Guidance on efficacy requirements for biocides: Hard-surface disinfection and sanitization

Types of claims

In general, there are 2 different types of biocidal claims:

specific claims
- direct claims
- indirect claims
general claims

Direct claims show a causal relationship. These claims require efficacy information that demonstrates your biocide is effective against specific microorganisms. An example of a direct claim would be "This biocide kills SARS-CoV-2 (the virus that causes COVID-19)".

Indirect claims show a relationship and are only accepted during a declared pandemic by the World Health Organization (WHO). These claims allow you to provide Health Canada with efficacy testing using another microorganism that's similar or harder to kill than the proposed microorganism. An example of an indirect claim would be "This biocide kills rhinovirus type 16 and is likely to kill SARS-CoV-2 (the virus that causes COVID-19)".

If your biocide qualifies for both a direct and an indirect claim, you should only include the direct claim on the label.

Learn more about claims against emerging pathogens.

General claims

Before proposing a general claim for your biocide, you should first demonstrate if your biocide is a general (broad-spectrum) biocide or a hospital or health care disinfectant. This applies to claims to destroy (kill), inactivate or reduce or control the number of:

germs
viruses
mycobacteria
specific bacterial spores
fungi, mould and mildew
other non-spore forming bacteria
- such as Escherichia coli

To propose a specific non-food contact surface sanitization claim, you should demonstrate that your biocide is a general non-food contact surface sanitizer. The same applies for specific food contact surface sanitization claims.

Studies to support your biocide application may be electronically signed by laboratories. This includes efficacy reports containing good laboratory practice (GLP) statements.

Hard-surface disinfectant claims

Disinfectants can destroy or irreversibly inactivate pathogenic (disease-causing) and potentially pathogenic (opportunistic) microorganisms, but not necessarily bacterial spores, on hard surfaces. This is because of the antimicrobial action of the active ingredients.

Hard-surface disinfectants against bacteria should show they're effective 10 minutes after application or sooner.

Limited, general (broad-spectrum) and hospital or health care disinfection

For limited, general (broad-spectrum) and hospital or health care disinfectant claims, we require:

3 samples of the biocide representing 3 separate batches per representative test organism for the bactericide
60 inoculated carriers per batch per bacterium

The testing requirements for limited, general (broad-spectrum) and hospital or health care disinfectants are listed in Table 3.

Use the following methods for testing these core bacteria for disinfection claims:

Staphylococcus aureus: AOAC 955.15 method
Pseudomonas aeruginosa: AOAC 964.02 method
Salmonella enterica: AOAC 955.14 method

You should conduct 3 independent tests on 3 different test days for Pseudomonas aeruginosa and Staphylococcus aureus. You should test for Salmonella enterica on different days.

Claims against bacteria allow you to label your biocide as a "bactericide" and a "bactericidal". You must provide efficacy data to show different levels of efficacy for your biocide.

Table 3: Bacteria disinfection on hard surfaces
Claim	Physical form, method of application	Recommended test methods	Test organisms	Number of batches per organism, carriers per batch	Inoculum or carrier count		Performance criteria for efficacy
Core bacteria test requirements
Limited disinfectant (effective against Gram-positive or Gram-negative, but not both)	Liquids (cloth, mop, immersion applications)^{Footnote 1} Water-soluble powders	AOAC use-dilution method 955.14, 955.15	Salmonella enterica (formerly Salmonella choleraesuis) (ATCC 10708) or Staphylococcus aureus (ATCC 6538)	3 LCL batches 60 carriers per batch	As prescribed in current test methods Unless otherwise prescribed AOAC use-dilution methods: S. enterica: 1.0 x 10⁵ to 1.0 x 10⁶ CFU per carrier (minimum mean 5 log₁₀ density) S. aureus and P. aeruginosa, each batch to be tested on a different day: 1.0 x 10⁶ to 1.0 x 10⁷ CFU per carrier (minimum mean 6 log₁₀ density) AOAC 961.02 and towelette methods: S. enterica: 1.0 x 10⁴ to 3.2 x 10⁵ CFU per carrier (minimum mean 4 log₁₀ density) S. aureus and P. aeruginosa: 1.0 x 10⁵ to 3.2 x 10⁶ CFU per carrier (minimum mean 5 log₁₀ density)		As prescribed in current test methods, per batch at proposed contact time Unless otherwise prescribed AOAC 955.14 testing against S. enterica: 59/60 carriers negative for growth AOAC 955.15 testing against S. aureus: 57/60 carriers negative for growth per batch AOAC 964.02 testing against P. aeruginosa: 54/60 carriers negative for growth per batch AOAC 961.02 and towelette methods: 59/60 carriers negative for growth
	Spray^{Footnote 1}	AOAC 961.02
	Single-use towelettes	Modified AOAC 961.02 ASTM E2362
General (broad-spectrum) disinfectant^{Footnote 2} (effective against Gram-positive and Gram-negative)	Liquids Water-soluble powders	AOAC use- dilution method 955.14 955.15 964.02	Staphylococcus aureus (ATCC 6538) and Salmonella enterica (formerly Salmonella choleraesuis) (ATCC 10708) or Pseudomonas aeruginosa (ATCC 15442)
	Spray	AOAC 961.02
	Single-use towelettes	Modified AOAC 961.02 ASTM E2362
Hospital and health care disinfectant^{Footnote 2} (effective against Gram-positive and Gram-negative)	Liquids Water-soluble powders	AOAC use- dilution method 964.02, 955.15	Pseudomonas aeruginosa (ATCC 15442) and Staphylococcus aureus (ATCC 6538)
	Spray	AOAC 961.02
	Single-use towelettes	Modified AOAC 961.02 ASTM E2362
Additional bacteria test requirements
Claim against additional bacterium (non-spore forming)	Liquids Water-soluble powders	AOAC use-dilution method	Specific bacterium claimed on the label in addition to the general (broad-spectrum) or hospital or health care disinfectant claim	2 NCL batches (separately compounded per representative test organism) 10 carriers per batch	As prescribed in current test methods Unless otherwise prescribed: 1.0 x 10⁴ to 1.0 x 10⁵ CFU per carrier		Unless otherwise prescribed: 10/10 carriers negative for growth per batch at proposed contact time
	Spray	AOAC 961.02
	Single-use towelettes	Modified AOAC 961.02 ASTM E2362
Confirmatory efficacy data requirements for bacteria disinfection
General (broad-spectrum) disinfectant	Same as core bacteria requirements Testing against additional bacteria not required			2 LCL batches 10 carriers per batch		Same as core bacteria requirements
Hospital or health care disinfectant	Same as core bacteria requirements Testing against additional bacteria not required			2 LCL batches 10 carriers per batch		Same as core bacteria requirements
Abbreviation: CFU: colony-forming units Footnote 1 The UDM method is also acceptable for biocides with both trigger spray and non-spray applications and biocides that contain non-volatile ingredients. Return to footnote 1 referrer Footnote 2 If tested on the same day, the performance standard 59/60 must be met. If batches are tested on different days, a performance standard 58/60 will also be considered, in line with US EPA's requirement described in SOP MB-05. Return to footnote 2 referrer

Mycobacteria disinfection

You will need efficacy data against a representative Mycobacterium species to claim your biocide as a:

mycobactericide
mycobactericidal

A representative species could be Mycobacterium bovis BCG (ATCC 35473) or Mycobacterium terrae (ATCC 15755).

To make specific claims against certain mycobacteria, you must provide supporting efficacy data.

Only include specific claims (for example, effective against or kills Mycobacterium fortuitum) on your label if you:

do not have efficacy data to support your biocide as a "mycobactericide" or "mycobactericidal" biocide
have efficacy data to support the specific claim

Note: Mycobacterium terrae (ATCC 15755) has only been validated with the:

ASTM quantitative carrier methods (ASTM E2111 and E2197)
OECD quantitative method

If you have quaternary ammonium compounds as the sole active ingredient in your biocide, you should perform verification testing to confirm the mycobactericidal label claim. To do so, you must test 1 of the original biocide lots using the same test procedures and under the same conditions. Testing must be done in either:

a different laboratory than the original one
the same laboratory using different laboratory personnel
- for example, different study director, technical staff and quality assurance (QA) auditor

Table 4: Mycobacteria disinfection on hard surfaces
Claim	Physical form, method of application	Recommended test methods	Test organisms	Number of batches per organism, carriers per batch	Inoculum or carrier count		Performance criteria for efficacy
Core mycobacteria test requirements
Mycobactericide and tuberculocide	Liquids Water-soluble powders	AOAC 965.12 US EPA QTB^{Footnote 1}	Choosing the appropriate Mycobacterium species depends on the test method: Mycobacterium bovis (BCG) (ATCC 35743) or Mycobacterium terrae^{Footnote 2} (ATCC 15755)	2 LCL batches 10 carriers per batch For the QTB method: 4 replicates per batch	As prescribed in current test methods Unless otherwise prescribed: QTB method: 1 x 10⁷ to 1 x 10⁸ CFU per mL Carrier test method: 1 x 10⁴ to 1.0 x 10⁶ CFU per carrier		As prescribed in current test methods and at proposed contact time Unless otherwise prescribed: Carrier test method: 10/10 carriers negative for growth per batch at specified contact time, no growth in inoculated subculture media QTB method: each of 4 replicates should demonstrate less than or equal to 1.0 x 10⁴ CFU (less than or equal to 4.0 log₁₀ kill, 99.99% reduction)
	Spray	AOAC 961.0 modified for mycobacteria
	Single-use towelettes	Modified AOAC 961.02 ASTM E2362
Additional mycobacteria test requirements
Claim against specific mycobacteria^{Footnote 1}	Liquids Water-soluble powders	AOAC 965.12 US EPA QTB1	Any specific mycobacteria claimed on the label	2 NCL batches, 10 carriers per batch QTB method: 4 replicates per batch	As prescribed in current test methods Unless otherwise prescribed: QTB method: 1 x 10⁷ to 1 x 10⁸ CFU per mL Carrier test method: 1 x 10⁴ to 1.0 x 10⁶ CFU per carrier		As prescribed in current test methods and at proposed contact time Unless otherwise prescribed: Carrier test method: 10/10 carriers negative for growth per batch at specified contact time, no growth in inoculated subculture media QTB method: Each of 4 replicates should demonstrate less than or equal to 1.0 x 10⁴ CFU (less than or equal to 4.0 log₁₀ kill, 99.99% reduction)
	Spray	AOAC 961.02 modified for mycobacteria
	Single-use towelettes	Modified AOAC 961.02 ASTM E2362
Confirmatory efficacy data requirements for mycobacteria disinfection
Mycobactericide and tuberculocide	Refer to core mycobacteria requirements			1 LCL batch 10 carriers per batch QTB method: 4 replicates per batch		Refer to core mycobacteria requirements
Abbreviation: CFU: colony-forming unit Footnotes Footnote 1 US EPA’s QTB method is recommended only for glutaraldehyde-based biocides. Return to footnote 1 referrer Footnote 2 Unless otherwise prescribed in the most recent version of 1 of the recommended test methods, efficacy testing using Mycobacterium terrae (ATCC 15755) has only been validated with the ASTM (ASTM E2111 and ASTM E2197) and OECD methods. You may choose to use these methods to conduct efficacy testing against mycobacteria, with target performance criteria of a mean 4 log₁₀ reduction in the viability of the test organism. Return to footnote 2 referrer

Fungi

To make claims against fungi for your biocide, you must conduct fungicidal testing using fungal conidia (spores). You can only label your biocide as a fungicide or fungicidal if your efficacy testing is against Trichophyton interdigitale (formerly Trichophyton mentagrophytes) (ATCC 9533).

If you want to make specific claims against certain fungi, you must provide supporting efficacy data. If you don't have efficacy data to support a general fungicide claim, you may indicate a specific fungus claim on your label.

Table 5: Fungi disinfection on hard surfaces
Claim	Physical form, method of application	Recommended test methods	Test organisms	Number of batches per organism, carriers per batch	Inoculum or carrier count	Performance criteria for efficacy
Core fungi test requirements
Fungicide	Liquids Water-soluble powders	AOAC 955.17/ AOAC use-dilution methods (UDM) modified for fungi	Trichophyton interdigitale (formerly Trichophyton mentagrophytes) (ATCC 9533)	2 LCL batches, 10 carriers (for carrier test methods) AOAC 955.17 suspension method: 2 LCL batches, 0 carrier	As prescribed in current test methods Unless otherwise prescribed: AOAC 955.17 suspension method: 5 x 10⁶ conidia to 5 x 10⁷ conidia per mL Carrier test method: 1 x 10⁴ conidia to 1 x 10⁵ conidia per carrier	As prescribed in current test methods and at proposed contact time AOAC 955.17 suspension method: test conducted at 10-min exposure time or less All fungal spores killed at 10 min or less to support a 10-min contact time Carrier test method: 10/10 carriers negative for growth per batch at prescribed contact time
	Spray	AOAC 961.02 modified for fungi
	Single-use towelettes^{Footnote 1}	Modified AOAC 961.02 ASTM E2362
Claim against specific fungus (for example, C. auris)^{Footnote 2}	Liquids Water-soluble powders	AOAC 955.17 AOAC use-dilution methods (UDM) modified for fungi	Any specific fungus claimed on the label	If the biocide also showed efficacy as a fungicide: 2 NCL^{Footnote 3} batches, 10 carriers (for carrier test method) AOAC 955.17 suspension method: 2 NCL batches, 0 carrier If the biocide has not been tested as a fungicide: 2 LCL^{Footnote 2} batches, 10 carriers (for carrier test method) AOAC 955.17 suspension method: 2 LCL batches, 0 carrier	As prescribed in current test methods Unless otherwise prescribed: AOAC 955.17 suspension method: 5 x 10⁶ conidia to 5 x 10⁷ conidia per mL Carrier test method: 1 x 10⁴ conidia to 1 x 10⁵ conidia per carrier	As prescribed in current test methods and at proposed contact time AOAC 955.17 suspension method: test conducted at 10-min exposure time or less All fungal spores killed at 10 min or less to support a 10-min contact time Carrier test method: 10/10 carriers negative for growth per batch at prescribed contact time
	Spray	AOAC 961.02 modified for fungi
	Single-use towelettes	Modified AOAC 961.02 ASTM E2362
Confirmatory efficacy data requirements for fungi disinfection
Fungicide	Refer to core microorganism requirements			2 LCL batches, 10 carriers (for carrier test method) AOAC 955.17 suspension method: 2 LCL batches, 1 carrier	Refer to core microorganism requirements
Abbreviation: CFU: colony-forming units Footnotes Footnote 1 A pass is defined as moving from 1 side of the carrier to the other with a single motion. One towelette used to treat 10 carriers. Return to footnote 1 referrer Footnote 2 If you are seeking to market disinfectants with a claim of efficacy against Candida auris, consult US EPA's Candida auris – Guidance and Methods for Antimicrobial Efficacy Testing for General Efficacy and Labelling Considerations for Claims against Candida auris. Testing against C. auris should always be conducted at LCL. Return to footnote 2 referrer Footnote 3 If you want to market your biocide as a fungicide, you must test T. interdigitale at LCL. Additional fungi claims, with the exception of C. auris, can be tested at NCL. If you want to market your biocide only with claims for specific fungi, you should test all specific fungi claims at LCL. Return to footnote 3 referrer

Virus disinfection

To have claims against viruses on your biocide label, you must show that your biocide is either a:

virucide
- may include claims against specific viruses
broad-spectrum virucide

For broad-spectrum virucide claims, you must:

test using 2 samples of the biocide representing 2 separately compounded batches per virus
test on 1 inoculated carrier or surface per batch per virus (except when using surrogate viruses)
test in quadruplicates at each dilution level

For all virus claims, you should use the ASTM E1053 test method with:

an assay to detect the presence of remaining viable virus after treating the virus with the biocide, using the appropriate virological technique, such as:
- cytopathic effect
- fluorescent antibody
- plaque count
4 determinants per dilution in the assay system in the:
- virus recovery (control carrier counts)
- cytotoxicity control
- neutralization controls
- treated carriers (test for disinfectant activity)

Note: For a modified ASTM E1053 method, use 1 towelette to wipe 1 test carrier (the carrier is large and the folded towelette is small). We recommend that you either:

treat carriers in 2 parallel sections using the same towelette or
use the wiping technique in ASTM E2896 for viral testing

Use an appropriate statistical method to calculate the 50% infectious dose (ID50) for each assay. Examples of appropriate methods include:

Reed and Munch
Most Probable Number
Spearman-Karber

The hardest-to-kill virus on the label should be tested at or below the LCL. This is any small and non-enveloped virus that's indicated on the label, if present (for example, feline calicivirus, rhinovirus).

All easier-to-kill viruses can be tested at or below the nominal concentration (NCL). If there are no small non-enveloped viruses on the label, then it's 1 of the large non-enveloped viruses on the label (for example, adenovirus type 5, rotavirus).

If the biocide indicates claims only against enveloped viruses, then testing should be conducted at LCL against any 1 of the enveloped viruses (for example, SARS-CoV-2).

Table 6: Virus disinfection on hard surfaces
Claim	Physical form, method of application	Recommended test	Test organism	Number of batches per organism, carriers and surfaces per batch	Inoculum or carrier count	Performance criteria for efficacy
Core virus test requirements
Virucide	Liquids Water-soluble powders	ASTM E1053	Specific virus claimed on label except for surrogate viruses for efficacy claims against human hepatitis B virus (HBV), human hepatitis C virus (HCV) and norovirus	2 LCL batches, 1 carrier per batch for hardest-to-kill virus on the label^{Footnote 1} Easier-to-kill viruses can be tested using 2 NCL batches, 1 carrier per batch	Unless otherwise prescribed: Minimum recoverable endpoint viral titer after drying between 10^4.80 (6.3 x 10⁴) and 10^6.3 (1.9 x 10⁶) per carrier	As prescribed in current test methods and at proposed contact time Host cell line should exhibit cytopathic effects (CPE) in the presence of the selected virus Neutralization control should demonstrate complete neutralization of the biocide Unless otherwise prescribed: If cytotoxicity is present, a minimum 3 log₁₀ reduction in viral titer beyond the cytotoxic level for all test carriers
	Spray	ASTM E1053
	Single-use towelettes	Modified ASTM E1053
Broad-spectrum virucide	Liquids Water-soluble powders	ASTM E1053	Testing against any small non-enveloped virus from the following families: Picornaviridae (such as rhinovirus) Parvoviridae (such as canine parvovirus) Caliciviridae (such as feline calicivirus) Astroviridae (such as human astrovirus) Polyomaviridae (such as human polyomavirus)	2 LCL batches, 1 carrier per batch^{Footnote 2}
	Spray	ASTM E1053
	Single-use towelettes	Modified ASTM E1053
Confirmatory efficacy data requirements for virus disinfection
Virucide	Refer to core microorganism requirements			2 LCL batches, 1 carrier or batch for hardest-to-kill virus on the label^{Footnote 1}	Refer to core microorganism requirements
Broad-spectrum virucide	Refer to core microorganism requirements			2 LCL batches 1 carrier in quadruplicate per batch^{Footnote 2}	Refer to core microorganism requirements
Footnote 1 The hardest-to-kill virus on the label is the small non-enveloped virus (if no small non-enveloped viruses on label, then it's any of the large non-enveloped viruses). Return to footnote 1 referrer Footnote 2 If you are testing a small non-enveloped virus for a general virucide claim that's also a surrogate virus (such as Feline calicivirus), you must test this virus with the carrier replication requirement for a surrogate virus (2 carriers per batch). Return to footnote 2 referrer

Surrogate efficacy

You may use the following surrogate viruses to generate efficacy data for some virucide claims:

duck hepatitis B virus (DHBV) for hepatitis B virus (HBV)
bovine viral diarrhoea virus (BVDV) for hepatitis C virus (HCV)
feline calicivirus for human norovirus

Note: Testing against 2 determinants per dilution in the assay system is acceptable for cytotoxicity and neutralization controls.

Table 7: Disinfection of viruses supported by surrogates on hard surfaces
Claim	Physical form, method of application	Recommended test methods	Test organisms	Number of batches per organism, carriers and surfaces per batch	Inoculum or carrier count	Performance criteria for efficacy
Core surrogate virus test requirements
Human hepatitis B virus (HBV) claim supported by surrogate efficacy	Liquids Water-soluble powders	US EPA's Protocol for testing the efficacy of disinfectants used to inactivate duck hepatitis B virus and to support corresponding label claims	Duck hepatitis B virus (DHBV)	2 LCL batches 2 carriers in quadruplicate or batch	Unless otherwise prescribed: Minimum recoverable endpoint viral titer after drying between 10^4.80 (6.3 x 10⁴) and 10^6.3 (1.9 x 10⁶) per carrier is required	As prescribed in current test methods and at proposed contact time Host cell line should exhibit cytopathic effects (CPE) in the presence of the selected virus Neutralization control should demonstrate complete neutralization of the biocide Unless otherwise prescribed: If cytotoxicity is present, a minimum 3 log₁₀ reduction in viral titer beyond the cytotoxic level for all test carriers
	Spray
	Single-use towelettes
Human hepatitis C virus (HCV) claim supported by surrogate efficacy	Liquids Water-soluble powders	US EPA's Virucidal effectiveness test using bovine viral diarrhea virus (BVDV) as surrogates for human hepatitis C virus	Bovine viral diarrhea virus (BVDV)
	Spray
	Single-use towelettes
Human norovirus claim supported by surrogate efficacy	Liquids Water-soluble powders	US EPA's Initial virucidal effectiveness test: Using feline calicivirus as surrogate for norovirus	Feline calicivirus
	Spray
	Single-use towelettes
Confirmatory efficacy data requirements for viruses supported by surrogates
Human norovirus^{Footnote 1}	Refer to core requirements			2 LCL batches, 2 carriers per batch	Refer to core requirements
Footnote 1 Confirmatory efficacy data is required for norovirus only when it's the hardest-to-kill virus on the label. Return to footnote 1 referrer

Residual disinfection claims

Testing for bacteria and viruses should be conducted at LCL for 2 separately compounded batches. If your biocide has virucidal claims, residual disinfection data is also required against the hardest-to-kill virus indicated on the label.

To make general residual bactericidal claims, you must show your biocide is effective against both:

Staphylococcus aureus (ATCC 6538) and
Pseudomonas aeruginosa (ATCC 15442)

For residual self-disinfecting claims and efficacy claims of dried chemical residues on hard, non-food contact surfaces, consult:

Guidance for products adding residual efficacy claims (US EPA)

That guidance supports residual bactericidal and virucidal claims on hard, non-food contact surfaces for biocides with 24-hour residual claims.

Note: This guidance does not address residual disinfection claims against mycobacterium, fungi, yeasts or bacterial spores. It's also limited to biocides for use on hard, non-porous, non-food contact surfaces and does not address biocides for use on textile (porous) surfaces. Contact us to discuss testing requirements for other types of residual claims.

Table 8: Residual bacteria disinfection on hard surfaces
Claim	Physical form, method of application	Recommended test methods	Test organisms	Number of batches per organism, replication or carrier surfaces per batch	Inoculum or carrier count	Performance criteria for efficacy
Core bacteria test requirements for residual bacteria disinfection
Up to 24 hr for bacteria^{Footnote 1}	Liquids Water-soluble powders Combination liquid/spray Spray Single-use towelettes	US EPA's Protocol for Residual Self-Sanitizing Activity of Dried Chemical Residuals on Hard, Non-Porous Surface (Protocol 01-1A)^{Footnote 2}	Staphylococcus aureus (ATCC 6538) and Pseudomonas aeruginosa (ATCC 15442)	2 LCL batches, 4 carriers per batch (plus 4 controls, 2 neutralization controls)	At least 1.0 x 10⁵ bacteria should have survived on inoculated control squares	99.999% mean reduction (minimum 5 log₁₀) is required for each bacterium following 24-hr wear and re-inoculation procedure and within a 10-min contact time after sanitization test
Bridging additional bacteria for residual disinfection
Up to 24 hr – claim for bridging additional bacteria	No additional data required if performance criteria against S. aureus (ATCC 6538) and P. aeruginosa have been met
Confirmatory efficacy data test requirements for residual bacteria disinfection
Up to 24 hr for bacteria	All test requirements are the same as for core bacteria residual disinfection
Footnote 1 Residual disinfectant testing against S. aureus and P. aeruginosa supports general residual disinfection (99.99% kill) claims for 24 hours against bacteria as well as specific claims against tested microorganisms. Biocides seeking to indicate residual disinfection claims should first demonstrate efficacy as a general (broad-spectrum) or hospital or health care disinfectant. Return to footnote 1 referrer Footnote 2 Durability testing should include 12 wear cycles consisting of abrasions (alternating wet and dry) and re-inoculations to support a 24-hr residual disinfectant claim. Each wear cycle consists of 2 passes (1 pass to the left and a return pass to the right) of the abrasion material over the surface followed by re-inoculation. The protocol should be modified for single-use towelettes: Where shorter residual claims are desired (no less than 4 hrs), the test may be ratiometrically reduced (for example, 6 wear cycles for a 12-hr residual claim). This protocol should be modified for towelette testing. If you want to add residual sanitization claims for towelettes, you should have the carriers treated using 1 towelette per lot to wipe 4 test carriers. Each carrier is 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. Return to footnote 2 referrer

Table 9: Residual virus disinfection on hard surfaces
Claim	Physical form, method of application	Recommended test methods	Test organisms	Number of batches per organism, replication or carrier surfaces per batch	Inoculum or carrier count	Performance criteria for efficacy
Core test requirements for residual virus disinfection
Up to 24 hr for virus^{Footnote 1}	Liquids Water-soluble powders Combination liquid/spray Spray Single-use towelettes	US EPA's Protocol for Residual Self-Sanitizing Activity of Dried Chemical Residuals on Hard, Non-Porous Surface (Protocol 01-1A) modified for viral testing using ASTM E1053^{Footnote 2}	Most difficult-to-kill virus indicated on the label (such as small non-enveloped virus)	2 LCL batches, 4 carriers per batch	At least 1.0 x 10^4.8 virus should have survived on the inoculated control squares	99.9% mean reduction (minimum 3 log₁₀) for each bacterium following 24-hr wear and re-inoculation procedure and within a 10-min contact time after sanitization test Biocide should also meet non-residual viral disinfection efficacy requirements for all claimed viruses for residual hard-surface disinfection
Bridging additional viruses for residual disinfection
Up to 24 hr – claims for bridging additional viruses	Liquids Water-soluble powders Combination liquid/spray Spray Single-use towelettes	No additional data required if performance criteria for residual hard-surface disinfection against S. aureus (ATCC 6538), P. aeruginosa and hardest-to-kill virus are met
Confirmatory efficacy data requirements for residual virus disinfection
Up to 24 hr for virus	All test requirements are the same as for core virus residual disinfection
Footnote 1 Biocides that have non-residual virucidal claims should also be tested for residual virus efficacy against the hardest-to-kill virus. Return to footnote 1 referrer Footnote 2 Durability testing should include 12 wear cycles consisting of abrasions (alternating wet and dry) and re-inoculations to support a 24-hr residual disinfectant claim. Each wear cycle consists of 2 passes (1 pass to the left and a return pass to the right) of the abrasion material over the surface, followed by re-inoculation. The protocol should be modified for single-use towelettes. For shorter residual claims (no less than 4 hrs), the test may be ratiometrically reduced (for example, 6 wear cycles for a 12-hr residual claim). Return to footnote 2 referrer

Table 10: Residual non-food contact sanitizers on hard surfaces
Claim	Physical form, method of application	Recommended test methods	Test organisms	Number of batches per organism, replication or carrier surfaces per batch	Inoculum or carrier count	Performance criteria for efficacy
Core test requirements for bacteria residual sanitization
Up to 24 hr^{Footnote 1}	Liquids Water-soluble powders Combination liquid and spray Spray Single-use towelettes	US EPA's Protocol for Residual Self-Sanitizing Activity of Dried Chemical Residuals on Hard, Non-Porous Surface (Protocol 01-1A)^{Footnote 2}	Staphylococcus aureus (ATCC 6538) and Klebsiella pneumoniae (ATCC 4352) or Klebsiella aerogenes (also known as Enterobacter aerogenes) (ATCC 13048)	3 LCL batches, 4 carriers per batch (plus 4 controls, 2 neutralization controls)	At least 1 x 10⁴ bacteria should have survived on the inoculated control squares	99.9% reduction (minimum 3 log₁₀) for each bacterium following 24-hr wear and re-inoculation procedure and within 5-min contact time following sanitization test
Efficacy requirements for bridging of additional bacteria residual sanitization
Up to 24 hr – bridging additional bacteria	No additional data required to bridge additional bacteria claims if the performance criteria for residual hard-surface self-sanitization against S. aureus (ATCC 6538) and K. aerogenes (ATCC 13048) or K. pneumoniae (ATCC 4352) are met
Footnote 1 Residual sanitization testing against S. aureus and either K. pneumonia or K. aerogenes supports general residual sanitization (99.9% kill) claims for 24 hrs against bacteria and specific claims against the tested microorganisms. If you want to indicate residual sanitization claims for your biocide, you should first demonstrate efficacy as a non-residual non-food contact sanitizer. Return to footnote 1 referrer Footnote 2 Durability testing should include 12 wear cycles consisting of abrasions (alternating wet and dry) and re-inoculations to support a 24-hr residual disinfectant claim. Each wear cycle consists of 2 passes (1 pass to the left and a return pass to the right) of the abrasion material over the surface, followed by re-inoculation. The protocol should be modified for single-use towelettes. Where shorter residual claims are desired (no less than 4 hrs), the test may be ratiometrically reduced (for example, 6 wear cycles for a 12-hour residual claim). If you want to add residual sanitization claims for towelettes, you should have the carriers treated using 1 towel per lot to wipe 4 test carriers. Each carrier is 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. Return to footnote 2 referrer

Spore and sterilant

You need efficacy testing against viable spore form (not vegetative cells) for all spore claims. If you want to label your biocide as a sporicide, sporicidal biocide or sterilant, you must have efficacy data using AOAC 966.04 against both:

Bacillus subtilis (ATCC 19659), with spores generated using method 2 of the AOAC 966.04 in nutrient agar treated with MnSO₄
Clostridium sporogenes (ATCC 3584) with spores generated using method 1 of the AOAC 966.04 in cooked meat medium treated with MnSO₄ for spore generation and inoculation of porcelain penicylinders and silk suture loops

For all sterilant and sporicide claims, use AOAC 966.04 with:

all tested carriers being negative for growth at the proposed contact time (unless otherwise prescribed in the test method)
a mean control count of 1 x 10⁵ to 1 x 10⁶ spores per carrier
a level of acid resistance outlined in the test
a confirmed neutralization using growth media and incubation conditions for each microbe according to method 2 (section C, subsection h)
verification testing using 1 batch showing all tested carriers are negative for growth at the proposed contact time

If you want to make specific claims against specific bacterial spores, you must provide supporting efficacy data. You only need to include a specific claim (for example, "inactivates spores of Clostridioides difficile" or "kills spores of Clostridioides difficile) on your label if you:

have efficacy data to support a specific claim
do not have the efficacy data to support your biocide as a sporicide or sporicidal biocide

For surface-specific claims against additional bacterial spores (for example, Clostridioides difficile, Bacillus anthracis, Bacillus cereus, Geobacillus stearothermophilus), we require:

evidence your biocide is a sporicide
2 samples of the biocide, representing 2 separately compounded batches per bacterium
30 inoculated carriers of 1 type (stainless steel penicylinders, porcelain penicylinders or silk suture loops, for example) per batch per additional bacterial spore
- for example: 1 carrier type per additional bacterial spore x 30 carriers per type = 60 carriers per type per batch

If there are no general sporicide or sterilant claims, you do not need to conduct verification testing when testing only against specific bacterial spores, such as:

Bacillus cereus (ATCC 14579)
Bacillus anthracis (ATCC 14578)
Clostridioides difficile (ATCC 43598)
Geobacillus stearothermophilus (ATCC 12980)

Table 11: Disinfection on hard surfaces against bacteria spores
Claim	Physical form, method of application	Recommended test methods	Test organisms	Number of batches per organism, carriers per batch	Inoculum or carrier count	Performance criteria for efficacy
Core bacteria spore test requirements
Sterilant	Liquids Water-soluble powders	AOAC 966.04 (method 1: silk suture loops, method 2: porcelain penicylinders)	Bacillus subtilis (ATCC 19659) (method 1 of AOAC 966.04 for spore production) and Clostridium sporogenes (ATCC 3584) (method 1 of AOAC 966.04 for spore production)	3 batches 2 types of carriers (suture loop and porcelain penicylinder, as prescribed in current version of test method) 60 carriers per type per batch^{Footnote 1}	Unless otherwise prescribed: Mean control count of 1 x 10⁵ to 1 x 10⁶ spores per carrier, for both microbes and carrier types	Unless otherwise prescribed: 720/720 carriers negative for growth at proposed contact time and for verification testing: 120/120 carriers negative for growth at proposed contact time
Sterilant	Sprays, mists, foams	A liquid biocide applied as a spray using a pump or trigger should be tested as a liquid A wetness test to demonstrate the biocide will achieve the required contact time when sprayed
Sporicide	Liquids Water-soluble powders	AOAC 966.04 (method 1: silk suture loops, method 2: porcelain penicylinders)	Bacillus subtilis (ATCC 19659) (method 2 of AOAC 966.04 for spore production) and Clostridium sporogenes (ATCC 3584) (method 1 of AOAC 966.04 for spore production)	3 batches at least 1 type of carrier for a surface- specific sporicide claim (stainless steel penicylinder, suture loop or porcelain penicylinder), as prescribed in current version of test method) 60 carriers per type per batch^{Footnote 2}	Unless otherwise prescribed: Mean control count of 1 x 10⁵ to 1 x 10⁶ spores per carrier, for both microbes and carrier types	Unless otherwise prescribed: 360/360 carriers negative for growth at proposed contact time and for verification testing: 60/60 carriers negative for growth at proposed contact time (for each microorganism tested using 30 carriers)
Sporicide	Sprays, mists, foams	A liquid biocide applied as a spray using a pump or trigger should be tested as a liquid A wetness test to demonstrate the biocide will achieve the required contact time when sprayed
Additional bacteria spore test requirements
Claim against additional bacterial spores (for example, Bacillus cereus (ATCC 14579), Bacillus anthracis (ATCC 14578), Clostridioides difficile (ATCC 43598), Geobacillus stearothermophilus (ATCC 12980)^{Footnote 3}	Liquids Water-soluble powders	AOAC 966.04 (method 1: silk suture loops, method 2: porcelain penicylinders) or a specific test method for the test organisms (such as C. difficile)	Any specific bacterial spore claimed on the label along with the general sporicidal claim	2 batches 1 type of carrier (stainless steel penicylinder, suture loop or porcelain penicylinder) 30 carriers per type per batch	Unless otherwise prescribed: Mean control count of 1 x 10⁵ to 1 x 10⁶ spores per carrier, for each bacterial spore and carrier type	Unless otherwise prescribed: 60/60 carriers negative for growth per carrier type at proposed contact time
	Sprays, mists, foams	Liquid biocide applied as a spray using a pump or trigger should be tested as a liquid
Footnote 1 Sterilant claims: 3 samples of the biocide (representing 3 separately compounded batches per bacterial spore (720 carriers tested when using AOAC 966.04, methods 1 and 2)) each compounded batch to include 2 different types of carriers, each type containing 60 inoculated carriers and 2 different test microorganisms verification testing to confirm the label claim, with 1 additional test conducted in a different laboratory from the original or in same laboratory using different laboratory personnel and same test procedure and conditions as in first laboratory test verification testing of 1 batch to use both types of carriers and 30 carriers per batch (for example, 2 carrier types x 2 test microorganisms x 60 carriers per type = 240 carriers per batch samples, or 3 biocide batches x 240 carriers per batch = 720 carriers) Return to footnote 1 referrer Footnote 2 Sporicide claims: 3 samples of the biocide (representing 3 separately compounded batches per bacterial spore) 60 inoculated carriers of 1 type (for example, stainless steel penicylinders, porcelain penicylinders or silk suture loops) per batch per bacterial spore, for surface-specific sporicide claim use same carrier type for both microbes (for example, 1 carrier type x 2 test microorganisms x 60 carriers per type = 120 carriers per batch sample, for total of 360 carriers tested in 3 batches) verification testing to confirm the label claim, with 1 additional test conducted in a different laboratory from the original or in same laboratory using different laboratory personnel and same test procedure and conditions as in first laboratory test verification testing of 1 batch to use 1 type of carrier and 30 carriers per batch Return to footnote 2 referrer Footnote 3 When testing for sporicide, sterilant claims or claims against additional bacterial spores, use: silk suture loops as a type of soft-surface carrier or Dacron loops (braided polyester) if testing with oxidative chemistries such as biocides containing peroxide, peracetic acid, chlorine dioxide or sodium hypochlorite Return to footnote 3 referrer

Claims supported by only Clostridioides difficile

For Clostridioides difficile (ATCC 43598)claims without a sporicide claim:

a carrier control count of 1.0 x 10⁶ to 1.0 x 10⁷ spores per carrier
a mean 6 log₁₀ reduction based on recoverable spores within proposed contact time for all carriers
10 inoculated hard non-porous carriers per batch
3 samples of the biocide representing 3 separately compounded batches:
- AOAC 2008.05 using glass slides
- ASTM E2197 using stainless steel disks
- US EPA's 2018 test methodology against Clostridiodes difficile using:
  - 430 stainless steel disks
  - 3 independent tests (for example, 1 batch tested per day)
5% organic soil or comparable alternatives 430 stainless steel carriers or 304 stainless steel carriers if testing biocides with oxidative chemistries (for example, containing peracetic acid and peroxides)

Non-food contact and food contact sanitizers on hard surfaces

Sanitizers significantly reduce the number of microbes on a non-living and non-liquid surface due to the antimicrobial action of the active ingredients. They do not destroy or eliminate all microorganisms.

Non-food contact hard non-porous surface sanitizer

You should use vegetative bacteria for your tests to demonstrate your biocide is a non-food contact surface sanitizer. Once the biocide is demonstrated to be a non-food contact surface sanitizer, you can make additional specific bacteria non-food contact surface sanitization claims on your biocide's label (for example, Escherichia coli).

To qualify to add the claim "non-food contact surface sanitizer" to the label, you must provide efficacy data against the following core bacteria:

Staphylococcus aureus (ATCC 6538) and Klebsiella pneumoniae (ATCC 4352) or
Staphylococcus aureus (ATCC 6538) and Klebsiella aerogenes (ATCC 13048)

Table 12: Non-food contact sanitizers on hard surfaces
Claim	Physical form, method of application	Recommended test methods	Test organisms	Number of batches per organism, carriers per batch	Inoculum or carrier count	Performance criteria for efficacy
Core test requirements for bacteria non-food contact sanitization
Non-food contact surface sanitizer	Liquids Water-soluble powders	ASTM E1153	Staphylococcus aureus (ATCC 6538) and Klebsiella pneumoniae (ATCC 4352) or Klebsiella aerogenes (previously known as Enterobacter aerogenes) (ATCC 13048)	3 LCL batches (separately compounded batches per bacterium) 5 carriers per batch per bacterium	Average of at least 7.5 x 10⁵ (5.88 log) bacteria should survive the inoculated control squares with an upper limit of 2.0 x 10⁶ (6.3 log) As prescribed in current test method and at proposed contact time	Unless otherwise prescribed: Less than or equal to 99.9% mean reduction (minimum 3 log₁₀) for each bacterium within 5-min contact
Non-food contact surface sanitizer	Spray Single-use towelettes	Modified ASTM E1153
Test requirements for additional bacteria non-food contact sanitization
Claim against additional bacteria	Liquids Water-soluble powders	ASTM E1153	Any specific bacteria claimed on label in addition to non-food contact surface sanitizer claim	2 NCL batches (separately compounded batches per bacterium) 5 carriers per batch per bacterium	Average of at least 2.5 x 10⁴ bacteria should survive the inoculated control squares with an upper limit of 2.0 x 10⁶ (4.4 to 6.3 log) As prescribed in current test method and at proposed contact time	Unless otherwise prescribed: Less than or equal to 99.9% mean reduction (minimum 3 log₁₀) for each bacterium within 5-min contact
Claim against additional bacteria	Spray Single-use towelettes	Modified ASTM E1153
Confirmatory efficacy data requirements for bacteria non-food contact sanitization
Non-food contact surface sanitizer	Same requirements as for core bacteria			2 LCL batches 5 carriers per batch	Same requirements as for core bacteria

Food contact surface sanitizer

To claim your biocide as a food contact surface sanitizer, you must provide efficacy data that relates to the chemical nature of your biocide (refer to Table 13). Once the biocide is demonstrated to be a food contact surface sanitizer, you may make additional specific bacteria food contact surface sanitization claims on your biocide label.

To qualify to add the claim “food contact surface sanitizer” to the label, you must provide efficacy data against the following core bacteria:

For non-halide biocides: Staphylococcus aureus (ATCC 6538) and Echerichia coli (ATCC 11229)
For halide biocides: Staphylococcus aureus (ATCC 6538) or Salmonella enterica (ATCC 6539)
For towelette biocides: Staphylococcus aureus (ATCC 6538) and Echerichia coli (ATCC 11229)

Table 13: Examples of halide and non-halide active ingredients
Non-halide	Halide
Peracetic acid Hydrogen peroxide Chlorinated trisodium phosphate Anionic detergent-acid formulants Quaternary ammonium compounds Hydrogen peroxide-related compounds	Iodophors Mixed halides Chlorine-bearing chemicals

Table 14: Food contact sanitizers on hard surfaces
Claim	Physical form, method of application	Recommended test methods	Test organisms	Number of batches per organism, carriers per batch	Inoculum or carrier count	Performance criteria for efficacy
Core test requirements for bacteria food contact sanitization
Food contact surface sanitizer	For non-halide biocides: Liquids Water-soluble powders Spray	AOAC 960.09	Escherichia coli (ATCC 11229) and Staphylococcus aureus (ATCC 6538)	3 LCL batches (3 separate compounded batches per bacterium) Use number of carriers and types of surfaces prescribed in current test method	The numbers control must fall between 1 x 10⁷ and 1 x 10⁸ (7.0 to 8.0 log)	Unless otherwise prescribed: A 5 log₁₀ reduction of each bacterium within 30 s
	For halide biocides: Liquids Water-soluble powders Spray	AOAC 955.16	Salmonella enterica (ATCC 6539) or Staphylococcus aureus (ATCC 6538)		As prescribed in current test method	Unless otherwise prescribed: Test results should demonstrate biocide concentrations equivalent in activity to 50, 100 or 200 ppm of available chlorine as labelled (reference standard is sodium hypochlorite)
	Single-use towelettes^{Footnote 1}	US EPA's Draft Interim Guidance for Non-Residual Sanitization of Hard Inanimate Food Contact Surfaces Using Pre- Saturated Towelettes	Escherichia coli (ATCC 11229) and Staphylococcus aureus (ATCC 6538)		As prescribed in current test method	Unless otherwise prescribed: minimum 99.999% (5 log₁₀) reduction of each bacterium within 30 s
Test requirements for food contact sanitization of additional bacteria
Claim against additional bacteria	For non-halide biocides: Liquids Water-soluble powders Spray	AOAC 960.09	Any specific bacteria claimed on the label in addition to food contact surface sanitizer claim	2 NCL batches (2 separately compounded batches per bacterium) Number of carriers and types of surfaces prescribed in current test method	Numbers control must fall between 1 x 10⁷ and 1 x 10⁸ (7.0 to 8.0 log)	A 5 log₁₀ reduction of each bacterium within 30 s or less
	For halide biocides: Liquids Water-soluble powders Spray	AOAC 955.16			As prescribed in current test method	Test results should demonstrate biocide concentrations equivalent in activity to 50, 100 or 200 ppm of available chlorine as labelled (reference standard is sodium hypochlorite)
	Single-use towelettes	US EPA's Draft Interim Guidance for Non-Residual Sanitization of Hard Inanimate Food Contact Surfaces Using Pre-Saturated Towelettes			As prescribed in current test method	Minimum 99.999% (5 log₁₀) reduction of each bacterium within 30 s or less
Confirmatory data requirements for food contact sanitization
Food contact surface sanitizer	Same as core bacteria sanitization requirements			2 LCL batches Number of carriers and types of surfaces prescribed in current test method	Same as core bacteria sanitization requirements
Abbreviation: ppm: parts per million Footnotes Footnote 1 For towelette biocides: size of test surface equal to 0.37 m² (4 square feet) with 1 towelette to wipe the number of carriers, which equals 0.37m² (4 square feet) at minimum, testing on surfaces, including both: stainless steel or glass a plastic with a rough surface (for example, plastic cutting board) Return to footnote 1 referrer

Residual disinfection and sanitization

Residual sanitization claims

For residual self-sanitizing claims (under 24 hours), you should test your biocide to confirm it continues to sanitize surfaces despite surface re-inoculation and abrasion. Depending on the sanitization claim, we recommend that you refer to US EPA's:

810.2300 and 810.2400 product performance test guidelines for general efficacy and labelling considerations for products with residual self-sanitizer efficacy or
Protocol 01-1A for supporting residual self-sanitizing efficacy claims of dried chemical residues on hard, non-food contact surfaces

Use on alternative porous surfaces

If your biocide is for use on other types of porous surfaces, such as wood, concrete or rubber, you should:

indicate the porous material (as a carrier) on the test protocol for the existing standard method
develop control data (for example, dried carrier microbial count neutralization confirmation, sterility controls)

Refer to the US EPA's 810 product performance test guidelines for general efficacy and labelling considerations for products with sanitization or disinfection claims on alternative surfaces.

Pre-soak laundry sanitizers

If you used an appropriate immersion test method (for example, ASTM E1153), you may use efficacy data from a hard, non-porous 1-step sanitizer to support laundry pre-soak sanitizer claims.

The test method should be modified to include a:

minimum of 5% organic soil
test carrier using unglazed ceramic tile

These data may be extrapolated for the sanitization of soiled fabrics by total immersion in the use solution before routine laundry operations. Therefore, testing against the same bacteria specified in the section on the requirements for non-food contact hard non-porous surface sanitizer claims is required.

Batch replication, microbial counts and performance standards

We require the same as what is required for non-food contact hard non-porous surface sanitizer claims. The test carriers used should be unglazed ceramic tiles.

We require:

a minimum mean of 7.5 x 10⁵ colony-forming units (CFU) per carrier (minimum 5.88 log₁₀ density)
a target range of 7.5 x 10⁵ (5.88 log) to 2.0 x 10⁶ (6.30 log) to improve standardization and reproducibility
results demonstrating a minimum reduction of 99.9% (3 log₁₀ minimum) for each bacterium for the same control count
- should be demonstrated against each test microorganism within the contact time claimed on the label

For claims against additional bacteria, you should use ASTM E1153 testing with :

an average of at least 2.5 x 10⁴ bacteria surviving the inoculated control squares
a less than or equal to 99.9% mean reduction (3 log₁₀ minimum) for each bacterium within a 5-minute contact time

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Date modified:: 2024-12-23

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Guidance on efficacy requirements for biocides: Hard-surface disinfection and sanitization

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Types of claims

General claims

Hard-surface disinfectant claims

Limited, general (broad-spectrum) and hospital or health care disinfection

Mycobacteria disinfection

Footnotes

Fungi

Footnotes

Virus disinfection

Surrogate efficacy

Residual disinfection claims

Spore and sterilant

Claims supported by only Clostridioides difficile

Non-food contact and food contact sanitizers on hard surfaces

Non-food contact hard non-porous surface sanitizer

Food contact surface sanitizer

Footnotes

Residual disinfection and sanitization

Residual sanitization claims

Use on alternative porous surfaces

Pre-soak laundry sanitizers

Batch replication, microbial counts and performance standards

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