RG-1 Regulatory Guidance:
Chapter 6 - Sampling and Laboratory Requirements
6.2 Good Laboratory Practices Protocol for Feed Ingredient Approvals
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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).
The method(s) must be current and in use by the submitting company. The following details must be provided for each method:
- a clear and concise method description
- identification of the method performance standards, that is to say,
- percent acceptable recovery
- analytical range (minimum and maximum)
- limit of quantitation
- limit of detection
- % coefficient of variation (CV) repeatability (within lab)
- % CV reproducibility (between labs), if available
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).
- identification of the critical control points and specifications, for example, evaporation temperature in step 5.3 to be 35°C ± 3°C.
- 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]
- results of method evaluation(s) to be in tabular form, with statistical analysis.
- proof of interference study of common drugs and feed additives, and identification of known interferences.
- 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.
- 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).
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.
- A minimum of five differently formulated complete feeds, that is to say, 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)
- 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 (that is to say, mass spectrometry, thin layer chromatography, et cetera)
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, et cetera.
- 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, et cetera, 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.
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