Transmissible Spongiform Encephalopathies:Two Models for Evaluating Effects of Cleaning and

Decontamination Measures on Residual Agent Infectivity

Food and Drug AdministrationTransmissible Spongiform Encephalopathies

Advisory Committee

17 July 2003Holiday Inn

Bethesda, MarylandDavid M. Asher, MD

Laboratory of Bacterial, Parasitic and Unconventional Agents

Division of Emerging and Transfusion-Transmitted Diseases

Office of Blood Research and ReviewCenter for Biologics Evaluation and ResearchUnited States Food and Drug Administration

e-mail address: asher@cber.fda.gov

Participants in CBER TSE Activities (partial list)

* = Participants in Decontamination Studies OBE

Steven Anderson OBRR Dot Scott (DH) Jaro Vostal & al (DH) Alan Williams (DBA) Pat McMahon* Olga Maximova* Pedro Piccardo* Kitty Pomeroy* Rolf Taffs* OCTGT Ruth Solomon

OCBQ Jerry Davis Maureen Knippen OD William Freas Diane Maloney OVRR Christine Anderson &

al Kostantin Chumakov &

al Alfred DelGrosso William Egan Theresa Finn Paul Richman

Other Participants in FDA TSE Activities

(partial list)* = Participants in Decontamination

Studies CDER Yuan-Yuan Chiu Gerald Feldman CDRH Stanley Brown* Charles Durfor Katharine

Merritt* Kiki Hellman Martha O’Lone Celia Witten

CFSAN Marilyn Ballmer Robert Brackett Karen Carson Elisa Elliott CVM George Graber Dragan

Momcilovic Burt Pritchett ORA Pete Cook Steven Solomon

Models to Evaluate Decontamination of TSE Agents Dried onto Test Surfaces:

Basic Methods Agent Strain: 263K hamster-adapted

scrapie, from Kimberlin R & Walker C: Characteristics of a short incubation model of scrapie in the golden hamster. J Gen Virol 1977;34:295

Glass slips: Modified “glass crushing spray method” from Chen JHS. Methods of testing virucides. In: Disinfection, Sterilization and Preservation, 4th edition, ed. Block SS. (Lea & Febiger, Malvern PA) 1991 p 1087

Steel needles: Modified from Flechsig E & al. Transmission of scrapie by steel-surface-bound prions. Mol Med 2001;7:679 and Zobeley E & al. Infectivity of scrapie prions bound to a stainless steel surface. Mol Med 1999;5:701

Dry Scrapie Agent Survives Autoclaving(263K hamster brain dried on glass)

Autoclaving Regimen

Control Scrapie

log10 LD50

Objects Infected (% of 10)

Animals Infected (% of 40)

121o C x 60’

>4

100%

100%

132o C x 60’

~7

90%

57%

134o C x 60’

~7

100%*

75%*

* Two objects assayed in 8 hamsters. Mean survival = 228 da ± 85 da. Brains of all dying hamsters had PrPSc by WB.

263K Scrapie Agent as a Suspensionof Hamster Brain Diluted in Phosphate-Buffered

Saline Dried on Steel Needles: Assay Results

DilutionScrapie+/

TotalAverage Scrapie Survival

(da)

Range of Scrapie Survival

(da)

10-1 2/2 103 96,110

10-2 2/2 97 97,97

10-3 2/2 106 103,109

10-5 1/2 101 101

10-7 0/2 na na

10-9 0/2 na na

Study to Evaluate WHO-RecommendedTSE Decontamination Regimens

1. Decontamination and cleaning procedures

Infected brain tissue dried on objects Saline suspension on glass slides Tissue paste on steel needles

Decontamination steps Chemical soak

»1N NaOH in autoclave x 30’»NaOCl 22oC x 60’

Autoclave (vacuum): 121oC x 30’ or 134oC x 90’ (in NaOH or in dH20 after NaOCl soak)

Cleaning: Ultrasound, detergent pH 9.45, >60oC, 90’

Rinse: H20 Terminal sterilization: Autoclave 121oC x 20’

Study to Evaluate WHO-RecommendedTSE Decontamination Regimens

2a. Assay of Residual Scrapie Infectivity: Glass Slips

Positive controls: Ten slips, each with 0.1 ml of dried-on 263K scrapie-infected 10% brain suspension, ground to powder in 1 ml PBS, glass let settle, fluids pooled, 10-fold dilutions in PBS, assayed (4 hamsters per dilution intracerebrally [ic] with 0.03 ml of each sample)

Tests: Each of 10 infected slips exposed to a decontamination regimen, ground to powder in PBS, fluid assayed as for control (0.03-ml samples of each ground slip into 4 hamsters ic), i.e., approx 12% of fluid assayed using a total of 40 hamsters per test, less any incidental deaths before 45 days.

Study to Evaluate WHO-RecommendedTSE Decontamination Regimens

2b. Assay of Residual Infectivity: Steel Needles

Positive control: Untreated samples as 10-fold dilutions of infected hamster brain in 10% (w/w) normal calf brain paste transferred to 4 wells of 96-well plastic tray, needles charged, dried, assayed (4 hamsters inoculated ic with separate needle—rotated quarter turn each direction)

Test: Each of 40 needles charged with 10% scrapie hamster brain in normal calf brain, dried, treated, then tested as for control above, i.e., a total of 40 hamsters for each test, less any incidental deaths before 45 days.

Positive-Control Titration of Scrapie 263K Agent as Hamster Brain Dried on Glass

(8.5 mo interim score)

Dilution Scrapie +/Total

Av Survival (da)

Range Survival

10-2 4/4 84 78-88

10-3 4/4 85 85-88

10-4 4/4 91 88-91

10-5 4/4 106 102-111

10-6 4/4 117 106-122

10-7 4/4 136 132-148

10-8 2/4 202 162,241

10-9 0/4 >250 >250

10-10 0/4 >250 >250

10-11 0/4 >250 >250

10-12 0/4 >250 >250

Positive-Control Titration of Scrapie Agent 263K in Hamster Brain, Diluted in Normal

Brain,Paste Dried on Steel Needles

(8.5 mo interim score)Dilution Scrapie +/Total Av Survival (da) Range Survival

Control Normal

0/3 >250 >250

10-1 3/4 88 87-88

10-2 4/4 93 88-95

10-3 4/4 93 82-101

10-4 4/4 107 101-111

10-5 4/4 110 107-111

10-6 4/4 125 113-135

10-7 2/4 152 150,153?

10-8 0/4 >250 >250

10-9 0/4 >250 >250

10-10 0/4 >250 >250

Conclusions

Methods devised to evaluate the effects of virucides are adaptable to evaluate decontamination of TSE agents.

Studies with two models both suggested that exposure of 263K scrapie agent dried on surfaces to solutions of NaOH or NaOCl with simultaneous (NaOH) or sequential (NaOCl) autoclaving and ultrasonic cleaning in hot alkaline detergent markedly reduced amounts of infectivity and the risk that objects would retain detectable amounts of agent.

Other chemical treatments may also be effective. Uncertainties remain:

Reliability of the decontamination procedures Predictive value of results from the models for

healthcare and manufacturing situations