Correlative and mechanistic QSAR models in toxicology.

An outline is provided on the development and use of correlative and mechanistic approaches to predictive toxicology, with particular emphasis on the experience at the U.S. EPA as applied to assessing the potential hazard posed by new industrial chemicals for which little or no test data are provided under the Toxic Substances Control Act. This information is presented with a historical perspective.

Comparison of the up-and-down, conventional LD50, and fixed-dose acute toxicity procedures.

The up-and-down procedure (UDP), fixed-dose procedure (FDP) and conventional LD50 tests were compared to determine their consistency in chemical hazard classification for acute oral toxicity according to the European Economic Community (EEC) system. There was consistent classification for 23 out of 25 cases between the UDP and the conventional LD50 results, in 16 out of 20 cases between the FDP and the conventional LD50, and in seven out of 10 cases between the UDP and the FDP. The UDP needed only between six and 10 animals of one sex (fewer than either the LD50 or the FDP). Available literature indicates that the sexes are usually similar in their acute toxicity responses and that of females are often more sensitive than males when acute toxicity differences do exist, thus obviating the need for both sexes to be tested in most cases. Unlike the FDP, the UDP also estimates an LD50, thus providing data directly applicable to all current hazard classification systems based on acute oral toxicity.

Framework for validation and implementation of in vitro toxicity tests.

The development and application of in vitro alternatives designed to reduce or replace the use of animals, or to lessen the distress and discomfort of laboratory animals, is a rapidly developing trend in toxicology. However, at present there is no formal administrative process to organize, coordinate, or evaluate validation activities. A framework capable of fostering the validation of new methods is essential for the effective transfer of new technologic developments from the research laboratory into practical use. This committee has identified four essential validation resources: chemical bank(s), cell and tissue banks, a data bank, and reference laboratories. The creation of a Scientific Advisory Board composed of experts in the various aspects and endpoints of toxicity testing, and representing the academic, industrial, and regulatory communities, is recommended. Test validation acceptance is contingent on broad buy-in by disparate groups in the scientific community--academics, industry, and government. This is best achieved by early and frequent communication among parties and agreement on common goals. It is hoped that the creation of a validation infrastructure composed of the elements described in this report will facilitate scientific acceptance and utilization of alternative methodologies and speed implementation of replacement, reduction, and refinement alternatives in toxicity testing.

Report of the Validation and Technology Transfer Committee of the Johns Hopkins Center for Alternatives to Animal Testing. Framework for validation and implementation of in vitro toxicity tests.

The development and application of in vitro alternatives designed to reduce or replace the use of animals, or to lessen the distress and discomfort of laboratory animals, is a rapidly developing trend in toxicology. However, at present there is no formal administrative process to organize, coordinate, or evaluate validation activities. A framework capable of fostering the validation of new methods is essential for the effective transfer of new technological developments from the research laboratory into practical use. This committee has identified four essential validation resources: chemical bank(s), cell and tissue banks, a data bank, and reference laboratories. The creation of a Scientific Advisory Board composed of experts in the various aspects and endpoints of toxicity testing, and representing the academic, industrial and regulatory communities, is recommended. Test validation acceptance is contingent upon broad buy-in by disparate groups in the scientific community-academics, industry and government. This is best achieved by early and frequent communication among parties and agreement upon common goals. It is hoped that the creation of a validation infrastructure composed of the elements described in this report will facilitate scientific acceptance and utilization of alternative methodologies and speed implementation of replacement, reduction and refinement alternatives in toxicity testing.

Nikolai Vasilyevich Lazarev, toxicologist and pharmacologist, comes in from the cold.

Important and pioneering work on the physicochemical properties underlying the biological activity of nonelectrolytes was performed 'behind the iron curtain' by Soviet scientist Nikolai Vasilyevich Lazarev. In this Special Feature, Robert Lipnick and Vladimir Filov examine the contribution of Lazarev's numerous observations to the knowledge base in pharmacology and toxicology, which has only recently gained recognition in the West.

Outliers: their origin and use in the classification of molecular mechanisms of toxicity.

Quantitative structure-activity relationships (QSARs) provide a useful tool for defining a mathematical relationship between chemical structure and toxicity, and for applying such statistically derived models for predicting the toxicity of untested chemicals. Outlier chemicals can be encountered both in the derivation of QSAR models as well as in their application. Information regarding the relationship between molecular descriptors and molecular mechanism of toxicity can provide insight into the origin of outlier behavior as well as guidance regarding the predictive limitations of such models. Comparison of measured toxicity data for fish and rats with baseline QSAR prediction provides a means of identifying outliers and for categorizing more specific molecular mechanisms.

A QSAR study of the toxicity of amines to the fathead minnow.

Simple and multiple linear regressions were applied to the development of fish toxicity QSAR models for the 96-h LC50 to the fathead minnow, Pimephales promelas. The data on unbranched saturated primary alkylamines as well as the complete data set were well-fitted to linear QSAR models using log P or the valence first-order connectivity index (1XV) as descriptors. Although adding data on other subclasses of amines in this data set yield acceptable QSARs, only the tertiary amine subclass provided a poor fit with both of these descriptors. The amines include both acyclic and cyclic derivatives, either with no additional functional groups, or with the hydroxyl, keto, methoxy, and propargyl moieties. The molecular mechanism for fish toxicity of these amines as well as the outliers in the study were investigated. Based upon the calculated log P value of -1.40, tripropargylamine has an apparent excess toxicity of 84 times; in contrast, the measured shake-flask log P for this compound was subsequently found to be 1.26, giving a predicted LC50 consistent with the observed value. An upward curvature of the QSAR plot for the most hydrophilic compounds suggests a shift in mechanism for the lowest members of the series.

Hans Horst Meyer and the lipoid theory of narcosis.

This year marks the 90th anniversary of the publication of Hans Horst Meyer's classic paper in which he proposed that the ability of a substance to produce narcosis or anesthesia is governed by its partition coefficient. In this article, Robert Lipnick describes the experiments carried out by Meyer and his colleagues which disproved the earlier theories that potency was determined by the presence of particular functional groups or their metabolites or by water or fat solubility, and which led to the formulation of the lipoid theory of narcosis.

The toxicity of acetylenic alcohols to the fathead minnow, Pimephales promelas: narcosis and proelectrophile activation.

1. The 96-h LC50 values for 16 acetylenic alcohols in the fathead minnow (Pimephales promelas) were determined using continuous-flow diluters. The measured LC50 values for seven tertiary propargylic alcohols agreed closely with the QSAR predictions based upon data for other organic non-electrolytes acting by a narcosis mechanism. 2. Four primary and four secondary propargylic alcohols were 7 to 4600 times more toxic than the respective narcotic toxicity estimated by QSAR. Metabolic activation to electrophilic alpha,beta-unsaturated propargylic aldehydes or ketones is proposed to account for the increased toxicity. 3. 3-Butyn-1-ol and 4-pentyn-2-ol, primary and secondary homopropargylic alcohols, were 320 and 160, respectively, times more toxic than predicted. In this case an activation step involving biotransformation to an allenic electrophile intermediate was proposed.

A QSAR study of the acute toxicity of some industrial organic chemicals to goldfish. Narcosis, electrophile and proelectrophile mechanisms.

1. A baseline toxicity QSAR model was derived for the 24-h LC50 to the goldfish, Carassius auratus. 2. The QSAR-predicted LC50 values for six epoxide derivatives were 2.8-985 times greater than measured. The excess toxicity of these epoxides and other compounds was ascribed to an electrophile molecular mechanism involving SN2 reaction with sulphydryl and other neucleophile groups present in enzymes and other biological macromolecules. 3. The excess toxicities of allyl alcohol and pentaerythritol triallyl ether were interpreted in terms of proelectrophile mechanisms. For the latter compound, this involves a monooxygenase-mediated free radical proton abstraction to a stable allyl radical. The allyl-free radical can undergo enzymic free radical hydroxylation to afford the corresponding acetal, which on decomposition yields the Michael acceptor electrophile acrolein.

Structure and conformation of pseudouridine analogues.

The structural and conformational features of the "anomeric" DL-trans- and DL-cis-5-(3-hydroxytetrahydrofuran-2-yl)uracils (3a, 4a) and five similar analogues were studied in order to determine their applicability as models of beta- and alpha-pseudouridine. The 270-MHz proton NMR spectra were measured for all analogues to define their ring geometries in solution and to estimate the solution population of model N, S conformers in a two-state dynamic equilibrium treatment. Two sets of calculations were employed to evaluate the relative contributions of these states to the observed vicinal coupling constants related to the C(3')-C(4') fragment. In the first, similar geometries were assumed for each pair of conformers, while in the second, limited to 3, the geometries were those derived from X-ray crystallographic data; both gave comparable results. The cis analogues 4a and 4b are excellent conformational models for alpha-pseudouridine. In the trans series (3a-c), the equilibrium is weighted toward the N conformer (approximately 80%), differing from that found in beta-pseudouridine for which each model conformer is equally populated. Possible implications of the conformational effects upon the pairing properties of pseudouridine in tRNA are discussed.

A comparative proton magnetic resonance conformational study of the tRNA "wobble" nucleosides 5-carboxymethyl-, 5-methoxycarbonylmethyl-, and 5-carbamoylmethyl-uridine.

The 270-MHz proton magnetic resonance spectra of 5-carboxymethyluridine, 5-methoxycarbonylmethyluridine, and 5-carbamoylmethyluridine have been obtained, and analyzed iteratively using LAOCOON 3. A standard treatment of the obtained data reveals that in each the 5-substituent produces no significant alteration in the furanose puckers, their equilibrium state, and the relative populations of the gg, tg, and gt exocyclic hydroxymethyl rotamers relative to uridine. The analogous similarity in furanose chemical shifts suggests that these derivatives favor the anti glycosyl state. The 5-substituent may act at the polynucleotide level in modulating the conformation and interaction(s) of the anticodon loop.

A comparative conformational study of certain 2'-deoxy-2'-fluoro-arabinofuranosylcytosine nucleosides.

The 270 MHz proton NMR spectra of 2'-deoxy-2'-fluoro-beta-D-arabinofurano-syl-5-iodocytosine as well as the respective 5-bromo and 5-chloro derivatives have been iteratively analyzed using LAOCOON 3. The derived parameters indicate that the fluorine substituent in an arabino configuration results in an approx. 50 : 50 C(3')-endo C(2')-endo conformer mixture. The C(4')--C(5') rotamer populations are 41% gg, 18% tg, and 41% gt. These equilibrium distributions, which are maintained within the series studied, are similar to that of other ara-C derivatives, but quite different from that of 2'-deoxycytidine. The agreement in chemical shift for each of the furanose protons within the series studied suggests a similar syn, anti distribution for each analog.