NA 0.06±0.08 Muscle 2 <LOQ NA 18, , <LOQ NA 11, , , <LOQ 1.29, NA 10, , <LOQ NA NA NA Mean ±S.D. NA 0.62±0.23 NA: concentration measured by GC-ECD residue method is below LOQ (20 µg/kg); ratio of alpha-cypermethrin to TRR could not be calculated. Bound residues. Radioactivity remaining in the PES liberated by 6N HCl hydrolysis were < 6% in omental fat, < 10% in back fat, 22-41% in liver and <11% in muscle (kidney data are missing). In the case of liver, enzyme and 6N HCl hydrolysates from PES were found to contain the same metabolites. Analysis of cis- and trans-forms. Normal phase HPLC-LSC analysis was used to analyse the cis:trans isometric ratio of the parent compound in a single fat and muscle sample at 2 days post treatment (higher residue contents). Cis-2 cypermethrin was the only residue component in sheep muscle and the major residue component in sheep fat (a minor component was detected with a retention time similar to cis-1 isomer but low concentrations did not permit its structural identity. There was no substantial evidence, therefore, of interconversion of the cis to the trans isomeric form resulting from metabolism following topical treatment. METHODS OF ANALYSIS FOR RESIDUES IN TISSUES Sheep (HE 355/20-01R): An analytical method for the determination of alpha-cypermethrin residues in sheep tissues (muscle, fat, kidney and liver) was submitted. It has been validated according to the requirements of the European Community. Alpha-cypermethrin residues are extracted from tissues (1g) by homogenisation with hexane:acetone (1+1) twice and organic phases separated. This combined extract was partitioned with dimethylformamide twice. A 1% sulphate salt solution was added to the separated dimethylformamide phase and was partitioned with hexane twice, the hexane extracts were passed through anhydrous sodium sulphate and evaporated to dryness under nitrogen. The dry residue is reconstituted in hexane:acetone (9+1v/v) and analyzed by GC-MS (1µl injection volume). GC was performed in a HP- 5MS column (30 x 0.25 mm ID, 0.25 µm) with a temperature gradient and MS detection (ionization mode) monitored at m/z 207 and 209 ions. Accuracy/recovery/precision (repeatability): Three fortification concentrations (20, 40 and 80 µg/kg) were used for recovery determinations (n=3 at each fortification level). For fat, recoveries and coefficient of variation (CV) at each

11 respective level were: 84.1% (10.2), 74.5% (4.7), 95.2% (5.6), respectively, with an overall mean 84.6% (12.3). Similarly, for muscle, recoveries were 80.4% (3.6), 73.8% (2.3), 86.6% (2.7), respectively, with an overall mean 80.3% (7.4). For liver, the recoveries were 84.8% (9.7), 73.1% (1.2), 84.9% (3.7), respectively, with an overall mean 80.9% (9.1). For kidney, the recoveries were 88.6% (3.3), 81.9% (2.7), 95.3% (2.5), respectively, with an overall mean 88.6% (7.0). Linearity: The linearity of detector response was assessed in the four tissues matched with standard solutions of 0.002, 0.006, and µg/ml. For fat, kidney and liver, the response ratio (peak response divided the amount of standard injected) for each standard injection was within 7% of the average response ratio of the four standards, correlation coefficient > For muscle, the response ratio was within 13%, correlation coefficient > Specificity: Sample extracts of one control matched with µg/ml standard and one fortified tissue sample at the corresponding LOQ (20 µg/kg) were injected. The ratio of the m/z 207 to the m/z 209 in the fortified samples agreed with that observed for the control : for fat and 1.441, for kidney and 1.530, for liver and 1509 and for muscle and (chromatograms are presented for control matched samples, but no raw data). Typical chromatograms of control tissue samples (n=20) shown to be free of interfering peaks. Limit of detection (LOD): It was determined by calculating the mean plus 3x SD of the mean from the analysis of 20 independently extracted control samples of tissues from at least 4 different animals. For fat, liver, kidney and muscle the LOD were estimated to be 2.60, 0.507, and µg/kg, respectively. Limit of quantitation (LOD): The protocol indicated that it would be demonstrated for each tissue by meeting the criteria for accuracy and precision. Fortified samples at 20 µg/kg were shown to satisfy this criteria in all tissues. Practicability: The analysis were performed using commercially available reagents and equipment. Sponsors declared that the methods were performed safely by a trained analyst and a large number of samples (n=20) were analyzed in a 48 hour period. Applicability: This method was used for the analysis of different types of sheep tissues. Note: The ion used for quantification is not specified, but in the chromatograms it appears the m/z 207 which it is mentioned as the base peak of the spectrum in the studies in cattle and sheep (confirmation of alpha-cypermetrhin as main residue by MS) Cattle (SAMS and SAMS 461-1) Two analytical methods for the determination of alpha-cypermethrin residues in cattle tissues (muscle, fat, kidney and liver) and milk were submitted. They have been validated as the sheep tissue method. Milk extraction (SAMS 456-1): Samples of milk (20 ml) are treated with potassium oxalate solution and ethanol and extracted with diethyl ether and hexane, solvent phase evaporated to dryness and redissolved in hexane. The extract is passed through a Chem Elut CE 1020 column. Further clean up is obtained by use of a cyano Bond Elut cartridge. Tissues extraction (SAMS 461-1): Samples of tissues (5g) are chopped or finely minced and extracted by boiling with a mixture of acetone: hexane (1+2 v/v), solvent evaporated and residue redissolved in hexane. For fat and muscle, a portion of the extract is partitioned with acetonitrile by using a Chem Elut CE 1020 column. A normal hexane/acetonitrile partition is used for all the liver and kidney extracts. Extracts are further cleaned up by liquid-solid chromatography on a Florisil cartridge. Residues of alpha-cypermethrin are determined by GC with ECD detection and confirmed by GC-MS (GC-ECD conditions: SE 54 column, 15 m x 0.32 mm ID, 0.25µm, gradient temperature, injection volume 5µl). GC-MS conditions are the same as the GC conditions. MS negative ion chemical ionization mode, ions monitored are m/z 207 and 209 amu. Accuracy/recovery /precision (repeatability): Three fortification concentrations (50, 100 and 200 µg/kg) were used for recovery determinations (n=3 at each fortification level). For fat, recoveries and coefficient of variation (CV) at each respective level were: 84.1% (10.2), 74.5% (4.7), 95.2% (5.6), with an overall mean 84.6% (12.3). Similarly, for muscle 97.1% (7.2), 108% (1.4), 99.9% (7.2), with an overall mean 102% (6.8). For liver, the recoveries were 81.6% (3.5), 100%(1.1), 91.5%(1.8), with an overall mean 91.0%(8.9). For kidney, the recoveries were 101% (4.2), 83.5% (1.9), 101% (3.6), with an overall mean 95.3% (9.7). In milk, fortification levels used were 10, 20 and 40 µg/kg (n=3). Recoveries and coefficient of variation (CV) at each respective level were 84.7% (1.3), 103%(1.5), and 110% (4.3), with an overall mean 99.0% (12.0). Linearity: The linearity of detector response was assessed with standard solutions of 0.002, 0.006, and µg/ml not matched to tissue matrix. The response ratio 8% of the average response ratio of the four standards, correlation coefficient > Specificity: The specificity was assessed by injecting the sample extracts of one control and one fortified sample at the LOQ (at 2x) for each matrix (100 µg/kg for tissues, 20 µg/kg for milk), using the GC-MS. Recoveries were 97.2%, 83.1%, 75.9%, 91.1%, 66.6% for muscle, fat, liver, kidney and milk, respectively (chromatograms were not presented) FAO FNP 41/14

12 Limit of detection (LOD): The LOD was determined by calculating the mean plus 3x SD of the mean from the analysis of 20 independently extracted control samples of tissues. For muscle, fat, liver, kidney and milk the limits of detection were 3.90 µg/kg, 3.13 µg/kg, 1.4 µg/kg*, 5.68 µg/kg, µg/kg, respectively. * The concentration of alphacypermethrin in liver samples was calculated (using baseline noise at the expected retention time). LOD was calculated by multiplying mean value by 3. Limit of quantification (LOQ): Fortified samples at 50 µg/kg for tissues and 10 µg/kg for milk have shown to satisfy this criteria in all tissues. Practicability: The practicability for cattle was demonstrated in similar way as in sheep. Applicability (SAMS 461-1): The method may be used for the analysis of different types of cattle tissues. Methods have been suitably validated for determining the LOQ values reported based upon the data provided. They proved to be suitable for determination of alpha-cypermethrin in fat tissues of cattle and sheep and milk of cattle (LOQ = 20 µg/kg for sheep tissues, 50 µg/kg for cattle tissues, 10 µg/kg for cattle milk). However, because of the low residue concentrations in muscle, liver and kidney, they are of limited value for determination of residues in liver, kidney or muscle (most values are below the LOQ). Analysis of the chromatograms suggests that lower limits of quantitation may be possible. APPRAISAL Two new radiolabel studies one each in sheep and cattle were submitted for evaluation by the Committee. Both were conducted in accordance with good laboratory practices. No new studies for poultry were provided. One additional report was provided on analytical methods. 14 C-Alpha-cypermethrin was formulated at a nominal concentration of 15 g/l and was topically administered to steers ( kg bw) and 8 lactating cows ( kg) along the region of the back between the shoulders and rump along the mid-dorsal line. Cows were treated following the morning milking. Mean doses achieved were 3 mg/kg BW. This was more than three times the recommended dose. The study was conducted in two phases In sheep alpha-cypermethrin was formulated at a nominal concentration of 12.5 g/l and was topically administered on either side of the spine and around the rump to 6 male and 6 female sheep (28-39 kg BW) at a dose level of 15 mg/kg BW. This was the maximum recommended dose. The metabolite profile in tissues and in cattle milk was defined. The ratio of parent drug to total 14 C-radiolabelled residues (TRR) in edible tissues was investigated at various withdrawal periods. In cattle, depletion rates of alphacypermethrin residues in edible tissues from steers and lactating cows were provided beyond the recommended 14 day withdrawal time for tissues and 0 day for milk following a single topical application. In sheep, the study was conducted with data points extending beyond the recommended withdrawal time of 7 days for pour-on treated sheep. Interconversion of the cis to trans isomeric form of alpha-cypermethrin was also studied. The identified metabolites indicate that alpha-cypermethrin in sheep, steers and lactating cows, following topical application, undergoes phase I oxidative hydroxylation at the phenyl ring and hydrolysis at the ester linkage to finally produce 3-phenoxybenzoic acid and its conjugates. The ester hydrolysis products are further oxidized to form 3- phenoxybenzoic acid (3-PBA) and 4-hydroxy-3-phenoxybenzoic acid (4 -OH-3-PBA). These compounds contain a free carboxylic and hydroxyl moiety, respectively, that undergo phase II metabolism to form glutamic acid and sulfate conjugates. Thus, the metabolic fate of alpha-cypermethrin is the same found in orally treated cow (Morrison and Richardson, 1994.) and the rat (Crawford and Hutson, 1977). Conversion from cis- to trans-configuration had not occurred in the milk or animal tissues. In cattle, identification and quantification of extracted radiolabelled residues showed that alpha-cypermethrin as the cis isomeric form was the main metabolite in both omental and back fat, milk and in the sample of muscle analyzed. Extensive metabolism has also been shown in liver and kidney, where the main metabolite identified was 3-PBA glutamate. Others metabolites tentatively identified were 3-PBA, 4-OH-3-PBA and 3-PBA-4-O-sulfate. A number of unknown extractable residues and bound residues were also detected in liver and kidney. In sheep, the cis isomeric form of alpha-cypermethrin was the main compound in fat tissues and muscle. Main metabolite in liver was the 4-OH-parent and in kidney was 3-PBA-glutamate. A number of unknown extractable residues and bound residues were also detected. In cattle, the results of the new study were consistent with those obtained in the previous limited studies, though residues are considerably higher. Alpha-cypermethrin shows a similar ratio to TRR as obtained in the orally dosed cows of the previous studies. In milk, maximum concentration and depletion follow the same pattern as in the earlier studies. Mean TRR are higher in fat tissues, followed by kidney, liver, and muscle. In fat tissues, TRR reached the maximum values at 21 days (not between 7 and 14 days as in the previous studies). In kidney, liver, and muscle, peak values occurred at 14, 7 and 7 days respectively, with low radioactivity residues. There are no marked differences in most cases for subsequent time points due to the high variability, but the TRR levels decreased with the post-treatment time. FAO FNP 41/

13 Efficiency of extraction of radiolabelled residues was: 70-99% TRR for fat, 47-84% TRR for liver, 50-66% TRR for kidney and 82-96% TRR in milk. Total radiolabelled residues were detected at all times points post treatment in all tissues. The maximum 14 C alphacypermethrin residues as analyzed by HPLC radio-analysis were <35µg/kg in kidney and muscle, 647 µg/kg in back fat and 421µg/kg in omental fat and 83 µg/kg in milk. No 14 C-alpha-cypermethrin was detected in liver tissue. The percentage of parent drug to total residues at different time points were: 84 ±17% for back fat, 91 ±10% for omental fat, 90% in muscle (only one sample) and 16 ±13% for kidney and 86 ±6% in milk. In a residue depletion study using a GC-ECD method, alpha-cypermethrin residues followed the same tendencies with time as the radiolabel measures. Concentrations of alpha-cypermethrin are below the LOQ (50 µg/kg) for kidney and muscle at three days and later sampling times. In milk, alpha-cypermethrin residues at different milking times ( h, n=13) are similar to those measured by radioanalysis. The GC-ECD maximum results were: <50 µg/kg for kidney, muscle and liver, 713 µg/kg for back fat, and 337 µg/kg for omental fat and 89 µg/kg for milk,. The percentage of alpha-cypermethrin to TRR calculated from GC-ECD analyses of alpha-cypermethrin were lower: 76 ±44%, 70 ±16% and 73 ±13% in back fat, omental fat and milk, respectively. For both types of fat, alpha-cypermethrin on average accounted for 71 ±33% of the TRR, lower than the 87 ±11% obtained from HPLC-radioanalysis of the fat samples. In sheep, mean TRR reached maximum values at 4 days for all tissues except muscle (2 days), then declined with time. Considering the high variability (SD) at each individual time point, results show no marked differences for subsequent time points in most cases. The depletion of 14 C-alpha-cypermethrin is slow in fats. Extraction of radiolabelled residues were 88-95% for fat, 85-99% for muscle, 49-82% for kidney and 43-90% for liver. The maximum 14 C alpha-cypermethrin residues as analyzed by HPLC radio-assay were 1323 µg/kg for back fat, 314 µg/kg for omental fat, 22 µg/kg for kidney and <20 µg/kg for muscle and liver. The percentage of parent drug to total residues at different time points were 85 ±5% for back fat, 83 ±17% for omental fat and 62 ±23% in muscle, 9 ±6% in liver and 6 ±8% in kidney. In a residue depletion study using a GC-ECD method, alpha-cypermethrin residues followed the same tendencies with time as in the radiolabel study. Concentrations of alpha-cypermethrin are below the LOQ (20 µg/kg) for kidney, liver and muscle at all sampling times. The GC-ECD maximum levels were: 1360 µg/kg for back fat, 218 µg/kg for omental fat, and <20 µg/kg for muscle, kidney and liver. The percentage of alpha-cypermethrin to TRR calculated from GC- ECD analyses of alpha-cypermethrin were lower, 85 ±20% for back fat and 59 ±18% for omental fat. For both types of fat, alpha-cypermethrin on an average (n=16) accounted for 72 ±23% of the fat TRR. In this study, fat residues are detected at longer times after dosing than in previous studies using non-radiolabelled alpha-cypermethrin in sheep pour-on treatments. Analytical methods have been validated according to data provided. They are applicable for determination of alphacypermethrin in fat tissues of cattle and sheep and milk of cattle. The LOQ for the GC-ECD method are 20 µg/kg for sheep tissues, 50 µg/kg for cattle tissues and 10 µg/kg for cattle milk. RECOMMENDED MAXIMUM RESIDUE LIMITS The following factors have been taken into account in recommending maximum residue limits for alpha-cypermethrin in cattle and sheep tissues and cattle milk: - An ADI of 0-20 µg/kg bw has been established, which is equivalent to a maximum theoretical intake of 1200 µg for a 60-kg person. - The metabolism of alpha-cypermethrin is similar in cattle and sheep tissues. - The new studies indicate that the parent drug, alpha-cypermethrin, is the only appropriate marker residue in tissues and in cow's milk. - The target tissue in cattle and sheep is fat. For calculation of the theoretical maximum residue intake, the ratios of alpha-cypermethrin to total residues in cattle and sheep are 0.75 in fat and muscle, 0.10 in liver and kidney and 0.95 in milk. - Suitable methods are available for determining residues in muscle and fat tissues and in cow's milk. - The MRLs for liver and kidney are based on the limits of quantitation of the GC-ECD method, which are 50 µg/kg for cattle and 20 µg/kg for sheep. Based on the available data, the following permanent MRLs of alpha-cypermethrin as parent drug in cattle and sheep tissues and cattle milk are recommended: muscle, liver and kidney 100 µg/kg; fat 1000 µg/kg and cattle milk 100 µg/kg measured as alpha-cypermethrin. Using the food consumption factors, the theoretical maximum intake of residues is 415 µg. The TMDI accounts for 35 percent of the ADI for alpha-cypermethrin FAO FNP 41/14