Development of the EpiOcular(TM) eye irritation test for hazard identification and labelling of eye irritating chemicals in response to the requirements of the EU cosmetics directive and REACH legislation

The recently implemented 7th Amendment to the EU Cosmetics Directive and the EU REACH legislation have heightened the need for in vitro ocular test methods. To address this need, the EpiOcular(TM) eye irritation test (EpiOcular-EIT), which utilises the normal (non-transformed) human cell-based EpiOcular tissue model, has been developed. The EpiOcular-EIT prediction model is based on an initial training set of 39 liquid and 21 solid test substances and uses a single exposure period and a single cut-off in tissue viability, as determined by the MTT assay. A chemical is classified as an irritant (GHS Category 1 or 2), if the tissue viability is ≤ 60%, and as a non-irritant (GHS unclassified), if the viability is > 60%. EpiOcular-EIT results for the training set, along with results for an additional 52 substances, which included a range of alcohols, hydrocarbons, amines, esters, and ketones, discriminated between ocular irritants and non-irritants with 98.1% sensitivity, 72.9% specificity, and 84.8% accuracy. To ensure the long-term commercial viability of the assay, EpiOcular tissues produced by using three alternative cell culture inserts were evaluated in the EpiOcular-EIT with 94 chemicals. The assay results obtained with the initial insert and the three alternative inserts were very similar, as judged by correlation coefficients (r2) that ranged from 0.82 to 0.96. The EpiOcular-EIT was pre-validated in 2007/2008, and is currently involved in a formal, multi-laboratory validation study sponsored by the European Cosmetics Association (COLIPA) under the auspices of the European Centre for the Validation of Alternative Methods (ECVAM). The EpiOcular-EIT, together with EpiOcular's long history of reproducibility and proven utility for ultra-mildness testing, make EpiOcular a useful model for addressing current legislation related to animal use in the testing of potential ocular irritants.     

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&A) for liquids, 100.0/68.4/ and 84.6% SS&A for solids, and 97.6/68.3/ and 83.1% for overall SS&A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.     

The bovine corneal opacity and permeability test in routine ocular irritation testing and its improvement within the limits of OECD test guideline 437

Data on eye irritation are generally needed for the hazard identification of chemicals. As the Bovine Corneal Opacity and Permeability (BCOP) test has been accepted by many regulatory agencies for the identification of corrosive and severe ocular irritants since September 2009 (OECD Test Guideline 437, TG 437), we evaluated this alternative method for routine testing at BASF. We demonstrated our technical proficiency by testing the reference standards recommended in TG 437, and 21 additional materials with published BCOP and in vivo data. Our results matched the published in vitro data very well, but with some intentionally selected false negatives (FNs) and false positives (FPs), the concordance was 77% (24/31), with FN and FP rates of 20% (2/10) and 24% (5/21), respectively. In addition, we tested 21 in-house materials, demonstrating the utility of the BCOP assay for our own test material panel. Histopathological assessment of the corneas by light microscopy was also conducted, as this was suggested as a means of improving the identification of FNs. The histopathology corrected the classification of some FNs, but also increased the number of FPs. Parallel to the test method evaluation, we compared three new opacitometer models with the current standard device. We recommend the use of an opacitometer developed in our BASF laboratory, which has certified components and electronic data storage, resulting in what we consider to be excellent sensitivity, stability and reproducibility.     

Determining the Depth of Injury in Bioengineered Tissue Models of Cornea and Conjunctiva for the Prediction of All Three Ocular GHS Categories

The depth of injury (DOI) is a mechanistic correlate to the ocular irritation response. Attempts to quantitatively determine the DOI in alternative tests have been limited to ex vivo animal eyes by fluorescent staining for biomarkers of cell death and viability in histological cross sections. It was the purpose of this study to assess whether DOI could also be measured by means of cell viability detected by the MTT assay using 3-dimensional (3D) reconstructed models of cornea and conjunctiva. The formazan-free area of metabolically inactive cells in the tissue after topical substance application is used as the visible correlate of the DOI. Areas of metabolically active or inactive cells are quantitatively analyzed on cryosection images with ImageJ software analysis tools. By incorporating the total tissue thickness, the relative MTT-DOI (rMTT-DOI) was calculated. Using the rMTT-DOI and human reconstructed cornea equivalents, we developed a prediction model based on suitable viability cut-off values. We tested 25 chemicals that cover the whole range of eye irritation potential based on the globally harmonized system of classification and labelling of chemicals (GHS). Principally, the MTT-DOI test method allows distinguishing between the cytotoxic effects of the different chemicals in accordance with all 3 GHS categories for eye irritation. Although the prediction model is slightly over-predictive with respect to non-irritants, it promises to be highly valuable to discriminate between severe irritants (Cat. 1), and mild to moderate irritants (Cat. 2). We also tested 3D conjunctiva models with the aim to specifically address conjunctiva-damaging substances. Using the MTT-DOI method in this model delivers comparable results as the cornea model, but does not add additional information. However, the MTT-DOI method using reconstructed cornea models already provided good predictability that was superior to the already existing established in vitro/ex vivo methods.     

Validation study of the in vitro skin irritation test with the LabCyte EPI-MODEL24

A validation study on an in vitro skin irritation assay was performed with the reconstructed human epidermis (RhE) LabCyte EPI-MODEL24, developed by Japan Tissue Engineering Co. Ltd (Gamagori, Japan). The protocol that was followed in the current study was an optimised version of the EpiSkin protocol (LabCyte assay). According to the United Nations Globally Harmonised System (UN GHS) of classification for assessing the skin irritation potential of a chemical, 12 irritants and 13 non-irritants were validated by a minimum of six laboratories from the Japanese Society for Alternatives to Animal Experiments (JSAAE) skin irritation assay validation study management team (VMT). The 25 chemicals were listed in the European Centre for the Validation of Alternative Methods (ECVAM) performance standards. The reconstructed tissues were exposed to the chemicals for 15 minutes and incubated for 42 hours in fresh culture medium. Subsequently, the level of interleukin-1 alpha (IL-1 α) present in the conditioned medium was measured, and tissue viability was assessed by using the MTT assay. The results of the MTT assay obtained with the LabCyte EPI-MODEL24 (LabCyte MTT assay) demonstrated high within-laboratory and between-laboratory reproducibility, as well as high accuracy for use as a stand-alone assay to distinguish skin irritants from non-irritants. In addition, the IL-1α release measurements in the LabCyte assay were clearly unnecessary for the success of this model in the classification of chemicals for skin irritation potential.     

The ECVAM international validation study on in vitro tests for acute skin irritation: report on the validity of the EPISKIN and EpiDerm assays and on the Skin Integrity Function Test

ECVAM sponsored a formal validation study on three in vitro tests for skin irritation, of which two employ reconstituted human epidermis models (EPISKIN, EpiDerm), and one, the skin integrity function test (SIFT), employs ex vivo mouse skin. The goal of the study was to assess whether the in vitro tests would correctly predict in vivo classifications according to the EU classification scheme, "R38" and "no label" (i.e. non-irritant). 58 chemicals (25 irritants and 33 non-irritants) were tested, having been selected to give broad coverage of physico-chemical properties, and an adequate distribution of irritancy scores derived from in vivo rabbit skin irritation tests. In Phase 1, 20 of these chemicals (9 irritants and 11 non-irritants) were tested with coded identities by a single lead laboratory for each of the methods, to confirm the suitability of the protocol improvements introduced after a prevalidation phase. When cell viability (evaluated by the MTT reduction test) was used as the endpoint, the predictive ability of both EpiDerm and EPISKIN was considered sufficient to justify their progression to Phase 2, while the predictive ability of the SIFT was judged to be inadequate. Since both the reconstituted skin models provided false predictions around the in vivo classification border (a rabbit Draize test score of 2), the release of a cytokine, interleukin-1alpha (IL-1alpha), was also determined. In Phase 2, each human skin model was tested in three laboratories, with 58 chemicals. The main endpoint measured for both EpiDerm and EPISKIN was cell viability. In samples from chemicals which gave MTT assay results above the threshold of 50% viability, IL-1alpha release was also measured, to determine whether the additional endpoint would improve the predictive ability of the tests. For EPISKIN, the sensitivity was 75% and the specificity was 81% (MTT assay only); with the combination of the MTT and IL-1alpha assays, the sensitivity increased to 91%, with a specificity of 79%. For EpiDerm, the sensitivity was 57% and the specificity was 85% (MTT assay only), while the predictive capacity of EpiDerm was not improved by the measurement of IL-1alpha release. Following independent peer review, in April 2007 the ECVAM Scientific Advisory Committee endorsed the scientific validity of the EPISKIN test as a replacement for the rabbit skin irritation method, and of the EpiDerm method for identifying skin irritants as part of a tiered testing strategy. This new alternative approach will probably be the first use of in vitro toxicity testing to replace the Draize rabbit skin irritation test in Europe and internationally, since, in the very near future, new EU and OECD Test Guidelines will be proposed for regulatory acceptance.     

The use of HET-CAM test in detecting the ocular irritation

Agrochemicals must undergo numerous toxicological tests before registration. One of these experiments is the examination of eye irritation potential. To get knowledge about eye irritation, recently only the in vivo Draize-test is accepted, which is one of the most criticized methods because of the injuries inflicted on the test animals. Several in vitro methods have been used to investigate the toxicity of potential eye irritants with a view to replacing in vivo eye irritation testing. In the HET-CAM test chemicals are placed in direct contact with chorioallantoic membrane of the hen's egg. The occurrence of vascular injury or coagulation in response to a compound is the basis for employing this technique as an indication of the likelihood that a chemical would damage mucous membranes (especially the eye) in vivo. In our studies comparative screening was performed with a set of agrochemicals to establish paralell data on in vitro (HET-CAM) and in vivo (Draize) results in case of 6 agrochemicals. The solutions to be tested are added to the membrane and left in contact for 5 minutes and the membrane is examined for vascular damage at set time periods. Irritancy is scored according to the severity and speed at which damage occurs providing an indication of the likely irritant effect of the compound. Our study showed good correlation between results obtained by the HET-CAM test and those of the Draize rabbit eye test most cases. The present form of the HET-CAM test can be proposed as a pre-screen method of eye irritation tests.     

The ECVAM international validation study on in vitro tests for acute skin irritation: selection of test chemicals

The ECVAM-funded skin irritation validation study (SIVS) was initiated in 2003, with the aim to evaluate whether the EpiDerm, EPISKIN and the SIFT alternative methods were able to reliably identify skin irritant and non-irritant chemicals, and could therefore be candidates for replacing the rabbit Draize test for skin irritation. The primary goal of the study was to evaluate the predictive capacity of the assays with regard to the EU classification system, which employs the risk phrases, "R38", for skin irritants, and "no label" for non-irritants. A secondary objective was the retrospective analysis of the data, to assess whether the in vitro tests would be able to discriminate between strong irritants (category 2), mild irritants (category 3) and non-irritants (no category), as defined by the OECD and United Nations proposal for a Globally Harmonised System (GHS) for the classification and labelling of dermal irritancy. A Chemicals Selection Sub-Committee (CSSC) was appointed to identify test chemicals to be used in the SIVS, for which existing, high quality in vivo data were available, with which to correlate the in vitro measurements. Since chemicals from the European Centre for the Ecotoxicology and Toxicology of Chemicals (ECETOC) database of reference chemicals for skin irritation/skin corrosion had been extensively used in preceding studies, the CSSC made use of novel sources for potential test chemicals. The first source of chemicals screened was the New Chemicals Database (NCD), which is the central archive within the EU notification scheme for 'new' commercial chemicals. Data registered in the NCD originate from standard assays, submitted in compliance with the legislation which regulates the marketing of industrial chemicals, and are subject to quality assurance by the competent authorities of the EU Member States. In addition, to obtain 'existing' chemicals which were readily available from major manufacturing and/or distribution sources, additional databases were surveyed, such as the Toxic Substance Control Act (TSCA) database maintained by the US Environmental Protection Agency (EPA), and the ECETOC database, with the exclusion of the chemicals used in the previous optimisation and prevalidation phases. A total of approximately 3500 chemicals from the NCD and 1600 from the additional databases were screened. Pre-determined selection criteria were applied, primarily to ensure the quality of the in vivo data and the practicability of their use in testing. Overall, the number of chemicals fulfilling the CSSC selection criteria was found to be limited, particularly in the case of GHS category 2 chemicals. However, a total set of 60 chemicals were selected and proposed to the Management Team of the SIVS for independent coding and supply to the participating laboratories. The selected chemicals: i) represented statistically justified sample sizes for distinguishing R38 from no-label chemicals; ii) provided a balanced representation of the three GHS categories, to allow for the post hoc evaluation of the performance of the assays for that classification system; and iii) acknowledged, to a certain degree, the large prevalence known to exist for chemicals which have oedema and erythema scores of 0. The selected chemicals represented a variety of molecular structures, functional chemical groups, and effect and use categories, as well as a wide range of physico-chemical properties. They represented a challenging set of chemicals, relevant to current industrial commerce, with which to validate the alternative methods.     

The EpiDerm test protocol for the upcoming ECVAM validation study on in vitro skin irritation tests--an assessment of the performance of the optimised test

During the past decade, several validation studies have been conducted on in vitro methods for discriminating between skin irritating and non-irritating chemicals. The reconstructed human skin models, EpiDerm and EPISKIN, provided the most promising results. Based on experience of the similar performance of the two skin models, it was suggested that a common test protocol and prediction model should be developed for the prediction of skin irritation potential with the two models. When the EPISKIN protocol was applied with the EpiDerm model, an acceptable specificity (80%) was achieved, whereas the sensitivity (60%) was low. In 2003, the EPISKIN protocol was further refined by extending the post-incubation period following exposure to test chemicals. This extension and additional technical improvements to the EpiDerm protocol were evaluated with 19 chemicals from the prevalidation study. With the new test design, high sensitivity (80%) and specificity (78%) were obtained. The statistical probability for correct classifications was high, so the test was considered to be ready for formal validation. However, since test optimisation had been conducted with the same test chemicals as were used in the ECVAM prevalidation study, it was decided that the optimisation of the protocol had to be verified with a new set of chemicals. Thus, in the current study, 26 additional chemicals (10 rabbit irritants and 16 non-irritants), which had previously been selected and tested by LOREAL with EPISKIN, were evaluated in three independent experiments with EpiDerm. With this unbalanced testing set, a specificity of 94%, and a sensitivity of 60% were obtained, while the positive and negative predictivity and accuracy remained almost unchanged (around 80%) in comparison to the in vivo rabbit data. Overall, 45 chemicals (20 irritants and 25 non-irritants) were tested according to the final protocol. The resulting high positive (82%) and negative predictive values (79%) confirmed the reliability (accuracy of 80%) of the improved test protocol of the EpiDerm model.     

Assessment of the eye irritating properties of chemicals by applying alternatives to the Draize rabbit eye test: the use of QSARs and in vitro tests for the classification of eye irritation

Huggins has reported on the current situation relating to the development of alternatives to the Draize eye irritation test with rabbits, and an ECVAM Working Group have reviewed the efforts needed in order to replace this animal test within the next 10 years by using the results of non-animal assessment methods. Our report reviews regulatory experience gained over the last 20 years with the EU chemicals notification procedure with respect to the assessment of eye lesions observed in Draize tests. The nature of eye lesions and their importance for classification and labelling of possible hazards to human eyes are evaluated and discussed, with a view to promoting the development of specific in vitro assays which are able to discriminate between eye damage, moderate eye irritation, and minor irritation effects which are completely reversible within a few days. Structural alerts for the prediction of eye irritation/corrosion hazards to be classified and labelled according to international classification criteria, are presented, which should be validated in accordance with internationally agreed (OECD) principles for (Q)SAR system validation. Physicochemical limit values for prediction of the absence of any eye irritation potential relevant for human health can make available a definition of the applicability domains of alternative methods developed for the replacement of the Draize eye irritation test.     

Development of a sensory neuronal cell model for the estimation of mild eye irritation

In an attempt to improve the in vitro test strategy for the estimation of eye irritation, a neuronal cell model has been developed, with cells expressing vanilloid receptor type 1 (VR1) nociceptors. The currently accepted method for measuring eye irritancy is the ethically and scientifically criticised Draize rabbit eye test, despite the fact that alternative in vitro methods are available which have proved to be reliable and reproducible for predicting severe ocular toxicity. However, no alternative tests for measuring neuronal stimulation have yet been developed, and the prediction of eye irritation in the mild range is therefore insufficient. VR1 is a nociceptor localised in C-fibre neurons innervating the cornea and the surrounding tissue, and it responds to potentially damaging stimuli by releasing Ca2+ into the cytoplasm. As a sensory endpoint, [Ca2+]i was measured in VR1 transfected cells, as well as in control cells. Short-term cell cytotoxicity studies (cell membrane rupture and morphological divergence) were used to determine the non-corrosive concentrations of the test chemicals. Preliminary results indicated that hygiene products used daily may induce eye irritation via VR1 nociceptors. The lowest toxic concentration (0.025%) of liquid hand soap, as determined by morphologic divergences of cells, generated an 80% increase in [Ca2+]i over the basal [Ca2+]i in VR1 transfected cells, whereas the non-specific [Ca2+]i increased by 33%. Furthermore, all the endpoints studied indicated that shampoo for children was less active than shampoo for adults. If this method is successfully validated with standardised reference chemicals, the model could complete the test battery of in vitro alternatives, resulting in the saving of thousands of laboratory animals.     

Sensory nerves, neurogenic inflammation and pain: missing components of alternative irritation strategies? A review and a potential strategy

The eyes and skin are highly innervated by sensory nerves; stimulation of these nerves by irritants may give rise to neurogenic inflammation, leading to sensory irritation and pain. Few in vitro models of neurogenic inflammation have been described in conjunction with alternative skin and eye irritation methods, despite the fact that the sensory innervation of these organs is well-documented. To date, alternative approaches to the Draize skin and eye irritation tests have proved largely successful at classifying severe irritants, but are generally poor at discriminating between agents with mild to moderate irritant potential. We propose that the development of in vitro models for the prediction of sensory stimulation will assist in the re-classification of the irritant potential of agents that are under-predicted by current in vitro strategies. This review describes the range of xenobiotics known to cause inflammation and pain through the stimulation of sensory nerves, as well as the endogenous mediators and receptor types that are involved. In particular, it focuses on the vanilloid receptor, its activators and its regulation, as these receptors function as integrators of responses to numerous noxious stimuli. Cell culture models and ex vivo preparations that have the potential to serve as predictors of sensory irritation are also described. In addition, as readily available sensory neuron cell line models are few in number, stem cell lines (with the capacity to differentiate into sensory neurons) are explored. Finally, a preliminary strategy to enable assessment of whether incorporation of a sensory component will enhance the predictive power of current in vitro eye and skin testing strategies is proposed.     

In vitro cytotoxicity test for estimating non-ocular irritation dose of ophthalmic solutions

The in vitro cytotoxicity test for estimating the non-ocular irritation dose of ophthalmic solutions was investigated. In the in vitro test, normal human epidermal keratinocytes (NHEK) in a confluent monolayer were incubated for 48hr in a medium with test compounds. The concentration of a test compound which causes a 50% reduction in NHEK viability was determined as IC₅₀ by MTT colorimetric assay. For comparison, the in vivo rabbit ocular irritation tests were carried out by the standard Draize method. The maximum concentration, which did not show any ocular irritation, was determined as DS₀. The results showed the correlation coefficient between the IC₅₀ values and the DS₀ values for 19 test compounds to be 0.82. However, the correlation coefficients for 10 compounds, which have IC₅₀ values of less than 300g/ml, and for 7 alcohols were 0.99. The IC₅₀-DS₀ correlation curves obtained could be utilized as the critical concentrations for ocular irritation. These results suggest that our in vitrolin vivo test can estimate non-ocular irritation dose of the ophthalmicpreparations in advance of the in vivo tests.Abbreviations DS₀ Draize Score 0 - KGM keratinocyte growth medium - NHEK normal human epidermal keratinocytes     

Repeated exposure to benzalkonium chloride in the Ex Vivo Eye Irritation Test (EVEIT): observation of isolated corneal damage and healing

The prediction of side-effects is a key issue in the REACH initiative on chemicals, in the production of cosmetics and in the preclinical testing of drugs. A new ex vivo test for repeated substance application is presented, that is able to identify corrosive and irritant effects on the eye by using crucial endpoints, such as cellular and morphological damage, and healing characteristics. The test is intended to replace the Draize eye test and to improve the preclinical testing of drugs and chemicals that are likely to come into direct contact with the cornea. The Ex Vivo Eye Irritation Test (EVEIT) is a self-healing system, involving living corneas obtained from abattoir rabbit eyes. The corneas are cultured in a similar way to the method used during the transplantation of corneal grafts. The corneas are exposed to multiple small, mechanical abrasions, and then test substances are repeatedly dropped onto the centres of the corneas. The test substances applied in this study were citrate-buffered hyaluronate eye drops and an artificial tear replacement, with increasing concentrations of up to 0.1% benzalkonium chloride. A dose-dependent inhibition of recovery and impairment of the lactate production mechanism in the cornea was observed with benzalkonium chloride treatment.     

鸡胚尿囊膜（HET-CAM）眼刺激替代试验对产品及原料评价的比较

目的比较鸡胚尿囊膜试验（HET-CAM）作为一种眼刺激替代方法对产品或原料的评价。方法采用HET-CAM和兔眼Draize试验方法,对19种原料和23种化妆品及家用洗涤产品眼刺激性进行检测,对体内体外试验的结果进行统计比较。结果比较体内体外试验,原料和产品的kappa系数分别为0.826,0.531;灵敏度分别为100%,81.8%;特异度分别为85.7%,77.8%。结论 HET-CAM可作为Draize试验的替代试验,HET-CAM系统更适合对单纯化学品原料进行眼刺激试验。     

Using in vitro prediction models instead of the rabbit eye irritation test to classify and label new chemicals: a post hoc data analysis of the international EC/HO validation study

The international validation study on alternative methods to replace the Draize rabbit eye irritation test, funded by the European Commission (EC) and the British Home Office (HO), took place during 1992-1994, and the results were published in 1995. The results of this EC/HO study are analysed by employing discriminant analysis, taking into account the classification of the in vivo data into eye irritation classes A (risk of serious damage to eyes), B (irritating to eyes) and NI (non-irritant). A data set for 59 test items was analysed, together with three subsets: surfactants, water-soluble chemicals, and water-insoluble chemicals. The new statistical methods of feature selection and estimation of the discriminant functions classification error were used. Normal distributed random numbers were added to the mean values of each in vitro endpoint, depending on the observed standard deviations. Thereafter, the reclassification error of the random observations was estimated by applying the fixed function of the mean values. Moreover, the leaving-one-out cross-classification method was applied to this random data set. Subsequently, random data were generated r times (for example, r = 1000) for a feature combination. Eighteen features were investigated in nine in vitro test systems to predict the effects of a chemical in the rabbit eye. 72.5% of the chemicals in the undivided sample were correctly classified when applying the in vitro endpoints lgNRU of the neutral red uptake test and lgBCOPo5 of the bovine opacity and permeability test. The accuracy increased to 80.9% when six in vitro features were used, and the sample was subdivided. The subset of surfactants was correctly classified in more than 90% of cases, which is an excellent performance.     

Irritative effects of some pesticides and a technical component on tissue structure of the chorioallantoic membrane

The chorioallantoic membrane (CAM) is a complete tissue that responds to injury with a complete inflammatory reaction, this process similar to that induced by chemicals in the conjunctival tissue of the rabbit eye. During the study chemicals are placed directly onto the chorioallantoic membrane and the occurrence of vascular injury or coagulation in response to a compound is as an indication of the potential of a chemical to damage mucous membranes. In our study irritant pesticides (Fusilade S, Karathane LC) and a technical pesticide component (Trend) were tested and their effects on the tissue structures of CAM were examined. After treatment with the test materials, first lysis and then haemorrhage were observed macroscopically on CAM. In histological pictures stained with H-E the rupture of the blood vessel wall was seen and blood was observed around the blood vessels in the middle layer. The histological findings correlated well with the macroscopic appearance in this study. In general a good correlation was found between the HET-CAM results and reported data from Draize test. The subjective nature of the evaluation is reduced through the histological examination of treated CAM. The HET-CAM test can be a useful component of a battery of tests needed for replacing the Draize rabbit eye irritation test.     

The use of bootstrap resampling to assess the variability of Draize tissue scores

The acute dermal and ocular effects of chemicals are generally assessed by performing the Draize skin and eye tests, respectively. Because the animal data obtained in these tests are also used for the development and validation of alternative methods for skin and eye irritation, it is important to assess the inherent variability of the animal data, since this variability places an upper limit on the predictive performance that can be expected of any alternative model. The statistical method of bootstrap resampling was used to estimate the variability arising from the use of different animals and time-points, and the estimates of variability were used to determine the maximal extent to which Draize test tissue scores can be predicted.     

A reassessment of the in vitro RBC haemolysis assay with defibrinated sheep blood for the determination of the ocular irritation potential of cosmetic products: comparison with the in vivo Draize rabbit test

We examined the correlation between results obtained from the in vivo Draize test for ocular irritation and in vitro results obtained from the sheep red blood cell (RBC) haemolytic assay, which assesses haemolysis and protein denaturation in erythrocytes, induced by cosmetic products. We sought to validate the haemolytic assay as a preliminary test for identifying highly-irritative products, and also to evaluate the in vitro test as alternative assay for replacement of the in vivo test. In vitro and in vivo analyses were carried out on 19 cosmetic products, in order to correlate the lesions in the ocular structures with three in vitro parameters: (i) the extent of haemolysis (H50); (ii) the protein denaturation index (DI); and (iii) the H50/DI ratio, which reflects the irritation potential (IP). There was significant correlation between maximum average scores (MAS) and the parameters determined in vitro (r = 0.752-0.764). These results indicate that the RBC assay is a useful and rapid test for use as a screening method to assess the IP of cosmetic products, and for predicting the IP value with a high level of concordance (94.7%). The assay showed high sensitivity and specificity rates of 91.6% and 100%, respectively.     

The luminescent bacteria toxicity test: Its potential as an in vitro alternative

During the past several years, the use of animals for toxicity testing has come under critical surveillance. For ethical and economic reasons, various techniques have been developed and proposed as potential alternatives for some of the whole animal toxicity assays. One assay proposed as an alternative to animal testing is the luminescent bacteria toxicity test (LBT), provided under the trade name of Microtox®. The sensitivity and specificity of the LBT was compared with two commonly used toxicity tests–-the L-929 Minimal Eùgle's Medium (MEM) elution cytotoxicity test and the Draize test. Cytotoxicity and LBT test data from 709 medical device and biomaterial extracts were compared using a positive/negative ranking system which provided a measurement of false positive and false negative results. These data were compiled from nine separate laboratories producing or using a wide variety of biomaterials and medical device products. The LBT was more sensitive than the tissue culture assay and displayed few false negatives. LBT EC₅₀ values were compared with eye irritancy categories for a group of 34 chemicals and 27 personal care products. As with tissue culture, the LBT was more sensitive and produced minimal false negatives. The data from this study indicate the LBT has potential as a rapid, simple method to screen biomaterials and personal care products for toxicity and irritancy.