Prevention, Pesticides

 and Toxic Substances

 (7101)

EPA 810-[insert number]

DRAFT 12-8-09

Product Performance

Test Guidelines

OPPTS 810.XXXX

Products with PrionRelated Claims
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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 (CWD), bovine spongiform encephalopathy (BSE), and 

various forms of Creutzfeldt-Jakob disease (CJD), 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, normal protein 

called the “cellular” prion protein (PrPC). This 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 creation of other misfolded 

copies of the prion protein. These abnormal prions resist proteosomal 

degradation, slowly accumulate in the brain, and further infect 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 PrP

res, PrPd

, PrPTSE, PrPCWD, and PrPCJD to designate the 

abnormal forms of PrP, either 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 normal prion protein (PrPC): they are 

usually insoluble in non-denaturing detergent-salt solutions and relatively 

resistant to digestion with the enzyme proteinase K. These properties are 

attributed to misfolding of PrPCto yield isoforms that are enriched in betasheeted secondary structure. Many scientific experts 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). 

1. Manuelidis L. 2007a. A 25 nm virion is the likely 

cause of transmissible spongiform encephalopathies. 

Journal of cellular biochemistry; 100(4):897-915.

2. 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 
4

Sciences of the United States of America; 

104(6):1965-70.

3. Prusiner SB. 1982. Novel proteinaceous infectious 

particles cause scrapie. Science; 216:136-44.

4. 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 pesticide 

products intended to reduce the infectivity of prions on inanimate, environmental 

surfaces (hereafter called “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) by (X) 

logs (insert log reduction number supported by data) based on a bioassay of 

the (insert prion type) in (insert type of organism in which the prions were 

tested).” 

For example, “Has been demonstrated to reduce infectivity of prions (TSE 

agents) by six (6) logs based on a bioassay of hamster adapted 263K 

scrapie prions in transgenic mice.”

(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 

bioassay 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: 

1. Lemmer K, Mielke M, Kratzel C, Joncic M, Oezel M, Pauli G, Beekes 

M. Decontamination of surgical instruments from prions. II. In vivo 

findings with a model system for testing the removal of scrapie infectivity 

from steel surfaces. J Gen Virol. 2008 Jan;89(Pt 1):348-58.
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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.

3. Weissmann C, Enari M, Klohn PC, Rossi P, Flechsig E. 2002. Transmission 

of prions. J Infect Dis 186 Suppl 2:S157-65.

4. 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.

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 

biosassay 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: 

1. 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 

generally 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. The former method is 

best suited to analyze suspension-based testing and the latter is best suited for

carrier-based testing. Further, incubation time interval assays are not as 

accurate as endpoint titration assays, and 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 given a 

thorough neuropathological examination that looks for signs of prion disease as 

described in section (4) below. The titer may be determined by several statistical 

methods (see references below). The titers of subsequent samples are 

determined in the same fashion.
6

The following are examples of end-point titration methods:

1. 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.%20fift

y%20p

ercent%20effective%20dose%20(ed50):%20spearman%20kärber%20me

thod

2. Reed LJ and Muench H. A simple method for estimating fifty percent 

endpoints. Amer J Hyg 1938;27:493-7.

3. Bliss CI. The method of probits. Science. 1934 Jan 12;79(2037):38-39

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 should be included 

to insure that model mean survival time has remained unchanged in the 

experimental assay.

The following are examples of incubation time interval assay methods:

1. 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.

2. 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 assays 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 

since it is faster, uses fewer animals, and provides 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 difference 

between the titer of the starting inoculum and the treated carrier is very high, very 

low or if one wants to directly compare two samples with similar titers. A 

comparison of the number of animals required for these assays is listed in 
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Appendix 1. The prion strain is the 263K strain of hamster-adapted scrapie is 

being provided as an example. The endpoint titration requires between two and

four times the animals as the incubation time interval assay. 

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

as Western blot, ELISA, Protein Misfolding Cyclic Amplification (PMCA), or cell 

culture assays), the resulting data may be submitted along with the suspensionbased 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 104to 1011

ID50 units/g of brain 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. The dynamic range of the test method’s ability to 

measure reduction in infectivity should be well established. In order for the test 

to be able to measure at least a six (6) log reduction in infectivity [see section (5) 

Evaluation of Success below], the titer of prions on the carrier or in a suspension 

(i.e., the titer to which test animals will be exposed) should be at least 107ID50

units/g brain homogenate (or about 109ID50 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). 

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 

animal-related (e.g., farm premises, farm equipment, veterinary clinics). A 

human-related 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.] Human-derived TSE materials 

with known contents of infectivity assayed in known susceptible rodents are 

available at 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, i.e., in the 

following age ranges; mice, 6-10 weeks old; hamsters, 5-8 weeks old; and 

guinea pigs, 5-7 weeks old. Historical mortality data on the selected strain of test 
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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 another animal) mice. If the animals are transgenic, the test protocol 

should identify the source of the transgenic gene material and available 

information that characterizes it. “Native species” of prions (i.e., those prions 

which occur naturally in specific species of animals) may also be tested in their 

natural animal host species.

Animal housing and environmental conditions: The number of animals per 

cage should be kept to a minimum and the cages should be compatible with the 

animals. Other animal housing issues should be addressed to avoid influencing 

the test results; for 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. The temperature 

in the experimental animal room should be 22°C (± 3°C). The relative humidity 

should be at least 30% and preferably not exceed 70% other than during room 

cleaning. Lighting should be artificial, the sequence being 12 hours light and 12 

hours dark.

Length of study: The study should continue up to the end of the animals’ 

normal life span (the point at which normal, age-related mortality begins to 

increase significantly for the particular animal species/strain being used). 

Historical mortality data on the selected strain of test animals should be provided 

to substantiate the selection of these critical lifespan points. Transgenic or wildtype mice usually can be kept for 500 days. Hamsters usually can be kept for 

450 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 appropriate to the strain of prion used, and at 

least one of the following: Western blot, immunological histochemistry or similar 

testing that confirms the presence or absence of prions in nervous system tissue. 

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. 
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(5) Evaluation of Success: The target criterion for success is no less than six (6)

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

(6) Submission of Draft Protocol: Due to the wide variety of available animal 

bioassays for measuring reduction of prion infectivity by a prion product on 

environmental surfaces, the Agency strongly recommends that the registrant 

submit a draft test protocol to EPA for review prior to conducting such a study. 

The Agency also recommends that the registrant request a pre-registration 

conference, prior to conducting any prion efficacy testing, to discuss registration 

data and labeling requirements for a prion product.
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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