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B. thur., Cry3Bb1 - Registration of YieldGard Rootworm™ Rootworm Protection 2/04

New York State Department of Environmental Conservation
Division of Solid & Hazardous Materials

Bureau of Pesticides Management
Pesticide Product Registration Section
625 Broadway, Albany, New York 12233-7257
Phone 518-402-8768     FAX 518-402-9024
Website: http://www.dec.state.ny.us/website/dshm/pesticid/pesticid.htm
E-Mail: ppr@gw.dec.state.ny.us

February 13, 2004

CERTIFIED MAIL
RETURN RECEIPT REOUESTED

Ms. Tann Schafer
State Registration Manager
Plant Biotech Products
Monsanto Company
800 N. Lindbergh Blvd (A2ND)
St. Louis, MO 63167

Dear Ms. Schafer:

Re: Registration of One New Pesticide Product, YieldGard Rootworm™ Rootworm Protection (EPA Reg. No. 524-528), Which Contains the New Active Ingredient Bacillus thuringiensis Cry3Bb1

    The New York State Department of Environmental Conservation (Department) has reviewed your application, received August 8, 2003, to register YieldGard Rootworm™ Rootworm Protection (EPA Reg. No. 524-528) in New York State.

    The application was deemed complete for purposes of review on September 23, 2003, and a registration decision is due by February 20, 2004. YieldGard Rootworm™ Rootworm Protection is a genetically engineered field corn modified to produce Bacillus thuringiensis, (Bt) Cry3Bb 1 insecticidal protein. The endogenously produced protein provides protection from certain rootworms.

    The registration of this product is based on the technical reviews received from the Department of Health, the Bureau of Habitat, and our own Bureau review for impacts to human health, non-target organisms, and the environment, respectively.

Bureau of Habitat (Bon)

    Bacillus thuringiensis is a common soil bacteria. It produces an endotoxin protein that is selectively toxic to a limited number of species from three orders of insects: lepidoptera (Cryl); lepidoptera and diptera (Cry2); coleoptera (Cry3) and diptera (Cry4). Numerous pesticide products have been produced by extracting and concentrating the endotoxin protein from killed bacteria, then registered and used to control blackflies, mosquitoes, and several other agricultural pests. This product is significantly different in that instead of extracting, concentrating and utilizing the endotoxin protein from the bacteria as a stand alone product, the genes for the Cry3Bb endotoxin protein were spliced into the corn DNA so that the plant itself would produce a dose (characterized as low to moderate) of the toxic protein. A similar transgenic corn product that produced a lepidopteran toxin (Mycogen Brand B.T. Cry1F Corn) has already been reviewed and registered in New York State in September 2002.

    Ecological risks associated with this type of product are as follows: primary and secondary toxicity to non-target insects, birds, fish, and mammals; environmental fate; risk of transgenic genes spreading to wild plants; risk of off-site toxicity primarily through dispersion of pollen; endangered species protection; potential for development of insect resistance. Data requirements to support registration of this type of product are quite different from the data requirements for a typical chemical pesticide.

    The environmental degradation of the Cry3Bb protein was tested in soil as well as in simulated intestinal fluid (SIF) and simulated gastric fluid (SGF) mammalian digestion models. In soil, the Cry3Bb protein was degraded in sandy loam soil with a half life of 2-3 days with 90% degradation being achieved in 8-9 days. In SGF, the Cry3Bb protein was completely digested in 15 - 30 seconds. When placed in SIF, however, a protein stable protein degradation product resulted after 30-60 minutes that did retain some insecticidal activity. Because insecticidal activity was retained, United States Environmental Protection Agency (USEPA) procedures require that the Cry3Bb protein be evaluated for it's potential to act as an allergen. To accomplish that, segments of the DNA that codes for the Cry3Bb protein were compared to similar DNA sequence segments of known allergens. The DNA sequences of the Cry3Bb protein were found to bear no similarity to known allergens, so the allergen potential of Cry3Bb was considered to be slight.

    The following table summarizes toxicity testing conducted by the applicant to support the registration of YieldGard Rootworm corn:
Species Tested Results Risk evaluated
Mice NOEC = 3780 mg/kg for
Cry3Bb1.11231 variety; 2980
mg/kg for Cry3Bb1.11098
Primary toxicity from
consumption of transgenic
corn tissue; secondary
toxicity from the
consumption of insects
Bobwhite quail NOEC = 100,000 ppm 10% of total diet was
transgenic corn for a five day
feeding exposure
Channel catfish No toxicity noted 35% of total diet was ground
transgenic corn for an eight
week exposure
Daphnia magna
48 hour NOEC = 120 mg/L transgenic corn pollen added
to test chambers at a
concentration of 120 mg/L
Honeybee Larvae single 3 ,uL dose of Cry3Bb
protein in deionized water;
NOEC =1790 ug/L
Direct toxicity to a non-target
beneficial insect
Honeybee Adults 360 ug/L Cry3Bb protein
administered in 30%
sucrose:deionized water
solution. NOEC = 360 ug/L
Direct toxicity to a non-target
beneficial insect
Ladybird beetle
Hippodamia convergens
Purified Cry3Bb protein mixed
with honey. NOEC = 8,000
ug/ml
Direct toxicity to a beneficial
non-target insect in the target
order (coleoptera)
Ladybird beetle adults
Coleomegilla maculata

Adults fed a diet consisting of
50% fruit fly eggs and 50%
transgenic corn pollen
containing 37.4 ug/g Cry3Bb
protein for 30 days. No toxicity noted.
Direct toxicity to a beneficial
non-target insect in the target
order (coleoptera)
Ladybird beetle larvae
Coleomegilla maculata
Larvae fed a diet consisting of
50% fruit fly eggs and 50%
transgenic corn pollen
containing 93 ug/g Cry3Bb
protein for 30 days. No toxicity
noted
Direct toxicity to a beneficial
non-target insect in the target
order (coleoptera)

Green lacewing larvae
Chrysoperla carnea
Purified Cry3Bb protein mixed
with moth eggs; NOEC = 8000
ppm
Secondary toxicity to a
beneficial insect predator

parasitic wasp
Nasonia vitripennis
Purified Cry3Bb protein mixed
with honey. NOEC = 8,000
ug/ml
Secondary toxicity to a
beneficial insect predator
earthworm
Eisenia fetida

Cry3Bb protein mixed into
artificial soil. NOEC = 57
mg/kg dry weight, equivalent to
estimated environmental
concentration
Direct toxicity to consumer
of Cry3Bb protein released
into soil from degradation of
plant material
monarch butterfly larvae
Danaus plexippus
Larvae fed milkweed leaves
treated with transgenic corn
pollen averaging 63.1 ug/g
Cry3Bb fresh weight at
concentrations as high as 3200
grains/cm2. No toxicity noted
after 10 days
Direct toxicity to non-target
insect known to be
susceptible to Cryl protein

These tests show that transgenic corn with Cry3Bb protein is not toxic at concentrations equal or higher than those likely to be encountered in the environment. The only test that did exhibit a positive toxic response was earthworms exposed to 570 mg/kg Cry3Bb protein in artificial soil. This concentration was 100X higher than the expected environmental concentration.

    In addition to the lab toxicity testing, the applicant conducted a two year field study in which invertebrate diversity and abundance was evaluated in corn field test plots consisting of non-BTi corn with no insecticides; non BTi-corn treated with an in-furrow, soil insecticide; non-BTi corn treated with a foliar insecticide; and BTi corn were compared. There was no statistically significant differences between the non-BTi corn and the transgenic (BU) corn. However, the ecological community in the plot containing non-BTi corn treated with a foliar insecticide was significantly degraded compared to the control plot and transgenic corn plot.

    An important concern with any transgenic plant is the potential for the spliced genes to be transferred to wild populations. This could occur if pollen from the transgenic plant was allowed to pollinate a genetically similar, that is, closely related, plant. This issue was discussed at great length by the applicant and their findings reviewed and assessed by the USEPA. The USEPA concurred with the finding that there was very little likelihood that the Cry3Bb gene could be transferred from corn to other plants. This finding was based on the genetic differences between corn and other related plants, and the limited viability and mobility of corn pollen.

    In their risk assessment, the applicant concluded that feral species related to domesticated, agricultural corn (Zea mays ssp. mays) cannot be pollinated by crop varieties because of differences in chromosome number, phenology (i.e., periodicity or timing of events within the plant's life cycle as related to climate, e.g. flowering time), and habitat. Zea mays has been domesticated for nearly 7200 years, and over that time has been modified considerably from its ancestral form by selective breeding for specific agronomic characteristics such as non-shattering rachis (cob), grain yield, and pest resistance. Modern corn is so different genetically from the original parent plant that cross fertilization with wild varieties is unlikely to occur. The applicant's risk assessment specifically examined the cross fertilization potential with related plants, including genus Tripsacum and Zea species teosintes. Those plants were evaluated in detail in order to estimate the potential for cross fertilization to occur. In all cases, the expert opinion was that successful cross fertilization was unlikely.

    Besides the genetic problems associated with cross fertilization, it is essential that a corn relative be located close to any field that was planted with transgenic corn. Corn pollen is heavy and is not dispersed widely. Studies have shown that only 1% of the pollen from a corn field would be moved 100 feet downwind. This percentage decreased to 0.1% at 130 feet and 0.03% at 160 feet. At 1000 feet, no corn pollen could be detected. Also, corn pollen only remains viable for 18-24 hours. Under conditions of high temperature or low humidity, it might only survive a matter of minutes. This low viability further limits the potential for pollen from transgenic corn to spread to wild plants.

    The potential for the development of insect resistance is the one topic that received the most attention in the applicant's data support package for the registration application. It is also the basis for the only restriction on the YieldGard Rootworm™ label. In order to minimize the potential for the development of insect resistance, the YieldGard Rootworm label requires that 20% of the area in which the transgenic corn is planted must be planted with non-transgenic corn to provide a refuge. Within the refuge, non-resistant corn rootworms would persist. The idea behind the refuge would be that if any BTi-resistant corn rootworm were to develop, the refuge would provide non-resistant stock that the resistant population would reproduce with, thus diluting the allele for resistance and produce non-resistant hybrids. The approach seems to be well-documented and supported with past experience with other transgenic crops. The other factor for minimizing the development of insect resistance is the use of low to moderate dose of the Cry3Bb protein. The dose is considered low to moderate because the survival of larvae feeding on the transgenic corn ranges from 17% - 62%. A low dose is described by the EPA as 50% survival (of the target pest) and a moderate dose is described as 30% survival. At those survival rates, enough non-resistant corn rootworm larvae should survive to reproduce with any individuals with alleles for resistance and dilute the influence of the resistance allele.

    The Cry3Bb protein appears to be specifically toxic to beetles in the family Chrysomelidae, which includes the corn rootworm beetle and the Colorado potato beetle. There are no species from the family Chrysomelidae family on the federal endangered species list. The applicant evaluated all endangered/threatened beetle species habitats in counties where corn was grown to evaluate. the potential for them to be exposed to corn pollen. Their habitat and breeding grounds were found not to overlap corn fields. The applicant also evaluated the risks to endangered birds and bats, to determine if the reduction of prey might impact them. Because insectivorous bats rely on flying insects and do not consume larvae, the likelihood that this transgenic corn would impact their food supply was deemed as low.

    The applicant has submitted well documented scientific evidence to support the registration of YieldGard Rootworm corn. The studies submitted were accompanied with USEPA DERS that thoroughly evaluated them, and generally concurred with the findings of the applicant's studies. The use of BTi as a pesticide product, and its low risk to non-target organisms is well studied. The integration of the Cry3Bb protein directly into the corn tissue appears to have little or no adverse impacts and results in fewer impacts to ecological communities than foliar insecticides.

NYS Department of Health DOH

    The USEPA required only a limited number of studies to assess the potential health effects from dietary exposure to the Cry3Bbl protein. The required studies included acute oral toxicity testing, an in vitro digestion assay to test whether or not the protein is stable to digestion in simulated gastric fluid, and amino acid sequence homology comparisons to show whether or not the protein is similar to known food allergens or toxins. These tests were conducted using Cry3Bb 1 protein from microbial cultures due to the impracticality of extracting sufficient quantities of protein from transgenic corn. The USEPA asserts that "the Cry3Bb 1 test material was biochemically and functionally similar to the protein produced by the plant-incorporated protectant ingredients in corn."

    The acute oral toxicity data on Cry3Bbl protein (tested in mice) indicated that this active ingredient was not very toxic by this route of exposure, even at doses up to 3,780 milligrams of Cry3Bbl protein per kilogram body weight. The USEPA did not require any other acute toxicity studies or 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 Cry3Bb 1 protein.

    Since Cry3Bbl 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. Based on the study data submitted, the USEPA determined that the Cry3Bb 1 protein is readily degraded by simulated gastric fluid (acidic solution containing the proteolytic 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 and digestion. In addition, a comparison of amino acid sequences of known allergens and protein toxins found no significant structural similarity with the Cry3Bbl protein. Based on the surmised lack of allergenic potential, and the low order of acute toxicity of the Cry3Bbl protein, 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.

    The data indicate that the Cry3Bbl protein produced in YieldGard Rootworm™ corn is neither acutely toxic to mammals, stable to digestion in simulated gastric fluid nor structurally similar to known protein allergens or the amino acid sequences of known toxins. Furthermore, the planting of YieldGard Rootworm™ corn has the potential to reduce the use of conventional chemical insecticides for control of corn rootworm that, in turn, could reduce worker risks and environmental loading. In their review of YieldGard Rootworm™ Rootworm Protection, the USEPA stated that YieldGard RootwormTM composed no unreasonable risks to humans from its production, handling and storage, and was unlikely to induce allergic responses.

Environmental Fate Review:

    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.

    Based on the above-mentioned information, the Department accepts for general use registration in New York State, Yieldgard Rootworm™ Rootworm Protection (EPA Reg. No. 524-528) which contains the active ingredient Bacillus thuringiensis Cry3Bb1. label.

    Enclosed is your Certificate of Registration and New York State stamped "ACCEPTED" The Monsanto Company 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 Manage
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