Evaluation of the plant protection product

Evaluation of the plant protection product

Milbeknock - milbemectin

regarding application for authorisation

The Norwegian Food Safety Authority, National Registration Section Scientific officers: Merete Dæhli, Roger Holten, Marit Randall, Erlend Spikkerud, Anna Mehl, Abdelkarim Abdellaue and Elisabeth Øya. For the Norwegian Scientific Committee on Food Safety October 2011

Milbeknock - milbemectin Page 2

Table of contents 1. Summary 1-1 1.1 Identity and physical/chemical data 1-1 1.2 Mammalian toxicology 1-2 1.3 Residues in food or feed 1-5 1.4 Environmental fate and ecotoxicological effects 1-6 1.5 Dossier quality and completeness 1-8 2. Product status 2-1 3. Efficacy 3-1 4. Identity and physical/chemical data 4-1 5. Mammalian toxicology 5-1 5.1 Milbemectin 5-1 5.1.1 Toksicokinetics 5-1 5.1.2 Acute toxicity 5-6 5.1.3 Irritation/sensitisation 5-8 5.1.4 Genotoxicity 5-10 5.1.5 Sub-chronic toxicity 5-11 5.1.6 Chronic toxicity and carcinogenicity 5-16 5.1.7 Reproductive toxicity 5-20 5.1.8 Teratology 5-22 5.1.9 Neurotoxicity 5-25 5.1.10 Special studies 5-27 5.1.11 Medical data 5-28 5.1.12 Classification and labelling 5-28 5.1.13 Reference values 5-28 5.2 Impurities and metabolites 5-28 5.3 Co-formulants 5-31 5.4 Milbeknock 5-31 5.4.1 Acute toxicity 5-31 5.4.2 Irritation/sensitisation 5-32 5.4.3 Classification and labelling 5-33 5.4.4 Dermal absorption 5-33 5.5 Operator, worker and bystander exposure 5-33 6. Residues in food or feed 6-1 7. Environmental fate and behaviour 7-1 7.1 Active substance 7-1 7.1.1 Degradation in soil 7-1 7.1.2 Sorption and mobility 7-5 7.1.3 Degradation in water 7-6 7.1.4 Fate and behaviour in air 7-7 7.2 Exposure assessment 7-7 8. Ecotoxicology 8-1 8.1 Active substance 8-1 8.1.1 Terrestrial organisms 8-1 8.1.2 Aquatic organisms 8-2 8.2 Co-formulants 8-4 8.3 Product 8-4 8.3.1 Terrestrial organisms 8-4 8.3.2 Aquatic organisms 8-7 8.4 Toxicity/exposure estimates 8-12 8.4.1 Terrestrial organisms 8-12 8.4.2 Aquatic organisms 8-13

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9. Dossier quality and completeness 9-1 References 9-1

Milbeknock - milbemectin Page 1-1

1. Summary

Milbeknock is a new product containing the new active substance milbemectin, which is consisting of the microbial fermentation products of Streptomyces. Milbeknock is an emulsifiable concentrate (EC) formulation containing 9.3 g/L of the active ingredient. The product is an acaricide/insecticide, and is applied for control of mites and leafminers. The intended use is as a foliar spray in fruits (apples/pears), strawberries (only after harvest) and in ornamental plants growing in glasshouses and outdoors. The Standardised Area Dose is 250 ml product (2.33 g milbemectin) per decare, and is based on the applied use in strawberries. The recommended maximum dose rate in fruits (apple/pear) is 190 ml product (1.77 g milbemectin) per decare depending of tree height. In ornamentals the recommended maximum dose rate is 200 ml product (1.86 g milbemectin) per decare.

The product is applied for spraying at a maximum frequency of up to two times in fruits and berries and up to four times in ornamentals.

Spin mites and Liriomyza species (leafminers) have in general high risk of developing resistance to chemical agents. To ensure maximum and prolonged effectiveness and to minimize the likelihood of resistant strains of pests developing, it is recommended that products with a different mode of action are incorporated into annual spray programs.

The product is harmful for several biological control agents used for mite control, and this should be instructed on the label.

1.1 Identity and physical/chemical data

Product name Milbeknock Active substance milbemectin Formulation EC formulation Concentration of active substance 9,3 g/Litre IUPAC-name Milbemectin consists of two milbemycin isomers: <30% milbemycin A3 (MA3):

(10E,14E,16E,22Z)-(1R,4S,5’S,6R,6’R,8R,13R,20R,21R,24S)-21,24-dihydroxy-

5’,6’,11,13,22-pentamethyl-3,7,19-trioxatetracyclo[15.6.1.14.8

.020,24

]pentacosa-10,14,16,22-tetraene-6-spiro-2’-tetrahydropyran-2-one ; and >70% milbemycin A4 (MA4): (10E,14E,16E,22Z (10E,14E,16E,22Z)-(1R,4S,5’S,6R,6’R,8R,13R,20R,21R,24S)-6’-ethyl-21,24-dihydroxy-5’,11,13,22-tetramethyl-3,7,19-

trioxatetracyclo[15.6.1.14.8

.020,24

]pentacosa-10,14,16,22-tetraene-6-spiro-2’-tetrahydropyran-2-one

CAS number MA3: 51596-10-2; MA4: 51596-11-3


Structural formula

Molecular weight Milbemycin A3 528.7 Milbemycin A4 542.7 Solubility in water Milbemycin A3: Moderate, 2.68 mg/l (20 °C) Milbemycin A4: Moderate, 4.55 mg/l (20 °C) Vapour pressure Milbemycin A3: Low, 9.7x10-12 Pa (20 °C) Milbemycin A4: Low, 4.3x10-10 Pa (20°C) Henrys law constant Milbemycin A3: Low, 2.56x10-3 Pa m3/mol Milbemycin A4: Low, 1.55x10-3 Pa m3/mol log Pow Milbemycin A3: Very high, 6.54 (25°C) Milbemycin A4: Very high, 7.0 (25°C) pKa -

1.2 Mammalian toxicology

Milbemectin

Toxicokinetics Absorption Based on the milbemycin A4 excretion in urine and bile, the absorption seems to be 47 % of a single low dose in both sexes and 30/40% in males/females at a single high dose. Thus absorption seems to be saturated at higher doses. Peak concentration in blood/plasma was reach after 2-3 hours. Distribution The concentration of substance was higher in tissues (except brain) than in blood/plasma at all time points. Most tissues had a residue peak at 2 hours, but reproductive fat had a peak at 6 hours after dosing. Tissue residues increased disproportionately more than the increase in dose. Repeated dosing gave the same tissue distribution as single dosing. There was no accumulation.


Metabolism Hydroxylation was the main metabolic pathway and different single-, di-, and trihydroxymetabolites were formed. The main metabolites were 13-hydroxy-MA3 and –MA4. MA3 seems to be more rapidly metabolised than MA4. There was a minor glucuronidation pathway. Elimination The main route of elimination was via bile, and a smaller amount was excreted via urine. There was a higher percentage of excretion of MA3 than MA4 in urine. Males had higher urine excretion than females, especially at low doses. There was rapid excretion the first 24 hours followed by a prolonged low excretion, and the elimination was more rapid at low dose than at high dose (reflected in the concentrations in blood). Repeated dosing gave the same elimination pattern as single doses. Acute toxicity Milbemectin is of moderate acute toxicity in the rat after oral and inhalation exposure and of low dermal toxicity. Milbemectin appears more toxic to dogs than to rats. Irritation/sensitisation Milbemectin was not found to be a skin- or eye irritant nor a skin sensitiser. Genotoxicity All in vitro and in vivo genotoxicity studies were negative. Milbemectin is not considered to be a genotoxic substance. Sub-chronic toxicity The dog was the most sensitive species with a LOAEL of 10 mg/kg bw/day and the lowest NOAEL of 3 mg/kg bw/day. In the short term studies effects on liver, kidney, central nervous system and body weight were seen in the rat, mouse and dog. Effects on the adrenals were seen in the rat, dog and rabbit (dermal study). Rats had in addition effects on the uterus, testes and immune system, but the most sensitive parameter was elevated cholesterol. Elevated cholesterol was also seen in dogs. The central nervous system seems relatively more vulnerable in the dog than in rodents, in which effects on other organs were seen at lower dose levels than effects on the CNS. Chronic toxicity/oncogenicity The long-term toxicity and carcinogenicity study in the rat gave systemic effects as increased kidney weight in males and effects on adrenals and uterus in females as the most sensitive parameters. At the highest dose level there was also effect on body weight and blood parameters. There was an increase in endometrial polyps and adenocarcinomas in the uterus. In the mouse elongated incisors, reduced body weight gain and reduced food consumption (females only), were seen at 2000 ppm in a 1.5-year oncogenicity study. The central nervous system, liver, kidney and adrenals were the target organs. There were no neoplastic changes. Reproductive toxicity and teratology The two-generation study in rats showed effects on parental body weights and food consumption in parental animals in the high dose group. The high dose level gave reduced litter size and live birth index in the F2 generation. Body weight and body weight gain in the lactation period was affected in both F1 and F2 pups. The F2 generation was more affected than the F1 generation. There were not seen structural abnormalities in the offspring. In the rat, the offspring is more sensitive for milbemectin than the mother. This effects can, however, be regarded as not relevant for humans. In the rat teratogenicity study, the maternal toxicity was manifested by a decrease in mean maternal body weight and food consumption. There were no effects on the foetuses. In the rabbit teratogenicity studies, there were seen clinical signs (bradypragia and piloerection), reductions in food intake and body weight, deaths, abortions, dead foetuses and reduced foetal weight. There were no teratogenic effects. Neurotoxicity Milbemectin may cause neurotoxic effects of concern. In an acute oral neurotoxicity study, some evidence for neurotoxicity was found. A decrease in motor activity was observed at all dose levels; at the lowest tested dose, this decreased motor activity was even observed


in the absence of overt systemic toxicity. It appears that the observations were not performed during peak time of the neurotoxic effects. Therefore a full evaluation of the neurotoxic potential of milbemectin cannot be performed. A NOAEL could not be derived in the acute neurotoxicity study. The LOAEL was 20 mg/kg body weight. Establishment of an ARfD can be based on this study. Repeated dose administration via the diet did not result in neurotoxic effects in rats at 4, 8, and 13 weeks of dosing. Based on the available data a NOAEL of 59 mg/kg bw/d was established for repeated dose neurotoxicity of milbemectin in rats. Special studies In a pharmacological study in male rats, mice and rabbits, the results were consistent with an action of milbemectin on the central nervous system and at the neuromuscular level. In another study, the abnormal growth of the incisors in rats was investigated. It was found that the treated rats moved very slowly and there was barely any attrition. The lack of gnawing was the cause of apparent elongation of the incisors. There was no clear explanation to why the animals did not gnaw, but many of the possibilities involve effects on the nervous system. In a third study the involvement of P-glycoprotein in the absorption of milbemectin through Caco-2 monolayers was determined. However, it was not possible to monitor the concentration of milbemectin due to high non-specific binding of MA3 and MA4 to the polystyrene and polypropylene plates in the experimental apparatus. An acute oral toxicity study in female CF-1 mice (3 animals /dose) was performed to investigate whether milbemectin is a substrate for the P-glycoprotein transporter. Two strains were compared: a wild type strain +/+ and a mutant type strain -/- for the expression of a functional mdr 1 P-glycoprotein. The study shows that milbemectin is much more toxic in the mice strain that lacks the mdr 1 P-glycoprotein transporter. Medical data There are no reports of clinical symptoms or poisoning from the manufacturing or use of milbemectin or Milbeknock. Impurities and metabolites Several impurities and metabolites were tested for acute toxicity in the mouse, and some clinical symptoms were observed. All in vitro genotoxicity studies were negative.

Milbeknock 1% EC (SI-9009EC)

Co-formulants The product contains aromatic hydrocarbons, thus the product may cause lung damage if swallowed. Acute toxicity Milbeknock 1 % EC was of low acute toxicity by the inhalation, oral and dermal exposure. Irritation and allergy Milbeknock 1 % EC is not irritating to the skin or eye, and it is not a dermal sensitiser Dermal absorption No data were submitted. However, based on the data provided on molecular weights (528.7 and 542.7) and the log Kow (6.43 and 7.00, for MA3 and MA4 respectively), and according to the guidance document on dermal absorption, dermal absorption of 10% is considered.

Operator, worker and bystander exposure

The exposure, estimated by the UK POEM, exceeds AOEL with 14% when spraying pomes without PPE. The use of PPE reduces the exposure to under the AOEL. For spraying in greenhouses and in strawberries the AOEL is not exceeded even without use of PPE. For bystanders and re-entry workers the estimated exposure was far below AOEL.


1.3 Residues

Residues are not discussed in this report.

1.4 Environmental fate and ecotoxicological effects

Environmental fate and behaviour

Degradation in soil The degradation rate of milbemycin A4 is medium to moderate, DT50: 21-82 days, geometric mean 36.5 days (arithmetic mean: 43 days). DT90: 69-271 days. Bound residue amounted to 40 % of AR at maximum and the mineralization to CO2 reached a maximum level of 35 % of AR. Two metabolites were identified > 10 % of applied radioactivity (AR); 27-hydroxy-milbemycin A3/A4 (max 14 % AR of A4) and 27-keto-milbemycin A3/A4 (max 12 % AR of A4).The degradation rate (DT50) of the metabolite 27-hydroxy-milbemycin A4 wascalculated to be 18 days with a DT90 estimated to be 59 days. The degradation rate is low under anaerobic conditions, DT50: 556 days in the soil phase. DT90 of 1835 days is extrapolated well beyond the study duration. Mineralisation and bound residues amounted to 1.9 and 22 % of AR after 363 days respectively. No metabolites > 5 % of AR.

weden.

At 10 °C the degradation rate of milbemycin A4 is moderate. DT50: 63 days, DT90: 208 days. Photolysis can be an important route of degradation for milbemycin A4. DT50 was 7.5 days in samples exposed to light and 27 days in dark control samples. Bound residues increased up to 29 % of AR at the end of the study. Mineralisation amounted to 12 % of AR. No metabolites detected > 10 % of AR. Two acceptable field studies performed in the US have been submitted. The degradation of milbemycin A3/A4 is medium to high with DT50: 8-13 days (geometric mean of 8.5 for milbemycin A3 and 11.4 for milbemycin A4). Weather conditions are not well described in the two studies and assessing the relevance toNorway is difficult. Swedish authorities have concluded that the studies were not performed under conditionsrelevant for S Sorption/mobility The sorption of milbemycin to soil can be classified as high to very high with Kd: 12-138 (average 61) and Koc: 1370-4059 (average 2817). 1/n varied from 0.92 to 1.04 with an average of 0.98. The sorption of the two metabolites, 27-hydroxy-milbemycin A4 and 27-keto-milbemycin A4, to soil can be classified as high to very high with Kf: 20-94 (average 55) and 59-246 (average 171) respectively. Koc: 1828-2462 (average 2111) and 5350-7444 (average 6718) respectively. 1/n varied from 0.80 to 0.85 for 27-hydroxy-milbemycin A4 and 0.95-1.05 for 27-keto-milbemycin A4. Based on the amount of radioactivity in the leachate in an aged column study (1.1-3.3 % of AR after 2 days), the mobility can be classified as medium to high in the four tested soils (sand, sandy loam, clay loam, silt loam), but neither milbemycin nor any of the major degradation products were detected in the leachate. Degradation in water Hydrolysis of 14C- milbemycin A4 was determined at 50 °C at pH 5, 7 and 9. DT50 at the different pH values were estimated to be 13, 318 and 241 days respectively. The regression coefficients indicate that the DT50 values at pH 7 and 9 are not reliable. 27-hydroxy-milbemycin A4 and 27-keto-milbemycin A4 were found at levels of 8.2 and 23 % of AR respectively at pH 5. Photolysis is an important degradation pathway for milbemycin A4 when comparing irradiated samples to the dark controls. The amount of initially applied radioactivity recovered was much higher in the dark controls (96-101 % AR) than in the irradiated samples (15-33 % AR). Three metabolites > 5 % AR were also identified. 14C- milbemycin A4 is not readily biodegradable. The degradation for the whole system can be classified as moderate with DT50system: 82-89 days, geometric mean 85 days (arithmetic mean 86 days). Bound residues amounted to about 30 % of AR after 100 days in


both systems and mineralization was low with only about 6 % after 100 days. The active substance quickly dissipated from the water phase to sediment. Metabolites were detected at levels < 5 % AR. Fate in air Hydroxyl reaction and ozone reaction half life were estimated to be 16.4 and 13.7 minutes respectively for milbemycin A3 and A4. Milbemycin A3 and A4 both have a vapour pressure of <1.3x10-5 Pa and a Henry’s law constant of 2.63x10-3 and 1.59x10-3 Pa m3 mol-1 respectively indicating that significant volatilization is unlikely to occur.

mycin A4.

Exposure

PIEC (predicted initial environmental concentration) in soil has been estimated in different crops after either one or two applications. Time Weighted Averages and PECplateau have also been estimated. Worst case PIEC and PECtwa was calculated to be 0.04 mg a.s./kg soil after two applications in strawberries. PECplateau was calculated with the Finish PEC calculator to be 0.07 mg a.s./kg soil. Only one application and applications in other crops gave lower PEC-values. Strawberries were used as a worst case culture in the assessment of groundwater exposure. The results of the modelling show that the tested Norwegian and Swedish scenarios gave a PECgw << 0.001 µg/l. The modelling was run by Mattilsynet using MACRO (4.4.2) Groundwater modelling performed in connection with the EU registration was done with PEARL v. 1.1.1 and all the relevant FOCUS scenarios (Hamburg-apples in Germany, Chateaudun for apples in France, Sevilla for apples in Spain and Piacenza for apples in Italy). PECgw was calculated to be << 0.001 µg/l in all scenarios for both milbemycin A4 and the two metabolites 27-hydroxy-milbemycin A4 and 27-keto-milbe Models developed by EU’s working group FOCUS estimates predicted environmental concentrations in surface water and sediment in different scenarios. The highest PECsw values were observed right after the second application, indicating that spray drift is the main route of exposure. The highest PIEC for the water and sediment phases are 0.18 µg a.s./l and 7.9 µg a.s./kg dw respectively in strawberries (leafy vegetables) and 1.3 µg a.s./l and 0.98 µg a.s./kg in the water and sediment phases respectively in pome fruit. Based on the need for setting buffer zones and the fact that drift seems to be the major route of exposure to surface water, PEC values estimated using the drift tables in Rautmann et al. 2001 were used in the risk assessment. In strawberries, with one application, PEC values ranged from 0.21 µg/L with a buffer zone of 1 meter to 0.01 µg/l with a zone of 20 meters. In Pome fruit PEC ranged from 0.5 to 0.03 µg/l at buffer zones of 5 and 30 meters respectively. In ornamentals PECs varied between 0.5 µg/l with a buffer zone of 3 meters and 0.01 µg/l with a zone of 30 meters.

Terrestrial organisms

The active substance milbemectin is a mixture of two microbial produced compounds: milbemycin A3 and milbemycin A4, naturally occurring at a ratio of approximately 3:7. All Annex II ecotoxicological studies have been conducted with technical milbemectin, containing the two components milbemycin A3 and milbemycin A4 in the appropriate relative amounts. Where there are indications that the plant protection product is more toxic than what can be explained by the content of active substance (or studies are only conducted with the product), or identified metabolites are more toxic than the active substance, these calculations are included in the summary below. If this is not the case, these values and calculations are omitted. Mammals Acute toxic to mammals (LD50: 456 mg/kg bw/d). TERacute for the indicator species in orchards is estimated as 156 and TERacute is estimated as 574 for the indicator species in strawberries. These values do not exceed the trigger (<10). Moderate reproductive toxicity, NOEC: 200 mg/kg. TERchronic is estimated to be 209 in orchards and 904 in strawberries. These values do not exceed the trigger (<5). Birds

Milbemectin is acutely toxic to birds (LD50: 347 mg/kg bw). TERacute for the indicator species in orchards is estimated as 363. For the indicator species in strawberries, TERacute values are estimated as 161 and 275 for herbivorous and insectivorous birds, respectively. These values do not exceed the trigger (<10). Milbemectin has moderate dietary toxicity (LC50: 1922 mg/kg feed), TERshort-term for all indicator species in all crops are estimated as >1000, which do not exceed the trigger (<10). Milbemectin also has a moderate


reproductive toxicity (NOEC: 150 mg/kg). TERchronic is estimated to be 281 for the indicator species in orchards, and 250 for herbivorous birds and 213 for insectivorous birds in strawberry fields. These values do not exceed the trigger (<5). Bees Very high contact toxicity to bees (LD50: 0.026 µg/bee). High oral toxicity to bees (LD50: 0.40 µg/bee). Hazard quotients for contact and oral exposure are estimated to be 680 and 44.2 for applications in orchards, 896 and 58 for applications in strawberries, and 731 and 47.5 for applications in ornamentals. The hazard quotients for contact exposure exceed the trigger value (>50) in all crops. In order to assess the risk of Milbeknock 1% EC, a semi-field (cage) test has been carried out. The results indicated no significant increase in mortality after application of 27.9 g a.s./ha (higher than the highest dose applied for in Norway), and no effects on flight intensity, behavior or brood. Non-target arthropods In Tier 1 laboratory acute contact toxicity studies, Milbeknock showed negligible effects on parasitoids and ground dwelling predators at relevant application rates. For foliage dwelling predators and predatory mites, the trigger of >30% effect is exceeded. Earthworms Milbemectin is acutely toxic to earthworms (LC50corr: 28.5 mg/kg d.w. soil). TERacute for orchard and ornamentals is estimated to be 1425 and 950, respectively. TERacute for the strawberry scenario is estimated to be 950. These values do not exceed the trigger (<10). Milbeknock has a high chronic toxicity to earthworms (NOECcorr: 0.11 mg/kg d.w. soil). TERchronic for orchards is estimated to be 11. TERchronic for ornamentals is estimated to be 6. These values do not exceed the trigger (<5). TERchronic for strawberries is estimated to be 3. This value exceeds the trigger (<5). TERchronic for strawberries recalculated based on a single application results in a TER of 6 which does not exceed the trigger. Microorganisms Neither mineralization nor nitrogen transformation by soil microflora of soils treated with milbemectin up to 75 g a.s./ha (3 x the maximum expected concentration) differed from untreated soils by greater than 25 % (trigger) after 28 days. Terrestrial plants Twenty tests are available for a number of crop species. In all treatments effects on emergence, shoot length and shoot weight were below the trigger of > 50% effect at the maximum application rate.

Aquatic organisms

All PEC-values below are based on single application drift values from Rautmann et al. (2001), since FOCUS modeling has shown that drift gives the highest PEC-values. TER calculations have been performed mostly on single species tests, but also with the microcosm study (for invertebrates). Fish Milbemectin showed extreme acute toxicity to fish (96h LC50: 4.4-35 µg a.s./L) and extreme chronic toxicity (ELS NOEC: 0.65 µg a.s./L). Milbeknock showed extreme acute toxicity to rainbow trout (96h LC50: 5.7 µg a.s./L). All TER calculations for milbemectin, both acute and chronic, pass the EU triggers (acute: 100, chronic: 10) with 5-30 meter buffer zones. Invertebrates Milbemectin showed extreme acute toxicity to Daphnia magna (48h EC50: 11 µg a.s./L) and extreme chronic toxicity to Daphnia magna (21d NOEC: 0.12 µg a.s./L). Milbeknock showed extreme acute toxicity to Daphnia magna (48h EC50: 3.43 µg a.s./L) and very high toxicity to other invertebrates (LC50: 49.3-187 µg a.s./L). All TER calculations for milbemectin pass the EU trigger with 3-20 meter buffer zones, except TERs for chronic exposure from use in ornamentals (TER:9) and pome fruit (TER:4) which fail the trigger (10) even with 30 meter buffer zones. Sediment dwelling organisms Milbemectin showed extreme chronic toxicity to Chironomus riparius larvae (28d NOEC: 6.3 µg/L (spiked water)). Milbeknock showed extreme acute toxicity to Chironomus riparius larvae (48h EC50: 30.1 µg a.s./L)


and medium acute toxicity to the oligochaeta Tubificidae (48h EC50: 1142 µg a.s./L). All TER calculations for milbemectin pass the EU trigger with 1-5 meter buffer zones. Aquatic plants Milbemectin showed high toxicity to duckweed (14d EC50: >620 µg a.s./L). All TER calculations for milbemectin pass the EU trigger with 1-3 meter buffer zones. Algae Milbemectin showed no effects on algae at the highest tested concentration (72h EC50: >2000 µg a.s./L, NOEC: 2000 µg a.s./L). Milbeknock showed very high toxicity to algae (72h EC50: 220 µg a.s./L). All TER calculations for milbemectin pass the EU trigger with 1-3 meter buffer zones. Microcosm studies A microcosm study representing a plankton-dominated community was submitted. The company suggests a NOEAEC of 3.68 µg a.s./L. KemI suggests a NOEC of 0.058 µg a.s./L, but argues that since it cannot be concluded that the most sensitive organisms were present in the microcosms, the study cannot be used to override the results from the single species tests. The Norwegian Food Safety Authority agrees that the NOEC should be 0.058 µg a.s./L. All TER calculations for milbemectin pass the Nordic/Baltic microcosm trigger of 3 with 20-30 meter buffer zones, except the TER for use in pome fruit (TER:1.8) which fail the trigger even with a 30 meter buffer zone. Bioconcentration Milbemectin shows a moderate potential for bioconcentration; in bluegill sunfish average whole fish BCF was 76 and 114 for milbemycin A3 and milbemycin A4, respectively. Rapid depuration occurred (CT50: 0.7-1.1 days).

1.5 Dossier quality and completeness

The dossier is complete and is adequate as a basis for an evaluation of the active substance, metabolites and product.

Milbeknock - milbemectin Side 2-1

2. Product status

Our reference 07/16932 Active substance milbemectin Product name Milbeknock Applicant Mitsui Chemicals Agro Inc Importer Profilering AS Concentration of active substance 9,3 g/L Formulation EC formulation (Emulsifiable concentrate) Packaging 1 Litre Function Acaricide, insecticide Application background A new product containing a new active substance. Application date 4.5.2007 Status in the EU Milbemectin was included on Annex I in 2005.

Status in Denmark and Sweden: Milbeknock is registered in Denmark for use in apples, pears, strawberry and ornamentals. In Sweden it has only been approved for use in ornamentals in glasshouses.


3. Efficacy

Teksten i dette kapitlet er hentet fra Bioforsk Plantehelse sin agronomiske vurdering samt etikettforslag fra importør.

3.1 Bruk/virkning

Søkt bruksområde Eple, pære, jordbær på friland, i plasttunnel og i veksthus, prydplanter i grøntanlegg og i veksthus og i planteskoler utendørs.

Virkeområde Spinnmidd, dvergmidd (bl.a. skuddtoppmidd, cyclamenmidd), frukttremidd,

jordbærmidd, og minerfluelarver. Preparatets virkning mot midd gjelder alle stadier. Med bakgrunn i et norsk forsøk mot bringebærbladmidd er det grunn til å tro at

Milbeknock har sideeffekt på bladmidd, men det er behov for mer dokumentasjon for å kunne anbefale bruk.

Virkemåte Milbemektin har både kontakt- og magevirkning, og virker på minerfluelarver samt på

egg, bladlevende larver/nymfer og voksne midd. Preparatet virker raskt (såkalt ”knock-down” effekt) og har også god langtidsvirkning.

Virkemekanisme Milbemektin tilhører den kjemiske gruppen avermektiner og milbemyciner (IRAC MoA Group 6 -kloridkanalaktivatorer). Midlet virker ved å hemme overføring av impulser

mellom nerveceller og mellom nerveceller og muskelceller slik at insektene lammes og dør.

Nytteorganismer/ Integrert plantevern Ifølge ”side-effects list” (http://side-effects.koppert.nl/#) så er Milbeknock svært skadelig

for flere nytteorganismer, men ikke for jordboende nyttenematoder. Ettervirkningstida er ikke oppgitt.

Preparatet er skadelig for flere nytteorganismer som brukes til middbekjempelse, og dette bør fruktdyrkere og andre som ønsker å tilrettelegge for naturlige fiender være klar over. Dette bør derfor redegjøres for på etiketten.

Resistens Milbeknock inneholder det aktive stoffet milbemektin, som tilhører den kjemiske

gruppen 6: avermektiner og milbemyciner (kloridkanalaktivatorer). Midlet er en GABA-agonist (virkning på γ-aminosmørsyre og glutamatregulerte kloridkanaler) med nerve- og muskelvirkning. Milbemektin har samme virkningsmekanisme som abamectin (Vertimec 018 EC). Kryssresistens mellom milbemektin og abamectin er påvist hos veksthusspinnmidd.

Av målorganismene for Milbeknock har veksthusspinnmidd og Liriomyza-artene (minérfluer) på karantenelista generelt høy risiko for å utvikle resistens mot kjemiske midler.

Resistens mot milbemektin er påvist hos feltpopulasjoner av veksthusspinnmidd og spinnmiddarten Oligonychus perseae i andre land. Resistens mot milbemektin er ikke undersøkt i Norge, og resistensmekanismen(e) er lite undersøkt. Det ser imidlertid ut som om resistensen mot både milbemektin og abamektin er ustabil, og kan gå tilbake i perioder når det ikke sprøytes med disse midlene. Det betyr at det er gode sjanser for å bevare virkningen av milbemektin (og abamectin) dersom de brukes i veksling med preparater som er gode resistensbrytere.

Vurdering av risiko for resistensutvikling mot Milbeknock i Norge

Eple og pære: Spinnmidd (frukttremidd): Apollo 50 SC og Envidor 240 SC er alternativer til Milbeknock. Begge er gode resistensbrytere for Milbeknock. Det er liten risiko for resistensutvikling mot Milbeknock dersom midlet bruks i veksling med de andre midlene.

http://side-effects.koppert.nl/


Jordbær: Jordbærmidd: Det eneste alternative midlet med annen virkningsmekanisme er Mesurol 500 SC, men midlet er trukket av importør og kan kun brukes ut 2011. I tillegg brukes forebyggende varmebehandling av småplanter før utplanting, og under tak brukes rovmidden Amblyseius cucumeris. Det er stor fare for ensidig bruk av Milbeknock og Vertimec, men det er uvisst hvor lett jordbærmidden utvikler resistens. Envidor 240 SC kan trolig virke som resistensbryter til en viss grad, da dette preparatet har sidevirkning mot jordbærmidd (men ikke god nok effekt til godkjenning).

Veksthusspinnmidd: Alternative midler med andre virkningsmekanismer er Apollo 50 SC og Nissorun (begge har samme virkningsmekanisme), samt Envidor 240 SC og Floramite. Under tak brukes også rovmidd. Det er lav risiko for resistensutvikling hos veksthusspinnmidd dersom preparatene brukes i et rotasjonsprogram.

Prydplanter i veksthus: Veksthusspinnmidd: Per i dag er det er midler med 5 andre virkningsmekanismer på markedet, som alle egner seg som resistensbrytere for Milbeknock. To av disse middelgruppene går ut i 2011. Det betyr at midler med 3 andre forskjellige virkningsmekanismer vil være tilgjengelig fra 2012 dersom det ikke blir godkjent nye preparater: Karate-preparatene (middelgruppe 3A), Apollo og Nissorun (middelgruppe 10A) og Floramite (ikke klassifisert-ukjent virkningsmekanisme). Det er påvist nedsatt følsomhet for Vertimec 018 EC hos noen populasjoner av veksthusspinnmidd, og det er fare for kryssresistens mellom Vertimec 018 EC og Milbeknock. Vertimec 018 EC har vært en del brukt, så risikoen for rask resistensutvikling er stor dersom Milbeknock brukes ensidig. Det er sannsynligvis liten fare for kryssresistens mellom Milbeknock og de andre midlene. I tillegg kan det brukes nyttedyr i mange kulturer. Risikoen for resistensutvikling mot Milbeknock kan dermed reduseres vesentlig dersom midlet brukes i veksling med midler med andre virkningsmekanismer og nyttedyr.

Amerikansk blomstertrips er ikke målorganisme for Milbeknock. Imidlertid er Vertimec 018 EC et viktig middel mot amerikansk blomstertrips, og brukes mye der det er problemer med sviktende virkning av andre tripsmidler. Bruk av Milbeknock vil føre til økt risiko for resistensutvikling mot Vertimec 018 EC hos tripsen, og dette er uheldig. Veksthusspinnmidd kan også bli eksponert ved sprøyting mot amerikansk blomstertrips. Derfor bør den samlede bruken av Milbeknock og Vertimec 018 EC begrenses.

Mot dvergmidd er det 1 alternativt middel (Thiovit Jet) med annen virkningsmekanisme, samt Amblyseius cucumeris. Dvergmidd er et sporadisk problem i prydplanter, og det sprøytes derfor lite. Det er derfor antakelig liten fare for resistensutvikling mot Milbeknock.

Når det gjelder minerfluer er det per i dag midler med 3 andre virkningsmekanismer på markedet som alle er gode resistensbrytere for Milbeknock og Vertimec 018 EC. Alle de kjemiske midlene har god virkning mot minerfluer (unntatt Liriomyza-artene på karantenelista), og det er derfor liten fare for ensidig bruk av Milbeknock.

Nyttedyr brukes en del mot veksthusspinnmidd og dvergmidd men kan ikke brukes i alle kulturer/situasjoner. Nyttedyr er også godkjent mot minerfluer, men brukes lite. Forskjellig toleranse hos prydplanter for de ulike kjemiske midlene gjør at det i enkelte situasjoner kan være færre midler å velge mellom enn det som totalt er godkjent i prydplanter.

Prydplanter i grøntanlegg og i planteskoler: Det sprøytes forholdsvis lite mot spinnmidd og minerfluer i prydplanter på friland. Det finnes 3 alternative kjemiske midler med andre virkningsmekanismer mot spinnmidd og midler med 2 andre virkningsmekanismer mot minerfluer. Milbeknock er en god resistensbryter for alle disse midlene. Det er liten risiko for resistensutvikling mot Milbeknock dersom midlet bruks i veksling med de andre midlene.

Resistensforebyggende tiltak

På grunn av stor fare for resistensoppbygging mot preparater i middelgruppe 6: Avermektiner og milbemyciner hos veksthusspinnmidd og amerikansk blomstertrips i veksthus, bør behandling med Milbeknock og Vertimec mot disse skadedyrene


begrenses til 2 behandlingsblokker (en behandlingsblokk = 1-2 behandlinger innen en generasjon av skadedyret) per år. Milbeknock må brukes i veksling med andre midler med andre virkningsmekanismer og/eller nytteorganismer. Virkningen av Milbeknock bør overvåkes, med spesielt fokus på veksthusspinnmidd siden det er påvist nedsatt følsomhet i noen populasjoner mot Vertimec.

3.2 Behandlingsmåte og dosering

Eple og pære: Mot frukttrespinnmidd og veksthusspinnmidd sprøytes det fra avblomstring og fram til frukten har ca. halv normal størrelse (behandlingsfrist = 14 dager). Dosering er avhengig av trestørrelsen: Trehøyde ≤ 2 m: 130 ml preparat/100 l vann, 50 l væskemengde per 100 m rad. Trehøyde > 2 m: 130 ml preparat/100 l vann, 75 l væskemengde per 100 m rad. Maks arealdose: 190 ml preparat/daa. Maksimalt 2 behandlinger per sesong. Jordbær i veksthus, plasttunnel og på friland: Mot spinnmidd og jordbærmidd sprøytes angrepne blader etter avhøsting: 250 ml preparat/minimum 120 l vann/1000 m rad (dekar) ved full bladmasse. Sprøyting må skje før dvalehunnenen er dannet, og mot jordbærmidd må sprøyting være avsluttet før første frost. Maksimalt 2 behandlinger per sesong. Prydvekster i veksthus, grøntanlegg og planteskoler utendørs: Mot veksthusspinnmidd og dvergmidd sprøytes det ved begynnende angrep. Konsentrasjon: 50 ml preparat/100 l vann, vannmengden avhenger av plantestørrelsen: Opp til 50 cm: 50 ml i 100 l vann pr. dekar 50-125 cm: 75 ml i 150 l vann pr. dekar Over 125 cm: 100 ml i 200 l vann pr. dekar Maks arealdose er 100 ml/daa. Mot minerfluelarver sprøytes det når angrepet konstateres. Konsentrasjon: 100 ml preparat/100 l vann, vannmengde avhenger av plantestørrelsen: Opp til 50 cm: 100 ml i 100 l vann pr. dekar 50 – 125 cm: 150 ml i 150 l vann pr. dekar Over 125 cm: 200 ml i 200 l vann pr. dekar Maks arealdose er 200 ml/daa. I prydplanter skal Milbeknock maksimalt brukes i 2 behandlingsblokker per år. Hver behandlingsblokk skal bestå av 1-2 behandlinger som skal utføres innenfor 1. generasjon av skadedyret. Det vil si maksimalt 4 behandlinger per år.

NAD Med bakgrunn i preparatets dosering i jordbær foreslås normert arealdose (NAD) fastsatt til 250 ml preparat per dekar. Dette tilsvarer 2,5 g v.s./daa.

Spredeutstyr Preparatet sprøytes på plantene med dysevanningssystemet, lavtrykks- eller høytrykkssprøyte, tåkesprøyte eller traktormontert sprøyteutstyr. Aktuelt

spredeutstyr, dysetype, antall dyser, trykk osv. er avhengig av hvilke kultur og skadegjører som skal behandles.


3.3 Anbefaling fra Bioforsk Plantehelse

Milbeknock har tilsvarende virkemekanisme og effektivitet, samt overlappende bruksområder som Vertimec. Preparatet vil derfor ikke ha betydning som resistensbryter for midler i andre middelgrupper i de bruksområdene der Vertimec også er godkjent

Eple og pære Milbeknock vil være viktig i forbindelse med bekjemping av frukttremidd i kjernefrukt, for å unngå ensidig bruk av Envidor 240 SC. I tillegg er også Apollo 50 SC godkjent, men har kun effekt mot egg og larver. Da Envidor 240 SC også har dokumentert effekt mot bladmidd, vil imidlertid dette preparatet mest sannsynlig bli foretrukket av dyrkerne. Envidor 240 SC er i tillegg vurdert å være noe mindre skadelig for nyttefaunaen enn Milbeknock. Jordbær Mot spinnmidd er det flere likeverdige preparater å velge imellom, men mot jordbærmidd er det kun Vertimec som er godkjent. Da begge preparatene tilhører samme middelgruppe og har lignende virkning vil det ikke få noen umiddelbare konsekvenser dersom Milbeknock ikke skulle bli godkjent i jordbær. Unntaket er veksthusjordbær, der det for tiden mangler kjemiske preparater mot jordbærmidd. Det agronomiske behovet her er imidlertid lite, siden det er begrenset mengde veksthusjordbær i Norge, og plantene brukes vanligvis bare en sesong. Rovmidd (Amblyseius cucumeris) fungerer dessuten bra mot jordbærmidd i veksthusjordbær. Prydplanter i veksthus Det finnes flere alternative preparater mot veksthusspinnmidd og minerfluer. Så lenge Vertimec er på markedet vil det derfor ikke være noe stort behov for Milbeknock. Prydplanter i grøntanlegg og planteskoler utendørs Vertimec er ikke godkjent i dette bruksområdet, så her vil Milbeknock være et bra middel med ny virkemekanisme og som kan forhindre resistensutvikling. På den annen side sprøytes det forholdsvis lite mot spinnmidd og minerfluer i prydplanter på friland. Det finnes 3 alternative kjemiske midler med andre virkningsmekanismer mot spinnmidd og 2 alternative kjemiske midler med andre virkningsmekanismer mot minerfluer. Milbeknock har også en viss sideeffekt mot bladmidd, som vil være en nyttig opplysning når dyrkerne skal velge mellom ulike middpreparater. Det er imidlertid behov for mer dokumentasjon for å kunne oppgi dette på etiketten.

Bioforsk Plantehelse anbefaler godkjenning av preparatet, men har gjort noen mindre

justeringer på det opprinnelige etikettforslaget fra importør.

Milbeknock – milbemectin Page 4-1

4. Identity and physical/chemical data IUPAC-name Milbemectin consists of two milbemycin isomers: <30% milbemycin A3 (MA3): (10E,14E,16E,22Z)-(1R,4S,5’S,6R,6’R,8R,13R,20R,21R,24S)-21,24-dihydroxy-5’,6’,11,13,22-pentamethyl-3,7,19-

trioxatetracyclo[15.6.1.14.8

.020,24

]pentacosa-10,14,16,22-tetraene-6-spiro-2’-tetrahydropyran-2-one ; and >70% milbemycin A4 (MA4): (10E,14E,16E,22Z (10E,14E,16E,22Z)-(1R,4S,5’S,6R,6’R,8R,13R,20R,21R,24S)-6’-ethyl-21,24-

dihydroxy-5’,11,13,22-tetramethyl-3,7,19-trioxatetracyclo[15.6.1.14.8

.020,24

]pentacosa-10,14,16,22-tetraene-6-spiro-2’-tetrahydropyran-2-one CAS number MA3: 51596-10-2; MA4: 51596-11-3 Structural formula

Molecular weight Milbemycin A3 528.7 Milbemycin A4 542.7 Solubility in water Milbemycin A3: Moderate, 2.68 mg/l (20 °C) Milbemycin A4: Moderate, 4.55 mg/l (20 °C) Vapour pressure Milbemycin A3: Low, 9.7x10-12 Pa (20 °C) Milbemycin A4: Low, 4.3x10-10 Pa (20°C) Henrys law constant Milbemycin A3: Low, 2.56x10-3 Pa m3/mol Milbemycin A4: Low, 1.55x10-3 Pa m3/mol log Pow Milbemycin A3: Very high, 6.54 (25°C) Milbemycin A4: Very high, 7.0 (25°C) pKa -


5. Mammalian Toxicology This assessment is based on documentation submitted by the applicant (referenced with author and year) as well as EUs Draft Assessment Report (DAR) of milbemectin (volume 3 – annex B) by The Netherlands (T1), Milbemectin, Volume 3 Annex B, Addendum VII (February 2005) (T2), position statement for ECB Meeting 21 March 2006 (T3) and ECB classification 2006 (T4).

5.1 Milbemectin

Milbemectin is a mixture of about 30% Milbemycin A3 (MA3) and 70% Milbemycin A4 (MA4).The onlydifference between the two milbemycins is a methyl (MA

3) versus an ethyl (MA4) side chain. The

developmental code for milbemectin is E-187.

5.1.1 Toxicokinetics

Study 1. Rat oral, single low and high dose, repeated low dose

Absorption, distribution, metabolism and excretion (including bile cannulation) of [14C)-Milbemycin A4 and Milbemycin A4

Study design: Sprague-Dawley rats (5/sex) were administrated as a single oral dose of 2.5 or 25 mg/kg bw C-14 MA4 by gavage in a 1% CMC suspension. Low dose of 2.5 mg/kg bw/day MA4 was given for 14 days followed by a single dose of 14C- MA4. Separate animal groups (4-9/sex) were used for studying pharmacokinetics in plasma, tissue distribution and bile cannulation after single low or high dose exposure. Elimination of radioactivity was monitored in urine and faeces for 7 days. Animals were sacrificed 7 days after dosing of the radiolabelled test compound. More details of the study design in tables B.6.1.1.1 and B.6.1.1.2 in T1. OECD 417and GLP. (Fathulla et al., 2000) Results: Absorption: The peak plasma concentration (Tmax) was reached 2 hours after administration of the high dose (both sexes) and in females in the low dose group. The males at low dose had Tmax in plasma after 3 hours. Plasma concentration (Cmax and AUC) was proportionally increased with increased dose. The plasma concentration was higher in females than in males. Plasma concentration rapidly declined the first 24 hours, and then there was a slow decline through 72 hours in the low dose and through 168 hours in the high dose. There was slower decline in females than in males. Table 5.1: Pharmacokinetic Parameters in Plasma Dose Sex Cmax

µg equiv·h/g tmax hours

t1/2 hours

AUC0 – t

µg equiv·h/g AUC0 - ∞

µg equiv·h/g Male 0.313 3.0 10.9 2.48 2.50 Single oral

2.5 mg/kg bw Female 0.255 2.0 13.0 3.14 3.19 Male 2.64 2.0 27.4 27.1 27.3 Single oral

25 mg/kg bw Female 2.29 2.0 31.7 37.9 38.3 The absorption of milbemycin seems to be at least 47 % in both sexes based on the bile cannulation study of single low dose (0-48 hours post dose). The single high dose gave an estimated absorption of 30/40 % for male/female. This may point to a saturated absorption of milbemectin at larger doses. Distribution: The gastrointestinal tract and the liver contained most of the radioactivity. The residue concentration in the adrenal glands, kidneys, pancreas, lymph nodes, bone marrow, heart, lungs muscle, skin, thymus, urinary bladder, and reproductive fat was higher than in the plasma at all time points. Radioactivity in blood and plasma was the same. Most tissues had the residue peak 2 hours post dosing (around tmax), but residues in reproductive fat had a peak 6 hours after dosing (tmax/2) in the high dose group and in females in the low dose group. The radioactivity was similar in blood aplasma. Tissue residues increased disproportionately more than the increase in dose.

nd


Table 5.2: Tissue distribution of milbemycin A4 Mean concentration of radioactivity (µg equivalents of 14C-milbemycin A4/g

Dose Sex Time point * Hours post dose

Adrenal glands

Liver Kidneys Pancreas Lymph nodes, mesenteric

Reproductive fat

3 1.88 4.09 0.894 1.14 1.13 1.55 6 0.762 1.86 0.436 0.735 0.802 0.994

Male

24 0.049 0.240 0.064 0.051 0.083 0.164 2 2.09 2.59 0.778 1.59 1.65 1.62 6 1.37 1.74 0.464 1.11 1.34 1.98

2.5 mg/kg bw single oral

Female

24 0.109 0.234 0.069 0.093 0.104 0.441 2 22.4 48.5 15.1 19.5 20.2 20.4 6 18.0 32.8 10.4 14.8 19.5 31.9

Male

24 1.33 4.51 1.41 1.40 2.28 9.21 2 25.4 48.1 13.7 22.7 17.5 14.4 6 26.0 35.0 14.3 22.6 27.2 33.7

25 mg/kg bw single oral

Female

24 3.60 5.31 2.30 3.59 4.36 13.9 * Time points are tmax, tmax/2 and tmax/10. Metabolism: The unchanged parent compound was detected in all liver and kidney samples of the high dose group and in male plasma at 2-6 hours and in female plasma at 2 hours post dose, but not in these tissues in samples from the low dose group. The parent compound was not found in bile samples from any group or in faeces from the low dose group. In the high dose group, the parent compound in faeces was detected as 31.0% (males) and 37.4% (females). The main metabolic pathway was hydroxylation at several positions in the molecule (mono-, di- and trihydroxylation). There were also some carboxylation (high dose) and a minor glucuronidation conjugation pathway. The major metabolite found in plasma, liver, and kidney was 13-hydroxy- M.A4, accounting for up to 60.2%, 52.4%, and 66.7% respectively. The metabolites found in bile were the same as in faeces fronon-bile cannulated rats. In urine there were found several dihydroxy-metabolites of milbemycin A

m

n.

4. Since all mother substance was transformed at the low dose, and not all can be accounted for in bileand urine, there seems to be another degradation process involved, presumably intestinal secretio Table 5.3: Tissue distribution, gastrointestinal tract, blood and blood related organs

Mean concentration of radioactivity µg equivalents of 14C-milbemycin A4/g (and % of dose)

Dose Sex Time point * Hours post dose

Stomach Stomach contents & wash

Intestinal tract

Intestinal tract contents & wash

Spleen Blood Bone marrow (femur)

3 23.9 (5.03)

3.74 (12.6)

6.72 (8.03)

1.60 (18.9)

0.608 (0.06)

0.359 (0.52)

0.777 (0.01)

6 4.04 6.49 2.96 3.45 0.307 0.182 0.358

Male

24 0.086 0.219 0.178 0.003 0.036 0.015 0.023 2 38.4 2.82 6.07 2.78 0.647 0.297 1.46 6 5.37 3.85 2.91 7.02 0.431 0.186 1.01

2.5 mg/kg bw single oral Female

24 0.116 0.983 0.161 0.006 0.043 0.023 0.046 2 217 40.8 59.3 45.1 6.95 2.70 11.7 6 18.9 4.6 35.7 78.4 5.07 2.16 9.47

Male

24 0.989 0.203 2.30 5.83 0.477 0.264 0.608 2 164 41.0 57.8 43.4 7.71 2.51 18.9 6 30.4 8.54 36.3 51.1 8.12 2.58 30.5


Female

24 2.35 0.133 5.94 12.9 1.21 0.494 3.41 Elimination: The main route of elimination was via bile, and a smaller amount was excreted via urine. The excretion was rapid the first 24 hours, but there was also a steady low excretion from 24 to 48 hours post dosing. There was more rapid excretion of the low dose as the terminal half life in blood was 10.9-13 hours (m-f) at the low dose compared to 27.4-31.7 hours (m-f) at the high dose.


Summary table 5.4: Excretion: Dose group 2.5 mg/kg bw,

single oral Male – female

2.5 mg/kg bw, repeated oral Male – female

25 mg/kg bw, single oral

Male – female

2.5 mg/kg bw, single oral, bile cannula

Male – female

25 mg/kg bw, single oral, bile cannula

Male – female Urine, (%) 9.29 – 4.92 7.22 – 4.48 7.19 – 3.28 6.18 – 3.35 4.68 – 2.46 Faeces, (%) 84.8 – 100 84.7 – 91.6 81.5 – 92.8 36.2 – 44.7 55.3 – 64.8 Bile, (%) Np Np Np 41.0 – 43.8 35.7 – 27.6 Cage cleaning, (%)

4.60 – 0.74 2.22 – 0.82 4.65 – 0.32 0.73 – 0.42 2.30 – 0.47

CO2, (%) – – – – – Total excreted, (%)

84.2 – 92.4 98.0 – 95.4

Residues in carcass + tissues, (%)

0.38 – 0.43 0.37 – 0.44 0.36 – 0.43 1.90 – 1.22 1.51 – 2.79

Total recovered, (% of dose)

99.1 – 106 94.5 – 97.4 93.7 – 96.8 86.1 – 93.5 99.6 – 98.0

Np = not performed

Study 2: Rat oral, single low and high dose, repeated low dose (supplementary)

Metabolic study of E-187 (milbemectin; [14C]-Milbemycin A4 and 5- [3H]-Milbemycin A3)

in rats

Study design: Fischer rats (3/sex) were administrated as a single oral dose of 2.5, 25 or 250 (MA only) mg/kg bw of C-MA4,

314 3H-MA3, (14C-MA3) or the combination E-187 by gavage. Low dose of 2.5

mg/kg bw/day MA4 was given for 10 days followed by a single dose of 14C-MA . Separate animal groups (animal number always not stated) were used for studying pharmacokinetics in plasma, tissue distribution and bile cannulation after single low or high dose exposure.

4

Elimination of radioactivity was monitored in urine and faeces for 7 days. Animals were sacrificed 7 days after dosing of the radiolabelled test compound. In addition the in vitro metabolism in presence of microsomal fraction from Fischer rats have been investigated, but no results (raw data or tables) are shown in the study report. More details of the study design in tables B.6.1.4.1 and B.6.1.4.2 of T1. No OECD guideline or GLP. (Sadakane et al., 1990). Results: Metabolism: The main metabolite in vitro was 13-hydroxy-A3 and –A4. This hydroxylation was faster in MA3, than in MA4, resulting in a more rapid metabolism of MA3. Minor metabolites detected was 5-keto-A4, 27-hydroxy-one and its furan-ring opening isomer, 14,15-epoxy-one, and dihydroxy-ones. In bile at 6 hours after administration (only male rats cannulated), the main metabolites were 13-hydroxyA4, dihydroxyA4s (some glucuronidated) and trihydroxyA4s. All these metabolites were also detected in the faeces. Unchanged MA4 was found in concentrations of 3 % in blood and 8 % in liver 6 hours after administration. After the very high single dose of 250 mg/kg of MA3, about 70 % was excreted as the mother substance in faeces during the first 2 days.

Distribution: The tmax in blood was around 3 hours after administration and then decreased rapidly. Half-life of elimination was 7-8 hours. MA3 was eliminated slightly faster than MA4 (ethyl-group). Liver and fat (hypodermic and intraperitoneal) had high levels of radioactivity. The high dose gave a MA3 concentration in tissues that were higher than in blood 6 hours after administration except for the concentration in bone (males) and brain (both sexes). High dose MA4 gave a lower tissue concentration than blood only in brain (both sexes). Repeated dosing gave the same tissue distribution as single dosing. There was no accumulation.


Table 5.5: Distribution of radioactivity in tissues after administration of E-187 (MA3/MA4 = 3/7)

Mean concentration of radioactivity (µg equivalents of 3H-milbemycin A3 /14C-milbemycin A4 per

gram tissue; ppm) Dose Sex Time

point Hours post dose

Blood Fat, hypo-dermic

Fat, intra-periton.

Liver Kidney Adrenal Stomach Small intestine

Caecum Contents in Caecum

6 A3 A4

0.04/ 0.16

0.11/ 1.55

0.14/ 1.99

0.29/ 1.74

0.068/ 0.58

0.072/ 0.80

0.30/ 1.55

0.16/ 1.16

1.01/ 3.42

15.3/ 47.0

24 0.01/ 0.03

0.009/ 0.17

0.011/ 0.22

0.079/ 0.46

0.017/ 0.10

0.027/ 0.10

0.079/ 0.35

0.020/ 0.14

0.016/ 0.62

3.19/ 13.0

Male

72 0.002/ 0.01

0.007/ 0.03

0.008/ 0.02

0.017/ 0.07

0.007/ 0.03

0.004/ <0.01

0.006/ 0.02

0.003/ 0.01

0.002/ 0.01

0.018/ 0.08

6 0.019/ 0.10

0.15/ 1.73

0.18/ 2.13

0.19/ 1.19

0.060/ 0.44

0.064/ 0.58

0.16/ 0.77

0.17/ 1.07

1.14/ 3.50

14.4/ 43.7

24 0.010/ 0.03

0.010/ 0.19

0.014/ 0.16

0.046/ 0.27

0.014/ 0.08

0.009/ 0.04

0.038/ 0.17

0.028/ 0.14

0.13/ 0.49

2.25/ 20.5

2.5 mg/kg bw single oral

Female

72 0.002/ 0.01

0.004/ 0.03

0.005/ 0.04

0.015/ 0.07

0.006/ 0.03

0.004/ <0.01

0.004/ 0.02

0.003/ 0.01

0.003/ 0.01

0.029/ 0.10

6 0.22/ 1.4

2.26/ 22.3

2.35/ 23.1

3.23/ 16.4

0.82/ 5.7

1.34/ 11.7

1.00/ 5.1

1.10/ 7.0

6.14/ 18.9

82.4/ 238.0

24 0.12/ 0.2

0.19/ 3.3

0.16/ 3.0

0.48/ 2.8

0.15/ 0.9

0.10/ 0.9

0.31/ 1.2

0.19/ 1.0

0.47/ 2.0

18.1/ 72.7

Male

72 0.03/ 0.1

0.06/ 0.8

0.08/ 0.4

0.18/ 0.8

0.06/ 0.3

0.07/ <0.1

0.05/ 0.2

0.03/ 0.1

0.05/ 0.7

0.59/ 3.4

6 0.37/ 1.5

4.30/ 22.0

5.10/ 19.9

3.93/ 15.0

1.60/ 6.6

2.66/ 12.9

1.70/ 6.2

2.31/ 10.0

4.96/ 13.3

79.7/ 221.0

24 0.15/ 0.3

0.48/ 5.2

0.36/ 5.7

0.41/ 2.7

0.16/ 1.2

0.15/ 1.3

0.16/ 0.8

0.21/ 1.5

1.05/ 2.9

14.8/ 55.9


Female

72 0.03/ 0.1

0.07/ 0.9

0.06/ 0.9

0.16/ 0.9

0.06/ 0.3

0.05/ <0.1

0.06/ 0.2

0.03/ 0.2

0.11/ 0.5

2.50/ 10.9

Elimination: More than 95% of the dose was excreted during the first 3 days after administration at all three dose levels. More than 98% was excreted in a week. There was a higher percentage of excretion of MA3 than MA4 in urine, and males had higher urine excretion than females. Percent excretion in urine was rather constant and unaffected by dose; 8-17 % in males and 5-13 % in females, except for the very high dose of 250 mg/kg of MA3 (3 % in both sexes). 42 % of the radioactivity was found in the bile from cannulated rats (low dose, male, collected for 24 hours). Repeated dosing gave the same elimination pattern as single doses.

Table 5.6: Excretion of MA3 and MA4 in urine and faeces (% radioactivity of dose administered)

MA3 Males Females Dose Excreta 1 day 2 days 3 days 1 day 2 days 3 days

Faeces 70.6 82.8 83.8 68.6 87.5 89.1 2.5 mg/kg E-187 3:7 5-3H-MA3

Urine 13.4 14.3 14.5 7.8 8.6 8.7

Faeces 58.8 78.0 80.7 48.6 77.4 83.1 25 mg/kg E-187 3:7 5-3H-MA3

Urine 16.0 17.0 17.2 11.3 12.7 13.0

Faeces 69.7 87.9 94.5 80.3 93.8 95.6 250 mg/kg 5-3H-MA3 Urine 2.1 2.7 2.8 1.8 2.3 2.4 MA4 Males Females Dose Excreta 1 day 2 days 3 days 1 day 2 days 3 days

Faeces 73.8 89.3 90.8 68.9 91.0 93.1 2.5 mg/kg E-187 3:7 14C-MA4

Urine 7.3 7.6 7.7 4.2 4.5 4.6

Faeces 60.4 84.7 89.1 43.8 80.2 88.8 25 mg/kg E-187 3:7 14C-MA4

Urine 7.5 8.1 8.3 5.1 6.0 6.2

Faeces 73.8 84.7 85.7 50.2 86.7 90.4 25 mg/kg 14C-MA4 Urine 11.4 12.1 12.2 5.9 6.6 6.7


Summary (toxicokinetics):

Figure 1. (From the DAR) Proposed metabolic pathway for milbemycin A4:

O

O O

H

OH

CH3H

OH

O

O

CH3

CH3

H

CH2CH3

CH2CH3

H

CH3

O

O

HOCH3

O

O O

OH

CH3

OH

H

O

O O

OH

CH3H

OH

CH3

H

CH2CH3

CH3HO

O

O

OH

Other dihydroxy M.A4

Other hydroxy M.A4

Dihydroxy M.A4 - Glu

Trihydroxy M.A4

Carboxylated M.A4m/z 572

M.A4

13 Hydroxy M.A4m/z 558

13,30-Dihydroxy-M.A4m/z 574

m/z 574m/z 590

m/z 542

CH3

H

CH3

Absorption: Based on the milbemycin A4 excretion in urine and bile, the absorption seems to be 47 % of a single low dose in both sexes and 30/40% in males/females at a single high dose. Thus absorption seems to be saturated at higher doses. Peak concentration in blood/plasma was reach after 2-3 hours. Distribution: The concentration of substance was higher in tissues (except brain) than in blood/plasma at all time points. Most tissues had a residue peak at 2 hours, but reproductive fat had a peak at 6 hours after dosing. Tissue residues increased disproportionately more than the increase in dose. Repeated dosing gave the same tissue distribution as single dosing. There was no accumulation. Metabolism: Hydroxylation was the main metabolic pathway and different single-, di-, and trihydroxymetabolites were formed. The main metabolites were 13-hydroxy-MA3 and –MA4. MA3 seems to be more rapidly metabolised than MA4. There was a minor glucuronidation pathway. Elimination The main route of elimination was via bile, and a smaller amount was excreted via urine. There was a higher percentage of excretion of MA3 than MA4 in urine. Males had higher urine excretion than females, especially at low doses. There was rapid excretion the first 24 hours followed by a prolonged


low excretion, and the elimination was more rapid at low dose than at high dose (reflected in the concentrations in blood). Repeated dosing gave the same elimination pattern as single doses.

5.1.2 Acute Toxicity

Acute Oral

Study 1: Oral gavage single dose, rat

Acute oral LD50 were 762 mg/kg bw in males and 465 mg/kg bw in females of 95.5% pure E-187 (A3:22.8%; A4:72.7%) in Fischer 344 rats (10/sex/dose group). This indicates a higher sensitivity to acute toxicity for female rats. Symptoms of toxicity was irregular respiration, crouching, abasi, staggering gait or loss of righting reflex, and lowered body temperature and body weight. Death occurred from days 1-4, and there were no clinical signs in surviving animals after days 3-4.There was no pathological changes. The substance warrant a classification with R22 Harmful if swallowed (Cat. 4 H302 Harmful if swallowed) in reflection of the acute toxicity in both sexes (200 mg/kg bw LD50 < 2000 mg/kg bw). GLP/QA/OECD 401. (Kimura 1988a)

Study 2: Oral gavage single dose, mouse

Acute oral LD50 were 324 mg/kg bw in males and 313 mg/kg bw in females of 92.9 % pure E-187 (A3: 26.7; A4:66.2%) in CD-1 mice (10/sex/dose group). This indicates a slightly higher sensitivity to acute toxicity for female rats. Symptoms of toxicity were sedation, disturbance of gait in all groups and sporadic findings of wetness of lower abdomen, lacrimation and filthy fur on the whole body. Females given 232 mg/kg bw of higher dose showed decreased body weight. Death occurred day 1 in females and up to day 6 in males. Symptoms ceased after days 1-5. Pathological changes in the lung were seen in one male and one female that died. One male had blotted fur and another had small body size. There were no pathological findings in surviving animals. The substance warrant a classification with R22 Harmful if swallowed (Cat. 4 H302 Harmful if swallowed) due to acute toxicity in both sexes (200 mg/kg bw LD50 < 2000 mg/kg bw). GLP/QA/OECD 401. (Ebino, 1986).

Study 3: Oral capsule single dose, dog

Capsules with E-187, purity 92.9 % (A3: 26.7; A4: 66.2%) were given to beagle dogs (2/sex/dose). The administration induced vomiting in all groups. Symptoms of toxicity were salivation, sedation and tremor. One female had somnolence and two males were found in coma. Body weight loss was seen mainly on day 3 after dosing. Death occurred day 2 in one male from the 400 mg/kg bw group and in one male from the 600 mg/kg bw group. Pathological changes in the two males included pulmonary lobes darkly reddened entirely with oedemas, darkly reddened gastric mucosa with pseudomembranous adhesives, congestion and urinary bladder distended with urine. The dead male in the 400 mg/kg bw group had in addition pale discolouration in the heart and spleen and dark red spots in the thymus. There were no pathological findings in surviving animals. An LD50 could not be calculated due to few animals. However, the lethal dose for males lies between 200 and 400 mg/kg bw/day and the minimal lethal dose for females is >600 mg/kg bw. The substance warrants a classification with R22 Harmful if swallowed (Cat. 4 H302 Harmful if swallowed) due to acute toxicity in both sexes. GLP/QA/OECD 401 (Ebino, 1987)

Acute Dermal

Acute dermal LD50 of 95.5% pure E-187 (A3:22.8%; A4:72.7%) in Fischer 344 rats (10/sex/dose) was found to be > 5000 mg/kg bw (limit test) for males and females. GLP/QA/OECD 402 (Kimura 1988b).

Acute Inhalation

Acute toxicity after exposure (whole-body exposure system) by inhalation to 97.6 % E-187* (A3: 21%; A4: 76.6%) aerosol analytical concentration for 4 hours in SPF Fischer rats (10/sex/dose) were seen in a dose dependent manner. The exposure aerosol was characterized by a mass median aerodynamic diameter (MMAD) of 5-6 μm with a geometric standard deviation of 1.9 μm. Due to the large particle size, the calculated LC50s (males: 1.9 mg/L; females: 2.8 mg/L) are somewhat uncertain. Clinical


symptoms seen in both treated and control groups were eye closure, slow and deep respiration and red brown stain around eyes, nose and mouth. The E-187 groups had additional symptoms as abnormal posture, decreased spontaneous movements, wets around the genitalia, mouth and nose, unsteady gait, darkened aye colour, lacrimation, filthy fur around genitalia, epilation around eyes and blotted fur around anus. All exposed groups had reduced weight or reduced body weight gain day 7 after exposure. Almost all animals had body weight gain one week later. Pathological findings in the animals found dead, were filthy fur around nose, mouth and genitalia, intratracheolaryngeal white content, sebum palpebrale or lacrimation and gas-filled stomach and small intestine. Terminally killed treated animals had eyelids with hair loss. The control group had no pathological findings. Although there are some limitations due to large particle size, milbemectin requires classification with R20 Harmful by inhalation (Cat. 4 H332 Harmful if inhaled) (1 mg/L/4 hr < LC50 < 5 mg/L/4 hr for aerosols/particulate matter). GLP/ OECD 403 (Yoshida, 1989). *E-187 was mixed with white carbon at a ratio of 90:10 (w/w) and ground thereafter. Table 5.7: Mortality in acute inhalation study, rats Dose level

(mg/L) No. dead/No. treated Time of death after dosing

2.97 9/10 day 0,5 - 1 (1 day 18#)

2.10 7/10 day 1

1.49 1/10 day 1

0.98 0/10 -

Male

0 0/10 -

2.97 6/10 day 0,5 - 1

2.10 2/10 day 1 -3

1.49 2/10 day 1

0.98 2/10 day 0 - 1

Female

0 0/10 -

# prolonged observation period

Summary (acute toxicity):

Milbemectin is of moderate acute toxicity after oral and inhalation exposure and of low dermal toxicity. The proposed classification is Xn, R20/22 Harmful by inhalation and if swallowed (Cat. 4 H332 Harmful if inhaled. Cat. 4 H302 Harmful if swallowed).

5.1.3 Irritation and sensitisation

Skin irritancy

Mean values (24, 48 and 72 h) were found to be 0 for erythema and 0 for oedema. The compound E-187 (97% purity) did not show skin irritation in six female New Zealand White Rabbits. GLP /OECD 404 (Liggett and McRae, 1990a).

Eye irritation

Mean values (24, 48 and 72 h) were 0.2 for conjunctival redness, 0.1 for corneal chemosis, 0.1 for corneal opacity and 0 for iritis in six female New Zealand White Rabbits. GLP/QA/OECD 405 (Liggett and McRae, 1990b).

Skin sensitisation

Study 1: Buehler test, supplementary


Milbemectin (E-187, 97% purity) (96.5% purity) was tested for skin sensitization with the Buehler method in female Dunkin-Hartley guinea pigs. Only 10 animals were used in the test group. The dosing regimen was 3 topical inductions with 70 % test substance in acetone (the highest obtainable practical concentration) and one topical challenge (occlusive, 6 hours). There was no positive response in the study. No conclusion can be drawn because of too few animals. GLP/ not in accordance with OECD 406. (Parcell and Denton, 1990)

Study 2: Maximisation test

Milbemectin (E-187, purity MA3 27.09%, MA4 71.84%) was tested and found not sensitizing in the Maximisation test in female Dunkin-Hartley guinea pigs. The dosing regimen was 5% intradermal induction and 55 % topical induction followed by 55% and 25% topical challenge (occlusive, 48 hours). Intradermal injection and topical induction caused slight to well defined erythema in the test animals. No dermal reaction was seen after topical challenge in the test animals. The positive control, α-hexyl cinnamic aldehyde was positive. GLP/ OECD 406. (Ruddock, 2001a)

Summary (irritation and sensitisation):

Milbemectin was not found to be a skin- or eye irritant nor a skin sensitiser. Table 5.8: Summary of acute toxicity, irritancy, and sensitisation studies with milbemectin.

# Not in accordance with OECD 406

Test Species Result Classification (93/21/EEC)

Reference

Acute oral Rat LD50 762 mg/kg bw in males LD50 465 mg/kg bw in females

R22 (Cat. 4 H302)

Kimura 1988a

Acute oral Mouse

LD50 324 mg/kg bw in males LD50 313 mg/kg bw in females

R22 (Cat. 4 H302)

Ebino, 1986

Acute oral Dog

200<LD50<400 mg/kg bw in males LD50>313 mg/kg bw in females

R22 (Cat. 4 H302)

Ebino, 1987

Acute dermal Rat LD50>5000 mg/kg bw None Kimura 1988b

Acute inhalation Rat LC50 1.9 mg/L in males LC50 2.8 mg/L in females

R20 (Cat. 4 H332)

Yoshida, 1989

Skin irritation Rabbit Not irritating to the skin None Liggett and McRae, 1990a

Eye irritation Rabbit Not irritating to eyes None Liggett and McRae, 1990b

Skin sensitisation (Buehler test)#

Guinea pig

No significant skin responses

None Parcell and Denton, 1990

Skin sensitisation (Maximisation test)

Guinea pig

No significant skin responses

None Ruddock, 2001a


5.1.4 Genotoxicity

Table 5.9: Summary of the genotoxicity studies: Study Test system Concentration/ dose

range tested Result Reference/

Guidelines/ GLP

In vitro: Point mutations in bacteria

Bacterial reverse mutation assay (Ames test)

S. typhimuriumTA98, TA100, TA1535, TA1537 E.coli WP2uvrA. E-187, 93% purity

5-5000 L/plate (+/- S9)Solvent: DMSO

Negative (+/- S9)

Ohta, 1986/ OECD 471/ GLP#1

Gene mutation in mammalian cells

Mouse lymphoma cell L5178Y gene mutation (TK)

Mouse lymphoma cell L5178Y Milbemectin, 98.01 % purity (A3: 26.22%; A4: 71.79%)

0.234 – 30µg/ml (-S9) 3.125 – 75 µg/ml (+S9) solvent DMSO

Negative (+/- S9)

Fellows, 1998/ OECD 476/ GLP

Cytogenetic assay in mammalian cells

CHL, chromosome aberration

Chinese hamster lung cells E-187, 93% purity

3.3x10-6 to 1.0x10-4M (-S9, 24 and 48h) 1.0x10-5 to 1.0x10-3M (+S9, 12 and 18h) solvent DMSO

Negative (+/- S9)

Sasaki, 1986/ OECD 473/ GLP

In vivo – somatic cells: Chromosomal aberration

Micronuclei (bone marrow)

Bone marrow cells of CD-1 mice, 5/sex/dose Milbemectin, 98.01 % purity (A3: 26.22%; A4: 71.79%)

25, 50 or 100 mg/kg bw/day (males) and 37.5, 75 or 150 bw/day (females) on 2 consecutive days by gavage. Sacrifice 24 or 48 h after last dose. Vehicle 0.5% CMC

Negative

Burman, 1988/ OECD 474/ GLP#2

#1 In the same report there was also described a DNA repair test (rec-assay) in B. subtilis, strains H17 and M45. Dose range 100-5000g/disk ±S9. With metabolic activation, no inhibitory zones, neither in DNA deficient nor in repair proficient strains, were noted. Without metabolic activation, inhibitory zones were comparable in DNA repair deficient and repair proficient strains. #2 At the highest dose level males and females showed clinical signs including abnormal gait and abnormal breathing. Females also showed lethargy and prostration and two dies prior to sampling. They were replaced by spare animals. PCN/NCE ratios were similar to vehicle controls at both sampling times at all dose levels.

Summary (genotoxicity):

All in vitro and in vivo genotoxicity studies were negative. Milbemectin is not considered to be a genotoxic substance.


5.1.5 Sub-chronic toxicity

Dog, 4 weeks, diet, dose-range determination study

Study design: Beagle dogs (1/sex/dose group) received 0, 10, 30, 100, 200 or 300 mg/kg bw/day respectively) of E-187purity 93.4% (A3:22.0%; A4 71.4%) in gelatine capsules for 4 weeks (7 days/week). No OECD guideline/GLP/QA. Results: All dogs at 200 and 300 mg/kg bw/ day died after the first administered dose day 1. The symptoms were salivation, staggering gait and coma. All these dogs had diffuse vacuolization of hepatocytes. The highest dose gave swelling of the stomach with its contents in both dogs. In the male there were also seen adhesion of the lung and pleura. In the female there was noted dark red lung, pulmonary oedema, dark red thymus and catarrh or congestion of the small intestinal mucosa. The 200 mg/kg bw –dose gave swelling of the stomach (with watery content), scattered dark red oedema of the lung, dark red thymus, and catarrh or congestion of the small intestinal mucosa in the male dog. In the female dog there was found swelling and the stomach with its contents, decoloured spleen, and congestion of the intestinal mucosa. In the 100 mg/kg bw/day group both sexes had vomiting of the diet, sedation, salivation and staggering gait. The female also had coma, somnolence, tremor and nebula. There was a decrease in food and water intake and body weight in both the male and female dog. In week two and after, there was a tendency to increased body weight. The liver weight was increased (hepatomegaly) in both sexes. The male dog that got 30 mg/kg bw/day showed vomiting of the diet, sedation, salivation and staggering gait. The female dog in this group had a tendency of decreased food and water intake and the body weight in week 4 was lower than in week 3. Both sexes had higher liver weight than the control group. The lowest dose, 10 mg/kg bw/day, gave higher liver weight in the male dog than in the male dog from the control group. Statistics evaluation is of no relevance due to the low number of animals used. NOAEL: Due to the nature as a range-finding study and limited study design a NOAEL is not set. Liver effects were seen at all dose levels. The study showed high toxicity of milbemectin. All dogs died of a single dose of 200 or 300 mg/kg bw. The dose of 100 mg/kg bw gave severe toxicity in the female dog, including coma. Presumably LD50 lies between 100 and 200 mg/kg bw in dogs. Thus milbemectin may warrant classification with R25 Toxic if swallowed (Cat. 3 H301 Toxic if swallowed) in reflection of the acute toxicity (25 mg/kg bw LD50 < 200 mg/kg bw) In CLP the limits are >50 - ≤300 mg/kg bw. The target organs are the central nervous system, GI-tract and liver. (Shirasu, 1987)

Rat, 13 weeks, diet,

Study design: F344 rats (10/sex/group) were administrated E-187, purity 92.9%, via dietary inclusion at levels of 0, 375, 750, 1500 or 3000 ppm in 90 days. The dose levels are equal to 0, 25, 49, 101, and 213 mg/kg bw/days for males and to 0, 28, 56, 116, and 231 mg/kg bw/day for females. OECD guideline 408 (1981) /GLP.

Results: There was no mortality in the study. Animals from the two highest dose levels had stained eyelids. The highest dose resulted also in clinical signs as hypersensitivity, calm by debility, staggering gait, piloerection, decreased movement and marked front teeth growth. The abnormal teeth growth was noted from week 3 and made it necessary to periodically cut the teeth. Body weight and food consumption was significantly decreased at the highest dose level. Urinalysis and ophthalmoscopy did not reveal any anomalies. Haematological parameters in females were affected at all dose levels, and in males at the three highest dose levels (see table). The decrease in haemoglobin, hematokrit, Mean


Corpuscular Haemoglobin concentration and Mean Corpuscular Volume were dose related in both sexes. The increase in red blood cell count and the reduction in MCHC were dose related in females. Regarding the increased white blood cell count, the increase in neutrophils was dose related in males at the two highest dose levels. Table 5.10: Haematological findings in rat, diet, 13 weeks Dose, ppm 0 375 750 1500 3000 Parameter male female male female male female male female male female Red blood cells, x106/µl

8.93 ± 0.04

8.22 ± 0.03

9.09 ± 0.06

8.25 ± 0.07

9.12 ± 0.06

8.64 ± 0.05*

9.56 ± 0.07*

9.27 ± 0.07*

9.27 ± 0.33

9.38 ±0.19*

Haemoglobin, g/dl

16.13 ± 0.04

16.32 ± 0.08

16.21 ± 0.08

16.07 ± 0.11

15.95 ± 0.08

15.67 ± 0.04*

15.71 ± 0.01*

15.21 ± 0.10*

14.60 ± 0.44*

14.47 ± 0.22*

Hematokrit, % 46.5 ± 0.1

46.8 ±0.2

46.7 ± 0.3

46.3 ± 0.3

46.3 ± 0.2

45.8 ± 0.2*

45.5 ± 0.3*

44.6 ± 0.2*

43.2 ± 0.9*

42.7 ± 0.4*

Reticulocytes, %

1.1 ± 0.1

1.1 ± 0.1

1.2 ± 0.1

1.3 ± 0.1

1.2 ± 0.1

1.5 ± 0.1

1.2 ± 0.1

1.3 ± 0.1

3.5 ± 0.8

2.7 ± 0.3*

MCV, µ3 52.1 ± 0.2

56.8 ± 0.1

51.4 ± 0.2

56.2 ± 0.1*

50.7 ± 0.2*

52.9 ± 0.2*

47.5 ± 0.2*

48.1 ± 0.2*

46.8 ± 1.0*

45.8 ± 0.6*

MCH, pg 18.1 ± 0.04

19.9 ± 0.1

17.8 ± 0.1

19.5 ± 0.1*

17.5 ± 0.04*

18.1 ± 0.1*

16.4 ± 0.1*

16.4 ± 0.1*

15.8 ±0.1*

15.5 ± 0.1*

MCHC, % 34.7 ± 0.1

34.9 ± 0.1

34.7 ± 0.1

34.7 ± 0.1

34.5 ± 0.1

34.2 ± 0.1*

34.6 ± 0.1

34.1 ± 0.1*

33.7 ± 0.4

33.9 ± 0.2*

Platelets, x104/µl

46.04 ± 1.21

57.11 ± 1.34

50.65 ± 1.96

56.08 ± 1.13

47.93 ± 1.80

62.38 ± 1.75

52.84 ± 1.10*

62.33 ± 1.52

66.54 ± 3.77*

64.76 ± 2.53

Fibrinogen, mg/dl

281.2 ± 5.5

225.7 ± 11.0

290.4 ± 6.4

203.4 ± 2.6

328.5 ± 10.7*

210.1 ± 7.1

322.0 ± 9.1*

216.4 ± 12.5

223.6 ± 6.6*

194.3 ± 5.3

White blood cells, x103/µl

3.52 ± 0.18

3.49 ± 0.27

3.56 ± 0.17

3.15 ± 0.18

3.69 ± 0.14

3.16 ± 0.16

4.40 ± 0.13*

4.83 ± 0.24*

5.24 ± 0.59

4.18 ± 0.22

* Significant different from control p<0.01. Clinical chemistry revealed statistically significantly elevated total cholesterol levels in both sexes at 750 and 1500 ppm, and in females at 3000 ppm. The other changes in clinical chemistry parameters were only seen at the highest dose level: significant increase in ALP, potassium and inorganic phosphate in both sexes; decrease in ALAT/ASAT, bilirubin and protein in males; decrease in A/G-ratio in females; and decrease in calcium in both sexes. There were seen increased weights of liver, kidney and adrenals of both sexes and of the spleen in males at the two highest dose levels. Males at highest dose level had also decrease in absolute organ weight of the thymus and decrease absolute weight with increased relative weight of the thyroid, testes, lung, brain and heart. Females at the highest dose level had also decreased absolute and relative weight of the uterus, decreased absolute weight of lung and brain and increased relative weight of the heart. Histopathological investigations showed hypertrophy of the liver of both sexes at the two highest dose levels. In the spleen there was seen acceleration of haematopoiesis at the highest dose level, most marked in males. There was seen atrophy of the thymus in animals of both sexes from the highest dose group. In the adrenals, there was hypertrophy in zona fasciculata cells in both sexes, but more pronounced in females. Acceleration of haematopoiesis in bone marrow and spleen was seen at the two highest dose levels in both males and females. NOAEL: 375 ppm (corresponding to 25/28 mg/kg bw/day in males /females respectively) based on changes in blood parameters and elevated cholesterol at 750 ppm,. Most sensitive parameters were cholesterol and blood parameters. Several organ systems were affected in the study. Clinical signs at the highest dose level probably results from effects on the central nervous system. Both males and females had signs of haemolytic anaemia reflected in effects on blood parameters and findings in the blood forming organs. The kidney, liver (hypertrophy) and adrenal weights were elevated in both sexes at the two highest dose levels. The adrenals production of glucocorticoids was affected as the adrenals were enlarged because of hypertrophy in the zona fasciculata. Males at the highest dose level had also lower weight of the thymus, although atrophy was seen in both males and females. Males had elevated levels of white blood cells, especially neutrophils,


at the two highest dose levels. Females at 1500 had increased level of white blood cells. (Masuda, 1986).

Mouse, diet, 13-week

Study design: ICR (Crj:CD-1) mice (12/sex/group) were administrated E-187, purity 93.1%, via dietary inclusion at levels of 0, 500, 1000, 2000 or 4000 ppm in 90 days. The dose levels are equal to 0, 57, 113, 226, and 439 mg/kg bw/days for males and to 0, 68, 138, 286, and 499 mg/kg bw/day for females. OECD guideline 408 (1981) except that no ophthalmological examination was performed and only a limited number of clinical biochemical parameters in blood (AP, ALAT,ASAT, total protein and cholesterol, glucose, urea nitrogen, calcium) was determined /GLP. Results: Transient decrease in food consumption and body weight were found in males the first week in the study at 2000 ppm. Both sexes had decrease in body weight and food consumption at 4000 ppm, although for males the decrease in food consumption was significant only for week 1-4. Females at the highest dose level had elongated incisors. Effects on blood parameters were mainly seen in females. At 2000 ppm they had decrease in haemoglobin (dose related), MCH and MCHV and at 4000 ppm decrease in haemoglobin, hematokrit, MCH and MCV. Males at 4000 ppm had decreased level of MCH. Clinical chemistry revealed decreased levels of protein and calcium in the blood of females at the highest dose level. Increased relative liver weight was seen in males at the two highest dose levels. The absolute weight of the liver was significantly decreased in females at 4000 ppm, although the relative liver weight was increased (not significantly). The relative kidney weight was significantly increased in both sexes at 4000 ppm and in females at 2000 ppm. For males at 2000 ppm, the absolute kidney weight was increased, but the increase in the relative kidney weight was not significant at this dose level. There were no histopathological changes. NOAEL: 1000 ppm (corresponding to 113/138 mg/kg bw/day in males and females respectively) based on statistically significant increase in kidney weight in both sexes at 2000 ppm. Males had also increased relative liver weight and females had significant reduced blood parameters at 2000 ppm. The blood, kidney and liver were target organs in this study. (Maita, 1987).

Dog, oral, 13 weeks

Study design: Beagle dogs (4/sex/group) were fed capsules with E-187, purity 93.7 % (A3: 21.3; A4: 72.4%) immediately after feeding, to give doses of 0, 3, 10, and 30 mg/kg bw/day for 90 days. OECD guideline 409 (1981)/GLP.) Results: There were no deaths in the study. Clinical signs at 10 mg/kg bw/day were salivation in males and increased incidence of vomiting of food in one male and one female. At the highest dose level there were seen sedation, staggering gait, tremor of the head, salivation, eye discharge and increased incidence of vomiting of food in both sexes. The symptoms were more pronounced in males which also showed signs of emaciation. There was seen decrease in body weight gain and food consumption in males and females of the high dose group, but this was significant only for the body weight gain in males that also showed body weight loss the first 4 weeks of treatment. No treatment-related findings were seen in the urinalysis, ophthalmoscopy, haematology or clinical chemistry investigations. The males at the highest dose level had significant decrease in weight of the brain and adrenals. There were no histopathological findings. NOAEL: 3 mg/kg bw/day in males and females based on clinical symptoms.


The most sensitive target organs in this study were the central nervous system followed by the adrenals (males). (Ebino, 1988.

Dog, oral, 52 weeks

Study design: Beagle dogs (6/sex/group) were fed capsules with E-187, purity of 95.6 % (A3: 21.8; A4: 73.8%) immediately after feeding, to give doses of 0, 3, 10, and 30 mg/kg bw/day for 12 months. OECD guideline 452 except that the haematological parameters were not assessed after 3 months /GLP. Results: There were no deaths in the study. One male had increased incidence of vomiting at 10 mg/kg bw/day. Clinical signs at the highest dose level were sedation, staggering gait and increased incidence of vomiting of food in both sexes. Salivation was seen in females at the highest dose level. Females had a non-significant decrease in body weight at the two highest dose levels, but food consumption was significantly decreased at the highest dose level. No treatment-related findings were seen in the urinalysis, ophthalmoscopy, or haematological investigations. Clinical chemistry revealed a significant increase in total cholesterol and calcium in males at the highest dose level. Males had a dose-related increase in relative and absolute weight of the liver at the two highest dose levels. Females at the highest dose level had increased relative brain weight. There were no histopathological findings. NOAEL: 3 mg/kg bw/day in males based on increased liver weight (25 %) and higher incidence of vomiting of food at 10 mg/kg bw/day, and 3 mg/kg bw/day in females based on decreased body weight from week 13 onwards. The target organs in this study were the central nervous system and the liver (males) followed by effects on cholesterol. There were also effects on body weight in females. (Ebino, 1989)

Rabbit, dermal, 4 weeks

Study design: New Zealand White rabbits (5/sex/group) were dermally exposed at a semi-occluded skin test site to 0, 100, 500 and 1000 mg milbemectin technical /kg bw/day, 6 h/day, 5 days a week for 4 consecutive weeks. Purity was 98.24 % (MA3: 30.35%; MA4: 67.89%). Satellite groups of 5/sex/group were added for the control and highest dose level groups and observed for a 2-week treatment-free period after the study. Terminal body weights were not measured. OECD guideline 410/GLP. Results: There were no deaths, clinical signs or effects on body weight and food consumption in the study. In females serum glucose levels were significantly decreased in a dose-dependent manner. Since most individual values were within normal limits, this was not regarded to be of toxicological relevance. White blood cell count were significantly decrease in females at the two highest dose levels (although not dose-related), and in males at 500 mg/kg bw/day (not significant). This finding was also evaluated as of no toxicological concern. Males in the highest dose level had significantly increased absolute adrenal weights that were still present 2 weeks after treatment. There were no pathological changes. As increased adrenal weight is seen in other studies with milbemectin, this is regarded as a substance-related effect. NOAEL (systemic): 500 mg/kg bw/day based on significantly increased absolute adrenal weight in males at 1000 mg/kg bw/day that were still present 2 weeks after treatment.(Kuhn, 2000).

Summary (sub-chronic toxicity):

The dog was the most sensitive species with a LOAEL of 10 mg/kg bw/day and the lowest NOAEL of 3 mg/kg bw/day. In the short term studies effects on liver, kidney, central nervous system and body weight were seen in the rat, mouse and dog. Effects on the adrenals were seen in the rat, dog and rabbit (dermal study). Rats had also effects on the uterus, testes and immune system. In rats the most


sensitive parameter was elevated cholesterol. Elevated cholesterol was also seen in dogs. The central nervous system seems relatively more vulnerable in the dog than in the rodents were effects in other organs were seen at lower dose levels than effects on the CNS. Table 5.11: Summary of short-term toxicity studies with milbemectin (E-187)

Study NOAEL

(mg/kg bw/d) Effects Reference

Oral studies

Dog, 4 weeks, diet, dose-range determination study

No NOAEL were proposed due to the limited study design (range-finding study,

lack of histopathological

examinations)

The study showed high toxicity of milbemectin. All dogs died of a single dose of 200 or 300 mg/kg bw. The dose of 100 mg/kg bw gave severe toxicity in the female dog, including coma. 10 mg/kg bw/day, gave higher liver weight in the male dog. The target organs are the central nervous system, GI-tract and liver.

Shirasu, 1987

M: 25

(375 ppm) Rat, 13

weeks, diet F: 28

(375 ppm)

Elevated cholesterol and effects on blood parameters were seen in both sexes at 750 ppm. Most sensitive parameters were cholesterol and blood parameters. Higher doses gave effects also on the liver, kidney, adrenals, spleen, immune system, central nervous system, uterus and testes.

Masuda, 1986

M: 113

(1000 ppm)

13-week mouse, diet

F: 138

(1000 ppm)

Males had increased weight of the liver and kidney and females had increased weight of the liver and effects on blood parameters at 2000 ppm. The liver, kidney and blood were the most sensitive target organs followed by the central nervous system.

Maita, 1987

13-week dog,

capsules

3

Clinical symptoms caused by effects on the central nervous system were seen in both sexes at 10 mg/kg bw/day. Higher dose levels also affected the body weight and the adrenal weight (males). The target organs were the central nervous system and the adrenals (males)

Ebino 1988

52-week dog, diet

3

NOAEL in males based on increased liver weight (25 %) and higher incidence of vomiting of food, and in females based on decreased body weight from week 13 onwards at 10 mg/kg bw/day. The target organs in this study were the central nervous system and the liver (males) followed by effects on cholesterol. There was also effect on body weight in females.

Ebino, 1989

Dermal study

4-week rabbit, dermal

500

Males in the highest dose level (1000 mg/kg bw) had significantly increased absolute adrenal weights that were still present 2 weeks after treatment. There were no pathological changes.

Kuhn, 2000


5.1.6 Chronic toxicity and carcinogenicity

Rat, diet, 104 weeks, combined chronic toxicity and carcinogenicity study

Study design: F344 rats (50/sex/group) were administrated E-187, several lots, purity 93.5-95.4 % via dietary inclusion at levels of 0, 15, 150, or 750-1500 ppm for 104 weeks. The dose of 1500 ppm was reduced to 750 after 7 weeks of administration due to abnormal growth of incisors that strongly interfered with food intake. Dose levels were equal to a mean intake of 0, 0.7, 6.8, and 33 mg/kg bw/day for males and 0, 0.9, 8.8, and 44 mg/kg bw/day for females. The high dose of 1500 ppm during week 1-7 gave a mean intake of 121 mg/kg bw/day for males and 132 mg/kg bw/day for females of E-187. There were no examinations after 3 or 6 months. Urinalysis was performed in week 51, 77 and 103. The interim sacrifices of the satellite groups of 15 animals/sex/dose were done after 52 and 78 weeks. Ophthalmological examinations was performed prior to start and in week 103 in control and high dose group animals. Results: The abnormal growth of incisors (mainly in females) in the high dose group at 1500 ppm disappeared after the dose was reduced to 750 ppm after 7 weeks. Most animals in the high dose group had stained eyelids. Food consumption was significantly increased at the two highest dose levels, except in the females at 150 ppm where it was significantly decreased. The body weight gain was decreased in females in the high dose group before the dose was reduced, and in some weeks at 750 ppm, especially at the end of the study. Thin hair was seen in animals from all groups (included control), but in the high dose group it was present in most or all animals. Enlarged hair follicles were detected microscopically in the high dose group. Measurement of haematological parameters revealed significantly increased red blood cell count and platelet concentration in females and decreased MCH and MCV in both sexes in the high dose group. Serum cholesterol was significantly elevated in both sexes in the high dose group. ALAT and ASAT were significantly decreased in females at the highest dose level. ASAT was also decreased in males, but not significantly. The high dose resulted in increased weight of the liver, kidney, adrenal glands (females) and the uterus. In the kidney there was increased incidence of rough surface and moderate chronic nephrosis in males, and decreased mineral deposition in females. There was seen increased incidence of nodules in the uterus and microscopy revealed also increased incidence of endometrial polyps, endometrial proliferation and adenocarcinoma.


Table 5.12: Effects on the uterus Dose, ppm 0 15 150 750/1500 Nodules in the uterus, incidence Week 52 0/15 2/15 0/15 0/15 Week 78 1/15 2/15 1/15 4/14 Week 104 6/36 6/40 12/37 11/34 Found dead or moribund

5/14 0/10 3/13 8/17

Total 12/80 10/80 16/80 23/80 Endometrial proliferation Week 104 0/36 0/40 0/37 2/34 Total 0/80 0/80 0/80 2/80 Endometrial polyps Week 52 0/15 2/15 0/15 0/15 Week 78 0/15 2/15 1/15 4/14 Week 104 5/36 5/36 10/37 8/34 Found dead or moribund

1/14 0/10 0/13 4/17

Total 6/80 9/80 11/80 16/80 Adenocarcinoma Week 52 0/15 0/15 0/15 0/15 Week 78 0/15 0/15 0/15 0/14 Week 104 0/36 1/40 2/37 3/34 Found dead or moribund

1/14 0/10 0/13 1/17

Total 1/80 1/80 2/80 4/80 The 150 ppm dose gave significantly reduced food consumption in females, but increased food intake in males. This had no effect on body weight and thus is regarded as of no toxicological relevance. Effects on organ weight were increased weight of the kidneys in males and increased weight of the adrenals in females (after 78 weeks only). There was also seen increased incidence of nodules and endometrial polyps in the uterus.


Table 5.13: selected organ weights Dose, 0 ppm 15 ppm 150 ppm 750/1500 ppm

Males Parameter Absolute

weight relative weight

Absolute weight

relative weight

Absolute weight

relative weight

Absolute weight

relative weight

Liver Week 52 12.15 ±

0.173 2.93 ± 0.023

12.33 ± 0.200

2.97 ± 0.042

12.51 ± 0.019

3.04 ± 0.023*

12.26 ± 0.263

3.01 ± 0.032

Week 78 12.61 ± 0.267

3.03 ± 0.041

12.72 ± 0.307

3.03 ± 0.073

12.22 ± 0.238

3.00 ± 0.036

13.80 ± 0.568

3.33 ± 0.175

Week 104 13.61 ± 0.402

3.55 ± 0.101

13.88 ± 0.309

3.53 ± 0.101

14.09 ± 0.289

3.72 ± 0.103

15.28 ± 0.328*

4.11 ± 0.089*

Kidney, left Week 52 1.37 ±

0.027 0.33 ± 0.004

1.38 ± 0.024

0.33 ± 0.005

1.44 ± 0.018

0.35 ± 0.004*

1.44 ± 0.021

0.35 ± 0.003*

Week 78 1.47 ± 0.023

0.35 ± 0.005

1.45 ± 0.018

0.35 ± 0.005

1.45 ± 0.025

0.36 ± 0.005

1.57 ± 0.050

0.38 ± 0.015

Week 104 1.57 ± 0.017

0.41 ± 0.009

1.59 ± 0.017

0.40 ± 0.007

1.65 ± 0.025*

0.44 ± 0.014

1.75 ± 0.022*

0.48 ± 0.011*

Adrenal, left Week 52 21.8 ±

0.51 5.25 ± 0.099

23.7 ± 0.64

5.71 ± 0.147

23.8 ± 0.47*

5.82 ± 0.129*

21.0 ± 0.85

5.16 ± 0.197

Week 78 24.5 ± 0.68

5.89 ± 0.141

24.3 ± 0.63

5.79 ± 0.152

25.4 ± 0.56

6.26 ± 0.175

26.8 ± 2.25

6.52 ± 0.648

Week 104 33 ± 0.68 8 ± 0.23 34 ± 1.49 9 ± 0.44 33 ± 0.92 9 ± 0.35 34 ± 0.69 9 ± 0.33

Females Liver Week 52 7.37 ±

0.173 3.28 ± 0.045

7.43 ± 0.161

3.31 ± 0.055

7.16 ± 0.141

3.27 ± 0.056

7.73 ± 0.136

3.51 ± 0.047*

Week 78 8.21 ± 0.176

3.15 ± 0.050

7.96 ± 0.234

3.19 ± 0.051

7.94 ± 0.138

3.21 ± 0.068

8.76 ± 0.117

3.41 ± 0.056*

Week 104 9.08 ± 0.278

3.48 ± 0.117

8.87 ± 0.253

3.57 ± 0.079

9.17 ± 0.194

3.58 ± 0.091

9.65 ± 0.256

3.91 ± 0.120

Kidney, left Week 52 0.89 ±

0.015 0.39 ± 0.006

0.90 ± 0.016

0.40 ± 0.005

0.87 ± 0.013

0.40 ± 0.006

0.90 ± 0.009

0.41 ± 0.006

Week 78 0.96 ± 0.014

0.37 ± 0.007

0.99 ± 0.027

0.40 ± 0.009

0.97 ± 0.014

0.40 ± 0.011

1.02 ± 0.018

0.40 ± 0.011

Week 104 1.08 ± 0.015

0.41 ± 0.009

1.06 ± 0.017

0.43 ± 0.009

1.08 ± 0.012

0.42 ± 0.008

1.19 ± 0.023*

0.48 ± 0.012*

Adrenal, left Week 52 32.8 ±

0.83 14.62 ± 0.348

33.2 ± 0.98

14.80 ± 0.375

32.2 ± 0.55

14.74 ± 0.268

33.3 ± 0.54

15.18 ± 0.395

Week 78 30.8 ± 0.81

11.83 ± 0.328

49.0 ± 18.20

18.93 ± 6.333

33.0 ± 0.78

13.33 ± 0.373*

35.3 ± 1.08*

13.78 ± 0.557*

Week 104 34 ± 1.03 13 ± 0.60 34 ± 0.91 14 ± 0.47 34 ± 0.67 13 ± 0.44 40 ± 0.83*

16 ± 0.63*

Uterus Week 52 1.00 ±

0.069 0.45 ± 0.031

0.99 ± 0.061

0.44 ± 0.027

0.97 ± 0.059

0.45 ± 0.029

1.07 ± 0.084

0.49 ± 0.038

Week 78 0.95 ± 0.098

0.37 ± 0.042

1.05 ± 0.097

0.42 ± 0.039

0.99 ± 0.069

0.41 ± 0.035

0.97 ± 0.080

0.38 ± 0.034

Week 104 0.86 ± 0.040

0.33 ± 0.016

1.06 ± 0.085

0.44 ± 0.040

1.07 ± 0.075

0.43 ± 0.038

1.16 ± 0.070*

0.48 ± 0.037*

* Significantly different from control p< 0.01 t-test NOAEL: 15 ppm corresponding to 0.7 and /0.9 mg/kg bw/day in males and females respectively can be set for systemic effects based on increased kidney weight in males and effects on adrenals and uterus in the female. The target organs were the liver, kidney, adrenals, uterus and blood.


There was an increase in endometrial polyps and adenocarcinomas in the uterus. There is no historical control data. It is referred to published articles in the monograph and historical control data was asked for from the ECCO-meeting. We have not received any historical control data. (Matsunuma, 1989)

Mouse, 96-weeks oncogenicity dietary study

Study design: ICR (Crj: CD-1) mice (60/sex/dose level) were fed diets containing E-187, purity 94% (A3:20.9%, A4 73.1%) at levels of 0, 20, 200 or 2000ppm for 96 weeks. Dose levels were equal to a mean intake of 0, 1.95, 18.9 and 193 mg/kg bw/day in males and 0, 1.97, 19.6 and 231 mg/kg bw/day in females. 10 animals/sex/group were sacrificed in week 52. Blood smears was analysed for this group, but not from animals after 18 months. At the termination of the study the weight of brain, kidneys, thymus, adrenals, and liver were weight in 10/animals/sex/dose only. Testes from 10 males were weight. Ophthalmoscopy, urinalysis and clinical chemistry were not performed. OECD guideline 451/GLP/QA. Results: The highest dose gave elongated incisors in both sexes, more marked in the females, in the mail study. Body weight gain was decreased in males and females but food consumption was significantly reduced only in females in the high dose group. Emaciation and small body size were significantly increased in the females. Emaciation was also noted in some males at the high dose level. The weights of the liver, kidney, and adrenals were significantly reduced in females at the high dose level. There was not found any treatment related neoplastic lesions or other histopathological changes in the study. NOAEL(18 months): 200 ppm, corresponding to 19.6 in males and 18.9 in females based on elongated incisors, reduced body weight gain and reduced food consumption (females only). E-187 (milbemectin) is not found oncogenic. The central nervous system, liver, kidney and adrenals were the target organs in this study. (Maita, 1989).

Summary (chronic toxicity and cancer):

Long-term toxicity and carcinogenicity study in the rat gave systemic effects as increased kidney weight in males and effects on adrenals and uterus in the female as the most sensitive parameters. At the highest dose level there was also effect on body weight and blood parameters. There was an increase in endometrial polyps and adenocarcinomas in the uterus. In the mouse elongated incisors, reduced body weight gain and reduced food consumption (females only), were seen at 2000 ppm in a 1.5-year oncogenicity study. The central nervous system, liver, kidney and adrenals were the target organs. There were no neoplastic changes.


Table 5.14: Summary of chronic toxicity and carcinogenicity studies with milbemectin: NOAEL

Study (mg/kg bw/d)

Effects Reference

M: 0.7 (15 ppm)

2- year chronic toxicity/oncogenicity rat, diet

F: 0.9 (15 ppm)

NOAEL is based on increased kidney weight in males and effects on adrenals and uterus in the female. The higher dose gave also effects on the blood and body weight. The target organs were the liver, kidney, adrenals, uterus and blood. There was an increase in endometrial polyps and adenocarcinomas in the uterus. There is no historical control data.

Matsunuma, 1989

M: <19.6 (200 ppm)

1.5- year oncogenicity mouse, diet

F: <18.9 (200 ppm)

Elongated incisors, reduced body weight gain and reduced food consumption (females only) were seen at 2000 ppm. The central nervous system, liver, kidney and adrenals were the target organs. No neoplastic changes were found.

Maita, 1989

5.1.7 Reproductive toxicity

Rat, two-generation study

Study design: Rats (Crj:CD) (24/sex/dose level) received concentrations of 0, 50, 200, or 800 ppm E-187, purity 93.2%, in the diet throughout the entire experimental period up to weaning of F2 litters (pre-mating, mating, gestation and rearing) to evaluate the potential for reproductive toxicity and effects on neonatal growth and survival. The doses were based on a preliminary study with a 3-week rearing and a subsequent breeding period. There was found toxic effects at 1000 ppm and higher doses in both parents and offspring, but no effects of 500 or 250 ppm. 416*/GLP/QA. * The food consumption of females during mating and on day 4 after parturition was not determined. Males were only weighted every second week during premating. Table 5.15: Mean daily dose of E-187 administrated per group in rat, 2 generation study:

0 50 200 800 ppm Males 0 3.75

(2.4-4.9) 12.4

(9.4-20.5) 53.3

(39.2-78.8) mg/kg bw/day (F0)

(mean min.- mean max.)) 0 4.17

(2.7-6.2) 17.4

(11.7-26.4) 65.6

(45.1-95.0) mg/kg bw/day (F1)

(mean min.- mean max.) Females 0 3.07

(2.9-5.2) 14.8 (11.6-

19.7) 60.5

(46.9-81.1) mg/kg bw/day (F0)

(mean min.- mean max.) 0 4.68

(3.5-6.8) 18.8

(14.0-27.4) 75.7

(59.3-104.3) mg/kg bw/day (F1)

(mean min.- mean max.) Results: There were no treatment related deaths of the parents during the study. Parental females in the high dose group had decrease body weight (F0) or body weight gain (F1) in the two first weeks of the study. The high dose gave reduced food consumption in the first two weeks in males (F1) and females (F0). F1 females had reduces food consumption during the lactation period. There was a dose related increase of blotted fur in the dorso-lumbar region in parental females (F0 and F1) at the two highest dose levels during the study. There was not found any histopathological changes in the skin.


The litter size at birth and the live birth index were significantly reduced for the F2-pups at the highest dose level. The F2-pups had reduced 14-day and 21-day survival indices, but not significantly. Data for F2-pups are presented in the table 5.16. Table 5.16: Litter size and survival indices (F2-pups) Dose (ppm) 0 50 200 800 Litter size1, pups delivered 13.6 13.0 13.6 11.3* Live birth index (%) 98.4 98.3 98.6 75.1** 4-day survival index (%) 96.9 93.6 94.7 81.8 21-day survival index (%) 98.6 100 97.2 92.3 1)including dead pups *) statistically significant difference (p<0.05) when compared to the control **) statistically significant difference (p<0.01) when compared to the control The highest dose level gave reduced body weight postnatal day 21 in the F1-generation pups (statistical significant for females only). In the F2-pups, significantly reduced body weight were seen in males (postnatal days 14 and 21) and in females (postnatal days 0, 14 and 21) in the 800 ppm group. The F2 generation was more affected than the F1 generation. Table 5.17: Body weights (F1 and F2-pups) Dose (ppm) 0 50 200 800 F1 -pups males females males females males females males females Lactation day 0 6.6 6.1 6.5 6.1 6.7 6.3 6.5 6.1 Lactation day 14 33.1 32.5 33.3 32.2 35.1** 33.6 34.4 33.4 Lactation day 21 54.5 52.9 54.8 52.7 57.9** 54.5 52.4 50.0** F2 -pups males females males females males females males females Lactation day 0 6.6 6.3 6.5 6.1 6.5 6.1 6.2 5.8* Lactation day 14 33.7 32.5 34.4 33.0 34.3 32.2 30.2* 28.1*

Lactation day 21 56.1 53.6 57.2 54.3 56.9 53.4 48.8** 45.4** *) statistically significant difference (p<0.05) when compared to the control **) statistically significant difference (p<0.01) when compared to the control NOAEL (parental toxicity): 200 ppm (12.4 mg/kg bw/day for males and 14.8 mg/kg bw/day for females) based upon decreased body weights, body weight gain and food consumption in parental animals in the high dose group. NOAEL (offspring): 200 ppm (12.4 mg/kg bw/day for males and 14.8 mg/kg bw/day for females) based upon reduced litter size, live birth index, and body weights at birth and at lactation days 14 and 21 in the high dose group. NOAEL (reproduction): 200 ppm (12.4 mg/kg bw/day for males and 14.8 mg/kg bw/day for females) based upon reduced litter size and live birth index in the F2 high dose group. (Kaneda, 1988a).


5.1.8 Teratology

Study 1: Rat

Study design: Pregnant rats (Crj:CD) (24/dose level) were administrated E-187, purity 93.2%, by gavage at dose levels of 0, 6, 20, or 60 mg/kg bw/day on days 6-15 of gestation. The vehicle was 1% aq. CMC. The dose levels were based on two preliminary studies. The first had dose levels of 0, 20, 40, 80 and 160. There were seen lowered food consumption and body weights from 80 mg/kg bw/day and up. At 160 mg/kg bw/day 8 out of 10 rats died during the dosing period. No effects on the foetuses were seen up to 80 mg/kg bw/day. The second preliminary study used dose levels of 0, 20, 40, 60, 80 and 120 mg/kg bw/day. Effects on body weight gain and food consumption were seen at dose level 60 mg/kg bw/day and higher, and mortality was observed at 120 mg/kg bw/day. OECD guideline 414/GLP. Results: There were no treatment related mortality, abortions or clinical signs in the study. The maternal food consumption was decreased during the dosing period in the high dose group, reaching significance days 6-9 and 9-12 of gestation. The body weight gain was also lowered at the highest dose level, being significant days 8-12 of gestation. No teratogenic effects were observed. NOAEL(maternal): 20 mg/kg bw/day based on decreased mean maternal body weight gain and significant decreased food consumption in the high dose group. NOAEL(developmental): >60 mg/kg bw/day based on no teratogenic effects in the study. (Kaneda, 1988b).

Study 2: Rabbit

Study design: Japanese White female rabbits (14-19 pregnant rabbits/dose level) were administrated E-187, purity 95.6% (A3:21.8%; A4: 73.8%) in aqueous methyl cellulose (0.5%) by oral gavage at dose levels of 0, 160, 400 or 1000 mg/kg bw/day on GD 6-18. OECD guideline 414*/GLP/QA. *The animals used in the study arrived at three different dates and were used in the study after 1 week of acclimatisation. The number of animals per set that were mated and the number of (pregnant) animals/dose per set and starting date were not reported. Results: There was no mortality in the study, but clinical sign (bradypragia and piloerection) were seen at all treatment doses. The two lowest doses each presented one abortion, but 4 abortions took place in the high dose group. All these dams had clinical signs. The abortion in the control group was regarded as spontaneous. There was no decrease in group mean of body weight or food consumption. Individuals (2, 3 and 5 dams treated with 160, 400 and 1000 mg/kg bw/day respectively) showed rapidly decrease in food intake, resulting in no food uptake, followed by decrease in body weight. The foetuses of these dams had lowered foetal weight on contrast to foetuses from the other dams. There was no reduction in the mean foetal weights. There were no teratogenic effects. NOAEL (maternal): <160 mg/kg bw/day based on clinical signs, reduced food consumption, reduced maternal body weights and abortions at all dose levels. NOAEL (developmental): <160 mg/kg bw/day due to treatment related reduced foetal weights and abortions at all dose levels. (Tanase, 1988).

Study 3: Rabbit

Study design: Japanese White female rabbits (15-20 pregnant rabbits/dose level) were administrated E-187, purity 95.7% (A3:22%; A4: 73.7%) in aqueous methyl cellulose (0.5%) by oral gavage at dose levels of 0, 5, 50 or 500 mg/kg bw/day on GD 6-18. OECD guideline 414/GLP/QA. Results: At the highest dose level decreased body weight and decreased food consumption was seen in 6 of 20 rabbits. One of these dams died, and two had abortions (including one still birth on GD 25).


At sacrifice one of affected rabbits had only dead foetuses while the other dams had foetuses with decreased body weight. There were no teratogenic effects. NOAEL (maternal): 50 mg/kg bw/day based on decreased maternal body weights and food consumption, death of 1 animal, two abortions, and clinical signs at the highest dose level. NOAEL (developmental): 50 mg/kg bw/day due to abortions, dead foetuses and reduced foetal weights in affected dams at the highest dose level. (Tanase, 1989).

Summary (reproductive toxicity and teratology):

Two-generation study in rats effects on parental body weights and food consumption in parental animals in the high dose group. The high dose level gave reduced litter size and live birth index in the F2 generation. Body weight and body weight gain in the lactation period was affected in both F1 and F2 pups. The F2 generation was more affected than the F1 generation. There were not seen structural abnormalities in the offspring. In the rat, the offspring is more sensitive for milbemectin than the mother. This effects is however, not regarded as relevant for humans. (See special studies and classification and labelling) In the rat teratogenicity study, the maternal toxicity was manifested by a decrease in mean maternal body weight and food consumption. There were no effects on the foetuses. In the rabbit teratogenicity studies, there were seen clinical signs (bradypragia and piloerection), reductions in food intake and body weight, death, abortions, dead foetuses and reduces foetal weight. There were no teratogenic effects. Table 5.18: Summary of reproductive toxicity studies Type of study Dose levels NOAEL

mg/kg b.w./day Findings Reference

Dietary 2-generation

study

Crj:CD Rats

0, 50, 200, and 500

ppm

parental 12.4

(200 ppm)

offspring 12.4

(200 ppm)

reproduction 12.4

(200 ppm)

Decreased body weights, weight gain and food consumption in parental animals. Reduced litter size, live birth index, and body weights at birth and at lactation days 14 and 21. Reduced litter size and live birth index.

Kaneda, 1988a

Teratogenicity study (oral by

(gavage)

Crj:CD rats

0, 6, 20 and 60 mg/kg b.w./day

maternal 20 mg/kg bw/day

foetal

>60 mg/kg bw/day

Maternal toxicity: Decrease in mean maternal body weight and food consumption. Embryo-/foetotoxicity: No effects.

Kaneda, 1988b


(gavage)

Japanese White Rabbits

0, 160, 400 and 1000

mg/kg b.w./day

maternal <160 mg/kg

b.w./day

foetal <160 mg/kg

b.w./day

Maternal toxicity: Decreased body weight and food consumption clinical signs, and abortions. Embryo-/foetotoxicity: Decreased foetal weights. No teratogenicity.

Tanase, 1988


Type of study Dose levels NOAEL mg/kg b.w./day

Findings Reference


(gavage)

Japanese White Rabbits

0, 50, 50 and 500 mg/kg

b.w./day

maternal 50 mg/kg b.w./day

foetal 50 mg/kg b.w./day

Maternal toxicity: Decreased maternal body weights and food consumption, clinical signs, death of 1 animal, and 2 abortions. Embryo-/foetotoxicity: Abortions, foetal deaths, decreased foetal weights. No teratogenicity.

Tanase, 1989

5.1.9 Neurotoxicity

In insects or mites, E-187 is a compound acting on the site where the nerve contacts with the muscle (neuromuscular synapse), especially on the inhibitory nervous system using γ-aminobutyric acid (GABA) as the neurotransmitter, which leads in insects and mites to death.

5.1.9.1 Acute neurotoxicity

Rat, oral, 15 days

Study design: Crl:CD®(SD)BR VAF/Plus® rats (10/sex/dose#1) received a single oral dose of 0, 20, 60, 100 and 500 mg/kg bw of milbemectin by gavage and were observed for 15 days. Due to mortality of all high dose females, 8 additional females were treated with 60 mg/kg bw. Functional observational battery (FOB) testing was done prior to treatment, on day 1 at approximately 1 h. postdose, and on day 8 and 15. FOB testing was followed by motor activity testing for 40 min. On day 1 motor activity testing was performed approximately 75-135 min postdose. Following macroscopic examination of all animals, neuropathological evaluation of central and peripheral nervous systems were conducted on 6 animals/sex of the control group, 6 males treated at 500 mg/kg bw and 6 females treated at 100 mg/kg bw. OECD guideline 424#2/QA/GLP. #1 10 animals/sex/dose were treated except at 60 mg/kg bw (8 females only) and 500 mg/k bw (10 males, 5 females). #2 FOB testing seems not to have been performed at peak concentration time. Results: The results are summarized in the table below:


Table 5.19: Results acute neurotoxicity: Dose

(mg/kg bw)

0

20

60

100

500

dr

m

f

m

f

f

m

f

m

f

Mortality

0/10

0/10

0/10

0/10

0/8

0/10

1/10

0/10

5/5

Clinical signs - tremors

+

+

+

+

+

- ataxia + + + ++ na m, f - hypoactivity + + + ++ na m, f - recumbency + + + ++ - irregular/laboured breathing

+ + + + ++ ++ m, f

- cold to touch + - pale (entire body) + + Body weight gain

dc1

na

Food consumption

no treatment-related findings

na

Functional observational battery - forelimb grip strength

dc

2

Motor activity measurements Day 1 0-10 minutes

dc

dc

dc

dc

dc

na

m, f 10-20 minutes dc na 20-30 minutes dc dc dc na 30-40 minutes na 0-40 minutes dc dc dc dc dc dc na m, f Days 8 and 15 no treatment-related findings na Macroscopy


Neuropathology


na

dr dose related dc/ic statistically significantly decreased/increased compared to the controls d/i decreased/increased, but not statistically significantly compared to the controls a/r absolute/relative organ weight na not applicable/observed/performed due to death of all animals on day of dosing + present in one/a few animals ++ present in most/all animals 1 statistically significant only in first week 2 findings in females included lethargy, recumbency, low carriage and prostrate position, ataxia, lower body temperature.

It cannot be excluded that these signs of neurotoxicity are caused by general toxicity. Clinical signs were induced at doses of 60, 100 and 500 mg/kg bw, and decreased motor activity were observed at all dose levels. The decreased motor activity (MA), in the absence of overt signs of systemic toxicity at lower dose levels, is considered to be a neurotoxic effect. The majority of clinical signs were seen on the day of dosing after the animals had completed their MA testing. This indicates that the effects of milbemectin peaked after and not during the neurotoxicity tests. If FOB and MA testing had been performed at peak concentration time, the observed neurotoxic effects might have been even more prominent. LOAEL: <20 mg/kg bw based on decreased MA, in the absence of overt signs of systemic toxicity at lower dose levels. No NOAEL could be established from this study (Weiler, 1998a). Establishment of an ARfD can be based on this study.

5.1.9.2 Subchronic neurotoxicity

Rat, oral diet, 13-weeks

Study design: Crl:CD®(SD)BR VAF/Plus® rats (10/sex/dose) received oral dose of 0, 150, 375 and 750 mg/kg food (equal to approximately 12, 32 and 59 mg/kg bw/d for males and 13, 36 and 72 mg/kg


bw/d for females) of milbemectin in the diet and were observed for 13 weeks. Detailed clinical observations and FOB testing were performed prior to treatment and once during weeks 4, 8 and 13. FOB testing was followed by motor activity testing for 40 min. Following macroscopic examination on all animals, neuropathological evaluations of central and peripheral nervous systems were conducted on 6 animals/sex from the control and high dose groups. No OECD guideline*/QA/GLP. * Not in accordance with OECD guideline 424. Derailed clinical observations, FOB testing and motor activity testing was not performed during the first or second week of exposure.

Results: The results are summarized in the table below: Table 5.20: Results semi-chronic neurotoxicity:

Dose (mg/kg food)

0

150

375

750

dr

m

f

m

f

m

f

m

f

Mortality

none

Clinical signs


Body weight gain

dc1

Food consumption


Functional observational battery


Motor activity measurements


Macroscopy


Neuropathology


dr dose related 1 over the first week only No neurotoxic effects were observed after 4, 8 and 13 weeks of repeated dose administration of the test substance. However, it can not be excluded that repeated dose administration of milbemectin would have resulted in neurotoxic effects within the first 2 weeks after dosing. NOAEL: 59 mg/kg bw/d based on no adverse effects. However, the possible effects that may have occurred in the first two weeks of the study must be taken into consideration.

Summary (neurotoxicity):

Milbemectin may cause neurotoxic effects of concern. In an acute oral neurotoxicity study, some evidence for neurotoxicity was found. A decrease in motor activity was observed at all dose levels; at the lowest tested dose, this decreased MA was even observed in the absence of overt systemic toxicity. It appears that the observations were not performed during peak time of the neurotoxic effects. Therefore a full evaluation of the neurotoxic potential of milbemectin cannot be performed. A NOAEL could not be derived in the acute neurotoxicity study. The LOAEL was 20 mg/kg bw. Establishment of an ARfD can be based on this study. Repeated dose administration via the diet did not result in neurotoxic effects in rats at 4, 8, and 13 weeks of dosing. Based on the available data a NOAEL of 59 mg/kg bw/d was established for repeated dose neurotoxicity of milbemectin in rats.

5.1.10 Special studies

Study 1: Kamoshita, 1988


In this pharmacological study in male rats, mice and rabbits, the results were consistent with an action of E-187 on the central nervous system and at the neuromuscular level. Study 2: Yamamoto et al., 1988 The abnormal growth of the incisors in rats was investigated. It was found that the treated rats moved very slowly and there was barely any attrition. The lack of gnawing was the cause of apparent elongation of the incisors. There was no clear explanation to why the animals did not gnaw, but many of the possibilities involve effects on the nervous system. Study 3: Dow and Burkhardt, 2006 The objective of this study was to determine the involvement of P-glycoprotein in the absorption of milbemectin through Caco-2 monolayers. However, it was not possible to monitor the concentration of milbemectin due to high non-specific binding of MA3 and Ma4 to the polystyrene and polypropylene plates in the experimental apparatus. Study 4: Smedley, 2006 (interim report) This acute oral toxicity study in female CF-1 mice (3 animals /dose) was performed to investigate whether milbemectin is a substrate for the P-glycoprotein transporter. Two strains were compared: a wild type strain +/+ and a mutant type strain -/- for the expression of a functional mdr 1 P-glycoprotein. (OECD 425) Results: The wild type mice died of 2700 mg/kg bw on the day of dosing, and there were no deaths of the doses 11, 33, 100, 300, or 900 mg/kg bw. The mutant mice died of a dose of 300 mg/kg bw on the day of dosing, but survived doses of 3.7, 11, 33 or 100 mg/kg bw. The Clinical symptoms were seen at 300 and 900 mg/kg/bw in the wild type strain: prostration, decreased activity, wobbly gait, rapid and laboured breathing, tremors, slow breathing, few faeces and urine stain. The symptoms were more severe in the higher dose and the 900 mg/kg bw-group had also symptoms cool to touch, eyelids closed, ocular discharge, decreased food consumption and body weight loss. There were no findings at necropsy day 2. In the high dose group, the necropsy revealed abnormal contents of the stomach. In the mutant mice strain, clinical symptoms similar to the ones seen in the wild type was seen at dose levels 33 and 100 mg/kg bw. Necropsy of the high dose group showed abnormal content of the jejunum. The study shows that milbemectin is much more toxic in the mice strain that lacks the mdr 1 P-glycoprotein transporter.

5.1.11 Medical data

There are no reports of clinical symptoms or poisoning from the manufacturing or use of milbemectin or Milbeknock.

5.1.12 Classification and labelling

Milbemectin was evaluated in ECB in 2006 for the 31th ATP of 91/414. It was regulated by 1. ATP to CLP (790/72009)(1272/2008) with the classification Xn;R20/22. A possible classification for developmental toxicity was discussed. Based on a statement from the notifier (T3), ECB decided not to classify for this effect (T4).

5.1.13 Reference values

ADI

An ADI of 0.03 mg/kg bw/day is proposed for milbemectin based on applying a 100-fold assessment factor to NOAEL of 3 /kg bw/day determined in the 90-days and 1-year dog studies (Ebino, 1988/1189). The critical effects in these studies were an increase in vomiting and in liver weigh. The uncertainty factor accounts for interspecies extrapolation (10X) and intraspecies variability (10X). The


chronic study in the rat has a lower NOAEL than the dog studies, but the NOAEL in the rat study was based on marginal effects.

AOEL

An AOEL of 0.014 mg/kg bw/day is proposed for milbemectin based on applying a 100-fold assessment factor to the NOAEL of 3 /kg bw determined in the 90-days and 1-year dog studies (Ebino, 1988/1189). A correction for incomplete oral absorption of 47 % is applied.

ARfD

An ARfD of 0.03 mg/kg bw/day is proposed for milbemectin based on reduced motor activity in the acute neurotoxicity study in the rat (Weiler, 1998) with a NOAEL of 3 mg/kg bw (extrapolated from a LOAEL of 20 mg/kg bw) and a 100 fold assessment factor. An ARfD of 0.03 mg/kg is also supported by the 90-days dog study with a NOAEL of 3 mg/kg/dag based on an increase in vomiting.

5.2 Impurities and metabolites

Acute toxicity Several impurities and metabolites were tested for acute toxicity in the mouse (Slc:ddY) with 6 or 10 animals/sex/group. Table 5.21: LD50 of impurities and metabolites of E-187 Test substance Purity, % Type LD50, males,

mg/kg bw LD50, females, mg/kg bw

Δ2,3-A4 97.2 impurity >5000 >5000 M.D. 99.4 impurity >5000 >5000 5-keto A3 98.0 Imp + metabolite >5000 >5000 5-keto A4 97.7 Imp + metabolite >5000 >5000 27-keto A3 100.0 Metabolite >5000 >5000 27-keto A4 100.0 metabolite 3880#1 3550#1 14,15-epoxy A3 100.0 metabolite >2000#2 ≥2000#2 14,15-epoxy A4 100.0 metabolite 204#3 176#3 8.9Z-M.A3 99.4 metabolite 490#4 520#4 8.9Z-M.A4 98.5 metabolite 1570#5 1520#5 #1 Clinical symptoms in the animals were inactivity, stagger, weakness, incontinence and reduction in respiration rate. Death was observed from the next morning up to four days after administration. #2 Clinical symptoms; suppression of voluntary action and then prone position, reduction in respiration rate and abnormal nasal noise, were observed from 500-2000 mg/kg bw. Two of six females died of 2000 mg/kg bw. #3 Clinical symptoms, suppression of voluntary action and then prone position, reduction in respiration rate and abnormal nasal noise, were observed from 10 minutes after administration. Systemic weakness was seen in severe cases. Deaths occurred from 10 minutes to 2 hours after administration. #4 Clinical symptoms were suppression of voluntary action followed by staggering gait, weakness and reduction in respiration rate. Deaths occurred from 1 to 4 hours after administration. #5 Clinical symptoms were action stop followed by weakness, prone position and reduction I respiration rate. Animals were found dead in the first or second day after administration. Summary (acute toxicity): Several impurities and metabolites were tested for acute toxicity in the mouse (Slc:ddY), and some clinical symptoms were observed. Genotoxicity The 10 impurities in the table above and 3 additional impurities (14-desmethyl A3, 14-desmethyl A4 and 5.methoxy A3) were tested in two genetic assays in bacteria. All tests were negative. The first


assay was the reverse mutagenicity study, with Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100) and E. coli (WP2hcr). The second assay was the DNA-repair study with Bacillus subtilis M-45 and H-17 strains. The photoisomers 8,9Z-MA3 and 8,9Z-MA4 were found to be of toxicological significance. They were further tested for genotoxicity in several studies. Table 5.22: Summary of the genotoxicity studies: Study Test system Concentration/ dose

range tested Result Reference/

Guidelines/ GLP

In vitro: Point mutations in bacteria 8,9Z-MA3


S. typhimurium TA98, TA100, TA1535 & TA1537. E.coli WP2 uvrA

Range: 15.8 - 5000 g/plate (+/- S9)

Negative (+/- S9)

Williams, 2004a/ OECD 471#1/ GLP/ QA

Point mutations in bacteria 8,9Z-MA4


S. typhimurium TA98, TA100, TA1535 & TA1537. E.coli WP2 uvrA

Range: 15.8 - 5000 g/plate (+/- S9)

Negative (+/- S9)

Williams, 2004b/ OECD 471#1/ GLP/ QA

Chromosomal aberration 8,9Z-MA3

Cytogenetic assay

Human peripheral blood lymphocytes

Dose range finding: 6.7 – 600 µg/ml. Main: -S9 experiment 1: 21.1, 26.4 and 33 µg/ml. +S9 experiment 1: 26.4, 64.4 and 80.5 µg/ml. -S9 experiment 2: 17.7, 24.5 and 33.9 µg/ml. +S9 experiment 2: 57.4, 70.9 and 78.7 µg/ml.

Negative Whitwell, 2004/ OECD 473#2/ GLP/ QA

Chromosomal aberration 8,9Z-MA4

Cytogenetic assay

Human peripheral blood lymphocytes

Dose range finding: 2.8 – 600 µg/ml. Main: -S9 experiment 1: 26.4, 33 and 41.2 µg/ml. +S9 experiment 1: 62.8, 69.8 and 77.5 µg/ml. -S9 experiment 2: 13.4, 21 and 32.8 µg/ml. +S9 experiment 2: 59.3, 73.2 and 81.3 µg/ml.

Negative Kumaravel, 2004/ OECD 473#3/ GLP/ QA


Study Test system Concentration/ dose range tested

Result Reference/ Guidelines/ GLP

Gene mutation in mammalian cells 8,9Z-MA3

Mutation assay

Mouse lymphoma L5178Y cells

Dose range finding: 31.3, 62.5, 125, 250, 500 and 1000 µg/ml. Main: -S9: 10 doses ranging from 2.5 – 60 µg/ml. Concentrations selected for mutation assessment range from 2.5 – 40 µg/ml. +S9: 10 doses ranging from 10 – 125 µg/ml. Concentrations selected for mutation assessment range from 10 – 90 µg/ml.

Negative (+/- S9)

Lloyd, 2004a/OECD 476/ GLP / QA

Gene mutation in mammalian cells 8,9Z-MA4

Mutation assay

Mouse lymphoma L5178Y cells

Dose range finding: 18.8, 37.5, 75, 150, 300 and 600 µg/ml. Main: -S9: 10 doses ranging from 5 – 60 µg/ml. Concentrations selected for mutation assessment range from 5 – 35 µg/ml.+S9: 10 doses ranging from 10 – 140 µg/ml. Concentrations selected for mutation assessment range from 10 – 90 µg/ml.

Negative Lloyd, 2004b/ OECD 476/ GLP / QA

#1 The study deviated from OECD 471 (1997). As the results of the first experiment (plate incorporation method) were negative, treatments in the presence of S-9 in experiment 2 included a pre-incubation step. #2 Toxicity –S9: 33 µg/ml and above (about 50% mitotic inhibition and above). Toxicity +S9: 78.7 µg/ml and above (about 50% mitotic inhibition and above). #3 Toxicity –S9: 32.8 µg/ml and above (about 50% mitotic inhibition and above). Toxicity +S9: 77.5 µg/ml and above (about 50% mitotic inhibition and above). Summary (genotoxicity): All in vitro genotoxicity studies were negative. The photoisomers 8,9Z-MA3 and 8,9Z-MA4 were tested in Ames test, a chromosome aberration test and a tk mutation test. The overall weight of evidence shows that both photoisomers are not of gentoxic concern. These studies of the photoisomers in combination with the studies and statement evaluated in addendum IV (December 2003), show that the risk for consumers and workers is acceptable.

5.3 Co-formulants

The product contains aromatic hydrocarbons and has to be labelled with R65: Harmful: may cause lung damage if swallowed.


5.4 Milbeknock 1 % EC (SI-9009EC)

5.4.1 Acute toxicity

Oral, rat Groups of ten fasted rats (five males and females) were given a single dose by gavage of the test substance at dose levels of 2000, 3200, 5000 and 8000 mg/kg bw. All animals surviving treatment were killed and examined macroscopically on day 15, the end of the observation period. LD50 in female rats is > 5000 mg/kg bw. There were deaths among females at 3200 mg/kg bw (1/5), 5000 mg/kg bw (2/5) and at 8000 mg/kg bw (4/5) and among males at 5000 mg/kg bw (2/5) and at 8000 mg/kg bw (5/5). Microscopic evaluation of these animals revealed congestion of blood vessels of the stomach and small intestine, or thickened, white stomach walls. Clinical signs of reaction to treatment included piloerection, hunched posture, waddling, lethargy, decreased respiratory rate, pallor of the extremities, and increased salivation. Recovery of surviving animals was complete at intervals between from day 3 and day 5. Slightly decreased body weight gain was observed on day 8 for male rats at 2000 (2/5), and 3200 mg/kg bw (2/4), and all surviving males that were exposed to 5000 or 8000 mg/kg bw, and on day 15 for 1 males at 2000 mg/kg bw. No macroscopic abnormalities were observed for animals killed on day 15 (Allan, 1992). Oral, mouse Groups of ten fasted mice (five males and females) were given a single dose by gavage of the test substance at dose levels of 2000, 3200, 5000 and 8000 mg/kg bw. All animals surviving treatment were killed and examined macroscopically on day 15, the end of the observation period. LD50 was 4700 and 5200 mg/kg bw respectively in females and males. Five males and 5 females (8000 mg/kg bw), 2 males and 2 females (5000 mg/kg bw), and 1 female (3200 mg/kg bw) died within 2 days after dosing. Piloerection was observed in all test animals. Hunched posture, waddling, lethargy, decreased respiratory rate, pallor of extremities were frequently observed in all groups, except in test animals receiving the lowest dose. Ptosis and prostrate were mainly noted in test animals exposed to 5000 or 8000 mg/kg bw. Recovery of surviving animals was complete by day 2 or 3 for the majority and by day 5 for female dosed at 3200 mg/kg bw. Among surviving mice, no change in bodyweight was recorded for one female at 3200 mg/kg bw; this animal achieved a satisfactory gain on day 15. All other mice achieved satisfactory bodyweight gains throughout the study. No macroscopic abnormalities were observed for animals killed on day 15 (Allan, 1992). Dermal, rat A group of ten rats (5 males and five females) was treated with 2000 mg/kg bw. LD50 in rats is >200 mg/kg body weight in both sexes. There were no deaths, systemic signs of toxicity. Except for 3 males showing slightly low body weight gain on day 8, body weight gain was considered to be normal. No pathological changes were noted. (Allan, 1992). Inhalation, rat LC50 in rats is > 6.75 mg/L air. Five rats/sex/group were exposed for 4 hours (nose-only) to an aerosol in air. The concentration tested was 6.75 mg/L (analytic). The MMAD of the groups were 2.18 ± 2.07 µm. There was no death during the course of the study. Substance-related clinical signs observed at day 1 included: lethargy, coldness, piloerection, hunched posture, semi-closed eyes, shallow


respiration, prostration, gripping the cage with paws, and noisy respiration. From day 2 till day 9 hunched posture, rough fur, and sneezing were observed (Mould, 1998). Summary (acute toxicity): Milbeknock 1 % EC was of low acute toxicity by the inhalation, oral and dermal exposure.

5.4.2 Irritation and sensitisation

Dermal, rabbit New Zealand rabbits (6 females) were used in the study. Mean dermal irritation index (24, 48 and 72 hours) was 1.5 for erythema and 0.2 for oedema. Desquamation of the stratum corneum was observed in 4 females starting 24 h after exposure, and lasting for 5 to 7 days. Milbeknock 1 % EC has not to be classified as irritating to skin (Liggett, 1992). Eye, rabbit New Zealand rabbits (6 females) were used in the study. Mean eye irritation index was 0.17 for corneal opacity, 0 for iritis, 0.3 for conjunctival chemosis, and 0.3 for conjunctival redness. Milbeknock 1% EC is not classified as irritating to the eye (Liggett, 1992). Sensitisation, mouse Milbeknock 1 % EC has not been found to induce a dermal sensitisation response using the Maximisation test in guinea pig. Intradermal injection caused slight erythema in the test animals, and topical induction caused well defined erythema in the test animals. Following challenge with 1 and 2% w/w, no dermal responses were observed in any of the test and control animals with the exception of desquamation seen in one test animal at the 48 hour observation only. The positive control (α-hexyl cinnamic aldehyde) gave a positive result. (Ruddock, 2001). Summary (irritation and allergy): Milbeknock 1 % EC is not irritating to the skin or eye, and it is not a dermal sensitizer.

5.4.3 Classification and labelling

Milbeknock 1 % EC contains aromatic hydrocarbons and has to be labelled with R65: Harmful: may cause lung damage if swallowed.

5.4.4 Dermal absorption

No data were submitted. However, based on the data provided on molecular weights (528.7 and 542.7) and the log Kow (6.43 and 7.00, for MA3 and MA4 respectively), and according to the guidance document on dermal absorption, dermal absorption of 10% is considered.

5.5 Operator, worker and bystander exposure

Operator The operator exposure, when using mechanical spraying in pomes and strawberries, was estimated based on the UK POEM and the German model. Exposure, when spraying in greenhouses, was estimated by the Dutch model. The results of the estimations are presented in table below:


Table 5.23: Exposure models Total absorbed dose (mg/kg bw/day)

% of AOEL without PPE

% of AOEL with PPE

Modell

Without PPE Gloves under mixing and loading

Gloves under mixing and loading + application

UK Poem Liquid formulation (mechanical spraying pomes) UK Poem Liquid formulation (mechanical spraying strawberries)

0,0149

0,01

0,002 106

71

14

German model, 75 th percentile (pomes) German model, 75 th percentile (strawberries)

0,011

0,004

78

28

Dutch model , manual spraying of ornamentals in greenhouses *

0,01

71

* Since only the Dutch model is available for estimating dermal and inhalation exposure during mixing/loading and application, these estimates will serve the present purpose. Dermal absorption of 10 % and a body weight of 60 kg were used.

The exposure exceeds AOEL when spraying pomes without PPE. The use of PPE reduces the exposure under the AOEL. For spraying in greenhouses and in strawberries the AOEL is not exceeded even without use of PPE. Bystander exposure For estimating bystander exposure the EUROPOEM II model will be used. These are for upward spraying: Dermal exposure = 0.05 x (0.00372 – 0.0188) kg/ha x 2 m2 x 0.01 (correction for kg/ha to mg/m2) = <0.01 mg a.s./day (<0.01 mg/kg bw/day). Inhalation exposure = 0.06 ml/m3 x 1 hour x 1.25 m3/hour x (0.009 – 0.013) mg/ml = <0.01 mg a.s./day (<0.01 mg/kg bw/day). Dermal exposure: a maximum of 5% of the application rate (kg/ha) on a assumed body surface of 2 m2

Inhalation exposure: 0.06 ml spraying liquid per m3 . For inhalation exposure, a duration of 1 hour and a ventilation rate of 1.25 m3/hour will be assumed.

Re-entry workers The exposure for re-entry workers harvesting fruits after spraying pomes was estimated based on the following equation: Estimated exposure (mg a.s.)/day = dosage (kg/ha) x (100 (taking care for the dimensions mg/m2)/LAI(m2/m2)) x TF (m2/hour) x duration of task (hours).


LAI = leaf area index (m2 foliar surface / m2 ground surface) TF = task specific transfer factor (m2/hour) Assuming an LA = 2 m2/m2 and TF = 1 m2/m2, a body weight of 60 kg and a dermal absorption of 10 %. = 0.00372 – 0.0188 x 100/2 x1 x 6 = 1.116-5.64 mg a.s/day (0.001-0.009 mg/kg/day) (<<AOEL) The exposure for re-entry workers in greenhouses was estimated using a Dutch model and were calculated as follows: Cutting Dermal exposure = 20 (mg/hr)/(kg/ha) x (0.002 – 0.028) kg/ha x 3 hr = 0.12 – 1.7 mg. Inhalation exposure = 0.2 (mg/hr)/(kg/ha) x (0.002 – 0.028) kg/ha x 3 hr = < 0.01 – 0.02 mg. Sorting and bundling Dermal exposure = 10 (mg/hr)/(kg/ha) x (0.002 – 0.028) kg/ha x 3 hr = 0.06 – 0.84 mg. Inhalation exposure = 0.02 (mg/hr)/(kg/ha) x (0.002 – 0.028) kg/ha x 3 hr = < 0.01 mg. Since both tasks are mostly performed by the same person during one day, total exposure estimates are: Cutting, sorting and bundling Dermal exposure = 0.18 – 2.5 mg a.s./day. Inhalation exposure = <0.01 – 0.02 mg a.s./day. With a body weight of 60 kg and a dermal absorption of 10 % the maximum exposure = 0,004 mg/kg/dag (<<AOEL)

Summary of exposure:

The exposure, estimated by the UK POEM, exceeds AOEL with 14% when spraying pomes without PPE. The use of PPE reduces the exposure to under the AOEL. For spraying in greenhouses and in strawberries the AOEL is not exceeded even without use of PPE. For bystanders and re-entry workers the estimated exposure was far below AOEL.


6. Rester i produkter til mat eller fôr

Vurderingen er basert på dokumentasjon fra tilvirker samt rapport fra EFSA (SANCO/10386/2002, 4.april 2005). Det foreligger også en vurdering fra Sverige/KemI, men denne dekker kun prydplanter i veksthus. Rapport fra EFSA konkluderer med at søkt bruk ikke vil medføre skadelige effekter ved inntak hos mennesker eller dyr. Rapporten er basert på bruk i epler (og prydplanter). Bruk i eple er med bakgrunn i følgende GAP (God Agronomisk Praksis): Dosering: 0,93 – 1,25 g v.s./100 l vann, og 0,3 – 1,8 g v.s./daa (avhengig av væskemengde). Maksimalt 2 behandlinger, behandlingsfrist 14 dager. Vurderingen dekker norsk bruk i eple, og hvor GAP er som følger: Dosering; 1,2 g v.s./100 l vann, maks arealdose 1,7 g v.s./daa. Maksimalt 2 behandlinger, behandlingsfrist 14 dager.

Metabolisme i planten Milbemektin er et produkt av to aktive stoffer, milbemycin A3 og milbemycin A4. I

forbindelse med en mikrobiologisk fermenteringsprosess oppstår de i et forhold på 3:7 (A3: A4). Stoffenes struktur er svært lik i karakteristikk og i hvordan de reagerer. Tilvirker har derfor kun utført studier basert på milbemycin A4, og mener dette bør være tilstrekkelig for å kunne evaluere milbemectin.

Det er utført metabolismestudier i 3 ulike kulturer innen frukt og bær (eple, jordbær og

appelsin). Dette oppfyller kriteriene mht forsøksomfang for bruk i kulturer innen gruppen frukt/bær.

Nedenfor er sammendraget på de ulike studiene, og som er hentet fra tilvirker sin

dokumentasjon (Summary Documentation, Tier II, Annex IIA, Section 4, Point 6): Eple: Radiomerket milbemycin A4 ble påført bladverket, og 7 dager etter påføring hadde

stoffet i stor utstrekning blitt nedbrutt. I frukten var mengden gjenfunnet radiomerket stoff 0,018 mg/kg (og 0,006 mg/kg i vasket frukt).

Jordbær: Radiomerket milbemycin A4 ble påført bladverket. I løpet av 72 timer (3 dager)

var gjenfunnet radiomerket stoff i bærene 0,037 mg/kg (og 0,028 mg/kg i vasket frukt). Appelsin: Radiomerket milbemycin A4 ble påført bladverket. Stoffet ble i stor utstrekning

nedbrutt i løpet av både 7 og 14 dager etter påføring. Totale radioaktive rester i fruktkjøttet var 0,003 mg/kg både 7 og 14 dager etter påføring.

I appelsin ble det også gjort forsøk med radioaktivt M.A3 og/eller M.A4 på blad og frukt.

Gjenfunnet rester var hovedsakelig morstoffet, men et antall mindre metabolitter ble også identifisert. Ingen av metabolittene ble gjenfunnet i mengder som oversteg 10% av totalt gjenfunnet radioaktivt stoff.

Nedbryting i jord:

Studier viser at nedbrytningen i jord er medium til moderat, jf. kapittel 7. Definisjon rester Mattilsynet har ikke gjort noen egen vurdering mht definisjon av rester, men forholder

seg til rådsforordning 396/2005/EF som er implementert i norsk regelverk (forskrift om rester av plantevernmidler i næringsmidler). Her er rester av milbemektin i forbindelse med overvåking definert som sum av MA4 + 8,9Z-MA4, uttykt som milbemektin.

Restdefinisjon i forbindelse med risikovurdering er sum av MA4 + 8,9Z- MA4 og MA3 +

8,9Z- MA3 (hentet fra list of endpoints, februar 2005).


Restdata Det er innlevert nordeuropeiske restforsøk i eple, pære og jordbær. Resultatene er gjengitt i tabellen nedenfor. (Resultatene er hentet fra vedlegg 10 i KemI sin produktrapport fra april 2010).

År, land

Kultur Dose (g v.s /daa)

PHI (dager)

Restnivå (mg/kg)

MRL Norsk kritisk GAP

N-FR/98

2 x 1,4 g v.s./100 l vann, 100 l væske/daa

eller 2 x 1,4 g v.s./daa (8 dager mellom behandlingene)

20

< 0,02

DE/98 2 x 1,4 g v.s./100 l vann, 150 l væske/daa


3, 7, 14,

21

< 0,02

DE/98 3, 7, 14,

21

< 0,02

DE/98



3, 7, 14,

21

< 0,02

DE/98 7, 14, 21, 28

< 0,02

DE/98



7, 14, 21, 28

< 0,02

DE/99 2 x 1,4 g v.s./100 l vann, 150 l væske/daa


7, 14 21

< 0,02

DE/99

Eple



14

< 0,02

0,05

2 x 1,2 g v.s./100 l

vann

2 x 1,76 g v.s./daa

PHI: 14

(minimum 5 -7 dager

mellom hver

sprøyting)

N-FR/00

Pære 2 x 1,4 g v.s./100 l vann, 100 l væske/daa

eller 1,4 + 1,5 g v.s./daa

(7 dager mellom behandlingene)

1, 3, 7,

14

< 0,02

0,05

2 x 1,2 g v.s./100 l

vann

2 x 1,76 g v.s./daa

PHI: 14

(minimum 5 -7 dager

mellom hver

sprøyting) DK/06

309 < 0,02

DK/06

Jordbær 1 x 2,33 g v.s./daa, påført etter høsting

309 < 0,02

0,05 1 x 2,5 g v.s./daa,

etter høsting


Oppsummering Eple og pære: Det er innlevert 8 restforsøk i eple og som er utført i Nord-Europa. Dette tilfredsstiller kravene til antall forsøk for ”major crops”. Forsøkene er utført med en GAP som dekker norsk bruk. I alle forsøkene er gjenfunnet restverdier < 0,02 mg/kg. Dette viser at det ikke vil være noen fare for overskridelse av gjeldende grenseverdi ved omsøkt bruk. I pære er det kun levert inn ett restforsøk. I følge EUs retningslinjer for ekstrapolering mellom kulturer (Sanco/7039/VI/, rev. 8) kan imidlertid restforsøk i eple ekstrapoleres til pære. Både forsøket i pære og forsøkene i eple viser at det ikke vil være noen fare for overskridelse av gjeldene grenseverdi ved omsøkt bruk. Jordbær: Bruk i jordbær vil skje etter høsting. Nedbrytningsstudier i jord viser at preparatets nedbryting i jord er medium til moderat. Det forventes at det ikke vil gjenfinnes rester i bær påfølgende sesong. Dette bekreftes av to innleverte restforsøk fra Danmark, forsøkene dekker norsk GAP. Omsøkt bruk ikke vil medføre fare for overskridelse av gjeldende grenseverdi.

Foreslåtte behandlingsfrister Eple og pære: 14 dager

Jordbær: Etter høsting


7. Fate and Behaviour in the Environment This assessment is based on documentation submitted by the applicant (referenced with author and year) as well as the EU Draft Assessment Report, DAR (E1) and Addendum II (E4), the European Commission’s Review report for the active substance milbemectin (04 April 2005) with List of Endpoints (E2) and a Swedish assessment (E3, Kemi, 2010). Application rate: 12.1-17.7 g a.s./ha in apple/pear, 23.3 g a.s./ha in Strawberry, 4.7-19 g a.s./ha in ornamentals. Number of applications pr season: 2 in all crops. Time of application: apple/pear: from the time after flowering to the time the fruit is of half size, strawberries: after harvest, ornamentals: at the outbreak of decease.

7.1 Milbemectin

7.1.1 Degradation in soil

Route of degradation in soil The substance milbemectin is a mixture of two compounds, milbemycin A and milbemycin A , occurring at a ratio of 3:7 in technical milbemectin. The environmental fate and behaviour of milbemycin A and milbemycin A have been concluded to be similar so their metabolism and degradation rates are regarded comparable (Addendum II to DAR, November 2002). Consequently this applies to the metabolites,

3 4

3 4

27-hydroxy-milbemycin A3/A4 (max 14 % Applied Radioactivity (AR) of A4) and 27-keto-milbemycin A3/A4 (max 12 % AR of A4), as well. Mineralisation of milbemycin A isbetween 14-35 % of Applied Radioactivity (AR). The proposed metabolic pathway is presented in Fig. 7.1.

4


*

O

O*

** C H 3

*H

*

C H 3

*

*CH 3 *

O

* *

*C H 3

O H

OO

H

H

HO H

C H 3

OH

*

O

O*

** C H 3

*H

*

C H 3

*

*CH 3 *

O

* *

*C H 3

O H

OO

H

H

HO H

C H 3

*

O

O*

** C H 3

*H

*

C H 3

*

*CH 3 *

O

* *

*C H 3

O H

OO

H

H

HO H

C H 3

O

m ilb e m y c in A 4

2 7 -h y d ro x y -m ilb e m y c in A 4

2 7 -k e to -m ilb e m y c in A 4

o th e r m in o rd e g ra d a t io n

p ro d u c ts

b o u n dre s id u e

C O 2

Fig. 7.1: Proposed metabolic pathway for the aerobic degradation of milbemycin A in soil.

4

Rate of degradation in soil - Aerobic degradation The degradation rate of milbemycin A is medium to moderate, DT50: 21-82 days, geometric mean 36.5 days (arithmetic mean: 43 days). DT90: 69-271 days. DT90 values were estimated on basis of DT50 values assuming first order kinetics and using a factor of 3.3. Bound residue amounted to 40 % of AR at maximum and the mineralization to CO reached a maximum level of 35 % of AR. The degradation rate (DT50) of the metabolite

4

2

27-hydroxy-milbemycin A4 was calculated to be 18 days with a DT90 estimated to be 59 days. Details of the study are summarised in table 7.1. DT50 values mentioned here were recalculated by RMS in DAR using data from the original report and by non-linear regression of first order kinetics. All regression coefficients were 0.97. At 10 °C the degradation rate of milbemycin A is moderate. DT50: 63 days, DT90: 208 days. 4

Anaerobic degradation The degradation rate is low under anaerobic conditions, DT50: 556 days in the soil phase. DT90 of 1835 days is extrapolated well beyond the study duration and must be regarded as uncertain. Mineralisation and bound residues amounted to 1.9 and 22 % of AR after 363 days respectively. No metabolites > 5 % of AR were reported. Details of the study are summarised in table 7.1.


Table 7.1: Summary of the degradation studies on milbemycin A4 under aerobic and anaerobic conditions in soil. Sandy loam (1) Sandy loam (1) Sandy loam (2) Silt loam Clay loam Sandy loam (1) Sandy loam (1) Substance 14C- milbemycin A4

14C-27-hydroxy-milbemycin A4

14C- milbemycin A4

Aerobic/ anaerobic

Aerobic Aerobic Anaerobic

Temperature (°C) 20 10 20 20 20 10 20 Study Duration, days

120 120 120 120 120 120 363

Sand (%) 54 54 77 35 38 54 54 Silt (%) 34 34 10 53 33 34 34 Clay (%) 11 11 13 12 29 11 11 pH 6.9 6.9 4.4 5.5 7.3 6.9 6.9 Organic Carbon (%)

1.7 1.7 1.1 2.4 3.4 1.7 1.7

MWHC (g/100 g soil)

- - - - - - -

% MWHC 45 45 45 45 45 45 Flooded Microbial biomass

Start: 201 End: 548

Start: 201 End: 298

Start: 132 End: 35

Start: 206 End: 29

Start: 767 End: 652

Start: 201 End: 548

-

DT50, days 21* 63* 47* 82* 22* 18* Water phase: 7.5 d. Soil phase: 556 d.

Whole system: 650 d. DT90, days 69** 208** 155** 271** 73** 59** 1835** CO (% AR) 2 35 (120 d.) 16 (120 d.) 18 (120 d.) 14 (120 d.) 35 (120 d.) - 1.9 (363 d.) Bound residue (% AR)

29 (120 d.) 25 (120 d.) 24 (120 d.) 13 (120 d.) 40 (91 d.) - 5.1 (120 d.) 22 (363 d.)

Metabolites > 5 %. Maximum within 120 days

27-hydroxy-milbemycin A4: 9 (46 d.)

27-keto-milbemycin A4: 5 (30 d.)







- None > 5 %

References Lewis, 1999 (E1, IIA 7.1.1.1.1/01) E1, IIA 7.1.1.2.1/03 * Values recalculated by RMS in DAR by non-linear regression of first order kinetics. ** Not calculated in original report or DAR, but estimated in LoEP by assuming first order exponential decay: 3.3xDT50


Photolysis in soil Photolysis can be an important route of degradation for milbemycin A4. A sandy loam soil was exposed to artificial sunlight for 21 days and DT50 was 7.5 days in the light samples and 27 days in the dark control samples. Bound residues increased up to 29 % of AR at the end of the study while mineralisation amounted to 12 % of AR. No metabolites were detected at levels > 10 % of AR (E1, IIA 7.1.1.1.2/02). Field Dissipation Three field studies performed in the US have been submitted, but only two were accepted by the EU as one of the studies was of poor quality. The degradation of milbemycin A /A3 4 is medium to high with DT50: 8-13 days (geometric mean of 8.5 for milbemycin A3 and 11.4 for milbemycin A4). Recovery was low in both studies (55-59 % on average). Even though the two metabolites were found at quantifiable levels, no statement could be made on their relevance due to the low recovery in the study. Weather conditions are not well described in the two studies and assessing the relevance to Norway is difficult. Weather conditions in New York might be expected to be more relevant than the conditions in Florida, but the content of organic carbon was very low in the New York soil in comparison to what is found in Norwegian agricultural soils. Swedish authorities have concludedthe two studies were not performed under conditions relevant for Sweden (E3). The two studies are

that

ummarised in table 7.2.

Summary of fi wit

by RMS to 20 °C by using old Q10 of 2.2. By using new Q10 of 2.58 DT50s are 10 and 15 days ectively.

able 7.3: Climate data from Ås and Værnes. Based rom 1961-1990.

(month erage)

s Table 7.2: eld dissipation studies performed h Milbeknock 1% EC.

* Recalculatedresp** T on data f

Annual ly av

April- Sept.

Oct. - March

Ås 11,8 age) -1,2 (average) Temp. (°C) 5,3 (aver Precipitation (mm) 785 4) (65, 421 364 Værnes Temp. (°C) 5,0 10,3 -0,3 Precipitation (mm) 892 (74,3) 464 428

Field site Wayne Count h Rose, New Lake County, Central Ridge, near Umatilla, Florida, USA

y, near NortYork, USA

Substance, application rate and application date(s)

Milbeknoc , 4x90 g

2 19 and 26 October, 1998

Milbekn a.s./ha

10 and 16 september

k 1 % ECa.s./ha,

1 and 28 September, 1998

ock 1 % EC, 4x90 g12 and 19 August

Duration (days) 321 439 soil type Sand Loa nd my saSand (%) 96 88.4 Silt (%) 3 7.3 Clay (%) 1 4.3 pH 5.9 6.9 Org. C (%) 0.7 0.99 Temp. (°C) range at 23.3-31.7 12.2-21.1 application dates Total precipitation

718 712

in one yearDT50, days

Milbemycin A : 13 (20 °C)* Milbemy A4: 10 Milbemycin A : 9 (20 °C)* 3

4

Milbemycin A : 8 3

cinDT90, days - - Relevance for No No Norway References Temple 1, IIA Temple et al., 1998, (E1, IIA 7.1.1.2.2/03) et al., 2000, (E

7.1.1.2.2/02)


7.1.2 Sorption and mobility in soil

Sorption The sorption of milbemycin to soil can be classified as high to very high with Kd: 12-138 (average 61) and Koc: 1370-4059 (average 2817). 1/n varied from 0.92 to 1.04 with an average of 0.98. All soils tested are of relevance to Norwegian conditions. From the submitted data there are no indications that certain factors are more important for the sorption than others. The sorption and desorption coefficients are in the same order of magnitude, indicating that the sorption is reversible. Details of the data on milbemycin are summarised in Table 7.4. The sorption of the two metabolites, 27-hydroxy-milbemycin A4 and 27-keto-milbemycin A4, to soil can be classified as high to very high with Kf: 20-94 (average 55) and 59-246 (average 171) respectively. Koc: 1828-2462 (average 2111) and 5350-7444 (average 6718) respectively. 1/n varied from 0.80 to 0.85 for 27-hydroxy-milbemycin A4 and 0.95-1.05 for 27-keto-milbemycin A4. All soils tested are of relevance to Norwegian conditions, even though the sandy loam has a very low pH and a low content of organic C. From the submitted data there are no indications that certain factors are more important for the sorption than others. Details of the data on the two metabolites are summarised in Table 7.5. Table 7.4: Sorption of 14C- milbemycin A4 in soil. Sand Clay loam Sandy

loam Silt loam

Sand (%) 90 38 54 24 Silt (%) 6 33 35 54

Clay (%) 4 29 11 22 pH 6.3 7.8 7.2 7.3 Org. C. (%) 0.6 3.4 1.7 2.0 Kd ds) 12 138 65 27 Koc (ads) 2033 4059 3806 1370 1/n (ads) 1.04 1.02 0.93 0.92 Kd(des) 13 99 68 40 Koc (des) 2217 2912 4006 1975 References Bashir, 1998 (E1, IIA, 7.1.2/01)

Table 7.5: Sorption of 27-hydroxy-milbemycin A4 and 27-keto-milbemycin A4 in soil. Clay loam Sandy loam Silty clay loam Substance 27-hydroxy-

milbemycin A4

27-keto-milbemycin

A4

27-hydroxy-milbemycin

A4

27-keto-milbemycin

A4

27-hydroxy-milbemycin

A4

27-keto-milbemycin

A4 Sand (%) - - - Silt (%) - - -

Clay (%) 34 10 19 pH 7.6 4.0 6.1 Org. C. (%) 4.6 0.8 2.8 Kf (ads) 94 246 20 59 51 208 Koc (ads) 2043 5350 2462 7360 1828 7444 1/n (ads) 0.82 0.95 0.85 0.97 0.80 1.05 References E4, IIA 7.1.2/02

Column, aged Based on the amount of radioactivity in the leachate (1.1-3.3 % of AR after 2 days), the mobility can be classified as medium to high in the four tested soils (sand, sandy loam, clay loam, silt loam), but neither milbemycin nor any of the major degradation products were detected in the leachate. The soils used in this study were aged for 14-27 days. Half lives of both milbemycin A4 and the metabolites 27-hydroxy-milbemycin A4 and 27-keto-milbemycin A4 were also estimated in this study. The respective half lives were 9-29 days for milbemycin A4, 2-13 days for 27-hydroxy-milbemycin A4 and 2-12 days for 27-keto-milbemycin A4 (E1, IIA, 7.1.3.2/01).


7.1.3 Degradation in water

Hydrolysis The hydrolysis of 14C- milbemycin A4 was determined at 50 °C and at pH 5, 7 and 9. DT50 at the different pH values was estimated to be 13, 318 and 241 days respectively (r2 values 0.97, 0.16 and 0.31). The regression coefficients indicate that the DT50 values at pH 7 and 9 are not reliable even though they indicate that hydrolysis at these pH values will be low. The degradation products 27-hydroxy-milbemycin A4 and 27-keto-milbemycin A4 were found at levels of 8.2 and 23 % of AR respectively at days 21-22 at pH 5 and at levels < 5 % at pH 7 (E1, AII, 7.2.1.1/01). Photolysis in water Photolysis is an important degradation pathway for milbemycin A4 compared to the dark control. The amount of initially applied radioactivity recovered was much higher in the dark controls than in the irradiated samples, indicating that photolysis may play an important role in the degradation of milbemycin A4. For details of the studies reviewed in the DAR (E1, AII, 7.2.1.2/01 and 7.2.1.2/02), see table 7.6. Three metabolites > 5 % AR have also been identified. For details see table 7.7. Table 7.6: Photolysis of 14C- milbemycin A4 and Milbemycin A4. Substance Duration

(days) Temp.

(°C) pH Light

source Wave length (nm)

Amount recovered of AR irradiated/dark

control (%)

DT50 photolysis (days)

14C- milbemycin A4 2 25 5 artificial sunlight

>290 15.4/96 0.93


>290 33/99 2.6


>290 26/101 4.1

Milbemycin A4 32.6 hours

22-25 6.9 artificial sunlight

304 - 7.8 hours (irradiated)

273 hours (dark control)

Table 7.7: Photolysis metabolites of 14C- milbemycin A4 and the highest measured values (/% of AR) at different pH. pH 8,9-Z-milbemycin A4 6,11.dihydroxy-8-

carboxy-milbemycin A4 14,15-epoxy-milbemycin

A4 5 4.9 9.8 6.2 7 13 - 7.4 9 11 - 6.1 Easily degradable 14C- milbemycin A4 is not readily biodegradable (E1, AII, 7.2.1.3.1/01). Water/sediment systems The degradation for the whole system can be classified as moderate with DT50system: 82-89 days, geometric mean 85 days (arithmetic mean 86 days). Bound residues amounts to about 30 % of AR after 100 days in both systems and mineralization is low with only about 6 % after 100 days. The active substance quickly dissipates from the water phase and adsorbs to sediment. Metabolites were detected and identified but at levels < 5 % AR in most cases. RMS recalculated all DT50 values by using non-linear fitting of first order kinetics. A detailed summary of the study and the results can be viewed in Table 7.8. Table 7.8: Degradation/dissipation of 14C- milbemycin A4 in water/sediment systems. Mill stream pond, Clay loam Emperor Lake, sandy clay loam Aerobic/anaerobic Aerobic Aerobic Temperature (°C) 20 20 Duration (days) 100 100 Sand (%) 22.1 60.7 Silt (%) 47.5 18.3 Clay (%) 30.4 21 pH 7.7 6.2 Organic C. (%) 8.1* 5.5*


DT50 (water) (days) 1.8 3.9 DT50 (sediment) (days) 98 56 DT50 (whole system) (days) 89 82 CO (% AR) after 100 days 2 6.0 5.9 Bound residue (% AR) after 100 days 30 32 Radioactivity in water phase (%) 71 (0 d.)

4.5 (100 d.) 79 (0 d.)

3.6 (100 d.) 14C- milbemycin A4 in water phase (% AR)

67 (0 d.) nd (59 d.)

76 (0 d.) 1.4 (59 d.)

Metabolites > 5 % AR Maximum within 100 days

27-hydroxy-milbemycin A4: 4.7 (30 d.)

27-keto-milbemycin A4: 3.1 (30 d.) d.)

27-hydroxy-milbemycin A4: 7.2 (100 d.)

27-keto-milbemycin A4: 4.5 (100

References Lewis, 1999 (E1, AII, 7.2.1.3.2/01) * Unknown amount of iron sulphide caused overestimation and excessive variation of carbon content.

7.1.4 Fate and behaviour in air

Photolysis in air No data submitted. Degradation in air Hydroxyl reaction and ozone reaction half life were estimated by modelling to be 16.4 and 13.7 minutes respectively for milbemycin A3 and A4 (E1, B.8.8). Vaporization Milbemycin A3 and A4 both have a vapour pressure of <1.3x10-5 Pa and a Henry’s law constant of 2.63x10-3 and 1.59x10-3 Pa m3 mol-1 respectively indicating that significant volatilization is unlikely to occur (E1, B.8.8)

7.2 Exposure assessment

7.2.1 Soil

PIEC (predicted initial environmental concentration) has been estimated in different crops after either one or two applications. Time Weighted Averages and PECplateau have also been estimated. The results are presented in Table 7.9. The calculations are based on the following assumptions; DT50: 82 days (worst case, lab.), number of applications: 1-2 with a minimum interval between applications of 5 days, 40-70 % interception). In the PECplateau calculations application dates were set to spring/early summer (late summer in strawberries), but changing the dates within the summer months did not influence the results. Fig. 2 shows the accumulation curve of milbemycin after several years of use. Table 7.8: PIEC, PECtwa and PECplateau for milbemycin A4 in soil. Crop Appl. rate

(g a.s./ha) Crop cover

(%)

No. of appl.

Days between

appl.

PIEC (mg

a.s./kg soil)

PECtwa (mg

a.s./kg soil)

PECplateau (mg a.s./kg

soil)*

Strawberry 23.3 40 2 5 0.04 0.04 0.07 Strawberry 23.3 40 1 - 0.02 0.02 0.04 Apple/pear (Trees >2 m)

17.7 70 2 5 0.01 0.01 0.03

Apple/pear (Trees >2 m)

17.7 70 1 - 0.0007 0.007 0.01

Ornamentals (outdoors, height >125 cm)

18.6 50 2 5 0.02 0.02 0.05

Ornamentals (outdoors, height >125 cm)

18.6 50 1 - 0.02 0.02 0.02

* Finish PEC-calculator


Fig. 7.2: Predicted concentration and PECplateau of milbemycin in soil after use over several years.

7.2.2 Groundwater

Strawberries were used as a worst case culture in the assessment of groundwater exposure due to the fact that this crop has the highest application rate of all the crops in the GAP. The most important input data in the MACRO 4.4.2 model has been summarised in Table 7.9. The results of the modelling is summarised in Table 7.10 showing that all scenarios gave a PECgw << 0.001 µg/l. The modelling was run by Mattilsynet with Swedish and Norwegian ground water scenarios. Modelling performed in connection with the EU registration is described in the List of endpoints (E2) The modelling was performed with PEARL v. 1.1.1 and all the relevant FOCUS scenarios (Hamburg for apples in Germany, Chateaudun for apples in France, Sevilla for apples in Spain and Piacenza for apples in Italy). PECgw was calculated to be << 0.001 µg/l in all scenarios for both milbemycin A4 and the two metabolites 27-hydroxy-milbemycin A4 and 27-keto-milbemycin A4. Input parameters in the modelling of milbemycin A4 are summarised in Table 7.11. Table 7.9: Input-parameters for FOCUS groundwater modelling with Norwegian and Swedish scenarios (PECgw). Application rate: 2x23.3 g a.s./hs Interval: 7 days First day of treatment: 15th may (day 196) Plant cover: 40 % Crop: Strawberries Plant uptake: 0.5 Active substance Milbemycin A4 Half life (d): 36.5 Koc: 2817 1/n: 0.98 Table 7.10: PECgw for Milbemycin A4 in strawberries estimated in Swedish and Norwegian ground water scenarios with MACRO 4.4.2. Scenario PECgw, 80th percentile (µg/l) Önnestad < 0.001 Krusenberg < 0.001 Heia < 0.001 Table 7.11: Input-parameters for milbemycin A4 in FOCUS groundwater modelling with relevant FOCUS scenarios (PECgw). Scenario and crop

Hamburg, apples Chateaudun, apples Sevilla, apples

Application rate: 2x0.0188 g a.s./ha 1x0.0174 g a.s./ha 2x0.0174 g a.s./ha

0,00

0,01

0,02

0,03

0,04

0,05

0,06

0,07

0,08 concentration (mg/kg) in soil 0-5 cm

substance

-79 -81 -83 -85 -87 -89 -91 -93 -95 -97


Interval: - 10 days 10 days Half life (d): 43* 43 43 Kom: 1670 (Koc: 2879) 1670 (Koc: 2879) 1670 (Koc: 2879) * Arithmetic mean

7.2.3 Surface water

Surface water modelling step 3 has been performed by Mattilsynet according to the Norwegian GAP. Application windows were chosen according to the crop development specified for each scenario as described in FOCUS guidance and adjusted to Norwegian conditions as much as possible with regard to crop growth stages and relevant application dates. Different approaches to this can be viewed in the DAR (E1), list of Endpoints (E2) and the Swedish report (E3) but none of these approaches completely fulfil Norwegian requirements. Leafy vegetables were chosen as a representative crop for strawberries in addition to pome fruit as these were the biggest and most relevant crops applied in Norway. In the modelling performed by Mattilsynet, the same input parameters as described by Swedish authorities were used. The highest PECsw values were observed right after the second application, indicating that spray drift is the main route of exposure. Modelling/PECsw, PECsed. Input parameters for the modelling with milbemycin A4 are summarized in Table 7.12 and Table 7.13, and the most relevant results are summarized in Table 7.14. Table 7.12: Data on the number of applications, application rates, crops etc, used in surface water modelling. Leafy vegetables were used as substitute for strawberries in the modelling as strawberries is not an official FOCUS crop.

Scenario D=Drainage R=Runoff

Crop (Nr. in season in brackets)

Water body Application rate

(g a.s./ha)

Nr. of applications

Min. interval between

applications (days)

Application windows (first and

last possible

day of application)

Actual application

dates (picked by SWASH)

D3, Vredepeel

Vegetables, leafy* (1st)

Ditch 23.3 2 5 15/6-20/7 14/6 25/6

D3 Vegetables, leafy* (2nd)

Ditch 23.3 2 5 5/8-20/10 17/9 22/9

D4, Skousbo

Vegetables, leafy*

Pond 23.3 2 5 10/5-26/9 16/5 21/5

D4 Vegetables, leafy*

Stream 23.3 2 5 10/5-26/9 16/5 21/5

D6, Thiva Vegetables, leafy*

Ditch 23.3 2 5 15/8-30-11 19/8 25/8

R1, Weiherbach


Pond 23.3 2 5 15/6-20/7 29/6 11/7

R1 Vegetables, leafy* (1st)

Stream 23.3 2 5 15/6-20/7 29/6 11/7

R1 Vegetables, leafy* (2nd)

Pond 23.3 2 5 27/9-1/11 6/10 11/10


Stream 23.3 2 5 27/9-1/11 6/10 11/10

R2, Porto Vegetables, leafy* (1st)

Stream 23.3 2 5 10/6-15/7 10/6 15/6


Stream 23.3 2 5 27/10-1/12 27/10 8/11

R3, Bologna


Stream 23.3 2 5 6/5-10/6 18/5 1/6


Stream 23.3 2 5 27/9-1/11 27/9 22/10

R4, Roujan Vegetables, leafy* (1st)

Stream 23.3 2 5 6/5-10/6 6/5 27/5


Stream 23.3 2 5 27/9-1/11 27/5 15/10


D3 Pome/stone fruit, early application

Ditch 17.7 2 5 15/4-1/7 20/4 4/5


Pond 17.7 2 5 20/4-5/7 20/4 30/5


Stream 17.7 2 5 20/4-5/7 20/4 30/5

D5, La Jailliere

Pome/stone fruit, early application

Pond 17.7 2 5 1/4-31/5 8/4 14/4


Stream 17.7 2 5 1/4-31/5 8/4 14/4

R1 Pome/stone fruit, early application

Pond 17.7 2 5 15/4-1/7 26/4 2/5


Stream 17.7 2 5 15/4-1/7 26/4 2/5


Stream 17.7 2 5 15/3-31/7 22/3 22/4


Stream 17.7 2 5 1/4-31/5 4/4 11/4


Stream 17.7 2 5 15/4-31/5 15/4 20/4

D3 Pome/stone fruit, late application

Ditch 17.7 2 5 15/4-1/7 20/4 4/5


Pond 17.7 2 5 20/4-5/7 20/4 30/5


Stream 17.7 2 5 20/4-5/7 20/4 30/5


Pond 17.7 2 5 1/4-31/5 8/4 14/4


Stream 17.7 2 5 1/4-31/5 8/4 14/4

R1 Pome/stone fruit, late application

Pond 17.7 2 5 15/4-1/7 26/4 2/5


Stream 17.7 2 5 15/4-1/7 26/4 2/5


Stream 17.7 2 5 15/3-31/7 22/3 22/4


Stream 17.7 2 5 1/4-31/5 4/4 11/4


Stream 17.7 2 5 15/3-31/5 15/4 20/4

Table 7.13: Input parameters for milbemycin A4 for the FOCUS step 3 surface water modelling (PECsw, PECsed) using SWASH.

FOCUS surface water step 3 Molecular weight [g mol-1] 542.7 Water solubility [mg L-1]; 20 °C 4.55


Vapour pressure [Pa] 4.3x10-10 Kfoc[L kg-1] 2817 1/n 0.98 DT50 whole system [days] 86

Table 7.14: Worst case PECsw-values for milbemycin A4 in surface water and sediment for applications in leafy vegetables (strawberries) and fruit, step 3, 2 applications.

PECSW (µg/L) PECSED (µg/kg dw)

Scenario Crop Waterbody

Max 28 d TWA Max 28 d TWA

R2, Porto Vegetables, leafy Stream 0.112 0.00878 7.928 7.296 R4, Roujan Vegetables, leafy Stream 0.180 0.0135 2.595 2.233 R3, Bologna Pome/stone fruit Stream 1.340 0.0356 0.416 0.220 D3, Vredepeel Pome/stone fruit Ditch 1.181 0.0936 0.975 0.506


8. Ecotoxicology This assessment is based on documentation submitted by the applicant (referenced with author and year), EUs Draft Assessment Report (DAR) (E1), the European Commission’s Review report for the active substance milbemectin (04 April 2005) with List of Endpoints (E2), and Swedish KemIs assessment from 2010 (E3). Applied application rates result in a release of 1.767 g a.s./decare (daa) to the environment in apple/pear, 2.33 g a.s./daa in strawberry, and up to 1.9 g a.s./daa in ornamentals. Number of applications pr season: 2. The active substance milbemectin is a mixture of two microbially produced compounds: milbemycin A and milbemycin A , naturally occurring at a ratio of approximately 3:7. All Annex II ecotoxicological studies have been conducted with technical milbemectin, containing the two components milbemycin A and milbemycin A in the appropriate relative amounts.

3

4

3 4

8.1. Milbemectin

8.1.1. Terrestrial organisms

8.1.1.1. Mammals Toxic to mammals (Acute LD50: 456 mg/kg bw/d). Low reproductive toxicity (NOEC: 200 mg/kg feed). Test substance Species Type of

study LD/LC50 (mg/kg

bw )

NOEL/ NOEC (mg/kg feed )

Reference

Milbemectin technical

Rat Acute 456 - E2


Rat Reproductive 200 E2

8.1.1.2. Birds Toxic to birds (Acute LD50: 347 mg/kg bw/d). Moderate dietary toxicity (LC50: 1922 mg/kg feed) and moderate reproductive toxicity (NOEC: 150 mg/kg bw/d). Test substance

Species Type of study

LD50 (mg/kg bw)

LC50 (mg/kg feed)

NOEC (mg/kg feed)

Reference


Anas platyrhynchos

Acute 347 - - E2


Anas platyrhynchos

Dietary - 1922* - E2


Anas platyrhynchos

Reproduction

- - 150 E2

*recalculated based on average measured concentrations in the feed (from 8-day LC50: 1969 mg/kg feed). 8.1.1.3. Bees Very high contact toxicity (LD50: 0.026 µg/bee) and high oral toxicity (LD50: 0.40 µg/bee) to bees. Test substance Species Contact LD50

(µg/bee) Oral LD50 (µg/bee)

Reference

Milbemectin technical Apis mellifera 0.026 0.40 E2


8.1.1.4. Non-target arthropods See the section on Plant Protection Product 8.1.1.5 Earthworms Toxic to earthworms (Acute LC50corr: 28.5 mg/kg d.w. soil). Moderate toxicity for the metabolites (LC50: >250->500 mg/kg d.w. soil). Test substance

Species Exposure LC50 (mg/kg dry soil

with standard 5% OM)*

Reference

Milbemectin Eisenia fetida Acute 28.5 E2 27-hydroxy-milbemycin A4

Eisenia fetida Acute >250 E2

27-keto-milbemycin A4

Eisenia fetida Acute >500 E2

*Corrected for the difference between the organic matter content of the test soil and that of the soils where the substance will be used since log Kow>2. 8.1.1.6. Microorganisms The effects of milbemectin technical on carbon mineralization and nitrogen transformation by soil microflora in three soil types (a common agricultural soil; a loam soil; and a humus sandy soil) were investigated in a laboratory test. Milbemectin technical was applied at a concentration equivalent to 75 g a.s./ha (3 x the maximum expected concentration). For each soil type, nitrogen transformation was evident from the decline in ammonium concentrations and increase in nitrate concentrations over the first 14 days. Although statistical evaluation of this data showed a significantly greater level (P<0.05) of ammonium in the sandy soil in Day 14, a significantly lower level (P<0.05) of nitrate in the sandy soil on Day 28, and a significantly greater level (P<0.01) of ammonium in the loam soil on Day 28 compared to the respective control soils, at the end of the 28 day study period the deviations in measured activity in the treated soils compared to the controls were less that 25% (the trigger is more than 25% after 100 days)) for both ammonium and nitrate concentrations for all soil types. For each soil type, carbon mineralization in the treated soils was evident from the cumulation of CO2 over the first 14 days. At the end of the 28 days study period, the deviations from the cumulative mean ppm C as CO2 /g soil for the treated soils compared to the control were less than 25% (trigger) at 10.3 % (sandy soil), 2.77% (loam soil) and 24.7% (humus soil). The results from this study indicate that carbon mineralization and nitrogen transformation by soil microflora in three soil types at three times the maximum expected concentration were comparable to the results obtained from the control soils, the deviations in measured activity at the end of the study period being less that the trigger of 25% for all parameters examined. (Carter & Jackson, 1995) 8.1.1.7. Terrestrial plants Twenty tests are available for a number of crop species with technical milbemectin. Non-crop data were not available. The rates used in the tests were at least 14 times higher than the single maximum dose in the field. Except for corn, the lowest NOEC (emergence, shoot length and shoot weight) was 90 g a.s/ha which is more than 4 times the maximum single dose in the field. With corn, a significant reduction of the shoot weight was measured at 30, 270, and 2430 g a.s./ha, although in all treatments this was not more than 20% (the trigger is more than 50% effect at the maximum application rate). In corn, the NOEC was considered to be <30 g a.s./ha. The results indicate that the potential effect of milbemectin is considered to be low. (E1)

8.1.2 Aquatic organisms

8.1.2.1. Fish Extreme acute toxicity to fish (96h LC50: 4.4-35 µg a.s./L). Extreme chronic toxicity (Early life stage test NOEC: 0.65 µg a.s./L).


Test substance

Species Exposure LC50 (µg a.s./L)

NOEC (µg a.s./L)

Reference

Milbemectin Rainbow trout Oncorhynchus mykiss

96 h 4.4 2.0 Graves & Swigert 1996

Milbemectin Bluegill sunfish Lepomis macrochirus

96 h 28 12 Graves & Swigert 1995

Milbemectin Fathead minnow Pimephales promelas


Milbemectin Sheepshead minnow Cyprinodon variegatus


Milbemectin Common carp Cyprinus carpio

96 h 35 25 Gries et al., 2003

Milbemectin Rainbow trout Oncorhynchus mykiss

Early Life Stage test

- 0.65 Graves et al., 1996

8.1.2.2. Bioconcentration Milbemectin shows a moderate potential for bioconcentration. In a bioconcentration study, bluegill sunfish (Lepomis macrochirus) were exposed for 28 days to 4.1-4.6 µg a.s. equivalents/L followed by a 14 days depuration period. The average whole fish BCF was 76 and 114 at steady state for milbemycin A3 and milbemycin A4, respectively. Rapid depuration occurred (CT50: 0.69-1.1 days). After the 14 days depuration period, 3.2-6.3 % (% of the steady state concentration) milbemectin was measured. (Drottar et al., 1998) 8.1.2.3. Invertebrates Extreme acute toxicity to Daphnia magna (48h EC50: 11 µg a.s./L). Extreme chronic toxicity to Daphnia magna (21d NOEC: 0.12 µg a.s./L). Test substance Species Exposure EC50

(µg a.s./L) NOEC

(µg a.s./L)Reference

Milbemectin Daphnia magna 48 h 11 0.90 Graves & Swigert 1996

Milbemectin Daphnia magna 21 d - 0.12 Graves et al., 1996

8.1.2.4. Sediment-dwelling organisms Extreme chronic toxicity to Chironomus riparius larvae (28d NOEC: 6.3 µg/L (spiked water)). Test substance Species Exposure EC50

(µg a.s./L) NOEC


Milbemectin Chironomus riparius

28 d 20.4 6.3 Mattock, 2001

8.1.2.5. Aquatic plants High toxicity to duckweed (14d EC50: >620 µg a.s./L). Test substance Species Exposure EC50

(µg a.s./L) NOEC


Milbemectin Lemna gibba 14 d >620 620 Thompson & Swigert, 1996

8.1.2.6. Algae No effects on algae at the highest tested concentration (72h EC50: >2000 µg a.s./L).


Test substance Species Exposure EC50 (µg a.s./L)

NOEC (µg a.s./L)

Reference

Milbemectin Selenastrum capricornutum

120 h >2000 (r) >2000 (b)

2000 Graeme et al., 1995

8.1.2.7. Microorganisms Milbemectin did not inhibit the respiration of activated sludge at concentrations up to and including 1000 mg/L, the highest concentration tested. (Bealing & Watson, 2001) 8.1.2.8. Micro-/mesocosm studies See section on plant protection product.

8.2 Co-formulants

One of the formulants is toxic to aquatic organisms, but it is not expected to increase the toxicity of the formulated product.

8.3 Plant Protection Product

8.3.1 Terrestrial organisms

Birds After exposure to the formulated product Milbeknock 1% EC, the LD50 for the mallard duck was found to be higher than the highest dose of 2250 mg product/kg bw (>23.4 mg as/kg bw). Test substance

Species Type of study

LD50 (mg product/kg bw)

(mg a.s./kg bw)

NOED (mg/kg bw)

Reference

Milbeknock 1% EC

Anas platyrhynchos

Acute >2250 (>23.4) 486 E1

Bees Very high contact (LD50: 0.047µg a.s./bee) and high oral (LD50:>0.65 µg a.s./bee) toxicity to bees. Test substance Species 48-hr Contact LD50

(µg product/bee) (µg a.s./bee)

48-hr Oral LD50 (µg product/bee)

(µg a.s./bee)

Reference

Milbeknock 1% EC Apis mellifera

- >65 >0.68

Hoxter et al., 1998a

Milbeknock 1% EC Apis mellifera

0.047

- Hoxter et al., 1998b

Semi-field test The acute toxicity of Milbeknock 1% EC to bees was tested under semi-field conditions in a tent cage test. The test was carried out according to EPPO Guideline 170 in Rossdorf, Germany from August 31 to September 15, 2003. The test comprised of 3 cages (tents) treated with Milbeknock at 27.9 g a.s./ha, applied shortly before bee flight (early morning application); 3 cages (tents) treated with 2.96 g a.s./ha (drift rate) during bee flight; 3 cages (tents) treated with 7.5 g a.s./ha during bee flight; three cages (tents) treated with water (controls); and 3 cages (tents) treated with a toxic standard (Perfekthion EC, 400 g/L dimethoate). The cages contained a plot of flowering Phacelia (2 x 3 m) and each cage contained one bee hive with a small healthy bee colony. Mortality and foraging activity of the bees were assessed before (5 days) and after (7 days) application. Dead bees were collected from a trap in front of the hive and from the side paths. Sublethal effects, such as changes in behaviour, were also monitored. Colony assessments (food stores incl. pollen, eggs, larvae, number of dead pupae etc) were made at the beginning and at the end of the study (10 days after treatment). During


the test, mean temperatures ranged from 12.4 to 18.6°C and mean relative humidity from 55.4% to 93%. Major rainfall events occurred on the evening day 1 after application (9.8 mm/day) and on day 3 after application (8.8 mm/day). However, these rainfall events occurred after the main exposure period. The results indicate similar natural mortality levels among the different treatment groups before application. After exposure of the bees to 27.9 g a.s./ha before bee flight there was a slightly higher mortality compared to the levels found in the water-treated control, but this was not significant. Mortality levels after direct spray application at rates of 2.96 g a.s./ha and 7.5 g a.s./ha were comparable to the mortality levels before treatment and after the control treatment. After treatment with toxic standard (Perfekthion), a strong increase of bee mortality was observed. A high number of foraging bees (ranging from 12-25 bees/m2 compared to the required >10/m2 by the protocol) were present shortly before the treatment. No reduction in flight intensity was observed after application in any Milbeknock treatment compared to the water-treated control. The application of the toxic standard led to a clear decrease in flight intensity. No abnormalities in behaviour were observed after any milbemectin treatment, but in the toxic control aggressiveness and trouble with coordinating movements were observed. Ten days after treatment there were no treatment related differences in brood development compared to the control. Not all brood stages could be found in each of the colonies at the end of the test. In all hives (also in the control), the level of brood was decreasing, but the development of the bees was normal for the time of the season. (Schmitzer, 2004) Good levels of exposure were obtained for the applications during bee flight as demonstrated by the high number of foraging bees observed before application and by the high mortality obtained with the toxic standard. There was no significant increase in mortality after application of 27.9 g a.s./ha (higher than the highest dose applied for in Norway), and no effects on flight intensity, behavior or brood. Non-target arthropods In Tier 1 laboratory acute contact toxicity studies, Milbeknock showed negligible effects on parasitoids and ground dwelling predators at relevant application rates. For foliage dwelling predators and predatory mites, the trigger of >30% effect is exceeded. The table below is from the EU Draft Assessment Report (E1)


Species Substrate and duration

Application dose [L product/ha]

Effects Risk classification

Predatory mites Typhlodromus pyri glass, 7 d 2.4 100% mortality high Typhlodromus pyri glass, 7 d 4 100% mortality high Typhlodromus pyri leaves, 7 d 0.24 and 0.48 96 and 100% mortality high Typhlodromus pyri leaves, 7 d 0.12 29% mortality, 77% reduction in fecundity medium Typhlodromus pyri leaves, 7 d =0.06 =9% mortality low Typhlodromus pyri leaves, 7 d LC50 0.14 kg product/ha (0.0014 kg as/ha) Typhlodromus pyri leaves, 14 d 4 100% mortality high Typhlodromus pyri leaves, 14 d 4 65% mortality medium Typhlodromus pyri leaves, 14 d 4 49% mortality medium Typhlodromus pyri Leaves, 7d 3.742 81,5 % mortality high Typhlodromus pyri Leaves, 7d 0.580 12,9% mortality, 1.6% reduction in

fecundity low

Typhlodromus pyri Leaves, 7d 0.0561 0% mortality, 45.3% reduction in fecundity medium Typhlodromus pyri Leaves, 7d 3 x 3.226 71.4% mortality high Typhlodromus pyri Leaves, 7d (residues

aged for 7 days) 3.742 8,8% mortality, 56.9% stimulation of

fecundity low

Typhlodromus pyri Leaves, 7d (residues aged for 7 days)

0.580 2,9% mortality, 19% stimulation of fecundity low


0.0561 2% mortality, 50% stimulation of fecundity low


3 x 3.226 7.4% mortality, 91.4% stimulation of fecundity

low

Phytoseiulus persimilis strawberry field, 20 d 2x2.25 kg/ha 93% decrease in # adults 86% decrease in # eggs

medium to high

Leaf dwelling predators Chrysoperla carnea leaves 1 8% mortality low Chrysoperla carnea leaves 3 13% mortality low Chrysoperla carnea leaves 3 21% mortality, no reduction in fecundity low Orius laevigatus glass, 10 d 2.4 100% mortality high Orius laevigatus glass, 10 d 4 100% mortality high Orius laevigatus leaves, 7 d 1 22% mortality low Orius laevigatus leaves, 7 d 3 41% mortality medium Orius laevigatus leaves, 10+10 d 1 0% mortality, no reduction in fecundity low Orius laevigatus leaves, 10+10 d 3 3% mortality, no reduction in fecundity low Orius laevigatus Leaves, 7d 3.742 0% mortality, 29% reduction in fecundity low Orius laevigatus Leaves, 7d 0.580 5,5% mortality, 25% reduction in fecundity low Orius laevigatus Leaves, 7d 0.0561 0% mortality, 23% reduction in fecundity low Orius laevigatus Leaves, 7d 3 x 3.226 0% mortality, 4% reduction in fecundity low Orius laevigatus Leaves, 7d (residues

aged for 7 days) 3.742 0% mortality low

Orius laevigatus Leaves, 7d (residues aged for 7 days)

0.580 1,8% mortality low


0.0561 9.2% mortality low


3 x 3.226 16.6% mortality low

Coccinella septempunctata glass, 39 d 2.4 100% mortality high Coccinella septempunctata glass, 39 d 4 100% mortality high Aphid parasitoids Aphidius rhopalosiphi glass, 48 h 2.4 100% mortality high Aphidius rhopalosiphi glass, 48 h 4 100% mortality high Aphidius rhopalosiphi plants, 48+24 1 4% mortality, no reduction in fecundity low Aphidius rhopalosiphi plants, 48+24 3 10% mortality, 12.5% (ns) reduction in

fecundity low

Aphidius rhopalosiphi Leaves, 7d 3.742 6,7% mortality, 2% reduction in fecundity low Aphidius rhopalosiphi Leaves, 7d 0.580 0% mortality, 21.8% stimulation of fecundity low Aphidius rhopalosiphi Leaves, 7d 0.0561 0% mortality, 17.8% stimulation of fecundity low Aphidius rhopalosiphi Leaves, 7d 3 x 3.226 0% mortality, 14.9% reduction in fecundity low Aphidius rhopalosiphi Leaves, 7d (residues

aged for 7 days) 3.742 0% mortality low

Aphidius rhopalosiphi Leaves, 7d (residues aged for 7 days)

0.580 0% mortality low


0.0561 0% mortality low


3 x 3.226 0% mortality low

Ground dwelling predators Poecilus cupreus sand, 14 d 3 no mortality, no effect on feeding low


Earthworms In the 14 day study with the formulation, the LC50 was higher than the highest tested concentration of 1067 mg product/kg (>11 mg as/kg). The effects of Milbeknock 1% EC on the mortality, body weight, feeding activity and reproduction of adult Eisenia fetida were investigated in the laboratory. Statistically significant effects on reproduction were observed at the two highest test concentrations resulting in a NOEC of 21.72 mg/kg dry soil = 0.2171 mg a.s./kg. A NOEC of 0.109 mg a.s./kg d.w. soil (corrected for 5% organic matter content) is used in the risk assessment.(E2) Test substance Species Exposure EC/LC50

(mg/kg) NOEC (mg/kg) Reference

Milbeknock 1% EC

Eisenia fetida 14 days >1067 mg product/kg

>11 mg a.s./kg

270 mg product/kg dry soil

E1

Milbeknock 1% EC

Eisenia fetida 8 weeks 21.72 mg product/kg (0.109 mg a.s./kg)*

E2

* corrected for 5% OM


Fish Extreme acute toxicity to rainbow trout (96h LC50: 5.7 µg a.s./L). Test substance Species Exposure LC50

(µg a.s./L) NOEC


Milbeknock EC (1.0 %)

Rainbow trout Oncorhynchus mykiss

96 h (static with sediment)

10.2 5.4 Gries & Purghart, 2003



96 h (static) 5.7 3.2 Gries et al., 2003



96 h (flow-through)

4.8 - End Points, Registration Report, 2005

Invertebrates Extreme acute toxicity to Daphnia magna (48h EC50: 3.43 µg a.s./L) and very high toxicity to other invertebrates (LC50: 49.3-187 µg a.s./L). Test substance Species Exposure LC50

(µg a.s./L) NOEC



Daphnia magna 48 h (static with sediment)

3.43 - Gries et al., 2003


Daphnia magna 96 h (static) 5.33 1.5 Gries et al., 2003

Milbeknock EC (1 %)

Gastropoda (Planorbidae)

48 h 187 100 Kroos, 2003


Ephemeroptera, Baetidae

48 h screening

461 125 Kroos, 2003


Chaoboridae 48 h screening

49.3 10 Kroos, 2003


Cladocera, Daphniidae

48 h screening

62.7 25 Kroos, 2003


Copepoda, Cyclopoidae

48 h screening

85.9 50 Kroos, 2003


Sediment-dwelling organisms Extreme acute toxicity to Chironomus riparius larvae (48h EC50: 30.1 µg a.s./L) and medium acute toxicity to the oligochaeta Tubificidae (48h EC50: 1142 µg a.s./L). Test substance Species Exposure LC50

(µg a.s./L) NOEC


Milbeknock EC (1 %)

Chironomidae 48 h 30.1 1.5 Kroos, 2003

Milbeknock EC (1 %)

Tubificidae 48 h 1142 125 Kroos, 2003

Aquatic plants No information. Algae Very high toxicity to algae (72h EC50: 220 µg a.s./L). Test substance Species Exposure EbC50

(µg a.s./L) NOEC


Milbeknock EC (1 %)

Selenastrum capricornutum

120 h 220 38 End Points, Registration Report, 2005

Micro-/mesocosm studies Microcosms consisting of three 18 L glass cylinders, each containing a sediment layer of approximately 0.02 m and a water layer of 0.3 m were prepared using sediment and water from an uncontaminated eutrophic ditch at the Sinderhoeve Experimental Station (van Wijngaarden 2006). The microcosms simulated a plankton-dominated nutrient-rich system. The planktonic communities contained algae, cladocerans, copepods, ostracods and rotifers. The seeding material originated from the plankton in the experimental ditches and from other uncontaminated experimental ponds also located at the Sinderhoeve Experimental Station. In order to increase the numbers of cladocerans, an additional collection was carried out and a zooplankton concentrate (mainly consisting of Daphnia longispina / pulex and Simocephalus vetulus) was added to each microcosm. Daphnia magna were additionally introduced into the microcosms, these were obtained from a temporary laboratory culture. Since a pilot study had indicated that the cladoceran Chydorus sphaericus was potentially sensitive 25 individuals were added to each microcosm. Milbeknock EC was introduced as two applications each at initial intended concentrations of 0.058, 0.23, 0.92, 3.68, 14.7 and 58.9 µg a.s./L. The interval between the applications was 10 days. There were 3 replicates of each treatment, including the controls. The test lasted 60 days after the second treatment. The concentrations of the active substance were monitored 3 and 9 days after the first application and 3 and 7 days after the second. Samples of the zooplankton were taken 8 days and 1 day before the first application, and then at 3, 8, 13, 21, 28 35, 42, 49, 56 and 70 days after the first application of Milbeknock EC. Dissolved oxygen, temperature and pH were measured at mid-water depth. The measurements were carried out around mid-day. Measurements were performed in each microcosm 7 days and 1 day before application, and then 2, 8, 15, 22, 29, 36, 43 50, 57 and 70 days after the first application. Conclusion from the company (Annex E5): Treatment (µg a.s./L)

Highest peak (µg a.s./L)

Response

0.058 0.068 No treatment-related effects observed. NOECpopulation for most sensitive group (calanoid copepods).

0.23 0.28 Clear short-term effects on 1 copepod taxon (Calanoida). No effects at the community level: NOECcommunity


0.92 1.17 Slight transient effect: reduction 1 copepod taxon (Cyclopoida) Longer term effect: reduction 1 copepod taxon (Calanoida) but low numbers and recovery limited by closed system. Slight transient effect at the community level (one sampling date, duration < 1 week). A more consistent NOEC for the community was at this treatment level.

3.68 4.67 Clear short-term (most sensitive cladocerans and copepods (Cyclopoida, nauplii, total)) with recovery by 32 days after 2nd application (except Calanoida where numbers low and recovery limited by closed system). Clear short-term effect at the community level.

14.7 19.9 Clear long-term and short-term effects: reductions in copepods and cladocera. Indirect effects (increases) on rotifers (mostly short-term) and community metabolism. Clear short-term effect at the community level.

58.9 74.3 Clear long-term effects: reduction in copepods and cladocerans. Indirect effects (increases) on rotifers. Consistently higher planktonic chl-a levels. Changed community metabolism and water chemistry endpoints. Clear long-term effect at the community level. In most cases, changes lasted up to and including the end of the experiment.

A consistent NOECpopulation for sensitive taxa: (Cladocera, total Copepoda): is 0.92 µg a.s./L, with effects at 3.68 µg a.s./L recovered within 32 days after 2nd application. A consistent NOECcommunity: is 0.92 µg a.s./L, with effects at 3.68 µg a.s./L showing recovery within 25 days after the 2nd application. On the basis of whole study results, it is considered that the critical endpoint for the microcosm study for use in the aquatic risk assessment is the NOEAEC of 3.68 μg a.s./L. In the case of the NOEAEC, a number of factors need to be taken into account, including the magnitude and duration of any effects seen, the range and significance of taxa affected and the causal nature (direct or indirect), as well as the general issues identified in the SANCO guidance document. In total, 46 zooplankton taxa were differentiated in the microcosm systems, indicating the presence of relatively diverse communities. With increased community diversity, the possibility of including outlier taxa i.e. from the tail of the susceptibility distribution is increased. Given this situation, the presence of 1 group (in this case the calanoid Copepoda), showing a higher level of effect is not surprising as it represents only about 2% of the total taxa identified. In the case of majority of the taxa (about 98% of those identified), a consistent picture was found with population NOECs of 0.92 µg a.s./L and short-term effects (i.e. recovery within 32 days after the 2nd application) at 3.68 µg a.s./L. There are a number of other reasons for considering the response of the calanoid Copepoda in the test system not to be representative of zooplankton communities when considering the risk assessment for aquatic invertebrates. Initial numbers of this group were low (less than 10 individuals/L in the controls), so that it would have been inherently unstable in the test system (vulnerable to perturbation), as pointed out in the SANCO guidance. Also, given the closed nature of the microcosm systems, recovery was limited to internal population growth with no natural recolonisation. Under these test conditions the recovery potential of numerically small group such as the calanoid Copepoda will have been greatly limited. This will have been compounded by indirect effects within the small test community e.g. competitive pressures. Thus, if they experienced an initial treatment effect resulting in reduced numbers, these pressures will have increased as other groups (e.g. other copepod groups and cladocerans) showed greater potential for increase. Thus, the longer recovery time for the calanoids is likely to have been due in part at least to these specific aspects related to the test design. This assessment is supported by the results of a preliminary (range-finding) study in which no effects on the Copepoda (cyclopods and calanoids) were seen at 0.23 µg a.s./L and only slight effects at 2.3 µg a.s./L. Taking the results of the microcosm study as a whole gives a clear picture. In the case of majority of the taxa (about 98% of those identified), there is a population NOEC of 0.92 µg a.s./L and short-term effects (i.e. recovery within 32 days after the 2nd application) at 3.68 µg a.s./L. This demonstrates that even if there are any initial effects in natural water bodies following the use of milbemectin, there will


be an acceptable level of recovery for both individual species and the community as a whole. On the basis of these results, it is considered that the critical endpoint for the microcosm study for use in the aquatic risk assessment is the NOEAEC of 3.68 µg a.s./L. Conclusion from the Swedish Chemicals Inspectorate (Annex E3): Performance of the test: As indicated in the checklist above the test was well performed and reported. Representativity of the test system: The test system represents a plankton dominated community and addresses uncertainty regarding effects on zooplankton and algal communities. The community was dominated by relatively few crustacean zooplankton species and several species of rotifers. From inspection of the raw data KemI interprets that it was only for three species of cladocerans and the copepod nauplii and Cyclopoids, that a statistical analysis is meaningful, i.e they were present in most enclosures in high enough numbers and low variability between ‘cosms. Each system only consisted of 14-18 L water, thus the ability to maintain a stable community with many species was limited. Nevertheless, RMS considers that the test system can be used to infer effects on the plankton community. However, from the information obtained from the single species tests it cannot be concluded that the zooplankton community is more sensitive than other invertebrates (e.g. insects and larger crustaceans). Static laboratory test indicate that Daphnia is less sensitive than the tested Chironomid and Chaoborus with a factor of 1.5-2, but more sensitive than a tested Ephemeroptera (see Tab. 1 below).

Tab. 1: Acute toxicity to aquatic invertebrates (Table IIIA 10.2.1.3-1. from the Tier2-IIIa-SS6.doc)

Test species Chemical Test sytem EC50 (g

a.s./L)*

NOEC (g a.s./L)

Report Authors

Daphnia magna Technical 48-hour flow-through

11 1.7 Graves, W.C. & Swigert, J.P., 1996c

Daphnia magna EC Formulation

48-hour flow-through

4.4 1.8 Graves, W.C. & Krueger, H.O., 1997b

Ephemeroptera, Baetidae

EC Formulation

48-hour static 461 125 Kroos, M., 2003a

Chaoboridae EC Formulation

48-hour static 49.3 10 Kroos, M., 2003b

Cladocera, Daphniidae

EC Formulation

48-hour static 62.7 25 Kroos, M., 2003c

Copepoda, Cyclopoidae

EC Formulation

48-hour static 85.9 50 Kroos, M., 2003d

Gastropoda (Planorbidae)

EC Formulation

48-hour static 187 100 Kroos, M., 2003e

Chironomidae EC Formulation

48-hour static 30.1 1.5 Kroos, M., 2003f

Tubificidae EC Formulation

48-hour static 1142 125 Kroos, M., 2003g

* Values as reported in Review Report (SANCO/10386/2002 -rev. final, 18 November 2005), where applicable Therefore in order to resolve the uncertainty relating to effects on the invertebrate aquatic community also insects and larger crustaceans (e.g. gammarids) should have been included in the test. Power to detect effect of the test system:


The power to detect effects were not reported, however it was probably rather low, given that only three replicates per treatment level was available and the relatively large variation between replicates. Conclusion, selection of endpoint and safety factor: The most sensitive group was Calanoid copepods which was present in very low numbers and with a high variability. KemI agrees with the authors that the results indicate a reduction at a treatment with 0.23 µg/L (statistically significant, William test between days 3-21). The applicant argues that the effect observed for calanoid copepods should not be regarded as ecologically significant and a NOAEC of 3.68 should be used for risk assessment. However, calanoid copepods are important constituents of aquatic ecosystems and in a system where these groups would have been present in higher numbers an effect might have been more marked. Therefore the NOEC from the ‘cosm study is 0.058 µg/L. In general Sweden applies an application factor of 2-3 on a NOEC from a well performed and representative ‘cosm study in order to derive an EAC (Ecologically Acceptable Concentration, i.e the concentration at which no unacceptable effects are likely to occur). However, the present study does not address the uncertainty relating to effects on insects and larger crustaceans. When deriving an EAC the results from single species tests should also be considered and since it cannot be concluded that the most sensitive organisms were present in the ‘cosms it cannot be used to override the results from the first tier test. Furthermore, even when using this non representative NOEC in a TER calculation the result is below 1, which cannot be considered acceptable. Conclusion from the Norwegian Food Safety Authority: The Norwegian Food Safety Authority agrees with KemI that the NOEC should be 0.058 µg a.s./L.

8.4 Toxicity/Exposure estimates

8.4.1 Terrestrial organisms

Mammals Acute toxic to mammals (LD50: 456 mg/kg bw/d). TERacute for the indicator species small herbivorous mammal in orchards is estimated as 156 and 574 in strawberries. These values do not exceed the trigger (<10). Moderate reproductive toxicity, NOEC: 200 mg/kg. TERchronic is estimated to be 209 in orchards and 904 in strawberries. These values do not exceed the trigger (<5). Birds Milbemectin is acutely toxic to birds (LD50: 347 mg/kg bw). The risk assessment is based on the Guidance Document on Risk Assessment for Birds and Mammals under Council Directive 91/414 (SANCO, 2002). The main route of exposure to birds from foliar applied pesticides, such as milbemectin, is to residues on food items such as treated vegetation, insects, earthworms etc. According to the Guidance Document on Terrestrial ecotoxicology, when pesticides are applied as a spray, the residues on food are better considered in terms of the active substance rather than the formulation. The recommended species for the orchard scenario is a small insectivorous bird (such as a 10 g wren) and for the leafy crop scenario (strawberries) the recommended species are a medium herbivorous bird (such as a 300 g pigeon) as well as the insectivorous bird. TERacute for the indicator species in orchards is estimated as 363, and in strawberry field the TERacute is estimated as 161 and 275 for herbivorous and insectivorous birds, respectively. These values do not exceed the trigger (<10). Milbemectin has moderate dietary toxicity (LC50: 1922 mg/kg feed), TERshort-term for all indicator species in all crops are estimated as >1000, which do not exceed the trigger (<10). Milbemectin also has a moderate reproductive toxicity (NOEC: 150 mg/kg). TERchronic is estimated to be 281 for the indicator species in orchards, and 250 for herbivorous birds and 213 for insectivorous birds in strawberry fields. These values do not exceed the trigger (<5). Bees Very high contact toxicity to bees (LD50: 0.026 µg/bee). High oral toxicity to bees (LD50: 0.40 µg/bee). Hazard quotients for contact and oral exposure are estimated to be 680 and 44.2, 896 and 58, 731 and 47.5, respectively for applications in orchard, strawberry and ornamentals. The hazard quotients for contact exposure exceed the trigger value (>50) in all crops and indicate that there is a potential for


exposure of bees from the use of milbemectin. This will primarily be through either direct exposure, resulting from foraging bees being oversprayed or indirect exposure, as a result of the foraging bees coming into contact with residues on treated surfaces such as flowering weeds or apple blossom. However, oral exposure, through the uptake of contaminated nectar or pollen represents a relatively low risk (with the exception of use on strawberries). In order to assess the risk of Milbeknock 1% EC, honey bees were exposed under realistic conditions of a semi-field (cage) test. Good levels of exposure were obtained for the applications during bee flight as demonstrated by the high number of foraging bees observed before application and by the high mortality obtained with the toxic standard. There was no significant increase in mortality after application of 27.9 g a.s./ha (higher than the highest dose applied for in Norway), and no effects on flight intensity, behavior or brood. Milbeknock will not be applied to apple orchards during flowering, but will be applied from after petal fall when there will be no foraging activity in the target apple tree themselves and as such there is only a low potential for exposure of bees through indirect contact (residues on flowering weeds in the orchard that are subject to drift from application to the apple trees). Similarly, Milbeknock will be applied to strawberries only after harvest. Non-target arthropods According to SETAC/ESCORT-guidance, representative species of parasitoids, predatory mites, ground dwelling predators and foliage dwelling predators should be included in risk assessment. For parasitoids, tests on Aphidius rhopalosiphi with a higher application rate than the proposed rates in Norway indicate a low risk with <25% effect on survival and fecundity. In laboratory tests, the trigger of effect is exceeded for the group of predatory mites (Phytoseiulus persimilis and Typhlodromus pyri). For T. pyri, 96 to 100% mortality was found after application to leaves at an application rate equivalent to 1/10th of the proposed application rate in Norway. After application of 2.25 kg product/ha on a strawberry field (the proposed rate in Norway), the decrease in the number of adult and juvenile P. persimilis amounted to 93%. This indicates that there is a high risk for predatory mite species. For ground dwelling predators, a test with the beetle Poecilus cupreus shows less than 30% effect. For foliage dwelling predators, laboratory tests with Coccinella septempunctata showed 100% mortality. This is above the trigger of 30% effect, which indicates that there is a potential risk for this species at the proposed use of Milbeknock. Additional information from an extended laboratory test or a (semi-) field test is considered necessary. For Chrysoperla carnea, tests on a natural substrate indicate a low risk for this species with less than 25% effect on mortality and fecundity. Laboratory tests with Orius laevigatus indicate that there is a low to medium risk after application of the highest intended dose on a natural substrate: in one test 41% mortality was found (above the trigger); in the other test, 3% mortality and no effect on fecundity was observed. Earthworms Milbemectin is acutely toxic to earthworms (LC50corr: 28.5 mg/kg d.w. soil). TERacute for orchard and ornamentals is estimated to be 1425 and 950, respectively. TERacute for the strawberry scenario is estimated to be 950. These values do not exceed the trigger (<10). Milbeknock has a high chronic toxicity to earthworms (NOECcorr: 0.11 mg/kg d.w. soil). TERchronic for orchards is estimated to be 11. TERchronic for ornamentals is estimated to be 6. These values do not exceed the trigger (<5). TERchronic for strawberries is estimated to be 3. This value exceeds the trigger (<5). The estimation is based on 2 applications (7 days apart). If the time between applications is increased to 21 days, TERchronic is estimated to be 4 which still exceed the trigger. TERchronic for strawberries recalculated based on a single application results in a TER of 6 which does not exceed the trigger. Microorganisms


Neither mineralization and nitrogen transformation by soil microflora of soils treated with milbemectin up to 75 g a.s./ha (3 x the maximum expected concentration) differed from untreated soils by greater than 25 % (trigger) after 28 days. Terrestrial plants Twenty tests are available for a number of crop species. In all treatments effects on emergence, shoot length and shoot weight were below the trigger of > 50% effect at the maximum application rate.


All PEC-values below are based on single application drift values from Rautmann et al. (2001), since FOCUS modeling has shown that drift gives the highest PEC-values. TER calculations have been performed mostly on single species tests, but also with the microcosm study (for invertebrates). Fish All TER calculations for milbemectin, both acute and chronic, pass the EU triggers with 5-30 meter buffer zones.

Test substance

Applications (number x

rate)

FOCUS Step

Time scale

Endpoint µg/L

PEC µg/L

Buffer (m)

TER Trigger

Milbemectin 1 x 2.325

Strawberries Drift acute 4.4 0.04 5 100 100


Ornamentals Drift acute 4.4 0.03 20 169 100

Milbemectin 1 x 1.767 Pome fruit

(late) Drift acute 4.4 0.03 30 138 100


Strawberries Drift chronic 0.65 0.04 5 15 10


Ornamentals Drift chronic 0.65 0.03 20 25 10


(late) Drift chronic 0.65 0.06 20 10 10

Invertebrates All TER calculations for milbemectin based on single species tests pass the EU trigger with 3-20 meter buffer zones, except chronic exposure from use in ornamentals and pome fruit where even 30 meters is not enough. TERs based on the microcosm NOEC and 20-30 meter buffer zones, pass the Nordic microcosm trigger, except for use in pome fruit (TER:1.8) which fail the trigger (2-3) even with a 30 meter buffer zone. Test substance

Applications (number x rate)

FOCUS Step

Time scale

Endpoint µg/L

PEC µg/L

Buffer (m)

TER Trigger

Milbemectin 1 x 2.325 Strawberries

Drift acute 11 0.07 3 149 100

Milbemectin 1 x 1.86 Ornamentals

Drift acute 11 0.08 10 144 100


(late)

Drift acute 11 0.06 20 171 100


Drift chronic 0.12 0.01 20 10 10


Drift chronic 0.12 0.01 30 9 10


(late)

Drift chronic 0.12 0.03 30 4 10

Milbemectin 1 x 2.325 Drift Microcosm 0.058 0.01 20 5 2-3


Strawberries chronic Milbemectin 1 x 1.86

Ornamentals Drift Microcosm

chronic 0.058 0.01 30 4.3 2-3


(late)

Drift Microcosm chronic

0.058 0.03 30 1.8 2-3

Sediment-dwelling organisms All TER calculations for milbemectin pass the EU trigger with 1-5 meter buffer zones. Test substance


FOCUS Step

Time scale

Endpoint µg/L

PEC µg/L

Buffer (m)

TER Trigger


Drift Chronic 6.3 0.21 1 29 10




(late)


Aquatic plants All TER calculations for milbemectin pass the EU trigger with 1-3 meter buffer zones. Test substance


FOCUS Step

Time scale

Endpoint µg/L

PEC µg/L

Distance (m)

TER Trigger


Drift - >620 0.21 1 2888 10


Drift - >620 0.50 3 1247 10


(late)

Drift - >620 0.93 3 669 10

Algae All TER calculations for milbemectin pass the EU trigger with 1-3 meter buffer zones. Test substance


FOCUS Step

Time scale

Endpoint µg/L

PEC µg/L

Distance (m)

TER Trigger


Drift - 220 0.21 1 1025 10


Drift - 220 0.50 3 442 10


(late)

Drift - 220 0.93 3 237 10

Bioconcentration Milbemectin shows a moderate potential for bioconcentration. In a bioaccumulation study, bluegill sunfish (Lepomis macrochirus) were exposed for 28 days to 4.1-4.6 µg a.s. equivalents/L followed by a 14 days depuration period. The average whole fish BCF was 76 and 114 at steady state for milbemectin A3 and milbemectin A4, respectively. Rapid depuration occurred (CT50: 0.7-1.1 days). After the 14 days depuration period, 3.2-6.3 % (% of the steady state concentration) milbemectin was measured. (Drottar et al., 1998)


9. Dossier quality and completeness Toxicology The submitted documentation is adequate for a risk evaluation of the mammalian toxicology of both the active substance and the formulation. However, a dermal absorption study, material safety data sheets on co-formulants and final report for study 4 under special studies are not available. Residues Residues are not discussed in this report. Ecotoxicology The submitted documentation is adequate to assess the environmental fate and behaviour and ecotoxicological effects of both the active substance and the formulation.

References T1: EU Draft Assessment Report (DAR) T2: Milbemectin, Volum 3 Annex B, Addendum VII (February 2005) T3: Position statement for ECB Meeting 21 March 2006 T4: ECB classification 2006 E1: EU Draft Assessment Report (DAR) E2: European Commission’s Review report for the active substance milbemectin (04 April 2005) with

List of Endpoints E3: Kemi, Produktrapport Milbeknock, April 2010 E4: Milbemectin, Volum 3 Annex B, Addendum II (November 2002) E5: Microcosm study summary (Mitsui Chemicals Agro Inc, September 2011). Rautmann et al. 2001. New basic drift values in the authorization procedure for plant protection products. Mitt. Biol. Bundesanst. Land- Forstwirtsch. 383, 2001.

Documents

Evaluation of the plant protection product