United States Environmental 

Protection Agency

 Chemical Safety and 

Pollution Prevention

 (7101)

EPA 712-[insert number]

DRAFT 6-29-11

Product Performance

Test Guidelines

OCSPP 810.2700:

Products with PrionRelated Claims
2

NOTICE

This guideline is one of a series of test guidelines established by the Office of Prevention, 

Pesticides and Toxic Substances (OPPTS), United States Environmental Protection Agency 

for use in testing pesticides and chemical substances to develop data for submission to the 

Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), 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 (FFDCA) (21 U.S.C. 346a).

The OPPTS test guidelines serve as a compendium of accepted scientific methodologies 

and protocols that are intended to provide data to inform regulatory decisions under TSCA, 

FIFRA and/or FFDCA. This document provides guidance for conducting the test, and is also 

used by EPA, the public and the companies that are subject to data submission 

requirements under TSCA, FIFRA and/or the FFDCA. As a guidance document, these 

guidelines are not binding on either EPA or any outside parties, and the EPA may depart 

from the guidelines where circumstances warrant and without prior notice. At places in this 

guidance, the Agency uses the word “should.” In this guidance, use of “should” with regard 

to an action means that the action is recommended rather than mandatory. The 

procedures contained in this guideline are strongly recommended for generating the data 

that are the subject of the guideline, but EPA recognizes that departures may be appropriate 

in specific situations. You may propose alternatives to the recommendations described in 

these guidelines, and the Agency will assess them for appropriateness on a case-by-case 

basis. 

For additional information about OPPTS harmonized test guidelines and to access the 

guidelines electronically, please go to http://www.epa.gov/oppts and select “Test Methods & 

Guidelines” on the left side navigation menu. You may also access the guidelines in 

http://www.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-

0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.

DRAFT DOCUMENT DISCLAIMER: This draft document is distributed solely for the 

purpose of external review. It has not been formally disseminated by the EPA and should 

not be construed to represent any Agency determination or policy. The information 

correction process under the Agency’s Information Quality Guidelines does not apply until 

this document is formally disseminated by the EPA in its final form. This draft document 

should only be cited or quoted in the context of providing comments. 
3

OPPTS 810.2400: Products with Prion-Related Claims.

(a) Scope—

(1) Applicability. This guideline describes test methods that EPA believes will 

generally satisfy certain 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, 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 infectious 

particles, which accumulate in brain tissue during the incubation periods of TSEs,

are comprised of abnormal folding conformations of a normal, ubiquitous protein 

called the “cellular” prion protein (PrPC). This normal cellular prion protein is 

synthesized and eventually degraded through normal metabolic processes. In 

certain instances, however, protein molecules may become misfolded. When 

prion proteins misfold (hereafter called “prions”), the resultant shape change 

serves as a template to induce the misfolding of the normal cellular prion protein

to produce more infectious prions and, thereby, propagate an infection. These

abnormal prions resist proteosomal degradation and slowly accumulate in the 

brain and infect brain tissue.

The first described prion disease was 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 specific, abnormal forms of PrP associated 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 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 beta-sheet 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. 
4

Proceedings of the National Academy of 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 guideline provides pesticide applicants and registrants guidance on 

test systems and performance standards that apply to pesticide products intended to 

reduce the infectivity of prions on inanimate, environmental surfaces (hereafter called 

“prion-related 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. Studies conducted 

under this guideline are to be completed under EPA’s Good Laboratory Practice 

regulations (40 CFR Part 160). 

(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. 

Assuming acceptable data were available, the following is the general format for

a claim that EPA may consider accepting :

“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).” 

An example of a claim that EPA could find to be acceptable depending on 

supporting data would be:

“Has been demonstrated to reduce infectivity of prions (TSE agents) by six

(6) logs based on a bioassay of the scrapie prion 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 reducing the 

infectivity of prions on inanimate 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: 
5

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.

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 

bioassay 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 of the 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 

may be best suited to analyze suspension-based testing and the latter may be 

best suited for carrier-based testing. Further, incubation time interval assays are 

generally not quite as accurate as endpoint titration assays (0.5 log difference1), 

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 (i.e., the concentration of the pathogen). To 

 

1

Prusiner SB, Cochran SP, Downey DE, Groth DF. Determination of scrapie agent titer from incubation 

period measurements in hamsters. Adv. Exp. Med. Biol. 1981;134:385-99.

Prusiner SB, Cochran SP, Groth DF, Downey DE, Bowman KA, Martinez HM. Measurement of the 

scrapie agent using an incubation time interval assay. Ann. Neurol. 1982 Apr;11(4):353-8.
6

begin with, a sample is serially diluted by a factor of 10 until less than 1 ID50 (the 

infectious dose which kills 50% of the pathogen) 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, and the 

symptoms are scored. 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 

before they would die of the prion disease 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.

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%20percent%20effective%20dose%20(ed50):%20spearman%20kärber

%20method

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 

pathogen in an 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 sample is determined by observing the incubation period of an 

inoculated animal group (usually at least four) and using this value to predict a 

corresponding titer based on the empirically determined relationship. The initial 

endpoint titration should not be greatly, if at all, separated in time from the 

experimental assay. If the initial endpoint titration is to be replaced with historical 

animal model data, then sufficient control animals should be included to ensure 

that the historical animal model data are relevant to 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.
7

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)

2

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 prions in the starting inoculum and the treated carrier is 

very high or 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 Appendix 1. 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. EPA welcomes data from screening tests (such as 

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

culture assays), but such tests are not a substitute for the suspension-based or 

carrier-based tests.

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

Titer of prions: Depending on the test method and type of prion used, the titer

of the prions in the initial inoculum may range from 104to 1011 ID50 units/g of 

brain homogenate, based on currently available studies. After dilution and drying 

of the inoculum on the carrier or in a suspension, the titer of the 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 the 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 

 

2

See references in footnote 1.
8

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 

of a known titer currently are available at only a few research laboratories.

Age of test animals at beginning of study: Animals should be old enough to 

tolerate intracerebral 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. The study should continue up to the end of the animals’ 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. Native species of prions may also be 

tested in relevant animal 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 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 450 days.
9

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 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. 

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.

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

bioassays for measuring reduction of prion infectivity by a prion-related 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 to discuss registration data and labeling requirements for a prionrelated product.
10

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