                                       
Federal Food, Drug, and Cosmetic Act (FFDCA) Considerations for VNT 1 Protein in Potato

                    Docket ID Number: EPA-HQ-OPP-2014-0457
                            Date: December 6, 2016
                                       
Section 408(c)(2)(A)(i) of FFDCA allows the U.S. Environmental Protection Agency (EPA) to establish an exemption from the requirement for a tolerance (the legal limit for a pesticide chemical residue in or on a food) only if the EPA determines that the exemption is "safe." Section 408(c)(2)(A)(ii) of FFDCA defines "safe" to mean that "there is a reasonable certainty that no harm will result from aggregate exposure to the pesticide chemical residue, including all anticipated dietary exposures and all other exposures for which there is reliable information." This includes exposure through drinking water and in residential settings but does not include occupational exposure. Pursuant to FFDCA section 408(c)(2)(B), in establishing or maintaining in effect an exemption from the requirement of a tolerance, the EPA must take into account the factors set forth in FFDCA section 408(b)(2)(C), which require the EPA to give special consideration to exposure of infants and children to the pesticide chemical residue in establishing a tolerance or tolerance exemption, and to "ensure that there is a reasonable certainty that no harm will result to infants and children from aggregate exposure to the pesticide chemical residue . . . ." Additionally, FFDCA section 408(b)(2)(D) requires that the EPA consider "available information concerning the cumulative effects of [a particular pesticide's] . . . residues and other substances that have a common mechanism of toxicity."
The EPA performs a number of analyses to determine the risks from aggregate exposure to pesticide residues. First, the EPA determines the toxicity of the pesticide. Second, the EPA examines exposure to the pesticide through food, drinking water, and other exposures that occur as a result of pesticide use in residential settings.
I.  Summary of Petitioned-for Tolerance Exemption
In the Federal Register of April 25, 2016 (Vol. 81, No. 79 FR 24047), the EPA issued a notice pursuant to FFDCA section 408(d)(3), 21 U.S.C. 346a(d)(3), announcing the filing of a pesticide tolerance petition (PP 5F8425) by J.R. Simplot Co., 5369 W. Irving St., Boise, ID 83706. The petition requested that 40 CFR part 180 be amended by establishing an exemption from the requirement of a tolerance for residues of VNT-1 protein in potatoes. That document referenced a summary of the petition prepared by the petitioner J.R. Simplot, which is available in Docket ID Number EPA-HQ-OPP-2014-0457 via http://www.regulations.gov.
II.  Toxicological Profile
Consistent with FFDCA section 408(b)(2)(D), the EPA reviewed the available scientific data and other relevant information on the VNT-1 protein, and considered its validity, completeness, and reliability, as well as the relationship of this information to human risk. The EPA also considered available information concerning the variability of the sensitivities of major identifiable subgroups of consumers, including infants and children.
Expression of Rpi-vnt1 in wild and cultivated potato confers broad-spectrum resistance to late blight strains caused by Phytophthora infestans.  Resistant potato parts include leaves, stems and tubers.  This resistance is based on the recognition of a pathogen-specific protein (effector protein) by a host-specific protein (resistance or R-protein).  Upon infection, AVR-VNT1, an effector protein produced by Phytophthora infestans, is recognized by the R-protein VNT1 expressed in potato (Pel, 2010).  Consequently, VNT1 activates a signal transduction pathway that leads to localized plant cell death or the hypersensitive response.  Death is restricted to a few plant cells and limits the growth and spread of P. infestans throughout the rest of the plant. Using Simplot Innate[TM] Technology, the Rpi-vnt1 gene has been transformed into several commercially important potato varieties successfully conferring late blight resistance.
The VNT1 protein is not detectable by current methodologies and attempts to isolate or produce the VNT1 protein were not successful.  Therefore, no toxicity testing was performed with either plant purified protein or protein produced in a surrogate organism. Rather, the Agency has reviewed a bioinformatics analysis of the allergenic and toxic potential of the VNT1 protein and on similar proteins to which humans are currently and regularly exposed through ingestion of edible plants.  Allergens were analyzed based upon searching for sequence identity with a curated database of allergens hosted by the Food Allergy Research and Resource Program (FARRP). A database of toxin-related proteins was generated using the Universal Protein Resource (uniprot). The databases used for all queries were the latest available resources on May 18, 2015.  The bioinformatics analyses showed that no significant similarity between VNT1 and known allergens or toxins was identified.
The Rpi-vnt1 resistance gene introduced into potato is similar to genes that have been bred into tomato (Solanum lycopersicum; a species closely related to potato) for resistance to tomato mosaic virus (ToMV).  Submitted data, including a nucleotide BLAST search, show that the Rpi-vnt1 gene shares greater than 90% genetic sequence identity with ToMV resistance genes (referred to as Tm-2 or Tm2[2]).  Also, the VNT1 protein is greater than 75% homologous to the Tm-2 To-MV protein from tomato at the amino acid level. Taken together, results from the bioinformatics analyses suggest that genes highly similar to Rpi-vnt1 at both the nucleic acid and protein levels are constituents of tomato.  
Given the large per capita consumption of fresh market tomato consumption in the United States, consumers are very likely to have been exposed to ToMV resistant tomatoes in their diet. ToMV-resistant tomato varieties are readily available and grown in the U.S. for fresh market tomato production. Some examples of tomatoes with the ToMV resistance gene are Amai (STM 7704) grape, Arbason F1, Atomic, and Big Beef F1 cultivars. Since the Rpi-vnt1 gene is substantially similar to the ToMV resistance genes (approximately 90% homologous), it is reasonable to conclude that U.S. consumers have dietary exposure to proteins that are substantially similar to the VNT1 protein without negative impacts to human health.
III.  Aggregate Exposure

In examining aggregate exposure, FFDCA section 408 directs the EPA to consider available information concerning exposures from the pesticide residue in food and all other non-occupational exposures, including drinking water from ground water or surface water and exposure through pesticide use in gardens, lawns, or buildings (residential and other indoor uses).

The Agency has considered available information on the aggregate exposure levels of consumers (and major identifiable subgroups of consumers) to the pesticide chemical residue and to other related substances. These considerations include dietary exposure under the tolerance exemption and all other tolerances or exemptions in effect for the plant-incorporated protectant chemical residue, and exposure from non-occupational sources. Exposure via the skin or inhalation is not likely since the plant-incorporated protectant is contained within plant cells, which essentially eliminates these exposure routes or reduces these exposure routes to negligible.

As discussed above, bioinformatics analyses and the similarity of the Rpi vntl gene with the tomato mosaic virus disease resistance gene (ToMV) has been used to demonstrate that it is unlikely that introduction of VNT1 potatoes would represent a safety risk related to toxicity.

IV.  Cumulative Effects from Substances with a Common Mechanism of Toxicity

Section 408(b)(2)(D)(v) of FFDCA requires that, when considering whether to establish, modify, or revoke a tolerance, the EPA consider available information concerning the cumulative effects of a particular pesticide's residues and other substances that have a common mechanism of toxicity.

The Rpi-vnt1 gene that expresses the VNT1 protein is not toxic and does not have a common mechanism of toxicity with other substances. Consequently, section 408(b)(2)(D)(v) does not apply. For information regarding EPA's efforts to determine which chemicals have a common mechanism of toxicity and to evaluate the cumulative effects of such chemicals, see EPA's website at http://www.epa.gov/pesticides/cumulative.

V.  Determination of Safety for the U.S. Population, Infants and Children

A.  U.S. Population

As discussed in section II above, the data submitted and cited regarding potential health effects for the VNT1 protein include the characterization that the Rpi-vnt1 gene shares greater than 90% sequence identity with tomato mosaic virus (ToMV) resistance genes.  Also, the VNT1 protein is greater than 75% homologous to Tm-2 ToMV proteins from Solanum lycopersicum (tomato) at the amino acid level. Taken together, results from the bioinformatics analyses suggest that genes highly similar to Rpi-vnt1 at both the nucleic acid and protein levels are already present in food as constituents of tomato.  Since, ToMV resistant tomato varieties are readily available and grown in the US for fresh market tomato production, it is reasonable to conclude that proteins similar to the VNT1 protein (as expressed by the Rpi-vnt1 gene) have already been consumed as part of the U.S. human diet.

B.  Infants and Children

FFDCA section 408(b)(2)(C) provides that EPA shall assess the available information about consumption patterns among infants and children, special susceptibility of infants and children to pesticide chemical residues and the cumulative effects on infants and children of the residues and other substances with a common mechanism of toxicity. 
In addition, FFDCA section 408(b)(2)(C) also provides that EPA shall apply an additional tenfold margin of safety for infants and children in the case of threshold effects to account for prenatal and postnatal toxicity and the completeness of the data base unless EPA determines that a different margin of safety will be safe for infants and children. 
In this instance, based on the available information discussed in section II above, the Agency concludes that there is a finding of no toxicity for the VNT1 protein. Thus, there are no threshold effects of concern and, as a result, the provision requiring an additional margin of safety does not apply. Further, the provisions of consumption patterns, special susceptibility, and cumulative effects do not apply.

VI.  Conclusions
The Agency concludes, there is reasonable certainty that no harm will result from aggregate exposure of the U.S. population, including infants and children, to the VNT1 protein produced from the Rpi-vnt1 gene. This includes all anticipated dietary exposures and all other exposures for which there is reliable information. The Agency has arrived at this conclusion because, as previously discussed, there is no indication of toxicity or allergenicity potential for the plant-incorporated protectant.




