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The basis for the prediction of toxicity from chemical structure is that the properties of a chemical are implicit in its molecular structure. Biological activity can be expressed as a function of partition and reactivity, that is, for a chemical to be able to express its toxicity, it must be transported from its site of administration to its site of action and then it must bind to or react with its receptor or target. This process may also involve metabolic transformation of the chemical. The application of these principles to the prediction of the toxicity of new or untested chemicals has been achieved in a number of different ways covering a wide range of complexity, from computer systems containing databases of hundreds of chemicals, to simple "reading across" between chemicals with similar chemical/toxicological functionality. The common feature of the approaches described in this article is that their starting point is a mechanistic hypothesis linking chemical structure and/or functionality with the toxicological endpoint of interest. The prediction of toxicity from chemical structure can make a valuable contribution to the reduction of animal usage in the screening out of potentially toxic chemicals at an early stage and in providing data for making positive classifications of toxicity.

The principles of quantitative structure-activity relationships (QSAR) are based on the premise that the properties of a chemical are implicit in its molecular structure. Therefore, if a mechanistic hypothesis can be proposed linking a group of related chemicals with a particular toxic end point, the hypothesis can be used to define relevant parameters to establish a QSAR. Ways in which QSAR and in vitro toxicology can complement each other in development of alternatives to live animal experiments are described and illustrated by examples from acute toxicological end points. Integration of QSAR and in vitro methods is examined in the context of assessing mechanistic competence and improving the design of in vitro assays and the development of prediction models. The nature of biological variability is explored together with its implications for the selection of sets of chemicals for test development, optimization, and validation. Methods are described to support the use of data from in vivo tests that do not meet today's stringent requirements of acceptability. Integration of QSAR and in vitro methods into strategic approaches for the replacement, reduction, and refinement of the use of animals is described with examples.

Virtually all current detergent formulations contain mixtures of surfactants. Our experience and test data on these formulations, which is in agreement with that of many others, has shown that in use the formulations exhibit lower acute irritation potential than predicted by simple summation of the irritation potential of the individual actives. Using the criteria of the Dangerous Preparations Directive (EC Directive 88/379/EEC), many of these formulations classify as irritant in the neat state, with consequent labelling requirements. Such classification is based on addition of irritant components giving a total concentration which exceeds a nominal threshold. In this study, mixtures of surfactants were tested by application to a panel of 31 human volunteers for up to 4 hr, using the technique established for the assessment of acute skin irritation potential. The positive control, sodium dodecyl sulfate (SDS) at 20% concentration, gave an 84% positive response. Dimethyl dodecyl amido betaine (DDAB) at the same concentration gave a 94% response. However, a combination of 20% of each of these surfactants in the same panellists gave a response of only 44%--a significant reduction in the irritation potential. A further test conducted with a mixture of 10% SDS and 10% DDAB in a second panel gave a 31% positive response compared with a 94% positive response to the 20% SDS control in that panel. These results clearly demonstrate that the acute irritation potential of mixed surfactants cannot be predicted by simple summation of the irritation potential of the component substances. Initial results of the mechanistic investigation indicate that the reduced irritation induced by the mixed surfactant systems correlates with a reduced critical micelle concentration (CMC). However, the reduced CMC itself seems not to be responsible for the lowered irritation, since these experiments were conducted at concentrations well above the CMC. It is proposed that the critical event leading to skin irritation is binding to skin protein and that in mixed surfactant systems, the individual surfactants exhibit less affinity for this protein.

For over half a century, guinea pig methods have dominated the field of toxicology concerned with the identification of skin sensitizers. Specific protocols, for example the guinea pig maximization test (GPMT), have been pre-eminent in the identification of skin sensitization hazard for regulatory purposes. However, there are increasingly several forces driving change, not least animal use/welfare considerations. In response to this and to address the need for a rapid screen for chemical allergens, an alternative strategy has been developed. In the first instance, a chemical is assessed by a computer-based expert system. This system is constructed from some 50 rules describing the key chemically reactive substructures of known skin sensitizers. The output from the expert system is also evaluated in the light of the understanding of the skin penetration characteristics of the chemical. In this way, and without use of animals, the likelihood that a chemical represents a skin sensitization hazard is assessed based on the two key characteristics of a skin sensitizer: (1) its direct or indirect ability to react with skin protein (i.e. does it contain a structural alert?); and (2) the ability of the chemical to partition into the appropriate epidermal compartment. When the chemical does possess a structural alert and has the capacity to penetrate skin sufficiently, then it may be regarded as a potential skin sensitizer. Subsequent to this screening phase, if necessary the chemical may be assessed in the murine local lymph node assay. This assay is quicker and cheaper than traditional guinea pig assays and importantly is less stressful to the fewer animals that it requires. The assay is well validated and produces objective results which are equivalent to the GPMT in terms of identifying significant skin sensitization hazard. In this paper, the above strategy is described in more detail, focusing on its relevance to hazard identification and its value in animal welfare terms. It is concluded that the strategy provides an important opportunity for both substantial reduction and refinement of animal use in a manner which will not compromise the existing standard of classification and labelling of skin sensitization hazard in the European Union.

The light-mediated effects of Rose Bengal and 3,3',4',5-tetrachlorosalicylanilide (T4CS) on porcine erythrocyte membranes have been investigated. Irradiation in the presence of Rose Bengal and oxygen causes extensive destruction of the unsaturated fatty acids of the erythrocyte membrane. This results in a decrease in the membrane flexibility as measured by a nitroxide spin probe. Irradiation in the presence of T4CS and oxygen had no measurable effect on the unsaturated fatty acids or on the membrane flexibility. Irradiation of erythrocyte membranes both in the presence of Rose Bengal and oxygen and of T4CS gave rise to polymerisation of the membrane proteins. This was evident by polyacrylamide gel electrophoresis and by freeze-fracture electron microscopy. Aggregation of membrane proteins could be observed with low levels of Rose Bengal and short irradiation times at which no loss of unsaturated fatty acids could be detected. Irradiation of the n-butanol-extracted apoprotein of porcine erythrocyte membranes both in the presence of Rose Bengal and of T4CS resulted in polymerisation of the protein as measured by gel electrophoresis and electron microscopy. The results obtained from the two photodynamic compounds are compared in terms of their different mechanisms of action on the membrane. The implications of the results in determining the molecular events leading to photohaemolysis are discussed.

The detailed molecular structure of the complex formed by the apoprotein from porcine high density lipoprotein and dimyristoly phosphatidylcholine (lecithin) has been investigated by a range of physical techniques. The complex, an oblate ellipsoid with major axis 11.0 nm and minor axis 5.5 nm (see the accompanying paper), is comprised of a section of lecithin bilayer with apoprotein at the surface. The main site of interaction between protein and lipid is in the lipid glycerophosphorylcholine group region; as with native high density lipoprotein the surface of the particle consists of a mosaic of lecithin polar groups and protein. The formation of this mosaic reduces the cooperativity of the lecithin chain motions and changes the curvature of the lipid-water interface, as compared to a bilayer. Otherwise, there are no major changes in lecithin motions indicating that no strong binding of lipid to protein occurs. The interaction involves the intercalation of amphipathic, 60% alpha-helical, apoprotein molecules among the lecithin molecules so that the protein residues at the lipid-water interface. The apoprotein has a high affinity for the lipid-water interface but specific lipid-protein interactions are not involved.

The photochemical reactions of the photoallergen fentichlor with soluble proteins have been studied. [35S]Fentichlor was shown to bind covalently to human serum albumin (HSA) when irradiated with UV light (313 nm). HSA had the ability to bind at least eight molecules of fentichlor per molecule of protein. Fractionation of fentichlor-HSA photoadducts after (a) treatment with cyanogen bromide and (b) reduction, carboxymethylation and digestion with trypsin showed that the bound fentichlor was distributed fairly evenly throughout the sequence of the HSA molecule. Fentichlor was also shown to form photoadducts with human gamma-globulin and with bovine insulin. Its binding to insulin was restricted to the B chain of the molecule. Fundamental differences between the photochemical reactions of the photo-allergens fentichlor and tetrachlorosalicylanilide (T4CS) with soluble proteins are discussed. The reactions of fentichlor with soluble proteins are not restricted to specific binding sites (unlike T4CS). Fentichlor has the potential to react photochemically with a wide range of proteins in the epidermis and dermis, to form antigens.