Transmissible Spongiform Encephalopathies: Two Models for Evaluating Effects of Cleaning and Decontamination Measures on Residual Agent Infectivity Food and Drug Administration Transmissible Spongiform Encephalopathies Advisory Committee 17 July 2003 Holiday Inn Bethesda, Maryland David M. Asher, MD Laboratory of Bacterial, Parasitic and Unconventional Agents Division of Emerging and Transfusion-Transmitted Diseases Office of Blood Research and Review Center for Biologics Evaluation and Research United States Food and Drug Administration address:

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, 4 th 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)

263K Scrapie Agent as a Suspension of Hamster Brain Diluted in Phosphate-Buffered Saline Dried on Steel Needles: Assay Results Dilution Scrapie+/ Total Average Scrapie Survival (da) Range of Scrapie Survival (da) /210396, /29797, / , / /2na /2na

Study to Evaluate WHO-Recommended TSE 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 22 o C x 60’  Autoclave (vacuum): 121 o C x 30’ or 134 o C x 90’  (in NaOH or in dH 2 0 after NaOCl soak) Cleaning: Ultrasound, detergent pH 9.45, >60 o C, 90’ Rinse: H 2 0 Terminal sterilization: Autoclave 121 o C x 20’

Study to Evaluate WHO-Recommended TSE 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-Recommended TSE 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) DilutionScrapie +/TotalAv Survival (da)Range Survival / / / / / / / , /4> /4> /4> /4>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) DilutionScrapie +/TotalAv Survival (da)Range Survival Control Normal0/3> / / / / / / / ,153? /4> /4> /4>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