Prevention, Pesticides and Toxic Substances (7101) EPA 8 1 0
- [insert number] DRAFT 1 2
- 8
- 0 9 

# Product Performance Test Guidelines OPPTS 810.XXXX Products w ith Prion
Related Claims2 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 developm ent 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, Subchap ter 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.3

OPPTS 810.XXXX : Products w ith Prion - Related Claims
. (a) Scope

— (1) Ap plicability
. 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 Cosme tic Act (FFDCA) (21 U.S.C., 301 et seq. ). (2) Background. Prion (“ proteinaceous 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 collective ly called the transmissible spongiform encephalopathies ( TSEs ) or prion diseases, including scrapie, chronic wasting disease (CW D) , bovine spongiform encephalopathy (BSE) , and various forms of Creutzfeldt
-Jakob disease (CJD) , etc. These isolated infectiou s 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 ( PrP C ). Th is normal cellular pri on protein is synthesized and eventually degraded thro ugh 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

. T he se abnormal prion s resist proteosomal degradation , slow ly 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 P rP Sc in all TSEs. Some scientists prefer to use other terms such as P r P res , PrP d , PrP TSE , PrP CWD , and PrP CJD 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 ( PrP C ) : they are usually insoluble in non - denaturing detergent
- salt s olutions and relatively 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
sheeted secondary structure

. Many scientific experts have concluded that the ab normal prion proteins themselves are the self
- replicating infectious agents causing TSEs (Prusiner 1982, 2004); however, t here 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 parti cles. Proceedings of the National Academy of4 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. Scien tific 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 in animate, 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 guida nce over time, as needed, to reflect advances in available test methods and scientific knowledge pertaining to prions. (c) Guidance

— (1) Label ing Claims
. Testing conducted according to c urrently available test methods are adequate for measuring reductio n 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 numb er 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 ina nimate 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 surfa ces. J Gen Virol. 2008 Jan;89(Pt 1):348
- 58.5 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. We issmann 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 W eissman C. 1999. Infectivity of scrapie prions bou nd 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 th e 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 d ose 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 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 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 perio d, 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 be low). 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 (ED 50 ): Spearman
- Kärber metho d. 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 estimati ng 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 in cubation 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 ti me 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 s crapie 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. 1 34:385
- 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 mo re widely used assay method for prions since it is faster, uses fewer animals , and provides results that are negligibl y 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 in7

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 te sts (such as W estern blot, ELISA, Protein Misfolding Cyclic Amplification (PMCA), 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 10 4 to 10 11 ID 50 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) E valuation 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 10 7 ID 50 units/g brain homogenate (or about 10 9 ID 50 units/g brain homogenate in the initial inocu lum). 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 u se 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.] H uman - 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) inocul ation 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 test8

animals should be provided to substantiate the se lection 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 a ltered 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
-ty pe 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 wild
type mice usually can be kept for 500 days. Hamsters usually can be kept for 4 5 0 days. Examination of animals at end of study or when animals die prematurely: Wh en animals reach the defined age limit or die prematu rely, 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 l east one of the following: W estern blot , immunological histochemistry or sim i lar 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 calc ulations should also be addressed in the statistical evaluation plan for the study.9

(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, t he Agency strongly recommends that the registrant submit a draft test proto col to EPA for review prior to conducting such a study. The Agency also recommends that the registrant request a pre
- registrati on conference , prior to conducting any prion efficacy testing, to discuss registration data and labeling requirements for a prio n product.10

Ap pendix 1. Animals used in an endpoint titration versus an incubation time interval assay under different sets of assumptions As suming:

 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 : T itration/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 dil utions) 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