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163 Evaluating the ocular irritation potential of 54 test articles using the epiocular™ human tissue construct model (OCL-200)
Poster Session P4. Alternative methods 163
EVALUATING THE OCULAR IRRITATION POTENTIAL OF 54 TEST ARTICLES USING THE EPIOCULAR™ HUMAN TISSUE CONSTRUCT MODEL (OCL-200)
J. Sheasgreen 1 , M. Blazka 2 , J. Harbell 3 , M. Klausner 1 , P. Hayden, J. Merrill 3 , J. Kubilus 1 , C. Kloos 2 , D. Bagley 2 . 1 MatTek Corporation, Ashland, MA; 2 Colgate-Palmolive Company, Piscataway, NJ; 3 The Institute for In Vitro Sciences, Inc., Gaithersburg, MD Colgate-Palmolive is sponsoring a research program to validate the use of the EpiOcular™ Model in evaluating the eye irritation potential of surfactants. Previously, in a study that demonstrated the reliability of the EpiOcular Model, four laboratories using a formal and detailed study protocol tested 19 test materials. In the current study, two laboratories (MatTek Corp. and Institute for In Vitro Sciences, Inc.) have tested 54 test articles using the same study protocol. EpiOcular is a commercially available three-dimensional in vitro model of the human corneal epithelium composed of normal human-derived epidermal keratinocytes. Test articles included a shampoo formulation and 30 different surfactants (10-cationic; 11-anionic; 7-nonionic; 1-amphoteric; 1zwitterionic) which were liquids, powders or creams. Multiple concentrations of 11 of the surfactants were tested, to evaluate the model’s ability to predict dose-related differences in a test article’s potential for ocular irritation. Testing was conducted in compliance with FDA GLPs. The laboratories were blinded to the identities of the test articles. Test results were compared to previously published animal eye irritation studies. In terms of reliability, the results were reproducible within and between the laboratories. In terms of relevance, the EpiOcular model correctly predicted the Draize score for a majority of the samples tested. The model also correctly predicted increasing irritation potential of surfactants with increased concentrations. These data provide additional +evidence that the EpiOcular model meets the validation criteria, as defined by the Interagency Coordinating Committee on the Validation of Alternative Methods (NIH Publication No. 97–3981), for assessing the ocular irritation potential of certain classes of surfactant and surfactant-based formulations. 164
EVALUATION OF THE EFFECTS OF CHRONIC SUBCYTOTOXIC SURFACTANT EXPOSURE TO A HUMAN CORNEAL CELL LINE
P.J. Wilkinson 1 , R.H. Clothier 1 . 1 FRAME Laboratories, School of Biomedical Sciences, University of Nottingham, Nottingham, United Kingdom In this study we aim to generate an in vitro model to investigate chronic corneal damage. Initially, the viability and barrier function integrity of SV40 human corneal epithelial cells (J-HCET) was monitored following chronic exposure to subcytotoxic concentrations of surfactants. Human Corneal Epithelial cells were cultured in a defined media containing non-cytotoxic concentrations of Tween 20 (T20: 25µgml−1 ), Sodium Dodecyl Sulphate (SDS: 4µgml−1 ), Benzalkonium chloride (BAK: 0.0025µgml−1 ) or Cocoamidpropylbetaine (CAPB: 6µgml−1 ). Pre-exposed and unexposed J-HCET were grown to confluency (n=8) in 96 well plate culture inserts and exposed to acute sub-lethal concentrations of T20 (150mgml−1 ), SDS (0.3mgml−1 ), BAK (0.15mgml−1 ) or CAPB (1.0mgml−1 ). Cell viability and barrier function, using the Resazurin reduction- and fluorescein leakage-assay, were determined prior to acute exposure and every 24 hour intervals over 96hrs. Acute surfactant exposure effects following pre-exposure were determined and compared statistically (repeat measures ANOVA) with non-pre-exposed cultures. Pre-exposure to BAK and SDS rendered J-HCET more susceptible (P<0.0001) to subsequent acute exposures of all classes of surfactants. CAPB pre-exposure render J-HCET more susceptible (P<0.0001) to acute CAPB and BAK exposure and moderately less susceptible (P<0.015) to acute SDS exposure. CAPB preexposure has no significant effect on subsequent acute T20 exposure. Pre-exposure to T20 reduced susceptibility to acute SDS, CAPB and BAK exposure (P<0.0001) and moderately less susceptible (P<0.025) to acute T20 exposure. Barrier function integrity was
s47
lost in all combinations of acute-chronic exposure with subsequent recovery over time. Pre-exposure to BAK (cationic), SDS (anionic) and CAPB (amphoteric), render J-HCET more susceptible to acute exposures of ionic surfactants. In contrast, pre-exposure to a non-ionic surfactant (T20) renders J-HCET less susceptible to subsequent acute exposures. Modulation via the intercellular junctions following acute exposure is therefore dependent on pervious exposure history. This work was funded by a grant from the FRAME research council. 165
ACUTE TOXICITY: ALTERNATIVES TO THE LD50 TEST
C. Longobardi, V. Pacelli, A. Argentino Storino. RTC, Research Toxicology Centre S.p.A., Pomezia - Rome, Italy The objectives of acute toxicity testing are to obtain information on the primary toxicological properties of chemicals, pharmaceuticals and biocidal products. This information is not only used in hazard identification and risk management during production, handling, packaging and labelling of chemical and biocidal products, but also for the identification of toxicological effects (in terms of clinical signs and lethality) of pharmaceutical products. The LD50 value, defined as statistically derived dose that is expected to cause death in 50% of the treated animals following a single administration in rats or mice has been considered, up until the end of 2001, as the basis for toxicological classification. The Fixed Dose, Toxic Class and Up and Down methods are valid alternatives to the LD50 test. However, although these methods have the advantage of using just a few animals, they only provide an estimation of the potential range of the LD50 value. The first version of these methods was adopted in 1996. Since then modifications have been made to further reduce the number of animals used (only females) and a new version of each method was adopted at the end of 2001. The previous and recent versions of these methods were applied to various products. The advantages and disadvantages, in terms of number of animals used, objectives and the time necessary to achieve them, have been analysed. The methods were compared in order to establish which was the most suitable for each type of product and to evaluate if the recent versions are an improvement on the previous methods. All three methods are suitable for the classification of chemicals. The further reduction in the number of animals and the use of females only (more sensitive sex) generally provides an estimation of the LD50 value which is towards the lower limit of the range. However, these procedures do not provide adequate toxicity data for pharmaceutical products to be used for further toxicological evaluations (i.e. maximum tolerated dose or minimal lethal dose). 166
PRELIMINARY (PHASE I) RESULTS OF A VALIDATION STUDY TO EVALUATE THE RELIABILITY AND RELEVANCE OF TWO IN VITRO CYTOTOXICITY ASSAYS FOR PREDICTING RODENT AND HUMAN ACUTE SYSTEMIC TOXICITY
S. Casati 1 , J.A. Strickland 2,3 , M.W. Paris 2,3 , W.S. Stokes 2 , R.R. Tice 2,3 , J. Haseman 4 , A.P. Worth 5 , H. Raabe 6 , C. Cao 7 , R. Clothier 8 , J. Harbell 6 , R. Curren 6 , M.L. Wenk 9 , M.K. Vallant 4 , G. Mun 6 , M. Clear 6 , G.O. Moyer 6 , J. Madren-Whalley 7 , C. Krishna 7 , M. Owen 8 , N. Bourne 8 . 1 European Centre for the Validation of Alternative Methods (ECVAM) JRC, Ispra Italy, 2 NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM), RTP, NC USA 27709, 3 ILS, Inc., RTP, NC USA, 4 National Institute of Environmental Health Sciences (NIEHS), RTP, NC USA, 5 European Chemicals Bureau (ECB) JRC, Ispra Italy, 6 Institute for In Vitro Sciences, Gaithersburg, MD USA, 7 U.S. Army Edgewood Chemical Biological Center, APG, MD USA, 8 Univ. of Nottingham, Nottingham, UK, 9 BioReliance Corp., Rockville, MD USA In order to assess the reliability and relevance of two in vitro basal cytotoxicity assays for predicting acute systemic toxicity in rodents and humans, NICEATM and ECVAM designed and started a joint validation study in July 2002. The 3T3 neutral red uptake (NRU) assay and the normal human keratinocyte (NHK) NRU
EVALUATING THE OCULAR IRRITATION POTENTIAL OF 54 TEST ARTICLES USING THE EPIOCULAR™ HUMAN TISSUE CONSTRUCT MODEL (OCL-200)
J. Sheasgreen 1 , M. Blazka 2 , J. Harbell 3 , M. Klausner 1 , P. Hayden, J. Merrill 3 , J. Kubilus 1 , C. Kloos 2 , D. Bagley 2 . 1 MatTek Corporation, Ashland, MA; 2 Colgate-Palmolive Company, Piscataway, NJ; 3 The Institute for In Vitro Sciences, Inc., Gaithersburg, MD Colgate-Palmolive is sponsoring a research program to validate the use of the EpiOcular™ Model in evaluating the eye irritation potential of surfactants. Previously, in a study that demonstrated the reliability of the EpiOcular Model, four laboratories using a formal and detailed study protocol tested 19 test materials. In the current study, two laboratories (MatTek Corp. and Institute for In Vitro Sciences, Inc.) have tested 54 test articles using the same study protocol. EpiOcular is a commercially available three-dimensional in vitro model of the human corneal epithelium composed of normal human-derived epidermal keratinocytes. Test articles included a shampoo formulation and 30 different surfactants (10-cationic; 11-anionic; 7-nonionic; 1-amphoteric; 1zwitterionic) which were liquids, powders or creams. Multiple concentrations of 11 of the surfactants were tested, to evaluate the model’s ability to predict dose-related differences in a test article’s potential for ocular irritation. Testing was conducted in compliance with FDA GLPs. The laboratories were blinded to the identities of the test articles. Test results were compared to previously published animal eye irritation studies. In terms of reliability, the results were reproducible within and between the laboratories. In terms of relevance, the EpiOcular model correctly predicted the Draize score for a majority of the samples tested. The model also correctly predicted increasing irritation potential of surfactants with increased concentrations. These data provide additional +evidence that the EpiOcular model meets the validation criteria, as defined by the Interagency Coordinating Committee on the Validation of Alternative Methods (NIH Publication No. 97–3981), for assessing the ocular irritation potential of certain classes of surfactant and surfactant-based formulations. 164
EVALUATION OF THE EFFECTS OF CHRONIC SUBCYTOTOXIC SURFACTANT EXPOSURE TO A HUMAN CORNEAL CELL LINE
P.J. Wilkinson 1 , R.H. Clothier 1 . 1 FRAME Laboratories, School of Biomedical Sciences, University of Nottingham, Nottingham, United Kingdom In this study we aim to generate an in vitro model to investigate chronic corneal damage. Initially, the viability and barrier function integrity of SV40 human corneal epithelial cells (J-HCET) was monitored following chronic exposure to subcytotoxic concentrations of surfactants. Human Corneal Epithelial cells were cultured in a defined media containing non-cytotoxic concentrations of Tween 20 (T20: 25µgml−1 ), Sodium Dodecyl Sulphate (SDS: 4µgml−1 ), Benzalkonium chloride (BAK: 0.0025µgml−1 ) or Cocoamidpropylbetaine (CAPB: 6µgml−1 ). Pre-exposed and unexposed J-HCET were grown to confluency (n=8) in 96 well plate culture inserts and exposed to acute sub-lethal concentrations of T20 (150mgml−1 ), SDS (0.3mgml−1 ), BAK (0.15mgml−1 ) or CAPB (1.0mgml−1 ). Cell viability and barrier function, using the Resazurin reduction- and fluorescein leakage-assay, were determined prior to acute exposure and every 24 hour intervals over 96hrs. Acute surfactant exposure effects following pre-exposure were determined and compared statistically (repeat measures ANOVA) with non-pre-exposed cultures. Pre-exposure to BAK and SDS rendered J-HCET more susceptible (P<0.0001) to subsequent acute exposures of all classes of surfactants. CAPB pre-exposure render J-HCET more susceptible (P<0.0001) to acute CAPB and BAK exposure and moderately less susceptible (P<0.015) to acute SDS exposure. CAPB preexposure has no significant effect on subsequent acute T20 exposure. Pre-exposure to T20 reduced susceptibility to acute SDS, CAPB and BAK exposure (P<0.0001) and moderately less susceptible (P<0.025) to acute T20 exposure. Barrier function integrity was
s47
lost in all combinations of acute-chronic exposure with subsequent recovery over time. Pre-exposure to BAK (cationic), SDS (anionic) and CAPB (amphoteric), render J-HCET more susceptible to acute exposures of ionic surfactants. In contrast, pre-exposure to a non-ionic surfactant (T20) renders J-HCET less susceptible to subsequent acute exposures. Modulation via the intercellular junctions following acute exposure is therefore dependent on pervious exposure history. This work was funded by a grant from the FRAME research council. 165
ACUTE TOXICITY: ALTERNATIVES TO THE LD50 TEST
C. Longobardi, V. Pacelli, A. Argentino Storino. RTC, Research Toxicology Centre S.p.A., Pomezia - Rome, Italy The objectives of acute toxicity testing are to obtain information on the primary toxicological properties of chemicals, pharmaceuticals and biocidal products. This information is not only used in hazard identification and risk management during production, handling, packaging and labelling of chemical and biocidal products, but also for the identification of toxicological effects (in terms of clinical signs and lethality) of pharmaceutical products. The LD50 value, defined as statistically derived dose that is expected to cause death in 50% of the treated animals following a single administration in rats or mice has been considered, up until the end of 2001, as the basis for toxicological classification. The Fixed Dose, Toxic Class and Up and Down methods are valid alternatives to the LD50 test. However, although these methods have the advantage of using just a few animals, they only provide an estimation of the potential range of the LD50 value. The first version of these methods was adopted in 1996. Since then modifications have been made to further reduce the number of animals used (only females) and a new version of each method was adopted at the end of 2001. The previous and recent versions of these methods were applied to various products. The advantages and disadvantages, in terms of number of animals used, objectives and the time necessary to achieve them, have been analysed. The methods were compared in order to establish which was the most suitable for each type of product and to evaluate if the recent versions are an improvement on the previous methods. All three methods are suitable for the classification of chemicals. The further reduction in the number of animals and the use of females only (more sensitive sex) generally provides an estimation of the LD50 value which is towards the lower limit of the range. However, these procedures do not provide adequate toxicity data for pharmaceutical products to be used for further toxicological evaluations (i.e. maximum tolerated dose or minimal lethal dose). 166
PRELIMINARY (PHASE I) RESULTS OF A VALIDATION STUDY TO EVALUATE THE RELIABILITY AND RELEVANCE OF TWO IN VITRO CYTOTOXICITY ASSAYS FOR PREDICTING RODENT AND HUMAN ACUTE SYSTEMIC TOXICITY
S. Casati 1 , J.A. Strickland 2,3 , M.W. Paris 2,3 , W.S. Stokes 2 , R.R. Tice 2,3 , J. Haseman 4 , A.P. Worth 5 , H. Raabe 6 , C. Cao 7 , R. Clothier 8 , J. Harbell 6 , R. Curren 6 , M.L. Wenk 9 , M.K. Vallant 4 , G. Mun 6 , M. Clear 6 , G.O. Moyer 6 , J. Madren-Whalley 7 , C. Krishna 7 , M. Owen 8 , N. Bourne 8 . 1 European Centre for the Validation of Alternative Methods (ECVAM) JRC, Ispra Italy, 2 NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM), RTP, NC USA 27709, 3 ILS, Inc., RTP, NC USA, 4 National Institute of Environmental Health Sciences (NIEHS), RTP, NC USA, 5 European Chemicals Bureau (ECB) JRC, Ispra Italy, 6 Institute for In Vitro Sciences, Gaithersburg, MD USA, 7 U.S. Army Edgewood Chemical Biological Center, APG, MD USA, 8 Univ. of Nottingham, Nottingham, UK, 9 BioReliance Corp., Rockville, MD USA In order to assess the reliability and relevance of two in vitro basal cytotoxicity assays for predicting acute systemic toxicity in rodents and humans, NICEATM and ECVAM designed and started a joint validation study in July 2002. The 3T3 neutral red uptake (NRU) assay and the normal human keratinocyte (NHK) NRU