On the Use of Mixture Toxicity Assessment in REACH and the Water Framework Directive: A Review

This review seeks to connect the scientific theory of mixture toxicity to its implementation within different regulatory frameworks. The aim is to demonstrate how mixture toxicity assessment can be more thoroughly integrated into the European chemical regulations, REACH, and the Water Framework Directive (WFD), using the experiences gained through other regulatory frameworks. The article consists of (1) an examination of the scientific underpinnings of the common mixture toxicity assessment methods; (2) a discussion of how these methods have been used in regulatory frameworks; and (3) a discussion of how the methods could be applied within REACH and the WFD. It is concluded that concentration addition should be applied as a default model for mixture toxicity assessment. Furthermore, it is concluded that REACH and the WFD only include mixture toxicity assessments in specific situations. However, it is shown that it is scientifically feasible and regulatorally practicable to integrate a more holistic mixture toxicity approach into both legislations. In this connection, the experience gained from the U.S. frameworks on mixture toxicity assessment could be useful. The construction of a database that includes data on chemicals in the European environment could be used for mixture toxicity assessment of the chemicals with individual PEC/PNECs > 0.1.     

On the quantitative genetics of mixture characters

Finite mixture models are helpful for uncovering heterogeneity due to hidden structure. Quantitative genetics issues of continuous characters having a finite mixture of Gaussian components as statistical distribution are explored in this article. The partition of variance in a mixture, the covariance between relatives under the supposition of an additive genetic model, and the offspring-parent regression are derived. Formulas for assessing the effect of mass selection operating on a mixture are given. Expressions for the genetic and phenotypic correlations between mixture and Gaussian traits and between two mixture traits are presented. It is found that, if there is heterogeneity in a population at the genetic or environmental level, then genetic parameters based on theory treating distributions as homogeneous can lead to misleading interpretations. Some peculiarities of mixture characters are: heritability depends on the mean values of the component distributions, the offspring-parent regression is nonlinear, and genetic or phenotypic correlations cannot be interpreted devoid of the mixture proportions and of the parameters of the distributions mixed.     

Use of the Brompton mixture in treating the chronic pain of malignant disease.

Physical, psychological, financial, interpersonal and spiritual factors all modify the appreciation of chronic pain. The Brompton mixture is a highly effective, flexible, safe and convenient means of controlling the chronic pain of malignant disease. The mixture is a solution containing morphine; the dose of narcotic can be varied with the need for analgesia. It is given regularly, usually every 4 hours, with a phenothiazine, the main aims of therapy being prevention of pain rather than treatment, an unclouded sensorium and a normal affect.     

A bias in ML estimates of branch lengths in the presence of multiple signals

Sequence data often have competing signals that are detected by network programs or Lento plots. Such data can be formed by generating sequences on more than one tree, and combining the results, a mixture model. We report that with such mixture models, the estimates of edge (branch) lengths from maximum likelihood (ML) methods that assume a single tree are biased. Based on the observed number of competing signals in real data, such a bias of ML is expected to occur frequently. Because network methods can recover competing signals more accurately, there is a need for ML methods allowing a network. A fundamental problem is that mixture models can have more parameters than can be recovered from the data, so that some mixtures are not, in principle, identifiable. We recommend that network programs be incorporated into best practice analysis, along with ML and Bayesian trees.     

Reliable predictive computational toxicology methods for mixture toxicity: toward the development of innovative integrated models for environmental risk assessment

A main objective in the field of mixture toxicity is to determine how well combined effects are predictable based on the known effects of mixture constituents. Conducting toxicity tests for all conceivable combinations of chemicals, to understand all mechanisms in the combined toxicity of environmental pollutants, is virtually unfeasible due to cost- and time-consuming procedures. Therefore, predictive tools for mixture toxicity are required to be developed within the applicable range of predictive toxicology. The concept of concentration addition (CA) model is often considered a general method for estimating mixture toxicity at the regulatory level. In the long run, however, the possibility of toxicological synergism between mixture components actually occurs, especially from the no-effect level or non-toxic substances. This is ignored under the CA concept, and needs to be examined and integrated into existing addition models at a scientific level, this paper reviews existing integrated models for estimating the toxicity of complex mixtures in literature. Current approaches to assess mixture toxicity and the need for new research concepts to overcome challenges which recent studies have confronted are discussed, particularly those involved in computational approaches to predict mixture toxicity in an environment risk assessment based on mixture components.     

ELISA-based method for determining the affinity of bivalent antibodies of two specificities in a mixture

The problem of the evaluation of the affinity for two types of bivalent antibodies in a mixture is considered. It is shown that the binding curve in appropriate coordinates can be used to compose either a system of four equations with four unknowns or a system of two equations with two unknown variables. The numerical solution of these equation systems yields affinity constants for both antibodies and the relationship between concentrations of antibodies studied in the mixture.     

Preparation of Mixtures for the Calibration of Gas Analysers

Differential gas analysers for use in photosynthesis experimentscan be calibrated with the same accuracy as that specified forthe single standard gas mixture that is required. With the useof only one gas mixing pump, the composition of a mixture canbe specified to better than ±1 per cent, compared with±5 per cent usually offered by commercial gas suppliers.     

Simultaneous determination of the individual concentration gradients of two solute species in a centrifuged mixture: application to analytical ultracentrifugation

A procedure for determining the absolute activity of 14C-labeled and 3H-labeled solutes in a mixture from the measured counts per minute in two scintillation energy windows is described. It is shown that the method described here provides a substantially more accurate determination of 3H activity in the presence of a larger 14C activity, and a more accurate determination of 14C activity in the presence of a larger 3H activity, than does the standard dual label analysis implemented in a Beckman LS 3801 scintillation counter. The new dual label procedure is combined with the automated fractionation procedure of Attri and Minton [(1986) Anal. Biochem. 152, 319-328] to permit the gradients of each of two differently radiolabeled solute species in a mixture to be individually determined following centrifugation. It is shown that the sedimentation coefficients of each of two differently labeled noninteracting proteins in a mixture may be readily determined in a sedimentation velocity experiment, and that the molecular weights of each of two such proteins in a mixture may be readily determined in a sedimentation equilibrium experiment.     

Dietary nucleotide requirements of the mosquito, Culex pipiens

Dietary nucleic acid, or certain constituents thereof, is essential for full development of the mosquito Culex pipiens. Yeast RNA, whole or hydrolysed, is fully effective. Sperm DNA is totally ineffective on its own, but becomes fully effective when supplemented with uridylic acid. RNA can be replaced with only partial success by a mixture of its component nucleotides (adenylic, guanylic, cytidylic, and uridylic acids), but with the addition of thymidylic acid (characteristic of DNA but present in RNA only as a minor component of transfer RNA), the resulting five-nucleotide mixture is a fully adequate replacement for yeast RNA. Single and multiple deletions from this five-nucleotide mixture showed a minimal requirement for three nucleotides: adenylic acid (a purine ribonucleotide) and thymidylic acid (a pyrimidine deoxyribonucleotide) were both specifically required; as the third nucleotide, either cytidylic or uridylic acid (both pyrimidine ribonucleotides) was equally satisfactory. Mixtures of the five nucleosides or five deoxynucleotides corresponding in base composition to the effective five-nucleotide mixture were only partially effective substitutes.     

Isolation and characterization of the proteolytic enzyme component from commercially available crude trypsins

A purification procedure for the isolation of a mixture of the major proteolytic pancreatic enzymes (trypsin, chymotrypsin, and elastase) from commercial crude trypsins is described. These enzymes are apparently the enzymes responsible for tissue dispersal in numerous cell culture systems. Materials toxic to cell cultures, present in certain crude trypsin samples, are removed during a purification involving centrifugation, dialysis, treatment with a cellulose ion-exchange resin, removal of salts, and lyophilization. While the fundamental use of this proteolytic mixture would be to prepare primary cell culture, the broad peptide bond specifleity of this mixture would suggest application in cases where a general protease, free of other enzymatic activities, is required.     

A differential scanning calorimetry study of the interaction of free fatty acids with phospholipid membranes

Mixtures of stearic, arachic, oleic and linoleic acids with dimyristoylphosphatidylcholine (DMPC) and distearylphosphatidylcholine (DSPC) have been studied by means of differential scanning calorimetry (DSC). The mixtures of stearic (SA) and arachic acids (AA) with DMPC and DSPC show phase diagrams of the peritectic type, with a region of solid phase immiscibility from 0 to 28.5 mol% of fatty acid. A pure component, with a stoichiometry fatty acid/phospholipid (2:1) seems to be formed except for the system AA/DSPC. The mixtures of oleic (OA) and linoleic acids (LA) show complex phase diagrams. In the case of OA, different regions where a phase separation exists can be observed and for the mixture of OA with DMPC, a pure component seems to be formed with a stoichiometry OA/DMPC (1:2). LA shows different behaviour in the mixtures with DMPC and with DSPC. For the mixture, LA/DMPC, a fluid phase immiscibility region is observed over the same range of concentration as for the mixture with OA, however, the mixture with DSPC shows a solid phase immiscibility for the samples containing 45 mol% or more of LA. The interaction of the different free fatty acids with equimolar mixtures of DMPC and DSPC, showing monotectic behaviour, has also been analyzed. From our results it can be clearly concluded that saturated fatty acids partition preferentially into solid-like domains, while cis-unsaturated fatty acids partition preferentially into fluid-like domains.     

Temperature dependence of the structural dimensions of the inverted hexagonal (HII) phase of phosphatidylethanolamine-containing membranes

The characteristic temperature dependence of the lattice basis vector length d of phospholipid-water systems in the inverted hexagonal (HII) phase has been investigated with X-ray diffraction. For 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), d falls sharply from 78.1 A at 10 degrees C to 62.5 A at 90 degrees C. When used in conjunction with the volume fractions of the constituents, d can be used to determine the dimensions within the lipid and water regions. These data showed that a reduction in the radius of the HII-phase water cylinders Rw accounted for most of the reduction in d. From geometrical relationships between the dimensions in the HII phase, it was shown that both d and Rw are sensitive functions of the thickness of the lipid monolayer dHII. The characteristic shape of d(T) could be parameterized with the small temperature dependence of dHII along with the ratio v/a, which is the ratio of the specific volume to the area per lipid molecule at the polar interface. The ratio v/a was found to be independent of temperature for the fully hydrated HII system. Additional measurements made with a mixture of DOPE and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), mole ratio 5.07:1, produced a similar parameterization of d(T). The larger basis vector lengths for this mixture compared to those for DOPE can be attributed to a smaller ratio of v/a, which was also found to be temperature independent for this mixture. The smaller value of v/a is due to the larger effective headgroup area of DOPC.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the Composition of “Viscol” and the Employment of Gum Arabic for Mounting Media

“Viscol”, a water soluble permanent mounting medium hardening through evaporation of water under the cover glass, has been analyzed. It proves to consist of a mixture of gum arabic, glycerol, phenol and water and is especially suitable for simple botanical preparations. The use of gum arabic for hardening permanent mounting media is reviewed. Instead of a glycerol-phenol-water mixture a lactophenol, potassium acetate or zinc chloride solution mixed with gum arabic may be used for a permanent mounting medium. However, gum arabic contains calcium, magnesium and potassium ions giving crystals with the solutions mentioned. In the case of lactophenol and potassium acetate, the calcium and magnesium ions must be removed beforehand, which is done by precipitation with sodium or potassium carbonate. In the case of zinc chloride the potassium ions must be removed, which is done by dialysis with zinc chloride. It is pointed out that the same principles may be used for a great variety of different mounting media.     

Estimating the occurrence of false positives and false negatives in microarray studies by approximating and partitioning the empirical distribution of p-values

MOTIVATION: The occurrence of false positives and false negatives in a microarray analysis could be easily estimated if the distribution of p-values were approximated and then expressed as a mixture of null and alternative densities. Essentially any distribution of p-values can be expressed as such a mixture by extracting a uniform density from it. RESULTS: The occurrence of false positives and false negatives in a microarray analysis could be easily estimated if the distribution of p-values were approximated and then expressed as a mixture of null and alternative densities. Essentially any distribution of p-values can be expressed as such a mixture by extracting a uniform density from it. AVAILABILITY: An S-plus function library is available from http://www.stjuderesearch.org/statistics.     

Non-canonical nucleotide exchange catalyzed by Escherichia coli DNA-polymerase I and the two-center model of the mechanism of action of this enzyme

It is shown, that DNA hydrolysis catalyzed by E. coli DNA polymerase I is inhibited, when a reaction mixture contains one type of deoxynucleoside 5'-triphosphate (dNTP). When the reaction mixture contains [32P]dNTP, then [32P] is incorporated into DNA and v. v. (32P) from DNA is transferred into dNTP. The nucleotide exchange between DNA and dNTP in the assay mixture is observed only in the case, when the chemical nature of nucleotide residue of dNTP and that of the 3'-terminus of DNA is the same. Analysis of products of DNA hydrolysis in the presence of one type of dNTP using electrophoresis in polyacrylamide gel shows that most of the DNA molecules are terminated at the 3'-termini by the dNMP residue of the same chemical nature as the dNTP in the assay mixture. However, in some cases DNA molecules contain one additional nucleotide residue. This phenomenon can be explained by incorporation of one additional dNMP residue originating from dNTP only in those cases, when a non-typical base pairing of this nucleotide residue with a template residue readily takes place. The above-mentioned facts can be interpreted within the model for DNA hydrolysis with involvement of two intermediate covalent forms of dNMP residues with DNA polymerase I; one dNMP-intermediate should be placed at the elongation center and the other--at the hydrolysis center. The DNA hydrolysis by 3'----5' exonuclease activity of DNA polymerase I proceeds through these two covalent forms. DNA polymerases alpha from calf thymus and T4 phage do not catalyze the nucleotide exchange between DNA and dNTP from the reaction media.     

A new method for analysis of components in a mixture without preseparation: evaluation of the concentration ratio and protein-protein interaction

A new method for evaluating the ratio of two structurally different proteins or other compounds in a mixture that does not require any preseparation steps and is based on using the aqueous two-phase partition technique is described. Mathematical analysis of the partitioning of a mixture of two solutes demonstrates its ability to quantitatively evaluate the ratio of the concentrations of the solutes in the initial mixture. It also provides the means for detecting interactions between the two solutes. Experimental results confirm the analysis to be correct for mixtures of low-molecular-weight compounds as well as for mixtures of structurally different proteins. An example of analysis of a clinically relevant mixture of two proteins-transferrin and carbohydrate-deficient transferrin-is provided. An analysis of interactions between two proteins and/or peptides is also illustrated using two examples.     

Analytical processing of binary mixture information by olfactory bulb glomeruli

Odors are rarely composed of a single compound, but rather contain a large and complex variety of chemical components. Often, these mixtures are perceived as having unique qualities that can be quite different than the combination of their components. In many cases, a majority of the components of a mixture cannot be individually identified. This synthetic processing of odor information suggests that individual component representations of the mixture must interact somewhere along the olfactory pathway. The anatomical nature of sensory neuron input into segregated glomeruli with the bulb suggests that initial input of odor information into the bulb is analytic. However, a large network of interneurons within the olfactory bulb could allow for mixture interactions via mechanisms such as lateral inhibition. Currently in mammals, it is unclear if postsynaptic mitral/tufted cell glomerular mixture responses reflect the analytical mixture input, or provide the initial basis for synthetic processing with the olfactory system. To address this, olfactory bulb glomerular binary mixture representations were compared to representations of each component using transgenic mice expressing the calcium indicator G-CaMP2 in olfactory bulb mitral/tufted cells. Overall, dorsal surface mixture representations showed little mixture interaction and often appeared as a simple combination of the component representations. Based on this, it is concluded that dorsal surface glomerular mixture representations remain largely analytical with nearly all component information preserved.     

Isolation of induced mutations in E. coli affecting the electrophoretic mobility of enzymes

A screening method was developed for detection of bacterial mutants having active enzymes with altered electrophoretic mobility. The method is based on the use of a mixture of several clones, and examination of an extract of the mixture electrophoretically. A variant enzyme will thus be detectable by its position apart from the mixture of wild-type enzymes.Following exposure to a mutagenic agent, five mutants of E. coli K12 were detected and isolated. Two of these have variant MDH (malate dehydrogenates), the others have variant forms of 6-phosphogluconate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and esterase.Preliminary mapping of the MDH locus has been performed.     

Stereochemical nature of the products of linoleic acid oxidation catalyzed by lipoxygenases from potato and soybean

When linoleic acid was incubated with the purified potato lipoxygenase under O2 atmosphere, a mixture of 9 and 13-hydroperoxyoctadecadienoic acids was formed. Stereochemical analysis of the respective methyl-hydroxyoctadecadienoic acids revealed that the 9-isomer was in S-configuration whereas 13-hydroxyoctadecadienoic acid was a mixture of S (39%) and R (61%). Exactly the opposite was the case with the soybean lipoxygenase products, where the 13-isomer was found to be in S-configuration and 9-hydroxyoctadecadienoic acid - a mixture of S (73%) and R (27%). A general scheme is proposed for the stereochemical nature of oxidation products of enzymes which are predominantly either [+2] or [-2] lipoxygenases.