Overall performance of Bovine Corneal Opacity and Permeability (BCOP) Laser Light-Based Opacitometer (LLBO) test method with regard to solid and liquid chemicals testing.

A prospective study of the Bovine Corneal Opacity and Permeability (BCOP) Laser Light-Based Opacitometer (LLBO) test method was conducted to evaluate its usefulness to identify chemicals as inducing serious eye damage (Cat. 1) or chemicals not requiring classification for eye irritation (No Cat.) applying United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS). The aim was to demonstrate the reproducibility of the BCOP LLBO protocol for liquids and solids and define its predictive capacity. Briefly, 145 chemicals were simultaneously tested with BCOP LLBO and OP-KIT (OECD TG 437), one to two times in one laboratory. When used to identify Cat. 1, the BCOP LLBO has a false negative rate (FNR) of 24.1% (N = 56) compared to 34.8% (N = 56) for the BCOP OP-KIT, with a comparable false positive rate (FPR, N = 89) of 18.5% and 20.8%, respectively. When used to identify chemicals not requiring classification (No Cat.) the BCOP LLBO and BCOP OP-KIT had a FNR (N = 104) of 6.2% and 7.2% and a FPR (N = 41) of 45.1% and 42.7%, respectively. The OP-KIT and LLBO devices are interchangeable at no cost to data quality and reliability. The OP-KIT and LLBO devices are interchangeable at no cost to data quality and reliability. The performance of the LLBO is at least as good as the OP-KIT, both methods can be used to identify UN GHS Cat. 1 and UN GHS No Cat. chemicals.

Development of a defined approach for eye irritation or serious eye damage for neat liquids based on cosmetics Europe analysis of in vitro RhCE and BCOP test methods.

The focus of Cosmetics Europe's ocular toxicity programme is on development of testing strategies and defined approaches for identification of ocular effects of chemicals in the context of OECD's Guidance Document on an Integrated Approach on Testing and Assessment (IATA) for Serious Eye Damage and Eye Irritation. Cosmetics Europe created a comprehensive database of chemicals for which in vitro data are available with corresponding historical in vivo Draize eye data and physicochemical properties. This database allowed further exploration of the initially proposed strategies from the CON4EI project and to identify opportunities for refinement. One key outcome of this project is that combining in vitro test methods (RhCE and BCOP LLBO) with physicochemical properties in a two-step Bottom-Up approach applicable to neat liquids, resulted in an improvement of the specificity, without reducing the sensitivity, when compared to the combination of in vitro methods alone. The Bottom-Up approach proposed here for neat liquids correctly predicted 58.3% (EpiOcular™ EIT followed by BCOP LLBO) to 62.6% (SkinEthic™ HCE EIT followed by BCOP LLBO) of No Cat., 59.1% to 68.7% of Cat. 2, and 76.5% of Cat. 1. Incorporating specific physicochemical properties with this Bottom-Up approach increased the correct identification of No Cat. neat liquids to between 72.7% and 79.7%.

Development of a defined approach for eye irritation or serious eye damage for liquids, neat and in dilution, based on cosmetics Europe analysis of in vitro STE and BCOP test methods.

The focus of Cosmetics Europe's programme on serious eye damage/eye irritation is on development of testing strategies and defined approaches for identification of ocular effects of chemicals in the context of OECD's Guidance Document on an Integrated Approach on Testing and Assessment (IATA) for Serious Eye Damage and Eye Irritation. Cosmetics Europe created a comprehensive database of chemicals for which in vitro data are available with corresponding historical in vivo Draize eye data. This database allowed further exploration of the initially proposed strategies from the CON4EI project and to identify opportunities for refinement. The current analysis focused on the development of a defined approach, applicable to liquid non-surfactant chemicals, neat and in dilution, that can distinguish between the three UN GHS categories (Cat. 1, Cat. 2, and No Cat.). Combining the modified-protocol Short Time Exposure (STE) test method (OECD TG 491 with extension to highly volatile substances) with the Bovine Corneal Opacity and Permeability Laser Light-Based Opacitometer (BCOP LLBO) test method in a Bottom-Up approach identified 81.2% Cat. 1, 56.3% Cat. 2, and 85.3% No. Cat correctly, with an NPV of 96.7% and a PPV of 68.6%. Therefore, the performance of the defined approach was better than the standalone test methods.

Assessment of cosmetic ingredients in the in vitro reconstructed human epidermis test method EpiSkin™ using HPLC/UPLC-spectrophotometry in the MTT-reduction assay.

Cosmetics Europe recently established HPLC/UPLC-spectrophotometry as a suitable alternative endpoint detection system for measurement of formazan in the MTT-reduction assay of reconstructed human tissue test methods irrespective of the test system involved. This addressed a known limitation for such test methods that use optical density for measurement of formazan and may be incompatible for evaluation of strong MTT reducer and/or coloured chemicals. To build on the original project, Cosmetics Europe has undertaken a second study that focuses on evaluation of chemicals with functionalities relevant to cosmetic products. Such chemicals were primarily identified from the Scientific Committee on Consumer Safety (SCCS) 2010 memorandum (addendum) on the in vitro test EpiSkin™ for skin irritation testing. Fifty test items were evaluated in which both standard photometry and HPLC/UPLC-spectrophotometry were used for endpoint detection. The results obtained in this study: 1) provide further support for Within Laboratory Reproducibility of HPLC-UPLC-spectrophotometry for measurement of formazan; 2) demonstrate, through use a case study with Basazol C Blue pr. 8056, that HPLC/UPLC-spectrophotometry enables determination of an in vitro classification even when this is not possible using standard photometry and 3) addresses the question raised by SCCS in their 2010 memorandum (addendum) to consider an endpoint detection system not involving optical density quantification in in vitro reconstructed human epidermis skin irritation test methods.

Use of HPLC/UPLC-spectrophotometry for detection of formazan in in vitro Reconstructed human Tissue (RhT)-based test methods employing the MTT-reduction assay to expand their applicability to strongly coloured test chemicals.

A number of in vitro test methods using Reconstructed human Tissues (RhT) are regulatory accepted for evaluation of skin corrosion/irritation. In such methods, test chemical corrosion/irritation potential is determined by measuring tissue viability using the photometric MTT-reduction assay. A known limitation of this assay is possible interference of strongly coloured test chemicals with measurement of formazan by absorbance (OD). To address this, Cosmetics Europe evaluated use of HPLC/UPLC-spectrophotometry as an alternative formazan measurement system. Using the approach recommended by the FDA guidance for validation of bio-analytical methods, three independent laboratories established and qualified their HPLC/UPLC-spectrophotometry systems to reproducibly measure formazan from tissue extracts. Up to 26 chemicals were then tested in RhT test systems for eye/skin irritation and skin corrosion. Results support that: (1) HPLC/UPLC-spectrophotometry formazan measurement is highly reproducible; (2) formazan measurement by HPLC/UPLC-spectrophotometry and OD gave almost identical tissue viabilities for test chemicals not exhibiting colour interference nor direct MTT reduction; (3) independent of the test system used, HPLC/UPLC-spectrophotometry can measure formazan for strongly coloured test chemicals when this is not possible by absorbance only. It is therefore recommended that HPLC/UPLC-spectrophotometry to measure formazan be included in the procedures of in vitro RhT-based test methods, irrespective of the test system used and the toxicity endpoint evaluated to extend the applicability of these test methods to strongly coloured chemicals.

Cosmetics Europe multi-laboratory pre-validation of the SkinEthic™ reconstituted human corneal epithelium test method for the prediction of eye irritation.

Cosmetics Europe, The Personal Care Association, known as Colipa before 2012, conducted a program of technology transfer and assessment of Within/Between Laboratory (WLV/BLV) reproducibility of the SkinEthic™ Reconstituted Human Corneal Epithelium (HCE) as one of two human reconstructed tissue eye irritation test methods. The SkinEthic™ HCE test method involves two exposure time treatment procedures - one for short time exposure (10 min - SE) and the other for long time exposure (60 min - LE) of tissues to test substance. This paper describes pre-validation studies of the SkinEthic™ HCE test method (SE and LE protocols) as well as the Eye Peptide Reactivity Assay (EPRA). In the SE WLV study, 30 substances were evaluated. A consistent outcome with respect to viability measurement across all runs was observed with all substances showing an SD of less than 18%. In the LE WLV study, 44 out of 45 substances were consistently classified. These data demonstrated a high level of reproducibility within laboratory for both the SE and LE treatment procedures. For the LE BLV, 19 out of 20 substances were consistently classified between the three laboratories, again demonstrating a high level of reproducibility between laboratories. The results for EPRA WLV and BLV studies demonstrated that all substances analysed were categorised similarly and that the method is reproducible. The SkinEthic™ HCE test method entered into the experimental phase of a formal ECVAM validation program in 2010.

Cosmetics Europe multi-laboratory pre-validation of the EpiOcular™ reconstituted human tissue test method for the prediction of eye irritation.

Cosmetics Europe, The Personal Care Association (known as Colipa before 2012), conducted a program of technology transfer and within/between laboratory reproducibility of MatTek Corporation's EpiOcular™ Eye Irritation Test (EIT) as one of the two human reconstructed tissue test methods. This EIT EpiOcular™ used a single exposure period for each chemical and a prediction model based on a cut-off in relative survival [ ≤60%=irritant (I) (GHS categories 2 and 1); >60%=no classification (NC)]. Test substance single exposure time was 30 min with a 2-h post-exposure incubation for liquids and 90 min with an 18-h post-exposure incubation for solids. Tissue viability was determined by tetrazolium dye (MTT) reduction. Combinations of 20 coded chemicals were tested in 7 laboratories. Standardized laboratory documentation was used by all laboratories. Twenty liquids (11 NC/9 I) plus 5 solids (3 NC/2 I) were selected so that both exposure regimens could be assessed. Concurrent positive (methyl acetate) and negative (water) controls were tested in each trial. In all, 298 independent trials were performed and demonstrated 99.7% agreement in prediction (NC/I) across the laboratories. Coefficients of variation for the% survival for tissues from each treatment group across laboratories were generally low. This protocol has entered in 2010 the experimental phase of a formal ECVAM validation program.

The red blood cell phototoxicity test (photohaemolysis and haemoglobin oxidation): EU/COLIPA validation programme on phototoxicity (phase II).

In the EU/COLIPA validation programme on "Photoirritation in vitro", two core tests and a number of mechanistically based tests were carried out to examine their suitability as regulatory tests for phototoxicity testing. In the meantime, one core test, the 3T3 neutral red uptake phototoxicity test (NRU PT) has been validated and has been accepted by ECVAM and the European Commission. The second core test, the red blood cell phototoxicity test (Photo-RBC test), has passed through a prevalidation process during this programme. This test protocol combines two endpoints, photohaemolysis and met-haemoglobin (met-Hb) formation. These endpoints are determined by measuring changes in the optical density of the haemoglobin spectrum at 525 nm and 630 nm, respectively. In addition, a prediction model was inserted into the Standard Operating Procedure (SOP) with two cut-off values: a photohaemolysis factor (PHF) > or = 3.0 for photohaemolysis, and a deltaOD(max) > or = 0.05 for met-Hb formation. Three laboratories agreed to implement the SOP and to perform the study by testing 30 selected test chemicals (25 phototoxicants and 5 non- phototoxic chemicals). The outcome of the study presents a good overall fit, including acceptable accuracy, sensitivity, and positive predictivity. The specificity and the negative predictivity are comparably low, due to the low number of non-phototoxic substances among the test chemicals. Further analysis of the data showed that the transfer of the SOP from between laboratories could have been more efficient. The results, especially of the lead laboratory, clearly indicate that an experienced laboratory can handle the SOP with high predictivity for phototoxicants and non-phototoxic substances. Finally, it was concluded that the combined Photo-RBC test can be considered as a second in vitro test, which can be used advantageously to obtain some mechanistic information, in particular on photodynamic effects on cellular proteins and biomembranes.

Performance of the Pollen Tube Growth Test in the COLIPA Validation Study on Alternatives to the Rabbit Eye Irritation Test.

In the present paper, we describe and analyse the performance and the results of the pollen tube growth (PTG) test applied to the COLIPA international validation study of in vitro alternatives to the Draize eye irritation test. The PTG test, based on photometric quantification of in vitro pollen tube mass production, was used by three independent laboratories to estimate the acute eye irritation potentials of 23 ingredients and 32 cosmetic formulations. Basing on historical Draize test data and on IC(50) values of previously tested cosmetic formulations, a mathematical formula was generated to predict rabbit eye irritation potentials from PTG test results. Statistical evaluation of the calculated modified maximal average scores (MMAS) revealed a high prediction capability of the PTG test in regard to the finished formulations but a relatively low one for alcohols, higher concentrated cationic surfactants, and acidic and alkaline materials. Furthermore, our results indicated that the PTG test was able to produce precise IC(50) values without any limitations from all of the 55 test substances with good intra- and interlaboratory reproducibility. From these findings we suggest that the PTG test is not a validated test at present but is considered to be a potent candidate for further validation processes. For this purpose an additional prediction model for ingredient classes as mentioned above must be generated.

COLIPA validation project on in vitro eye irritation tests for cosmetic ingredients and finished products (phase I): the red blood cell test for the estimation of acute eye irritation potentials. Present status.

The red blood cell test (RBC test) is part of the COLIPA Validation Project on Alternatives to Draize Eye Irritation. It shows good intra- and interlaboratory reproducibility (reliability) and represents one of the promising in vitro alternatives of this project with a good fit to prediction models (relevance) for the assessment of acute ocular irritancy caused by certain classes of chemicals (mainly surfactants) and formulations. Results obtained during the period of test development, prevalidation, and validation are summarized. The method is based on that of Pape et al. (1987), Pape and Hoppe (1990) and Lewis et al. (1993). The protocol has two endpoints: cellular lysis and changes in protein conformation which can be correlated with initial events in tissue injury inducing inflammatory responses as assessed by Draize eye irritation scoring. Both endpoints are detected by spectrophotometric changes in the haemoglobin absorption at 541nm. The protocol also includes a set of prediction models (PM). One PM is designed to predict three classes of irritancy (classification model) based on both endpoints and the three other PMs are designed to predict modified maximum average scores (MMAS) by algorithms based on data from cellular lysis only. These three PMs [with prediction intervals (PIs)] are: (i) for surfactant ingredients, (ii) for surfactant containing finished products, and (iii) for both groups together. The three PMs are based on a common algorithm derived from historic data. It is shown that PMs derived from historic data from several laboratories, by the same procedure, also produce a good fit with the presented data. Therefore, participating laboratories concluded that the protocol as used in this formal validation study can be considered to be validated for the estimation of acute eye irritation potential of surfactant-containing finished products.

The International EU/COLIPA In Vitro Phototoxicity Validation Study: Results of Phase II (Blind Trial). Part 1: The 3T3 NRU Phototoxicity Test.

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 Study on UV Filter Chemicals from Annex VII of European Union Directive 76/768/EEC, in the In Vitro 3T3 NRU Phototoxicity Test.

In 1996, the Scientific Committee on Cosmetology of DGXXIV of the European Commission asked the European Centre for the Validation of Alternative Methods to test eight UV filter chemicals from the 1995 edition of Annex VII of Directive 76/768/EEC in a blind trial in the in vitro 3T3 cell neutral red uptake phototoxicity (3T3 NRU PT) test, which had been scientifically validated between 1992 and 1996. Since all the UV filter chemicals on the positive list of EU Directive 76/768/EEC have been shown not to be phototoxic in vivo in humans under use conditions, only negative effects would be expected in the 3T3 NRU PT test. To balance the number of positive and negative chemicals, ten phototoxic and ten non-phototoxic chemicals were tested under blind conditions in four laboratories. Moreover, to assess the optimum concentration range for testing, information was provided on appropriate solvents and on the solubility of the coded chemicals. In this study, the phototoxic potential of test chemicals was evaluated in a prediction model in which either the Photoirritation Factor (PIF) or the Mean Photo Effect (MPE) were determined. The results obtained with both PIF and MPE were highly reproducible in the four laboratories, and the correlation between in vitro and in vivo data was almost perfect. All the phototoxic test chemicals provided a positive result at concentrations of 1µ/ml, while nine of the ten non-phototoxic chemicals gave clear negative results, even at the highest test concentrations. One of the UV filter chemicals gave positive results in three of the four laboratories only at concentrations greater than 100µ/ml; the other laboratory correctly identified all 20 of the test chemicals. An analysis of the impact that exposure concentrations had on the performance of the test revealed that the optimum concentration range in the 3T3 NRU PT test for determining the phototoxic potential of chemicals is between 0.1µg/ml and 10µg/ml, and that false positive results can be obtained at concentrations greater than 100µg/ml. Therefore, the positive results obtained with some of the UV filter chemicals only at concentrations greater than 100µg/ml do not indicate a phototoxic potential in vivo. When this information was taken into account during calculation of the overall predictivity of the 3T3 NRU PT test in the present study, an almost perfect correlation of in vitro versus in vivo results was obtained (between 95% and 100%), when either PIF or MPE were used to predict the phototoxic potential. The management team and participants therefore conclude that the 3T3 NRU PT test is a valid test for correctly assessing the phototoxic potential of UV filter chemicals, if the defined concentration limits are taken into account.

A summary report of the COLIPA international validation study on alternatives to the draize rabbit eye irritation test.

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.

Combined in vitro assay for photohaemolysis and haemoglobin oxidation as part of a phototoxicity test system assessed with various phototoxic substances.

12 well-known phototoxicants used in the EC/COLIPA (The European Cosmetic, Toiletry and Perfumery Association) interlaboratory validation trial on 'Photoirritation in vitro' have been analysed in view of their ability to induce photohaemolysis. As well as, or instead of, this effect, oxidative reactions involving oxyhaemoglobin have been monitored, by assessing intra- and extracellular methaemoglobin formation, as an additional parameter in the screening of deleterious photoeffects on the cellular protein of red blood cells. A combination of both photohaemolysis and photo-oxidation of haemoglobin is proposed for the investigation of light-induced toxicity in an in vitro test system. Piroxicam and 8-methoxypsoralen did not produce any marked phototoxic alterations in this system. Rose bengal and amiodarone demonstrated predominantly membrane effects, whereas the antibiotics tetra- and doxycycline damaged exclusively the intracellular haemoglobin. All other phototoxins caused both photohaemolysis and photo-oxidation of haemoglobin.

Validation of the red blood cell test system as in vitro assay for the rapid screening of irritation potential of surfactants.

An in vitro red blood cell assay (RBC assay) is presented that allows one to estimate irritation potentials of tensides and detergents. The estimation is based on the differentiation between cell membrane lysis and cell protein denaturation. Both effects are measured photometrically by use of the inherent native dye oxyhemoglobin (HbO2). Besides hemolysis (H50) as a test parameter, the denaturation index DI is introduced, which is defined to be equal to 100% at a concentration of 30 mol sodium dodecylsulfate (SDS) per mole HbO2 as internal standard. The HbO2 release (H50), its denaturation (DI), and the ratio of both parameters (L/D ratio) are used to characterize the in vitro effects of surfactants. All data, including the L/D ratio are compared with in vivo data on eye irritancies, evaluated according to OECD Guideline #405 for testing chemicals, and with other published results from in vivo experiments. The good correlation of the L/D ratio of a broad range of 100 marketable shampoos with their corresponding Draize data (r = .806, p less than .0001) allows one to predict eye irritation potentials from another 20 commercially available shampoos in a blind trial with highly significant rank correlations to their in vivo irritancies (rs = .911, p less than .0001). Nearly similar good correlations were obtained by comparing ranks of in vivo and in vitro data of 16 anionic surfactants. All correlations found were significant (rs greater than .80 and p less than .0001). The RBC assay is an inexpensive, rapid, irritancy screening test that provides reliable results with good reproducibility. The test helps to reduce or even avoid animal testing in this application.