Prevention, Pesticides

and Toxic Substances (7101) EPA 712- [insert number]

DRAFT 2-23-09



# Product Performance

Test Guidelines

OPPTS 810.XXXX

Products with PrionRelated ClaimsINTRODUCTION

This guideline is one of a series of test guidelines that have been developed by the Office of Prevention, Pesticides and Toxic Substances (OPPTS), United States Environmental Protection Agency for use in the testing of pesticides and toxic substances, and the development of test dat a to meet the data requirements of the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601), the Federal Insecticide, Fungicide and Rodentic ide Act (FIFRA) (7 U.S.C. 136, et seq. ), and section 408 of the Federal Food, Drug and Cosmetic Act (FFDCA) (21 U.S.C. 346a).

OPPTS developed this guideline thr ough a process of harmonization of the testing guidance and requirements that exist ed for the Office of Pollution Prevention and Toxics (OPPT) in Title 40, Chapter I, Subchapt er R of the Code of Federal Regulations (CFR), the Office of Pesticide Programs (OPP) in publications of the National Technical Information Service (NTIS) and in the gui delines published by the Organization for Economic Cooperation and Development (OECD).

For additional information about OPPTS harmonized guidelines and to access this and other guidelines, please go to http://www.epa.gov/oppts and select “Test

Methods & Guidelines” on the left side menu.

2OPPTS 810.XXXX: Products with Prion-Related Claims

.

(a) Scope —

(1) Applicability. This guideline is intended to m eet testing requirements of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et

seq. ), and the Federal Food, Drug, and Co smetic Act (FFDCA) (21 U.S.C., 301
et seq. ).

(2) Background. Prion (“ pr oteinaceous in fectious particle”) is a term often used to designate an infectious agent that causes progressive degenerative diseases of the central nervous system ( CNS), which are collectively called the transmissible spongiform encephalopathies (TSEs or prion diseases, including scrapie, chronic wasting disease, bovine spongiform encephalopathy, and various forms of Creutzfeldt-Jakob disease, etc.). These is olated infectious particles, which accumulate in brain tissue during the in cubation periods of TSEs, are comprised of abnormal folding conformations of a ubiquitous protein called the “cellular” prion protein (PrP C ). The normal cellular prio n protein is synthesized and eventually degraded through normal metabolic processes; however, in certain instances protein molecules may become misfolded. The resultant shape change serves as a template for recruitm ent of other misfol ded copies of the prion protein and accounts for the re sistance of the abnormal prions to proteosomal degradation and their slow accumu lation in infected brain tissue.

Prions were first detected in animals with scrapie, and so such infectious proteins are often designated collectively as PrP Sc in all TSEs. Some scientists prefer to

use other terms such as P r P

P res , PrP d , PrP TSE , PrP CWD

P , and PrP CJD to designate the abnormal forms of PrP associated with prion diseases in general or with specific prion diseases.

For purposes of this guidance, the terms “prions” and “TSE agents” are synonymous. These abnormal prion pr oteins, although somewhat variable, share properties that dist inguish them from PrP C : they are usually insoluble in detergent-salt solutions and relatively resistant to digestion with the enzyme proteinase K. These properties are attri buted to misfolding of alpha-helical PrP C

to yield isomers that are predominantly beta-sheeted. Many authorities have

concluded that the abnormal prion proteins themselves are the self-replicating infectious agents causing TSEs (Prusiner 1982, 2004); however, there remain notable reservations, as outlined by Manuelidis (2007a,b).

Manuelidis L. 2007a. A 25 nm vi rion is the likely cause of transmissible spongiform encephalopat hies. Journal of cellular biochemistry; 100(4):897-915.

Manuelidis L, Yu ZX, Barquero N, Mullins B. 2007b. Cells infected with scrapie and Creutzfeldt-Ja kob disease agents produce intracellular 25-nm virus-like particles. Proceedings of the National Academy of Sciences of the United States of America; 104(6):1965-70.

3Prusiner SB. 1982. Novel proteinac eous infectious particles cause scrapie. Science; 216:136-44.

Prusiner SB. 2004. Detecting mad cow disease. Scientific American;291(1):86-93.

(b) Purpose. This document provides pesticide applicants and registrants guidance on labeling claims, test system s and performance standards that apply to prion products. EPA encourages pesticide applicants and registrants to follow this guidance and submit a draft test protocol to EPA for review prior to conducting any studies. EPA may revise this guidance over time, as needed, to reflec t advances in available test methods and scientific knowledge pertaining to prions.

(c) Guidance —

(1) Labeling Claims. Testing conducted according to currently available test methods are adequate for measuring reduction in prion infectivity, but not for demonstrating complete destruction or inactivation of prions. Accordingly, claims such as “inactivates,” “destroys,” “ denatures” and “eliminates ” are not supported by currently available test methods. Fu rther, a prion-related claim should include the type of prion against which the pr oduct has been successfully tested. An example of an acceptable claim would be, “Has been demonstrated to reduce infectivity of prions (TSE agents) based on testing using (insert type of organism in which the prions were raised) (inser t prion type).” Fo r example, “Has been demonstrated to reduce infectivity of pr ions based on testing using hamsteradapted scrapie.”

(2) Selecting a Test System. The test system should be appropriate to the uses that appear on the proposed label.

a. Carrier-based Method . If the intended uses of a product are for treating

inanimate items or envir onmental surfaces, then a carrier-based, animal infectivity test method should be used to measure the amount of prion infectivity reduction that is achieved by the product when used according to label directions

. Examples of published, carrier-b ased test methods include:

Chen JHS. 1975. Comparison of virus recovery techniques for assessing the virucidal activity of disinfectant spray products. JAOAC 1975; 58(1):130-2

.

Peretz D, Supattapone S, Giles K, Vergara J, Freyman Y, Lessard P, Safar JG, Glidden DV, McCulloch C, Nguyen H-OB, Scott M, DeArmond SJ, and Prusiner SB. 2006. Inactivation of prions by sodium dodecyl sulfate. J. Virol. 80:322-331.

Weissmann C, Enari M, Klohn PC, Rossi P, Flechsig E. 2002. Transmission of prions. J Infect Dis 186 Suppl 2:S157-65.

Zobeley E, Flechsig E, Cozzio A, Enari M, and Weissman C. 1999. Infectivity of scrapie prions bound to a stainless steel surface. Molecular Medicine 5:240-243.

4b. Suspension-based Test Method. If the intended use of a product includes only treating liquids (e.g., liquid wast es), then a suspension-based, animal infectivity test method should be used to measure the amount of prion infectivity reduction that is achieved by the product when used according to label directions. An example of a published, suspension-based test method is:

Peretz D, Supattapone S, Giles K, Vergara J, Freyman Y, Lessard P, Safar JG, Glidden DV, McCulloch C, Nguyen H-OB, Scott M, DeArmond SJ, and Prusiner SB. 2006. Inactivation of prions by sodium dodecyl sulfate. J. Virol. 80:322-331.

(3) Methods of Estimating the Re duction of Prion Infectivity. Prion diseases are characterized by a long asymptomatic incubation period followed by the rapid onset of symptoms followed by death. The length of the incubation period is reproducible upon repeated passages of a given prion and is inversely proportional to the log infectious dose of that prion although at both very high and very low concentrations of infectivity t he relationship is no longer linear. Two methods of estimating the reduction of prion infectivity have been employed: endpoint titration and incubation time interval assay. Either method is suited to analyze suspension-based or carrier-based approaches. However, incubation time assays may be less reliable for quantifying very low levels of infectivity.

a. End-point Titration. An end-point titration is a classical method of determining the titer of a sample. To begin with, a sample is serially diluted by a

factor of 10 until less than 1 ID 50 remains in the final dilution. The resulting set of

serial dilutions is used to inoculate a corresponding set of experimental animals, typically at least four animals per 10-fold dilution. The experimental animals are observed for the appearance of symptoms. The symptoms are scored and the experimental animals are humanely euthani zed before they would die of the prion disease. The animals are obser ved for a predetermined period, usually 450 days for hamsters or 500 days for mice . At the end of the predetermined period, any surviving animals would be humanely euthaniz ed and examined for signs of prion disease (e.g., presence in the brain of prion pr otein characteristic of the disease in the test organism). The titer may be determined by several statistical methods (see references below ). The titers of subsequent samples

are determined in the same fashion.

Andersen J, Barrett T, Scott GR. 1996. Appendix 3. Fifty percent effective dose (ED 50 ): Spearman-Kärber method. In Manual of the Diagnosis of Rinderpest. (FAO Animal Health Manual – 1) Food and Agriculture Organization of the

United Nations. Rome. Available on-line: http://www.fao.org/docrep/w0049e/w0049e07.htm#appendix%203.%20fifty%20p ercent%20effective%20dose%20(ed50):%20spearman%20kärber%20method Reed LJ and Muench H. A simple method for estimating fifty percent endpoints. Amer J Hyg 1938;27:493-7. Bliss CI. The method of probits. Science. 1934 Jan 12;79(2037):38-39

5b. Incubation Time Interval Assay. An incubation time interval assay exploits the relationship between the length of the incubation pe riod and the titer of the inoculum. An endpoint titration of a starting brain homogenate is used to establish an empirical relationship betw een prion titer and length of incubation period. Once the relationship is establis hed, the titer of any subsequent samples is determined by observing the incubation period of an inoculated animal group (usually at least four) and using this value to calculate a corresponding titer based on the empirically determined relationship. The model mean survival time study should not be greatly, if at all, s eparated in time from the experimental assay. If the mean survival performance in the model is to be predicted largely on the basis of past studies, then sufficient control animals to insure that model mean survival time has remained unchanged should be included in the experimental assay.

Prusiner SB, Cochran SP, Groth DF, Downey DE, Bowman KA, Martinez HM. 1982. Measurement of the scrapie agent using an incubation time interval assay. Ann. Neurol. April;11(4):353-8.

Prusiner SB, Cochran SP, Downey DE, Groth DF. 1981. Determination of scrapie agent titer from incubation period measurements in hamsters. Adv Exp

Med Biol. 134:385-99.

The endpoint titration and incubation time in terval assay yield similar results, however, the endpoint titrati on requires many more anim als. As a result, the incubation time interval assay is the more widely used assay method for prions, for it is faster, uses fewe r animals with results that ar e negligibly different (± 0.5 log) compared to the endpoint titration. However, an endpoint titration assay may be more useful than an incubation time assay if the titer of the starting inoculum is very high or if one wants to directly compare two samples with similar titers. A comparison of the number of animals required for theses assays is listed below. The prion strain is the 263K strain of hamster-adapted scrapie. The endpoint titration requires between tw o to four times the animals to accomplish the same goal. Appendix 1 co mpares the typical number of animals used for an endpoint titration versus an incubation time interval assay.

c. Other Relevant Tests. If an applicant has conduct ed screening tests (such as Western blot, ELISA, or cell cult ure assays), the resulting data may be submitted along with the suspension-based or carrier-based test data, if desired.

(4) Other important aspects of the t est system include the following:

Concentration (titer) of prions : Depending on the test method and type of prion used, the titer of prions in the initial inoculum may range from 10 4 to 10 11

ID 50 units/g of homogenate. After dilution and drying of the inoculum on the carrier or in a suspension, the titer of prions may be further reduced by 1-2 logs. The study should have a reliable method for determining the concentration of prions in the initial inoculum and the c oncentration of prions dried on the carrier or mixed in a suspension. In order for t he test to be able to measure a six (6) log reduction in infectivity (see Performance Standard below), the titer of prions on

6the carrier or in a suspension (i.e., the ti ter to which test animals will be exposed)

should be at least 10 6 ID 50 units/g brain homogenate (or about 10 8 ID 50 units/g brain homogenate in the initia l inoculum). Finally, t he protocol should address and balance such issues as the preparation of the initial inoculum, the order in which animals are inoculated, and how the animals are housed (see Animal Housing below). If a test system would not be abl e to achieve a log 6 ID 50 units inoculum at baseline, the applicant s hould provide a scientific justification supporting the proposed test system.

Prion type: Several types of prions are available for use in infectivity tests, such as scrapie prions (hamster adapted 263K), BSE prions (mouse adapted 301V, 310C and 6BP1), and sCJD prions (human). An animal-related prion (e.g., scrapie, CWD) should be selected for te sting if the proposed use sites are agricultural (e.g., farm premises, fa rm equipment, veterinary clinics). A humanrelated prion (e.g., sCJD) should be sele cted if the proposed use sites are related to humans (e.g., surgical instruments, hospital rooms, laboratories). [Note: Prospective registrants should consult with the U.S. Food and Drug Administration (FDA) for uses that are al so under its jurisdiction (e.g., medical devices or adjuncts to medical devices). The FDA considers claims of activity against prions (TSE agents) to be unique and unclassified, and recommends that product manufacturers seeking a product cl aim of reducing t he infectivity of prions (TSE agents) for any healthcare us e meet with FDA to learn the FDA’s recommendations for seeking product appr oval.] Unfortunately, human-derived

TSE materials with known contents of in fectivity assayed in known susceptible rodents are not readily available except in a few research laboratories.

Age of test animals at beginning of study : Animals should be old enough to tolerate intracerebral (IC) inoculation wit hout excessive mortalit y (e.g., mice 6-10 weeks old; hamsters 5-8 weeks old; guinea pigs 5-7 weeks old) up to the end of their normal life span (the point at whic h normal mortality begins to increase significantly). Historical mortality data on the selected strain of test animals should be provided to substantia te the selection of these critical lifespan points.

Number of animals : The numbers of animals in the treatment and control groups should be sized based on statis tical validity, and the calculations supporting the proposed animal group sizes s hould be included. The number of animals typically used is between 4 and 24.

Types/Species of animals : The type/species of animal is determined by the

test method selected. The test animal s are either genetically homogeneous mice or hamsters, or transgeni c (genetically altered to express the prion protein— PrP— of the host animal) mice or hamster s. If the animals are transgenic, the test protocol should ident ify the source of the transgenic gene material and

available information that characterizes it.

Animal housing : The number of animals per cage should be kept to a minimum (e.g., 1-2) and the cages should be com patible with the animals. Other animal housing issues should be addressed to avoi d influencing the test results; for

7example, such as which groups may be hous ed next to each other, and rotating cages to balance exposure to heat, light, noise, etc. Transgenic or wild-type mice can be housed 5-8 per cage, depending on the size of the cage, and hamsters are generally housed two per cage.

Length of study : The study should be extended as long as possible, taking into account the historical mortality data fo r the particular animal type/species used. Transgenic or wild-type mice should be kept for 500 days. Hamsters should be kept for 400 days.

Examination of animals at end of st udy or when animals die prematurely:

When animals reach the def ined age limit or die prem aturely, tests should be performed on all members of both the treated and control groups, in order to ascertain whether they are infected with prions. Such tests should include a neuropathological examination and at least one of the following: Western blot, immunological histochemistry or “blin d passage.” The method for dealing with premature deaths and how to place them into the statistical calculations should

also be addressed in the statistica l evaluation plan for the study.

(5) Evaluation of Success: The target criterion for succe ss is generally six (6) logs of reduction of infectivity in the tr eated versus untreated (control) groups.

8Appendix 1. Animals used in an endpoint titration versus an incubation time interval assay under different sets of assumptions

Assuming:

• Initial brain titer of 10 10 ID 50 /g

• 10 treatments are evaluated.

• 6 animals are used per log dilution.

• Each treatment reduces infectivity by 10 4

.

Endpoint titration:

Initial titration (10 log dilutions) 10 x 6 = 60 10 x Titration of each treatment (7 log dilutions) 10 x 7 x 6 = 420

Total 480

Incubation time interval assay:

Titration/calibration curve (10 log dilutions) 10 x 6 = 60 10 x 1 group per treatment 10 x 6 = 60

Total 120

Same assumptions except:

• Each treatment reduces infectivity by 10 6

.

Endpoint titration:

Initial titration (10 log dilutions) 10 x 6 = 60 10 x Titration of each treatment (5 log dilutions) 10 x 5 x 6 = 300

Total 360

Incubation time interval assay:

Titration/calibration curve (10 log dilutions) 10 x 6 = 60 10 x 1 group per treatment 10 x 6 = 60

Total 120

Same assumptions except:

• Each treatment reduces infectivity by 10 8

.

Endpoint titration:

Initial titration (10 log dilutions) 10 x 6 = 60 10 x Titration of each treatment (3 log dilutions) 10 x 3 x 6 = 180

Total 240

Incubation time interval assay:

Titration/calibration curve (10 log dilutions) 10 x 6 = 60 10 x 1 group per treatment 10 x 6 = 60

Total 120

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