Guidance on efficacy requirements for biocides: Planning your test product and testing

On this page

• Formulation-specific testing
• Replicating the formulation
• Replicating the physical form and method of application
• Testing efficacy
• Acceptable test methods
• Sourcing test organisms
• Batch replication, microbial counts and performance standards
• Re-testing
• Confirmatory data requirements

Formulation-specific testing

The efficacy information you include in your application should:

• be specific to the formulation at in-use concentration
  • with the exception of dyes and fragrances under or equal to 1% weight per weight (w/w)
• be specific to the proposed physical form and method of application, for example:
  • towelettes should be tested using towelette methods
  • sprays should be tested using spray methods
• have tests showing that your specific biocide is able to meet the proposed claims
• have tests that reflect the recommended conditions for use that are on your biocide's label

The method you use to test efficacy should be relevant to the:

• label claims and target microorganisms
• settings in which the biocide is intended to be used
• achievable contact times for the proposed use
  • the biocide is able to evenly coat all of the surface for the entire contact time without re-application to achieve efficacy
  • the contact time should be 10 minutes or less for non-immersion biocides and less time for volatile formulations

For biocides that come in different dilutions, you may use the same in-use formulation and methods of application to develop efficacy information to support your application.

Replicating the formulation

Your test formulation should be representative of your biocide. For example, it should have the same:

• ingredients and in-use concentrations
• diluent use (if applicable)
• activator use (if applicable)

Ingredients

The ingredients and shelf life used in the test sample should be the same as to those in the proposed biocide. The only differences that we accept without confirmatory data are differences in dyes and fragrances under or equal to 1% w/w. Other alternative formulations should be supported by confirmatory efficacy data.

The test product you use to test efficacy for each sample or batch should be formulated at the lower certified limits (LCL) for all representative microorganisms within each microbial category. LCLs (described in Table 1) are calculated using the biocide's labelled nominal active ingredient concentrations.

As there are potential difficulties associated with generating test samples exactly at the LCL, we accept testing up to, and not above, its upper limit.

Testing against representative test organisms in each microbial category should be done using your biocide's LCL. If you want your biocide to have a narrower LCL (approved by us), you should provide quality and stability information that supports the proposed limits.

Refer to quality requirements for biocides for more information.

Additional microorganisms that are not considered representative microorganisms for a microbial class can be tested at or below the nominal concentration. Refer to Table 2 for examples of:

• common microbial categories when determining which microorganisms can be tested at or below the nominal concentration
• representative microorganisms that must be tested at LCL

For more information on efficacy data requirements for other microbial categories, refer to the following sections in this guidance:

• hard-surface disinfection and sanitization
• soft-surface disinfection and sanitization
• other types of claims
• confirmatory data

You should provide a certificate of analysis for the tested batches to demonstrate that testing has been done at the appropriate concentration. If you use a method that cannot differentiate between active ingredients in a formulation, you should submit analytical testing for the combined ingredients (for example, quaternary ammonium compounds (QACs)). The LCL can be determined for the sum of these ingredients.

To test your biocide at the LCL, you may either produce a test batch with an active ingredient at the LCL or dilute your biocide with water. Biocides that may be reactive or less stable in water may be diluted with a primary solvent already present in the formulation. Avoid using emulsifiers or surfactants as diluents even if they're present in the formulation, as these may alter the biocide's efficacy.

Biocides in a solid physical form (powder or tablets) should be diluted with more water than indicated on the label to achieve an in-use concentration at LCL. For example, before efficacy testing:

• dilute a 10 g tablet containing 50% active ingredient with an indicated use at 0.5% w/w (1 tablet diluted in 1 L of water) to an LCL level of 0.485% w/w (N minus 3%, 20% less than N less than or equal to 100%) with 1.04 L

Sometimes it may not be feasible to test a biocide formulated at the exact LCL. If this is the case, provide a rationale in your efficacy summary to support why your efficacy testing was not conducted at the exact lower limits.

If it's not possible to dilute the biocide, you may choose other ways to test at the LCL, for example, testing:

• batches past the shelf life or
• a biocide that has been stored at accelerated stability test conditions and duration

For more information on acceptable accelerated stability conditions, consult:

• Guidance on quality requirements for biocides

Diluent or activator use

If your biocide is to be diluted or activated (or both) before using, your test formulation should be representative of the biocide's labelled directions for use. This means it should, for example:

• use the same diluent (distilled tap or hard water, for example) or activator
• have the same dilution ratio

All microorganisms in the same microbial category (bacteria, viruses, fungi) should be tested at the same level of water hardness. Hard water tolerance levels may differ with the level of antimicrobial activity claimed for a biocide. Unless your biocide label says otherwise, we recommend using water with a minimum hardness of 200 ppm (parts per million), expressed as the amount of calcium carbonate (CaCO3) present in your testing.

If your biocide indicates efficacy claims specifically in hard water for any of the microbial categories, testing should be conducted with water at a higher hardness level for all microorganisms within that microbial category. For example, if your biocide makes claims of food contact surface sanitization efficacy in 500 ppm (parts per million) of hard water, all microorganisms for that claim should be tested with 500 ppm water.

We may allow you to use distilled water as a diluent in your test product if you provide an acceptable justification. An example would be if the devices used in testing are sensitive to calcium carbonate buildup. If this is the case, your biocide label should indicate to dilute with distilled water.

Replicating the physical form and method of application

The method of application and conditions of use for your test product and your biocide should be the same. This applies to biocides with the following physical forms and methods of application:

• biocides applied as liquids
• towelette biocides
• reusable biocides
• biocides for soft surfaces
• diluted or activated biocides
• vapour, mist, gas, dry gas or charged spray biocides

Liquid biocides

Immersion, mopping or using a cloth or sponge are methods of application that involve evenly applying a sufficient amount of a liquid biocide to a surface. Biocides with these methods of application can be tested using immersion methods, such as the "use dilution method" for bacteria.

Liquid biocides that are intended to be sprayed on a surface should be using spray methods such as the "germicidal spray test" for bacteria.

Non-volatile biocides are expected to evenly cover a treated surface for the entire contact time. Biocides that have both spray and non-spray applications may be tested using an immersion method.

Volatile biocides (containing isopropyl alcohol or ethanol as an active ingredient, for example) should be tested using spray methods. This is because immersion methods do not accurately simulate the way in which volatile biocides perform on environmental surfaces.

Conduct a spray test on your biocide. If it evenly covers the surface of the carrier for the entire contact time, then a spray method can be used for efficacy testing.

Towelette biocides

If your biocide is a single-use pre-saturated or impregnated towelette, you should use the towelette to test the biocide's efficacy. Your test method should include detailed procedures for:

• wiping the surface of inoculated test carriers with the towelette and
• sub-culturing the carriers after the specified contact time

You can also use the liquid expressed from a towelette to conduct your testing. In your application include analytical data on the concentration levels of the active ingredients in the expressed liquid.

To test with a towelette, use 1 towelette to treat either:

• 10 carriers or
• 1 carrier with a surface area equal to 10 1-by-1-inch (2.54-by-2.54-centimetres) per carrier set per batch

Each carrier should be wiped back and forth up to 3 times, for a total of 6 passes, before moving to the next carrier. A pass is defined as moving from 1 side of the carrier to the other with a single motion. The contact time begins after 6 passes.
For disinfectant claims against bacteria, mycobacteria and fungi, we recommend either of the following test methods:

• Modified AOAC 961.02 Germicidal Spray Products as Disinfectants or
• ASTM E2362 Standard practice for evaluation of pre-saturated or impregnated towelettes for hard surface disinfection

We recommend the ASTM E1053 method for virucidal claims. Use 1 towelette to wipe 1 test carrier.

You can bridge data from a liquid biocide for the same biocide in towelette form using confirmatory data. All representative microorganisms in each microbial category should be tested using the biocide in towelette form. If the towelette biocide has the same efficacy claims as the biocide in liquid form, you can bridge data if you:

• provide a certificate of analysis for the liquid used to saturate the towelettes and the liquid expressed from the towelettes
• have tests that show the active ingredient is delivered to the target surface and does not bind to the towelette material
• use the same towelette material for the tests

Extended-use and reusable biocides

You should provide data to support your biocide's effectiveness at the end of its shelf life:

• after being diluted or activated beyond 24 hours
• when reused for a certain number of days

For example, a biocide that is used for a certain number of days for immersion application after repeated soaking of contaminated instruments.
The data should reflect simulated use, soiling and dilution as applicable.

Vapour or gas biocides

If your biocide is a vapour, mist or gas, you should test your biocide under similar conditions and settings (for example, simulated-use testing). This includes the type and size of the areas the biocide is for use in. Examples of vapour or gas biocides include those:

• used in a fogging or misting machine
• applied by a specific device

When testing vapour or gas biocides, be sure that all key parameters for efficacy are monitored throughout the use area and reflect the labelled conditions, such as:

• temperature
• contact time
• relative humidity
• active ingredient concentration

When planning your testing, you should:

• use appropriate controls
• demonstrate the biocide's efficacy on surfaces that have a mild to moderate soil load
  • for example, 5% fetal bovine serum
• describe how you monitor and determine the disinfection process is effective
• determine and test the total mass of vapour, mist or gas released into the area and the maximum volume of space that can be disinfected
• demonstrate that the active ingredient concentration is maintained throughout the entire area and for the duration of the contact time that's indicated on the label
• place carriers in different parts of the room to demonstrate that the vapour, mist or gas will reach all parts of the room, including:
  • every corner
  • various locations on the wall faces
  • centre of the floor
  • underneath horizontal surfaces
  • various heights, including the maximum height specified in the fogging system instructions

For guidance on the test method for vapour or gas biocides, consult:

• Protocol for room sterilization by fogger application (US Environmental Protection Agency)

Testing efficacy

In addition to replicating your biocide's formulation, physical form and conditions of use, you should use the same conditions and parameters as the referenced test method (unless test method does not prescribe the conditions and parameters). Conditions and parameters include:

• use-dilution
• contact time
• number of carriers
• water hardness level
• temperature being used
• organic burden level (soil load)
• type of carriers (for soft, porous surfaces)

The number of carriers should be based on the most recent version of the test method being referenced.

All microorganisms should be tested in similar conditions to how the biocide will be used. If the biocide will not be used in a conventional way, test modifications or alternative test methods may be needed to support the efficacy of the biocide for its proposed use. Contact us to discuss the appropriate alternative test methods or modifications for your biocide.

Contact time

Your efficacy testing should use the same (or a shorter) contact time as the time indicated on your proposed biocide label.

Note: Any change in contact time in efficacy testing may be restricted by the limitations of the test method or the proposed method of application. For instance, an exposure period greater than 10 minutes for a biocide that will likely evaporate from a treated hard surface within 10 minutes. Shorter contact times may be required for some uses where the 10-minute contact time is not achievable.

Your biocide should meet the performance standard set for the test method and target microorganism within a 10-minute contact time. Exceptions may be given to disinfectants that direct users to immerse objects in the solution for a specified time.
You will not be able to propose contact times above 10 minutes unless either:

• your biocide is meant for immersion application
• you provide a wetness test showing the biocide will not evaporate in 10 minutes

Contact us for a pre-submission meeting if you require a longer contact time.
For liquid or spray biocides with volatile active ingredients, you can determine the maximum contact time by testing your biocide's evaporation over the proposed contact period using a wetness test.

For spray-only biocides, do not use a test method that immerses your test organism in the biocide fluid. Immersion does not accurately represent the way biocides perform on surfaces. Instead, use 1 of the following test methods:

• AOAC 961.02
• ASTM E1053
• ASTM E1153 (modified for a spray application)

You may use an immersion test method for non-volatile biocides that are applied by both spray and non-spray methods. Volatile formulations should be tested using spray methods as this accurately represents the way volatile biocides perform on surfaces.

For biocides in towelette forms, you may use a wetness test to determine the maximum contact time. This test will:

• assess the amount of liquid left on the surface treated (wiped) when used according to the label
• for example, surface remains evenly wet before the end of the contact period
• determine the contact time you should use in your efficacy testing

Temperature

Your efficacy testing should have the same temperature conditions as those indicated your biocide's label. Otherwise, conduct your test between 18°C and 25°C, including the steps to:

• inoculate carriers
• confirm neutralization
• expose carriers to the test substance

Neutralization

Neutralization inactivates your test product's antimicrobial activity. For all efficacy testing, follow procedures to neutralize your biocide at the end of all contact times. This will prevent residual effects in the subculture medium.

Confirm how you will neutralize the product when you test for efficacy or as required by the test method. The neutralization procedure should align with your test method.
You may neutralize your biocide by:

• physical means
• filtration, dilution, secondary subculture
• chemical means
• adding sodium thiosulfate to the diluent

If you are unable to validate the neutralizers used for all biocide tests against microorganisms (except for viruses) using standard test methods, refer to the:

• ASTM E1054 method to validate the neutralizers
• ASTM E1482 method for virucidal tests

Organic burden

If your biocide will be used in light to moderate amounts of soil, your organic soil load should be:

• a minimum of 5% v/v (volume-per-volume) or
• the amount required by the test method (for example, 3-part soil)

Examples of these types of biocides are:

• a 1-step cleaner and disinfectant
• a 1-step cleaner and sanitizer
• fogging and misting biocides for whole room treatments
  • used to treat all room surfaces, including hard-to-reach places that may not be possible to pre-clean
• residual sanitizers and disinfectants
• biocides for use on soft surfaces, such as:
  • sanitizers
  • disinfectants
  • laundry biocides
  • pre-soak treatments
  • carpet treatments
• biocides for use against C. difficile and C. auris

Biocides for use on soft surfaces and for treating an entire room require a 5% soil load. For these applications, we recommend you use:

• blood
• animal serum or
• another scientifically accepted equivalent (for example, 3-part soil)

Acceptable test methods

In general, Health Canada recognizes biocide test methods and protocols published by international standards organizations or other regulators. You should use the current official version of the test method.

You may not apply requirements from different test methods unless you have a suitable rationale for doing so. An example would be modifying test methods commonly recommended by other regulators or international organizations to test alternate organisms or product forms.

You should follow all prescribed information outlined for the test method you use. If the method does not outline certain information (for example, microbial count or performance criteria), you should meet the requirements outlined in this guidance.
In general, we recognize test methods published by:

• Association of Official Analytical Collaboration International
• American Society for Testing and Materials International
• Australian Therapeutic Goods Administration's Guideline for the evaluation of sterilants and disinfectants
• European Chemical Agency's Guidance on the Biocidal Products Regulations: Volume II – Efficacy (Parts A, B, C)
• European Committee for Standardization
• Organisation for Economic Co-operation and Development's Quantitative test method for hard surface disinfectants
• US Environmental Protection Agency's 810.2000, 810.2100, 810.2200, 810.2300 and 810.2400 product performance guidelines
  • includes additional stand-alone methods, procedures and guidance documents for surface disinfectants

Note: When using European (EN) test methods, phase 1 test methods are only considered acceptable as secondary supporting evidence of efficacy. While they cannot be used solely to support a proposed claim and are not accepted for biocide authorization, they can be used to help develop a proposed biocide. You should conduct all testing using at least 2 batches and phase 2 test methods.

Sourcing test organisms

Your efficacy testing for each microbial category claimed on your biocide's label should use representative test organisms.

We recommend that you use test organisms available for purchase through the American Type Culture Collection (ATCC). You may use microorganism stocks purchased from other suppliers if they:

• meet quality assurance standards and
• are appropriate for the chosen efficacy test method

Batch replication, microbial counts and performance standards

To show that efficacy results are reproducible and statistically sound, you should have:

• separately compounded batches for testing
• carriers for each batch tested against the same microorganism, as required by the method
• replicates for each batch tested for each carrier used, as required by the method

Find more information, consult:

• hard-surface disinfection and sanitization
• soft-surface disinfection and sanitization
• other types of claims

The microbial count of a sample (inoculum counts or carrier counts) determines the microbial challenge level for the test. For a test result to be valid, the microbial levels should be within the prescribed limits for the test method.

The way you determine the microbial count should be in line with the test method. For suspension test methods, you should determine the microbial count of the sample. For carrier test methods, you should determine the microbial counts on untreated dried control carriers after applying the sample.

In general, it's acceptable if the microbial counts exceed the prescribed levels outlined in the test requirements as long as the biocide meets the prescribed performance criteria.

Re-testing

A biocide may fail to meet the performance standard required for the efficacy claim due to contamination or not meeting the prescribed carrier count.

If a laboratory test indicates that the failure is due to a false positive, you may re-test that particular batch.

If you decide to re-test, you do not need to use:

• a different laboratory
• different laboratory personnel
  • for example, study director, technical staff, quality assurance auditor

You may conduct up to 2 re-tests for a contamination or population control failure. You do not need to re-test for carrier population controls that are greater than the acceptance criteria (unless otherwise specified in the test methods).

If the issue has not been resolved after 2 re-tests, we will not accept the efficacy information.

When re-testing, conditions must be identical to the original test.

Failure due to contamination

Contaminants are microorganisms that are not the test organism in the study. Contamination in the test system may invalidate the assay. We recommend that you follow the US EPA's 810.2000 product performance test guideline to help you determine whether you need to retest the biocide and if so, how.

Use 1 of the following methods to identify the contaminant and presence or absence of the test organism:

• Gram staining
• colony morphology
• biochemical assays

If contamination happens again, the laboratory may consider a quality plan to correct the issue. Quality plans can include mitigation strategies such as:

• replacing the stock tube
• purchasing a new lyophilized stock
• performing a facility or equipment cleaning

Failed re-testing

If you re-tested and your results continue to fail to meet performance standards, you may wish to do a different test by changing the test conditions. This will affect your labelled claim for your biocide. Examples of changes include:

• removing soil load
• using a higher concentration
• using an increased contact time

Confirmatory data requirements

You may use confirmatory data with reduced batch replication requirements to support situations that may affect your biocide's efficacy, such as:

• formulation variations
• major changes to formulants
• bridging liquid biocide data for a towelette biocide
• different conditions of use for your biocide
  • for example, adding a new contact time

We accept reduced batch replication requirements for confirmatory data for:

• formulation variations
• significant changes to formulants
• different conditions of use for your biocide

For more information, visit:

• hard-surface disinfection and sanitization
• soft-surface disinfection and sanitization
• other types of claims

Formulation variations

If you include formulation variations in your application, make sure they are substantially similar to the basic formulation.

All variations should be supported by a single benefits (efficacy) package and label text to demonstrate that they:

• meet every label claim
• have physical and chemical properties that are within product specifications
• are as safe as the basic formulation
• do not negatively affect the stability and quality of the biocide

When proposing formulation variations, you should include both:

• confirmatory data to demonstrate the alternate formulation is as effective and
• a scientific rationale to demonstrate the formulation has the same efficacy, safety and quality

The confirmatory data that you include in the application should:

• have the same or more stringent testing conditions
• be produced using the same lower certified limit (LCL) as your basic formulation
• include data according to the batch replication requirements against each representative test organism for each microbial category on your biocide's label

For more information, visit the:

• hard-surface disinfection and sanitization
• soft-surface disinfection and sanitization
• other types of claims

You do not need to provide confirmatory efficacy data when:

• only the concentration of a fragrance or dye is increased, substituted or decreased in a formulation and either
• the concentration of fragrances does not exceed 1% of the total formulation, expressed as a percentage on a weight-per-weight basis (% w/w) or
• the total percentage of changes in dyes does not exceed 1% of the total formulation, expressed as % w/w

For more information, consult:

• Guidance on application pathways and general requirements

Towelette biocides

Confirmatory testing allows you to bridge additional specific claims approved for your liquid biocide to the same biocide in towelette form. This confirmatory data should include the representative test organisms for each microbial category claimed on the label of your towelette biocide and be tested using towelettes. If acceptable, you do not need efficacy data to support all other additional claims.

When conducting confirmatory testing, you should:

• use the same testing conditions (same contact time, soil load, temperature) or more stringent conditions
  • an example of more stringent conditions would be to include soil for a towelette test if the original test did not include soil
• meet the batch replication requirements outlined in this guide for the representative test organisms for each microbial category claimed on your label