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March 1-2 , 2005 Meeting Agenda
DRAFT
February 9, 2005
FIFRA SCIENTIFIC ADVISORY PANEL (SAP)
OPEN MEETING
MARCH 1-2, 2005
FIFRA SAP WEB SITE https://www.epa.gov/scipoly/sap/
OPP Docket Telephone: (703) 305-5805
Docket Number: OPP-2004-0395
SCIENTIFIC ISSUES ASSOCIATED WITH THE HUMAN HEALTH ASSESSMENT OF THE CRY 34Ab1 PROTEIN
TUESDAY, MARCH 1, 2005
Holiday Inn - National Airport
2650 Jefferson Davis Highway
Arlington , VA 22202
Telephone: (703) 684-7200
8:30 AM Introduction and Identification of Panel Members - Stephen Roberts, PhD. (FIFRA SAP Session Chair)
8:45 AM Administrative Procedures by Designated Federal Official - Paul Lewis, PhD.
8:50 AM Welcome - Clifford Gabriel, PhD. (Director, Office of Science Coordination and Policy, EPA)
8:55 AM Opening Remarks - Janet Andersen, PhD. (Director, Biopesticides and Pollution Prevention Division, Office of Pesticide Programs, EPA)
9:00 AM Scientific Issues Associated with the Human Health Assessment of the Cry34Ab1 Protein - Rebecca Edelstein, PhD. (Office of Pesticide Programs, EPA)
10:15 AM BREAK
10:30 AM Public Comments
12:30 PM LUNCH
1:30 PM Panel Discussion
Protocols for Digestibility Assays
1) Dow has stated that enzyme kinetic theory predicts first order kinetics for pepsin hydrolysis under conditions of high enzyme and low substrate concentrations and has demonstrated that the rate of substrate disappearance under these conditions follows first-order kinetics for a number of proteins. However, for several proteins, initial time points were omitted to achieve a good fit to the model. Dow states that the data were not included "based on theoretical considerations, which include: potential zero-order or mixed order kinetics due to high substrate concentration, possible presence of denatured and highly digestible protein contaminating the native protein preparation, or the possibility of an initial burst phase or transient phase preceding the first-order phase of digestion (Schnell and Maini, 2000; Milgrom et al., 1998). "
The Panel is requested to comment on whether the explanation justifies omitting early time points or whether the poor fit of early time points indicates a problem with the model.
2) Dow has asserted that first-order decay is predicted based on enzyme theory as long as the pepsin concentration is high and the substrate concentration is low (<< K m ) and that the first-order rate constant determined under these conditions is equal to V max /K m . Dow has also stated that as long as first-order conditions are met, first-order rate constants and half-lives are unaffected by changes in substrate protein concentration and that first-order rate constants can be used to predict relative digestion efficiencies for proteins even if the protein concentration is varied among experiments. In addition, Dow has stated that at the USP concentration for pepsin of 0.32%, the enzyme concentration is saturating and can also be varied between experiments without affecting the first-order rate constant.
The Panel is asked to comment on these statements. How much can the pepsin or protein substrate concentrations vary without affecting the kinetics of pepsin digestion and first-order rate constants?
3:00 PM BREAK
3:15 PM Panel Discussion (continued)
3) Typically, for comparing the in vitro digestibility of different proteins, researchers have used fixed concentrations of pepsin and substrate protein on a weight basis (mg/mL) rather than adjusting for molecular weight of the substrate protein, presumably because larger proteins likely have more potential pepsin cleavage sites. However, Dow states that "while multiple pepsin-labile sites may occur within a protein, a single site is often responsible for limiting digestion rates, and thus the number of molecules, rather than total weight, is most often more influential in determining the kinetics that describe decay."
The Panel is asked to comment on Dow's statement. To compare the rate of pepsin digestion of different proteins, is it more appropriate for the concentration of test protein to be constant on a weight basis (mg/mL) or a mole basis (mol/L)?
4) Typically, researchers have looked at the effect of pepsin to substrate ratio rather than concentrations on digestion (Karamac, et al. , 2002). How do varying the ratios and/or concentrations affect the rate of hydrolysis?
4 :30 PM ADJOURNMENT
FIFRA SCIENTIFIC ADVISORY PANEL (SAP)
OPEN MEETING
MARCH 1-2, 2005
FIFRA SAP WEB SITE https://www.epa.gov/scipoly/sap/
OPP Docket Telephone: (703) 305-5805
Docket Number: OPP-2004-0395
SCIENTIFIC ISSUES ASSOCIATED WITH THE HUMAN HEALTH ASSESSMENT OF THE CRY 34Ab1 PROTEIN
WEDNESDAY, MARCH 2, 2005
Holiday Inn - National Airport
2650 Jefferson Davis Highway
Arlington , VA 22202
Telephone: (703) 684-7200
C 8:30 AM Introduction and Identification of Panel Members - Stephen Roberts, Ph.D. (FIFRA SAP Session Chair)
8:35 AM Administrative procedures by Designated Federal Official - Paul Lewis, PhD.
8:40 AM Follow-up from Previous Day's Discussion - Rebecca Edelstein, PhD. (Office of Pesticide Programs, EPA)
9:00 AM Panel Discussion (continued)
5) Different assays exist for determining pepsin activity. A pepsin activity assay based on measuring the trichloracetic acid-soluble products of pepsin hydrolysis of hemoglobin is provided in USP, 2004 under the entry for pepsin. However, the entry in USP, 2004 for "gastric fluid, simulated" references the Food Chemicals Codex for pepsin activity, which provides an assay that measures pepsin digestion of egg albumen.
The Panel is asked to comment on the appropriateness of using a fixed concentration of pepsin versus using a fixed specific activity of pepsin in digestibility protocols. How would the use of different pepsin activity assays affect the measured pepsin activity units?
6) Typically, scientists have used SDS-PAGE with staining or western blot analysis for monitoring digestion reactions. HPLC is also sometimes used.
The Panel is asked to comment on the pros and cons of the different methods that could be used for monitoring digestion reactions.
10:30 AM BREAK
12:00 PM LUNCH
1 :00 PM Panel Discussion (continued)
7) Some researchers have used one digestion reaction and removed aliquots at various times for monitoring, while others have set up separate reactions for each of the time points.
What are the pros and cons of these approaches?
8) Under the current protocol, Dow's kinetic approach is only applicable to moderately digestible proteins (i.e., using Dow's protocol, many proteins digest too quickly and some too slowly to obtain an adequate number of data points for quantitative kinetic analysis).
Please comment on the usefulness of the kinetic approach for proteins that are not rapidly degraded.
3:00 AM BREAK
3 :15 PM Panel Discussion (continued)
Allergenicity Assessment
9) The 2001 FAO/WHO report and 2003 Codex guidelines both recommend using in vitro digestibility in assessing the allergenicity potential of a protein. The FAO/WHO report provides a "decision tree" approach, while the Codex guidelines suggest a weight of evidence approach. Codex guidelines state "resistance of a protein to degradation in the presence of pepsin under appropriate conditions indicates that further analysis should be conducted to determine the likelihood of the newly expressed protein being allergenic," and "it should be taken into account that a lack of resistance to pepsin does not exclude that the newly expressed protein can be a relevant allergen." The Codex guidelines, however, don't specify how a protein should be further evaluated if it is "resistant" to degradation, and "resistant" is not defined.
a) What weight should in vitro digestibility studies be given in the overall assessment compared with other criteria such as sequence homology?
b) The Panel is asked to comment on the appropriateness of setting acceptable/unacceptable limits for digestibility in assessing the safety of a protein.
10) Stable digestion fragments are often formed during pepsin digestion of proteins, and Dow has used the kinetic approach to estimate the half-lives of several digestion fragments.
Please comment on the significance of the rate of digestion of protein fragments for allergenicity assessments.
Cry34Ab1 and Cry35Ab1 Assessment
11) Cry34Ab1 appears to be moderately digested in SGF, rather than rapidly digested. Considering all of the available information- Cry34Ab1 originates from a non-allergenic source, has no sequence similarity with known allergens, is not glycoslyated, is inactivated by heat, is moderately digested in SGF, and will only be present at low levels in food- EPA has concluded that Cry34Ab1 is unlikely to be a food allergen.
Please comment on the Agency's conclusions regarding the allergenicity of Cry34Ab1.
5 :00 PM ADJOURNMENT
Please be advised that agenda times are approximate. For further information, please contact the Designated Federal Official for this meeting, Paul Lewis via telephone: (202) 564-8450; fax: (202) 564-8382; or email: lewis.paul@epa.gov