Animals >
Livestock Feeds >
Trade Memoranda
T-3-148s - Guidelines for the evaluation of methods to
determine the activity of enzyme ingredients used in animal feeds
December 1, 1996
Part of the process for registration of feeds containing enzymes by the
Canadian Food Inspection Agency 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:
2.1 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.
2.1.1 Specify analytical range (maximum and
minimum)
- specify limit of quantitation,
- specify limit of detection.
2.1.2 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 (ie. correlation coefficient).
2.1.3 Provide a statistical comparison of the 3
standard curves.
2.2 Enzyme Assay.
Generally, a multi-point enzyme assay should be used for the
determination of activity (ie. a range of enzyme concentrations).
2.2.1Provide 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.
2.2.2Where a known enzyme standard is not available, provide
statistical data in support of the use of an "in-house" standard.
(ie. provide previous assay results (with statistical comparison).
2.2.3Provide 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.
2.2.4Specify the percentage acceptable for the recovery of
enzyme activity.
2.2.5Indicate percent coefficient of variation of
repeatability (within laboratory).
2.2.6Indicate 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. Canadian Food Inspection Agency Laboratory Evaluation
4.1 When the above requirements are met, Lab Services
Division (LSD) will proceed with the laboratory testing of the method. For LSD
to test the method, the following materials must be supplied by the
company.
4.1.1Appropriate amount of pure standard enzyme or compound
to be measured for constructing the calibration curve.
4.1.2Appropriate 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.
4.1.3Appropriate amount of substrate for the analyses.
4.1.4Any 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 CV of 5%
6. Report
Upon request, the Canadian Food Inspection Agency 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 laboratory evaluation
- Comparison of submission data and LSD laboratory evaluation
- Raw Data, Statistics, Tables
- Discussions
- Conclusions
7. Changes to Methodology
The Canadian Food Inspection Agency 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.
Questions regarding this memorandum may be directed to:
Canadian Food Inspection Agency
Animal Health and Production Division
Feed Section
59, Camelot Drive
Nepean, Ontario
K1A 0Y9
tel: (613) 225-2342
fax: (613) 228-6614
|