Chapter 6 - Sampling and Laboratory Requirements

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6.3 Guidelines for the Evaluation of Methods to determine the Activity of Enzyme Ingredients used in Animal Feeds

Part of the process for registration of feeds containing enzymes by the CFIA is the provision by the applicant of an acceptable method of analysis of the enzyme in carrier/filler. The applicant is also required to provide the necessary data to support reliability of the method. The following protocol is intended to ensure that the method, data and test materials provided by the applicant are adequate to allow for expeditious evaluation, validation and approval of the methodology.

1. Method Format

The purpose of the method submission is to allow the evaluator and analyst to understand and perform the method exactly as the originator intended. It is therefore necessary that the method contains all the necessary information presented in a detailed step by step standard operating procedure.

The method submitted must be clear and concisely written; currently in use by the submitting company; and proven to be specific, selective, reliable, and accurate for the active ingredient (in formulated products). The method must detail instructions for the preparation of a Standard Curve, and include detailed equations required to calculate activity (include Units). Critical control points and specifications for quality control and familiarization must be included in the methodology. In the event that the method in use requires highly specialized, expensive or automated equipment, an alternate manual and basic method should also be provided.

The following supporting documents for method performance standards must be included in the submission to properly evaluate the method(s). (Please provide data in tabular form):

• Scope and principal of methodology
• References
• Description of the reagents and equipment, along with specific ordering information and performance criteria for the reagents and equipment
• Standard/reagent preparation, storage, stability
• Sample preparation, stability
• Instrument operating parameters and expected performance
• Procedure for analysis of samples, controls, and standards
• Equations to calculate concentration or potency, instructions for preparation of standard curves and demonstration of calculations using the equations.
• Unit definition
• Information on collaborative studies
• System suitability information (statistical criteria)
• Interfering substances
• Safety information and precautions

2. Supporting Data

The applicant must provide the following information in support of the proposed methodology, performance standards, critical points, sample storage, stability etc.:

Standard Curve

The applicant must provide information on the statistical confidence of the standard curve which is used (directly or indirectly) to measure enzyme activity.

Specify analytical range (maximum and minimum)

• specify limit of quantitation,
• specify limit of detection.

Provide three sets of standard curve calibration data and graphs (minimum 5 points). For each set, provide statistical data which supports reliability in the curve.

• indicate slope, intercept of the calibration line,
• indicate determination of linearity (i.e. correlation coefficient).

Provide a statistical comparison of the 3 standard curves.

Enzyme Assay

Generally, a multi-point enzyme assay should be used for the determination of activity (i.e. a range of enzyme concentrations).

Provide a minimum of three (3) sets of enzyme assays graphs (minimum 5 points) for the determination of linearity, and analytical range. Specify the region of linearity.

Where a known enzyme standard is not available, provide statistical data in support of the use of an "in-house" standard. (i.e. provide previous assay results (with statistical comparison).

Provide analytical results from 3 batches (indicate lot number) of enzyme product (each tested in triplicate) for the determination of accuracy, precision, specificity, coefficient of variation, percent error.

Specify the percentage acceptable for the recovery of enzyme activity.

Indicate percent coefficient of variation of repeatability (within laboratory).

Indicate percent coefficient of variation of reproducibility (between laboratories) if possible.

3. Description of Experimental Design For Method Evaluation

Provide a description of the experimental design used by the company or other laboratory for the method evaluation(s). Include raw data and statistical analyses of method evaluation(s).

4. CFIA Laboratory Evaluation

When the above requirements are met, CFIA laboratories will proceed with the laboratory testing of the method. For CFIA laboratories to test the method, the following materials must be supplied by the company.

• Appropriate amount of pure standard enzyme or compound to be measured for constructing the calibration curve.
• Appropriate amount of enzyme sample for five, duplicate runs of analyses for each level of guarantee (formulation). Provide sample from same lot that was used to generate supporting data whenever possible.
• Appropriate amount of substrate for the analyses.
• Any uncommon reagent that is required by the methodology.

5. Evaluation Criteria

The following criteria will be applied as a guide to the evaluation of methodology:

• Regression of at least 0.995 for standard curves
• Recoveries of 95-110%
• Within laboratory coefficient of variation of 5%

6. Report

Upon request, the CFIA will prepare a report that includes the following information:

• Title Cover showing title, applicant, laboratory identification, evaluator's name and titles, date of report.
• Summary statement that includes acceptance or rejection of the method with signatures and dates
• Evaluation results of data provided by applicant
• Evaluation results of CFIA laboratory evaluation
• Comparison of submission data and CFIA laboratory evaluation
• Raw Data, Statistics, Tables
• Discussions
• Conclusions

7. Changes to Methodology

The CFIA must be informed of any modifications or new methodology. The new or modified methods must be verified. The degree of verification will depend on the changes.

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