Human T47D-ERß breast cancer cells with tetracycline-dependent ERß expression reflect ERα/ERß ratios in rat and human breast tissue.

T47D-ERß breast cancer cells with tetracycline-dependent ERß expression and constant ERα expression can be used to investigate effects of varying ERα/ERß ratios on estrogen-induced cellular responses. This study defines conditions at which ERα/ERß ratios in T47D-ERß cells best mimic ERα/ERß ratios in breast and other estrogen-sensitive tissues in vivo in rat as well as in human. Protein and mRNA levels of ERα and ERß were analyzed in T47D-ERß cells exposed to a range of tetracycline concentrations and compared to ERα and ERß levels found in breast, prostate, and uterus from rat and human origin. The ERα/ERß ratio in T47D-ERß cells exposed to >150ng/ml tetracycline is comparable to the ratio found in rat mammary gland and in human breast tissue. The ERα/ERß ratio of other estrogen-sensitive rat and human tissues can also be mimicked in T47D-ERß cells. The ERα/ERß ratio found in MCF-7 and native T47D breast cancer cell lines did not reflect ratios in analyzed rat and human tissues, which further supports the use of T47D-ERß cells as model for estrogen-responsive tissues. Using 17ß-estradiol and the T47D-ERß cells under the conditions defined to mimic various tissues it could be demonstrated how these different tissues vary in their proliferative response.

Safety evaluation of chemical mixtures and combinations of chemical and non-chemical stressors.

Recent developments in hazard identification and risk assessment of chemical mixtures are reviewed. Empirical, descriptive approaches to study and characterize the toxicity of mixtures have dominated during the past two decades, but an increasing number of mechanistic approaches have made their entry into mixture toxicology. A series of empirical studies with simple chemical mixtures in rats is described in some detail because of the important lessons from this work. The development of regulatory guidelines for the toxicological evaluation of chemical mixtures is discussed briefly. Current issues in mixture toxicology include the adverse health effects of ambient air pollution; the application of such modern, sophisticated methodologies as genomics, bioinformatics, and physiologically based pharmacokinetic modeling; and databases for mixture toxicity. Finally, the state of the art of our knowledge on the potential adverse health effects of combined exposures to chemicals and non-chemical stressors (noise, heat/cold, microorganisms, immobilization, restraint, or transportation), research initiatives in these fields, and the development of an indicator for the cumulative health impact of multiple environmental exposures are discussed.

In vitro intestinal lead uptake and transport in relation to speciation.

Children might be exposed substantially to contaminants such as lead via soil ingestion. In risk assessment of soil contaminants there is a need for information on oral bioavailability of soilborne lead. Oral bioavailability can be seen as the result of four steps: (1) soil ingestion; (2) mobilization from soil during digestion, i.e., bioaccessibility; (3) transport across the intestinal epithelium; and (4) first-pass effect. Lead bioaccessibility and speciation in artificial human small intestinal fluid, i.e., chyme, have been investigated in previous studies. In the present study, transport of bioaccessible lead across the intestinal epithelium was investigated using the Caco-2 cell line. Cell monolayers were exposed to (diluted) artificial chyme. In 24 h, approximately 27% of the lead were associated to the cells and 3% were transported across the cell monolayer, without signs of approaching equilibrium. Lead associated to the cells showed a linear relationship with the total amount of lead in the system. Bile levels did not affect the fraction of lead associated to Caco-2 cells. Extrapolation of the lead flux across the Caco-2 monolayer to the in vivo situation indicates that only a fraction of the bioaccessible lead is transported across the intestinal epithelium. Furthermore, the results indicate that as the free Pb(2+) concentration in chyme was negligible, lead species other than the free metal ion must have contributed to the lead flux toward the cells. On the basis of lead speciation in chyme, this can be attributed to dissociation of labile lead species, such as lead phosphate and lead bile complexes, and subsequent transport of the released free metal ions toward the intestinal membrane.

Toxicological evaluation of chemical mixtures.

This paper addresses major developments in the safety evaluation of chemical mixtures during the past 15 years, reviews today's state of the art of mixture toxicology, and discusses challenges ahead. Well-thought-out tailor-made mechanistic and empirical designs for studying the toxicity of mixtures have gradually substituted trial-and-error approaches, improving the insight into the testability of joint action and interaction of constituents of mixtures. The acquired knowledge has successfully been used to evaluate the safety of combined exposures and complex mixtures such as, for example, the atmosphere at hazardous waste sites, drinking water disinfection by-products, natural flavouring complexes, and the combined intake of food additives. To consolidate the scientific foundation of mixture toxicology, studies are in progress to revisit the biological concepts and mathematics underlying formulas for low-dose extrapolation and risk assessment of chemical mixtures. Conspicuous developments include the production of new computer programs applicable to mixture research (CombiTool, BioMol, Reaction Network Modelling), the application of functional genomics and proteomics to mixture studies, the use of nano-optochemical sensors for in vivo imaging of physiological processes in cells, and the application of optical sensor micro- and nano-arrays for complex sample analysis. Clearly, the input of theoretical biologists, biomathematicians and bioengineers in mixture toxicology is essential for the development of this challenging branch of toxicology into a scientific subdiscipline of full value.

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.

Availability of polychlorinated biphenyls (PCBs) and lindane for uptake by intestinal Caco-2 cells.

Children may ingest contaminated soil from hand to mouth. To assess this exposure route, we need to know the oral bioavailability of the contaminants. Two determining steps in bioavailability of soil-borne contaminants are mobilization from soil during digestion, which is followed by intestinal absorption. The first step has been investigated in previous studies that showed that a substantial fraction of PCBs and lindane is mobilized from soil during artificial digestion. Furthermore, almost all contaminants are sorbed to constituents of artificial human small intestinal fluid (i.e., chyme), whereas only a small fraction is freely dissolved. In this study, we examine the second step using intestinal epithelial Caco-2 cells. The composition of the apical exposure medium was varied by addition of artificial chyme, bile, or oleic acid at similar or increasing total contaminant concentrations. The uptake curves were described by rate constants. The uptake flux seemed to be dose-dependent. Furthermore, different exposure media with similar total contaminant concentrations resulted in various uptake rates. This can be attributed to different freely dissolved concentrations and carrier effects. In addition, the large fractions of contaminants in the cells indicate that PCBs and lindane sorbed to bile, oleic acid, and digestive proteins contributed to the uptake flux toward the cells. These results can be extrapolated qualitatively to in vivo conditions. Because the sorbed contaminants should be considered available for absorption, the first step of mobilization from soil is the most important step for oral bioavailability of the presently investigated soil-borne contaminants.

Toxicology of simple and complex mixtures.

Humans are exposed to mixtures of chemicals, rather than to individual chemicals. From a public health point of view, it is most relevant to answer the question of whether or not the components in a mixture interact in a way that results in an increase in their overall effect compared with the sum of the effects of the individual components. In this article, options for the hazard identification and risk assessment of simple and complex chemical mixtures will be discussed. In addition, key research needed to continue the development of hazard characterization of chemical mixtures will be described. Clearly, more collaboration among toxicologists, model developers and pharmacologists will be necessary.

Cryopreservation of precision-cut rat liver slices using a computer-controlled freezer.

Precision-cut liver slices are frequently used to study hepatic toxicity and metabolism of xenobiotics in vitro. Successful cryopreservation techniques will enhance an efficient and economic use of scarcely available (human) liver tissue. For primary hepatocytes, slow freezing has been accepted as the best approach towards successful cryopreservation. For slices, however, no agreement exists on the optimal way of cryopreservation and both slow and fast freezing techniques have been reported. The aim of the present study was to determine the applicability of a computer-controlled slow freezing technique for the cryopreservation of (rat) liver slices. Thus far, this technique has not been described in detail. Our studies confirmed that slow freezing was most successful in the cryopreservation of primary rat hepatocytes. Based on this observation, the slow freezing technique was applied to the cryopreservation of rat liver slices. Directly after thawing, slice viability was between 60 and 100% of fresh values, depending on the parameter determined. However, after additional culturing, slice viability was reduced. This decrease in slice viability was more pronounced in comparison to primary hepatocytes. In conclusion, the slow freezing technique was confirmed to be a successful approach for the cryopreservation of primary rat hepatocytes, and was found to be of limited use for the cryopreservation of rat liver slices.

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.

Intercellular communication and cell proliferation in precision-cut rat liver slices: effect of medium composition and DDT.

Gap junctional intercellular communication (GJIC) and cell proliferation were studied in control and 1,1'-bis(p-chlorophenyl)-2, 2,2,-trichloroethane (DDT) treated precision-cut liver slices of rat by evaluating connexin 32 (Cx32) expression and 5-bromo-2'-deoxyuridine (BrdU) incorporation. In addition, the effect of different culture media (RPMI and WME) on control and DDT influenced Cx32 expression and cell proliferation was determined. Cx32 expression in control precision-cut liver slices was maintained during 8 h of culturing, but decreased after prolonged culturing. Control cell proliferation was significantly higher when WME was used as culture medium than when RPMI was used. In slices treated with DDT Cx32 expression was decreased. In slices cultured in RPMI medium, this decrease preceded a dose-dependent increase in cell proliferation. These results show the usefulness of precision-cut liver slices in studying cellular proliferation and intercellular communication.

Role of metallothionein in cisplatin sensitivity of germ-cell tumours.

Cisplatin (CDDP) is an extremely active drug in the treatment of germ-cell tumours. Earlier, we found an unexpected inverse correlation between the total amount of sulfhydryl groups and CDDP sensitivity in a panel of 3 human germ-cell-tumour and 3 colon-carcinoma cell lines. Major components of the sulfhydryl groups are glutathione and metallothionein (MT). We further investigated a possible role of MT in the CDDP sensitivity of germ-cell tumours. MT levels and functionality of the germ-cell-tumour and colon-carcinoma cell lines were found to be inversely correlated with CDDP sensitivity. No difference in sub-cellular localization of MT could be observed among the types of cell lines. In agreement with the in vitro data, immunohistochemical detection of MT was high in 11/14 primary human germ-cell tumours and low in 7/7 human colon carcinomas. MT-protein expression in primary germ-cell tumours did not discriminate between responding and non-responding patients. As compared with the primary tumours, MT-protein expression decreased in 5/7 post-chemotherapy residual vital tumours or remained undetectable (2/7). MT-protein expression in the germ-cell tumours was not related to total p53-protein expression. In summary, over-expression of MT was found in germ-cell tumours, both in cell lines and in human tumours. Although MT-protein over-expression seems to be associated with the CDDP sensitivity of germ-cell tumours, MT-protein expression in primary germ-cell tumours did not differ between responding and non-responding patients and therefore cannot be used to predict response to chemotherapy.

Mixtures and interactions.

Drinking water can be considered as a complex mixture that consists of tens, hundreds or thousands of chemicals of which the composition is qualitatively and quantitatively not fully known. From a public health point of view it is most relevant to answer the question of whether chemicals in drinking water interact in a way that results in an increased overall response as compared to the sum of the responses to the individual chemicals in the mixture, or indeed in an effect that is simply a summation of the expected effects of the individual chemicals. Present methods for risk assessment of mixtures rely heavily on some form of additivity model, unless data are adequate for a direct risk assessment of the mixture of concern in its entirety. The "dose-addition" concept ("simple similar action") is the most common approach to risk assessment of mixtures and it is applicable over the whole range of exposure levels from low non-toxic to toxic levels when all chemicals in the mixture act in a similar way. However, in toxicity studies at environmentally relevant exposure scenarios the mixtures that meet such conditions are the exception rather than the rule. In that case the "effect addition" model has to be followed assuming "independent joint action". For these compounds now experimental data have indicated that the results at low exposure levels are probably difficult to predict based on response additivity found at higher dose levels. Thus, although the additivity models are mathematically simple, they require assumptions about the mechanisms of action and the high-to-low dose extrapolation. Therefore, theoretical considerations in risk assessment of chemical mixtures should be verified by simple case studies. Up till now, the number of environmentally relevant mixtures to which a direct risk assessment has been devoted is limited. Even if toxicity data on individual compounds are available, we are still facing the immense problem of extrapolation of findings obtained at relatively high exposure concentration in laboratory animals to man being exposed to (much) lower concentrations. Therefore the prioritization of compounds for further research and the extrapolation to low doses should be considered as key issues in the assessment of possible health risks from exposure to chemical mixtures such as drinking water.

An analysis of the possibility for health implications of joint actions and interactions between food additives.

The possibility that structurally unrelated food additives could show either joint actions or interactions has been assessed based on their potential to share common sites and mechanisms of action or common pathways of elimination. All food additives approved in the European Union and allocated numerical acceptable daily intake values were studied, initially based on the reports by the FAO-WHO Joint Expert Committee for Food Additives. Target organs were identified based on the effects reported at doses above the no-observed-adverse-effect level (NOAEL) in animal and human studies. The descriptions of the pathological and other changes reported were used to assess whether different additives, sharing the same target organ, would produce a common toxic effect. In all but a very few cases, the possibility of joint actions or interactions could be excluded on scientific grounds. The exceptions were on the liver (curcumin, thiabendazole, propyl gallate, and BHT), the kidney (diphenyl, o-phenylphenol, and ferrocyanide salts), the blood (azorubine and propyl gallate), and the thyroid (erythosine, thiabendazole, and nitrate). Toxicokinetic interactions were considered unlikely because of the low dosages involved, the diverse nature of the routes of metabolism and elimination, and the fact that enzyme induction or inhibition would have influenced selection of the NOAEL. Many of those additives which could not be excluded from showing joint actions or interactions would have low intakes; in some cases they were alternatives for the same application, thereby further lowering the combined intake. In consequence, joint actions or interactions between additives do not represent a significant health concern.

Effects of cadmium chloride on the paracellular barrier function of intestinal epithelial cell lines.

In the present study we characterized the functional and structural disruption of the paracellular barrier of intestinal epithelium in vitro in relation to cytotoxicity after apical Cd2+ exposure. For that purpose filter-grown Caco-2 and IEC-18 cells were apically exposed to 5 to 100 microM CdCl2 for 4 or 14 h. It was found that the effects of Cd2+ on the epithelial barrier were concentration- and time-dependent. The first detected effects of Cd2+ in Caco-2 cells after 4 h exposure were a decrease in transepithelial electrical resistance, increased permeabilities of mannitol and PEG-4000, and changes in intercellular localization of ZO-1, occludin, and e-cadherin. The effects were far more pronounced after prolonged exposure. The disruption of the paracellular barrier by 5 to 30 microM Cd2+ was detected without a significant loss of viability of the Caco-2 cells. In the IEC-18 cells, Cd2+ concentrations affecting the barrier (50 and 100 microM) also affected cell viability. In both cell lines the effects on the cell layers continued to develop after removal of extracellular Cd2+. This correlated with the cellular retention of Cd2+, which was high for the 12 h following 4 h accumulation. This study showed that the decreased epithelial barrier function of intestinal epithelial cells is accompanied by tight junction disruption. It is concluded that Cd2+ causes increased paracellular permeability by disruption of junctional function and structure. The initial junctional effects of Cd2+ suggest that Cd2+ increases its own bioavailability by causing disruption of the intestinal paracellular barrier.

Toxicology of chemical mixtures: international perspective.

This paper reviews major activities outside the United States on human health issues related to chemical mixtures. In Europe an international study group on combination effects has been formed and has started by defining synergism and antagonism. Successful research programs in Europe include the development and application of statistically designed experiments combined with multivariate data analysis and modeling in vitro and in vivo studies on a wide variety of chemicals such as petroleum hydrocarbons, aldehydes, food contaminants, industrial solvents, and mycotoxins. Other major activities focus on the development of safety evaluation strategies for mixtures such as the use of toxic equivalence factors or alternatives such as the question-and-answer approach, fractionation followed by recombination of the mixture in combination with a mixture design, and quantitative structure-activity relationship analysis combined with lumping analysis and physiologically based pharmacokinetic/pharmacodynamic modeling for studying complex mixtures. A scheme for hazard identification and risk assessment of complex mixtures and a consistent way to generate total volatile organic compound values for indoor air have also been developed. Examples of other activities are carcinogenicity studies on complex mixtures (petroleum middle distillates, foundry fumes, pesticides, heterocyclic amines, diesel exhaust, solid particles), neurotoxicity studies of mixtures of solvents alone or in combination with exposure to physical factors, and toxicity studies of outdoor air pollutants, focusing on particulates. Outside the United States, toxicologists and regulators clearly have a growing interest in the toxicology and risk assessment of chemical mixtures.

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.

Absorption enhancement, structural changes in tight junctions and cytotoxicity caused by palmitoyl carnitine in Caco-2 and IEC-18 cells.

Palmitoyl carnitine chloride (PCC) has been shown to be an effective enhancer of intestinal transport of hydrophilic molecules. The exact mechanism by which the epithelial barrier function is decreased is not clear. In an attempt to elucidate the mechanism of action of PCC, we studied the relationship among absorption enhancement, cell viability and tight junction protein localization in the human colonic Caco-2 cell line and the rat small intestinal cell line IEC-18. Filter-grown cells were exposed to 0 to 1 mM PCC for 30 min, and the efficacy of PCC treatment was determined by assessing the transepithelial electrical resistance and the apparent permeability for mannitol and PEG-4000. Membrane lysis and cytotoxicity were assessed by measurement of lactate dehydrogenase leakage and uptake of propidium iodide and neutral red. The immunolocalization of the tight junctional protein ZO-1 was quantified using CSLM and image-processing software. In both cell lines, PCC caused a dose-dependent decrease in transepithelial electrical resistance and a concomitant increase in the permeability for mannitol and PEG-4000. The transport enhancement was accompanied by an increase in apical membrane permeability and a reduction in cell viability. At higher PCC concentrations (>/=0.4 mM), the distribution of the tight junctional protein ZO-1 was changed and cells were unable to recover viability. PCC is effective as an absorption enhancer for hydrophilic macromolecules. However, lytic effects on the cell membrane and reduced cell viability were concomitant with transport enhancement.

Toxicological evaluation and risk assessment of chemical mixtures.

A major objective of combination toxicology is to establish whether a mixture of chemicals will result in an effect similar to that expected on the basis of additivity. This requires understanding of the basic concepts of the combined toxicological action of the compounds of the mixture: simple similar action (dose addition), simple dissimilar action (effect or response addition), and interaction (synergism, potentiation, antagonism). The number of possible combinations of chemicals is innumerable, and in vivo testing of these mixtures is unattainable from an ethical, economical, or pragmatic perspective. Prediction of the effect of a mixture based on the knowledge of each of the constituents requires detailed information on the composition of the mixture, exposure level, mechanism of action, and receptor of the individual compounds. Often, such information is not or is only partially available and additional studies are needed. Research strategies and methods to assess joint action or interaction of chemicals in mixtures such as whole mixture testing, physiologically based toxicokinetic modeling and isobologram and dose response surface analyses are discussed. Guidance is given for risk assessment of both simple and complex mixtures. We hypothesize that, as a rule, exposure to mixtures of chemicals at (low) non-toxic doses of the individual constituents is of no health concern. To verify the hypothesis is a challenge; to timely detect exceptions to the rule is the real challenge of major practical importance.

Exposure of humans to complex chemical mixtures: hazard identification and risk assessment.

A complex chemical mixture is defined as a mixture that consists of tens, hundreds or thousands of chemicals, and of which the composition is qualitatively and quantitatively not fully known. In contrast, a simple mixture consists of a relatively small number of chemicals, say ten or less, and the composition of which is fully known. In the present paper a number of options for hazard identification and risk assessment of complex chemical mixtures is discussed, and a scheme aimed at selecting the most appropriate approach for each (type of) complex mixture is presented. A conspicuous element of this scheme is the dichotomy of complex mixtures into mixtures that are readily available and mixtures that are virtually unavailable for testing in their entirety. Another characteristic aspect of the scheme is the inclusion of the "top-ten" and "pseudo top-ten" approaches, which in essence are ways to select the, say ten, most risky chemicals or pseudocomponents to be dealt with as a simple chemical mixture.

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.