VB-GL-3.19: Licensing veterinary diagnostic test kits in Canada

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1. Introduction

The purpose of this document is to inform veterinary biologics (VB) researchers, manufacturers and Canadian importers of the requirements for in vitro diagnostic test kits for the diagnosis of infectious diseases in animals. The Canadian Centre for Veterinary Biologics (CCVB) of the Canadian Food Inspection Agency (CFIA) is responsible for licensing veterinary biologics in Canada and for regulating their manufacture, testing, importation, serial release, and use.

In addition to the present guideline, manufacturers that intend to submit an application for regulatory approval of an in vitro diagnostic test kit as a veterinary biologic should review VB-GL-3.1: Preparation of new product licensing (registration) submissions for veterinary biologics, for additional information regarding licensing applications and a checklist of required material, and VB-GL-3.7: Preparation of Outline of Production, special outlines and summary of changes for guidance on preparing the required documentation.

This guideline is not intended to address in vivo diagnostic test kits, which would have additional data requirements. Manufacturers of in vivo diagnostic kits should consult the CCVB directly.

1.1 Legal Authority

2. Products regulated as veterinary biologics

As defined in the Health of Animals Act, veterinary biologics include any product that is manufactured, sold, or represented for use in the diagnosis of a disease, disorder, or abnormal physical state in animals. Consequently, diagnostic test kits that are intended for use in the diagnosis of infectious diseases in animals are regulated as veterinary biologics by the CCVB.

Regulated kits include those based on antibody-antigen interactions, for example, enzyme-linked immunosorbent assays [ELISA], lateral flow assays, as well as diagnostic test kits based on other platforms, including the polymerase chain reaction (PCR), aptamers, and nanotechnology.

In vitro tests that are developed and used "in-house" by a diagnostic laboratory are considered veterinary biologics, and thus must conform to the principles and technical standards outlined in the Health of Animals Act and regulations, and in CCVB guidance documents. The CCVB does not typically review and approve in-house in vitro diagnostic tests, unless the diagnostic test is assembled into a kit for sale or distribution to other laboratories.

Microbial antigens, animal sera, antibodies, PCR primers, and other individual reagents that are sold without any claims regarding their potential use as a reagent in diagnostic tests are not considered veterinary biologics for regulatory purposes. Individuals who wish to import these products should contact their CFIA Area Health Office for guidance on whether an import permit is required for other reasons.

Although in vitro diagnostic test kits for detecting physiological parameters (for example, pregnancy tests), organ function, blood chemistry, hormone levels, or non-infectious diseases (for example, cancer) might be considered to fit the definition of a veterinary biologic, they could also fit the definition of a veterinary drug under the Food and Drugs Act. According to the current Memorandum of Understanding between the CFIA and Health Canada regarding regulatory control of veterinary biologics and veterinary drugs, these types of veterinary diagnostic kits fall under Health Canada's regulatory jurisdiction.

3. Requirements to support product licensing

The CCVB uses a risk-based approach to evaluate veterinary diagnostic test kits. The level of regulatory review depends on multiple factors, including the kit's intended purpose (herd surveillance versus individual animal certification), the disease being diagnosed (for example, commonly occurring treatable indigenous diseases versus notifiable or reportable diseases subject to federal disease control programs), and the kit's final user (for example, accredited diagnostic laboratory versus practising veterinarian). In general, to license a diagnostic kit in Canada, the manufacturer must demonstrate that the kit is pure and safe for use, and that it is sufficiently potent and efficacious to accurately detect its intended disease agent.

The following information is required as part of the licensing application for an in vitro veterinary diagnostic test kit:

3.1 Master seed and cell line quality control

Master seeds and cell lines used in the production of a diagnostic test kit should be tested to confirm identity and purity (that is, freedom from adventitious agents such as extraneous bacteria, fungi, mycoplasmas, and viruses).

3.2 Reference panel selection and potency testing

The manufacturer must develop a reliable potency test for the product. Potency tests are performed on each batch (serial) of a kit to ensure that kit components are functioning as expected, and to qualify a serial as suitable for release. This is accomplished by testing a panel of well-characterized samples of varying reactivity (strong positive, weak positive, and negative) and verifying that the results meet some pre-defined acceptance criteria for a satisfactory serial.

Section V.C. of the Outline of Production (OP) for a kit must provide a description of the potency test, the identity of the reference panel members, and the criteria for a satisfactory potency test result. The potency test should be performed, employing the test procedures recommended to end-users in the product labelling. Appendix 1 of this guideline provides more information on reference panel selection.

The potency test criteria should be sufficiently stringent to reliably differentiate serials with satisfactory versus sub-standard potency. Whenever possible, manufacturers should develop objective, quantitative, or semi-quantitative methods to interpret the potency test results. For example, rather than simply interpreting the results of a lateral flow device as visually positive or negative, the intensity of coloured bands or spots should be measured, using a densitometer or given a value by comparison to a monochromatic colour scale. Within the OP, the manufacturer must define criteria for an acceptable result for each panel member (for example, optical density between 0.80 and 1.00), as well as criteria for a satisfactory potency test for the serial, based on the proportion of reference panel members that achieve their acceptable result (100%, unless otherwise justified).

The potency test procedure, samples, and criteria should be established prior to the production of the serials used in the key efficacy and performance studies.

3.3 Batch-to-batch consistency

The manufacturer must submit the results of the potency tests performed on at least 3 pre-licensing serials of the kit, which were produced and tested according to the OP approved by the CCVB, to demonstrate the manufacturer's ability to maintain batch-to-batch (serial-to-serial) consistency in product quality.

3.4 Efficacy and performance

Note: Whenever possible, test samples used in efficacy studies should be coded so that the individuals who perform the analyses are unaware of the identity of the samples.

3.4.1 Minimum detection limit – The manufacturer should conduct a study using test samples of varying concentration (serial dilutions of a sample of known concentration) to estimate the minimum amount of antibody, protein antigen, or nucleic acid template that the kit can detect.

Manufacturers should also consider conducting temporal studies to establish the kit's ability to detect a disease agent or an antibody response over a specified number of days post-infection.

3.4.2 Cross-reactivity – When validating a new diagnostic test kit, manufacturers must consider the limitations of the assay and determine whether any factors exist that would lead to inaccurate results. For example, for kits that detect the presence of pathogen constituents, manufacturers should perform a database and/or literature search to identify any related antigenic proteins or homologous DNA/RNA sequences from other pathogens, which could yield false positives. The conclusions of such theoretical analyses should be communicated to the CCVB. If a related agent is identified, and there is a possibility of its presence in samples from Canada, experiments to examine whether the kit also detects the related agent should be conducted. The manufacturer should also test samples known to be positive for the most common pathogens affecting the target species in Canada.

Where appropriate, kits detecting the presence of antibody should similarly be tested against sera from animals exposed to, or vaccinated against, related pathogens in order to detect cross-reactivity.

In addition to demonstrating the absence of unwanted cross-reactivity, when distinct strains or serotypes of a pathogen exist (for example, bovine viral diarrhea virus types 1 and 2, canine parvovirus types 2, 2a, 2b, and 2c), the manufacturer must determine which strains or serotypes that the test kit can accurately detect and present these data to the CCVB. If not all strains or serotypes can be detected by the kit, this information should be disclosed in the product labelling.

3.4.3 Diagnostic threshold – For kits with a quantitative result, the manufacturer will need to define diagnostic threshold (cut-off) values to direct the interpretation of the results (for example, categorize as negative, suspicious, or positive). An analysis, such as a receiver operating characteristic (ROC) curve analysis, should be performed to determine the optimal cut-off values for the kit. Consideration should be given to the intended purpose of the kit when selecting cut-off values. In some instances, it might be desirable to choose cut-off values that maximize sensitivity (when false negatives are especially undesirable) or maximize specificity (when false positives are especially undesirable), rather than simply picking the cut-off values that yield the highest overall accuracy (those corresponding to the upper-left corner of the ROC curve).

3.4.4 Repeatability/reproducibility – Data must be provided that demonstrate the repeatability and reproducibility of a diagnostic test kit. Typically, this involves examining the consistency of reactivity of positive, weak positive, and negative samples when:

  • samples are assayed in multiple wells of the same multi-sample apparatus (for an ELISA or quantitative PCR in plate format), or on a series of equivalent single-sample devices (such as multiple lateral flow devices from the same serial) in the same testing session
  • samples are tested separately on at least 3 different days
  • samples are assayed independently by at least 3 different operators
  • samples are tested, using kits of different serials (at least 3 pre-licensing serials)

For kits with quantitative results, the manufacturer should calculate the mean, standard deviation, and coefficient of variation for each sample. It is recommended that samples from the potency test reference panel be used in the repeatability/reproducibility evaluation.

3.4.5 Sensitivity and specificity – A study must be performed to allow for an estimation of the sensitivity and specificity of the kit. If the product labelling indicates that multiple sample types (for example whole blood or serum) or samples from multiple animal species (for example chickens or turkeys) can be analyzed using the kit, sensitivity and specificity data should be provided for each combination.

Sensitivity

means the proportion of diseased (or exposed) animals that test positive. This is calculated by dividing the number of true positive results by the sum of the true positive and false negative results.

Specificity

means the proportion of non-diseased (or unexposed) animals that test negative. This is calculated by dividing the number of true negative results by the sum of the true negative plus false positive results.

As the above definitions suggest, samples must be tested by a reference method to determine the "true" positive or negative status of the samples. Ideally, this will be accomplished by using a pathognomonic test that is an absolute predictor of the disease state (for example, culture of the micro-organism) or a widely accepted "gold standard" test as the reference test to which the results of the kit under evaluation are compared.

If a pathognomonic or gold standard test does not exist or is unavailable for use as a reference, the manufacturer may estimate the relative sensitivity (positive percent agreement) and the relative specificity (negative percent agreement) of the kit under evaluation by comparing the test kit results to those generated by another relevant test kit. In cases where potential comparison tests are known to be imperfect, it may be preferable to use a battery of tests as the reference test method. In this situation, the manufacturer must clearly pre-define the criteria that will be used for determining the positive/negative status of each sample by the reference method (for example, only samples that test positive by all test methods are considered positive).

To accurately estimate the sensitivity of a kit, samples covering a range of reactivities, from strong positive to weak positive, should be evaluated. Similarly, samples should be obtained from uninfected animals originating from various sources, and should include animals that have received commonly used immunizing products to properly estimate the specificity.

When designing studies to estimate sensitivity and specificity, manufacturers should give consideration to minimizing bias and sources of error. Persons who interpret the test results should not be aware of the expected positive/negative result of the sample. Samples should be tested concurrently with the kit under evaluation and the reference test to eliminate differences in the storage and handling of samples as a potential factor for discrepant results. In addition, the study should employ at least some previously untested samples, rather than only samples that have been previously tested by the reference test. This helps avoid the exclusion of borderline positive samples from the sensitivity and specificity study, and inclusion of only samples that have been well-characterized to test strongly positive or clearly negative. Diseases with low prevalence will likely necessitate including samples based on a prior positive test result. If samples are being chosen from a bank of positive samples, samples of varying levels of positivity on the initial test should be selected.

The studies used to estimate the sensitivity and specificity of a kit should be performed, using kits at the minimum acceptable level of potency, as defined in the OP for the release of a kit serial for sale. The studies are usually performed with at least 1 of the pre-licensing serials.

3.4.6 Suitability (field performance) – The manufacturer must evaluate the suitability of the kit for use in typical laboratory settings outside of the manufacturer's establishment. Participating laboratories (usually at least 2) should be independent from the manufacturer who has developed the kit, and should be experienced in using the proposed technology. They should follow only those instructions provided in the product labelling when testing samples. Samples for testing should include known positive and negative samples supplied by the manufacturer (typically including the reference panel members used for serial release potency testing), as well as samples provided by the participating laboratory, which have been characterized as positive or negative according to its preferred testing method. Suitability testing is arranged by the manufacturer and usually involves at least 1 of the pre-licensing serials.

3.5 Stability

Studies must be performed to establish the stability and to confirm the proposed expiration dating (shelf life) for a kit at its recommended storage temperature. This is typically achieved by repeating the serial release potency test at multiple time points post-release (post-manufacture) to document the level of reactivity of the kit over time.

Expiration dates for veterinary biologics are typically computed from the date that the first potency test is initiated for that serial. For multi-component diagnostic kits, the expiration date of the assembled kit shall not exceed the expiration date of the shortest-dated component.

In circumstances where real-time stability assays cannot be completed by the time all other licensing requirements have been met, the CCVB could license a diagnostic kit with an interim shelf life (typically no more than 12 months), provided a stability monitoring program is in place to detect whether the potency of any released serials drops below the minimum potency level required for a serial.

3.6 Materials of animal origin

To allow the CCVB to assess whether the manufacture or importation of a diagnostic kit would present a risk for the introduction and/or spread of foreign animal diseases, transmissible spongiform encephalopathy (TSE) agents, or other contaminants, the CCVB requires manufacturers to identify all materials of animal origin (MAO) used in the production of a kit. Manufacturers must submit a list with the name of each MAO, the supplier, the animal species, and tissue type from which the MAO is derived, and the country where the source animals originated.

Manufacturers must submit a signed Declaration of Compliance, certifying that all MAO used in the production of the kit originate from sources considered to be safe from animal TSE infection or contamination, and that the kit does not contain any ingredients defined as specified risk material originating from a country not considered free from bovine spongiform encephalopathy.

More information on MAO requirements, as well as sample text for the Declaration of Compliance, are provided in VB-GL-3.32: Minimising the risk of introducing transmissible spongiform encephalopathy prions and other infectious agents through veterinary biologics.

3.7 Pre-licensing testing

The CCVB may require up to 3 pre-licensing serials of a diagnostic kit to be independently tested by a CFIA laboratory, or another laboratory selected by CCVB, to confirm the manufacturer's potency test results and to verify the absence of technical problems when the kit is used under typical laboratory conditions. The CCVB notifies the manufacturer if such testing is required, and if so, communicates the number of kits and volume of each potency test panel member sample for submission.

4. Label requirements

Copies of the labels for each component of the kit and the product monograph (package insert) must be submitted to CCVB for approval prior to licensing, and if any changes are sought for the labels post-licensing. Refer to VB-GL-3.3: Labelling of veterinary biologics for detailed information on labelling requirements for all veterinary biologics.

In addition to the standard information that must be present in the labelling of all veterinary biologics (serial number, expiration date, manufacturer identification, and so on), the labelling for diagnostic kits should include the following:

  • the principles of the test
  • whether its use is to detect antibody, antigen, DNA, RNA, or another marker of the disease agent
  • the sample types that can be used for testing (blood, serum, chicken, turkey, and so on)
  • the equipment and reagents making up the kit
  • a list of materials required for the test but not included in the kit
  • the number of samples (or animals) that can be tested
  • a description of the test procedure and methods of interpretation
  • a hypothetical example of the results and interpretation
  • a list of any precautions and test limitations
  • instructions and precautions for the handling and disposal of the test kit

Note: the CCVB allows the use of certain internationally recognized symbols in the labelling of diagnostic kits in situations wherein the use of text is impractical. The symbols, however, must be defined elsewhere in the product labelling. Please contact the CCVB for more details.

The CCVB strongly encourages manufacturers to include a summary of the results from sensitivity and specificity studies in product labelling, to give consumers an indication of the expected kit performance. Table 1 suggests a format for presenting this information.

Table 1: Recommended format for presenting sensitivity and specificity summary data.
Study ID Results Kit/ReferenceTable notea Sample type Relative sensitivity (95% CI) Relative specificity (95% CI)
+/+ -/+ +/- -/-
1 39 6 1 91 Serum 87% (74%-94%) 99% (94%-100%)
2 44 1 3 89 Serum 98% (88%-100%) 97% (91%-99%)
3 53 2 2 101 Whole blood 96% (88%-99%) 98% (93%-99%)

CI = confidence interval

Study 1. Kit results compared with results from an in-house PCR assay for virus DNA.
Study 2. Kit results compared with results from a commercially available antigen detection ELISA.
Study 3. Kit results compared with results from a commercially available antigen detection ELISA.

5. Post-licensing serial release

Following licensure, Canadian manufacturers and manufacturers from foreign countries, other than those from the U.S., usually require the CCVB's authorization to release a serial of their kit for sale in Canada. To qualify a serial as suitable for release, the manufacturer must perform the serial release testing described in section V of the OP approved by CCVB. For diagnostic kits, this typically only involves a potency test. The manufacturer submits the results of its serial release testing to the CCVB by completing a Manufacturer's serial release test report (MSRTR). The CCVB examines the results of the manufacturer's testing of the serial, and then informs the manufacturer whether the submission of samples for confirmatory testing is necessary. The manufacturer may release the serial for sale in Canada only upon receipt of a copy of the MSRTR for the serial, bearing the CCVB's "Released" stamp and the signature of a CCVB reviewer.

6. Special considerations for test kits licensed in the United States

U.S. manufacturers and "Permittees" in the U.S. may apply to license in Canada veterinary biologic products already licensed by the US Department of Agriculture (USDA) Center for Veterinary Biologics (CVB). Canadian licensing requirements for veterinary biologics are largely harmonized with those of the USDA-CVB. When assembling the validation information to include in the Canadian licensing application, the manufacturer or permittee should include all of the validation studies provided to the USDA-CVB to license the product in the U.S., as well as copies of all relevant correspondence between the manufacturer or permittee and the USDA-CVB. The CCVB will review this information, then notify the manufacturer if any additional information or data are required.

Master seeds and pre-licensing serials of in vitro diagnostic test kits tested by the USDA-CVB are not typically re-tested by the CFIA. However, under certain circumstances, the CCVB may require U.S. manufacturers or permittees to submit samples for confirmatory testing – for instance, to verify that the kit can diagnose Canadian strains of the infectious agent in question. Copies of the USDA-CVB Biologics Test Reports for the pre-licensing serials and any master seeds tested by the USDA should be included in the Canadian licensing application.

The CCVB generally waives the requirement for U.S. manufacturers and permittees to apply to release individual serials of their kits in Canada post-licensing, provided these serials are tested and released by the USDA-CVB in a manner that is acceptable to the CCVB. When such an exemption is granted, only serials released by the USDA-CVB may be shipped to Canada.

7. Test kits for use in a federal disease control program

Obtaining a Veterinary Biologics Product Licence or a Permit to Import Veterinary Biologics for a diagnostic test kit does not necessarily qualify a product for use in a CFIA federal disease control program. Approval for use in such a program is made by the groups within the CFIA who are responsible for administering the animal health diagnostic or surveillance program, and may necessitate the manufacturer submitting additional data, kits, and reference test samples.

8. Diagnostic test kits manufactured in Canada for export only

Manufacturers in Canada may decide to license a diagnostic kit for export only (FEO). The general requirements concerning facility, equipment, personnel, production, and testing are the same for FEO products as those for products manufactured for distribution in Canada. Any specific requirements or exceptions will be determined on a case-by-case basis. The licensing application for a FEO product should clearly indicate the name of the importing country and any specific requirements or exceptions for approving or distributing the diagnostic kit in that country. It is the responsibility of the Canadian manufacturer to ensure compliance with the regulatory requirements of each importing country.

Appendix 1: Reference panel preparation

During the development of an in vitro diagnostic kit, the CCVB requires manufacturers to compile a reference panel of test samples for use in serial potency testing (among other tests). Details regarding the identification, history, and attributes of individual reference panel members should be fully described in the Outline of Production (OP) for the product or in a Special Outline (SO) submitted to CCVB. The samples making up a panel are generally not part of the kit. Addition or removal of test samples from a panel must be documented and approved by the CCVB, and would necessitate a revision to the OP or relevant SO. The information on panel members, which should be provided to the CCVB, and the factors for consideration in their preparation, are as follows:

Identification – The following information concerning the test samples in the reference panel should be provided in the OP or the SO:

  • panel member identification by means of a unique code
  • dates of collection and preparation for the reference panel
  • sample storage history
  • type of sample
  • source of sample (geographical location, including country and province or state)
  • disease status of the animal
  • method used to reach the diagnosis (clinical signs, post-mortem lesions, culture, serological tests)
  • information on factors that could affect the outcome of the diagnostic test (vaccination status)
  • information specifically relating to the label claim (for example, age of the animal, species, clinical form of the disease, antibody titre)

Volume – Reference panel members must be of sufficient volume to be available over a sufficient period of time to evaluate the consistency of the kit from serial to serial, and through the end of stability testing. Samples from the reference panel must also be available for testing on request by CCVB.

Sample size – The manufacturer should include in the panel a sufficient number of relevant samples to detect changes in the potency (level of reactivity) of the various components of the kit. Ideally, a reference panel should consist of at least 20 samples.

Type – A separate reference panel of test samples should be prepared to evaluate each of the different types of samples (for example, blood and serum) and animal species (for example, chickens and turkey) described in the label claims.

Range of reactivity – The reference panel should be made up of different samples that give "strong positive, "weak positive," and "negative" reactions. The "positive" samples should consistently give a reaction above a pre-established positive diagnostic threshold, and should cover the full range of positive reactions described by the manufacturer in the product monograph, including some representative "weak positive" samples. The "negative" samples should consistently give a reaction that falls below the pre-established negative threshold. Each sample should be tested using the kit, multiple times, by more than 1 analyst, on different days, to determine the limits of their range of reactivity.

Specificity – Reference panels should include samples produced from:

  • experimentally infected animals and/or culture positive and/or serologically confirmed positive or negative animals
  • animals infected with the relevant strains of the infectious disease agent from different geographical locations
  • animals harbouring related agents or antigens
  • animals that have received commonly used immunizing products

Method of storing the reference panel – The method used to store the panel members (frozen, lyophilized) should be described. For frozen samples, a maximum permitted number of thaws per aliquot should be specified.

Adding or removing a test sample from the reference panel – The test procedures used to add or remove samples from the reference panel should be defined.

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