Species differences in the hepatotoxicity of coumarin: a comparison of rat and Mongolian gerbil.

The acute hepatic effects of coumarin (2H-1-benzopyran-2-one) in male Wistar rats and Mongolian gerbils has been compared. A single dose of coumarin (125 mg/kg, intraperitoneally (i.p.)) was hepatotoxic to rats within 24 h as assessed by its effects on a variety of hepatic parameters. Coumarin-induced hepatotoxicity was associated with significant increases in relative liver weight, plasma alanine and aspartate aminotransferase activities and hepatic non-protein sulphydryl groups. Cytochrome P-450 content and 7-ethoxycoumarin O-deethylase and glucose 6-phosphatase activities were significantly lower in coumarin-treated compared with control rats. Centrilobular necrosis was only observed in two out of six rats at this dose, but was present in all four coumarin-treated rats when the dose was increased to 150 mg/kg. In contrast to the effects observed in the rat, no evidence was found for coumarin-induced hepatotoxicity in gerbils following a single i.p. dose of 125 mg/kg. These data indicate that the gerbil is less sensitive to the hepatotoxic effects of coumarin than the rat.

Why, when and how in vitro tests should be accepted into regulatory toxicology.

The use of in vitro techniques in toxicological research is widespread, but, up to now, relatively little progress has been made in applying the knowledge gained in regulatory toxicity testing. In vitro tests should be accepted into regulatory toxicology for at least five reasons: scientific, humanitarian, legislative, logistical and economic. In particular, in vitro tests have the potential to provide a mechanistic basis for toxicity testing, and they may permit the use of tissues from more-appropriate target species and individuals, including humans. The relevance and reliability of the in vitro test, with regard to its use for a particular purpose and with particular types of chemicals, should have been adequately demonstrated (i.e. it should have been validated) prior to regulatory acceptance. There are several obstacles to this, including whether validation should be based on comparisons between in vitro data and animal data, and whether the in vitro tests should be expected to provide regulators with the same kinds of predictions and classification criteria that they currently obtain from animal tests. Regulatory incorporation should be a permissive process, rather than a restrictive one. Any scientifically defensible in vitro test which has been properly validated and independently recommended, should be acceptable for the specific purposes for which its use would be appropriate.

The validation and acceptance of alternatives to animal testing.

Validation is the key to the regulatory status of alternative methods. A series of questions are put, to which answers are given, including the following: What is validation? What is meant by "relevance", "reliability" and "purpose"? Why and when is formal validation necessary? What comes before and after a formal validation study? How have validation criteria been defined, and to what extent have they been harmonized internationally? How are validation studies set up, managed and funded? What is a test? Do prediction models have to be validated? What is prevalidation? What is acceptance, and who is responsible for acceptance? How are validation studies reported? How should a validated test be defined and recognized? Must all new tests be validated? Are the same standards being applied to new in vitro and new in vivo tests? Has validation been successful so far? What can be done to improve validation? Is validation helping or hindering the development of in vitro toxicology and the implementation of the 'Three Rs' of Russell & Burch?

In vitro cytotoxicity tests for the prediction of acute toxicity in vivo.

Investigations of the use of in vitro cytotoxicity tests for the prediction of acute toxicity in vivo have been reviewed with particular emphasis on those studies that have been published during the past 5 years. Numerous cell types, endpoints and exposure periods have been used in cytotoxicity tests, although these appear generally to have little effect on the resulting correlation between in vitro IC(50) values and in vivo LD(50) values. The in vitro data correlate better with rodent parenteral (ip or iv) LD(50) values than with oral LD(50) values due to kinetic considerations. For certain groups of related chemicals (e.g. antitumour compounds, metal salts), and for some sets of unrelated chemicals, the in vitro data correlate very well with LD(50) values. However, while cytotoxicity tests are useful for screening chemicals for their intrinsic and relative toxicities, it is impossible to tell whether predictions based on cytotoxicity data alone would be sufficiently accurate for labelling and classifying a new chemical according to its likely acute toxicity in vivo. The in vitro endpoints need to be of greater relevance to the possible mechanisms of chemically-induced acute toxicity in vivo than most of those that are used at present.

Toxicity of coumarin and various methyl derivatives in cultures of rat hepatocytes and V79 cells.

The toxicity of coumarin and various simple methyl derivatives in rat hepatocyte and V79 cell cultures was studied to investigate further the mechanism of coumarin hepatotoxicity. Coumarin was six times more toxic in hepatocyte cultures from phenobarbitone (PB)-treated rats than in those from untreated rats. At concentrations below 3 mm, coumarin did not affect the survival of V79 lung fibroblasts. SKF-525A inhibited coumarin-induced toxicity in hepatocytes cultured from PB-treated rats, whereas depletion of hepatocyte glutathione (GSH) levels with buthionine sulphoximine (BSO) significantly increased toxicity. Dihydrocoumarin (DHC) had little effect on the survival of cultured hepatocytes, indicating that the 3,4-double bond is an important determinant of coumarin toxicity. In general, the toxicity of coumarin in hepatocyte cultures was reduced by substitution with one or more methyl groups. 3-Methylcoumarin (MeC), however, was more toxic than coumarin itself in hepatocyte cultures from untreated rats. Except for 3,4-diMeC, the methyl derivatives were markedly more toxic in rat hepatocytes than in V79 cell cultures. The data obtained for coumarin and 4-MeC, and possibly 6-MeC and 7-MeC, are consistent with hepatocyte toxicity being due to the cytochrome P-450-dependent formation of one or more toxic metabolites that may be detoxified by reacting with GSH. This was less apparent for 3-MeC and 3,4-diMeC, although depletion of GSH levels significantly increased the hepatocyte toxicity of both compounds.

Biliary excretion of fluorescent cholephiles in hepatocyte couplets: An in vitro model for hepatobiliary and hepatotoxicity studies.

Hepatocyte couplets (26.8% of cell preparation with >85% viability) were prepared from rat livers. The preparation maintained the activity of 7-ethoxycoumarin-O-deethylase throughout an 8-hr incubation. Couplets extensively secreted the fluorescent cholephiles, fluorescein and cholyl-lysyl-fluorescein isothiocyanate, into sealed canalicular spaces within 2 hr and 15-30 min, respectively, which is indicative of functional tight junctions. The time scale for biliary secretion of the cholephiles correlated well with the relative kinetics of biliary excretion reported in vivo. Hepatocyte couplets may prove useful for studies on toxicity directed towards the hepatobiliary system.

Gene expression analysis of EpiDerm following exposure to SLS using cDNA microarrays.

There is a need to investigate the mechanistic basis of the human skin irritation response if relevant in vitro test systems for the predictive identification of skin irritation hazards are to be developed. Recent progress in genomics technologies mean that tools for the identification and investigation of important biochemical events in the processes of skin irritation are now available. The aim of this work was to identify genes (for further mechanistic investigation) which may be regulated in response to skin irritation, following exposure of the EpiDerm skin model to the known skin irritant sodium lauryl sulphate (SLS). EpiDerm cultures were treated in triplicate with a non-cytotoxic dose of SLS (0.1 mg/ml, as determined by the MTT assay and histological examination) for 15 min, 30 min, 1 h, 2 h, 3 h, 4 h and 24 h. Total RNA was extracted from the pooled EpiDerm cultures and used to probe Atlas human arrays (Clontech) covering approximately 3600 genes. Preliminary data indicated an up-regulation at early time points (15-30 min) of a number of genes involved in transportation (e.g. the sodium and chloride dependent taurine transporter) and receptors (e.g. ZAP70 and protocadherin 42 precursor). The gene encoding the UV excision repair protein and other DNA repair genes (e.g. DNA-directed RNA polymerase II) were up-regulated after 1-3 h, along with TGF beta 3 and other tumour suppressors, which play a role in cellular development and wound healing. At the later time points of 4-24 h, genes involved in protein translation (e.g. Cathepsin D receptor) and metabolism (e.g. CYP27A) were up-regulated. In addition, a number of genes were down-regulated in response to treatment with SLS, although these followed less of a time dependent pattern. These results indicate the differential regulation of a number of genes in response to treatment with SLS, some of which may provide additional clues to the molecular events underpinning the irritation response to this particular surfactant and possibly to other chemical irritants.

Validation-Lessons learned from practical experience.

With regard to the problems encountered and the experience gained in validation studies conducted in the past, suggestions have been made concerning criteria for the selection of the tests and laboratories to be included in a validation study, the selection and distribution of test chemicals, and procedures for the handling, analysis and interpretation of the resulting data. In particular, tests should have been developed to the extent that detailed protocols and standard operating procedures have been produced and evaluated. The laboratories should be chosen on the basis of evidence of their appropriate experience, competence and ability to comply with good laboratory practice (GLP) requirements. The choice of test chemicals depends primarily on the goals of the validation study and on the availability of reliable in vivo toxicity data of high quality. A biostatistician should be involved in the initial design of the validation study as well as in the analysis of the resulting data. The quality of the in vivo and in vitro data must be ensured, prior to determining the reproducibility and predictivity of the alternative test.

The ECVAM International Validation Study on In Vitro Tests for Skin Corrosivity. 1. Selection and Distribution of the Test Chemicals.

An international validation study on in vitro tests for skin corrosivity was conducted during 1996 and 1997 under the auspices of the European Centre for the Validation of Alternative Methods (ECVAM). The main objectives of the study were to assess the performances of selected in vitro tests in discriminating between: (a) corrosives (C) and non-corrosives (NC), for selected groups of chemicals (e.g. organic acids, phenols) and/or for all chemicals (single chemical entities only); and (b) known R35 (UN packing group I) and R34 (UN packing groups II & III) chemicals. Each test was evaluated for reliability and relevance by using a test set of 60 coded chemicals. In this paper, the test chemicals used in the validation study are identified; they include organic acids (6C/5NC), organic bases (7C/3NC), neutral organics (9NC), phenols (2C/3NC), inorganic acids (6C/1NC), inorganic bases (2C/2NC), inorganic salts (1C/2NC), electrophiles (3C/5NC) and soaps/surfactants (3NC). The in vivo classifications and important physicochemical properties (e.g. logP, pKa) of the test chemicals are given. The main criterion for including chemicals in the test set was that their corrosivity classifications were based on unequivocal animal data. Where available, structure-activity information was also used to support the corrosivity classifications. Despite the small numbers of chemicals in some of the categories, it was felt that the test set chosen represented the best possible for evaluating the performances of the in vitro tests for predicting skin corrosivity, given the limited availability of unequivocal animal data. The prediction of skin corrosivity from pH data was also investigated for those chemicals with extreme pH values (i.e. pH2 or 11.5). Nine of the 12 strongly acidic or alkaline chemicals in the test set, which were predicted to be C on the basis of their pH values, had also been found to be C in vivo.

The ECVAM International Validation Study on In Vitro Tests for Skin Corrosivity. 2. Results and Evaluation by the Management Team.

As a follow-up to a prevalidation study on in vitro tests for replacing the in vivo rabbit test for skin corrosivity, an international validation study was conducted during 1996 and 1997 under the auspices of ECVAM. The main objectives of the study were to: (a) identify tests capable of discriminating corrosives from non-corrosives for selected types of chemicals and/or all chemicals; and (b) determine whether these tests could identify correctly known R35 (UN packing group I) and R34 (UN packing groups II & III) chemicals. The tests evaluated were the rat skin transcutaneous electrical resistance (TER) assay, CORROSITEX(TM), the Skin(2TM) ZK1350 corrosivity test and EPISKIN(TM). Each test was conducted in three independent laboratories. 60 coded chemicals were tested. All of the tests evaluated showed acceptable intralaboratory and interlaboratory reproducibilities, and the TER, Skin(2) and EPISKIN tests proved applicable to testing a diverse group of chemicals of different physical forms, including organic acids, organic bases, neutral organics, inorganic acids, inorganic bases, inorganic salts, electrophiles, phenols and soaps/surfactants. Two of the four tests evaluated, the TER assay and EPISKIN, met the criteria agreed by the Management Team concerning acceptable underprediction and overprediction rates for them to be considered scientifically validated for use as replacements for the animal test for distinguishing between corrosive and non-corrosive chemicals for all of the chemical types studied [objective (a)]. EPISKIN was the only test able to distinguish between known R35 (UN packing group I) and R34 (UN packing groups II & III) chemicals, for all of the chemical types included, on an acceptable number of occasions [objective (b)]. The corrosive potentials of about 40% of the test chemicals could not be assessed with CORROSITEX, and the assay did not meet all of the criteria for it to be considered acceptable as a replacement test. However, CORROSITEX may be valid for testing specific classes of chemicals, such as organic bases and inorganic acids. The Skin(2) assay did not meet the criteria for it to be considered scientifically validated. Thus, the validities of (i) the TER and EPISKIN assays for discriminating corrosives from non-corrosives, and (ii) the EPISKIN assay for identifying correctly known R35/I and R34/II & III chemicals, have been demonstrated in this study. CORROSITEX appears to be valid when used only with certain types of chemicals.

A prevalidation study on in vitro tests for acute skin irritation. results and evaluation by the Management Team.

A prevalidation study on in vitro tests for acute skin irritation was conducted during 1999 and 2000. The overall objective of validation in this area, of which this prevalidation study is an initial stage, is to identify tests capable of discriminating irritants (I) from non-irritants (NI), as defined according to European Union (EU) risk phrases ("R38"; no classification) and the harmonised OECD criteria ("Irritant"; no label). This prevalidation study specifically addressed aspects of: protocol refinement (phase I), protocol transfer (phase II), and protocol performance (phase III), in accordance with the prevalidation scheme defined by the European Centre for the Validation of Alternative Methods (ECVAM). The tests evaluated were: EpiDerm (phases I, II and III), EPISKIN (phases I, II and III), PREDISKIN (phases I and II, and additional protocol refinement), the non-perfused pig ear method (phases I and II, and additional protocol refinement), and the mouse skin integrity function test (SIFT; phases I and II). Modified, standardised test protocols and well-defined prediction models were available for each of the tests at the end of phase I. The results of phase I (intralaboratory reproducibility) were sufficiently promising for all of the tests to progress to phase II. Protocol transfer between the Lead Laboratory and Laboratory 2 was undertaken for all five tests during phase II, and additional refinements were made to the test protocols. For EpiDerm, EPISKIN and the SIFT, the intralaboratory and interlaboratory reproducibilities were acceptable; however, better standardisation of certain aspects of the test protocols was needed prior to commencing phase III. Neither PREDISKIN nor the pig ear test performed sufficiently well in phase II to progress to phase III. The PREDISKIN protocol was overly sensitive, resulting in the prediction of all the NI chemicals as I. The variability in the pig ear test results was too great, indicating that the test would show limited predictive ability. In additional studies (a repeat of phase I), further modification of the PREDISKIN protocol and a change in the prediction model considerably improved the ability of the test to distinguish I from NI chemicals. However, attempts to improve the intralaboratory reproducibility of the pig ear test were unsuccessful. In phase III an initial assessment of the reproducibility and predictive ability, in three independent laboratories per test, was undertaken for the EpiDerm and EPISKIN tests (the SIFT was a late inclusion in the prevalidation study, and is being evaluated in a separate phase III study). A set of 20 coded chemicals (10 I, 10 NI) were tested with the final, refined, test protocols. The intralaboratory reproducibility was acceptable for both EpiDerm and EPISKIN. The interlaboratory reproducibility was considered to be acceptable for EPISKIN; however, for EpiDerm, analysis of variance (ANOVA) indicated that there was a statistically significant laboratory effect on the overall variability, suggesting that the interlaboratory transferability of the test needs to be improved. The EpiDerm test had an overall accuracy of 58%, with an over-prediction rate of 37% and an under-prediction rate of 47%. The EPISKIN test had an overall accuracy of 58%, showing an under-prediction rate of 23% and an over-prediction rate of 60%. It is concluded that, as yet, none of the tests evaluated in this prevalidation study are ready for inclusion in a formal validation study on in vitro tests for acute skin irritation. Overall protocol performance of the SIFT is currently being evaluated in a phase III study. Further studies are also in progress to improve the test protocols and prediction models for EpiDerm and EPISKIN.

Non-animal testing strategies for assessment of the skin corrosion and skin irritation potential of ingredients and finished products.

The dermatotoxicologist today is faced with a dilemma. Protection of workers and consumers from skin toxicities (irritation and allergy) associated with exposure to products, and the ingredients they contain, requires toxicological skin testing prior to manufacture, transport, or marketing. Testing for skin corrosion or irritation has traditionally been conducted in animals, particularly in rabbits via the long established Draize test method. However, this procedure, among others, has been subject to criticism, both for its limited predictive capacity for human toxicity, as well as for its use of animals. In fact, legislation is pending in the European Union which would ban the sale of cosmetic products, the ingredients of which have been tested in animals. These considerations, and advancements in both in vitro skin biology and clinical testing, have helped drive an intensive effort among skin scientists to develop alternative test methods based either on in vitro test systems (e.g. using rat, pig or human skin ex vivo, or reconstructed human skin models) or ethical clinical approaches (human volunteer studies). Tools are now in place today to enable a thorough skin corrosion and irritation assessment of new ingredients and products without the need to test in animals. Herein, we describe general testing strategies and new test methods for the assessment of skin corrosion and irritation. The methods described, and utilized within industry today, provide a framework for the practicing toxicologist to support new product development initiatives through the use of reliable skin safety testing and risk assessment tools and strategies.

Structural requirements for the direct and cytochrome P450-dependent reaction of cyclic alpha,beta-unsaturated carbonyl compounds with glutathione: a study with coumarin and related compounds.

The interaction of glutathione (GSH) with coumarin, or one of a series of compounds related to coumarin, was assessed in the absence and presence of liver microsomes (direct reaction and indirect reaction, respectively) to determine the structural requirements for direct and mono-oxygenase-mediated reaction of cyclic alpha,beta-unsaturated carbonyls with GSH. Acrolein was used as a positive control for the direct reaction, and produced complete or nearly complete depletion of GSH under all assay conditions. 5,6-Dihydro-2H-pyran-2-one and 2-cyclohexen-1-one also produced substantial depletion of GSH in the direct reaction, which was not increased by the addition of liver microsomes. Coumarin, 2H-pyran-2-one and precocene I (a substituted pyran lacking the 2-one structure) were not substrates for the direct reaction but did cause depletion of GSH when incubated in the presence of rat or human liver microsomes. These depletions were dependent on a functioning mono-oxygenase system as judged by the effects of omission of cofactors, addition of competitive or inactivating inhibitors of cytochrome P450, and induction. Dihydrocoumarin, delta-valerolactone, cyclohexanone and 4H-pyran-4-one were not substrates for either the direct or indirect reaction. These findings are rationalized on the basis of a direct nucleophilic attack of GSH on the alpha,beta-centre of the alpha,beta-unsaturated carbonyl compounds, which is hindered by benzenoid resonance in coumarin and 2H-pyran-2-one, for which enzyme-mediated reaction with GSH, probably via a 3,4-epoxide, is the favoured mechanism.

Progress Toward the Validation of Alternative Tests.

ECVAM's role in the practical validation of replacement alternative methods for use in regulatory testing is reviewed, including an outline of the criteria which have been used in determining ECVAM's priorities. Some of the difficulties which have arisen in validation studies are discussed, and solutions to these are proposed, with particular emphasis on ensuring that methods are sufficiently well-developed to enter the validation process, and on the ECVAM prevalidation scheme for encouraging protocol optimisation and the prior assessment of interlaboratory transferability. Comments are made on problems encountered in selecting test materials backed by adequate in vivo data and in undertaking appropriate in vivo/in vitro comparisons.

Comparative evaluation of five in vitro tests for assessing the eye irritation potential of hair-care products.

This study compared five methods, the isolated rabbit eye (IRE), bovine corneal opacity and permeability (BCOP), EpiOcular, fluorescein leakage (FL) and neutral red release (NRR) assays, for predicting the eye irritation potential of hair-care formulations. Ten shampoo and seven conditioner formulations of known ocular irritation potential were tested. Each group included a market-acceptable formulation as a comparative benchmark. Predictions of ocular irritation were made by using classification models (IRE, BCOP and EpiOcular assays) or by direct comparison with benchmarks (IRE, EpiOcular, FL and NRR assays). The BCOP assay was less sensitive than the IRE test in discriminating between formulations of different irritation potentials, and did not perform as well as the other assays in identifying mild formulations. All of the assays appeared to be better at discriminating correctly between the shampoos than between the conditioners. The EpiOcular assay showed the closest concordance between the in vivo results and the in vitro data from cell-based assays (particularly for shampoos). The FL assay also showed a high concordance (particularly for conditioners). There was a tendency for these in vitro assays to over-predict eye irritation potential, but there was no under-prediction and they were particularly successful at identifying mild formulations. The NRR assay was less predictive with both shampoos and conditioners. The results from this comparative evaluation fully support the continued use of the IRE test as a suitable alternative to in vivo eye irritation testing in rabbits, although it also over-predicted the irritancies of several of the formulations. The value of using concurrent benchmarks (reference standards), appropriate to the materials being tested, in interpreting the data obtained from in vitro tests, was also demonstrated. Overall, the results indicate that further comparisons of the IRE, EpiOcular and FL assays are warranted using much larger numbers of test materials.

An evaluation of the proposed OECD testing strategy for skin corrosion.

The use of testing strategies which incorporate a range of alternative methods and which use animals only as a last resort is widely considered to provide a reliable way of predicting chemical toxicity while minimising animal testing. The widespread concern over the severity of the Draize rabbit test for assessing skin irritation and corrosion led to the proposal of a stepwise testing strategy at an OECD workshop in January 1996. Subsequently, the proposed testing strategy was adopted, with minor modifications, by the OECD Advisory Group on Harmonization of Classification and Labelling. This article reports an evaluation of the proposed OECD testing strategy as it relates to the classification of skin corrosives. By using a set of 60 chemicals, an assessment was made of the effect of applying three steps in the strategy, taken both individually and in sequence. The results indicate that chemicals can be classified as corrosive (C) or non-corrosive (NC) with sufficient reliability by the sequential application of three alternative methods, i.e., structure-activity relationships (where available), pH measurements, and a single in vitro method (either the rat skin transcutaneous electrical resistance (TER) assay or the EPISKIN™ assay). It is concluded that the proposed OECD strategy for skin corrosion can be simplified without compromising its predictivity. For example, it does not appear necessary to measure acid/alkali reserve (buffering capacity) in addition to pH for the classification of pure chemicals.

Eye Irritation Testing: The Way Forward. The Report and Recommendations of ECVAM Workshop 34.

This is the report of the thirty-fourth of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM's main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine or replace the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of procedures which would enable it to become well-informed about the state-of-the-art of non-animal test development and validation, and the potential for the possible incorporation of alternative tests into regulatory procedures. It was decided that this would be best achieved by the organisation of ECVAM workshops on specific topics, at which small groups of invited experts would review the current status of various types of in vitro tests and their potential uses, and make recommendations about the best ways forward (1). The workshop on Eye Irritation Testing: The Way Forward was held in Egham, UK, on 15-17 June 1998, under the chairmanship of Michael Balls (ECVAM, Italy). The workshop had two aims, the first of which was to review some of the previous multi-laboratory validation studies on alternatives to the Draize eye test and assess why many promising alternative methods were not successful in these studies. The second aim was to discuss strategies for making progress toward the short-term reduction, refinement, and eventual replacement, of the Draize test, including: a new approach to the validation of in vitro tests for eye irritancy, based on the use of reference standards, which promises to overcome some of the problems encountered in previous studies; the use of stepwise testing strategies which reduce and refine the use of animals in eye irritation testing; the use of multivariate and other statistical techniques for the further analysis of data generated in previous validation studies; and a programme of research aimed at understanding the underlying mechanisms of eye irritation.