Non-animal Replacements for Acute Toxicity Testing.

Current approaches to predicting adverse effects in humans from acute toxic exposure to cosmetic ingredients still heavily necessitate the use of animals under EU legislation, particularly in the context of the REACH system, when cosmetic ingredients are also destined for use in other industries. These include the LD50 test, the Up-and-Down Procedure and the Fixed Dose Procedure, which are regarded as having notable scientific deficiencies and low transferability to humans. By expanding on previous in vitro tests, such as the animal cell-based 3T3 Neutral Red Uptake (NRU) assay, this project aims to develop a truly animal-free predictive test for the acute toxicity of cosmetic ingredients in humans, by using human-derived cells and a prediction model that does not rely on animal data. The project, funded by Innovate UK, will incorporate the NRU assay with human dermal fibroblasts in animal product-free culture, to generate an in vitro protocol that can be validated as an accepted replacement for the currently available in vivo tests. To date, the project has successfully completed an assessment of the robustness and reproducibility of the method, by using sodium lauryl sulphate (SLS) as a positive control, and displaying analogous results to those of the original studies with mouse 3T3 cells. Currently, the testing of five known ingredients from key groups (a surfactant, a preservative, a fragrance, a colour and an emulsifier) is under way. The testing consists of initial range-finding runs followed by three valid runs of a main experiment with the appropriate concentration ranges, to generate IC50 values. Expanded blind trials of 20 ingredients will follow. Early results indicate that this human cell-based test holds the potential to replace aspects of in vivo animal acute toxicity testing, particularly with reference to cosmetic ingredients.

Comparative studies of apoptosis in Xenopus laevis and mouse thymoma cell lines.

With the use of in vitro methods and cell lines, functional aspects of apoptosis in the Xenopus laevis B3/B7 and mouse EL4 thymoma cell lines are revealed. Moreover, by using information gleaned from digital imaging and immunocytochemistry, changes in locations of key proteins implicated in apoptotic anti-cancer responses, e.g. p53 and Mdm2, are shown. Suggestions are offered as to what these results might mean with respect to the evolutionary conservation of the function and structure of these two molecules and to cancer resistance in amphibians. Finally, studies are described on resveratrol as an anti-cancer therapeutic reagent in the two thymoma cell lines and in normal X. laevis thymocytes.

Resistance to cancer in amphibians: a role for apoptosis?

The rarity of spontaneous cancer in amphibians, and the difficulty of inducing cancer in these lower vertebrates, suggest that they possess an effective system for resistance to the development of cancer. The first part of this narrative presents evidence for cancer resistance in amphibians, and then a variety of studies designed to help understand the physiological basis for this resistance are reviewed. Here, our emphasis is on evidence with regard to the role that apoptosis might play.

A FRAME response to the Draft Report on Alternative (Non-animal) Methods for Cosmetics Testing: Current Status and Future Prospects--2010.

This response on behalf of FRAME to the European Commission's consultation on the five chapters of the Draft Report on Alternative (Non-animal) Methods for Cosmetics Testing: Current Status and Future Prospects--2010, is via a Comment in ATLA, rather than via the template supplied by the Commission. This is principally so that a number of general points about cosmetic ingredient testing can be made. It is concluded that the five draft chapters do not provide a credible basis for the Commission's forthcoming report to the European Parliament and the European Council on the five cosmetic ingredient safety issues for which the 7th Amendment to the Cosmetic Directive's ban on animal testing was postponed until 2013. This is mainly because there is insufficient focus in the draft chapters on the specific nature of cosmetic ingredients, their uses, their local effects and metabolism at their sites of application, and, in particular, on whether their possible absorption into the body would be likely to lead to their accumulation in target sites at levels approaching Thresholds of Toxicological Concern. Meanwhile, there continues to be uncertainty about how the provisions of the Cosmetics Directive should be applied, given the requirements of the REACH system and directives concerned with the safety of other chemicals and products.

The development and evaluation of in vitro alternative assays: a personal perspective.

Over the past 30 years, FRAME has actively participated in the development, evaluation and validation of in vitro alternative methods through the FRAME Alternatives Laboratory (FAL) in the University of Nottingham Medical School. Much has been learned through collaboration with industry (especially the cosmetics industry), other organisations (especially ECVAM), and certain individuals (notably Dr Björn Ekwall), particularly in relation to the need to use human cell cultures and to obtain, wherever possible, high-quality human data for use in in vivo/in vitro comparisons. Reference is made to the author's experience as Director of the FAL, notably in the development of in vitro assays for basal cytotoxicity, phototoxicity and the effects of repeated dosage.

A proposed eye irritation testing strategy to reduce and replace in vivo studies using Bottom-Up and Top-Down approaches.

In spite of over 20 years of effort, no single in vitro assay has been developed and validated as a full regulatory replacement for the Draize Eye Irritation test. However, companies have been using in vitro methods to screen new formulations and in some cases as their primary assessment of eye irritation potential for many years. The present report shows the outcome of an Expert Meeting convened by the European Centre for the Validation of Alternative Methods in February 2005 to identify test strategies for eye irritation. In this workshop test developers/users were requested to nominate methods to be considered as a basis for the identification of such testing strategies. Assays were evaluated and categorized based on their proposed applicability domains (e.g., categories of irritation severity, modes of action, chemical class, physicochemical compatibility). The analyses were based on the data developed from current practice and published studies, the ability to predict depth of injury (within the applicable range of severity), modes of action that could be addressed and compatibility with different physiochemical forms. The difficulty in predicting the middle category of irritancy (e.g. R36, GHS Categories 2A and 2B) was recognized. The testing scheme proposes using a Bottom-Up (begin with using test methods that can accurately identify non-irritants) or Top-Down (begin with using test methods that can accurately identify severe irritants) progression of in vitro tests (based on expected irritancy). Irrespective of the starting point, the approach would identify non-irritants and severe irritants, leaving all others to the (mild/moderate) irritant GHS 2/R36 categories.

An evaluation of the chick cardiomyocyte micromass system for identification of teratogens in a blind trial.

The chick micromass culture system has advantages over the validated rat system - ready availability and non-culling of the donor parent - but needs to give comparable results. This study confirmed comparability and the ability to extend the system to cover cardiac effects. It was also compared with the validated embryonic stem cell cardiomyocyte model. A teratogen and paired non-teratogen with known in vivo effects were used. Differential effects were measured via changes in cell protein content, cell viability (resazurin reduction and neutral red uptake), and cell contractility. Results showed that teratogens [L-ethionine, 5-fluorouracil and sulphisoxazole] could be distinguished from non-teratogens [DL-methionine, 6-methyluracil and sulphanilamide respectively]. Dichloroacetone and dichloropropanol affected embryonic stem cells but not the micromass; dichloropropanol had a greater effect than dichloroacetone. This approach revealed differential effects on contractility independent of effects on activity/viability, whilst the total cell protein remained unchanged. We suggest that pre-validation of this system should be examined.

FRAME and the validation process.

FRAME's historical involvement in the development of the principles of validation, whereby the reliability and relevance of a procedure are established for a specific purpose, and in the practical application of the process, is summarised, and examples of participation in various validation studies on in vitro tests are reviewed. Emphasis is placed on the need for a parallel invalidation process, and on the role of ATLA as a forum for objective reporting and discussion on all aspects of the validation process.

A database of IC50 values and principal component analysis of results from six basal cytotoxicity assays, for use in the modelling of the in vivo and in vitro data of the EU ACuteTox project.

The main aim of the ACuteTox project (part of the EU 6th Framework programme) is to demonstrate that animal tests for acute systemic toxicity can be replaced by alternative in vitro assays. In this project, data for 97 reference chemicals were collected in the AcuBase database, designed to handle deposited in vitro and in vivo (human and animal) data. To demonstrate the applicability of in vitro basal cytotoxicity tests and in vitro-in vivo modelling, it was deemed necessary to obtain data that were generated via defined standard operating procedures. The molar basal cytotoxicity IC50 values (the 50% inhibitory concentrations for the endpoint measured) for a mouse fibroblast cell line (3T3), a human hepatic cell line (HepG2), a rat hepatic cell line (Fa32), and a human neutrophil cell line (HL-60), were compared, and gave an R(2) correlation of 0.83. To identify chemicals that showed differential cytotoxicity to the various cell types involved, principal component analysis (PCA) was undertaken independently, once all the results had been returned. This showed that colchicine, cycloheximide, digoxin, 5-fluorouracil and hexachlorobenzene gave the lowest correlations with the first score vector of the PCA. The results presented are to be used to identify outliers that need to be further studied via the use of tissue-specific in vitro assays.

Comparative evaluation of cytotoxicity and phototoxicity of mono and diacylglycerol amino acid-based surfactants.

Extensive efforts have been made, recently, to find surfactants with lower irritancy potential than those presently commercially employed in pharmaceutical and cosmetic preparations. Cytotoxic and phototoxic effects of novel mono and diacylglycerol amino acid-based surfactants (glutamic acid, or arginine) were evaluated. All tested surfactants showed a clear concentration-response relationship to two immortalized cell lines, murine fibroblast cell line, 3T3, and one human keratinocyte cell line, HaCaT, demonstrated by and decrease of NR uptake. Concentrations resulting in 50% inhibition of NR uptake (IC(50)) range from 30 to 300microgmL(-1). The potential phototoxicity which could result in irritant products, was determined by modulated cytotoxicity via the resazurin reduction to resorufin and neutral red uptake (NRU) endpoints. Surfactants with two chains showed, in general, less cytotoxic but higher phototoxic effect than surfactants with only one chain.

Estimation of human blood LC50 values for use in modeling of in vitro-in vivo data of the ACuteTox project.

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 effects of terahertz radiation on human keratinocyte primary cultures and neural cell cultures.

Terahertz (THz) frequencies are found in a previously underexploited region of the radiation spectrum. This non-ionising energy is now being employed in medical imaging, so the possibility of adverse effects on human skin was evaluated. Primary cultures of normal human keratinocytes (NHKs) express adhesion molecules that comprise part of the natural barrier function of the skin. The effects of exogenous agents on this barrier function can be measured. The ND7/23 cell line, which displays the characteristics of sensory neurones, can proliferate in the undifferentiated state, but can be induced to differentiate and develop neurite-like projections. Previous studies with NHK and neural cell cultures produced no evidence of the inability of these cells to differentiate and form a barrier following THz exposure. The cells were exposed to 0.14THz radiation for times varying from 10 minutes to 24 hours. For each 80-nanosecond pulse, the cells were exposed to a peak power of between 24 and 62mW/cm(2), i.e. a total energy at peak power of 345J, or 86J at average power over 24 hours. No changes in cell activity occurred, as monitored with the resazurin reduction assay, or with the barrier function of the human corneal cells, as measured with the fluorescein leakage assay. The monitoring of differentiation by using an assay for cornified envelope formation, revealed no adverse effects. Glutathione (GSH) and heat shock protein 70 levels were examined before and after differentiation, to determine the degree of the stress response, with the effects of UVB radiation as a control. UVB induced a stress response, as did heat shock treatment at 43 degrees C, whilst 0.15THz radiation, even after 24 hours of exposure, did not. Repeated exposure to THz radiation at this level, also resulted in no detectable adverse reactions.

Cancer resistance in amphibians.

While spontaneous tumours may occasionally develop in inbred and isogenic strains of Xenopus laevis, the South African clawed toad, they are extremely rare in natural and laboratory populations. Only two amphibian neoplasms, the renal adenocarcinoma of Rana pipiens and the lymphosarcoma of Xenopus laevis, have been extensively explored. Amphibians are resistant to the development of neoplasia, even following exposure to "direct-acting" chemical carcinogens such as N-methyl-N-nitrosourea, that are highly lymphotoxic, thus diminishing immune reactivity. Regenerative capacity in adults, and a dramatic metamorphosis which remodels much of the larval body to produce the adult form, are unique to amphibian vertebrates, and the control mechanisms involved may protect against cancer. For example, naturally rising corticosteroid titres during metamorphosis will impair some T-cell functions, and the removal of T-regulatory (suppressor) functions inhibits the induction of altered-self tolerance. Altered-self tolerance is not as effectively induced in adult Xenopus laevis as in mammals, so cancer cells with new antigenicity are more likely be rejected in amphibians. Amphibian immunocytes tend to undergo apoptosis readily in vitro, and, unlike mammalian immunocytes, undergo apoptosis without entering the cell cycle. Cells not in the cell cycle that die from nuclear damage (apoptosis), will have no opportunity to express genetic instability leading to cell transformation. We suggest that all these factors, rather than any one of them, may reduce susceptibility to cancer in amphibians.

An evaluation of the effects of photoactivation of bithionol, amiodarone and chlorpromazine on human keratinocytes in vitro.

Human skin is a continual target for chemical toxicity, due to its constant exposure to xenobiotics. The skin possesses a number of protective antioxidant systems, including glutathione and enzymic pathways, which are capable of neutralising reactive oxygen species (ROS). In combination with certain chemicals, the presence of ROS might augment the levels of toxicity, due to photoactivation of the chemical or, alternatively, due to an oxidatively-stressed state in the skin which existed prior to exposure to the chemical. Bithionol is a phototoxic anti-parasitic compound. The mechanism of its toxicity and the possible methods of protection from its damaging effects have been explored. The capacity of keratinocytes to protect themselves from bithionol and other phototoxic chemicals has been investigated. In addition, the potential of endogenous antioxidants, such as vitamin C and E, to afford protection to the cells, has been evaluated. The intracellular glutathione stores of HaCaT keratinocytes were reduced following treatment with biothionol. Following photoactivation, both bithionol and chlorpromazine had similar effects, which suggests that glutathione is important in the detoxification pathway of these chemicals. This was confirmed by means of the visual identification of fluorescently-labelled glutathione. Endogenous antioxidants were unable to protect the HaCaT keratinocytes from bithionol toxicity or chlorpromazine phototoxicity. Amiodarone was shown to have no effect on cellular glutathione levels, which suggests that an alternative mechanism of detoxification was occurring in this case. This was supported by evidence of the protection of HaCaT cells from amiodarone phototoxicity via endogenous antioxidants. Thus, it appears that amiodarone toxicity is dependent on the levels of non-gluathione antioxidants present, whilst bithionol and chlorpromazine detoxification relies on the glutathione antioxidant system. This type of approach could indicate the likely mechanisms of phototoxicity of chemicals in vitro, with relevance to potential effects in vivo.

Development of an innervated model of human skin.

Neuronal cell responses and interactions with the epithelial and fibroblastic cells of the skin are a key factor in the production in vivo of the irritation/inflammatory response. Currently, few in vitro models are available that contain dermal, epidermal and the relevant neuronal components. The primary objective of this study was to produce and maintain a 3-D in vitro model of human skin containing these elements. The relevant neuronal component was supplied by adding sensory neurons derived from the dorsal root ganglion (DRG). Since adult neuronal cells do not grow significantly in vivo or in vitro, and since it is very difficult to obtain such cells from humans, it was necessary to employ embryonic rat DRG cells. The ultimate purpose of this model is to improve prediction of the in vivo skin irritancy potential of chemicals and formulations, without the need to use animal models. In addition, this approach has also been applied to the in vitro human eye and bronchial 3-D models being developed in the FRAME Alternatives Laboratory.

A review of in vitro modelling approaches to the identification and modulation of squamous metaplasia in the human tracheobronchial epithelium.

Squamous metaplasia in the tracheobronchial epithelium (TBE) involves the replacement of the normal pseudostratified mucociliary epithelium with a stratified squamous epithelium. Squamous metaplasia is considered to be an adaptive response that protects the lumen from the effects of inhaled airborne pollutants, but which might also feature as a pre-neoplastic lesion preceding squamous cell carcinoma. With the exception of transglutaminase I, involucrin, and cytokeratins 5, 6 and 13, few markers that contribute to the squamous phenotype have been identified in human TBE that can be used in diagnosis or to monitor its development in laboratory investigations, and current models are inadequate to provide statistically meaningful data. Therefore, new predictive markers have been identified, and new techniques established, in epithelial in vitro models capable of expressing squamous characteristics, which will be used to identify hazardous exposures and elucidate the mechanisms by which they induce their effects. A protocol for the quantitative detection of transglutaminase activity has been standardised in keratinocytes, based on the enzymatic incorporation of fluorescein-cadaverine (FC) into bis(gamma-glutamyl) polyamine cross-links. The specificity of this compound as a transglutaminase substrate was demonstrated by using a range of competitive transglutaminase inhibitors, and by modulation of the squamous pathway. FC incorporation was localised to the cell membrane of terminally differentiating cells, and was not visible in basal, proliferating cells. High calcium-containing medium, nicotine and cigarette smoke condensates (CSC) induced an increase in FC incorporation, providing evidence of their role in enhancing the squamous pathway. Analysis by flow cytometry was used to provide a quantitative assessment of a range of optimised squamous differentiation markers, identified in normal human bronchial epithelia and in a bronchial cell line. Transglutaminase I was induced in a time-dependent manner, in post-confluent cells induced to differentiate down the squamous pathway, whereas involucrin was ubiquitously expressed and the levels of cytokeratins 5, 6 and 18 were reduced. The response of these and other differentiation markers to squamous-inducing conditions is being explored.

An evaluation of a novel chick cardiomyocyte micromass culture assay with two teratogens/embryotoxins associated with heart defects.

This study was aimed at determining whether the chick cardiomyocyte micromass (MM) system could be employed to predict the teratogenicity/embryotoxicity of exogenous chemicals. Two documented teratogens/embryotoxins, sodium valproate (the sodium salt of valproic acid; VPA) and all-trans retinoic acid (tRA), were used in the initial phase of the study. White Leghorn 5-day-old embryo hearts were dissociated to produce a cardiomyocyte suspension in Dulbecco's Modified Eagle's Medium. Cultures were incubated at 37 degrees C in 5% CO(2) in air, and observations were made every 24 hours over 5 days, for the detection of beating. Culture viability was assessed by using the resazurin reduction assay for determining culture activity and the kenacid blue assay for determining cell number. It was found that tRA significantly reduced cell activity and beating, whilst not affecting total cell number. VPA up to 500 microM induced no cytotoxicity in the MM cardiomyocyte cultures, whilst all the VPA concentrations tested reduced beating. The results demonstrate the potential of the chick cardiomyocyte MM culture assay to identify teratogens/embryotoxins that alter functionality, which may result in a teratogenic outcome, whilst not causing cytotoxicity (direct embryotoxicity). This could form part of a screen for developmental toxicity related to cardiac function, whilst limb cultures and brain cultures based on the same system could be relevant to teratogenic effects on those tissues.

Phototoxicity and acute toxicity studies conducted by the FRAME Alternatives Laboratory: a brief review.

FRAME and the University of Nottingham have been in association for the past 25 years. During this time, the research in the FRAME Alternatives Laboratory (FAL) at the University of Nottingham, which is partly funded by FRAME and also, more recently, by ECVAM, has involved participation in a number of international validation studies. Validation has become a pre-requisite for the regulatory acceptance of in vitro alternative test procedures, and a number of key lessons have been learned from these studies. The directors of validation studies need to ensure that standard operating procedures (SOPs) are fully complied with, and that the equipment used is certified to be of an acceptable standard. Database managers need to be able to check the original data, and to ensure adherence to procedures agreed before the study began. When the validation study is part of an integrated EU Framework Project, such as ACute-Tox, the Workpackage Leader must have the ability to understand and evaluate the data to be presented for inclusion in the study analysis, and to check that it complies with acceptance criteria. The potential to relate observed cellular biochemical changes to morphological endpoints also increases the level of understanding of the relevance and/or limitations of an assay. For example, exposure to a surfactant can induce the temporary loss of adhesion junctions between adjacent epithelial cells, resulting in the loss of barrier integrity and other effects on cell culture activity, which can potentially be restored over time. Unexpected results from the NRU phototoxicity assay with human keratinocytes instead of 3T3 cells, stimulated research into the ability of the in vitro assay, not only to identify phototoxins, but also to identify their possible mechanisms of action and mechanisms underlying the protective capacity within human primary keratinocytes in vitro. The protective effects of UV-filters can also be used to ascertain their effects on the photoactivation of drugs.

Cannabinoid receptor agonists are mitochondrial inhibitors: a unified hypothesis of how cannabinoids modulate mitochondrial function and induce cell death.

Time-lapse microscopy of human lung cancer (H460) cells showed that the endogenous cannabinoid anandamide (AEA), the phyto-cannabinoid Delta-9-tetrahydrocannabinol (THC) and a synthetic cannabinoid HU 210 all caused morphological changes characteristic of apoptosis. Janus green assays of H460 cell viability showed that AEA and THC caused significant increases in OD 595 nm at lower concentrations (10-50 microM) and significant decreases at 100 microM, whilst HU 210 caused significant decreases at all concentrations. In rat heart mitochondria, all three ligands caused significant decreases in oxygen consumption and mitochondrial membrane potential. THC and HU 210 caused significant increases in mitochondrial hydrogen peroxide production, whereas AEA was without significant effect. All three ligands induced biphasic changes in either mitochondrial complex I activity and/or mitochondrial complex II-III activity. These data demonstrate that AEA, THC, and HU 210 are all able to cause changes in integrated mitochondrial function, directly, in the absence of cannabinoid receptors.