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The article highlighted in this issue is "A PBPK Modeling-Based Approach to Account for Interactions in the Health Risk Assessment of Chemical Mixtures" by Sami Haddad, Martin Béliveau, Robert Tardif, and Kannan Krishnan (pp. 125-131).
Sublinear dose-response relationships are often seen in toxicity testing, particularly with bioassays for carcinogenicity. This is the result of a superimposition of various effects that modulate and contribute to the process of cancer formation. Examples are saturation of detoxification pathways or DNA repair with increasing dose, or regenerative hyperplasia and indirect DNA damage as a consequence of high-dose cytotoxicity and cell death. The response to a combination treatment can appear to be supra-additive, although it is in fact dose-additive along a sublinear dose-response curve for the single agents. Because environmental exposure of humans is usually in a low-dose range and deviation from linearity is less likely at the low-dose end, combination effects should be tested at the lowest observable effect levels (LOEL) of the components. This principle has been applied to combinations of genotoxic agents in various cellular models. For statistical analysis, all experiments were analyzed for deviation from additivity with an n-factor analysis of variance with an interaction term, n being the number of components tested in combination. Benzo[a]pyrene, benz[a]anthracene, and dibenz[a,c]anthracene were tested at the LOEL, separately and in combination, for the induction of revertants in the Ames test, using Salmonella typhimurium TA100 and rat liver S9 fraction. Combined treatment produced no deviation from additivity. The induction of micronuclei in vitro was investigated with ionizing radiation from a 137Cs source and ethyl methanesulfonate. Mouse lymphoma L5178Y cells revealed a significant 40% supra-additive combination effect in an experiment based on three independent replicates for controls and single and combination treatments. On the other hand, two human lymphoblastoid cell lines (TK6 and WTK1) as well as a pilot study with human primary fibroblasts from fetal lung did not show deviation from additivity. Data derived from one cell line should therefore not be generalized. Regarding the testing of mixtures for deviation from additive toxicity, the suggested experimental protocol is easily followed by toxicologists.
1. Glutathione (GSH) depletion is often reported as an early cytotoxic effect, caused by many reactive organic chemicals. In the present study, GSH depletion in primary rat hepatocytes was used as an in vitro effect-equivalent to measure the toxic potency of alpha,beta-unsaturated esters (acrylates and methacrylates). 2. When these compounds were administered as a mixture, GSH depletion was dose additive. The result of the mixture study shows that GSH depletion may be a useful effect-equivalent for the risk assessment of mixtures of alpha,beta-unsaturated esters. 3. To get more insight in the underlying mechanisms of GSH depletion, the metabolism of two esters was investigated in greater detail. One of them, allyl methacrylate, was metabolized to acrolein. This metabolic pathway can explain the high potency of allyl methacrylate to deplete GSH despite its low intrinsic chemical reactivity.
Quantitative structure-activity relationships were calculated for the inhibition of bioluminescence of Photobacterium phosphoreum by 22 nonreactive organic chemicals. The inhibition was measured using the Microtox test and correlated with the partition coefficient between n-octanol and water (Poct), molar refractivity (MR), and molar volume (MW/d). At log Poct less than 1 and greater than 3, deviations from linearity were observed. Introduction of MR and MW/d improved the quality of the relationships. The influences of MR or MW/d may be related with an interaction of the tested chemicals to the enzyme system which produces the light emission. The sensitivity of the Microtox test to the 22 tested compounds is comparable to a 14-day acute mortality test with guppies for chemicals with log Poct less than 4. The inhibition of bioluminescence by a mixture of the tested compounds was slightly less than was expected in case of concentration addition. The Microtox test can give a good estimate of the total aspecific "minimum toxicity" of polluted waters. When rather lipophilic compounds or pollutants with more specific modes of action are present, this test will underestimate the toxicity to other aquatic life.
This study was undertaken to observe the type of interaction that exists between polychlorinated biphenyls (Clophen A60) and diethyl phthalate (DEP) on the adrenal and thyroid glands of male and female Wistar rats. Animals were divided into four groups of six animals each, group I male and female rats were fed on a normal diet and water ad libitum. Groups II, III and IV male and female rats were given Clophen A60, DEP, or mixture of Clophen A60 and DEP, respectively, each dissolved in corn oil mixed with the diet at 50mg/kg of the diet/day. One hundred days after treatment, females were mated with males for 10 days. Exposure to the pollutants was continued throughout mating, gestation (21 days) until termination at weaning (21 days), which was 150 days of total treatment period of the parental generation. When the F1-generation pups (six males and six females of each group) were 75-100g in weight, they were treated in a similar manner to the parental generation, again for a period of 150 days, with the dose reduced to 25mg/kg of the diet/day in all treated groups. After 150 days of treatment, animals were sacrificed and histology of the adrenal and thyroid glands was asessed. An antagonistic interactive effect of treatment was seen in male parental and F1-generation rats, while an inhibitory type of interactive effect was observed in female rats. In the zona fasciculata region of the adrenal cortex of treated rats of both generations, vacuolations and degeneration were seen in samples from male animals and intracellular vacuolations in samples from females. A synergistic interactive toxic effect to the thyroid gland was observed in treated parental generation male rats, and mild changes in F1-generation-treated male rats, showing follicular shrinkage, loss of thyroglobulin and fibrosis of the interfollicular epithelium. In females, an antagonistic effect to the thyroid gland was observed in both parental and F1-generation-treated rats, showing similar effects as observed in males. From this study, we can conclude that combined administration of Clophen A60 and DEP shows an enhanced toxic effect on adrenal glands of F1-generation male and female rats, but the effect is much more marked in the thyroid gland of F1-generation male rats, and seen to a lesser extent in F1-generation female rats.
Trichloroacetic acid (TCA) and trifluoroacetic acid (TFA) have been detected together in environmental water samples throughout the world. TCA may enter into aquatic systems via rainout as the degradation product of chlorinated solvents, herbicide use, as a by-product of water disinfection and from emissions of spent bleach liquor of kraft pulp mills. Sources of TFA include degradation of hydrofluorocarbons (HFCs) refrigerants and pesticides. These substances are phytotoxic and widely distributed in aquatic environments. A study to assess the risk of a binary mixture of TCA and TFA to macrophytes in aquatic microcosms was conducted as part of a larger study on haloacetic acids. M. spicatum and M. sibiricum were exposed to 0.1, 1, 3 and 10 mg/l of both TCA and TFA (neutralized with sodium hydroxide) in replicate (n = 3) 12000 l aquatic microcosms for 49 days in an one-way analysis of variance design. Each microcosm was stocked with 14 individual apical shoots per species. The plants were sampled at regular intervals and assessed for the somatic endpoints of plant length, root growth, number of nodes and wet and dry mass and the biochemical endpoints of chlorophyll-a, chlorophyll-b, carotenoid content and citric acid levels. Results indicate that there were statistically significant effects of the TCA/TFA mixture on certain pigment concentrations immediately after the start of exposure (2-7 days), but the plants showed no signs of stress thereafter. These data suggest that TCA/TFA mixtures at environmentally relevant concentrations do not pose a significant risk to these aquatic macrophytes.
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In order to identify nonadditive effects on development, three compounds were combined using five dosages of each agent (a 5 x 5 x 5 full-bacterial design). Trichloroethylene (TCE), di(2-ethylhexyl) phthalate (DEHP), and heptachlor (HEPT), in corn oil, were administered by gavage to Fischer-344 rats on Gestation Days 6-15. Dose levels were 0, 10.1, 32, 101, and 320 mg/kg/day for TCE; 0, 24.7, 78, 247, and 780 mg/kg/day for DEHP; and 0, 0.25, 0.8, 2.5, and 8 mg/kg/day for HEPT. The dams were allowed to deliver and their pups were weighed and examined postnatally. Maternal death showed no main effects but DEHP and HEPT were synergistic. For maternal weight gain on Gestational Days 6-8, main effects for all three agents were observed, as well as 6-8 main effects for all three agents were observed, as well as TCE-DEHP synergism, and DEHP-HEPT antagonism. Maternal weight gain on Gestational Days 6-20 adjusted for litter weight showed main effects for TCE and HEPT, but no interactions. Main effects for all three agents were evident for full-litter resorptions and prenatal loss. The HEPT main effects were unexpected and were interpreted as reflecting potentiation by HEPT of the other agents. For full-litter loss, the TCE-HEPT and DEHP-HEPT interactions were antagonistic, perhaps due to a "ceiling" effect. For prenatal loss, the TCE-DEHP interaction was synergistic. Postnatal loss showed DEHP and HEPT main effects but no interaction. Analysis of pup weights on Day 1 revealed TCE and DEHP main effects and DEHP-HEPT antagonism; on Day 6, DEHP and HEPT main effects, DEHP-HEPT antagonism, and TCE-DEHP synergism were evident. Microphthalmia and anophthalmia incidences revealed TCE and DEHP main effects but no interactions. This extensive examination of a full-factorial design elucidates the complexities of studying and interpreting mixture toxicity. The data are available for further analysis.
Two different concepts, termed concentration addition (CA) and independent action (IA), describe general relationships between the effects of single substances and their corresponding mixtures allowing calculation of an expected mixture toxicity on the basis of known toxicities of the mixture components. Both concepts are limited to cases in which all substances in a mixture influence the same experimental endpoint, and are usually tested against a "fixed ratio design" where the mixture ratio is kept constant throughout the studies and the overall concentration of the mixture is systematically varied. With this design, interaction among toxic components across different mixture ratios and endpoints (i.e. lethal versus sublethal) is not assessed. In this study lethal and sublethal (feeding) responses of Daphnia magna individuals to single and binary combinations of similarly and dissimilarly acting chemicals including the metals (cadmium, copper) and the pyrethroid insecticides (lambda-cyhalothrin and deltamethrin) were assayed using a composite experimental design to test for interactions among toxic components across mixture effect levels, mixture ratios, lethal and sublethal toxic effects. To account for inter-experiment response variability, in each binary mixture toxicity assay the toxicity of the individual mixture constituents was also assessed. Model adequacy was then evaluated comparing the slopes and elevations of predicted versus observed mixture toxicity curves with those estimated for the individual components. Model predictive abilities changed across endpoints. The IA concept was able to predict accurately mixture toxicities of dissimilarly acting chemicals for lethal responses, whereas the CA concept did so in three out of four pairings for feeding response, irrespective of the chemical mode of action. Interaction effects across mixture effect levels, evidenced by crossing slopes, were only observed for the binary mixture Cd and Cu for lethal effects. The analysis of regression residuals showed that interaction effects across mixture ratios were restricted to feeding responses in binary mixtures that included Cu. These results indicate that the ability of the CA and IA concept to predict mixture toxicity effects varies from lethal to sublethal endpoints irrespective of their primary mode of action. This suggests that when considering complex responses, the pharmacological notion of mode of action should be extended to encompass an ecotoxicological mode of action, based on the concentration at which various toxicological effects become operative in the biological system under consideration.
A mixture toxicity investigation was conducted using the bioluminescent marine bacterium Vibrio harveyi as the test organism for dual combinations of nitrobenzene and dinitrobenzene. Change in bioluminescence was used for determination of toxicity. Combination toxicity was evaluated using statistical comparisons, isopleths (isobologram and isobole plot), an additive index, and a mixture toxicity index. Both isopleths and mixture toxicity indices suggest that various combinations are additive, while the additive index value suggests antagonism. All evaluations were conducted as equitoxic mixtures. Statistical determination was performed using the z test. Numerous comparisons were different at the 1% level. Slope of line associated with isobole plot was suggested to be an important factor, resulting in statistical differences among comparisons. Distribution, using the Shapiro-Wilk test, was determined for both individual combination groups and solution composition in isopleths. All distributions evaluated were normal. These results suggest that the V. harveyi toxicity test is useful for mixture toxicity studies.
2,4-dichlorophenoxyacetic acid and monosodium methanearsonate are often sold in commercial herbicide mixtures. Toxicity studies have been performed for each herbicide individually, but there is a dearth of information concerning the toxicity of these herbicides in a mixture. The following study examined the toxicity of a mixture of these two herbicides in the red swamp crawfish, Procambarus clarkii. 96-hour acute toxicity assays were performed to determine whether surfactant significantly altered the toxicity of these herbicides individually or in combination. Markings additive index was calculated to identify the interactions of the herbicide mixture. Surfactant was observed to significantly increase the toxicity of 2,4-dichlorophenoxyacetic acid and the toxicity of the herbicide mixture. The herbicide mixture alone displayed half the toxicity of the individual herbicides, but the mixture with surfactant was twice as toxic as the individual herbicides. The synergistic action of surfactant may be attributed to increased pesticide absorption across biological membranes. 2,4-dichlorophenoxyacetic acid and surfactant may also compromise gill function, increasing the sensitivity of the crawfish to herbicide toxicity. The antagonistic effects of the herbicide mixture in the absence of surfactant may be caused by competition of both herbicides for the same sites of activity.
An analysis of current research on mixture toxicity was conducted by critically reviewing published journal articles. The scope was limited to complex mixtures (more than two components) where at least one component was a chlorinated organic chemical. Although the basics of dose-response are widely accepted for mixtures, a number of technical issues, including dose metrics and the unquantified influence of toxicity modifying factors, confound data interpretation and restrict the ability to establish reliable determinations of the presence, nature, and extent of additivity. Lack of knowledge about dose level influences and species-specific variations contribute further interpretational limitations. Within this context, available data indicates that most tested mixtures are near or below simple dose/concentration additivity. Exceptions (both positive and negative) tend to occur when tested mixtures have only a few components or where sensitive whole organism or sub-organismal changes are used as the response metric. Available information does not routinely identify the presence of chlorine as a marker either of a particular type of toxicity or consistently greater potency. The most profound difficulty is the problem of clearly defining when and why similarity and dissimilarity of toxic action is expected for a particular mixture. This impediment largely results from the lack of a generally accepted, technical classification for mode/mechanism of toxic action coupled with the lack of a generally accepted classification scheme for mode/mechanism of toxicity interactions.
The individual and joint toxicities of binary mixtures of 2,4-Dinitrotoluene (2,4-DNT) and 8 nitrobenzenes and anilines to V. fischeri were determined respectively and their mixture toxicity effects were assessed using the Additivity Index (Al). The energy of the lowest unoccupied molecular orbital (E(LUMO)) of studied compounds was calculated and was used to develop QSARs. The prediction models for the individual and mixture toxicity data were respectively: log 1/EC50=3.213-0.716 E(LUMO), n=9, r=0.830, s=0.545 and log 1/EC50=3.672-0.882 E(LUMO), n=8, r=0.925, s=0.406. The joint toxic effects derived from AI values show that the mixture toxicity of 2,4-DNT and the other nitrobenzenes is synergistic (AI>0), whilst the mixture toxicity of 2,4-DNT and the anilines is antagonistic (AI<0). These results confirm that the mixture toxicity is mainly related to intracellular oxidation and reduction. -E(LUMO) values of nitrobenzenes are higher. The nitrobenzenes when mixed with 2,4-DNT make the mixture systems easily reduced; the mixture toxicity effect is synergism. On the other hand, -E(LUMO) values of anilines are lower. The anilines when mixed with 2,4-DNT can make the mixture systems less easily reduced, and the mixture toxicity effect is antagonism.
An analysis of current mixture theory, policy, and practice was conducted by examining standard reference texts, regulatory guidance documents, and journal articles. Although this literature contains useful theoretical concepts, clear definitions of most terminology, and well developed protocols for study design and statistical analysis, no general theoretical basis for the mechanisms and interactions of mixture toxicity could be discerned. There is also a poor understanding of the relationship between exposure-based and internal received dose metrics. This confounds data interpretation and limits reliable determinations of the nature and extent of additivity. The absence of any generally accepted classification scheme for either modes/mechanisms of toxic action or of mechanisms of toxicity interactions is problematic as it produces a cycle in which research and policy are interdependent and mutually limiting. Current regulatory guidance depends heavily on determination of toxicological similarity concluded from the presence of a few prominent constituents, assumed from a common toxicological effect, or presumed from an alleged similar toxic mode/mechanism. Additivity, or the lack of it, is largely based on extrapolation of existing knowledge for single chemicals in this context. Thus, regulatory risk assessment protocols lack authoritative theoretical underpinnings, creating substantial uncertainty. Development of comprehensive classification schemes for modes/mechanisms of toxic action and mechanisms of interaction is needed to ensure a sound theoretical foundation for mixture-related regulatory activity and provide a firm basis for iterative hypothesis development and experimental testing.
The toxicities of selected oil shale components and of mixtures of these components to a mixed marine bacterial culture have been determined. The toxicities of mixtures whose components were members of the same homologous series were found to be additive. In all other cases, even when the compounds were as closely related as structural isomers, synergism was observed. Simple and multiple linear regression equations utilizing measures of toxicity and molecular descriptors were used successfully to predict the enhancement of toxicity due to interaction of components in mixtures regardless of the mode of interaction.