Biocide efficacy testing: What does it all mean?

With an increasing number of cleaning products on the market claiming to have a range of biocidal properties, we thought it was about time we explored the topic of efficacy testing and explained what the difference is between all the different standards and testing methodologies.

Testing Arena: Laboratory Testing vs Field Testing

In most countries biocidal products (disinfectants, antimicrobials, etc.) are heavily regulated. This ensures that not only are they safe to use, requiring clear operating instructions and warnings on the bottle, but they also perform effectively for the task they are marketed and sold for. This means that all products in this category must have undertaken some laboratory (lab) based testing to back up and validate any efficacy claims made in relation to the product.

In this article I mainly go into the “ins and outs” of lab testing, but its important to note that although there are some standards that aim to replicate “real world” scenarios, they can never truly replicate the randomness of testing outside of lab conditions. 

The popular way the effectiveness of biocides are tested “in the field” is by using something called an ATP meter. This is a swab test that is done before and after cleaning to measure the amounts of Adenosine Triphosphate (ATP – which is the universal energy carrier in all living things) on a surface. Although calculating the percentage difference between the ATP levels before and after cleaning may seem like a good way of calculating the efficacy of a biocide, this method has some key flaws. 

  1. ATP is only found in living things such as bacteria, but is not found in other pathogens such as viruses which need a living host cell to replicate. 
  2. ATP levels on a surface could be lowered by just using a cloth / friction rather than any biocidal effect of the product. 
  3. This method doesn’t work well for long life coatings like Protect Professional which use a physical kill method rather than a chemical leaching. This is because a high ATP reading maybe because of other living organisms left on the surface such as skin cells.

At InfectProtect we have solved this by using our “Blue Test” to prove that our market leading protection is still present and active on a surface. 

Lab Test Type: Suspension vs Surface Testing

When it comes to lab based testing, there are many test methodologies that can be used to demonstrate the efficacy of a biocide. But usually these are broken down to two main types – Suspension Tests and Surface Tests.

Suspension Tests are when a biocide is directly added to a “suspension” (liquid mix) of the organism to be tested against. Suspension tests are traditionally used for testing disinfectants which need to prove they can kill certain pathogens when the disinfected is sprayed on to it. Because traditional disinfectants only need to work when they are wet, this is seen as a fair methodology to prove their effectiveness as long as the contact time is short enough to be practical (i.e. shorter than the duration of the cleaning process). That said it should also be noted that pathogens like viruses are unlikely to ever be found “in suspension” so it doesn’t fully replicate real world scenarios.

When it comes to long life antimicrobial coatings, such as Protect Professional, the results from Suspension tests should only be considered in relation to its effectiveness when it is being applied to a surface, irrelevant of how long the experiment “contact time” is. This is because once the antimicrobial coating is dry and bonded to a surface it will never be in a liquid form again, meaning suspension test methodology is no longer relevant. This is where Surface Tests come in.

Surface Tests are when a surface (either fabric or hard surface) is pre-coated with the biocide and then has the test organisms applied directly to it. This is the perfect test type for antimicrobial coatings as it more closer replicates a real world scenario. It is important to note that unless the lab has stored the pre-coated surface for a set time prior to the test, no longevity claims can be made (see below the difference between contact time and storage time).

Test Speed: Contact Time vs Storage Time

When it comes to using lab tests to make claims around the speed or longevity of a biocide this is where we see the most misrepresentation misunderstanding of the data by our competitors. This is in part because each test comes with a number of time based parameters;

  1. Contact Time – This is the time the pathogen (or suspension) is in “contact” with the biocide. The shorter the time here, the fastest you can state your biocide starts to kill / achieves its efficacy. Warning: Some companies have tests with 24hr contact times and try to pass this off as showing 24hr protection. Put simply this is a lie and actually shows their product probably doesn’t kill very quickly so would be impractical / useless in most situations.
  2. Incubation Time – Depending on the test methodology and the organism being used, the incubation time usually relates to the time after the “contact time” where the recovered organism is incubated (usually for days) to allow for the calculation of the surviving units.
  3. Storage Time – For “real time” long life tests this is the length of time a treated surface is stored for in ambient temperatures prior to the experiment being undertaken.
  4. Accelerated Ageing Time – This is the time a treated surface is stored at raised temperatures in an attempt to “artificially age” the surface (usually weeks instead of years). At InfectProtect this is something we don’t believe in, as heat doesn’t adversely effect our coating (infact it makes the bond stronger) and it goes against the general scientific assumption that disinfectants are less effective in low temperatures compared to higher temperatures. 

Test Organisms: SARS-CoV-2 vs Human Coronavirus vs Vaccinia Virus

Depending on what types of pathogen the biocide targets, be it bacteria, yeast, mould, viruses or all of the above, commercial labs have a full sample library that can be directly tested against. But just because a bottle of disinfectant may claim it “kills all known viruses”, doesn’t mean its actually been tested against every single known virus in the world. Instead Scientists have already done the heavy lifting and have grouped each pathogen together in a hierarchy based on their physical characteristics (i.e. enveloped viruses) and have selected model virus for each group which accurately represents all the others in the group, while being the hardest to neutralise.

The Vaccinia Virus is one of these model enveloped viruses, meaning a successful test against this would allow the product to confidently be able to claim that it kills all enveloped viruses including coronaviruses such as SARS-CoV-19 (Covid 19).  It’s important to note that a lot of products that were rushed to the market in the wake of the Covid-19 pandemic just wanted test results that they could represent as working against covid-19, because of this they went for tests against feline coronavirus or “human coronaviruses”. Unfortunately because neither of these are model enveloped viruses they can only say for sure that it kills that exact type of virus and nothing further.

To summarise the claims that can be confidently be made;

  • Test against Vaccinia Virus = all known enveloped viruses (inc. SARS-CoV-19). 
  • Test against feline coronavirus = feline coronavirus only (including close mutations)
  • Test against “human coronavirus” = only the specific virus tested (i.e NOT SARS-CoV-19)
  • Test against SARS-CoV-19 = The only way to make a direct “Covid 19” claim (including mutations / variants)

The good thing about this is if the product has been tested against the model pathogens for each group you can be confident the product will effectively naturalise any new pathogen which turns up.

Test Validation: Standardisation & Accreditation

In an attempt to ensure that there is a level playing field for each test, standards organisations such as International Organization for Standardization (ISO) and European Committee for Standardization (EN) define a the method for each test and the criteria for passing. Although ISO & EN produce some of the most globally recognised standards, there are many other regional standards organisations such as The British Standards Institution (BS) who sometimes create competing standards (think the superior UK plug) or have standards which eventually get adopted / duplicated by ISO or EN.

It’s important to note that anybody is able to go online, pay a small fee and download the method for each standard. This is where the slightly complicated and some would say unnecessary world of Accreditation comes in. Accreditation bodies such as United Kingdom Accreditation Service (UKAS) or Perry Johnson Laboratory Accreditation (PJLA) charge commercial labs a fee to come in and audit the lab each year to assess that they were able to correctly follow the downloaded method for each standard. Which sounds great, but the problem is that because they charge a fee for each standard most labs can’t afford to get accredited for each one even though they are all well within their capabilities.

A more pragmatic approach, and one set out in the Biocidal Product Regulations (BPR) is that all the lab tests, irrelevant of standard followed, must be carried out by a professional lab which in itself must follow a range of standards to operate as one.

The Popular Biocide Standards:

EN 14476 - Quantitative suspension test for the evaluation of virucidal activity of disinfectants intended for use in the medical area.

This is a suspension test which must have a contact time of no longer that 60 minutes and be tested against against Poliovirus, Norovirus, Influenza A and adenovirus if an "kills all known viruses" claim is made or Vaccinia Virus for a full enveloped virus (inc. Covid 19) claim is made. Tests against any other organisms will only allow for a claim against that exact virus.

Strengths: This method is able to test both concentrate and diluted concentrations within the same test as it requires three different dilutions.

Weaknesses: As with all suspension tests, this method only shows the efficacy of a biocide when it is in a liquid form and may not translate to efficacy against dried virus films on hard, non-porous surfaces

EN 1276 - Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic and institutional areas.

This is a suspension test which must have a contact time of no longer that 5 minutes and be tested against against four different bacteria - Staphylococcus aureus, Escherichia coli Pseudomonas aeruginosa and Enterococcus hirae for a "kills all known bacteria" claim. Tests against any other organisms will only allow for a claim against that exact bacteria and technically doesn't achieve the standard.

Strengths This method has parameters for replicating both "clean" and "dirty" environments by adjusting the amount of organisms added to the suspension. Please Note: If a test has been carried out in a clean environment then it should only be used after a pre-clean.

Weaknesses: As with all suspension tests, this method only shows the efficacy of a biocide when it is in a liquid form.

ISO 21702 - Measurement of antiviral activity on plastics and other non-porous surfaces

This test involves loading a pre-determined concentration of virus onto a pre-treated test surfaces and a reference surface, which are then left in a humidified chamber at room temperature for up to 24 hours. This test can be used on any virus, but model viruses should be used to make group viral claims.

Strengths Like most surface tests, this method more closely emulates real world scenarios of viruses landing on a surface and has multiple reference and controls to ensure the result is accurate.

Weaknesses: This method involves actually measuring how many host cells are alive at the end of an incubation period (less cells = more virus present). Because of this if the surface is a leaching chemicals it could potentially kill the host cells too making the test look failed when it was actually a success.

PAS 2424 - Quantitative Surface Test for the Evaluation of Residual Antimicrobial (Bactericidal and/or Yeasticidal) Efficacy of Chemical Disinfectants on Hard Non-Porous Surfaces Involving Abrasive Action

This test was developed to test the increasing number of cleaning products which claim to leave a 24hr protection. This surface test also adds in abrasion cycles throughout the test to more closely replicate real world scenarios. To achieve the precise standard a product should only be tested for 24hrs against the same model bacteria as the EN 1276, at InfectProtect we commissioned a lab to follow this methodology for 90 days allowing us to show our products effectiveness over an elongated time period.

Strengths Unlike suspension tests and most other surface tests, this method adds in abrasion cycles to try and replicate real world scenarios within a lab based setting.

Weaknesses: This methodology is only able to be used against bacteria and yeast so can't support a full antimicrobial claim. Its also a reasonably new and less well known as other standards.

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Picture of Michael Howe

Michael Howe

Michael is the MD of InfectProtect. He has over 19 years worth of experience bringing innovations to market and is focused on identifying solutions that will truly have an impact in the "new normal"

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