4.15 S-Methoprene (147)

RESIDUE AND ANALYTICAL ASPECTS

Methoprene, an insect growth regulator originally evaluated by the JMPR in 1984 and re-evaluated for residues several times up to 1989, is included in the CCPR periodic review programme. At the 30^th session of the CCPR (ALINORM 99/24, Appendix VII), methoprene was originally scheduled for periodic residue review by the 2003 JMPR but this was postponed to 2005.

The manufacturer supplied information on identity; metabolism and environmental fate; residue analysis; use pattern; residues resulting from supervised trials on wheat, maize, rice, sorghum, barley, and oats; and the fate of residues on wheat, maize and rice during storage and in processing. GAP information and enforcement method were supplied by the manufacturer and the government of Australia. In addition, methoprene is also recommended by WHO for treatment of drinking water.

Animal metabolism

The Meeting received information on the fate of orally-dosed methoprene in steers, lactating cows and laying hens.

S-methoprene is the biologically active enantiomer in the racemic methoprene and constitutes 50% of methoprene. Investigations into the metabolism and fate of methoprene could be accepted as supporting metabolism and fate requirements of S-methoprene.

The metabolism of methoprene in laboratory animals (mice, rats, guinea pigs, rabbits and dogs) was evaluated by the WHO panel of the 2001 JMPR. It was concluded that, after administration of single oral doses of methoprene, the radiolabel was relatively rapidly absorbed and excreted in urine, faeces and expired air. In most species investigated, the bulk of the radiolabel was extensively metabolized by O-demethylation and hydrolysis to polar conjugates and excreted within 5 days or less, and the [5-¹⁴C]-molecule underwent rapid á and â oxidation to produce CO₂ and acetate, which was incorporated into natural products.

[5-¹⁴C]-methoprene was administered orally in gelatin capsules to a Hereford steer as a single dose of 2 g (corresponding to 7.2 mg/kg bw). The administered radiolabel was quantitatively excreted during a 2 week post-treatment period, exclusive of unquantified respiratory losses and other minor losses; 22% of the dose was excreted in the urine, 39% in faeces. In faeces, the major extractable radioactive compound was unchanged methoprene. Approximately 13% of the administered radiolabel remained in the animal tissues.

At sacrifice 2 weeks after treatment, the levels of radioactivity in edible tissues were: liver (5.0 mg/kg), kidney (4.4 mg/kg) and fat (3.2 mg/kg). All the principal meat tissues had less than 1 mg/kg wet tissue. No primary methoprene metabolites could be characterized, but the major identified radiolabeled compound in liver, muscle and fat was cholesterol (16% TRR, 28% TRR and 88% TRR, respectively. TRR = total radioactive residue).

[5-¹⁴C]-methoprene was administered orally in gelatin capsules to a Jersey lactating cow as a single dose of 208 mg (corresponding to 0.61 mg/kg bw). After 7 days, 73% of the radiolabel had been eliminated, with 20% in urine, 30% in faeces, 15% in expired air and 8% in the milk. Only about 0.08% of the applied dose was excreted as methoprene and no detectable primary metabolites occurred in milk. About 27% of the administered radiolabel remained in the cows' tissues. The concentrations of radiolabel in expired air, urine, faeces and milk peaked about 24-48 h after treatment. By day 7 after treatment, the concentrations of radiolabel in edible tissues were: liver (0.49 mg/kg), kidney (0.37 mg/kg) and omental fat (0.25 mg/kg). Muscle tissues of the cow had less than 0.1 mg/kg of total radioactive equivalents.

In whole milk, peak radioactivity occurred at 30-h post-treatment. After 7 days, the amount present was only about 10% of the maximum value. The total recovery of radioactive material in the milk was 8% of the applied dose. [5-¹⁴C]-methoprene was extensively metabolized by the lactating dairy cow to acetate. Radioactive acetate incorporated into milk fat was degraded to radiolabeled saturated, monoenoic, and dienoic fatty acids. Radioactive lactose (11% TRR), lactalbumin (3.8% TRR) and casein (2.5% TRR) were also isolated from milk.

[5-¹⁴C]-methoprene was administered orally in gelatin capsules to colostomized or intact laying White Leghorn hens, as single oral doses of 0.6-77 mg/kg bw. The average percentage elimination of ¹⁴C in the 0-48 hr period via respiration was 37% when chickens were given low doses of methoprene (0.6-3.4 mg/kg bw) and was 24% when chickens were given high doses (31-64 mg/kg bw).

Over 14 days after administration, up to 19% of the radiolabel was eliminated in eggs, mainly in the yolk. At doses of 0.6 to 77 mg/kg, methoprene contributed only 1-2% of the total ¹⁴C in yolk and primary metabolites were only detectable (< 0.1 mg/kg) at the 77 mg/kg dose rate. For the range of doses tested, the majority of radiolabeled products in meat were natural triglycerides (20% TAR at a rate of 59 mg/kg, TAR = total applied radioactivity). Radiolabeled natural products were by far the main ¹⁴C residues in tissues and eggs, particularly at the lower dose of 0.6 mg/kg where cholesterol and normal fatty acids (as triglyceride) contributed 8% and 71% of the total radiolabel in egg yolk. The high initial doses resulted in methoprene residues in muscle (0.01 mg/kg), fat (2.1 mg/kg) and egg yolk (8.0 mg/kg), which represented 39 and 2% of the total ¹⁴C label in fat and egg yolk, respectively. After 48 h, chicken liver contained about 1% of the applied ¹⁴C from methoprene.

The metabolism of methoprene in laboratory animals was qualitatively similar to that in farm animals.

Plant metabolism

The Meeting received plant metabolism studies for methoprene on wheat in storage, alfalfa and rice.

Individual wheat grains were exposed to the vapour of [5-¹⁴C]-methoprene at 20^oC for 1 day, or were topically treated with [5-¹⁴C]-methoprene, using aqueous emulsions or solutions in cyclohexane. Two days after treatment or exposure, highest residue of intact methoprene was found in the aleurone layers, much less in the germ and virtually none in the endosperm or outer seed coats. There was no significant amount of ¹⁴C-activity associated with the high molecular weight fraction after either 1 week or 3 weeks storage at 20 ^o C and 18% moisture content.

Forty 25 g lots of wheat samples were dosed in screw-capped jars with 10 mL of a solution of methoprene in hexane at a rate of 10 mg/kg. The jars were sealed and stored in the dark at 20 ^o C. The residual half-life of methoprene in freshly-harvested wheat of 19% moisture was 2-3 weeks. In the older wheat at 12% and 18% moisture contents, the respective half lives were 6-7 weeks and 3-4 weeks. The main metabolic change observed was ester cleavage. Detectable metabolism was almost entirely to the free acid and could account for only 20-40% of the degradation.

Leaves of potted alfalfa were painted with the diluted [5-¹⁴C]-methoprene emulsifiable concentrate at a rate equivalent to 1.1 kg ai/ha. Parent methoprene disappeared in approximate first-order decay with a half-life of about 2 days for alfalfa. Volatility was a minor pathway for loss. The concentration of nonpolar metabolites maximized after 3 days in alfalfa. The primary nonpolar metabolites in alfalfa after 7 days constituted only approximately 1% of TAR. The aglycones in alfalfa after enzymic cleavage constituted approximately 10% of TAR as identifiable metabolites. A large amount (56%) of the radioactivity in chloroform extract fraction was associated with high molecular weight products (mol weight > 600). Further analysis of GPC fractions supported the incorporation of ¹⁴C label into naturally occurring plant pigments and other higher molecular weight plant constituents. After 30 days in alfalfa, 1% of the applied methoprene was retrieved as methoprene.

Leaves of potted rice were painted with the diluted [5-¹⁴C]-methoprene emulsifiable concentrate at a rate equivalent to 1.1 kg ai/ha. Parent methoprene disappeared in an approximate first-order decay with a half-life of about 0.5 day for rice. A total of 30% of the applied dose of methoprene on rice was isolated as condensed vapours after 1 week, which proved that volatility was a major path of elimination. The concentration of nonpolar metabolites maximized after 1 day in rice. The primary nonpolar metabolites in rice after 3 days constituted approximately 2% of TAR. The aglycones in rice after enzymic cleavage constituted approximately 1.5% of TAR. After 15 days in rice, 0.4% of the applied methoprene was retrieved as methoprene.

In both animals and plants, methoprene undergoes ester hydrolysis, O-demethylation, and oxidative scission of the 4-ene double bond. Further metabolism results in the corporation of methoprene-derived fragments into natural products.

Environmental fate in soil

The Meeting received information on aerobic degradation in soil.

The aerobic degradation of [5-¹⁴C]-methoprene was studied in sandy loam and silt loam soils for 60 days at dose rates of 0.7, 1.0 and 10 kg ai/ha. The residual half-life of methoprene in sandy loam was about 10 days at a surface treatment rate of 1 kg ai/ha. By day 14 only 0.7% of TAR could be identified as known metabolites of methoprene.

Environmental fate in water/sediment systems

The Meeting received information on sterile aqueous hydrolysis, photolysis, thin film photolysis and metabolism in pond water.

Sterile aqueous solutions of methoprene (0.5 mg/L), buffered at various pH values (pH5, 7 and 9), were found to be extremely stable to hydrolysis over four weeks at 20°C in the dark. No degradation was seen for the duration of the experiment in sterile water buffered at pH 7 and 9, and similar stability was observed in pH 5 buffer for 21 days.

In the first study of photolysis, photodecomposition of [5-¹⁴C]-methoprene was investigated in the autoclaved phosphate buffer (0.05 M, pH 7) at 0.01 mg/kg and 0.50 mg/kg. Methoprene was rapidly decomposed with both concentrations giving half-lives of apparently less than 1 day. In a second study of photolysis after 1 week, four photoproducts (24% yield overall) were characterized as metabolites of methoprene. Parent methoprene was not detectable and there were at least 46 other photoproducts but none represented more than 2% yield.

Photolysis on glass was investigated at a rate corresponding to 11 ìg/cm² (1.1 kg ai/ha) and film thickness of 0.1 ìm. Methoprene was rapidly degraded when a thin film on glass was exposed to sunlight through glass. The half-life for photochemical breakdown under these conditions was 6 h. The recovery of only 72% of TAR after 27 h suggested photolysis of methoprene to volatile products which were lost by vaporization. Collection of vapours above the photolysate resulted in recovery of 13% of TAR, of which only 0.2% was methoprene and 6%, ¹⁴CO₂. Resolution of the crude photolysate after exposure of methoprene to sunshine for 4 days gave methoprene (7%, equal mixture of 2E and 2Z) and at least 50 other metabolites and photoproducts, but none represented more than 6% of TAR.

In the first study, the degradation studies of methoprene labelled in the 10-³H (purity > 99%) and the 5-¹⁴C (purity 97.9%) were performed in the pond water. The half-lives of [10-³H]methoprene at 0.001 mg/kg and at 0.01 mg/kg were approximately 30 h and 40 h, respectively.

Methods of analysis

The Meeting received information on several methods for the determination of parent methoprene and/or S-methoprene residues in cereal grains, related processed products, stored grain and corn, milk, eggs, poultry and cattle tissues using GC-FID and HPLC, and on a method for the detection of methoprene residues in wheat grain with ELISA.

Residues of methoprene are first extracted with solvents (acetonitrile, acetone/hexane, hexane, methanol, and iso-octane). Fatty extracts are subjected to cold-temperature precipitation and filtration to remove fat. Solvent partitioning and/or column chromatography (florisil, alumina and silica column) are used for clean-up. Methoprene was analysed by GC with FID or HPLC-UV. The identity of suspected residues was confirmed by alternative GC column, GC-MS, and [¹⁴C]-methoprene. The lower limits of quantification (LOQs) are: soils, blood and urine, 0.001-0.01 mg/kg; forage grasses, forage legumes and rice foliage, 0.005 mg/kg; milk, eggs, stored grain and corn kernels, fish, shellfish, poultry and cattle tissues and faeces, 0.01 mg/kg; cereal grains and processed products 0.01-0.2 mg/kg. The LOQ's and recoveries were validated by analysis of laboratory and field samples fortified with [¹⁴C]methoprene in some methods. Two methods (LOQ: 0.008 mg/kg and 0.05 mg/kg, respectively) are considered suitable for enforcement for grain and grain products.

A rapid enzyme immunoassay was used as a screening test for methoprene in animal feed grains, and sensitive enough to detect methoprene at 0.5 ppm in the grain. This assay can be used as a screening test, but cannot be used for quantitative detection of methoprene.

Stability of pesticide residues in stored analytical samples

The Meeting received information on the stability of methoprene in milk, and supplemental information on the stability of S-hydroprene in bologna, chicken, bread and hamburger.

Information on storage stability of methoprene in cereal grains was not submitted. However, field residue samples were stored at -20°C until needed for analysis (storage time not stated). Numerous lab studies and field trials have shown long-term stability of methoprene in stored grains, not only at -20°C but even at room temperature.

Stability of S-hydroprene was demonstrated in hamburger, chicken, bread, apples and lettuce at -15°C for 7 to 24 days. S-hydroprene is a compound with very similar structure and properties to S-methoprene. It is therefore likely that S-methoprene was also stable in animal commodities at -15°C

The Meeting concluded that methoprene would be stable in cereal grains and animal commodities when stored frozen.

Definition of the residue

Methoprene was rapidly and extensively metabolized by animals and plants. There was 8% of TRR in whole cow milk. 0.015 mg/kg of methoprene was detected, but primary metabolites were not detected (< 0.01 mg/kg). [5-¹⁴C]-methoprene was extensively metabolized to acetate by the lactating dairy cow.

In steer tissues, no primary methoprene metabolites were found, and the major identified radioactivity (16-88%, depending on tissue) was [¹⁴C]-cholesterol.

At doses of 0.6 to 77 mg/kg, methoprene contributed only 1-2% of the total ¹⁴C in yolk and primary metabolites were only detectable (< 0.1 mg/kg) at the 77 mg/kg dose rate. Higher initial doses resulted in detectable residues of methoprene in muscle, fat and egg yolk. Radiolabeled natural triglycerides and cholesterol also contributed major portions of the total ¹⁴C residue in fat.

In the animal metabolism studies, the concentration of residue was substantially higher in fat and egg yolk than that in muscle and egg white. The values of log P_ow (4 for methoprene, approximate 6 for S-methoprene) also indicate that methoprene is a fat-soluble compound. However, methoprene was metabolized quickly and extensively by animals, so its accumulation in fat was just temporary.

After the pre-harvest treatment of alfalfa and rice, five primary non-polar metabolites were found. Methoprene which remained in alfalfa and rice was a minor part of the residue. However, after post-harvest treatment of wheat grains, the residue consisted mainly of methoprene.

The primary metabolites were not toxicologically significant compounds, which were evaluated by the WHO panel of 2001 JMPR. The Meeting agreed that methoprene is suitable for enforcement in plant and animal commodities and is also the compound of interest for estimation of dietary risk.

Definition of residue (for compliance with the MRL and for estimation of dietary intake): methoprene.

The residue is fat-soluble.

Results of supervised trials on crops

The Meeting received information on supervised trials of post-harvest treatments of methoprene/S- methoprene in wheat grain, shelled corn, rice, sorghum grain, barley grain and oats grain in USA, Australia and Thailand. This data was generated from large-scale storage trials with the exception of four laboratory studies on S-methoprene in 2003; most of the trials were conducted in Australia and the USA.

Methoprene

Wheat

Thirty-one trials were conducted on wheat in Australia (GAP: 0.50~1.0 g ai/t) in 1982~89. In twenty-four trials conducted at the maximum GAP, the highest concentrations during sampling were 0.38, 0.50 (2), 0.59, 0.60, 0.63, 0.70 (3), 0.72, 0.74 (3), 0.78, 0.79, 0.80 (2), 0.85, 0.90, 1.0, 1.1, 1.2, 1.9 and 2.0 mg/kg.

Four trials on wheat were conducted in the USA (GAP: 5.0 g ai/t) in 1982~85. Two USA trials and two Australian trials were conducted against the maximum GAP (USA), and the highest concentrations during sampling were 2.1, 4.0, 5.1 and 8.0 mg/kg.

Maize

Seventeen trials were conducted on maize in the USA (GAP: 5.0 g ai/t) in 1982~85. In three trials conducted at the maximum GAP, the highest concentrations during sampling were 3.9, 4.2 and 4.6 mg/kg.

Rice

Eight trials on rice were conducted in the USA (GAP: 5.0 g ai/t) in 1984~1985, and in Thailand (no GAP; uses that of the USA) in 1984. In three trials conducted at the maximum GAP (USA), the highest concentrations were 2.9, 6.8, and 8.1 mg/kg.

Sorghum

Two trials were conducted on sorghum at GAP in Australia (GAP: 0.50~1.0 g ai/t) in 1984. The highest concentrations of methoprene residues found during storage were 0.93 and 0.98 mg/kg.

Two trials were conducted on sorghum at the maximum in the USA (GAP: 5.0 g ai/t) in 1985. The highest concentrations of methoprene residues found during storage were 7.5 and 7.8 mg/kg.

Barley

Four trials on barley were conducted on barley grain in Australia (GAP: 0.50~1.0 g ai/t) in 1985. In three trials conducted at the maximum GAP, the highest concentrations were 0.60, 0.63, 0.65 and 1.1 mg/kg.

Oats

Four trials were conducted on oats grain in Australia (GAP: 0.50-1.0 g ai/t) in 1985. The highest concentrations of methoprene residues found during storage were 0.77, 0.96 and 1.0 (2) mg/kg.

The Meeting considered the combined data sufficient for cereal grains. The data from Australia and the USA were considered to represent different populations. The Meeting decided to evaluate the USA trials and other trials against the critical GAP in USA (5.0 g ai/t). The concentrations of residues in trials conducted (4 trials on wheat, 3 trials on maize, 3 trials on rice and 2 trials on sorghum) were, in ranked order: 2.1, 2.9, 3.9, 4.0, 4.2, 4.6, 5.1, 6.8, 7.5, 7.8, 8.0 and 8.1 mg/kg. The Meeting estimated an STMR value of 4.85 mg/kg, a highest residue of 8.1 mg/kg and a maximum residue level of 10 mg/kg for cereal grains. The recommendation for a maximum residue level of 10 mg/kg for cereal grains replaces the previous recommendation of 5 mg/kg.

S-Methoprene

Wheat

Two trials were conducted on wheat grain at the maximum GAP in Australia (GAP: 0.60 g ai/t) in 1986. The highest concentrations of S-methoprene residues found during storage were 0.33 and 0.54 mg/kg.

One trial and four laboratory studies were conducted on wheat grain in the USA (GAP: 0.60-4.4 g ai/t) in 1985 and 2003, but none of trials were conducted at the maximum GAP.

As residues arising from S-methoprene were covered by those from methoprene, the Meeting agreed not to recommend a maximum residue level for S-methoprene in wheat after post-harvest treatment.

Fate of residues during processing

The Meeting received information on the fate of residue of methoprene and S-methoprene during simulated processing of stored wheat (milling), stored rice (husking and polishing) and stored maize (extraction and refinement of maize oil).

In processing

Wheat with various storage times after post-harvest treatment with methoprene, was milled. The parent compound was determined in processed products. Processing factors derived from stored wheat were comparable. Calculated processing factors were 0.13 - 0.56 for flour; 0.43 - 1.1 for wholemeal; 1.5 - 4.1 for bran; 1.7-7.0 for germ; 1.4 - 4.3 for pollard.

In the USA, a processing study was conducted in 1985 on milling products, generated from whole maize that was previously treated with 5.3 g ai/t methoprene. At 30 day intervals, grain composites were removed from the granary and were extracted for crude and refined oil. The parent compound was determined in processed products. Calculated processing factors were 0.81 - 1.4 for maize meal; 3.9 - 44 for crude oil; < 0.06 (3) and < 0.05 (3) for edible oil. The refining processes converting crude to refined oil evidently removed or destroyed all methoprene residues.

In the USA, a processing study was conducted in 1985 on rice that was previously treated with 5.3 g ai/t methoprene. At 30 day intervals, rice was removed from the granary and milled and polished. The parent compound was determined in processed products. Calculated processing factors were 0.12 - 0.26 for husked rice; 4.6 for hulls; < 0.01, < 0.02 (3), and < 0.03 (3) for polished rice. Polished rice produced by hulling followed by polishing of the exterior bran layers virtually removed all methoprene residues.

The processing factors for wheat, maize and rice commodities are summarized in Table 14. All processing data on maize crude oil were generated with samples coming from the same trial at various intervals. Because of the large variability in the same processing study, the use of the median processing factor for the calculation of highest residue-Ps and STMR-Ps for maize crude oil is more suitable than using the maximum processing factor. The Meeting decided to take the median processing factor for the calculation of highest residue-Ps and STMR-Ps.

Table 14. Processing factors for wheat, maize and rice commodities.

From the highest residue and STMR for cereal grains (8.1 mg/kg and 4.85 mg/kg respectively) and the processing factors for wheat bran (unprocessed), flour and wholemeal, the Meeting estimated STMR-P values of 13.6 mg/kg in bran (unprocessed), 1.72 mg/kg in flour, and 4.51 mg/kg in wholemeal, 18.9 mg/kg in pollard, 23.3 mg/kg in germ and a maximum residue level of 25 mg/kg in bran (unprocessed), which replace the previous estimate of 10 mg/kg in unprocessed bran. The Meeting also recommended withdrawal of the existing CXL for wheat flour of 2 mg/kg and for wheat wholemeal of 5 mg/kg because the processing factors are less than 1.

No residues of methoprene were found at levels above the LOQ of 0.2 mg/kg in refined oil prepared from maize in the processing studies. The Meeting recommended withdrawal of the existing CXL for edible oil of 0.2 mg/kg PoP and estimated STMR-P values of 87.3 mg/kg in crude oil, 0 mg/kg in edible oil, and a maximum residue level of 200 mg/kg in crude oil.

From the STMR for cereal grains (4.85 mg/kg) and the processing factors for husked rice, hulls and polished rice indicated above, the Meeting estimated STMR-Ps of 1.07 mg/kg in husked rice, and 22.3 mg/kg in hulls, and a maximum residue level of 40 mg/kg in hulls. No residues of methoprene were found at levels above the LOQ of 0.1 mg/kg in polished rice prepared from rice in the processing studies. The STMR-P for polished rice was estimated to be 0.1 mg/kg.

Farm animal dietary burden

The Meeting estimated the dietary burden of methoprene residues in farm animals from the diets listed in Appendix IX of the FAO Manual (FAO, 2002). Calculation from the highest residues and STMR-P values provided the concentrations in feed suitable for estimating MRLs for animal commodities, while calculation from the STMR values for feed was suitable for estimating STMR values for animal commodities.

Estimation of maximum farm animal dietary burdens.

¹ Use of wheat bran.

Estimation of median farm animal dietary burdens.

¹ Use of wheat bran.

Farm animal feeding studies

The Meeting received information on residues in the tissues of several steers and a cow, in the milk of lactating cows, and in the egg of laying hens orally administered with [5-¹⁴C]methoprene through the feed.

Lactating dairy cows have been fed methoprene in their feed for 28 days at the levels of 0.1, 0.3 and 1.0 ppm. No residues were found in the muscle tissues at any of the three treatment levels at the limits of detection (0.01 mg/kg). The residues found in kidney, liver, fat (subcutaneous, renal and omental) ranging from < 0.01-0.096 mg/kg. No residues of methoprene were found in the milk at the limits of quantitation (0.01 mg/kg) 2 to 28 days after beginning the feeding. No residue data in cream were provided.

A lactating dairy cow was administered methoprene at a rate of 83 ppm daily in feed for 4 months. Residues were found in milk ranging from 0.29-0.72 mg/kg (mean 0.47 mg/kg).

A steer was administered methoprene at a rate of 33 ppm daily in feed for 14 days. Residues were found in fat, muscle and edible offal (liver, kidney, spleen and heart) (1.3-2.3 mg/kg in fat, 0.05-0.10 mg/kg in muscle, 0.01-0.06 mg/kg in edible offal).

Three groups of two steers were administered methoprene at rates of 16.7, 33.3 and 167 ppm in feed for 14 days. No residues were found in liver at any of the three treatment levels at the LOQ (0.01 mg/kg). The residues found in edible offal, muscle, fat ranging from <0.01-0.92 mg/kg, <0.01-0.39 mg/kg and 0.17 -7.9mg/kg, respectively.

Laying hens were fed methoprene at 25, 50 and 100 ppm in the diet for varying periods between 14 and 63 days. At these three administered rates, residues found in poultry meat ranged from < 0.01-0.032 mg/kg, < 0.01-0.074 mg/kg, and < 0.01-0.302 mg/kg, respectively. The residues found in egg ranging from < 0.01-0.045 mg/kg, < 0.01-0.054 mg/kg and < 0.01-0.201 mg/kg, respectively. In all of the studies, there was also a withdrawal period of varying duration. At all three treatment levels, residues in poultry meat and egg decreased rapidly as withdrawal periods increased.

Animal commodity maximum residue levels

The dietary burden for the dairy cow was 7.73 mg/kg, below the feeding level (83 ppm) and the dietary burden for the steers was 6.18 mg/kg, below the lowest level in the feeding study (16.7 ppm in the feed). Therefore the resulting residues in milk and steer tissues were calculated by applying the respective transfer factors (transfer factor = residue level in tissue or milk ÷ residue level in feed) to the estimated dietary burden. In the feeding study the highest residue levels in tissues were used to calculate the highest likely mammal commodity residue levels and mean residue levels in milk and tissues were used to estimate the mammal commodity STMRs (Table 15).

Table 15. Calculation of MRLs and STMRs for milk and animal tissues.

¹ Residue values in parentheses in italics are extrapolated from residues found at the feeding level in the cattle metabolism study.

² Values in italics are the estimated dietary burdens. Values in normal font are feeding levels in the cattle metabolism study.

³ Highest is the residue level calculated from that found in the feeding study and the estimated maximum dietary burden.

⁴ Mean is the residue level calculated from that found in the feeding study and the estimated STMR dietary burden.

⁵ Exclude 0 day residue value.

The dietary burden for laying hens was 7.73 mg/kg, below the lowest level in the feeding study (25 ppm in the feed) and therefore the resulting residues in eggs and hen meats (including edible offal) were calculated by applying the respective transfer factors (transfer factor = residue level in egg or tissue ÷ residue level in feed) to the estimated dietary burden. In the feeding study the highest residue levels in meat and egg were used to calculate the highest likely poultry commodity residue levels, and mean residue levels in meat and egg were used to estimate the poultry commodity STMRs (Table 16).

Table 16. Calculation of MRLs and STMRs for poultry meat and eggs.

¹ Residue values in parentheses in italics are extrapolated from residues found at the feeding level in the hen metabolism study.

² Values in italics are the estimated dietary burdens. Values in normal font are feeding levels in the hen metabolism study.

³ High is the residue level calculated from that found in the feeding study and the estimated maximum dietary burden.

⁴ Mean is the residue level calculated from that found in the feeding study and the estimated STMR dietary burden.

The concentration of residues in milk when dairy cows were "fed through" with up to 1ppm showed no residues in milk which were lower than those calculated from dietary burden and animal feeding studies. The recommended MRLs were therefore based on the dietary burden of farm animals and animal feeding studies.

The Meeting estimated maximum residue levels of 0.2 mg/kg for methoprene in meat (fat) from mammals, other than marine mammals, 0.02 mg/kg in edible offal from mammals and 0.1 mg/kg for milk. The Meeting also recommended withdrawal of the existing CXL for cattle milk of 0.05 mg/kg F, for edible offal (mammalian) except cattle of 0.1 mg/kg; and for meat from mammals other than marine mammals and cattle of 0.2 mg/kg (fat). The Meeting could not estimate maximum residue levels for methoprene in milk fat without data submission on cream.

The Meeting estimated STMRs of 0.007 mg/kg for muscle, 0.092 mg/kg for fat, 0.014 mg/kg for edible offal and 0.044 mg/kg for milk.

The Meeting estimated a maximum residue level of 0.02 mg/kg and STMR of 0.007 mg/kg for methoprene in poultry meat and edible offal from poultry, a maximum residue level of 0.02 mg/kg and STMR of 0.006 mg/kg for methoprene in eggs.

DIETARY RISK ASSESSMENT

Long-term intake

The International Estimated Daily Intakes (IEDIs) of methoprene, based on the STMRs estimated for seven commodities, were 20-40% of the maximum ADI 0.09 mg/kg bw for the five GEMS/Food regional diets. The Meeting concluded that the long-term intake of residues of methoprene resulting from its uses that have been considered by JMPR is unlikely to present a public health concern.

Short-term intake

The 2001 JMPR decided that an ARfD is unnecessary. The Meeting therefore concluded that the short-term intake of methoprene residues is unlikely to present a public health concern.

4.16 Novaluron (217)

TOXICOLOGY

Novaluron is the provisionally approved ISO common name for (±)-1-[3-chloro-4-(1,1,2-trifluoro-2-trifluoromethoxyethoxy) phenyl]-3-(2,6-difluorobenzoyl)urea, a racemic compound. Novaluron is an insecticide of the benzoylphenyl urea class that inhibits chitin synthesis, affecting the moulting stages of insect development. It acts by ingestion and contact, and causes abnormal endocuticular deposition and abortive moulting. Novaluron has not been evaluated previously by the JMPR.

For technical novaluron, the FAO specification was established by the FAO/WHO Joint Meeting on Pesticide Specifications (JMPS) and published as FAO Specification 672/TC (December 2004).

All pivotal studies with novaluron were certified to be compliant with GLP.

Biochemical aspects

After oral administration in rats, [chlorophenyl-¹⁴C (U)]-novaluron was poorly absorbed (? 7%) after a single low dose (2 mg/kg bw) and about tenfold less after a single high dose (1000 mg/kg bw), with maximum plasma concentrations occurring at 5-8 h or 2-5 h, respectively. Novaluron was widely distributed. The tissue concentrations of radioactivity were highest in fat, liver and kidneys and were about three- to fivefold higher after 14 repeated daily doses than after a single dose, with a terminal half-life of 52-56 h in fat after the final dose. Excretion was rapid, primarily via the faeces (> 94%; via bile £ 1%) and to a lesser extent via urine (about 5%), with most of the administered dose being excreted within 48 h.

Absorbed novaluron was extensively metabolized, mainly by cleavage of the urea bridge between the chlorophenyl and difluorophenyl moieties. In urine and bile, up to 15 metabolites were detected, and individual metabolites accounted for £ 1% of a low dose of [chlorophenyl-¹⁴C (U)]-novaluron. Most of the faecal radioactivity consisted of unchanged novaluron, which was also the major component present in fat, liver and kidneys. The aniline metabolite of novaluron, 3-TFA, (3-chloro-4-(1,1,2-trifluoro-2-trifluoromethoxyethoxy)aniline) was identified at low levels (? 0.7%) in the urine, bile, liver and kidneys.

Toxicological data

Novaluron had low acute toxicity in rats, causing no mortality at limit doses after oral (LD₅₀ > 5000 mg/kg bw), dermal (LD₅₀ > 2000 mg/kg bw) or inhalation (LC₅₀ > 5.15 mg/L air) exposure. Novaluron was not irritating to the skin and eyes of rabbits and not sensitizing not sensitizing to guinea-pig skin.

In short-term and long-term studies of toxicity, the erythrocyte was identified as the primary target of toxicity attributable to novaluron, with secondary effects apparent in the spleen and less commonly in liver and kidneys. The spectrum of effects was essentially similar in mice, rats and dogs, and the underlying mechanism was considered to be the same. Although the mechanism of the effects on erythrocytes has not been elucidated, it was considered to be most likely that the aniline metabolite of novaluron, 3-TFA, caused oxidative damage to the mature erythrocyte, resulting in increased concentrations of methaemoglobin (caused by accelerated oxidation of haemoglobin from the ferrous to the ferric state) and increased numbers of erythrocytes containing Heinz bodies (which are formed when damaged haemoglobin precipitates onto the cell membrane). The presence of Heinz bodies led to early destruction of erythrocytes by the spleen, with the consequence of increased erythrocyte turnover, characterized by stimulated erythropoiesis in both normal sites (sternum, femur) and in functional reserve sites (spleen, liver) and increased deposition of the products of haemoglobin catabolism (haemosiderin) in the spleen, liver and kidneys. After cessation of treatment, the adverse effects regressed, although incompletely, over a 4-week period after treatment in rats and dogs, and completely within 8 weeks in mice.

In 28-day and 90-day studies of toxicity in mice treated orally, the overall NOAEL was 30 ppm (equal to 4.2 mg/kg bw per day) on the basis of haematological changes (decrease in erythrocyte volume fraction and erythrocyte counts, increase in Heinz bodies and sulfhaemoglobin) at dietary concentrations of 100 ppm (equal to 12.8 mg/kg bw per day) and above, while changes in the spleen (increased weight, increased haematopoiesis and haemosiderosis) were evident at 700 ppm (equal to 114.7 mg/kg bw per day) and above.

In 28-day and 90-day studies in rats treated orally, the overall NOAEL was 50 ppm (equal to 4.2 mg/kg bw per day) on the basis of haematological changes (decrease in haemoglobin, erythrocyte volume fraction and erythrocyte counts) and histopathological changes in the spleen and liver (increased haemopoiesis and haemosiderosis) at dietary concentrations of 100 ppm (equal to 8.3 mg/kg bw per day) and above. By week 4 of the reversibility period there was full recovery for most changes, except for increased concentrations of methaemoglobin, spleen weights and splenic haemosiderosis at dietary concentrations of 20 000 ppm (equal to 1667 mg/kg bw per day).

In 90-day and 1-year studies in dogs treated orally, the overall NOAEL was 10 mg/kg bw per day on the basis of haematological changes (decrease in haemoglobin, erythrocyte volume fraction and erythrocyte counts; increase in reticulocytes, Heinz bodies and Howell-Jolly bodies), increased serum concentrations of bilirubin and changes in the spleen and liver (increased weight; increased red pulp congestion, increased haemosiderin in Kupffer cells) at doses of 100 mg/kg bw per day or greater, while increased concentrations of methaemoglobin were evident at doses of 300 mg/kg bw per day or greater. By week 4 of a reversibility period there was full recovery for most changes, except for increased liver weights in female dogs at 1000 mg/kg bw per day.

In a 28-day study in rats treated dermally, the NOAEL for systemic toxicity was 75 mg/kg bw per day on the basis of increased concentrations of methaemoglobin at doses of 400 mg/kg bw per day or greater.

Novaluron gave negative results in an adequate battery of studies of genotoxicity in vitro and in vivo.

The Meeting concluded that novaluron was unlikely to be genotoxic.

Long-term studies of toxicity and carcinogenicity were conducted in mice and rats. In the study of carcinogenicity in mice, the NOAEL was 30 ppm (equal to 3.6 mg/kg bw per day) on the basis of increased body-weight gain (in the first 4 or 26 weeks in males or females, respectively), haematological changes (decrease in haemoglobin concentration, erythrocyte volume fraction and erythrocyte counts; increase in reticulocytes, sulfhaemoglobin, and Heinz bodies) and changes in spleen (increased weight, increased incidence of extramedullary haemopoiesis, haemosiderosis and congestion) and kidneys (increase in cortical tubular pigment) at dietary concentrations of 450 ppm (equal to 53.4 mg/kg bw per day) and greater. There was no evidence of carcinogenicity in mice at dietary concentrations of up to 7000 ppm (equal to 800 mg/kg bw per day), the highest dose tested.

In the long-term study of toxicity and carcinogenicity in rats, the NOAEL was 25 ppm (equal to 1.1 mg/kg bw per day) on the basis of haematological changes (decreases in haemoglobin concentration, erythrocyte volume fraction and erythrocyte counts; increases in methaemoglobin formation and reticulocytes) and changes in the spleen (increase in weight, haemosiderosis) and kidneys (increase in cortical tubular pigment) at dietary concentrations of 700 ppm (equal to 30.6 mg/kg bw per day) and greater. There was no evidence of carcinogenicity in rats at dietary concentrations of up to 20 000 ppm (equal to 884.2 mg/kg bw per day), the highest dose tested.

In view of the absence of a carcinogenic potential in rodents and the lack of genotoxic potential in vitro and in vivo, the Meeting concluded that novaluron is unlikely to pose a carcinogenic risk to humans.

In a two-generation study of reproductive toxicity in rats, the NOAEL for effects on fertility was 12 000 ppm (equal to 894.9 mg/kg bw per day), the highest dose tested. The NOAEL for systemic toxicity in parental animals and offspring could not be identified since there were secondary changes in spleen and liver relating to increased erythrocyte damage at all doses tested. The LOAEL for systemic toxicity was 1000 ppm (equal to 74.2 mg/kg bw per day) on the basis of increased spleen weights in adults and increased spleen and liver weights in offspring.

In a study of prenatal developmental toxicity in rats, the NOAEL for maternal and for developmental toxicity was 1000 mg/kg bw per day, the highest dose tested. The increases in body-weight gain and food consumption in all treated groups were not considered to be adverse effects.

In a study of prenatal developmental toxicity in rabbits, the NOAEL for both maternal and developmental toxicity was 1000 mg/kg bw per day, the highest dose tested. In the absence of any other evidence for an effect on fetal development, the slight increase in incidence of incompletely ossified fifth sternebrae at 300 mg/kg bw per day and 1000 mg/kg bw per day was not considered to be adverse. The finding of absent implantation or high rates of pre-implantation loss in two dams at 1000 mg/kg bw per day was considered to be incidental and not related to treatment.

The Meeting concluded that novaluron is not a developmental toxicant.

In a study of acute neurotoxicity in rats, non-specific clinical signs (fast respiration, piloerection) of minor toxicological relevance were seen in all groups treated at doses of 200 mg/kg bw and greater. The NOAEL for neurotoxic effects was 2000 mg/kg bw, the highest dose tested.

The manufacturing impurity MCW RI 458 had low acute oral and dermal toxicity in rats (LD₅₀ > 5000 and > 2000 mg/kg bw, respectively) and was not mutagenic in an assay for gene mutation in bacteria. The manufacturing intermediate MCW I was not mutagenic in an assay for gene mutation in bacteria.

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

Toxicological evaluation

The Meeting established an ADI of 0-0.01 mg/kg bw on the basis of the NOAEL of 1.1 mg/kg bw per day for erythrocyte damage and secondary splenic and liver changes in a 2-year dietary study in rats, and a safety factor of 100.

The Meeting concluded that it was not necessary to establish an ARfD for novaluron in view of its low acute toxicity, the absence of relevant developmental toxicity in rats and rabbits that could have occurred as a consequence of acute exposure, and the absence of any other toxicological effect that would be elicited by a single dose.

A toxicological monograph was prepared.

Levels relevant to risk assessment

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

Estimate of acceptable daily intake for humans

0-0.01 mg/kg bw

Estimate of acute reference dose

Unnecessary

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 novaluron

RESIDUE AND ANALYTICAL ASPECTS

Novaluron, or (±)-1-[3-chloro-4-(1,1,2-trifluoro-2-trifluoromethoxy ethoxy) phenyl]-3-(2,6-difluorobenzoyl)urea, is an insect growth regulator. Novaluron inhibits chitin synthesis, affecting the moulting stages of insect development. It acts by ingestion and contact and causes abnormal endocuticular deposition and abortive moulting. It is being evaluated for the first time by the 2005 JMPR.

Animal metabolism

The metabolism of novaluron uniformly radiolabeled in the difluorphenyl ring and separately in the chlorophenyl ring was studied in goats and chickens. Lactating goats were dosed with the radiolabled compounds at rates equivalent to 11-12 ppm in the feed for five consecutive days. Most of the radioactivity was eliminated in the faeces, 52% of the administered dose for the [difluorophenyl-¹⁴C(U)]-novaluron and 72% for the [chlorophenyl-¹⁴C(U)]- novaluron. The Total Radioactive Residue (TRR) did not reach a plateau in milk during the five days, with the final concentration being 0.23-0.24 mg/kg. TRR concentrations in the tissues resulting from administration of the two radiolabelled compounds were similar: peritoneal fat, 1.4-1.9 mg/kg; kidney, 0.14-0.16 mg/kg; liver, 0.34-0.43 mg/kg, muscle, 0.09-0.16 mg/kg. Methanol extraction released 80 100% of the TRR from the various tissues, and greater than 90% of the TRR was extracted from milk with hexane/methanol.

Novaluron was the only residue identified in milk (93-95% TRR), peritoneal fat (96- 100% TRR), and foreleg muscle (98% TRR). It was the major component in kidney (73-83% TRR) and liver (80-84% TRR). The metabolite 2,6-difluorobenzoic acid was found in kidney (5.1% TRR), and 1-[3-chloro-4-(1,1,2-trifluoro-2-trifluoro methoxyethoxy)phenyl]urea was identified in liver, at 7.3% TRR (0.025 mg/kg). In faeces, 3-chloro-4-(1,1,2-trifluoro-2-trifluoro methoxyethoxy)aniline was tentatively identified. Very little degradation of the parent novaluron occurred, and the metabolites found are consistent with cleavage at the benzoyl-urea linkage.

Even less metabolism/degradation of novaluron was observed in poultry. [Difluorophenyl] ¹⁴C-Labelled novaluron was administered orally to five laying hens for fourteen consecutive days at a nominal rate of 10 ppm in the diet. The TRR concentrations were as follows: liver, 0.39 mg/kg; kidney, 0.39 mg/kg; muscle, 0.061-0.30 mg/kg; fat, 3.6 mg/kg; eggs (day 14), 0.50 mg/kg. Novaluron was the only TRR component detected and identified, accounting for 90-107% of the TRR.

The results of the ruminant metabolism studies compare favourably to those of a rat metabolism study. The ruminant metabolites 1-[3-chloro-4-(1,1,2-trifluoro-2-trifluoromethoxyethoxy)phenyl] urea and 3-chloro-4-(1,1,2-trifluoro-2-trifluoro methoxyethoxy) aniline were also found in the rat. Additionally, 2,6-difluorobenzamide was found in rat kidney (7% TRR).

The Meeting concluded that novaluron undergoes only minor metabolism in goats and hens, and that the limited metabolism is consistent with a cleavage of the benzoyl urea bond.

Plant metabolism

The metabolism of difluorophenyl-¹⁴C- or chlorophenyl-¹⁴C-labelled novaluron in apples, cabbages, potatoes, and cotton following foliar application(s) was reported to the Meeting. Novaluron, radiolabelled in either the [chlorophenyl-¹⁴C(U)] or [difluorophenyl-¹⁴C(U)] ring was formulated as a 10% EC and sprayed onto trees growing in outdoor pots in a netted tunnel. Either 2 (4 trees per radiolabelled form) or 3 applications (2 trees per radiolabelled form) were made to trees at a rate of 2.5-2.7 mg/tree/application. The applications were made 110 days, 90 days, and 60 days (3 applications only) before harvest. Novaluron comprised >90% TRR in all fruit samples from all applications and sampling intervals. No metabolite (HPLC) comprised more than 1% (< 0. 01 mg/kg) of the TRR.

Novaluron, radiolabelled in either the [chlorophenyl-¹⁴C(U)] or difluorophenyl-¹⁴C(U)] ring was prepared as a 10% EC formulation and sprayed onto two groups of cabbage plants growing in outdoor pots. Two applications (either, 8 and 6 weeks before harvest or 5 and 2 weeks before harvest) were made to replicate a rate of 30-45 g ai/ha. Residues (TRR) were 0.23-0.35 for the 6 week PHI application and 0.32-0.45 mg/kg for the 2 week PHI application. An acetonitrile wash removed 81-90% of the TRR at final harvest. Acetonitrile/water extraction released an additional 9-15% TRR, the majority of which was on the outer cabbage leaves. About 96-100% of the TRR on cabbage heads at final harvest (and at earlier harvest intervals) was identified as novaluron.

Novaluron, radiolabelled in either the [chlorophenyl-¹⁴C(U)] or [difluorophenyl-¹⁴C(U)] ring was prepared as a 10% EC formulation and sprayed onto potato plants growing in outdoor field plots. Two applications (43 and 29 days before harvest) were made to replicate plants at a rate of 91-100 g ai/ha. Whole plant samples were taken after each application and also at 22, 10 and 0 days before harvest. For both radiolabels, the TRR on tubers at all intervals was < 0.001 mg/kg. At harvest (29 days after the second application) the TRR on plants was 9.9 mg/kg for the [difluorophenyl-¹⁴C(U)] and 5.9 mg/kg for the [chlorophenyl-¹⁴C(U)]novaluron. An acetonitrile wash removed 82% of the TRR, and an acetonitrile/water extraction released an additional 17% TRR. Novaluron comprised 97% of the TRR for both labelled compounds. An unknown (1.3% TRR, 0.074 mg/kg) was found with the [chlorophenyl-¹⁴C(U)]novaluron.

Cotton plants grown outdoors were treated with [chlorophenyl-¹⁴C]novaluron or [difluorophenyl-¹⁴C]novaluron at an application rate equivalent to 50 g ai/ha/treatment. Two treatment regimes were used; Regime 1 consisted of two applications, 14 days apart with a 90 day PHI and Regime 2 consisted of two applications 14 days apart with a 30 day PHI. Samples from plants treated according to Regime 1 were taken for analysis after each application and at 60 and 30 days before the normal harvest. The maximum TRR on undelinted seed (for both treatment regimes) was 0.005 mg/kg, and no isolation and characterization of the residue was attempted. The TRR on cotton gin trash at harvest ranged from 0.27 mg/kg (90 day PHI) to 0.85 mg/kg (30 day PHI). Acetonitrile extraction released 91-97% TRR from the various final harvest gin trashes. Novaluron constituted 88-95% TRR. Total unidentified components in the extracts were <4% TRR (< 0.012 mg/kg)

The Meeting concluded that novaluron is stable when used as a foliar spray on various food crop plants. There is no appreciable metabolism or degradation under typical GAP conditions.

Environmental fate

Novaluron is stable in water at pH 5 and pH 7. At pH 9 (25°C), however, novaluron degraded with a first-order DT₅₀ of about 100 days. At 50 and 70 °C, first order DT₅₀s were 1.2 and 0.09 days, respectively (pH 9). Major metabolites exceeding 10% of applied radioactivity were identified as the chlorophenyl ring urea (1-[3-chloro-4-(1,1,2-trifluoro-2-trifluoromethoxyethoxy)phenyl]urea) and 2,6-difluorobenzoic acid. These degradates are also the metabolites observed in livestock metabolism.

In a confined rotational crop study, six containers of soil were treated with chlorophenyl-¹⁴C(U)]novaluron at a rate of 100 g ai/ha (approximately 3.5 mg ai/container). Rotational crops of spinach, turnips, and spring wheat were planted into separate containers (one container per crop at each plantback interval of 30 and 120 days). Crop and soil samples were taken at times after sowing that was representative of immature harvest, early harvest, and final harvest. At the 30 day plantback interval, all crops contained only very low levels of TRR, 0.001-0.004 mg/kg. Soil samples were extracted and analysed. Novaluron declined from 98-99% of the TRR on the day of application to 32-49% TRR at final harvest (127-195 days after application). Degradates identified in soil at final harvest were 1-[3-chloro-4-(1,1,2-trifluoro-2-trifluoromethoxyethoxy)phenyl]urea (10-14% TRR) and 3-chloro-4-(1,1,2-trifluoro-2-trifluoromethoxyethoxy)aniline (21-30% TRR).

The Meeting concluded that the accumulation of novaluron, or its degradates, in rotational crops from use on primary crops, under typical GAP conditions, is unlikely.

Methods of analysis

The Meeting concluded that adequate analytical methods exist both for the monitoring/enforcement of MRLs and for data gathering in supervised field trials and processing studies. Two methods were developed and validated for the determination of novaluron in plant and animal commodities.

A gas chromatography (GC) method with electron capture detection (ECD) may be used for various plant commodities (apple, cabbage, potato, apple processed commodities, broccoli, tomato, orange processed commodities) and animal commodities (fat, kidney, liver, muscle, milk, egg). Homogenized samples are extracted into aqueous methanol and portioned with hexane. The hexane extract is purified with a solid phase extraction cartridge prior to GC determination. A variation of the method uses a mass selective detector (MSD; ions m/z 337 and m/z 335). The method and its variations have been validated at 0.01 or 0.05 mg/kg for plant commodities and at 0.01 mg/kg for animal commodities.

The GC method was radiovalidated for animal commodities (but not for plant commodities). Samples of liver, fat (mesenteric/abdominal), thigh muscle and eggs (final day sample only) from the nature of the residue in poultry study (see above) were extracted and analysed according to the GC method. To radiovalidate this method, samples of extracts were radioassayed by LSC and the final post-SPE samples were analysed by TLC with radiodetection. Both methods of analysis gave similar results, with the GC method giving 110-120% of the recovery and detection of the metabolism results.

A HPLC reverse phase with ultraviolet detection (UV, 252 nm or 264 nm) method may be used for various plant commodities (apple, pear, peach, maize forage, soya plant and seeds, undelinted cotton seed, cotton foliage, tomato, potato). The method was validated at 0.01 or 0.05 mg/kg. A macerated sample is extracted with acetone and methylene chloride, and the organic layer is exchanged to acetonitrile. A gel permeation step may be used for high oil/fat content samples (e.g., cotton seed). The acetonitrile is extracted with hexane, and the residual acetonitrile extract is purified sequentially on Florisil and silica/Rumsil.

A variation of the HPLC method with tandem mass spectrometer detection (MS/MS) may be used for plant and animal matrices. Matrices are extracted with methanol/water and cleaned-up with hexane extraction and SPE. Analysis is by LC-MS/MS in the negative electrospray ionization mode. Novaluron m/z 491 > 471 is monitored. The method was validated at 0.05 mg/kg for apples and at 0.01 mg/kg for potatoes. An independent laboratory validation showed adequate recoveries of novaluron from milk, muscle, and liver at 0.02, 0.02, and 0.05 mg/kg respectively. Recovery from fat in another study was acceptable at a 0.1 mg/kg fortification

Stability of pesticide residues in stored analytical samples

The stability of novaluron in plant commodities under frozen storage conditions (-18^oC) for periods of at least 3 to 12 months was demonstrated. The periods of stability adequately cover the storage intervals for all supervised field trials reported. The following minimum intervals of frozen storage stability were determined: apple, 12 months; pear fruit, 158 days; apple juice, 99 days; potato, 12 months; undelinted cotton seed, 160 days; broccoli, 6 months; tomato, 12 months; orange processed fractions, 8 months.

No storage stability data was presented for animal products. The information in the livestock feeding study indicates that all analyses were completed within 53 days of the first sacrifice. The metabolism studies in ruminants and poultry indicate very little metabolism or degradation of novaluron occurs. The Meeting concluded that the relatively short interval of frozen storage (< 53 days) of the animal feeding study commodities should not have resulted in loss of novaluron residues.

Definition of the residue

The results of the radiolabeled novaluron plant metabolism studies on apple, cabbage, cotton, and potato indicate that novaluron does not metabolize or degrade under typical foliar application conditions. Greater than 90% of the TRR is recovered as novaluron, and no significant metabolites/degradates are found.

In ruminants, orally administered radiolabled novaluron (equivalent to 11-12 ppm in the diet) undergoes limited metabolism to 2,6-difluorobenzoic acid and 1-[3-chloro-4-(1,1,2-trifluoro-2-trifluoro methoxyethoxy)phenyl]urea, each < 10% TRR. The major component of the TRR was novaluron,? 93% TRR in milk, fat, and muscle and ³ 73% TRR in liver and kidney. In poultry orally administered novaluron (equivalent to 10 ppm in the diet) for 14 days, virtually no metabolism/degradation of novaluron occurred.

The log of the octanol/water partition coefficient, 4.3, suggests a preferential solubility in fat. In both ruminants and poultry, novaluron accumulated preferentially in fat as opposed to muscle (12-16:1 for ruminant; 12:1 for poultry).

The analytical methods determine only novaluron.

The Meeting noted that the residue definition in Australia and in the United States for monitoring/enforcement and for risk assessment purposes is novaluron.

Given the results of the metabolism studies and the capability of the analytical methods, the Meeting concluded that the residue definition for both enforcement and dietary intake considerations for both plant and animal commodities is novaluron. The Meeting also decided that novaluron is fat-soluble.

Results of supervised trials on crops

Supervised trials were presented for the foliar treatment of a variety of crops worldwide.

Apple and Pear

Trials on apples were conducted in Chile (GAP of foliar applications using a 100 g/L EC formulation at a rate of 0.07 kg ai/hL and a PHI of 14 days), USA and Canada (GAP foliar applications, at a rate of 0.37 kg ai/ha using a 75 g/kg WG formulation and a PHI of 14 days). The number of applications was not specified. One trial was not within 30% of GAP (0.005 kg ai/hL and 11 day PHI) with a residue of 0.17 mg/kg.

The GAP for apples in the USA is foliar application of a 75 g/kg WG formulation at 0.37 kg ai/ha. No more than 4 applications may be made per season and no more than 1.1 kg ai/ha may be applied per season. The rate per hectare is maintained regardless of water volume or tree size with a maximum spray concentration of 0.05 kg ai/hL for trees over 3 metre in height and a maximum of 0.08 kg ai/hL for trees less than 3 metres in height. The PHI is 14 days.

Many of the USA trials and all of the Canadian trials were conducted with three early season trials (commencing at petal fall) each at 0.38 kg ai/ha plus three late season trials (commencing at about 30 days before harvest) each at 0.38 kg ai/ha, for a total of 2.2-2.4 kg ai/ha (2 × concentration). The early season applications started at petal fall and continued at 7 day intervals. The time from the final early season application to harvest is 60-160 days. There were no apple residue decline studies upon which to estimate the residue attributable to the early season applications. However, several side-by-side trials were conducted in which 6 applications (3 early season plus 3 late season) and 3 applications (3 late season) were applied. It was found that the residues from 6 applications were comparable to those from 3 applications: Michigan: 0.81 mg/kg (2.2 kg ai/ha total) and 0.73 mg/kg (1.1 mg/kg ai/ha total); New York, 0.55 and 0.77 mg/kg; Oregon, 0.37 and 0.50 mg/kg; Virginia, 0.65 and 0.67 mg/kg respectively. Therefore, the trials conducted with 6 applications were considered to be at the approximate maximum GAP. The residues from 27 trials at GAP in ranked order were: 0.23, 0.27, 0.35, 0.37, 0.44, 0.44, 0.49, 0.49, 0.50, 0.50, 0.54, 0.55, 0.60, 0.65, 0.67, 0.67, 0.68, 0.71, 0.71, 0.73, 0.75, 0.77, 0.81, 0.86, 0.93, 0.96, and 1.1 mg/kg.

The GAP for pears in the USA is identical to that for apples (above). In eight trials conducted in the USA and four trials conducted in Canada, 3 early season applications each at 0.38 kg ai/ha were followed by 3 late season applications each at 0.38 kg ai/ha, for a total seasonal application of about 2.2 kg ai/ha, or 2 × the maximum GAP. However, side-by-side trials with apples (above) indicated that the early season use did not contribute to the final residue. Assuming a translation to pears, ten trials were conducted at the approximate maximum GAP, and the residues in ranked order are: 0.18, 0.42, 0.46, 0.47, 0.59, 0.91, 1.0, 1.3, 1.6, and 1.8 mg/kg.

The Meeting decided that the apple and pear residue data, resulting from identical application patterns, were from the same population and combined the data to give the following residues in ranked order: 0.18, 0.23, 0.27, 0.35, 0.37, 0.42, 0.44, 0.44, 0.46, 0.47, 0.49, 0.49, 0.50, 0.50, 0.54, 0.55, 0.59, 0.60, 0.65, 0.67, 0.67, 0.68, 0.71, 0.71, 0.73, 0.75, 0.77, 0.81, 0.86, 0.91, 0.93, 0.96, 1.0, 1.1, 1.3, 1.6, and 1.8 mg/kg. The Meeting estimated an STMR of 0.65 mg/kg and a maximum residue level of 3 mg/kg for pome fruit.

Fruiting vegetables, other than cucurbits

Tomatoes

Supervised field trials for the foliar application of novaluron to tomatoes were reported from Argentina and Brazil. The GAP for Argentina specifies foliar application of a 100 g/L EC foliar application at 0.005 kg ai/hL, 4 applications, and a 1 day PHI. Two trials were conducted in Argentina, but none were at GAP. Twelve trials were reported from Brazil, where the GAP is for the foliar application of a 100 g/L EC formulation at 0.002 kg ai/hL (0.02 kg ai/ha), with repeat applications as needed and a PHI of 7 days. The residues in ranked order are: < 0.01 (4) and < 0.02 (8). The Meeting estimated an STMR of 0.02 mg/kg and a maximum residue level of 0.02 (*) mg/kg.

Soya bean (immature seeds)

Field trials were reported for the foliar application of novaluron to soya beans (immature seeds) in Brazil. The GAP in Brazil specifies a foliar application of a 100 g ai/l EC formulation at a rate of 0.01 kg ai/ha with a PHI of 53 days. The number of applications is not specified. Eleven trials were conducted at the maximum GAP, and the residues on soya beans in ranked order were: < 0.01 (11) mg/kg. The Meeting estimated an STMR of 0.01 mg/kg and a maximum residue level of 0.01 (*) mg/kg.

Potato

Field trials were reported the EU, Mexico, and the USA for the foliar application of novaluron to potatoes. The GAP for use in Switzerland is a maximum of 2 applications (foliar) of a 100 g ai/L EC formulation at a single application rate of 0.02 kg ai/ha with a 21 day PHI. This GAP may be applied to trials conducted in Europe (Switzerland, Germany, France, Italy and Spain). Fourteen trials were at the GAP of Switzerland, and the residues in ranked order are: < 0.01 (14) mg/kg.

The GAP of Mexico specifies one foliar application of a 100 g/L EC formulation at a rate of 0.015 kg ai/ha with a PHI of 30 days. Two trials were conducted at 0.028 kg ai/ha (about 2 ×) and a PHI of 14 days, but may be considered as no quantifiable residues were found. The residues in ranked order were: < 0.01 (2) mg/kg.

The GAP of the USA specifies a maximum of 2 applications per season of a 100 g/L EC formulation at a rate of 0.087 kg ai/ha (0.17 kg ai/ha/season) with a PHI of 7 days. Two trials were reported from the USA, where two applications were made at a rate of 0.28 kg ai/ha each (3 ×). The trials may be considered as no quantifiable residues were found. The residues in ranked order were: < 0.05 (2) mg/kg. The analytical method (GC/ECD) was validated by concurrent fortified sample recoveries at 0.05 mg/kg. However, the same method was validated elsewhere at 0.01 mg/kg, including the method used for the European trials. The limit of quantitation was not adequately established for these USA trials.

The Meeting agreed to combine the non-quantifiable residues for the EU and Mexico, which in ranked order are: < 0.01 (16) mg/kg. The Meeting estimated an STMR of 0.01 mg/kg and a maximum residue level of 0.01 (*) mg/kg.

Oilseeds

Cotton seed

Supervised field trials for the foliar application of novaluron to cotton were conducted in Brazil, Mexico, South Africa, and the USA. The GAP of Brazil specifies foliar application of a 100 g/L EC formulation at a rate of 0.01 kg ai/ha (0.005 kg ai/hL) with a 93 day PHI. Four trials were conducted, three of which were at an exaggerated rate (2 ×) or a substantially shorter PHI. However, all residues on the cottonseed were below the limit of quantitation. The ranked order of residues found were: < 0.01 (4) mg/kg.

The GAP of Mexico specifies foliar application of a 100 g/L EC formulation at a rate of 0.015 kg ai/ha with a 30 day PHI. Only 1 application is allowed. Two trials were reported, but both were at an exaggerated rate (3 ×) with quantifiable residues.

The GAP of South Africa specifies the foliar application of a 100 g/L EC formulation at a rate of 0.035 kg ai/ha (0.007 kg ai/hL for ground equipment and 0.12 kg ai/hL for aerial equipment) with no specified PHI and a maximum of 3 applications per season. Two trials are reported, but the PHI is 71 days.

The GAP of the USA specifies the foliar application of a 100 g/L EC formulation at a rate of 0.1 kg ai/ha (0.53 kg ai/hL for aerial equipment and 0.21 kg ai/hL for ground equipment) with a PHI of 30 days. No more than 4 applications and a maximum application of 0.3 kg ai/ha are to be used per season. The re-treatment interval is a minimum of 7 days. The majority of the trials involved 5 applications with a total application of 0.42 kg ai/ha, or 140% of the maximum seasonal rate. The last three applications were made late in the season (3 × 0.1 kg ai/ha, 100% seasonal rate), with a 7 day retreatment interval and with 44-80 days between the first two and these three applications. The first two applications were made early season (3 to 4 weeks after crop emergence and 14 days later) at a nominal rate of 0.058 kg ai/ha/application. As the majority of the residue would result from the three late season applications, the trials may be considered to be at GAP.

The residues in ranked order for 16 trials at GAP are: < 0.05 (5), 0.060, 0.066, 0.067, 0.069, 0.10, 0.19, 0.21, 0.22, 0.25, 0.34, and 0.40 mg/kg. The trials from Brazil are considered not to be from the same population as the USA trials. The Meeting estimated an STMR of 0.068 mg/kg and a maximum residue level of 0.5 mg/kg.

Primary animal feed commodities of plant origin

Cotton gin trash

Eleven supervised field trials conducted in the USA were considered to be consistent with the GAP of the USA (see cotton above). The residues in ranked order are: 3.7, 4.0, 4.5, 5.4, 6.7, 7.3, 10, 11, 17, 20, and 27 mg/kg. The Meeting estimated a median of 7.3 mg/kg and a high residue of 27 mg/kg.

Fate of residues during processing

Commercial-type processing studies were reported for apple and cottonseed, and the processing factors and resulting STMR-P values are summarized as follows:

¹ Only one processing study was available for each raw agricultural commodity.

² Water content (%) was not reported.

³ 40 mg/kg for apple pomace dry based on a default dry matter content of 40%.

⁴ 12 mg/kg for apple pomace dry based on a default dry matter content of 40%.

Farm animal dietary burden

The Meeting estimated the dietary burden of novaluron residues in farm animals on the basis of the diets listed in Appendix IX of the FAO Manual. Calculation from MRLs, highest residues (HR) and STMR-P values provides the levels in feed suitable for estimating MRLs for animal commodities, while calculation from STMR and STMR-P values for feed is suitable for estimating STMR values for animal commodities. The percentage of dry matter is taken as 100% when MRLs and STMR values are already expressed as dry weight.

Estimated maximum dietary burden of farm animals

The calculated maximum dietary burdens for beef cattle, dairy cows, and poultry are 11, 8.5, and 0.01 ppm, respectively.

Estimated STMR dietary burden of farm animals

The STMR dietary burdens for beef cattle, diary cows, and poultry are 6.3, 4.0, and 0.01 mg/kg, respectively.

Farm animal feeding studies

A feeding study was conducted with Friesian cows in which groups received the equivalent of 0, 0.35, 2.6, 8.0, or 26 ppm in the feed for 42-44 consecutive days. Average novaluron residues in whole milk on day 42 at the 8 and 26 ppm feeding levels were 0.38 and 1.7 mg/kg; in cream, 6.6 and 14 mg/kg; and in skimmed milk, 0.03 and 0.12 mg/kg. The novaluron maximum residue levels in tissues at the 8 ppm feeding level were: muscle, 0.34 mg/kg; kidney, 0.35 mg/kg; liver, 0.41 mg/kg; subcutaneous fat, 4.4 mg/k; peritoneal fat, 6.8 mg/kg. The novaluron maximum residue levels in tissues at the 26 ppm feeding level were: muscle, 0.56 mg/kg; kidney, 1.2 mg/kg; liver, 1.4 mg/kg; subcutaneous fat, 8.2 mg/kg; peritoneal fat, 13 mg/kg.

Residues were quantifiable at the lowest feeding level (0.35 ppm): milk, 0.04 mg/kg; muscle, 0.05 mg/kg; kidney, 0.06 mg/kg; liver, 0.05 mg/kg; subcutaneous fat, 0.43 mg/kg; and peritoneal fat, 0.56 mg/kg.

Novaluron total residues, mg/kg

A poultry feeding study was not provided. The nature of the residue in poultry was conducted for fourteen consecutive days at a rate equivalent to 10 ppm in the diet. Novaluron residues in eggs, fat, muscle, kidney, and liver were 0.45, 3.5, 0.31, 0.37 and 0.41 mg/kg, respectively. Residues would most likely be non-quantifiable at the calculated dietary burden level of 0.01 ppm (1/1000 ×).

Maximum residue levels

The Meeting estimated maximum residue levels of 10 mg/kg for meat (fat), 0.7 mg/kg for edible offal, 7 mg/kg for milk fat, and 0.4 mg/kg for milk. The Meeting also estimated the following STMR values: muscle 0.19 mg/kg, fat 4.1 mg/kg, edible offal 0.26 mg/kg, whole milk 0.20 mg/kg, and cream 4.3 mg/kg.

The Meeting estimated maximum residue levels of 0.01 (*) mg/kg for eggs, poultry meat, and poultry edible offal, based on the demonstrated limit of quantification for poultry commodities by the GC/ECD method. Also estimated were STMRs of 0 for eggs, meat, and edible offal and 0.005 mg/kg for poultry fat.

DIETARY RISK ASSESSMENT

Long-term intake

The International Estimated Daily Intakes of novaluron, based on the STMRs estimated for 17 commodities, for the five GEMS/Food regional diets were in the range of 7% to 40% of the ADI (Annex 3). The Meeting concluded that the long-term intake of residues of novaluron resulting from its uses that have been considered by JMPR is unlikely to present a public health concern.

Short-term intake

The 2005 JMPR decided that an ARfD is unnecessary. The Meeting therefore concluded that the short-term intake of novaluron residues is unlikely to present a public health concern.