Recommendations on dose level selection for repeat dose toxicity studies.

Prior to registering and marketing any new pharmaceutical, (agro)chemical or food ingredient product manufacturers must, by law, generate data to ensure human safety. Safety testing requirements vary depending on sector, but generally repeat-dose testing in animals form the basis for human health risk assessments. Dose level selection is an important consideration when designing such studies, to ensure that exposure levels that lead to relevant hazards are identified. Advice on dose level selection is provided in test guidelines and allied guidance documents, but it is not well harmonised, particularly for selection of the highest dose tested. This paper further builds on concepts developed in a technical report by the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) which recommends pragmatic approaches to dose selection considering regulatory requirements, animal welfare and state of the art scientific approaches. Industry sectors have differing degrees of freedom to operate regarding dose level selection, depending on the purpose of the studies and the regulatory requirements/legislation, and this is reflected in the overall recommended approaches. An understanding of systemic exposure should be utilised where possible (e.g., through toxicokinetic approaches) and used together with apical endpoints from existing toxicity studies to guide more appropriate dose level selection. The highest dose should be limited to a reasonable level, causing minimal but evident toxicity to the test animals without significantly compromising their well-being. As the science of predictive human exposure further develops and matures, this will provide exciting and novel opportunities for more human-relevant approaches to dose level selection.

An in vitro approach for comparative interspecies metabolism of agrochemicals.

The metabolism and elimination of a xenobiotic has a direct bearing on its potential to cause toxicity in an organism. The confidence with which data from safety studies can be extrapolated to humans depends, among other factors, upon knowing whether humans are systemically exposed to the same chemical entities (i.e. a parent compound and its metabolites) as the laboratory animals used to study toxicity. Ideally, to understand a metabolite in terms of safety, both the chemical structure and the systemic exposure would need to be determined. However, as systemic exposure data (i.e. blood concentration/time data of test material or metabolites) in humans will not be available for agrochemicals, an in vitro approach must be taken. This paper outlines an in vitro experimental approach for evaluating interspecies metabolic comparisons between humans and animal species used in safety studies. The aim is to ensure, where possible, that all potential human metabolites are also present in the species used in the safety studies. If a metabolite is only observed in human in vitro samples and is not present in a metabolic pathway defined in the toxicological species already, the toxicological relevance of this metabolite must be evaluated.

Investigation into feed preparation for regulatory fish metabolism studies.

BACKGROUND: Test diets used in fish metabolism studies for regulatory purposes must be homogenously fortified with the radiolabelled test substance and stable with respect to leaching. Standard fish food, as used in commercial fish farming, should also be used in fish metabolism studies. Therefore, suitable spiking and coating procedures are required to ensure the correct dosing of the fish during the experiment. RESULTS: Methods for the homogeneous, safe and efficient application of radiolabelled test items to the surface of commercial feeding pellets were developed. Leaching studies showed that test items of low lipophilicity applied to feeding pellets need to be stabilised, to reduce the risk of significant losses prior to ingestion by the fish. Coating of solvent-spiked pellets with calcium alginate was shown to reduce leaching losses of water-soluble test items significantly. Alternatively, commercial feeding pellets can be coated with alginate or vegetable oil fortified with a radiolabelled test item also leading to sufficiently stabilised test diets. Experimental conditions, such as the water temperature, may have a significant effect on the leaching behaviour of the test items. CONCLUSIONS: A detailed description of different spiking and coating procedures for the preparation of experimental diets suitable for fish metabolism studies is provided.

Development of a regulatory testing procedure to study the metabolism of pesticides in farmed fish.

BACKGROUND: Diets used in commercial fish farming use significant proportions of crop-derived commodities, and it is important to understand the potential for transfer of any pesticide residues on the crop into edible tissues in fish. It is a current requirement in the EU that fish metabolism studies must be performed when a pesticide is used in crops where commodities or processed fractions are fed to farmed fish. Fish metabolism studies in both rainbow trout and common carp have been carried out, following the new working document on the nature of pesticide residues in fish using (14) C-labelled pesticide. RESULTS: The ingestion of experimental diets by rainbow trout and common carp resulted in the uptake and metabolism of the test item, as shown by liquid scintillation counting combined with radio-thin-layer chromatography. The metabolite profiles for trout and carp were qualitatively similar regarding the main residue. However, species-specific differences were found regarding the remaining residue with rainbow trout showing additional metabolites in comparison to carp. CONCLUSIONS: Metabolism studies for regulatory purposes can be carried out with both fish species under laboratory conditions. The experimental design reported is suitable for quantifying the transfer of residues to edible tissues and enables characterisation of the chemical nature of residues.