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The main aim of the ACuteTox project, under EU 6th Framework programme, is to investigate whether animal toxicity tests for acute systemic toxicity could be replaced by a combination of alternative assays. Data for 97 reference chemicals was collected in the ACuteTox database (Acutoxbase), designed to handle invitro and invivo (human and animal) lodged data. The principal basis for demonstration of the applicability of invitro tests is the invitro-invivo modeling, by using statistical correlation between invitro IC50 molar values (the 50% inhibitory concentration for the endpoints measured) and human blood molar concentrations LC50 (50% lethal concentrations). The LC50 values were calculated from time-related sub-lethal and lethal blood concentrations determined from human acute poisoning cases. The 3T3 standard NRU assay (3T3 NRU) was chosen, among the various basal cytotoxicity assays, applied in the ACuteTox project, to demonstrate the applicability of the IC50/LC50 values for invitro-invivo modeling. Linear regression analysis between IC50 (x) and LC50 (y) gave an explained variance R2=0.56 for the 67 reference chemicals, for which both sets of data were available. The results demonstrated usefulness of human LC50 values for invitro-invivo evaluation of the predictability of basal cytotoxicity assays for human acute systemic toxicity. The R2 value of 0.56 shows, as in the MEIC study, that additional organ-specific and biokinetic tests are needed in order to improve the predictability.

The European Commission White Paper, “Strategy for a future chemicals policy” (EC, 2001) is estimated to require the testing of approximately 30,000 ‘existing’ chemicals by 2012. Recommended in vitro tests require validation. As the White Paper (EC, 2001) requires neurotoxic data, this study evaluated an in vitro testing strategy for predicting in vivo neurotoxicity. The sensitivities of differentiated PC12 cells and primary cerebellum granule cells (CGC) were compared to undifferentiated PC12 cells which can indicate basal cytotoxicity. Cytotoxicants and neurotoxicants selected for testing covered a range of mechanisms and potencies. Neurotoxicants were not distinguished from cytotoxicants despite significantly different cell system responses using all endpoints; cell viability/activity, ATP depletion, MMP depolarisation, ROS production and cytoskeleton modifications. For all chemicals tested, neuronal-like cell systems were generally less sensitive than undifferentiated PC12 cells. Acute oral rodent LD50 values correlated with cytotoxicity IC50 values for the respective chemicals tested in each cell system. This study concluded that although simple non-specific assays are required to distinguish basal cytotoxicity from specific neurotoxicity by using different cell systems with different states of neuronal differentiation, further work is required to determine suitable combinations of cell systems and endpoints capable of distinguishing neurotoxicants from cytotoxicants.

1,3-Dichloro-2-propanol (1,3-DCP) is a chlorinated compound used in the fabrication of industrial products such as hard resins, celluloid or paints. It has also been detected in instant soups and soy sauce. 1,3-DCP has been associated with major necrosis of the liver in humans [Chem.-Bio. Interact. 80 (1991) 73]. In humans and laboratory animals, 1,3-DCP is metabolised to dichloroacetone (1,3-DCA) by cytochromes P450 2E1 and 1A2 [J. University Occup. Environ. Health 14 (1992) 13]. 1,3-DCA is a hepatotoxin. We suggest that 1,3-DCA could be embryotoxic at doses that do not cause adverse maternal hepatic damage. To investigate the embryotoxic effects of 1,3-DCA, we have adapted a micromass culture method from Atterwill and colleagues [1992. A tiered system for in vitro neurotoxicity testing. In: Zbinden, G. (Ed.), The Brain in Bits and Pieces. Verlag M.T.C., Vollikon, pp. 89-91], using chick midbrain cells and from Wiger et al. [Pharmacol. Toxicol. 62 (1988) 32] using chick mesenchymal cells. The basis of the micromass system is that embryotoxins in vitro are likely to affect development and differentiation of disaggregated neuronal and limb bud micromass cultures. The endpoints chosen for the midbrain assay are resazurin reduction (viability), total protein content (cell number), morphological quantification of neuronal cultures (neuronal projection number) and of limb bud cultures (cartilage nodule number). Preliminary results using chick whole embryo cultures indicated that 1,3-DCA had an inhibitory effect on whole chick embryo development. We also found that embryonic derived cells were sensitive to 1,3-DCA but not 1,3-DCP at concentrations above 1 microM, suggesting a potential teratogenic effect of 1,3-DCA. The exposure to 1,3-DCP is not limited to industrial settings, and hence a better knowledge of its effects and tissue specific actions on embryonic-derived cells would be beneficial.

Embryonic stem cells, which resemble the undifferentiated cells of the epiblast in a blastocyst, are able to differentiate into derivatives of the primary germ layers, including cardiomyocytes. The effects of embryotoxic/teratogenic compounds on the differentiation of cells was examined by semi-quantification of the mRNA expression using the RT-PCR protocol. Alpha and beta myosin heavy chain (alpha-MHC and beta-MHC, respectively) mRNA expression were chosen as tissue-specific markers, characteristic of early cardiac muscle development. Nine chemicals were investigated, chosen according to their in vivo embryotoxic/teratogenic potential in mice. The teratogens all-trans retinoic acid (RA), 5-fluorouracil (5-FU), hydroxyurea (HU), diphenylhydantoin (DPH) and caffeine (Caff) caused a significant reduction in MHC mRNA expression at a dose lower than that required for cytotoxicity. Saccharin (Sacc) had a similar effect, while penicillin G (PenG), isoniazid (Iso) and cytarabine (Ara-C) only showed an effect on MHC mRNA expression when the cells had a significant loss in viability. The inability to identify the strong teratogen Ara-C is related to the inappropriate target tissue.

Imipramine is metabolized in vitro by rat, mouse or human microsomes to several metabolites including 2-hydroxyimipramine, 2-hydroxydesipramine, desipramine, didesmethylimipramine and imipramine-N-oxide. Heat treatment (which inhibits FMO), verified the role of this group of enzymes in the oxidation of imipramine to imipramine N-oxide. These studies demonstrate that while rats and humans showed similar metabolic profiles, producing several minor metabolites in addition to the 2-hydroxyimipramne and desipramine, differences were seen in the mouse microsomes. In these samples, few minor metabolites were produced. In addition, imipramine N-oxide was the major metabolite in mouse, whereas none was detected in human samples. To conclude, the metabolic profile of imipramine can be used to reveal a number of cytochrome P450 enzymes active in microsomal fractions.

The ability of Madin–Darby canine kidney cells (MDCK ) to form cell tight-junctions and orientate correctly on porous membranes has been exploited to model corneal barrier function. While the ability to monitor recovery profiles has not yet been included in a prevalidation trial, its inclusion facilitates the prediction of potential adverse reactions. Combining a viability assay (Alamar blue reduction) with the fluorescein leakage assay, the chemical effects on cell membrane and adhesion molecules can be distinguished. The dose–response curves obtained with Tween 20, isopropanol and benzalkonium chloride indicate that Tween 20 at 200 mg/ml caused a 50% decline in Alamar blue reduction and fluorescein retention, while 100 mg isopropanol/ml and 0.1 mg benzalkonium chloride/ml cause similar effects. The more severe the initial damage the longer the recovery period. If a 20% increase in fluorescein leakage gave recovery in 48 hours, 50% gave marginal recovery in 96 hours. Comparable effects were noted for the restoration of the ability to reduce the Alamar blue dye. Resolution of the damaging effects of benzalkonium chloride and isopropanol took longer than for Tween 20, as is the case in the rabbit in vivo.

Increased permeability of the microvascular endothelium is a component of the inflammatory response. Inflammatory mediators such as histamine contribute to this permeability change. Modulation of cytoskeletal F-actin has been implicated as part of the cellular mechanism involved. Permeability changes occur predominantly at the microvascular level while the majority of current knowledge stems from research on cells from large vessels. We have therefore utilized an immortalized human dermal microvascular cell line, HMEC-1. Confluent monolayers were exposed to histamine (10 mum, 100 mum) for 1, 5, 10 or 15 minutes. F-Actin changes were detected by labelling with FITC-conjugated phalloidin. Histamine exposure resulted in the rounding of cells with the formation of intercellular gaps. The percentage of rounded cells and the number of gaps increased with exposure time. F-actin was redistributed from a peripheral band in control cultures to a perinuclear zone. Continual presence of the agonist was required for these phenotypic changes to occur. Removal of histamine caused reversal of these observations. Cells exposed to histamine for 1 minute needed 15 minutes to recover their normal morphology and F-actin distribution. These reversible effects suggest that F-actin redistribution maybe part of the microvascular cell response to histamine.

To date, no standardized international guideline for the testing of chemicals for phototoxic potential has been accepted for regulatory purposes. In 1991, the European Commission (EC), represented initially by the Directorate General XI and later by ECVAM (the European Centre for the Validation of Alternative Methods) and COLIPA (the European Cosmetic, Toiletry and Perfumery Association), agreed to establish a joint EU/COLIPA programme on the development and validation of in vitro phototoxicity tests. The first phase (phase I, 1992-93) was designed as a prevalidation study, to identify in vitro test procedures and test protocols for a formal validation trial under blind conditions. In the second phase (phase II, 1994-95), the formal validation study, the most promising in vitro phototoxicity tests were validated with 30 carefully selected test chemicals in 11 laboratories in a blind trial. The 3T3 mouse fibroblast neutral red uptake phototoxicity test (3T3 NRU PT) was performed as a core test in nine laboratories, since it provided the best results in phase I of the study. The purpose of phase II was to confirm the reliability and relevance of the in vitro tests for predicting phototoxic effects and for identifying phototoxic chemicals. In phase II the phototoxic potential of test chemicals in the 3T3 NRU PT test was either assessed by determining the phototoxicity factor (PIF) by using a cut-off value of 5 as in phase I of the study, or by determining the mean photo effect (MPE) by using a cut-off value of 0.1, as recently proposed by Holzhütter (1997). Results obtained with both approaches in the 3T3 NRU PT test in phase II were reproducible in the nine laboratories, and the correlation between in vitro and in vivo data was very high. Therefore, ECVAM and COLIPA conclude from this formal validation trial under blind conditions that the 3T3 NRU PT test is a scientifically validated in vitro test which is ready to be considered for regulatory purposes for assessing the phototoxic potential of chemicals. A draft OECD Guideline for "In Vitro Phototoxicity Testing", incorporating the standard protocol of the 3T3 NRU PT test, will be submitted to the OECD test guidelines programme in due course.

A specific, mechanistic, in vitro approach for the assessment of human skin irritation potential is outlined for the evaluation of surfactants and the results compared with in vivo human patch test data. The level of free available surfactant monomer and the solubilization of the corn protein zein in vitro were confirmed to be related to surfactant in vivo human skin irritation potential. In vitro cytotoxicity to monolayer keratinocyte cultures could not discriminate between the moderate human skin irritant sodium dodecyl sulfate (SDS) and the mild irritants cocamidopropylbetaine (CA) and Polysorbate 20 (P20). An in vitro stratified differentiated human epidermal equivalent (HEE) exhibited reduced cytotoxicity to the test chemicals, compared with monolayer culture responses, and was able to discriminate between the toxic potential of SDS and CA. Stimulation of interleukin-1α release from the A431 human keratinocyte cell line reflected in vivo erythema scores more closely than cytotoxic potential, and coincided with nitric oxide production by macrophages upon exposure to A431-conditioned medium. Combination of these mechanistic assays has allowed a profile of likely in vivo human responses to be approximated. Additional knowledge of skin penetrability and rate of recovery from toxic damage would affirm these predictions.

The Fluorescein Leakage (FL) test, a short-term in vitro assay measuring damage on exposure to eye irritants to a transepithelial barrier permeability system, has been assessed as an alternative to the Draize rabbit eye irritation assay in the EU/Home Office international validation trial. The assay is based on the degree of leakage of sodium fluorescein through a confluent layer of Madin-Darby canine kidney epithelial (MDCK) cells grown on a tissue culture insert following exposure to a potential irritant. Several different types of tissue culture inserts with different membrane growth surfaces, pore sizes and pore densities have been employed in the past. Problems with chemical binding and other incompatibilities with insert membranes can affect the sensitivity of MDCK cells. Cationic surfactants in particular appear to interfere with the passage of fluorescein, leading to underestimation of damage to MDCK cellular function. A blind comparison between MDCK cells cultured on Anopore() and the Millicell-HA inserts using the EU/Home Office set of surfactants indicate that the sensitivity of cells cultured on Anopore inserts appeared to correlate more closely with in vivo data. The ability to microscopically evaluate the status of MDCK cultures through Anopore further recommends this membrane as possibly the better choice for the FL assay.

Rhodamine B has been used as a histopathological stain for keratinization and cornification. Its ability as an in vitro indicator of the degree of epidermal keratinization was investigated in these preliminary studies. An immortalized human keratinocyte cell line, SVK-14, was evaluated as an alternative to primary human keratinocytes. The influence of extracellular calcium levels was evaluated alongside the effects of exposure to 1,25 (OH)2 vitamin D3 in serum-free and serum-containing media. Alamar blue (AB) conversion was used to measure changes in cellular reductive potential, and the amount of bound Rhodamine B relative to total protein per well was taken as an indicator of keratinization. Exposure to 1,25 (OH)2 vitamin D3 for 7 or 10 days did not increase Rhodamine B binding to confluent SVK-14 cultures, regardless of calcium concentration. Variation in Rhodamine B dye-binding to cells made it difficult to interpret the data. In addition, concern regarding the ability of SVK-14 cells to differentiate suggests that further studies need to be performed using normal human keratinocytes to validate this in vitro endpoint, with epidermal growth factor, insulin and hydrocortisone removed from the media to enhance epidermal differentiation.

A strategy for the examination of repeat chronic exposure employed chemicals in the MEIC scheme. Repeat exposure to a non-cytotoxic concentration of certain chemicals leads to changes in sensitivity to a subsequent acute challenge. Six sequential exposures to 3T3-L1 cells, maintained in exponential growth, to ethylene glycol results in an enhanced resistance to cytotoxic damage from a subsequent challenge with 2-propanol. 2-propanol pre-exposure did not alter sensitivity to ethylene glycol. Ethanol or methanol pre-exposure reduces the apparent toxicity to a subsequent challenge with methanol or ethanol. Paracetamol or aspirin pretreatment reduces the toxic effects of paracetamol or aspirin. Hence, repeat exposure can result in a modulation of the cellular responses noted in vitro to cytotoxic concentrations of chemicals. This method demonstrates one approach to examining in vitro the relevance of chronic toxicity in vivo.

The principal goal of this study was to determine whether the results from a set of selected currently available alternative methods as used by cosmetics companies are valid for predicting the eye irritation potential of cosmetics formulations and ingredients and, as a consequence, could be valid replacements for the Draize eye irritation test. For the first time in a validation study, prediction models (PMs) that convert the in vitro data from an assay to a prediction of eye irritation were developed for each alternative method before the study began. The PM is an unequivocal description of the relationship between the in vitro and the in vivo data and allows an objective assessment of the reliability and relevance of the alternative methods. In this study, 10 alternative methods were evaluated using 55 test substances selected as representative of substances commonly used in the cosmetics industry (23 ingredients and 32 formulations). Twenty of the single ingredients were common to the European Commission/British Home Office (EC/HO) eye irritation validation study (Balls et al., 1995b). The test substances were coded and supplied to the participating laboratories. The results were collected centrally and analysed independently, using statistical methods that had been agreed before the testing phase began. Each alternative method was then evaluated for reliability and relevance in assessing eye irritation potential. Using the criteria of both reliability and relevance as defined in the study, the preliminary results indicate that none of the alternative methods evaluated could be confirmed as a valid replacement for the Draize eye irritation test across the full irritation scale. However, three alternative methods—the fluorescein leakage test, the red blood cell assay (classification model) and the tissue equivalent assay—each satisfied one criterion of reliability or relevance. Further investigation of the decoded data from this study to explore more fully the relationship between the in vitro data and the in vivo data is recommended. Such a review may allow the development of new prediction models to be tested in a subsequent validation study.

Nitric oxide is produced in dermal inflammatory reactions; its role as an irritant mediator was therefore examined. Nitrite accumulation (an indicator of nitric oxide synthesis) was measured in human keratinocytes, NCTC 2544 cells and RAW 264 macrophages, following stimulation by lipopolysaccharide (LPS), sodium dodecyl sulfate (SDS) or ultraviolet irradiation (UVA). Nitrite synthesis was low in primary cultured keratinocytes (passage 3) at 0.36 +/- 0.16 nmol/well. This was minimally affected by LPS (0.58 +/- 0.1 nmol/well). SDS and UVA failed to modify keratinocyte nitrite production. No nitrite synthesis was detected in NCTC 2544 cells. RAW 264 macrophages responded to LPS (0-10 mug/ml) by increasing nitrite production from 2 +/- 0.6 to 22 +/- 4.5 nmol/well in keratinocyte serum-free media. Responses of macrophages to LPS were diminished in Dulbecco's media containing serum or dialysed serum. Human interleukin-1 (0-30 pg/ml) increased macrophage nitrite production and l-N-monomethyl arginine (l-NMMA) (0-1 mm) inhibited nitrite synthesis. Media conditioned by keratinocytes for 24 hr following treatment with SDS (0-100 mug/ml for 0-8 hr) or UVA (1.6 mW/cm(2) for 0-4 hr) enhanced macrophage nitrite synthesis. Conditioned media from UVA-treated NCTC 2544 cells failed to stimulate macrophage nitrite synthesis.

The 1980s saw widespread acceptance of the Three Rs (reduction, refinement, replacement) concept of alternatives, and a great deal of effort was put into the development of non-animal toxicity tests. Yet, although new methods are gaining acceptance as pre-screens and as adjunct methods, there is little sign that any of them will be accepted as genuine replacements for current animal test procedures. This is partly because, until recently, the question of scientific validation for relevance, reproducibility and transferability had not been properly addressed, and partly because, despite national and international laws that require that replacement alternatives be used wherever possible, little attention had been paid to promotion of their formal acceptance into regulatory toxicology. Reference is made to the recommendations of two workshops organized by members of ERGATT (European Research Group for Alternatives in Toxicity Testing) early in 1990-on validation (with the Johns Hopkins Center for Alternatives to Animal Testing) and on regulatory acceptance (with the support of the Commission of the European Communities)-which could provide a basis for meeting this challenge in the 1990s. It is argued, however, that primary responsibility for the effective and orderly development, validation, independent assessment and regulatory acceptance of non-animal toxicity tests should rest with in vitro toxicologists themselves.

A number of in vitro systems have been put forward as potential alternative methods for testing chemicals for teratogenic potential. The most promising of these systems, for example mammalian whole embryo culture and the micromass technique, are currently undergoing further interlaboratory validation. However, such tests involve the use of a considerable number of animals. It was therefore decided to investigate the possible use of a permanent cell line that possessed many of the properties of embryonic cells, that is a differentiating cell line, F9 (derived from a mouse teratocarcinoma), in the development of an in vitro teratogenicity test. In a preliminary study, six chemicals were tested for their modulating effects on differentiation in undifferentiated, differentiating and differentiated F9 cells. These effects were assessed morphologically and by measuring the production of laminin (a biochemial marker of F9 differentiation). The use of the F9 cell line in in vitro teratogenicity testing shows promise, but further work is necessary before its potential can be fully evaluated.

A battery of confirmatory and complementary in vitro assays was used to investigate a series of surfactants with different irritancy potentials, as confirmed by human skin patch testing. This was found to distinguish between sodium lauryl sulfate and its analogues, and when further tested on formulations the battery of assays was again capable of predicting the human data.

A new in vitro test for phototoxicity has been developed. Nine substances (eight photosensitizers and one non-photosensitizer) were screened for their ability to activate human complement in the presence of UV light, and all photosensitizers were found to be active. Complement activation was quantified by means of ELISA. In all cases activation was mediated by alternative, but not by classical, pathways. The results of a cell culture test performed with 5-methoxypsoralen suggest the existence of two different mechanisms of phototoxicity. This indicates that the battery of in vitro assays for phototoxicity prediction must contain at least two different tests.

The use of the MultiScreen filtration plate enables fluids to be removed efficiently from the plate without disturbing cells, or formazan crystals formed during the MTT test. Using a 1:1 mix of dimethyl sulfoxide and ethanol as solvent allows optical densities to be measured directly on the Multiscreen plate. The possibility of washing cells without losses in their number appears to be advantageous especially for in vitro studies on cytokines and on the cytotoxicity of coloured substances, such as dyes. In this study the results of cytokine assessment using the Multiscreen plate were compared with those obtained using a normal flat-bottomed plate. Two methods of assessment of the cytotoxicity of the dye rose bengal were also compared.

To answer the question, “How toxic is this material?”, rather than the question more usually asked in in vitro toxicology, “At what concentration does this material produce a given degree of toxicity?”, a fixed-dose approach has been developed with the FRAME fluorescein leakage (FL) test. Recovery from the initial cytotoxic effect was also noted sometimes, after the cells had been maintained in fresh medium for a further 72 hr. Chemicals rated R41 or R36 in vivo gave a level of FL above 20% immediately, and 66% or more 72 hr later (i.e. there was further deterioration, rather than recovery). Acetaldehyde, when duplicated and tested either in Hanks' balanced salt solution or oil, showed greater toxicity in the oil because of its high volatility. The chemicals classified as no-label gave FL values below 12% immediately after exposure, except Brij-35, which showed no further deterioration during the 72-hr period in fresh medium.

The ability of in vitro cytotoxicity to predict in vivo skin irritation was examined using three cell types and a set of previously examined chemicals with in vivo skin irritation data. There was concordance in ranking of the test chemicals according to their cytotoxicity between primary human keratinocytes (NHEK), NCTC 2544 and MDCK cells. When compared against rabbit in vivo skin irritation data, cytotoxicity to NHEK in serum-free media showed a better correlation (r2 = −0.833) than the cell lines or NHEK in serum-containing medium. These are encouraging results for the use of cytotoxicity as an endpoint for the in vitro prediction of skin irritation.

In vitro toxicity tests must be properly developed and scientifically validated for relevance and reliability before they are independently evaluated for possible inclusion in toxicity testing schemes and promoted for regulatory and legal acceptance. Some lessons learned in the administration of the FRAME Alternative Test Validation Scheme are reported, using as an example correlations between the results obtained in cell growth inhibition tests, both with each other and with rat oral and mouse intraperitoneal LD 50 values. Some recommendations are given for consideration for the design of future validation schemes.

A V79 cell incubation, incorporating rat or hamster liver 9000 g supernatant (S-9) or microsomal fraction and used previously to detect the toxicity of reactive metabolites of cyclophosphamide and bromobenzene, has been used to examine the toxicity of short-lived reactive metabolites of paracetamol. Cytotoxicity was observed in the absence of an activating system and did not increase when an activating system was included in the incubation, even when this was derived from the livers of hamsters treated with β-naphthoflavone (to increase the activity of the cytochrome P-450 form responsible for paracetamol activation) and diethyl maleate (to deplete protective glutathione stores). The failure to detect metabolism-mediated cytotoxicity of paracetamol in this assay system may be related to the high reactivity of the toxic metabolite. This, in turn, suggests that other systems, based on activation within target cells or on the detection of an endpoint closer to the activation event, are required for the in vitro detection of short-lived cytotoxic metabolites.