A2LA. R231 Specific Requirements: Threat Agent Testing Laboratory Accreditation Program. December 6, 2017

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1 Laboratory Page 1 of 17 Laboratory December 6, by A2LA All rights reserved. No part of this document may be reproduced in any form or by any means without the prior written permission of A2LA.

2 Laboratory Page 2 of 17 Table of Contents 1.0 SCOPE REFERENCES TERMS AND DEFINITIONS SPECIFIC CRITERIA... 5 DOCUMENT REVISION HISTORY... 15

3 Laboratory Page 3 of SCOPE This document describes the requirements for threat agent testing organizations seeking A2LA accreditation. For the purposes of this document threat agent testing refers to testing performed on submitted or collected chemical materials and/or biological organisms or products intended for harm and where the result of that testing could be used in criminal or civil litigation. Participation in this program is voluntary. Organizations seeking accreditation for threat agent testing must also meet A2LA policy and requirements documents: P102a Policy on Reference Material Traceability for Life Sciences Testing Laboratories.doc P103b Annex-Policy on Estimating Measurement Uncertainty for Life Sciences Testing Labs.doc P113 A2LA Policy on Measurement Traceability for Life Sciences Testing Laboratories R101 General Requirements: Accreditation of ISO/IEC laboratories R102 Conditions of Accreditation R103 General Requirements: Proficiency Testing for ISO/IEC Laboratories R103a Annex: Proficiency Testing for ISOIEC Laboratories R105 Requirements When Making Reference to A2LA Accredited Status When testing is performed outside the organization s permanent facility, R104 General Requirements: Accreditation of Field Testing and Field Calibration Laboratories applies. GENERAL CRITERIA: The general criteria for accreditation are contained in ISO/IEC 17025:2005, General requirements for the competence of testing and calibration laboratories, as referenced in Part A of R101 General Requirements: Accreditation of ISO/IEC Laboratories. PROFICIENCY TESTING: Please refer to R103 General Requirements: Proficiency Testing for ISO/IEC Laboratories for the proficiency testing requirements applicable to this program. 2.0 REFERENCES Food and Drug Administration ORA-LAB ORA Laboratory Procedure Volume II - Methods, Method Verification and Validation. Integrated Consortium of Laboratory Networks (ICLN) Guidelines for Comparison of Validation Levels Between Networks. 3.0 TERMS AND DEFINITIONS The following definitions were drawn from the ICLN s Guidelines for Comparison of Validation Levels Between Networks. Accuracy: A measure of the degree of conformity of a value generated by a specific procedure to the assumed or accepted true value, and includes precision and bias.

4 Laboratory Page 4 of 17 Certified Reference Material (CRM): reference material, accompanied by a certificate, one or more of whose property values are certified by a procedure which establishes metrological traceability to an accurate realization of the unit in which the property values are expressed, and for which each certified value is accompanied by an uncertainty at a stated level of confidence (slightly modified from VIM04). NOTE: The term "Standard Reference Material" (SRM) is the name of a certified reference material (CRM), which is the trademark name of a certified reference material that has been certified and is distributed by the National Institute of Standards and Technology (NIST). Limit of Quantification (LOQ): Lowest amount or concentration of analyte that can be quantitatively determined with an acceptable level of uncertainty, also referred to as the limit of determination. Linearity: Defines the ability of the method to obtain test results proportional to the concentration. Method Detection Limit (MDL): Lowest amount or concentration of analyte that a specific method can statistically differentiate from analyte-free sample matrix. This is dependent on sensitivity, instrumental noise, blank variability, sample matrix variability, and dilution factor. Minimum Detectable Concentration (MDC): An estimate of the minimum true concentration of analyte that must be present in a sample to ensure a specified high probability (usually 95%) that the measured response will exceed the detection threshold (i.e., critical value), leading one to conclude correctly that the analyte is present. Minimum Quantifiable Concentration (MQC): The smallest concentration of analyte whose presence in a laboratory sample ensures the relative standard deviation of the measurement does not exceed a specified value, usually 10 percent. Precision: Degree of agreement of measurements under specified conditions. The precision is described by statistical methods such as a standard deviation or confidence limit. See also Random Error. Repeatability expresses the precision under the same operating conditions over a short period of time. Intermediate precision expresses within-laboratory variations, such as different days, different analysts, and different equipment. Reproducibility expresses the precision between laboratories. Reference Material: A material or substance, one or more of whose property values are sufficiently homogenous and well established to be used for the calibration of an apparatus, the assessment of a measurement method, or for assigning values to materials. Repeatability: The closeness of the agreement between the results of successive measurements of the same measurand carried out under the same conditions of measurement. Sensitivity: The lowest concentration that can be distinguished from background noise or the smallest amount of a substance or organism that can accurately be measured by a method or test system is the analytical sensitivity. However, sensitivity is commonly defined as the slope of the calibration curve at a level near the LOQ. For assays that will be used to test human clinical specimens, the method's analytical sensitivity is distinct from the method's clinical diagnostic sensitivity. Clinical diagnostic sensitivity is the percentage of persons who have a given condition who

5 Laboratory Page 5 of 17 are identified by the method as positive for the condition (high analytical sensitivity does not guarantee acceptable diagnostic sensitivity). Specificity: Analytical specificity is the ability of a method to measure one particular analyte in the presence of components which may be expected to be present. For methods that will be used to test human clinical specimens, the method's analytical specificity is distinct from the method's clinical diagnostic specificity. Clinical diagnostic specificity is the percentage of persons who do not have a given condition who are identified by the method as negative for the condition. Trueness: The degree of agreement of the expected value from a measurement with the true value or accepted reference value. This is related to systematic error (bias). 4.0 SPECIFIC CRITERIA Specific criteria are used to augment the general criteria applicable to a certain field of testing, testing technology, or specific test. Along with the requirements of ISO/IEC 17025:2005, the specific requirements for the Threat Agent Testing laboratory program are outlined below. 4.0 Management Requirements 4.13 Control of Records : Records of original observations, derived data and sufficient information to establish an audit trail are required in order to enable the test to be repeated under conditions as close as possible to the original. These records include, but may not be limited to: Incubator temperature (start/end or at least twice daily); Oven temperature (start/end or at least twice daily); Time incubation started and ended (if no timer set); Extraction time started and ended (if no timer set); Initials of analyst involved in each step of the preparation and testing process; Lot numbers of reagents used (commercial and prepared); Lot numbers of media used (commercial and prepared); Reagent & media preparation records (including lot numbers of all components); Lot numbers of materials critical to the process (ex. extraction filters, petri dishes, etc.); Lot numbers and Certificates of Analysis of Certified Reference Materials (CRMs) and Reference Materials (RMs) (also see );

6 Laboratory Page 6 of 17 Identification of equipment used that is critical to the process (ex. pipettors, ovens, incubators, HPLC instruments, balances, etc.); Purchasing records, including POs and vendor evaluation/selection records; and QC of materials received critical to the test process. 5.0 Technical Requirements 5.3 Accommodation and Environmental Conditions 5.3.2: The laboratory shall monitor, control and record environmental conditions as required by the relevant specifications, methods and procedures or where they influence the quality of the results. This includes the following: Air exposure plates - daily (when plating is performed); Environmental swabs (contamination control swabs) of the test area to ensure cross-contamination has not occurred - daily (when testing performed) these swabs may be tested with daily samples, but are required to be tested if suspect target is identified; Restricted access to the test area when testing is in process, restricted access of unauthorized personnel, procedures to handle breach in access; and Evaluation of previous use of the area before new testing activities commence. 5.4 Test Methods and Method Validation The requirements for method validation are drawn from the minimal guidelines for the ICLN to validate methods which shall be used by the respective laboratory : Prior to developing the proposed methods: : The intended use of the method shall be defined; : The intended use should be aligned with current capabilities; : A written protocol which includes all of the following elements shall be prepared: Intended use and criteria for use; Assay principle and safety precautions; Acceptable sample types, sample collection method, preparation, preservation, storage and transportation conditions; Description of reagents supplied or directions for preparation and QC;

7 Laboratory Page 7 of 17 Description of quality controls to be used; Detailed instructions on how to perform the method; Detailed instructions on how to interpret and report the result; Any limitations of the method that are known or suspected; A summary of the performance characteristics of the method; and A statement on the target uncertainty of measurements for each analyte (analytical goal for accuracy) in order for the values to be fit for their intended purpose : Methods shall be validated whenever any of the following occur: : Development of a proposed new official method; : Expansion of the scope of an existing method to include additional analytes or new matrices; : Modification of a method s range beyond validated levels; : Modification of a method that may alter its performance specifications. All but the most trivial of changes shall be evaluated for effects on method performance. This includes: Changes to the fundamental science of an existing method and Equivalence issues such as substitutions of reagents/apparatus, or changes to some instrumental parameters since it is difficult to predict the results of any change : Performance specifications required to validate a method include the following: : Performance specifications that should be determined to validate a method will vary depending on the intended use, the type of method being validated, and the degree to which it has previously been validated. All methods submitted shall have all the proper controls and the parameters for calibrating and operating the method instrumentation included in the written procedure : Typical validation characteristics which shall be considered are the following: : Characteristics of quantitative methods: Method uncertainty at range of use; Minimum quantifiable concentration; Applicable analyte concentration range;

8 Laboratory Page 8 of 17 Accuracy (trueness); Precision (repeatability); Analytical specificity; Linearity; and Ruggedness/robustness : Characteristic for qualitative methods: Evaluate for reliable identification of an analyte at or below some target level; Sensitivity; Specificity; Limit of detection (See Detection limit, MDL, MDC); and Ruggedness/robustness : The following validation tools shall be used to demonstrate the ability to meet method specifications of performance: Blanks: Use of various types of blanks enables assessment of how much of the result is attributable to the analyte in relation to other causes; Reference materials and certified reference materials with the typical interferences expected. Use of known materials can be incorporated to assess the accuracy of the method, as well as for obtaining information on interferences; Fortified (spiked) materials and solutions: Recovery determinations can be estimated from fortification or spiking with a known amount of analyte. (Note: Understand that spiked recovery may not be truly representative of recovery from naturally incurred analytes); Repeatability: Replicate analyses provide a means of checking for changes in precision in an analytical process which could adversely affect the results; Statistics: Statistical techniques are employed to evaluate accuracy, trueness and precision, linear range, limits of detection and quantification, and measurement uncertainty. NOTE 1: Please see ORA-LAB in section 2.0 for reference : General Validation Protocol : The following provides practices that shall be used to determine method performance characteristics: Quantitative measurements, e.g. determine limit of quantification (LOQ), linear response.

9 Laboratory Page 9 of 17 Minimally need LOD; Prepare and analyze spiked blanks, matrix samples of known concentration utilizing one to three different concentration levels: low, medium, high based on the intended use of the method. These samples are carried through the complete sample preparation procedure, extraction and analytical steps of a particular method. Matrix effects shall be assessed with these samples. Accuracy or bias and precision are calculated from these results; data will also evaluate robustness of the method resulting from changes in the sample matrix. (Note: Proper certified reference materials and reference standards are used when available.); Assure that adequate sample replicates are performed and that results from replicate measurements of each analyte are compared; Analyze blanks (reagent and matrix) and compare these results to the reported limit of detection; and Evaluate interferences: spectral, physical, chemical or memory by analyzing samples containing various suspected interferences in the presence of the measurand : Validation of Methods (Original, New or Modified) : The following practices shall include, but not be limited to, matrix extensions and platform changes: In cases where the sample preparation and/or the extraction procedure/analytical method is modified from the existing test procedure and protocol, the new method shall demonstrate that the modifications do not adversely affect the precision and accuracy or bias of the data obtained; In order to implement the modified method, the standard or existing method is first performed. The modified method is then verified against the original method validation protocol; For original or new methods the authors shall pick a validation level that is suitable for their situation; Statistical methods are employed to verify performance between the original validated and new method sample means and to determine the degree of accuracy. (Note, for example: The t-test assesses whether the means of two groups are statistically different. The t-test is to be less than or equal to the t-critical value. The F-test is used to determine the significance of difference between two sample variances. The F value is to be less than or equal to the F-critical value). Note: See Tables for details : Estimation of Uncertainty of Measurement : Measurement Uncertainty is required to be estimated for tests with a qualitative reported result, if the result is based on quantitative endpoint, such as presence of a compound above a threshold value. 5.6 Measurement Traceability

10 Laboratory Page 10 of : In the event that CRMs are not available from an Asia Pacific Laboratory Accreditation Cooperation (APLAC) recognized, ISO/IEC accredited source, the laboratory shall provide records of material evaluation and traceability consistent with A2LA Policy P102a, Policy on Reference Material Traceability for Life Sciences Testing Laboratories. This includes the following records: : When Reference Materials are used as process controls: Unique identifier or lot number of the material (required); Description including (as appropriate) Origin, (e.g. inorganic, human or animal, vegetable, or microbial); Who and where manufactured (may be generated in-house or provided by supplier); Other pertinent information; and Records of use and process control results (required) : When Reference Materials are used to validate methods: Unique identifier or lot number (required); Origin (if applicable) (e.g. inorganic, human or animal, vegetable, or microbial); Who and where manufactured (may be generated in-house or provided by supplier); Issuing authority (if applicable) (e.g. WHO, BCR, IRMM, USP, ATCC); Records of characterization (if applicable); Molecular form(s) of or surrogate for the analyte (e.g. steric isomer for an amino acid, or glycerol for glycerol ester); Matrix (e.g. buffered bovine albumin solution); State(s) of aggregation (gas, liquid, solid); Phase(s) (solution, suspension, lyophilized); Records of use (required); and Records of Materials validation activities including an appropriate validation plan, acceptance criteria, and results (as appropriate) : When Reference Materials used are obtained from an authoritative source or prepared by a published standard procedure (to demonstrate operational traceability): Unique identifier or lot number (required);

11 Laboratory Page 11 of 17 Origin (if applicable) (e.g. inorganic, human or animal, vegetable, or microbial); Who and where manufactured (may be generated in-house or provided by supplier); Issuing authority (e.g. WHO, BCR, IRMM, USP, ATCC); Records of characterization (if applicable); Molecular form(s) of, or surrogate for the analyte (e.g. steric isomer for an amino acid, or glycerol for glycerol ester); Method used to establish value (AL); Matrix (e.g. buffered bovine albumin solution, analyte concentration in water, or soil, analyte concentration in air); State(s) of aggregation (gas, liquid, solid); Phase(s) (solution, suspension, lyophilized); and Records of use (required). 5.9 Assuring the Quality of Testing and Calibration Results 5.9.1: When commercial proficiency testing programs are available, laboratories shall participate in these programs annually for all targets, unless the programs are not relevant to the testing performed. Often, proficiency testing is not available for specific threat agents tested. In the event an external proficiency testing program is not available, the laboratory shall demonstrate competency annually, using blind spiked samples (spiked with target or surrogate). Prior to achieving initial accreditation, the laboratory shall have successfully participated in one of the two requirements listed above for each method on their desired Scope.

12 Laboratory Page 12 of 17 Table 1. Validation Recommendations for Chemistry Methods Originating Laboratory Study Level One: Urgent Usage/ Matrix Extension Level Two: Single Lab Validation Level Three: Independent Lab Validation Level Four: Collaborative Study a,b # of matrices 1 or more as the situation At least as the 3-5 as the 3-5 as the 3-5 as the # of sources situation situation situation situation # participating laboratories to 10 (qualitative) # minimum analyte level 1 spike level (spike at LOD) and 1 matrix blank 2 spike levels (spike at LOD and mid level) and 1 matrix blank 2 spike levels (spike at LOD and mid level) and 1 matrix blank 3 spike levels and 1 matrix blank Replicates per matrix tested at each level 2 2 (qualitative) 2 4 (qualitative) 2 6 (qualitative) 2 6 (qualitative) a Pure and Applied Chemistry, 67, No. 2, 1995,

13 Laboratory Page 13 of 17 b AOAC International, AOAC Peer-Verified Methods Program Manual on Policies and Procedures, AOAC international 1998.

14 Laboratory Page 14 of 17 Table 2. Validation Recommendations for Microbiological Methods Originating Laboratory Study Level One: Urgent usage Level Two: Single lab Validation Level Three: Independent Lab Validation Level Four: Collaborative Study i,j # of strains of target organism 1 to 5b 30b 30b 50b (inclusivity) a # of strains of non-target organism (exclusivity) 1 to 5 c 30 c 30 c 30 c # of matrices 1 or more d 1 or more d 1 or more d Up to 20 matrices d # analyte levels/matrix Set level based on intended use and uninoculated control One inoculated level e, one at 1 log higher, and uninoculated control One inoculated level e, one at 1 log higher, and uninoculated control One inoculated level e, one at 1 log higher (optional), and uninoculated control Replicates per matrix f 2 or more 6 or more 6 or more at least 10 Aging of inoculated samples prior to testing No No Yes g Yes g

15 Laboratory Page 15 of 17 Originating Laboratory Study Level One: Urgent usage Level Two: Single lab Validation Level Three: Independent Lab Validation Level Four: Collaborative Study i,j In 1 matrix at +1 In 1 matrix at +1 In 1 matrix at +1 Addition of competitor strain h Normal background flora log >analyte at fractional positive d analyte log >analyte at fractional positive d analyte log >analyte at fractional positive d analyte level level level Comparison to recognized method No Yes, if available Yes, if available Yes, if available a For bacteriological methods at 10 3 CFU/ ml (g) following the method protocol. b Select agent organisms may have limited strain availability for inclusion and exclusion studies. An attempt should be made to obtain necessary strains when possible. c For bacteriological methods at 10 3 CFU/mL for non-target organisms grown in a non-selective rich medium d Depends on applicability of method e Must be adjusted to achieve fractional positive results (one or both methods give 40-90% positive results) f Except for Level 1, must run six replicates per matrix, with as many different matrix sources as possible, up to six. Additional replicates may be required based on the specific method requirements for bias, precision, uncertainty, etc. g Period of aging depends on material being tested h An appropriate competitor is one that gives similar reactions in enrichment and detection systems i Pure and Applied Chemistry, 67, No. 2, 1995, j AOAC International, AOAC Peer-Verified Methods Program Manual on Policies and Procedures, AOAC international 1998.

16 Laboratory Page 16 of 17 Table 3. Validation Recommendations for Radiological Methods Originating Laboratory Study Level One: Urgent Usage Matrix Extension Level Two: Single Lab Validation Level Three: Independent Lab Validation Level Four: Collaborative Study a,b 1 or more as 1 or more as 1 or more as 1 or more as # of matrices the situation the situation the situation the situation 3-5 as the 3-5 as the 3-5 as the # of sources situation situation situation At least 5 # participating laboratories to 10 (qualitative) # minimum analyte levels/ matrix 1 spike level and 1 matrix blank 2 spike levels and 1 matrix blank 2 spike levels and 1 matrix blank 3 spike levels and 1 matrix blank Replicates per matrix tested at each level 2 2 (qualitative) 2 6 (qualitative) 2 6 (qualitative) 2 6 (qualitative) a Pure and Applied Chemistry, 67, No. 2, 1995, b AOAC International, AOAC Peer-Verified Methods Program Manual on Policies and Procedures, AOAC international 1998.

17 Laboratory Page 17 of 17 Document Revision History Date Description 4/1/2016 Initial version released. 12/6/2017 Section 4.0: Addition of C231 clauses. See C231 revision history from August 23, 2017 for further details. Formatting changes (throughout document) by A2LA All rights reserved. No part of this document may be reproduced in any form or by any means without the prior written permission of A2LA.