EN 14675 specifies a quantitative suspension test for evaluating the virucidal activity of chemical antiseptics and disinfectants used in the veterinary area. It is a phase 2, step 1 test, designed to assess product efficacy under controlled conditions that simulate certain aspects of practical use.
Unlike a basic screening test, EN 14675 incorporates variables such as temperature, contact time, organic load and product concentration. These conditions make it possible to evaluate how a disinfectant performs when exposed to factors that may reduce its efficacy.
The method uses Bovine enterovirus Type 1 (ECBO) as the reference virus and establishes a minimum reduction in viral titre of 4 log₁₀ as the main efficacy criterion.
For laboratories performing biocidal efficacy testing, correctly understanding the method is important not only to obtain a compliant result. It is also essential for ensuring test reproducibility and demonstrating technical competence through interlaboratory comparisons.
What Is EN 14675?
EN 14675:2015 is the European standard specifying a quantitative suspension test for evaluating the virucidal activity of chemical antiseptics and disinfectants used in the veterinary area.
It is a phase 2, step 1 test, which means that virucidal activity is evaluated in suspension under conditions intended to reproduce certain aspects of the product’s practical use.
At this stage, the method already incorporates variables that can affect disinfectant efficacy, including:
- Product concentration.
- Contact time.
- Interfering substances.
- The test organism.
- Product preparation and dilution conditions.
This differentiates EN 14675 from phase 1 tests, which are more basic and are not linked to a specific area of application.
For a broader explanation of the different test phases, see our article on procedures for claiming activity in biocide efficacy testing.
Which Products Does EN 14675 Apply To?
EN 14675 applies to chemical antiseptics and disinfectants intended for use in the veterinary area.
This includes products used in areas such as:
- Animal breeding.
- Livestock production.
- Animal husbandry.
- Veterinary facilities.
- Animal accommodation.
- Disposal and management of animals.
The standard does not apply once animals have been slaughtered and have entered the food processing chain.
It is therefore important not to confuse EN 14675 with standards used to evaluate virucidal activity in medical, food or industrial applications.
The appropriate standard must always correspond to the intended area of use and the activity claim that needs to be supported.
For a wider overview of European efficacy standards, you can also consult our guide to the effectiveness evaluation of antiseptics and disinfectants.
EN 14675 Is A Phase 2, Step 1 Test
Its classification as a phase 2, step 1 test is important for correctly interpreting what the method demonstrates.
At this stage, the product comes into contact with the virus in a liquid suspension under defined experimental conditions.
This allows variables such as the following to be precisely controlled:
virus + interfering substance + product concentration + temperature + contact time
The objective is to determine whether the disinfectant maintains sufficient virucidal activity under these conditions.
However, the method does not directly reproduce a contaminated surface, piece of equipment or veterinary facility.
Phase 2, step 2 tests subsequently introduce conditions that are even closer to particular practical applications.
Therefore, passing EN 14675 demonstrates virucidal activity under the defined suspension test conditions, but the result must always be interpreted within the specific scope of the method.
Which Virus Is Used In EN 14675?
The mandatory test organism is:
Bovine enterovirus Type 1 (ECBO), ATCC VR-248.
The standard selects this virus as a model organism because of its resistance and relevance to veterinary applications.
ECBO belongs to the picornavirus group, which includes viruses characterised by considerable resistance to certain physical and chemical influences.
Among other characteristics, these viruses may show high resistance to many chemical products and do not rely on a lipid envelope that can be readily disrupted by certain disinfectants.
For this reason, Bovine enterovirus Type 1 provides a demanding challenge when evaluating the virucidal activity of a product.
A model virus is used because testing every potentially relevant virus against every disinfectant formulation would not be practically or economically feasible.
Mandatory EN 14675 Test Conditions
EN 14675 establishes mandatory experimental conditions to allow results to be obtained within a common standardised framework.
The reference conditions include:
| Parameter | Condition |
| Test Virus | Bovine enterovirus Type 1 (ECBO) |
| Temperature | 10 °C |
| Contact Time | 30 minutes |
| Low-Level Soiling Condition | 3.0 g/l bovine albumin |
| High-Level Soiling Condition | 10 g/l bovine albumin + 10 g/l yeast extract |
| Required Virucidal Reduction | ≥ 4 log₁₀ |
The interfering substance should be selected according to the practical conditions in which the product is intended to be used.
This is important because organic matter can significantly affect disinfectant performance.
A product that is effective under very clean conditions may behave differently when proteins or other organic materials interfere with the activity of its active substances.
Additional Conditions: Temperature And Contact Time
The standard allows additional conditions to be used when other practical applications need to be represented.
In addition to the mandatory 10 °C, additional test temperatures can include:
- 4 °C
- 20 °C
- 40 °C
Additional contact times may also include:
- 1 minute
- 5 minutes
- 60 minutes
These conditions should be selected according to the practical use that the test result is intended to support.
For example, a product intended for rapid disinfection should not base its instructions for use solely on a result obtained using a contact time that cannot realistically be respected during normal application.
For this reason, virucidal efficacy should always be interpreted together with the concentration, temperature, contact time and organic load used during testing.
Product Concentrations: Why Are Several Tested?
EN 14675 requires product test solutions to be prepared at at least three different concentrations.
The test should include:
- A concentration within the active range.
- A concentration within the non-active range.
- One or more additional concentrations allowing the product response to be appropriately characterised.
There is also an important technical point.
During the test, the product is mixed with the viral suspension and the interfering substance, resulting in dilution.
Consequently, the highest final product concentration that can be tested is 80%, even when the product is initially used as supplied.
For concentrated products, test dilutions are normally prepared using hard water.
For ready-to-use products, water is used according to the conditions established by the method.
How Is The EN 14675 Test Performed?
The basic principle consists of exposing a viral suspension to the disinfectant under controlled conditions.
In simplified terms, the procedure involves the following stages.
1. Preparation Of The Viral Suspension
A suspension of Bovine enterovirus Type 1 (ECBO) is prepared with a sufficiently high viral titre to allow a reduction of at least 4 log₁₀ to be demonstrated.
The virus is multiplied using an appropriate cell line.
The standard describes the use of MDBK cells, although other suitable cell lines may be used if equivalence can be demonstrated.
2. Addition Of The Interfering Substance
The viral suspension is combined with the appropriate interfering substance.
This step reproduces the presence of organic matter that may occur during practical use of the disinfectant.
Depending on the intended application, the low-level or high-level soiling conditions specified in the standard are used.
3. Addition Of The Disinfectant Product
The disinfectant product is then added to the mixture.
The combination is maintained for the selected contact time at the specified temperature.
Under the mandatory test conditions, the exposure is performed at 10 °C for 30 minutes.
4. Suppression Of Product Activity
At the end of the selected contact time, disinfectant activity must be stopped immediately.
In the procedure described in EN 14675, this can be achieved by rapid dilution in cold culture medium, followed by the corresponding serial dilutions.
When dilution alone cannot adequately suppress the residual activity of the disinfectant, other detoxification techniques may be required.
This stage is critical.
If the disinfectant were allowed to continue acting after the official contact time had ended, the measured viral reduction could be overestimated.
5. Determination Of Residual Viral Infectivity
The amount of infectious virus remaining after treatment is then determined.
EN 14675 includes cell-culture-based methods such as:
- TCID₅₀ determination.
- Quantal tests based on observation of the cytopathic effect.
- Plaque assay.
The viral titre after treatment is compared with the corresponding control to calculate the logarithmic reduction produced by the disinfectant.
TCID₅₀, Cytopathic Effect And Plaque Assay
Virucidal activity testing differs considerably from conventional bactericidal testing.
Viruses do not form colonies on microbiological culture media in the same way as bacteria.
Their infectivity must therefore be detected using cell cultures that are susceptible to the virus.
One of the parameters used in EN 14675 is TCID₅₀ — Tissue Culture Infectious Dose 50% —, which represents the viral dose capable of producing a cytopathic effect in 50% of inoculated cell cultures.
The standard provides for calculation using the Spearman-Kärber method.
Another option is the plaque assay, in which viral infection produces visible areas of lysis within a cell monolayer.
In both cases, the objective is the same:
to determine the amount of infectious virus before and after exposure to the disinfectant.
The Key Criterion: A Reduction Of At Least 4 Log
To demonstrate virucidal activity according to EN 14675, the product must achieve a reduction in viral titre of at least:
4 log₁₀
This corresponds to reducing the amount of infectious virus by a factor of at least 10,000 compared with the relevant control.
However, mathematically obtaining this value is not sufficient on its own.
The result can only be interpreted when the validation criteria established by the method are also satisfied.
Controls Are As Important As The Final Result
An EN 14675 test must demonstrate that the experimental system is functioning correctly and that the observed result is genuinely due to the virucidal activity of the product.
The aspects that must be controlled include:
- The initial viral suspension must make it possible to demonstrate a 4 log₁₀ reduction.
- The reference system must behave as expected.
- Product cytotoxicity must not prevent detection of residual virus.
- Treatment of the cells must not significantly reduce their susceptibility to viral infection.
- Disinfectant activity must be adequately suppressed at the end of the contact time.
- At least one tested concentration must achieve a reduction equal to or greater than 4 log₁₀.
- At least one concentration must also demonstrate a reduction below 4 log₁₀.
These controls are essential because an apparently excellent result might not be valid if, for example, the disinfectant damages the cells used to detect residual virus.
In such a case, the absence of a cytopathic effect could be incorrectly interpreted as an absence of infectious virus when the detection system itself has actually been compromised.
Cytotoxicity: One Of The Main Challenges Of The Test
Cytotoxicity is one of the most important technical aspects of virucidal activity testing.
A disinfectant can affect not only the virus but also the cells subsequently used to determine viral infectivity.
If residual product concentrations damage the cells, it may become impossible to determine accurately whether viable virus is still present.
EN 14675 therefore establishes specific controls to confirm that cytotoxicity does not compromise demonstration of a 4 log₁₀ reduction.
When dilution alone cannot sufficiently reduce product toxicity, the standard describes additional detoxification approaches, including molecular sieving or ultrafiltration techniques.
This illustrates why virucidal activity testing requires particularly careful methodological control.
EN 14675 And Laboratory Competence
Correct application of EN 14675 requires control of numerous variables:
- Virus preparation and storage.
- Cell culture.
- Viral suspension titre.
- Product concentrations.
- Preparation of interfering substances.
- Contact time.
- Suppression of disinfectant activity.
- Viral titration.
- Calculation of logarithmic reduction.
Each of these stages can introduce variability.
As a result, two laboratories apparently applying the same standard may obtain different results if deviations occur at any of these points.
Proficiency Testing provides a way to evaluate laboratory competence through interlaboratory comparison.
More information about the role of these schemes is available on the SHAPYPRO Proficiency Testing Programs page.
Proficiency Testing For EN 14675
In a PT scheme based on EN 14675, participating laboratories receive a test item prepared under defined conditions and perform the evaluation using the corresponding method.
The results are subsequently compared to provide information on each participant’s performance.
This type of exercise can help identify:
- Systematic deviations.
- Problems related to viral titration.
- Differences in test preparation.
- Interpretation issues.
- Errors in reporting results.
- Areas where internal procedures may need improvement.
Participation in interlaboratory comparisons provides laboratories with independent evidence about their performance when applying a technically demanding method.
The SHAPYPRO EN 14675 Program
SHAPYPRO organises Proficiency Testing programs for the evaluation of virucidal activity according to EN 14675, aimed at laboratories performing efficacy testing of chemical antiseptics and disinfectants.
The program is registered in the international EPTIS database as the SHAPYPRO EN 14675:2015 scheme.
The property evaluated is the virucidal activity of chemical antiseptics and disinfectants used in the veterinary area, using Bovine enterovirus Type 1 as the reference organism.
Participation can provide laboratories with an external evaluation of their performance and useful information for:
- Monitoring method performance.
- Detecting trends or deviations.
- Investigating unsatisfactory results.
- Demonstrating technical competence.
- Supporting accreditation and quality-system requirements.
Laboratories interested in taking part can check the upcoming SHAPYPRO Proficiency Testing Programs to review available rounds and registration dates.
Another relevant veterinary efficacy method is EN 1656, which evaluates bactericidal activity rather than virucidal activity. You can read more in our article on EN 1656 and bactericidal activity in veterinary disinfectants.
EN 14675 Is More Than Obtaining A Result ≥ 4 Log
One of the main mistakes when interpreting a virucidal test is reducing the entire method to a single number.
EN 14675 is not simply about checking whether the product reaches 4 log₁₀.
For that reduction to be meaningful, the laboratory must demonstrate that:
- The viral system was suitable.
- The test had sufficient sensitivity.
- Cytotoxicity was controlled.
- Residual disinfectant activity was correctly suppressed.
- The controls complied with the established criteria.
- The tested conditions correspond to the intended application.
The logarithmic reduction is the final result of a much broader experimental process.
For this reason, laboratory technical competence plays a fundamental role in the reliability of the conclusion.
Conclusion
EN 14675 establishes a phase 2, step 1 method for evaluating the virucidal activity of chemical antiseptics and disinfectants used in the veterinary area.
The test uses Bovine enterovirus Type 1 (ECBO) as the reference virus and combines controlled conditions of temperature, contact time, concentration and organic load to determine the reduction in viral titre achieved by the product.
To demonstrate virucidal activity, a reduction of at least 4 log₁₀ must be achieved, but the result is valid only when the corresponding controls for cytotoxicity, infectivity, reference inactivation and suppression of product activity satisfy the established criteria.
Correctly applying EN 14675 therefore involves much more than following a sequence of laboratory steps.
It requires control of a complex experimental system in which cell culture, viral preparation, test conditions, controls and result calculation must all work consistently together.
In this context, SHAPYPRO Proficiency Testing programs provide laboratories with a tool for comparing their performance, identifying possible deviations and strengthening confidence in the correct application of the method.