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1 EPA Responses to Comments by the FIFRA Scientific Advisory Panel Concerning 1

“Scientific Information Co ncerning the Issue of Wheth er Prions Are a ’Pest’ 2 under the Federal Insecticide, Fungic ide, and Rodenticide Act (FIFRA)” 3 (2-17-10 DRAFT) 4

5 On March 31 and April 1, 2009, the FIFRA Scientific Advisory Panel (SAP) held 6 an open, public meeting to address a set of sc ientific issues being considered by the EPA 7 pertaining to the issue of whet her a prion is considered to be a “pest” under FIFRA. 8 Subsequent to the meeting, the SAP issued mi nutes, including its comments on the issues 9 and charge questions that EPA presented at the meeting (SAP Minutes No. 2009-05; 10

available at http://www.epa.gov/scipoly/sap ). 11

12 The purpose of this document is to pr ovide EPA’s responses to the SAP’s 13 comments as listed in the meeting minutes. First, the verbatim Summary of the SAP’s 14 comments are presented, followed by Charge Qu estion 1 and the SAP’s detailed response 15 to Charge Question 1. Following the SAP’s de tailed response to Charge Question 1 is 16 EPA’s response to the SAP’s detailed re sponses (beginning on page 17). EPA’s 17 responses to Charge Questions 2-4 fo llow and are similarly presented. 18

19 SAP Summary of Panel Discussion and Recommendations (quoted verbatim from 20 the SAP’s meeting summary) : 21

22 As charged by the EPA, the Panel revi ewed the interagency White paper titled 23 “Scientific Information Concerning the Issue of Whether Prions Are a ‘Pest’ under the 24 Federal Insecticide, Fungicide, and Rodentic ide Act (FIFRA),” and discussed the nature 25 of prions with respect to th eir potential classification as a “Pest” under FIFRA; guidance 26 on testing issues for assessing efficacy of pe sticides for reduction of prion infectivity; 27 efficacy performance and labeling guidance for such pesticides; and lastly, scientific 28 evidence for prion hierarchy in resistance to pesticide action. The prion terminology 29 defined in the White paper will be used in these meeting minutes: “...‘prion’ refers to a 30 misfolded isoform of any pri on protein; ‘prion protein’ can refer to both the normal and 31

misfolded isoforms.” 32

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1. Interagency White paper issue: Does the interagency White paper on prions 34 adequately identify and summarize available, relevant scientific studies and do these 35 support designation of prions as “Pests”? The White paper summarizing the prion 36 literature with respect to description of transmissibl e spongiform encephalopathies 37 (TSEs) and prions, key charac teristics of prions, the pri on hypothesis and normal prion 38 protein, neurotoxic mechanisms in TSEs, TSE diseases, history of TSE diseases, 39 transmission and routes of exposure for TS Es, occupational and safety procedures and 40 decontamination methods was accepted as adequate by the majority of the Panel. 41 However, many statements in the White paper need to be revised to reflect accurately 42 what is definitively known about prions and TSEs and that which is reasonably 43 hypothesized based on scientific observations. 44

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2 A major issue discussed by the Panel was the prion hypothesis for the infective nature of 1 TSEs. A majority of the Panel accepted the prion hypothesis for TSE infectivity with the 2 caveat that cofactors may also be involve d. The prion hypothesis, although considered 3 unproven by some Panel members, provides a l ogical explanation of the infective nature 4 of TSEs and the apparent lack of nucleic acid. The strength of the support for the prion 5 hypothesis by experimental inf ection of transgenic mice with synthetic prion fibrils was 6 questioned because spontaneous neurodegenera tive disease in unexposed mice is also 7 observed. A minority opinion contended that prion accumulation in TSEs is a pathologic 8 change in response to an as yet unidentif ied TSE etiologic agent, possibly a virus. 9

10 The penultimate scientific question from the EPA to the Panel relative to the character 11 and nature of prions is whether the science in the White paper suppor ts the classification 12 of prions as a “Pest” under the FIFRA. Th e regulatory basis (not reviewed by the Panel) 13 on which this science question is assesse d is whether prions are a form of a 14 microorganism, i.e., prions are microorganis ms other than bacteria or viruses, and 15 whether prions are living entitie s. A majority of the Panel concluded that prions can be 16 included in the category of “other microor ganisms.” A minority of Panel members 17

contended on scientific basi s that prions lack the signi ficant features typical of 18 microorganisms. 19

20 In conclusion, a majority of Panel members agreed that once revised as suggested by the 21 Panel the White paper can adequately identify and summarize the scientific literature 22 concerning prions. Furthermore, a majority of the Panel accept that prions are “other 23 microorganisms” exhibiting some features of living entities. 24

25 2. Efficacy Guidance Test Method Issue: Are the specific test systems 26 recommended in the EPA draft guidance documen t scientifically appropriate to support 27 the registration of pesticide produc ts with prion-related claims? 28

29

The consensus of the Panel was that animal bioassays are necessary to adequately assess 30 reduction of prion infectivity by prion pest icide products. The Panel was asked to 31 comment specifically on the three requested areas. 32

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a. Carrier-based, animal infectivity ass ays for environmental surfaces (e.g., 34 hard, nonporous surfaces). 35 The Panel had reservations about the appropr iateness of application of carrier-based 36 animal bioassays for assessing pesticide produc t reduction of prion in fectivity. In the 37 case of carrier-based assays, e nd-point titrations pose the pr oblem of the practicality of 38 doing serial dilutions of the treated surface, i. e., treated stainless st eel wire or beads of 39 various compositions, where the presentation of absorbed prions to the bioassay may be 40 limited by the surface area of the carrier exposed to the brain. There may be substantial 41 problems in quantifying reduction of infectivity in carrier-based models as well, because 42 absorption of diluted brain homogenates on to the carrier may not pr oportionately reflect 43 the infectivity present in the original samp le. However, it was also suggested that 44 disproportionate absorption can be assessed by use of a positive standard dose-response 45 curve for carrier-bound infectivity. 46EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

3 1

b. Suspension-based, animal infectivity assays used in liquid media (e.g., 2 wastewater). 3 The Panel agreed that the suspension-based end point dilution animal bioassay is the 4

most reliable method to be used for final validation of reduction of prion infectivity. 5 Furthermore, the Panel considered incubation interval animal bioassays as a screening 6 method to better focus the range of test dilu tions necessary for more definitive end point 7 dilution animal bioassays, with the advantag e of reducing cost and use of animals for 8 testing; however, an end point animal bioa ssay is necessary to calculate directly a 9

reduction in infectious titers and cannot be substituted by an incubation interval animal 10 bioassay. Some Panel members supported de signation of a standard animal bioassay 11 such as a rodent-adapted scrapie strain in a rodent model system for use with all 12 registrant prion pesticide test ing, whereas others considered that testing needed to be 13 conducted with native prion strains in releva nt animal hosts. The Panel urged EPA to 14 monitor closely the particulars of testing me thods for uniformity of treatment of control 15 and experimental animals, preparation of pr ion inocula, sufficiency of animal numbers 16

used relative to the variance of the bioa ssay model system, and determination of the 17 bioassay end points. Necropsies of all test animals for the presence of TSE disease based 18

on Western blot detection of protease resistant prion a nd neuropathologic changes 19 consistent with TSE are required. 20

21

c. Any other known test methods for eval uating the efficacy of pesticide 22 products used on either environmental surfaces or in liquid media. 23 The majority of the Panel concluded that in vitro immunodetection assays or cell-based 24 bioassays are not adequate for use in asse ssing reduction in inf ectivity for evaluating 25 efficacy of pesticide products with prion re lated claims (hereafter referred as prion 26 pesticide products). In the case of imm unodetection assays, these do not measure 27 infectivity; and in the case of cell-based bi oassays, these have not been adapted for the 28 application of assessing reduction in infectiv ity. A minority contended that in vitro 29 amplification immunodetection assays su ch as protein misfolding by cyclical 30 amplification (PMCA) and cell-based bioassays can serve as a complementary assay to 31 animal bioassays allowing assessment of ma ny more prion strains and prion substrates 32 than is practical with animal bioassays. 33

34 The Panel recommends to EPA that end point di lution animal bioassays be used to assess 35 reduction in infectious titer by treatment of samples with prion pesticide products. 36

37 3. Efficacy Guidance Perf ormance Criterion Issue: Are the product performance 38 criteria specified in the EPA draft guidance document to support the registration of 39 pesticide products with prion-rela ted claims scientifically sound? In particular, the draft 40 efficacy guidance document recommends a target efficacy criterion of six (6) logs of 41 reduction of infectivity in the treated versus untreated (control) groups. 42

43 The Panel was in general agreement that some widely used model systems, such as the 44 263K or Sc237 laboratory rodent-adapted scra pie prion strains have sufficiently high 45 infectivity titers of brain ti ssue, making it possible to det ect a six (6) log reduction in 46EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

4 infectivity. However, several factors ma y reduce the dynamic range of detection for 1 prions from particular prion st rains or natural hosts to much less than six (6) logs. These 2 factors include 1) limited volume of about 25μ l for intracranial injection in laboratory 3 rodents, 2) dilution of inoculum by inactiv ation or other reagents prior to animal 4 bioassays, and 3) lower infec tivity of naturally occurring prion strains from the natural 5 host in the range of 10 5 to 10 8 ID 50 /g resulting in a dynamic range for animal bioassays of 6 three (3) or four (4) logs. 7

8 Prion pesticide registrant’s claims of efficaci ous log reductions in infectivity will need to 9 be accompanied by a statistical methodology, assessment of assay variability for a 10 desired level of confidence, and regressi on analysis models for endpoint animal 11 bioassays. Although suspension-based methods have a rational basis for calculating log 12 reductions in prion infectivity, carrier-bas ed methods provide further issues for 13 calculating log reduction. Inf ectious brain material seri ally diluted and dried onto 14 carriers may not retain infectivity proportiona l to that in the original sample thereby 15 negating the endpoint animal bioassay calcu lation of log reduction in infectivity. 16

17 In addition, the target degree of reduction in infectivity may need to be considered in 18 light of the proposed applicati on or use, risk to human and animal health, and what is 19 achievable and can be validated for a particul ar prion pesticide. A reduction of six (6) 20 logs of infectivity in waste water may be ad equate to reduce the infectivity compared to 21 the risk posed by chronic wasting disease (C WD) prions to human health, whereas the 22 efficacy of a six (6) log reduction for Creutz feldt-Jakob disease (CJD) prions may not be 23 adequate to reduce the potential that a human patient might contract CJD from a surgical 24 instrument. Risk assessments may be availabl e to help formulate expected standards of 25 performance, but a one-size fits all product performance guidance may be neither valid 26

nor desirable. 27

28 In conclusion, the Panel recommends that targ et reduction in infectiv ity be assessed in a 29 relevant animal model using the naturally occu rring prion strain from the natural host to 30 support the specific label claim made by the ma nufacturer of the pr ion pesticide. To 31 demonstrate target efficacy, the level of reduction should be set at the maximum 32 measurable reduction with the designated na tural host prion strain in the designated 33 model. No consensus was reached on accepta bility of target efficacy of a six (6) log 34 reduction in a widely used model such as the hamster-adapted 263K or Sc237 scrapie 35 prion strains as the sole test for pesticide e fficacy of reduction of in fectivity for a target 36 prion strain; however, there wa s consensus that a widely us ed model could be used in 37 addition to the target prion st rain to show the possibility of higher reduction levels of 38 prion infectivity. 39

40 4. Efficacy Guidance Labeling Claim Issue: Are the labeling clai ms described in the 41 EPA draft guidance document sc ientifically appropriate ba sed on the recommended test 42 systems and product performance standard? Th is guidance: “Has been demonstrated to 43 reduce infectivity of prions (TSE agents) base d on testing using (ins ert type of organism 44 in which the prions were raised) (insert pri on type)” is careful to avoid claims normally 45EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

5 applied to microorganisms, e.g., “destroy,” “m itigate,” “eliminate,” “control” which may 1 be misleading when applied to prions.” 2

3 The Panel agrees with EPA’s proposal to fr ame the label claim statement as “reduce 4 infectivity,” but even with this relatively conservative claim, there are several caveats. 5 From a scientific perspective, the state of “complete elimination of infectivity” is 6 probably impossible to achieve in practice, because minimal infective doses of prions are 7 not known, particularly for humans. In addi tion, experimental obser vations suggest that 8 treatments which reduce TSE infectivity may le ad to incubation periods that fall outside 9 the linear range of animal bioassays. The phr ase “reduce infectivity” is valid only as long 10 as the method used to measure infectivity has an acceptable dynamic range of detection 11 in a relevant animal bioassay system. 12

13

Care will need to be taken to state accura tely prion pesticide product performance to 14 guide user understanding and expectations for the prion pesticide product. Users in 15 hospitals concerned about CJD may expect something closer to “elimination of 16 infectivity” on environmental su rfaces, whereas the veterinary and wildlife management 17 community may be more comfortable with th e “reduced infectivity” approach. EPA 18 might require registrants to discuss the imp lications of the “reduce vs. elimination” 19 approach in sufficient detail so that users can understand and apprec iate what the product 20

can and cannot do in relation to intended uses, e.g., decontamination of hospital 21 environmental surfaces, decontamination of laboratory working surfaces, application on 22 porous materials such as fences, etc. 23

24 With respect to specificity of the labeling, the Panel suggests that the prion strain and 25 original host, bioassay animal host, and observe d log reduction in infectivity be present in 26 the label as well as the dynamic range of th e animal bioassay used. Concerning whether 27 a six (6) log reduction in infectivity is needed to receive approval as efficacious, it was 28 noted that even a five (5) log reduction would be acceptable for a product with a prion29 related claim under the above conditions spec ified on the label for prion strains and 30 natural hosts for which five (5) log is the ma ximum attainable reduction in infective titer 31 possible in the animal bioassay. EPA is urged to have scientific advisement to seek a 32 consensus compilation of acceptable prion strain /animal bioassay models. In particular, 33 this activity might help guide registrants away from choosing model systems with low 34 levels of prion infectivity to test their product, thereby pr eventing inappropriate testing 35 for prion pesticide products. 36

37 In conclusion, there was gene ral consensus among the Panel members that the label give 38 sufficiently detailed and accurate informati on to support the manufacturer’s label claim 39 for reduction in prion infectivity and to enab le the product user to assess how efficiently, 40 and under what usage, the prion pestic ide product reduces prion infectivity. 41

42 5. Efficacy Guidance Hierarchy Issue: Do different prion types exhibit variation in the 43 degree of resistance to inac tivation by pesticide chemical s and can a hierarchy of 44 resistance by prion ty pe be reliably determined at this time? 45

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6 There is clear scientific basi s for distinguishing various pr ion strains based on distinct 1 incubation time and lesion profiles in a give n host as well as biophysical and chemical 2 properties. However, there are limited data on the relative resistance of prions from 3 various species and strains to chemical inac tivation. Furthermore, a simplified discussion 4 of chemical reduction of prions infectivity is complicated because different prion 5 pesticide products may act by differing mechan isms causing reduction of infectivity in 6 different and unpredictable ways. With the unde rstanding that there ma y be variability in 7 assessment of reduction in infectivity based on the prion strain and animal bioassay used 8 by registrants, the Panel considered the advi sability of using one animal bioassay or a 9 combination of two animal bioassays to assess prion pesticide efficacy. For the 10 combined assay approach, one of these animal bioassays could be specific to the samples 11 proposed for specific use of the prion pesticid e, whereas the other could be a standard, 12 widely-used model, e.g. 263K scrapie prion in hamsters. The majority of the Panel 13 members favored using only one animal bioassa y in an end-point titration format. The 14 single animal bioassay needs to be robust and specific to the specified decontamination 15 objectives. 16 In conclusion, the consensus from the Panel is that no single hierarchy of strain resistance 17 to prion pesticide action can be established at this time. 18

19 EPA’s Responses to the SAP’s Comments 20

21 EPA’s responses to the SAP’s comments (both in the Executive Summary and in 22 the body of its meeting summary) concerning Charge Questions #1-5 are found after the 23 “Panel Response” for each Issue and Charge Question below. 24

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Issue 1. Whether EPA’s draft review paper, “S cientific Information Concerning the 28

Issue of Whether Prions Are a ‘Pest’ unde r the Federal Insecticide, Fungicide, and 29 Rodenticide Act (FIFRA),” adequately identi fies and summarizes available, relevant 30

scientific studies. 31

32 Charge Question 1. Please comment on the accuracy of the characterization of the 33 nature of prions, and the adequacy of the revi ew of the relevant scientific information to 34 support that characterization, as presented in EPA’s draft pa per, “Scientific Information 35 Concerning the Issue of Whether Prions Ar e a ‘Pest’ under the Fe deral Insecticide, 36 Fungicide, and Rodenticide Act (FIFRA).” 37

38 Panel Response 39

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The Panel discussed the nature of prions described in the in teragency White paper for the 41 accuracy of its scientif ic characterization of prions a nd adequacy of the scientific 42 information cited. The White paper is divide d into three Parts with Part I concerning 43 EPA’s reasoning for reviewing the prion litera ture with respect to declaring prions as 44 ”Pests” under FIFRA, Part II 45EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

7 summarizing the prion literature with respec t to description of TSEs and prions, key 1 characteristics of prions, the prion hypot hesis and normal pri on protein, neurotoxic 2 mechanisms in TSEs, TSE diseases, history of TSE diseases, transmission and routes of 3 exposure for TSEs, occupational safety pro cedures and decontamination methods, and 4 Part III examining the science of prions in relation to the FIFRA definition of “Pests”. 5 The authors of the White paper have done a tremendous amount of work assembling this 6 information and are commended for summarizing a great deal of prion literature. In 7 general, the Panel found the White paper to be comprehensive and most of its content to 8 be accurate. As a review of available scientific literature on TSEs and prions, the White 9 paper was accepted as adequate by the major ity of the Panel, pending incorporation of 10 suggested revisions presented herein. It was noted by seve ral Panel members that there 11 are many statements in the White paper needi ng revision to accurately reflect what is 12 known and what is reasonably speculated a bout prions and TSEs. One Panel member 13 found the White paper to be strongly biased fo r the prion hypothesis re lative to the viral 14 hypothesis and lacking in relevant information. 15

16 The Panel questioned EPA representa tives at the Meeting about Part I. Introduction 17 and Background to better understand the EPA’s intent for the Panel’s re view process. 18

There are some inconsistencies in the material presented in this Part compared to Part III 19 of the White paper. One Panel member c oncluded that Part I read like there is a 20 presumption that prions will be recognized as “pests” with the assertion that prions share 21 key characteristics with other microorganisms , but in Part III the authors of the White 22 paper did not appear to be entirely convinced that their “pest” ar gument would withstand 23 scientific scrutiny. This member thought th at Part I might be more correctly titled 24 “Prions and Consequences for Public Health.” 25

26 For the peer review of White paper Parts II. The Science of Prions and III. The 27

Science of Prions as It Relates to the FIFRA Definition of “Pests” the Panel’s 28 comments are provided as general comments immediately below and specific comments 29 in the subsequent section. 30

31

General Comments 32

For Part II. The Science of Prions , one Panel member commented that in multiple areas 33 of the White paper too much credence is gi ven to the Legname paper as proof of the 34 prion hypothesis (Legname, Baskakov et al. 2004). First, the synthetic fibrils accelerated 35 neurodegenerative disease only in transgenic animals that overexpress (by 16-fold) the 36 mutated prion transgene (PrP90- 231), and not in wild type mice; the transgenic mice that 37 were used become spontaneously ill even wit hout any inoculation whatsoever, albeit at a 38 slower rate. Moreover, this paper and the subseque nt literature lacks the negative control 39 most critical to establishing that the synthe tic amyloid contained pr ions, i.e., proof that 40 prions are not spontaneously accumulating in mock-inoculated control transgenic mice. 41 Thus, it remains unclear whether the synthetic amyloid initiated, or merely accelerated, a 42 spontaneous, ongoing pathogenic process that was due to massive overexpression of the 43 transgene. Infectious agents, i.e. prions, shoul d be initiators rather than mere accelerants 44 of disease. A much more important study of the primacy of pri on conversion in prion 45 propagation that is not appropr iately emphasized in the White paper is the Deleault study 46EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

8 (Deleault, Harris et al. 2007) in which prions were amplified by PMCA containing 1 normal prion protein (purified from brai n) and polyanions, a study which strongly 2 indicates the importance of non-prion polyanions , i.e., RNA or heparan sulfates, in prion 3 replication. 4

5 A. Description is a good review of the “protein only” hypothesis. Although the “virus” 6 hypothesis is mentioned, one Panel member thought that this needs to be discussed in 7 more detail. Also, there was general agr eement with the hypothesis that cofactors 8 (polyanion/sugar/nucleic acids) pl ay a critical role in the pr ocess and also need to be 9 described. A Panel member suggested that Griffith (Griffith 1967) , not Alper (Alper, 10 Cramp et al. 1967) or Prusiner (Prusiner 1982) , should be more prominently portrayed as 11 the originator of the protein only hypothesis and that this section should be based on a 12 more accurate history. 13

14 B. Key Characteristics 15

16 1. Prion Protein and Terminology 17 One Panel member suggested that including a brief summary of yeast prions could be 18 beneficial to emphasize the more general scope of the prion phenomenon, and another 19 thought that the definition of prion must emphasize infectivity rather than the mere 20 misfolding of prion protein; many misfolded forms of prion protein are not (infectious) 21 prions. 22

23

2. Current Status of “Protein Only” Hypothesis 24 One Panel member commented that the “prote in only” hypothesis for prions, although it 25 does provide a logical explanation for many of the characteristics of this agent, does not 26 fit exactly with “infectivity” when the contex t of the discussion centers on the generation 27 of increasing amounts of the abnormal isoform. The “recruitment and conversion” 28 explanation appears to be a mo re logical description of the process. Another Panelist 29 disagreed that the prion hypot hesis is a logical or ade quate explanation of TSE 30 infectivity, but rather is circular reasoning. 31

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3. Normal Prion Protein 33

One Panel member commented that the disc ussion of the expre ssion (homozygosity vs. 34 heterozygosity) at codon 129 of th e prion protein is an importa nt topic. This could be 35 expanded with regards to vCJD, where there is good epidemiologic information from the 36 United Kingdom on apparent susceptibility or resistance and its impact on the incidence 37 of the disease in that populati on. It should also be expanded to scrapie, where it is well 38 known that certain genotypes of sheep are mo re resistant or susceptible to developing 39 scrapie. Another Panel member indicated that the physiologica l role of the prion protein 40 is not discussed sufficiently (synaptic tran smission, T cell activation, circadian rhythm, 41 olfactory physiology). 42

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4. Neurotoxic Mechanism 44 Although this topic is interesting, one Panel me mber agreed with the White paper authors 45 that we need to learn more about the pa thology of TSEs. Despite some in depth 46EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

9 knowledge about important aspects of the neurot oxic mechanism, we have an incomplete 1 understanding of the process as a whole. Also in this section, the au thors exclusively talk 2 about degradation of prions in “proteas omes”, but do not mention “lysosomes” as 3 important intracellular sites of prion degradation. In addi tion, it was noted that prion 4 amyloid cannot be considered responsible fo r neurotoxicity as there is no correlation 5 between prion amyloid plaques and neuronal death, and furthermore, one Panel member 6 noted that the requirement for endogenous norma l prion protein for toxicity has not been 7 proven, as is implied in the White paper. 8

9

C. TSE Diseases 10

11 The Panel generally agreed that this is a good discussion and review of the known TSE 12 diseases in animals and humans. However, one Panel member noted that the statement 13 that most prion diseases in humans are “ac quired” needs to be rephrased. When one 14 considers the epidemiology of CJD, the overwhelming majority of cases are identified as 15 sporadic, with the next largest category being familial or inherited CJD. Rather, the 16 statement can be clarified to say that cer tain TSEs (kuru and vCJD) appear to be 17 “acquired” as a result of “exposur e” via ingestion of the prions. There also needs to be 18 some discussion about the period between expos ure and the onset of symptoms in this 19

section of the document. 20

21 D. History of TSE Disease 22

23 This section of the review presents a reasona ble summary of the history of TSE diseases. 24

25 E. Transmission and the Potential Routes of Exposure 26

27 One Panel member felt that an importan t point missing from the discussion of 28 transmission is the differing lengths of tim e between “exposure” and onset of symptoms 29 as related to the portal of entry. Direct inoc ulation of prion-contam inated material into 30 the central nervous system (CNS) results in a relatively shortened time interval, whereas 31 there is a considerably longer interval between exposure and onset when the prions enter 32 at a site considered as “peripheral.” 33

34 F. Occupational Safety Procedures 35

36 One Panel member thought that this section ne eds to be discussed in more depth. This 37 section also provides a good opportunity to di scuss the “Chain of Infection,” a concept 38 that explains why infectious diseases occur “when and where” they do. For example, just 39 because there is infectivity pot entially present in some source at some time, this does not 40 automatically mean that transmission will occu r and infection will ensue. There is very 41 little evidence to support the notion that prions can be acquired from environmental 42 surfaces. In spite of this, it is prudent to keep work areas and surfaces clean and 43 decontaminated, especially if one is wo rking with known infectious materials. 44

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10 With regard to the six cases associated with neurosurgical instruments and devices, there 1 is only strong evidence of prion transmission in those cases associated with the use of 2 stereotactic depth electrodes. In the opinion of one Panel member, the four cases 3 presumably associated with neurosurgical in struments are not very well described, and 4 there are very few details on the methods used to clean and reprocess the instruments. It 5 is also important to note that the last of the instrument or device cases was reported in 6

1976. 7

8

G. Decontamination Methods 9

10 One Panel member noted that the history of development of decontamination methods is 11 interesting and provides a logical explanation of why experiments were designed as they 12 were. Veterinary scientists attempted to deactivate prion infectivity while retaining 13 immunogenicity such that thes e “disinfected” prion prepar ations could be used to 14 immunize sheep against scrapie. Clearly, the objective was very different than modern 15 day objectives of eliminating prion bioma ss by decontamination. This Panel member 16

indicated that there is increas ing interest in exploring pri on decontamination methods that 17 more closely resemble reprocessing methods currently used in modern hospitals and 18 laboratories for which the objective of ster ilization is to remo ve patient derived 19 biomaterials and bioburdens as completely as possible, making medical instruments safe 20 for use with subsequent patient s. New technologies are bein g evaluated in this regard, 21

and some of these should be discussed here. An additional complicat ing factor is that 22 strain diversity in natural populations and its impact on decontamination is not known. 23

24 It was noted that the susceptibility of humans to infection with CWD prions is not known. 25 Currently, there is no epidemiological ev idence to suggest th at CWD has been 26 transmitted to humans. Experimentally, CW D has not been successfully transmitted to 27 transgenic mice expressing human prion protei n. Human prion protei n can be converted 28 to a proteinase K resistant form in vitro by CWD prions; however, this conversion is 29 much less efficient compared to species with known susceptibility to CWD. Finally, 30 non-human primates are susceptible to intrace rebral CWD infection. Recent evidence 31 suggests that more than one strain of CWD may exist, complicating the interpretation of 32 previous work involving human susceptibility to CWD infection. 33

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The Panel discussed Part III. Science of Prions as It Re lates to the FIFRA Definition 35

of a “Pest” extensively and had a divided opini on about whether the scientific 36 understanding of the nature of pr ions supports its classification as a “Pest” as specified in 37 FIFRA Sections 2(t) or 25(c)(1). The major opinion was that there is sufficient scientific 38 evidence to support this clas sification, whereas a minority of Panel members did not 39 support this view. 40

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A. How Do Prions Relate to Common Definitions of “Microorganisms”? 42

43 The Panel was in general agreement that pri ons are pathogenic and infectious, may have 44 some similarity to viruses, are present in the environmen t resulting in human exposures, 45EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

11 might have a non-host encoded component, i nduce innate immune responses and are 1 transmissible infectious agents. 2

3 A minority of Panel members concluded that prions do not possess features typical of 4 microorganisms. 5

6 One Panel member agreed that prions are de leterious to animal and human health, but 7 disagreed that prions can be classified as “o ther microorganisms.” In order to do this, the 8 White paper authors would need to emphasi ze the “unknown virus” hypothesis. This 9 Panel member stated that prions do not re produce and they do not grow. The normal 10 prion protein exhibits turnover as a normal function, but prions accumulate. Are prions 11 alive? This panel member thought the answer was “no.” 12

13 Another Panel member suggested that whether scientific studies s upport classification of 14 prions as pests depends on the in terpretation of what “pests” ar e. In particular the second 15 part of the definition of “pests” as “any ot her form of ... virus, bacteria or other 16 microorganism....” is problematic because prions lack the characteri stics of viruses or 17 bacteria and the meaning of “other microorga nisms” implies a living microbe, which this 18 discussant believed was not scientifically supported. Prions are primary proteinaceous 19

substances and have some characteristics in common with self-assembling structural 20 components of bacteria such as the multimeric extracellular curli pr otein of the bacteria 21

Escherichia coli (Wang and Chapman 2008), but prions lack the overall multi-component 22 complexity and organization that are hallmarks of microorganisms. 23 Instead of being microorganisms, prions coul d be classified as “infectious substances” 24 under the definition of “biological agent” in Code of Federal Regulations Title 42 Public 25 Health Section 73.1 (42CFR). Biological agen ts are defined as “any microorganism or 26 infectious substance capable of causing deat h, disease, or other bi ological malfunction in 27 a human, an animal, a plant, or another living organism; of deleterious alterations of the 28 environment”. “Infectious substances” of Title 42 consist of microbial, plant and animals 29 toxins, and many of these, like prions, are en tirely proteinaceous substances (7CFR). 30 Protein toxins do many things that prions do. Protein toxi ns exhibit host and cell-type 31 specificity of intoxication, use host cellular machinery for their intoxication mechanisms, 32 and can resist endosomal protein degrad ation pathways (Watson and Spooner 2006). 33 Some protein toxins are stab le in the environment and can enter the host via ingestion 34 (Wobeser, Baptiste et al. 1997). Protein toxi ns recruit normal host ce ll proteins to adopt 35 different conformations in a way that cause s injury to cells a nd organs (Fabbri, 36 Travaglione et al. 2008). I ngestion of protein toxin inf ected tissues can result in 37 transmission of infection to new susceptible hosts (Wobeser, Baptiste et al. 1997). Prions 38

do not share extensive similarities to bacteria or viruses, but prions share many distinctive 39 features of protein toxins. It appears that prions might be best classified as “infectious 40 substances” rather than “other microorganis ms”. This Panel member concluded that 41 attempts to classify prions as “other mi croorganisms” as a means to gain regulatory 42 oversight is not supported scientifically. 43

44 A majority of Panel members supported the view that prions can be categorized as “other 45 microorganisms.” 46EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

12 1

One Panel member commented that when consid ering the issue of whether or not prions 2 fit the definition of a microorganism, the White paper concludes that prions do not 3 reproduce, adapt, grow or move. However, wh en viewed from the point of view of TSE 4 biology, these conclusions are inaccurate. Rese arch spanning the last 50 years leaves no 5 doubt that the agents which cause prion dis ease can reproduce, adapt, grow and move 6

within the host. 7

8

Another Panel member commented that re plication of prions has been amply 9 demonstrated by the fact that they can be ti tered and that animals infected with a low 10 level of prion infectiv ity eventually accumulate exponentia lly higher levels of infectivity. 11 This can only occur if prions can replicate th emselves. As demonstrated by the isolation 12

and careful characterization of multiple mouse and hamster-adapted sheep scrapie strains, 13 prions can adapt to new species. Peripheral inoculation of TSE in fectivity eventually 14 leads to infectivity in the brain, something that can only occur if the infectious agent 15 moves within the host. Finally, prions most de finitely exhibit a stable steady state over a 16 long period of time (maintenance). Establ ished stocks of TSE infectivity can be 17 maintained for decades with no change in either their titer or their biological 18

characteristics. 19

20 A third Panel member said that defining bact eria and viruses as microorganisms does not 21 depend upon the very different mechanisms they use to replicate, spread, and adapt. 22 Likewise, defining TSE agents as mi croorganisms should not depend upon the 23 mechanisms underlying their replication a nd spread. Rather, it should be based upon 24 whether or not their general biological char acteristics are consistent with that of a 25 microorganism. It is only in trying to r econcile the proposed pr otein-only mechanisms 26 underlying TSE pathogenesis with the uni versally accepted nucleic acid based 27

mechanisms of more conventional infectio us organisms that defining prions as 28 microorganisms becomes more complex. 29

30 Another Panel member suggested re-writing Part III to better reflect th e characteristics of 31 prions similarity to other microorganisms, such as viruses. Prions gr ow, replicate, adapt, 32 maintain, move and are organized. The discussion should be centered around Koch’s 33 postulates, instead of a dictionary definition of microorganism or life. 34

35 Another Panel member stated that TSE agents , i.e., prions, are environmental pathogens 36 that infect mammals, replicate virus-like, cau se disease according to Koch’s postulates, 37 though the infectious agent has no t been sufficiently purified. 38

39 One Panel member stated that the virus hypothe sis of TSE infectivity is in accord with a 40 classification of the TSE agent as a microorga nism. It was suggested that Part III needs 41 to discuss Koch’s postulates as they relate to prions. Important considerations for FIFRA 42 being: 43

• Infectivity: Kuru epidemic stopped with end of ritual cannibalism, United 44 Kingdom BSE epidemic markedly decr eased with removal of external 45 source, endemic scrapie, & environmental CWD 46EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

13 • TSE agents as environmental pathog ens, not spontaneous generation of 1 infectivity 2

• Strains of TSE agents as: 3

o Encode individual properties: obvio us in controlled host genotype 4 background 5

o Classified by incubation time and by neuropathology (lesion 6 profile) 7

o Agents often maintain strain identity even after passage in different 8 species, e.g., sCJD to mice 9

o Most individual agents not se parated by abnormal prion band 10 profiles on blots 11

12

B. Are Prions Alive? 13

14

One Panel member concluded that instead of focusing so much on the vague question of 15 whether prions are living microorganisms, it seems that the prions as pests discussion 16 might be more fruitfully oriented towards th e discrimination between prions as pathogens 17 (which propagate in the host) or toxins (which don’t propagate). 18

19 In conclusion, a majority of the Panel agreed that once revised to incorporate revisions 20 suggested by the Panel the White paper wi ll adequately identify and summarize the 21

scientific literature concer ning prions and that prions are “other microorganisms” 22 exhibiting some features of living entities. 23

24 Specific Comments 25 With respect to specific comments and editorial remarks on the White paper, the Panel 26 noted the following specifics: 27

1. Please add Castilla et al., 2005; Delault et al., 2007 and Sigurdson et al., 2009 to 28 the literature list in section II, A: 29 Castilla, J., P. Saa, C. Hetz, and C. So to. 2005. In vitro generation of infectious 30 scrapie prions. Cell 121:195-206. 31 Deleault, N. R., B. T. Harris, J. R. R ees, and S. Supattapone. 2007. Formation of 32 native prions from minimal components in vitro. Proceedings of the National 33 Academy of Sciences of the Unite d States of America 104:9741-6. 34 Sigurdson, C. J., K. P. Nilsson, S. Horn emann, M. Heikenwalder, G. Manco, P. 35 Schwarz, D. Ott, T. Rulicke, P. P. Liberski, C. Julius, J. Falsig, L. Stitz, K. 36 Wuthrich, and A. Aguzzi. 2009. De novo generation of a transmissible 37 spongiform encephalopathy by mouse transg enesis. Proc Natl Acad Sci USA 38

106:304-9. 39

2. Page 2, 1 st paragraph: 40

a. Delete “Alper et al., 1966, 1967” when referring to the prion-only hypothesis 41

b. “bovine spongiform encephalitis” should be “bovine spongiform 42 encephalopathy. 43

3. Page 2, 2 nd paragraph: 44

a. The “by-passing of the proteasome” does NOT produce TSE clinical features. 45 Please rephrase. 46EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

14 b. TSE clinical features depend on “species AND TSE strain used for infection.” 1 Please modify. 2

c. The statement that “the property of inf ectivity appears to reside in the beta 3 sheet-rich conformation” is a great ov ersimplification. In fact many beta4 sheet-rich forms of prion have been generated that have no infectivity. 5

4. Page 3, 1 st paragraph: 6

a. The use of the word transcript in the sentence “misfolded transcripts recruit 7 and convert normal prion transcripts” is incorrect. Prion protein would be 8

more correct. 9

b. “low level of recruitment and c onversion...” This is incorrect. 10

c. It is not the “recru it and convert” mechanism that separates prions from other 11 protein misfolding diseases ; it is their relative transmissibility between 12

individuals. 13

d. Plaques are not linked to cell death 14

5. Page 3, 2 nd paragraph: 15

a. Replace plaque-forming isoform” by “aggregation-prone isoform” 16

b. Correct spelling of “plaque” in line 8 17 c. Take out sentence on Legname et al., 2005 18

6. Page 3, 3 rd paragraph: There are still cases of bovine spongiform encephalopathy 19 (BSE) in the UK. Wrong statement, th at BSE “appears to have effectively 20

ended.” 21

7. Page 3, last paragraph: The apparent molecular wei ght of prion-associated 22 fragments can range from ~17,000 to 37,000. 23

8. Page 4, first paragraph: 24

a. The term PrP d is prion that is present in diseas ed animal and is not detected in 25 uninfected animals using immunohistoche mistry or immunoelectron microscopy. 26

b. PrP-res has been used to connote a “purified protein or recombinant 27 polypeptide fragment” resistant to PK. This is incorrect. 28 c. It needs to be stated that “pri on is infectious, not only “misfolded.” 29

e. Replace “disease isoform” by “agent strain”; 30

f. Replace “infectious conformation of transcripts” by “misfolded 31 PrP” and also “prion” should refer to “infectious particle” whether it is 32 constituted solely of prion or not 33

9. Page 4, 2 nd paragraph: 34 a. Reference “Colby et al, 2007” is wrong 35 b. Wrong statement regarding Legname manuscript: Fibrils consisting of 36 recMoPrP(89-230) were inoculated intrac erebrally into transgenic (Tg) mice 37 expressing MoPrP(89-231). 38 b. The explanation regarding the doma ins of interactions between PrP C and PrP Sc 39 is very narrowly focused. Other studies, such as those by Horiuchi et al., 2001 40 and Chabry et al., 1998 on the localized interactions between the two prion 41

isoforms should be included. 42

10. Page 4, 4 th paragraph: 43 a. The statement "The Prnp gene appears to be conserved across species from 44 yeast to primates" is incorrect. Prnp is conserved among mammals and also to 45 amphibians and reptiles. More distantly rela ted proteins are present in fish. Yeast 46EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

15 and fungal prions are unrelated to mammalia n prions on a sequence level, but are 1 so named because they share the charact eristic of being able to adopt two 2 different conformations, one of which leads to aggregation. 3 b. The word “homology” is used incorrec tly. This should be replaced with 4 “sequence identity” or in the case with doppe l to indicate if normal prion protein 5 and doppel are homologous. 6

11. Page 5, 2 nd paragraph: There is just as much evidence that copper inhibits prion 7 protein conversion as there is th at copper stimulates conversion. 8

12. Page 5, 4 th paragraph: Prion function involve s olfactory physiology (Le Pichon et 9 al., 2009) etc. 10

13. Page 5, 5 th paragraph: 11

a. It is incorrect that prions kill neurons by disrupting the function of the normal 12 prion protein. 13

b. Delete “development of pr ion diseases requires the presence of normal PrP” 14

14. Page 5, last paragraph and page 6, first 4 paragraphs: 15 a. The literature interpretation in rela tion with the neurodegenerative mechanism 16 is incorrect. Plaques or immunohistochemically detectab le prion aggregates are 17 not neurotoxic. Several groups have shown that peptide models of prion (Haik et 18 al. 1998) and small prion oligomers are neurotoxic (Kazlaus kaite et al. 2005, 19 Novitskaia et al. 2006, Simoneau et al. 2007) in the presence or absence of prion 20 expression. Therefore the current view is that prion-related neurodegeneration 21 might have two components: a loss of f unction of prion protein, and a gain of 22 toxicity of prion oligomers (independ ent of neuronal prion expression). 23 b. In the three references cited (Brandner, Mallucci and Chesebro), the form of 24 prion that was detected was large, non- toxic aggregates and does not demonstrate 25 that the absence of prion protein expre ssion is responsible for the absence of 26 toxicity. In Brandner, the absence of prion expression by the mice prevented the 27 generation of toxic prion oligomers. In Mallucci, prion was la rgely sequestered in 28 astrocytes, and no reactive prion oligomers were genera ted, again because of the 29 absence of prion expression by the mice. In Chesebro, prion plaques are not 30 toxic, but again it has noth ing to do with the absence of cell surface prion protein 31 expression. It is suggested to remove these three references and cite the two 32 currently accepted components of neur odegenerative mechanism, with the 33 appropriate references. 34

15. Page 6, 4 th paragraph: “GTI” should be changed to “GPI”. It should also be noted 35 that these mice have approximately 1 log lo wer brain titers and that the disease is 36 altered not prevented. 37

16. Page 7, Table 1: The table should be di vided into “infectious’, “sporadic”, and 38 “inherited” prion diseases. Also, the di sease is called “fatal familial insomnia, 39 FFI”, Chronic wasting disease of “deer, elk and moose.” 40

17. Page 8, 1 st paragraph: 41

a. Please mention also transmissibl e mink encephalopathy (TME) outbreaks. 42

b. Please add “France” to the other contamin ated European countries. In France, 43 13 cases of BSE were already diagnosed in 1995. 44

c. Canada and USA had their fi rst indigenous cases in 2003 and 2004, 45 respectively. 46EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

16 d. Canada not only has importe d but also indigenous cas es of BSE. Should be 1

deleted from last sentence. 2

18. Page 8, 2 nd paragraph: The second US BSE animal was from Texas. 3

19. Page 9, 3 rd paragraph: 4

a. CWD was found in moose (Baeten et al., 2007). 5

b. Epidemiology/Prevalence of cervids in captivity can be rather high (up to 6 90%). 7

20. Page 10, 1 st paragraph: add “intrali ngual, intranasal and intrasciated” to the route 8 of applications. 9

21. Page 10, 2 nd paragraph: 10 a. Consumption of contaminated food is generally not considered to be involved 11 in transmission of scrapie. Mention that both vertical and hor izontal routes are 12 likely natural scrapie transmission. 13 b. “prionuria”: remove Ligios et al., 2007

─ add Gabizon and Gregori literature. 14

22. Page 10, 4 th paragraph: Maternal transmi ssion of BSE and CWD is rather 15 questionable. 16

23. Page11, 1 st paragraph: 17

a. The interpretation of the study by Heaton et al, 2008 is inco rrect. The study 18 concludes that the prevalence for the E211K variant is extremely low (less 19 than 1:2000 cattle) and, therefore, additio nal atypical BSE cases with K211 in 20 the US will be very low. 21

b. Citation “Chesebro, 2003” is incorrect. The Chesebro review actually states 22

that it has been suggested that the feeding of scrapi e or BSE-infected tissue to 23

mink caused BSE. It was not a statement of fact. 24

24. Page 11, 3 rd paragraph: So far, 4 cases of vCJD blood transfusion have been 25 reported. 26

25. Page 12, 4 th paragraph: The citation for the Guideline for Handwashing and 27 Hospital Environmental Control is incorrect . This guideline ha s different authors 28

and was released in 1985. 29

26. Page 14, 2nd paragraph: There is no ev idence to support the statement "prions 30 bind tightly to stainless steel and are most difficult to inactivate on this surface." 31 Prions do indeed appear to bind tightly to stainless steel and are difficult to 32 remove, but there are very limited comp arative studies of other surfaces. 33

27. Page 14, last paragraph- to page 15: In the context of decontamination a 34 limitation can be the intrinsic limited sensitivity of the bioassay model, rather than 35 a problem of the lifespan of the animals. 36

28. Page 15, 3 rd paragraph: BSE prions “readily ” transmit to felines, but not to 37 humans. So far, 210 people out of the millions that were presumably exposed to 38 BSE have gotten vCJD. 39

29. Page 17, 1 st paragraph: “solely of prot eins” needs to be modified. 40

30. Page 18, 2 nd paragraph: correct spelling of reference from “Leninger” to 41 Lehninger. 42

31. Throughout manuscript the authors use regul arly “mad cow disease”; they should 43 use “Bovine Spongiform Encephalopathy, BSE” instead. 44

32. It is not clear if 263K or 301V reca pitulate the scrapie and BSE agent, 45 respectively. 46EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

17 33. In general, the word “plaque” is not used correctly. The generalization of TSEs as 1 diseases where neuronal plaques are made is not precisely correct. In fact, 2 plaques are not the primary phenotype in the majority of cases of the most 3 common human TSE, sporadic CJD. It would be more accurate to define TSE 4 diseases as diseases of protein misf olding where, depending upon the type of 5 TSE, prion can be found in either diffus e “non-amyloid deposits” or in “amyloid 6 plaques.” 7

8 EPA Response to the SAP’s Response to Charge Question #1 9

10 Charge question #1 related to the EPA’s White Paper’s review and summary of 11 the available, relevant scientific literature. A majority of the Panel accepted the EPA’s 12 White Paper as adequate. However, the Pa nel cautioned that many statements in the 13 White Paper need to be revised to reflect accurately what is definitively known about 14 prions and the TSEs and that which is reasonably hypothesized based on scientific 15

observations. In addition to several General Co mments related to individual sections of 16 the White Paper, the SAP provided 33 Specifi c Comments, which in some cases reflected 17 the opinions of individual panel members rather than a majority or a consensus of the full 18 Panel. The specific comments identified mi scellaneous editing mistakes in the White 19 Paper, offered guidance on subjects on whic h the White Paper’s discussion could be 20 further elaborated, identified issues that so me investigators believe remain unresolved, 21 and offered ways to capture a full spectrum of different scientific points of view. 22

23 In various overarching comments, th e SAP’s key conclusions were that: 24

25

1. A majority of the Panel accepted the pri on (or “protein only”) hypothesis with the 26 caveat that unknown cofactors may also be involved. EPA has made several 27 revisions in the White Paper that acknowledge this alternative co factor hypothesis. 28

29

2. A majority of the Panel conc luded that prions could be included in the category of 30 “other microorganism” under FIFRA. EPA ha s made revisions in the White Paper to 31 acknowledge the SAP’s point of view on this issue. 32

33 General SAP Comments: Suggestions for Revi sion: White Paper Part I. Introduction and 34 Background. 35

36 SAP page 16, 2 nd paragraph . One panel member thought that Part I of the White Paper 37 might be more correctly titled “ Prions and Consequences for Public Health .” 38

39

EPA believes that the Part I title—“Introduction and Bac kground”—is appropriate and, 40 therefore, will remain unchanged. 41

42 This same panel member also suggested that Part I presumes that prions will be 43 recognized as “pests,” but noted that in Part III the White Pa per does not appear 44 “convinced that the ‘pest’ argument woul d withstand scientif ic scrutiny.” 45

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18 EPA’s response is that Part I summari zed EPA’s rationale for its September 2003 1 decision that a prion should be considered a “pest” under FIFRA and presumes only that 2 past policy decisions will remain in effect until action is taken to change them. Part III of 3 the White Paper addressed the available scient ific information on prions (summarized in 4 Section II) in regard to the terms “microorga nism”, “organism” and “living” as those 5 terms are used by biologists and the medical community. While Part I remains largely 6 unchanged, Part III has been changed to incl ude most of the SAP’s comments on this 7

issue. 8

9 General SAP Comment: White Paper Part II. The Science of Prions. 10

11 SAP Page 17. One panel member commented that too much credence was given to the 12 Legname et al. (2004) paper as proof of the pr ion hypothesis. Specifica lly, the paper left 13 unclear whether the synthetic amyloid [pri on protein produced by recombinant DNA in a 14 cell free, bacterial system] initiates or merely accelerat es a spontaneous pathogenic 15 process in transgenic mice. Furthermore, th e panel member did not believe that the study 16 of Deleault et al. (2007) wa s sufficiently emphasized. The Deleault et al. study was 17 thought by the panelist to pr ovide strong evidence of the importance of non-prion 18 polyanions in the cell free replication of prions. 19

20

EPA has evaluated individual instances where the reference Legname et. (2004) was used 21 in the White Paper. In particular, EPA ha s added the suggested De leault et al. (2007) 22

citation to section II.A ., and has modified the related text to deemphasize “purified” prion 23 protein in these experimental cel l free systems. In addition, the alternative point of view 24 that cofactors may be involved has also been added. 25

26 General SAP Comment: White Paper Part II. The Science of Prions. A. Description. 27

28 SAP page 18. The SAP found that the review of the “protein only” hypothesis is good. 29 One panel member thought that the virus hypothesis should be discussed and expanded to 30

include more detail. EPA has added this al ternative hypothesis to the White Paper. One 31 panel member noted that the prion only hypothesi s should not be attribut ed to Alper et al. 32 (1967). EPA agrees with the panelist, a nd has modified the text as suggested. 33

34 General SAP Comment: White Paper Part II. The Science of Prions. B. Key 35

Characteristics. Individual general comments are o ffered individually for each of the 36 four sections of II.B., as follows: 37

38 #1 Prion terminology: One panel member suggested that a review of yeast prions could 39 be beneficial. EPA also believes that signi ficant research is, in fact, underway using 40 yeast “prion” protein and recombinant models to resolve the mechanisms of misfolding, 41

such as described in recent papers by Lindquist (Tessier and Lindquist, 2007; Alberti et 42 al., 2009). Based on the panelist’s recommenda tion, references to the Lindquist papers 43 have been added to Section II.B.2 and Section III (in reference to non-Mendelian 44 inheritance of phenotypes by “prion ogenic proteins” in yeast. 45

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19 #2. Current Status of the “Protein Only” Hypothesis. 1

2 Two panel members offered slightly different comments that the protein-only hypothesis 3 does not fully account for the critical property of infectivity attribut ed to prions. Their 4 concerns were repeated in other secti ons of the SAP meeting summary, and these 5 comments appear to be at odds with both the SAP’s General support of the prion 6 hypothesis and especially of the Panel’s comment immediately above under Section 7 II.B.1. Prion Terminology. In response to th ese comments, EPA notes that a summary of 8 recent yeast work (eg, Tessier and Lindquist, 2007; Alberti et al., 2000) specifically 9 addresses the separate issues of misfoldi ng and infectivity, concludes that the prion 10 hypothesis accounts for both mechanisms of disease, and emphasizes a growing belief 11 that the prion phenomenon has a larger genera l scope than previously realized, as has 12 been recognized also in the case of “prionoi d” behavior in amyloid plaque diseases 13 (Aguzzi, 2009). EPA has added this information to Section II.A. 14

15

#3. Normal Prion Protein. 16

17 One panel member suggested expanding the discussion of genetic polymorphisms for 18 codon 129 in the human PRNP gene and extendi ng the discussion to include scrapie. 19 Another panel member suggested that the nor mal physiological role of prion protein is 20 not discussed sufficiently. EPA believes th at while both suggestions have merit, 21 appropriate revision of the White Paper woul d add considerable length without lending 22 weight to the decision of whether prions are “pests” or not. 23

24

#4. Neurotoxic Mechanism. 25

26 One panel member agreed with the White Pa per that resolution of the mechanisms of 27 neurotoxicity is incomplete. The panel memb er further noted that the White Paper failed 28 to mention the role of lysosomes and that there was no correlation between prion amyloid 29 plaques and neuronal cell death. Both sugge stions have merit; however, introduction of a 30 lysosomal hypothesis would add considerable length without lending weight to the 31 decision of whether prio ns are “pests.” 32

33 On the other hand, it is a sign ificant issue and EPA agrees that plaque formation and 34 neuronal cell death are not proven to be cau sally related in a ny of the “amyloid” 35 neurodegenerative diseases. To avoid a misi nterpretation that ne uronal cell death from 36 scrapie protein is caused by plaque formation, the mention of killing neurons has been 37 deleted altogether. The text has been modified to focus on hypotheses about “disrupting” 38 normal cell surface protein function as pa rt of the mechanism of disease. 39

40 Another panel member noted that the require ment for endogenous normal prion protein in 41 toxicity has not been proven as implied in the White Paper. In response, EPA points out 42 that the role of normal PrP in the progression of TSE disease is discussed in response to 43 Specific Comment #13, below. 44

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20 General SAP Comment: White Paper Part II. The Science of Prions. C. The TSE 1

Diseases. 2

3 One panel member suggested that the term “acquired” needs to be rephrased and 4 suggested that the relationship between ex posure and onset of clinical symptoms 5

discussed. EPA has modified the text as suggested regarding “spor adic” and “acquired” 6 incidents. The relationship between expos ure and onset of clinical symptoms is 7 discussed more fully below in response to section E. Transmission and Routes of 8 Exposure. 9

10 General SAP Comment: White Paper Part II. The Science of Prions. D. History of 11

Prion Disease. 12

13 EPA accepts the SAP’s description of th is section as a reasonable summary. 14

15 General SAP Comment: White Paper Part II. The Science of Prions. E. 16 Transmission and Potential Routes of Exposure. 17

18

One Panel member noted that discussion of preclinical incubation time between exposure 19 and onset of symptoms is missing from the di scussion. EPA agrees that the often 20 observed and strikingly “slow” progression of disease from exposure to clinical 21 symptoms is an important fact, and has noted it Section II.A. of the White Paper. EPA 22 agrees that the relationsh ip between exposure and onset of symptoms is profoundly 23 important to diagnosis and treatment of a ny TSE, but not necessarily to determining 24 whether a prion may be regarded as a “pest” , which is the primary focus of the White 25 Paper. EPA also notes that the relationshi p between titer, expos ure and progression of 26 disease is discussed in the companion document on efficacy guidance. . 27

28 General SAP Comment: White Paper Part II. The Science of Prions. F. 29 Occupational Safety Procedures. 30

31 One Panel member suggested that this sectio n needs to be discussed in more depth, 32 including the “chain of infec tion.” EPA believes that while this section could be 33 expanded, it probably would not make an a dditional contribution of determining if a 34 prion is a “pest.” 35

36

One Panel member offered a diffe rent opinion on the six cases in the literature of disease 37 associated with surgical instruments. EPA’ s response is that the White Paper only notes 38

various rationales under which the CDC has i ssued guidance and – in this instance – the 39 White Paper simply states there are six documented cases of CJD where an iatrogenic 40 cause associated with “neurosurgical inst ruments and devices” has been suspected. 41

42 General SAP Comment: White Paper Part II. The Science of Prions. G. 43

Decontamination Methods. 44

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21 One Panel member wanted EPA to add some background information on the history and 1 development of decontamination methods to the White Paper. EPA does not believe that 2 adding such information would be us eful or germane to the paper. 3

4

One Panel member noted that there is no known transmission of CWD to humans. EPA 5 notes that the White Paper alr eady states this (see discussi on of CWD, page 10, paragraph 6 1). 7

8 General SAP Comment: White Paper Part III. Th e Science of Prions as it Relates to 9

the FIFRA Definition of a “Pest.” 10

11 Documented by the comments of several indivi dual panelists, the majority of the SAP 12 supported the view that prions can be categor ized as “other microorganisms.” A minority 13 of the Panel concluded that prions do not posse ss the typical features of microorganisms. 14 For example, one Panel member suggested that prions might better fit the classification of 15

“infectious substances” under the definition of “biological agent” (42 CFR Part 73.1), 16 that prions share many distinctive features of protein toxins, and that prions do not share 17

extensive similarities to bacteria or viruses. EPA recognizes that there is a range of 18 opinions on the issue of whether prions ar e microorganisms, and has revised its White 19 Paper to include altern ative points of view. 20

21 At least one Panel member suggested that Part III.A. needs to discuss Koch’s postulates 22 as they relate to prions. EPA agrees that ap plying Koch’s postulates to the characteristics 23 of prions provides another approach to determ ining whether prions should be considered 24 to be microorganisms. Accordingly, EPA has revised Part III.A. of the White Paper to 25 include a description of Koch’s postulates and a discussion of how the characteristics of 26 prions relate to each of Koch’s postulates. 27

28 One Panel member concluded that part III. should discriminate between prions as 29 pathogens (which propagate in the host) or toxins (which don’t propagate). EPA has 30 added this point to part III.C. 31

32 Specific Comments: 33

34

1. SAP comment: Please add Castilla et al. (2005), Delault et al (2007) and Sigurdson et 35 al. (2009) to II.A. 36

37 Response: Castilla et al. (2005) is already cite d in II.A. Delault et al. (2007) has 38 been added to the pertinent citation, as sugge sted. Sigurdson et al. (2009) has not been 39 added, as this paper does not appear to be directly related to prion hypothesis. 40

41

2. SAP comment: Delete Alper reference when refe rring to the protein-only hypothesis. 42

43 Response: The comment is correct and the Alper reference has been deleted. 44

45

3. a. SAP comment: Please rephrase “by-passing the proteasome.” 46EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

22 1 Response: Text modified. 2

3

3. b. SAP comment: Please modify use of strain and species. 4

5 Response: Text modified. 6

7

3. c. SAP comment: Oversimplification. 8

9 Response: Text modified. 10

11

4. a. SAP comment: Transcript misuse. 12

13 Response: Correction accepted. 14

15

4.b. SAP comment: Usage of “low level of recruitmen t and conversion” is incorrect. 16

17 Response: What modification the comment inte nds is not clear. The recent paper 18 of Aguzzi (2009) is consistent with the White Paper and stat es the case more specifically: 19 “Like the growth of crystals, PrP Sc propagates by recruiting monomeric PrP C into its 20 aggregates . . . . ”. Furthermore, Aguzzi (2009) suggests that this process of recruitment 21 may be a more genearalized phenomena among the amyloid proteins gi ving rise to “hints 22 of prion-like behavior” descri bed as “prionoids.” Thus, Agu zzi (2009) has been added to 23 the White Paper in reference to a “low leve l of recruitment and conversion” in White 24 Paper II.A. 25

26

4. c. SAP comment: Infectivity is the discriminating principle that separates prion 27 diseases from other plaque-formi ng neurodegenerative diseases. 28

29 Response: The White Paper is already consis tent with this point, but EPA has 30 nonetheless modified the text to specify “infectious” conformations to clarify any 31 possible confusion. 32

33

4. d. SAP comment: Plaques are not linked to cell death. 34

35 Response: Plaques are a typical feature coll ectively of all the “amyloid plaque” 36 diseases, all of which also feature cell death: Alzheimer’s, Parkinson’s, etc. It is probably 37 true, also, that not all individual cases of TSEs feature definitive plaques. Hence, use of 38 the word “linked” seems appropriate as a general comment, as linkage does not imply 39

causation. 40

41

5. a. SAP comment : Replace text with aggregation-prone isoform. 42

43 Response: Modification accepted. 44

45

5. b. SAP comment : Correct spelling. 46EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

23 1 Response: Spelling corrected. 2

3

5. c. SAP comment : Delete sentence. 4

5 Response: Sentence deleted. 6

7

6. SAP comment : BSE has not ended. 8

9 Response: The intent was that the epidemic has effectively ended in cows, which 10

seems to be correct. Text has been modi fied to state that the potential for long, 11 unresolved dormancy of prion diseases in hum ans is a matter of great public concern, 12 with the risk that prion disease in humans resultant from the BSE epidemic, such as the 13 corresponding peak of vCJD in 2000, might persist in hu man populations long after the 14 consumption of contaminated food ended

. . 15

16

7. SAP comment : The molecular weight can range from 17,000 to 37,000. 17

18 Response: This comment seems to make a statement of fact based on the full 19 range of original and current literature. However, the comment is directed to an 20 introductory historical note. Specifically, the characterizatio n of molecular weight given 21 on page 3 [27,000 to 30,000 daltons] is the original identification of protein that purifies 22 with the scrapie prion, as specified by Bolton et al. (1982). Furthermore, this molecular 23 weight range is specified as “i n the original description.” 24

25 8. SAP comments 8.a. though 8.f. are derived from a single Panelist [see SAP General 26 Comments, p. 18]. 27

28

8. a. SAP comment : The terms PrP d and PrP res are defined 29

30 Response: The Panelist’s definition and the more general statement in the White 31 Paper appear to be consistent. Specifically, Brown (2005) defines PrP d as any misfolded 32

isoform that is “disease-associated”. For cl arity, this Brown citation has been inserted. 33

34

8. b. SAP comment : The comment states that usage of PrP res is incorrect. 35

36 Response: The Panelist may be incorrect. Brown (2005) defines PrP res as “a 37 proteolytic cleavage product of PrP d . . . when generated follo wing incomplete proteolytic 38 digestion in Western bl otting.” The White Paper states that “PrP res has been used to 39

connote a PrP d polypeptide fragment that features the resistance to proteinase K 40 digestion.” The text has been modified by adding attribution to Brown (2005). 41

42

8. c. SAP comment : Needs to be stated that prion is “infectious,” not only “misfolded.” 43

44 Response: paragraph begins with and repeats the premise of prion infectivity six 45 times in this one paragraph. Possibly, the Pa nelist’s statement refers to the distinction 46EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

24 between the word “prion” the phrase “prion protein,” as used in the White Paper. To 1 achieve consistency, the White Paper employs the word “prion” refers to a misfolded 2 isoform of any prion protein; “prion protein” can refer to both the normal and misfolded 3

isoforms. That the abnormal mi sfolded isoforms are also infectious is implied in the 4 context of the paragraph. The text has been modified for further clarity. 5

6

8. e. SAP comment : Replace “disease isoform” with “agent strain”. 7

8 Response: In his definitions, Br own (2005) made no mention of “agent strain.” 9 Rather, he speaks in terms of polypeptide “isoforms;” however, it seems pertinent that 10 Brown states that “nomenclature of the PrP sp ecies is confusing.” In Brown’s context, 11 protein chemistry would suggest th at his use of “species,” i.e., isoforms, is in reference to 12 structural “species” of biochemically homol ogous polypeptide molecules, not the disease 13

agent. 14

15

8. f. SAP comment : There are two suggested changes: 1) replace “infectious 16 conformation of transcripts” with “misfolded PrP” and 2) “prion” should refer to as “an 17 ‘infectious particle’ whether it is constituted of protein or not.” 18

19 Response: first comment makes the assumption that all misfolded PrP species are 20 infectious, which may not be correct a nd can never be known. Regarding the 2 nd 21 comment, “prion” is a word coined and defined by Prusiner (1982) and in many 22 additional papers by Prusiner a nd others. Prion is a “proteinace ous infectious particle.” 23 Specifically, Prusiner has never used the word prion to connote any “‘ infectious particle’ 24

whether it is constituted sole ly of prion or not,” as comme nted by the Panelist. The 25 White Paper conforms to Prusiner’s defin ition of the word “prion” and the convention 26 used for the word “prion” and the phrase “pri on protein” has been addressed above in 27 response to comment 8.c. 28

29

9. a. SAP comment : The reference to Colby et al. (2007) is wrong. 30

31 Response: The sentence is paraphrased dir ectly from Colby et al. (2007): 32 “Polymerization of recombinan t prion protein (recPrP), whic h was produced in bacteria, 33 into amyloid fibers was accompanied by acqui sition of prion infectivity.” The White 34 Paper states, “Amyloid polymerization of reco mbinant prion protein pr oduced in bacteria 35 has been shown to accompany the acquisition of prion infectivity (L egname et al. 2004; 36 Colby et al. 2007) that is char acteristic of the TSEs.” 37

38

9. b. SAP comment : The statement attributed to the Legname manuscript is wrong. 39

40 Response: The Panelist’s comment is correct, and the suggested sentence has 41 been added to the White Paper. 42

43 9. b. SAP comment: (2 nd 9. b.). Interactions between PrP C and PrP Sc are very narrowly 44 focused and could be expanded. 45

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25 Response: The intent of the White Paper is to account for the principle of 1 homologous interaction that would explain re cruitment and conversion, as described in a 2 recent publication (Lau et al., 2007). While it is necessary for the White Paper to 3 document the history of the pri on concept, in some detail, a comprehensive review of the 4

literature on each additional issu e raised in the White Paper is not feasible for its intended 5 purpose. Additionally, the examples provided by the SAP [Horiuchi et al., 2001; Chabry 6 et al., 1998] predate the more current Lau et al., 2007. The text has been modified to 7 clarify the context of Lau et al. (2007) without expanding the scope of the discussion. 8

9

10. a. SAP comment : The White Paper statement that Prnp gene appears to be conserved 10 from yeast to primates is incorrect. 11

12 Response: There are two responses to this comment. 13

14 In the first response, the Panelist’s comment that stating that Prnp gene “appears to be 15 conserved across species from yeast to pr imates” [White Paper] contradicts the 16 conclusions of Westaway and Pr usiner (1986), which is the citation and interpretation in 17 question. Westaway and Prusiner (1986) us e a DNA-hybridization adaptation of classic 18 methodology in comparative and evolutiona ry biology and reach a conventional 19 conclusion that PrPs of living species from yeast to mammals probably “may” 20 [Westaway and Prusiner] all arise by homol ogous descent with evolutionary modification 21 from authentic PrP ancestral genes. A specific cDNA was employed by Westaway and 22 Prusiner (1986) to show homologous binding with stringency across representative 23

mammals and - with declini ng stringency - hybridization across representative 24 invertebrates as well, such as nematode, Dros ophilia, yeast, with declining stringency in 25 that order. The lowest level of stringency was between mammal and yeast, which is what 26 evolutionary biology and stringe ncy of homologous binding w ould predict. Hence, the 27 hybridization data do evid ence that the “prion” genes from yeast to primates possibly do 28 [or may] share a common ancestral origin (homology) from ancestral DNA, described by 29 the authors as “authentic PrP genes.” 30

31 Furthermore, SAP comment 10.a. asserts that “yeast and fungal proteins are unrelated 32 to mammalian on a sequence level, but are so named because they shar e the characteristic 33 of being able to adopt two different conformations . . . .” This assertion is an 34 interpretation that discount s the opposite interp retation evidenced in the data of 35 Westaway and Prusiner (1986). Specificall y, Westaway and Prusiner (1986) show a 36 quantitative, deterministic relationship in which the stringency of hybridization is 37 inversely proportional to evolutionary distance, i.e., the larger the evolutionary distance 38 the lower the stringency of DN A hybridization. Furthermore, Westaway and Prusiner 39 (1986) did not measure and did not comment on conservation of “being able to adopt two 40 different conformations” [SAP comment 10.a.]. Additionally, the abi lity of “being able 41 to adopt different conformations” is not a reas onable application of evolutionary theory, 42 DNA hybridization, or amino acid sequence da ta. Evolutionary biology and protein 43 chemistry predict that the more distant the stem origin of compared sequences, the less 44 stringent is the sequence homology . Specifically, the structural conservation of genes 45 and proteins is based on se quence data and cannot be base d on the status of protein 46EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

26 conformations, as would be required in SA P comment 10.a. The status of protein 1 conformation is a phenotype, no t a genotypic concept. In co ntrast, the comparative DNA 2 hybridization data in Westaway and Prusiner (1986) is a genetic evolutionary concept 3

based on the core values of 4

5 1) phylogenetic relatedness [Nuttall, 1904, Blood immunity and blood relationships, 6 Cambridge Univ. Press] 7

8 2) and of descent with modification of conserved genes and their encoded amino acid 9 sequences (Wilson and Kaplan, 1964, Enzy me structure and its relation to 10 taxonomy, Taxonomic biochemistry and sero logy, Ronald Press; Zuckerkandl 11 and Pauling, 1965, Mutations, trends, and random processes in the evolution of 12 informational macromolecules, J. Mol. Bioil., 59:473-490). 13

14 The concept of a shared ability to cha nge conformations unrel ated to sequence 15 conservation, as suggested by commen t 10.a., would (if it were true) be analogy , not 16 homology and would not be an example of “cons ervation.” The scenario described in 17 comment 10.a. would be an example of e volutionary convergence, for which no 18 explanation is offered. Specifically, evolutionary convergence of structures (or of protein 19 conformations) is achieved by population selection pressures for a particular phenotype, 20 in which case the correct terminology is analogy, not conservation. 21

22 In the second response, the White Pa per has added notation that Susan Lindquist 23 (recent personal communication) believes he r most recent study of the yeast prions 24 (Alberti et al., 2009) is best interpreted as evidence of evolutionary convergence of 25 sequence similarities to conserve “phenotypic variation” and allows the yeast “prions” to 26

act as “non-Mendelian” elements of inheritance. In other words, sh e agrees with the SAP 27 comment that the yeast prnp ge ne is not an example of c onservation of homology across 28 phyla to the living vertebrates. This update to the literature on the yeast prions has been 29 added to the White Paper. 30

31

10. b. SAP comment : the word “homology” is used incorrectly – replace with “sequence 32 identity.” 33

34 Response: Conservation of sequence identi ty across phyla and evolutionary 35 distance is by definition e volutionary homology, except in the theoretical case of 36 potential evolutionary convergence of sequences, which is not the conventional 37 interpretation of the classic picture of des cent with modification evidenced in Westaway 38 and Prusiner (1986). In the case of yeast pr ions and other vertebra te proteins such as 39 Shadoo and Doppel, the real question is, at what point is sequence dissimilarity evidence 40 of a non-homologous, non-evolutionary rela tionship? In the case of possible 41 convergence of sequences in the yeast prions , as in the more recent interpretation of 42 Alberti et al. (2009, above), the broadly representative comparative data would not have a 43 quantitative deterministic relationship with the time scale of phylogeny, but rather would 44 have a stochastic relationship with th e time scale of phylogeny. The prion gene 45 comparative sequence data in Westaway and Prusiner (1986) does have a predictable 46EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

27 quantitative deterministic relationship with time scale, such that less stringency is 1 observed with increasing evolutionary distance. That is, within vert ebrates, a higher level 2 of stringency of binding is observed than th e stringency of binding between yeast and 3 vertebrates. In the case of amino acid sequen ce identity in the vert ebrate prion proteins, 4 based on the consistent trends in the limited sequ ence data obtained to date, in this and in 5 other papers, “homology” is the correct word in the White Paper, as applied to vertebrate 6 prion proteins. However, the comment rais es the speculative nature studies to date 7 stretching across larger phylogene tic distances from yeast to vertebrates, and the word 8 “homology” has been deleted with reference to yeast prions a nd with reference to the less 9 studied Shadoo and Doppel proteins. As s uggested by the SAP, in these instances, 10 “homology” has been replaced with “sequence similarity.” 11

12

11. SAP comment : There is just as much evidence that copper binding inhibits prion 13 protein conversion as there is that copper stimulates. 14

15 Response: No citation was offered and the co mment is not exclusive from what 16 the paragraph says. The White Paper cites Mc Kenzie et al. (1998) a nd only asserts that 17 copper binding status may be an importa nt cofactor in conversion of PrP C and the 18 stability of the PrP Sc infectious conformation. No a ssertion was made as to whether 19 copper stimulates or inhibits any particular conformati on. Brown (2005) also links 20 copper binding status to PrP status. 21

22

12. SAP comment : Prion function involves olfactory physiology.... 23

24 Response: A survey of the literature s hows numerous functions now being 25

attributed to normal PrP C in the brain. The White Paper is already principally focused on 26 abnormal PrP, not normal PrP. 27

28

13. a. SAP comment : It is incorrect that prions kill neurons by disruption of normal 29

PrP.... 30

31 Response: In the series of the SAP’s specific comments #13-14, the SAP 32 correctly notes that the mechanisms of prion neurotoxicity and infectivity remain 33 unresolved in detail and that for every prominent and widely acclaimed study published 34 on the problem, rigorous analysis shows there can be found in the literature seemingly 35 inconsistent results. The White Paper has b een modified to provide a larger overview of 36 the incomplete state of prion research as it is related to the specific mechanisms in 37 neurotoxicity and specific changes have been made as well to address these issues and 38 suggestions These changes are all consistent with the SAP’s general endorsement of the 39 prion hypothesis and with the other recurren t SAP view that much remains unknown in 40

detail. 41

42

The SAP’s assertion is attributed in th e White Paper to Brandner et al. (1996), 43 who conclude, “normal host prion protei n [is] necessary for scrapie-induced 44 neurotoxicity.” In response, an additional citation has been added to the text, Mallucci et 45 al. (2003), which states that “depleting neuronal PrP in pri on infection prevents disease 46EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

28 and reverses spongiosis.” Both of these pape rs both appear to suppo rt the concept that 1 normal host prion protein is necessary for neurotoxicity. 2

3

13. b. SAP comment : Delete “development of prion disease requires the presence of 4

normal PrP.” 5

6 Response: The sentence in the White Pape r is supported by Brandner et al. 7 (2005) in subsequent text. On the surface, the title of the Brandner et al. paper is 8 “Normal host protein necessary for scrapie-in duced disease.” Furthermore, Mallucci et 9 al. (2003) states that “depleting neuronal Pr P in prion infection prevents disease and 10 reverses spongiosis.” 11

12 14. General comments related to Specific Co mment #14 indicate the concerns are of one, 13 or only a small number, of the Panelists. 14

15

14. a. SAP comment : The current view is that pri on-related neurodegeneration might 16 have two components . . . 17

18 Response: The comment has merit. Accordi ngly, the assertion that scrapie prion 19 “kills” neurons has been deleted and replaced by “ . . . the pathology of scrapie prion in 20 neurons features disrupting the function . . . .” and a brief discussion and suggested 21 additional references concerning possible m echanisms for neurodegeneration have been 22 added to the white paper. 23

24

14. b. SAP comment : Some Panelists suggested deleti ng the Brandner et al., Mallucci et 25 al., and Chesebro et al., citations and acco mpanying text and cited the two currently 26 accepted components of neurodegeneration . . . 27

28 Response: The White Paper citations chos en (Brandner et al . 1996, Mallucci et 29 al. 2003, and Chesebro et al. 2005) were select ed because of their placement in such 30 prominent journals as Science and Nature. As in SAP comment 14a, the expansion of the 31 White Paper as the literature continues to e volve would not further support the intended 32 purpose of the White Paper. The only mo re recent study recommended by the SAP is 33 Simoneau (2007), which is already included in the discussion along with Piccardo et al. 34 (2007), which both point to the confusion in wh at intermediate steps might prove critical 35 in the neurotoxicity pathway of prions. Further expansion of the neurotoxicity 36 information would not significantly improve the White Paper’s utility for informing the 37 Agency’s decision as to whether pr ions should be considered pests. 38

39

15. SAP comment : The acronym is GPI, not GTI. It is noted the tite rs are altered and 40 that disease is altered, not prevented. 41

42 Response: The text has been modified. 43

44

16. SAP comment : Redesign Figure 1. 45

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29 Response: Figure 1 does have an internally inconsistent format although the 1 suggested fix (“infectious, spor adic, and inherited) also give s rise to problems. Fatal 2

familial insomnia has been corrected. Moose have been included. 3

4

17. a. SAP comment : Add a mention of mink TME outbreak. 5

6 Response: Page 8 is about human outbreak s of disease, including the acquisition 7

of vCJD as an extension of BSE. TME would not seem to fit in this context. A better 8 location to include TME is in sequence with cats (FSE), page 9-10; the text has been 9

modified. 10

11

17. b. SAP comment : Add France to other European countries. 12

13 Response: Additional information has been inserted. 14

15

17. c. SAP comment : Canada and USA also have cases. 16

17 Response: Canada and the USA have been added. 18

19

17. d. SAP comment : Delete Canada as a case of imported only. 20

21 Response: Text has been corrected. 22

23

18. SAP comment : Comment mentions additional information on USA’s 2 nd case. 24

25 Response: Texas has been added. 26

27

19. a. SAP comment : Comment mentions CWD in moose. 28

29 Response: Additional information has been added. 30

31

19. b. SAP comment : Comment mentions prevalence in captivity. 32

33 Response: Prevalence in captive animals is no t discussed in the text related to 34 any of the susceptible species. No expansio n of the text to include epidemiology of 35 species in captivity is needed. 36

37

20. SAP comment : Add other routes to the list. 38

39 Response: Suggested routes have been added. 40

41

21. a. SAP comment : Food is not likely; both vertical and horizontal are likely by the 42

natural route. 43

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30 Response: The text of the White Paper does not mention food as a route of 1 scrapie transmission. The paragr aph specifies in the lead sent ence that the di scussion is 2

in reference to natural transmission. 3

4

21. b. SAP comment : Remove reference to Ligios et al. and insert reference to Gabizon 5 and Gregori. 6

7 Response: No reason was given for the directiv e to delete Ligios et al.; however, 8 the text has been modified by adding one of the early papers on prionuria by Gabizon. 9

10

22. SAP comment : Maternal transmission of BSSE and CWD is questionable. 11

12 Response: The text was already consistent with the comment; however, further 13

clarification has been added. 14

15

23. a. SAP comment : Heaton et al. was misinterpreted . Additional “cases with K211 in 16 the US will be very low.” 17

18 Response: The text states “that BSE cases with the K211 allele that occurred in 19 Alabama would be unlikely.” Presumably, the comment takes issue with “unlikely” 20 [White Paper] versus “very low” [SAP comment]. No te: the background nomenclature 21 here is the Prnp gene allele E211K denot es a DNA mutation in which the normal amino 22 acid glutamate (E) at position 211 is replaced by lysine (K), resulting in BSE. The 23

authors did not find an other instance of E211K in a su rvey of 6062 cattl e, so that – 24 statistically – the upper limit for possible othe r occurrence is less than 1 in 2000 cattle. A 25 text modification has been suggested to captu re the nuances. The authors use the phrase 26 “vanishingly low” to describe the probability of another atypical BSE case in the U.S. 27

from the E211K mutati on; the panelist’s suggestion (le ss than 1:2000) could imply that 28 the frequency is much higher than it really is. 29

30

24. SAP comment : Currently, there are four known cas es of vCJD by blood transfusion. 31

32 Response: The text cites 2004 references, at which time there were only two 33 known cases. No citation is provided fo r the occurrence of four to date. 34

35

25. SAP comment : The citation for Guideline for hand washing . . . . is incorrect; it was a 36 1985 report. 37

38 Response: A correct citation is not provided although the White Paper uses two 39 other citations. The comment is probably referencing: Garner and Favero, 1985, 40 guideline for handwashing and hospital environm ental control, 1985. This original report 41 seems to be an internal report of the CD C, now available on the internet. The 42 corresponding actual pub lication seems to be Garner and Favero (1986), which has been 43 added the text. Also, Section F. of the White Paper does not have a corresponding 44 References list, and there is no citation to Boly ard in the References section. A section F. 45 has been added to the References with the single entry of Garner and Favero. 46EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

31 1

26. SAP comment : There is no evidence that prions “b ind tightly to stainless steel and 2

most difficult to inactivate on this surface.” 3

4 Response: Text has been modified. 5

6

27. SAP comment : There is a context issue about sensi tivity of bioassay versus lifespan. 7

8 Response: The comment does not seem to conflict with the White Paper text, 9 which assumes the bioassay test method is appr opriate to the goal. The same issues are 10 dealt with at length in th e Efficacy Guidance document. 11

12

28. SAP comment : The commenter takes issue with BSE “readily” transmits to humans. 13

14 Response: The Panelist seems to be volunteering that BSE “readily” transmits to 15 felines, but not to humans. The White Paper did not mention felines in this context; 16 however, the intent seems to deny that BSE readily transmits to humans” which are 17 separate events. In order to keep the discu ssion at a general level, the text has been 18 modified to be both more precise and still brief. 19

20

29. SAP comment : Modify “solely of protein.” 21

22 Response: The comment continues the theme that the TSE disease process might 23

involve other unknown cofactors besides the prion itself. While SAP presented no 24 evidence that the prion itsel f is anything other than prot ein encoded by a gene, EPA has 25 included the cofactor hypothesis as an alternative point of view. 26

27

30. SAP comment : Spelling error identified. 28

29 Response: Spelling error corrected. 30

31

31. SAP comment : White Paper should use BSE instead of “mad cow.” 32

33 Response: Except for instances where “mad cow” is appropriate to the context, 34 references to the disease have been replaced with BSE. 35

36

32. SAP comment : The commenter asserts that it is not clear if 263K or 301V 37 recapitulate scrapie and BSE. 38

39 Response: The comment was not di rected at any specific text, and the issue of 40

263K versus 301V in the mechanisms of TSEs is not discussed in the White Paper. 41

42

33. SAP comment : The White Paper tends to misuse the word “plaque.” 43

44 Response: The text has been modified in se veral places to capture the nuance that 45 not all TSEs produce “amyloid plaques.” 46EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

32 1

========================= ==================== ================== 2

3

Issue 2. Whether the specific test systems r ecommended in the draft guidance document 4 are scientifically appropriate to support the registration of pesticide products with prion5

related claims. 6

7 Charge Question 2. Please comment on the scientific appropriateness of: 8

9

a. Carrier-based, animal infectivity assa ys recommended by EPA’s guidance for 10 evaluating the efficacy of pesticide pr oducts used on environmental surfaces 11 (e.g., hard, nonporous surfaces). 12

13

b. Suspension-based, animal infectivity assays recommended by EPA’s guidance 14 for evaluating the efficacy of pesticid e products used in liquid media (e.g., 15 wastewater). 16

17

c. Any other known test methods for ev aluating the efficacy of pesticide 18 products used on either environmental surfaces or in liquid media. 19

20 Panel Response 21

22

a. Carrier-based, animal infectivity a ssays recommended by EPA’s guidance 23 for evaluating the efficacy of pesticide products used on environmental surfaces 24 (e.g., hard, nonporous surfaces). 25

26 One Panel member commented that there are substantial problems in quantifying 27 reduction of infectivity in carrier-based mode ls. Wires (or other ca rriers) incubated in 28 diluted brain homogenate may not effectiv ely represent proportionately the dilution 29 made. Proportional dilution would only occur if all components of brain homogenate had 30 exactly the same affinity for the surface. If prions bind more efficiently than other 31 components the endpoint would be an overestim ate, if prions bind less efficiently the 32 endpoint would be an underestimate. Thus, dilution of brain homogenate and absorption 33

to a carrier cannot then be used as a basi s for incubation time bioassay. Determinations 34 of log reduction would therefor e be invalid. According to this Panel member, there is 35 one published stainless steel wire based endpoi nt titration (hamster 263K prions serially 36 diluted in normal hamster brain) which gave an endpoint of 5.6 (five point six) log ID 50 37 (Fichet, Comoy et al. 2004). Dr. David As her, Laboratory of B acterial, Parasitic & 38 Unconventional Agents, Division of Emergi ng and Transfusion-Transmitted Diseases, 39 FDA, reported at the open Panel meeti ng an endpoint of 7 (seven) log ID 50 when 263K 40 was diluted in calf brain paste and mentioned a difficulty in getting prions to stick to the 41 wires when using PBS as diluent. This highl ights the potential problem and could lead a 42 potential applicant for EPA approval to choos e a formulation for which it is easiest to 43 reach a defined log reduction, but which is no t valid. Conversely, it is not possible to do 44 an endpoint titration with a treated wire to determine remaining titer. 45

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33 Another Panel member said that full end-poi nt titrations, or at least titer reduction 1 experiments including the end-point, are the test me thod of choice for both the 2 suspension-based and the carrier-based assa ys. Indeed, it has been shown in several 3 studies that various physical (e .g. heat) or chemical treatments of prion-infected material 4 can increase the incubation period by bioassay (when compared to the untreated sample) 5 disproportionately to the titer reduction meas ured by end-point titration. Therefore, 6 incubation period assays can overestimate the log reduction of a given treatment 7 (Somerville and Carp 1983; Taylor and Fernie 1996). 8

9 In the case of carrier-based assays, end- point titrations pose the problem of the 10 practicality of doing serial dilu tions of the treated surface (pr actically, the treated wire or 11 beads) to which prions are attached. If th e treated surface, once “i noculated” induces an 12 incomplete attack rate, it means that the amount of infectivity corresponds to the end13 point and the log reduction can be calculated directly. If not, the only available method 14 so far is the glass adsorption and crushi ng method, published by Chen and validated for 15 prions by Dr. Asher (Chen 1975). A standa rd dose-response curve for carrier-bound 16 infectivity can be provided by the adsorption onto carriers of seri ally diluted brain 17 material (positive control). This Panel member commented on the potential lack of 18 proportionality of adsorption of diluted samp le on to carriers. Although this theoretical 19 possibility is worthwhile cons idering, there is so far no e xperimental evidence for this. 20 More data are needed to clarify this point. In any case, this point can be overcome by 21 performing the same carrier crushing met hodology to generate the standard curve. 22

23

b. Suspension-based, animal infectiv ity assays recommended by EPA’s 24 guidance for evaluating the efficacy of pest icide products used in liquid media (e.g., 25 wastewater). 26

27 The Panel agreed that suspension-based an imal bioassays are the only validated and 28 broadly accepted ways to determine infectious titer. Additionally, all agreed on the final 29 requirement for a quantitative and direct anim al bioassay of infectivity and for a test 30 system specific for each of the different ag ents, e.g., sCJD, scrapie, CWD, vCJD, and 31 BSE agents. The Panel discussed in detail the potential limitations of the incubation-time 32 bioassay and the potential hi gh cost and animal usage of the end-point bioassay. 33

34

One Panel member stated that an incubati on time assay done at >5 readable (each one 35 log-significant difference) poi nts, including the end-point, wo uld be most acceptable. 36 This would reduce costs of a complete end- point dilution assay. A few animals (~5) 37

inoculated with uninfected mate rial from the same tissue type should also be included to 38 rule out non-specific and toxic effects. In addition, clinical illne ss is not sufficient for 39

readout because animals die from other cau ses, especially during prolonged end-point 40 incubations. A full necropsy inspection of organs to rule out such non TSE causes of 41 death, as well as western blot ting or neuropathology of all animals, is required to 42 confirm the infectious agent is causal. Su ch criteria are important for a statistically 43 limited group of animals. Furthermore, the agent-type can be determined by the 44 distribution of spongiform changes as well as prion staining in brain and spleen. 45EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

34 Methods are available to evaluate both spongiform changes and prion deposits 1 simultaneously (Manuelidis, Fritch et al. 1997). 2

3 Another Panel member disagreed stating that the end point dilution assay was the best 4 method for final validation of any dec ontamination method, although the incubation 5 interval assay could be used as a screeni ng method to focus the dilution groups to be 6 performed with end point dilution. This Pa nel member suggested that additional detail 7 should be sought by EPA to provide guidance to registrants in testing their products. 8

9 The end point titration assay is the only mean s of directly calculating titer. The numbers 10 of animals per group should be based on the expe cted variance in the parental strain that 11 is tested. The time post inoculation the an imals are monitored should be based on the 12 incubation period of the strain examined and should be at least 50% greater than the 13 incubation period of the highest dilution that causes disease. Clinical signs should be 14 monitored a minimum of three times per week. The monitoring of clinical signs can be 15 by subjective observation of clinical signs or quantifica tion of behavioral changes and 16

should be consistent between control and e xperimental groups. All animals in the study 17

should be examined for evidence of infec tion (Western blot for PrPres and/or 18 neuropathology). Only differences in calculate d titers that are equal to or greater than 19 one log are considered signifi cant (Mould, Dawson et al. 1967). 20

21 The incubation time interval assay has seriou s limitations that need to be addressed. 22

Please note that the incubation time interval assay provides an estimate of titer and is not 23

a direct calculation of titer. Although the incubation time interval assay can yield similar 24 results as endpoint titration, seri ous limitations of this assay have been reported. Several 25 reports have indicated that tr eatment of prions with heat and/or detergent can alter the 26 relationship between titer and incubation period resulting in large errors in estimating 27 titer based on incubation period alone (Dic kinson and Fraser 1969; Lax, Millson et al. 28 1983; Somerville and Carp 1983; Taylor a nd Fernie 1996). Additionally, it has been 29 shown that the relationship between the dose of the agent and incubation period can be 30 tissue specific, precluding using incubati on interval assays when comparing the 31 dissimilar tissues (Robinson, Cheevers et al . 1990). Finally, there are numerous 32 examples in the literature of 10-fold differences in titer of the agent that have similar 33 incubation periods that redu ce the precision of the incubation time interval assay 34 (Kimberlin and Walker 1977; Kaeser, Klein et al. 2001; Kincaid and Bartz 2007). This is 35 in contrast to endpoint diluti on analysis that is accurate to 0.5 (zero point five) log ID 50 36 and is highly reproducible (M ould, Dawson et al. 1967; Tayl or, McConnell et al. 2000). 37

38 Inoculum preparation should be consistent be tween control and experimental groups with 39 the only variable being the treatment. The bi ological matrix used should mimic the likely 40 form the agent would be present for the intended use. It is important to note that brain 41 homogenate and preparations that are enriched for infectivity can differ in the response to 42

inactivation. 43

44 c. Any other known test methods for e valuating the efficacy of pesticide 45 products used on either environmental surfaces or in liquid media. 46EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

35 1

Most of the Panel was not enthusiastic about using protease resistan t prion quantification 2 or amplification (PMCA) as a measure of in fectivity because infectious titer in many 3 cases does not correspond to protease resistan t prion. Nevertheless, an applicant may 4 wish to use such an approach in initial pi lot studies to save time and reduce costs. 5

6

There are also stable tissue culture models that can be used to assess infectivity for a 7 number of TSE agents, including vCJD, Japa nese CJD, 22L, Chandler (RML) and ME7 8 scrapie (Manuelidis, Liu et al . 2009) (Arjona, Simarro et al. 2004; Nishida, Katamine et 9 al. 2005; Mahal, Baker et al. 2007). These can speed the first choice of best pesticide 10 reagents, especially for materials dried on stai nless steel or glass su rfaces. These treated 11 surfaces can be cultured with susceptible cells for titer readout in as few as twenty-one 12 days. Experiments with some of these agen ts grown in culture, such as Japanese CJD 13 and 22L scrapie, have shown faithful reprodu ction of the original TSE by re-inoculation 14

of animals. Such cultured cells carry higher brain-like titers of infectivity than most other 15

cultures. 16

17 The majority of the Panel concluded that in vitro immunodetection assays or cell-based 18 bioassays are not adequately developed for us e in assessing reducti on in infectivity for 19 evaluation of efficacy of pesticide products with prion related claims because in the case 20 of immunodetection assays thes e do not measure infectivity a nd in the case of cell-based 21 bioassays these have not been adapted for the application of a ssessing reduction of 22 infectivity. The Panel recommends to EPA th at end point dilution animal bioassays be 23

used to assess reduction in inf ectious titer by treatment of samples with prion pesticide 24 products. 25

26 EPA Response to the SAP’s Response to Charge Question #2 27

28 The SAP responded to the three sub-questions (a., b., c.) under Charge Question #2, and 29 EPA’s responses follow the same format: 30

31

a. Carrier-based, animal infectivity ass ays recommended by EPA’s guidance for 32 evaluating the efficacy of pesticide pr oducts used on environmental surfaces 33 (e.g., hard, nonporous surfaces). 34

35 In its summary statement, the SAP expresse d “reservations” about the use of carrier36 based animal bioassays. Those reservations appeared to focus on the potential for diluted 37 brain homogenates not to adsorb on to the carrier in proportion to their concentrations. In 38 its detailed comments, the SAP attributed this concern to one Panel member. The SAP 39 also cited one (?) other Panel member who asse rted that full end-poi nt titrations are the 40 “method of choice” for both suspension-based a nd carrier-based assays, asserting that 41 incubation time bioassays can overestimate th e log reduction of a given treatment. 42 However, the majority (?) of the SAP also re cognized that end-point titrations cannot be 43 done with solid carriers (e.g., steel wires) because the carriers cannot usually be ground 44 up to make a dilution. The SAP noted that only a glass adsorption/crushing method is 45 available (Chen 1975). 46EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

36 1 In response to these comments, EPA interprets the SAP as indicating that while it would 2 prefer the use of an end-point titration bioassay to an inc ubation time interval assay for 3 carrier-based testing, the SAP recognizes that it is physically impo ssible to do such a 4 bioassay with a hard, nonporous carrier such as steel. Further, although the SAP reported 5

the concerns of one Panel member about th e incubation time interval assay (i.e., the 6 potential for disproportionate adsorption of brai n homogenate to a carrier), it later stated, 7 “there is so far no experimental evidence for this.” 8

9 EPA firmly believes that a disinfectant should be tested in a way that represents the way 10 the product is used. Most uses of disinfect ants involve applicati on to a hard, nonporous, 11 inanimate surface. For this reason, EPA current ly requires carrier-based efficacy testing 12 for all sanitizers, disinfectants and sterilan ts. The fact that the end-point titration 13 bioassay cannot be performed on a hard mate rial (except one method that uses glass, 14 which can be ground up and diluted as requi red by the end-point titration method) 15 precludes it from being used. Therefore, EPA’s guidance on prion disinfectant efficacy 16 testing will continue to recommend the us e of an incubation time interval assay for 17 testing of disinfectants on carriers that represent har d, nonporous, inanimate surfaces. 18

19

b. Suspension-based, animal infectivity assays recommended by EPA’s guidance 20 for evaluating the efficacy of pestic ide products used in liquid media (e.g., 21 wastewater). 22

23 In its summary statement, the SAP agreed that the suspension-based end-point dilution 24 bioassay is the most reliable method for meas uring reduction of pr ion infectivity. The 25

Panel also considered the in cubation time interval assay to be a screening method. The 26 Panel further asserted that an end-point ti tration assay is “necessary” to calculate 27

reduction in infectious titers and “cannot be substituted” by an incubation time interval 28 assay. Also, some Panel members supported us ing one rodent model system for testing 29 all products, but others thought that testing s hould be done with na tive prion strains in 30 their corresponding animal hosts. Finally, the Panel stated that necropsies of all test 31 animals using Western blot detection a nd neuropathologic changes are required. 32

33 In its detailed comments, the SAP reiterated its opinion that that the suspension-based 34 end-point dilution bioassay is the most relia ble method for measuring reduction of prion 35 infectious activity. On the issue of which prion strains should be tested, the SAP’s 36

detailed comments stated that all Panel memb ers agreed that tests should be conducted 37 for each of the different prion types, wh ereas in the summary statement the SAP 38

indicated that some Panel members favored using rodent models systems while others 39 favored using native prion strains. The SAP then delved further into the debate of 40 incubation time interval bioassay versus th e end-point dilution bioassay. One Panel 41

member stated that an incubation time interval assay with more than five readable points, 42 including the end point, would be acceptable. A few control animals inoculated with 43 uninfected material from the same tissue type should also be included to rule out non44 specific and toxic effects. Finally, a full necropsy shoul d be done. A second Panel 45 member disagreed with the first Panel member , asserting that the en d-point dilution assay 46EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

37 is the best method for final validation of a decontamination method. This member’s main 1 reason appeared to be that the end-point dilu tion assay provides a direct calculation of 2 prion titer whereas the incubation time interv al assay provides an estimate of titer. 3 Another reason given by the Panel was the difference in test accuracy—the end-point 4 method is accurate within 0.5 log ID 50 in contrast to 1 log for the incubation time interval 5

assay. 6

7 In response to the SAP’s summary comments, EPA observes that the SAP’s 8 recommendation for an end-point dilution bi oassay, which is suspension-based, is 9 consistent with EPA’s guidance that re gistrants use a suspension-based bioassay for 10 evaluating the efficacy of prion products us ed in liquid media (e.g., wastewater). 11 Regarding the SAP’s comment that an end- point dilution assay is “necessary,” EPA 12 interprets that this statement is applicable to suspension-based test s and not carrier-based 13 tests since Charge Question #2b only pertains to suspension-based testing. Accordingly, 14 EPA will revise its guidance to recommend that an end-point dilution assay be used for 15 evaluating prion products applied to liquid media such as wastewater. 16

17

EPA further observes that the SAP did not reach a consensus on whether prion efficacy 18 testing should be conducted with rodent-adapted prions (in place of native prions) or on 19 native prion strains in a relevant animal host. Since each approach has been used 20 successfully by various researchers, EPA has decided to allow use of either testing 21 approach. Hence, EPA’s draft guidance will a llow for use of either a rodent model or a 22 native strain of prion in a relevant animal host. 23

24 Finally with regard to the SAP’s summary comments, the Panel recommended necropsies 25 along with Western blot detec tion to determine whether surv iving animals are infected 26 with prions. Consistent with this reco mmendation, EPA’s draft guidance recommends 27 that at least one prion-specifi c test (Western blot, immunolog ical histochemistry or “blind 28 passage”) be performed to confirm whether either the surviving or dying animals are 29 infected with prions. 30

31 In response to the SAP’s detailed comments, EPA observes (as mentioned above) that the 32 SAP recommendation for an end-point diluti on bioassay, which is suspension-based, is 33 consistent with EPA’s guidance that regist rants use a suspension-based bioassay. EPA 34 notes that with respect to th e issue of which prion strain s should be tested, the SAP’s 35 detailed comments conflicted with its summary co mments. The latter stated that all Panel 36 members agreed that tests should be conducted for each of the different native prion 37 types, whereas the former indicated that some Panel members favored using a rodent 38 model system. EPA assumes that since conf licting opinions were e xpressed on this issue 39

that no consensus was reached. As noted above , EPA’s guidance allows for use of either 40

a rodent model or a native strain of prion in relevant animal hosts. 41

42 Regarding the differences of opinion among Panel members on the issue of end-point 43 dilution assay versus incubation time inte rval assay, EPA appreciates the SAP’s 44 discussion of the advantages and disadvantag es of each type of assay. However, EPA 45 presumes that the Panel’s recommendation that an end-point dilution assay is “necessary” 46EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

38 is applicable to suspension-based tests and not carrier-based tests since Charge Question 1 #2b only pertains to suspension-based testing. 2

3 c. Any other known test methods for e valuating the efficacy of pesticide 4 products used on either environmental surfaces or in liquid media. 5

6 In its summary statement and detailed comment s, a majority of the Panel concluded that 7 in vitro immunodetection assays or cell-bas ed bioassays are not adequate for use in 8 assessing reduction in prion inf ectivity. A minority of the Pa nel contended that in vitro 9 amplification immunodetection assays su ch as protein misfolding by cyclical 10 amplification (PMCA) and cell-based bioassays can serve as a complementary assay to 11 animal bioassays. The Panel also reiterat ed that end-point dilution animal bioassays 12

should be used to assess reduc tion in infectious titer. 13

14 In response to these comments, EPA agrees wi th the Panel that in vitro immunodetection 15 assays are not adequate for assessing reducti on in prion infectivity, and that in vitro 16 amplification immunodetection assays should only complement but not substitute for 17 animal bioassays. EPA presumes that the Panel’s recommendation for using an end-point 18 dilution assay is applicable to suspension-ba sed tests and not carri er-based tests since 19 Charge Question #2b only pertains to suspension-based testing. 20

================== ================== ================== ========= 21

22

Issue 3. Whether the product performance crite rion specified in the draft guidance 23 document to support the registra tion of pesticide products with prion-related claims is 24 scientifically sound. 25

26 Charge Question 3. Please comment on the scientif ic soundness of the product 27 performance criterion recommended in th e draft guidance docum ent to support the 28 registration of pesticide pr oducts with a prion claim. 29

30 Panel Response 31

32 The Panel was in general agreement that some widely used model systems, such as the 33 263K or Sc237 rodent-adapted prion strains, have titers up to ~10 11 ID 50 /g of brain tissue 34 and make it possible to detect a six (6) log re duction in infectivity. However, bioassays 35 in rodents are usually performed by intracereb ral inoculation of a small volume such as 36 25μl. For a 10% brain homoge nate, 25μl represents 2.5 x 10 8 ID 50 , making an 8 (eight) 37 log reduction measurable for bioassays in rodents with model prion strains. The 38 treatment methodology may further reduce the initial infectious dose, for example, 39 mixing a brain homogenate with an inactiva tion reagent would d ilute the level of 40 infectivity. It may then be necessary to furt her dilute the sample to reduce any potential 41 toxic effects of the inactivation reagent on the bioassay animals. In such cases it may not 42 be possible to measure such a high reduction. 43

44 The Panel considered the important caveat that for naturally occurring prion strains from 45 natural hosts, titers are much lower in the available animal bioassay models: only in the 46EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

39 range of 10 5 to 10 8 ID 50 /g. In such cases it may only be possible to measure a three (3) or 1 four (4) log reduction in infectivity. 2

3 Furthermore, any claim of a log reduction in infectivity needs to be accompanied by a 4 statistical methodology, and some understa nding of assay va riability, e.g., 95% 5 confidence interval, in attaining such a give n log reduction. End point animal bioassays 6 should utilize Kaplan-Meier and/or Cox regression analys is models to account for 7 observed censoring, e.g., from animals dying prematurely, in the estimation of mean 8 times to outcome, e.g., onset of clinical si gns, death, etc. In this way, confidence 9 intervals for these estimates can also be provi ded. In addition, sp ecific tests (test for 10 equality of appropriate Cox regression pa rameters, or non-parametric tests for equal 11 survival curves via the Kaplan-Meier curves) and parameter/characteristic to be used for 12 comparing controls to treatment groups need to be specified. 13

14

One Panel member added that carrier-bas ed methods provide further issues for 15 calculating log reduction in in fectivity. Firstly, it is not possible to use endpoint based 16 methods, with the possible exception of the ground glass surface (Chen 1975). This is 17 not an established method for quantification of prion titer, although preliminary data were 18 reported to the Panel by Dr. Asher of the FDA. Also, there is no basis to assume all non19 porous surfaces are equal in terms of prion binding. One published value for stainless 20

steel wires incubated in se rially diluted 263K-infected brain homogenate gave an 21 endpoint of 5.6 log ID 50 (Fichet, Comoy et al. 2004). A similar value was reported in a 22 recent paper (Lemmer et al. 2008). 1 Researchers have found that the composition of the 23

steel makes a differen ce (Luhr et al. 2009). 2 However, there is a po ssibility that carriers 24 incubated in serially diluted brain homogenate do not accurately bind infectivity at the 25 same proportion to the dilution. If this is th e case, then the endpoint calculation is not 26 valid. Conversely, concerns were also e xpressed about the incubation period animal 27 bioassay, because it has been shown that estim ation of titer of physically or chemically 28 treated samples by this method does not al ways correspond with titer calculated by 29 endpoint titration (Somerville and Ca rp 1983; Taylor and Fernie 1996). 30

31 Another Panel member noted that the targ et amount of reduction may need to be 32 considered in light of the propos ed application or use, risk an d what is achievable and can 33 be validated for a particular chemical or phys ical treatment. For example, treatment of 34 potentially CWD contaminated waste water from an area associated with deer housing or 35 processing may have a different standard for re duction in infectivity compared to that for 36 surgical instruments in a human hospital whic h have potentially been exposed to CJD. A 37 reduction of six (6) log in inf ectivity of CWD in waste water may be adequate to reduce 38 the infectivity compared to the risk posed by CWD prions to human health, whereas the 39 efficacy of a six (6) log reduc tion for CJD may not be adequa te to reduce the potential 40 that a human patient might contract CJD from an instrument with a six (6) log reduction 41

1 Lemmer K, Mielke M, Kratzel C, Joncic M, Oezel M, Pauli G, Beekes M. 2008. Decontamination of surgical instruments from prions. II. In vivo find ings with a model system for testing the removal of scrapie infectivity from steel surfaces. J Gen Virol. 2008 Ja n;89(Pt 1):348-58.

2 Luhr KM, Löw P, Taraboulos A, Bergman T, Kristensso n K. 2009. Prion adsorption to stainless steel is promoted by nickel and molybdenum. J Gen Virol. 2009 Jul 15. [Epub ahead of print]EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

40 in CJD prions. Risk assessments may be avai lable to help formulate expected standards 1 of performance, but a one-size fits all approach may not be valid. 2

3 The Panel recommends that if it is not possibl e to measure to six (6) log reduction in a 4 relevant model (one using the naturally occu rring prion strain from the natural host for 5 which the inactivation method is designed to protect against), then the maximum 6 measurable reduction should be required to de monstrate target effi cacy. However, it was 7 not generally agreed whether this should al so be accompanied by demonstration of at 8 least a six (6) log redu ction in a widely used model, su ch as the hamster-adapted scrapie 9 prion 263K or Sc237 strains. 10

11 EPA Response to the SAP’s Response to Charge Question #3 12

13 In its summary comments, the SAP’s overall recommendation was that target reduction 14 in infectivity should be assessed in a relevant animal model using the naturally occurring 15 prion strain from the natural host to support the specific label claim. The SAP stated that 16

the level of reduction should be set at the maximum measurable reduction achievable for 17 the target strain and for the efficacy test model used. 18

19 In both its summary comments and detailed comments, the SAP generally agreed that 20 some widely used model systems, such as the 263K or Sc237 rodent-adapted prion 21 strains, can detect six (6) l og reduction in infectivity. The Panel also noted that for 22 naturally occurring prion strains from natural hosts, it may only be possible to measure a 23 three (3) or four (4) log reduction in infec tivity. In addition, the SAP stated that any 24 claim of reduction in infectivity needs to be accompanied by a statistical methodology. 25

One Panel member raised a concern about th e possibility that ca rriers incubated in 26 serially diluted brain homogenate do not accurately bind infectivity at the same 27 proportion to the dilution, which could affect the accuracy of the log reduction estimate 28 from an incubation time interval assay. Anot her Panel member suggest ed that the target 29 amount of reduction may need to be considered in light of the pr oposed application or 30 use, risk, and what is achievable and can be validated. 31

32 EPA’s response to the SAP’s comments is as follows. The central issue of Charge 33 Question #3 regards the scientific soundne ss of the product performance criterion 34 recommended in the draft guidance document (i.e., a six (6) log reduction in prion 35 infectivity) to support the regi stration of pesticide products with a prion claim. EPA 36 agrees with the SAP’s assessment that some te st models can achieve this target criterion 37 for success and others cannot, but does not agree with the SAP’s recommendation that a 38 lower target criterion (e.g., 4 or 5 log reducti on) should necessarily be acceptable if an 39 efficacy test method is only capable of meas uring a lower reduction in prion infectivity 40 for a particular prion strain, Because prions are very difficult to inactivate and present a 41 serious health risk (i.e., irre versible neurological disease and death) to humans, EPA has 42 decided to maximize protection of human health by setting a target cr iterion that is very 43 stringent but achievable. Accordingly, EPA will keep its general target performance 44 criterion at six (6) logs of reduction of inf ectivity as demonstrated by acceptable efficacy 45 test methods. However, since EPA’s document provides guidance, EPA may consider 46EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

41 alternative test methods and/or a different target performa nce criterion if persuasive, 1 valid, scientific data and/or arguments are submitted to EPA for review. 2

3 In response to the SAP’s recommendation in this section that infectivity should be 4 assessed in a relevant animal model using th e naturally occurring prion strain from the 5 natural host, EPA is aware that it is extremely difficult to obtain the naturally occurring 6 strain of certain prions (e.g., BSE, sCJD), that this approach may be cost prohibitive, and 7 that rodent adapted strains are readily avai lable for testing. Accordingly, EPA believes 8 that while use of naturally occurring prion st rains from the natural host might be optimal 9 from a purely scientific standpoint, use of rode nt adapted prion strains would be far more 10 practical and feasible. Nevert heless, EPA may consider test ing with a native strain of 11 prion in a relevant animal hos t if a registrant submits a pr oposed test protocol along with 12 persuasive scientific arguments as to why EPA should accept that protocol. 13

14 Finally, EPA agrees with the SAP comment that that any claim of reduction in infectivity 15 needs to be accompanied by a statistical me thodology. EPA’s guidanc e already specifies 16

the need for a statistical evaluation plan for each study. 17

18

========================= ==================== ================== 19

20

Issue 4. Whether the labeling claim described in the draft guidance document is 21 scientifically appropriate based on th e recommended test systems and product 22 performance standard. 23

24 Charge Question 4. Please comment on the scientific appropriateness of the term 25 “reduce infectivity” in a label claim to re flect the action of a pesticide on prions. 26

27 Panel Response 28

29 The Panel agreed with the EPA’s proposal to frame the label claim statement as “reduce 30 infectivity.” From a scientific perspective, the state of “complete elimination of 31 infectivity” is probably impossible to achieve in practice, let alone from a probability 32 standpoint. One Panel member asked why th e EPA was hesitant to claim complete 33 elimination of infectivity given that techniques to measure TSE infectious titer are similar 34 to those of other infectious agents. The EP A replied that with other microorganisms the 35 target reduction is also less than 100%, and they want to be consistent with 36 recommendations from other Federal agencies, for example the recommendations of the 37 FDA Transmissible Spongiform Ence phalopathies Advisory Committee. 38

39

Another Panel member stated that no one knows what constitutes a minimal infective 40 dose of prions and that there is no way to measure this in humans. Infective dose can 41 also be influenced by the immune and othe r systems and how these systems react to 42 different doses of prions is unknown. It is very difficult to estimate the dose of prions 43 needed to transmit TSE infectivity to humans. As a result, there is great reluctance to 44

claim that all TSE infectious doses have b een removed following treatment with a prion 45 decontaminating reagent. Additionally, one Panel member noted that due to the 46EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

42 prolonged disease incubation times and the lack of understanding as to what constitutes 1 an infectious dose, reduced infectivity is the only term that can be used to assess a 2

solution used to decrease the impact of prion disease. 3

4 One Panel member noted that different expectations drive the reluctance to use a word 5

like “inactivation”. There was concern that registrants and the user communities will 6 have differing expectations of the prion pe sticide product’s performance. Users in 7 hospitals concerned about CJD may expect something closer to “elimination of 8 infectivity” on environmental su rfaces, whereas the veterinary and wildlife management 9 community may be more comfortable with th e “reduced infectivity” approach. One 10 Panel member recommended that the label clearly indicate the implications of the 11 “reduce vs. elimination” approach in sufficien t detail so that the user can understand and 12 appreciate what the product can and cannot do. Furthermore, the label of the registered 13 product should follow current EPA labeling recommendations in providing use condition 14 details for each general app lication of the product, e.g., decontamination of hospital 15 environmental surfaces, decontamination of laboratory working surf aces, or application 16 on porous materials such as fences, etc. The Panel asked EPA represen tatives if the label 17 was intended to be understood by the genera l public, and an EPA representative 18

confirmed this. 19

20 One Panel member noted that when considering the phrase “reduce infectivity” it is 21 important to note 1) that it does not refer to the complete elimination of infectivity with 22 absolute confidence and, 2) that any product ha s to be tested in a way that supports the 23 labeling claim. Thus, the phrase “reduce inf ectivity” is valid only as long as the method 24 used to measure infectivity de monstrates a log reduction in a valid model system. Use of 25

the word reduce would therefore be model de pendent. A claim to “reduce infectivity” 26 also implies that the efficacy testing will i nvolve a measurable endpoint, so the reduction 27 can be quantified. 28

29

One Panel member stated that incubation time animal bioassays used to measure 30 infectivity after treatment of pr ions may not be the best assays to use as there are several 31 examples in the literature that suggest tr eatments which reduce prions may lead to 32 incubation periods that fall outside the linear range of the assay . There was a general 33 consensus among Panel members that endpoint t itration animal bioassay is the best way 34 to measure log reduction in TSE infectivity. 35

36 The issue of whether or not to qualify the phr ase “reduce infectivity” was discussed. The 37 phrases “maximally reduced” or “maximally achievable reduction of infectivity” were 38 introduced as was the idea that the label could note that infectivity was reduced to below 39 the level of detection in a specific assay. An EPA representative noted that EPA was 40 reluctant to put adjectives in front of “reduced.” 41

42 It was suggested that, if th e testing is based on a specifi c animal bioassay model, the 43

details of that model be included in the la bel. One suggestion was to separate the 44 description of the prion strain and host of origin from the species of host in which the 45 prion strain had been assayed. Another s uggestion was to precisely define the log 46EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

43 reduction as well as the dynamic range of the assay (range of infective titer for which the 1 bioassay yields precise and accurate results) so that the reader could understand whether 2 the reduction of infectivity corresponded to an exact value or a lower limit. Two 3 examples were read to the Panel: 4

5 Example 1 : Has been demonstrated to reduce inf ectivity of prions (TSE agents) by five 6 (5) logs or more based on bioassay of th e bovine BSE strain in transgenic mice 7 expressing the bovine prion gene in (specify transgenic mice). The dynamic range of the 8 bioassay is five (5) logs. 9

10 Example 2 : Has been demonstrated to reduce inf ectivity of prions (TSE agents) by six 11 (6) logs based on bioassay of the hamster Sc 237 strain in Syrian hamsters. The dynamic 12 range of the bioassay is nine (9) logs. 13

14 Given the variety of different prion animal bioassay systems, the Panel discussed whether 15 or not a specific log reduction of infectivity should be specified by EPA. It was brought 16 out that even a six (6) log reduction would not be enough to completely inactivate high 17

titer brain tissues such as a hamster brain in fected with the Sc237 strain which could have 18 as much as eleven (11) logs of infectivity per gram of brain. However, a six (6) log 19 reduction is thought to be within the range of inactivation for human sporadic CJD or 20 cattle BSE prions as these gene rally contain around six (6) logs of infectivity per gram of 21 brain. One Panel member brought up the important caveat that limited sensitivity of 22 these test models could account for the appa rent lower infectivity TSE-infected human 23 and bovine brain tissue, rather than a smaller number of infec tious particles. One Panel 24 member suggested that even a five (5) l og reduction might be acceptable for a product 25 with a prion-related claim unde r the condition that the labeli ng specified the infectivity 26 reduction and the dynamic range of the animal bioassay. 27

28

Another Panel member was concerned with th e notion that the regi strant provide the 29 agency with the details of the product test ing methods, thereby presenting EPA with 30 potentially a wide variety of methods requi ring a great deal of time on EPA’s part to 31

review. Other Panel members raised concer ns that registrants might choose a model 32 system with low levels of TSE infectivity to test their product. It was suggested that EPA 33 poll members of this SAP and/or members of the interagency TSE working group to 34 review the literature and gather the detail s of various animal bioassay designs, seek a 35 consensus on these details and compile a list of acceptable prion strain/animal bioassay 36 models. This would facilitate agency review of registrant methods by establishing an 37 array of acceptable efficacy test methods. 38

39 There was general consensus among the Panel members that the label should give 40 sufficiently detailed and accurate informati on to enable the product user to assess how 41 efficiently, and under what usage, the prion pesticide product inactiv ates TSE infectivity. 42

43 EPA Response to the SAP’s Response to Charge Question #4 44

45EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

44 In its summary and detailed comments, the SA P agreed with EPA’s proposal to frame the 1

label claim statement as “reduce infectiv ity,” but stated that the phrase “reduce 2 infectivity” is valid only as long as the method used to measure infectivity has an 3 acceptable dynamic range of detection in a re levant animal bioassay system. The Panel 4 also suggested that the label statement include the prion strain and original host, bioassay 5 animal host, and observed log reduction in inf ectivity as well as the dynamic range of the 6 animal bioassay used. The SAP provided exampl es of what it considered to be desirable 7

label statements. 8

9 In response to the SAP’s comments, EPA will c ontinue with its proposal to use the term 10 “reduces infectivity” provided the method used to measure infectivity has a dynamic 11 range that measures at least six (6) logs of prion infectiv ity reduction. In addition, EPA 12

will add to the label statement the amount of log reduction demonstrated by the product 13 and basic information about the prion strain tested and the test organism. EPA does not 14

see the need to add another statement about th e dynamic range of the test since that is 15 implicit in the amount of log reduction stated on the label. 16

17

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19

Issue 5. Whether different prion types exhibit va riation in the degr ee of resistance to 20 inactivation by pesticide chemicals and whethe r a hierarchy of resi stance by prion type 21 can be reliably determined at this time. 22

23 Charge Question 5. Please comment on whether a hierarchy of resistance among prion 24 types can be reliably demonstrated for different pesticide chemicals based on the 25

available data. 26

27 Panel Response 28

29 There is currently little data on the relative resistance to inactivation of prions from 30 various species and strains, even though some st udies report strain sp ecific variations in 31 resistance to inactivation (Peretz, Supatta pone et al. 2006; Fernie , Steele et al. 2007; 32 Giles, Glidden et al. 2008). However, it is clear that prions from different species and 33 strains within the same species have distinct incubation time and lesi on profiles in a given 34

host as well as biochemical and biophysical properties. Given the fact that different prion 35 pesticide products might act in different ways, it is likely that strains may respond 36 differently and unpredictably to inactivati on procedures. Therefore, the Panel’s 37 consensus is that no single hierarchy of strains to inactivation procedures can be 38

established at this time. 39

40 Efficiency of a particular prion inactivati on procedure must be measured by a relevant 41 and robust infectivity bioassay, depending on th e prion agent for which the procedure is 42

intended to be used. There was some deba te among Panel members regarding the need 43 relative to the prion strain hier archy of resistance issue for us e of two bioassay tests, with 44 one of them done in a standard widely-use d model, e.g. scrapie prion strain 263K in 45 hamsters, and the other being specific to th e samples under study. This will enable a 46EO 12866 REVIEW DRAFT DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE

45 more robust proof-of-concept validation and the possibility to compare results from 1 different prion pesticide products. However, the limitation is that some procedures may 2 not work as well in the standard assay and st ill be useful for specific applications as 3 supported by an animal bioassay in a releva nt species. After discussion, and based on 4 practical considerations, the majority of the Panel members recommend using end-point 5

titrations in a relevant and robust animal bioassay depending on the samples to be 6 decontaminated. Although, there was some de bate, the majority of the Panel members 7

think that decontamination st udies should be performed on tissues from the natural host 8 and that rodent adapted models of natural pr ion disease are not reliable substitutes. The 9 experiments should be done with the natural sa mples, e.g. CJD, scrapie, or CWD, in the 10 host such as either wild type animals or tr ansgenic that provides the best practical and 11 dynamic range for the study. One Panel member expressed concern th at transgenic mice 12 overexpressing prion protein may not be a more appropriate model to assess the problem 13 than mice with wild type prion protei n (J. Cell. Biochem. 106: 220-231; 2009). 14

15 The Panel recommends that the experimental protocol to study efficacy of a given 16 procedure be reviewed in advance by a group of experts to decide th e best animal model 17 and sample preparation to conduct the study. 18

19 There was also discussion that experiment s done with one source of material, e.g. one 20 strain of sCJD, in one animal model, may not extrapolate to related strains of the agent, 21 e.g. vCJD, fCJD or even othe r strains of sCJD (although one Panel member did not think 22

there is substantial evidence of the existence of sCJD strains), so registration based on 23 positive results with one strain may not guarant ee that the product works even in related 24 strains of the same species. One suggestion to approach this problem in a practical way 25 was to complement results obtained in the in vivo infectivity assay with in vitro or cell 26 based assays that can analyze a large diversity of strains. 27

28 In conclusion, the consensus from the Panel is that no single hierarc hy of strain resistance 29 to prion pesticide action can be established at this time. 30

31 EPA Response to the SAP’s Response to Charge Question #5 32

33 In its summary and detailed comments, the SA P concluded that no si ngle hierarchy of 34 strain resistance to pesticide action ca n be established at this time. 35

36 In response to the SAP’s comments, EPA agrees that inadequate data exist with which to 37 establish a hierarchy of resistance to inact ivation by pesticides. EPA’s question was 38 aimed at determining whether testing one strain of prions that has been documented to be 39 significantly more resistant to inactivation by pesticides than any other strain, and, if so, 40 could that one prion strain be tested rather than every in dividual strain tested. The 41 answer provided by the SAP c onfirms EPA’s belief that individual prion strains need to 42 be tested and that label claims should be speci fic to reduction of infectivity of each prion 43

strain. 44