Statistically designed experiments to screen chemical mixtures for possible interactions.

For the accurate analysis of possible interactive effects of chemicals in a defined mixture, statistical designs are necessary to develop clear and manageable experiments. For instance, factorial designs have been successfully used to detect two-factor interactions. Particularly useful for this purpose are fractionated factorial designs, requiring only a fraction of all possible combinations of a full factorial design. Once the potential interaction has been detected with a fractionated design, a more accurate analysis can be performed for the particular binary mixtures to ensure and characterize these interactions. In this paper this approach is illustrated using an in vitro cytotoxicity assay to detect the presence of mixtures of Fusarium mycotoxins in contaminated food samples. We have investigated interactions between five mycotoxin species (Trichothecenes, Fumonisins, and Zearalenone) using the DNA synthesis inhibition assay in L929 fibroblasts. First, a central composite design was applied to identify possible interactive effects between mycotoxins in the mixtures (27 combinations from 5(5) possible combinations). Then two-factor interactions of particular interest were further analyzed by the use of a full factorial design (5 x 5 design) to characterize the nature of those interactions more precisely. Results show that combined exposure to several classes of mycotoxins generally results in effect addition with a few minor exceptions indicating synergistic interactions. In general, the nature of the interactions characterized in the full factorial design was similar to the nature of those observed in the central composite design. However, the magnitude of interaction was relatively small in the full factorial design.

Statistical designs in combination toxicology: a matter of choice.

Combination toxicological studies with statistically designed experiments do not often elaborate on the qualities of the design. Therefore, it may seem that the design used is the only one to meet the objectives of the study. This article demonstrates, on the contrary, that there are often several possibilities. Three recent toxicological experiments, featuring economy of experimentation, robustness against inhomogeneity of experimental material and increasing precision of results, respectively are used for illustration. The alternatives differ statistically in the number of distinct effects for which information can be obtained or in the safeguards to counter both inhomogeneity of material and incorrect models. Practically they differ in complexity of experimental conduct. It is concluded that the choice between the alternatives is a result of balancing practical complexity of the design against informational gain. Continuous co-operation between toxicologists and statisticians is needed to strike the balance again and again.

Use of factorial designs in combination toxicity studies.

The use of factorial designs, in which n chemicals are studied at xn dose levels (x treatment groups), has been put forward as one of the valuable statistical approaches for hazard assessment of chemical mixtures. Very recently a "2(5) study' was presented to describe interactions between the carcinogenic activity of five polycyclic aromatic hydrocarbons and a "5(3) study' was used to identify the non-additive effects of three compounds on developmental toxicity. Full factorial designs, however, lead to very costly experiments and, even if only two dose levels are used, it is not always possible to perform conventional toxicity tests using 2n test groups to identify possible interactions between all chemicals of interest. One way to deal with this problem is the use of fractionated factorial designs. These fractionated designs still identify most of the interactions between the compounds and determine which compounds are important in causing effects, but have the advantage that the number of test groups is manageable. Fractional factorial designs have been shown to be an efficient (i.e. cost-effective) approach to: (a) identify interactive effects between seven trace elements and cadmium accumulation in the body; and (b) describe cases of non-additivity in a mixture of nine chemicals tested in a 4-wk toxicity study in rats.

Statistically designed experiments in a tiered approach to screen mixtures of Fusarium mycotoxins for possible interactions.

This paper presents a test strategy to detect interactive effects between several mycotoxins using a DNA synthesis inhibition assay in L929 cells. The joint action of the Fusarium mycotoxins T-2 toxin (T2), deoxynivalenol (DON), nivalenol (NIV), zearalenone (ZEA) and fumonisin (FB1) was studied in a tiered approach. In the first stage, the mycotoxins were tested either jointly in a five-compound mixture, or individually. At the highest dose level, the mixture showed a clear less than additive action of the mycotoxins, as compared to the effects of the five individual compounds, whereas at lower dose levels the mycotoxins behaved additive. In the second stage, the non-additivity as established in the first experiment was further analyzed with a central composite design to detect interactions between specific mycotoxins in the mixture. This experiment confirmed less than additivity for five of the mixes tested. However, it also revealed four significant synergistic interactions between mycotoxins. Finally, two interactions that were established in stage 2 were further studied in full factorial designs involving two mycotoxins. One of the interactions observed in the central composite design was retrieved whereas the other two-factor interaction was not. It was concluded that several classes of mycotoxins when present simultaneously in a mixture might show interaction. The effect of the mixture cannot be predicted solely on the basis of the effect of the individual compounds.

Haematology and blood-chemistry for predicting abscesses and other abnormalities in slaughtered pigs.

Blood values of slaughtered pigs were determined in an attempt to relate the blood profile with the prevalence of abscesses. To this end 319 pigs were selected and grouped into classes on the basis of pathological ante- and post-mortem findings. The classes thus distinguished were (i) no pathology, (ii) pleurisy or pneumonia and (iii) abscesses occurring singly, metastasised or in combination with other abnormalities. By stepwise discriminant analysis it was established that the blood variables ln(fibrinogen) and albumin were particularly suitable for the detection of abscesses in slaughtered pigs. In this way a correct classification of 89.3% of affected pigs is possible. The numbers of false-positives and false-negatives were 19.3% and 10.7%, respectively. For meat-inspection purposes application of blood analyses is promising. For application in meat-inspection practice rapid on-line methods need to be devised.

Optimizing the generation of random amplified polymorphic DNAs in chrysanthemum.

Many conditions of the RAPD reaction procedure may influence the result. This paper presents rapid detection of influential factors with a fractional factorial experiment. A more extensive study of these factors is also presented. Polymerase brand, thermal cycler brand, annealing temperature, and primer, are important factors in obtaining good DNA yields and optimal fragment patterns. Each primer has its optimal annealing temperature, and this is not correlated with the GC content of the primer. Optimal species-primer combinations have to be found by trial and error.

Determination of concentration-time-mortality relationships versus LC50s according to OECD guideline 403.

The effect of decreasing the number of animals on the accuracy of estimated concentration-time-mortality relationships and the LC50 values derived from these was investigated by means of simulation methods. Studies of NH3, Cl2, H2S, and COCl2 were made using 5 rats per sex per group. Mortality rates were analysed in 500 new data sets obtained by randomly removing 4 rats/sex/group from the original data sets. It was concluded that the concentration-time-mortality relationship and the LC50 values can be determined over a 5-10-fold time range using one rat/sex/group. The resulting 5 and 95 percentiles compare favourably with the 90% confidence limits when determining a LC50 according to OECD guideline 403. When using concentration-time-mortality relationships, additional information can be obtained which can be used in inhalation hazard risk assessment.

Statistical analysis of growth limitations in Paracoccus denitrificans: an experiment with a completely randomized two-way factorial design with replications.

Cultivation of microorganisms under growth limitation is a widely used technique in microbiology. The relevant investigations, though seemingly based on straightforward experiments, have generated conflicting results, e.g., concerning molar growth yields. The purpose of this paper is to show that discrepancies in the literature could be solved if more attention were paid to methodology, especially statistics. New experimental results, concerning growth limitations in Paracoccus denitrificans, will exemplify this. They include the following items. Two limitations, one of them being succinate limitation, were established in the present study. Molar growth yields on succinate were 41 g X mol-1 for succinate limitation (95% confidence limits were 38 and 44) and 32 g X mol-1 for the other limitation (95% confidence limits were 29 and 35). The latter result is compatible with sulphate limitation, but the present experimental design does not really permit this conclusion.

A prospective, double-blind, split-subject study on local skin reactions after administration of human menopausal gonadotrophin preparations to healthy female volunteers.

This study was designed to investigate local reactions after the intracutaneous (i.c.) administration of two human menopausal gonadotrophin preparations. For this purpose, 20 healthy female volunteers received six i.c. injections simultaneously, viz. three different batches of both Humegon (Organon, Oss, The Netherlands) and Pergonal (Serono, Geneva, Switzerland) at six different sites on their bodies. Local pain, induration and erythema were registered at 2, 4 and 24 h after administration. No pain was observed. At 4 h after administration, Pergonal-treated sites showed more induration (P = 0.008) and greater surfaces of erythema (P < 0.001) than Humegon-treated sites. Batches of Pergonal showed variation in the surface of erythema induced (P < 0.001), indicating heterogeneity of the batches tested.

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.

Assessment of some critical factors in the freezing technique for the cryopreservation of precision-cut rat liver slices.

A number of studies on the cryopreservation of precision-cut liver slices using various techniques have been reported. However, the identification of important factors that determine cell viability following cryopreservation is difficult because of large differences between the various methods published. The aim of this study was to evaluate some important factors in the freezing process in an effort to find an optimized approach to the cryopreservation of precision-cut liver slices. A comparative study of a slow and a fast freezing technique was carried out to establish any differences in tissue viability for a number of endpoints. Both freezing techniques aim at the prevention of intracellular ice formation, which is thought to be the main cause of cell death after cryopreservation. Subsequently, critical variables in the freezing process were studied more closely in order to explain the differences in viability found in the two methods in the first study. For this purpose, a full factorial experimental design was used with 16 experimental groups, allowing a number of variables to be studied at different levels in one single experiment. It is demonstrated that ATP and K(+) content and histomorphology are sensitive parameters for evaluating slice viability after cryopreservation. Subsequently, it is shown that freezing rate and the cryopreservation medium largely determine the residual viability of liver slices after cryopreservation and subsequent culturing. It is concluded that a cryopreservation protocol with a fast freezing step and using William's Medium E as cryopreservation medium was the most promising approach to successful freezing of rat liver slices of those tested in this study.