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Bacillus thuringiensis var. aizawai, Cry1F Protein - Registration of Herculex I 9/02

New York State Department of Environmental Conservation
Division of Solid & Hazardous Materials
Bureau of Pesticides Management
625 Broadway, Albany, New York 12233-7257
Phone: 518-402-8788 FAX: 518-402-9024
Website: http://www.dec.state.ny.us/website/dshm/pesticid/pesticid.htm
E-Mail: ppr@gw.dec.state.ny.us

September 5, 2002

CERTIFIED MAIL
RETURN RECEIPT REQUESTED


Raymond S. Brinkmeyer, Ph.D.
State Regulatory Leader
Dow AgroSciences Mycogen Seeds Division
9330 Zionsville Road
Indianapolis, Indiana 46268

Dear Dr. Brinkmeyer:

Re: Registration of One New Pesticide Product, Herculex I (EPA Reg. No. 68467-2), Which Contains the New Active Ingredient: Bacillus Thuringiensis var Aizawai Cry1F Insect Control Protein as Expressed in Maize, contained in Mycogen Brand BT Cry1F Corn

The New York State Department of Environmental Conservation (Department) has reviewed the application, received December 6, 2001, submitted by Dow AgroSciences, to register Herculex I (EPA Reg. No. 68467-2) in New York State.

The application was deemed complete for purposes of review on April 19, 2002, and a registration decision is due by September 16, 2002. Herculex I is a genetically engineered field corn modified to produce Bacillus thuringiensis, (Bt) subspecies aizawai Cry1F insecticidal protein. The endogenously produced protein provides protection from the lepidopteran pests European corn borer (Ostrinia nubilalis), Southwestern corn borer (Diatraea grandiosella), fall armyworm (spodeptera frugiperda), and black cutworm (Agrotis ipsilon). Herculex I has also been modified with a second inserted gene that codes for production of phosphinothricin acetyltransferase (PAT) conferring resistance to glufosinate ammonium herbicides.

ECOLOGICAL EFFECTS:

Herculex I exhibits a benign nontarget toxicity profile similar to traditional microbial Bt products. Results from toxicity studies conducted for federal registration can be summarized as follows:

Mice-Acute Oral: No effect observed at 576 mg Cry1F protein/kg body weight. All mammalian subchronic and chronic toxicity studies were waived.

Northern Bobwhite-Dietary: No effect observed in juvenile quail when Cry1F corn grain constituted ten percent (10%) of their diet for five days.

No other avian studies were reported.

Daphnia-Acute: Both the EC50 and NOEC are > 100ppm for both purified bacterially produced Cry1F delta-endotoxin and pollen from Cry1F expressing corn.

All other aquatic organism toxicity testing, including algae and aquatic macrophytes, was waived.

Honey bee-Dietary: No mortality, behavioral changes, or developmental effects were observed in a honey bee brood cell feeding study when larvae were fed either Cry1F corn pollen, or bacterially produced Cry1F protein at rates higher than expected field exposures.

While many of the toxicity studies normally conducted for a more traditional pesticide product were waived or not triggered by the results of the coarse screening work summarized above, studies with a number of nontarget beneficial invertebrates not required under United States Environmental Protection Agency (USEPA) guidelines were submitted.

Earthworm-Acute: No apparent treatment related effects or mortality reported during a 14-day study. LC50 > 2.26 mg Cry1F/kg soil, NOEC = 2.26mg Cry1F/kg soil which is roughly 100X the estimated environmental concentration, EEC, in the top six inches of soil following incorporation of 25,000 senescent corn plants per acre.

Collembola-Chronic: The LC50 and NOEC were both > 12.5 mg Cry1F/kg soil in a 28-day chronic study with Folsomia candida. The highest exposure level, 12.5 mg/kg, is roughly 1560X the soil EEC.

Green Lacewing Larvae-Dietary: The LC50 and NOEC were both > 480 ppm Cry1F protein. No mortality, abnormal behavior, or signs of toxicity were observed when Chrysoperla carnae larvae were fed Cry1F protein for 13 days at approximately 15X the concentration in corn pollen.

Parasitic Hymenoptera-Diet: The LC50 and NOEC were both > 320 ppm Cry1F protein in a 12-day Nasonia vitripennis feeding study. No treatment related mortality, signs of toxicity, abnormal appearance or behavior were observed.

Ladybird Beetle-Diet: The LC50 and NOEC were both > 480 ppm Cry1F protein in a 29-day feeding study with Hippodamia convergens. There was no observed mortality or signs of toxicity in adult beetles fed Cry1F at a rate approximating what they would ingest when feeding on aphids in the field (15X the concentration found in pollen).

Monarch Butterfly-Diet: The LC50 was > 10 ug Cry1F protein/ml diet and the NOEC was < 10 ug Cry1F protein/ml diet in a seven-day feeding study with first instar larval Danaus plexippus. There was no mortality at the 10 ug/ml rate, the highest rate tested, but there was some growth inhibition.

Field Survey of Beneficial Arthropods: A scientifically sound six-week sticky trap and visual survey study showed no consistent pattern of differences between beneficial arthropod numbers in two Cry1F corn isolines, 1507 and 1360, and nontransgenic control plantings. Visual counts showed no significant difference between Cry1F and control plantings with two exceptions. There was a significantly greater number of lady beetles in the 1507 line and a significantly greater number of spiders in the 1306 line than in the nontransgenic control plantings. Sticky trap counts showed no significant differences between the numbers of most arthropod species in Cry1F and control corn.

However, the average number of parasitic Hymenoptera and pirate bugs, Orius sp., was significantly greater at all sampling dates in the 1507 line than in the non-transgenic plantings.

In a tobacco budworm larvae, Heliothis virescens, bioassay based study purified Cry1F protein in powder form degraded in a bulk soil with a half-life of 3.13 days. The August 2001 USEPA Cry1F corn Biopesticide Registration Action Document, BRAD, states that several published studies indicate that Cry proteins expressed in transgenic corn degrade more rapidly in soil than purified Cry protein and that microbial populations in the rhizosphere are commonly 100X higher than bulk soils. Since Cry1F environmental dissipation is primarily microbially mediated, the submitted study is likely a conservative approximation of Cry1F field degradation rates.

RISK ASSESSMENT:

Herculex I corn is not likely to adversely affect fish or wildlife resources. In addition to the toxicity studies summarized previously, the USEPA BRAD contains summaries of studies showing that Cry1F and PAT proteins are readily digestible. In simulated gastric fluid, PAT protein degraded to small peptide fragments within ten seconds. Cry1F degraded within five minutes in a similar study. Cry1F was also shown to be labile to heat at 75 degrees C or greater. In addition, it has been shown that conventional processes used in the commercial preparation of fish food inactivate any Cry1F protein present in corn grain. No differences in levels of fatty acids, ash, vitamins, fiber, moisture, amino acids, or minerals were observed between the 1507 Cry1F hybrid and a genetically similar non Cry1F hybrid. In organisms not susceptible to Bt endotoxins, it appears that Cry1F protein is simply a digestible dietary protein.

In both the August 2001 Cry1F corn BRAD and the October 15, 2001 USEPA reassessment of Bt Plant-Incorporated Protectants (corn, cotton, and potatoes), there are substantial sections addressing the risks to nontarget lepidopterans, Monarch and Karner blue butterflies specifically. An analysis of corn pollen toxicity and dispersal, spatial overlap of host plants and corn fields, and temporal overlap of pollen shed and oviposition suggests minimal risk is posed to either species from Herculex I.

The avian dietary and soil degradation studies reviewed in the Cry1F BRAD were both classified as supplemental. To confirm the results of the Quail study, the USEPA has required a longer-term feeding study at a higher dose rate, six weeks with 60% to 70% Cry1F corn in the diet, as a condition of registration. They also required Cry1F soil concentration studies to be conducted over a range of corn culture climate conditions and locations in fields where Bt corn has been grown continuously for at least three years. The beneficial arthropod field study submitted was a single season, small plot study. A larger-scale, longer-term study has been required.

The Environmental Assessment Branch of the USEPA does not require an environmental exposure assessment on biological active ingredients unless the Toxicology Branch has a problem with the information presented by the registrant. In this case, no problem was noted; therefore, environmental fate data were not required. Since environmental fate data were not necessary, there is no impact to the groundwater to be assessed. Therefore, groundwater staff have no objection to the registration of this product as labeled.

HEALTH ASSESSMENT:

The USEPA required only a limited number of studies to assess the potential health effects from dietary exposure to the Cry1F protein. Specifically, the data generated from these studies were intended to show whether this protein displays any significant acute oral toxicity when administered at high doses and whether it is likely to be a food allergen.

An acute oral toxicity study on the end-use product Herculex I was not conducted since plant tissue such as corn is not considered an appropriate component of a laboratory animal's (rat or mouse) diet. The submitted acute oral toxicity data on Cry1F protein (from testing in mice) showed that this active ingredient, even at relatively high dose levels (576 milligrams Cry1F protein per kilogram body weight), was not toxic by this route of exposure. The USEPA did not require any other acute toxicity studies nor subchronic, chronic/carcinogenicity, genotoxicity, reproductive or developmental toxicity studies. Our search of the toxicological literature did not find any information on adverse reactions from exposure to the Cry1F protein.

Since Cry1F is a bacterial protein that could be introduced (either inadvertently or intentionally) into the human diet, the potential for this protein to act as a food allergen needs to be considered. The USEPA, in its review of the Cry1F protein, determined that this protein is readily degraded by simulated gastric (acidic) fluid containing the digestive enzyme pepsin, and also that it is unstable to heat (such as that produced during cooking and food processing). By contrast, common food allergens tend to be resistant to heat, acid and digestive (gastric) enzymes. In addition, a comparison of amino acid sequences of known allergens uncovered no evidence of linear sequence homology with the Cry1F protein. Finally, USEPA concluded that studies conducted in laboratory animals using B. thuringiensis or the endotoxin protein did not suggest any potential for allergic reactions to the Cry1F protein. Based on the apparent lack of allergenicity and toxicity by the Cry1F protein expressed in genetically modified corn, the USEPA exempted it and the genetic material necessary for its production in corn from the requirement of a tolerance when used as a plant pesticide in the food and feed commodities of field corn, sweet corn and popcorn.

An additional issue to consider is whether the Cry1F protein can be present in the milk, meat or meat by-products from animals fed field corn grown with Herculex I seed. While no data were available to directly address this potential, the available information suggests that the protein would not survive digestion in animals that were fed Herculex I corn. The data also suggest that the Cry1F protein would be removed by cooking products from such animals. If, however, an individual were to consume animal products containing the Cry1F protein, the protein would be subject to digestive processes and would not likely survive intact.

Although the data indicate that the Cry1F insect control protein produced in Herculex I corn possesses neither acute toxicity nor allergenic properties, some uncertainties remain. It is reasonable that the lack of heat stability and lack of resistance to enzymatic digestion of the Cry1F protein would prevent it from being an allergen by dietary exposure, but it is not entirely certain that this is so. Whereas these properties are useful as a screening tool, they cannot absolutely predict that a protein will or will not be an allergen. In addition, since the sequences for all allergens are not known, there is uncertainty that the lack of amino acid homology of the Cry1F protein with other known allergens ensures that dietary exposure to the Cry1F protein would not evoke an allergic response. Consequently, we cannot know with certainty whether the labeled use of Herculex I corn seed will not result in an increase in allergic reactions from food. As stated by the USEPA Scientific Advisory Panel (SAP Report No. 2000-07c, March 12, 2001, on Bt Plant Pesticides Risk and Benefits Assessments), "[O]nly surveillance and clinical assessment of exposed individuals will confirm the allergenicity of Bt products or for any other novel protein introduced into the diet of consumers." The USEPA states in their BRAD "...there is a reasonable certainty that no harm will result from aggregate exposure to the U.S. population, including infants and children, to the Cry1Ab and Cry1F proteins and the genetic material necessary for their production. The USEPA concludes that there are no adverse effects on human health from the use of Cry1Ab or Cry1F proteins expressed in corn."

According to the Herculex I Product Use Guide, the grower must have a valid executed Grower Agreement on file with Dow AgroSciences to legally obtain, plant and grow Herculex I Insect Protection corn. The grower must comply with the terms of the Grower Agreement and the Product Use Guide to prevent forfeiting the privilege to grow Herculex I corn. One of the terms of the agreement is the implementation of Insect Resistance Management (IRM). Not implementing Insect Resistance Management plans could lead to adaptation by the target pest populations to the Bt toxin and loss of efficacy of the product. IRM is mandated by the USEPA and is the responsibility of each grower using Bt corn protection technology.

As stated in the Product Use Guide, a key element in IRM is the use of a refuge. This would allow certain target insects to survive without exposure to Bt insecticidal proteins and sufficient target insects susceptible to Bt insecticidal proteins nearby to mate with any rare insects naturally resistant to Bt proteins that may emerge from the Herculex I corn fields. The susceptibility to Bt proteins is then passed on to their offspring.

In a corn belt growing area, up to 80 percent (80%) of the corn acres may be planted with Herculex I corn and at least 20 percent (20%) with non-Bt corn. The refuge requirements state that at least 20 percent (20%) of the total corn acres are planted with non-Bt corn. Insecticides may be applied to the refuge area only if economic thresholds are reached for one or more of the target pests. Microbial Bt insecticide products must not be used on the non-Bt corn refuge. The refuge may be arranged in different patterns but accurate records must be maintained of where the Bt and non-Bt crops are planted. Models from the USEPA BRAD predict that the European corn borer will not evolve resistance for at least 99 years if a 20 percent (20%) refuge is implemented.

As corn is a naturally cross-pollinated crop, the Herculex Product Use Guide suggests maintaining a non-corn buffer between Herculex I corn fields, not growing Herculex I upwind (based on the prevailing wind directions) of non-Herculex I corn fields, and discussing crop planting plans with neighbors in advance.

The following table outlines the confirmatory data that must be provided to USEPA as a condition of the amendment to the Cry1Ab and Cry1F corn product registrations including the due dates for the protocols and the data.

DATA
DESCRIPTION
DUE DATE
Residue Analytical Methods Analytical method including characterization of the antisera and independent laboratory validation required June 1, 2002
Protein Expression Expression data provided for initial registration; confirmatory data required to provide consistency across Bt crops March 15, 2003
For Bt11: Amino Acid Sequencing Comparison of AA sequence to known toxins and allergens; Stepwise 8 amino acid analysis March 15, 2003
For MON810: Amino Acid Sequencing and Heat Stability Stepwise 8 amino acid analysis and processing and/or heat stability study March 15, 2003
Cry1Ab Protein Levels in Soil Supplemental studies; protocol to be submitted before studies are initiated Protocol by March 15, 2002; interim report 12 months after protocol approved; final report
    24 months after protocol approved
Cry1F Protein Levels in Soil Supplemental studies; protocol to be submitted before studies are initiated Protocol by March 15, 2002; final report due no later than March 15, 2008
Nontarget Insects Either existing studies or protocol and studies Existing studies or protocol by March 15, 2002; interim report 12 months after protocol approved; final report 36 months after protocol approved
Monarch Long-Term Exposure for Cry1Ab Examine fitness and reproductive costs to monarchs from subchronic exposure to Bt corn January 31, 2003 or earlier
Chronic Avian Study Test a diet for chronic exposure to high levels of Bt corn that may occur in the field Existing studies or protocol by March 15, 2003; final report 18 months after approval of protocol
IRM - North/South Movement of Corn Earworm Potential for north to south movement of corn earworm Protocol by March 15, 2002; interim report 12 months after protocol approved; final report 24 months after protocol approved
IRM - Insecticide Impact on Effectiveness of Refuge Studies in areas where the refuge is commonly treated with insecticides Protocol March 15, 2002; interim report in 12 months; final report in 24 months after protocol submitted or approved
IRM - Discriminating Concentration for Cry1F Development of discriminating concentration for ECB, CEW, SWCB Studies must be submitted on or before March 15, 2003


Based on the above-mentioned information, the Department accepts for general use registration in New York State, Herculex I (EPA Reg. No. 68467-2) which contains the active ingredient Bacillus Thuringiensis var Aizawai Cry1F Corn Protein as expressed in maize, contained in Mycogen Brand BT Cry1F Corn.

Enclosed is your Certificate of Registration and New York State stamped "ACCEPTED" label.

Dow AgroSciences Mycogen Seeds Division is reminded that if New York State registration is requested for this product which contains an expansion of use patterns, the product will be considered a Major Change in Labeling and the Department will require an extensive review.

If you have any questions, please contact Samuel Jackling, Chief of our Pesticide Product Registration Section, at (518) 402-8768.

Sincerely,

Maureen P. Serafini
Director, Bureau of Pesticides Management
Division of Solid & Hazardous Materials

Enclosures
cc: w/enc. - N. Kim/D. Luttinger - NYS Dept. of Health
R. Zimmerman/R. Mungari - NYS Dept. of Ag. & Markets
G. Good/W. Smith- Cornell University, PMEP