1 MEMORANDUM SUBJECT: Review of Non Target Insect Studies, Select Non target Organism Studies, and Test Substance Analysis Studies Submitted for Registration of Event MIR604 Which Contains the Modified Cry3A Protein ( mCry3A) in Maize. DP # 303605 MRID Numbers: 46265602 through 46265612, 46265615 and 46155618 FROM: Mika J. Hunter, Biologist Microbial Pesticides Branch Biopesticides and Pollution Prevention Division ( 7511C)

THROUGH: Tessa Milofsky, M. S., Agronomist Microbial Pesticides Branch Biopesticides and Pollution Prevention Division ( 7511C)

Zigfridas Vaituzis, Ph. D, Microbiologist Microbial Pesticides Branch, Biopesticides and Pollution Prevention Division ( 7511C)

TO: Mike Mendelsohn Senior Regulatory Action Leader Microbial Pesticides Branch Biopesticides and Pollution Prevention Division ( 7511C)

Background Syngenta Seeds, Inc. submitted data to the EPA in support of registration of a new genetically modified maize, Event MIR604. MIR604 maize contains a modified Cry3A protein ( mCry3A) that targets the western corn rootworm, Diabrotica virgifera. This memorandum contains reviews of Syngenta's submitted non target insect studies for ladybird beetle ( Coccinella septempunctata), carabid beetle ( Poecilus cupreus), rove beetle ( Aleochara bilineata), insidious UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D. C. 20460 OFFICE OF PREVENTION, PESTICIDES AND TOXIC SUBSTANCES 2 flower bug ( Orius insidiosus), and honeybee ( Apis mellifera). In addition, this document also includes reviews of the earthworm toxicity study ( Eisenia fetida), analysis of soil used in the earthworm study, fish feed analysis study, and broiler chicken study ( Gallus domesticus). Data evaluation records are attached at the end of this document.

This document also includes discussion of information provided in the submission titled " Environmental Safety Assessment of Modified Cry3A Protein and Event MIR604 Corn to Non Target Organisms" ( MRID 46265601). A complete review and DER for this study is included in the Ecological Risk Assessment ( Milofsky, 2006). Only information pertinent to this document was included.

Conclusions and Recommendations The submitted studies were all scientifically sound and are classified as acceptable. However, several studies contained deviations from the OPPTS testing guidelines. Such deviations included, but are not limited to, length of observation period, control group mortality, and actual dose of test substance ingested by test organisms. Specific aspects of particular studies that were significant to note are described in detail below. It was confirmed for each species, except for honeybee, that bioactive mCry3A ( MCRY3A 0102) was present in the ladybird beetle, carabid, rove beetle, insidious flower bug, and earthworm test diets. Event MIR604 maize does not express mCry3A in pollen. Overall, the submitted studies show that direct dietary exposure to mCry3A protein at levels encountered in the field, because not all tests were done at 10X the concentration in maize leaves, results in no significant adverse effects on ladybird beetles, carabid beetles, rove beetles, insidious flower bugs, honeybees, earthworms, and chickens.

Ladybird Beetle Testing ( Coccinellidae: Coccinella septempunctata)

MRID 46265603 A Tier I non target insect study on ladybird beetles was submitted based on OPPTS Guideline 885.4340. The study looked at both larvae and adult ladybird beetles. In addition, developmental time from larvae to adult was also reported. In the study, administered between September 27, 2003 and October 26, 2003, 4 day old ladybird beetle larvae were fed daily with live pea aphids ( Acyrthosiphon pisum). Three treatments were established ( test treatment, positive and negative control) and each treatment was replicated 40 times. Each replication contained one ladybird beetle. The test substance used was MCRY3A 0102, a purified preparation of microbially expressed mCry3A. The test substance was formulated so that a solution was created that consisted of 50 µ g MCRY3A 0102/ mL Agral solution ( 5.5 mg MCRY3A 0102 in 100 mL of 0.004 v/ v Agral 90 solution). This concentration is approximately 10X the amount of mCry3A protein expressed in transgenic maize leaves. Aphids fed to the negative control group were immersed in the Agral 90 solution only. Aphids fed to the positive control group were immersed in a solution of 0.5 mL of Nemolt ( teflubenzuron)/ L of 0.004% v/ v Agral 90. Agral 90 is a non ionic surfactant. Aphids were immersed in the appropriate solution for approximately 30 seconds and allowed to dry for 30 minutes. Freshly treated live aphids were provided daily until larvae pupated. The number of aphids fed to the larvae increased with 3 age of larvae ( table provided in Date Evaluation Record). No other food or water source was provided. The test was conducted in a room maintained at 21 27 ° C, 35 92% relative humidity, and 16 hours light. After adult beetles emerged, they were moved into clean dishes ( 1 male and 1 female per dish, where numbers allowed). Adult beetles were fed approximately 50 treated pea aphids 3 times a week for 14 days. The condition of beetle larvae was assessed daily, while the condition of the adult beetles was assessed at each feeding. In addition, it should be noted that the study was compliant with OECD and UK Good Laboratory Practice ( GLP) regulations.

There were no statistically significant differences in pre imaginal or adult survival between the negative control and MCRY3A 0102 treatments. All larvae in the positive control died in the pre imaginal stage. Results for individual treatments were compared to the control using a Chisquare test.

There was no significant difference in the mean number of days for pupae to form in the negative control and MCRY3A 0102 treatments. However, the mean number of days from larvae to adult emergence was significantly lower in the MCRY3A 0102 treatment ( no P value provided in study report). Mean developmental time was compared between the test treatment and control treatment using t tests for unmatched pairs.

An additional study ( MRID 46265604) was conducted to analyze the concentration of mCry3A that was present on the surface of immersed aphids. An ELISA analysis showed that treated aphids contained 9 µ g MCRY3A 0102/ g of aphid ( 9 ng mCry3A/ aphid). This concentration is 1.8X greater than the concentration of mCry3A expressed in maize leaves. The diet analysis study also showed, through Western blot analysis, that intact proteins were present on the aphids.

Discussion: The diet analysis study ( 46265604) showed that the actual amount of MCRY3A 0102 protein consumed by larvae and adult ladybird beetles was below the targeted concentration of 50 µ g/ g aphid. The actual concentration of protein ingested was 9 µ g MCRY3A 0102/ g aphid. Although this study did not dose the ladybird beetles at the targeted concentration of 10X the concentration expressed in maize leaves, the study shows that this ingestion of MCRY3A 0102 protein does not result in negative effects to ladybird beetles. The decrease in development time from larvae to adult does not indicate a substantial negative adverse effect. Because each larva was considered a separate replicate, the statistical analyses might have been exaggerated. In the results section of the study, the author reports that statistical significance was observed with a P value < 0.05. The author does not indicate how close to 0.05 the value was which would have provided a better indication of significance. In addition, the mean numbers of days to pupae formation was not statistically different. However, it would have been beneficial to report if there were significant differences in development from pupae to adult.

A discussion of the margin of exposure provided by the registrant ( MRID 46265601) presents information regarding the estimated concentration of Cry1Ab protein in ladybird beetle prey. Researchers estimate that aphids contain at most a concentration of Cry1Ab that is 250 times less than the corn on which they are feeding. In addition, the concentration of Cry1Ab in mites has been shown to be 1.4 times lower than the corn they were feeding on. Additionally, the author 4 states that a worst case exposure scenario for ladybird beetles would be a diet consisting of 15% mites and 85% aphids. The author expects that the concentration of mCry3A that ladybird beetle prey contains is substantially lower than 10X the concentration expressed in maize leaves. BPPD agrees with the rationale the registrant submitted.

It is important to take into consideration the likely route of exposure, as well as the likely concentration of exposure that the non target insects will be subject to. However, it is important that the study sponsor and study author include explanations of anticipated and appropriate dose concentrations within the study submission. In the submitted ladybird beetle study, the sponsor does not include an explanation why they were not dissatisfied that the ladybird beetles were not dosed with the intended concentration of MCRY3A 0102 ( 50 µ g). It is recommended that if the sponsor thought that it was not appropriate to dose the ladybird beetles at 10X the concentration expressed in the maize leaves, the sponsor should have provided that information along with the submitted study. Therefore, whether adverse effects would have been seen at 10X concentrations has not been determined.

Conclusion: This study is considered acceptable. Although the test was not conducted at the targeted 10X concentrations, the results do show that no hazard to ladybird beetles is expected at mCry3A concentrations that could be encountered in the field.

Ground Beetle Testing ( Carabidae: Poecilus cupreus)

MRID 46265605 A Tier I non target insect study on carabid beetle larvae was submitted based on OPPTS guideline 885.4340. The submitted study was OECD and UK GLP compliant. The study was conducted between November 21, 2003 and January 8, 2004. During the study, larvae of Poecilus cupreus were fed daily with blowfly pupae ( Calliphora vomitoria) that had been injected with MCRY3A 0102 solution. The MCRY3A 0102 was a sample of modified Cry3A protein ( mCry3A) that was purified from cells of recombinant E. coli. The solution that was injected into the blowfly pupa contained 50 µ g MCRY3A 0102/ g of pupa. The negative control carabid larvae were fed blowfly pupa that were injected with only deionized water. A positive control included blowfly pupa that were injected with teflubenzuron. After being injected, fly pupae were frozen to preserve for later use. Each treatment was replicated 40 times, and each replicate contained one carabid larva. 24 to 48 hour old beetle larvae were put in glass tubes filled with 25 g ( dry weight) of sandy soil maintained at approximately 35% of its water holding capacity. One fly larva was placed in each container, and capped with a ventilated stopper. Tubes were maintained at 19 º to 22 º ( 64% 90% relative humidity) and were placed upright in plastic boxes with ventilated lids in constant darkness. The fly pupae were replaced daily with freshly defrosted pupae until the beetle began pupation. The beetle larvae were assessed 3 times per week for the first two weeks of the study, and two times per week thereafter until pupation. Starting at day 32, the test containers were checked daily for emerging adult beetles. The emerged adults were weighed and sexed, and overall mortality and final adult weights were analyzed statistically. 5 In another study ( 46265606), the diet of the carabid beetles was analyzed and tested for activity of MCRY3A 0102. The study determined that the actual concentration of MCRY3A 0102 was 12 µ g/ g fly pupae. This amount is less than 10X the expressed concentration in maize leaves ( 50 µ g). The study also showed, through Western blot analysis, that active MCRY3A 0102 was likely present in the fly pupa.

Discussion: Although carabid beetle larvae were not dosed at 10X the concentration expressed in maize leaves, the study was scientifically sound and the beetle larvae were dosed at a level greater than the concentration expressed in maize leaves.

The registrant submitted supporting information regarding EEC's in a separate document ( MRID 46265601) that is important to note in this review. In the document the registrant presents rationale for their expected maximum EEC. The author states that the main route of exposure of non target carabids to mCry3A will be through eating prey that has eaten tissue of MIR604 hybrids. One common prey organism for carabid beetles is larvae of the black cutworm. The author states that black cutworm larvae contain a concentration of Cry1Ab that is 10 times less than the concentration expressed in the plant. The registrant further discusses EECs and routes of exposure, stating that an extreme worst case scenario is to assume that the concentration of MCRY3A 0102 in the cutworm larvae is 1.4 times lower than in the MIR604 hybrids. This conversion factor is derived from the concentration of Cry1Ab in spider mites relative to their diet. Therefore, the registrant concludes that the maximum concentration that carabid beetles could be exposed to is much less than 10X the concentration in maize leaves. BPPD agrees with the rationale the registrant provided.

Conclusion: This study is considered acceptable. This study shows that at a concentration of 12 µ g/ g fly pupae, no significant adverse effects were observed with regards to carabid beetle mortality or adult weight. There was no significant difference in the percent of pre imaginal mortality or mean weight of emerged adults between the MCRY3A 0102 treatment and the negative control group. The positive treatment resulted in 100% pre imaginal mortality Rove Beetle Testing ( Staphylindae: Aleochara bilineata)

MRID 46265607 A Tier I non target insect study using rove beetles was submitted based on OPPTS Guideline 885.4340. The 76 day study looked at effects of mCry3A protein on adult beetle mortality and their larval offspring. The study was conducted between July 2, 2003 and October 1, 2003 and was OECD and UK GLP compliant. The rove beetles were obtained in advance of the bioassay as parasitized pupae of the onion fly, Delia antique. After emergence, adult beetles were placed in a dark cold room ( 0 8 ° C) for six days to delay their physiological development. Three days before the study commenced, beetles were removed from the cold room and maintained at 23 25 ° C and 64 79% relative humidity. For the remainder of the study beetles were maintained at 6 this temperature and humidity ( with minor fluctuations) with a 16 hour photoperiod. Beetles were physiologically 4 days old at the start of the study. For the first 7 days of the study, beetles ( 10 female/ 10 male) were kept in round, plastic pots ( 9 cm in diameter and 5 cm deep). From day 7 to day 35, test arenas comprised polystyrene boxes ( 17 cm x 11.1 cm x 6 cm deep). The boxes were filled with at least 4 cm of quartz sand that was maintained at a constant wet weight. Beetles were provided one of three treatments of cooked minced beef. The mCry3A test substance was derived from E. coli producing mCry3A ( MCRY3A 0102). The MCRY3A 0102 treatment beetles were given approximately 0.2 g of treated minced beef. The beef for the MCRY3A 0102 test treatment was mixed with a solution containing MCRY3A 0102 that resulted in a concentration of 50 µ g mCry3A protein/ g meat. The MCRY3A 0102 beef was frozen to maintain freshness and defrosted as needed. Negative control beetles were fed cooked minced meat that had been mixed with deionozed water ( 90 g meat/ 10 mL deionized water). Positive control beetles were fed cooked minced beef that was mixed with teflubenzuron at a rate of 0.01 mg a. i. / g diet. Food was replaced daily in each treatment. Each treatment of 20 beetles was replicated four times. To assess the fecundity of the beetles, host fly pupae ( D. antique) were incorporated beneath the surface of the sand in each test box on day 14, 21, and 28. On each occasion, approximately 500 fly pupae were placed in each box. The boxes were uncovered on day 35 and any live beetles were removed. The sand was allowed to dry for approximately one week, after which the fly pupae were separated from the sand and placed in plastic pots according to treatment. The base of each pot acted as a sieve through which emerging beetles could crawl.

The condition of the beetles was assessed on days 1, 7 and 35 as being alive, moribund, dead, or missing. For the statistical analyses, any moribund insects were grouped with the dead and missing beetles for the calculation of percent mortality. The number of F1 beetles that developed from the onion fly pupae was recorded every 1 4 days up to day 76. The test ended when the mean number of beetles emerging per replicate fell to below 2 per day in the control treatment.

The control group resulted in 34% mortality of the parental generation on day 35, compared to 31% in the MCRY3A 0102 treatment and 35% in the positive control treatment. When corrected for the control mortality, the percentage mortalities were 0% and 2% in the test treatment and positive control, respectively. These results did not differ significantly from the control treatment. The author expected the low mortality of the positive control group. Teflubenzuron is a chitin synthesis inhibitor that is known to affect the fertility of adult Coleoptera. The study author notes that extended laboratory tests with A. bilineata about 20 40% of the introduced adult beetles are not recovered alive in the negative control treatment after 28 days. In the reproduction assessment, the mean number of F1 progeny in the control was 647 compared with 663 beetles in the MCRY3A 0102 treatment. These results did not differ significantly. In the positive control treatment, only 3 beetles emerged.

An additional study ( MRID 46265608) analyzed the diet fed to the rove beetles and tested MCRY3A 0102 bioactivity using Western Blot Analysis as well as a bioassay using Colorado Potato Beetle ( CPB). The study found that 91.7% of the nominal 50 µ g MCRY3A 0102/ g diet was recovered from the test diet. In addition, the Western Blot Analysis found that the diet contained bioactive protein. The activity was confirmed in the CPB bioassay, resulting in 57% 7 and 77% mortality after 96 hours for the CPB test diets prepared with 10% and 20% rove beetle test diet.

Discussion: The beetles were dosed daily at a concentration that exceeds the concentration expressed in maize leaves. The approximate concentration of MCRY3A 0102 protein in the beetle diet was 45.85 µ g/ g diet. However, this study does not draw any conclusions regarding the fitness of the test beetle's offspring or other factors associated with the second generation. In addition, the study author states that long term bioassays using this particular species of rove beetle usually results in high numbers of control beetle mortality. In the submitted study, 34% mortality was observed in the negative control. Current OPPTS guidelines state that control treatment mortality should not exceed 20%. Although it is unlikely that the high mortality of the control group impacted the ability to observe effects in the test group, it is noted in this review.

The registrant submitted supporting information regarding EEC's in a separate document ( MRID 46265601) that is important to note in this review. In the document the registrant presents rationale for their expected maximum EEC. They state that a worst case EEC would be based on consumption of soil invertebrates that have been exposed to mCry3A via protein expression in the maize roots. The author states that the concentration of mCry3A in maize roots is equivalent to 3.7 µ g/ g fresh weight. The author states that the insect with the highest concentration of Cry1Ab is the spider mite, which generally contains a concentration that is 1.4 times less than the amount expressed in the plant. Therefore, the registrant concludes that the actual concentration ingested by rove beetles would be much less than 10X the concentration in maize leaves. BPPD agrees with the rationale that the registrant submitted.

Conclusion: This study is classified as acceptable. The dose of 45.85 µ g/ g diet did not result in an increase in beetle mortality or negatively impact their ability to produce offspring.

Predatory Hemiptera ( Orius insidiosus)

MRID 46265609 A Tier I non target insect study on insidious flower bugs ( O. insidiosis) was submitted based on OPPTS Guideline 885.4340. The study was carried out between July 22, 2003 and August 13, 2003 and was compliant with OECD and UK GLP guideline requirements. The aim of the study was to determine under laboratory conditions whether mCry3Aprotein fed to O. insidiosis would result in adverse effects. Again, the protein test substance was purified from cells of E. coli. The mCry3A protein was administered to nymphal O. insidiosis during their development for 21 days. A diet consisting of beef, liver, yeast, honey, egg, sugar, water, and Nipagin ( preservative) was fed to the insects. The diet was properly heated so that raw ingredients were cooked. The MCRY3A 0102 test diet was formulated to contain a concentration of 50 µ g/ g of diet. The control diet was formulated by mixing 20 mL deionized water with 180 g diet. The positive control contained teflubenzuron at a concentration of 0.01 mg a. i. / g diet. Approximately 0.2 g of diet was placed into a small plastic cup and a short length of parafilm was stretched over the 8 cup. The insects were capable of piercing the parafilm to reach the diet. Diet cups were replaced daily and additional diet cups were frozen until needed. Insects were contained in clear, plastic pots ( 35 mm deep x 35 mm internal diameter at base x 30 mm internal diameter at top). The sides of the containers were treated with a 1.5 cm deep strip of Fluon ® suspension around the top sidewalls that created a frictionless surface to deter insects from crawling up the sides. The study was conducted in a controlled environment room maintained at 25 26 ° C and 61 87% relative humidity with 16 hours of light. The condition of the nymphs was assessed each day until they became adults, or until 21 days after test initiation. Insects were assessed as being alive, moribund, dead, or missing/ squashed. It was assumed that any bugs noted as missing that were not found in subsequent assessments had escaped from their test containers. Such missing bugs, or any that were accidentally squashed or injured during the study, were excluded from any data analysis. The percentage of pre imaginal mortality of insects was calculated for each treatment before and after correction for control mortality.

Mortality in the control treatment was 23% while the mortality of the MCRY3A 0102 treatment was 18%. The positive control treatment resulted in 98% mortality. The study author did not calculate the average pre imaginal development time. The average development time in the MCRY3A 0102 and negative control treatment was calculated as part of this review and was found to not be significantly different ( P > 0.5).

A non guideline study ( MRID 46265610) analyzed the diet that was used in the Orius study. The study used ELISA following extraction of MCRY3A 0102 from the test diet and recovered 95.6% of the nominal concentration put into the diet ( 50 µ g / g diet). Therefore, the test diet contained a concentration of 47.8 µ g ( MCRY3A 0102)/ g diet. Western blot analysis indicated that the mCry3A protein did not degrade when the Orius test diet was prepared. In addition, a bioassay was conducted that exposed Colorado potato beetles ( CPB) to a diet containing mCry3A and the Orius test diet. The bioassay resulted in 83% and 90% mortality of CPB larvae following exposure to diet containing 10% and 20% Orius diet, respectively.

Discussion: EPA guidelines state that control treatment mortality should not exceed 20%. In this study, control treatment mortality was 23%, and treatment mortality was 18%. Although it is unlikely that the high mortality of the control group impacted the ability to observe effects in the test group, it should be noted.

The registrant submitted supporting information regarding EEC's in a separate document ( MRID 46265601) that is important to note. In the document the registrant presents rationale for their expected maximum EEC. They state that a worst case EEC would be based on the consumption of only spider mites, which have been shown to accumulate the most Cry1Ab protein compared to other tested insects ( 1.4X less than the concentration in plants). Therefore, the registrant concludes that the actual EEC that Orius would be exposed to is much less than the concentration expressed in maize plants. BPPD agrees with this rationale. 9 Conclusion: This study is acceptable. This study shows that when Orius are fed a test diet containing 47.8 µ g MCRY3A 0102/ g diet during their development from nymphs to adults no negative adverse effects are observed Honey Bee Testing ( Hymenoptera: Apis mellifera)

MRID 46155618 A semi field study was conducted to fulfill OPPTS guideline 885.4380. The aim of the study was to determine whether mCry3A protein has harmful effects on brood development and adult worker bee survival in hives of the honeybee, Apis mellifera ( Hymenoptera: Apidae). The study was carried out between July 15 and August 11, 2003 in a private garden in the United Kingdom. The honeybees were exposed to microbially produced mCry3A protein via in hive feeders. The MCRY3A 0102 protein test substance was purified from cells of recombinant E. coli. The feeders were filled daily for five days with one of three sucrose solutions. The MCRY3A 0102 protein was suspended in 200 mL sucrose solution ( at a concentration of 50 µ g MCRY3A 0102 protein/ g sucrose solution). This concentration was approximately equal to 10X the concentration of mCry3A protein expressed in maize leaves. A negative control treatment contained only sucrose, and a positive control treatment contained a solution of sucrose and an insect growth regulator ( Dimilin Flo). The sucrose solution was completely consumed each day. Each treatment was replicated 4 times. In each hive, 100 eggs and 100 1 3 day old larvae were located and recorded by marking a transparent acetate sheet over the hive frames and tracing the cells. Therefore, each replicate followed the development of 200 bees. To assess brood success, the relative proportions of brood and food ( pollen and nectar) on the frames within each hive were recorded before the treatments were administered. At the end of the study, this assessment was repeated. To assess adult bee mortality, dead bee traps were placed at the opening of each hive. After the five day feeding period, bee development and brood success was monitored for 21 days. Egg cells were assessed at days 4, 6, 12, 19, and 21. Larval cells were assessed at days 4, 6, 12, and 19. The dead bee traps were assessed at the start of the study and every 1 to 3 days thereafter. The hives were assessed for general condition on days 0 and 22. This study was UK and OECD GLP compliant.

Egg cell mortality in the negative control, MCRY3A 0102 treatment, and positive control was 28.5%, 27.3%, and 100%, respectively. Larval cell mortality was 6.0%, 6.8%, and 100%, respectively. Mortality was not significantly different between the negative control and MCRY3A 0102 treatment. In addition, there was no significant difference in pre and post test hive condition between negative control and MCRY3A 0102 treatments. The number of dead adult bees found during the test was too low for statistical comparison among treatments ( 36 in negative control, 27 in mCry3A treatment, and 29 in positive control).

Discussion: Certain aspects of the study were inconsistent with OPPTS guideline 885.4380. However, these inconsistencies likely do not affect the study's results but are noted in this review. OPPTS 10 guideline 885.4380 states that the control and treated bees should be observed for at least 30 days after dosing. In the submitted study, bees were only observed for 21 days. However, the 21 days was sufficient to observe bee development. In addition, the bees were only dosed for five days. The study author states that the study site did not have any areas of flowering crops within foraging distance; however, no distance was given. Honey bees are known to travel far distances in search of food. Although it is unlikely that this absence impacted the results, a numerical distance should have been included. Along those lines, the distance between hives was not stated in the study report or indicated on the submitted map of the test area. Further, the negative control group resulted in more than 20% mortality ( 28.5%). OPPTS guidelines state that for a study to be valid, control mortality should not exceed 20%. The negative control mortality was similar to the treatment mortality. The study did not analyze the diet that was used, but it was observed that the sucrose solutions were completed consumed each day.

Conclusion: This study is classified as acceptable. The results of the study showed that there were no adverse effects on honeybee development as a result of exposure to MCRY3A 0102 protein.

Characterization of Fish Feed Test Substance MRID 46265602 This study was submitted to supplement the 28 day fish toxicity study ( MRID 46155617) that was submitted to support the experimental use permit for MIR604. The fish toxicity study was reviewed in a memo titled Ecological risk assessment for 67979 EUP U, from Syngenta, Inc. for corn containing Bacillus thuringiensis Cry3A protein for control of corn root worm ( Diabrotica spp.), dated February 15, 2005 ( Tomimatsu, 2005).

In the current study, the corn and feed pellets that were fed to the fish were analyzed to determine the actual amount of mCry3A present. This study was US EPA GLP compliant. The grain that was used to produce the fish feed was derived from two field maize hybrids. One hybrid was transgenic, expressing the mCry3A protein. The other hybrid was non transgenic, and was a similar hybrid to the transgenic maize. Both hybrids were grown concurrently in Bloomington, IL according to local agronomic practices. After harvest, the grain was shipped to Syngenta and was ground to a powder suitable for making fish feed pellets. A sample of transgenic and non transgenic powder was saved for mCry3A quantification. The powder was sent to Zeigler Brothers Inc. where it was formulated into feed pellets. The feed pellets were formulated to contain 50% maize powder. The remaining 50% was formulated with several fish feed items, including soybean meal, fish meal, vitamins and minerals. Samples of transgenic and non transgenic feed pellets were saved for further analysis. The mCry3A protein was extracted from the feed pellets and powder using standard operating procedures and quantified using ELISA. For the fish feed, three separate, random samples of the retained pellets were analyzed, to check for homogeneity.

No mCry3A protein was detected in the control maize powder, or in the control non transgenic fish feed. The measured mCry3A concentration in the transgenic maize powder was 0.3 µ g 11 mCry3A/ g fresh weight. The mean concentration in the fish feed was 0.09 µ g mCry3A/ g fresh weight. The mCry3A was uniformly distributed in the feed pellets at a concentration of 0.09 ± 005 µ g mCry3A/ g fresh weight.

Discussion/ Conclusion: This study is acceptable. The submitted study adequately characterizes the amount of mCry3A present in the fish feed used in the 28 day fish toxicity study Earthworm Toxicity ( Eisenia fetida)

MRID 46265611 A laboratory study was conducted to determine the effect on earthworms when exposed to MCRY3A 0102 in the soil for 14 days. The study took place between October 14, 2003 and November 5, 2003 and was OECD and EPA GLP compliant. Earthworms may be exposed to mCry3A protein when they forage around the roots of the transgenic maize. To expose the earthworms to mCry3A, an artificial soil substrate was used in which a concentration of MCRY3A 0102 was added. The MCRY3A 0102 protein was purified from cells of recombinant E. coli. The artificial soil was composed of 10% peat, 20% clay, and 70% sand with 1.5 g CaCo3 added to adjust the pH to 6.0 ± 0.5. During the test the soil was maintained at about 50% water holding capacity. To formulate the test soil, a solution of MCRY3A 0102 in deionized water was added to the soil at a concentration of 370 µ g/ g dry soil. This is equivalent to 334 µ g MCRY3A 0102 protein/ g dry weight soil. This concentration is approximately 10X the maximum concentration of mCry3A expressed in maize roots. A positive control treatment contained 2 choroacetamide at several concentrations ( 10, 20, 30, 40, or 50 mg/ kg of dry soil) to determine a dose response. The negative control treatment contained deionized water. The test chambers for this study were 1 liter glass jars with hinged glass lids. Each jar contained 10 adult earthworms. The test material and negative control treatments were replicated four times, while the positive control treatments were replicated three times. The test was conducted in a controlled environment room maintained at 18 20 º C and 57 89% relative humidity with constant overhead light. Before being placed in the glass containers, worms were placed in a large container of artificial soil to acclimate to the conditions of the study. Only adult worms, with clitellum, and weighing between 300 and 600 mg were selected for the study. On day 0 of the study worms were removed from the artificial soil, washed and dried, and weighed on an analytical balance. Within 30 minutes of treatment incorporation into the soil, worms were placed on the surface of the soil in the test containers and the lids were closed. Worm mortality was assessed on days 7 and 14. Worms were recorded as being alive, dead, or missing. It was assumed that any worms that were not found were dead. Worms classified as ` dead' did not respond to mechanical stimulus. On day 14, living worms were washed and dried before being re weighed.

Earthworm mortality and mean weight loss were comparable between the MCRY3A 0102 and negative control treatments. The positive control resulted in 100% mortality on day 7 for all groups receiving greater than or equal to 30 mg 2 chloroacetamide/ kg soil. The LC50 for the toxic reference treatment was calculated to be 18 g/ kg dry weight of artificial soil. In a 12 supporting study ( MRID 46265612), the artificial soil was analyzed for presence and activity of MCRY3A 0102. Soil samples were collected on days 0, 3, 7, and 14. Extraction of MCRY3A 0102 from treated soil samples were18.1%, 14.8%, 6.3%, and 7.6% of the nominal concentration, respectively. The study author comments that the declining recovery of the protein indicates degradation of the protein. In addition, the author comments that the actual concentration of MCRY3A 0102 in the test soil is likely higher than detected, and the difficulty in extracting the protein from the soil is caused by the physico chemical properties of the soil matrix. In the study, Western blot analysis detected a band of immunoreactive material corresponding to the predicted molecular weight of MCRY3A 0102. To further confirm activity of the protein, a bioassay was conducted using Colorado potato beetle ( CPB) larvae. The samples of mCry3A treated soil showed bioactivity at all sampling times, resulting in CPB larvae mortality of 60% and 83% after 120 hours for the CPB diets prepared with 5% and 10% w/ w treated soil.

Discussion: The mortality of the positive control group indicates that the earthworms were ingesting soil during the study and the study was an adequate model to assess earthworm toxicity. It should be noted that the actual concentration of MCRY3A 0102 in the artificial soil was less than the anticipated concentration. However, throughout the study the extracted concentration of MCRY3A 0102 was greater than ( days 0 3) or near the concentration ( days 7 14) expressed in maize roots. The study author comments that the investigators were unable to effectively extract and measure the MCRY3A 0102 from the soil due to physico chemical soil properties. The study author does not elaborate on this conclusion, or describe properties of the artificial soil that would lead to a reduction in extraction. In addition, EPA guideline 850.6200 recommends that earthworm toxicity studies are conducted over 28 days.

In addition, the registrant provided supporting information regarding EEC's in a separate document ( MRID 46265601) that is important to note in this review. In the document the registrant presents rationale for their expected maximum EEC. The most likely route of exposure for earthworms would be through ingestion of senescent plant materials that are incorporated into the soil. The registrant states that the average concentration of mCry3A in senescent roots of MIR604 plants is 5.5 µ g/ g fresh weight. Therefore, the registrant states that the maximum EEC that earthworms could be exposed to is much less than the concentration of mCry3A in the artificial soil. BPPD agrees with this rationale.

Conclusion: This study is classified as acceptable. The results show that no hazard to earthworms is expected at mCry3A concentrations that could be encountered in the field.

Broiler Chicken Toxicity Study ( Gallus domesticus)

MRID 46265615 A long term feeding study using broiler chickens was conducted to determine the toxicity of MIR604 maize grain to chickens. The submitted study was not USEPA GLP compliant, but was 13 conducted according to accepted scientific methods. Chickens were fed formulated diets containing either transgenic MIR604 corn with the mCry3A protein, a MIR604 isoline ( nontransgenic or a non transgenic commercial corn grown in North Carolina in 2003 ( NC 2003). The concentration of mCry3A in the transgenic diets was reported to be 0.04 µ g/ g dry weight, 0.06 µ g/ g dry weight, and 0.08 µ g/ g dry weight of the starter, grower, and finisher diets, respectively. The test diets were formulated to be similar in nutrient composition, so that available nutrition did not influence the test results. Following the standardization of the different corn types to the same nutrient content, three standard base diets ( starter, grower, and finisher) were formulated to contain 57.5%, 63.0%, and 67.5% of the standardized corn. The chickens were fed for 49 days. Broiler chicks were hatched from eggs produced and incubated at the North Carolina State University. The parent stock was a commercial strain of Ross 344 males and feather sexable Ross 308 females. Chicks were maintained on coccidostat but were not given any vaccinations. Chicks were sexed and placed into pens that measured 4 x 12 x 8 feet high. Each pen contained 25 birds of the same sex. Each treatment group contained 12 cages of birds, 6 cages of males and 6 cages of females. The pens were housed in a curtain sided poultry house. The chickens were provided with continuous access to feed and water. The birds were given access to 2.5 lbs of starter diet per bird at the start of the study. The grower diet was provided at day 16, and the finisher diet was introduced at day 31. Access to feed was discontinued on the 50th day of the experiment. Total pen weights were collected at hatch ( 1 day), 16 days, 31 days, and 49 days of age. At the later three times, feed consumption per pen was determined in order to calculate feed conversion ratios and adjusted feed conversion ratios. At 51 days, two birds from each pen were sacrificed and processed in order to determine carcass meat yield. The remaining birds that had been fed the transgenic corn containing feed were killed and buried in a secure landfill along with the remaining unused feed and corn. Body weight, feed conversion, and survival data were analyzed in order to determine statistical differences between corn diets and sex. Statistical analyses were performed using the General Linear Model ( GLM) procedure of SAS with sex and corn source as independent variables in a two way analysis of variance within a randomized complete block design. Carcass data on a gross and adjusted to body weight basis were analyzed for effects due to corn source within sex using a one way analysis of variance as effects due to sex are well known.

The report of the quantification and characterization of mCry3A in the test diets was not submitted to EPA. The study author does report that the amount of mCry3A in the MIR604 transgenic corn grain was determined through ELISA to be 0.23 ± 0.035 µ g/ g dry weight. In addition, the amount of mCry3A protein in the three diets as determined by ELISA were as follows: a) starter: 0.04 ± 0.005 µ g/ g dry weight; b) grower: 0.06 ± 0.005 µ g/ g dry weight; c) finisher: 0.08 ± 0.013 µ g/ g dry weight.

The results showed that at 16 days of age sex effects became evident with the males weighing significantly more than the females. These effects were also observed at 35 and 49 days of age. There was statistically significant body weight difference due to corn source at 31 and 49 days of age in the NC 2003 treatment, where lower body weights were observed at both dates. In addition, corn source had a significant effect on feed conversion during the grower period ( 16 31 days) in the NC 2003 group that resulted in cumulative effects at 31 and 49 days of age. There were significant differences in survival due to sex, with faster growing males exhibiting higher 14 mortality during the finisher period ( 31 49 days) and cumulatively ( 0 49), as is expected under normal conditions. Carcass analysis showed that among the female chickens of the MIR604 transgenic treatment produced greater relative thigh meat than the MIR604 non transgenic or NC 2003. Among the males, the NC 2003 group had larger Pectoralis minor than either MIR604 group. Overall carcass yield was not significantly different among treatment groups.

Discussion: However, the measured concentrations of mCry3A in the formulated diets provided in the study report could not be verified. The study report references a Syngenta report that quantifies and characterizes mCry3A in the chicken diet as being in progress, but BPPD has not received such a report.

Conclusion: This study is acceptable. The study shows that feed made from MIR604 transgenic corn does not adversely affect broiler chickens. 15 References Milofsky, T., " Environmental Risk Assessment for modified Cry3A ( mCry3A) Bacillus thuringiensis protein and the genetic material necessary for its production in Event MIR604 corn", February 2006.

Tomimatsu, G. S., " Ecological risk assessment for 67979 EUP U, from Syngenta, Inc. for corn containing Bacillus thuringiensis Cry3A protein for control of corn root worm ( Diabrotica spp.)", February 15, 2005. 16 DATA EVALUATION RECORD Primary Reviewer: Eric B. Lewis EPA Secondary Reviewer: Mika J. Hunter STUDY TYPE: Nontarget Insect Testing, Tier I ( 885.4340)

MRID NO: 46265603 DP BARCODE: DP302786 CASE NO: Not provided SUBMISSION NO: Not provided TEST MATERIAL: MCry3A 0102 STUDY NO: Mambo Tox Study/ Report No. SYN 03 50 Syngenta Project No. 2033032 SPONSOR: Syngenta Seeds, Inc., Research Triangle Park, NC TESTING FACILITY: Mambo Tox Ltd., Southampton, UK TITLE OF REPORT: A Laboratory Test of the Toxicity of Modified Cry3A Protein ( MCRY3A 0102) to Larvae and Adults of the Ladybird Beetle, Coccinella septempunctata ( Coleoptera: Coccinellidae)

AUTHOR: Waterman, L.

STUDY COMPLETED: April 6, 2004 CONFIDENTIALITY CLAIMS: None GOOD LABORATORY PRACTICE: A signed GLP statement was provided. The study was compliant with OECD and UK GLP regulations. 17 STUDY SUMMARY: In a laboratory study, 4 day old ladybird beetle larvae ( Coccinella septempunctata) were fed daily with live pea aphids ( Acyrthosiphon pisum) that had been immersed for 30 seconds in diluted wetting solution containing approximately 5.5 mg of MCRY3A 0102 ( equivalent to 50 mg of mCry3A protein). This amount is approximately equivalent to 10X the amount of mCry3A expressed in maize leaves. The test item was a sample of mCry3A protein purified from cells of recombinant E. coli ( MCRY3A 0102). Negative control larvae received aphids dipped in wetting solution only, and positive control larvae received aphids dipped in teflubenzuron. The larvae were allowed to pupate, and adult beetles that emerged were fed treated aphids for 14 days. There were no statistically significant differences in pre imaginal or adult survival between the negative control and MCRY3A 0102 treatments; all larvae in the positive control treatment died in the pre imaginal stage. There was no significant difference in the mean number of days for pupae to form in the negative control and MCRY3A 0102 treatments, but the mean number of days to adult emergence was significantly lower in the MCRY3A 0102 group.

CLASSIFICATION: Acceptable Test Material The test substance, MCRY3A 0102, active ingredient mCry3A protein, Batch Number MCRY3A 0102, was supplied by the sponsor with a reported purity of 90.3% w/ w and an expiration date of December 3, 2003. The test substance was purified from cells of recombinant E. coli. The test substance was stored at  30E to  20EC prior to use. The study was conducted prior to the test substance expiration date. The MCRY3A 0102 concentration in the treated aphids used in this test was determined in a separate study ( MRID 46265604) to be 9 Fg mCry3A/ g of aphid ( 9 ng mCry3A/ aphid).

Test Methods Four day old ladybird beetle larvae ( Coccinella septempunctata, from an in house colony), nominally in the second instar, were placed in ventilated, 5 cm diameter plastic Petri dishes ( 1/ dish) at the start of the test. The larvae were fed daily with live pea aphids ( Acyrthosiphon pisum) that had been immersed for 30 seconds in a solution containing 5.5 mg of the test material (~ 50 mg MCRY3A 0102) in100 mL of 0.004% v/ v Agral 90 ( a non ionic surfactant) in deionized water and allowed to dry for 30 minutes. This amount ( 50 Fg mCry3A 0102) is approximately equivalent to 10X the concentration of mCry3A expressed in maize leaves. Aphids fed to the negative control group were immersed in the Agral 90 18 solution only, those fed to the positive control group were immersed in a solution of 0.5 mL of Nemolt ( teflubenzuron)/ L of 0.004% v/ v Agral 90. Each treatment was replicated 40 times. Each replication consisted of one larvae/ adult. Freshly treated live aphids were provided daily until the larvae pupated. The number of aphids provided increased with age of the larvae ( Table 1). No other food or water source was provided. The test was conducted in a room at 21 27EC, 35% 92% relative humidity, and 16 hrs light/ 8 hrs darkness.

TABLE 1. Number of treated aphids supplied to ladybeetle larvae Age of larvae Approximate number ( and instar) of aphids provided daily 4 days 20 ( I and II)

5 and 6 days 30 ( III and IV)

7 and 8 days 40 ( IV and adult)

Data from p. 14, MRID 46265603 After adult beetles emerged from the pupae, they were moved into clean dishes ( 1 male and 1 female/ dish, where numbers allowed) and fed approximately 50 treated pea aphids 3 times/ week for 14 days. The adults were assessed for condition at each feeding.

Results Summary There were no statistically significant differences in pre imaginal or adult survival between the negative control and mCry3A treatments. All larvae in the positive control treatment died in the pre imaginal stage. There was no significant difference in the mean number of days for pupae to form in the negative control and mCry3A treatments, but the mean number of days to adult emergence was significantly lower in the mCry3A group ( Table 3). The study authors speculated that the faster development might have been related to the additional dietary protein provided by the test substance.

TABLE 2. Mortality Results Pre Imaginal Mortality Adult Mortality Treatment Concentration % Mortality a Corrected % Mortality % Mortality Corrected % Mortality Control Agral 90 0  7.5  MCRY3A 0102 50 µ g mCry3A/ mL Agral solution 0 0 15.0 8.1 Nemolt 0.5 mL/ L Agral solution 100*** 100

a) Results for individual treatments were compared to the control using Chi Square test and means marked with asterisks differed significantly from the control (*** P < 0.001) 19 TABLE 3. Developmental time ( days) of 4 day old C. septempunctata larvae Treatment Larvae to pupae Mean ± std deviation Larvae to adult Mean ± std deviation Negative control 5.48 ± 0.50 9.80 ± 0.68 MCRY3A 0102 5.33 ± 0.47 9.48* ± 0.59 Mean development time was compared between the test item and control treatment using t tests for unmatched pairs * Significantly different from control P< 0.05 Study Author's Conclusions The study author concluded that prolonged dietary exposure to MCRY3A 0102 via application to aphids at a nominal rate equivalent to 50 Fg mCry3A/ mL of Agral solution was not harmful to C. septempunctata larvae or adults 20 DATA EVALUATION RECORD Primary Reviewer: Eric B. Lewis EPA Secondary Reviewer: Mika J. Hunter STUDY TYPE: Nonguideline MRID NO: 46265604 DP BARCODE: DP303605 CASE NO: Not provided SUBMISSION NO: Not provided TEST MATERIAL: MCRY3A 0102 STUDY NO: SSB 004 04 SPONSOR: Syngenta Seeds, Inc., Research Triangle Park, NC TESTING FACILITY: Syngenta Biotechnology, Inc., Research Triangle Park, NC TITLE OF REPORT: Analysis of Test Diet Used to Expose Coccinella septempunctata to Modified Cry3A Protein: Supplement to Report Titled " A Laboratory Test of The Toxicity of Modified Cry3A Protein ( MCRY3A 0102) to Larvae and Adults of the Ladybird Beetle Coccinella septempunctata ( Coleoptera: Coccinellidae)"; Mambo Tox Study/ Report No. SYN 03 50 AUTHOR: Graser, G.

STUDY COMPLETED: April 26, 2004 CONFIDENTIALITY CLAIMS: None GOOD LABORATORY PRACTICE: A signed GLP statement was provided. The study was EPA GLP compliant. 21 STUDY SUMMARY: Samples of pea aphids ( Acyrthosiphon pisum) that had been immersed in a wetting agent solution containing MCRY3A 0102 at a concentration nominally equivalent to 50 Fg MCRY3A 0102 / mL and provided as diet in a ladybird beetle ( Coccinella septempunctata) toxicity study ( MRID 46265603) were analyzed for MCRY3A 0102 protein using an enzyme linked immunosorbent assay ( ELISA). The integrity of the mCry3A in the treated pupae was determined using Western blot analysis. ELISA results showed that Cocccinella larvae and adults in the toxicity study were exposed to 9 Fg MCRY3A 0102 / g of aphid ( 9 ng MCRY3A 0102 / aphid), and the Western blot analysis showed that intact mCry3A was present in the aphids.

CLASSIFICATION: Acceptable Test Material The test substance was a sample of pea aphids ( Acyrthosiphon pisum) that had been treated in the same manner as, and in parallel with, pea aphids that were used as treated diet in a previous non target insect toxicity study with Coccinella septempunctata ( MRID 46265603). The pea aphids in that study were immersed in a wetting agent solution containing MCRY3A 0102 at a concentration nominally equivalent to 50 Fg MCRY3A 0102 / mL and provided as diet to the coccinnelids. A sample of control aphids not treated with MCRY3A 0102 was also submitted for analysis. The samples were frozen immediately after preparation in the original study and remained frozen until analysis.

Test Methods The test substance was quantitatively analyzed for MCRY3A 0102 using an enzyme linked immunosorbent assay ( ELISA), and the integrity of the mCry3A was determined using Western blot analysis. Standard operating procedures were referenced in the report. Two mL of extraction buffer were added to 0.156 g of the mCry3A treated aphids ( 150 aphids) and to 0.176 g of the untreated aphids ( 140 aphids). Each sample was mixed, incubated on ice for one hour, homogenized, centrifuged for 15 minutes at 2600 x g, and the supernatants were collected. The pellets were extracted a second time using the same procedure, and a third extraction was done in an overnight incubation at 4EC. The supernatants from the second and third extractions were combined and centrifuged again for 10 minutes at 13,000 x g prior to analysis. These extracts were used for the ELISA, while only the first extract of each sample was used for the Western blot analysis.

For the ELISA, the extracts were analyzed for MCRY3A 0102 using immuno affinity purified rabbit anti mCry3A polyclonal antibodies and immuno affinity purified goat anti Btt ( native Cry3A from Bacillus thuringiensis subsp. tenebrionis) polyclonal antibodies. 22 For the Western blot analysis, two aliquots of the treated aphid extract, containing 6 and 12 ng mCry3A, respectively ( as determined by ELISA), were subjected to SDS PAGE using an 8 16% polyacrylamide gradient gel. Two aliquots of a solution of MCRY3A 0102 ( positive control) containing 6 and 12 ng of mCry3A, respectively, and one aliquot of the untreatedaphid extract ( negative control) were run concurrently on the same gel. After electroblotting, the membrane was probed with immuno affinity purified goat anti Btt polyclonal antibodies. Donkey anti goat IgG linked to alkaline phosphatase, diluted in blocking buffer, was used to bind to the primary antibody and visualized by development with alkaline phosphatase substrate solution. The Western blot was examined for the presence of intact immunoreactive mCry3A ( ca. 67,000 molecular weight) and immunoreactive MCRY3A 0102 polypeptides of lower molecular weight.

Results Summary ELISA of the treated aphid extract indicated a recovery of 9 Fg MCRY3A 0102 / g of aphid ( 9 ng MCRY3A 0102 / aphid). No mCry3A was detected in the untreated aphid extract. Western blot analysis of the treated aphid extract showed a single immunoreactive band corresponding to the predicted molecular weight of MCRY3A 0102 ( ca. 67,700 Da). No immunoreactive material was detected in the untreated aphids.

Study Author's Conclusions The study author concluded that the ELISA results showed that Coccinella larvae and adults in the toxicity study were exposed to about 9 Fg MCRY3A 0102 / g of aphid diet throughout the larval and adult feeding stages, and the Western blot analysis showed that intact MCRY3A 0102 was present. 23 DATA EVALUATION RECORD Primary Reviewer: Eric B. Lewis EPA Secondary Reviewer: Mika J. Hunter STUDY TYPE: Nontarget Insect Testing, Tier I ( 885.4340)

MRID NO: 46265605 DP BARCODE: DP303605 CASE NO: Not provided SUBMISSION NO: Not provided TEST MATERIAL: MCry3A 0102 STUDY NO: Mambo Tox Study/ Report No. SYN 03 54 Syngenta Project No. 2033034 SPONSOR: Syngenta Seeds, Inc., Research Triangle Park, NC TESTING FACILITY: Mambo Tox Ltd., Southampton, UK TITLE OF REPORT: A Laboratory Toxicity Test of the Modified Cry3A Protein ( MCRY3A 0102) to Larvae of the Ground Dwelling Beetle, Poecilus cupreus ( Coleoptera: Carabidae)

AUTHOR: Vinall, S.

STUDY COMPLETED: April 7, 2004 CONFIDENTIALITY CLAIMS: None GOOD LABORATORY PRACTICE: A signed GLP statement was provided. The study was compliant with OECD and UK GLP regulations. 24 STUDY SUMMARY: In a laboratory study, ground dwelling beetle ( Poecilus cupreus) larvae were fed daily with blowfly ( Calliphora vomitoria) pupae that had been injected with MCRY3A 0102 at a nominal rate equivalent to 50 Fg mCry3A protein/ g of fly pupa. This nominal amount is approximately equivalent to 10X the amount of mCry3A protein expressed in maize leaves. The test item, MCRY3A 0102, was purified from cells of recombinant E. coli. A negative control of fly pupae injected with deionized water only, and a positive control of fly pupae injected with teflubenzuron ( 0.664 ng a. i./ g of fly pupa) were also used in the test. When the P. cupreus larvae pupated, feeding stopped and adult emergence was monitored. There was no statistically significant difference in the percent pre imaginal mortality or mean weight of emerged P. cupreus adults between the MCRY3A 0102 and negative control groups. Treatment with the positive control produced 100% pre imaginal mortality. An additional study analyzed the actual amount of mCry3A in fly pupa. This amount was determined to be 12 Fg MCRY3A 0102 / g pupae.

CLASSIFICATION: Acceptable Test Material The test substance, MCRY3A 0102, active ingredient mCry3A protein, Batch Number MCRY3A 0102, was supplied by the sponsor with a reported purity of 90.3% w/ w and an expiration date of July 15, 2004. The MCRY3A 0102 test substance was purified from cells of recombinant E. coli. The test substance was stored at  30E to  20EC prior to use. The study was conducted prior to the test substance expiration date. The mean concentration of MCRY3A 0102 in the fly pupae used in this test was determined in a separate study ( MRID 46465606) to be 12 Fg MCRY3A 0102 / g pupa, or approximately 0.82 Fg mCry3A/ pupa ( 24% of nominal, calculated by primary reviewer).

Test Methods Larvae of the ground dwelling beetle ( Poecilus cupreus, obtained from Bio Test Labor GmbH, Sagerheide, Germany) were fed blowfly ( Calliphora vomitoria) pupae that had been injected with the test substance at a rate equivalent to 50 Fg mCry3A/ g of fly pupa. The test substance solution was prepared by diluting 0.0399 g of MCRY3A 0102 (~ 0.03601 g mCry3A) to 10 mL with deionized water to give a final solution concentration of 3.601 Fg MCRY3A 0102 / 0.072 g fly pupa. Each pupa was injected with 1 FL of the test solution. A negative control of fly pupae injected with deionized water only, and a positive control of fly 25 pupae injected with teflubenzuron ( 0.664 ng a. i./ g of fly pupa) were also used in the test. At test start, the beetle larvae were 24 to 48 hours old.

The test containers were glass tubes filled with 25 g ( dry wt) of LUFA 2.1 standard sandy soil maintained at approximately 35% of its water holding capacity. One fly pupa was placed in each container, which was capped with a ventilated polyethylene stopper. The remaining treated pupae were frozen at  20E to  30EC until needed each day. Each treatment was replicated 40 times ( each replication consisting of one beetle). The tubes were kept upright in plastic boxes with ventilated lids and maintained in an environmental chamber at 19E to 22EC and 64% to 90% relative humidity in constant darkness. The fly pupae were replaced daily with freshly defrosted pupae until the beetle larvae were seen to have prepared and entered a pupating chamber in the soil, at which point the feeding was stopped. The condition of the beetle larvae was assessed 3 times/ week for the first two weeks of the study, and 2 times/ week thereafter until day 32 following treatment. At day 32 and afterward, the test containers were checked daily and the number of emerging adults was recorded. The emerged adults were weighed and sexed, and overall mortality and final adult weights were analyzed statistically.

Results Summary There was no significant difference in the percent pre imaginal mortality or mean weight of emerged adults between the MCRY3A 0102 and the negative control groups ( Table 1). Treatment with the positive control produced 100% pre imaginal mortality.

TABLE 2. Larval mortality and adult weight of P. cupreus fed treated and untreated fly pupae.

Treatment % Mortality % Corrected mortality a Mean weight of adults ( mg) b MCRY3A 0102 10 0 82.9 Negative control 20  81.5 Positive control 100 100

a) A Chi square test was used to compare mortality in the negative control and mCry3A 0102 treatment. b) A t test was used to compare the mean weight of the emerging beetles between the negative control and mCry3A 0102 treatment. No significant differences occurred ( P > 0.05) Data from p. 16, MRID 46265605 Study Author's Conclusions The study author concluded that continuous oral exposure to MCRY3A 0102 during development did not affect survival of P. cupreus larvae or affect the weight of adult beetles that emerged after pupation of the treated larvae. 26 DATA EVALUATION RECORD Primary Reviewer: Eric B. Lewis EPA Secondary Reviewer: Mika J. Hunter STUDY TYPE: Nonguideline MRID NO: 46265606 DP BARCODE: DP303605 CASE NO: Not provided SUBMISSION NO: Not provided TEST MATERIAL: MCRY3A 0102 STUDY NO: SSB 007 04 SPONSOR: Syngenta Seeds, Inc., Research Triangle Park, NC TESTING FACILITY: Syngenta Biotechnology, Inc., Research Triangle Park, NC TITLE OF REPORT: Analysis of Test Diet Used to Expose Poecilus cupreus to Modified Cry3A Protein: Supplement to Report Titled  " A Laboratory Toxicity Test of the Modified Cry3A Protein ( MCRY3A 0102) to larvae of the Ground Dwelling Beetle Poecilus cupreus ( Coleoptera: Carabidae)"; Mambo Tox Study/ Report No. SYN 03 54 AUTHOR: Graser, G.

STUDY COMPLETED: April 26, 2004 CONFIDENTIALITY CLAIMS: None GOOD LABORATORY PRACTICE: A signed GLP statement was provided. The study was EPA GLP compliant. 27 STUDY SUMMARY: Samples of blowfly ( Calliphora vomitoria) pupae that had been injected with MCRY3A 0102 at a concentration nominally equivalent to 50 Fg mCry3A/ g pupae and used as diet in a ground dwelling beetle ( Poecilus cupreus) toxicity study ( MRID 46265605) were analyzed for mCry3A protein using an enzyme linked immunosorbent assay ( ELISA). The integrity of the MCRY3A 0102 in the treated pupae was determined using Western blot analysis. ELISA results showed that Poecilus larvae in the toxicity study were exposed to about 12 Fg MCRY3A 0102 / g pupae throughout their development and the Western blot analysis showed that intact MCRY3A 0102 was present in the pupae.

CLASSIFICATION: Acceptable Test Material The test substances were samples of blowfly ( Calliphora vomitoria) pupae that had been treated in the same manner as, and in parallel with, treated pupae that were used as diet in a previous non target insect toxicity study with Poecilus cupreus ( MRID 46265605). The pupae in that study were injected with MCRY3A 0102 at a nominal concentration equivalent to 50 Fg MCRY3A 0102 / g fly pupae. Samples of control pupae ( injected with deionized water instead of MCRY3A 0102) used in that study were also submitted for analysis. The samples were frozen immediately after preparation in the original study and remained frozen until analysis.

Test Methods The test substance was quantitatively analyzed for MCRY3A 0102 using an enzyme linked immunosorbent assay ( ELISA), and the integrity of the MCRY3A 0102 was determined using Western blot analysis. Standard operating procedures were referenced in the report. The pupae were ground to a fine powder in liquid nitrogen using a mortar and pestle. Two mL of extraction buffer were added to 0.208 g of the MCRY3A 0102  treated pupae ( 3 pupae) and to 0.213 g of the untreated pupae ( 3 pupae). Each sample was mixed, incubated on ice for one hour, homogenized, centrifuged for 15 minutes at 2600 x g, and the supernatants were collected. The pellets were extracted a second time using the same procedure, and a third extraction was done in an overnight incubation at 4EC. The supernatants from the second and third extractions were combined and centrifuged again for 10 minutes at 13,000 x g prior to analysis. These extracts were used for the ELISA, while only the first extract of each sample was used for the Western blot analysis. 28 For the ELISA, the extracts were analyzed for MCRY3A 0102 using immuno affinity purified rabbit anti mCry3A polyclonal antibodies and immuno affinity purified goat anti Btt ( native Cry3A from Bacillus thuringiensis subsp. tenebrionis) polyclonal antibodies.

For the Western blot analysis, five aliquots of the fly pupae extracts, containing 0.075, 0.150, 0.375, 0.750, and 1.500 ng mCry3A, respectively ( as determined by ELISA), were subjected to SDS PAGE using an 8 16% polyacrylamide gradient gel. Three aliquots of a solution of MCRY3A 0102 ( positive control), containing 0.2, 0.4, and 0.8 ng of mCry3A, respectively, and three aliquots of the extract from the untreated fly pupae ( negative control) were run concurrently on the same gel. After electroblotting, the membrane was probed with immunoaffinity purified goat anti Btt polyclonal antibodies. Rabbit anti goat IgG linked to horseradish peroxidase, diluted in blocking buffer, was used to bind to the primary antibody and visualized by development with SuperSignal ® West Pico Chemiluminescent Substrate ( Pierce) solution. The Western blot was examined for the presence of intact immunoreactive MCRY3A 0102 ( ca. 67,000 molecular weight) and immunoreactive MCRY3A 0102 polypeptides of lower molecular weight.

Results Summary ELISA of the treated blowfly pupae extracts recovered approximately 12 Fg mCry3A/ g of pupae ( 0.82 Fg MCRY3A 0102 / pupa), for about 24% recovery of mCry3A that had been injected into the pupae ( nominally 50 Fg MCRY3A 0102 / g pupae). No mCry3A was detected in the untreated blowfly pupae.

Western blot analysis of the treated blowfly pupae showed a single immunoreactive band corresponding to the predicted molecular weight of MCRY3A 0102 ( ca. 67,700 Da). No immunoreactive material was detected in the untreated blowfly pupae.

Study Author's Conclusions The study author concluded that the ELISA results showed that Poecilus larvae in the toxicity study were exposed to about 12 Fg MCRY3A 0102 / g of pupae diet throughout their development, and Western blot analysis showed that intact MCRY3A 0102 was present. 29 DATA EVALUATION RECORD Primary Reviewer: Eric B. Lewis EPA Secondary Reviewer: Mika J. Hunter STUDY TYPE: Nontarget Insect Testing, Tier I ( 885.4340)

MRID NO: 46265607 DP BARCODE: DP303605 CASE NO: Not provided SUBMISSION NO: Not provided TEST MATERIAL: MCRY3A 0102 STUDY NO: Mambo Tox Study/ Report No. SYN 03 53 Syngenta Project No. 2033036 SPONSOR: Syngenta Seeds, Inc., Research Triangle Park, NC TESTING FACILITY: Mambo Tox Ltd., Southampton, UK TITLE OF REPORT: A Laboratory Toxicity Test of Modified Cry3A Protein ( MCRY3A 0102) to the Rove Beetle, Aleochara bilineata ( Coleoptera: Staphlinidae)

AUTHOR: Vinall, S.

STUDY COMPLETED: November 26, 2003 CONFIDENTIALITY CLAIMS: None GOOD LABORATORY PRACTICE: A signed GLP statement was provided. The study was compliant with OECD and UK GLP regulations. 30 STUDY SUMMARY: In a laboratory study, mortality was determined for adult rove beetles ( Aleochara bilineata) given continuous access to beef paste diet containing MCRY3A 0102 at a nominal rate equivalent to 50 Fg mCry3A/ g of diet for 35 days. The test substance, MCRY3A 0102, was purified from cells of recombinant E. coli. In an additional study ( 46265608) it was determined that 91.7% of the MCRY3A 0102 protein mixed into the test diet was recovered and it was found to be bioactive. Negative control beetles were fed diet mixed with deionized water only, and positive control beetles were fed diet mixed with teflubenzuron ( 0.01 mg a. i./ g of diet). To assess the effects of treatment on fecundity, approximately 500 onion fly ( Delia antiqua) pupae, which are parasitized by rove beetles, were placed in the beetle habitat on days 14, 21, and 28 of the study. During days 35 to 76, emergence of adult rove beetles from the onion fly pupae was monitored. There was no statistically significant difference in day 35 mortality among any of the rove beetle groups, and no statistically significant difference in the mean number of progeny produced by the negative control and MCRY3A 0102 groups from days 35 to 76. Reproduction in the positive control group was significantly reduced.

CLASSIFICATION: Acceptable Test Material The test substance, MCRY3A 0102, active ingredient mCry3A protein, Batch Number MCRY3A 0102, was supplied by the sponsor with a reported purity of 90.3% w/ w and an expiration date of December 3, 2003. The test substance was purified from cells of recombinant E. coli. The test substance was stored at  30E to  20EC prior to use. The study was conducted prior to the test substance expiration date. The concentration, stability, and activity of the treated diet used in the test were determined in a separate study ( MRID 46265608). Recovery of mCry3A from the treated diet was 91.7% of the nominal concentration.

Test Methods The test insects ( A. bilineata) were obtained from De Groene Vlieg, Nieuwe Tonge, The Netherlands in advance of the bioassay as parasitized pupae of the onion fly ( D. antiqua). Adult beetles that had emerged over a 24 hour period were individually confined and provided with a pellet of raw minced beef for food. The beetles were placed in a dark cold room ( 0 8

C) for six days to delay their development. Three days before the bioassay started, beetles were moved back to a controlled environment room maintained at 23 25 º C 31 and 64 79% relative humidity, under a 16 hour photoperiod. At the start of the bioassay, beetles were physiologically 4 days old. Groups of 10 male and 10 female adult rove beetles were fed beef paste diet containing the test material at a rate equivalent to 50 Fg MCRY3A 0102 / g of diet. The treated diet was prepared by diluting 0.0554 g of MCRY3A 0102 in 100 mL of deionized water and mixing 10 mL of the resulting solution into 90 g of previouslycooked pureed beef. Negative control beetles were fed diet mixed with deionized water only, and positive control beetles were fed diet mixed with teflubenzuron at 0.01 mg a. i./ g of diet. Diets were prepared at one time, with the remainder frozen at  20EC until use. Each treatment of 20 beetles was replicated 4 times.

For the first 7 days of the test the beetles were housed in plastic pots with ventilated, clip on lids in order to encourage regular contact with the diet. The beetles were then transferred to polystyrene boxes with ventilated lids for the remaining 28 days of the test. The boxes contained a 4 cm layer of washed and graded sand that was kept wet with distilled water. Approximately 0.2 g of the treated diet was placed in the boxes and replaced daily with diet allowed to defrost for one hour. Condition of the beetles was assessed on days 1 and 7 of the test. The test occurred in a controlled environment room maintained at 20E to 24EC and 61% to 88% relative humidity ( with minor fluctuations) with a photoperiod of 16 hrs light/ 8 hrs darkness.

To assess the effects of treatment on fecundity, approximately 500 host onion fly pupae were placed under the surface of the wet sand on days 14, 21, and 28 of the study. On day 35, live beetles were removed and assessed for condition. The sand was allowed to dry for approximately one week, after which the onion fly pupae were sieved out and transferred to plastic emergence chambers with ventilated lids. The bottom of the chambers contained netting that acted as a sieve through which emerging adult beetles would fall into a collecting chamber below. The emergence chambers were maintained in a controlled environment room for a further 34 days to permit the F1 generation of rove beetles to emerge, and the number of emergents recorded every 1 to 4 days. The test ended when the number of emergents/ replicate fell below 2.0/ day in the control treatment.

Results Summary There was no statistically significant difference in day 35 mortality among the groups ( Table 1). Although the control mortality was 34%, the study author stated that A. bilineata mortality in similar 28 day lab tests is generally 20 to 40%. There was also no statistically significant difference in the mean number of progeny produced by the negative control and the treated groups. Reproduction in the positive control group was significantly reduced. 32 TABLE 1. Mortality and number of progeny of A. bilineata supplied MCRY3A 0102 for 35 days Treatment % Mortality at 35 daysa % Corrected Mortalityb Mean no. of F1 Progenyc % Effect on Reproductiond MCRY3A 0102 ( 50 Fg mCry3A/ g diet) 31 0 663 ± 219  2.5 Negative control 34  647 ± 169

Teflubenzuron ( 10 Fg/ g diet) 35 2 3*** ± 5 99.5 a) The overall number of adult beetles that were found dead or were missing at 35 DAT were compared by Fisher's Exact test. None of the treatment results different significantly from the control ( P > 0.05) b) Corrected using Abbotts formula c) The numbers of progeny emerging per replicate were compared by one way ANOVA and Dunnett's test ( prior to analysis, a log 10 ( n+ 1) transformation was applied to the data). d) The percentage reduction/ increase in numbers of F1 progeny, relative to the control, was calculated using the formula R= ( 1( Rt/ Rc)) x 100, where Rt and Rc are the numbers of offspring observed in the treatment and control groups, respectively. A negative value indicates an increase and a positive value a decrease. *** Signficantly different from control p# 0.001 Data from pp. 18 19, MRID 46265607 Study Author's Conclusions The study author concluded that MCRY3A 0102 did not adversely affect survival or reproductive capacity of A. bilineata when offered continuously in the diet at a rate equivalent to 50 Fg mCry3A/ g of diet. 33 DATA EVALUATION RECORD Primary Reviewer: Eric B. Lewis EPA Secondary Reviewer: Mika J. Hunter STUDY TYPE: Nonguideline MRID NO: 46265608 DP BARCODE: DP303605 CASE NO: Not provided SUBMISSION NO: Not provided TEST MATERIAL: MCRY3A 0102 STUDY NO: SSB 006 04 SPONSOR: Syngenta Seeds, Inc., Research Triangle Park, NC TESTING FACILITY: Syngenta Biotechnology, Inc., Research Triangle Park, NC TITLE OF REPORT: Analysis of Test Diet Used to Expose Aleochara bilineata to Modified Cry3A Protein: Supplement to Report Titled  " A Laboratory Toxicity Test of Modified Cry3A Protein ( MCRY3A 0102) to the Rove Beetle, Aleochara bilineata ( Coleoptera: Staphylinidae)"; Mambo Tox Study/ Report No. SYN 03 53 AUTHOR: Graser, G.

STUDY COMPLETED: April 23, 2004 CONFIDENTIALITY CLAIMS: None GOOD LABORATORY PRACTICE: A signed GLP statement was provided. The study was EPA GLP compliant. 34 STUDY SUMMARY: Samples of formulated diet treated with MCRY3A 0102 and used in a rove beetle ( Aleochara bilineata) toxicity study ( MRID 46265607) were analyzed for MCRY3A 0102 protein using an enzyme linked immunosorbent assay ( ELISA). The integrity of the MCRY3A 0102 in the treated diet was determined using Western blot analysis, and the bioactivity of MCRY3A 0102 in the treated diet was determined in a bioassay using Colorado potato beetle ( CPB) larvae. ELISA of the treated test diet recovered 91.7% of the nominal mCry3A diet concentration used in the original study, indicating MCRY3A 0102 was present at the intended concentration. Western blot analysis of the treated diet indicated that mCry3A did not degrade under conditions of the original test diet preparation or storage, and the CPB bioassay confirmed that the original nontarget insect test group ( A. bilineata) had been exposed to active MCRY3A 0102.

CLASSIFICATION: Acceptable Test Material The test substances were samples of a formulated beef paste diet containing MCRY3A 0102 ( active ingredient, mCry3A protein) that had been used in a previous non target insect toxicity study with Aleochara bilineata ( MRID 46265607). The nominal active ingredient concentration in the diet for that study was 50 Fg MCRY3A 0102 / g of diet. Samples of control diet ( with deionized water instead of MCRY3A 0102) used in that study were also submitted for analysis. The samples were frozen immediately after preparation in the original study and remained frozen until analysis.

Test Methods The test substance was quantitatively analyzed for MCRY3A 0102 using an enzyme linked immunosorbent assay ( ELISA), and the integrity of the MCRY3A 0102 was determined using Western blot analysis. Standard operating procedures were referenced in the report. The bioactivity of MCRY3A 0102 in the test substance was determined in a bioassay using Colorado potato beetle ( CPB) ( Leptinotarsa decemlineata) larvae.

For the ELISA and Western blot analysis, 2 mL of extraction buffer were added to approximately 0.35 g of the Aleochara test or control diet. Each sample was mixed, incubated on ice for one hour, homogenized, centrifuged for 10 minutes at 2600 x g, and the supernatants were collected. The pellets were extracted a second time using the same procedure, and a third extraction was done in an overnight incubation on ice. All three extracts of each sample were centrifuged again for 10 minutes at 13,000 x g prior to analysis. 35 These extracts were used for the ELISA, while only the first extract of each sample was used for the Western blot analysis.

For the ELISA, the extracts were analyzed for MCRY3A 0102 using immuno affinity purified rabbit anti mCry3A polyclonal antibodies and immuno affinity purified goat anti Btt ( native Cry3A from Bacillus thuringiensis subsp. tenebrionis) polyclonal antibodies.

For the Western blot analysis, two aliquots of the Aleochara test diet extract, containing 15 and 49 ng mCry3A, respectively ( as determined by ELISA), were subjected to SDS PAGE using an 8 16% polyacrylamide gradient gel. Two aliquots of a solution of MCRY3A 0102 ( positive control), containing 15 and 50 ng of MCRY3A 0102, respectively, and one aliquot of the Aleochara control diet ( negative control) were run concurrently on the same gel. After electroblotting, the membrane was probed with immuno affinity purified goat anti Btt polyclonal antibodies. Donkey anti goat IgG linked to alkaline phosphatase, diluted in blocking buffer, was used to bind to the primary antibody and visualized by development with alkaline phosphatase substrate solution. The Western blot was examined for the presence of intact immunoreactive MCRY3A 0102 ( ca. 67,000 molecular weight) and immunoreactive MCRY3A 0102 polypeptides.

For the CPB bioassay, diets containing 10% and 20% of the Aleochara diet were prepared by adding 0.522 and 0.464 g, respectively, of stock CPB diet to 0.058 and 0.116 g, respectively, of the Aleochara diet. Control CPB diet was prepared in the same manner using the Aleochara control diet. Positive control diets were also prepared by adding MCRY3A 0102 to stock CPB diet at concentrations equivalent to 12.5 and 50 Fg MCRY3A 0102 / g. As a control for the positive control diet, CPB diet was prepared using the same volume of water as was used to add the positive control solution to the CPB diet. An untreated CPB diet control was also included. A piece of the supplemented diets ( 10% or 20% Aleochara test diet or 10% or 20% Aleochara control diet), was placed in a Petri dish with 10 newlyhatched CPB larvae. Similarly, groups of 10 larvae were provided with stock CPB diet ( untreated control), stock CPB diet treated directly with MCRY3A 0102 ( positive control), or stock CPB diet treated with water ( control for the positive control). Each treatment was replicated 3 times. The dishes were covered and maintained at ambient conditions for 96 hours. Diets were replaced after approximately 24 and 48 hrs with fresh diet prepared in the same manner. Mortality was assessed daily.

Results Summary ELISA of the Aleochara test diet extract showed a recovery of 91.7% of the nominal 50 Fg MCRY3A 0102 / g of diet concentration used in the original Aleochara study. No mCry3A was detected in the Aleochara control diet.

Western blot analysis of the Aleochara test diet showed a single immunoreactive band corresponding to the predicted molecular weight of MCRY3A 0102 ( ca. 67,700 Da). No immunoreactive material was detected in the Aleochara control diet. 36 In the CPB bioassay, the diets prepared with 10% and 20% of the original Aleochara test diet produced 57% and 77% larval mortality, respectively, after 96 hours. The positive controls produced similar mortality. The Aleochara control diet produced 10% mortality, as did the control CPB diets.

Study Author's Conclusions The study author concluded that a) the high proportion of MCRY3A 0102 recovered by ELISA ( 91.7%) indicated MCRY3A 0102 was present in the Aleochara test diet at the intended concentration, b) the Western blot analysis showed that MCRY3A 0102 did not degrade under conditions of the Aleochara test diet preparation or storage, and c) the bioassay results confirmed that the Aleochara test group was exposed to active MCRY3A 0102. 37 DATA EVALUATION RECORD Primary Reviewer: Eric B. Lewis EPA Secondary Reviewer: Mika J. Hunter STUDY TYPE: Nontarget Insect Testing, Tier I ( 885.4340)

MRID NO: 46265609 DP BARCODE: DP303605 CASE NO: Not provided SUBMISSION NO: Not provided TEST MATERIAL: MCRYA 0102 STUDY NO: Mambo Tox Study/ Report No. SYN 03 52 Syngenta Project No. 2033033 SPONSOR: Syngenta Seeds, Inc., Research Triangle Park, NC TESTING FACILITY: Mambo Tox Ltd., Southampton, UK TITLE OF REPORT: A Laboratory Toxicity Test of Modified Cry3A Protein ( MCRY3A 0102) to the Predatory Bug, Orious insidiosus ( Heteroptera: Anthocoridae)

AUTHOR: Vinall, S.

STUDY COMPLETED: November 19, 2003 CONFIDENTIALITY CLAIMS: None GOOD LABORATORY PRACTICE: A signed GLP statement was provided. The study was compliant with OECD and UK GLP regulations. 38 STUDY SUMMARY: In a laboratory study, predatory bug ( Orius insidiosus) nymphs were provided artificial diet containing MCRY3A 0102 at a nominal rate equivalent to 50 Fg mCry3A/ g diet for 21 days. The test substance was purified from cells of recombinant E. coli. A negative control group received artificial diet mixed with deionized water only, and a positive control group received diet containing teflubenzuron ( 0.01 mg a. i./ g of diet). At the end of the test, there was no statistically significant difference in the percent mortality of the MCRY3A 0102 and negative control groups. Mortality in the positive control group was significantly increased.

CLASSIFICATION: Acceptable Test Material The test substance, MCRY3A 0102, active ingredient modified Cry3A protein, Batch Number MCRY3A 0102, was supplied by the sponsor with a reported purity of 90.3% w/ w and an expiration date of December 3, 2003. The test substance was purified from cells of recombinant E. coil. The test substance was stored at  30E to  20EC prior to use. The study was conducted prior to the test substance expiration date. The concentration and activity of MCRY3A 0102 in the treated diet were determined in a separate study ( MRID 46265610). Recovery of MCRY3A 0102 from the test diet was 95.6% of the nominal concentration.

Test Methods Predatory bug nymphs( Orius insidiosus, reared in house from adults obtained from Koppert UK Ltd., Suffolk, UK) were provided artificial diet containing MCRY3A 0102 at a rate equivalent to 50 Fg MCRY3A 0102/ g diet for 21 days. The base diet was a paste composed of beef, lamb, yeast, and honey. Diet for the test substance group was prepared by diluting 0.0554 g (~ 0.05 g mCry3A) of MCRY3A 0102 to 100 mL with deionized water, and mixing 20 mL of this preparation with 180 g of diet. The negative control group received the artificial diet mixed with deionized water only, and the positive control group received diet containing teflubenzuron ( 0.01 mg a. i./ g of diet). The prepared diet was stored frozen until needed.

The test chambers were plastic pots with ventilated lids. The tops of the cylinders were coated with Fluon to prevent the bugs from crawling upwards. One 2 to 3 day old nymph was placed in each chamber with approximately 0.2 g of treated diet in a plastic cup covered with Parafilm. The nymphs pierced the Parafilm to feed. Fresh diet was provided daily after being allowed to defrost for approximately one hour. A cotton ball moistened with tap water provided drinking water. 39 The study was carried out in an environment controlled room maintained at 25 to 26EC and 61 to 87% relative humidity. The photoperiod was 16 hrs light/ 8 hrs darkness. The nymphs were monitored for condition daily until they became adults or until 21 days after test initiation, whichever came first. At the end of the study, the percent pre imaginal mortality for each treatment was calculated and the data were analyzed statistically.

Results Summary There was no significant difference in the percent pre imaginal mortality between the MCRY3A 0102 and negative control groups ( Table 1). Mortality in the positive control group was significantly increased.

TABLE 1. Mortality of O. insidiosus supplied with treated diet Treatment % Pre imaginal Mortalitya % Corrected Mortalityb MCRY3A 0102 ( 50 Fg mCry3A/ g diet) 18 0 Negative control 23

Teflubenzuron ( 10 Fg/ g diet) 98*** 97 a) Results for individual treatments were compared to the control by Fisher's Exact Test b) Corrected using Abbotts formula *** Signficantly different from control p# 0.001 Data from p. 16, MRID 46265609 Study Author's Conclusions The study author concluded that MCRY3A 0102 was not harmful to O. insidiosus when offered continuously in the diet at a rate equivalent to 50 Fg MCRY3A 0102/ g of diet. 40 DATA EVALUATION RECORD Primary Reviewer: Eric B. Lewis EPA Secondary Reviewer: Mika J. Hunter STUDY TYPE: Nonguideline MRID NO: 46265610 DP BARCODE: DP303605 CASE NO: Not provided SUBMISSION NO: Not provided TEST MATERIAL: MCRY3A 0102 STUDY NO: SSB 005 04 SPONSOR: Syngenta Seeds, Inc., Research Triangle Park, NC TESTING FACILITY: Syngenta Biotechnology, Inc., Research Triangle Park, NC TITLE OF REPORT: Analysis of Test Diet Used to Expose Orius insidiosus to Modified Cry3A Protein: Supplement to Report Titled  " A Laboratory Toxicity Test of Modified Cry3A Protein ( MCRY3A 0102) to the Predatory Bug, Orius insidiosus ( Heteroptera: Anthocoridae)"; Mambo Tox Study/ Report No. SYN 03 52 AUTHOR: Graser, G.

STUDY COMPLETED: April 23, 2004 CONFIDENTIALITY CLAIMS: None GOOD LABORATORY PRACTICE: A signed GLP statement was provided. The study was EPA GLP compliant. 41 STUDY SUMMARY: Samples of formulated meat diet treated with MCRY3A 0102 and used in a predatory bug ( Orius insidiosus) toxicity study ( MRID 46265609) were analyzed for MCRY3A 0102 protein using an enzyme linked immunosorbent assay ( ELISA). The integrity of the mCry3A in the treated diet was determined using Western blot analysis, and the bioactivity of MCRY3A 0102 in the treated diet was determined in a bioassay using Colorado potato beetle ( CPB) larvae. ELISA of the treated diet recovered 95.6% of the nominal MCRY3A 0102 diet concentration used in the original study ( 50 Fg/ g), indicating MCRY3A 0102 was present at the intended concentration. Western blot analysis of the treated diet indicated that MCRY3A 0102 did not degrade under conditions of the original test diet preparation or storage, and the CPB bioassay confirmed that the original nontarget insect test group ( O. insidiosus) had been exposed to active MCRY3A 0102.

CLASSIFICATION: Acceptable Test Material The test substances were samples of a formulated meat based diet containing MCRY3A 0102 ( active ingredient, mCry3A protein) that had been used in a previous non target insect toxicity study with Orius insidiosus ( MRID 46265609). The nominal active ingredient concentration in the diet for that study was 50 Fg MCRY3A 0102/ g of diet. Samples of control diet ( with deionized water instead of MCRY3A 0102) used in that study were also submitted for analysis. The samples were frozen immediately after preparation in the original study and remained frozen until analysis.

Test Methods The test substance was quantitatively analyzed for MCRY3A 0102 using an enzyme linked immunosorbent assay ( ELISA), and the integrity of the MCRY3A 0102 was determined using Western blot analysis. Standard operating procedures were referenced in the report. The bioactivity of mCry3a in the test substance was determined in a bioassay using Colorado potato beetle ( CPB) ( Leptinotarsa decemlineata) larvae.

For the ELISA and Western blot analysis, 3 mL of extraction buffer were added to approximately 1 g of the Orius test or control diet. Each sample was mixed, incubated on ice for one hour, homogenized, centrifuged for 10 minutes at 2600 x g, and the supernatants were collected. The pellets were extracted a second time using the same procedure, and a third extraction was done in an overnight incubation on ice. All three extracts of each sample 42 were centrifuged again for 10 minutes at 13,000 x g prior to analysis. These extracts were used for the ELISA, while only the first extract of each sample was used for the Western blot analysis.

For the ELISA, the extracts were analyzed for MCRY3A 0102 using immuno affinity purified rabbit anti mCry3A polyclonal antibodies and immuno affinity purified goat anti Btt ( native Cry3A from Bacillus thuringiensis subsp. tenebrionis) polyclonal antibodies.

For the Western blot analysis, two aliquots of the Orius test diet extract, containing 19 and 57 ng MCRY3A 0102, respectively ( as determined by ELISA), were subjected to SDS PAGE using an 8 16% polyacrylamide gradient gel. Two aliquots of a solution of MCRY3A 0102 ( positive control), containing 20 and 60 ng of MCRY3A 0102, respectively, and one aliquot of the Orius control diet ( negative control) were run concurrently on the same gel. After electroblotting, the membrane was probed with immuno affinity purified goat anti Btt polyclonal antibodies. Donkey anti goat IgG linked to alkaline phosphatase, diluted in blocking buffer, was used to bind to the primary antibody and visualized by development with alkaline phosphatase substrate solution. The Western blot was examined for the presence of intact immunoreactive mCry3A ( ca. 67,000 molecular weight) and immunoreactive MCRY3A 0102 polypeptides.

For the CPB bioassay, diets containing 10% and 20% of the Orius diet were prepared by adding 0.9 and 0.8 g, respectively, of stock CPB diet to 0.1 and 0.2 g, respectively, of the Orius diet. Control CPB diet was prepared in the same manner using the Orius control diet. Positive control diets were prepared by adding MCRY3A 0102 to stock CPB diet at concentrations equivalent to 12.5 and 50 Fg MCRY3A 0102/ g. As a control for the positive control diet, CPB diet was prepared using the same volume of water as was used to add the positive control solution to the CPB diet. An untreated CPB diet control was also included. A piece of the supplemented diet ( 10% or 20% Orius test diet or 10% or 20% Orius control diet), was placed in a Petri dish with 10 newly hatched CPB larvae. Similarly, groups of 10 larvae were provided with stock CPB diet ( untreated control), stock CPB diet treated directly with MCRY3A 0102 ( positive control), or stock CPB diet treated with water ( control for the positive control). Each treatment was replicated 3 times. The dishes were covered and maintained at ambient conditions for 96 hours. Diets were replaced after approximately 24 and 48 hrs with fresh diet prepared in the same manner. Mortality was assessed daily.

Results Summary ELISA of the Orius test diet extract showed a recovery of 95.6% of the nominal 50 Fg MCRY3A 0102/ g of diet concentration used in the Orius study. No MCRY3A 0102 was detected in the Orius control diet.

Western blot analysis of the Orius test diet showed a single immunoreactive band corresponding to the predicted molecular weight of MCRY3A 0102 ( ca. 67,700 Da). No immunoreactive material was detected in the Orius control diet. 43 In the CPB bioassay, the diets prepared with 10% and 20% of the original Orius test diet produced 83% and 90% larval mortality, respectively, after 96 hours. The positive controls produced similar mortality. The Orius control diet produced 13% mortality, similar to the control CPB diets ( 10%).

Study Author's Conclusions The study author concluded that a) the high proportion of MCRY3A 0102 recovered by ELISA ( 95.6%) indicated mCry3A was present in the Orius test diet at the intended concentration, b) the Western blot analysis showed that MCRY3A 0102 did not degrade under conditions of the Orius test diet preparation or storage, and c) the bioassay results confirmed that the Orius test group was exposed to active mCry3A. 44 DATA EVALUATION RECORD Primary Reviewer: Eric B. Lewis EPA Secondary Reviewer: Mika J. Hunter STUDY TYPE: Honey Bee Testing, Tier I ( 885.4380)

MRID NO: 46155818 DP BARCODE: DP300338 CASE NO: Not provided SUBMISSION NO: Not provided TEST MATERIAL: MCry3A 0102 STUDY NO: SYN 03 57 SPONSOR: Syngenta Seeds, Inc., Research Triangle Park, NC TESTING FACILITY: Mambo Tox Ltd., Southampton, UK TITLE OF REPORT: A Semi Field Test to Evaluate the Effects of the Modified Cry3A Protein ( MCRY3A 0102) on Brood Development of the Honeybee, Apis mellifera ( Hymenoptera: Apidae)

AUTHOR: Halsall, N.

STUDY COMPLETED: December 2, 2003 CONFIDENTIALITY CLAIMS: None GOOD LABORATORY PRACTICE: A signed GLP statement was provided. The study was OECD and UK GLP compliant. 45 STUDY SUMMARY: Honeybees ( Apis mellifera) were exposed via in hive feeders to either a nominal concentration of 50 Fg MCRY3A 0102/ g of sucrose solution, 480 g of diflubenzuron insect growth regulator/ L of sucrose solution, or 50% w/ v sucrose solution alone for 5 days and monitored for 21 days. There were no statistically significant differences in egg cell mortality, larval cell mortality, or pre and post test hive condition between the test and sucrose solution only groups. The diflubenzuron positive control produced 100% mortality in both egg and larval cells, and significantly reduced post treatment hive condition. Adult bees were generally not affected by any of the treatments.

CLASSIFICATION: Acceptable Test Material The test substance, MCRY3A 0102, active ingredient modified Cry3A protein, Batch Number MCRY3A 0102, was supplied by the sponsor with a reported purity of 90.3%. For the test, the test substance was suspended in 200 mL of 50% w/ v sucrose solution at a concentration of 50 Fg mCry3A protein/ g of sucrose solution. The test substance was purified from cells of recombinant E. coli.

Test Methods The test occurred in a private garden in Chilworth, Hampshire, UK, with no areas of flowering crops within foraging distance ( distance not specified). Three days prior to the study, 16 healthy hives of honeybees ( Apis mellifera) in similar condition were transferred to the test site. The distance between test hives was not noted in the study write up. At the test start, a sufficient number of frames were removed from each hive to record the position of 100 cells containing eggs and 100 cells containing 1 to 3 day old larvae. The cells were mapped on an acetate sheet overlaid on each frame. A dead bee trap was fitted to the entrance of each hive.

The treatment solutions were supplied to each treated hive using a 1 L capacity commercial bee feeder. Hives received either 50 Fg of the test material/ g of sucrose solution, a negative control of 50% w/ v sucrose solution, or a positive control of 480 g/ L diflubenzuron insect growth regulator in sucrose solution. Each treatment was replicated 4 times. The treatment solutions were prepared fresh daily and replaced in each hive daily over 5 days. The diet was not analyzed in any additional studies. 46 Brood development was monitored for 21 days after the beginning of treatment. Egg cells were assessed at days 4, 6, 12, 19, and 21. Larval cells were assessed at days 4, 6, 12, and 19. The dead bee traps were assessed at study start and every 1 to 3 days thereafter for the duration of the study. Dead adults, larvae, and pupae were discarded after counting. The hives were assessed for general condition [ the amount of brood and food ( pollen and nectar reserves)] on days 0 and 22.

The percent mortality among treatments was compared using a Chi square test, both before and after applying Abbott's formula to correct for control mortality. Hive condition before and after treatments was compared using ANOVA. The level of significance used was 5%.

Results Summary All the treatment solutions were consumed each day. Egg cell mortality in the negative control, test, and positive control groups was 28.5%, 27.3%, and 100%, respectively. Larval cell mortality was 6.0%, 6.8%, and 100%, respectively. There was no significant difference in mortality between the test and negative control groups. There was also no significant difference in pre and post test hive condition between the test and negative control treatments. Results for the positive control treatment were significantly different from the other treatments for both mortality and hive condition. The number of dead bees found during the test was too low for meaningful comparison among treatments.

Table 1. Summary of Development of Egg Cells, Larval Cells, and of Brood Count Per Frame Treatment Brood Development Assessmentsa Mean % Brood per Frame b( before and after treatment) Egg Cells Larvae Cells Before After % Mortality % Corrected Mortality % Mortality % Corrected Mortality Treatment 28.5  6.0  36.4 49.6 MCRY3A 0102 27.3 0 6.8 0.9 34.2 40.7 Dimilin Flo 100.0*** 100.0 100.0 100*** 34.5 27.7** a) For the brood development assessments, mortality in individual treatments was compared to the control using Chi square tests. Results marked with asterisks differed significantly from the control (*** P < 0.01) b) For the brood percent assessments, a one way ANOVA was used to compare mean percent brood on each side of each frame between treatments. Prior to analysis an angular transformation was applied to the data. No significant differences occurred between any of the treatments before treatment ( P > 0.05). After treatment, however, the mean percentage area of each frame occupied by brood was significantly lower in the Diminlo Flo treatment when compared to the control Study Authors' Conclusions The study authors concluded that MCRY3A 0102 fed to honeybees for 5 days at a concentration nominally equivalent to 50 Fg MCRY3A 0102 protein/ g of sucrose solution did not adversely affect condition or survival of larvae in brood cells, and had no effect on survival of exposed adult worker bees. 47 DATA EVALUATION RECORD Primary Reviewer: Eric B. Lewis EPA Secondary Reviewer: Mika J. Hunter STUDY TYPE: Nonguideline MRID NO: 46265602 DP BARCODE: DP303605 CASE NO: Not provided SUBMISSION NO: Not provided TEST MATERIAL: MCry3A 0102 STUDY NO: Brixham Environmental Laboratory Report Project ID 03 0294/ A Syngenta Seeds Biotechnology Report No. SSB 001 04 SPONSOR: Syngenta Seeds, Inc., Research Triangle Park, NC TESTING FACILITY: Brixham Environmental Laboratory, Brixham, Devon TQ5 8BA, UK TITLE OF REPORT: Characterization of Fish Feed Test Substance ( FFMIR604 0103) Prepared From Event MIR604 Derived Maize Grain AUTHOR: Graser, G.

STUDY COMPLETED: April 26, 2004 CONFIDENTIALITY CLAIMS: None GOOD LABORATORY PRACTICE: A signed GLP statement was provided. The study was USEPA GLP compliant.

STUDY SUMMARY: Formulated fish feed containing 50% by weight transgenic maize ( Event MIR604) and used as diet in a 28 day rainbow trout feeding study was analyzed for mCry3A protein content by enzyme linked immunosorbent assay. The mCry3A was uniformly distributed in the formulated feed at a concentration of 0.09 ± 005 Fg/ g fresh weight.

CLASSIFICATION: Acceptable 48 Test Material The test substance, FFMIR604 0103, was a formulated fish feed ( Zeigler Bros., Inc.) prepared from Event MIR604 maize grain, which contains mCry3A protein. The nutritionally balanced feed contained 50% w/ w maize grain, and was " cold" pelleted to minimize thermal degradation of mCry3A. A fish feed control substance, FFMIR604 0103C, was prepared in the same manner, using grain from a non transgenic isoline. The test and control substances were used in a previous 28 day rainbow trout feeding study ( MRID 46155617), and samples were frozen until analysis. The ingredients and nutritional composition of the formulated fish feeds are provided in Appendix A of MRID 46465602, and the method used to prepare the feeds is provided in Appendix B. A sample of the ground transgenic maize grain ( KMIR604 0103) used to prepare the test substance, and a sample of the non transgenic maize grain ( KMIR604 0103C) used in the control feed, were also analyzed.

Test Methods The test substances were quantitatively analyzed for mCry3A using an enzyme linked immunosorbent assay ( ELISA). Standard operating procedures were referenced in the report. Three mL of extraction buffer were added to approximately 1 g samples of the ground maize grains and to three separate, random aliquots of the fish feed samples. The samples were then mixed, incubated on ice for 30 minutes, and homogenized prior to centrifugation for 15 minutes at 2500 x g. The supernatants were quantitatively analyzed by ELISA using immuno affinity purified rabbit anti mCry3A polyclonal antibodies and immuno affinity purified goat anti Btt ( native Cry3A from Bacillus thuringiensis subsp. tenebrionis) polyclonal antibodies. The homogeneity of the fish feed test substance FFMIR604 0103 was assessed by comparing the mCry3A concentrations in the three samples.

Results Summary The mCry3A concentration in the ground transgenic grain ( KMIR604 0103) was 0.3 Fg mCry3A/ g fresh weight. The mean mCry3A concentration in the fish feed test substance ( FFMIR604 0103) was 0.09 ± 0.005 Fg mCry3A/ g fresh weight. The mCry3A concentrations in the three random fish feed test substance samples were 0.09, 0.09, and 0.10 Fg mCry3A/ g fresh weight, indicating that mCry3A was present in the feed at a uniform concentration. No mCry3A was detected in the ground control maize grain ( KMIR604 0103C) or in the control fish feed ( FFMIR604 1013C). It should be noted that the concentration in the fish food was significantly less than 10 Fg. It was not stated the concentration of mCry3A expressed in the corn itself.

Study Author's Conclusions The study author concluded that mCry3A protein was present in the fish feed test substance ( FFMIR604 0103) that was used as test diet in the previous 28 day rainbow trout feeding 49 study. The mCry3A concentration in the feed was 0.09 Fg/ g fresh weight, and mCry3A appeared to be uniformly distributed in the feed. 50 DATA EVALUATION RECORD Primary Reviewers: Lewis, E. B., A. Q. Armstrong, R. H. Ross, L. A. Wilson EPA Secondary Reviewers: G. S. Tomimatsu, Ph. D, Mika J. Hunter STUDY TYPE: Earthworm Subchronic Toxicity Test; OECD Guideline Study No. 207 MRID NO: 462656011 DP BARCODE: DP303605 TEST MATERIAL: mCry3A 0102 STUDY NO: Mambo Tox Study/ Report No. SYN 03 55 Syngenta Project No. 2033037 SPONSOR: Syngenta Seeds, Inc., Research Triangle Park, NC TESTING FACILITY: Mambo Tox Ltd., Southampton, UK TITLE OF REPORT: Determination of Acute Toxicity of Modified Cry3A Protein ( MCRY3A 0102) to the Earthworm Eisenia fetida in an Artificial Soil Substrate AUTHOR: Vinall, S. STUDY COMPLETED: April 7, 2004 CONFIDENTIALITY CLAIMS: None GOOD LABORATORY PRACTICE: A signed GLP statement was provided. The study was compliant with OECD and UK GLP regulations.

STUDY SUMMARY: In a laboratory test, earthworms ( Eisenia fetida) in artificial soil were exposed to MCRY3A 0102 at a nominal concentration of 370 Fg/ g dry soil ( 334 Fg MCRY3A 0102 protein/ g dry soil) for 14 days. A negative control of deionized water and a positive control of 10, 20, 30, 40, or 50 mg 2 chloroacetamide/ kg of dry soil were also used in the test. At test end, MCRY3A 0102 treated worms had a mortality rate of 5% and a mean weight loss of 5.8%. Negative control worms had a mortality rate of 0% and a mean weight loss of 11.4%. Positive control worms exposed to   30 mg 2 chloroacetamide/ kg of dry soil had a mortality rate of 100%. CLASSIFICATION: Acceptable Test Material The test substance, MCRY3A 0102, active ingredient modified Cry3A protein, Batch Number MCRY3A 0102, was supplied by the sponsor with a reported purity of 90.3% w/ w and an expiration date of July 15, 2004. The test substance, MCRY3A 0102, was purified from cells of recombinant E. coil. The test substance was stored at  30E to  20EC prior to use. The study was conducted prior to the test substance expiration date. The presence and activity of mCry3A in the artificial soil used in the test were confirmed in a separate study 51 ( MRID 46265612). Extraction of mCry3A from treated soil samples collected on days 0, 3, 7, and 14 of the study recovered 18.1%, 14.8%, 6.3%, and 7.6% of the nominal concentration, respectively.

Test Methods Adult earthworms with clitellum ( Eisenia fetida, from Blades Biological, Edenbridge, Kent, UK) were exposed to MCRY3A 0102 in artificial soil , a negative control ( deionized water) or a positive control ( 2 chloroacetamide) for 14 days. The test material was prepared by adding 184.6 mg of MCRYC3A 0102 to127.5 mL of deionized water and mixing it with 500 g ( dry wt) of artificial soil, for a concentration of 370 Fg/ g dry soil ( 334 Fg MCRY3A 0102 protein/ g dry soil) 21; as clarified in a detailed report, " Characterization of Modified Cry3A Test Substance ( MCRY3A 0102) and Certificate of Analysis ( Joseph and Graser, 2003; Syngenta Seeds Biotechnology Report No. SSB 025 03). The negative control group received 127.5 mL of deionized water in 500 g of dry soil. The positive control was a rate response test using concentrations equivalent to 10, 20, 30, 40, or 50 mg of 2 choroacetamide/ kg of dry soil. Worms with a wet weight of 300 to 600 mg each was used for testing and was acclimated for about 24 hrs.

The artificial soil in each test chamber was composed of 10% peat, 20% clay, and 70% sand ( total of 498.5 g) with 1.5 g CaCO3 added to adjust the pH to 6.0 ± 0.5. During the test the soil was maintained at approximately 50% water holding capacity. The test chambers were 1 L glass jars with hinged glass lids. At study start, 10 adult earthworms ( wet weight 300 to 500 mg each) were placed on the surface of the treated soil in each test chamber and the lids were fitted. The test material and negative control treatments were replicated 4 times; the positive control treatments were replicated 3 times. Mortality was assessed at days 7 and 14, and surviving worms in the test material and negative control treatments were individually weighed at test end. The test was conducted in a controlled environment of 18 to 20EC and 57% to 89% relative humidity, with constant overhead light ( 485 569 lux; ELE Single Channel Light Measuring System, Skye Instruments Ltd., Powys, UK). The soil pH was measured at study start and end ( Hydrus 300 pH meter).

Frozen samples of treated soils were shipped to Syngenta Biotechnology ( SBI) for confirmation of the presence/ absence of MCRY3A 0102 protein.

Results Summary Mortality ( based on motility in response to a mechanical stimulus) and mean weight loss were comparable between the mCRY3A 0102 and negative control treatments ( Table 1). Day 7 mortality in the positive control treatment was 100% for all groups receiving $ 30 mg 1Based on the sponsor's determination that the average concentration of Event MIR604 hybrids was ~ 0.25Fg/ g dry weight roots; and the extraction efficiency for roots was 75% and after correcting for purity ( 90.3%). 52 2 chloroacetamide/ kg soil, indicating the assay was appropriate. The 14 day LC50 for 2 chloroacetamide was calculated by probit analysis to be 18 mg/ kg.

TABLE 1. Mortality and weight loss of E. fetida Cumulative mortality (%) Treatment Day 7 Day 14 14 Day Weight loss (%)

MCRY3A 0102 2.5 5.0 5.8 Negative control 0 0 11.4 Data from p. 16, MRID 46265611 TABLE 2. Cumulative Percent Mortality of Worms for Each Rate of the Toxic Reference at the 7 and 14 DAT Assessments Concentration of 2 chloroacetamide ( mg/ kg[ dry weight] or artificial soil Assessment Time 10 20 30 40 50 Estimate of LC50 a 7 DAT 0 60 100 100 100  14 DAT 3 60 100 100 100 18 mg/ kg a Probit analysis was performed on 14 day mortality for the 2 chloroacetamide treatment rates to derive the LC50. Prior to analysis, the dose rates were log10 transformed.

Range of pH values were recorded and appeared consistent throughout the test: pH 6.0 ± 0.2 amongst untreated and treated soils.

Study Author's Conclusions The study author concluded that a 14 day exposure of earthworms to the test material at a rate equivalent to 334 Fg MCRY3A 0102/ g dry soil did not adversely affect earthworm weight or survival. 53 DATA EVALUATION RECORD Primary Reviewer: Eric B. Lewis EPA Secondary Reviewer: Mika J. Hunter STUDY TYPE: Nonguideline MRID NO: 46265612 DP BARCODE: DP303605 CASE NO: Not provided SUBMISSION NO: Not provided TEST MATERIAL: MCRY3A 0102 STUDY NO: SSB 008 04 SPONSOR: Syngenta Seeds, Inc., Research Triangle Park, NC TESTING FACILITY: Syngenta Biotechnology, Inc., Research Triangle Park, NC TITLE OF REPORT: Analysis of Artificial Soil Used to Expose Eisenia fetida to Modified Cry3A Protein: Supplement to Report Titled " Determination of Acute Toxicity of Modified Cry3A Protein ( MCRY3A 0102) to the Earthworm Eisenia fetida in an Artificial Soil Substrate"; Mambo Tox Study/ Report No. SYN 03 55 AUTHOR: Graser, G.

STUDY COMPLETED: April 23, 2004 CONFIDENTIALITY CLAIMS: None GOOD LABORATORY PRACTICE: A signed GLP statement was provided. The study was US EPA GLP compliant. 54 STUDY SUMMARY: Samples of artificial soil treated with MCRY3A 0102 at a rate nominally equivalent to 334 Fg MCRY3A 0102 protein/ g dry soil and used in an earthworm ( Eisenia fetida) toxicity study ( MRID 46265611) were analyzed for MCRY3A 0102 protein using an enzyme linked immunosorbent assay ( ELISA). The integrity of the MCRY3A 0102 in the treated soil was determined using Western blot analysis, and the bioactivity of MCRY3A 0102 in the treated soil was determined in a bioassay using Colorado potato beetle ( CPB) larvae. ELISA of the treated soil recovered about 18% of the nominal MCRY3A 0102 soil concentration on day 0. Extraction on days 3, 7, and 14 resulted in 14.8%, 6.3%, and 7.6% of the nominal concentration mixed in the soil. The author attributed the low recovery to the physico chemical characteristics of the soil. Western blot analysis of treated soil indicated that MCRY3A 0102 was present in the soil throughout the 14 day study, and that some degradation to lower molecular weight polypeptides occurred, beginning at day 3. The CPB bioassay confirmed that MCRY3A 0102 treated soil was bioactive at all sampling times, resulting in larvae mortality of 60% and 83% after 120 hours for the CPB diets prepared with 5% and 10% treated soil.

CLASSIFICATION: Acceptable Test Material The test substance was artificial soil prepared in parallel in the same manner and maintained under the same conditions as soil treated with MCRY3A 0102 ( active ingredient, mCry3A protein) that was used in a previous non target organism toxicity study with Eisenia fetida ( MRID 46265611). For that study, the artificial soil was treated with MCRY3A 0102 at a nominal rate equivalent to 334 Fg MCRY3A 0102 / g dry weight. Samples of the treated soil were collected directly after preparation ( day 0), and at 3, 7, and 14 days after treatment. A sample of untreated artificial soil from the same batch was used as a control. All samples were frozen and remained frozen until analysis.

Test Methods The test substance was quantitatively analyzed for MCRY3A 0102 using an enzyme linked immunosorbent assay ( ELISA), and the integrity of the MCRY3A 0102 was determined using Western blot analysis. Standard operating procedures were referenced in the report. The bioactivity of mCry3a in the test substance was determined in a bioassay using Colorado potato beetle ( CPB) ( Leptinotarsa decemlineata) larvae. 55 For the ELISA and Western blot analysis, 10 mL of extraction buffer were added to approximately 2 g of treated or control soil. Each sample was mixed, incubated on ice for one hour, centrifuged for 15 minutes at 2500 x g, and the supernatants were collected. The pellets were extracted a second and a third time for 30 minutes under constant rotation. A fourth extraction was done overnight in the same way. All four extracts for each sample were centrifuged again for 10 minutes at 13,000 x g prior to analysis. These extracts were used for the ELISA, while only the first extract of each sample was used for the Western blot analysis.

For the ELISA, the extracts were analyzed for mCry3A using immumo affinity purified rabbit anti mCry3A polyclonal antibodies and immuno affinity purified goat anti Btt ( native Cry3A from Bacillus thuringiensis subsp. tenebrionis) polyclonal antibodies.

For the Western blot analysis, two aliquots of the treated soil extract, containing approximately 8.5 and 17 ng MCRY3A 0102 respectively ( as determined by ELISA), were subjected to SDS PAGE using an 8 16% polyacrylamide gradient gel. Two aliquots of a solution of MCRY3A 0102 ( positive control), containing 10 and 20 ng of MCRY3A 0102, respectively, and one aliquot of the control soil extract ( negative control) were run concurrently on the same gel. After electroblotting, the membranes were probed with immuno affinity purified goat anti Btt ( native Cry3A from Bacillus thuringiensis subsp. tenebrionis) polyclonal antibodies. Donkey anti goat IgG linked to alkaline phosphatase, diluted in blocking buffer, was used to bind to the primary antibody and visualized by development with alkaline phosphatase substrate solution. The Western blot was examined for the presence of intact immunoreactive MCRY3A 0102 ( ca. 67,000 molecular weight) and immunoreactive MCRY3A 0102 polypeptides.

For the CPB bioassay, diets containing 10% and 5% ( w/ w) treated artificial soil were prepared by adding 4.50 and 4.75 g, respectively, of stock CPB diet to 0.50 and 0.25 g, respectively, of the MCRY3A 0102  treated soil. Control CPB diet was prepared in the same manner using the untreated artificial soil, to which 250 FL of deionized water/ g of soil was added. Positive control diets were also prepared by adding MCRY3A 0102 to stock CPB diet at concentrations equivalent to 12.5 and 50 Fg MCRY3A 0102 / g. As a control for the positive control diet, CPB diet was prepared using the same volume of water as was used to add the positive control solution to the CPB diet. An untreated CPB diet control was also included. A piece of the supplemented diets ( 5% or 10% control soil or 5% or 10% MCRY3A 0102  treated soil) was placed in a Petri dish with 10 newly hatched CPB larvae. Similarly, groups of 10 larvae were provided with stock CPB diet ( untreated control), stock CPB diet treated directly with MCRY3A 0102 ( positive control), or stock CPB diet treated with water ( control for the positive control). Each treatment was replicated 3 times. The dishes were covered and maintained at ambient conditions for 120 hours. Diets were replaced every 24 hrs with fresh diet prepared in the same manner. Mortality was assessed daily. 56 Results Summary ELISA of the treated artificial soil extracts showed recoveries of 18.1%, 14.8%, 6.3%, and 7.6% for the 0, 3, 7, and 14 day samples, respectively. No MCRY3A 0102 was detected in the untreated control soil.

Western blot analysis of the MCRY3A 0102 treated artificial soil collected at all sampling times showed a single immunoreactive band corresponding to the predicted molecular weight of mCry3A ( ca. 67,700 Da). Beginning with the 3 day samples, one or more bands representing MCRY3A 0102 degradation products were seen; these were more prominent in the 7 and 14 day samples. The major degradation products appeared as a doublet band at ca. 57,000 Da. No immunoreactive material was detected in extracts of the untreated control artificial soil.

In the CPB bioassay, the MCRY3A 0102  treated soil showed bioactivity at all sampling times, producing 60 83% larval mortality after 120 hrs for the diets prepared with 5% and 10% treated soil. The positive controls produced 87% and 97% mortality. The negative control soils produced 13 and 20% mortality, similar to the control CPB diets ( 20%).

Study Author's Conclusions The study author concluded that active MCRY3A 0102 was present in the artificial soil used in the Eisenia fetida toxicity study. While only about 18% of the nominal mCry3A concentration in the day 0 treated soil was recovered, the study author attributed the low recovery to the physico chemical properties of the soil matrix. Western blot analysis indicated that intact MCRY3A 0102 was present in the soil throughout the study, and that some degradation to lower molecular weight polypeptides occurred during the study. Finally, the study author concluded that MCRY3A 0102  treated soil samples collected at days 0, 3, 7, and 14 of the CPB bioassay were highly bioactive against CPB larvae. 57 DATA EVALUATION RECORD Primary Reviewer: Eric B. Lewis EPA Secondary Reviewer: Mika J. Hunter STUDY TYPE: Nonguideline MRID NO: 46265615 DP BARCODE: 303605 CASE NO: Not provided SUBMISSION NO: Not provided TEST MATERIAL: MIR604 ( Bacillus thuringiensis mCry3A protein)

STUDY NO: MIR604 SPONSOR: Syngenta Seeds, Inc., Research Triangle Park, NC 27709 TESTING FACILITY: North Carolina State University, Chicken Educational Unit, Raleigh, NC 27603 TITLE OF REPORT: Evaluation of Transgenic Corn ( Maize) MIR604 in Broiler Chickens AUTHOR: Brake, J. T.

STUDY COMPLETED: April 14, 2004 CONFIDENTIALITY CLAIMS: None GOOD LABORATORY PRACTICE: A signed GLP statement was provided stating the study was not GLP compliant, but was conducted according to accepted scientific methods. 58 STUDY SUMMARY: Chickens were fed formulated diets containing either transgenic MIR604 corn with mCry3A protein, an MIR604 isoline non transgenic corn, or a non transgenic NC 2003 commercial corn for 49 days. The concentration of mCry3A protein in the transgenic MIR604 diets was reported to be 0.04 Fg/ g dry wt, 0.06 Fg/ g dry wt, and 0.08 Fg/ g dry wt in the starter, grower, and finisher diets, respectively. Chicken mortality in all treatments was comparable. Mean body weight and feed conversion ratios for birds in the NC 2003 treatment were significantly lower than those for birds in either MIR604 treatment at days 31 and 49, probably as a result of a slightly lower protein concentration in the NC 2003 diet. Overall carcass yield was similar for all treatments, although thigh weight was significantly lower in the NC 2003 treatment males.

CLASSIFICATION: Supplemental Test Material The test substance was MIR604 Positive ( maize grain containing Bacillus thuringiensis mCry3A protein), supplied by the sponsor and formulated into broiler diet. The concentration of mCry3A protein in MIR604 Positive grain was reported to be 0.23 ± 0.035 Fg/ d dry wt. The concentration of mCry3A protein in the formulated diets used in the test were reported to be 0.04 ± 0.005 Fg/ g dry wt in the starter diet, 0.06 ± 0.005 Fg/ g dry wt in the grower diet, and 0.08 ± 0.013 Fg/ g dry wt in the finisher diet.

Test Methods Chickens ( Gallus domesticus, Ross 344 males and Ross 308 females, from eggs produced and incubated in house) were fed diets containing grain from a transgenic corn hybrid ( MIR604 Positive), a non transgenic isoline corn hybrid ( MIR604 Negative), or a nontransgenic locally grown commercial corn ( NC 2003) for 49 days. Day old chicks were randomly assigned to the treatments. Each pen contained 25 birds of the same sex. Each treatment group contained 12 cages of birds, 6 cages of males and 6 cages of females. ( 900 birds total in the study). The pens were 4 ft x 12 ft x 8 ft wood frames with plastic coated wire mesh and were maintained in a curtain sided house that was heated or ventilated as necessary. Illumination was from incandescent lighting for 23 hrs/ day for days 1 7, 21 hrs/ day from days 8 21, and from natural daylight thereafter. The house was checked twice/ day for temperature and lighting, and the birds were checked twice/ day for clinical signs and mortality.

Diets formulated with the different corn types were standardized to be of relatively similar nutrient composition ( nutrient analyses were provided in the report). Starter, grower, and finisher diets were formulated with 57.5%, 63.0%, and 67.5% corn, respectively. Starter diet 59 was provided until day 16, at which time grower diet was added to any remaining starter in the feeders and provided until day 31. On day 31, any remaining grower diet was discarded and finisher diet was provided until 16 hours prior to slaughter on day 50. Diet and well water were provided ad libitum. Pen weights were collected on days 1, 16, 31, and 49. Feed consumption data/ pen was determined on days 16, 31, and 49. On day 51 a random sample of 2 birds/ pen was collected and processed to determine carcass yield.

Body weight, feed conversion, and survival data were analyzed using the General Linear Model procedure by SAS, with a significance level of 5%. These variables were analyzed for the starter period ( days 0 16), grower period ( days 16 31), finisher period ( days 31 41), and a cumulative period ( 0 49 days).

Results Summary There were no significant adverse clinical signs noted, and no statistically significant difference in mortality was found among any of the treatments. Mean body weight and feed conversion ratios for birds in the MIR604 Positive and MIR604 Negative treatments were comparable ( Tables 1 and 2). Mean body weight and feed conversion ratios for birds in the NC2003 treatment were significantly lower than those for birds in either MIR604 treatment at days 31 and 49, probably as a result of a slightly lower protein concentration ( 6 8%, calculated by reviewer) in the NC2003 grower and finisher diets. Overall carcass yields for males and females of all treatments were comparable, although thigh weights of males in both MIR604 treatments were significantly higher than those of NC2003 males ( Table 3). When part weights were considered as a percent of live weight, there were some significant differences among the treatments for thighs in females and pectoralis minor in males. However, differences between the MIR604 Positive and NC2003 treatments were not significant in either sex.

Table 1. Mean Body Weight and Standard Error of Each Corn Diet and Sex Treatment Body weight ( hatch)

Grams Body weight ( day 16)

Grams Body weight ( day 31)

Grams Body weight ( day 49)

Grams MIR604 Positive 44.60 ± 0.19 547.8 ± 9.6 1684.1 ± 50.6a 3468.4 ± 129.5a MIR604 Negative 44.66 ± 0.18 563.0 ± 5.4 1690.5 ± 40.2a 3469.4 ± 113.1a NC 2003 44.70 ± 0.19 551.1 ± 9.6 1634.0 ± 44.3b 3365.2 ± 117.6b Male 44.70 ± 0.17 574.0 ± 5.07 A 1814.0 ± 13.27 A 3825.9 ± 26.65 A Female 44.54 ± 0.13 534.0 ± 4.93 B 1525.1 ± 9.51 B 3042.7 ± 15.56 B MIR604 Negative Male 44.76 ± 0.17 576.4 ± 6.38 1817.7 ± 23.00 3831.3 ± 58.73 MIR604 Negative Female 44.56 ± 0.21 549.7 ± 3.95 1563.2 ± 10.72 3107.5 ± 16.98 MIR604 Positive Male 44.53 ± 0.33 573.4 ± 10.42 1848.1 ± 19.11 3895.5 ± 22.93 60 MIR604 Positivefemale 44.67 ± 0.21 522.3 ± 6.24 1520.1 ± 11.86 3041.3 ± 34.15 NC 2003   Male 45.01 ± 0.25 572.1 ± 10.52 1776.2 ± 19.55 3750.9 ± 34.15 NC 2003   Female 44.40 ± 0.25 530.0 ± 10.66 1491.9 ± 12.30 2979.5 ± 13.34 a, b Means with different superscripts differ significantly ( p# 0.05) A, B Means that possess different superscripts differ significantly ( P # 0.01) Data from p. 23, MRID 46265615 TABLE 2. Mean feed conversion ratio1 ( g: g) of surviving birds Treatment Starter FCR ( days 0 16) Grower FCR ( days 16 31) Finisher FCR ( days 31 49) Cumulative FCR ( days 0 31) Cumulative FCR ( days 0 49)

MIR604 Positive 1.32 ± 0.0 1.55 ± 0.0b 2.00 ± .0 1.48 ± 0.0b 1.75 ± 0.0b MIR604 Negative 1.31 ± 0.0 1.56 ± 0.0b 2.01 ± 0.0 1.48 ± 0.0b 1.75 ± 0.0b NC 2003 1.35 ± 0.0 1.60 ± 0.0a 2.05 ± 0.0 1.52 ± 0.0a 1.79 ± 0.0a 1Total feed consumed/ total body weight a, bMeans with different superscripts differ significantly ( p# 0.05) Data from p. 24, MRID 46265615 61 TABLE 3. Carcass and parts yield at day 51 Males Treatment Dressed carcass1 ( g) Thighs ( g) Pectoralis major ( g) Pectoralis minor ( g)

MIR604 Positive 2947.2 ± 57.44 526.3 ± 14.05a 638.8 ± 19.18 150.8 ± 4.63 MIR604 Negative 2936.7 ± 56.88 484.3 ± 15.10ab 609.6 ± 16.73 145.7 ± 4.57 NC 2003 2812.9 ± 61.49 454.6 ± 20.30b 605.1 ± 23.09 151.3 ± 4.97 Dressed carcass2 (%) Thighs (%) Pectoralis major (%) Pectoralis minor (%)

MIR604 Positive 74.08 ± 0.50 13.23 ± 0.26 16.06 ± 0.39 3.80 ± 0.11ab MIR604 Negative 74.97 ± 0.27 12.37 ± 0.34 15.57 ± 0.33 3.71 ± 0.07b NC 2003 75.14 ± 0.37 12.12 ± 0.42 16.14 ± 0.45 4.03 ± 0.07a Females Dressed carcass ( g) Thighs ( g) Pectoralis major ( g) Pectoralis minor ( g)

MIR604 Positive 2248.3 ± 45.53 380.8 ± 7.6 515.4 ± 18.9 126.2 ± 3.3 MIR604 Negative 2291.0 ± 41.4 361.9 ± 9.2 511.8 ± 13.8 128.0 ± 3.5 NC 2003 2204.2 ± 54.8 366.2 ± 11.3 487.1 ± 22.0 125.9 ± 3.4 Dressed carcass (%) Thighs (%) Pectoralis major (%) Pectoralis minor (%)

MIR604 Positive 75.16 ± 0.41 12.74 ± 0.12a 17.22 ± 0.48 4.23 ± 0.11 MIR604 Negative 75.05 ± 0.51 11.87 ± 0.28b 16.76 ± 0.35 4.19 ± 0.10 NC 2003 75.27 ± 0.34 12.51 ± 0.25ab 16.59 ± 0.53 4.30 ± 0.06 1Fresh carcass without head, neck, feet, feathers, viscera, blood 2Expressed as percent of live body weight a, bMeans with different superscripts differ significantly ( p# 0.05) Data from pp. 27 and 28, MRID 46265615 Study Author's Conclusions The study author concluded that transgenic MIR604 hybrid diets supported broiler chicken growth at low mortality rates and very good feed conversion ratios without large differences in overall carcass yield. The study author also concluded that while it was unclear if the small differences seen could be attributed to the corn source per se or to slight differences in the formulated diets, the transgenic corn clearly had no deleterious effects in this study.