EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

1

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

2 “Scientific Information Concerning the Issue of Whether Prions Are a ’Pest’ 

3 under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA)” 

4 (2-17-10 DRAFT)

5 

6 On March 31 and April 1, 2009, the FIFRA Scientific Advisory Panel (SAP) held 

7 an open, public meeting to address a set of scientific issues being considered by the EPA 

8 pertaining to the issue of whether a prion is considered to be a “pest” under FIFRA. 

9 Subsequent to the meeting, the SAP issued minutes, including its comments on the issues 

10 and charge questions that EPA presented at the meeting (SAP Minutes No. 2009-05; 

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

12 

13 The purpose of this document is to provide EPA’s responses to the SAP’s 

14 comments as listed in the meeting minutes. First, the verbatim Summary of the SAP’s 

15 comments are presented, followed by Charge Question 1 and the SAP’s detailed response 

16 to Charge Question 1. Following the SAP’s detailed response to Charge Question 1 is 

17 EPA’s response to the SAP’s detailed responses (beginning on page 17). EPA’s 

18 responses to Charge Questions 2-4 follow and are similarly presented. 

19 

20 SAP Summary of Panel Discussion and Recommendations (quoted verbatim from 

21 the SAP’s meeting summary):

22 

23 As charged by the EPA, the Panel reviewed the interagency White paper titled 

24 “Scientific Information Concerning the Issue of Whether Prions Are a ‘Pest’ under the 

25 Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA),” and discussed the nature 

26 of prions with respect to their potential classification as a “Pest” under FIFRA; guidance 

27 on testing issues for assessing efficacy of pesticides for reduction of prion infectivity; 

28 efficacy performance and labeling guidance for such pesticides; and lastly, scientific 

29 evidence for prion hierarchy in resistance to pesticide action. The prion terminology 

30 defined in the White paper will be used in these meeting minutes: “...‘prion’ refers to a 

31 misfolded isoform of any prion protein; ‘prion protein’ can refer to both the normal and 

32 misfolded isoforms.” 

33 

34 1. Interagency White paper issue: Does the interagency White paper on prions 

35 adequately identify and summarize available, relevant scientific studies and do these 

36 support designation of prions as “Pests”? The White paper summarizing the prion 

37 literature with respect to description of transmissible spongiform encephalopathies 

38 (TSEs) and prions, key characteristics of prions, the prion hypothesis and normal prion 

39 protein, neurotoxic mechanisms in TSEs, TSE diseases, history of TSE diseases, 

40 transmission and routes of exposure for TSEs, occupational and safety procedures and 

41 decontamination methods was accepted as adequate by the majority of the Panel. 

42 However, many statements in the White paper need to be revised to reflect accurately 

43 what is definitively known about prions and TSEs and that which is reasonably 

44 hypothesized based on scientific observations. 

45 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

2

1 A major issue discussed by the Panel was the prion hypothesis for the infective nature of 

2 TSEs. A majority of the Panel accepted the prion hypothesis for TSE infectivity with the 

3 caveat that cofactors may also be involved. The prion hypothesis, although considered 

4 unproven by some Panel members, provides a logical explanation of the infective nature 

5 of TSEs and the apparent lack of nucleic acid. The strength of the support for the prion 

6 hypothesis by experimental infection of transgenic mice with synthetic prion fibrils was 

7 questioned because spontaneous neurodegenerative disease in unexposed mice is also 

8 observed. A minority opinion contended that prion accumulation in TSEs is a pathologic 

9 change in response to an as yet unidentified TSE etiologic agent, possibly a virus. 

10 

11 The penultimate scientific question from the EPA to the Panel relative to the character 

12 and nature of prions is whether the science in the White paper supports the classification 

13 of prions as a “Pest” under the FIFRA. The regulatory basis (not reviewed by the Panel) 

14 on which this science question is assessed is whether prions are a form of a 

15 microorganism, i.e., prions are microorganisms other than bacteria or viruses, and 

16 whether prions are living entities. A majority of the Panel concluded that prions can be 

17 included in the category of “other microorganisms.” A minority of Panel members 

18 contended on scientific basis that prions lack the significant features typical of 

19 microorganisms. 

20 

21 In conclusion, a majority of Panel members agreed that once revised as suggested by the 

22 Panel the White paper can adequately identify and summarize the scientific literature 

23 concerning prions. Furthermore, a majority of the Panel accept that prions are “other 

24 microorganisms” exhibiting some features of living entities. 

25 

26 2. Efficacy Guidance Test Method Issue: Are the specific test systems 

27 recommended in the EPA draft guidance document scientifically appropriate to support 

28 the registration of pesticide products with prion-related claims?

29 

30 The consensus of the Panel was that animal bioassays are necessary to adequately assess 

31 reduction of prion infectivity by prion pesticide products. The Panel was asked to 

32 comment specifically on the three requested areas. 

33 

34 a. Carrier-based, animal infectivity assays for environmental surfaces (e.g., 

35 hard, nonporous surfaces). 

36 The Panel had reservations about the appropriateness of application of carrier-based 

37 animal bioassays for assessing pesticide product reduction of prion infectivity. In the 

38 case of carrier-based assays, end-point titrations pose the problem of the practicality of 

39 doing serial dilutions of the treated surface, i.e., treated stainless steel wire or beads of 

40 various compositions, where the presentation of absorbed prions to the bioassay may be 

41 limited by the surface area of the carrier exposed to the brain. There may be substantial 

42 problems in quantifying reduction of infectivity in carrier-based models as well, because 

43 absorption of diluted brain homogenates on to the carrier may not proportionately reflect 

44 the infectivity present in the original sample. However, it was also suggested that 

45 disproportionate absorption can be assessed by use of a positive standard dose-response 

46 curve for carrier-bound infectivity. 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

3

1 

2 b. Suspension-based, animal infectivity assays used in liquid media (e.g., 

3 wastewater). 

4 The Panel agreed that the suspension-based end point dilution animal bioassay is the 

5 most reliable method to be used for final validation of reduction of prion infectivity. 

6 Furthermore, the Panel considered incubation interval animal bioassays as a screening 

7 method to better focus the range of test dilutions necessary for more definitive end point 

8 dilution animal bioassays, with the advantage of reducing cost and use of animals for 

9 testing; however, an end point animal bioassay is necessary to calculate directly a 

10 reduction in infectious titers and cannot be substituted by an incubation interval animal 

11 bioassay. Some Panel members supported designation of a standard animal bioassay 

12 such as a rodent-adapted scrapie strain in a rodent model system for use with all 

13 registrant prion pesticide testing, whereas others considered that testing needed to be 

14 conducted with native prion strains in relevant animal hosts. The Panel urged EPA to 

15 monitor closely the particulars of testing methods for uniformity of treatment of control 

16 and experimental animals, preparation of prion inocula, sufficiency of animal numbers 

17 used relative to the variance of the bioassay model system, and determination of the 

18 bioassay end points. Necropsies of all test animals for the presence of TSE disease based 

19 on Western blot detection of protease resistant prion and neuropathologic changes 

20 consistent with TSE are required. 

21 

22 c. Any other known test methods for evaluating the efficacy of pesticide 

23 products used on either environmental surfaces or in liquid media. 

24 The majority of the Panel concluded that in vitro immunodetection assays or cell-based 

25 bioassays are not adequate for use in assessing reduction in infectivity for evaluating 

26 efficacy of pesticide products with prion related claims (hereafter referred as prion 

27 pesticide products). In the case of immunodetection assays, these do not measure 

28 infectivity; and in the case of cell-based bioassays, these have not been adapted for the 

29 application of assessing reduction in infectivity. A minority contended that in vitro 

30 amplification immunodetection assays such as protein misfolding by cyclical 

31 amplification (PMCA) and cell-based bioassays can serve as a complementary assay to 

32 animal bioassays allowing assessment of many more prion strains and prion substrates 

33 than is practical with animal bioassays. 

34 

35 The Panel recommends to EPA that end point dilution animal bioassays be used to assess 

36 reduction in infectious titer by treatment of samples with prion pesticide products. 

37 

38 3. Efficacy Guidance Performance Criterion Issue: Are the product performance 

39 criteria specified in the EPA draft guidance document to support the registration of 

40 pesticide products with prion-related claims scientifically sound? In particular, the draft 

41 efficacy guidance document recommends a target efficacy criterion of six (6) logs of 

42 reduction of infectivity in the treated versus untreated (control) groups. 

43 

44 The Panel was in general agreement that some widely used model systems, such as the 

45 263K or Sc237 laboratory rodent-adapted scrapie prion strains have sufficiently high 

46 infectivity titers of brain tissue, making it possible to detect a six (6) log reduction in 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

4

1 infectivity. However, several factors may reduce the dynamic range of detection for 

2 prions from particular prion strains or natural hosts to much less than six (6) logs. These 

3 factors include 1) limited volume of about 25μl for intracranial injection in laboratory 

4 rodents, 2) dilution of inoculum by inactivation or other reagents prior to animal 

5 bioassays, and 3) lower infectivity of naturally occurring prion strains from the natural 

host in the range of 105

 to 108 6 ID50/g resulting in a dynamic range for animal bioassays of 

7 three (3) or four (4) logs. 

8 

9 Prion pesticide registrant’s claims of efficacious log reductions in infectivity will need to 

10 be accompanied by a statistical methodology, assessment of assay variability for a 

11 desired level of confidence, and regression analysis models for endpoint animal 

12 bioassays. Although suspension-based methods have a rational basis for calculating log 

13 reductions in prion infectivity, carrier-based methods provide further issues for 

14 calculating log reduction. Infectious brain material serially diluted and dried onto 

15 carriers may not retain infectivity proportional to that in the original sample thereby 

16 negating the endpoint animal bioassay calculation of log reduction in infectivity. 

17 

18 In addition, the target degree of reduction in infectivity may need to be considered in 

19 light of the proposed application or use, risk to human and animal health, and what is 

20 achievable and can be validated for a particular prion pesticide. A reduction of six (6) 

21 logs of infectivity in waste water may be adequate to reduce the infectivity compared to 

22 the risk posed by chronic wasting disease (CWD) prions to human health, whereas the 

23 efficacy of a six (6) log reduction for Creutzfeldt-Jakob disease (CJD) prions may not be 

24 adequate to reduce the potential that a human patient might contract CJD from a surgical 

25 instrument. Risk assessments may be available to help formulate expected standards of 

26 performance, but a one-size fits all product performance guidance may be neither valid 

27 nor desirable. 

28 

29 In conclusion, the Panel recommends that target reduction in infectivity be assessed in a 

30 relevant animal model using the naturally occurring prion strain from the natural host to 

31 support the specific label claim made by the manufacturer of the prion pesticide. To 

32 demonstrate target efficacy, the level of reduction should be set at the maximum 

33 measurable reduction with the designated natural host prion strain in the designated 

34 model. No consensus was reached on acceptability of target efficacy of a six (6) log 

35 reduction in a widely used model such as the hamster-adapted 263K or Sc237 scrapie 

36 prion strains as the sole test for pesticide efficacy of reduction of infectivity for a target 

37 prion strain; however, there was consensus that a widely used model could be used in 

38 addition to the target prion strain to show the possibility of higher reduction levels of 

39 prion infectivity. 

40 

41 4. Efficacy Guidance Labeling Claim Issue: Are the labeling claims described in the 

42 EPA draft guidance document scientifically appropriate based on the recommended test 

43 systems and product performance standard? This guidance: “Has been demonstrated to 

44 reduce infectivity of prions (TSE agents) based on testing using (insert type of organism 

45 in which the prions were raised) (insert prion type)” is careful to avoid claims normally 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

5

1 applied to microorganisms, e.g., “destroy,” “mitigate,” “eliminate,” “control” which may 

2 be misleading when applied to prions.” 

3 

4 The Panel agrees with EPA’s proposal to frame the label claim statement as “reduce 

5 infectivity,” but even with this relatively conservative claim, there are several caveats. 

6 From a scientific perspective, the state of “complete elimination of infectivity” is 

7 probably impossible to achieve in practice, because minimal infective doses of prions are 

8 not known, particularly for humans. In addition, experimental observations suggest that 

9 treatments which reduce TSE infectivity may lead to incubation periods that fall outside 

10 the linear range of animal bioassays. The phrase “reduce infectivity” is valid only as long 

11 as the method used to measure infectivity has an acceptable dynamic range of detection 

12 in a relevant animal bioassay system. 

13 

14 Care will need to be taken to state accurately prion pesticide product performance to 

15 guide user understanding and expectations for the prion pesticide product. Users in 

16 hospitals concerned about CJD may expect something closer to “elimination of 

17 infectivity” on environmental surfaces, whereas the veterinary and wildlife management 

18 community may be more comfortable with the “reduced infectivity” approach. EPA 

19 might require registrants to discuss the implications of the “reduce vs. elimination” 

20 approach in sufficient detail so that users can understand and appreciate what the product 

21 can and cannot do in relation to intended uses, e.g., decontamination of hospital 

22 environmental surfaces, decontamination of laboratory working surfaces, application on 

23 porous materials such as fences, etc. 

24 

25 With respect to specificity of the labeling, the Panel suggests that the prion strain and 

26 original host, bioassay animal host, and observed log reduction in infectivity be present in 

27 the label as well as the dynamic range of the animal bioassay used. Concerning whether 

28 a six (6) log reduction in infectivity is needed to receive approval as efficacious, it was 

29 noted that even a five (5) log reduction would be acceptable for a product with a prion30 related claim under the above conditions specified on the label for prion strains and 

31 natural hosts for which five (5) log is the maximum attainable reduction in infective titer 

32 possible in the animal bioassay. EPA is urged to have scientific advisement to seek a 

33 consensus compilation of acceptable prion strain/animal bioassay models. In particular, 

34 this activity might help guide registrants away from choosing model systems with low 

35 levels of prion infectivity to test their product, thereby preventing inappropriate testing 

36 for prion pesticide products. 

37 

38 In conclusion, there was general consensus among the Panel members that the label give 

39 sufficiently detailed and accurate information to support the manufacturer’s label claim 

40 for reduction in prion infectivity and to enable the product user to assess how efficiently, 

41 and under what usage, the prion pesticide product reduces prion infectivity. 

42 

43 5. Efficacy Guidance Hierarchy Issue: Do different prion types exhibit variation in the 

44 degree of resistance to inactivation by pesticide chemicals and can a hierarchy of 

45 resistance by prion type be reliably determined at this time? 

46 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

6

1 There is clear scientific basis for distinguishing various prion strains based on distinct 

2 incubation time and lesion profiles in a given host as well as biophysical and chemical 

3 properties. However, there are limited data on the relative resistance of prions from 

4 various species and strains to chemical inactivation. Furthermore, a simplified discussion 

5 of chemical reduction of prions infectivity is complicated because different prion 

6 pesticide products may act by differing mechanisms causing reduction of infectivity in 

7 different and unpredictable ways. With the understanding that there may be variability in 

8 assessment of reduction in infectivity based on the prion strain and animal bioassay used 

9 by registrants, the Panel considered the advisability of using one animal bioassay or a 

10 combination of two animal bioassays to assess prion pesticide efficacy. For the 

11 combined assay approach, one of these animal bioassays could be specific to the samples 

12 proposed for specific use of the prion pesticide, whereas the other could be a standard, 

13 widely-used model, e.g. 263K scrapie prion in hamsters. The majority of the Panel 

14 members favored using only one animal bioassay in an end-point titration format. The 

15 single animal bioassay needs to be robust and specific to the specified decontamination 

16 objectives. 

17 In conclusion, the consensus from the Panel is that no single hierarchy of strain resistance 

18 to prion pesticide action can be established at this time. 

19 

20 EPA’s Responses to the SAP’s Comments

21 

22 EPA’s responses to the SAP’s comments (both in the Executive Summary and in 

23 the body of its meeting summary) concerning Charge Questions #1-5 are found after the 

24 “Panel Response” for each Issue and Charge Question below. 

25 

26 =============================================================== 

27 

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

29 Issue of Whether Prions Are a ‘Pest’ under the Federal Insecticide, Fungicide, and 

30 Rodenticide Act (FIFRA),” adequately identifies and summarizes available, relevant 

31 scientific studies. 

32 

33 Charge Question 1. Please comment on the accuracy of the characterization of the 

34 nature of prions, and the adequacy of the review of the relevant scientific information to 

35 support that characterization, as presented in EPA’s draft paper, “Scientific Information 

36 Concerning the Issue of Whether Prions Are a ‘Pest’ under the Federal Insecticide, 

37 Fungicide, and Rodenticide Act (FIFRA).”

38 

39 Panel Response 

40 

41 The Panel discussed the nature of prions described in the interagency White paper for the 

42 accuracy of its scientific characterization of prions and adequacy of the scientific 

43 information cited. The White paper is divided into three Parts with Part I concerning 

44 EPA’s reasoning for reviewing the prion literature with respect to declaring prions as 

45 ”Pests” under FIFRA, Part II 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

7

1 summarizing the prion literature with respect to description of TSEs and prions, key 

2 characteristics of prions, the prion hypothesis and normal prion protein, neurotoxic 

3 mechanisms in TSEs, TSE diseases, history of TSE diseases, transmission and routes of 

4 exposure for TSEs, occupational safety procedures and decontamination methods, and 

5 Part III examining the science of prions in relation to the FIFRA definition of “Pests”. 

6 The authors of the White paper have done a tremendous amount of work assembling this 

7 information and are commended for summarizing a great deal of prion literature. In 

8 general, the Panel found the White paper to be comprehensive and most of its content to 

9 be accurate. As a review of available scientific literature on TSEs and prions, the White 

10 paper was accepted as adequate by the majority of the Panel, pending incorporation of 

11 suggested revisions presented herein. It was noted by several Panel members that there 

12 are many statements in the White paper needing revision to accurately reflect what is 

13 known and what is reasonably speculated about prions and TSEs. One Panel member 

14 found the White paper to be strongly biased for the prion hypothesis relative to the viral 

15 hypothesis and lacking in relevant information. 

16 

17 The Panel questioned EPA representatives at the Meeting about Part I. Introduction 

18 and Background to better understand the EPA’s intent for the Panel’s review process. 

19 There are some inconsistencies in the material presented in this Part compared to Part III 

20 of the White paper. One Panel member concluded that Part I read like there is a 

21 presumption that prions will be recognized as “pests” with the assertion that prions share 

22 key characteristics with other microorganisms, but in Part III the authors of the White 

23 paper did not appear to be entirely convinced that their “pest” argument would withstand 

24 scientific scrutiny. This member thought that Part I might be more correctly titled 

25 “Prions and Consequences for Public Health.” 

26 

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

28 Science of Prions as It Relates to the FIFRA Definition of “Pests” the Panel’s 

29 comments are provided as general comments immediately below and specific comments

30 in the subsequent section. 

31 

32 General Comments

33 For Part II. The Science of Prions, one Panel member commented that in multiple areas 

34 of the White paper too much credence is given to the Legname paper as proof of the 

35 prion hypothesis (Legname, Baskakov et al. 2004). First, the synthetic fibrils accelerated 

36 neurodegenerative disease only in transgenic animals that overexpress (by 16-fold) the 

37 mutated prion transgene (PrP90-231), and not in wild type mice; the transgenic mice that 

38 were used become spontaneously ill even without any inoculation whatsoever, albeit at a 

39 slower rate. Moreover, this paper and the subsequent literature lacks the negative control 

40 most critical to establishing that the synthetic amyloid contained prions, i.e., proof that 

41 prions are not spontaneously accumulating in mock-inoculated control transgenic mice. 

42 Thus, it remains unclear whether the synthetic amyloid initiated, or merely accelerated, a 

43 spontaneous, ongoing pathogenic process that was due to massive overexpression of the 

44 transgene. Infectious agents, i.e. prions, should be initiators rather than mere accelerants 

45 of disease. A much more important study of the primacy of prion conversion in prion 

46 propagation that is not appropriately emphasized in the White paper is the Deleault study 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

8

1 (Deleault, Harris et al. 2007) in which prions were amplified by PMCA containing 

2 normal prion protein (purified from brain) and polyanions, a study which strongly 

3 indicates the importance of non-prion polyanions, i.e., RNA or heparan sulfates, in prion 

4 replication. 

5 

6 A. Description is a good review of the “protein only” hypothesis. Although the “virus” 

7 hypothesis is mentioned, one Panel member thought that this needs to be discussed in 

8 more detail. Also, there was general agreement with the hypothesis that cofactors 

9 (polyanion/sugar/nucleic acids) play a critical role in the process and also need to be 

10 described. A Panel member suggested that Griffith (Griffith 1967), not Alper (Alper, 

11 Cramp et al. 1967) or Prusiner (Prusiner 1982), should be more prominently portrayed as 

12 the originator of the protein only hypothesis and that this section should be based on a 

13 more accurate history. 

14 

15 B. Key Characteristics 

16 

17 1. Prion Protein and Terminology

18 One Panel member suggested that including a brief summary of yeast prions could be 

19 beneficial to emphasize the more general scope of the prion phenomenon, and another 

20 thought that the definition of prion must emphasize infectivity rather than the mere 

21 misfolding of prion protein; many misfolded forms of prion protein are not (infectious) 

22 prions. 

23 

24 2. Current Status of “Protein Only” Hypothesis 

25 One Panel member commented that the “protein only” hypothesis for prions, although it 

26 does provide a logical explanation for many of the characteristics of this agent, does not 

27 fit exactly with “infectivity” when the context of the discussion centers on the generation 

28 of increasing amounts of the abnormal isoform. The “recruitment and conversion” 

29 explanation appears to be a more logical description of the process. Another Panelist 

30 disagreed that the prion hypothesis is a logical or adequate explanation of TSE 

31 infectivity, but rather is circular reasoning. 

32 

33 3. Normal Prion Protein

34 One Panel member commented that the discussion of the expression (homozygosity vs. 

35 heterozygosity) at codon 129 of the prion protein is an important topic. This could be 

36 expanded with regards to vCJD, where there is good epidemiologic information from the 

37 United Kingdom on apparent susceptibility or resistance and its impact on the incidence 

38 of the disease in that population. It should also be expanded to scrapie, where it is well 

39 known that certain genotypes of sheep are more resistant or susceptible to developing 

40 scrapie. Another Panel member indicated that the physiological role of the prion protein 

41 is not discussed sufficiently (synaptic transmission, T cell activation, circadian rhythm, 

42 olfactory physiology). 

43 

44 4. Neurotoxic Mechanism 

45 Although this topic is interesting, one Panel member agreed with the White paper authors 

46 that we need to learn more about the pathology of TSEs. Despite some in depth 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

9

1 knowledge about important aspects of the neurotoxic mechanism, we have an incomplete 

2 understanding of the process as a whole. Also in this section, the authors exclusively talk 

3 about degradation of prions in “proteasomes”, but do not mention “lysosomes” as 

4 important intracellular sites of prion degradation. In addition, it was noted that prion 

5 amyloid cannot be considered responsible for neurotoxicity as there is no correlation 

6 between prion amyloid plaques and neuronal death, and furthermore, one Panel member 

7 noted that the requirement for endogenous normal prion protein for toxicity has not been 

8 proven, as is implied in the White paper. 

9 

10 C. TSE Diseases 

11 

12 The Panel generally agreed that this is a good discussion and review of the known TSE 

13 diseases in animals and humans. However, one Panel member noted that the statement 

14 that most prion diseases in humans are “acquired” needs to be rephrased. When one 

15 considers the epidemiology of CJD, the overwhelming majority of cases are identified as 

16 sporadic, with the next largest category being familial or inherited CJD. Rather, the 

17 statement can be clarified to say that certain TSEs (kuru and vCJD) appear to be 

18 “acquired” as a result of “exposure” via ingestion of the prions. There also needs to be 

19 some discussion about the period between exposure and the onset of symptoms in this 

20 section of the document. 

21 

22 D. History of TSE Disease 

23 

24 This section of the review presents a reasonable summary of the history of TSE diseases. 

25 

26 E. Transmission and the Potential Routes of Exposure 

27 

28 One Panel member felt that an important point missing from the discussion of 

29 transmission is the differing lengths of time between “exposure” and onset of symptoms 

30 as related to the portal of entry. Direct inoculation of prion-contaminated material into 

31 the central nervous system (CNS) results in a relatively shortened time interval, whereas 

32 there is a considerably longer interval between exposure and onset when the prions enter 

33 at a site considered as “peripheral.” 

34 

35 F. Occupational Safety Procedures 

36 

37 One Panel member thought that this section needs to be discussed in more depth. This 

38 section also provides a good opportunity to discuss the “Chain of Infection,” a concept 

39 that explains why infectious diseases occur “when and where” they do. For example, just 

40 because there is infectivity potentially present in some source at some time, this does not 

41 automatically mean that transmission will occur and infection will ensue. There is very 

42 little evidence to support the notion that prions can be acquired from environmental 

43 surfaces. In spite of this, it is prudent to keep work areas and surfaces clean and 

44 decontaminated, especially if one is working with known infectious materials. 

45 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

10

1 With regard to the six cases associated with neurosurgical instruments and devices, there 

2 is only strong evidence of prion transmission in those cases associated with the use of 

3 stereotactic depth electrodes. In the opinion of one Panel member, the four cases 

4 presumably associated with neurosurgical instruments are not very well described, and 

5 there are very few details on the methods used to clean and reprocess the instruments. It 

6 is also important to note that the last of the instrument or device cases was reported in 

7 1976. 

8 

9 G. Decontamination Methods 

10 

11 One Panel member noted that the history of development of decontamination methods is 

12 interesting and provides a logical explanation of why experiments were designed as they 

13 were. Veterinary scientists attempted to deactivate prion infectivity while retaining 

14 immunogenicity such that these “disinfected” prion preparations could be used to 

15 immunize sheep against scrapie. Clearly, the objective was very different than modern 

16 day objectives of eliminating prion biomass by decontamination. This Panel member 

17 indicated that there is increasing interest in exploring prion decontamination methods that 

18 more closely resemble reprocessing methods currently used in modern hospitals and 

19 laboratories for which the objective of sterilization is to remove patient derived 

20 biomaterials and bioburdens as completely as possible, making medical instruments safe 

21 for use with subsequent patients. New technologies are being evaluated in this regard, 

22 and some of these should be discussed here. An additional complicating factor is that 

23 strain diversity in natural populations and its impact on decontamination is not known. 

24 

25 It was noted that the susceptibility of humans to infection with CWD prions is not known. 

26 Currently, there is no epidemiological evidence to suggest that CWD has been 

27 transmitted to humans. Experimentally, CWD has not been successfully transmitted to 

28 transgenic mice expressing human prion protein. Human prion protein can be converted 

29 to a proteinase K resistant form in vitro by CWD prions; however, this conversion is 

30 much less efficient compared to species with known susceptibility to CWD. Finally, 

31 non-human primates are susceptible to intracerebral CWD infection. Recent evidence 

32 suggests that more than one strain of CWD may exist, complicating the interpretation of 

33 previous work involving human susceptibility to CWD infection. 

34 

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

36 of a “Pest” extensively and had a divided opinion about whether the scientific 

37 understanding of the nature of prions supports its classification as a “Pest” as specified in 

38 FIFRA Sections 2(t) or 25(c)(1). The major opinion was that there is sufficient scientific 

39 evidence to support this classification, whereas a minority of Panel members did not 

40 support this view. 

41 

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

43 

44 The Panel was in general agreement that prions are pathogenic and infectious, may have 

45 some similarity to viruses, are present in the environment resulting in human exposures, 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

11

1 might have a non-host encoded component, induce innate immune responses and are 

2 transmissible infectious agents. 

3 

4 A minority of Panel members concluded that prions do not possess features typical of 

5 microorganisms. 

6 

7 One Panel member agreed that prions are deleterious to animal and human health, but 

8 disagreed that prions can be classified as “other microorganisms.” In order to do this, the 

9 White paper authors would need to emphasize the “unknown virus” hypothesis. This 

10 Panel member stated that prions do not reproduce and they do not grow. The normal 

11 prion protein exhibits turnover as a normal function, but prions accumulate. Are prions 

12 alive? This panel member thought the answer was “no.”

13 

14 Another Panel member suggested that whether scientific studies support classification of 

15 prions as pests depends on the interpretation of what “pests” are. In particular the second 

16 part of the definition of “pests” as “any other form of ... virus, bacteria or other 

17 microorganism....” is problematic because prions lack the characteristics of viruses or 

18 bacteria and the meaning of “other microorganisms” implies a living microbe, which this 

19 discussant believed was not scientifically supported. Prions are primary proteinaceous 

20 substances and have some characteristics in common with self-assembling structural 

21 components of bacteria such as the multimeric extracellular curli protein of the bacteria 

22 Escherichia coli (Wang and Chapman 2008), but prions lack the overall multi-component 

23 complexity and organization that are hallmarks of microorganisms. 

24 Instead of being microorganisms, prions could be classified as “infectious substances” 

25 under the definition of “biological agent” in Code of Federal Regulations Title 42 Public 

26 Health Section 73.1 (42CFR). Biological agents are defined as “any microorganism or 

27 infectious substance capable of causing death, disease, or other biological malfunction in 

28 a human, an animal, a plant, or another living organism; of deleterious alterations of the 

29 environment”. “Infectious substances” of Title 42 consist of microbial, plant and animals 

30 toxins, and many of these, like prions, are entirely proteinaceous substances (7CFR). 

31 Protein toxins do many things that prions do. Protein toxins exhibit host and cell-type 

32 specificity of intoxication, use host cellular machinery for their intoxication mechanisms, 

33 and can resist endosomal protein degradation pathways (Watson and Spooner 2006). 

34 Some protein toxins are stable in the environment and can enter the host via ingestion 

35 (Wobeser, Baptiste et al. 1997). Protein toxins recruit normal host cell proteins to adopt 

36 different conformations in a way that causes injury to cells and organs (Fabbri, 

37 Travaglione et al. 2008). Ingestion of protein toxin infected tissues can result in 

38 transmission of infection to new susceptible hosts (Wobeser, Baptiste et al. 1997). Prions 

39 do not share extensive similarities to bacteria or viruses, but prions share many distinctive 

40 features of protein toxins. It appears that prions might be best classified as “infectious 

41 substances” rather than “other microorganisms”. This Panel member concluded that 

42 attempts to classify prions as “other microorganisms” as a means to gain regulatory 

43 oversight is not supported scientifically. 

44 

45 A majority of Panel members supported the view that prions can be categorized as “other 

46 microorganisms.” 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

12

1 

2 One Panel member commented that when considering the issue of whether or not prions 

3 fit the definition of a microorganism, the White paper concludes that prions do not 

4 reproduce, adapt, grow or move. However, when viewed from the point of view of TSE 

5 biology, these conclusions are inaccurate. Research spanning the last 50 years leaves no 

6 doubt that the agents which cause prion disease can reproduce, adapt, grow and move 

7 within the host. 

8 

9 Another Panel member commented that replication of prions has been amply 

10 demonstrated by the fact that they can be titered and that animals infected with a low 

11 level of prion infectivity eventually accumulate exponentially higher levels of infectivity. 

12 This can only occur if prions can replicate themselves. As demonstrated by the isolation 

13 and careful characterization of multiple mouse and hamster-adapted sheep scrapie strains, 

14 prions can adapt to new species. Peripheral inoculation of TSE infectivity eventually 

15 leads to infectivity in the brain, something that can only occur if the infectious agent 

16 moves within the host. Finally, prions most definitely exhibit a stable steady state over a 

17 long period of time (maintenance). Established stocks of TSE infectivity can be 

18 maintained for decades with no change in either their titer or their biological 

19 characteristics. 

20 

21 A third Panel member said that defining bacteria and viruses as microorganisms does not 

22 depend upon the very different mechanisms they use to replicate, spread, and adapt. 

23 Likewise, defining TSE agents as microorganisms should not depend upon the 

24 mechanisms underlying their replication and spread. Rather, it should be based upon 

25 whether or not their general biological characteristics are consistent with that of a 

26 microorganism. It is only in trying to reconcile the proposed protein-only mechanisms 

27 underlying TSE pathogenesis with the universally accepted nucleic acid based 

28 mechanisms of more conventional infectious organisms that defining prions as 

29 microorganisms becomes more complex. 

30 

31 Another Panel member suggested re-writing Part III to better reflect the characteristics of 

32 prions similarity to other microorganisms, such as viruses. Prions grow, replicate, adapt, 

33 maintain, move and are organized. The discussion should be centered around Koch’s 

34 postulates, instead of a dictionary definition of microorganism or life. 

35 

36 Another Panel member stated that TSE agents, i.e., prions, are environmental pathogens 

37 that infect mammals, replicate virus-like, cause disease according to Koch’s postulates, 

38 though the infectious agent has not been sufficiently purified. 

39 

40 One Panel member stated that the virus hypothesis of TSE infectivity is in accord with a 

41 classification of the TSE agent as a microorganism. It was suggested that Part III needs 

42 to discuss Koch’s postulates as they relate to prions. Important considerations for FIFRA 

43 being: 

44 • Infectivity: Kuru epidemic stopped with end of ritual cannibalism, United 

45 Kingdom BSE epidemic markedly decreased with removal of external 

46 source, endemic scrapie, & environmental CWD 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

13

1 • TSE agents as environmental pathogens, not spontaneous generation of 

2 infectivity 

3 • Strains of TSE agents as: 

4 o Encode individual properties: obvious in controlled host genotype 

5 background 

6 o Classified by incubation time and by neuropathology (lesion 

7 profile) 

8 o Agents often maintain strain identity even after passage in different 

9 species, e.g., sCJD to mice 

10 o Most individual agents not separated by abnormal prion band 

11 profiles on blots 

12 

13 B. Are Prions Alive? 

14 

15 One Panel member concluded that instead of focusing so much on the vague question of 

16 whether prions are living microorganisms, it seems that the prions as pests discussion 

17 might be more fruitfully oriented towards the discrimination between prions as pathogens 

18 (which propagate in the host) or toxins (which don’t propagate). 

19 

20 In conclusion, a majority of the Panel agreed that once revised to incorporate revisions 

21 suggested by the Panel the White paper will adequately identify and summarize the 

22 scientific literature concerning prions and that prions are “other microorganisms” 

23 exhibiting some features of living entities. 

24 

25 Specific Comments

26 With respect to specific comments and editorial remarks on the White paper, the Panel 

27 noted the following specifics: 

28 1. Please add Castilla et al., 2005; Delault et al., 2007 and Sigurdson et al., 2009 to 

29 the literature list in section II, A: 

30 Castilla, J., P. Saa, C. Hetz, and C. Soto. 2005. In vitro generation of infectious 

31 scrapie prions. Cell 121:195-206. 

32 Deleault, N. R., B. T. Harris, J. R. Rees, and S. Supattapone. 2007. Formation of 

33 native prions from minimal components in vitro. Proceedings of the National 

34 Academy of Sciences of the United States of America 104:9741-6. 

35 Sigurdson, C. J., K. P. Nilsson, S. Hornemann, M. Heikenwalder, G. Manco, P. 

36 Schwarz, D. Ott, T. Rulicke, P. P. Liberski, C. Julius, J. Falsig, L. Stitz, K. 

37 Wuthrich, and A. Aguzzi. 2009. De novo generation of a transmissible 

38 spongiform encephalopathy by mouse transgenesis. Proc Natl Acad Sci USA 

39 106:304-9. 

2. Page 2, 1st 40 paragraph: 

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

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

43 encephalopathy. 

3. Page 2, 2nd 44 paragraph: 

45 a. The “by-passing of the proteasome” does NOT produce TSE clinical features. 

46 Please rephrase. 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

14

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

2 Please modify. 

3 c. The statement that “the property of infectivity appears to reside in the beta 

4 sheet-rich conformation” is a great oversimplification. In fact many beta5 sheet-rich forms of prion have been generated that have no infectivity. 

4. Page 3, 1st 6 paragraph: 

7 a. The use of the word transcript in the sentence “misfolded transcripts recruit 

8 and convert normal prion transcripts” is incorrect. Prion protein would be 

9 more correct. 

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

11 c. It is not the “recruit and convert” mechanism that separates prions from other 

12 protein misfolding diseases; it is their relative transmissibility between 

13 individuals. 

14 d. Plaques are not linked to cell death 

5. Page 3, 2nd 15 paragraph: 

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

17 b. Correct spelling of “plaque” in line 8 

18 c. Take out sentence on Legname et al., 2005 

6. Page 3, 3rd 19 paragraph: There are still cases of bovine spongiform encephalopathy 

20 (BSE) in the UK. Wrong statement, that BSE “appears to have effectively 

21 ended.” 

22 7. Page 3, last paragraph: The apparent molecular weight of prion-associated 

23 fragments can range from ~17,000 to 37,000. 

24 8. Page 4, first paragraph: 

a. The term PrPd 25 is prion that is present in diseased animal and is not detected in 

26 uninfected animals using immunohistochemistry or immunoelectron microscopy. 

27 b. PrP-res has been used to connote a “purified protein or recombinant 

28 polypeptide fragment” resistant to PK. This is incorrect. 

29 c. It needs to be stated that “prion is infectious, not only “misfolded.” 

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

31 f. Replace “infectious conformation of transcripts” by “misfolded 

32 PrP” and also “prion” should refer to “infectious particle” whether it is 

33 constituted solely of prion or not 

9. Page 4, 2nd 34 paragraph: 

35 a. Reference “Colby et al, 2007” is wrong 

36 b. Wrong statement regarding Legname manuscript: Fibrils consisting of 

37 recMoPrP(89-230) were inoculated intracerebrally into transgenic (Tg) mice 

38 expressing MoPrP(89-231). 

b. The explanation regarding the domains of interactions between PrPC and PrPSc 39 

40 is very narrowly focused. Other studies, such as those by Horiuchi et al., 2001 

41 and Chabry et al., 1998 on the localized interactions between the two prion 

42 isoforms should be included. 

10. Page 4, 4th 43 paragraph: 

44 a. The statement "The Prnp gene appears to be conserved across species from 

45 yeast to primates" is incorrect. Prnp is conserved among mammals and also to 

46 amphibians and reptiles. More distantly related proteins are present in fish. Yeast 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

15

1 and fungal prions are unrelated to mammalian prions on a sequence level, but are 

2 so named because they share the characteristic of being able to adopt two 

3 different conformations, one of which leads to aggregation. 

4 b. The word “homology” is used incorrectly. This should be replaced with 

5 “sequence identity” or in the case with doppel to indicate if normal prion protein 

6 and doppel are homologous. 

11. Page 5, 2nd 7 paragraph: There is just as much evidence that copper inhibits prion 

8 protein conversion as there is that copper stimulates conversion. 

12. Page 5, 4th 9 paragraph: Prion function involves olfactory physiology (Le Pichon et 

10 al., 2009) etc. 

13. Page 5, 5th 11 paragraph: 

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

13 prion protein. 

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

15 14. Page 5, last paragraph and page 6, first 4 paragraphs: 

16 a. The literature interpretation in relation with the neurodegenerative mechanism 

17 is incorrect. Plaques or immunohistochemically detectable prion aggregates are 

18 not neurotoxic. Several groups have shown that peptide models of prion (Haik et 

19 al. 1998) and small prion oligomers are neurotoxic (Kazlauskaite et al. 2005, 

20 Novitskaia et al. 2006, Simoneau et al. 2007) in the presence or absence of prion 

21 expression. Therefore the current view is that prion-related neurodegeneration 

22 might have two components: a loss of function of prion protein, and a gain of 

23 toxicity of prion oligomers (independent of neuronal prion expression). 

24 b. In the three references cited (Brandner, Mallucci and Chesebro), the form of 

25 prion that was detected was large, non-toxic aggregates and does not demonstrate 

26 that the absence of prion protein expression is responsible for the absence of 

27 toxicity. In Brandner, the absence of prion expression by the mice prevented the 

28 generation of toxic prion oligomers. In Mallucci, prion was largely sequestered in 

29 astrocytes, and no reactive prion oligomers were generated, again because of the 

30 absence of prion expression by the mice. In Chesebro, prion plaques are not 

31 toxic, but again it has nothing to do with the absence of cell surface prion protein 

32 expression. It is suggested to remove these three references and cite the two 

33 currently accepted components of neurodegenerative mechanism, with the 

34 appropriate references. 

15. Page 6, 4th 35 paragraph: “GTI” should be changed to “GPI”. It should also be noted 

36 that these mice have approximately 1 log lower brain titers and that the disease is 

37 altered not prevented. 

38 16. Page 7, Table 1: The table should be divided into “infectious’, “sporadic”, and 

39 “inherited” prion diseases. Also, the disease is called “fatal familial insomnia, 

40 FFI”, Chronic wasting disease of “deer, elk and moose.” 

17. Page 8, 1st 41 paragraph: 

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

43 b. Please add “France” to the other contaminated European countries. In France, 

44 13 cases of BSE were already diagnosed in 1995. 

45 c. Canada and USA had their first indigenous cases in 2003 and 2004, 

46 respectively. 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

16

1 d. Canada not only has imported but also indigenous cases of BSE. Should be 

2 deleted from last sentence. 

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

19. Page 9, 3rd 4 paragraph: 

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

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

7 90%). 

20. Page 10, 1st 8 paragraph: add “intralingual, intranasal and intrasciated” to the route 

9 of applications. 

21. Page 10, 2nd 10 paragraph: 

11 a. Consumption of contaminated food is generally not considered to be involved 

12 in transmission of scrapie. Mention that both vertical and horizontal routes are 

13 likely natural scrapie transmission. 

14 b. “prionuria”: remove Ligios et al., 2007 ─ add Gabizon and Gregori literature. 

22. Page 10, 4th 15 paragraph: Maternal transmission of BSE and CWD is rather 

16 questionable. 

23. Page11, 1st 17 paragraph: 

18 a. The interpretation of the study by Heaton et al, 2008 is incorrect. The study 

19 concludes that the prevalence for the E211K variant is extremely low (less 

20 than 1:2000 cattle) and, therefore, additional atypical BSE cases with K211 in 

21 the US will be very low. 

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

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

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

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

26 reported. 

25. Page 12, 4th 27 paragraph: The citation for the Guideline for Handwashing and 

28 Hospital Environmental Control is incorrect. This guideline has different authors 

29 and was released in 1985. 

30 26. Page 14, 2nd paragraph: There is no evidence to support the statement "prions 

31 bind tightly to stainless steel and are most difficult to inactivate on this surface." 

32 Prions do indeed appear to bind tightly to stainless steel and are difficult to 

33 remove, but there are very limited comparative studies of other surfaces. 

34 27. Page 14, last paragraph- to page 15: In the context of decontamination a 

35 limitation can be the intrinsic limited sensitivity of the bioassay model, rather than 

36 a problem of the lifespan of the animals. 

28. Page 15, 3rd 37 paragraph: BSE prions “readily” transmit to felines, but not to 

38 humans. So far, 210 people out of the millions that were presumably exposed to 

39 BSE have gotten vCJD. 

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

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

42 Lehninger. 

43 31. Throughout manuscript the authors use regularly “mad cow disease”; they should 

44 use “Bovine Spongiform Encephalopathy, BSE” instead. 

45 32. It is not clear if 263K or 301V recapitulate the scrapie and BSE agent, 

46 respectively. 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

17

1 33. In general, the word “plaque” is not used correctly. The generalization of TSEs as 

2 diseases where neuronal plaques are made is not precisely correct. In fact, 

3 plaques are not the primary phenotype in the majority of cases of the most 

4 common human TSE, sporadic CJD. It would be more accurate to define TSE 

5 diseases as diseases of protein misfolding where, depending upon the type of 

6 TSE, prion can be found in either diffuse “non-amyloid deposits” or in “amyloid 

7 plaques.” 

8 

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

10 

11 Charge question #1 related to the EPA’s White Paper’s review and summary of 

12 the available, relevant scientific literature. A majority of the Panel accepted the EPA’s 

13 White Paper as adequate. However, the Panel cautioned that many statements in the 

14 White Paper need to be revised to reflect accurately what is definitively known about 

15 prions and the TSEs and that which is reasonably hypothesized based on scientific 

16 observations. In addition to several General Comments related to individual sections of 

17 the White Paper, the SAP provided 33 Specific Comments, which in some cases reflected 

18 the opinions of individual panel members rather than a majority or a consensus of the full 

19 Panel. The specific comments identified miscellaneous editing mistakes in the White 

20 Paper, offered guidance on subjects on which the White Paper’s discussion could be 

21 further elaborated, identified issues that some investigators believe remain unresolved, 

22 and offered ways to capture a full spectrum of different scientific points of view. 

23 

24 In various overarching comments, the SAP’s key conclusions were that: 

25 

26 1. A majority of the Panel accepted the prion (or “protein only”) hypothesis with the 

27 caveat that unknown cofactors may also be involved. EPA has made several 

28 revisions in the White Paper that acknowledge this alternative cofactor hypothesis. 

29 

30 2. A majority of the Panel concluded that prions could be included in the category of 

31 “other microorganism” under FIFRA. EPA has made revisions in the White Paper to 

32 acknowledge the SAP’s point of view on this issue. 

33 

34 General SAP Comments: Suggestions for Revision: White Paper Part I. Introduction and 

35 Background. 

36 

SAP page 16, 2nd 37 paragraph. One panel member thought that Part I of the White Paper 

38 might be more correctly titled “Prions and Consequences for Public Health.” 

39 

40 EPA believes that the Part I title—“Introduction and Background”—is appropriate and, 

41 therefore, will remain unchanged. 

42 

43 This same panel member also suggested that Part I presumes that prions will be 

44 recognized as “pests,” but noted that in Part III the White Paper does not appear 

45 “convinced that the ‘pest’ argument would withstand scientific scrutiny.” 

46 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

18

1 EPA’s response is that Part I summarized EPA’s rationale for its September 2003 

2 decision that a prion should be considered a “pest” under FIFRA and presumes only that 

3 past policy decisions will remain in effect until action is taken to change them. Part III of 

4 the White Paper addressed the available scientific information on prions (summarized in 

5 Section II) in regard to the terms “microorganism”, “organism” and “living” as those 

6 terms are used by biologists and the medical community. While Part I remains largely 

7 unchanged, Part III has been changed to include most of the SAP’s comments on this 

8 issue. 

9 

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

11 

12 SAP Page 17. One panel member commented that too much credence was given to the 

13 Legname et al. (2004) paper as proof of the prion hypothesis. Specifically, the paper left 

14 unclear whether the synthetic amyloid [prion protein produced by recombinant DNA in a 

15 cell free, bacterial system] initiates or merely accelerates a spontaneous pathogenic 

16 process in transgenic mice. Furthermore, the panel member did not believe that the study 

17 of Deleault et al. (2007) was sufficiently emphasized. The Deleault et al. study was 

18 thought by the panelist to provide strong evidence of the importance of non-prion 

19 polyanions in the cell free replication of prions. 

20 

21 EPA has evaluated individual instances where the reference Legname et. (2004) was used 

22 in the White Paper. In particular, EPA has added the suggested Deleault et al. (2007) 

23 citation to section II.A., and has modified the related text to deemphasize “purified” prion 

24 protein in these experimental cell free systems. In addition, the alternative point of view 

25 that cofactors may be involved has also been added. 

26 

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

28 

29 SAP page 18. The SAP found that the review of the “protein only” hypothesis is good. 

30 One panel member thought that the virus hypothesis should be discussed and expanded to 

31 include more detail. EPA has added this alternative hypothesis to the White Paper. One 

32 panel member noted that the prion only hypothesis should not be attributed to Alper et al. 

33 (1967). EPA agrees with the panelist, and has modified the text as suggested. 

34 

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

36 Characteristics. Individual general comments are offered individually for each of the 

37 four sections of II.B., as follows: 

38 

39 #1 Prion terminology: One panel member suggested that a review of yeast prions could 

40 be beneficial. EPA also believes that significant research is, in fact, underway using 

41 yeast “prion” protein and recombinant models to resolve the mechanisms of misfolding, 

42 such as described in recent papers by Lindquist (Tessier and Lindquist, 2007; Alberti et 

43 al., 2009). Based on the panelist’s recommendation, references to the Lindquist papers 

44 have been added to Section II.B.2 and Section III (in reference to non-Mendelian 

45 inheritance of phenotypes by “prionogenic proteins” in yeast. 

46 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

19

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

2 

3 Two panel members offered slightly different comments that the protein-only hypothesis 

4 does not fully account for the critical property of infectivity attributed to prions. Their 

5 concerns were repeated in other sections of the SAP meeting summary, and these 

6 comments appear to be at odds with both the SAP’s General support of the prion 

7 hypothesis and especially of the Panel’s comment immediately above under Section 

8 II.B.1. Prion Terminology. In response to these comments, EPA notes that a summary of 

9 recent yeast work (eg, Tessier and Lindquist, 2007; Alberti et al., 2000) specifically 

10 addresses the separate issues of misfolding and infectivity, concludes that the prion 

11 hypothesis accounts for both mechanisms of disease, and emphasizes a growing belief 

12 that the prion phenomenon has a larger general scope than previously realized, as has 

13 been recognized also in the case of “prionoid” behavior in amyloid plaque diseases 

14 (Aguzzi, 2009). EPA has added this information to Section II.A. 

15 

16 #3. Normal Prion Protein.

17 

18 One panel member suggested expanding the discussion of genetic polymorphisms for 

19 codon 129 in the human PRNP gene and extending the discussion to include scrapie. 

20 Another panel member suggested that the normal physiological role of prion protein is 

21 not discussed sufficiently. EPA believes that while both suggestions have merit, 

22 appropriate revision of the White Paper would add considerable length without lending 

23 weight to the decision of whether prions are “pests” or not. 

24 

25 #4. Neurotoxic Mechanism.

26 

27 One panel member agreed with the White Paper that resolution of the mechanisms of 

28 neurotoxicity is incomplete. The panel member further noted that the White Paper failed 

29 to mention the role of lysosomes and that there was no correlation between prion amyloid 

30 plaques and neuronal cell death. Both suggestions have merit; however, introduction of a 

31 lysosomal hypothesis would add considerable length without lending weight to the 

32 decision of whether prions are “pests.” 

33 

34 On the other hand, it is a significant issue and EPA agrees that plaque formation and 

35 neuronal cell death are not proven to be causally related in any of the “amyloid” 

36 neurodegenerative diseases. To avoid a misinterpretation that neuronal cell death from 

37 scrapie protein is caused by plaque formation, the mention of killing neurons has been 

38 deleted altogether. The text has been modified to focus on hypotheses about “disrupting” 

39 normal cell surface protein function as part of the mechanism of disease. 

40 

41 Another panel member noted that the requirement for endogenous normal prion protein in 

42 toxicity has not been proven as implied in the White Paper. In response, EPA points out 

43 that the role of normal PrP in the progression of TSE disease is discussed in response to 

44 Specific Comment #13, below. 

45 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

20

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

2 Diseases.

3 

4 One panel member suggested that the term “acquired” needs to be rephrased and 

5 suggested that the relationship between exposure and onset of clinical symptoms 

6 discussed. EPA has modified the text as suggested regarding “sporadic” and “acquired” 

7 incidents. The relationship between exposure and onset of clinical symptoms is 

8 discussed more fully below in response to section E. Transmission and Routes of 

9 Exposure. 

10 

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

12 Prion Disease. 

13 

14 EPA accepts the SAP’s description of this section as a reasonable summary. 

15 

16 General SAP Comment: White Paper Part II. The Science of Prions. E. 

17 Transmission and Potential Routes of Exposure. 

18 

19 One Panel member noted that discussion of preclinical incubation time between exposure 

20 and onset of symptoms is missing from the discussion. EPA agrees that the often 

21 observed and strikingly “slow” progression of disease from exposure to clinical 

22 symptoms is an important fact, and has noted it Section II.A. of the White Paper. EPA 

23 agrees that the relationship between exposure and onset of symptoms is profoundly 

24 important to diagnosis and treatment of any TSE, but not necessarily to determining 

25 whether a prion may be regarded as a “pest”, which is the primary focus of the White 

26 Paper. EPA also notes that the relationship between titer, exposure and progression of 

27 disease is discussed in the companion document on efficacy guidance. . 

28 

29 General SAP Comment: White Paper Part II. The Science of Prions. F. 

30 Occupational Safety Procedures.

31 

32 One Panel member suggested that this section needs to be discussed in more depth, 

33 including the “chain of infection.” EPA believes that while this section could be 

34 expanded, it probably would not make an additional contribution of determining if a 

35 prion is a “pest.” 

36 

37 One Panel member offered a different opinion on the six cases in the literature of disease 

38 associated with surgical instruments. EPA’s response is that the White Paper only notes 

39 various rationales under which the CDC has issued guidance and – in this instance – the 

40 White Paper simply states there are six documented cases of CJD where an iatrogenic 

41 cause associated with “neurosurgical instruments and devices” has been suspected. 

42 

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

44 Decontamination Methods.

45 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

21

1 One Panel member wanted EPA to add some background information on the history and 

2 development of decontamination methods to the White Paper. EPA does not believe that 

3 adding such information would be useful or germane to the paper. 

4 

5 One Panel member noted that there is no known transmission of CWD to humans. EPA 

6 notes that the White Paper already states this (see discussion of CWD, page 10, paragraph 

7 1). 

8 

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

10 the FIFRA Definition of a “Pest.”

11 

12 Documented by the comments of several individual panelists, the majority of the SAP 

13 supported the view that prions can be categorized as “other microorganisms.” A minority 

14 of the Panel concluded that prions do not possess the typical features of microorganisms. 

15 For example, one Panel member suggested that prions might better fit the classification of 

16 “infectious substances” under the definition of “biological agent” (42 CFR Part 73.1), 

17 that prions share many distinctive features of protein toxins, and that prions do not share 

18 extensive similarities to bacteria or viruses. EPA recognizes that there is a range of 

19 opinions on the issue of whether prions are microorganisms, and has revised its White 

20 Paper to include alternative points of view. 

21 

22 At least one Panel member suggested that Part III.A. needs to discuss Koch’s postulates 

23 as they relate to prions. EPA agrees that applying Koch’s postulates to the characteristics 

24 of prions provides another approach to determining whether prions should be considered 

25 to be microorganisms. Accordingly, EPA has revised Part III.A. of the White Paper to 

26 include a description of Koch’s postulates and a discussion of how the characteristics of 

27 prions relate to each of Koch’s postulates. 

28 

29 One Panel member concluded that part III. should discriminate between prions as 

30 pathogens (which propagate in the host) or toxins (which don’t propagate). EPA has 

31 added this point to part III.C. 

32 

33 Specific Comments:

34 

35 1. SAP comment: Please add Castilla et al. (2005), Delault et al (2007) and Sigurdson et 

36 al. (2009) to II.A. 

37 

38 Response: Castilla et al. (2005) is already cited in II.A. Delault et al. (2007) has 

39 been added to the pertinent citation, as suggested. Sigurdson et al. (2009) has not been 

40 added, as this paper does not appear to be directly related to prion hypothesis. 

41 

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

43 

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

45 

46 3. a. SAP comment: Please rephrase “by-passing the proteasome.” 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

22

1 

2 Response: Text modified. 

3 

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

5 

6 Response: Text modified. 

7 

8 3. c. SAP comment: Oversimplification. 

9 

10 Response: Text modified. 

11 

12 4. a. SAP comment: Transcript misuse. 

13 

14 Response: Correction accepted. 

15 

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

17 

18 Response: What modification the comment intends is not clear. The recent paper 

19 of Aguzzi (2009) is consistent with the White Paper and states the case more specifically: 

“Like the growth of crystals, PrPSc propagates by recruiting monomeric PrPC 20 into its 

21 aggregates . . . . ”. Furthermore, Aguzzi (2009) suggests that this process of recruitment 

22 may be a more genearalized phenomena among the amyloid proteins giving rise to “hints 

23 of prion-like behavior” described as “prionoids.” Thus, Aguzzi (2009) has been added to 

24 the White Paper in reference to a “low level of recruitment and conversion” in White 

25 Paper II.A. 

26 

27 4. c. SAP comment: Infectivity is the discriminating principle that separates prion 

28 diseases from other plaque-forming neurodegenerative diseases. 

29 

30 Response: The White Paper is already consistent with this point, but EPA has 

31 nonetheless modified the text to specify “infectious” conformations to clarify any 

32 possible confusion. 

33 

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

35 

36 Response: Plaques are a typical feature collectively of all the “amyloid plaque” 

37 diseases, all of which also feature cell death: Alzheimer’s, Parkinson’s, etc. It is probably 

38 true, also, that not all individual cases of TSEs feature definitive plaques. Hence, use of 

39 the word “linked” seems appropriate as a general comment, as linkage does not imply 

40 causation. 

41 

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

43 

44 Response: Modification accepted. 

45 

46 5. b. SAP comment: Correct spelling. 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

23

1 

2 Response: Spelling corrected. 

3 

4 5. c. SAP comment: Delete sentence. 

5 

6 Response: Sentence deleted. 

7 

8 6. SAP comment: BSE has not ended. 

9 

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

11 seems to be correct. Text has been modified to state that the potential for long, 

12 unresolved dormancy of prion diseases in humans is a matter of great public concern, 

13 with the risk that prion disease in humans resultant from the BSE epidemic, such as the 

14 corresponding peak of vCJD in 2000, might persist in human populations long after the 

15 consumption of contaminated food ended.. 

16 

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

18 

19 Response: This comment seems to make a statement of fact based on the full 

20 range of original and current literature. However, the comment is directed to an 

21 introductory historical note. Specifically, the characterization of molecular weight given 

22 on page 3 [27,000 to 30,000 daltons] is the original identification of protein that purifies 

23 with the scrapie prion, as specified by Bolton et al. (1982). Furthermore, this molecular 

24 weight range is specified as “in the original description.” 

25 

26 8. SAP comments 8.a. though 8.f. are derived from a single Panelist [see SAP General 

27 Comments, p. 18]. 

28 

8. a. SAP comment: The terms PrPd and PrPres 29 are defined 

30 

31 Response: The Panelist’s definition and the more general statement in the White 

Paper appear to be consistent. Specifically, Brown (2005) defines PrPd 32 as any misfolded 

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

34 

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

36 

Response: The Panelist may be incorrect. Brown (2005) defines PrPres 37 as “a 

proteolytic cleavage product of PrPd 38 . . . when generated following incomplete proteolytic 

digestion in Western blotting.” The White Paper states that “PrPres 39 has been used to 

connote a PrPd 40 polypeptide fragment that features the resistance to proteinase K 

41 digestion.” The text has been modified by adding attribution to Brown (2005). 

42 

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

44 

45 Response: paragraph begins with and repeats the premise of prion infectivity six 

46 times in this one paragraph. Possibly, the Panelist’s statement refers to the distinction 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

24

1 between the word “prion” the phrase “prion protein,” as used in the White Paper. To 

2 achieve consistency, the White Paper employs the word “prion” refers to a misfolded 

3 isoform of any prion protein; “prion protein” can refer to both the normal and misfolded 

4 isoforms. That the abnormal misfolded isoforms are also infectious is implied in the 

5 context of the paragraph. The text has been modified for further clarity. 

6 

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

8 

9 Response: In his definitions, Brown (2005) made no mention of “agent strain.” 

10 Rather, he speaks in terms of polypeptide “isoforms;” however, it seems pertinent that 

11 Brown states that “nomenclature of the PrP species is confusing.” In Brown’s context, 

12 protein chemistry would suggest that his use of “species,” i.e., isoforms, is in reference to 

13 structural “species” of biochemically homologous polypeptide molecules, not the disease 

14 agent. 

15 

16 8. f. SAP comment: There are two suggested changes: 1) replace “infectious 

17 conformation of transcripts” with “misfolded PrP” and 2) “prion” should refer to as “an 

18 ‘infectious particle’ whether it is constituted of protein or not.” 

19 

20 Response: first comment makes the assumption that all misfolded PrP species are 

infectious, which may not be correct and can never be known. Regarding the 2nd 21 

22 comment, “prion” is a word coined and defined by Prusiner (1982) and in many 

23 additional papers by Prusiner and others. Prion is a “proteinaceous infectious particle.” 

24 Specifically, Prusiner has never used the word prion to connote any “‘infectious particle’ 

25 whether it is constituted solely of prion or not,” as commented by the Panelist. The 

26 White Paper conforms to Prusiner’s definition of the word “prion” and the convention 

27 used for the word “prion” and the phrase “prion protein” has been addressed above in 

28 response to comment 8.c. 

29 

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

31 

32 Response: The sentence is paraphrased directly from Colby et al. (2007): 

33 “Polymerization of recombinant prion protein (recPrP), which was produced in bacteria, 

34 into amyloid fibers was accompanied by acquisition of prion infectivity.” The White 

35 Paper states, “Amyloid polymerization of recombinant prion protein produced in bacteria 

36 has been shown to accompany the acquisition of prion infectivity (Legname et al. 2004; 

37 Colby et al. 2007) that is characteristic of the TSEs.” 

38 

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

40 

41 Response: The Panelist’s comment is correct, and the suggested sentence has 

42 been added to the White Paper. 

43 

9. b. SAP comment: (2nd 9. b.). Interactions between PrPC

 and PrPSc 44 are very narrowly 

45 focused and could be expanded. 

46 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

25

1 Response: The intent of the White Paper is to account for the principle of 

2 homologous interaction that would explain recruitment and conversion, as described in a 

3 recent publication (Lau et al., 2007). While it is necessary for the White Paper to 

4 document the history of the prion concept, in some detail, a comprehensive review of the 

5 literature on each additional issue raised in the White Paper is not feasible for its intended 

6 purpose. Additionally, the examples provided by the SAP [Horiuchi et al., 2001; Chabry 

7 et al., 1998] predate the more current Lau et al., 2007. The text has been modified to 

8 clarify the context of Lau et al. (2007) without expanding the scope of the discussion. 

9 

10 10. a. SAP comment: The White Paper statement that Prnp gene appears to be conserved 

11 from yeast to primates is incorrect. 

12 

13 Response: There are two responses to this comment. 

14 

15 In the first response, the Panelist’s comment that stating that Prnp gene “appears to be 

16 conserved across species from yeast to primates” [White Paper] contradicts the 

17 conclusions of Westaway and Prusiner (1986), which is the citation and interpretation in 

18 question. Westaway and Prusiner (1986) use a DNA-hybridization adaptation of classic 

19 methodology in comparative and evolutionary biology and reach a conventional 

20 conclusion that PrPs of living species from yeast to mammals probably “may” 

21 [Westaway and Prusiner] all arise by homologous descent with evolutionary modification 

22 from authentic PrP ancestral genes. A specific cDNA was employed by Westaway and 

23 Prusiner (1986) to show homologous binding with stringency across representative 

24 mammals and - with declining stringency - hybridization across representative 

25 invertebrates as well, such as nematode, Drosophilia, yeast, with declining stringency in 

26 that order. The lowest level of stringency was between mammal and yeast, which is what 

27 evolutionary biology and stringency of homologous binding would predict. Hence, the 

28 hybridization data do evidence that the “prion” genes from yeast to primates possibly do 

29 [or may] share a common ancestral origin (homology) from ancestral DNA, described by 

30 the authors as “authentic PrP genes.” 

31 

32 Furthermore, SAP comment 10.a. asserts that “yeast and fungal proteins are unrelated 

33 to mammalian on a sequence level, but are so named because they share the characteristic 

34 of being able to adopt two different conformations . . . .” This assertion is an 

35 interpretation that discounts the opposite interpretation evidenced in the data of 

36 Westaway and Prusiner (1986). Specifically, Westaway and Prusiner (1986) show a 

37 quantitative, deterministic relationship in which the stringency of hybridization is 

38 inversely proportional to evolutionary distance, i.e., the larger the evolutionary distance 

39 the lower the stringency of DNA hybridization. Furthermore, Westaway and Prusiner 

40 (1986) did not measure and did not comment on conservation of “being able to adopt two 

41 different conformations” [SAP comment 10.a.]. Additionally, the ability of “being able 

42 to adopt different conformations” is not a reasonable application of evolutionary theory, 

43 DNA hybridization, or amino acid sequence data. Evolutionary biology and protein 

44 chemistry predict that the more distant the stem origin of compared sequences, the less 

45 stringent is the sequence homology. Specifically, the structural conservation of genes 

46 and proteins is based on sequence data and cannot be based on the status of protein 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

26

1 conformations, as would be required in SAP comment 10.a. The status of protein 

2 conformation is a phenotype, not a genotypic concept. In contrast, the comparative DNA 

3 hybridization data in Westaway and Prusiner (1986) is a genetic evolutionary concept 

4 based on the core values of 

5 

6 1) phylogenetic relatedness [Nuttall, 1904, Blood immunity and blood relationships, 

7 Cambridge Univ. Press] 

8 

9 2) and of descent with modification of conserved genes and their encoded amino acid 

10 sequences (Wilson and Kaplan, 1964, Enzyme structure and its relation to 

11 taxonomy, Taxonomic biochemistry and serology, Ronald Press; Zuckerkandl 

12 and Pauling, 1965, Mutations, trends, and random processes in the evolution of 

13 informational macromolecules, J. Mol. Bioil., 59:473-490). 

14 

15 The concept of a shared ability to change conformations unrelated to sequence 

16 conservation, as suggested by comment 10.a., would (if it were true) be analogy, not 

17 homology and would not be an example of “conservation.” The scenario described in 

18 comment 10.a. would be an example of evolutionary convergence, for which no 

19 explanation is offered. Specifically, evolutionary convergence of structures (or of protein 

20 conformations) is achieved by population selection pressures for a particular phenotype, 

21 in which case the correct terminology is analogy, not conservation. 

22 

23 In the second response, the White Paper has added notation that Susan Lindquist 

24 (recent personal communication) believes her most recent study of the yeast prions 

25 (Alberti et al., 2009) is best interpreted as evidence of evolutionary convergence of 

26 sequence similarities to conserve “phenotypic variation” and allows the yeast “prions” to 

27 act as “non-Mendelian” elements of inheritance. In other words, she agrees with the SAP 

28 comment that the yeast prnp gene is not an example of conservation of homology across 

29 phyla to the living vertebrates. This update to the literature on the yeast prions has been 

30 added to the White Paper. 

31 

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

33 identity.” 

34 

35 Response: Conservation of sequence identity across phyla and evolutionary 

36 distance is by definition evolutionary homology, except in the theoretical case of 

37 potential evolutionary convergence of sequences, which is not the conventional 

38 interpretation of the classic picture of descent with modification evidenced in Westaway 

39 and Prusiner (1986). In the case of yeast prions and other vertebrate proteins such as 

40 Shadoo and Doppel, the real question is, at what point is sequence dissimilarity evidence 

41 of a non-homologous, non-evolutionary relationship? In the case of possible 

42 convergence of sequences in the yeast prions, as in the more recent interpretation of 

43 Alberti et al. (2009, above), the broadly representative comparative data would not have a 

44 quantitative deterministic relationship with the time scale of phylogeny, but rather would 

45 have a stochastic relationship with the time scale of phylogeny. The prion gene 

46 comparative sequence data in Westaway and Prusiner (1986) does have a predictable 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

27

1 quantitative deterministic relationship with time scale, such that less stringency is 

2 observed with increasing evolutionary distance. That is, within vertebrates, a higher level 

3 of stringency of binding is observed than the stringency of binding between yeast and 

4 vertebrates. In the case of amino acid sequence identity in the vertebrate prion proteins, 

5 based on the consistent trends in the limited sequence data obtained to date, in this and in 

6 other papers, “homology” is the correct word in the White Paper, as applied to vertebrate 

7 prion proteins. However, the comment raises the speculative nature studies to date 

8 stretching across larger phylogenetic distances from yeast to vertebrates, and the word 

9 “homology” has been deleted with reference to yeast prions and with reference to the less 

10 studied Shadoo and Doppel proteins. As suggested by the SAP, in these instances, 

11 “homology” has been replaced with “sequence similarity.” 

12 

13 11. SAP comment: There is just as much evidence that copper binding inhibits prion 

14 protein conversion as there is that copper stimulates. 

15 

16 Response: No citation was offered and the comment is not exclusive from what 

17 the paragraph says. The White Paper cites McKenzie et al. (1998) and only asserts that 

copper binding status may be an important cofactor in conversion of PrPC 18 and the 

stability of the PrPSc 19 infectious conformation. No assertion was made as to whether 

20 copper stimulates or inhibits any particular conformation. Brown (2005) also links 

21 copper binding status to PrP status. 

22 

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

24 

25 Response: A survey of the literature shows numerous functions now being 

attributed to normal PrPC 26 in the brain. The White Paper is already principally focused on 

27 abnormal PrP, not normal PrP. 

28 

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

30 PrP.... 

31 

32 Response: In the series of the SAP’s specific comments #13-14, the SAP 

33 correctly notes that the mechanisms of prion neurotoxicity and infectivity remain 

34 unresolved in detail and that for every prominent and widely acclaimed study published 

35 on the problem, rigorous analysis shows there can be found in the literature seemingly 

36 inconsistent results. The White Paper has been modified to provide a larger overview of 

37 the incomplete state of prion research as it is related to the specific mechanisms in 

38 neurotoxicity and specific changes have been made as well to address these issues and 

39 suggestions These changes are all consistent with the SAP’s general endorsement of the 

40 prion hypothesis and with the other recurrent SAP view that much remains unknown in 

41 detail. 

42 

43 The SAP’s assertion is attributed in the White Paper to Brandner et al. (1996), 

44 who conclude, “normal host prion protein [is] necessary for scrapie-induced 

45 neurotoxicity.” In response, an additional citation has been added to the text, Mallucci et 

46 al. (2003), which states that “depleting neuronal PrP in prion infection prevents disease 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

28

1 and reverses spongiosis.” Both of these papers both appear to support the concept that 

2 normal host prion protein is necessary for neurotoxicity. 

3 

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

5 normal PrP.” 

6 

7 Response: The sentence in the White Paper is supported by Brandner et al. 

8 (2005) in subsequent text. On the surface, the title of the Brandner et al. paper is 

9 “Normal host protein necessary for scrapie-induced disease.” Furthermore, Mallucci et 

10 al. (2003) states that “depleting neuronal PrP in prion infection prevents disease and 

11 reverses spongiosis.” 

12 

13 14. General comments related to Specific Comment #14 indicate the concerns are of one, 

14 or only a small number, of the Panelists. 

15 

16 14. a. SAP comment: The current view is that prion-related neurodegeneration might 

17 have two components . . . 

18 

19 Response: The comment has merit. Accordingly, the assertion that scrapie prion 

20 “kills” neurons has been deleted and replaced by “ . . . the pathology of scrapie prion in 

21 neurons features disrupting the function . . . .” and a brief discussion and suggested 

22 additional references concerning possible mechanisms for neurodegeneration have been 

23 added to the white paper. 

24 

25 14. b. SAP comment: Some Panelists suggested deleting the Brandner et al., Mallucci et 

26 al., and Chesebro et al., citations and accompanying text and cited the two currently 

27 accepted components of neurodegeneration . . . 

28 

29 Response: The White Paper citations chosen (Brandner et al. 1996, Mallucci et 

30 al. 2003, and Chesebro et al. 2005) were selected because of their placement in such 

31 prominent journals as Science and Nature. As in SAP comment 14a, the expansion of the 

32 White Paper as the literature continues to evolve would not further support the intended 

33 purpose of the White Paper. The only more recent study recommended by the SAP is 

34 Simoneau (2007), which is already included in the discussion along with Piccardo et al. 

35 (2007), which both point to the confusion in what intermediate steps might prove critical 

36 in the neurotoxicity pathway of prions. Further expansion of the neurotoxicity 

37 information would not significantly improve the White Paper’s utility for informing the 

38 Agency’s decision as to whether prions should be considered pests. 

39 

40 15. SAP comment: The acronym is GPI, not GTI. It is noted the titers are altered and 

41 that disease is altered, not prevented. 

42 

43 Response: The text has been modified. 

44 

45 16. SAP comment: Redesign Figure 1. 

46 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

29

1 Response: Figure 1 does have an internally inconsistent format although the 

2 suggested fix (“infectious, sporadic, and inherited) also gives rise to problems. Fatal 

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

4 

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

6 

7 Response: Page 8 is about human outbreaks of disease, including the acquisition 

8 of vCJD as an extension of BSE. TME would not seem to fit in this context. A better 

9 location to include TME is in sequence with cats (FSE), page 9-10; the text has been 

10 modified. 

11 

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

13 

14 Response: Additional information has been inserted. 

15 

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

17 

18 Response: Canada and the USA have been added. 

19 

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

21 

22 Response: Text has been corrected. 

23 

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

25 

26 Response: Texas has been added. 

27 

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

29 

30 Response: Additional information has been added. 

31 

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

33 

34 Response: Prevalence in captive animals is not discussed in the text related to 

35 any of the susceptible species. No expansion of the text to include epidemiology of 

36 species in captivity is needed. 

37 

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

39 

40 Response: Suggested routes have been added. 

41 

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

43 natural route. 

44 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

30

1 Response: The text of the White Paper does not mention food as a route of 

2 scrapie transmission. The paragraph specifies in the lead sentence that the discussion is 

3 in reference to natural transmission. 

4 

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

6 and Gregori. 

7 

8 Response: No reason was given for the directive to delete Ligios et al.; however, 

9 the text has been modified by adding one of the early papers on prionuria by Gabizon. 

10 

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

12 

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

14 clarification has been added. 

15 

16 23. a. SAP comment: Heaton et al. was misinterpreted. Additional “cases with K211 in 

17 the US will be very low.” 

18 

19 Response: The text states “that BSE cases with the K211 allele that occurred in 

20 Alabama would be unlikely.” Presumably, the comment takes issue with “unlikely” 

21 [White Paper] versus “very low” [SAP comment]. Note: the background nomenclature 

22 here is the Prnp gene allele E211K denotes a DNA mutation in which the normal amino 

23 acid glutamate (E) at position 211 is replaced by lysine (K), resulting in BSE. The 

24 authors did not find another instance of E211K in a survey of 6062 cattle, so that – 

25 statistically – the upper limit for possible other occurrence is less than 1 in 2000 cattle. A 

26 text modification has been suggested to capture the nuances. The authors use the phrase 

27 “vanishingly low” to describe the probability of another atypical BSE case in the U.S. 

28 from the E211K mutation; the panelist’s suggestion (less than 1:2000) could imply that 

29 the frequency is much higher than it really is. 

30 

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

32 

33 Response: The text cites 2004 references, at which time there were only two 

34 known cases. No citation is provided for the occurrence of four to date. 

35 

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

37 1985 report. 

38 

39 Response: A correct citation is not provided although the White Paper uses two 

40 other citations. The comment is probably referencing: Garner and Favero, 1985, 

41 guideline for handwashing and hospital environmental control, 1985. This original report 

42 seems to be an internal report of the CDC, now available on the internet. The 

43 corresponding actual publication seems to be Garner and Favero (1986), which has been 

44 added the text. Also, Section F. of the White Paper does not have a corresponding 

45 References list, and there is no citation to Bolyard in the References section. A section F. 

46 has been added to the References with the single entry of Garner and Favero. 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

31

1 

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

3 most difficult to inactivate on this surface.” 

4 

5 Response: Text has been modified. 

6 

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

8 

9 Response: The comment does not seem to conflict with the White Paper text, 

10 which assumes the bioassay test method is appropriate to the goal. The same issues are 

11 dealt with at length in the Efficacy Guidance document. 

12 

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

14 

15 Response: The Panelist seems to be volunteering that BSE “readily” transmits to 

16 felines, but not to humans. The White Paper did not mention felines in this context; 

17 however, the intent seems to deny that BSE readily transmits to humans” which are 

18 separate events. In order to keep the discussion at a general level, the text has been 

19 modified to be both more precise and still brief. 

20 

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

22 

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

24 involve other unknown cofactors besides the prion itself. While SAP presented no 

25 evidence that the prion itself is anything other than protein encoded by a gene, EPA has 

26 included the cofactor hypothesis as an alternative point of view. 

27 

28 30. SAP comment: Spelling error identified. 

29 

30 Response: Spelling error corrected. 

31 

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

33 

34 Response: Except for instances where “mad cow” is appropriate to the context, 

35 references to the disease have been replaced with BSE. 

36 

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

38 recapitulate scrapie and BSE. 

39 

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

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

42 

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

44 

45 Response: The text has been modified in several places to capture the nuance that 

46 not all TSEs produce “amyloid plaques.” 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

32

1 

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

3 

4 Issue 2. Whether the specific test systems recommended in the draft guidance document 

5 are scientifically appropriate to support the registration of pesticide products with prion6 related claims. 

7 

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

9 

10 a. Carrier-based, animal infectivity assays recommended by EPA’s guidance for 

11 evaluating the efficacy of pesticide products used on environmental surfaces 

12 (e.g., hard, nonporous surfaces). 

13 

14 b. Suspension-based, animal infectivity assays recommended by EPA’s guidance 

15 for evaluating the efficacy of pesticide products used in liquid media (e.g., 

16 wastewater). 

17 

18 c. Any other known test methods for evaluating the efficacy of pesticide 

19 products used on either environmental surfaces or in liquid media. 

20 

21 Panel Response 

22 

23 a. Carrier-based, animal infectivity assays recommended by EPA’s guidance 

24 for evaluating the efficacy of pesticide products used on environmental surfaces 

25 (e.g., hard, nonporous surfaces). 

26 

27 One Panel member commented that there are substantial problems in quantifying 

28 reduction of infectivity in carrier-based models. Wires (or other carriers) incubated in 

29 diluted brain homogenate may not effectively represent proportionately the dilution 

30 made. Proportional dilution would only occur if all components of brain homogenate had 

31 exactly the same affinity for the surface. If prions bind more efficiently than other 

32 components the endpoint would be an overestimate, if prions bind less efficiently the 

33 endpoint would be an underestimate. Thus, dilution of brain homogenate and absorption 

34 to a carrier cannot then be used as a basis for incubation time bioassay. Determinations 

35 of log reduction would therefore be invalid. According to this Panel member, there is 

36 one published stainless steel wire based endpoint titration (hamster 263K prions serially 

37 diluted in normal hamster brain) which gave an endpoint of 5.6 (five point six) log ID50

38 (Fichet, Comoy et al. 2004). Dr. David Asher, Laboratory of Bacterial, Parasitic & 

39 Unconventional Agents, Division of Emerging and Transfusion-Transmitted Diseases, 

40 FDA, reported at the open Panel meeting an endpoint of 7 (seven) log ID50 when 263K 

41 was diluted in calf brain paste and mentioned a difficulty in getting prions to stick to the 

42 wires when using PBS as diluent. This highlights the potential problem and could lead a 

43 potential applicant for EPA approval to choose a formulation for which it is easiest to 

44 reach a defined log reduction, but which is not valid. Conversely, it is not possible to do 

45 an endpoint titration with a treated wire to determine remaining titer.

46 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

33

1 Another Panel member said that full end-point titrations, or at least titer reduction 

2 experiments including the end-point, are the test method of choice for both the 

3 suspension-based and the carrier-based assays. Indeed, it has been shown in several 

4 studies that various physical (e.g. heat) or chemical treatments of prion-infected material 

5 can increase the incubation period by bioassay (when compared to the untreated sample) 

6 disproportionately to the titer reduction measured by end-point titration. Therefore, 

7 incubation period assays can overestimate the log reduction of a given treatment 

8 (Somerville and Carp 1983; Taylor and Fernie 1996). 

9 

10 In the case of carrier-based assays, end-point titrations pose the problem of the 

11 practicality of doing serial dilutions of the treated surface (practically, the treated wire or 

12 beads) to which prions are attached. If the treated surface, once “inoculated” induces an 

13 incomplete attack rate, it means that the amount of infectivity corresponds to the end14 point and the log reduction can be calculated directly. If not, the only available method 

15 so far is the glass adsorption and crushing method, published by Chen and validated for 

16 prions by Dr. Asher (Chen 1975). A standard dose-response curve for carrier-bound 

17 infectivity can be provided by the adsorption onto carriers of serially diluted brain 

18 material (positive control). This Panel member commented on the potential lack of 

19 proportionality of adsorption of diluted sample on to carriers. Although this theoretical 

20 possibility is worthwhile considering, there is so far no experimental evidence for this. 

21 More data are needed to clarify this point. In any case, this point can be overcome by 

22 performing the same carrier crushing methodology to generate the standard curve. 

23 

24 b. Suspension-based, animal infectivity assays recommended by EPA’s 

25 guidance for evaluating the efficacy of pesticide products used in liquid media (e.g., 

26 wastewater). 

27 

28 The Panel agreed that suspension-based animal bioassays are the only validated and 

29 broadly accepted ways to determine infectious titer. Additionally, all agreed on the final 

30 requirement for a quantitative and direct animal bioassay of infectivity and for a test 

31 system specific for each of the different agents, e.g., sCJD, scrapie, CWD, vCJD, and 

32 BSE agents. The Panel discussed in detail the potential limitations of the incubation-time 

33 bioassay and the potential high cost and animal usage of the end-point bioassay. 

34 

35 One Panel member stated that an incubation time assay done at >5 readable (each one 

36 log-significant difference) points, including the end-point, would be most acceptable. 

37 This would reduce costs of a complete end-point dilution assay. A few animals (~5) 

38 inoculated with uninfected material from the same tissue type should also be included to 

39 rule out non-specific and toxic effects. In addition, clinical illness is not sufficient for 

40 readout because animals die from other causes, especially during prolonged end-point 

41 incubations. A full necropsy inspection of organs to rule out such non TSE causes of 

42 death, as well as western blotting or neuropathology of all animals, is required to 

43 confirm the infectious agent is causal. Such criteria are important for a statistically 

44 limited group of animals. Furthermore, the agent-type can be determined by the 

45 distribution of spongiform changes as well as prion staining in brain and spleen. 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

34

1 Methods are available to evaluate both spongiform changes and prion deposits 

2 simultaneously (Manuelidis, Fritch et al. 1997). 

3 

4 Another Panel member disagreed stating that the end point dilution assay was the best 

5 method for final validation of any decontamination method, although the incubation 

6 interval assay could be used as a screening method to focus the dilution groups to be 

7 performed with end point dilution. This Panel member suggested that additional detail 

8 should be sought by EPA to provide guidance to registrants in testing their products. 

9 

10 The end point titration assay is the only means of directly calculating titer. The numbers 

11 of animals per group should be based on the expected variance in the parental strain that 

12 is tested. The time post inoculation the animals are monitored should be based on the 

13 incubation period of the strain examined and should be at least 50% greater than the 

14 incubation period of the highest dilution that causes disease. Clinical signs should be 

15 monitored a minimum of three times per week. The monitoring of clinical signs can be 

16 by subjective observation of clinical signs or quantification of behavioral changes and 

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

18 should be examined for evidence of infection (Western blot for PrPres and/or 

19 neuropathology). Only differences in calculated titers that are equal to or greater than 

20 one log are considered significant (Mould, Dawson et al. 1967). 

21 

22 The incubation time interval assay has serious limitations that need to be addressed. 

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

24 a direct calculation of titer. Although the incubation time interval assay can yield similar 

25 results as endpoint titration, serious limitations of this assay have been reported. Several 

26 reports have indicated that treatment of prions with heat and/or detergent can alter the 

27 relationship between titer and incubation period resulting in large errors in estimating 

28 titer based on incubation period alone (Dickinson and Fraser 1969; Lax, Millson et al. 

29 1983; Somerville and Carp 1983; Taylor and Fernie 1996). Additionally, it has been 

30 shown that the relationship between the dose of the agent and incubation period can be 

31 tissue specific, precluding using incubation interval assays when comparing the 

32 dissimilar tissues (Robinson, Cheevers et al. 1990). Finally, there are numerous 

33 examples in the literature of 10-fold differences in titer of the agent that have similar 

34 incubation periods that reduce the precision of the incubation time interval assay 

35 (Kimberlin and Walker 1977; Kaeser, Klein et al. 2001; Kincaid and Bartz 2007). This is 

36 in contrast to endpoint dilution analysis that is accurate to 0.5 (zero point five) log ID50

37 and is highly reproducible (Mould, Dawson et al. 1967; Taylor, McConnell et al. 2000). 

38 

39 Inoculum preparation should be consistent between control and experimental groups with 

40 the only variable being the treatment. The biological matrix used should mimic the likely 

41 form the agent would be present for the intended use. It is important to note that brain 

42 homogenate and preparations that are enriched for infectivity can differ in the response to 

43 inactivation. 

44 

45 c. Any other known test methods for evaluating the efficacy of pesticide 

46 products used on either environmental surfaces or in liquid media. 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

35

1 

2 Most of the Panel was not enthusiastic about using protease resistant prion quantification 

3 or amplification (PMCA) as a measure of infectivity because infectious titer in many 

4 cases does not correspond to protease resistant prion. Nevertheless, an applicant may 

5 wish to use such an approach in initial pilot studies to save time and reduce costs. 

6 

7 There are also stable tissue culture models that can be used to assess infectivity for a 

8 number of TSE agents, including vCJD, Japanese CJD, 22L, Chandler (RML) and ME7 

9 scrapie (Manuelidis, Liu et al. 2009) (Arjona, Simarro et al. 2004; Nishida, Katamine et 

10 al. 2005; Mahal, Baker et al. 2007). These can speed the first choice of best pesticide 

11 reagents, especially for materials dried on stainless steel or glass surfaces. These treated 

12 surfaces can be cultured with susceptible cells for titer readout in as few as twenty-one 

13 days. Experiments with some of these agents grown in culture, such as Japanese CJD 

14 and 22L scrapie, have shown faithful reproduction of the original TSE by re-inoculation 

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

16 cultures. 

17 

18 The majority of the Panel concluded that in vitro immunodetection assays or cell-based 

19 bioassays are not adequately developed for use in assessing reduction in infectivity for 

20 evaluation of efficacy of pesticide products with prion related claims because in the case 

21 of immunodetection assays these do not measure infectivity and in the case of cell-based 

22 bioassays these have not been adapted for the application of assessing reduction of 

23 infectivity. The Panel recommends to EPA that end point dilution animal bioassays be 

24 used to assess reduction in infectious titer by treatment of samples with prion pesticide 

25 products. 

26 

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

28 

29 The SAP responded to the three sub-questions (a., b., c.) under Charge Question #2, and 

30 EPA’s responses follow the same format: 

31 

32 a. Carrier-based, animal infectivity assays recommended by EPA’s guidance for 

33 evaluating the efficacy of pesticide products used on environmental surfaces 

34 (e.g., hard, nonporous surfaces). 

35 

36 In its summary statement, the SAP expressed “reservations” about the use of carrier37 based animal bioassays. Those reservations appeared to focus on the potential for diluted 

38 brain homogenates not to adsorb on to the carrier in proportion to their concentrations. In 

39 its detailed comments, the SAP attributed this concern to one Panel member. The SAP 

40 also cited one (?) other Panel member who asserted that full end-point titrations are the 

41 “method of choice” for both suspension-based and carrier-based assays, asserting that 

42 incubation time bioassays can overestimate the log reduction of a given treatment. 

43 However, the majority (?) of the SAP also recognized that end-point titrations cannot be 

44 done with solid carriers (e.g., steel wires) because the carriers cannot usually be ground 

45 up to make a dilution. The SAP noted that only a glass adsorption/crushing method is 

46 available (Chen 1975). 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

36

1 

2 In response to these comments, EPA interprets the SAP as indicating that while it would 

3 prefer the use of an end-point titration bioassay to an incubation time interval assay for 

4 carrier-based testing, the SAP recognizes that it is physically impossible to do such a 

5 bioassay with a hard, nonporous carrier such as steel. Further, although the SAP reported 

6 the concerns of one Panel member about the incubation time interval assay (i.e., the 

7 potential for disproportionate adsorption of brain homogenate to a carrier), it later stated, 

8 “there is so far no experimental evidence for this.” 

9 

10 EPA firmly believes that a disinfectant should be tested in a way that represents the way 

11 the product is used. Most uses of disinfectants involve application to a hard, nonporous, 

12 inanimate surface. For this reason, EPA currently requires carrier-based efficacy testing 

13 for all sanitizers, disinfectants and sterilants. The fact that the end-point titration 

14 bioassay cannot be performed on a hard material (except one method that uses glass, 

15 which can be ground up and diluted as required by the end-point titration method) 

16 precludes it from being used. Therefore, EPA’s guidance on prion disinfectant efficacy 

17 testing will continue to recommend the use of an incubation time interval assay for 

18 testing of disinfectants on carriers that represent hard, nonporous, inanimate surfaces.

19 

20 b. Suspension-based, animal infectivity assays recommended by EPA’s guidance 

21 for evaluating the efficacy of pesticide products used in liquid media (e.g., 

22 wastewater). 

23 

24 In its summary statement, the SAP agreed that the suspension-based end-point dilution 

25 bioassay is the most reliable method for measuring reduction of prion infectivity. The 

26 Panel also considered the incubation time interval assay to be a screening method. The 

27 Panel further asserted that an end-point titration assay is “necessary” to calculate 

28 reduction in infectious titers and “cannot be substituted” by an incubation time interval 

29 assay. Also, some Panel members supported using one rodent model system for testing 

30 all products, but others thought that testing should be done with native prion strains in 

31 their corresponding animal hosts. Finally, the Panel stated that necropsies of all test 

32 animals using Western blot detection and neuropathologic changes are required. 

33 

34 In its detailed comments, the SAP reiterated its opinion that that the suspension-based 

35 end-point dilution bioassay is the most reliable method for measuring reduction of prion 

36 infectious activity. On the issue of which prion strains should be tested, the SAP’s 

37 detailed comments stated that all Panel members agreed that tests should be conducted 

38 for each of the different prion types, whereas in the summary statement the SAP 

39 indicated that some Panel members favored using rodent models systems while others 

40 favored using native prion strains. The SAP then delved further into the debate of 

41 incubation time interval bioassay versus the end-point dilution bioassay. One Panel 

42 member stated that an incubation time interval assay with more than five readable points, 

43 including the end point, would be acceptable. A few control animals inoculated with 

44 uninfected material from the same tissue type should also be included to rule out non45 specific and toxic effects. Finally, a full necropsy should be done. A second Panel 

46 member disagreed with the first Panel member, asserting that the end-point dilution assay 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

37

1 is the best method for final validation of a decontamination method. This member’s main 

2 reason appeared to be that the end-point dilution assay provides a direct calculation of 

3 prion titer whereas the incubation time interval assay provides an estimate of titer. 

4 Another reason given by the Panel was the difference in test accuracy—the end-point 

5 method is accurate within 0.5 log ID50 in contrast to 1 log for the incubation time interval 

6 assay. 

7 

8 In response to the SAP’s summary comments, EPA observes that the SAP’s 

9 recommendation for an end-point dilution bioassay, which is suspension-based, is 

10 consistent with EPA’s guidance that registrants use a suspension-based bioassay for 

11 evaluating the efficacy of prion products used in liquid media (e.g., wastewater). 

12 Regarding the SAP’s comment that an end-point dilution assay is “necessary,” EPA 

13 interprets that this statement is applicable to suspension-based tests and not carrier-based 

14 tests since Charge Question #2b only pertains to suspension-based testing. Accordingly, 

15 EPA will revise its guidance to recommend that an end-point dilution assay be used for 

16 evaluating prion products applied to liquid media such as wastewater. 

17 

18 EPA further observes that the SAP did not reach a consensus on whether prion efficacy 

19 testing should be conducted with rodent-adapted prions (in place of native prions) or on 

20 native prion strains in a relevant animal host. Since each approach has been used 

21 successfully by various researchers, EPA has decided to allow use of either testing 

22 approach. Hence, EPA’s draft guidance will allow for use of either a rodent model or a 

23 native strain of prion in a relevant animal host. 

24 

25 Finally with regard to the SAP’s summary comments, the Panel recommended necropsies 

26 along with Western blot detection to determine whether surviving animals are infected 

27 with prions. Consistent with this recommendation, EPA’s draft guidance recommends 

28 that at least one prion-specific test (Western blot, immunological histochemistry or “blind 

29 passage”) be performed to confirm whether either the surviving or dying animals are 

30 infected with prions. 

31 

32 In response to the SAP’s detailed comments, EPA observes (as mentioned above) that the 

33 SAP recommendation for an end-point dilution bioassay, which is suspension-based, is 

34 consistent with EPA’s guidance that registrants use a suspension-based bioassay. EPA 

35 notes that with respect to the issue of which prion strains should be tested, the SAP’s 

36 detailed comments conflicted with its summary comments. The latter stated that all Panel 

37 members agreed that tests should be conducted for each of the different native prion 

38 types, whereas the former indicated that some Panel members favored using a rodent 

39 model system. EPA assumes that since conflicting opinions were expressed on this issue 

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

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

42 

43 Regarding the differences of opinion among Panel members on the issue of end-point 

44 dilution assay versus incubation time interval assay, EPA appreciates the SAP’s 

45 discussion of the advantages and disadvantages of each type of assay. However, EPA 

46 presumes that the Panel’s recommendation that an end-point dilution assay is “necessary” 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

38

1 is applicable to suspension-based tests and not carrier-based tests since Charge Question 

2 #2b only pertains to suspension-based testing. 

3 

4 c. Any other known test methods for evaluating the efficacy of pesticide 

5 products used on either environmental surfaces or in liquid media. 

6 

7 In its summary statement and detailed comments, a majority of the Panel concluded that 

8 in vitro immunodetection assays or cell-based bioassays are not adequate for use in 

9 assessing reduction in prion infectivity. A minority of the Panel contended that in vitro 

10 amplification immunodetection assays such as protein misfolding by cyclical 

11 amplification (PMCA) and cell-based bioassays can serve as a complementary assay to 

12 animal bioassays. The Panel also reiterated that end-point dilution animal bioassays 

13 should be used to assess reduction in infectious titer. 

14 

15 In response to these comments, EPA agrees with the Panel that in vitro immunodetection 

16 assays are not adequate for assessing reduction in prion infectivity, and that in vitro 

17 amplification immunodetection assays should only complement but not substitute for 

18 animal bioassays. EPA presumes that the Panel’s recommendation for using an end-point 

19 dilution assay is applicable to suspension-based tests and not carrier-based tests since 

20 Charge Question #2b only pertains to suspension-based testing. 

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

22 

23 Issue 3. Whether the product performance criterion specified in the draft guidance 

24 document to support the registration of pesticide products with prion-related claims is 

25 scientifically sound. 

26 

27 Charge Question 3. Please comment on the scientific soundness of the product 

28 performance criterion recommended in the draft guidance document to support the 

29 registration of pesticide products with a prion claim. 

30 

31 Panel Response 

32 

33 The Panel was in general agreement that some widely used model systems, such as the 

263K or Sc237 rodent-adapted prion strains, have titers up to ~1011 34 ID50/g of brain tissue 

35 and make it possible to detect a six (6) log reduction in infectivity. However, bioassays 

36 in rodents are usually performed by intracerebral inoculation of a small volume such as 

25μl. For a 10% brain homogenate, 25μl represents 2.5 x 108 37 ID50, making an 8 (eight) 

38 log reduction measurable for bioassays in rodents with model prion strains. The 

39 treatment methodology may further reduce the initial infectious dose, for example, 

40 mixing a brain homogenate with an inactivation reagent would dilute the level of 

41 infectivity. It may then be necessary to further dilute the sample to reduce any potential 

42 toxic effects of the inactivation reagent on the bioassay animals. In such cases it may not 

43 be possible to measure such a high reduction. 

44 

45 The Panel considered the important caveat that for naturally occurring prion strains from 

46 natural hosts, titers are much lower in the available animal bioassay models: only in the 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

39

range of 105

 to 108 1 ID50/g. In such cases it may only be possible to measure a three (3) or 

2 four (4) log reduction in infectivity. 

3 

4 Furthermore, any claim of a log reduction in infectivity needs to be accompanied by a 

5 statistical methodology, and some understanding of assay variability, e.g., 95% 

6 confidence interval, in attaining such a given log reduction. End point animal bioassays 

7 should utilize Kaplan-Meier and/or Cox regression analysis models to account for 

8 observed censoring, e.g., from animals dying prematurely, in the estimation of mean 

9 times to outcome, e.g., onset of clinical signs, death, etc. In this way, confidence 

10 intervals for these estimates can also be provided. In addition, specific tests (test for 

11 equality of appropriate Cox regression parameters, or non-parametric tests for equal 

12 survival curves via the Kaplan-Meier curves) and parameter/characteristic to be used for 

13 comparing controls to treatment groups need to be specified. 

14 

15 One Panel member added that carrier-based methods provide further issues for 

16 calculating log reduction in infectivity. Firstly, it is not possible to use endpoint based 

17 methods, with the possible exception of the ground glass surface (Chen 1975). This is 

18 not an established method for quantification of prion titer, although preliminary data were 

19 reported to the Panel by Dr. Asher of the FDA. Also, there is no basis to assume all non20 porous surfaces are equal in terms of prion binding. One published value for stainless 

21 steel wires incubated in serially diluted 263K-infected brain homogenate gave an 

22 endpoint of 5.6 log ID50 (Fichet, Comoy et al. 2004). A similar value was reported in a 

recent paper (Lemmer et al. 2008).1 23 Researchers have found that the composition of the 

steel makes a difference (Luhr et al. 2009).2 24 However, there is a possibility that carriers 

25 incubated in serially diluted brain homogenate do not accurately bind infectivity at the 

26 same proportion to the dilution. If this is the case, then the endpoint calculation is not 

27 valid. Conversely, concerns were also expressed about the incubation period animal 

28 bioassay, because it has been shown that estimation of titer of physically or chemically 

29 treated samples by this method does not always correspond with titer calculated by 

30 endpoint titration (Somerville and Carp 1983; Taylor and Fernie 1996). 

31 

32 Another Panel member noted that the target amount of reduction may need to be 

33 considered in light of the proposed application or use, risk and what is achievable and can 

34 be validated for a particular chemical or physical treatment. For example, treatment of 

35 potentially CWD contaminated waste water from an area associated with deer housing or 

36 processing may have a different standard for reduction in infectivity compared to that for 

37 surgical instruments in a human hospital which have potentially been exposed to CJD. A 

38 reduction of six (6) log in infectivity of CWD in waste water may be adequate to reduce 

39 the infectivity compared to the risk posed by CWD prions to human health, whereas the 

40 efficacy of a six (6) log reduction for CJD may not be adequate to reduce the potential 

41 that a human patient might contract CJD from an instrument with a six (6) log reduction 

 

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 findings with a model system for testing the removal of 

scrapie infectivity from steel surfaces. J Gen Virol. 2008 Jan;89(Pt 1):348-58. 2

 Luhr KM, Löw P, Taraboulos A, Bergman T, Kristensson 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

1 in CJD prions. Risk assessments may be available to help formulate expected standards 

2 of performance, but a one-size fits all approach may not be valid. 

3 

4 The Panel recommends that if it is not possible to measure to six (6) log reduction in a 

5 relevant model (one using the naturally occurring prion strain from the natural host for 

6 which the inactivation method is designed to protect against), then the maximum 

7 measurable reduction should be required to demonstrate target efficacy. However, it was 

8 not generally agreed whether this should also be accompanied by demonstration of at 

9 least a six (6) log reduction in a widely used model, such as the hamster-adapted scrapie 

10 prion 263K or Sc237 strains. 

11 

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

13 

14 In its summary comments, the SAP’s overall recommendation was that target reduction 

15 in infectivity should be assessed in a relevant animal model using the naturally occurring 

16 prion strain from the natural host to support the specific label claim. The SAP stated that 

17 the level of reduction should be set at the maximum measurable reduction achievable for 

18 the target strain and for the efficacy test model used. 

19 

20 In both its summary comments and detailed comments, the SAP generally agreed that 

21 some widely used model systems, such as the 263K or Sc237 rodent-adapted prion 

22 strains, can detect six (6) log reduction in infectivity. The Panel also noted that for 

23 naturally occurring prion strains from natural hosts, it may only be possible to measure a 

24 three (3) or four (4) log reduction in infectivity. In addition, the SAP stated that any 

25 claim of reduction in infectivity needs to be accompanied by a statistical methodology. 

26 One Panel member raised a concern about the possibility that carriers incubated in 

27 serially diluted brain homogenate do not accurately bind infectivity at the same 

28 proportion to the dilution, which could affect the accuracy of the log reduction estimate 

29 from an incubation time interval assay. Another Panel member suggested that the target 

30 amount of reduction may need to be considered in light of the proposed application or 

31 use, risk, and what is achievable and can be validated. 

32 

33 EPA’s response to the SAP’s comments is as follows. The central issue of Charge 

34 Question #3 regards the scientific soundness of the product performance criterion 

35 recommended in the draft guidance document (i.e., a six (6) log reduction in prion 

36 infectivity) to support the registration of pesticide products with a prion claim. EPA 

37 agrees with the SAP’s assessment that some test models can achieve this target criterion 

38 for success and others cannot, but does not agree with the SAP’s recommendation that a 

39 lower target criterion (e.g., 4 or 5 log reduction) should necessarily be acceptable if an 

40 efficacy test method is only capable of measuring a lower reduction in prion infectivity 

41 for a particular prion strain, Because prions are very difficult to inactivate and present a 

42 serious health risk (i.e., irreversible neurological disease and death) to humans, EPA has 

43 decided to maximize protection of human health by setting a target criterion that is very 

44 stringent but achievable. Accordingly, EPA will keep its general target performance 

45 criterion at six (6) logs of reduction of infectivity as demonstrated by acceptable efficacy 

46 test methods. However, since EPA’s document provides guidance, EPA may consider 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

41

1 alternative test methods and/or a different target performance criterion if persuasive, 

2 valid, scientific data and/or arguments are submitted to EPA for review. 

3 

4 In response to the SAP’s recommendation in this section that infectivity should be 

5 assessed in a relevant animal model using the naturally occurring prion strain from the 

6 natural host, EPA is aware that it is extremely difficult to obtain the naturally occurring 

7 strain of certain prions (e.g., BSE, sCJD), that this approach may be cost prohibitive, and 

8 that rodent adapted strains are readily available for testing. Accordingly, EPA believes 

9 that while use of naturally occurring prion strains from the natural host might be optimal 

10 from a purely scientific standpoint, use of rodent adapted prion strains would be far more 

11 practical and feasible. Nevertheless, EPA may consider testing with a native strain of 

12 prion in a relevant animal host if a registrant submits a proposed test protocol along with 

13 persuasive scientific arguments as to why EPA should accept that protocol. 

14 

15 Finally, EPA agrees with the SAP comment that that any claim of reduction in infectivity 

16 needs to be accompanied by a statistical methodology. EPA’s guidance already specifies 

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

18 

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

20 

21 Issue 4. Whether the labeling claim described in the draft guidance document is 

22 scientifically appropriate based on the recommended test systems and product 

23 performance standard. 

24 

25 Charge Question 4. Please comment on the scientific appropriateness of the term 

26 “reduce infectivity” in a label claim to reflect the action of a pesticide on prions.

27 

28 Panel Response 

29 

30 The Panel agreed with the EPA’s proposal to frame the label claim statement as “reduce 

31 infectivity.” From a scientific perspective, the state of “complete elimination of 

32 infectivity” is probably impossible to achieve in practice, let alone from a probability 

33 standpoint. One Panel member asked why the EPA was hesitant to claim complete 

34 elimination of infectivity given that techniques to measure TSE infectious titer are similar 

35 to those of other infectious agents. The EPA replied that with other microorganisms the 

36 target reduction is also less than 100%, and they want to be consistent with 

37 recommendations from other Federal agencies, for example the recommendations of the 

38 FDA Transmissible Spongiform Encephalopathies Advisory Committee. 

39 

40 Another Panel member stated that no one knows what constitutes a minimal infective 

41 dose of prions and that there is no way to measure this in humans. Infective dose can 

42 also be influenced by the immune and other systems and how these systems react to 

43 different doses of prions is unknown. It is very difficult to estimate the dose of prions 

44 needed to transmit TSE infectivity to humans. As a result, there is great reluctance to 

45 claim that all TSE infectious doses have been removed following treatment with a prion 

46 decontaminating reagent. Additionally, one Panel member noted that due to the 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

42

1 prolonged disease incubation times and the lack of understanding as to what constitutes 

2 an infectious dose, reduced infectivity is the only term that can be used to assess a 

3 solution used to decrease the impact of prion disease. 

4 

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

6 like “inactivation”. There was concern that registrants and the user communities will 

7 have differing expectations of the prion pesticide product’s performance. Users in 

8 hospitals concerned about CJD may expect something closer to “elimination of 

9 infectivity” on environmental surfaces, whereas the veterinary and wildlife management 

10 community may be more comfortable with the “reduced infectivity” approach. One 

11 Panel member recommended that the label clearly indicate the implications of the 

12 “reduce vs. elimination” approach in sufficient detail so that the user can understand and 

13 appreciate what the product can and cannot do. Furthermore, the label of the registered 

14 product should follow current EPA labeling recommendations in providing use condition 

15 details for each general application of the product, e.g., decontamination of hospital 

16 environmental surfaces, decontamination of laboratory working surfaces, or application 

17 on porous materials such as fences, etc. The Panel asked EPA representatives if the label 

18 was intended to be understood by the general public, and an EPA representative 

19 confirmed this. 

20 

21 One Panel member noted that when considering the phrase “reduce infectivity” it is 

22 important to note 1) that it does not refer to the complete elimination of infectivity with 

23 absolute confidence and, 2) that any product has to be tested in a way that supports the 

24 labeling claim. Thus, the phrase “reduce infectivity” is valid only as long as the method 

25 used to measure infectivity demonstrates a log reduction in a valid model system. Use of 

26 the word reduce would therefore be model dependent. A claim to “reduce infectivity” 

27 also implies that the efficacy testing will involve a measurable endpoint, so the reduction 

28 can be quantified. 

29 

30 One Panel member stated that incubation time animal bioassays used to measure 

31 infectivity after treatment of prions may not be the best assays to use as there are several 

32 examples in the literature that suggest treatments which reduce prions may lead to 

33 incubation periods that fall outside the linear range of the assay . There was a general 

34 consensus among Panel members that endpoint titration animal bioassay is the best way 

35 to measure log reduction in TSE infectivity. 

36 

37 The issue of whether or not to qualify the phrase “reduce infectivity” was discussed. The 

38 phrases “maximally reduced” or “maximally achievable reduction of infectivity” were 

39 introduced as was the idea that the label could note that infectivity was reduced to below 

40 the level of detection in a specific assay. An EPA representative noted that EPA was 

41 reluctant to put adjectives in front of “reduced.” 

42 

43 It was suggested that, if the testing is based on a specific animal bioassay model, the 

44 details of that model be included in the label. One suggestion was to separate the 

45 description of the prion strain and host of origin from the species of host in which the 

46 prion strain had been assayed. Another suggestion was to precisely define the log 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

43

1 reduction as well as the dynamic range of the assay (range of infective titer for which the 

2 bioassay yields precise and accurate results) so that the reader could understand whether 

3 the reduction of infectivity corresponded to an exact value or a lower limit. Two 

4 examples were read to the Panel: 

5 

6 Example 1: Has been demonstrated to reduce infectivity of prions (TSE agents) by five 

7 (5) logs or more based on bioassay of the bovine BSE strain in transgenic mice 

8 expressing the bovine prion gene in (specify transgenic mice). The dynamic range of the 

9 bioassay is five (5) logs. 

10 

11 Example 2: Has been demonstrated to reduce infectivity of prions (TSE agents) by six 

12 (6) logs based on bioassay of the hamster Sc237 strain in Syrian hamsters. The dynamic 

13 range of the bioassay is nine (9) logs. 

14 

15 Given the variety of different prion animal bioassay systems, the Panel discussed whether 

16 or not a specific log reduction of infectivity should be specified by EPA. It was brought 

17 out that even a six (6) log reduction would not be enough to completely inactivate high 

18 titer brain tissues such as a hamster brain infected with the Sc237 strain which could have 

19 as much as eleven (11) logs of infectivity per gram of brain. However, a six (6) log 

20 reduction is thought to be within the range of inactivation for human sporadic CJD or 

21 cattle BSE prions as these generally contain around six (6) logs of infectivity per gram of 

22 brain. One Panel member brought up the important caveat that limited sensitivity of 

23 these test models could account for the apparent lower infectivity TSE-infected human 

24 and bovine brain tissue, rather than a smaller number of infectious particles. One Panel 

25 member suggested that even a five (5) log reduction might be acceptable for a product 

26 with a prion-related claim under the condition that the labeling specified the infectivity 

27 reduction and the dynamic range of the animal bioassay. 

28 

29 Another Panel member was concerned with the notion that the registrant provide the 

30 agency with the details of the product testing methods, thereby presenting EPA with 

31 potentially a wide variety of methods requiring a great deal of time on EPA’s part to 

32 review. Other Panel members raised concerns that registrants might choose a model 

33 system with low levels of TSE infectivity to test their product. It was suggested that EPA 

34 poll members of this SAP and/or members of the interagency TSE working group to 

35 review the literature and gather the details of various animal bioassay designs, seek a 

36 consensus on these details and compile a list of acceptable prion strain/animal bioassay 

37 models. This would facilitate agency review of registrant methods by establishing an 

38 array of acceptable efficacy test methods. 

39 

40 There was general consensus among the Panel members that the label should give 

41 sufficiently detailed and accurate information to enable the product user to assess how 

42 efficiently, and under what usage, the prion pesticide product inactivates TSE infectivity. 

43 

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

45 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

44

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

2 label claim statement as “reduce infectivity,” but stated that the phrase “reduce 

3 infectivity” is valid only as long as the method used to measure infectivity has an 

4 acceptable dynamic range of detection in a relevant animal bioassay system. The Panel 

5 also suggested that the label statement include the prion strain and original host, bioassay 

6 animal host, and observed log reduction in infectivity as well as the dynamic range of the 

7 animal bioassay used. The SAP provided examples of what it considered to be desirable 

8 label statements. 

9 

10 In response to the SAP’s comments, EPA will continue with its proposal to use the term 

11 “reduces infectivity” provided the method used to measure infectivity has a dynamic 

12 range that measures at least six (6) logs of prion infectivity reduction. In addition, EPA 

13 will add to the label statement the amount of log reduction demonstrated by the product 

14 and basic information about the prion strain tested and the test organism. EPA does not 

15 see the need to add another statement about the dynamic range of the test since that is 

16 implicit in the amount of log reduction stated on the label. 

17 

18 =============================================================== 

19 

20 Issue 5. Whether different prion types exhibit variation in the degree of resistance to 

21 inactivation by pesticide chemicals and whether a hierarchy of resistance by prion type 

22 can be reliably determined at this time. 

23 

24 Charge Question 5. Please comment on whether a hierarchy of resistance among prion 

25 types can be reliably demonstrated for different pesticide chemicals based on the 

26 available data. 

27 

28 Panel Response 

29 

30 There is currently little data on the relative resistance to inactivation of prions from 

31 various species and strains, even though some studies report strain specific variations in 

32 resistance to inactivation (Peretz, Supattapone et al. 2006; Fernie, Steele et al. 2007; 

33 Giles, Glidden et al. 2008). However, it is clear that prions from different species and 

34 strains within the same species have distinct incubation time and lesion profiles in a given 

35 host as well as biochemical and biophysical properties. Given the fact that different prion 

36 pesticide products might act in different ways, it is likely that strains may respond 

37 differently and unpredictably to inactivation procedures. Therefore, the Panel’s 

38 consensus is that no single hierarchy of strains to inactivation procedures can be 

39 established at this time. 

40 

41 Efficiency of a particular prion inactivation procedure must be measured by a relevant 

42 and robust infectivity bioassay, depending on the prion agent for which the procedure is 

43 intended to be used. There was some debate among Panel members regarding the need 

44 relative to the prion strain hierarchy of resistance issue for use of two bioassay tests, with 

45 one of them done in a standard widely-used model, e.g. scrapie prion strain 263K in 

46 hamsters, and the other being specific to the samples under study. This will enable a 
EO 12866 REVIEW DRAFT 

DRAFT, DELIBERATIVE, DO NOT CITE OR RELEASE 

45

1 more robust proof-of-concept validation and the possibility to compare results from 

2 different prion pesticide products. However, the limitation is that some procedures may 

3 not work as well in the standard assay and still be useful for specific applications as 

4 supported by an animal bioassay in a relevant species. After discussion, and based on 

5 practical considerations, the majority of the Panel members recommend using end-point 

6 titrations in a relevant and robust animal bioassay depending on the samples to be 

7 decontaminated. Although, there was some debate, the majority of the Panel members 

8 think that decontamination studies should be performed on tissues from the natural host 

9 and that rodent adapted models of natural prion disease are not reliable substitutes. The 

10 experiments should be done with the natural samples, e.g. CJD, scrapie, or CWD, in the 

11 host such as either wild type animals or transgenic that provides the best practical and 

12 dynamic range for the study. One Panel member expressed concern that transgenic mice 

13 overexpressing prion protein may not be a more appropriate model to assess the problem 

14 than mice with wild type prion protein (J. Cell. Biochem. 106: 220-231; 2009). 

15 

16 The Panel recommends that the experimental protocol to study efficacy of a given 

17 procedure be reviewed in advance by a group of experts to decide the best animal model 

18 and sample preparation to conduct the study. 

19 

20 There was also discussion that experiments done with one source of material, e.g. one 

21 strain of sCJD, in one animal model, may not extrapolate to related strains of the agent, 

22 e.g. vCJD, fCJD or even other strains of sCJD (although one Panel member did not think 

23 there is substantial evidence of the existence of sCJD strains), so registration based on 

24 positive results with one strain may not guarantee that the product works even in related 

25 strains of the same species. One suggestion to approach this problem in a practical way 

26 was to complement results obtained in the in vivo infectivity assay with in vitro or cell 

27 based assays that can analyze a large diversity of strains. 

28 

29 In conclusion, the consensus from the Panel is that no single hierarchy of strain resistance 

30 to prion pesticide action can be established at this time. 

31 

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

33 

34 In its summary and detailed comments, the SAP concluded that no single hierarchy of 

35 strain resistance to pesticide action can be established at this time. 

36 

37 In response to the SAP’s comments, EPA agrees that inadequate data exist with which to 

38 establish a hierarchy of resistance to inactivation by pesticides. EPA’s question was 

39 aimed at determining whether testing one strain of prions that has been documented to be 

40 significantly more resistant to inactivation by pesticides than any other strain, and, if so, 

41 could that one prion strain be tested rather than every individual strain tested. The 

42 answer provided by the SAP confirms EPA’s belief that individual prion strains need to 

43 be tested and that label claims should be specific to reduction of infectivity of each prion 

44 strain.