Prevention, Pesticides 

 and Toxic Substances 

 (7101) 

EPA 712-[insert number]

DRAFT 2-23-09 

Product Performance

Test Guidelines

OPPTS 810.XXXX 

Products with PrionRelated Claims
INTRODUCTION 

 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 data 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 Rodenticide 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 through a process of harmonization of the 

testing guidance and requirements that existed for the Office of Pollution Prevention and 

Toxics (OPPT) in Title 40, Chapter I, Subchapter 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 guidelines 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. 

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

(a) Scope— 

(1) Applicability. This guideline is intended to meet testing requirements of the 

Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et 

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

et seq.).

(2) Background. Prion (“proteinaceous infectious 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 isolated infectious particles, which 

accumulate in brain tissue during the incubation periods of TSEs, are comprised 

of abnormal folding conformations of a ubiquitous protein called the “cellular” 

prion protein (PrPC). The normal cellular prion 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 recruitment of other misfolded copies of the 

prion protein and accounts for the resistance of the abnormal prions to 

proteosomal degradation and their slow accumulation in infected brain tissue. 

Prions were first detected in animals with scrapie, and so such infectious proteins 

are often designated collectively as PrPSc in all TSEs. Some scientists prefer to 

use other terms such as PrPP

res, PrPd

, PrPTSE, PrPCWDP , and PrPCJD 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 proteins, although somewhat variable, 

share properties that distinguish them from PrPC: they are usually insoluble in 

detergent-salt solutions and relatively resistant to digestion with the enzyme 

proteinase K. These properties are attributed to misfolding of alpha-helical PrPC

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 virion is the likely cause of 

transmissible spongiform encephalopathies. Journal of cellular 

biochemistry; 100(4):897-915. 

Manuelidis L, Yu ZX, Barquero N, Mullins B. 2007b. Cells infected 

with scrapie and Creutzfeldt-Jakob 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. 

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Prusiner SB. 1982. Novel proteinaceous 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 systems 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 reflect 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. Further, a prion-related claim should include 

the type of prion against which the product 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) (insert prion type).” For example, “Has been 

demonstrated to reduce infectivity of prions 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 environmental 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-based 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. 

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b. Suspension-based Test Method. If the intended use of a product includes 

only treating liquids (e.g., liquid wastes), 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 Reduction 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 the 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 ID50 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 euthanized before they would die of the 

prion disease. The animals are observed 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 euthanized and examined for 

signs of prion disease (e.g., presence in the brain of prion protein 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 

 (ED50): 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 

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b. Incubation Time Interval Assay. An incubation time interval assay exploits 

the relationship between the length of the incubation period and the titer of the 

inoculum. An endpoint titration of a starting brain homogenate is used to 

establish an empirical relationship between prion titer and length of incubation 

period. Once the relationship is established, 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, separated 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 interval assay yield similar results, 

however, the endpoint titration requires many more animals. As a result, the 

incubation time interval assay is the more widely used assay method for prions, 

for it is faster, uses fewer animals with results that are 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 two to four times the animals to 

accomplish the same goal. Appendix 1 compares the typical number of animals 

used for an endpoint titration versus an incubation time interval assay.

c. Other Relevant Tests. If an applicant has conducted screening tests (such 

as Western blot, ELISA, or cell culture 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 test 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 104 to 1011

ID50 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 concentration of prions dried on the carrier 

or mixed in a suspension. In order for the test to be able to measure a six (6) log 

reduction in infectivity (see Performance Standard below), the titer of prions on 

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

should be at least 106

 ID50 units/g brain homogenate (or about 108 ID50 units/g 

brain homogenate in the initial inoculum). Finally, the 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 able to achieve a log 6 ID50 units 

inoculum at baseline, the applicant should 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 testing if the proposed use sites are 

agricultural (e.g., farm premises, farm equipment, veterinary clinics). A humanrelated prion (e.g., sCJD) should be selected 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 also 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 claim of reducing the infectivity of 

prions (TSE agents) for any healthcare use meet with FDA to learn the FDA’s 

recommendations for seeking product approval.] Unfortunately, human-derived 

TSE materials with known contents of infectivity 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 without excessive mortality (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 which normal mortality begins to increase 

significantly). Historical mortality data on the selected strain of test animals 

should be provided to substantiate 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 statistical validity, and the calculations 

supporting the proposed animal group sizes should 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 animals are either genetically homogeneous mice 

or hamsters, or transgenic (genetically altered to express the prion protein—

PrP— of the host animal) mice or hamsters. If the animals are transgenic, the 

test protocol should identify 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 compatible with the animals. Other animal 

housing issues should be addressed to avoid influencing the test results; for 

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example, such as which groups may be housed 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 for 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 study or when animals die prematurely: 

When animals reach the defined age limit or die prematurely, 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 “blind passage.” The method for dealing with 

premature deaths and how to place them into the statistical calculations should 

also be addressed in the statistical evaluation plan for the study. 

(5) Evaluation of Success: The target criterion for success is generally six (6) logs 

of reduction of infectivity in the treated versus untreated (control) groups. 

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Appendix 1. Animals used in an endpoint titration versus an incubation time interval 

assay under different sets of assumptions

Assuming: 

• Initial brain titer of 1010 ID50/g 

• 10 treatments are evaluated. 

• 6 animals are used per log dilution. 

• Each treatment reduces infectivity by 104. 

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 106

. 

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 108

. 

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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