United States Environmental Protection Agency Chemical Safety and

Pollution Prevention (7101) EPA 712-C-13-001

December 2012


# Product Performance

Test Guidelines

OCSPP 810.2700:

Products with PrionRelated Claims2 NOTICE

This guideline is one of a series of test guidelines established by the United States Environmental Protection Agency’s Office of Chemical Safety and Pollution Prevention (OCSPP) 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 Act (FFDCA) (21 U.S.C. 346a). Prior to April 22, 2010, OCSPP was known as the Office of Prevention, Pesticides and Toxic Substances (OPPTS). To distinguish these guidelines from guidelines issued by other organizations, the numbering convention adopted in 1994 specifically included OPPTS as part of the guideline’s number. Any test guidelines developed after April 22, 2010 will use the new acronym (OCSPP) in their title.

The OCSPP harmonized 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 doc ument 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, the 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 these test guidelines and to access these guidelines electronically, please go to http://www.epa.gov/ocspp and select “Test Methods & Guidelines” on the 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-2009-0576.3 OCSPP 810.2700: Products with Prion-Related Claims

.

(a) Scope —

(1) Applicability. This guideline describes test methods that EPA believes will generally satisfy certain testing require ments 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 (“ 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, which are collectively called the transmissible spongiform encephalopathies (TSEs or pri on diseases), including scrapie, chronic wasting disease (CWD), bov ine spongiform encephalopathy (BSE), and

various forms of Creutzfeldt-Jakob disease (CJD), etc. These infectious particles,

which accumulate in brain tissue duri ng the incubation periods of TSEs, are comprised of abnormal folding conformati ons of a normal, ubiquitous protein

called the “cellular” prion protein (PrP C ). 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 calle d “prions”), the resultant shape change serves as a template to induce the misfol ding of the normal cellular prion protein to produce more infectious prions and, thereby, propagate an infection. These abnormal prions resist proteosomal degr adation and slowly accumulate in the

brain and infect brain tissue.

The first described prion disease was scrapi e, and so such infectious proteins are often designated collectively as PrP Sc in all TSEs. Some scientists prefer to

use other terms such as PrP res , PrP d , PrP TSE , PrP CWD , and PrP CJD to designate the specific, abnormal forms of PrP asso ciated 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 distinguis h them from normal PrP C : they are usually insoluble in non-denaturing detergent-salt solutions and re latively resistant to digestion with the enzyme proteinase K. These properties are attributed to misfolding of PrP C to 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 reservati ons, 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 biochemistr y; 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 Na tional Academy of Sciences of the

United States of Am erica;104(6):1965-70.

3. Prusiner SB. 1982. Nove l proteinaceous infectious particles cause scrapie. Science; 216:136-44.

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

(b) Purpose. This guideline provides pesticide appl icants and registrants guidance on test systems and performance standards that apply to pesticide products intended to reduce the infectivity of pri ons on inanimate, environmenta l surfaces (hereafter called “prion-related products”). EPA encourages pestic ide applicants and registrants to follow this guidance and submit a draft test protoc ol 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 knowled ge pertaining to prions. Studies conducted under this guideline are to be complet ed 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 inacti vation of prions. Accordingly, claims such as “inactivates,” “destroys,” “ denatures” and “eliminates ” are not supported by currently available test methods. Furt her, 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 infectiv ity of prions (TSE agents) by (X) logs (insert log reduction number suppor ted 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 infectiv ity 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 i nanimate 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 in struments from prions. II. In vivo findings with a model system for testi ng the removal of scrapie infectivity

from steel surfaces. J Gen Vi rol. 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 wast es), 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 acco rding 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 Re duction of Prion Infectivity. Prion diseases are generally characterized by a long asympt omatic incubation period, followed by the rapid onset of symptom s, followed by death. The length of the incubation period is reproducible upon r epeated passages of a given prion and is inversely proportional to the log of the infectious dose of that pr ion, although at both very high and very low concentrations of infectiv ity 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 difference 1 ), and may be less reliable for quantifying ve ry low levels of infectivity.

a. End-point Titration. An end-point titration is a cl assical method of determining the titer of a sample (i.e., the concent ration of the pathogen). To begin with, a

1 Prusiner SB, Cochran SP, Downey DE, Groth DF. Dete rmination of scrapie agent titer from incubation period measurements in hamsters. Ad v. 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 interv al assay. Ann. Neurol. 1982 Apr;11(4):353-8.6 sample is serially diluted by a factor of 10 until less than 1 ID 50 (the infectious dose which kills 50% of the pathogen) remain s in the final dilution. The resulting

set of serial dilutions is used to i noculate 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 t he end of the predet ermined period, any surviving animals would be humanely eutha nized before they would die of the prion disease and given a thorough neuropathol ogical 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 (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.%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 pe riod and the titer of the pathogen in an inoculum. An endpoint titrat ion of a starting brain homogenate is used to establish an empirical relations hip between prion titer and length of incubation period. Once the relationshi p 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 empiricall y 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 contro l animals should be included to ensure

that the historical animal model data ar e 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.

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. 134:385-99.7 The endpoint titration and incubation time in terval assays yield similar results; however, the endpoint titration requires m any 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 titr ation. 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 star ting inoculum and the treated carrier is very high or very low, or if one want s 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 stra in 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, Prot ein Misfolding Cyclic Ampl ification (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 t est 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 10 4 to 10 11 ID 50 units/g of brain homogenate, based on currently availa ble studies. After dilution and drying

of the inoculum on the carrier or in a su spension, the titer of the prions may be further reduced by 1-2 logs. The st udy should have a reliable method for determining the concentration of prions in the initial inoculum and the concentration of prions dried on the ca rrier 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 t he 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 10 7 ID 50 units/g brain homogenate (or

about 10 9 ID 50 units/g brain homogenate in the initial inoculum). Finally, the protocol should address and balance such i ssues as the preparation of the initial inoculum, the order in which animals ar e 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 te sting 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 al so under its jurisdiction (e.g., medical

2 See references in footnote 1.8 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.] Human-derived TSE materials of a known titer currently are availabl e at only a few research laboratories.

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

tolerate intracerebral inoculation without ex cessive 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 mortalit y begins to increase significantly). Historical mortality data on the selected st rain 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 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 animals are either genetically homogeneous mice or hamsters, or transgenic (genet ically altered to expre ss the prion protein—PrP— of the host animal) mice or hamsters. If the animals are tr ansgenic, the test protocol should identify the source of the transgenic gene material and available

information that characterizes it. Native species of pri ons 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 ex posure 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 ar e generally housed two per cage. The temperature in the experimental anima l 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 fo r the particular animal type/species used. Transgenic or wild-type mice should be k ept for 500 days. Hamsters should be kept for 450 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 to9 ascertain whether they are infected wit h prions. Such tests should include at least one of the following: Western blot, immunological histochemistry or “blind passage.” The method for dealing with prem ature deaths and how to place them

into the statistical calculations shoul d also be addressed in the statistical evaluation plan for the study.

Evaluation of Success: The target criterion for su ccess is no less than six (6) logs of reduction of infectivity in t he treated versus untr eated (control) groups.

Submission of Dr aft 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 st rongly recommends that the registrant submit a draft test protocol to EPA fo r review prior to conducting such a study. The Agency also recommends that the r egistrant 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 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