The Health and Consumer Protection Directorate General of the European Commission (DG-SANCO) is mandated to “ensure food and consumer goods sold in the European Union (EU) are safe, that the EU’s internal market works for the benefit of consumers and that Europe helps protect and improve its citizens’ health.” In the context of regulatory toxicology, DG-SANCO is responsible for developing frameworks for the safety assessment and regulation of:
Additionally, DG-SANCO is the lead Commission service for coordinating the development of a Community Action Plan on the Protection and Welfare of Animals.
Pesticides are authorized at EU level for all member states according to the provisions of Directive 91/414/EEC, as amended, concerning the placing of plant protection products on the market. This regulation applies to manufactured chemical (e.g., organophosphate), bio-chemical (e.g., pheromones), and biological (e.g., microorganisms) pesticides for agricultural use, as well as to non-pesticidal ingredients of formulated products. Non-agricultural pesticides (also known as “biocides”) are subject to a separate regulatory regime established by DG-Environment.
Animal testing requirements specified in Directive 91/414/EEC call for dozens of human health and ecotoxicological evaluations of each “active ingredient” in a pesticide formulation, including:
In addition, each formulated pesticide product is required to undergo separate acute toxicity testing via the oral, dermal, and inhalation routes, skin and eye irritation, and skin sensitization (known as the acute toxicity “six-pack”) for labeling purposes. Recognized testing methods include the internationally harmonized OECD Test Guidelines, as well as methods published in Annex V to Council Directive 67/548/EEC. In some cases, independent scientific advice may be sought regarding pesticide risk assessments and data needs, in which case the Standing Committee on Health and Environmental Risks (SCHER) and/or the European Food Safety Authority Panel on plant protection products and their residues (EFSA PPR Panel) may be consulted.
It is estimated that upwards of 12,000 animals may be consumed in the toxicological evaluation of each pesticide product on the EU market. Such high costs prompted the creation of a multi-stakeholder technical committee under the auspices of the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) with a mandate to develop integrated testing strategies for pesticide safety assessments that minimize redundancies, use fewer animals, and use resources more wisely. The EFSA PPR Panel has reviewed and endorses the strategies proposed by the HESI technical committee.
Work is underway to revise both the legislative text of Directive 91/414/EEC as well as the testing requirements, which may lead to a reduction in animal testing if certain requirements are eliminated (e.g., mouse cancer bioassay, 1-year dog study, etc.), and an increase in others, as new requirements (e.g., developmental neurotoxicity, endocrine effects, etc.) are established.
Under EU legislation, additives – including colorants, sweeteners, preservatives and emulsifiers – must be explicitly authorized at the EU level before they can be used in foods. The overall legal framework for the regulation of these substances is provided by Directive 89/107/EEC, together with implementing Directives 95/2/EC, 94/35/EC and 94/36/EC. With respect to safety assessments, the framework directive provides that: “Food additives can be approved only provided that…they present no hazard to the health of the consumer at the level of use proposed, so far as can be judged on the scientific evidence available.” Moreover, “To assess the possible harmful effects of a food additive or derivatives thereof, it must be subjected to appropriate toxicological testing and evaluation. The evaluation should also take into account, for example, any cumulative, synergistic or potentiating effect of its use and the phenomenon of human intolerance to substances foreign to the body.”
Guidance prepared by the European Commission’s former Scientific Committee on Food – which has since been replaced by the European Food Safety Authority (EFSA) Food Additives, Flavorings and Food Contact Materials (EFSA AFC) Panel – outlines a battery of “core tests,” including the following:
With respect to food flavorings, the core legislation, Directive 88/388/EEC, provides that, “Member States shall take all measures necessary to ensure that … flavorings do not contain any element or substance in a toxicologically dangerous quantity…” Neither directive specifies toxicological testing requirements. Consequently, data needs are left to the discretion of the EFSA AFC Panel. In the case of smoke flavoring, however, specific testing requirements are imposed pursuant to Regulation (EC) 2065/2003. These include:
Food contact materials are regulated under the framework Regulation No. 1935/2004, which establishes general requirements for all food contact materials, as well as a number of specific directives authorizing the use of individual substances and groups of substances. The framework regulation requires that any materials coming into contact with food must be sufficiently inert to preclude contamination of the food or endangerment of human health. Guidance concerning toxicological testing requirements for food contact materials has been prepared by the EFSA AFC Panel and provides for a tiered approach based on a substance’s potential to be transferred into food. In the worst case, for “high-migration” substances, the core set of required toxicological tests includes the following:
The EU defines supplements as “concentrated sources of nutrients or other substances with a nutritional or physiological effect whose purpose is to supplement the normal diet.” Directive 2002/46/EC relating to food supplements establishes harmonized rules for the labeling of food supplements and introduces rules with respect to vitamins and minerals in food supplements. The directive does not specify toxicological testing requirements for food additives and flavorings; however, the Scientific Committee on the Food Chain and Animal Health committee may be consulted with respect to toxicological findings and data needs for individual substances.
Foods of animal or plant origin may present intrinsic hazards due to microbiological contamination (e.g., Salmonella), for which general EU-wide criteria have been established under Regulation (EC) No 2073/2005. Separate criteria have also been established under Regulation (EC) 2074/2005 and Regulation (EC) No 1664/2006 for testing of oysters, scallops, and other shellfish for toxins that can cause sickness ranging from indigestion to paralysis and death. These regulations require independent member state laboratories to regularly test shellfish beds for the presence of toxins, and establish upper limits on contamination in the sale of live shellfish. The longstanding test for shellfish toxins is known as the “mouse bioassay,” and involves the injection of a shellfish extract into the abdomen of mice and timing how long it takes for them to die. The death of 2/3 or more of the animals within 24 hours is believed to indicate the presence of a toxin.
EU regulations above also permit the use of non-animal methods, once they have been specifically validated for a particular class of toxin. For example, physicochemical techniques such as high performance liquid chromatography have been accepted in some countries (e.g., Germany and New Zealand) as complete replacements for the mouse bioassay for the detection of shellfish toxins, while many other countries have begun to use these techniques in tandem with the mouse test.
Today, chemical chromatography has become the default approach EU-wide for detecting amnesic shellfish toxins (although Regulation (EC) No 2074/2005 also allows use of “any other recognized method,” which could include animal tests). In the case of paralytic shellfish toxins, the Lawrence method of high performance liquid chromatography has been endorsed as scientifically valid by the Association of Official Analytical Chemists, and has recently been taken up under Regulation (EC) No 1664/2006 for the detection of paralytic toxins. However, the regulation continues to require the use of the mouse bioassay if the non-animal results are challenged.
With regard to diarrhetic shellfish toxins, the mouse bioassay continues to be the default method at EU level. Yet in the opinion of the German Federal Institute for Risk Assessment, “the physico-chemical analytical procedures have proved to be superior to the Mouse-Bioassay and to represent the more appropriate methods for safeguarding consumer protection.”
Genetically Modified Organisms
Through modern biotechnology, numerous plants, animals, and microorganisms have been modified genetically to produce characteristics desirable for agriculture (e.g., increased productivity, resistance to inclement conditions, and endogenous pesticide/toxin production). Numerous EU regulations have been established to control the creation, study, release, and transport of these genetically modified organisms (GMOs). Among the most significant from a toxicological testing standpoint are Directives 90/220/EC on the deliberate release into the environment of GMOs, and Regulation (EC) 1829/2003 concerning the marketing of GMOs intended for food or feed and of food or feed products containing, consisting of, or produced from GMOs.
The framework Directive requires the submission of a technical dossier of information including a full health and environmental risk assessment, which in turn should include information on potential “pathogenicity: infectivity, toxigenicity, virulence, allergenicity, carrier (vector) of pathogen, possible vectors, host range including non-target organism,” as well as “toxic or allergenic effects of the non-viable GMOs and/or their metabolic products.” Interpretive guidance developed by the EFSA GMO Panel states that requirements of toxicological testing in the safety assessment of food/feed derived from GMOs “must be considered on a case-by-case basis and will be determined by the outcome of the assessment of the differences identified between the GM product and its conventional counterpart, including available information on intended changes.”
In the case of newly expressed proteins, toxicological assessments generally emphasize short-term effects (e.g., 1-month subacute toxicity studies in rodents, allergenicity), whereas the core set of tests for new constituents other than proteins normally consists of the following:
In some cases, testing of whole GM foods may be required, generally using a modified subchronic (3 month) feeding study in rodents. In addition, where doubt exists regarding wholesomeness and nutritional value of a GM food, separate feeding studies in fast-growing farmed animal species (e.g., broiler chickens or lambs) may be carried out.
Current EU legislation does not contain specific provisions regarding nanomaterials; however, nano-forms of substances still fall within the scope of existing regulatory frameworks (e.g., chemicals, pharmaceuticals and cosmetics), which require that the assessment of potential health and/or ecological hazards. The difficulty, however, is that most existing toxicity testing methods are generally considered to be inadequate for the evaluation of substances in nano-form. Thus, it is anticipated that the EU will need to prepare technical guidance to support the implementation of existing regulatory requirements in relation to nanomaterials.
The following multi-stage risk assessment framework for nanomaterials has been proposed by the Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR):
Although no validated nano-specific test methods of any kind are available at this time, rapid, non-animal in vitro tests are widely regarded as a critical cornerstone in nanosafety testing. For example, the SCENIHR has reported that: “In vitro testing has provided mechanistic data on particle toxicology in general and many in vitro assays demonstrate convincing differences between low and high toxicity particles; it is therefore considered appropriate that in vitro testing is used in situations involving nanoparticles. There is however, a clear need for validated in vitro assays for nanoparticle evaluation, including assays with meaningful endpoints for genotoxicity tests. In vitro tests should address key properties of the nanoparticles such as biopersistence, free radical generation, cellular toxicity, cell activation and other generic endpoints and provide target cell-specific endpoints.”
Alternatives Policies & Actions
Directive 86/609/EEC regarding the protection of animals used for experimental and other scientific purposes stipulates that “[a]n experiment shall not be performed if another scientifically satisfactory method of obtaining the result sought, not entailing the use of an animal, is reasonably and practicably available,” and further, “[i]n a choice between experiments, those which use the minimum number of animals, involve animals with the lowest degree of neurophysiological sensitivity, cause the least pain, suffering, distress or lasting harm and which are most likely to provide satisfactory results shall be selected.” The 3Rs mandate imposed by Directive 86/609 applies to all Commission services, EU member state authorities, and animal users throughout Europe. Accordingly, new and revised Community and member state legislation must conform to the letter and spirit of this directive.
The Research Framework Programmes coordinated by DG-Research provide extramural funding for the development and validation of 3Rs methods. As 3Rs methods are standardized and optimized, they may be eligible for pre-validation and validation through the European Centre for the Validation of Alternative Methods (ECVAM), a division of DG-Joint Research Centre. The results of validation studies may be subject to independent peer review by the ECVAM Scientific Advisory Committee (ESAC) and, if endorsed, may be considered for regulatory acceptance at the EU level and/or internationally.
Given the EU’s centralized processes for the development, validation, and acceptance of 3Rs methods, individual DGs tend to have little direct involvement in these activities, apart from being represented on ESAC. DG-SANCO is an exception to this rule, as the lead Commission service for coordinating the development of a Community Action Plan on the Protection and Welfare of Animals. The current Commission working document includes a section on application of the 3Rs principles for animals used for experiments, stating: “Further work will need to be carried out to reinforce a full implementation of the 3Rs in all areas of animal use, ensure coherence between Directive 86/609/EEC and legislation requiring animal experiments, as well as examining in more detail the mutual acceptance of data and mutual recognition agreements as a means of reducing the numbers of animals used in experiments. The installation of a Community Reference Laboratory for the Validation of Alternative Testing Methods should further enhance the quality of alternative testing methods and speed up the validation process.”