IIVS is happy to partner with MatTek, manufacturers of 3-D reconstructed human epidermis models, to provide special discounted pricing for select OECD approved testing methods. We will be discounting $750 off the total study cost for assays conducted on groups of at least 5 test materials in the Skin Irritation Test (SIT) (OECD TG 431) or the Skin Corrosion Test (OECD TG 439). Follow the link above to read about the program in MatTek's recent newsletter. Please contact one of our study directors today to discuss how this special pricing may be applied to your projects. (MatTek terms and conditions apply.)
The DB-ALM of the European Union Reference Laboratory on Alternatives to Animal Testing (EURL ECVAM) wants to remind you, as previously announced by the European Partnership for Alternative Approaches to animal testing (EPAA), that a second survey has been launched on the use of alternative methods in the industry for the assessment of reproductive toxicity of compounds and/or formulations covering Reduction and Refinement (2Rs) methods applied for both regulatory and non-regulatory purposes. Follow the link above to the EPAA website and download the EPAA questionnaire. The deadline for responding has been extended to May 18th.
The survey has been launched in the context of the EPAA thematic review project to provide an overall picture on available alternative methods at all stages of development, validation and/or regulatory acceptance covering the 3Rs in the topic area of reproductive toxicity testing. This project was based on an extensive bibliographic review and the full content published in 2011 on the DB-ALM together with the first survey results.This second survey, in fact, follows and complements the first one that covered Replacement methods providing in this way a full picture on the use of all the 3Rs methods in industry for reproductive toxicity testing. For more information, please visit the EPAA website and the ECVAM DB-ALM website
ESTIV is welcoming the submission of abstracts for presentation as posters or oral papers during their annual meeting October16-19 in Lisbon, Portugal. Authors wishing to submit an abstract are requested to follow the abstract submission guidelines available from the ESTIV2012 website (linked above). Please consider the preliminary program to make sure your paper falls within the scope of this event. Authors will be informed about the acceptance of their abstracts to be presented as an oral or poster presentation by the 30th June 2012. All accepted abstracts will be included in the ESTIV2012 Abstract Book and distributed to all participants. Please note that presenters (poster & oral) will be required to pay registration fees. Please review the attached pdf for additional information and the preliminary program.
British American Tobacco’s (BAT) Group Research & Development (GR&D) Centre has become the newest member of the Scientific Advisory Panel of the Institute for In Vitro Sciences (IIVS). “We rely on the expertise of our panel members to help determine the direction and focus of our scientific activities,” said Dr. Rodger Curren, President of IIVS. “As companies such as BAT dedicate significant resources to the implementation and use of alternative methods, we assist them in evaluating the technology and introducing these methods to the regulatory community.” “We fully support the development and application of in vitro methods as alternatives to limit the use of in vivo studies,” said Dr. Marianna Gaca, BAT’s IIVS Scientific Advisory Panel representative. “ We hope the in vitro models we are developing will help facilitate the understanding of the biological effects of tobacco smoke and, in the future, help support the assessment of conventional and modified risk tobacco products,” she said. To read the entire press release, open the document above.
The DB-ALM of the European Union Reference Laboratory on Alternatives to Animal Testing (EURL ECVAM) wants to remind you, as previously announced by the European Partnership for Alternative Approaches to animal testing (EPAA), that a second survey has been launched on the use of alternative methods in the industry for the assessment of reproductive toxicity of compounds and/or formulations covering Reduction and Refinement (2Rs) methods applied for both regulatory and non-regulatory purposes. Please click on the pdf document above to download and answer the survey.
This questionnaire survey is part of an EPAA-initiated activity to review the state-of-the-art of development, acceptance and use of 3Rs in Reproductive Toxicity testing. A previous survey conducted in 2010 covered Replacement or non-animal in vitro methods used in industry. The present questionnaire follows and complements that survey and covers the 2Rs (Reduction and Refinement) as applicable to in vivo methods used either as stand-alone methods or as part of Integrated Testing Strategies (ITS) using a combination of in vivo and in vitro methods. It addresses the ways that the 2Rs are applied for both regulatory and non-regulatory purposes. Therefore, together with the previous questionnaire, this survey aims to provide a full picture of all the 3Rs methods used in the assessment of Reproductive Toxicity of compounds (chemicals, drugs, etc.) and/or formulations (including biologicals, i.e. proteins and vaccines).
Deadline to respond to this survey is April 13th.
The U.S. Department of Agriculture (USDA) Center for Veterinary Biologics (CVB) regularly seeks public comment on drafts of proposed guidance documents concerning all aspects of veterinary vaccines production, testing, and distribution. Draft Notice 465, which is currently available for comment, provides proposed guidance on the use of humane endpoints and methods in animal testing of biological products. The draft document includes specific guidance regarding the use of humane endpoints in biological products testing, including guidance on humane endpoints for the rabies challenge test. The draft guidance also strongly encourages the use of anesthesia for intracerebral inoculation of mice during rabies vaccine testing.
The draft guidance incorporates recommendations for refinement of rabies vaccine testing made by participants at the October 2011 NICEATM-ICCVAM workshop on alternative methods for rabies vaccine potency testing. Information about the workshop is available on the NICEATM-ICCVAM website. A summary of the workshop is posted here.
Comments on the draft proposed guidance document should be submitted by April 23, 2012, via email to cvb@aphis.usda.gov. Draft proposed CVB guidance documents and additional information about submitting comments are available on the USDA website above.
/RabiesVaccineWkspSumm-30Nov11.pdf
The US Department of Transportation Pipeline and Hazardous Materials Safety Administration (PHMSA) Office of Hazardous Materials Safety has updated all guidance documents and references on its webpage to reflect the currently accepted practice of using in vitro corrosion test methods wherever possible. Due in large part to efforts by PETA, PHMSA's website, letters of interpretation and all other materials on this issue have been updated to reflect recent amendments made to the United Nations Recommendations on the Transport of Dangerous Goods Model Regulation and OECD guidance documents. Previously-issued letters and positions have been replaced by the current position which promotes to the extend possible the use of in vitro skin corrosion test methods.
Shaping science and driving innovation: 2011 NC3Rs Annual Report
The NC3Rs has recently published its Annual Report for 2011. The report describes the work the NC3Rs as done within the scientific community to support the 3Rs and the Coalition Government's pledge to work to reduce the use of animals in science. To read the report please visit the link above.
Abstracts are currently being accepted for poster and oral presentations during the 2012 EUSAAT/Linz conference. Topics to be covered include:
21st century non-animal tools for basic and biomedical research (e. g. humane disease pathways, specific disease models, transgenic animals, xenotransplantation, teratoma assays to prove pluripotency of stem cells)
Implementing of the Directive 2010/63/EU on the protection of animal used for scientific purposes (incl. position of the EU Commission, of EU member states and of animal welfare organisations, examples for the implementation)
Progress in 3Rs research: EU FP6 & FP7 projects on alternatives and member state research funding. (e. g. status national and EU: Research topic overview and future funding policy)
7th amendment of EU Cosmetics Directive: Cosmetics and animal testing - an end in sight (e. g. skin regulatory acceptance, eye regualtory accaptance, sensitisation, skin models and open source)
Chemicals: REACH and animal welfare (e. g. report on use of alternatives, views of companies, animal welfare, methods, search engines)
3Rs progress in other sectors (e. g. vaccine testing, replacing mouse bioassays)
Inhalation toxicology & toxicology of nanomaterials
3R goes 3D! Implementation of 3D methods in toxicity testing
GCCP - Good cell culture practice
Ethical and & legal issues
*Free communications
To submit your lecture or your poster please use the ONLINE SUBMISSION FORM! The deadline for the submission of lectures is April 30th and the deadline for the submission of posters is May 31st.
The US Occupational Safety and Health Administration (OSHA) has released the final rule establishing its new Hazard Communication Standard that incorporates provisions of the Globally Harmonized System (GHS) of Classification and Labeling of Chemicals specifically in the workplace. The updated standard that includes GHS labeling, hazard categorization, and safety data sheet requirements, will be phased in over the next four years. It is due to be published in the Federal Register on 26 March. Read a summary of the news in Chemical Watch or click on the link above for information directly from the US Department of Labor website.
Source: SOT 2012
Authors: G. Mun; N. Wilt; D. A. Donahue; J. Avalos; K. Norman; A. Hilberer; F. A. Simion; H. Raabe
In vitro assays evaluating ocular irritation potential are routinely used by personal care companies. Two of these in vitro assays include the Chorioallantoic Membrane Vascular Assay (CAMVA) and the Bovine Corneal Opacity and Permeability Assay (BCOP). These assays do not require the use of live animals, provide reliable predictive data and are rapid to conduct. The BCOP uses excised bovine corneas to predict ocular irritation. The CAMVA uses the vascular network of fertilized chicken eggs as a conjunctival model to predict eye irritation. Both BCOP and CAMVA have been used for over fifteen years for product development, worker safety, and safety claims substantiation.
This poster describes procedures and considerations for demonstrating the inter-laboratory reliability of the BCOP and CAMVA. It is important to have a valid assay that can be implemented consistently at several different laboratories. For Kao Brands Company, a large BCOP and CAMVA database exists that covers multiple consumer product categories such as hair shampoos, skin cleansers, and hair styling sprays (containing ethanol). Therefore, a proper review of candidate laboratories is important for seamlessly generating consistent results that can be used for assessing potential ocular irritation of new products. First, a candidate laboratory should be audited for proper facility operation and personnel training. Second, the laboratory’s use of Good Laboratory Practices (GLPs) should be reviewed. Third, reference materials with known BCOP and CAMVA data (one irritant and two non-irritants for initial assessment) should be tested at each new laboratory for verification of proper assay performance.
Source: SOT 2012
Authors: Kimberly Norman, Arnhild Schrage, Susanne N. Kolle, Maria C. Rey Moreno, Bennard van Ravenzwaay, Robert Landsiedel, Hans Raabe
The Bovine Corneal Opacity and Permeability Assay (BCOP) is an ex vivo assay, which may be used to assess the eye irritation potential of new chemicals and finished products. The BCOP assay has been accepted by several regulatory agencies for the identification of severe and corrosive ocular irritants, replacing the rabbit eye test. According to OECD Test Guideline 437 adopted in September 2009, two treatment protocols may be used; one for liquids and one for solids. Solids are tested as 20% (w/v) solutions or suspensions in deionized water. Freshly excised bovine corneas are mounted in special corneal holders and are treated with the 20% (w/v) test material dilutions for four hours at approximately 32°C. Changes in corneal opacity are measured using an opacitometer, and impairment of the corneal barrier function is determined by measuring fluorescein passage through the corneas. Histological evaluation of the treated corneas may be used to determine the degree and depth of injury at the tissue level. In this study, the reference standard solids recommended in the OECD TG 437 were tested in an inter-laboratory study. Overall, the results from the evaluation of solids were highly congruent between the two laboratories and to the historical data and for several substances histological evaluation improved the understanding of eye irritation effect. However, for chlorhexidine and dibenzoyl-L-tartaric acid there were inter-laboratory differences, which were further evaluated. For chlorhexidine, differences in results were attributed to different sources of the chemical. This study demonstrates the reproducibility of the BCOP assay when evaluating solid test substances. In parallel, the study compared the opacity scores from a newly developed opacitometer (BASF-OP2.0) to those of the standard device (OP-KIT). The comparison between the BASF-OP2.0 showed very little variability and overall corresponded very well with the OP-KIT values.
Source: SOT 2012
Authors: Monica Krcha, Lindsay Krawiec, Kimberly Norman
Topical antioxidants, which have the capacity to neutralize reactive oxygen species (ROS), have been shown to prevent skin damage and improve the appearance of sun-damaged skin. Accordingly, we have developed an in vitro method capable of evaluating the antioxidant performance of ingredients and final formulations which may be applied to the skin. For assessment of antioxidant potential, NHEKs (normal human epidermal keratinocytes) were subjected to UVA-irradiation to induce oxidative stress and then protection from oxidative stress was evaluated in cells incubated with antioxidants. Cells were seeded in 96-well plates and incubated with the fluorescent ROS-detecting probe, 5-(and-6)-chloromethyl-2?,7?-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA), for 40 minutes. Next, the cells were exposed to a serial dilution of antioxidants/antioxidant-containing formulations for 1 hour. ROS were generated by exposing the cultures to UVA light for 50 minutes (5 Joules/cm2), and then detected by fluorescence measurements of the cells. Cytotoxicity was assessed concurrently using the neutral red uptake (NRU) assay. Using this method, we evaluated several antioxidants and antioxidant-containing formulations for their ROS-reducing capabilities, including ascorbic acid, quercetin, resveratrol, kinetin, nicotinamide, and EGCG. Based on our results, several antioxidants and antioxidant-containing formulations noticeably reduced ROS levels compared to untreated controls. The greatest reduction was observed in cells treated with ascorbic acid, which has been qualified as the positive control for the assay. Other antioxidants which did not show this reduction were likely not water soluble and/or poorly bioavailable to cells. Overall, our results indicate that this method may provide a valuable in vitro tool for assessing antioxidant performance in a biologically relevant model.
Source: SOT 2012
Authors: Costin, Gertrude-Emilia; Barroso, Joao; Raabe, Hans; Curren, Rodger; Nash, Jennifer R.; Kong, Amanda; Zuang, Valerie
Improvement of ocular irritancy prediction using a modified (shortened) 3-minute exposure in the BCOP assay was proposed for alcohols and ketones, which have been identified by ICCVAM as responsible for the most over-predictions in the BCOP. Eight alcohols and six ketones were tested using both the modified and the standard 10-minute exposure, and the data were compared with the GHS categories from the ICCVAM database. The evaluation of the 3-minute exposure data revealed that five of the 6 over-predicted alcohols showed an improved prediction, and of the 2 correctly predicted alcohols, one became an under-prediction and one remained the same. Two of the five over-predicted ketones showed an improved prediction, with the three other remaining the same. The one correctly predicted ketone remained the same. The results of the evaluation of the modified BCOP assay using the 3-minute exposures for alcohols and ketones suggest that improvements in the predictive capacity of the assay can be achieved by reducing the over-prediction of these small molecule, solvent-type chemicals, without an adverse impact upon the rate of under-prediction of similar chemicals. It is our recommendation that a) additional small molecule alcohols and ketones exhibiting solvent-like physical characteristics should be tested in the BCOP assay using the 3-minute (or shorter) and 10-minute exposures, and b) prior to any additional testing a more thorough evaluation of the supporting rabbit ocular irritation data be conducted to ascertain whether the correct standards are being used to calibrate the assay.
The draft recommendations for three Cell Transformation Assays (CTAs), the Syrian Hamster Embryo (SHE) CTA at pH 6.7, the SHE CTA at pH 7.0, and the BALB/c 3T3 CTA for in vitro carcinogenicity was developed following the ESAC (ECVAM Scientific Advisory Committee) opinion (No. 2011.01). Please follow the link above to view the recommendation in it's entirety. IIVS President, Dr. Rodger Curren, is currently serving on the ESAC and participated in the formation of the published opinion document that has lead to this recommendation.
The U.S. Environmental Protection Agency and L’oréal cosmetic company announced a research collaboration designed to determine if EPA’s chemical toxicity forecaster (ToxCast) can be used in systemic toxicity tests. EPA is using ToxCast to screen chemicals to understand their potential impact on processes in the human body that lead to adverse health effects. L’oréal is providing EPA $1.2 million in collaborative research funding plus robust safety data from a set of representative substances from the cosmetic sector, expanding the types of chemical use groups assessed by ToxCast. EPA will compare the ToxCast results to the L’oréal data to determine if the reliability and the relevance are appropriate for use in the safety assessment of chemicals in cosmetics. Please follow the link above to view the entire article.
A new paper published in Regulatory Toxicology and Pharmacology elaborates on guiding principles for a non-animal safety assessment concept for skin sensitization of cosmetic ingredients. to purchase the complete article, follow the link above. Please visit the skin sensitization section of our website for information specifically on the KeratinoSens Assay for Identification of Skin Sensitizers. We also have several KeratinoSens posters and manuscripts available on our Publications page.
Characterisation of skin sensitisation potential is a key endpoint for the safety assessment of cosmetic ingredients especially when significant dermal exposure to an ingredient is expected. At present the mouse local lymph node assay (LLNA) remains the ‘gold standard’ test method for this purpose however non-animal test methods are under development that aim to replace the need for new animal test data. COLIPA (the European Cosmetics Association) funds an extensive programme of skin sensitisation research, method development and method evaluation and helped coordinate the early evaluation of the three test methods currently undergoing pre-validation. In May 2010, a COLIPA scientific meeting was held to analyse to what extent skin sensitisation safety assessments for cosmetic ingredients can be made in the absence of animal data. In order to propose guiding principles for the application and further development of non-animal safety assessment strategies it was evaluated how and when non-animal test methods, predictions based on physico-chemical properties (including in silico tools), threshold concepts and weight-of-evidence based hazard characterisation could be used to enable safety decisions. Generation and assessment of potency information from alternative tools which at present is predominantly derived from the LLNA is considered the future key research area.
The next time you use shampoo, air freshener, or moisturizing cream, consider this: How do you know it's safe? In all likelihood, whatever toxicologic screening its component ingredients were subjected to involved laboratory animals, the method of choice for decades and the industry's reigning "gold standard." Yet as Bob Dylan once put it, the times, they are a-changing. Animal-based testing is expensive and time-consuming, morally and ethically troubling, and most significantly, often a poor predictor of human toxicity. Animals aren't going anywhere just yet. But their numbers are dropping. Driven both by legislative mandate and scientific need, a new suite of in vitro and cell culture-based animal-free methods are gaining a foothold in toxicology labs. Read the full article at sciencemag.org by clicking on the link above.
SkinEthic is running two training sessions covering the use of their tissue and the validated methods they are used in. Their training sessions are build up to allow in one hand, any new users to be able to feel comfortable in the 3D screening tool approaches and possibilities,and on the other hand, already trained professionals in the field of in vitro alternatives, to be updated on current validated protocols. SkinEthic’s one-day training sessions will run on March 27 and April 24. One-on-one interaction is fostered by limiting the maximum participants to 5.These sessions will be held in Lyon, France and cost € 1000 – (lunch included) VAT excluded. Please click on the link above for the registration form. Contact us Alain Alonso with questions or to register at aalonso@skinethic.com.
Cambridge research that created liver cells from stem cells has been recognized with a national prize by the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs). These cells, known as human induced pluripotent stem cells (hIPSCs), have already attracted attention for the possibilities they offer to regenerate damaged tissues and organs. But it is their potential to reduce the number of animals used for screening potential drug treatments that led to Dr. Vallier receiving the Centre’s 3Rs prize for 2011.
The prize, sponsored by GlaxoSmithKline, of a £2,000 personal award and a £18,000 research grant, is for the scientific paper published in the last three years that contributes most to the advancement of the 3Rs (Replacement, Reduction and Refinement). Dr Vallier’s winning paper was published in The Journal of Clinical Investigation in 2010. He received his prize from Professor Paul Matthews OBE of GlaxoSmithKline at the NC3Rs Annual Science Review Meeting in London on 28 February.
London – Seeking to maximise the value of computational modelling in avoiding animal testing for the European Union's Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), the People for the Ethical Treatment of Animals (PETA) Foundation has produced a free resource for potential registrants, identifying sources of information and expertise on the use of Quantitative Structure Activity Relationships (QSARs). The short brochure "QSARs and REACH: A Guide to Sources of Information and Advice" was produced in consultation with leading experts in the field and lists publicly available online resources and selected contact points for individuals and organisations that can offer support to REACH registrants and consultants on the use of QSARs.
QSARs predict chemical behaviour directly from chemical structure and simulates adverse effects in cells, tissues and lab animals, minimising the need to use animal tests to comply with regulatory requirements for human health and ecotoxicology endpoints. The REACH regulation promotes the use of alternative methods and states that animal testing should be a last resort. The use of QSAR is specifically encouraged. However, while QSARs have already been used in many registrations, it is clear from the European Chemicals Agency's (ECHA) 2011 report, "The Use of Alternatives to Testing on Animals for the REACH Regulation", that many opportunities to use them have been missed and that, in some cases, registrants have not submitted QSAR data in accordance with REACH's requirements, leading to potential failure at the REACH compliance check, additional costs, and increased animal testing.
"QSARs, used in the context of intelligent testing strategies and in combination with a chemical category approach, have the potential to replace many animal tests for REACH – but registrants must feel confident both with the use of the technology and with integrating it into weight-of-evidence arguments that ECHA will accept", says PETA policy adviser Alistair Currie. "It's clear from our discussions with companies that those less familiar with its use feel cautious about using it to replace testing. This resource answers the need to link the experts in QSAR use for REACH with the registrants who need that expertise." The list was compiled by PETA in consultation with PETA US and contacts within industry and academia, and selection and inclusion was based entirely on expert judgment – the list contains no paid advertising. The resource is currently being distributed gratis to chemical companies, consultants and other stakeholders and is available online at the link above.
The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) recommended to Federal agencies that the murine local lymph node assay, or LLNA, may be used to categorize the potency of chemicals causing allergic contact dermatitis (ACD) in humans. Specifically, ICCVAM recommended that the LLNA may be used to categorize some substances as strong sensitizers, thus identifying those substances considered to have a significant potential for causing skin hypersensitivity resulting in ACD.
In today's Federal Register, the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) announced availability of Federal agency responses to the ICCVAM recommendations. Regulatory agencies, including FDA, EPA, CPSC, and OSHA, have indicated that they will take actions in response to the ICCVAM recommendations to encourage use of the LLNA for this purpose where appropriate.
According to the U.S. Bureau of Labor Statistics, skin diseases are the most common type of occupational illness. Many of these cases arise from repeated exposures to skin-sensitizing substances, which can lead to ACD, an immunologically mediated hypersensitivity reaction. Studies have shown that ACD has a significant adverse impact on quality of life in affected individuals.
For over 10 years, the LLNA has been accepted worldwide as a valid alternative to traditionally accepted guinea pig test methods for assessing ACD hazard potential for most testing applications. The new ICCVAM recommendation provides guidance on how to use the LLNA to categorize some chemicals and products as strong skin sensitizers. However, since only half of the known strong human skin sensitizers can be identified in this way (52% or 14 out of 27), additional testing or information will be necessary to conclude that substances are not strong skin sensitizers.
Substances with the potential to cause ACD can also be categorized with the traditional test methods using guinea pigs. However, the LLNA uses fewer animals than guinea pig test methods, requires less time to perform, provides dose-response information, and, in most cases, eliminates the potential for pain and distress in the test animal. In accordance with Animal Welfare Act regulations and the Public Health Service Policy on Humane Care and Use of Laboratory Animals, the LLNA should be routinely considered when planning animal studies that evaluate whether chemicals and products are strong sensitizers in order to minimize animal use and to avoid pain and distress.
The ICCVAM evaluation is detailed in a report entitled ICCVAM Test Method Evaluation Report: Usefulness and Limitations of the Murine Local Lymph Node Assay for Potency Categorization of Chemicals Causing Allergic Contact Dermatitis in Humans (NIH Publication No. 11-7709). In June 2011, ICCVAM forwarded recommendations to Federal agencies and made these recommendations available to the public (76 FR 18639). In accordance with the ICCVAM Authorization Act of 2000 (42 U.S.C. 285l-3), agencies have notified ICCVAM in writing of their findings, and ICCVAM is making these responses available to the public.
NICEATM and ICCVAM are also currently evaluating several in vitro and in chemico methods for their potential to further reduce and eventually replace the use of animals for ACD safety testing.
The Federal agency responses to the ICCVAM recommendations and more information about the ICCVAM evaluation of the LLNA for potency categorization can be found on the NICEATM-ICCVAM website. The ICCVAM Test Method Evaluation Report is also available. In addition, the Federal Register notice announcing the availability of Federal agency responses to the ICCVAM recommendations is available.
Allergan is pleased to announce the company has received two positive opinions regarding its fully in vitro, cell-based assay for use in the stability and potency testing of BOTOX(R) and VISTABEL(R) (Allergan's botulinum toxin type A products). he first positive opinion, from Agence Francaise de Securite Sanitaire des Produits de Sante (AFSSAPS), relates to VISTABEL(R) and paves the way for approval in 29 countries in the European Union. The second positive opinion was granted by the Irish Medicines Board (IMB) for BOTOX(R) and covers 14 European Union countries involved in the Mutual Recognition Process. The Medicines and Healthcare products Regulatory Agency (MHRA) have already approved the assay for BOTOX(R) vials sold in the UK. Once approval is finalized, the new assay will be utilized to release the product for sale in the relevant countries.
The China State Food and Drug Administration have issued a draft proposal for an alternative method to animal experiments when testing cosmetic ingredients for acute phototoxic effects on the skin.
ECHA (the European Chemical Agency) has announced that it is in favor of accepting the Extended One-Generation Reproductive Toxicity Study (EOGRTS) for reproductive toxicity. This test allows just one generation of animals to be used, with additional tests on a second generation required only if the first round raised concerns. The agency says that the streamlined test will, “under certain conditions”, provide sufficient safety information to replace the two-generation reproductive toxicity study. It says it has already received around 230 proposals from companies to carry out the new test.
The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) recently evaluated the usefulness of a non-animal test method that can identify substances with the potential for interacting with the estrogen receptor in vitro. As announced in today's Federal Register, ICCVAM recommended to Federal agencies that this test method, the BG1Luc ER TA, can be used as a screening test to identify substances with in vitro estrogen agonist and antagonist activity.
This issue includes how to use use in vitro methods as a pre-screen for clinical testing, international educational and outreach activities in Russia, China, and Brazil and more.
This issue includes how to use in vitro methods as a pre-screen for clinical testing, international educational and outreach activities in Russia, China, and Brazil and more.
Recognizing the urgent need to drive regulatory change in those countries that still require animal testing for cosmetic and personal care products, the Institute for In Vitro Sciences, Inc (IIVS) is expanding its international outreach program. The expanded program will be designed to demonstrate to regulators and industry in these countries how alternative testing strategies for cosmetic products can be integrated into a regulatory acceptance program.
NICEATM announces availability of the report on the "International Workshop on Alternative Methods To Reduce, Refine, and Replace the Use of Animals in Vaccine Potency and Safety Testing: State of the Science and Future Directions." The report was published as a dedicated issue of the journal Procedia in Vaccinology (Volume 5, pp 1-266, 2011) and is publicly available online.
The European Coalition to End Animal Experiments (ECEAE) has issued a statement suggesting that the European Commission is likely to propose an exemption to the animal testing ban for products that contain "significant added value".
Dr. Patric Amcoff is the new Operational Manager of the European Union Reference Laboratory for Alternative Methods to Animal Testing (EURL ECVAM) as of mid-November. Before joining the European Commission, Dr. Amcoff worked for nine years at the Organization for Economic Co-operation and Development (OECD) in Paris, where he was responsible for driving the regulatory acceptance at international level of alternative testing methods and approaches for chemical and nano-safety. The mission of ECVAM is to promote the development and use of alternatives to animal testing, for use as research tools and for supporting regulatory safety assessment. ECVAM will continue to coordinate validation studies at European level and act as a focal point for exchange of information on alternative methods to animal testing in the EU. Follow the link above fore more information on Dr. Amcoff available on the ECVAM website.
ECVAM fully endorses the ESAC Opinion of these methods and has additionally provided some further suggestions concerning the CTAs.The ECVAM Recommendation is now out for public consultation until 31 December 2011. The documents that are open for public commenting are divided into two separate parts. Please note that the ESAC Opinion (Annex 1 of Part 1) and the documents in Part 2 are finalised and will not be revised or changed following this open commenting round. Please visit the IHCP Website above for instructions on submitting comments.
Dr. Gertrude-Emilia Costin, Study Director at IIVS, has been recently elected as a member of the PanAmerican Society for Pigment Cell Research (PASPCR) Council for the 2012-2014 term. More info on PASPCR Council and its activities can be found at http://paspcr.med.umn.edu/paspcr.htm.
Dr. Gertrude-Emilia Costin, IIVS Study Director, serves as editor of the PanAmerican Society for Pigment Cell Research (PASPCR) Newsletter. The Newsletter is published three times a year and is intended to serve as a regular means of communication for the members of the Society.
The latest issue of the AltTox newsletter is now available for review online. IIVS and American Society of Cellular and Computational Toxicology (ASCCT) president Rodger Curren as well as Kate Willett, Director, Regulatory Toxicology, Risk Assessment and Alternatives for the HSUS, have been named new members of the AltTox Management Team. The newsletter also includes links to recent forum postings and upcoming events. Please click on the link above to view the full newsletter.
Proteome Sciences announces that it has developed a number of novel in vitro tests for chemical compounds and substances that induce allergies when they come into contact with the skin or the respiratory system. Testing for these allergens in products including chemicals, pharmaceuticals, cosmetics and detergents will become mandatory under EU legislation. Proteome Sciences presented data from eight assays at the Sens-it-iv Scientific Congress in Brussels, 24-25 November 2011. Sens-it-iv is a European Commission project which began in 2005 and brings together companies and academic institutions with the aim of developing in vitro alternatives to animal tests, which are currently used for the risk assessment of potential skin or lung sensitizers. As well as reducing the use of animal testing, the program is aimed at improving consumer safety and benefiting the environment.
As of 28 November 2011, Novo Nordisk will no longer use living animals to test the quality of the batches of medicine coming out of Novo Nordisk's production lines. These tests have for years been required by health authorities as part of their approval of the products. "Today's achievement is a milestone in our ongoing commitment to animal ethics in Novo Nordisk. We have been working for more than a decade, in close collaboration with regulatory authorities around the world, to eliminate obsolete tests or develop and certify new laboratory assays that can be used instead of animals to evaluate the consistent quality of our marketed products," says Executive Vice President and Chief Science Officer, Mads Krogsgaard Thomsen. Novo Nordisk has a commitment to the 3R principles to 'Reduce', 'Refine' or 'Replace' the use of animal testing within the pharmaceutical industry. Therefore, a task force was established more than ten years ago with the ambitious aim to eliminate all redundant product control tests in living animals or replace them with other test methods that would guarantee the same product safety. To read more, please click on the link above.
Click the link above for links to both free and subscription downloadable content including: Editorial: Toxicity Testing: The Need for New Maps for the Future, News & Views, and articles including "Assessing the Search for Information on Three Rs Methods, and their Subsequent Implementation: A National Survey among Scientists in The Netherlands" and "A Critical Evaluation of the 2011 ECHA Reports on Compliance with the REACH and CLP Regulations and on the Use of Alternatives to Testing on Animals for Compliance with the REACH Regulation".
The NIEHS and the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) request public comments that can be considered by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and agencies' program offices in updating The NICEATM-ICCVAM Five-Year Plan (2008-2012) (ICCVAM, 2008). The current plan addresses: (1) Identification of areas of high priority for new and revised non-animal and alternative assays to reduce, refine (enhance animal well-being and lessen or avoid pain and distress), and replace the use of animals in testing and (2) research, development, translation, and validation of new and revised non-animal and other alternatives assays for integration of relevant and reliable methods into Federal agencies' testing programs. Please follow the link above for more information and for information on how to submit your comments.
Source: http://www.ncbi.nlm.nih.gov/pubmed/22082478
Authors: Costin GE, Birlea SA, Norris DA
Chronic ulceration of the leg represents a major, underestimated problem of modern health care, involving physical and cosmetic impairment and social stigma along with high community costs for patients’ treatment. The increasing prevalence of chronic ulcers, currently reported to be as much as 0.3% in the general population, should stimulate identification of more efficacious therapeutic approaches to achieve complete healing. The strategies of regenerative medicine based on small molecules, biomimetic scaffolds, gene or cell therapy, and electromagnetic field manipulation represent some of the modern therapeutic alternatives for wound healing. Here we review in an integrated, interdisciplinary approach the modern cellular and molecular mechanistic concepts regarding the involvement of extremely low frequency electromagnetic fields (ELF-EMF) in the complex process of tissue repair, with particular focus on chronic wounds. The data analysis supports three main effects of electromagnetic fields on the wound healing pathways: 1) an anti-inflammatory effect, by modulation of cytokine profile that induces the transition of the healing process from a chronic pro-inflammatory to an anti-inflammatory state; 2) a neo-angiogenic effect, by increased endothelial cells proliferation and tubulization and production of fibroblast growth factor (FGF)-2; and 3) a re-epithelialization effect, by stimulation of collagen formation. We believe that utilization of ELF-EMF in larger clinical trials designed to optimize these functional parameters would facilitate a better understanding of ELF-EMF-induced healing mechanisms and lead to improved therapeutic outcomes for this disabling condition which is often totally resistant to treatment.
Fifteen years ago, animal protection organizations launched the Coalition for Consumer Information on Cosmetics (CCIC), which administers the Leaping Bunny Program in the United States and Canada. CCIC was created to provide consumers with accurate information on cruelty-free cosmetics, personal care, and household products. The leaping bunny logo has made it easier for consumers to quickly identify which products on the shelf have met standards for being identified as cruelty free. Please read the link above for more information.
SEURAT-1 is a research initiative involving nearly 100 scientists from over 70 European organizations. In October it completed the first of six annual reports titled: "Towards the replacement of in vivo repeated dose systemic toxicity testing". The report aims to aims to inform policy makers about scientific progress relevant to the implementation of European Directives and Regulations and about essential gaps in knowledge corresponding to research needs. It contains detailed information about the scientific progress, the strategic development, and the evolution of the legislative and regulatory context in the field of repeated dose systemic toxicity testing. You can access the report by clicking on the link above.
AXLR8 is funded by the European Commission Directorate General for Research & Innovation (Health Directorate; Advanced Therapies and Systems Medicine Unit) under the 7th European RTD Framework Programme Health Theme. The newly released report includes:
An introduction to the AXLR8 project
An update on the activities and achievements of 3Rs-oriented projects funded under the health and environment themes of the 6th and 7th EU Research Framework Programs during the year 2010
The AXLR8-2 workshop report, including contributions from workshop presenters and reports from break-out groups
AXLR8 Scientific Panel recommendations for future research and innovation funding
A directory of key projects and coordinators
To read the report, please click on the news link provided.
Woodcliff Lake, N. J. (November 07, 2011)—Dr. Anne Marie Api, Vice President of Human Health Science at the Research Institute for Fragrance Materials, Inc. (RIFM), co-wrote with Dr. Matthias Vey, Scientific Director of the International Fragrance Association (IFRA) “Regulatory and safety aspects of natural fragrance ingredients,” Chapter 6, 89-106, in the new book, Formulating, Packaging, and Marketing of Natural Cosmetic Products, edited by N. Dayan and L. Kromidas, New Jersey and Canada, John Wiley & Sons, Inc., 2011.
The chapter defines the role of natural ingredients in perfumery. It explains how natural fragrance ingredients are characterized and evaluated and the roles that RIFM and IFRA have in the evaluation process. The chapter also provides details on how natural fragrance ingredients are regulated in the framework of the IFRA Standards.
The book is available from the RIFM web site through the RIFM Online Store.
Content includes the announcement of our next webinar on the BCOP assay, strategies for identification of skin irritants and corrosives in vitro, a special workshop in Brazil, information on our January Practical Methods for In Vitro Toxicology Workshop and much more.
The testing of chemicals for skin irritation after an acute exposure is considered to be among the simplest of toxicology endpoints to evaluate. Historically, this process simply involved putting the chemicals of interest on the skin of a rabbit, according to the procedures described by Draize and Woodard, and observing the result. However, animal welfare concerns and uncertainty of the relevance of extrapolating rabbit test data to the human experience have brought the relevance of the rabbit test into question. With the introduction and validation of a variety of non-animal methods to identify and classify skin irritants and non-irritants, the study design process has become slightly more complex. But by considering what the project goals are, the following guidance can make the process very manageable and the resulting data extremely useful.
Martin Stephens, Ph.D., a founding member of the AltTox management team, editorial board, and moderator group, has moved from the Humane Society of the United States to the Johns Hopkins Center for Alternatives to Animal Testing, where his primary responsibility will be advancing evidence-based toxicology. Please read the attached pdf for additional information.
On 2 October 2011, Prof. Dr. med. Horst Spielmann was honored by the Deutschen Tierschutzbund (German Animal Protection Society) and was awarded with their Goldene Ehrennadel (Golden Badge of Honor) for his commitment and services towards animal protection and welfare.
Contents of the latest edition of Alternatives to Laboratory Animals with links to free articles and abstracts:
Editorial
*The Three Rs and Animal Experimentation — Documented Signs of Advancement, or of Stagnation? - Michael Balls
*News & Views : CAAT News & Views : IIVS News & Views
Comment
*The Latest Statistics of Scientific Procedures on Living Animals Reveal Little Three Rs Progress in Great Britain in 2010 - Michelle Hudson
Letter to the Editor
*Biomedical Research Involving Chimpanzees - Jarrod Bailey
Articles
*Vaginal Irritation Models: The Current Status of Available Alternative and In Vitro Tests - Gertrude-Emilia Costin, Hans A. Raabe, Robert Priston, Eric Evans and Rodger D. Curren
*Development of the EpiOcular™ Eye Irritation Test for Hazard Identification and Labelling of Eye Irritating Chemicals in Response to the Requirements of the EU Cosmetics Directive and REACH Legislation - Yulia Kaluzhny, Helena Kandárová, Patrick Hayden, Joseph Kubilus, Laurence d’Argembeau-Thornton and Mitchell Klausner
*In-house Validation of the EpiOcular™ Eye Irritation Test and its Combination with the Bovine Corneal Opacity and Permeability Test for the Assessment of Ocular Irritation - Susanne N. Kolle, Helena Kandárová, Britta Wareing, Bennard van Ravenzwaay, and Robert Landsiedel
*The Tenth Anniversary of the Björn Ekwall Memorial Foundation - Erik Walum, Hanna Tähti and Ada Kolman
Source: http://www.ncbi.nlm.nih.gov/pubmed/21942546
Authors: Gertrude-Emilia Costin, Hans A. Raabe, Robert Priston, Eric Evans and Rodger D. Curren
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.
Many of the studies that use animals to model human diseases are too small and too prone to bias to be trusted, says Malcolm Macleod, author of the article titled "Why animal research needs to improve".
"...The most reliable animal studies are those that: use randomization to eliminate systematic differences between treatment groups; induce the condition under investigation without knowledge of whether or not the animal will get the drug of interest; and assess the outcome in a blinded fashion. Studies that do not report these measures are much more likely to overstate the efficacy of interventions. Unfortunately, at best one in three publications follows these basic protections against bias. This suggests that authors, reviewers and editors accord them little importance..."
To read more from the article, follow the "read source" link above.
PETA has added a third category to their "do test" and "don't test" list naming companys that perform animal testing and those that don't perform any tests using animals. The new category, "Working for Regulatory Change", recognizes companies that conduct tests on animals only if they're required by government agencies, that are actively working for the replacement of animals in these tests, and that annually disclose to PETA what tests have been done and why. New York–based Colgate-Palmolive Company is the first company to meet PETA's stringent requirements and will head the "Working for Regulatory Change" list. Please follow the link for the full article.
A new ‘Dragon’s Den’-style funding scheme launched by the UK’s NC3Rs is offering public- and private-sector researchers grants totalling £4.25 million from industrial sponsors to find solutions that will contribute to the replacement, refinement and reduction of animal research in the life sciences. The National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) has worked with pharmaceutical and other sponsors including AstraZeneca, Roche and Eli Lilly to come up with six initial CRACK IT challenges, spanning research areas such as developing in vitro human-cell based models, novel non-invasive measurement techniques and whole-system in vitro testing.
The CRACK IT scheme also includes a web-based research engine for problem-solving and idea-sharing between small companies, academic researchers and industry. Follow the link for a more detailed article or view the CRACK IT website directly.
Authors: Robert Priston, Eric Evans, Gertrude-Emilia Costin, Hans A. Raabe, Rodger D. Curren
The vaginal mucosa provides an effective barrier against numerous pathogens as one of the body’s host defense and immune surveillance components. However, some feminine-care and cosmetic products may induce irritation of the vaginal epithelium, consequently making the tissues more susceptible to infections. Therefore, it is important that the compatibility of newly developed cosmetic or personal care products with the human mucosal surface be assessed before the product is marketed. The most frequently used test to screen for vaginal mucosal irritation is the in vivo rabbit vaginal irritation model. However, the current emphasis and preference in toxicology is to use alternative, in vitro methods that Reduce, Refine, or Replace the use of animals in testing programs. To that end, a clear understanding of the current status, applicability, and limitations of available alternative tests for vaginal irritation assessment is critical when companies are building their safety testing strategies. We present an overview of the available alternative and in vitro techniques for vaginal irritation assessment, from simple cell cultures to more complex explants and reconstructed tissues. We further assess their advantages and disadvantages compared to whole animal test systems and their role in the safety assessment strategy used for a wide array of active ingredients or final formulations.
Paul Locke of the Center for Alternatives for Animal Testing at Johns Hopkins University said more must be done to require agencies including U.S. EPA, the National Institutes of Health and the Food and Drug Administration along with the chemical industry to end animal testing during a congressional briefing on Capitol Hill on Tuesday September 13th. Speaking at the briefing was Representative James Moran, who is ranking member of the House Appropriation Committee's subcommittee on Interior, Environment and Related Agencies and co-chair of the Congressional Animal Protection Caucus (CAPC). CAPC is a group staffed by both parties that seeks to raise awareness of animal welfare issues in Congress. CAPC replaced the "Friends of Animals Caucus" that had existed in previous Congresses. For more information on the briefing, follow the link to CAAT's website.
This issue of AltTox summarizes happenings at the 8th World Congress, recent forum postings, and updates to the calendar of events.
The report published on September 13th stresses the continued commitment in Europe and worldwide to find alternative approaches. It is based on the findings of scientific experts who have been assessing the availability of alternative methods and prospects for the future. The Commission's report states that full replacement of animal test methods for all endpoints will not be possible by the 2013 deadline. The lack of full alternatives does not mean that the Commission will propose postponing the deadline. Instead, the European Commission is currently assessing the impacts of the implementation of the full marketing ban by 2013 (environmental, animal welfare, economic and social) and on the basis of that assessment will decide whether or not to make a proposal in relation to the marketing ban. The Commission will announce its final decision by the end of 2011.
Authors: Chantra Eskes, Erin Hill
With the recent advancements and adoption of alternative methods to animal testing , and the global harmonization of the world commerce, the importance of providing education, throughout the world, on alternative methods with regulatory standing is becoming increasingly necessary. In particular, 1) the specifics of the in vitro test method protocols, 2) the reliability and relevance of the method for regulatory purposes, 3) the importance of ensuring good laboratory practices, and 4) the necessity for a laboratory to demonstrate proficiency, calls for education preferentially based on practical demonstrations and/or hands-on-training. Such training is essential to scientists performing in vitro tests, and is also key for regulators to make critical assessments of the in vitro data applied to their specific needs. Furthermore, such training could favor standardization of regulatory assessment and decisions on hazard properties of chemicals across the world. A critical step is the examination and interpretation of the resulting data for regulatory purposes. It is expected that when familiarized to the biological basis and relevance of alternative methods and the way in which the resulting data can be interpreted and utilized, the more acceptance will these methods gain.
Content includes the announcement of a new member to our Science Advisory Panel, a scientific article on the cytosensor microphysiometer assay, announcement of our September webinar on skin sensitization and the KeratinoSens assay, a synopsis of our recent trip to the California EPA, and more.
Authors: Mun, G, Wilt, N, Donahue, DA, Avalos, J, Norman, K, Hilberer, A, Simion, FA, and Raabe, H
In vitro assays evaluating ocular irritation potential are routinely used by personal care companies. Two of these in vitro assays include the Chorioallantoic Membrane Vascular Assay (CAMVA) and the Bovine Corneal Opacity and Permeability Assay (BCOP). These assays do not require the use of live animals, provide reliable predictive data and are rapid to conduct. The BCOP uses excised bovine corneas to predict ocular irritation. The CAMVA uses the vascular network of fertilized chicken eggs as a conjunctival model to predict eye irritation. Both BCOP and CAMVA have been used for over fifteen years for product development, worker safety, and safety claims substantiation.
This poster describes procedures and considerations for demonstrating the inter-laboratory reliability of the BCOP and CAMVA. It is important to have a valid assay that can be implemented consistently at several different laboratories. For Kao Brands Company, a large BCOP and CAMVA database exists that covers multiple consumer product categories such as hair shampoos, skin cleansers, and hair styling sprays (containing ethanol). Therefore, a proper review of candidate laboratories is important for seamlessly generating consistent results that can be used for assessing potential ocular irritation of new products. First, a candidate laboratory should be audited for proper facility operation and personnel training. Second, the laboratory’s use of Good Laboratory Practices (GLPs) should be reviewed. Third, reference materials with known BCOP and CAMVA data (one irritant and two non-irritants for initial assessment) should be tested at each new laboratory for verification of proper assay performance.
Authors: Sarah S. Willems, Amy M. Sheppard, Jaime L. Treichel, Hans Raabe, Roger Curren
The purpose of this analysis was to evaluate the Reconstructed Human Epidermis (RhE) model as an in vitro method to predict skin corrosivity (OECD 431) for acid products with extreme pH (?2) when compared with in vivo data and the AISE Method (The Worst Case Table) of classification. Extreme pH can be a useful predictor of irritation but may lead to over classification in weakly buffered systems. Our objective was to determine whether the RhE method could accurately identify corrosive and non-corrosive acid products. When compared with the in vivo data, 4/7 products tested using the RhE method predicted the same skin classification. The skin classification of the remaining three formulas was over-predicted when compared with the in vivo data. There were no products for which the RhE under-predicted the skin classification when compared to the in vivo results. When compared with The AISE Method (which considers the results of the EU conventional method calculation and pH/acid reserve), 8/23 products tested using a RhE method predicted the same skin classification. The skin classification of the remaining fifteen formulas was over-predicted when compared with the AISE Method. There were no products in which the RhE under-predicted the skin classification when compared to the AISE method. Overall, the RhE did not reliably identify non-corrosive formulations when compared to either the in vivo data or the AISE Method. This presents significant challenges under hazard classification guidelines such as the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), which requires testing with a validated in vitro method to confirm a non-corrosive classification for an extreme pH product.
Via the Doctor Hadwen Trust for Humane Research, a UK based non-animal research charity, we would like to remind you that the Home Office public consultation on the transposition of the new EU Directive 2010/63/EU, on the protection of animals used for scientific purposes, into UK Law is closing soon. Comments will be accepted until September 5. For an in-depth look at the transposition and for more details on how to get involved with the public consultation, please click on the link above to be directed to the Doctor Hadwen Trust website.
PLEASE SAVE THE DATE: September 20, 10:00 AM when ASCCT will present a webinar which will be a first look at the Effectopedia: The Online Encyclopedia of Adverse Effect Pathways. Effectopedia was conceived by the International QSAR Foundation of Duluth, MN to gather and make available sources and information on what have come to be known as Adverse Outcome Pathways (AOPs). An AOP is a description of the known linkages between molecular initiation and adverse manifestation at the organism or even population level. It is similar to, but more comprehensive than, the concept of the "toxicity pathway" described in the National Academy of Sciences report "Toxicity Testing in the 21st Century: A Vision and Strategy."
Effectopedia is becoming recognized as one of the key tools necessary to implement a transition to "21st Century Toxicity". It is an open knowledge aggregation and collaboration tool that provides a means of describing adverse outcome pathways (AOPs) in an encyclopedic manner. Effectopedia is currently in the development stage, and IQF expects a first public release in April 2012. Effectopedia will be entirely open to the public and is intended to be populated by both targeted grants and voluntary contributions by the scientific community. Effectopedia has two main interfaces. The user interface provides Wiki-like search engine-optimized articles of the AOPs in a relational manner. For each AOP, there is a major overview wiki article linked to in-depth descriptions of the biological response sequences that link a chemical-induced molecular effect to the adverse outcomes needed for safety assessment. Effectopedia will also provide threshold values or dose-response linkages between intermediate biological effects and assessment endpoints.
The contributer interface of Effectopedia supplies tools for editing the content, building AOPs, and participating in discussions - one of the more important aspects for building new social networks among specialists. Please feel free to inform your colleagues about the event, and suggest they join the ASCCT in order to participate in and help support programs like this.
Contents include an editorial on Animal Experiments and Alternatives: Matters of Belief and Trust, News and Views from IIVS, ECVAM and CAAT, comments on Integrated Testing Strategies for Toxicity Employing New and Existing Technologies and Is a Compromise Possible in Russia Between Animal Advocates and Researchers Who Use Animals in Harmful Experiments?, and several articles. Click on the link above to view the full text of several of these articles and to purchase others.
ECHA has recently published two reports required by REACH Regulations: - one on the operation of REACH & CLP and one on the use of alternatives to testing chemicals on vertebrate animals. These reports, along with video interviews of ECHA staff, can be viewed at the ECHA website. The report on the use of alternatives to testing on animals was met with criticims by US and European animal protection groups who claim, among other misuses of animals, that over 100 animal experiments were conducted automatically when they should have been subjected to public consultation. Statements from ICAPO (The International Council on Animal Protection in OECD Programmes), PCRM, (the Physicians Committee for Responsible Medicine), and PETA (People for the Ethical Treatment of Animals) can be found by clicking on the links.
A recent article in Nature News describes the analysis of 200 dossiers by R. Corvida (on behalf of CAAT EU). According to Corvida significant data gaps exist in the submissions and regulators have done little to pressure industry to supply missing information. Corvida will present her findings of over 800 dossiers at the World Congress on Alternatives in August.
Source: http://www.ncbi.nlm.nih.gov/pubmed/21473931
Authors: Pfuhler S, Fellows M, van Benthem J, Corvi R, Curren R, Dearfield K, Fowler P, Frötschl R, Elhajouji A, Le Hégarat L, Kasamatsu T, Kojima H, Ouédraogo G, Scott A, Speit G.
Abstract
Improving current in vitro genotoxicity tests is an ongoing task for genetic toxicologists. Further, the question on how to deal with positive in vitro results that are demonstrated to not predict genotoxicity or carcinogenicity potential in rodents or humans is a challenge. These two aspects were addressed at the 5th International Workshop on Genotoxicity Testing (IWGT) held in Basel, Switzerland, on August 17-19, 2009. The objectives of the working group (WG) were to make recommendations on the use of cell types or lines, if possible, and to provide evaluations of promising new approaches. Results obtained in rodent cell lines with impaired p53 function (L5178Y, V79, CHL and CHO cells) and human p53-competent cells (peripheral blood lymphocytes, TK6 and HepG2 cells) suggest that a reduction in the percentage of non-relevant positive results for carcinogenicity prediction can be achieved by careful selection of cells used without decreasing the sensitivity of the assays. Therefore, the WG suggested using p53- competent - preferably human - cells in in vitro micronucleus or chromosomal aberration tests. The use of the hepatoma cell line HepaRG for genotoxicity testing was considered promising since these cells possess better phase I and II metabolizing potential compared to cell lines commonly used in this area and may overcome the need for the addition of S9. For dermally applied compounds, the WG agreed that in vitro reconstructed skin models, once validated, will be useful to follow up on positive results from standard in vitro assays as they resemble the properties of human skin (barrier function, metabolism). While the reconstructed skin micronucleus assay has been shown to be further advanced, there was also consensus that the Comet assay should be further evaluated due to its independence from cell proliferation and coverage of a wider spectrum of DNA damage.
Copyright © 2011 Elsevier B.V. All rights reserved.
The International Council on Animal Protection in OECD Programmes (ICAPO)is calling for immediate action by companies and regulatory authorities worldwide to replace the traditional “two-generation” animal test for reproductive toxicity with a new “extended one-generation” method that has just been adopted by the Organisation for Economic Co-operation and Development. Although still an animal test, the new one-generation test uses approximately half the number of animals as the old two-generation method (1,400 rats per test vs. 2,600). The new OECD method was adopted on 28 July, just in time for a large number of reproductive toxicity proposals under the European chemicals regulation “REACH,” the revision of the EU testing requirements for pesticides and biocides, and increased U.S. testing of certain pesticides and industrial chemicals. To read the full press release on the ICAPO website, please click on the link above.
Dr. Gertrude-Emilia Costin, Study Director at IIVS, serves as editor of the PanAmerican Society for Pigment Cell Research (PASPCR) Newsletter which is published three times a year and is intended to serve as a regular means of communication for the members the Society. The August number is now available at the link above.
The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) recently evaluated the usefulness of the murine local lymph node assay, or LLNA, for categorizing the potency of chemicals causing allergic contact dermatitis in humans. As announced in today's Federal Register, ICCVAM recommended to Federal agencies that the LLNA may be used to categorize some substances as strong sensitizers. Strong sensitizers are those substances considered to have a significant potential for causing skin hypersensitivity resulting in allergic contact dermatitis.
According to the U.S. Bureau of Labor Statistics, occupational skin diseases are the most common type of occupational illness. Many of these cases arise from repeated exposures to skin-sensitizing substances, which can lead to allergic contact dermatitis or ACD, an immunologically mediated hypersensitivity reaction. Studies have shown that ACD has a significant adverse impact on quality of life in affected individuals.
ICCVAM concluded that the LLNA can correctly categorize some substances as strong sensitizers using a criterion published in the Globally Harmonized System of Classification and Labelling of Chemicals (GHS). However, nearly half of the known human strong sensitizers evaluated by ICCVAM were not identified using the GHS criterion. ICCVAM concluded that additional information would need to be considered to confirm whether substances that do not meet this criterion are or are not strong sensitizers.
Substances with the potential to cause ACD can also be categorized with the traditional test methods using guinea pigs. However, the LLNA uses fewer animals than guinea pig test methods, requires less time to perform, provides dose-response information, and, in most cases, eliminates the potential for pain and distress in the test animal. In accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals, the LLNA should be routinely considered when planning animal studies to evaluate whether chemicals and products are strong sensitizers in order to minimize animal use and to avoid pain and distress.
The ICCVAM evaluation is detailed in a report entitled "ICCVAM Test Method Evaluation Report: Usefulness and Limitations of the Murine Local Lymph Node Assay for Potency Categorization of Chemicals Causing Allergic Contact Dermatitis in Humans" (NIH Publication No. 11-7709). The ICCVAM report and recommendations have been transmitted to Federal agencies for their review and response to ICCVAM in accordance with the provisions of the ICCVAM Authorization Act of 2000, which requires agencies to review the recommendations and respond to ICCVAM within 180 days.
ICCVAM and the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) are also currently evaluating several in vitro and in chemico methods for their potential to further reduce and eventually replace the need for animals for ACD safety testing. More information about the ICCVAM evaluation of the use of the LLNA for potency categorization can be found on the NICEATM-ICCVAM Web at the link provided.
The IHCP has now one vacancy open for a senior scientist. A position description is available. If interested, applications are being accepted through August 5.
In a study commissioned by the European arm of the Center for Alternatives to Animal Testing (CAAT) at the University of Konstanz, Germany, consultant chemist Costanza Rovida has analyzed summaries of 200 dossiers submitted under the REACH program and found gaps in the data. The study states that regulators have done little to pressure industry to fill these data gaps. Some submissions relied on studies performed over 20 years ago that do not adhere to current quality standards. Of the 200 summaries, only 2 contained data from non-animal test methods. To read the full article in Nature News, please click on the link above. Also, full results on the evaluation of 800 summaries will be presented at the World Congress on Alternatives to Animal Use in the Life Sciences meeting next month in Canada.
This issue contains commentary on the recent FDA approval of Allergan's in vitro assay for botox safety and potency testing as well as links to recent forum postings and upcoming events.
The Home Office has released their annual report on animal use in the UK titled "Statistics of Scientific Procedures on Living Animals Great Britain 2010". Some points from the report are summarized below:
Please review the news link above for access to a pdf summary of the document as well as supplementary tables.
One June 24, Allergan received US FDA approval for their in vitro test for the safety and potency of Botox and Botox Cosmetic. This in vitro assay will replace the use of the LD50 test here in the US for each lot or batch of the botox products. It is hoped that total animal testing on the products can be reduced by 95% as additional agencies accept Allergan's new assay. Please take a look at the AltTox forum posting on the topic or in the news link above for Allergan's press release.
Introduced into Congress on June 24, the Safe Cosmetics Act of 2011 has now been uploaded onto the Library of Congress' website where the text can be viewed in its entirety. Comments on the bill from the Campaign for Safe Cosmetics and the Personal Care Products Council can be found on their websites. Section 624 outlines steps to limit the use of animals for testing ingredients and cosmetics. It also encourages funding for the validation of alternative test methods.
The European Chemicals Association (ECHA) has gathered data on the use of non-animal test methods in dossier submissions under REACH from June 1, 2008 thru February 28, 2011. The report is the first provided by ECHA on the use of alternatives to testing on animals since REACH came into effect. The report shows that data sharing between companies making the same material effectively reduced the amount of animal studies that would have otherwise been performed. Existing studies and non-animal (in vitro) test methods were also used to minimize the need for the performance of new animal tests under REACH. The report shows that so far very few new animal studies were conducted for the purpose of registering phase-in substances. The full report titled "The Use of Alternative to Testing On Animals For the Reach Regulation 2011" can be found by following the link above. A summary and video are available on the ECHA website.
Includes information on the new David Sainsbury Fellowship Scheme and a call for submissions to CRACK-IT® Challenges.
Several hundreds of key representatives from the European cosmetics industry gathered in Brussels to discuss the importance of the industry on the European economy and how it can build for a sustainable future. Sirpa Pietikainen, chair of Globe EU and a Member of European Parliament said, "The challenge is exponentially speeding up and that means we have more serious threats, sooner and faster, and we have to react, sooner and faster." Closing the session, Bertil Heerink, Colipa's Director-General concluded that innovation and sustainable development go hand-in-hand. “We can be proud that sustainability in our industry is clearly articulated within our Association. We can also be proud of our performance, but there is no room for complacency. Our industry needs to continue to make sustainability a reality." Select the option above to read the full article.
The Personal Care Products Council (the Council) announced today that Halyna Breslawec, Ph.D., will become the organization’s chief scientist, replacing John Bailey, Ph.D., who officially retires on July 29 after nine years of service. In her new role, Breslawec will oversee the Council’s scientific programs, technical committees and publications and will serve as the industry’s scientific liaison for several domestic and international organizations.
“Halyna’s scientific credentials and background are impressive, and we are so pleased that she will soon be managing our scientific programs,” said Lezlee Westine, Council President & CEO. “She brings a great scientific foundation and proven leadership skills to the Council and will be a wonderful asset in her work on behalf of an industry that is committed to product safety, quality, and innovation.”
Breslawec worked at the U.S. Food and Drug Administration (FDA) for more than 14 years in numerous leadership roles directing the review and approval of medical devices at both the office and division levels. She assessed clinical trials as well as the organization and structure of operational units at the Center for Devices and Radiological Health. While working in the FDA Commissioner’s office Breslawec also developed a program to implement FDA's human subject protection regulations for the clinical and academic communities.
Breslawec currently serves as Deputy Director for the Cosmetic Ingredient Review (CIR), an independent, non-profit panel of scientific and medical experts that assesses the safety of cosmetic ingredients used in the U.S. She is responsible for planning and executing CIR activities while serving as liaison between the expert panel and the CIR professional staff.Prior to joining CIR, Breslawec worked as a consultant where she was a recognized expert in FDA regulation. She helped medical device companies develop regulatory policies, evaluated clinical trials and prepared medical device applications for FDA submission.She earned a Ph.D. in pharmacognosy (medicinal chemistry) and a B.S. in biochemistry from the University of Minnesota.
For more information on cosmetic and personal care products and their ingredients, visit the link above.
Including a feature article titled "Independent or Coordinated Revolutions?", a summary of recent forum postings, and upcoming events.
Dr Andrew Bennett, Director of the FRAME Alternatives Laboratory (FAL), has been appointed a FRAME Trustee. As a result of his interest in finding human-based alternative models he was appointed Director of the FAL in 2006, where he and colleagues continue to work on primary human cell culture. Chairman of the FRAME Trustees Prof Michael Balls said: “We are all very pleased that Andrew has accepted our invitation to be a Trustee.”
The Interagency Coordinating Committee for the Validation of Alternative Methods (ICCVAM) is asking for input and data on both animal and in vitro methods used for botulinum toxin testing and non-endotoxin pyrogen testing. Please follow the link to read the full notice in the Federal Register for more details.
Content of the June 2011 newsletter includes information on a non-animal test method for skin sensitization (KeratinoSens), a summary of our recent training meetings in China, and registration for and details on the expanded program for the 2012 Practical Methods for In Vitro Toxicology Workshop.
An article written by a number of industry scientists and published in Regulatory Toxicology and Pharmacology evaluated eight surfactants in the local lymph node assay, guinea pig maximization test, and a number of in vitro assays.
A summary article indicated that "with one exception, the chemicals are non-sensitising skin irritants. However, one particular method, the local lymph node assay (LLNA), was shown to overestimate the sensitisation potential of surfactants relative to the other methods. The scientists conclude that, as results obtained from LLNAs are considered as the gold standard for the development of new nonanimal alternative test methods, there is a need to carefully evaluate the applicability domains of test methods in order to develop reliable non-animal alternative testing strategies for sensitisation testing."
To read more about skin sensitization testing available at IIVS, please review information on the KeratinoSens Assay for Skin Sensitization and contact Dr. Kimberly Norman, knorman@iivs.org, for more information.
The European Chemicals Agency has expanded its acceptance of GLP data to include countries that are not part of the OECD Mutual Acceptance of Data system, provided that the laboratories producing the data have been inspected by a recognized GLP monitoring authority and have been found to be in compliance. Please review the news link above for additional detail.
Developed by Givaudan, the KeratinoSens assays offers a cost effective way to screen ingredients for their potential as skin sensitizers. The assay is based on the Nrf2-Keap1-ARE toxicity pathway and utilizes a reporter cell line with a luciferase gene to detect electrophilic chemicals - a feature of all skin sensitizers. The assay showed promising reproducibility and predictivity during a recent mulit-laboratory study (Natsch, e. al, 2011) and is currently under review by ECVAM. To learn more about the assay please review the attached document and contact Dr. Kimberly Norman at knorman@iivs.org.
WORKSHOP DATE: June 21, 2011
REGISTRATION DEADLINE: June 15, 2011, 4:00pm ET
The purpose of the workshop is to discuss the scientific advancements that are needed, and the technical and regulatory challenges that must be overcome, for the development of human-relevant preclinical platforms for efficacy and toxicology testing. Please see the attached pdf for additional information.
Source: ATLA
Authors: Arnhild Schrage, Susanne N. Kolle, Maria C. Rey Moreno, Kimberly Norman, Hans Raabe, Rodger Curren, Bennard van Ravenzwaay and Robert Landsiedel
Data on eye irritation are generally needed for the hazard identification of chemicals. As the Bovine Corneal Opacity and Permeability (BCOP) test has been accepted by many regulatory agencies for the identification of corrosive and severe ocular irritants since September 2009 (OECD Test Guideline 437, TG 437), we evaluated this alternative method for routine testing at BASF. We demonstrated our technical proficiency by testing the reference standards recommended in TG 437, and 21 additional materials with published BCOP and in vivo data. Our results matched the published in vitro data very well, but with some intentionally selected false negatives (FNs) and false positives (FPs), the concordance was 77% (24/31), with FN and FP rates of 20% (2/10) and 24% (5/21), respectively. In addition, we tested 21 in-house materials, demonstrating the utility of the BCOP assay for our own test material panel. Histopathological assessment of the corneas by light microscopy was also conducted, as this was suggested as a means of improving the identification of FNs. The histopathology corrected the classification of some FNs, but also increased the number of FPs. Parallel to the test method evaluation, we compared three new opacitometer models with the current standard device. We recommend the use of an opacitometer developed in our BASF laboratory, which has certified components and electronic data storage, resulting in what we consider to be excellent sensitivity, stability and reproducibility.
The NC3Rs is currently seeking to appoint three new members of staff:
*Program Manager - Innovation and Translation
*Communications and Media Manager
*Web Manager
For more information on these vacancies, including details of personal requirements, salary and how to apply, please visit the link provided.
The closing date for applications is 1 June 2011.
In 2010, the European Commission appointed a committee to examine the current status and future prospects of replacement test methods that could be used to comply with the European 2013 ban on using animal tests for certain toxicological endpoints. The experts concluded that there are considerable scientific hurdles that need to be overcome prior to being able to fully replace a number of animal tests. They predict that 5 specific animal test methods will not be replaceable by alternative test methods by the 2013 deadline. The full report is available in the journal "Archives of Toxicology" Volume 85, Number 5, 367-485, and can be accessed through the link. A summary of the major findings is attached to this news article as a pdf document.
US Agencies released a statement on April 14th endorsing the ICCVAM recommendations for in vitro ocular testing methods previously communicated in September 2010. ICCVAM recommends that the Cytosensor microphysiometer (CM) test method can be used as a screening test to identify some types of substances that do not need hazard labeling for eye irritation. This is the first in vitro eye safety testing method adopted for use in what is referred to as a bottom-up approach to testing. ICCVAM also recommended that the CM test method can be used to identify some types of substances that can cause severe or irreversible eye damage. Please click on the link above to read and comment on the the full article on the AltTox website.
This issue includes commentary on the Toxicity Testing in the 21st Century initiative, recent forum postings, and newly announced events. Please follow the source link for the full content.
Includes NC3Rs/Society of Biology Symposium, Charles River/NC3Rs Toxicology Workshop, NC3Rs Primate Welfare Meeting, NC3Rs Studentship Scheme and NC3Rs Roadshows. Click on the link above to view the newsletter on the NC3rs site.
Dr. Gertrude-Emilia Costin, Study Director at IIVS, serves as Editor of the PanAmerican Society for Pigment Cell Research (PASPCR) Newsletter which is published three times a year and is intended to serve as a regular means of communication for the members the Society. The April number is now available by clicking the "Read Source" link above.
Abstract submission has been extended to April 28th for the 8th World Congress on Alternatives and Animal Use in the Life Sciences. The meeting will be held August 21-25, 2011. The organizers are seeking abstracts on the following topics: Safety & Efficacy Testing of Chemicals, Pharmaceuticals & Biologicals, Policy/law on Animal Use, Public Engagement & Ethics Review, Incorporation of the Three Rs in Education & Training, Animal Welfare for Refinement & High Quality Science, and Replacement & Reduction in Basic Research. Please visit the World Congress website for more information.
R. Curren will give a talk titled "Routine Use of Alternatives in the Cosmetic and Personal Care Industries in the US" at the International Symposium on Technology and Application of Alternatives to Animal Testing held at the Guangdong CDC (China) on April 14. More information on this and other recent meetings in China on the use and implementation of alternative methods will be featured in the May issue of the IIVS e-newsletter. If you would like to receive the IIVS Update, please continue to the contact page and provide us with your information.
A pdf of the November 2010 EPAA annual conference "Reduction and Refinement: Combining Excellence in Science and Animal Welfare" is available on their website.
The European Chemicals Agency will organise its Sixth Stakeholders’ Day on 17-18 May 2011, in Helsinki. The main event consisting of plenary and one-to-one sessions will take place on 18 May at the Helsinki Exhibition and Convention Centre (Messukeskus). A registration form for the main event will be published on the ECHA website shortly. The event is open to everyone and participation is free of charge. In conjunction with the event, a day of training will take place at ECHA on 17 May focusing on in-depth training on the Chemical Safety Assessment and Reporting tool (Chesar). The training session is open to all stakeholders with some experience in chemical safety assessment.
Includes NC3Rs Research Portfolio, ARRIVE guidelines adopted by Nature, and information on the 8th World Congress on Alternatives
Source: http://www.ncbi.nlm.nih.gov/pubmed?term=KeratinoSens
Authors: Natsch A., Bauch C., Foertsch L., Gerberick F., Norman K., Hilberer A., Inglis H., Landsiedel R., Onken S., Reuter H., Schepky A. and Emter R.
Due to regulatory constraints and ethical considerations, research on alternatives to animal testing to predict the skin sensitization potential of novel chemicals has gained a high priority. Accordingly, different in vitro, in silico and in chemico approaches have been described in the scientific literature to achieve this goal. To replace regulatory approved animal tests, these alternatives need to be transferable to other labs, their within and between laboratory reproducibility must be assured, and their predictivity should be high. The KeratinoSens assay is a cell-based reporter gene assay to screen substances with a full dose-response assessment. It is based on a stable transgenic keratinocyte cell line. The induction of a luciferase gene under the control of the antioxidant response element (ARE) derived from the human AKR1C2 gene is determined. Here we report on the results of a ring-study with five laboratories performing the KeratinoSens assay on a set of 28 test substances. The assay was found to be easily transferable to all laboratories. Overall both the qualitative (sensitizer/non-sensitizer categorization) and the quantitative (concentration for significant gene induction) results were reproducible between laboratories. A detailed analysis of the transferability, the within- and between laboratory reproducibility and the predictivity is presented.
Non-clinical CRO CIT will distribute Stemina’s hES-based toxicity testing platform to pharmaceutical and cosmetics developers in Europe under an agreement announced in late February The deal, financial terms of which were not disclosed, will see Stemina’s DevTox human embryonic stem cell (hES) based technology used to test the toxicity of candidate compounds as an alternative to animal testing.
Developmental neurotoxicity (DNT) is an issue of growing concern in children's health worldwide. The developing human nervous system is susceptible to many toxicants, and chemical exposure during development that may cause lasting neurological deficits. Such damage can range from subtle to severe, and may impose substantial burdens on affected individuals, their families, and society. This DNT3 meeting will provide a strong platform for comprehensive discussion among diverse stakeholders from around the globe, including research scientists, government scientists, regulators, policy analysts, industry representatives, academics, and advocacy groups concerned with children's health, animal welfare, and environmental protection. The following topics will be discussed:
We encourage your participation in the Third International Conference on Alternatives for Developmental Neurotoxicity Testing (DNT3). We also invite you to submit an abstract on a topic relevant to the conference program for consideration as an oral presentation (deadline 31st March 2011). Taking into consideration your expertise and published papers, we are convinced that your participation and contribution will accelerate and promote the development of alternative methods for DNT assessment.
Source: Society of Toxicology Meeting 2011
Authors: Natsch, Andreas; Bauch, Caroline; Ellis, Graham; Foertsch, Leslie; Gerberick, Frank; Norman, Kimberly; Landsiedel, Robert; Onken, Stefan; Reuter, Hendrik; Schepky, Andreas and Emter, Roger
Skin sensitizers in general are small, electrophilic, reactive molecules. The Keap1-Nrf2-ARE pathway is a known cellular toxicity pathway responding to reactive chemicals and it is increasingly being recognized as a key toxicity pathway induced by skin sensitizers. The in vivo relevance has been confirmed in Nrf2-knockout mice3.
The KeratinoSens assay is based on a stable reporter cell line with a luciferase gene under the control of the antioxidant response element (ARE) of the AKR1C2 gene in a HaCaT background. Luciferase induction and cytotoxicity are measured to test for skin sensitization potential.
Standard operating procedure of the assay *Chemicals are added at 12 concentrations to triplicate assay plates. *After 48 hours, induction of luciferase activity and cell viability is evaluated. *Chemicals with > 50% induction of Luciferase above solvent control at non-cytotoxic concentrations are rated positive. *EC 1.5 and EC3 values (Concentration for > 50% and > 200% induction of Luciferase) and IC50 values are calculated for potency estimations. *High throughput possible with full dose-response curve measurement
Source: Society of Toxicology Meeting 2011
Authors: Gandolfi, Lisa; Tierney, Neena; Johnson, Donzel; Walters, Russel; Fevola, Michael; Gunn, Euen; Martin, Katharine; Kong, Amanda; Hilberer, Allison; Barnes, Nicole; Wilt, Nathan; Nash, Jennifer R.; Inglis, Heather; Raabe, Hans; Costin, Gertrude-Emilia
A goal of personal care products manufacturers is to develop increasingly milder formulations. To this end, reproducible in vitro systems can accurately assess the irritation potential of the products, thus avoiding the use of animals for testing. The three-dimensional EpiDerm model (MatTek Corp.) provides a testing platform for Johnson & Johnson’s skin irritation assessment program targeting raw ingredients and final formulations. The testing program we have developed evaluated the potential dermal irritation of over 150 amphoteric and/or anionic surfactant-containing candidate formulations or individual raw ingredients. The formulations were diluted to 10% in water and were applied onto the surface of the 3-D tissues for 1 h, followed by 24 h post-exposure analysis for cytokine expression. The potential dermal irritation of the candidates was evaluated by MTT viability and IL-1? release. Another goal of the program was to qualify two representative benchmark materials with known skin irritation potential for use as references for the skin irritation evaluation of formulations with new surfactant ingredients. We have developed a database and established a range of irritation responses of the benchmarks using the IL-1? endpoint. Comparison of the potential dermal irritation of new formulations to existing mild formulations guides formulation development for new mild cleansing products. Most recently, we have demonstrated the reliability of the test system for the assessment of irritation potential of final formulations. We are currently expanding our database and work towards establishing a correlation between the in vitro pre-screening approach and clinical testing. This testing platform integrates the efforts made to meet the mildness testing needs of global manufacturers of personal care products that focus on developing increasingly milder lines of formulations to be applied to the skin.
Source: Society of Toxicology Meeting 2011
Authors: Kimberly Norman, Heather Inglis, Greg Mun
Environmental exposure of the skin to ultraviolet radiation makes it a primary target for UVA-generated reactive oxygen species (ROS). An overabundance of ROS causes oxidative stress which may result in the oxidation of proteins, lipids, and nucleic acids. This oxidative damage is a major contributor to photoaging and is associated with skin carcinogenesis. In order tomitigate the damaging effects of ROS, antioxidants are increasingly being added to formulations of consumer skincare products. The goal of this study was to develop an in vitro method capable of evaluating the antioxidant potential of ingredients and formulations in primary human keratinocytes (NHEKs). NHEK cells were seeded in 96-well plates and incubated with the fluorescent ROS-detecting probe, 5-(and-6)-chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA), for 40 minutes, then exposed to a serial dilution of antioxidants/antioxidant - containing formulations for 1 hour. ROS were generated by exposing the cultures to UVA light for 50 minutes (1.6-1.8 mW/cm2), and then detected by fluorescence measurements (ex/em 485/530 nm) of the cells. Cytotoxicity was assessed concurrently using the neutral red uptake assay. Using this method, we evaluated several known antioxidants and antioxidant-containing formulations for their ROS-reducing capabilities. Based on our results, several antioxidants significantly reduced ROS levels compared to untreated controls. Other antioxidants which did not show this reduction were likely not water soluble and/or poorly bioavailable to cells. Overall, our results indicate that this method may provide a valuable in vitro tool for assessing antioxidant capacity in a biologically relevant model.
Source: Society of Toxicology Meeting 2011
Authors: A. Schrage, H. Raabe, K. Norman, R. Curren, S.N. Kolle, M.-C. Rey-Moreno, B. van Ravenzwaay, and R. Landsiedel
Data on eye irritation are generally needed for hazard identification of chemicals. Since the bovine corneal opacity and permeability (BCOP) test has been accepted by many regulatory agencies for the identification of corrosive and severe ocular irritants since September 2009 (OECD Test Guideline 437), we evaluated this alternative method using a broad variety of chemicals and formulations. Ten reference standards recommended in the TG437 and 21 substances (including historical false negatives [FN] and false positives [FP]) with published BCOP and in vivo data were tested in two labs (BASF and IIVS). Our results matched the published in vitro data very well, the concordance was 77% (24/31) with FN and FP rates of 20% (2/10) and 24% (5/21). However, one of the solid test substances, Dibenzoyl-L-tartaric acid, had a high variability in both labs resulting in false negative (FN) classifications in about half of the BCOP assays performed (n=8). Additionally as suggested by the guideline, we used histopathological evaluation of cross sections of treated corneas to identify false FN. This endpoint corrected the classification of some FN, but increased the number of false positives at the same time. Thus it did not increase of the overall predictivity. In parallel, we compared the opacitometer which was originally used in the development of the BCOP in the 1980s to a new opacitometer model with certified components, electronic data processing and calibration standards (glass filters). The data obtained with the new instrument correlated almost perfectly with the traditionally machine. Regardless of the utilized equipment, we recommend the use of calibration filters for stability, reproducibility and comparability of BCOP results from different laboratories.
Source: Society of Toxicology Meeting 2011
Authors: Wurzburger, L., P. Kazmi, T. Re, A. Alonso, B. Bertino, N. Barnes, A. de Brugerolle de Fraissinette, A. Hilberer, H. Raabe, N. Wilt, and V. Srinivasan
Assuring the safety of personal care products without testing in animals is a goal common to many personal care products manufacturers, due to both ethical concerns for animal welfare, as well as the limited relevancy of animal models to predict human responses. Towards this goal, the cosmetics and personal care industry has increasingly relied upon human cell-based 3–dimensional reconstructed tissues to evaluate the safety of their product candidates in various target tissues. Accordingly, we have developed a program for screening the potential irritancy of teeth whitening products in oral mucosal tissues using commercially-available oral buccal (SkinEthic RHO) and gingival (SkinEthic RHG) models. Four formulations containing H2O2 at various concentrations and one sodium bicarbonate were tested at four exposure times to determine ET50 values. Three irritancy endpoints were measured after each exposure: viability using the MTT conversion assay, the amount of IL-1? released from the tissues, and histological changes. Both models presented the same rank order of the five materials, with increases in the ET50 values correlating with decreases in H2O2 concentration. The formula containing sodium bicarbonate, however, was non-toxic in both models. Histological analysis confirmed the MTT results and provided evidence of the chemical impact upon cellular and tissue morphology. IL-1? release did not appear to be as sensitive as the MTT assay at shorter exposure times, although it may be useful to differentiate among formulations predicted by the MTT assay to be of low irritation potential. Our results suggest that the MTT viability and histology endpoints in 3–D human oral reconstructed tissues can provide useful predictive information to support an oral care products safety program.
Source: Society of Toxicology Meeting 2011
Authors: G. Mun; N. Wilt; D. A. Donahue; J. Avalos; K. Norman; A. Hilberer; F. A. Simion; H. Raabe
In vitro assays evaluating ocular irritation potential are routinely used by personal care companies. Two of these in vitro assays include the Chorioallantoic Membrane Vascular Assay (CAMVA) and the Bovine Corneal Opacity and Permeability Assay (BCOP). These assays do not require the use of live animals, provide reliable predictive data and are rapid to conduct. The BCOP uses excised bovine corneas to predict ocular irritation. The CAMVA uses the vascular network of fertilized chicken eggs as a conjunctival model to predict eye irritation. Both BCOP and CAMVA have been used for over fifteen years for product development, worker safety, and safety claims substantiation. This poster describes procedures and considerations for demonstrating the inter-laboratory reliability of the BCOP and CAMVA. It is important to have a valid assay that can be implemented consistently at several different laboratories. For Kao Brands Company, a large BCOP and CAMVA database exists that covers multiple consumer product categories such as hair shampoos, skin cleansers, and hair styling sprays (containing ethanol). Therefore, a proper review of candidate laboratories is important for seamlessly generating consistent results that can be used for assessing potential ocular irritation of new products. First, a candidate laboratory should be audited for proper facility operation and personnel training. Second, the laboratory’s use of Good Laboratory Practices (GLPs) should be reviewed. Third, reference materials with known BCOP and CAMVA data (one irritant and two non-irritants for initial assessment) should be tested at each new laboratory for verification of proper assay performance.
IIVS goes paperless with its first digital, e-newsletter. Content includes an overview of our re-designed website, details on our assay designed to look at topical antioxidant materials, our participation in the ECVAM ESAC committee, and more. Please let us know what you think of the new format.
Dr. Rodger Curren will speak at the International Forum on Cosmetic Technology and Applications, the first meeting discussing alternatives to animal models in toxicology to be held in China. The meeting is being organized by the China Cosmetics Research Centre of the Beijing Technology and Business University and many interested industry parties such as Mark Kay, P&G, Colgate-Palmolive, and Johnson & Johnson. Please read the attached link for more information and details on registration.
Currently there is no widely accepted method to evaluate the antioxidant properties of skincare products. IIVS recently undertook a project to develop an in vitro method capable of evaluating the antioxidant potential of antioxidants and antioxidant skincare products in human cells. NHEKs (normal human epidermal keratinocytes) were subjected to UVA-irradiation to induce oxidative stress and then protection from oxidative stress was evaluated in cells incubated with antioxidants. Cytotoxicity was assessed concurrently using the neutral red uptake assay. Using this method, several known antioxidants and antioxidant-containing formulations were evaluated for their ROS-reducing capabilities.
The FDA published a call for comments in December on proposed changes to be made to the GLPs. The call for comments, linked to above, lists 9 specific areas which the FDA is considering for a future amendment. IIVS, along with many other companies and organizations, has submitted comments. Please read the attached document to see our perspective.
Abstract submission for this DNT3 conference has been extended until March 31, 2011. Please take the time to submit your abstracts. For more information on this event, please visit the IIVS event page.
Speaking at a workshop at the Department of Endocrinology in the University of Madras, Dr. M A Akbarsha (Director of the Mahatma Gandhi-Doerenkamp Center (MGDC), Bharatidasan University, India) summarized the IdMOC (Integrated Discrete Multiple Organ Co-Culture) procedure. The technology uses a large interconnecting chamber that contains multiple inner wells. Multiple cell types are first individually cultured in each well. Later the chamber is filled with a single universal medium. The test material is then added to this medium and its reactions to each cell type are subsequently analyzed. This technology, developed by Dr. Albert Li (managing director of Advanced Pharmaceutical Sciences, Columbia) will allow researchers to gather information on how test substances and their metabolites react with and interact with multiple cell types at once.
The National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) presented two workshops on "Best Practices for Regulatory Safety Testing" in January 2011. The workshops, "Assessing the Potential for Chemically Induced Eye Injuries" and "Assessing the Potential for Chemically Induced Allergic Contact Dermatitis," were organized by NICEATM and the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). Materials from the workshops, which were co-sponsored by the Society of Toxicology and the Society for Risk Analysis, are now available on the NICEATM-ICCVAM website at the source link above.
Materials from the workshops available on the web page include: * Program, goals, objectives, and background information for each workshop * Slides from presentations given at the workshops * Links to archived webcasts of the workshops * Abstracts of poster session presentations
About the Workshops These one-day workshops, which were each attended by over 70 scientists from government and industry, provided a practical understanding of the theory and application of available methods that can evaluate the hazard potential of chemicals and products while minimizing animal use and avoiding pain and distress. The workshops brought together scientific experts from relevant stakeholder organizations to discuss available alternative test methods for assessing chemicals and products for their ocular and allergic contact dermatitis hazard potential. Participants learned the strengths and weaknesses of available alternative test methods, became familiar with the types of data they provide, and learned how to use these data in regulatory safety assessments.
InVitro International (IVRO) is a customer and technology driven provider of non-animal testing methods. It is engaged in the development and marketing of both test kits and laboratory services. The IAS is a plate reader based computer driven upgrade from the former Eytex/Skintex methodology which has helped reduce industry costs of animal testing for safety and efficacy of ocular, skin care, personal care, and chemical products. In addition, the company also offers a series of services including protocol development (if necessary), customized contract testing services, and direct kit sales in support of product claims and compound or workplace safety. Click on the link above to read more.
U.S. Federal agencies have responded to recommendations on alternative safety testing methods to determine if chemicals and products may cause allergic skin reactions, also known as allergic contact dermatitis (ACD). The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) recently evaluated and recommended the methods, which are new versions and applications of the murine local lymph node assay (LLNA).
Responses are provided from agencies that include the Environmental Protection Agency and the Food and Drug Administration. The agency responses are being made available to the public as required by the ICCVAM Authorization Act of 2000, and as recently announced in the Federal Register (76 FR 2388).
The Humane Society of the United States presents the Russell and Burch Award to scientists who have made outstanding contributions to the advancement of alternative methods in the areas of biomedical research, testing, or higher education. Alternative methods—also known as the Three Rs—are methods that can replace or reduce the use of animals in specific procedures, or refine procedures so that animals experience less pain or suffering. Candidates for the award are judged on the scientific merit of their contribution to the alternatives field, as well as their impact and professional commitment to this field. Applicants should have a history of laboratory work that is above reproach on humane grounds. Send nominations by April 10, 2011 to ari@humanesociety.org. No special forms are necessary.
Persons nominating themselves should submit a cover letter explaining their suitability for the award, a curriculum vitae, and three published articles (preferably in PDF format) representative of their alternatives work. Persons nominating others should submit a letter explaining the nominee’s suitability for the award, and arrange to have supporting documents forwarded.
The IIVS Fall/Winter 2010 newsletter contains a wrap-up of the October In Vitro Alternatives Forum, IIVS' skin sensitization program, the Axlr8 program, the founding of the American Society for Cellular and Computational Toxicology, and more.
The journal ATLA (Alternatives to Laboratory Animals), which was awarded the William and Eleanor Cave Award for achievements in developing alternatives, is searching for new methods to subsidize its publishing and distribution costs. ATLA is an important journal not only for the countries leading the way in replacement of animal models in testing, but also in countries in which this field is still in its infancy. Please read the attached letter and visit FRAME's website for more information on what you can do to help this journal to keep running.
In recognition of its unique technology, rapid development, and management team, Epithelix has been awarded the 2011 innovation trophy by the Swiss and French Commercial and Industrial Chamber (CFSCI).
In light of recent concerns over the potential extension of the 2013 ban on the sale of cosmetics developed with the help of animal experimentation, members of the UK parliament are calling for the EU to uphold the original deadline. Read the full article at the source above.
Under the REACH regulation in Europe, companies are required to test many substances that had not previously been tested and submit the results in the form of a dossier to the European Chemicals Agency (ECHA). ECHA has announced that the process of dossier evaluation will become more open. They are taking steps such as publishing conclusions drawn from third party evaluations, allowing case-owners and stakeholders to be present during the evaluation discussions, and publishing Practical Guide 12 to explain to industry what the dossier evaluation process is and how they are processed.
The rule titled, "Hazardous Materials: Harmonization With the United Nations Recommendations, International Maritime Dangerous Goods Code, and the International Civil Aviation Organization Technical Instructions for the Safe Transport of Dangerous Goods by Air" brings the US regulations into line with international standards and calls for the use of In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER) (OECD guideline 430), In Vitro Skin Corrosion: Reconstructed Human Epidermis (RhE) Test Method (OECD guideline 431), and the In Vitro Membrane Barrier Test Method for Skin Corrosion (OECD guideline 435) where it previously only called for use of the rabbit test (OECD guideline 404). An excerpt from the document states:
“In our latest harmonization effort, we received over 2,200 comments in response to the NPRM (75 FR 52070, August 24, 2010). The majority of the comments received were from individuals in support of adoption of corrosivity testing methods not based on the results of live animal testing...We received over 2,200 comments additional to that received from PETA, in response to the NPRM supporting the adoption of in vitro testing methods to determine corrosivity and urging PHMSA to stop the requirement for use of methods based on live animal testing. Therefore, in this final rule we are adopting the OECD in vitro testing methods as proposed. See Section 173.137 for further discussion of such methods.”
Read the rule in the Federal Register by choosing the "Read Source" button above.
Source: Mutation Research/Genetic Toxicology and Environmental Mutagenesis
Authors: Erica L. Dahl, Rodger Curren, Brenda C. Barnett, Zubin Khambatta, Kerstin Reisinger, Gladys Quedraogo, Brigitte Faquet, Anne-Claire Ginestet, Greg Mun, Nicola J. Hewitt, Greg Carr, Stefan Pfuhler and Marilyn J. Aardema
Abstract
The European Cosmetic Toiletry and Perfumery Association (COLIPA), along with contributions from the European Centre for the Validation of Alternative Methods (ECVAM), initiated a multi-lab international prevalidation project on the reconstructed skin micronucleus (RSMN) assay in EpiDerm™ for the assessment of the genotoxicity of dermally applied chemicals. The first step of this project was to standardize the protocol and transfer it to laboratories that had not performed the assay before. Here we describe in detail the protocol for the RSMN assay in EpiDerm™ and the harmonized guidelines for scoring, with an atlas of cell images. We also describe factors that can influence the performance of the assay. Use of these methods will help new laboratories to conduct the assay, thereby further increasing the database for this promising new in vitro genotoxicity test.
Although the full text of this article is not available here, you may visit the website for the Mutation Research/ Genetic Toxicology and Environmental Mutagenesis to purchase a copy.
Authors: Evans, Eric; Priston, Robert; Inglis, Heather; Barnes, Nicole; Raabe, Hans; Costin, Gertrude-Emilia
Three dimensional (3D) human vaginal-ectocervical tissue constructs may be a relevant in vitro model to rapidly screen for vaginal mucosal irritation potential of raw materials and final formulations. Here we report results from a study with an in vitro vaginal tissue construct (EpiVaginalTM from Mattek Corporation, USA), used to predict irritation responses of a variety of benchmark ingredients (including surfactants and fragrances) and product formulations. Test products were representative body washes, personal care wipes, etc., tested at concentrations relevant to use in final product form. The time-to-toxicity (exposure time to reduce viability of the tissues to 50% of the controls, or ET50) for each test sample was determined. Samples were applied topically to the surface of the tissue constructs for various exposure times. Vaginal irritation expressed as ET50 values showed a close correlation with the expected irritation potential based on previous work, published research results and product market history. In general, our results demonstrated a shortening of the ET50 values with increasing sample concentration; as such, the ET50 values for a dilution series of the positive control (Triton-X-100) ranged from 8.77 to 4.00 h (for a dilution of 0.1%), from 3.83 to 1.53 h (for a dilution of 0.3%) and from 1.69 to 0.64 h for the 1% Triton-X-100. Furthermore, the ET50 of a currently marketed vaginal cream containing 20% benzocaine ranged from 3.03 to 1.50 h. Three categories of personal care wipes were tested and the ET50 values ranged from 4.64 to 6.64 to >24 h, correlating with their expected irritation potential based on other data. In conclusion, our findings suggest that the time-totoxicity assay using EpiVaginal tissues can be used to screen raw ingredients and final products for potential vaginal irritation.
The IIVS Summer 2010 newsletter contains valuable information on the proposed TSCA reform, the 2010 In Vitro Alternatives Forum, a formation of a new scientific society and much more.
Authors: Aardemaa, Barnetta, Khambattaa, Reisingerb, Ouedraogo-Arrasc, Faquetc, Ginestetc, Mun, Dahl, Hewitte, Corvif, Curren
After a new, comprehensive evaluation of the prior available data, the ECVAM scientific advisory committee (ESAC) has recently accepted the CytosensorMicrophysiometer as capable of identifying non-irritants for testing limited to water-soluble surfactants and water-soluble surfactant-containing mixtures. This 25-year development is remarkable and instructive in many respects. The authors see this as opening the door, at last, for an end to the use of animals as a standard requirement for eye irritation. Here, several of the people critically involved in this processes have summarized the important aspects of this history.
This newsletter contains information about: * The Mouse Embryonic Stem Cell Test: Technical Challenges and Recent Advances * 2010 In Vitro Alternatives Forum * IIVS Training Workshops * and much more!
Source: SOT
Authors: Pilar Prieto, Hans Raabe, Rodger Curren, Allison Hilberer, Maurice Whelan, Sandra Coecke, Rosemary Gibson (et el.)
The purpose of this validation study is to assess the capacity of the 3T3/NRU cytotoxicity assay to give a simple yes/no answer in order to discriminate between toxic/hazardous (LD50 < 2000 mg/kg) and not classified (LD50 >2000 mg/kg) substances. High (87%) prevalence of substances with acute oral LD50 > 2000 mg/kg. Registry of Cytotoxicity showed that the prediction from cytotoxicity data of low systemic toxicity is much better than the prediction of high systemic toxicity.
Source: SOT
Authors: Cater, Kathleen; Cerven, Daniel; Curren, Rodger; Wilt, Nathan; Raabe, Hans A.
The Bovine Corneal Opacity and Permeability assay (BCOP), an internationally recognized alternative to the Draize eye irritation test, uses excised bovine corneas to predict ocular irritation. Originally developed by Gautheron (1992) and utilizing the irritation class prediction established by Sina (1994), BCOP has been used independently at MB Research and at the Institute for in Vitro Sciences (IIVS) for over fifteen years for product development, worker safety, and safety claims substantiation. The assay has recently been included in an 18-month pilot evaluation program for use for eye irritation labeling of cleaning products with antimicrobial claims (EPA Office of Pesticide Programs, May 2009). In addition, the Organization for Economic Co-Operation and Development (OECD) adopted Test Guideline 437 describing the use of the assay for identifying ocular corrosives and severe irritants (Sept. 2009). Since MB Research and IIVS have extensive experience performing the BCOP assay utilizing a variety of protocols, they agreed to develop and evaluate the reproducibility of a standard harmonized protocol for regulatory labeling. Nine blind-coded chemicals, primarily comprised of surfactant dilutions, as well as imidazole and pyridine, were tested in three independent GLPcompliant trials using exactly the same protocol. The resulting In Vitro Scores were compared to Draize MMAS results (ECETOC, 1998). Intralaboratory and inter-laboratory reproducibility evaluations showed that both laboratories obtained the same irritation class predictions (except for cetyl pyridinium bromide). Some of the surfactant dilutions (sodium dodecyl sulfate, cetyl pyridinium bromide) were found to be under-predicted using the standard BCOP protocol for liquid test chemicals. Accordingly, the testing of certain classes of surfactants for regulatory safety using extended exposure times may be scientifically justified.
Source: SOT
Authors: Burrows-Sheppard, Amy M.; Willems, Sarah S.; Heitfeld, Fred; Treichel, Jamie; Raabe, Hans; Curren, Rodger
The purpose of this study was to evaluate the EpiDerm™ Corrosivity (OECD 431) and Corrositex® Time Monitor (OECD 435) as in vitro methods to predict skin corrosivity for extreme pH (? 11.5) products. Extreme pH can be a useful predictor of irritation but may lead to over classification in weakly buffered systems. Hazard classification guidelines such as the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) recommend testing with a validated in vitro method to confirm a non-corrosive classification for an extreme pH product. Our objective was to identify a method that could accurately identify corrosive and non-corrosive alkaline products. 8/12 products tested on the EpiDerm™ assay predicted the same skin classification when compared with the in vivo data. The remaining four formulas overpredicted the skin classification when compared with the in vivo data. There were no products in which the EpiDerm™ under-predicted the skin classification when compared to the in vivo results. The Corrositex® assay was able to accurately predict 3/7 formulas, four products were over predicted as corrosive by Corrositex® ; none were under predicted. EpiDerm™ results were also compared to classifications made under JDIs internal hazard assessment process which bridges new formulas to classification guidelines based on an extensive database of historical in vivo data on specific chemical and formulation categories. This weight of evidence approach is consistent with GHS guidance on use of professional judgment to classify a product. JDIs process accurately predicted the in vivo data 8/14 times. Four products were predicted to be more hazardous when compared with in vivo results and two were predicted to be less hazardous. This approach results in >85% accuracy in providing appropriate hazard classification for these materials without confirmatory testing to support the classification. The EpiDerm™ assay is a promising alternative to the use of animals to confirm non-corrosive classifications; however limitations were seen with higher solvent levels. The Corrositex® assay did not reliably identify non-corrosive formulations.
Authors: Srinivasan, Vinayak; Alonso, Alain; Bertino, Beatrice; Costin, Gertrude-Emilia; de Brugerolle de Fraissinette, Anne; et al.
A common goal of many personal care companies is to assure the safety of their products without animal testing, due to concerns about ethical and animal welfare issues as well as the relevancy of the animal model to humans. To address these issues, we have developed an in vitro testing program to support the safety evaluation of potential vaginal irritation in a number of bath and shower cleanser products. A series of surfactant-containing formulations, diluted to 10% in water to mimic the maximum concentration expected in bath water, were tested. The formulations were applied topically onto the surface of commercially-available SkinEthic HVE three-dimensional human vaginal epithelium tissues over various exposure times. The ET50 (i.e. the exposure time expected to reduce relative viability of the tissues to 50% of controls) for each candidate was determined. The test results were compared to reference formulations and available human clinical data. The vaginal irritancy evaluation and ranking of bath and body wash products based on ET50 values showed a good correlation with the expected irritation potential of individual ingredients. Histology analysis confirmed the overall MTT viability results and provided additional information regarding the effect of the tested products on the tissue’s integrity. However, IL-1? release did not appear to be as sensitive a marker as the MTT viability assessment at the short exposure times used (20 minutes, 1, 2, and 4 hours). This in vitro safety screening approach shows promise for predicting the vaginal irritancy of tested products and in meeting the typical needs of product development groups charged with developing increasingly milder products.
Authors: Steve Bryce, Erica Dahl, Greg Mun, Svetlana Avlasevich, Stephen Dertinger, Rodger Curren
Starting in March of 2009, in vivo genotoxicity testing for cosmetics sold in the European Union was banned by the 7th Amendment to the Cosmetics Directive. In vivo tests were used to confirm the results of in vitro tests, which produce a high number of false positives. The Reconstructed Skin Micronucleus Assay (RSMA) was developed as a possible replacement for in vivo tests in this tiered testing strategy. The RSMA uses a metabolically active 3-dimensional reconstructed human skin model with a functional stratum corneum (Figure 1). Test articles are applied topically, mimicking exposure of cosmetics. Cells from the basal layer are assessed for micronucleus induction by microscopy. Though this assay has performed well in prevalidation trials, scoring the micronuclei is labor intensive, which may limit widespread use of the assay. We have investigated the feasibility of automating the scoring of micronuclei from the basal cell layer using the In Vitro MicroFlow® system developed by Litron Laboratories.
Authors: Demetrulias, Janis; Acuff, Karen; Aust, Louise; Avalos, Javier; Burdick, Joel; Cater, Kathleen; Raabe, Hans, et al
Procedures for preparing barrier-compromised human donor skin for use in in vitro percutaneous penetration studies as a model of damaged or disease-state skin, where a reduced barrier function due to loss or abnormal development of the stratum corneum is characteristic, were developed. Two methods for disrupting the stratum corneum were evaluated, namely, tape stripping of the stratum corneum with 10, 20, or 30 serial tape strips, and topical exposures to sodium lauryl sulfate (SLS) at concentrations of 1, 5, and 10% SLS. The skin samples in the tape strip treatment groups were tape stripped prior to mounting in in-line diffusion cells, while the skin samples in the SLS treatment groups were mounted in in-line diffusion cells prior to the 4-hour exposures to SLS. A pair of untreated tissues were included as normal tissue controls. At least four valid trials using human skin from four different donors were tested. The barrier integrity of both normal and barrier-compromised skin samples was evaluated by applying an infinite dose of 3H2O topically and evaluating the tritiated water passage (% of applied dose of 3H2O). The 1, 5, and 10% SLS dilution series induced 8.0, 21.2, and 29.3-fold increases in 3H2O passage relative to the normal undamaged control tissues (0.179% of applied dose), while the 10, 20, or 30 serial tape strips induced 3.8, 6.6, and 15.6-fold increases in 3H2O passage. By comparing available in vivo measures of compromised barrier function in disease-state skin to these in vitro results, one can select the relevant in vitro method for preparing a barrier-compromised human skin model for percutaneous penetration studies.
Authors: Nash, Jennifer R.; Wilt, Nathan; Kong, Amanda; Raabe, Hans; Costin, Gertrude-Emilia
Long Term Corneal Culture has been proposed as an in vitro alternative to evaluate potential eye irritation and to measure chemical toxicity and post-exposure recovery up to 22 days. Excised porcine corneas mounted on 1% agar-gelatin mixture were cultured in two types of culture media for up to 21 days. Previous culture procedures resulted in significant corneal swelling, particularly at the endothelial layer, which was reduced when corneas were cultured in base medium Dulbecco Modified Eagle Media (DMEM) containing 4mM L-Glutamine and 10% Fetal Bovine Serum (FBS). Here we report a media refinement based on a concentration of 5% Dextran used as a deswelling agent evaluated to limit corneal swelling during long term culture. Corneas were cultured either in the base medium only (Group A) or in the base medium containing 5% Dextran (Group B) for the entire culture period. Corneas were collected upon arrival (Day 0), Day 1, Day 8, Day 15, and Day 22, were fixed in 10% neutral buffered formalin and hematoxylin & eosin sections were examined for histology analysis. Digital photography was used to present relative cloudiness of the corneas at the specified time points and corneal stromal thickness measurements were taken across the entire cornea and compared among the two groups. The thickness of the corneas increased with the time in culture, regardless of the type of media used (with or without Dextran). However, group A corneas (without Dextran) were significantly thicker than Group B corneas (with Dextran) at all time points. Our data show the ability to successfully culture corneas for 22 days with reduced swelling in DMEM media with 5% Dextran.
The Institute's Fall/Winter 2009 Newsletter contains information on: * Mucosal Irritation * Upcoming Events * Draize Replacement * Considering Alternatives Meeting * ECHA Clarification * 5th IWGT Workshop * ZEBET Anniversary * SAP and contributors, and * Eye Irriation Update!
Authors: Erin H. Hill; Hans A. Raabe; and Rodger D. Curren
The validation of in vitro methods is a lengthy process encompassing multiple phases. It progresses from initial test development, through test optimization and prevalidation, to a formal validation assessment, and eventually to regulatory acceptance. Each of these phases relies heavily on the outcome of laboratory activities - even the regulatory acceptance step involves careful inspection of the data to determine their applicability to the regulatory need under consideration. The competence and experience of laboratories participating in each phase have a significant effect on the efficiency of the entire process. History has shown that the process is never as fast as we would like; however, it can be even slower if technical errors are made along the way. High-quality laboratory work is required to maximize the opportunity for success at each stage. This emphasizes the need for a group of experienced, competent laboratories (reference laboratories) capable of readily participating in any of the phases. Such laboratories should be able to conduct assays under GLP-compliant conditions, and should optimally be independent from the developers. Reference laboratories experienced in each of the phases are particularly valuable to the process since they will be able to help test developers at an early stage to design robust protocols that can withstand the rigors of validation and subsequent routine usage. They will also be able to support the successful implementation of assays to naive laboratories post-validation, and assist the regulatory agencies in training reviewers to correctly interpret data from newly approved in vitro assays.
Authors: Hans A. Raabe, Angela M. Sizemore, Erica L. Dahl and Daniel M. Bagley
The EST has been formally validated by the European Centre for the Validation of Alternative Methods (ECVAM) as an acceptable in vitro embryotoxicity assay. During the prevalidation trials, the technology was easily transferred between laboratories in the European Union. However, transferring the assay to the Institute for In Vitro Sciences (IIVS) was problematic. Though we were able to provide good quality data, we experienced significant difficulty consistently running assays that passed our quality control criteria, which specify that at least 87.5% of the negative control cultures in the differentiation assay must contain well differentiated, beating myocytes. We began a research program to examine the parameters and technical factors which may have impacted our ability to consistently run this assay.
Authors: Cater, K; Cerven, D; Curren, R; Donovan, A; Wilt, N; Raabe, H
The Bovine Corneal Opacity and Permeability assay (BCOP), an internationally recognized alternative to the Draize eye irritation test, uses excised bovine corneas to predict ocular irritation. Originally developed by Gautheron (1992) and utilizing the irritation class prediction established by Sina (1994), BCOP has been used independently at MB Research and at the Institute for in Vitro Sciences (IIVS) for over fifteen years for product development, worker safety, and safety claims substantiation. The assay is currently under regulatory review by EPA, ECVAM and ICCVAM, and has recently been endorsed for prediction and labeling of severe/corrosive eye irritants. Since MB Research and IIVS have extensive experience performing the BCOP assay utilizing a variety of specific protocols to discriminate among mild and moderate, as well as severe/corrosive eye irritants, they agreed to develop and evaluate the reproducibility of a standard harmonized protocol for regulatory labeling. Nine blind-coded chemicals, primarily comprised of surfactant dilutions, as well as imidazole and pyridine, were tested in three independent GLP-compliant trials using exactly the same protocol. Intra-laboratory and inter-laboratory reproducibility evaluations showed that both laboratories typically obtained the same irritation class predictions. The resulting In Vitro Scores were compared to Draize MMAS results (ECETOC, 1998). Some of the surfactant dilutions (sodium dodecyl sulfate, cetyl pyridinium bromide) were found to be under-predicted using the standard BCOP protocol for liquid test chemicals.
Authors: Raabe, Hans; Burdick, Joel; Hanlon, Elizabeth; Hilberer, Allison; Hyder, Matthew; Inglis, Heather; Kong, Amanda; Majewski, Sh
In vitro eye and skin model assays are typically used to assure safety prior to consumer use by employing them in product development to support the creation of products with minimal irritation potential. As part of a high quality program, the test systems are constantly monitored for applicability, investigated for potential limitations, and continuously improved to ensure accurate and reliable data and information to support safety assessments. The applied research presented here evaluates an additional endpoint (ATP assay) of consideration in special cases where potential confounders may skew interpretation of results in core standard model systems (MTT assay). Viability assessments in 3-D in vitro eye and skin constructs have historically been assessed using the MTT assay. The MTT conversion assay measures the NAD(P)H-dependent microsomal enzyme and succinate dehydrogenase reduction of MTT to a blue formazan precipitate in viable cells (Berridge, 1996). Two factors can affect the accuracy of the MTT assay. First, since the MTT assay measured the mean metabolic rate of a cell population, subtoxic exposures may induce hormesis, where increased metabolism in response to cell damage incorrectly suggests high viability. Second, chemicals that directly reduct MTT (e.g., α-tocopherol) may overestimate tissue viability if these chemicals persist in the tissue model after rinsing. Freeze-killed tissue controls (KC) are tested in parallel to the viable tissues to determine the extent, if any, of the direct reduction by the test chemical. The ATP endpoint is an alternative to MTT reduction which would not be affected by hormesis since the endpoint measures cellular ATP content, rather than metabolic rate. The ATP endpoint may also be more appropriate for chemicals that are strong reducers of MTT, including many that are commonly used in personal care products. The ViaLight® Plus ATP assay kit utilizes the bioluminescent measurement of ATP (Crouch, et al., 1993). Since cytotoxicity is expressed as a reduction in the bioluminescent measurement of ATP, the assay provides a direct measure of the number of viable cells present. Upon cell stress or cell death, the amount of ATP is rapidly depleted or hydrolyzed.
Current mammalian cell in vitro genotoxicity assays induce a high level of false positive results leading to a large number of costly and time consuming followup in vivo genotoxicity studies. As of March 2009, the 7th Amendment to the EU Cosmetics Directive prohibits the use of in vivo genotoxicity tests in safety assessments for cosmetics, greatly impacting the assessment of genotoxicity of new ingredients. To address this, the European Cosmetic Toiletry and Perfumery Association (COLIPA) initiated an international project to establish and evaluate more predictive in vitro genotoxicity assays using 3D human tissues. One focus has been on the 3D human skin micronucleus assay (RSMN) in EpiDermTM. Since skin is the first site of contact with maximum exposure to many different products including cosmetics, the RSMN assay offers the potential for a more realistic application/metabolism of test compounds for evaluating genotoxicity (1,2,3). The COLIPA RSMN project is a multi-lab initiative involving Procter & Gamble (US), LOreal (France), Henkel (Germany), and the Institute for In Vitro Sciences (IIVS, US). Intra-laboratory and inter-laboratory reproducibility have been investigated with model genotoxins mitomycin C and vinblastine sulfate as well as a variety of chemicals that require metabolic activation. In addition studies with coded chemicals are in progress. This model is a promising new in vitro method for detecting micronuclei induction in human skin. This work is funded by the European Cosmetic Industry Association COLIPA.
Authors: Nash, Jennifer R.; Curren, Rodger; Hanlon, Elizabeth; Hilberer, Allison; Hyder, Matthew; Mun, Greg; Wilt, Nathan; Raab
The bovine corneal opacity and permeability (BCOP) assay Gautheron, 1992 & Sina, 1995), is used as an in vitro eye irritation screen for industrial hygiene, product development, and safety testing by measuring changes in corneal opacity, and permeability to fluorescein after chemical exposure. Histopathology has been used in BCOP studies to detect potential corneal injury, where the mode of chemical action might not induce opacity and permeability changes (Curren and Evans, 2000). Artifactual changes in the cornea associated with the collection, transportation, or BCOP methodology of the enucleated eyes have not been evaluated; therefore, corneas were excised and fixed in 10% buffered formalin at various steps in the assay process, paraffin embedded, H&E stained and evaluated using light microscopy. Stromal thickness and the thickness of the Descemet Membrane (DM) were measured along the entire length of the cornea. The epithelium, endothelium, and stroma were similar histologically among all groups. The normalized stromal thickness of the whole globe corneas (903.8 ?m ± 122.9 ?m), excised corneas immediately after enucleation (876.7 ?m ± 84.2 ?m), after the refrigerated transport (829.8 ?m ± 63.4 ?m), and at the end of the BCOP assay (721.2 ?m ± 17.2 ?m) suggest corneas undergo minimal artifactual changes as a result of refrigerated transport and the BCOP assay procedures.
Authors: Vavilikolanu, P; Lazaro, C; Davis, D; Wilt, N; Kong, A; Hanlon, E; Inglis, H; Mun, G; Costin, E; Raabe, H; and Curren, R
The Alberto-Culver Company uses a topical application time-to-toxicity protocol in in vitro 3-D human ocular and epidermal tissue constructs to assess the irritancy of a wide range of product formulations and ingredients. The irritation of some products with VOCs (e.g. hair sprays, mousses, etc.) has been over-predicted by these methods relative to clinical results. Since there is evidence that small chain organic solvents may induce excessive toxicity, we compared the irritation potential of a series of hair sprays with varying VOC concentrations using the standard 100 ?L “infinite” dose and a modified 30 ?L dose (the minimum volume assuring full coverage of the tissue surface). The rationale was that a lesser dose volume would result in similar or lesser irritation prediction, and more accurately model typical incidental exposure of eye and skin to alcohol-containing hair care products. Eight hair sprays containing 13%-93% VOC along with 3 reference materials (ethanol at 80%, 55%, and 6%), were evaluated. In the EpiOcular™ assay, these materials resulted in ET50 values ranging from <1 minute to 60 minutes when dosed with 100 ?L, and 1.1 minutes to 3.3 hours when dosed with 30 ?L; in the EpiDerm™ assay, these materials resulted in ET50 values between 2 to 20 hours when dosed with 100 ?L, and >24 hours when dosed with 30 ?L, indicating a significant dependence of cytotoxicity on dose volume. The effectiveness of the system has been assessed by comparing the in vitro results with clinical data and consumer experience information.
This newsletter contains information about: * Alternatives Highlights - 1st Half 2009 * FRAME Celebrates 40 Years and More * OECD Draft Guideline for Skin Irritation * Meeting report - Forinvitox: from innovation to market success * and more!
Authors: H. Raabe
The irritation potential of formulations and ingredients for industrial screening and product development is often conducted using in vitro 3-D human ocular and epidermal tissue constructs. To predict irritation potential after chemical exposure, tissue viability is typically determined by the ability of live cells to reduce MTT. Toxic exposures result in decreases in relative MTT reduction. However, two issues may contribute to inaccurate viability assessment: subtoxic exposures that induce higher metabolic rates typically greater than controls (i.e., hormesis) and chemicals that directly reduce MTT causing an overestimation of tissue viability (e.g., NaOH, α-tocopherol (α-t), ascorbic acid). For such chemicals, residues left on the tissues may increase the total MTT signal, so freeze-killed tissues are used to estimate chemical-mediated reduction of MTT. However, alternative methods of measuring tissue viability, such as amount of adenosine triphosphate (ATP) may be used. We compared these two methods by testing a series of model mild skin care formulations in 3-D human eye and skin constructs. The formulations were spiked with various concentrations of Triton® to induce a range of toxic effects, and were prepared with and without α-t, a MTT reducer. For formulations with α-t, freeze-killed tissues were tested in parallel in both the MTT and ATP assays. The results showed the same irritancy predictions for the 4 formulations containing α-t as for the 4 control formulations without α-t (e.g., formula with highest Triton conc.: ET50 eye = 172 and 157 min, ET50 skin = 778 and 772 min, with and w/o α-t). The ATP assay provided the same rank order of irritancy as did the MTT assay although the relative viability values from the ATP assay at each exposure were overall lower (e.g., formula with highest Triton conc.: ET50 eye = 14.5 and 12.9 min, ET50 skin = 202 and 231 min, with and w/o α-t). In summary, the MTT assay of formulas capable of MTT reduction should include freeze-killed tissues, and the ATP assay can confirm the relative rank order of the irritancy predictions.
Authors: J. Nash
The bovine corneal opacity and permeability (BCOP) assay, originally developed by Gautheron (1992) and Sina (1995) has been used as an in vitro eye irritation screen for industrial hygiene, product development and safety testing. It has recently been validated as a screen for corrosive or severe irritation by ICCVAM and ECVAM. The assay measures changes in corneal opacity, and increases in permeability to fluorescein after chemical exposure. Since Curren and Evans (2000) proposed the use of histopathology to detect potential corneal injury, where the mode of chemical action might not induce opacity and permeability changes, histopathology has been used in BCOP studies for nearly a decade. Although the state of the negative control corneas at the end of the BCOP assay has been characterized histologically, no studies have been conducted to determine if there are artifactual changes in the cornea associated with the collection and storage of the enucleated eyes, or the BCOP methodology. Corneas were excised and fixed at various steps in the assay process, at the time of collection of freshly enucleated eyes, after refrigerated transport, and at the end of the BCOP assay. Corneas were fixed in 10% buffered formalin, embedded in paraffin, H&E stained and the corneas evaluated using light microscopy. Stromal thickness was measured primarily at the central cornea. No remarkable artifacts were observed in the corneal epithelium and endothelium as a result of the various conditions, and the corneal stroma appeared very similar histologically in all cases. The thickness of the corneas collected immediately after enucleation were approx. 600 to 650 µm; corneas collected after the refrigerated transport prior to the BCOP assay were approx. 675 to 775 µm, and typical negative control corneas at the end of the BCOP assay range from 680 to 800 µm. These results show that the corneas undergo minimal artifactual changes as a result of refrigerated transport and the BCOP assay procedures.
Topics covered in this Institute Update include: the importance of conducting work according to GLPs, the profile of a new IIVS contributor, information on the upcoming SOT meeting and Practical Methods Workshop, and a look at the potentially promising year ahead.
View the pdf to read about recent skin irritation events, highlights from the 2008 In Vitro Alternatives Forum meeting (Spotlight on Ingredients) and the EPAA annual meeting, current progress for eye irritation models, and more.
The summer edition of IIVS Update has been mailed. Take a look at the pdf file to see our improved layout and information on the October Spotlight on Ingredients Forum, the June BCOP Histopathology Workshop, the annual Practical Methods for In Vitro Toxicology Workshop, and much more!
The IIVS Newsletter contains information on our current outreach programs, technical notes on the phototoxicity assay and information on future Institute activities. The Institute Update is published 3 times per year.
Source: Presented at the 47th Annual Society of Toxicology Meeting
Authors: Vavilikolanu, P.; Lazaro, C.; Mun, G.; Hilberer, A.; Hyder, M.; Raabe, H.; and Curren, R.
Assuring the safety of cosmetics and personal care products without testing in animals is a primary goal for Alberto-Culver Company. In addition, the Seventh Amendment to the Cosmetics Directive requires that after 2009, animal testing cannot be used to assess the eye or skin irritation potential of either cosmetic formulations or ingredients. To address these issues, we have developed an in vitro irritation assessment program to support the ocular safety evaluation of multiple surfactant systems used in shampoos and facial cleansers. This is particularly important as eye irritation is a foreseeable occurrence in the use of these cosmetics and personal care products. The program relies on the results of a topical application of formulations to the surface of a three-dimensional, human cell-derived model of the corneal epithelium (EpiOcular™, MatTek Corp., Ashland, MA, USA) and monitoring time to toxicity. 35 finished products and 15 prototype formulations with a range of multiple surfactant systems have been tested at dilutions of 2% and 10% (w/v in water). Two surfactant reference standards with well established safety profiles in commerce were tested along with these materials at same dilutions of 2% and 10%. The irritation potential of materials was then assessed by comparison to these benchmark materials. At these dilutions, we determined that the irritancy potential for most of the prototype shampoos fell in the mild to no irritation range shown as similar and less cytotoxic responses compared to the Reference materials. The effectiveness of this in vitro test system was evaluated by comparing the in vitro test results with consumer experience information.
Source: Presented at the 47th Annual Society of Toxicology Meeting
Authors: Lazaro, C.; Vavilikolanu, P.; Mun, G.; Hilberer, A.; Hyder, M.; Raabe, H.; and Curren, R.
Assuring the safety of cosmetics and personal care products without testing in animals has long been the goal of many international companies. This concern has become even more important with the requirement of the Seventh Amendment to the Cosmetics Directive that after 2009 animal testing cannot be used to assess the eye or skin irritation potential of either cosmetics formulations or ingredients. To address this problem, the Alberto-Culver Company has developed a program to support the ocular safety evaluation of certain hair conditioners. This program relies on the results of a topical application of formulations to the surface of a three-dimensional, human cell-derived model of the corneal epithelium (EpiOcularTM, MatTek Corp., Ashland, MA, USA) and monitoring time to toxicity (ET50; MTT activity reduced to 50% of the control condition). Twenty-eight different formulations primarily based on either single or dual quaternary ammonium compound (quats) systems utilizing various combinations of seven different quats have been evaluated in the model. Potential safety of the materials was assessed by comparison to a benchmark material having a well established safety profile in commerce. Twenty-seven of the materials, including the benchmark, had ET50 values of 24 hours or greater, indicating that they were quite mild. The effectiveness of the system has been assessed by comparing the in vitro results with consumer experience information.
Source: 6th World Congress on Alternatives and Animal Use in the Life Sciences, Tokyo, Japan 2007
Authors: Raabe, Hans A.; Hilberer, Allison; Kelly, Jeffrey; Moyer, Gregory O.; Powers, Mark; Curren, Rodger D.,
Ekwall et al. (ATLA 17:83-100, 1989) have proposed that ˜80% of chemical-induced systemic toxicity is the result of disruption of basic cellular processes common to most cell types in the body, and that systemic toxicity for many chemicals could be estimated in in vitro cultures. Strickland et al (The Toxicologist, Abs#761, 2003) reported on a joint European/USA validation to evaluate two cytotoxicity assays in NHEK and BALB/c 3T3 cells to predict acute systemic toxicity using a neutral red uptake (NRU) viability endpoint. We report on a two-lab validation study to evaluate the ATP viability endpoint using the optimized protocol from the aforementioned validation. Cytotoxicity was measured as a dose-dependent reduction in ATP from which an IC50 was determined. Initially, 20 chemicals from the Multicenter Evaluation of In Vitro Cytotoxicity (MEIC) program were tested to determine the relationship between the ATP IC50 values in vitro and the human LC50 values [log LC50 µM = 0.794(log IC50 µM)+0.176; r2 = 0.887]. Subsequently, 50 chemicals from Strickland et al were tested to determine the relationship between the ATP IC50 values and the rodent oral LD50 values [log LD50 mmol/kg = 0.495 (log IC50 mM)+0.413; r2 = 0.399] and is similar to the prediction model published in the Registry of Cytotoxicity (Halle, 1998). A high correlation (r2 = 0.930) was demonstrated between ATP IC50 values and NRU50 values obtained in the cytotoxicity validation study. The results demonstrate that the NHEK assay with the ATP endpoint may be used to predict systemic toxicity, or rodent oral LD50 doses, and that the ATP endpoint is an acceptable alternative to the NRU endpoint.
This quarters newsletter includes information on:
* ICCVAM 5 year plan,
* Moscow Seminar,
* Technical Notes,
* QA Initiatives,
* ECEAE Workshop,
* Alternatives Forum,
* SAP Member Highlight,
* Two New IIVS Contributors,
* What’s New at Our House
Take a look at our March 2007 newsletter: * 3-D Human Skin Models – The Next Generation of Tools for the In Vitro Toxicologist * Society of Toxicology Annual Meeting, * SAP Member Highlight - Dr. Marilyn Aardema, * SkinInVitro 2007 Meeting, * Development of Genotoxicity Assays in 3D Human Skin Models, * CELEBRATING 10 YEARS!, * POM Wonderful Supports IIVS’ Mission to Develop In Vitro Methods to Replace Animal Research.
Checkout our November newsletter: The Importance of Outreach, INVITOX 2006, Remembering William Russell, CAAT 25th Anniversary, Doris Day Animal League Merges with HSUS.
Source: Practical Training Workshop on In Vitro Eye Irritation Methods, August 26, 2005, Berlin, Germany
Authors: Rodger Curren
Practical Training Workshop on In Vitro Eye Irritation Methods Co-sponsored by: Institute for In Vitro Sciences, IIVS (Gaithersburg, USA) Beiersdorf AG (Hamburg, Germany) BfR-ZEBET (Berlin, Germany) SCOPE: The Workshop aims at practical training in most frequently used in vitro methods for prediction of eye irritation potential. Primary focus is a training in methods that have gained regulatory acceptance for specific purposes in Europe, namely the Bovine Cornea Opacity and Permeability Test (BCOP), the Chicken Enucleated Eye Test (CEET), the Isolated Rabbit Eye Test (IRE) and the Hen’s Egg Test on the Chorio-Allantois Membrane (HETCAM). In addition, as an example, the use of a reconstituted human corneal tissue model will be demonstrated. For logistical reasons, only the HET-CAM training will be a real-life, hands-on laboratory exercise, where participants perform the test, the outcome is beamed on a screen, and discussed with all participants. The other assays will be covered with technically orientated presentations given by experts routinely using these assays.
Source: Presented at the Society of Toxicology Annual Meeting, 2006
Authors: J.W. Harbell, G. Mun, and R.D. Curren
The BCOP assay was optimized to its present form by Drs. Pierre Gautheron and Joe Sina to address ocular irritation potential of pharmaceutical intermediates and is now widely applied across industries and chemical/formulation classes. Membrane lysis, protein coagulation, and saponification are common modes of action that lead to ocular irritation. In our experience, the opacity and permeability endpoints (generally combined into an “in vitro score”) have been able to identify the epithelial and stromal changes associated with these types of damage. However, chemicals that react with nucleic acids, mitochondrial proteins, or other cellular targets, that do not lead to immediate loss of cellular integrity or protein precipitation, have proven more difficult to identify without the addition of histological evaluation of the treated corneas. In this study, a series of reference chemicals (i.e., surfactants, solvents, acids, alkalis and oxidizers) were exposed to the corneas under standard assay conditions. The opacity and permeability to fluorescein were measured and the corneas fixed for histological evaluation. The test substances were chosen to induce corneal lesions consistent with the various modes of action. Histological evaluation was performed on the three layers of the cornea to provide a direct measure of the depth of injury. This evaluation was used to compare depth of injury with the opacity and permeability scores and to identify lesions that were not fully expressed in opacity or permeability changes. For materials inducing membrane lysis, coagulation, and saponification, the opacity and permeability scores paralleled the depth of injury and histology provided data on specific changes in the tissue. Furthermore, the action of reactive materials (i.e., peroxide) was detected with histology and its selective action on the stromal keratocytes demonstrated. Histological evaluation of the treated corneas can be useful in directly evaluating lesions and enhancing the interpretation of the opacity and permeability endpoints.
Source: Presented at the Society of Toxicology Annual Meeting, 2006
Authors: K. Cater, C. Reyes, and J. Harbell
An exploratory in vitro eye irritation study of marketed solid and liquid air fresheners was conducted to investigate the impact of fragrance/fragrance type on overall eye irritation for specific product forms. The Bovine Corneal Opacity and Permeability (BCOP) assay was selected to evaluate eye irritation potential due to its robustness and ability to test both solids and liquids by direct corneal application. Fragrance concentration, formula ingredients and product delivery system influence degree of impact on eye irritation potential. Six different air fresheners containing a representative “watery-type” fragrance were initially compared to respective un-fragranced product bases. The BCOP assay-testing scheme was optimized following several trials using neat solid and liquid air fresheners at 3- and 10-minute exposures. The greatest changes were noted in the in vitro scores, after 10-minute exposures. In vitro scores ranged from 0.0 to 97.1, reflecting a wide range of epithelial and stromal damage. The “watery-type” fragrance had greatest impact on eye irritation potential of gel electrics, non-aerosol sprays and scented oils compared to un-fragranced bases. These products were selected for additional investigations on impact of fragrance type on eye irritation potential. Citrus, floral and spice-type fragrances were evaluated for each product form. In vitro scores ranged from 5.7 to 110.4. Different fragrance types appeared to have observable impacts on eye irritation potential for specific product forms. Floral, citrus and spice-type fragrances had greatest impact on gel electric, non-aerosol spray, and scented oil product forms, respectively. Histological evaluation of corneas treated with selected solid and liquid air freshener products further supports the correlation of tissue damage (e.g., epithelial effects) with in vitro scores. Based on the BCOP assay and histology results, the recommended testing protocol for a solid or liquid air freshener product is to test neat product for a 10-minute exposure and use the in vitro score as the endpoint for evaluation of eye irritation potential.
Source: Presented at the Society of Toxicology Annual Meeting, 2006
Authors: Curren, Rodger D.; Aardema, Marilyn J.; Hayden, Patrick J.; Mun, Greg; Hu, Ting; Wilt, Nathan; Gibson, Dave
For many chemicals and products, most notably cosmetics, skin is the highest exposed tissue; however, very few assays are available to directly address potential genotoxicity to this tissue. Although rodent models are being developed to measure micronucleus induction in the skin, European legislation such as the 7th amendment to the Cosmetics Directive precludes the use of in vivo assays for genotoxicity assessments of cosmetic ingredients after 2009. To provide a useful alternative, we are developing an in vitro human skin micronucleus assay using the 3-D EpiDerm™ skin model (MatTek Corp, Ashland, MA). Theoretically such a model could approximate the complexities typical of in vivo exposures, e.g. absorption, tissue specificity, metabolism, etc., and at the same time reflect human-specific responses in these parameters.
Source: Presented at the Society of Toxicology Annual Meeting, 2006
Authors: E. Choi; C. Danilo; I. Rybina; R. Samia; H. Raabe; G. Moyer; J. Harbell
Ekwall et al (ATLA 17:83-100, 1989) have proposed that ˜80% of chemical-induced systemic toxicity is the result of disruption of basic cellular processes common to most cell types in the body, and that systemic toxicity for many chemicals could be estimated in in vitro cultures. The Multicenter Evaluation of In Vitro Cytotoxicity (MEIC) was established to test this hypothesis initially on 50 chemicals for which human systemic toxicity data were available. Subsequent studies have also shown relationships between cytotoxicity in vitro and systemic toxicity in vivo. Strickland et al (The Toxicologist, Abs#761, 2003) reported on a joint European/USA validation study initiated to evaluate two cytotoxicity assays using normal human epidermal keratinocytes (NHEK) and BALB/c 3T3 with a neutral red uptake (NRU) viability endpoint to predict acute systemic toxicity. Recently, we have conducted a two-lab proof of concept study to evaluate the ATP endpoint to assess viability of NHEK using the same cell culture and test chemical exposure protocol optimized during the aforementioned validation. The ATP assay is more rapid and utilizes fewer steps than the NRU endpoint. Since the amount of ATP in each metabolically-functional cell is uniform, the ATP assay provides a direct measure of the number of viable cells. Cytotoxicity was measured as a dose-dependent reduction in ATP from which an IC50 was determined. This provided a sensitive measure of both growth inhibition and cell loss. Twenty of the 50 chemicals from the MEIC program were coded and tested in 3 valid assays. Sodium lauryl sulfate was used as the positive control. The relationship between the IC50 values in vitro and human 50% lethal serum concentrations (LC50) reported by the MEIC program was determined. The resulting relationship was log LC50 µM = 0.774(log IC50 µM) + 0.353 [r2 = 0.887]. These data suggest that the NHEK assay with the ATP endpoint shows promise in the early evaluation of potential systemic toxicity.
Source: Presented at the Society of Toxicology Annual Meeting, 2006
Authors: A.K. Ulrey, R.D. Curren, J.W. Harbell
Pre-clinical assays, including in vitro assays, rely heavily on suppliers who provide specific products or services essential to the proper conduct of the study. The overall credibility of the assay and the results obtained from the assay are highly dependent on the quality of the supplies used. Variable results for the same control material over time could indicate that there is high lot to lot variability for a critical component of the assay. Monitoring positive and negative control results is one useful retrospective technique to help identify supplier quality. Instituting a supplier qualification program provides a prospective way to document that suppliers of critical products (such as the test system, critical media, or whole test kits) consistently adhere to the high standards necessary to support work performed in compliance with the Good Laboratory Practice (GLP) guidelines. While some suppliers provide products manufactured utilizing Good Manufacturing Practices (GMP) and International Organization for Standardization (ISO 9001) standards, many suppliers of in vitro test systems and test kits are smaller, more specialized companies that may have their origins in academic research.
Source: Presented at the 5th World Congress on Alternatives and Animal Use in the Life Sciences, Berlin, Germany, August 21-26, 2005
Authors: ME Blazka, M Diaco, JW Harbell, H Raabe, A Sizemore, N Wilt, and DM Bagley
The ability of the EpiOcular™ construct to predict the eye irritation potential of surfactants has been the subject of a formal validation program. EpiOcular™ correlates a test article’s potential for ocular irritation with the time it takes to reduce tissue viability by 50% (ET(sub>50) as measured by MTT. This study investigated whether the histological changes following exposure to the surfactants are in agreement with the MTT results. Eight surfactants were selected and applied to the EpiOcular™ tissue using exposure times that bracketed the ET50 values established in the validation study. Upon completion of the exposure half of the tissues (2/timepoint) were set aside for histological examination while the viability of the remaining tissues was assessed. For all surfactants, the results showed a good relationship between the degree of histological damage with changes in tissue viability. An increase in the depth and severity of tissue damage was associated with a decrease in tissue viability. Histological changes ranged from subtle cellular changes such as vacuolization and punctate chromatin condensation to overt tissue loss and cell necrosis. Loss of or damage to the surface squamous epithelium was associated with <20% decrease in viability, while the degree of damage to the central squamous epithelium was directly related to a 20-80% decrease in viability. In conclusion, the nature and severity of the histological changes were in agreement with the MTT results. Understanding the progression and types of cellular changes associated with tissue damage may be able to help distinguish the degrees of ocular irritation.
Have you wondered how IIVS provides reliable, reproducible in vitro testing services? Or how IIVS fosters scientific optimization, validation and implementation of alternatives to animal testing or who sponsors these activities? Learn about our Test System Monitoring and Good Laboratory Practices programs, some of our scientific outreach program activities and our sponsors in the March 2006 Newsletter.
Source: IIVS
Authors: Rodger Curren
The February issue of The Scientist carried an Opinion piece critical of the 3Rs. The response from IIVS in terms of a letter to the editor, and the original article, are available below.
IIVS progress on optimization of Alternative Tests, ICCVAM Expert Panel Reviews BRD Addenda, Standards for In Vitro Tests, and more.
Source: Practical Training Workshop on In Vitro Eye Irritation Methods, August 26, 2005, Berlin, Germany
Authors: John Harbell
Practical Training Workshop on In Vitro Eye Irritation Methods Co-sponsored by: Institute for In Vitro Sciences, IIVS (Gaithersburg, USA), Beiersdorf AG (Hamburg, Germany), BfR-ZEBET (Berlin, Germany). SCOPE: The Workshop aims at practical training in most frequently used in vitro methods for prediction of eye irritation potential. Primary focus is a training in methods that have gained regulatory acceptance for specific purposes in Europe, namely the Bovine Cornea Opacity and Permeability Test (BCOP), the Chicken Enucleated Eye Test (CEET), the Isolated Rabbit Eye Test (IRE) and the Hen’s Egg Test on the Chorio-Allantois Membrane (HETCAM). In addition, as an example, the use of a reconstituted human corneal tissue model will be demonstrated. For logistical reasons, only the HET-CAM training will be a real-life, hands-on laboratory exercise, where participants perform the test, the outcome is beamed on a screen, and discussed with all participants. The other assays will be covered with technically orientated presentations given by experts routinely using these assays.
Source: Presented at the 5th World Congress on Alternatives and Animal Use in the Life Sciences, Berlin, Germany, August 21-26, 2005
Authors: Raabe H, L Bruner, T Snyder, N Wilt, and J Harbell
The long-term culture of corneas has been proposed as an in vitro model to evaluate potential eye irritation and post-treatment recovery following chemical exposures. Porcine eyes were btained within 24 hours of sacrifice and disinfected prior to excising corneas. Corneas were filled with an agar/gelatin gel in M199 medium to support the corneas, and were cultured at 37ºC, 5% CO2, 90% RH in M199 medium to the limbus. The corneas were moistened by brief immersion in medium every 2.7 hours using a modified plate rocker. Corneas were treated with either SLS, EtOH, or H2O (controls). The corneas were rinsed with PBS, cultured for a pre-determined post exposure time, and fixed in buffered formalin. H&E-stained control corneas showed normal morphology after 4 days, similar to excised/immediately fixed corneas. Controls were characterized by an intact epithelium with viable squamous, wing, and basal cells. The stroma showed minimal swelling with frequent viable keratocytes. The endothelium was typically intact. Some stromal swelling near the sclera was noted after 5 to 7 days. Corneas treated with 3% SLS or EtOH showed complete epithelial cell damage or loss 24 hours after treatment, as well as loss of viable keratocytes in the upper stroma. After 48 hours, epithelial cell sheet migration was observed into the damaged zone. After 120 hours, the regeneration of a stratified epithelium was observed. These results confirm the ability to culture porcine corneas for at least 120 hours, as well as demonstrate the potential for further optimization of evaluating recovery of damaged corneas.
Source: Presented at the 5th World Congress on Alternatives & Animal Use in the Life Sciences, Berlin Germany, Aug. 21-26, 2005
Authors: Marilyn Aardema, David Gibson, Ting Hu, Greg Mun, Rodger Curren, Patrick Hayden
To meet the requirements of the EU 7th Amendment to the Cosmetics Directive, manufacturers of cosmetics products will need to ascertain the safety of ingredients using non-animal methods. Starting in 2009, in vivo genotoxicity tests for cosmetic ingredients will not be allowed. Skin is one of the target areas of interest for many cosmetic products because it is generally the tissue with the highest exposure. Therefore we have begun development of a micronucleus assay using a commercially available 3-D engineered human skin model, EpiDerm™ (MatTek Corp, Ashland, MA). We first evaluated whether a population of binucleated cells sufficient for a micronucleus assay could be obtained by exposing the tissue to 1-3 ug/ml cytochalasin B (Cyt B). The frequency of binucleated cells increased both with time and with increasing concentration of Cyt B. Cyt B at 3 ug/ml allowed us to reliably obtain 40–50% binucleated cells at 48 h and was used in future studies. The background frequency of micronuclei in this model is low (~0.1%) and reproducible. Studies with model genotoxins including mitomycin C, vinblastine sulfate and methylmethane sulfonate demonstrated that micronuclei can be reproducibly induced in this 3-D skin model. This is the first step in developing a routine “in vivo-like” assay for chromosomal damage in human tissue.
Source: Presented at the 5th World Congress on Alternatives and Animal Use in the Life Sciences, Berlin, Germany, August 21-26, 2005
Authors: Amanda K Ulrey, Rodger D Curren, John W Harbell, Greg Mun, and Hans A Raabe
The steady increase in industry use and regulatory acceptance of in vitro test methods has resulted in an increased need to apply Good Laboratory Practice (GLP) regulations to these systems. The original GLP regulations, developed to address the conduct of animal studies, are concerned with many special conditions that apply to animal housing and care, and the relatively long duration of animal studies, that are not present in the shorter in vitro studies. In animal studies, for example, emphasis is placed on the isolation of species and periodic analysis of feed and water; whereas in non-animal studies, there is increased importance on the justification of the test system. Recently, the Organization for Economic Cooperation and Development (OECD) has published advisories (No. 7, The Application of the GLP Principles to Short-term Studies, 1999; No. 14 The Application of the Principles of GLP to in vitro Studies, 2004) to clarify the application of the GLP principles to both short-term and in vitro studies. This poster outlines the approach applied at the Institute for In Vitro Sciences, Inc. (IIVS) to the conduct of in vitro GLP-compliant studies. We describe the translation of the OECD guidance documents into a framework for conducting assays which use ex vivo tissues, monolayer cell cultures, reconstructed skin constructs, and manufactured test kits. We are grateful to auditors from numerous study sponsors and regulatory agencies who have helped us develop what we feel is a best practices approach.
Source: Presented at the 5th World Congress on Alternatives and Animal Use in Life Sciences, Berlin, Germany, August 21-25.
Authors: Hans Raabe, Rodger Curren, Sherry Ward, John Harbell
The Institute for In Vitro Sciences (IIVS), Gaithersburg, Maryland, USA, hosted a workshop on in vitro percutaneous absorption (PA) methods for a small group of international stakeholders in July 2005. The purpose of the workshop was to provide a forum where stakeholders and method experts could come together to discuss the various OECD-approved guidance on in vitro PA methods (OECD Test Guideline 428, April 2004; and Guidance Document for the Conduct of Skin Absorption Studies, March 2004) and how this guidance may be practically applied to the protocols in current use. The workshop participants compared and contrasted specific components of different in vitro protocols, and made recommendations on protocol components that are essential for obtaining useful toxicological data from the in vitro PA methods. A major goal of this workshop was to provide industry, contract research laboratories and the regulatory community with practical information to facilitate successful, wider, and earlier use of in vitro PA data in regulatory submissions. Detailed conclusions and recommendations from the workshop will be presented.
Source: Presented at the 5th World Congress on Alternatives and Animal Use in the Life Sciences, Berlin, Germany, August 21-26, 2005
Authors: J.W. Harbell, H.A. Raabe, A. Ulrey, K.J. Trouba, G. Mun and R.D. Curren
In vitro test systems offer the potential for high consistency and resulting high predictive capacity. This promise comes from the ability to control many of the independent variables that can lead to the variability of responses in vivo. Furthermore, most of the in vitro tests currently employed, have undergone some level of formal validation or at least multi-center evaluation to establish formal or informal standards of performance. Those standards generally include methods for test system qualification, data acceptance criteria based on positive and negative control responses, and routine use of benchmark materials. These efforts have allowed many companies to use certain assays as full replacements for in vivo studies in their product development/safety programs. A goal of many developers and users of in vitro assays has been the formal acceptance of these tests by the regulatory community. The regulatory community needs to be able to evaluate data, from a wide range of laboratories, against common standards so that reviewers can make equitable decisions. In some cases, the regulatory agencies have established performance standards that are designed to link the validated test methods with the specific assay used for regulatory submission. This presentation will focus on the tools that allow this to be accomplished: test system qualification, selection and use of benchmark materials, and the importance of concurrent controls and associated assay acceptance criteria.
Authors: JE Swanson, N Cuellar, U Vedula and JW Harbell
SC Johnson is a global consumer products company that manufactures a variety of household products that must be evaluated for human health hazards. For over 10 years, SC Johnson has conducted the necessary research to spearhead efforts to reduce the use of animals in the hazard assessment process. We routinely use alternative approaches such as the eye and skin irritation assays in a weight-of-evidence approach for hazard classification and labeling purposes for a variety of products. Assay choice and protocol considerations are defined so as to address possible modes of action on the target tissues. Specific benchmark formulations have been employed with each study to facilitate interpretation of the results. The Bovine Corneal Opacity and Permeability Assay (BCOP) has been used to assess ingredient synergies and the impact of various formulation components on the irritancy potential of the end-use products. The overall safety evaluation approach will be illustrated using two case studies. Alternative assays, especially the BCOP, are indispensable tools for assessing the potential irritancy of our products distributed worldwide while reducing the use of animals.
IIVS news on
Source: IIVS announcement
Authors: IIVS
Join us at the 2005 Practical Methods in In Vitro Toxicology Workshop in June.
Source: Presented at the 44th Annual Meeting of the Society of Toxicology, New Orleans, Louisiana, March 6-10, 2005
Authors: R D Curren, G Mun, D P Gibson, and M J Aardema
The rodent in vivo micronucleus assay is an important part of a tiered testing strategy in genetic toxicology. However, this assay, in general, only provides information about materials available systemically, not at the point of contact, e.g. skin. Although in vivo rodent skin micronucleus assays are being developed, the results will still require extrapolation to the human. Furthermore, to fully comply with recent European legislation such as the 7th Amendment to the Cosmetics Directive, non-animal test methods will be needed to assess new chemicals and ingredients. Therefore we have begun development of a micronucleus assay using a commercially available 3-D engineered skin model of human origin, EpiDerm ™ (MatTek Corp, Ashland, MA). We first evaluated whether a population of binucleated cells sufficient for a micronucleus assay could be obtained by exposing the tissue to 1-3 ìg/ml cytochalasin B (Cyt B). The frequency of binucleated cells increased both with time (to at least 120 h) and with increasing concentration of Cyt B. Three ìg/ml Cyt B allowed us to reliably obtain 40-50% binucleated cells at 48h. Mitomycin C (MMC) was then used (in the presence of 3 ìg/ml Cyt B) to investigate toxicity and micronuclei formation in EpiDerm™. Exposing the tissue directly through the growth medium for 48h gave a dose response for toxicity between 0.03 and 0.6 ìg/ml. Maximum micronuclei induction (~5%) occurred at 0.1-0.3 ìg/ml MMC. Experiments conducted with and without Cyt B indicated higher frequencies in the presence of Cyt B as expected. A topical application protocol was then developed using two 10 &3956l (per 0.64 cm2 tissue) applications of MMC in ethanol 24 and 48h prior to harvest. Maximum micronucleus response (~8%) and toxicity occurred with applications of 6-60 ìg/ml MMC. The background frequency of micronuclei was very low (~0.1%). These studies show that micronuclei can be reproducibly induced in a 3-D skin model and are the first steps in developing a routine “in vivo-like” assay for chromosomal damage in human tissue.
Source: Presented at the 44th Annual Meeting of the Society of Toxicology, New Orleans, Louisiana, March 6-10, 2005
Authors: ME Blazka, M Diaco, JW Harbell, H Raabe, A Sizemore, N Wilt, and DM Bagley
The ability of the EpiOcular™ construct to predict the eye irritation potential of surfactants and surfactant-based formulations has been the subject of a formal validation program. EpiOcular™ correlates a test article’s potential for ocular irritation with the time it takes to reduce tissue viability by 50% (ET50) as measured by the tissue’s ability to reduce MTT. An algorithm is used to convert the ET50 value to a ‘predicted Draize’ score which can then be compared to in vivo data. This study investigated whether the histological changes following exposure are in agreement with the MTT results. Eight surfactants were selected from the validation study; 4 surfactants whose in vitro ocular irritation potential agreed with the in vivo data (cetyl alcohol; 3% sodium lauryl sulfate; 50% didecyldimonium chloride; sodium sulfolaurate mixture) and 4 whose in vitro data differed from the in vivo data (10% cetylpyridinium bromide; 3.2% benzethonium chloride; C10-12 alcohol ethoxylate; quaternium-18). Exposure times used in this study bracketed ET50 values established in the validation study. For all surfactants, the results showed a good relationship between the degree of histological damage with changes in tissue viability. An increase in the depth and severity of tissue damage was associated with a decrease in tissue viability. Histological changes ranged from subtle cellular changes such as vacuolization and punctate chromatin condensation to overt tissue loss and cell necrosis. Loss of or damage to the surface squamous epithelium was associated with <20% decrease in viability, while the degree of damage to the central squamous epithelium was directly related to a 20-80% decrease in viability. In conclusion, the nature and severity of the histological changes were in agreement with the MTT results. Understanding the progression and types of cellular changes associated with tissue damage may be able to help distinguish the degrees of ocular irritation.
Source: Presented at the 44th Annual Meeting of the Society of Toxicology, New Orleans, Louisiana, March 6-10, 2005
Authors: Raabe H, L Bruner, T Snyder, N Wilt, and J Harbell
The long-term culture of corneas has been proposed as an in vitro model to evaluate potential eye irritation and post-treatment recovery following chemical exposures. Techniques used were modifications of those of Foreman, DM (1996), Xu, KP (2004) and Boulton, M (2003). Porcine eyes were obtained from Sioux-Preme within 24 hours of sacrifice. The eyes were disinfected by immersion in 1% povidone iodine followed by 0.1% Gentamicin in PBS. Excised corneas were filled with an agar/gelatin gel in M199 medium to support the corneas, and were cultured at 37ºC, 5% CO2, 90% RH in M199 medium to the limbus, leaving the epithelium exposed to air. The corneas were moistened by brief immersion in medium every 2.7 h using a modified plate rocker. To induce damage, corneas were treated with either 1 or 3% SLS, or H2O (controls) for exposures of 2, 5 or 10 min. The corneas were rinsed with PBS, cultured for 6, 24 or 48 h, and then fixed in buffered formalin to determine their responses to damage and potential recovery. H&E-stained preparations of control corneas showed normal morphology throughout the 3-day assay, and were comparable to excised/immediately fixed corneas. Controls were characterized by an intact epithelium with viable squamous, wing, and basal cells. The stroma showed frequent viable keratocytes, and minimal swelling indicative of a functional endothelium. The endothelium was typically intact. Corneas treated with 1% SLS for 2 min showed no pathological changes, while exposure to 3% SLS for 2 min induced just slight hyper-eosinophilia in the squamous epithelium. However, corneas treated with 3% SLS for 5 or 10 min showed complete epithelial cell damage or loss 24 h after treatment, as well as loss of viable keratocytes in the upper stroma. By 48 h after treatment, evidence of epithelial cell sheet migration along the basal membrane into the damaged zone was observed. These results confirm the ability to culture porcine corneas for at least 72 hours, and demonstrate the potential for further optimization for the evaluation of recovery after induction of chemical damage.
Source: ATLA 23, 219-255
Authors: Phillip A. Botham, Mark Chamberlain, Martin D Barratt, Rodger D. Curren, David J. Esdaile, John Gardner et al.
The Report and Recommendation of ECVAM Workshop 6.
Source: Presented at the Environmental Mutagen Society Meeting, October 2-6, 2004, Pittsburgh, PA
Authors: R. Curren , G. Mun , D. Gibson , and M. Aardema
To meet the requirements of the EU 7th Amendment to the Cosmetics Directive, manufacturers of cosmetics products will need to ascertain the safety of ingredients using non-animal methods. Starting in 2009, in vivo genotoxicity tests for cosmetic ingredients will not be allowed. Skin - because of its generally high exposure - is a target area of interest for many cosmetic products. It would be beneficial to have a skin-based micronucleus assay that did not require live animals, and even more appropriate if it utilized human skin in vitro. We describe preliminary work on a human tissue-based micronucleus assay using EpiDerm
Authors: J. E. Swanson, W. M. Rees, D. S. Hilgers, J. C. Merrill and J. W. Harbell
Consumers continue to seek products that will both clean and combat mold and mildew stains on various surfaces in the home. Such products are commonly formulated as alkaline hypochlorite and surfactant-containing aqueous solutions and usually have significant ocular irritation potential. Previous studies have shown the applicability of the BCOP Assay to evaluate this class of cleaners (Rees et al., 2001). In this study, a standard cleaner containing 0.25% sodium hypochlorite, anionic surfactant and alkali (pH 13, 0.24% active chlorine content) was compared to a 2.5% stabilized hypochlorite cleaner containing sodium N-chlorosulfamate salts, the same anionic surfactant and citrate buffer (pH 5, 2.4% active chlorine content). A worst possible case human exposure time of 5 minutes was utilized in the assay. Treatment and scoring followed the standard BCOP protocol (Sina et al., 1995). However, longer post-exposure incubation times of 4 and 20 hours were found to be necessary for detecting the irritation potential of oxidizing/reactive chemistries in earlier work. Results show that the pH 5, 2.5% stabilized hypochlorite cleaner is much less irritating than the standard alkaline 0.25% sodium hypochlorite cleaner. The BCOP In Vitro Scores were 5.2 and 4.3 for the stabilized hypochlorite cleaner compared to 55.6 and 59.7 for the sodium hypochlorite cleaner with 4 and 20 hour exposure times, respectively. Histology confirmed these results. These data suggest that ocular irritation and tissue damage potential for active chlorine-containing cleaners may be markedly reduced when alternative chemical forms of hypochlorite are employed, relative to conventional sodium hypochlorite-based compositions.
Source: Presented at Society of Toxicology 2004
Authors: Pugh, George, Jr.; Moyer, Gregory O.; Raabe, Hans A.; Harbell, John W.; Bagley, Daniel M.
The reference material, caffeine (prepared in ethanol), was evaluated in 3 in vitro models to compare the rates of skin penetration in each of the models. The models were human donor skin, an engineered skin construct (MatTek Corporation, Model EPI-606X) and slaughterhouse-derived pig skin. The tissues were mounted in flow-through diffusion cells (PermeGear, Inc., 0.64 cm
Source: Presented at Society of Toxicology, 2004, Baltimore, MD
Authors: H. Raabe, G. Moyer, G. Mun, M. Clear, and J.W. Harbell
Multi-well plates provide an efficient format for cell-based bioassays. As the number of wells increases per plate, the surface area and number of cells per well decreases. Use of the small-well format can present some problems. We have observed cell cultures in 96-well plates where significant populations of cells begin to die shortly after refeeding the cell cultures (dumping the spent medium and adding fresh medium). The zones or areas of cell death appear to occur within a predictable ring around the edges of the multi-well plate wells, coinciding with the formation of a meniscus formed by the residual medium in the well following removal of the spent culture medium. This effect appears in larger well (e.g., 24-well plates) but has more impact in smaller well formats where the ratio of wall circumference to cell area is greater. The impact of these effects tend to be more pronounced when cultures are refed at relatively low confluence, resulting in areas devoid of cells. The zone is also more evident with cell types where cell migration is limited. Cells cultured in the presence or absence of serum show qualitatively similar results. When uniform, 30% confluent lawns of human keratinocytes in 96-well plates were refed, the cells within the zone rapidly lost the ability to take up neutral red and showed nuclear condensation. After 48 hours, the neutral red uptake (OD ) was significantly reduced in the 550 refed wells (0.830 ± 0.058, mean ± standard deviation, n=6 assays) compared to wells that were not refed (1.066 ± 0.034 where p < 0.0001). These results were confirmed in 3T3 cells cultured in serumcontaining medium. These observations show the impact of the medium change in the 96-well plate format, and suggest that protocols should be designed to minimize this cell loss.
Source: Presented at the Society of Toxicology Annual Meeting, Baltimore, MD, 2004
Authors: K. Cater, E. Patrick, J. Harbell, J. Merrill, and S. Schilcher
The BCOP assay can be used to predict relative eye irritation potential of surfactant-based personal care formulations relative to a corporate benchmark. (Toxicologist, March 2001). The Human Eye Sting test is typically used to evaluate product claims of no tears/no stinging for children’s bath products. A preliminary investigation was conducted to test a hypothesis that the BCOP assay could be used as a prediction model for relative ranking of human eye irritation responses under conditions of a standard Human Eye Sting test to surfactant-based formulations. BCOP assays and Human Eye Sting tests were conducted on four commercial and one prototype body wash (BW) developed specifically for children or as mild bath products. In the human eye sting assay 10 µl of a 10% dosing solution is instilled into one eye of each panelist (n=20), and the contra-lateral eye is dosed with sterile water as a control. Bulbar conjunctival erythema responses of each eye are graded at 30 seconds by an ophthalmologist. The BCOP assay permeability values (OD490) for the five BWs ranged from 0.438 1.252 (i.e., least to most irritating). By comparison, the number of panelists exhibiting erythema responses (mild to moderately pink) ranged from 3/20 panelists for the least irritating BW to 10/20 panelists for the most irritating BW tested. The relative ranking of eye irritation potential of the five BWs in the BCOP assay compares favorably with the relative ranking of the BWs in the Human Eye Sting test. Based on these findings, the BCOP assay as described for surfactant-based formulations shows promise as a prediction model for relative ranking of conjunctival erythema responses in the human eye. Consequently, screening of prototype formulations in the BCOP assay allows for formula optimization of mild bath products prior to investment in a Human Eye Sting test.
Source: Presented at the International Investigative Dermatology 2003
Authors: Raabe, Hans; Klausner, Mitchell; Pugh, George; Kubilus, Joe; Moyer, Gregory; Bagley, Daniel; Harbell, John
The rates of absorption of chemicals in human skin can be modeled in the in vitro percutaneous absorption assay. Moreover, the impact of various vehicles on the permeation rates of chemicals can also be evaluated. Benzoic acid (BA) was prepared in two vehicles (acetone and petrolatum) and was tested in duplicate trials in an engineered human skin construct (EPI-606X, MatTek Corp.) and in ex vivo human skin. The methods used are based upon guidance by the US Food and Drug Administration, with the exception that absorption was evaluated at 3 hours after test material application. Tissues were mounted in flow-through diffusion cells (0.64 cm2 area) and tested for barrier function by 3H2O passage, followed by a finite dose (9 µL) of 14C-labeled BA applied at 4 µg/cm2. Barrier function was tested by applying 200 µL of 3H2O onto each tissue for 20 minutes. Non-absorbed 3H2O was removed after 20 minutes, and the tissues were maintained in the diffusion cells for an additional 60 minutes. The amount of 3H2O absorbed into the receptor fluid was measured during the 80-minute test. Results of the barrier tests showed that 1.71±0.33% (n=16) and 0.36±0.13% (n=6) of the applied 3H2O dose permeated through the engineered and human donor skin, respectively. Results of the percutaneous absorption of BA showed that in both skin models, the permeation rates for BA prepared in petrolatum were significantly higher than for BA prepared in acetone. At 3 hours, permeation rates for BA prepared in petrolatum were 80.9% and 51.9% of applied dose, for engineered skin and human skin, respectively (1.6 fold higher in engineered skin vs. human skin). Permeation rates for BA prepared in acetone were 38.1% and 12.5% of applied dose, for engineered skin and human skin, respectively (3.0 fold higher in engineered skin vs. human skin). These results show that both the engineered and human skin can be used to evaluate the impact of vehicles on the permeation rates of test chemicals.
Authors: J.D. Burdick, Y. Gao, B. Kanengiser, J.C. Merrill, and J.W. Harbell
Evaluating ocular irritation of eye-area cosmetics is essential to their safety assessment. Although eye-area formulations are designed to be mild, subjective/objective ocular responses can limit theiracceptability. This study evaluated two similar formulations by comparing alternative assays, selected for their ability to distinguish milder effects, to human objective and subjective endpoints assessed by a sub-acute human eye irritation design. Ocular irritation alternative assays included Hen’s Egg Test -Chorioallantoic Membrane (HETCAM) assay, Cytosensor Microphysiometer Bioassay (CMB), and EpiOcular® (EPO) tissue construct assay. HETCAM evaluates inflammatory responses in a complete tissue model. CMB uses L929 cells to evaluate cytotoxicity through metabolic rate measurements. EPO uses differentiated human epidermal keratinocytes, having stratified into a squamous epithelium similar to corneal tissue. Human ocular irritation was assessed through subjective and objective measures at post-application intervals. Ocular exams included evaluation of the area/density of fluorescein staining of all tissues utilizing Kanengiser’s 13-point scale. Each formulation was assessed simultaneously by randomized, periorbital application to paired contralateral eye areas in an exaggerated use design. Clinical results indicated formulation A elicited a slightly greater magnitude and frequency of subjective and objective findings compared to formulation B. Results of alternative assays predicted both formulations were mild but were not entirely consistent in terms of rank ordering relative to the human responses. This study demonstrates the need to consider a battery of alternative assays when screening formulations for distinguishing mild ocular effects and the potential of this clinical protocol in predicting differences in eye irritation potential of mild formulations.
Source: Presented at the Annual Meeting of the Society of Toxicology, 2003
Authors: M E Blazka, J W Harbell, M Klausner, J Merrill, J Kubilus, C Kloos, D M Bagley
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 (Institute for In Vitro Sciences, Inc. and MatTek Corp.) 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; 1-zwitterionic) 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.
Source: Society of Toxicology Annual Meeting, 2003
Authors: R. Curen, G Moyer, N. Wilt, M. Clear, A. Sizemore and G. Mun
The NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) and the European Center for the Evaluation of Alternative Methods (ECVAM) are currently sponsoring a study to determine the usefulness of two in vitro basal cytotoxicity methods [employing BALB/c 3T3 (3T3) cells and normal human keratinocytes (NHK)] for predicting rodent and human acute toxicity. Although cytotoxicity protocols for these two cell types were available (and were used to qualify these cell types with the Registry of Cytotoxicity prediction model), it was thought prudent to reevaluate certain variables before starting a formal study. We examined acceptable solvent concentrations, the effect of exposure time on correct toxicity predictions, and the appropriate seeding density to match the desired exposure time. For solvent concentration, we concluded, based on cytotoxicity at higher doses, that 0.5% should be the highest concentration for both ethanol and DMSO for both cell types. To assess exposure time, we tested 6 chemicals whose cytotoxicity had been reported to increase significantly at exposures greater than 24 h [Riddell, et al. (1986) ATLA 14:86-92]. After testing the chemicals using 24, 48 and 72 h exposures, we concluded that a 48 h exposure was optimal for either 3T3 or NHEK cells. Finally, we found that the appropriate cell seeding densities that permitted just subconfluent growth 3 in the solvent-alone control wells after a 48 h exposure period were 2.5X10 cells/well 3 (96-well plates; 24 h prior to treatment) for 3T3 cells and 2.5X10 cells/well (96-well plates; 48-72 h prior to treatment) for NHEK cells. These new parameters have been incorporated into the protocols that are now being used in the ICCVAM/ECVAM sponsored multi-laboratory study to evaluate in vitro cytotoxicity assays.
Source: Presented at the Annual Meeting of the Society of Toxicology, 2003.
Authors: K. Cater, G. Min, G. Moyer, J. Merrill, and J. Harbell
An exploratory in vitro eye irritation study of 11 currently marketed alkaline dry laundry (ADL) detergents was conducted to investigate the correlation between an in vitro biological endpoint and pH/reserve alkalinity (RA) ranges of ADL detergents. Marketed products can be considered “safe benchmarks”, since they are produced by industry leaders and are assumed to have acceptable pH/RA characteristics. Based on performance in previous eye irritation studies with surfactants and the potential to measure depth of injury, the bovine corneal opacity and permeability (BCOP) assay was selected as an in vitro endpoint to evaluate biological effects. Based on preliminary studies, a 10% (w/v) aqueous suspension of each detergent was applied to the corneas for a 30-minute exposure. The degree of epithelial damage is reflected in the increase in fluorescein permeability value. Permeability values (OD490) for the 11 ADL detergents ranged from 0.267 to 0.856 reflecting a moderate range of epithelial damage. The range of opacity scores was more variable (0.3-21.5) and showed little consistent change with the permeability values. Anionic/nonionic surfactant formulations often produce little in vitro opacity. The pH of each dosing suspension (10% w/v) was measured and ranged from 11.0 to 12.0. The RA was determined for a 0.2% (v/v) aqueous suspension of the supernatant from each dosing suspension (i.e., 20% dilution of supernatant from 10% dosing suspension) titrated to a target pH of 9.5. Titration values ranged from 1.7 to 5.2 ml of 1N HCl. Neither pH nor RA values correlated with the epithelial damage as measured by permeability changes. These data suggest characteristics of the formulation other than pH or RA are responsible for the epithelial damage produced in the BCOP. This protocol, using a 10% (w/v) aqueous suspension with a 30-minute exposure, the permeability endpoint (with histological confirmation) and benchmark formulations, shows promise for evaluating ADL detergents.
Source: Human & Experimental Toxicology, 2002; 6: 313-323
Authors: L.H. Bruner, G.J. Carr, J.W. Harbell and R. D. Curren
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An area that requires further research is how best to measure test method performance in validation studies and how to set criteria that should be used to judge the adequacy of this performance. The studies reported here were designed to begin an investigation of these questions. Computer simulations were used to generate data sets similar to those that might be obtained from a large validation study. These data were then analysed using three procedures including determination of the 96%predidction interval (PI), calculation of Pearson’s correlation coefficient and calculation of the contingent probability statistics (CPS), sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV).The results of this work suggest that of the three approaches examined, quantitative measurements with calculation of the 95% PI provided the most information to allow discrimination between the performance of several different NTMs. The results also suggest that dividing data sets into positive and negative toxicity classifications followed by the calculation of CPS leads to considerable information loss. This loss of information may be so significant that it is not possible in certain circumstances to distinguish between NTMs that are adequate and those that are not.
Source: Human & Experimental Toxicology, 2002; 6: 325-335
Authors: L.H. Bruner, G.J. Carr, J.W. Harbell and R. D. Curren
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Often, the only measures of toxicity test performance provided in validation studies are the contingent probability statistic (CPS) sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Sensitivity and specificity are generally used in preference to NPV and PPV since NPV and PPV are assumed to vary with changes in prevalence while sensitivity and specificity are assumed to be independent of changes in prevalence. The purpose of the studies reported here was to test whether or not sensitivity an specificity are actually independent of changes in prevalence. Results derived from these studies indicate that sensitivity and specificity vary significantly depending on the prevalence of toxic substances in the set of chemicals being tested. This means sensitivity and specificity should not always be considered constant indicators of toxicity test performance.
Source: Human & Experimental Toxicology, 2002; 6: 305-312
Authors: L.H. Bruner, G.J. Carr, J.W. Harbell and R. D. Curren
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An approach commonly used to measure new toxicity test method (NTM) performance in validation studies is to divide toxicity results into positive and negative classifications, and then identify true positive (TP), true negative (TN), false positive (FP) and false negative (FN) results. After this step in completed, the contingent probability statistic (CPS), sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) are calculated. Although these statistics are widely used and often the only statistics used to assess the performance of toxicity test methods, there is little specific guidance in the validation literature on what values for these statistics indicate adequate performance. The purpose of this study was to begin developing data-based answers to this question by characterizing the CPS obtained from an NTM whose data have a completely random association with a reference test method (RTM). Determining the CPS of this worst-case scenario is useful because it provides a lower baseline from which performance of an NTM can be judged in future validation studies. It also provides an indication of relationships in the CPS that help identify random or near-random relationships in the data. The results from this study of randomly associated tests show that the values obtained from the statistics vary significantly depending on the cut-offs chosen, that high values can be obtained for individual statistics, and that the different measures cannot be considered independently when evaluating the performance of an NTM. When the association between results of an NTM and RTM is random the sum of the complementary pairs of statistics (sensitivity + specificity, NPV + PPV) is approximately 1, and the prevalence (i.e., the portion of toxic chemicals in the population of chemicals) and PPV are equal. Given that combinations of high sensitivity – low specificity or low specificity - high sensitivity (i.e., the sum of the sensitivity and specificity equal approximately 1) indicate lack of predictive capacity, an NTM having these performance characteristics should be considered no better for predicting toxicity than by chance.
Source: Presented at the 41 Annual Meeting of the Society of Toxicology, Nashville, Tennessee, March 17-21, 2002
Authors: J. Burdick , J. Merrill , T. Spangler , G. Moyer and J. Harbell
Fragrances are complex mixtures containing numerous chemical compounds and are referred to generically in the context of consumer product formulations. Aside from the inherently complex contribution of fragrance to a formulation’s ocular irritation potential, it is also difficult to elucidate the role of other Components in the mixture. This study was undertaken to evaluate the use of histological analysis to identify ocular damage not detectable by BCOP alone. Formulations were grouped according to base and fragrance type/concentration and comparisons made between base-only and a series of fragranced formulations using the same base. Ocular irritation was measured using a standard BCOP assay with isolated corneas exposed to test material for 10 minutes, followed by opacity and permeability determination and histological analysis. Ethanol was used as a concurrent positive control. Within a series, BCOP scores varied significantly across fragrance type, ranging from 0.1 to 44.8, due mainly to changes in permeability. Histological analysis confirmed the relative degree of damage. For a given base, predicted irritation potential was dependent on the particular fragrance mixture. This study reinforces the need to determine the ocular irritation potential of formulations and not rely solely on component information. Understanding the nature and degree of ocular irritation potential to complex mixtures may help to improve the selection process for base and fragrance components of consumer products.
Source: Annual Meeting of the Society of Toxicology, 2002
Authors: R. D. Curren, J. Harbell and L.H. Bruner
New toxicity tests, especially in vitro tests, are often used as screens, i.e. they are used to preliminarily divide a test set of chemicals into positive or negative subsets. One of two strategies can then be applied. Either 1) the negatives can be accepted as correct and the presumed positives evaluated in a second tier test, or 2) the positives can be accepted and the negatives exposed to a second tier. There has been a tendency to assume that validation does not have to be as stringent for a screen as it does for a stand-alone test because the screen is only a preliminary assessment. However, both approaches 1) and 2) above use screens to make some firm decisions; the first approach to label a chemical as non-toxic and the second approach to label the chemical as toxic. It is often thought that sensitivity (fraction of known positives correctly identified) is the most important factor and that specificity (fraction of known negative materials correctly identified) has much less significance. It follows that sensitivity alone could be characterized by using predominately positive chemicals in the validation test. We present data to show that this reasoning can lead to major errors. In approach 1), choosing a tier I test with low specificity will result in a large number of false positive materials passing through to the more expensive or animal intensive tier two test, possibly negating the advantage of having two tiers. This is especially relevant when several mechanistic specific tests are combined into a tier I battery. In approach 2), a low specificity tier I test means that many useful chemicals may be discarded, without necessarily decreasing the prevalence of positives in the subset of negative chemicals identified by the screen. Finally we show that using predominately positive test materials to validate a screen can result in a serious overestimation of the test’s sensitivity. For any test to be useful as a screening test or as a stand-alone test, it must it must be both sensitive and specific for the endpoint of interest.
Source: ATLA, 30, suppliment 2, 69-74, 2002
Authors: Rodger D. Curren and John W. Harbell
Ocular irritation testing has been one of the animal test methods most criticised by animal welfare advocates. Additional criticism has arisen from within the scientific community, based on the variability of the animal test results and the questionable relevance of the extremely high dose levels employed. As a result, the Draize eye irritation test has been one of the main targets for in vitro replacement. Despite extensive efforts, however, there is still no in vitro method that is fully validated as a regulatory replacement. In spite of this, many individual companies are using diverse in vitro ocular irritation tests to gain important safety and efficacy information about their products and raw materials, eliminating the need for animal testing in the process. This is done in a safe fashion by applying intelligent testing paradigms. ECVAM has played a major role in this success, through its many programmes that have emphasised the importance of understanding the true toxicological need, and then using in vitro tests to provide that information. Thus, even in the absence of a successfully validated regulatory assay, the desired result of reducing animal testing is being met.
Source: Toxicology In Vitro, Vol. 15, pp. 57-93, 2001
Authors: J.H. Fentem et al.
A prevalidation study on in vitro tests for acute skin irritation was conducted during 1999 and 2000. The overall objective of validation in this area, of which this prevalidation study is an initial stage, is to identify tests capable of discriminating irritants (I) from non-irritants (NI), as defined according to European Union (EU) risk phrases (“R36; no classification) and the harmonised OECD criteria (“Irritant”; no label).
Source: Toxicology In Vitro, Vol. 15, p. 95-103, 2001
Authors: K.J. Cooper, L.K. Earl, J. Harbell and H. Raabe
The isolated rabbit eye (IRE) test and bovine corneal opacity and permeability (BCOP) assay were evaluated for their ability to predict the eye irritation potential of a range of hair shampoo formulations, some containing a novel non-surfactant ingredient known to be an ocular irritant. The additional endpoints of corneal swelling and histological examination were incorporated into the standard BCOP protocol. Historic Draize data were available for several of the formulations and served as a reference. The standard BCOP assay (without histology) failed to distinguish between shampoos of low and high irritant potential, when exposure times of 10 and 60 min were employed (for undiluted and 10% dilution of the shampoos, respectively) and the in vitro score classified the majority of formulations as mild. The incorporation of the histological endpoint to the BCOP protocol allowed discrimination between formulations of differing irritancy, and should be included to augment the standard BCOP protocol. Corneal swelling values did not, however, correlate with the irritant potential of the shampoos tested. The IRE which includes the endpoints of corneal swelling and histopathological scoring produced classifications of irritancy that were fairly consistent with in vivo data and distinguished between the high and low irritant potential shampoos.
Source: Toxicology In Vitro, Vol. 13, pp. 313-323, 1999
Authors: J. W. Harbell, R. Osborne, G.J. Carr and A. Peterson
The Cytosensor
Source: Environmental Health Perspectives 106 (suppl.2), 477-484
Authors: Leon H. Bruner, Gregory J. Carr, Rodger D. Curren, and Mark Chamberlain
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 deonstration of the reliability and relevane of a test method for a particular purpose. This paper provides a brief review of the development of the theoretical aspets of the validation process and updates current thinking about objectively testing the preformance 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.
Source: Environ Health Perspec 106 (suppl 2):485-492 (1998)
Authors: R.D. Curren and J. W. Harbell
The necessity of using animals to test whether new chemicals and products are eye irritants has been questioned with increasing frequency and fervor over the last 20 years. During this time many new nonanimal methods have been proposed as reliable alternatives to the traditional rabbit (Draize) test. To date, however, none of these nonanimal (in vitro) tests have become universally accepted as a complete replacement for the Draize test. To understand why a complete replacement has not been found, one has to first understand the reasonably complex structure of the eye, the standard Draize scoring scale - which is based on a qualitative evaluation of three different tissues- the differences between human and rabbit eyes, the intrinsic variability of the animal test and the details of the different in vitro tests that have been proposed as replacements. The in vitro tests vary from relatively simple assays using single cells to more sophisticated assays that use discarded animal tissue or artificially constructed human tissue. It is clear that appropriately designed in vitro tests will eventually give more useful mechanistic information about ocular injury from which we can more comfortably predict the risk of human eye irritation from new products and ingredients.
Source: Environmen Health Perspect 106 (suppl 2):419-425 (1998)
Authors: R. Curren, L. Bruner, A Goldberg, and E. Walum.
Scientific principles demand that before newly developed alternative methods for safety testing are fully embraced by the regulatory community, they reliably and reproducibly predict the designated toxic end point. The process used to determine reliability and reproducibility is termed validation, and it generally culminates with a highly controlled, blinded study using multiple chemicals and laboratories. It is imperative that the validation study is designed to confirm the previous established reproducibility and predictive power of the assay. Much has been learned recently about the practical aspects of validation through investigation of alternative methods for acute toxicity testing, i.e., those methods that assess acute systemic toxicity, skin irritation, and eye irritation. Although considerable progress has been made - many alternative tests are now commonly used in various industrial settings - there have been few tests that have successfully passed a complete validation. Some of the barriers to successful validation have been a) lack of high-quality, reproducible animal data; b) insufficient knowledge of the fundamental biologic processes involved in acute toxicity; and c) the development of truly robust in vitro assays that can accurately respond to materials with a wide range of chemical and physical characteristics. It is recommended that to progress in the areas of eye and skin irritation we need to expand our knowledge of toxic markers in humans and the biochemical basis of irritation; progress in the area of acute systemic toxicity will require the development of in vitro models to determine gastrointestinal uptake, blood-brain barrier passage, and biotransformation.
Source: Toxicology In Vitro, Vol. 12, pp. 483-524, 1998
Authors: J. H. Fentem, G.E.B. Archer, M. Balls, P.A. Botham, R.D. Curren, L.K. Earl, D.J. Esdaile, H.-G. Holzhutter and M. Liebsch
As a follow-up to a prevalidation study on in vitro tests for replacing the in vivo rabbit test for skin corrosivity, an international validation study was conducted during 1996 and 1997 under the auspices of ECVAM. The main objectives of the study were to: (a) identify tests capable of discriminating corrosives from non-corrosives for selected types of chemicals and/or all chemicals; and (b) determine whether these tests could identify correctly known R35 (UN packing group I) and R34 (UN packing groups II & III) chemicals. The tests evaluated were the rat skin transcutaneous electrical resistance (TER) assay, CORROSITEX, the SKIN
Source: Dermatotoxicology Methods: The Laboratory Worker's Vade Mecum. F. N. Marzulli and H. I. Maibach., Washington, D.C. , Taylor & Francis:331-375
Authors: L. H. Bruner, G. J. Carr, M. Chamberlain, and R. D. Curren
Toxicologists have developed an array of animal-based test that have been used to assess the toxicity of chemicals and mixture of chemical during the last half century. These methods have been adopted by regulatory agencies throughout the world to provide data that are ultimately used to protect public health and to warn chemical users of potential health dangers.
The use of animals for routine acclivity testing is now questioned by a growing segment of society. New test procedures, called alternative methods, are now being develop to meet ethical concerns as well as to provide improved toxicological information. It is of critical important to determine whether such alternative methods are valid for use in the safety assessment process.
Source: ATLA 25, 505-516
Authors: Graeme Archer, Michael Balls, Leon Bruner, Rodger D. Curren, Julia H. Fentem et. al.
An alternative method is shown to consist of two parts; The test system itself; and a prediction model for converting in vitro endpoints into predictions of in vivo toxicity. For the alternative method to be relevant and reliable, it is important that its prediction model component is of high predictive power and is sufficiently robust against sources of data variability. In other words, the prediction model must be subjected to criticism, leading successful models to the sate of confirmation. It is shown that there are certain circumstances in which a new prediction model may be introduced without the necessity to generate new test system data.
Source: Comments Toxicology, 1997, Vol. 6, No. I, pp. 37-51
Authors: L. Bruner, G. Carr, R. Curren, M. Chamberlain
The purpose of this paper is to review a practical process that can be used to assess the validity of alternative methods for in vivo safety tests. The important role of a clearly stated prediction model, which defines how to use the results from an alternative method to predict an in vivo toxicity endpoint, has been discussed. Computer simulations have been used to demonstrate that data-based guidance can be developed to assist in judging the performance of alternative methods assessed in a validation study. Additionally, statistical procedures have been used in order to provide guidance on choosing the appropriate number of reference test substances and number of participating laboratories to include in a validation study. The validation of alternative methods for eye irritation testing is used as a specific example to illustrate important concepts. Although the focus of the discussion is on the validation of alternative methods intended to replace current in vivo tests, the procedures can be used to assess the performance of alternative methods intended for other uses.
Source: Toxicology In Vitro, Vol. 11, pp. 141-179, 1997
Authors: P. G. Brantom et al.
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.
Source: Comments Toxicology 1997. Vol. 6, No. 1, pp. 71-85
The eye is a very intricate organ that is susceptible to injury because of its exposure to the external environment. It is therefore important that manufacturers of chemicals and consumer products assess the eye safety of their products. In the past, eye safety has been assessed through the use of in vivo tests. More recently, there has been a considerable effort to develop and validate non-animal alternatives that may replace the in vivo test. This review provides a brief overview of the important anatomical structures of the eye that must be considered in an eye safety assessment, the methods that have been used to assess eye irritancy in vivo, and the approaches that are being taken to develop and validate non-animal alternatives for eye irritation testing. Much has been learned in the last few years about both the performance of alternative methods and the process of validation itself. This prngress has led to the identification of several in vitro ocular irritation assays that show considerable promise for validation within the foreseeable future.
Source: ATLA 24, 139-142
Authors: L. Bruner, G. Carr, et al.
There has recently been increasing interest in the development and validation of alternative methods that could be used in the place of in vivo toxicity tests. The goal is that a toxicologist will be able to test a substance by an alternative method, convert the results into correct predictions of toxic hazard and, ultimately, use the predictions for making decisions about the safety of a test substance. If a toxicologist can be assured that the predictions obtained from an alternative method will lead to the correct assessment decisions, the method may replace the in vivo test.
Source: ATLA 23, 398-409
Authors: Michael Balls, Walter De Klerck, Frank Baker et. al.
This is a report of the seventh of a series of workshops organised by the European Center for the Validation of Alternative Methods (ECVAM). This particular workshop was a joint initiative of ECVAM and the Consumer Policy Service (CPS).
Source: ATLA 23, 211-217, 1995
Authors: R.D. Curren, J.A. Southee, H. Spielmann, M. Liebsh, J.H. Fentem and M. Balls
Experience has shown that the outcome of large and expensive validation studies on alternative methods can be compromised if their managers do not insist that optimised test protocols and proof of their performance are submitted ,i>before the start of the formal validation study. One way for the sponsors of validation studies to confirm both the likely relevance of a method for its stated purpose and its readiness for validation would be to require a prevalidation study before formal validation was contemplated. This process would involve the developers (or other proponents for the method) and selected independent laboratories in protocol refinement (Phase I) and protocol transfer (Phase II). The optimised protocol would then be assessed in a protocol performance phase (Phase III), which would involve the testing of a relevant set of coded test materials and an evaluation of a proposed prediction model. In certain circumstances, a successful outcome of Phase III might be sufficient for promotion of the regulatory acceptance of the method. Normally, however, the method would proceed to a formal validation study. The European Center for Validation of Alternative methods, a recognised validation authority, no proposes to introduce this prevalidation scheme into its validation strategy.
Authors: John W. Harbell, Rodger D. Curren
In our 6 years of performing the bovine corneal opacity and permeability (BCOP) assay, a wide variety of chemicals or product classes have been evaluated. These include surfactants and surfactant-based formulations, industrial cleaners, air care products, pharmaceutical intermediates, organic solvents, and others. Furthermore, the application of the resulting data differed among assay users. Data were used to evaluate product safety, to assist in product development (comparing a range of potential formulations), and to determine industrial hygiene requirements for the workplace. To serve these ends, several standard protocols have been developed that differ in the duration of test material exposure to the corneas. In addition, many protocols now add histopathology to confirm negative responses and evaluate the specific tissue changes induced by the test material exposure. Specific protocol variations and the reasons for their use are described below
Source: Toxicology In Vitro, Vol. 8, No. 5, pp.889-891, 1994
Authors: C.L. Cannon, P.J. Neal, J.A. Southee, J. Kubilus and M. Klausner
An interlaboratory comparison of EpiDerm™, a new model of human epidermis, was conducted using a range of anionic and non-ionic surfactants and surfactant-containing final formulations.
The toxicity of the materials was estimated by MTT conversion, using both concentration (EC-50) and time (ET-50) protocols.
A range of 16 compounds were tested on different production lots of EpiDerm following 1 and 2 day storage periods (post-shipping) at MatTek and at two independent testing laboratories (Microbiological Associates, US and Scotland).
The EC-50’s and ET-50’s were compared and the least squares fit lines with resulting correlation coefficients (r) calculated. Correlation of in vitro results to human clinical chamber irritation and repeat handwash testing gave r values ranging from 0.977 to 0.993 and comparison of the results obtained in the independent laboratories with the site of manufacture was good (MA, US, r=0.84; MA, UK, r=0.90).
The EpiDerm model appears to have utility in predicting clinically observed dermal irritation in vitro which appears to be reproducible in different laboratories even after transatlantic shipping, such that it is worthy of further investigation.
Source: Toxicology In Vitro, vol. 6, No. 4, pp.367-371, 1992
Authors: K.A. Wallace, J.W. Harbell, N. Acomando, A. triana, S. Valone and R.D. Curren
Two methodologies used in vitro to estimate cytotoxicity in cell culture systems were compared: these were the neutral red uptake assay (NRU), which is used to measure toxicity caused by an extended (48-hr) exposure to the test material, and the neutral red release assay (NRR), which is used to measure toxicity caused by a short-term (1-min) exposure to the test material. Both methodologies used the normal human epidermal keratinocyte (NHEK)-based NeutralRed Bioassay supplied by Clonetics Corporation (San Diego, CA, USA). 10 materials (paracetamol, acetylsalicylic acid, ferrous sulphate, diazepam, amitriptyline, digoxin, ethylene glycol, methanol, ethanol and isopropanol), which are part of the Multicenter Evaluation of In Vitro Cytotoxicity (MEIC) panel, were tested. NRU(50) values for the 10 compounds covered more than an eight-log range from 0.004 mum (digoxin) to 1.0 x 10(6) mum (methanol). Because of solubility limits, NRR(50) values for diazepam, digoxin, ferrous sulphate and paracetamol could not be determined. NRR(50) values for the remaining six compounds covered approximately a three-log range from 3.2 x 10(3) to 7.1 x 10(6) mum. When compared with documented values for either the human acute oral lethal dose or the human acute lethal blood concentration, the NRU assay was found to be much more useful in predicting human acute toxicity than was the NRR assay.