&EPA
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 Prion-
Related Claims
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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 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, 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.
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OCSPP 810.2700: Products with Priori-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, etseq.), and the Federal
Food, Drug, and Cosmetic Act (FFDCA) (21 U.S.C., 301 etseq.).
(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 PrPres, 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 PrPc 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 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 intracellular25-nm virus-like particles.
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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:
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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 2:5157-65.
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 begin with, a
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.
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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-Karber 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.htmtfappendix%203.%20fift
v%20percent%20effective%20dose%20(ed50):%20spearman%20karber
%20method
2. Reed LJ and Muench H. A simple method for estimating fifty percent
endpoints. Amer J Hyg 1938;27:493-7.
3. Bliss Cl. 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.
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.
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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 107 ID50 units/g brain homogenate (or
about 109 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).
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,
31OC 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
2 See references in footnote 1.
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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.
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
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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 prion-
related 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) 10x6= 60
10 x Titration of each treatment (7 log dilutions) 10x7x6 = 420
Total 480
Incubation time interval assay:
Titration/calibration curve (10 log dilutions) 10x6= 60
10x1 group per treatment 10x6= 60
Total 120
Same assumptions except:
• Each treatment reduces infectivity by 106.
Endpoint titration:
Initial titration (10 log dilutions) 10x6= 60
10 x Titration of each treatment (5 log dilutions) 10x5x6 = 300
Total 360
Incubation time interval assay:
Titration/calibration curve (10 log dilutions) 10x6= 60
10x1 group per treatment 10x6= 60
Total 120
Same assumptions except:
• Each treatment reduces infectivity by 108.
Endpoint titration:
Initial titration (10 log dilutions) 10x6= 60
10 x Titration of each treatment (3 log dilutions) 10x3x6= 180
Total 240
Incubation time interval assay:
Titration/calibration curve (10 log dilutions) 10x6= 60
10x1 group per treatment 10x6= 60
Total 120
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