To that purpose, non sensitizing botanical extracts were tested alone and spiked with a known sen... more To that purpose, non sensitizing botanical extracts were tested alone and spiked with a known sensitizer. Results were different among the four different tests, depending on the nature of the read-out. The LC-MS assay yields a non-interpretable result due to the complexity of the mixtures. Dose-response analysis in Kerati-noSens did identify low levels of the spiked sensitizers in different extracts of low cytotoxicity, but the evaluation was more difficult in a highly cytotoxic extract. NCTC test and Epidermal Equivalent test showed reliable results which lead to conclude that the proofof-concept was established for these two tests.
Atla-alternatives To Laboratory Animals, Dec 1, 2009
The Principles of Humane Experimental Technique by William Russell and Rex Burch provides excepti... more The Principles of Humane Experimental Technique by William Russell and Rex Burch provides exceptional value, both to practitioners of alternative methodologies and to bibliophiles who enjoy the pursuit of an out-of-print book. I was stimulated to find an original edition by my first visits to FRAME and its Chairman, Michael Balls. The search continued for several years, until I was able to find an edition suitable for presentation to him. During this hunt, I was able to learn a great deal about the Three Rs of Replacement, Reduction and Refinement, and the programmes that FRAME initiated to educate scientists and the general public about alternative approaches. Very importantly, I was able to use many of FRAME's successful strategies to build up a similar organisation in the United States, the Institute for In Vitro Sciences. The double value of this book is now revealed-not only are its concepts scientifically and ethically valuable, but the price now being charged by secondhand booksellers means a more than tenfold increase in the value of my investment!
As part of the Interagency Regulatory Alternatives Group (IRAG) program to evaluate the state of ... more As part of the Interagency Regulatory Alternatives Group (IRAG) program to evaluate the state of the art in the development of alternative (non-whole animal) eye irritation tests, academic and industrial organizations were invited to submit in vitro eye irritation data generated in their laboratories to one of several working groups for review. The assays reviewed in this report (from Working Group 5, “Other Assays”) were the EYTEX™ assay, tissue equivalent assay, a cytotoxicity assay using three-dimensional human fibroblast constructs, the Microtox™ assay, and other miscellaneous assays. Each submission consisted of raw data for chemicals and products tested, a description of the methodology, and an analysis (generally by regression analysis and Pearson's correlation coefficient) for the performance of the in vitro test relative to its ability to predict individual ocular tissue scores or total ocular score. In vivo data were generated according to the scoring methods proposed by Draize. Working Group 5 evaluated the submissions and commented on the utility of the assays. The variability of the in vivo data made conclusions difficult in many situations. Most of these assays were deemed useful (within limited chemical classes) for screening purposes or for use in conjunction with other toxicological information.
The development and application of in vitro alternatives designed to reduce or replace the use of... more The development and application of in vitro alternatives designed to reduce or replace the use of animals, or to lessen the distress and discomfort of laboratory animals, is a rapidly developing trend in toxicology. However, at present there is no formal administrative process to organize, coordinate, or evaluate validation activities. A framework capable of fostering the validation of new methods is essential for the effective transfer of new technological developments from the research laboratory into practical use. This committee has identified four essential validation resources: chemical bank(s), cell and tissue banks, a data bank, and reference laboratories. The creation of a Scientific Advisory Board composed of experts in the various aspects and endpoints of toxicity testing, and representing the academic, industrial and regulatory communities, is recommended. Test validation acceptance is contingent upon broad buy-in by disparate groups in the scientific community-academics, industry and government. This is best achieved by early and frequent communication among parties and agreement upon common goals. It is hoped that the creation of a validation infrastructure composed of the elements described in this report will facilitate scientific acceptance and utilization of alternative methodologies and speed implementation of replacement, reduction and refinement alternatives in toxicity testing.
Atla-alternatives To Laboratory Animals, Sep 1, 2011
-Mucosal surfaces, such as the vaginal epithelium, are natural barriers to infection that are con... more -Mucosal surfaces, such as the vaginal epithelium, are natural barriers to infection that are constantly exposed to bacteria and viruses, and are therefore potential sites of entry for numerous pathogens. The vaginal epithelium can be damaged mechanically, e.g. by the incorrect use of objects such as tampons, and by chemicals that are irritating or corrosive. Consequently, this can lead to an increase in susceptibility to further damage or infection. Pharmaceutical, cosmetic and personal care products that are specifically formulated for application onto human external mucosae can occasionally induce undesirable local or systemic side-effects. Therefore, the compatibility of applied materials with this mucosal surface represents a key issue to be addressed by manufacturers. The most frequently used method for assessing vaginal mucosal irritation is the in vivo rabbit vaginal irritation test. However, the current emphasis in the field of toxicology is to use alternative in vitro methods that reduce, refine, and replace the use of animals, and which model and predict human, not animal, responses. Such an approach is of particular interest to the personal care and cosmetic industries in their effort to comply with European legislative measures, such as the 7th Amendment to the EU Cosmetics Directive that does not permit the marketing of cosmetic products if they, or their ingredients, have been tested for irritation responses in animals. The focus of this review is to provide an overview of the alternative and in vitro tests that are currently available for vaginal mucosal irritation assessment, and which are already used, or may become useful, to establish the safety of newly-designed products for human use.
Before nonanimal toxicity tests may be officially accepted by regulatory agencies, it is generall... more Before nonanimal toxicity tests may be officially accepted by regulatory agencies, it is generally agreed that the validity of the new methods must be demonstrated in an independent, scientifically sound validation program. Validation has been defined as the demonstration of the reliability and relevance of a test method for a particular purpose. This paper provides a brief review of the development of the theoretical aspects of the validation process and updates current thinking about objectively testing the performance of an alternative method in a validation study. Validation of alternative methods for eye irritation testing is a specific example illustrating important concepts. Although discussion focuses on the validation of alternative methods intended to replace current in vivo toxicity tests, the procedures can be used to assess the performance of alternative methods intended for other uses.
Abstract China and Brazil are two of the largest international economies now attempting to incorp... more Abstract China and Brazil are two of the largest international economies now attempting to incorporate non-animal, in vitro , testing methods into their regulatory systems. While Brazil has a government mandate to replace certain animal tests with alternative methods in a stated time-frame, China has no such requirement, but is none-the-less pursuing a similar goal. China lacks the internal infrastructure to provide in vitro testing and easy access to validated human tissue constructs, and Brazil lacks financial support to develop new alternative methods and has difficulty in commercialising human-based materials, e.g. cells and reconstructed tissues. However, both countries are embracing offers for training and other technical assistance, when needed. It is expected that formal regulatory acceptance of non-animal test results will continue and expand in both China and Brazil.
Advances in Experimental Medicine and Biology, 2016
New toxicology test methods, especially those using in vitro methods, are continually being devel... more New toxicology test methods, especially those using in vitro methods, are continually being developed. Some are used by industry for screening purposes; others are eventually validated for regulatory use. However, for a new test method to be firmly adopted by industry it must be readily available, generally through an in-house industry laboratory, an academic laboratory, or a contract research organization. Regardless of the type of laboratory which intends to implement the test method, certain steps must be taken to ascertain that the method that is put into place is reproducible and performs identically to the test method that was published or has undergone validation. This involves developing protocols and standard operating procedures, training staff, developing historic positive and negative control data, establishing acceptable performance with proficiency chemicals, and addressing all the safety concerns that may accompany the assay. From experience within a contract research laboratory, we provide guidance on how to most efficiently accomplish these tasks.
Introduction: The Institute for In Vitro Sciences is sponsoring a workshop series to identify, di... more Introduction: The Institute for In Vitro Sciences is sponsoring a workshop series to identify, discuss, and develop recommendations for optimal scientific and technical approaches for conducting in vitro assays-focusing first on genotoxicity assays-used for assessing potential toxicity within and across tobacco and electronic nicotine delivery product categories (hereafter referred to as tobacco products). Materials and Methods: Workshops provide a unique opportunity for invited expert stakeholders to share experiences and to develop recommendations that may serve as a resource for developing optimal testing approaches and data interpretation. It is envisioned that some recommendations would form the basis for the generation of guidance documents and/or serve as authoritative reference publications to support regulatory submissions. Results and Discussion: During the first workshop (November 27-28, 2018), workgroup members identified important issues for using in vitro genotoxicity assays for evaluating tobacco products. These issues were triaged into three priority categories that will provide the basis for selecting high-priority topics for subsequent workshops. To provide background for future workshops and to serve as a scientific community resource, the workgroup developed a tabulated referenced summary of the types of tobacco product test samples that have been evaluated using the regulatory genotoxicity assays and the types of regulatory questions that have been addressed. A touch-base meeting was held March 7, 2019, and a second workshop June 4-5, 2019 to discuss ongoing issues and to further organize workgroup activities. Conclusion: The current publication lists priority topics and background summary information for using regulatory genotoxicity assays to evaluate tobacco products.
... Methodologies for Organ Toxicity Horst Spielmann,1 Nikolay P. Bochkov,2 Lucio Costa,3 Laura G... more ... Methodologies for Organ Toxicity Horst Spielmann,1 Nikolay P. Bochkov,2 Lucio Costa,3 Laura Gribaldo,4 Andre Guillouzo,5 Jerrold J. Heindel,6 Meryl Karol,7 Ralph Parchment,8 Walter Pfaller,9 Pilar Prieto Peraita,4 and Tim Zacharewski10 ...
Atla-alternatives To Laboratory Animals, Jul 1, 2017
In 2009, the passing of the Family Smoking Prevention and Tobacco Control Act facilitated the est... more In 2009, the passing of the Family Smoking Prevention and Tobacco Control Act facilitated the establishment of the FDA Center for Tobacco Products (CTP), and gave it regulatory authority over the marketing, manufacture and distribution of tobacco products, including those termed 'modified risk'. On 4-6 April 2016, the Institute for In Vitro Sciences, Inc. (IIVS) convened a workshop conference entitled, In Vitro Exposure Systems and Dosimetry Assessment Tools for Inhaled Tobacco Products, to bring together stakeholders representing regulatory agencies, academia and industry to address the research priorities articulated by the FDA CTP. Specific topics were covered to assess the status of current in vitro smoke and aerosol/vapour exposure systems, as well as the various approaches and challenges to quantifying the complex exposures in in vitro pulmonary models developed for evaluating adverse pulmonary events resulting from tobacco product exposures. The four core topics covered were: a) Tobacco Smoke and E-Cigarette Aerosols; b) Air-Liquid Interface-In Vitro Exposure Systems; c) Dosimetry Approaches for Particles and Vapours/In Vitro Dosimetry Determinations; and d) Exposure Microenvironment/Physiology of Cells. The 2.5-day workshop included presentations from 20 expert speakers, poster sessions, networking discussions, and breakout sessions which identified key findings and provided recommendations to advance these technologies. Here, we will report on the proceedings, recommendations, and outcome of the April 2016 technical workshop, including paths forward for developing and validating non-animal test methods for tobacco product smoke and next generation tobacco product aerosol/vapour exposures. With the recent FDA publication of the final deeming rule for the governance of tobacco products, there is an unprecedented necessity to evaluate a very large number of tobacco-based products and ingredients. The questionable relevance, high cost, and ethical considerations for the use of in vivo testing methods highlight the necessity of robust in vitro approaches to elucidate tobacco-based exposures and how they may lead to pulmonary diseases that contribute to lung exposure-induced mortality worldwide.
Introduction The BCOP assay has gained widespread usage since it was first described in its curre... more Introduction The BCOP assay has gained widespread usage since it was first described in its current form by Gautheron, et al. in 1992. The assay combines two mechanistic endpoints – corneal opacity and epithelial permeability – to create an In Vitro Score that is then used to predict ocular irritation potential. However not all eye irritants are detected by these two endpoints, e.g. several strongly irritating materials used in the European Commission/British Home Office study (EC/HO study) did not induce a commensurately high In Vitro Score in the BCOP. Subsequently we were able to show that that these underpredicted materials did induce damage that was detectable using histopathology. Rationale Although the BCOP assay utilizes “living” (freshly excised; still viable) corneas, we hypothesized that some of the damage seen in in vivo treatments could be a consequence of inflammation subsequent to the initial injury. This type of irritation would most likely not be seen in an excised tissue since there would be no opportunity for the recruitment of inflammatory cells from outside the cornea. Jester et al. (1998) showed that the depth and area of injury occurring immediately after the initial ocular exposure to irritants was generally predictive of the extent of the final injury and especially of the potential recovery. Therefore we began studies to determine if there was damage to the epithelial, stromal, or endothelial layers of the excised cornea which were not being expressed either in the opacity or permeability scores. Results Initially we assessed the results of the EC/HO study on ocular irritation. Although the BCOP assay was arguably the best performing of all the in vitro assays, there were still several materials that were under predicted, e.g. parafluoroaniline, quinacrine, and sodium oxalate. When we retested these materials and added a histopathological examination of the corneas, we found that all three induced damage that could be observed microscopically. Further studies over the next five years revealed additional cases where histopathology of the bovine corneas was important in predicting eye irritation. As a result, the ICCVAM review of “In Vitro Test Methods for Identifying Ocular Corrosives and Severe Irritants”, suggested creating 1) a reference atlas of histopathology for chemically-induced ocular lesions and 2) a standardized scoring scheme to be used in conjunction with the histopathology atlas to identify decision criteria that could be used in hazard classification. We have now developed an atlas of ocular lesions observed in vitro and have convened a meeting of ocular histopathology experts to standardize the evaluation and lesion nomenclature for bovine corneas. Examples from this atlas and a summary of the histopathology meeting will be presented.
To that purpose, non sensitizing botanical extracts were tested alone and spiked with a known sen... more To that purpose, non sensitizing botanical extracts were tested alone and spiked with a known sensitizer. Results were different among the four different tests, depending on the nature of the read-out. The LC-MS assay yields a non-interpretable result due to the complexity of the mixtures. Dose-response analysis in Kerati-noSens did identify low levels of the spiked sensitizers in different extracts of low cytotoxicity, but the evaluation was more difficult in a highly cytotoxic extract. NCTC test and Epidermal Equivalent test showed reliable results which lead to conclude that the proofof-concept was established for these two tests.
Atla-alternatives To Laboratory Animals, Dec 1, 2009
The Principles of Humane Experimental Technique by William Russell and Rex Burch provides excepti... more The Principles of Humane Experimental Technique by William Russell and Rex Burch provides exceptional value, both to practitioners of alternative methodologies and to bibliophiles who enjoy the pursuit of an out-of-print book. I was stimulated to find an original edition by my first visits to FRAME and its Chairman, Michael Balls. The search continued for several years, until I was able to find an edition suitable for presentation to him. During this hunt, I was able to learn a great deal about the Three Rs of Replacement, Reduction and Refinement, and the programmes that FRAME initiated to educate scientists and the general public about alternative approaches. Very importantly, I was able to use many of FRAME's successful strategies to build up a similar organisation in the United States, the Institute for In Vitro Sciences. The double value of this book is now revealed-not only are its concepts scientifically and ethically valuable, but the price now being charged by secondhand booksellers means a more than tenfold increase in the value of my investment!
As part of the Interagency Regulatory Alternatives Group (IRAG) program to evaluate the state of ... more As part of the Interagency Regulatory Alternatives Group (IRAG) program to evaluate the state of the art in the development of alternative (non-whole animal) eye irritation tests, academic and industrial organizations were invited to submit in vitro eye irritation data generated in their laboratories to one of several working groups for review. The assays reviewed in this report (from Working Group 5, “Other Assays”) were the EYTEX™ assay, tissue equivalent assay, a cytotoxicity assay using three-dimensional human fibroblast constructs, the Microtox™ assay, and other miscellaneous assays. Each submission consisted of raw data for chemicals and products tested, a description of the methodology, and an analysis (generally by regression analysis and Pearson's correlation coefficient) for the performance of the in vitro test relative to its ability to predict individual ocular tissue scores or total ocular score. In vivo data were generated according to the scoring methods proposed by Draize. Working Group 5 evaluated the submissions and commented on the utility of the assays. The variability of the in vivo data made conclusions difficult in many situations. Most of these assays were deemed useful (within limited chemical classes) for screening purposes or for use in conjunction with other toxicological information.
The development and application of in vitro alternatives designed to reduce or replace the use of... more The development and application of in vitro alternatives designed to reduce or replace the use of animals, or to lessen the distress and discomfort of laboratory animals, is a rapidly developing trend in toxicology. However, at present there is no formal administrative process to organize, coordinate, or evaluate validation activities. A framework capable of fostering the validation of new methods is essential for the effective transfer of new technological developments from the research laboratory into practical use. This committee has identified four essential validation resources: chemical bank(s), cell and tissue banks, a data bank, and reference laboratories. The creation of a Scientific Advisory Board composed of experts in the various aspects and endpoints of toxicity testing, and representing the academic, industrial and regulatory communities, is recommended. Test validation acceptance is contingent upon broad buy-in by disparate groups in the scientific community-academics, industry and government. This is best achieved by early and frequent communication among parties and agreement upon common goals. It is hoped that the creation of a validation infrastructure composed of the elements described in this report will facilitate scientific acceptance and utilization of alternative methodologies and speed implementation of replacement, reduction and refinement alternatives in toxicity testing.
Atla-alternatives To Laboratory Animals, Sep 1, 2011
-Mucosal surfaces, such as the vaginal epithelium, are natural barriers to infection that are con... more -Mucosal surfaces, such as the vaginal epithelium, are natural barriers to infection that are constantly exposed to bacteria and viruses, and are therefore potential sites of entry for numerous pathogens. The vaginal epithelium can be damaged mechanically, e.g. by the incorrect use of objects such as tampons, and by chemicals that are irritating or corrosive. Consequently, this can lead to an increase in susceptibility to further damage or infection. Pharmaceutical, cosmetic and personal care products that are specifically formulated for application onto human external mucosae can occasionally induce undesirable local or systemic side-effects. Therefore, the compatibility of applied materials with this mucosal surface represents a key issue to be addressed by manufacturers. The most frequently used method for assessing vaginal mucosal irritation is the in vivo rabbit vaginal irritation test. However, the current emphasis in the field of toxicology is to use alternative in vitro methods that reduce, refine, and replace the use of animals, and which model and predict human, not animal, responses. Such an approach is of particular interest to the personal care and cosmetic industries in their effort to comply with European legislative measures, such as the 7th Amendment to the EU Cosmetics Directive that does not permit the marketing of cosmetic products if they, or their ingredients, have been tested for irritation responses in animals. The focus of this review is to provide an overview of the alternative and in vitro tests that are currently available for vaginal mucosal irritation assessment, and which are already used, or may become useful, to establish the safety of newly-designed products for human use.
Before nonanimal toxicity tests may be officially accepted by regulatory agencies, it is generall... more Before nonanimal toxicity tests may be officially accepted by regulatory agencies, it is generally agreed that the validity of the new methods must be demonstrated in an independent, scientifically sound validation program. Validation has been defined as the demonstration of the reliability and relevance of a test method for a particular purpose. This paper provides a brief review of the development of the theoretical aspects of the validation process and updates current thinking about objectively testing the performance of an alternative method in a validation study. Validation of alternative methods for eye irritation testing is a specific example illustrating important concepts. Although discussion focuses on the validation of alternative methods intended to replace current in vivo toxicity tests, the procedures can be used to assess the performance of alternative methods intended for other uses.
Abstract China and Brazil are two of the largest international economies now attempting to incorp... more Abstract China and Brazil are two of the largest international economies now attempting to incorporate non-animal, in vitro , testing methods into their regulatory systems. While Brazil has a government mandate to replace certain animal tests with alternative methods in a stated time-frame, China has no such requirement, but is none-the-less pursuing a similar goal. China lacks the internal infrastructure to provide in vitro testing and easy access to validated human tissue constructs, and Brazil lacks financial support to develop new alternative methods and has difficulty in commercialising human-based materials, e.g. cells and reconstructed tissues. However, both countries are embracing offers for training and other technical assistance, when needed. It is expected that formal regulatory acceptance of non-animal test results will continue and expand in both China and Brazil.
Advances in Experimental Medicine and Biology, 2016
New toxicology test methods, especially those using in vitro methods, are continually being devel... more New toxicology test methods, especially those using in vitro methods, are continually being developed. Some are used by industry for screening purposes; others are eventually validated for regulatory use. However, for a new test method to be firmly adopted by industry it must be readily available, generally through an in-house industry laboratory, an academic laboratory, or a contract research organization. Regardless of the type of laboratory which intends to implement the test method, certain steps must be taken to ascertain that the method that is put into place is reproducible and performs identically to the test method that was published or has undergone validation. This involves developing protocols and standard operating procedures, training staff, developing historic positive and negative control data, establishing acceptable performance with proficiency chemicals, and addressing all the safety concerns that may accompany the assay. From experience within a contract research laboratory, we provide guidance on how to most efficiently accomplish these tasks.
Introduction: The Institute for In Vitro Sciences is sponsoring a workshop series to identify, di... more Introduction: The Institute for In Vitro Sciences is sponsoring a workshop series to identify, discuss, and develop recommendations for optimal scientific and technical approaches for conducting in vitro assays-focusing first on genotoxicity assays-used for assessing potential toxicity within and across tobacco and electronic nicotine delivery product categories (hereafter referred to as tobacco products). Materials and Methods: Workshops provide a unique opportunity for invited expert stakeholders to share experiences and to develop recommendations that may serve as a resource for developing optimal testing approaches and data interpretation. It is envisioned that some recommendations would form the basis for the generation of guidance documents and/or serve as authoritative reference publications to support regulatory submissions. Results and Discussion: During the first workshop (November 27-28, 2018), workgroup members identified important issues for using in vitro genotoxicity assays for evaluating tobacco products. These issues were triaged into three priority categories that will provide the basis for selecting high-priority topics for subsequent workshops. To provide background for future workshops and to serve as a scientific community resource, the workgroup developed a tabulated referenced summary of the types of tobacco product test samples that have been evaluated using the regulatory genotoxicity assays and the types of regulatory questions that have been addressed. A touch-base meeting was held March 7, 2019, and a second workshop June 4-5, 2019 to discuss ongoing issues and to further organize workgroup activities. Conclusion: The current publication lists priority topics and background summary information for using regulatory genotoxicity assays to evaluate tobacco products.
... Methodologies for Organ Toxicity Horst Spielmann,1 Nikolay P. Bochkov,2 Lucio Costa,3 Laura G... more ... Methodologies for Organ Toxicity Horst Spielmann,1 Nikolay P. Bochkov,2 Lucio Costa,3 Laura Gribaldo,4 Andre Guillouzo,5 Jerrold J. Heindel,6 Meryl Karol,7 Ralph Parchment,8 Walter Pfaller,9 Pilar Prieto Peraita,4 and Tim Zacharewski10 ...
Atla-alternatives To Laboratory Animals, Jul 1, 2017
In 2009, the passing of the Family Smoking Prevention and Tobacco Control Act facilitated the est... more In 2009, the passing of the Family Smoking Prevention and Tobacco Control Act facilitated the establishment of the FDA Center for Tobacco Products (CTP), and gave it regulatory authority over the marketing, manufacture and distribution of tobacco products, including those termed 'modified risk'. On 4-6 April 2016, the Institute for In Vitro Sciences, Inc. (IIVS) convened a workshop conference entitled, In Vitro Exposure Systems and Dosimetry Assessment Tools for Inhaled Tobacco Products, to bring together stakeholders representing regulatory agencies, academia and industry to address the research priorities articulated by the FDA CTP. Specific topics were covered to assess the status of current in vitro smoke and aerosol/vapour exposure systems, as well as the various approaches and challenges to quantifying the complex exposures in in vitro pulmonary models developed for evaluating adverse pulmonary events resulting from tobacco product exposures. The four core topics covered were: a) Tobacco Smoke and E-Cigarette Aerosols; b) Air-Liquid Interface-In Vitro Exposure Systems; c) Dosimetry Approaches for Particles and Vapours/In Vitro Dosimetry Determinations; and d) Exposure Microenvironment/Physiology of Cells. The 2.5-day workshop included presentations from 20 expert speakers, poster sessions, networking discussions, and breakout sessions which identified key findings and provided recommendations to advance these technologies. Here, we will report on the proceedings, recommendations, and outcome of the April 2016 technical workshop, including paths forward for developing and validating non-animal test methods for tobacco product smoke and next generation tobacco product aerosol/vapour exposures. With the recent FDA publication of the final deeming rule for the governance of tobacco products, there is an unprecedented necessity to evaluate a very large number of tobacco-based products and ingredients. The questionable relevance, high cost, and ethical considerations for the use of in vivo testing methods highlight the necessity of robust in vitro approaches to elucidate tobacco-based exposures and how they may lead to pulmonary diseases that contribute to lung exposure-induced mortality worldwide.
Introduction The BCOP assay has gained widespread usage since it was first described in its curre... more Introduction The BCOP assay has gained widespread usage since it was first described in its current form by Gautheron, et al. in 1992. The assay combines two mechanistic endpoints – corneal opacity and epithelial permeability – to create an In Vitro Score that is then used to predict ocular irritation potential. However not all eye irritants are detected by these two endpoints, e.g. several strongly irritating materials used in the European Commission/British Home Office study (EC/HO study) did not induce a commensurately high In Vitro Score in the BCOP. Subsequently we were able to show that that these underpredicted materials did induce damage that was detectable using histopathology. Rationale Although the BCOP assay utilizes “living” (freshly excised; still viable) corneas, we hypothesized that some of the damage seen in in vivo treatments could be a consequence of inflammation subsequent to the initial injury. This type of irritation would most likely not be seen in an excised tissue since there would be no opportunity for the recruitment of inflammatory cells from outside the cornea. Jester et al. (1998) showed that the depth and area of injury occurring immediately after the initial ocular exposure to irritants was generally predictive of the extent of the final injury and especially of the potential recovery. Therefore we began studies to determine if there was damage to the epithelial, stromal, or endothelial layers of the excised cornea which were not being expressed either in the opacity or permeability scores. Results Initially we assessed the results of the EC/HO study on ocular irritation. Although the BCOP assay was arguably the best performing of all the in vitro assays, there were still several materials that were under predicted, e.g. parafluoroaniline, quinacrine, and sodium oxalate. When we retested these materials and added a histopathological examination of the corneas, we found that all three induced damage that could be observed microscopically. Further studies over the next five years revealed additional cases where histopathology of the bovine corneas was important in predicting eye irritation. As a result, the ICCVAM review of “In Vitro Test Methods for Identifying Ocular Corrosives and Severe Irritants”, suggested creating 1) a reference atlas of histopathology for chemically-induced ocular lesions and 2) a standardized scoring scheme to be used in conjunction with the histopathology atlas to identify decision criteria that could be used in hazard classification. We have now developed an atlas of ocular lesions observed in vitro and have convened a meeting of ocular histopathology experts to standardize the evaluation and lesion nomenclature for bovine corneas. Examples from this atlas and a summary of the histopathology meeting will be presented.
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Papers by Rodger Curren