Subacute toxicity of a mixture of nine chemicals in rats: detecting interactive effects with a fractionated two-level factorial design.

The present study was intended (1) to find out whether simultaneous administration of nine chemicals at a concentration equal to the "no-observed-adverse-effect level" (NOAEL) for each of them would result in a NOAEL for the combination and (2) to test the usefulness of fractionated factorial models to detect possible interactions between chemicals in the mixture. A 4-week oral/inhalatory study in male Wistar rats was performed in which the toxicity (clinical chemistry, hematology, biochemistry, and pathology) of combinations of the nine compounds was examined. The study comprised 20 groups, 4 groups in the main part (n = 8) and 16 groups in the satellite part (n = 5). In the main study, the rats were simultaneously exposed to mixtures of all nine chemicals [dichloromethane, formaldehyde, aspirin, di(2-ethylhexyl)phthalate, cadmium chloride, stannous chloride, butyl hydroxyanisol, loperamide, and spermine] at concentrations equal to the "minimum-observed-adverse-effect level" (MOAEL), NOAEL, or 1/3NOAEL. In the satellite study the rats were simultaneously exposed to combinations of maximally five compounds at their MOAEL. These combinations jointly comprise a two-level factorial design with nine factors (=9 chemicals) in 16 experimental groups (1/32 fraction of a complete study). In the main part many effects on hematology and clinical chemistry were encountered at the MOAEL. In addition, rats of the MOAEL group showed hyperplasia of the transitional epithelium and/or squamous metaplasia of the respiratory epithelium in the nose. Only very few adverse effects were encountered in the NOAEL group. For most of the end points chosen, the factorial analysis revealed main effects of the individual compounds and interactions (cases of nonadditivity) between the compounds. Despite all restrictions and pitfalls that are associated with the use of fractionated factorial designs, the present study shows the usefulness of this type of factorial design to study the joint adverse effects of defined chemical mixtures at effect levels. It was concluded that simultaneous exposure to these nine chemicals does not constitute an evidently increased hazard compared to exposure to each of the chemicals separately, provided the exposure level of each chemical in the mixture is at most similar to or lower than its own NOAEL.

Exposure to mixtures of chemical substances: is there a need for regulations?

The setting of standards for individual substances is an important tool in the protection of human health. However, it has been a topic of discussion for many years whether only this type of standard-setting will meet common goals for health protection, since humans are exposed to a large variety of chemical substances from many different sources in variable concentrations and by different routes of exposure. The complexity of this problem makes it difficult to answer this question and almost impossible to quantify the answer. It is common knowledge that combined exposure to chemical substances may cause synergism. However, these examples are referring to relatively high levels of exposure. In present environmental and occupational practice, exposure to individual chemicals has usually been reduced to acceptable levels. The key question is whether exposure to mixtures at levels of the single components near or below no-observed-adverse-effect levels can still cause adverse effects. Few countries have incorporated procedures concerning combination toxicity in their policy regarding chemical substances. If so, the uncertainty in these procedures is considerable because of lack of relevant data. This usually leads to a conservative approach. Roughly two approaches can be distinguished for systemic toxicants: introduction of an (extra) uncertainty factor or application of the additivity principle. In the Netherlands, for systemic toxicants a safety factor was introduced in 1989 to account for combination effects. Problems related to this approach have led to adaptations in procedures and a reconsideration of the chosen safety factor.

Toxicity studies in rats of simple mixtures of chemicals with the same or different target organs.

Mixtures of chemicals with different target organs or the same target organ but different target sites or different modes of action did not appear to be distinctly more hazardous than the individual chemicals, provided the dose level of each chemical in the mixture did not exceed its own 'No-Observed-Adverse-Effect Level'. Clearly, for such mixtures and exposure conditions the additivity assumption did not hold. However, the additivity rule appeared to be applicable to mixtures of chemicals with the same target organ and the same mechanism of action or receptor. Fractional 2-factorial study designs were found to be promising tools for examining possible combined actions or interactions of chemicals in a mixture.

Dioxins and related compounds: Status and regulatory aspects.

Several countries have put much effort in dioxin research programmes with the aim to assess the risks of dioxin exposure, to identify dioxin sources and to quantify their contribution to environmental emissions and human exposure. In this review, the accent has been put on: sources of dioxins and their contribution to the total dioxin emissions, sources of human exposure and their contribution to exposure and measures to reduce emission and exposure. The information from countries with existing dioxin regulations is reviewed and compared. Uncertainties which hamper comparability of data are indicated. Results of measures and expectations for the near future are presented.