Challenge tests are laboratory tests in which a known number of microorganisms (such as Listeria monocytogenes, Salmonella, Bacillus cereus, yeasts or fungi) are deliberately added to a food product. The product is then stored under realistic storage conditions (such as temperature, packaging and time) to observe whether and how quickly the microorganism grows, survives or dies. They aim to assess the microbiological safety or shelf life of a specific product in its real matrix and packaging. Challenge tests are best known in the context of Listeria control but have numerous other applications and assets.
Challenge tests are not a nice-to-have, but a critical part of food safety management. They provide concrete data about the behavior of microorganisms in your specific products, allowing you to make informed choices about safety, shelf life and quality.
Learn all about Listeria monocytogenes in 2 days at Wageningen Campus: risk management, sampling, sequencing, analysis and challenge testing. You will get practical examples, workshops and interaction with specialists, and conclude with a certificate.
Challenge tests provide insight into the behavior of microorganisms in the specific product. Each food product has a unique composition (pH, water activity, preservatives, etc.). Challenge tests show how a pathogen or spoilage organism actually behaves in that product, under those specific conditions (e.g., during shelf life, refrigerated transport).
Challenge testing provides validation of shelf life and safety. Tests help validate shelf life, especially for refrigerated, ready-to-eat products. They can confirm that a product remains safe throughout its shelf life, even if contamination were to occur. Whereas a classic shelf-life test only tests the status of microorganisms at a given time in a given product. The latter is therefore a sample, whereas a challenge test does provide information on the shelf life of all products within a given product range.
They also assess the effectiveness of barriers and preservation strategies. How effective are salt, acid, MAP packaging or heat treatment? A challenge test specifically shows the effects of these techniques in your products. Furthermore, they can show that a combination of mild preservation techniques (hurdle technology) can work effectively against microorganisms such as Salmonella spp., E.coli, B.cereus, and yeasts and molds.Hurdle technology is a strategy in food safety where multiple barriers (hurdles) are applied simultaneously to inhibit or prevent the growth of microorganisms. Instead of one harsh operation (such as sterilization), you combine mild techniques so that they are effective together without greatly affecting the taste, texture or nutritional value of the product. These techniques by themselves are often insufficient to ensure food safety. As a result, there are also no theoretical models to support safety. A challenge test can therefore provide you with sufficient substantiation in this regard. It thus allows you to balance safety, shelf life and quality.
The tests also provide validation of the management systems within the HACCP (GIRA) plans. The results can help inform decisions such as temperature control or product reformulation. By thoughtfully composing product groups for a challenge test, the results can be used for multiple products. This allows you to directly capture the shelf life of new products and get products to market faster, without having to wait for end-of-life analyses. This is certainly also useful for products with long shelf lives.
Challenge testing gives you information on quality maintenance and spoilage prevention. Challenge tests can also be performed with spoilage organisms, such as yeasts, fungi and lactic acid bacteria. You test the organoleptic shelf life (taste, smell, appearance) and microbiological stability.
Finally, challenge testing can help prevent recalls and brand damage. By identifying risks in advance, companies can prevent recalls and food incidents. This saves costs, protects the consumer and the image of your brand.
Challenge tests provide legal substantiation within the framework of the standards for Listeria monocytogenes in Regulation (EU) 2073/2005 in ready-to-eat meals. Please note that the tests here are the final piece of the puzzle in a fully substantiated Listeria study. Learn more about this in our Listeria white paper.
Challenge tests can also be very useful for validating the behavior of microorganisms other than Listeria monocytogenes.
B.cereus forms spores that can survive heat and can form toxins both before and after consumption. Especially a risk in starchy products, for example, cooked rice or pasta. This is often kept hot or cooled slowly. This can cause outgrowth and toxin formation. Dairy products such as desserts and puddings can become contaminated after heating. Cold storage is crucial here. A challenge test here will tell you if and how quickly the bacteria can outgrow and when toxin production will occur under your storage temperature and duration.
Salmonella spp. is found in raw animal ingredients (meat, eggs, milk) and can multiply at certain temperatures. In chocolate or dry products such as spices, low water activity protects against growth. However, Salmonella can survive for a very long time. The combination of low acidity and moisture pose a risk of outgrowth in refrigerated, ready-to-eat meals with raw vegetable components. A challenge test here helps assess the risks and validate shelf life.
Clostridium botulinum produces life-threatening neurotoxins in anaerobic environments (vacuum, MAP-packed), especially with inadequate preservation. Thus, under mild storage conditions and preservation, it can pose a risk in MAP-packed fish and meat products. Outgrowth is also possible in refrigerated, ready-to-eat meals with low acidity and preservatives. A challenge test can then show whether a combination of pH, aw, packaging and temperature is sufficient to prevent growth and toxin formation.
Escherichia coli (Shiga-toxin-producing, STEC) has a very low infectious dose. This makes it dangerous when consuming raw or only slightly processed products. Raw milk cheeses and cold-smoked meats are examples. There is also a risk of outgrowth in vegetable juices and smoothies because of the relatively neutral pH. Validation of the storage conditions against the specific matrix of the products is therefore essential to ensure the safety of these products.
These are not always pathogenic but cause spoilage and loss of sensory quality. Juices and sauces containing natural sugars are particularly susceptible to fermentation and gas formation. Mould may also occur in bread and pastries, especially in the case of longer shelf life without preservatives. A challenge test can then show which spoilage organisms dominate, how quickly they grow and whether your preservation is adequate.
Although predictive microbiological models (such as ComBase or FSSP) provide useful insights, they are no substitute for challenge testing in practice.A challenge test measures what is really happening with microorganisms in your specific product. Whereas a model is based on standard matrices (lab media or generic foods), which may differ greatly from your recipe. So a challenge test offers realistic results that reflect the real risks.Ingredient interactions, fat content, storage technique, packaging (MAP, vacuum), et cetera influence growth. Models often only consider pH, aw and temperature. A challenge test considers all product and process factors simultaneously.During a challenge test, packaging and environmental conditions are also tested, such as oxygen consumption in MAP-packed products or temperature fluctuations during distribution. These conditions can be exactly simulated during a challenge test.
Challenge tests provide empirical evidence that your preservation measures (e.g., low pH, salt, pasteurization) are effective. Models can only estimate.You get specific data about your unique product, not an estimate based on assumptions. This is especially important for borderline or innovative products that therefore do not fall within the standard matrices about which models provide information. A challenge test also reveals the effects of microbial interactions such as competitive flora present that suppresses pathogens or stimulates growth. Models rarely take natural contaminants or flora into account.Challenge tests also help you inform decisions such as when a shelf life extension of products is needed. Having a challenge test allows you to make quick and safe decisions in such circumstances and prevents food waste and loss of revenue.
So can't you use models at all? Sure. Often a hybrid approach where you use both predictive models and challenge tests is the most efficient and substantiated way to validate food safety and shelf life. Both methods have their strengths and together they form a powerful tool for risk assessment, product development and validation.
Predictive models are used as a quick and low-level initial assessment. They help determine if and when microorganisms can grow in a particular product formulation - for example, will Listeria monocytogenes grow at pH 5.2 and 9°C?
In addition, models are ideal for comparing different formulations or process settings before working in the lab. This way, during product development or changes, you can quickly assess whether your product is safe on paper. Is the answer "yes?" Then you can move on. Is the answer "no?" Then you know that adjustment or further research is needed.
Once a model indicates that the risk seems manageable, challenge testing comes into play. This serves to verify whether the predicted behavior actually occurs in your specific product matrix.
During a challenge test you observe in realistic conditions how fast a microorganism grows, whether toxins occur and whether your product conditions (such as pH, aw, packaging and storage temperature) are really sufficient to inhibit growth. This is also how you meet the requirements of legislation or audits, such as those of the NVWA/FAVV or BRCGS/IFS/FSSC22000.
Predictive models can remain useful even after a challenge test. If the test shows that the behavior of the bacteria is predictable, you can use models to calculate scenarios, such as the effect of temporarily increased temperature during transport.
Using dynamic models (such as ComBase or PMP), you can then simulate what happens with temperature fluctuations or small formulation changes, without having to retest each time.
A hybrid approach allows for smart use of resources. By using models as preselectors, you avoid unnecessary challenge testing in low-risk products. At the same time, you know exactly where you do need to invest in a comprehensive test, for example, when a model offers insufficient certainty or when legislation requires it.
This allows you to make the right choices in an efficient and informed way: models for quick go/no-go decisions and challenge tests where it really counts.
Our experts are happy to think with you and help you quickly on your way.
Do you want to know more about the dangers of listeria and how to prevent infection? Then enroll in our Listeria course: a course from science and practice. Especially pregnant women and people with a reduced immune system are at risk of this food infection from the listeria bacteria.
On November 20 and 21, 2025 Normec Foodcare, together with Wageningen Academy, is again organizing the two-day course Food Legislation. A practical course in which you will learn how European and national regulations around food are structured - from labeling to food safety.
Microbiological food safety remains one of the leading causes of recalls in the European food industry. Despite stricter controls, improved process control, and comprehensive HACCP systems, recent incidents show that microbiological risks still pose a real and evolving challenge.
Recalls remain an important measure of how robust food safety systems actually function. European authorities such as the FASFC in Belgium, the NVWA in the Netherlands and the corresponding regulators in France and Germany record recalls piece by piece, while the RASFF platform aggregates these signals on a European level. This creates a rich picture of where processes are vulnerable and where companies can still make gains in prevention and quality control. If you compare the recent figures from Belgium, the Netherlands, Germany and France, you see clear similarities in risk categories, but also clear differences in transparency, reporting culture and process errors. It is precisely these insights that help QA professionals sharpen their own systems.
