Toxicological Test Methods

When epidemiological studies form the basis for the risk assessment of a single chemical or even complex mixtures, such as various combustion emissions, it may be stated that in those cases the effects of combined action of chemicals have been incorporated. Examples can, for instance, be found in the updated WHO Air Quality guidelines (WHO 2000). Thus, the guideline value for, e.g., ozone was derived from epidemiological studies of persons exposed to ozone as part of the total mixture of chemicals in polluted ambient air. In addition, the risk estimate for exposure to polycyclic aromatic hydrocarbons was derived from studies on coke-oven workers heavily exposed to benzo[fl]pyrene as a component of a mixture of PAH and possibly many other chemicals at the workplace. Therefore, in some instances the derivation of a tolerable intake for a single compound can be based on studies where the compound was part of a complex chemical mixture. 

However, for most compounds the risk assessment has to be based on results from in vitro and in vivo studies. 

The in vitro and in vivo test methods available to study combined actions and toxicological and biochemical interactions of chemicals in mixtures are essentially the same as those used for the study of single chemicals in order to examine their potential general toxicity and special effects such as mutagenicity, carcinogenicity, and reproductive toxicity. 

One especially successful method of testing complex mixtures is bioassay-directed fractionation followed by chemical identification of active compounds. Until now this method has mainly been used for the testing and identification of genotoxic compounds in environmental mixtures such as extracts of air particulates, exhaust condensates, and cooked foods. In this approach, each fraction is bioassayed untd the major class of specihc chemical(s) responsible for the activity can be isolated and chemically characterized, which make a risk assessment of the mixture possible. 

The advantage of fractionation includes the separation of active constituents from inactive or otherwise toxic components. Disadvantages include the limited amount of sample available for testing following processing, the likelihood of spillover of chemical classes between fractions, and the possible loss or modification of components with fractionation. 

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The major FDA concern came to be better comprehension of diethylene glycol s toxicology. The imminent trial in court required this. In a more basic sense, the crisis made FDA scientists aware of inadequacies in the state of the discipline. In constant contact with their peers at the AMA and at the University of Chicago and Johns Hopkins, a team of FDA scientists launched a project that "developed the first valid process for determining the comparative toxicity of compounds, a statistically based and legally defensible process that opened the door to modern toxicological testing methods" (77). 

The main emphasis is paid to the identification of the basic principles for combined actions and interactions of chemicals (Section 10.2), and to the current knowledge on effects of exposures to mixtures of industrial chemicals, including pesticides and environmental contaminants. Test strategies to assess combined actions and interactions of chemicals in mixtures (Section 10.3) as well as toxicological test methods (Section 10.4) are addressed, approaches used in the assessment of chemical mixtures are presented (Section 10.5), and examples of experimental studies using simple, well-defined mixtures are given (Section 10.6). 

National Standard of the People s Republic of China (1995) Toxicological test methods of pesticides for registration, GB 15670-1995. National Technology Supervision Bureau... 

Adams WJ, Rowland CD. 2003. Aquatic toxicology test methods. In Hoffman DJ, Rattner BA, Burton GA, Cairns J, Jr, editors. Handbook of ecotoxicology. Boca Raton (FL) Lewis Publishers, p 19-43. 

OECD (1996). Final Report of the OECD Workshop on Harmonization of Validation and Acceptance Criteria for Alternative Toxicological Test Methods. Document ENV/MC/TG(96)9 (http //www. oecd. org/ehs/test/background. htm). 

Other inhalation toxicology test methods that are less invasive or potentially harmful include ... 

Final Report of the OECD Workshop on Harmonization of Validation and Acceptance Criteria for Alternative Toxicological Test Methods (2002). 

Several of the validated in vitro methods aim to be validated as full replacement methods to offer an alternative for in vivo test guideline method(s). However, for integrated testing strategies, validated mechanistic tools targeting key events in adverse outcome pathways are necessary to generate state-of-the-art alternative approaches to conventional toxicological test methods based on laboratory animals. 

Endocrine and immunologic related adverse effects of pesticides are another example of an area where we need toxicologic testing methods that will detect both acute and chronic toxicity and provide a reliable basis for predicting human effects. Before we add new protocols to the current methodology, however, we need to evaluate the answers provided by our current methodology. 

Hartung et al., 2013). However, they emphasize validating in vitro systems to ensure reliability and suitability for humans thus, the data can be invaluable and effective. Schechtman (2002) reviewed the implementation of the 3Rs (refinement, reduction, and replacement) validation and regulatory acceptance considerations for alternative toxicological test methods and suggested pros and cons of in vitro testing. 

Current toxicological test methods address the need to evaluate chemicals for both acute and chronic exposure with respect to a variety of effects on humans and organisms in the environment. Laboratory tests begin with acute tests. Chronic tests are performed in cases where high exposure or releases take place and/or where there is an indication from acute studies, chemical structure, or chemical and physicochemical properties indicating that further study is needed. 

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