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 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.

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%.

CON4EI: Slug Mucosal Irritation (SMI) test method for hazard identification and labelling of serious eye damaging and eye irritating chemicals.

Assessment of ocular irritancy is an international regulatory requirement in the safety evaluation of industrial and consumer products. Although many in vitro ocular irritation assays exist, alone they are incapable of fully categorizing chemicals. The objective of CEFIC-LRI-AIMT6-VITO CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project was to develop tiered testing strategies for eye irritation assessment that can lead to complete replacement of the in vivo Draize rabbit eye test (OECD TG 405). A set of 80 reference chemicals was tested with seven test methods, one method was the Slug Mucosal Irritation (SMI) test method. The method measures the amount of mucus produced (MP) during a single 1-hour contact with a 1% and 10% dilution of the chemical. Based on the total MP, a classification (Cat 1, Cat 2, or No Cat) is predicted. The SMI test method correctly identified 65.8% of the Cat 1 chemicals with a specificity of 90.5% (low over-prediction rate for in vivo Cat 2 and No Cat chemicals). Mispredictions were predominantly unidirectional towards lower classifications with 26.7% of the liquids and 40% of the solids being underpredicted. In general, the performance was better for liquids than for solids with respectively 76.5% vs 57.1% (Cat 1), 61.5% vs 50% (Cat 2), and 87.5% vs 85.7% (No Cat) being identified correctly.

CON4EI: Development of testing strategies for hazard identification and labelling for serious eye damage and eye irritation of chemicals.

Assessment of acute eye irritation potential is part of the international regulatory requirements for safety testing of chemicals. In the last decades, many efforts have been made in the search for alternative methods to replace the regulatory in vivo Draize rabbit eye test (OECD TG 405). Success in terms of complete replacement of the regulatory in vivo Draize rabbit eye test has not yet been achieved. The main objective of the CEFIC-LRI-AIMT6-VITO CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project was to develop tiered testing strategies for serious eye damage and eye irritation assessment that can lead to complete replacement of OECD TG 405. A set of 80 reference chemicals (e.g. balanced by important driver of classification and physical state), was tested with seven test methods. Based on the results of this project, three different strategies were suggested. We have provided a standalone (EpiOcular ET-50), a two-tiered and three-tiered strategy, that can be used to distinguish between Cat 1 and Cat 2 chemicals and chemicals that do not require classification (No Cat). The two-tiered and three-tiered strategies use an RhCE test method (EpiOcular EIT or SkinEthic™ EIT) at the bottom (identification No Cat) in combination with the BCOP LLBO (two-tiered strategy) or BCOP OP-KIT and SMI (three-tiered strategy) at the top (identification Cat 1). For our proposed strategies, 71.1% - 82.9% Cat 1, 64.2% - 68.5% Cat 2 and ≥80% No Cat chemicals were correctly identified. Also, similar results were obtained for the Top-Down and Bottom-Up approach.

CON4EI: Evaluation of QSAR models for hazard identification and labelling of eye irritating chemicals.

Assessment of ocular irritation is a regulatory requirement in safety evaluation of industrial and consumer products. Although a number of in vitro ocular irritation assays exist, none are capable of fully categorizing chemicals as stand-alone assays. Therefore, the CEFIC-LRI-AIMT6-VITO CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project was developed to assess the reliability of eight in vitro test methods and computational models as well as establishing an optimal tiered-testing strategy. For three computational models (Toxtree, and Case Ultra EYE_DRAIZE and EYE_IRR) performance parameters were calculated. Coverage ranged from 15 to 58%. Coverage was 2 to 3.4 times higher for liquids than for solids. The lowest number of false positives (5%) was reached with EYE_IRR; this model however also gave a high number of false negatives (46%). The lowest number of false negatives (25%) was seen with Toxtree; for liquids Toxtree predicted the lowest number of false negatives (11%), for solids EYE_DRAIZE did (17%). It can be concluded that the training sets should be enlarged with high quality data. The tested models are not yet sufficiently powerful for stand-alone evaluations, but that they can surely become of value in an integrated weight-of-evidence approach in hazard assessment.

CON4EI: SkinEthic™ Human Corneal Epithelium Eye Irritation Test (SkinEthic™ HCE EIT) for hazard identification and labelling of eye irritating chemicals.

Assessment of ocular irritancy is an international regulatory requirement and a necessary step in the safety evaluation of industrial and consumer products. Although a number of in vitro ocular irritation assays exist, none are capable of fully categorizing chemicals as a stand-alone assay. Therefore, the CEFIC-LRI-AIMT6-VITO CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project was developed with the goal of assessing the reliability of eight in vitro/alternative test methods as well as establishing an optimal tiered-testing strategy. One of the in vitro assays selected was the validated SkinEthic™ Human Corneal Epithelium Eye Irritation Test method (SkinEthic™ HCE EIT). The SkinEthic™ HCE EIT has already demonstrated its capacity to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage (No Category). The goal of this study was to evaluate the performance of the SkinEthic™ HCE EIT test method in terms of the important in vivo drivers of classification. For the performance with respect to the drivers all in vivo Cat 1 and No Cat chemicals were 100% correctly identified. For Cat 2 chemicals the liquids and the solids had a sensitivity of 100% and 85.7%, respectively. For the SkinEthic™ HCE EIT test method, 100% concordance in predictions (No Cat versus No prediction can be made) between the two participating laboratories was obtained. The accuracy of the SkinEthic™ HCE EIT was 97.5% with 100% sensitivity and 96.9% specificity. The SkinEthic™ HCE EIT confirms its excellent results of the validation studies.

CON4EI: Short Time Exposure (STE) test method for hazard identification and labelling of eye irritating chemicals.

Assessment of ocular irritancy is an international regulatory requirement in the safety evaluation of industrial and consumer products. Although many in vitro ocular irritation assays exist, alone they are incapable of fully categorizing chemicals. Therefore, the CEFIC-LRI-AIMT6-VITO CON4EI consortium was developed to assess the reliability of eight in vitro test methods and establish an optimal tiered-testing strategy. One assay selected was the Short Time Exposure (STE) assay. This assay measures the viability of SIRC rabbit corneal cells after 5min exposure to 5% and 0.05% solutions of test material, and is capable of categorizing of Category 1 and No Category chemicals. The accuracy of the STE test method to identify Cat 1 chemicals was 61.3% with 23.7% sensitivity and 95.2% specificity. If non-soluble chemicals and unqualified results were excluded, the performance to identify Cat 1 chemicals remained similar (accuracy 62.2% with 22.7% sensitivity and 100% specificity). The accuracy of the STE test method to identify No Cat chemicals was 72.5% with 66.2% sensitivity and 100% specificity. Excluding highly volatile chemicals, non-surfactant solids and non-qualified results resulted in an important improvement of the performance of the STE test method (accuracy 96.2% with 81.8% sensitivity and 100% specificity). Furthermore, it seems that solids are more difficult to test in the STE, 71.4% of the solids resulted in unqualified results (solubility issues and/or high variation between independent runs) whereas for liquids 13.2% of the results were not qualified, supporting the restriction of the test method regarding the testing of solids.

Reprint of "CON4EI: Selection of the reference chemicals for hazard identification and labelling of eye irritating chemicals".

Assessment of the acute eye irritation potential is part of the international regulatory requirements for testing of chemicals. In the past, several prospective and retrospective validation studies have taken place in the area of serious eye damage/eye irritation testing. Success in terms of complete replacement of the regulatory in vivo Draize rabbit eye test has not yet been achieved. A very important aspect to ensure development of successful alternative test methods and/or strategies for serious eye damage/eye irritation testing is the selection of appropriate reference chemicals. A set of 80 reference chemicals was selected for the CEFIC-LRI-AIMT6-VITO CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project, in collaboration with Cosmetics Europe, from the Draize Reference Database published by Cosmetics Europe based on key criteria that were set in their paper (e.g. balanced by important driver of classification and physical state). The most important goals of the CON4EI project were to identify the performance of eight in vitro alternative tests in terms of driver of classification and to identify similarities/differences between the methods in order the build a successful testing strategy that can discriminate between all UN GHS categories. This paper provides background on selection of the test chemicals.

CON4EI: Selection of the reference chemicals for hazard identification and labelling of eye irritating chemicals.

Assessment of the acute eye irritation potential is part of the international regulatory requirements for testing of chemicals. In the past, several prospective and retrospective validation studies have taken place in the area of serious eye damage/eye irritation testing. Success in terms of complete replacement of the regulatory in vivo Draize rabbit eye test has not yet been achieved. A very important aspect to ensure development of successful alternative test methods and/or strategies for serious eye damage/eye irritation testing is the selection of appropriate reference chemicals. A set of 80 reference chemicals was selected for the CEFIC-LRI-AIMT6-VITO CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project, in collaboration with Cosmetics Europe, from the Draize Reference Database published by Cosmetics Europe based on key criteria that were set in their paper (e.g. balanced by important driver of classification and physical state). The most important goals of the CON4EI project were to identify the performance of eight in vitro alternative tests in terms of driver of classification and to identify similarities/differences between the methods in order the build a successful testing strategy that can discriminate between all UN GHS categories. This paper provides background on selection of the test chemicals.

Multi-laboratory evaluation of SkinEthic HCE test method for testing serious eye damage/eye irritation using solid chemicals and overall performance of the test method with regard to solid and liquid chemicals testing.

A prospective multicentre study of the reconstructed human corneal epithelial tissue-based in vitro test method (SkinEthic™ HCE) was conducted to evaluate its usefulness to identify chemicals as either not classified for serious eye damage/eye irritation (No Cat.) or as classified (Cat. 1/Cat. 2) within UN GHS. The aim of this study was to demonstrate the transferability and reproducibility of the SkinEthic™ HCE EITS protocol for solids and define its predictive capacity. Briefly, 60 chemicals were three times tested (double blinded) in 3 laboratories and 35 additional chemicals were tested three times in one laboratory. Good within laboratory reproducibility was achieved of at least 95% (57/60) and 96.8% (92/95) for the extended data set. Furthermore, the overall concordance between the laboratories was 96.7% (58/60). The accuracy of the SkinEthic™ HCE EITS for the extended dataset, based on bootstrap resampling, was 81.0% (95% CI: 78.9% to 83.2%) with a sensitivity of 90.5% (95% CI: 88.1% to 92.9%) and specificity of 73.6% (95% CI: 71.7% to 75.5%). Overall, 200 chemicals were tested (105 liquids (EITL protocol) and 95 solids (EITS protocol)) resulting in a sensitivity of 95.2%, specificity of 72.1% and accuracy of 83.7%, thereby meeting all acceptance criteria for predictive capacity.

Phosphorylation of nucleosides and nucleoside analogs by mammalian nucleoside monophosphate kinases.

Nucleoside monophosphate kinases catalyze the reversible phosphotransferase reaction between nucleoside triphosphates and monophosphates, i.e., monophosphates are converted to their corresponding diphosphate form. These enzymes play an important role in the synthesis of nucleotides that are required for a variety of cellular metabolic processes, as well as for RNA and DNA synthesis. Human tissues contain a thymidylate kinase, a uridylate-cytidylate kinase, five isozymes of adenylate kinase, and several guanylate kinases. Nucleoside monophosphate kinases are also required for the pharmacological activation of therapeutic nucleoside and nucleotide analogs. This overview is focused on the substrate specificity, tissue distribution, and subcellular location of the mammalian monophosphate kinases and their role in the activation of nucleoside and nucleotide analogs.

Phosphorylation of deoxycytidine analog monophosphates by UMP-CMP kinase: molecular characterization of the human enzyme.

Phosphorylation of deoxycytidine analogs by cellular enzymes is a prerequisite for the activity of these compounds. We have investigated the kinetic parameters for the phosphorylation of 1-beta-D-arabinofuranosylcytosine (araC) and 2', 2'-difluorodeoxycytidine (dFdC) to their diphosphate forms catalyzed by human UMP-CMP kinase. We cloned the cDNA of this enzyme to enable characterization of the recombinant protein, determine its expression in different tissues, and determine the chromosome location of the gene. We showed that the recombinant UMP-CMP kinase phosphorylated CMP, dCMP, and UMP with highest efficiency and dUMP, AMP, and dAMP with lower efficiency. The monophosphates of araC and dFdC were shown to be phosphorylated with similar efficiency as dCMP and CMP. We further showed, in a combined enzymatic assay, that human deoxycytidine kinase and UMP-CMP kinase together phosphorylated araC, dFdC, and 2',3'-dideoxycytidine to their diphosphate forms. Northern blot analysis showed that the UMP-CMP kinase mRNA was ubiquitously present in human tissues as a 3.9-kb transcript with highest levels in pancreas, skeletal muscle, and liver. The human UMP-CMP kinase gene was localized to chromosome 1p34.1-1p33 by radiation hybrid analysis. We further expressed the UMP-CMP kinase as a fusion protein to the green fluorescent protein in Chinese hamster ovary cells, and showed that the fusion protein was located in the cytosol and nucleus.

Identification of a novel human adenylate kinase. cDNA cloning, expression analysis, chromosome localization and characterization of the recombinant protein.

Adenylate kinases have an important role in the synthesis of adenine nucleotides that are required for cellular metabolism. We report the cDNA cloning of a novel 22-kDa human enzyme that is sequence related to the human adenylate kinases and to UMP/CMP kinase of several species. The enzyme was expressed in Escherichia coli and shown to catalyse phosphorylation of AMP and dAMP with ATP as phosphate donor. When GTP was used as phosphate donor, the enzyme phosphorylated AMP, CMP, and to a small extent dCMP. Expression as a fusion protein with the green fluorescent protein showed that the enzyme is located in the cytosol. Northern blot analysis with mRNA from eight different human tissues demonstrated that the enzyme was expressed exclusively in brain, with two mRNA isoforms of 2.4 and 4.0 kb. The gene that encoded the enzyme was localized to chromosome 1p31. Based on the substrate specificity and the sequence similarity with the previously identified human adenylate kinases, we have named this novel enzyme adenylate kinase 5.

Phosphorylation of uridine and cytidine nucleoside analogs by two human uridine-cytidine kinases.

Uridine-cytidine kinases (UCK) have important roles for the phosphorylation of nucleoside analogs that are being investigated for possible use in chemotherapy of cancer. We have cloned the cDNA of two human UCKs. The approximately 30-kDa proteins, named UCK1 and UCK2, were expressed in Escherichia coli and shown to catalyze the phosphorylation of Urd and Cyd. The enzymes did not phosphorylate deoxyribonucleosides or purine ribonucleosides. UCK1 mRNA was detected as two isoforms of approximately 1.8 and approximately 2.7 kb. The 2.7-kb band was ubiquitously expressed in the investigated tissues. The band of approximately 1.8 kb was present in skeletal muscle, heart, liver, and kidney. The two isoforms of UCK2 mRNA of 1.2 and 2.0 kb were only detected in placenta among the investigated tissues. The genes encoding UCK1 and UCK2 were mapped to chromosome 9q34.2-9q34.3 and 1q22-1q23.2, respectively. We tested 28 cytidine and uridine nucleoside analogs as possible substrates of the enzymes. The enzymes phosphorylated several of the analogs, such as 6-azauridine, 5-fluorouridine, 4-thiouridine, 5-bromouridine, N(4)-acetylcytidine, N(4)-benzoylcytidine, 5-fluorocytidine, 2-thiocytidine, 5-methylcytidine, and N(4)-anisoylcytidine. The cloning and recombinant expression of the two human UCKs will be important for development of novel pyrimidine ribonucleoside analogs and the characterization of their pharmacological activation.

Cloning and characterization of the multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster.

A Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) was reported to phosphorylate all four natural deoxyribonucleosides as well as several nucleoside analogs (Munch-Petersen, B., Piskur, J., and Sondergaard, L. (1998) J. Biol. Chem. 273, 3926-3931). The broad substrate specificity of this enzyme together with a high catalytic rate makes it unique among the nucleoside kinases. We have in the present study cloned the Dm-dNK cDNA, expressed the 29-kDa protein in Escherichia coli, and characterized the recombinant enzyme for the phosphorylation of nucleosides and clinically important nucleoside analogs. The recombinant enzyme preferentially phosphorylated the pyrimidine nucleosides dThd, dCyd, and dUrd, but phosphorylation of the purine nucleosides dAdo and dGuo was also efficiently catalyzed. Dm-dNK is closely related to human and herpes simplex virus deoxyribonucleoside kinases. The highest level of sequence similarity was noted with human mitochondrial thymidine kinase 2, and these enzymes also share many substrates. The cDNA cloning and characterization of Dm-dNK will be the basis for studies on the use of this multisubstrate nucleoside kinase as a suicide gene in combined gene/chemotherapy of cancer.