Chapter 6 - Sampling and Laboratory Requirements

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Table of Contents

6.1 Sampling Protocol for Feed Products

This page is currently under review. For more information on its availability, please contact:

Anna Lamberti
Feed Evaluation and Nutrition Section
Animal Feed Division

6.2 Good Laboratory Practices Protocol for Feed Ingredient Approvals

Part of the process for clearance of new ingredients/guarantees by the CFIA is the provision by the registrant of an acceptable method of analysis in complete feeds. The following protocol was developed to ensure that the method and test materials provided by the registrant are adequate to allow for expeditious approval of the methodology.

To be included with each submission is a method proven to be specific, selective, reliable, and accurate for the active ingredient in technical products, premixes and formulated products. The following items must be included in the submission to properly evaluate the method(s).

1.

The method(s) must be current and in use by the submitting company. The following details must be provided for each method:

  1. a clear and concise method description
  2. identification of the method performance standards, i.e.
    1. percent acceptable recovery
    2. analytical range (minimum and maximum)
    3. limit of quantitation
    4. limit of detection
    5. % coefficient of variation (CV) repeatability (within lab)
    6. % CV reproducibility (between labs), if available

    Definitions
    Limit of detection = X + 3o
    Limit of quantitation = X + 10o
    where X is the average signal of a blank and o is the standard deviation (assumed to be constant at relatively low levels of X).

  3. identification of the critical control points and specifications, e.g. evaporation temperature in step 5.3 to be 35°C ± 3°C.
  4. description of the experimental design used for the method evaluation(s) [evaluation(s) should include recovery studies on a variety of blank feeds at the intended formulation level, with the method of fortification being identified]
  5. results of method evaluation(s) to be in tabular form, with statistical analysis.
  6. proof of interference study of common drugs and feed additives, and identification of known interferences.
  7. chromatograms (if using HPLC or GC) of standards and technical product, and all blanks, feed samples, and premixes used in the method of evaluation(s), and any pertinent comments.
  8. raw data and calculated results for the accuracy and precision evaluation of premixes and the various formulated levels of complete feeds that will be manufactured (feeds to contain a variety of ingredients appropriate to the intended species).

2.

When the above requirements have been met, the CFIA usually will test the method shown to be most acceptable. When the method is to be tested, the following materials must be supplied.

  1. A minimum of five differently formulated complete feeds, i.e. containing a variety of feed ingredients (for each feed, supply a blank and fortified sample), and three premixes in which the ingredient is added (at least 1 kg of each). The amount of ingredient in the samples submitted is to be verified through analysis by the submitting company (the method of analysis used is to be indicated)
  2. Five (5) grams of analytical standard and ten (10) grams of 2 lots of technical product, with % purity indicated on the labels.

3. It is desirable that an alternate method be provided to confirm the results (i.e. mass spectrometry, thin layer chromatography, etc.)

4. A trace level procedure that will quantitate 1% of the lowest formulation level of the component must be included when an ingredient/active ingredient is know to be toxic to certain livestock species.

Once the method(s) have been evaluated, it is desirable to collaboratively study the method.

The method(s) will be incorporated into the Laboratory Services Method Manual and will therefore be available upon request to the public, once the ingredient/guarantee is accepted by the proper authorities.

The Animal Feed Division must be informed of any modifications or new methodology. The new or modified methods must be verified by our Science Branch, however the degree of verification would depend on the effect and changes.

Guidelines for the Evaluation of Methods to Determine the Viability of Direct Fed Microbials and Forage Additives containing Viable Microorganisms

Part of the process for registration of direct fed microbials or forage additives containing viable microorganisms by the CFIA is the provision by the applicant of an acceptable method of analysis for the microorganisms in the carrier or 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 permit the verification of label claims and 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 enumeration of viable microorganisms in formulated products. Critical control points, specifications for quality control and familiarization must be included in the methodology.

The following supporting documents for method performance standards must be included in the submission to properly evaluate the method:

  • Description of the microorganism(s) added in the product with the strain identification and source.
  • Specifications on the level(s) of microorganism(s) guaranteed in the product, expressed in colony forming units (CFU) per g or ml.
  • Enunciation of the scope and principles of the methodology.
  • List of references supporting the methodology.
  • Description of the reagents and equipment, along with specific ordering information and performance criteria.
  • Description of the experimental design with complete standard operating procedures used for the method evaluation. The description should include information on sample preparation and procedures for the analysis of samples, controls and standards.
  • Identification of the critical control points and specifications, including information on stability of the reagents and samples, and specification on potentially existing interfering substances such as non-target organism, components of the sample matrix, chemical additives, drugs etc.
  • Safety information and precautions.
  • Any existing information on collaborative studies.
  • Any other pertinent information.
  • When the submitted method is an alternative to those based on standard plate counts, such as methods based on ELISA, DNA probing, bioluminescence etc., additional information including standard curves, specificity and sensitivity data may be requested depending on the need.

2. Supporting Data

The applicant must provide the following information in support of the proposed methodology, which includes raw data and statistical analyses from their own method evaluation (data should be presented in a tabular form):

  • Provide analytical results from three production batches, each tested in triplicate (indicate lot numbers), including calculations of CFU / g or ml and coefficients of variation for precision assessment.
  • Provide analytical results from various days, analysts, reagent lots or from various laboratories, including calculations of CFU / g or ml and coefficients of variation for repeatability and reproducibility assessments.
  • When a reference method is available, provide analytical results, including calculation of CFU / g or ml from the reference method, to specify the accuracy of the submitted method in comparison with the reference method.
  • Provide analytical data confirming the selectivity of the method resulting from the proper isolation of the target microorganism. Adequate identification may be obtained from data on gram stain, cell morphology, biochemical analyses, patterns on utilization of carbohydrates, or any other valid confirmation tests.
  • When the purpose of the submitted method is the analysis of microbial contaminants or microorganisms detected at low levels, provide analytical results establishing limits of detection.

3. CFIA Laboratory Evaluation

When the above requirements are met, CFIA will proceed with a desk review followed by a laboratory evaluation of the method. For the laboratory evaluation, the following materials must be supplied by the company.

  • An appropriate amount of pure culture or culture mix.
  • An appropriate amount of non-inoculated sample matrix, carrier or filler (minimum 250 g).
  • An appropriate amount of product sample (for 5 analyses per batch) from the same three production batches analysed previously by the company (see 2. Supporting data).
  • Any uncommon media, reagents or supplies that are required in the methodology.

4. Evaluation Criteria

An adequate method for the enumeration of the microorganism(s) will be approved following criteria based on specificity, accuracy, precision, repeatability and reproducibility, taking into consideration the analytical results provided by the company and those generated by CFIA.

5. CFIA Report

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

  • Title, name of applicant, identification of the CFIA laboratory and the evaluators, and date of report.
  • Summary statement indicating acceptance or rejection of the method, with signatures and dates.
  • Results of the evaluation of the data provided by the applicant and the data generated by the CFIA laboratory, with comparisons.
  • Tables including raw data, calculations and statistical analysis for the determination of specificity, accuracy, precision, repeatability and reproducibility of the submitted method.
  • Discussion and conclusion.

6. Changes to Methodology

The CFIA must be informed of any modifications of the methodology. The new or modified method must be submitted to a desk review. The degree of verification will depend on the changes.

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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