A Listeria challenge test: how does it work?

The mandatory study required under NVWA Information Sheet 85 or FASFC circular PCCB/S3/1763165 on the growth of Listeria monocytogenes may indicate that your product requires a challenge test. This test determines the risk of Listeria monocytogenes outgrowing in a ready-to-eat food product. But how does a challenge test actually work? Our specialist Sylvia Mol provides a look behind the scenes.

A challenge test is the cornerstone of a risk analysis: it validates the shelf life of a food product under specified storage conditions. Through artificial inoculation, you gain insight into the behavior of Listeria monocytogenes in your product—such as its growth, survival, or decline. The growth potential must not exceed 2 log CFU/g to be considered controllable. The dividing line between categories 1.2 and 1.3 is set at 0.5 log CFU/g. However, this is neither a simple test nor a routine task; every challenge test is custom-made.

Essential data required first

“That is why we need a substantial amount of information from a food manufacturer before getting started,” explains Sylvia Mol, Microbiology Project Leader at Eurofins Food Safety Solutions. “What is the target shelf life? Is the product MAP-packaged? What does the production process look like, and have any Listeria-inhibiting compounds been used? What is the intended target audience and market? What are the $a_w$ (water activity) and pH values? All of this is critical data that shapes the setup of the challenge test. The challenge test is only valid for that specific recipe. If the recipe changes, the study becomes invalid and a new one must be initiated. With this information, we can determine during preparation which cultures to grow and which temperature profile to follow.”

The journey to and inside the laboratory

Within 48 hours of production, product samples must be set up at the predetermined temperature. “We often handle transport as well, unburdening our clients,” says Mol. “Upon arrival at the lab, we immediately prepare multiple samples. We inoculate several with Listeria monocytogenes and several with an equal volume of sterile liquid to serve as process controls.”

“Do we inoculate the exterior, or could a gap form in the crust requiring us to inoculate the interior as well? We think this through carefully together with the client.”

How do we inoculate an empanada?

Inoculation is less straightforward than it might seem. Mol: “Every product is different and requires a tailored inoculation strategy. Take a pre-baked empanada that the consumer only needs to reheat: it has a sealed crust. Recontamination cannot occur on the inside. However, inoculating the interior produces a completely different growth profile compared to inoculating the exterior, where Listeria grows less readily. Do we inoculate the surface, or could a fissure appear in the crust requiring us to inoculate the inside too? We evaluate these scenarios carefully together with the client.”

Battered fish pieces: exterior and interior?

The same principle applies to products like kibbeling (battered deep-fried fish pieces). “Battered fish has a crust, but not all pieces remain intact. Therefore, we also include the interior during inoculation. Sometimes our precision comes down to the smallest details. Take a pack of sliced deli meats: consumers rarely finish it in one sitting. You open the pack and place it back into the refrigerator. For sliced meats, we therefore also assess: what happens 4 days after opening? And what happens at the end of shelf life? What is the worst-case scenario? That is what we must identify. The production process also plays a role. Does a fried product contact a conveyor belt on its underside? Then that is precisely where we must inoculate the product.”

Temperature-controlled study

Once everything is inoculated, we initiate a predetermined temperature profile. The storage temperature during a challenge test varies according to the shelf-life stage being simulated.

The 3 shelf-life stages are:

• Manufacturer stage: Storage at the production facility and during transport.
  In the Netherlands, Belgium, and Europe, a temperature of 7 °C applies.
• Retail stage: Display and storage in the store.
  In the Netherlands, Belgium, and Europe, a temperature of 7 °C applies.
• Consumer stage: Storage in the consumer's home refrigerator.

Legally, the standard is 7 °C; however, in the consumer stage of a challenge test, we account for potentially elevated domestic storage temperatures. In the Netherlands and Belgium, this is set at 9 °C; across Europe, it is set at 10 °C.

“If you have conducted a challenge test using 9 °C for the consumer stage, the test will not be valid for the wider European market.”

Targeting the European market? Choose 10 °C

“If you plan to distribute your product across the European market beyond the Netherlands or Belgium in the future, opt for 10 °C in the consumer stage. In the Netherlands and Belgium, average domestic refrigerator temperatures are well documented as being sufficiently cold. In other EU member states, this is not always the case. Once you carry out a challenge test at 9 °C in the consumer stage, it will not be valid for the broader European market. Keep in mind that Regulation (EC) No 2073/2005 will change as of July 2026. The regulations currently in place in the Netherlands and Belgium will then apply across all of Europe,” Mol advises.

Storage duration and temperature

The duration of each stage during the challenge test depends on the availability of storage time data as well as total shelf life.

Table 1: Temperature allocation across the 3 supply chain stages

“You may deviate from these parameters with proper scientific justification. However, the assigned temperatures for the retail and consumer stages are non-negotiable. I am currently running a challenge test where the producer stated the product is stored at their facility for 4 days. The total shelf life is 18 days, leaving 14 days split between retail and consumer stages. If the producer cannot specify average storage time, we default to one-third of the total shelf life: for an 18-day shelf life, that translates to 6 days at the manufacturer and 12 days across retail and consumer stages,” Mol notes.

“The level of Listeria monocytogenes in a product can even decline, ending up lower at the end of shelf life than at interim test points.”

Mapping the Listeria growth curve

Throughout the challenge test, we evaluate the growth potential of Listeria monocytogenes. This represents the difference between the baseline inoculation level—simulating the post-production stage—and the highest recorded growth level. Listeria monocytogenes often does not grow linearly. In fact, levels can decrease over time, ending up lower at the end of the shelf life than at mid-points.

“That is why we test across 5 distinct time points to capture peak growth accurately. We sample at baseline, at 3 interim intervals, and at the end of shelf life. At baseline, we analyze 3 separate samples to determine inoculation dispersion, in line with NVWA Information Sheet 85. We strategically select the 3 intermediate time points because I want to capture data right in the middle of that growth curve!”

“Results for some analyses are available within 48 hours, whereas yeasts and molds take up to 5 days.”

Types of analyses

This involves an intensive testing schedule. “Analyses must take place at strict, predefined time slots. In addition, a challenge test encompasses several tests, each with its own turnaround time.”

Analyses performed during a challenge test include:

• Listeria monocytogenes enumeration (count)
• Limit evaluation (presence/absence in 25 g)
• Background microflora (including total viable count, Enterobacteriaceae, yeasts and molds, or lactic acid bacteria)
  pH, a_w (water activity), moisture content, salt, and organic acids

“Some results are available within 48 hours, whereas yeasts and molds take 5 days. For products in MAP (Modified Atmosphere Packaging) that consumers may not use up at once, we include extra test units to measure growth potential after breaking MAP conditions. This accurately mimics the opening of the pack during the consumer stage,” says Mol.

“Our client decided to shorten the shelf life based on the same challenge test. They didn't have to restart the study, saving costs.”

Collaborative solutions: adjusting shelf life

“We maintain close communication with our clients throughout testing. Last week, for example, a producer wanted a 14-day shelf life, but Listeria growth began rising significantly around day 10. The client decided to shorten the shelf life using the data already collected in that same challenge test. This avoided having to restart the study from scratch and saved costs. We monitor the entire process, consult at every stage, and make adjustments where possible. That proactive collaboration is our core strength.”

“In such cases, I am candid and explain that a challenge test isn't strictly necessary.”

Not necessary? No test required

Helping clients optimize costs goes both ways. If a challenge test is not strictly required, Mol and her team will explicitly say so. “Under NVWA or FASFC guidelines, you are required to determine which category (1.2 or 1.3) your ready-to-eat product falls into, whether it supports the growth of Listeria monocytogenes, and which supporting studies are necessary. Challenge tests or predictive modeling can be part of this, but they are not always required. Recently, I received an inquiry for a challenge test on a product with a sufficiently low $a_w$ value and favorable pH. I was upfront with the client that a challenge test wasn't strictly necessary. We actively think along and work collaboratively with our clients.”