FDA recommendations for sampling and testing yellow corn and dry-milled yellow corn shipments intended for human food use for Cry9C protein residues
Final Guidance
This guidance represents FDA's current thinking on sampling and testing yellow corn and dry-milled yellow corn shipments for Cry9C protein residues. It does not create or confer any rights for or on any person and does not operate to bind FDA or the public. 21 CFR 10.115(d). The agency is now issuing final guidance for implementation in accordance with FDA's regulation on administrative practices and procedures for Good Guidance Practices, 21 CFR 10.115. (65 FR 56468; September 19, 2000).
GIPSA originally validated the TraitCheck Bt9 Lateral Flow Test Kit manufactured by Strategic Diagnostics, Inc., at a detection level of 1 in 400 kernels and the Cry9C QuickStixTM Test Kit manufactured by EnviroLogix, Inc., at a detection level of 1 in 500 kernels. GIPSA recently revalidated both test kits at a detection level of 1 in 800 kernels. Users of the Strategic Diagnostics test kit should be aware that the company has issued a new version of the test kit (part number 7000003) and that the earlier version of the kit (part number 700034) requires some protocol modifications to obtain valid results at a detection level of 1 in 800 kernels. Users of test kits with part number 700034 who wish to rely on a detection level of 1 in 800 kernels should consult with Strategic Diagnostics prior to using the kit.
As an overall example, a mill using a test validated by GIPSA to detect 1 kernel in 800 on a batch of yellow corn for which a 24-probe sample is recommended could use the following protocol: take 24 samples of corn according to GIPSA's sampling protocols, combine all 24, mix thoroughly, reduce to 2400 kernels using equipment that subdivides in a random manner, and separate the 2400 kernels into three 800-kernel subsamples. Each analytical subsample should be tested separately using a validated immunoassay kit. If any of the analytical subsamples tests positive, the entire corn lot should be diverted to animal feed use or industrial use (such as ethanol production).
This testing protocol is based on an analysis of the probability of rejecting corn lots containing a range of percentages of StarLinkTM corn. The operating characteristic curves and tables from this analysis are provided in reference 1. The curves and tables show the likelihood that a lot of corn containing StarLinkTM kernel(s) would be mistakenly accepted given different numbers of kernels tested and various levels of StarLinkTM present in the lot (0% to 2.5%). For example, the table shows that a lot containing 0.19% StarLinkTM corn would be rejected 99% of the time when 2400 kernels are tested according to these recommendations. A lot containing 0.29% StarLinkTM corn would be rejected 99.9% of the time.
In addition, the recommendations are not intended to apply to corn intended for animal feed use. Cry9C protein and the cry9c DNA are lawful in animal feed due to an EPA- established exemption from tolerance since the introduction of StarLinkTM corn in 1998.
This sampling plan was developed by Dr. Thomas B. Whitaker (USDA/ARS, North Carolina State University). The operating characteristic (OC) curves, attached, were developed based on a binomial distribution that assumes a random distribution of the Cry9C-containing kernels throughout a lot of corn. This distribution is believed to reasonably reflect field conditions, provided a representative sample is obtained using the procedures in the Grain Inspection Handbook for sampling grains published by the Grain Inspection, Packers, and Stockyards Administration (GIPSA). The OC curves represent the effect on lot acceptance of testing a given number of kernels, where a lot with any positive test is rejected. The statistical treatment assumes that the variability associated with the sample preparation (i.e., grinding and homogenizing) and analytical steps are negligible or zero, and that the only variability associated with the procedure, given a specific number of tested kernels, results from the sampling step. It also assumes that no biases are associated with the sample selection process, i.e., a representative sample is obtained. The accompanying table shows the probability that a lot actually containing Cry9C kernels will test negative and be mistakenly accepted, given different numbers of tests and actual percentages of Cry9C kernels.
GIPSA has conducted studies using yellow corn to validate the test kits manufactured by Strategic Diagnostics, Inc. (Newark, DE) and EnviroLogix, Inc. (Portland, ME). GIPSA has found that the data obtained indicate that the test kits are capable of successfully detecting the presence of the Cry9C protein in corn contaminated with Cry9C-containing corn at a detection level of 1 Cry9C-containing kernel in 800, provided appropriate protocols are followed.
Manufacturers who wish to use different tests that are developed in the future should consult with GIPSA (202-720-0252, www.usda.gov/gipsa) about the validity of the tests.
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Probability that a corn lot containing
Cry9C kernels will test negative and be mistakenly accepted given different numbers of kernels tested and actual percentages of Cry9C kernels |
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|---|---|---|---|---|---|
| % StarLink | Number of kernels tested | ||||
| 400 | 800 | 1200 | 2400 | 4800 | |
| 0.005 | 98.020% | 96.079% | 94.176% | 88.692% | 78.662% |
| 0.010 | 96.079% | 92.311% | 88.692% | 78.662% | 61.877% |
| 0.015 | 94.176% | 88.691% | 83.526% | 69.766% | 48.673% |
| 0.020 | 92.311% | 85.213% | 78.661% | 61.875% | 38.286% |
| 0.025 | 90.483% | 81.871% | 74.079% | 54.877% | 30.115% |
| 0.030 | 88.690% | 78.660% | 69.764% | 48.670% | 23.688% |
| 0.035 | 86.934% | 75.575% | 65.700% | 43.165% | 18.632% |
| 0.040 | 85.212% | 72.610% | 61.872% | 38.282% | 14.655% |
| 0.045 | 83.524% | 69.762% | 58.268% | 33.951% | 11.527% |
| 0.050 | 81.869% | 67.025% | 54.873% | 30.110% | 9.066% |
| 0.055 | 80.247% | 64.396% | 51.676% | 26.704% | 7.131% |
| 0.060 | 78.657% | 61.869% | 48.665% | 23.683% | 5.609% |
| 0.065 | 77.099% | 59.442% | 45.829% | 21.003% | 4.411% |
| 0.070 | 75.571% | 57.110% | 43.158% | 18.626% | 3.469% |
| 0.075 | 74.073% | 54.869% | 40.643% | 16.519% | 2.729% |
| 0.080 | 72.606% | 52.716% | 38.275% | 14.649% | 2.146% |
| 0.085 | 71.167% | 50.647% | 36.044% | 12.992% | 1.688% |
| 0.090 | 69.756% | 48.659% | 33.943% | 11.521% | 1.327% |
| 0.095 | 68.374% | 46.750% | 31.965% | 10.217% | 1.044% |
| 0.100 | 67.019% | 44.915% | 30.101% | 9.061% | 0.821% |
| 0.105 | 65.690% | 43.152% | 28.347% | 8.035% | 0.646% |
| 0.110 | 64.388% | 41.458% | 26.694% | 7.126% | 0.508% |
| 0.115 | 63.112% | 39.831% | 25.138% | 6.319% | 0.399% |
| 0.120 | 61.861% | 38.267% | 23.672% | 5.604% | 0.314% |
| 0.125 | 60.634% | 36.765% | 22.292% | 4.969% | 0.247% |
| 0.130 | 59.432% | 35.322% | 20.992% | 4.407% | 0.194% |
| 0.135 | 58.254% | 33.935% | 19.768% | 3.908% | 0.153% |
| 0.140 | 57.098% | 32.602% | 18.615% | 3.465% | 0.120% |
| 0.145 | 55.966% | 31.322% | 17.530% | 3.073% | 0.094% |
| 0.150 | 54.856% | 30.092% | 16.508% | 2.725% | 0.074% |
| 0.155 | 53.769% | 28.911% | 15.545% | 2.416% | 0.058% |
| 0.160 | 52.702% | 27.775% | 14.638% | 2.143% | 0.046% |
| 0.165 | 51.657% | 26.684% | 13.784% | 1.900% | 0.036% |
| 0.170 | 50.632% | 25.636% | 12.980% | 1.685% | 0.028% |
| 0.175 | 49.628% | 24.629% | 12.223% | 1.494% | 0.022% |
| 0.180 | 48.644% | 23.662% | 11.510% | 1.325% | 0.018% |
| 0.185 | 47.679% | 22.733% | 10.839% | 1.175% | 0.014% |
| 0.190 | 46.733% | 21.840% | 10.206% | 1.042% | 0.011% |
| 0.195 | 45.806% | 20.982% | 9.611% | 0.924% | 0.009% |
| 0.200 | 44.897% | 20.157% | 9.050% | 0.819% | 0.007% |
| 0.210 | 43.133% | 18.605% | 8.025% | 0.644% | 0.004% |
| 0.220 | 41.438% | 17.171% | 7.115% | 0.506% | 0.003% |
| 0.230 | 39.810% | 15.848% | 6.309% | 0.398% | 0.002% |
| 0.240 | 38.245% | 14.627% | 5.594% | 0.313% | 0.001% |
| 0.250 | 36.742% | 13.500% | 4.960% | 0.246% | 0.001% |
| 0.260 | 35.298% | 12.459% | 4.398% | 0.193% | 0.000% |
| 0.270 | 33.910% | 11.499% | 3.899% | 0.152% | 0.000% |
| 0.280 | 32.577% | 10.612% | 3.457% | 0.120% | 0.000% |
| 0.290 | 31.296% | 9.794% | 3.065% | 0.094% | 0.000% |
| 0.300 | 30.065% | 9.039% | 2.718% | 0.074% | 0.000% |
| 0.310 | 28.883% | 8.342% | 2.409% | 0.058% | 0.000% |
| 0.320 | 27.747% | 7.699% | 2.136% | 0.046% | 0.000% |
| 0.330 | 26.655% | 7.105% | 1.894% | 0.036% | 0.000% |
| 0.340 | 25.607% | 6.557% | 1.679% | 0.028% | 0.000% |
| 0.350 | 24.599% | 6.051% | 1.489% | 0.022% | 0.000% |
| 0.360 | 23.631% | 5.584% | 1.320% | 0.017% | 0.000% |
| 0.370 | 22.701% | 5.154% | 1.170% | 0.014% | 0.000% |
| 0.380 | 21.808% | 4.756% | 1.037% | 0.011% | 0.000% |
| 0.390 | 20.950% | 4.389% | 0.919% | 0.008% | 0.000% |
| 0.400 | 20.125% | 4.050% | 0.815% | 0.007% | 0.000% |
| 0.410 | 19.333% | 3.738% | 0.723% | 0.005% | 0.000% |
| 0.420 | 18.572% | 3.449% | 0.641% | 0.004% | 0.000% |
| 0.430 | 17.840% | 3.183% | 0.568% | 0.003% | 0.000% |
| 0.440 | 17.138% | 2.937% | 0.503% | 0.003% | 0.000% |
| 0.450 | 16.463% | 2.710% | 0.446% | 0.002% | 0.000% |
| 0.460 | 15.814% | 2.501% | 0.396% | 0.002% | 0.000% |
| 0.470 | 15.192% | 2.308% | 0.351% | 0.001% | 0.000% |
| 0.480 | 14.593% | 2.130% | 0.311% | 0.001% | 0.000% |
| 0.490 | 14.018% | 1.965% | 0.275% | 0.001% | 0.000% |
| 0.500 | 13.466% | 1.813% | 0.244% | 0.001% | 0.000% |
| 0.600 | 9.006% | 0.811% | 0.073% | 0.000% | 0.000% |
| 0.700 | 6.021% | 0.363% | 0.022% | 0.000% | 0.000% |
| 0.800 | 4.024% | 0.162% | 0.007% | 0.000% | 0.000% |
| 0.900 | 2.688% | 0.072% | 0.002% | 0.000% | 0.000% |
| 1.000 | 1.795% | 0.032% | 0.001% | 0.000% | 0.000% |
| 1.100 | 1.198% | 0.014% | 0.000% | 0.000% | 0.000% |
| 1.200 | 0.799% | 0.006% | 0.000% | 0.000% | 0.000% |
| 1.300 | 0.533% | 0.003% | 0.000% | 0.000% | 0.000% |
| 1.400 | 0.355% | 0.001% | 0.000% | 0.000% | 0.000% |
| 1.500 | 0.237% | 0.001% | 0.000% | 0.000% | 0.000% |
| 1.600 | 0.158% | 0.000% | 0.000% | 0.000% | 0.000% |
| 1.700 | 0.105% | 0.000% | 0.000% | 0.000% | 0.000% |
| 1.800 | 0.070% | 0.000% | 0.000% | 0.000% | 0.000% |
| 1.900 | 0.047% | 0.000% | 0.000% | 0.000% | 0.000% |
| 2.000 | 0.031% | 0.000% | 0.000% | 0.000% | 0.000% |
| 2.100 | 0.021% | 0.000% | 0.000% | 0.000% | 0.000% |
| 2.200 | 0.014% | 0.000% | 0.000% | 0.000% | 0.000% |
| 2.300 | 0.009% | 0.000% | 0.000% | 0.000% | 0.000% |
| 2.400 | 0.006% | 0.000% | 0.000% | 0.000% | 0.000% |
| 2.500 | 0.004% | 0.000% | 0.000% | 0.000% | 0.000% |
To Corn Millers and Corn Food Products Manufacturers:
As you know, recent investigations have detected the presence of DNA from StarLink corn in several food products made from dry-milled and masa processed yellow corn. StarLink is a variety of corn that has been genetically engineered to produce the pesticidal protein Cry9C. In certain limited cases, the Cry9C protein has also been detected in corn seeds of a non-StarLink variety or in corn from such seeds. Unlike other varieties of genetically engineered corn, the Environmental Protection Agency authorized the use of StarLink corn only for animal feed use, not for human food use. However, some Cry9C-containing corn was mistakenly commingled with yellow corn intended for human food use. FDA is now issuing recommendations to the dry-milling and masa industry on sampling yellow corn to prevent the introduction of Cry9C-containing corn into the food supply. The FDA believes that the best strategy for keeping Cry9C protein out of the food supply is to focus intervention as early as possible in the preparation of yellow corn products for human food use, i.e., at the level of corn milling rather than at the level of production of finished foods.
This letter and the enclosed guidance document outline the FDA's current recommendations to corn dry millers and masa producers on appropriate procedures for sampling yellow corn intended for human food use for Cry9C-containing corn. Copies of the guidance document and reference material will be posted under biotechnology on CFSAN's web site (www.cfsan.fda.gov). This letter and the guidance document do not create or confer any rights for or on any person and do not operate to bind the FDA or the public. A notice of availability for this guidance document will be published shortly in the Federal Register, in accordance with the FDA's policy on Good Guidance Practices for issuance of guidance documents, as set out in the Federal Register of September 19, 2000 (65 FR. 56468).
As described in the enclosed guidance document, the FDA is recommending that dry milling and masa operations take representative samples from incoming shipments of yellow corn, test 2400 kernels for the presence of the Cry9C protein using validated immunoassay testing procedures, and divert any lots that test positive for Cry9C protein to animal feed or industrial uses.
The FDA recognizes that some dry milling and masa operations may have inventories of stored grain or meal that have not been tested or have not been tested as described in the enclosed guidance. The FDA recommends that this sampling guidance be phased in over a period of no more than 30 days dating from December 27, 2000.
In this letter and the enclosed guidance document, the FDA is not setting an action level for the Cry9C protein, which is an unapproved pesticide in human food. Rather, the guidance recommends analytical testing protocols for dry milling and masa operations that the FDA believes will identify those lots of corn that contain the StarLink variety (or other varieties with the Cry9C trait) commingled with other yellow corn. Because corn containing StarLink is adulterated if intended for use in human food, lots containing the Starlink variety should be diverted to animal feed use or industrial use. As an additional layer of protection, the FDA is continuing its compliance oversight at dry milling and masa operations, e.g., by inspecting milling operations and testing cornmeal samples.
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Sincerely yours,
Janice F. Oliver Deputy Director Center for Food Safety and Applied Nutrition |
Hypertext updated by dav/dms 2001-JAN-19