Comment submitted by H. Quemada

I thank EPA for the opportunity to comment on the proposed rule EPA-HQ-OPP- 2006-0642. I commend EPA for taking this significant step forward in regulatory policy. The attempt to move away from an event-by-event approval of transgenic plants will be welcomed by many developers, especially those in the public sector who have been urging such a step (Bradford et al., 2005). Request for comment #1: As part of the request for comment #1, EPA requests information on the weediness potential of squash (Cucurbita pepo) or wild or weedy relatives that could acquire the a PVCP-PIP through gene flow. By this request I assume EPA is asking for information on the weediness potential of cultivated squash (C. pepo ssp. pepo var. ovifera) itself, and the weediness potential of wild or weedy (referred to collectively in this comment as free-living) relatives (C. pepo ssp. pepo var. ozarkana and var. texana). Clearly, the crop cannot meet proposed criterion 174.27(a)(1)(i), since the crop is known to exchange genes with its free-living relatives. However, the crop does fit proposed criterion 174.27(a)(2)(ii), as contained in proposed options 1-3. Cultivated squash is not a weedy or invasive species, either in or outside of agriculture (Federal and State Noxious Weeds in GRIN, http://www.ars-grin.gov/cgi-bin/npgs/html/taxweed.pl). Furthermore, free- living squash has been reported as a weed only in agriculture (Oliver et al 1983; Boyette et al 1984; Smith et al 1992; Bryson and Byrd 1998). Therefore, even the free-living populations of C. pepo fit the proposed exemption criterion 174.27(a)(2) (ii). The current squash crop containing PVCP-PIPs has shown its lack of weediness or invasiveness in over 10 years of commercial deployment. This history is the best evidence that the crop containing the PIP is unlikely to establish weedy or invasive populations outside of agricultural fields, and validates the conclusion by APHIS that the PVCP-PIP containing squash "is no more likely to become a weed than a virus-resistant plant developed by traditional breeding techniques" (USDA 1994, 1996). Therefore, cultivated squash fits the proposed criterion for exemption 174.27(a)(iii). With respect to free-living C. pepo that might acquire the PVCP-PIP, USDA/APHIS in its determinations of nonregulated status for virus resistant squash has concluded that the PVCP-PIP containing squash is "unlikely to increase the weediness potential for any other cultivated plant or native wild species with which it can interbreed" (USDA 1994, 1996). Subsequent research on the possible consequences of outcrossing of PVCP-PIPs from cultivated squash to free-living relatives has confirmed the validity of the risk assessment. Fuchs et al. (2004),showed that PVCP-PIPs conferring resistance to CMV, WMV2 and ZYMV conferred no fitness benefit under low disease pressure. Further research (Quemada et al., manuscript in preparation; futher details can be made available to EPA if the agency requests them) has shown that the incidence and severity of virus infection in free-living populations is more consistent with the low disease pressure described by Fuchs et al. (2004). Furthermore, our experiments similar in design to that of Fuchs et al. (2004), but with a different free-living genotype,showed no fitness advantage to hybrids containing the PVCP- PIP under high disease pressure (Quemada and Strehlow, manuscript in preparation; futher details can be made available to EPA if the agency requests them. Finally, we have followed the fluctuation of population size and virus infection in thirteen free-living populations over three subsequent seasons and found no correlation between population size and virus infection, demonstrating that virus infection does not determine the size of these populations. This indicates that the PVCP-PIP is unlikely to significantly change the population size or distribution of the species containing the PVCP-PIP outside of agricultual fields or the population size or distribution of any wild or weedy species in the United States that could acquire the PVCP-PIP through gene transfer. Therefore, squash would qualify for exemption under 174.27(a)(2) option 4. In view of this evidence, EPA should add to the final rule for this docket an exemption under 174.27(a)(2) for any PVCP-PIP containing plants that have already received a determination of nonregulated status from USDA/APHIS, since these plants have been subjected to a regulatory review where the issues of weediness have been considered. These plants have already been determined to present a low-risk. Furthermore, they are not likely to cause unreasonable adverse effects to the environment even in the absence of regulatory oversight under FIFRA, since they have been or (for future submissions) will be assessed by USDA/APHIS for any characteristics that may lead to weediness or invasiveness of the crop, or free-living relatives with which they can interbreed. Request for comment #2: As the only option which does not require the lack of crossing with free-living relatives as a condition, I agree with EPA that option 4 proposed for 174.27(a)(2) is the option most likely to identify the largest number of low-risk products that qualify for exemption. It is therefore the most preferred, if EPA is seeking regulatory relief from duplicative oversight for variety developers (both public and private), while not removing any potentially high risk plants from regulatory oversight under the Coordinated Framework for the Regulation of Biotechnology. I agree with EPA that the information that would be compiled to address weediness issues under option 4 would be the same as that which would be submitted to USDA/APHIS. In this regard, it should be noted that no proposed exemption under 174.27(a) removes any plant expressing a PVCP-PIP from the requirements for a determination of nonregulated status from USDA/APHIS. Plants expressing a PVCP-PIP that would require a registration under FIFRA if it were not exempt under the proposed rule, would require deregulated status before commercialization as well. Therefore, categorically exempting all PVCP-PIPs from consideration of weediness issues in plants containing them will not change the regulatory reality for developers of PVCP-PIP containing crop varieties. EPA should therefore exempt from requirements under FIFRA all PVCP-PIP containing plants that have received deregulated status from USDA/APHIS (previously or in the future), since these would have been determined by that agency to present a low risk and, because of that low risk, would be unlikely to cause unreasonable adverse effecte to the environment even in the absence of regulatory oversight under FIFRA. Request for comment #6: In parallel to the comments made in response to the request for comment #2 above, viral interactions, including recombination and transencapsidation are issues that must be addressed in USDA/APHIS petitions for deregulation. Therefore, all PVCP-PIPs, whether or not they met the conditions for exemption of proposed 174.17(b) would continue to be subject to regulatory review, where the issues of concern discussed in the present proposed rule are addressed. Therefore, EPA could simply exempt all PVCP-PIP containing plants that have received deregulated status from USDA/APHIS (previously or in the future), since those would have been determined to present a low risk and, because of that low risk, would be unlikely to cause unreasonable adverse effects to the environment even in the absence of regulatory oversight under FIFRA. Request for comment #7: EPA asks whether the 174.27(c)(1) should be modified to include constucts that would ensure lack of protein production. However, it is not clear why the lack of protein production should be a condition in the first place. Apparently, the concern is that recombination between genes encoding viral coat proteins or any other viral proteins might create novel proteins with new toxicological or allergenic properties. However, EPA must remember that, as amply supported by their own analysis in this proposed rule, the evidence for the lack of toxicity or allergenicity of viral coat proteins is very compelling. Therefore, the likelihood of creating novel toxic or allergenic sequences by recombining manifestly safe sequences is extremely low, whether that is done intentionally, as in a construct with chimeric PVC-PIPs (or any other viral gene for that matter), or as a result of recombination between a transgene and an infecting virus that had not previously had the opportunity to recombine. The likelihood of such an event would be on the order of the probablility that a random string of amino acids of typical viral coat protein length could produce toxic or allergenic proteins, and can be estimated by considering the number of known toxic or allergenic proteins isolated from plant or viral sources, relative to the total number of protein sequences known. Thus, EPA should reconsider whether preventing the production of a viral protein should be a prerequisite for exemption under 174.27(c)(1). Request for comment #8: EPA could extend the proposed exemption from FIFRA requirements to other PIPs that confer virus resistance, regardless of whether they produce a protein. None of the issues of concern, nor the exemptions proposed by EPA, nor the exemptions proposed in the response to request for comments #2 and #6, are limited to genes encoding viral coat proteins or to genes that encode any protein derived from a virus. EPA should remember that the weediness issues discussed in the present proposed rule are related to the trait of virus resistance, not to the gene that confers the trait. Therefore, an exemption based on a determination of low risk of weediness (either as proposed by EPA or as proposed in these comments) is applicable equally to any viral sequence. With regard to viral interactions, the issues of concern and regarding recombination are equally applicable to any viral sequence as to viral coat protein genes. It is RNA or DNA that undergoes recombination. Therefore,the considerations with respect to recombination risk do not apply to genes that encode viral coat proteins alone, or even to sequences that can be translated into proteins. Furthermore, the present proposed rule's discussion of transencapsidation, a question unique to coat proteins, is prima facie a low risk concern, based on the information presented in this proposed rule. It is therefore a sound decision to consider exemptions for viral sequence ("VS")-PIPs, rather than just PVCP-PIPs. Request for comment #9: I support EPA's proposal to exempt the listed marker genes from FIFRA when used as inert ingredients with any exempt PIP. I know of no cases where these listed genes would cause any effects that would be a concern under FIFRA. In particular, NPTII and GUS have been the subject of extensive risk assessment worldwide (including by EPA) and, in validation of those risk assessments, have had a long history of safe use. Thank you for the opportunity to comment. Hector Quemada Adjunct Professor, Department of Biological Sciences, Western Michigan University hector.quemada@wmich.edu ------------- References Boyette, G., Templeton, E., Oliver, L. (1984) Texas gourd (Cucurbita texana) control. Weed Science 32, 649-655. Bradford, K.J., Van Deynze, A., Gutterson, N., Parrott, W., Strauss, S.H. (2005) Regulating transgenic crops sensibly: lessons from plant breeding, biotechnology and genomics. Nature Biotechnology 23: 439-444. Bryson, C., Byrd, J. Jr. (1998) Texas gourd (Cucurbita texana) management in cotton. Proceedings of the Southern Weed Science Society 51, 265?266. Fuchs M., Chirco E.M., Mcferson J., Gonsalves D. (2004) Comparative fitness of a wild squash species and three generations of hybrids between wild X virus- resistant transgenic squash. Environmental Biosafety Research 3: 17-28. Oliver, L., Harrison, S., McClelland, M. (1983) Germination of Texasgourd (Cucurbita texana) and its control in soybeans (Glycine max). Weed Science 31, 700-706. Smith, B.D. Cowan, C.W., Hoffman, M.P. (1992) Is it an indigene or a foreigner? In: Smith, B.D. (ed.) Rivers of change: essays on the orgins of agriculture in eastern North America. Smithsonian Institution Press, Washington, D.C. pp. 67- 100. USDA (1994). Availability of determination of nonregulated status for virus resistant squash. 59 FR 64187 USDA (1996). Asgrow Seed Co.; Availability of determinatino of nonregulated status for squash line genetically engineered for virus resistance.