	     Prevention, Pesticides

     and Toxic Substances

     (7101)	EPA 810-[insert number]

DRAFT 12-8-09

	

Product Performance

Test Guidelines





OPPTS 810.XXXX

	Products with Prion-Related Claims



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   HYPERLINK
"http://www.epa.gov/oppts"  http://www.epa.gov/oppts  and select “Test
Methods & Guidelines” on the left side menu.  

OPPTS 810.XXXX: Products with Prion-Related Claims.

Scope—

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.).

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  SEQ CHAPTER \h \r 1  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 PrPres, 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
PrPC to yield isoforms that are enriched in beta-sheeted 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).  

Manuelidis L.  2007a. A 25 nm virion is the likely cause of
transmissible spongiform encephalopathies.  Journal of cellular
biochemistry; 100(4):897-915.

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 Sciences of
the United States of America; 104(6):1965-70.

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

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.

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.

	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: 	  HYPERLINK
"http://www.fao.org/docrep/w0049e/w0049e07.htm#appendix%203.%20fifty%20p
ercent%20effective%20dose%20(ed50):%20spearman%20kärber%20method" 
http://www.fao.org/docrep/w0049e/w0049e07.htm#appendix%203.%20fifty%20p
ercent%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 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 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 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 104 to 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 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, 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 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 wild-type 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.  

(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. 

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

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