Guideline for data to support efficacy of disinfectants for veterinary use

Introduction
Efficacy criteria
Efficacy tests
References

1. Introduction

Applicants seeking to register a disinfectant product for veterinary use should submit efficacy and safety data to support all label claims. This guideline is intended to provide advice on what data you should submit for the registration of disinfectants for veterinary use, based on generic label claims. We will consider applications for the registration of disinfectants for veterinary use with specific claims on a case-by-case basis. Irrespective of whether efficacy claims for a veterinary disinfectant are generic or specific, we encourage you to seek pre-application assistance from the APVMA before submitting your application.

In addition to providing efficacy and safety data, an application for a disinfectant for veterinary use should be accompanied by the relevant information on chemistry and manufacture, public health, environmental chemistry and fate, and occupational health and safety. You should therefore read this document in conjunction with:

the data guidelines (parts 1–10)
the Veterinary Labelling Code
the Standard for the Uniform Scheduling of Medicines and Poisons (SUSMP)
FAISD handbook—Handbook of first aid instructions, safety directions, warning statements and general safety precautions for agricultural and veterinary chemicals.

1.1. What are disinfectants for veterinary use?

For the purpose of this guideline, veterinary disinfectants are products that are used on hard, inanimate objects or surfaces to destroy a range of microorganisms.

For registration purposes, we recognise two general categories of veterinary disinfectants that are based on where disinfectants will be used:

areas that generally have a lower level of contamination, such as waiting rooms, consulting rooms and surgical suites in veterinary clinics or hospitals
areas that generally have a higher level of contamination, such as preparation areas and animal cages or runs within a veterinary clinic or hospital; animal housing facilities, such as dog kennels, catteries, horse stables, poultry houses, cattle and sheep facilities, and piggeries. This category of veterinary disinfectants may also be suitable for use on animal transport vehicles.

A third, albeit less common category of disinfectants for veterinary use, is those with specific label claims; for example, virucidal activity only. This category of veterinary disinfectant is considered on a case-by-case basis by the APVMA for registration.

For the purpose of this guideline, veterinary disinfectants do not include products that are:

used on surfaces that come into contact with food (sanitisers)
used on animals, either externally or internally (antiseptics).

1.2. Classification of veterinary disinfectants and label efficacy claims

The label claims for veterinary disinfectants should reflect the fitness-for-purpose of the product, regardless of where the product is used within a veterinary context. The minimum expectations for registration purposes are the demonstration of efficacy against the infectious agents described as critical in this guideline (refer to Table 1). Demonstrable efficacy against these organisms will provide for a generic claim on the product label. You may also elect to demonstrate efficacy against additional infectious agents. In this respect, the product label may list a generic claim and one or more specific claim(s) that have been substantiated by data.

Veterinary disinfectant products for use in veterinary clinics or hospitals should be suitable for disinfecting facilities such as waiting rooms, consulting rooms and surgical suites, and for disinfecting surgical instruments. The latter requires that the product is non-corrosive.

Veterinary disinfectant products for use in animal housing facilities as well as preparation areas and animal cages or runs within veterinary clinics or hospitals should satisfy the efficacy criteria as per Table 1.

1.3. Efficacy claims against exotic diseases

In February 2008, the APVMA gazetted an operational notice stating that disinfectant labels may not make claims for efficacy against diseases that are exotic to Australia. Efficacy claims should be for endemic diseases only.

Therefore, the label disclaimer should note that the disinfectant product does not protect against exotic diseases.

The APVMA deals with exotic diseases separately, on a case-by-case basis; this may involve an emergency permit being issued following the outbreak of an exotic disease.

2. Efficacy criteria

Before registering a veterinary disinfectant product, we evaluate the data derived from efficacy studies that are relevant to the claims being made.

The efficacy data you submit in support of the registration of a veterinary disinfectant should be technically sound, reproducible and reliable. To ensure that this is the case, you should ensure that the efficacy tests are conducted by laboratories that are accredited either by the National Association of Testing Authorities (NATA) or by another equivalent accreditation body. Efficacy tests conducted by these laboratories comply with the OECD principles of good laboratory practice (GLP).

The APVMA has endorsed the tests shown under the heading Efficacy tests, but will also consider, on a case-by-case basis, efficacy data that have been generated using other tests, including internationally accepted test standards and new technologies. Table 4 provides guidance about other tests that are available. When using tests other than those shown under Efficacy tests, you should submit scientific justification to support such data so that we can determine whether the methods used are equivalent to those endorsed in this guideline.

2.1. Test parameters for label claims for bactericidal, virucidal, fungicidal and sporicidal efficacy

You should demonstrate the efficacy of the proposed product by using acceptable test methods to support label claims. Appropriate methods should address:

fitness-for-purpose of the product—the method protocol should reflect the intended use pattern of the product and this will dictate the test parameters you should investigate (for example, growth on a solid surface or in a liquid suspension, contact times, microbiological species and strains). Some flexibility with regard to the substrate used to test the microorganism is acceptable, as shown in Table 4.
interference or confounders—efficacy validated in the presence of known interferences such as soiled environments (see Test conditions, hard water)
controls or validation—adequate controls to validate the protocol, which includes stopping disinfectant action, cytotoxicity, and other controls as listed in this guideline (see Test conditions and Validation of test methods)

Additional information on the efficacy tests we prefer is described under Efficacy tests in this document. Section 2 includes a detailed description of methodology based on the Therapeutic Goods Administration (TGA) Disinfectant Test, with modifications to suit veterinary situations. It also provides additional details on the testing methodologies for assessing virucidal activity.

If you decide to use a test methodology other than those described under Efficacy tests, you should submit a proposal to the APVMA for consideration. In this case, you should:

use high-level soiling conditions when it is necessary to simulate the conditions of animal housing—if your methodology does not provide specific instruction about interfering substances applicable to the veterinary field, you should use the test conditions described under Test conditions
use the performance standards described under Performance standards when values are not specified in the description of the test methodology
use the organisms described under Test organisms with all test protocols (bactericidal, fungicidal, sporicidal or virucidal)
use the validation steps described in the chosen method as well as those described under Validation of test methods.

2.2. Test conditions

You should describe the test conditions as well as the positive and negative controls used in the efficacy tests performed.

Efficacy tests for disinfectants should simulate a contaminated environment that mimics preparation areas and animal cages or runs within veterinary clinics or hospitals and animal housing. The test should be performed in the presence of organic and inorganic matter, such as killed yeast, serum, or serum albumin in order to simulate in vitro contaminated conditions. In the absence of a specification for contamination, a mixture of serum and killed yeast should be used such that the final test concentrations are:

0.5 per cent weight per volume of killed yeast, and
5 per cent volume per volume of sheep serum.

All efficacy tests should be conducted:

using hard water (342 parts per million [ppm], calculated as calcium carbonate [80:20 Ca²⁺:Mg²⁺]) as a diluent for those products that are diluted before use. A method for preparing sterile hard water is described under Efficacy tests. You should note that the degree of hardness of water (that is, the presence of Ca²⁺ and Mg²⁺) used to dilute the disinfectant may affect its performance. Generally, the harder the water the less effective the diluted disinfectant.
at the simulated in-use conditions in respect of pH, temperature, exposure time – concentration (dilution) combinations, and single or repeated applications. Surfaces should also be identical to the directions for use on the product label. Note that generally, disinfection performance increases with temperature. This applies to disinfection against all microorganisms, though the effect on individual species differs. Further, the prevailing degree of acidity or alkalinity during disinfection can affect the performance of a disinfectant. Generally, disinfectants are more active as undissociated molecules than as ionised molecules.
immediately prior to the end of the proposed shelf life of the product.

2.3. Performance standards

The performance standards described in this section apply only if you choose to use a test method that differs from the preferred efficacy tests described under Efficacy tests. You should describe the test protocols in detail to facilitate a valid assessment of the data.

For bacterial testing methodologies not included in Table 2, the product should be challenged with a minimum of 108 colony forming units (CFU). The acceptance criterion for passing the test is a 106-fold reduction in microorganisms (other than viruses) in a suspension or on an inanimate surface at a specified contact time and temperature.

Viruses should be tested in the appropriate cell culture and using appropriate controls and neutralisation of the disinfectant under test (refer to Validation of test methods). The cell cultures and growth conditions used in these studies should be validated. For the product to pass the test, a minimum of a 104-fold reduction in virus titre should be demonstrated from an initial inoculum of approximately 108 TCID50. When cytotoxicity is evident, a 103-fold reduction, or greater, beyond the cytotoxic level should be demonstrated. This is in accordance with the cytotoxicity testing protocol of the United States Environmental Protection Agency. Testing methodologies to assess virucidal activity are described under the heading Efficacy tests and in Table 4.

2.4. Test organisms

Test organisms should be selected from a recognised culture collection and their phenotypic and genotypic characteristics should be known and checked regularly. Examples of recognised culture collections include the American Type Culture Collection (ATCC) and the National Collection of Type Cultures (NCTC). If the test organisms have not been classified at a reference centre, you should submit detailed characterisation of the test organisms.

Table 1 lists the test organisms for disinfectant products for use in various situations. Table 1 should be read across the matrix, not down. Except in exceptional circumstances, the APVMA will not normally register a veterinary disinfectant with bactericidal, virucidal, fungicidal, or sporicidal activity only. However, claims for these individual activities are possible, provided you substantiate the specific claims of efficacy with efficacy data.

The selection of test organisms for efficacy testing should be relevant to the intended use and claim of the veterinary disinfectant product, and:

for a label claim for the use of a disinfectant in any veterinary clinic or hospital and all categories of animal housing (cats, dogs, horses, cattle, sheep, pigs and poultry) as a minimum, you should demonstrate the efficacy against all the organisms listed in the first row of Table 1
for a label claim for an individual category of animal housing, you should demonstrate efficacy against the microorganisms in Table 1 for the particular animal species. For example, if proposing a claim for a ‘dog kennel, cattery or piggery only’, you should demonstrate efficacy against the organism in the second row of Table 1.

The test protocols are designed to simulate an environment mimicking the level of contamination in preparation areas and animal cages or runs in veterinary clinics or hospitals and animal housing facilities.

The test organisms shown in Table 1 have been selected on the basis that they are more resistant than other microorganisms to the effect of disinfectants. Demonstrable efficacy to these test organisms will be accepted as evidence to support a generic claim in those situations described in Table 1.

Examples include:

disinfectants that demonstrate efficacy against canine parvovirus—the test virus in the first row of Table 1—will be considered an effective virucidal for use in Australian veterinary clinics or hospitals
a human rotavirus (for example, rotavirus Wa strain) is recommended as a surrogate test virus for veterinary rotaviruses
the vaccinia WR strain is recommended as a surrogate test virus for veterinary poxviruses
canine parvovirus is recommended as a surrogate test virus for feline and porcine parvoviruses.

These examples reflect the availability of standard protocols for these surrogate test viruses and the relative ease with which these surrogate test viruses can be grown and titrated in the laboratory. Other viruses within the specified virus families (rotaviruses, poxviruses, parvoviruses) may also be suitable for use as test viruses; however, their use should be justified scientifically.

Table 1: Test organisms for veterinary disinfectant products for use in veterinary clinics or hospitals and all categories of animal housing¹ or individual categories of animal housing facilities²
Situations	Test organisms
Situations	Bacterium	Virus	Fungus	Spore-forming organism
Veterinary clinics or hospitals and all categories of animal housing	Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli	Canine parvovirus Cornell strain	Trichophyton mentagrophytes	Spores of Bacillus subtilis
Dog kennel, cattery or piggery only	Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli	Canine parvovirus Cornell strain	Trichophyton mentagrophytes	Not applicable
Horse stable only	Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli	Rotavirus Wa strain, Vaccinia WR strain³	Trichophyton mentagrophytes	Spores of Bacillus subtilis
Poultry house only	Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli	Canine parvovirus Cornell strain⁴	Aspergillus niger	Spores of Bacillus subtilis
Cattle or sheep facility only	Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli	Rotavirus Wa strain, Vaccinia WR strain³	Not applicable	Spores of Bacillus subtilis

_{¹ Disinfectants for use in veterinary clinics or hospitals must be non-corrosive.}

_{² A disinfectant product for use in animal housing may be approved for use in animal housing for one or more animal species.}

_{³ Two test viruses (a poxvirus and a rotavirus) are recommended in horse stables and cattle or sheep facilities. This is important because poxviruses can demonstrate a higher level of resistance to some types of disinfectants, whereas rotaviruses can demonstrate a higher level of resistance to other disinfectant types.}

_{⁴ The recommendation to use canine parvovirus as a test virus to demonstrate virucidal activity in poultry houses takes into account the difficulties associated with using chicken anaemia virus and other circoviruses as test viruses. Applicants are, however, still able to submit a proposed protocol to assess virucidal activity against circoviruses for consideration.}

2.5. Number of batches to be tested

You should submit the results of efficacy tests in triplicate from at least three separate batches. All samples should pass the efficacy tests in order for the results to be accepted.

2.6. Validation of test methods

You should validate the efficacy test methods and use appropriate controls and reference organisms. Where reference materials are specified in a test method, they should be used at the nominated concentration. It may be appropriate to validate test methods using a reference substance such as:

benzalkonium chloride (the chain length and concentration as per the United States Pharmacopeia) for bactericidal tests
0.05 per cent phenol (see AOAC 955.11, AOAC International 2005) for bactericidal and fungicidal tests
2 per cent glutaraldehyde for sporicidal tests
hypochlorite for virucidal tests.

Some test methods include a neutralisation step to stop the action of the disinfectant at a specified time. A neutralisation step is critical when validating these test methods. Other test methods (for example, ASTM E 2197-11, ASTM International 2011b) include a filtration or washing step after contact of the infectious agent with the disinfectant. A neutralisation study is optional with these test methods.

When demonstrating effective neutralisation of the disinfectant under the test, you should conduct a study in which a neutralising agent is added to the samples of both treated and untreated control groups. The neutralising agent should completely inhibit the activity of the disinfectant without being toxic to the indicator organisms. In addition, the neutralising agent and active constituent should not combine to form a toxic compound. Some examples of neutralising agents are 3 grams per litre (g/L) lecithin, 30 g/L polysorbate 80, and 5 g/L sodium thiosulphate. You may wish to refer to ASTM E 1054–13 (ASTM International 2013), which describes a neutraliser validation test.

2.7. Good Manufacturing Practice

The disinfectants described in this guideline are agricultural chemical products. As such, they are exempt from the requirement of the Agricultural and Veterinary Chemicals Code Act 1994 (Agvet Code) to be manufactured in premises licensed by the APVMA under the Australian Code of Good Manufacturing Practice for Veterinary Chemical Products.

3. Efficacy tests

This document provides guidance on veterinary disinfectants with bactericidal, virucidal, fungicidal and sporicidal activities. The three sections below provide additional detail on the data relating to microbicidal activities for a veterinary disinfectant that demonstrates efficacy against bacteria, viruses, fungi and spores:

Section 1—Tests to determine bactericidal, virucidal, fungicidal and sporicidal efficacy
Section 2—The TGA Disinfectant Test to determine bactericidal efficacy
Section 3—Testing methodologies for assessing virucidal activity.

Although Section 2 refers solely to bactericidal activity and Section 3 refers solely to virucidal activity, these two sections are not intended to provide guidance on data submissions for a veterinary disinfectant demonstrating bactericidal activity only, or virucidal activity only.

The APVMA has endorsed the tests described in this section as suitable for demonstrating efficacy in support of label claims. Efficacy claims against specific microorganisms should be supported by data generated using the test organisms and parameters described under Test parameters for label claims for bactericidal, virucidal, fungicidal and sporicidal activity.

The test protocols described in this guideline do not assess the efficacy of disinfectants against microbes associated with biofilms. The latter are formed when bacteria and fungi adhere to surfaces and their cell surface properties are altered. This can result in reduced penetration and efficacy of disinfectants against the microbes.

The infectious agents provided in Table 1 have been selected for their resistance to veterinary disinfectants and the practicalities of being able to grow them in the laboratory. The testing of these microorganisms overcomes the need to test infectious agents that are less resistant to disinfectants. For example, if a product is demonstrably efficacious against canine parvovirus, we will grant a label claim against all canine viruses.

3.1. Section 1: Tests to determine bactericidal, virucidal, fungicidal and sporicidal efficacy

This section summarises the main test methodologies available for evaluating the bactericidal, virucidal, fungicidal and sporicidal efficacy of veterinary disinfectants. You should use the test protocols described in Table 2 in combination with the test organisms shown in Table 1 of this guideline. The label claim of the product will determine the appropriate test organisms you should use from Table 1. We will consider claims for efficacy against infectious agents in addition to the critical test organisms shown in Table 1 on the basis of demonstrable efficacy.

Table 2: Tests for bactericidal, virucidal, fungicidal or sporicidal claims of disinfectants
Label claim	Test method	Organism to be used
Bactericidal, virucidal, fungicidal or sporicidal	ASTM E 2197-11, Standard quantitative disk carrier test method for determining the bactericidal, virucidal, fungicidal, mycobactericidal and sporicidal activities of liquid chemical germicides (ASTM International 2011b) Use soiling conditions as recommended	Staphylococcus aureus (ATCC 6538) Pseudomonas aeruginosa (ATCC 15442) Salmonella typhimurium (ATCC 10708) Escherichia coli (NCTC 8196) canine parvovirus Cornell strain (ATCC VR-2017) Rotavirus Wa strain (ATCC VR-2018) Vaccinia WR strain (ATCC VR-119) Trichophyton mentagrophytes (ATCC 9533) Aspergillus niger (ATCC 16404) Bacillus subtilis (ATCC 19659)
Bactericidal	Option 1: The efficacy testing requirements described in ‘TGA Disinfectant Test’ for Option B (ref: Section 2)	Staphylococcus aureus (ATCC 6538) Pseudomonas aeruginosa (ATCC 15442) Salmonella typhimurium (ATCC 10708) Escherichia coli (NCTC 8196)
Bactericidal	Option 2: EN 1656, Chemical disinfectants and antiseptics—Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in veterinary field—Test method and requirements (phase 2, step 1) (DIN 2000) Use high-level soiling (10 g/L bovine albumin and 10 g/L yeast extract) as recommended	Staphylococcus aureus (ATCC 6538) Pseudomonas aeruginosa (ATCC 15442) Salmonella typhimurium (ATCC 10708) Escherichia coli (NCTC 8196)
Virucidal	Option 1: ASTM E 1053-11—Standard test method of virucidal agents intended for inanimate environmental surfaces (ASTM International 2011a) Use soiling conditions as recommended	Canine parvovirus Cornell strain (ATCC VR-2017) Rotavirus Wa strain (ATCC VR-2018) Vaccina WR strain (ATCC VR-119)
Virucidal	Option 2: EN 14675, Chemical disinfectants and antiseptics—Quantitative suspension test for the evaluation of virucidal activity of chemical disinfectants and antiseptics used in the veterinary area—Test method and requirements (phase 2, step 1) (DIN 2006) Use high level soiling (10 g/L bovine albumin and 10 g/L yeast extract) as recommended	Canine parvovirus Cornell strain (ATCC VR-2017) Rotavirus Wa strain (ATCC VR-2018) Vaccina WR strain (ATCC VR-119)
Fungicidal	EN 1657, Chemical disinfectants and antiseptics—Quantitative suspension test for the evaluation of fungicidal or yeasticidal activity chemical disinfectants and antiseptics used in the veterinary area—Test method and requirements (phase 2, step 1) (DIN 2005) Use high level soiling (10 g/L bovine albumin and 10 g/L yeast extract) as recommended	Trichophyton mentagrophytes (ATCC 9533) Aspergillus niger (ATCC 16404)
Sporicidal	EN 13704, Chemical disinfectants—Quantitative suspension test for the evaluation of sporicidal activity of chemical disinfectants used in food, industrial, domestic and institutional areas—Test method and requirements (phase 2, step 1) (DIN 2002) Use a high level soiling (10 g/L bovine albumin and 10 g/L yeast extract)	Spores of Bacillus subtilis (ATCC 19659 or ATCC 6633)

_{ATCC = American Type Culture Collection, EN = European standard, NCTC = and the National Collection of Type Cultures}

_{Note: Test conditions are described under Test conditions}

3.2. Section 2: The TGA Disinfectant Test to determine bactericidal efficacy

This section describes the test methods for determining bactericidal efficacy for the two general categories of veterinary disinfectants; namely, disinfectants for use in waiting rooms, consulting rooms and surgical suites in veterinary clinics or hospitals, and disinfectants for use in the preparation areas and animal cages or runs in veterinary clinics or hospitals and in animal housing facilities. The test conditions mimic worst-case environments. The methodology described below is based on the TGA Disinfectant Test with modifications to suit veterinary situations.

3.2.1. The principle of the TGA Disinfectant Test

The TGA Disinfectant Test, also known as a Kelsey-Sykes Capacity In Use Test, assesses the capacity of the disinfectant to destroy successive additions of a bacterial culture. The conditions of this test are an attempt to reproduce some of the conditions that disinfectants must overcome in normal use.

The disinfectant is tested at the dilution recommended on the product label. The test consists of:

challenging the diluted disinfectant with bacterial inoculum
withdrawing a sample after a given time and culturing the sample in a suitable recovery medium
challenging the mixture again with a second inoculum
after a second interval, sampling the mixture again for culturing.

The sample is passed or failed according to the extent of growth in the cultures sampled.

3.2.2. Test conditions

The bactericidal efficacy tests for veterinary disinfectants should be conducted using the organisms and conditions in Table 3. In all cases, sterile hard water should be used as the diluent. The test conditions are simulated by the addition of organic matter in the form of a mixture of killed yeast suspension and sheep serum.

Table 3 shows the test parameters to demonstrate a general bactericidal effect for veterinary disinfectants.

Table 3: Selection of test parameters for veterinary disinfectants
Test organisms	Test option for re-suspension of centrifuged organisms	Number of challenges	Inoculum density
Staphylococcus aureus Salmonella typhimurium Pseudomonas aeruginosa Escherichia coli	TGA Option B combination of killed yeast suspension and sheep serum and using hard water as the diluent	2	1 x 10⁸ to 1 x 10⁹

3.2.3. Methodology

The methodology for the TGA Disinfectant Test to determine bactericidal efficacy is outlined below.

3.2.3.1. Media

All media should be contained in capped glass containers. If media are stored, the containers should be sealed tightly or refrigerated.

3.2.3.1.1. Recommendations for sterile hard water

To prepare sterile hard water:

Dissolve 0.304 g of anhydrous calcium chloride and 0.065 g anhydrous magnesium chloride in glass-distilled water, and make up to one litre. The final concentration is 2.7 mM CaCl², 0.7 mM MgCl².
Dispense into glass containers and sterilise by autoclaving at 121 ± 1º C for 15 minutes.

3.2.3.1.2. Killed yeast suspension to simulate ‘contaminated’ conditions

To prepare the killed yeast suspension:

Weigh 200 g of moist, compressed baker's yeast. Cream by the gradual addition of sterile hard water using a heavy glass rod for stirring. Decant the creamed portion into a flask, add more water to any lumpy residue remaining and repeat the creaming and decantation until no residue remains and 500 mL of water has been used.
Shake the contents of the flask vigorously and strain through a 100-mesh sieve, breaking down any remaining lumps.
Add 500 mL of sterile hard water, shake vigorously and adjust the pH to 6.9–7.1 with 1N sodium hydroxide.
Transfer 50 mL, 100 mL or 200 mL of the yeast solution into screw-capped bottles.
Autoclave at 121 ± 1ºC for 15 minutes and allow the autoclave to cool without releasing pressure. Store at 4–8ºC.
Dry two Petri dishes to constant weight. Into each, pipette 25 mL of sterilised yeast suspension, and dry to constant weight at 100ºC. Calculate the average solids content of the suspension.
Before use, pipette 25 mL of the sterilised yeast suspension into a beaker. Determine the pH using the glass electrode, and determine the volume of 1N sodium hydroxide solution needed to adjust the pH to within the range 6.9 to 7.1.
Immediately before use, add to each bottle of sterilised yeast, a volume of sterile hard water to adjust the concentration of dry yeast to 5.0%, and a pre-determined volume of 1N sodium hydroxide to adjust the pH to within the range 6.9–7.1. Discard the prepared yeast 3 months after preparation.

3.2.3.1.3. Medium for growth of test organisms

To prepare the medium for growth of test organisms:

Prepare a 10% weight per volume (w/v) dextrose solution in distilled water, and sterilise by autoclaving at 121 ± 1ºC for 15 minutes. Cool to room temperature.
Prepare a Wright and Mundy medium, following the author's procedure (Wright & Mundy 1960) or use a commercial product of the same composition (see Supplementary note A), and sterilise by autoclaving at 121 ± 1ºC for 15 minutes. Cool to room temperature.
To each litre of Wright and Mundy medium prepared in step 2, add 10 mL sterile dextrose solution.
Aseptically dispense the mixture in either 10 mL or 15 mL amounts, as preferred. This medium is referred to as Wright and Mundy dextrose medium.

3.2.3.1.4. Recovery medium

To prepare the recovery medium:

Prepare a nutrient broth as follows or use a commercial product of the same composition (see Supplementary note A). Add the following to 970 mL of water and dissolve by heating
- beef extract powder, 10 g
- peptone, 10 g
- sodium chloride, 5 g
Adjust the pH to 8.0–8.4 using 1N sodium hydroxide. Boil for 10 minutes and filter. Cool.
To each litre of nutrient broth solution prepared in step 1–2, add 30 g polysorbate 80 (see Supplementary note B).
Adjust the pH to 7.2–7.4, using 1N sodium hydroxide.
Autoclave at 121 ± 1ºC for 15 minutes and immediately shake well to disperse the polysorbate 80.
Dispense aseptically, in 10 mL amounts, into sterile, capped glass tubes.

3.2.3.2. Test inoculum

3.2.3.2.1. Test organisms

Staphylococcus aureus (ATCC 6538 or NCTC 4163)
Salmonella typhimurium (ATCC 13311)
Pseudomonas aeruginosa (ATCC 15442 or NCTC 6749)
Escherichia coli (NCTC 8196)

3.2.3.2.2. Preparation of inoculum

To prepare the inoculum:

Incubate the contents of an ampoule of freeze-dried culture overnight at 37 ± 1ºC in the Wright and Mundy dextrose medium.
Inoculate the incubated culture onto nutrient agar slopes in McCartney bottles. Store for up to 3 months at 4 ± 1ºC.
At a suitable period before the test is to be conducted, sub-culture from an agar slope into 10 mL or 15 mL quantities of Wright and Mundy dextrose medium. Incubate at 37 ± 1ºC for 24 ± 2 hours.
Sub-culture from the medium in step 3 into fresh medium, using an inoculating loop of 4 mm diameter. Incubate at 37 ± 1ºC for 24 ± 2 hours.
Repeat step 4 daily. For the test procedure use only those cultures that have been sub-cultured at least 5, but not more than 14, times.
Filter the test cultures of P. aeruginosa and S. aureus through sterile Whatmans No. 4 filter paper to remove cell debris and clumps. Use the flow-through as the test culture suspension.
Centrifuge all test cultures until the cells are compacted, and remove the supernatant with a Pasteur pipette.
Re-suspend test organisms in the original volume of liquid (that is, 10 mL or 15 mL of sterile hard water containing 2% w/v killed yeast and 20% v/v sheep serum), and shake for 1 minute with a few sterile glass beads.

3.2.3.2.3. Enumeration of inoculum

Immediately before testing, take an aliquot of the re-suspended inoculum and prepare a series of 10-fold dilutions in a suitable diluent such as quarter-strength Ringer's solution.
Inoculate 1 mL from the dilution 10^–6, 10^–7 and 10^–8 in duplicate, using the pour-plate technique. Retain the tube containing the 10^–7 dilution for use as a control (see Fertility and inactivator efficacy tests).
Incubate the plates overnight and calculate the titre of the suspension. The number subsequently counted must represent not less than 10⁸ or more than 10⁹ colony forming units per millilitre (CFU/mL) or the test is considered invalid.

3.2.3.2.4. Disinfectant dilutions

Dilute a sample of the disinfectant to the specified extent, using sterile hard water as diluent. Use not less than 10 mL or 10 g of sample for the first dilution, and not less than 1 mL of any dilution to prepare subsequent dilutions. Make all dilutions in glass containers on the day of testing. The glass containers must be twice rinsed in glass-distilled water, and sterilised.

3.2.3.2.5. Temperature

Where air-conditioning does not maintain the test solutions at 21 ± 1ºC, hold the containers in which the test is to be carried out in a water bath at this temperature.

3.2.3.2.6. Test procedure

Perform the following test using each of the four test organisms listed under Test organisms above. It is not necessary to test with all organisms simultaneously.

Add 3 mL of diluted disinfectant to a capped glass container.
Start a timing device. Immediately inoculate disinfectant with 1 mL of re-suspension (see Preparation of inoculum, point 8) and mix by swirling.
At 8 minutes, subculture one drop (0.02 ± 0.002 mL) into each of 5 tubes containing recovery broth. To ensure delivery of 0.02 mL into the first tube of recovery broth at exactly 8 minutes, it will be necessary to withdraw a suitable amount from the disinfectant test mix shortly beforehand. This must be immediately preceded by vortexing. Surplus sample must be returned to the test mix. (See Supplementary note B.)
At 10 minutes, inoculate disinfectant with a further 1 mL of culture, and mix by vortexing.
At 18 minutes, proceed as in step 3.
Mix the contents of all tubes of recovery broth by vortexing. Incubate at 37 ± 1ºC for 48 ± 2 hours.
Examine for growth and record results.
For each test organism, repeat all the above steps on each of two subsequent days using a fresh disinfectant dilution and a freshly prepared bacterial suspension.

3.2.3.3. Controls

3.2.3.3.1. Recovery broth contamination

Incubate one un-inoculated tube of recovery broth at 37 ± 1ºC for 48 ± 2 hours and examine for growth.

If growth occurs, the test is considered invalid due to contamination of the recovery broth.

3.2.3.3.2. Disinfectant contamination

To 1 tube of recovery broth, add 0.02 mL of diluted disinfectant. Incubate at 37 ± 1ºC for 48 ± 2 hours.

If growth occurs, the test is considered invalid. Growth in this step, but not in the above step, indicates contamination of the disinfectant test solution.

3.2.3.3.3. Fertility test

To 1 tube of recovery broth, add 1.0 mL of the 10^–7 dilution retained in the enumeration of inoculum step. Incubate at 37 ± 1ºC for 48 ± 2 hours and examine for growth.

If no growth occurs, the test is considered invalid.

3.2.3.3.4. Inactivator efficacy

To 1 tube of recovery broth, add 0.02 mL of diluted disinfectant and 1.0 mL of the 10^–7 dilution retained in the enumeration of inoculum step. Incubate at 37 ± 1ºC for 48 ± 2 hours, and examine for growth.

If no growth occurs, the test is considered invalid. Growth in the fertility test, but not in the inactivator efficacy test, indicates inadequate inactivation of the disinfectant.

3.2.3.3.5. Procedure in case of invalid controls

When any control renders the test invalid, the test is to be repeated. Fresh recovery broth is to be used if growth occurred in the control ‘recovery broth contamination’ or if no growth occurred in the control ‘fertility test’ and ‘inactivator efficacy’.

Should disinfectant contamination be indicated on both occasions, the disinfectant is considered to have failed the test.

Should inadequate inactivation of the disinfectant be indicated by the control ‘inactivator efficacy’ on both occasions, the test is considered invalid (see Supplementary note C).

3.2.3.4. Results

The dilution test passes if there is no apparent growth in at least two of the five recovery broths specified in the test procedure, on all three occasions and using all four organisms.

The test fails if the test for one of the four organisms fails.

3.2.3.5. Supplementary notes

Wright and Mundy medium is commercially available as ‘Bacto Synthetic Broth’, AOAC Code No. 0352 (Difco Limited). The nutrient broth to be used is available as ‘Nutrient Broth – No. 2’ (Oxoid Limited).
The Oxford P-7000 sampler system with disposable plastic tips is recommended for the withdrawal of samples for sub-culturing.
Where inadequate inactivation is indicated, investigations should be conducted to find an effective inactivator.

3.2.3.6. Acknowledgement

This method has been reproduced with the permission of the TGA and includes some modification.

3.3. Section 3: Testing methodologies for assessing virucidal activity

This section elaborates on the testing methodologies for assessing virucidal activity, as described in ASTM E 1053–11 (ASTM International 2011a).

3.3.1. The principle of the ASTM Virucidal Disinfectant Test

The laboratory test method described in ASTM E 1053–11 (ASTM International 2011a) is designed to evaluate the virucidal efficacy of disinfectants on inanimate surfaces. This protocol uses the surface of glass Petri plates to represent environmental surfaces. An alternative protocol, using small carrier disks of stainless steel, is described in ASTM International Designation E 2197–11 (ASTM International 2011b). Both protocols are suitable for use with any of the recommended test viruses described in Table 1.

Briefly, in ASTM E 1053–11 (ASTM International 2011a), a high titre of virus suspension is prepared (10⁷–10⁸ infectious units/mL) and the virus suspension is dried on the test surface. The disinfectant is then applied to the dried film for the recommended time, as indicated on the instructions for use on the label of the disinfectant. The virus-disinfectant mixture is then resuspended and the virus titre is determined in cell culture. The extent of virus inactivation by the antimicrobial agent is determined and is recorded as the log₁₀ reduction in viral titre. The virus-disinfectant mixture is assayed at a dilution just beyond the cytotoxicity range of the disinfectant, as determined by an LD₅₀ method, to assess cytotoxicity. In addition to this virucidal test, the protocol also requires control parameters in the form of cell culture control, virus control, cytotoxicity control and neutralisation control.

3.3.2. Test conditions and methodology

Please refer to ASTM E 1053–11 (ASTM International 2011a), or alternatively E 2197-11 (ASTM International 2011b), for a detailed testing methodology and testing conditions for canine parvovirus, human rotavirus and vaccinia virus.

These ASTM protocols do not specify the requirements for passing the virucidal test. Therefore, this guideline applies the requirements of the United States Environmental Protection Agency (that is, a 10⁴-fold reduction in titre, or a 10³-fold reduction in viral titre beyond any disinfectant dilutions that exhibit cell culture cytotoxicity) for passing the virucidal test.

3.4. Guidelines for disinfectant product labels

3.4.1. Purpose and efficacy claims

There should be no ambiguity regarding each of the intended uses of the product. The proposed label wording should clearly identify the purpose of the product (for example, use in veterinary hospitals, use in dog kennels, use in horse stables) and the scope of the efficacy being claimed, so that the user will clearly understand the product’s intended use. The latter must align with the general categories defined under Disinfectants for veterinary use. The lowest use-dilution recommended on the label must induce a microbiocidal effect at the log reduction of the test organism specified in this guideline.

3.4.2. Product label information

The proposed label wording should specify the instructions for use, including the following:

use pattern
site and method of application
area and type of surfaces (for example, steel, porcelain, aluminium, glass, chrome, vinyl, rubber, plastic, polymeric flooring) on which the veterinary disinfectant may be applied
contact time
temperature
pH
concentration
mixing directions
mode of application and dose rates for the intended use.

The proposed label wording should:

inform the user how long a diluted and/or mixed disinfectant solution may be kept, reused and stored
indicate that the disinfectant should not be used on surfaces that come into contact with food for human consumption
indicate that no animal should come into contact with the disinfectant—animals, feed, water, bedding, litter and fomites should be removed from the area during the treatment
clearly specify the pre-cleaning requirements of surfaces to remove gross contamination, prior to use of the disinfectant product
indicate that the efficacy of the disinfectant against microbes associated with biofilms has not been assessed.

3.4.3. Rinsing and waste disposal

The proposed label wording should provide instructions about when a rinsing step is required to remove the disinfectant from a surface to which it has been applied. Not until after the surface with applied disinfectant product has been thoroughly cleaned with soap or detergent to remove residues and allowed to dry should animals, feed, water, bedding, litter and fomites be reintroduced to animal housing, and the use of feeding and watering appliances recommence.

The proposed label wording should provide a waste disposal statement for those disinfectants intended to be used in large volumes.

3.4.4. Warning statements

The proposed label wording should provide safety instructions for personnel and target animals, including any necessary precautionary measures.

3.4.5. Certain label statements are generally not used

Non-specific label statements such as ‘non-corrosive’, ‘non-toxic’, ‘non-irritant’, ‘safe’, ‘non-caustic’, ‘harmless’ are generally not used for disinfectant products, unless supported by appropriate data.

Table 4: Guidelines in the EN Series (European), AOAC Series (US) and ASTM Series (International) that describe efficacy test methodologies for veterinary disinfectants
Application	Interfering substances	Substrate used for test microorganism	Species of microorganism	Titre of microorganism	Temperature and time	Performance standard	Additional information	Reference
Bactericidal	5% v/v serum option in additional guidance	Surface carrier (stainless steel)	Staphylococcus aureus (ATCC 6538)	1 x 10⁶; use a 48–54 hour liquid culture in broth	20°C, 10 min	Liquid culture of carriers: less than 1 positive out of 60 tested	Controls: – viability controls – verification of positive carriers – neutralisation confirmation – quantitation of test organisms on carrier	AOAC International 2005b
Bactericidal	5% v/v serum option in additional guidance	Surface carrier (stainless steel)	Pseudomonas aeruginosa (ATCC 15442)	1 x 10⁶; use a 48–54 hour liquid culture in broth	20°C, 10 min	Liquid culture of carriers: less than 1 positive out of 60 tested	Controls: – viability controls – verification of positive carriers – neutralisation confirmation – quantitation of test organisms on carrier	AOAC International 2005c
Bactericidal veterinary use	Low-level soiling: 3 g/L bovine albumin High-level soiling: 10 g/L bovine albumin and 10 g/L yeast extract	Suspension	Enterococcus hirae (ATCC 10541) Proteus vulgaris (ATCC 13315) Pseudomonas aeruginosa (ATCC 15442) Staphylococcus aureus (ATCC 6538) Additional organisms possible	1.5 x 10⁸ to 5 x 10⁸; from a liquid culture in broth	10°C, 30 min Additional temperatures and times possible	At least 5 log reduction	Controls: – experimental conditions – neutraliser or filtration – method validation	European Standard (DIN 2000)
Fungicidal veterinary use	Low-level soiling: 3 g/L bovine albumin High-level soiling: 10 g/L bovine albumin and 10 g/L yeast extract	Suspension	Candida albicans (ATCC 10231) Aspergillus niger (ATCC 16404) (fungicidal) Additional organisms possible	1.5 x 10⁷ to 5 x 10⁷; suspension prepared from a culture on solid medium	10°C, 30 min Additional temperatures and times possible	At least 4 log reduction	Controls: – experimental conditions – neutraliser or filtration – method validation	European Standard (DIN 2005)
Sporicidal	Low-level soiling: 3 g/L bovine albumin	Suspension	Bacillus subtilis subsp. spizizenii (ATCC 6633) Additional organisms possible	1.5 x 10⁶ to 5 x 10⁶; suspension prepared from a culture on solid medium	20°C, 60 min Additional temperatures and times possible	At least 3 log reduction	No EN sporicidal test is available for veterinary use. If this protocol (EN 13704) is used, the soiling condition should be adjusted to high-level (10 g/L bovine albumin and 10 g/L yeast extract)	European Standard (2002)
Virucidal	Calf serum, other serum or pancreatic digest of casein (concentration not specified)	Surface (glass Petri plates)	Vaccinia WR strain (ATCC VR-119) rotavirus Wa strain (ATCC VR-2018) Additional viruses possible	10⁷ to 10⁸ TCID50	22°C ± 2°C Additional temperatures possible Variable times	At least 4 log reduction, or 3 log reduction of viral titre beyond any disinfectant dilutions that exhibit cell culture cytotoxicity	Controls: – cell culture control – virus control – cytotoxicity control – neutralisation control – other controls as required	ASTM Standard (2011a)
Virucidal	Low-level soiling: 3 g/L bovine albumin High-level soiling: 10 g/L bovine albumin and 10 g/L yeast extract	Suspension	Bovine enterovirus Type 1 (ATCC VR-248)	10^7.5 TCID50	10°C, 30 min	At least 4 log reduction		European Standard (DIN 2006)
Virucidal, fungicidal, or sporicidal	0.07% (w/v)Tryptone 0.05% (w/v) BSA 0.016% (w/v) Mucin	Surface carrier (stainless steel)	Staphylococcus aureus (ATCC 6538) Pseudomonas aeruginosa (ATCC 15442) Trichophyton mentagrophytes (ATCC 9533) Bacillus subtilis (ATCC 19659) rotavirus Wa strain (ATCC VR-2018) canine parvovirus Cornell strain (ATCC VR-2017) Additional organisms possible	S. aureus 7 x 10⁶, P. aeruginosa 7 x 10⁶ T. mentagro-phytes 7 x 10⁴ B. subtilis 7 x 10⁶	Variable	Variable performance standards apply	Elution from carrier, membrane filtration, incubation of filter on plate and colony count (Note: alternative protocol for viruses) Controls: – carrier load – use of neutraliser optional – additional controls required in virucidal tests	ASTM Standard (2011b)

ATCC = American Type Culture Collection, EN = , g/L = gram per litre, TCID50 = the tissue culture infective dose required to infect 50 per cent of the cell culture inoculated, v/v = volume per volume, w/v = weight per volume

4. References

Antimicrobials Division US EPA—Initial virucidal effectiveness test using feline calicivirus as surrogate for norovirus, available at www2.epa.gov/sites/production/files/2015-09/documents/fcv1_initial_surf_pcol.pdf

AOAC International 2005a, Official methods of analysis, 18th edn, Official Method 955.11, AOAC International, Gaithersburg, MD, United States.

AOAC International 2005b, Official Methods of Analysis, 18th edn, Official Method 955.15, AOAC International, Gaithersburg, MD, United States.

AOAC International 2005c, Official Methods of Analysis, 18th edn, Official Method 964.02, AOAC International, Gaithersburg, MD, United States.

APVMA 2008, Operational Notice—Label claims for efficacy against pests and diseases which are exotic to Australia, Australian Pesticides and Veterinary Medicines Authority.

ASTM International 2011a, ASTM Standard E 1053-11, Standard test method of virucidal agents intended for inanimate environmental surfaces, ASTM International, available at www.techstreet.com/products/1781458.

ASTM International 2011b, ASTM Standard E 2197-11, Standard quantitative disk carrier test method for determining the bactericidal, virucidal, fungicidal, mycobactericidal and sporicidal activities of liquid chemical germicides. ASTM International, available at www.techstreet.com/products/1781464.

ASTM International 2013, ASTM Standard E 1054-08(2013), Standard test methods for evaluation of inactivators of antimicrobial agents. ASTM International, available at www.techstreet.com/products/1857159.

DIN 2000, European Standard EN 1656, Chemical disinfectants and antiseptics—Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in veterinary field—Test method and requirements (phase 2, step 1).

DIN 2002, European Standard EN 13704, Chemical disinfectants—Quantitative suspension test for the evaluation of sporicidal activity of chemical disinfectants used in food, industrial, domestic and institutional areas—Test method and requirements (phase 2, step 1).

DIN 2005, European Standard EN 1657, Chemical disinfectants and antiseptics—Quantitative suspension test for the evaluation of fungicidal or yeasticidal activity of chemical disinfectants and antiseptics used in the veterinary area—Test method and requirements (phase 2, step 1).

DIN 2006, European Standard EN 14675, Chemical disinfectants and antiseptics—Quantitative suspension test for the evaluation of virucidal activity of chemical disinfectants and antiseptics used in the veterinary area—test method and requirements (phase 2, step 1).

Kelsey, JC & Maurer, IM 1974, ‘An improved (1974) Kelsey-Sykes test for disinfectants’, Pharmaceutical Journal (United Kingdom), vol. 213, pp. 528–530.

MacKinnon IH 1974, ‘The use of inactivators in the evaluation of disinfectants’, Journal of Hygiene (London), vol. 73, pp.189–195.

Office of Chemical Safety 2013, FAISD handbook—Handbook of first aid instructions, safety directions, warning statements and general safety precautions for agricultural and veterinary chemicals, Australian Government Department of Health, available at www.health.gov.au/internet/main/publishing.nsf/Content/ocs-faisd-handbook.htm

Therapeutic Goods Administration 2009, Therapeutic Goods Order No. 54 - Standard for Disinfectants and Sterilants, available at www.comlaw.gov.au/Details/F2009C00327

Therapeutic Goods Administration 2013, Standard for the Uniform Scheduling of Medicines and Poisons (SUSMP), available at: www.tga.gov.au/industry/scheduling-poisons-standard.htm.

Wright, ES & Mundy, RA 1960, ‘Defined medium for phenol coefficient tests with Salmonella typhosa and Staphylococcus aureus’, Journal of Bacteriology, vol. 80, pp. 279–280.

Content last updated: 1 July 2014

Content last reviewed: 10 July 2023