Mixtures study design

In our opinion, the data are sufficiently clear to suggest that when it is not feasible to test the mixture in question, mixture extrapolation is the preferred option compared to no extrapolation. Indeed, all literature observations suggest that applying mixture extrapolation is to be preferred over not applying mixture extrapolation. Technical options for extrapolation are concentration addition, response addition, and the mixed-model approach, of which concentration addition is most often applied. Exceptions may apply in cases that are more specific. For example, when it is clear that 2 compounds precipitate (a situation of no exposure due to chemical interactions in the environment), one should acknowledge this prior to assessing mixture risks by mixture extrapolation approaches. When the data of a study allow, refined conclusions are possible. For example, when the study design is appropriate and the mathematical models are appropriate, researchers are able to discriminate between concentration addition and response addition, and (with sufficient experiment efforts) between these models and the mixed-model approach. 

As reviewed in Kortenkamp et al. (2007), an essential requirement for experimental studies intended to address the issue of mixture effects at doses or concentrations below NOAELs is that NOAELs are estimated for each mixture component by using the same assay system (and endpoint) that is chosen for the mixture study, ideally under identical experimental conditions. Ignoring this requirement can lead to the inadvertent administration of some or all mixture components at doses higher than NOAELs, and would undermine the aim of the experiment. On the other hand, delivery of doses or concentrations smaller than the NOAEL, either by design or by accident, might present problems if the experimental system lacks the statistical power to detect effects. For example, it would be futile to attempt an experiment where 2 agents are combined at 1/100 of their individual NOEL. The resulting mixture effect, if it exists, would be too small to be detectable in most cases, and the experiment would be inconclusive. 

A) Study Design 3 (Fixed Sequence Nonoverlapping Panel Design-Mixture)... 

Study Design 9 (Mixture with Three-Panel Overlap)... 

Skin painting experiments ofa mixture of anthracene, chrysene, fluoranthene, phenanthrene, and pyrene were conducted on groups of 20 male C3H/HeJ mice (Warshawsky et al. 1993). Compounds dissolved in toluene were applied to shaved skin twice weekly for 6 months. Tumor incidence was determined at the end of the study. Treatment included solutions of a mixture of the five noncarcinogenic PAHs at 0.05 mg, or the same compounds in solution with 0.0005 mg benzo[a]pyrene, a dose known to be noncarcinogenic in a similar study design. Forthe mixture of the 5 PAHs at 0.05 mg each, administration alone produced papillomas and malignant tumors in 3 of 13 animals (23%), with a mean latency period of 73 weeks. With coadministration of benzo[a]pyrene, 8 of 17 (47%) had tumors (papillomas and malignant), with a mean latency period of 66 weeks. 

Definitive conclusions regarding human hepatotoxiciy are hampered by limitations in study design of available studies, such as exposure misclassificafion, lack of controls, lack of correction for common confounding variables (e.g., age and alcohol consumption), and natural partitioning of PCBs to serum lipids. The lack of unequivocal evidence in humans that is seen in laboratory animals may result from many factors, including species differences in susceptibility or sensitivity to PCBs, and dissimilarities in exposure levels, durations, and mixture compositions. 

Release of hydrocortisone. Initial exploratory in vitro studies designed to ascertain the usefulness of this polymer system have been carried out with a polymer based on 1,2,6-hexanetriol and various alkyl ortho esters. In these studies 2 wt% hydrocortisone was physically mixed into the ointment and the mixture placed into an erosion cell. A pH 7.4 buffer solution was then pumped across the cell at 9.5 ml/h, samples collected using an automatic fraction collector and analyzed for hydrocortisone by HPLC. The same study was also carried out by mixing into the ointment 2 wt% hydrocortisone and 2 wt% adipic acid. Results of these studies are shown in Fig. 32 [49]. The data clearly shows that without the incorporation of an acidic excipient erosion rate of the polymer is so slow that no hydrocortisone is released for two days. However, when an acidic excipient is mixed into the polymer, a fairly constant release is obtained. 

There are some excellent review articles on various aspects of the toxicology of mixtures of chemicals (Calabrese, 1995 Kri.shnan and Brodeur, 1991), pesticides (lyaniwura, 1990 Murphy, 1980), or OP compounds (Cohen, 1984 DuBois. 1961 Murphy, 1969. 1980). Many of these reviews, although not recent, provide a comprehensive overview of the possible mechanisms underlying interactions of chemicals in a mixture and, specifically, interactions of OP compounds in a mixture. This chapter does not provide an in-depth discussion of the mechanisms underlying interactions of OP or CM pesticides in a mixture. Rather, this chapter is designed to (i) address issues of experiment design of mixture studies (ii) summarize the available literature on OP pesticide mixtures, CM... 

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