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APPENDIX A Safety Tests In 1985, as was mentioned in the introduction, the Environmental Protection Agency approved a commercial neem-based insecticide for certain nonfood uses. Called Margosan-O®, the product is currently available in limited quantities, but demand for it is said to be high and its use is increasing quickly. Currently, Margosan-O® is registered for control of whiteflies, thrips, mealy bug, leafminers, looper, caterpillars, beet armyworms, aphids, ants, flies, cockroaches, fleas, weevils, psyllids, webworms, hornworms, spruce budworms, pin sawflies, and gypsy moths in greenhouses, commercial nurseries, forests, and homes. This is based on the results of toxicity studies required by the Environmental Protection Agency (EPA).1 Margosan-O® is an ethanolic neem extract concentrate having 3,000 ppm azadirachtin (± 10 percent) and is based on the original process developed by the U.S. Department of Agriculture (USDA) in Beltsville, Maryland. In a collaborative effort, Vikwood, Ltd. of Sheboygan, Wisconsin, undertook the challenge to stabilize and enhance the extract and attempt to bring it to the commercial market. In pilot plant runs, stability has been achieved up to I year under ambient conditions, and in excess of 3 years under refrigeration. Efficacy tests run by the USDA-Beltsville, Maryland, show activity in excess of 21 days. EPA registration has been granted for use on nonfood crops, and a U.S. patent has been granted on the product. Following are the toxicity tests ordered by the EPA to gain registration for Margosan-O® to be used on nonfood crops. Test 1— Avian Single-Dose Oral LD50. Margosan-O® was administered to mallard ducks in order to determine a dose lethal to 50 1 Information in this appendix courtesy Robert Larson.
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percent of the duck population. Dose levels of Margosan-O® ranged at 1-16 ml/kg of body weight. Observations showed no negative effects and all ducks remained active and healthy throughout the 14-day experimental period. The acute LD50 of Margosan-O® to mallard ducks is in excess of 16.0 ml/kg. Test 2— Avian Dietary LC50 (lethal concentration) with bobwhite quail. The birds were given their basal diet, with additions of Margosan-O® ranging from 1,000 to 7,000 ppm. Observations showed no negative effects and the quail were active and healthy throughout the 5-day test period and 3-day recovery phase. The acute oral LC50 of the Margosan-O® to bobwhite quail is therefore in excess of 7,000 ppm. Test 3— Avian Dietary LC50 Study with mallard ducks. The ducks were given a basal diet, plus Margosan-O® ranging from 1,000 to 7,000 ppm concentrate for 5 days. No mortalities resulted. The ducks were active and healthy throughout the test and recovery phases. The acute LC50 of the test material to mallard ducks is therefore in excess of 7,000 ppm. Test 4— (No. 1), Acute Toxicity of Margosan-O® to rainbow trout. This test involved a 96-hour LC50 of Margosan-O® at various concentrations. The LC50 was 8.8 ml of Margosan-O® per liter of water, and the 96-hour no-observed-effect concentration was 5 mg/l. Test 4— (No. 2), Acute Toxicity of Margosan-O® to bluegill sunfish. The results showed a 96-hour LC50 of 37 mg/l and a no-observed-effect 96-hour level of 20 mg/l. In the fish bioassay tests with trout and sunfish there were behavioral responses in static water that could probably not occur in moving water. Test 5— Acute Toxicity of Margosan-O® to Daphnia magna, the water flea. The test was done on newly molted in stars less than 20 hours old which were placed in a fresh aquatic habitat for up to 48 hours. The LC 50 was 13 mg/l and the no-observed-effect concentration at 48 hours was less than 10 mg/l. Therefore, the toxicity value obtained was well within the expected range, but it indicates that Margosan-O® will affect primitive aquatic invertebrates under static conditions. Test 6— Acute Oral Toxicity. Rats were dosed once and then observed for 14 days for abnormal behavior or mortality. No negative effects were observed and the acute oral toxicity of the test material was in excess of 5 ml/kg, the limit of the required test. Test 7— (No. 1), Acute Dermal Toxicity. A nonpermeable patch containing 2 ml/kg body weight of Margosan-O® was placed over small shaved areas on a group of albino rabbits. No
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mortality resulted and acute dermal toxicity (LC50) of Margosan-O ® was in excess of 2 ml/kg. Test 7— (No. 2), Primary Skin Irritation. Albino rabbits were treated with Margosan-O® applied under patches on shaved areas and on abraded areas. The results showed low to moderate primary irritation to the shaved area patch and high to moderate irritation to the abraded area. Test 8— Acute Inhalation Study. Albino rats were exposed to a total of 15.8 g of test material (estimated concentration of 43.9 mg/ l/hr) for 4 hours. (This test was recently repeated and reported in terminology more acceptable to the EPA.) The LC50 for the Margosan-O® in the inhalation test was in excess of 43.9 mg/ l/hr, the limit of the test. Test 9— Modified Eye Irritation. Margosan-O® was administered to one washed and one unwashed eye of albino rabbits. Over 7 days both eyes showed minimal irritation. Test 10— Immune Response. The effect of Margosan-O® on the hematology and serum electrophoretic pattern of rats, strain Sprague-Dawley, was determined. Eight male and eight female rats, each weighing between 200 and 250 g, were anesthetized by means of CO2 and weighed. A 3-ml sample of blood was taken via cardiac puncture and the blood studied. Five male and five female rats received 0.5 ml of Margosan-O® by intraperitoneal injection. The remaining six (control) rats were left untreated. The rats were maintained until the 14th day after substance administration. At that time, they were again anesthetized and weighed and a blood sample was taken as on day 0. Blood samples were submitted to repeat the analyses conducted at the study initiation, that is, complete blood counts with differential and serum protein electrophoresis. Body weights on day 0 and day 14 were combined for each of the four groups and a mean and standard deviation were calculated. All surviving rats gained weight and appeared active and healthy. No differences were evident between test and control animals. There were no significant changes in the hematology of the treated rats between day 0 and day 14, although statistical analysis of the electrophoretic pattern showed differences (P<0.05) in the globulin fractions during that period. The differential count showed a statistically significant change in the polymorphonuclear count, but none of the other differential counts differed statistically. Comparison of the changes in blood values of the control rats over 14 days with those in the treated rats did not uncover significant differences among any of the parameters measured.
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This suggests that the changes noted above were not treatment-related, but rather that they were normal and not unusual. The results of this study suggest that the test material does not cause an adverse immune response. Test 11— Sensitization. This test was done on guinea pigs, which were shaved and patched with Margosan-O® test material for 6 hours. The procedure was repeated on alternate days for a total of nine applications. A retest dose was applied after 14 days with duplicate patches, and the reaction was read 24 hours later. Margosan-O® does not produce sensitization. Test 12— Mutagenicity. This is the traditional Ames mutagenicity study used in the United States on five strains of Salmonella typhimurium. Results of this test indicate that the MargosanO® concentrate is nonmutagenic. Test 13— Bee Adult Toxicity Test. This test was done voluntarily and was not ordered by the EPA. With the assistance of the University of California's Apiary at Riverside, California, Vikwood, Ltd. ordered a "Bee Adult Toxicity Test" on honeybee worker adults. Margosan-O® was administered as a direct contact chemical using field dosages up to 4,478 ppm A.I./ha. It was found to be benign to honeybees at well above the recommended dosage of 20 ppm (diluted, as a foliar spray) for a common pest, the gypsy moth, Lymantria dispar (L.).
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