Toxic potency of complex chemical mixtures in diet, environment and products

A limited number of industrial chemicals is tested for their potential toxic properties, while even less is known about the vast number of natural chemicals. Nevertheless, we are mostly exposed to complex mixtures of all of those compounds. Biological effects by mixtures are typically hard to predict from data on the limited number of well-characterized chemicals, since these cover only a part of the compounds actually present. Direct measurement of the total potency of toxic, or bioactive components in the mixture using CALUX® reporter assays solves these issues in the following ways:

  • CALUX assays directly measure the combined effects by the total mixture that is analysed. Results are therefore independent of prior toxicological knowledge on the individual constituents of a mixture.
  • Specificity of the assays and extensive quality controls avoid assay interferences that may typically occur in less well defined and validated assays.
  • If desired, a combination of chemical analytical and biology-based techniques can be used to identify whether observed effects can be attributed to specific compounds or fractions.
  • BDS works together with key stakeholders to incorporate assays in relevant legislation, including the definition of standard operational procedures and thresholds of adversity.

In the next sections several important applications of CALUX methods are described in brief.

Dioxins and PAHs in food and feed

BDS offers analyses that specifically target dioxins, dioxin-like PCBs, non-dioxin-like PCBs, PAHs and PFASs. In addition to the targeted screening for these toxic compounds, CALUX® assays are also used in screenings for potentially adverse biological activities, like endocrine- or genotoxic effects. The presence of dioxins and PCBs in food and feed is subjected to international regulations. The EU has laid down general requirements for the determination of dioxins and dioxin-like PCBs in food and feed using cell-based bioassays. The DR CALUX bioassay (dioxins / furans and dioxin-like-PCBs) fully complies with these stringent requirements and is ISO 17025 accredited (registration number RvA L401). For samples that are suspected based on the DR CALUX results, we offer confirmative analysis by  GC-MS/MS. Analysis of non-dioxin-like PCBs is performed by chemical analysis (GC-MS/MS) only (ISO 17025 accredited; RvA L401).

Packaging & consumer product safety

Our health may not only be affected by chemical contaminants present in our food- or environment, but also by compounds that contaminate consumer products and/or food packaging materials, and may migrate under specific conditions. Examples of consumer product-related chemicals that are under suspicion for their potential health effects are certain plasticizers, PFAS,  brominated dioxins and fire-retardants. CALUX assays can be used to control and monitor consumer safety e.g. in combination with migration studies for monitoring release of bioactive compounds from a packaging material, or for consumer product safety testing, with a focus on endocrine activity, geno- and cytotoxicity related effects.

Water quality

Current water quality monitoring practices mainly rely on targeted chemical analysis of a selected set of compounds (e.g. priority compounds). This compound-oriented approach, however, does not cover the entire load of pollution and may overlook the presence of the vast majority of biologically active chemicals. Effect-oriented testing approaches can overcome this limitation by measuring the effect of the total mixture of compounds that are present in a sample. The CALUX assay panel is particularly suitable for this purpose since it detects several key adverse effects in relation to water quality. Specific trigger values have been defined allowing routine monitoring of water quality and water processing steps.


 BDS offers various tests that specifically target important groups of environmental health-damaging pollutants, such as dioxins, PCBs, PAHs and PFASs. In addition, various assays are available for the screening of environmental samples on effects relating to adverse health effects such as endocrine disruption, genotoxicity and reproductive toxicity. CALUX reporter assays are suitable for a wide range of applications regarding environmental safety assessment. Our assays have been used for identifying pollution hotspots, profiling health hazard related potencies on polluted sites, effect-directed identification of pollutants, ecological distribution of pollutants and the identification of hazards by emerging compounds. CALUX analysis is compatible with a range of environmental sampling strategies. Advantages of using CALUX bioassays in environmental screenings is that the measurement of effects directly relate to human health and very often to that of other higher vertebrates.

Cosmetic formulations

The EU cosmetics regulation prohibits testing of cosmetic formulations on animals. To assure safety of new formulations non-animal tests like CALUX, with their proven robustness and applicability to complex matrices, therefore are of key importance. BDS has been involved in several successful initiatives together with key industries and OECD to validate and demonstrate the applicability of such tests for cosmetics testing OECD approval was obtained for the (anti)estrogens test with ERalpha CALUX (TG-455) and for (anti)androgens with AR CALUX (TG 458), while the (anti)thyroid CALUX assay panel is under OECD review. Focus of in vitro testing of cosmetic formulations is on endocrine disruptors, via EATS testing.

Bio-based & circular economy applications

With shrinking resources of fossil fuels for production of energy and chemicals alternatives are needed, one of which is the use of biomass. The use of biomass provides interesting options to generate a circular economy in which waste is non-existent since it is reused, following a cradle-to-cradle principle. Future biorefineries can be used to optimally use biomass in value chains with various food- and non-food products. BDS designs methods to identify novel high value biobased specialty products, while assuring safety. Safe disposal of end-of-life products and residues, allowing agricultural or other applications is essential to allow closure of the circular process.  In addition, Green Chemistry principles need to be rigorously included already in the design of new chemicals and processes. BDS provides bio-based solutions to identify new bioactivities, and to assure safety and quality control to meet market requirements.

Human health impact assessment

Human health can be affected by exposure to a wide variety of chemicals both of synthetic and natural origin (i.e. the exposome). Only when present at sufficiently high levels this may lead to adverse health effects. Classical targeted approaches measuring a relatively small set of these chemicals are very limited in their scope. By focussing on central processes that govern cellular functioning and homeostasis, CALUX reporter assays can be used to integrate the combined effects of the exposome and relate these to adverse health effects such as endocrine disruption, obesity and metabolic diseases, carcinogenesis and reproductive- and developmental toxicity. The sensitivity and high-throughput compatibility of these assays makes them suitable both for mechanistic studies as well as screenings of large sample panels. In many cases, epidemiological studies and monitoring of human health effects are hampered by the limited amount of available sample material. In addition, many analytical techniques are costly and time-consuming to perform. High-throughput CALUX bioassays provide solutions to all these issues. Another important benefit of using CALUX assays is that they often give mechanistic insight in health effects that may otherwise only be correlated to a certain exposure from a statistical point of view. We are experienced to analyse a wide range of human tissues, such as blood plasma, breast milk, and urine.

Non-animal safety assessment of single chemicals, active ingredients

Chemicals and pharmaceutics are indispensable in modern daily life. Correct safety evaluation of chemicals is essential to protect users. The use of experimental animals in chemical safety evaluation is under pressure, not only because of ethical reasons, but also because these tests are expensive, time consuming, low in throughput and often give unsatisfactory predictions of human toxicity. For these reasons, we have limited toxicological knowledge of many chemicals. Our modern mechanistic CALUX bioassays provide insight in the modes of action of toxicants and can be used as alternative methods to assess chemical safety, particularly when coupled to so-called adverse outcome pathways. These methods are particularly suited for use in integrated testing and read across procedures as described by ECHA and OECD.

Chemicals & pharmaceuticals

Chemical safety assessment is important to assure consumer safety and confidence. Delays in testing and evaluation of industrial chemicals are being addressed using innovative risk assessment approaches that include novel approach methods (NAMs) that are independent of animal experimentation. To enhance confidence and reduce uncertainties integrated testing methods that use all available date, including data on structurally related chemicals using so-called read-across methods are increasingly used.  Mechanistic understanding is important when applying these integrated testing methods. Our specific mechanistic in vitro CALUX bioassays provide precise insight in the modes of action of toxicants and have been proven to be  powerful methods in such NAM-based risk assessment, as exemplified by several case studies, including two OECD case studies on Integrated Approaches for Testing and Assessment (IATA). Major additional applications for chemical-and pharmaceutical safety assessment include screening and bioactivity profiling to select new candidate drugs and chemicals with promising profiles with respect to safety using a safe design/green chemistry approach, profiting from the high throughput of our testing batteries. All tests can be carried out using metabolising systems and methods to assess pharmacokinetics.  In these evaluations additional elements like physico-chemical properties, intended application and exposure scenario, and structural similarities and alerts can also be taken into account in order to further improve predictions. With our extensive knowledge in this area, we can provide independent consultancy, while we also are happy to work with your safety officers and risk assessors.

Cosmetic ingredients

The EU cosmetics regulation prohibits testing of cosmetic ingredients on animals. To assure safety of new ingredients non-animal tests like CALUX therefore are of key importance to assure innovation in the industry. BDS has been involved in several important initiatives together with key industries and OECD to validate and demonstrate the applicability of such tests for cosmetics testing.