The European Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) has published its recommendation on the performance of the 3T3 Neutral Red Uptake (NRU) in vitro test method for acute oral toxicity.The recommendation says that 3T3 NRU could be used to identify chemicals that do not require classification as toxicants, so called "negative" substances. This, EURL ECVAM says, will be useful for industrial chemicals under REACH, where information on acute oral toxicity is one of two health effects requiring animal tests at low production levels.The recommendation summarzses the overall performance of the 3T3 NRU test method, its applicability and limitations, and provides guidance for proper scientific use. It also suggests ways of tackling some remaining gaps to provide more complete characterisation of substances using the test method. For more information, please follow the link and visit the EURL ECVAM website.
Register to see a presentation and participate in discussions on a scientific roadmap for the future of animal-free systemic toxicity testing, at a workshop on May 30-31, 2013 at the U.S. Food and Drug Administration's Wiley Building in College Park, Maryland. The scientific roadmap was the product of an October 2011 workshop held under the auspices of the transatlantic think tank for toxicology (t4). The program includes an opportunity for public discussion of the roadmap, as well as its possible updating in light of more recent developments. Please visit the link above for the agenda, registration information, and a list of the organizing committee.
An appeal has overturned the European Chemicals Agency’s (ECHA) request for additional animal toxicity testing on the automotive air-conditioning refrigerant 2,3,3,3-tetrafluoropropene (HFO-1234yf). ECHA requested that Honeywell, manufacturers of HFO-1234yf, conduct extended safety studies in rabbits. Honeywell countered that such studies would not provide the agency with the information they were looking for and that safety could be confirmed by analyzing the current data. ECHA's board of appeal has rulled that the requested test would oppose the ECHA’s responsibility under the Reach (registration, evaluation, authorisation and restriction of chemicals) regulation to ensure that animal tests are only undertaken as a last resort, and that any testing involves the minimum number of animals. Read more by following the link above.
The OECD has revised the test guidelines for the Bovine Corneal Opacity and Permeability (BCOP) test and the Isolated Chicken Eye (ICE) test to extend the applicability domain of two in vitro methods. This is the first time that in vitro methods are accepted for the identification of non-irritant chemicals in the field of eye irritation. The revision of the two test guidelines was adopted at the OECD meeting of the Working Group of National Coordinators of the Test Guideline Programme held in Paris on 9 to 11 April 2013. They had originally been adopted in 2009 for the identification of serious eye damage/eye irritation of chemicals further to retrospective validation by the US Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) in collaboration with EURL ECVAM and the Japanese Center for the Validation of Alternative Methods (JaCVAM). Additional validation of the two tests showed their usefulness also for the identification of chemicals not requiring classification for serious eye damage/eye irritation (i.e. non-irritant chemicals), thus leading to the revision of the test guidelines under the co-lead of EURL ECVAM and the Netherlands. Please follow the link above to read the full story on the EURL ECVAM website.
Chemicals company BASF has teamed up with life sciences company Promega to jointly develop an alternative method to animal testing to detect allergic reactions in the skin to certain substances. Dr. Robert Landsiedel, head of the short term Toxicology unit at BASF states, "Combining the new method with two additional alternative methods to investigate skin sensitization allows us not only to significantly reduce the number of animal studies, but also to predict a possible allergic potential more reliably than before." This new method has been submitted to ECVAM (The European Center for the Validation of Alternative Methods) for evaluation.
Swedish researchers at Lund University have developed various in vitro test strategies to replace animal testing when determining skin allergens, thanks to a special special gene expression analysis software. To read the article on Cosmetics Design Europe's website, please follow 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 April number is now available by following the link above.
PETA is presenting a new award—the Laurie and Carlee McGrath Award, which includes a $5,000 cash prize—to the MatTek Corporation for its development of sophisticated non-animal tests. The award is named in honor of Laurie McGrath and her mother, Carlee McGrath, whose San Diego–based McGrath Family Foundation supports PETA's work to replace animals in laboratories with non-animal methods. IIVS and MatTek have collaborated over many years and we congratulate MatTek for being recognized for their work in developing 3-dimensional human cell based tissue constructs that can be used in place of animals for various types of toxicity tests. Please follow the link above to read the full article.
The Alternatives Research & Development Foundation is currently soliciting research proposals for its 2013 Alternatives Research Grant Program. For nearly 20 years, this program has created opportunities for scientists who have interest and expertise in alternatives research.
Up to $40,000 in funding available to support individual projects
Preference given to U.S. universities and research institutions
Preference given to projects that use pathway-based approaches as exemplified by the 2007 National Academy of Sciences report, Toxicity Testing in the Twenty-first Century: A Vision and A Strategy
Deadline: Applications must be electronically submitted or postmarked by Tuesday, April 30, 2013
Recipients notified: Tuesday, July 16, 2013
Criteria:
Potential to significantly replace or reduce laboratory animals
Scientific merit and feasibility
To apply, please download the Guidelines for submission and Application cover page Applicants must notify ARDF via email of intent to submit a proposal by April, 23, 2013. Please see instructions in Grant Guidelines.
As the final animal testing ban deadline went into effect in Europe last week, Chinese regulatory bodies in their efforts to follow closely behind the West, have organized a workshop on in vitro alternative methods to further educate industry professionals on the Asia Pacific region. IIVS, along with two Chinese agencies, served as organizers for the event taking place March 19, 2013. Follow the link above to read more detail on the Cosmetics Design Asia website.
Source: Presented at the 2013 Society of Toxicology Annual Meeting
Authors: Costin, Gertrude-Emilia; Jeong, Yo-Chan; Anderson, Donna; Bader, Jackie E.; Krawiec, Lindsay; Nash, Jennifer R.; Raabe, Hans
In compliance with OECD Test Guideline 437 for eye irritation (BCOP assay), non-surfactant solid materials are typically tested as 20% dilutions prepared in 0.9% sodium chloride solution, distilled water, or other solvent that has been demonstrated to have no adverse effects on the test system. However, the limited solubility of some chemicals adds technical challenges in finding a vehicle that would ensure the material’s availability to the excised corneas and that itself would not affect the test system. In this study, we evaluated five solvents frequently used in the BCOP assay: distilled water, mineral oil, corn oil, polyethylene glycol (PEG)-400, and methocel solution (0.5%). Based on the available classification systems, our preliminary data showed that water, methocel, mineral oil and corn oil were predicted as non-irritants, while PEG-400 was predicted as a mild irritant. To demonstrate the influence of the type of solvent on the outcome/prediction of the BCOP assay for solid materials, we tested a 20% suspension of benzoic acid (BA) prepared in these solvents. BA has a non-polar benzoic ring that would preferably dissolve in non-polar solvents and a polar acidic group with affinity for polar solvents, thus making it a good model for testing its effect on corneas when dissolved in various solvents. Previous animal tests reported moderate to severe eye irritation induced by BA. Our results demonstrated that when mixed in water, mineral oil, corn oil, or methocel, BA was predicted to be a corrosive/severe irritant, while it was predicted to be a moderate irritant when mixed in PEG-400. These results support the need for further investigation of the solvent’s influence in the BCOP assay to allow the correct prediction of the irritation potential of solid materials.
Source: Presented at the 2013 Society of Toxicology Annual Meeting
Authors: D. Gan, K. Norman, N. Barnes, H. Raabe, C. Gomez, and J. Harbell
An essential step in the safety review of cosmetic/personal care ingredients is hazard assessment for a series of endpoints, including dermal sensitization potential. In vitro methods have been developed to identify allergic (haptenic) potential for individual chemicals based on electrophilic interaction with marker peptides or cellular target systems. These assays generally use a specific molar ratio of the test chemical to the test system. Botanical extracts are used increasingly in formulas and, as mixtures, specific molar ratios cannot be determined for these assays. Often, the botanical extract portion is a relatively small portion of the complete ingredient. To assess these mixtures, the KeratinoSens assay was selected because it operates over a wide dose range and sets cytotoxicity limits on doses used to measure marker gene expression (Emter et al, 2010 [7]). In the KeratinoSens assay, the induction of a luciferase gene, under the control of the antioxidant response element (ARE) derived from the human gene AKR1C2 gene, is measured. In parallel, cytotoxicity is assessed by both Neutral Red Uptake (NRU) and MTT assays. Test concentrations ranged up to 1000 μg/mL (of complete ingredient) and a test concentration was considered positive if the relative viability was ≥ 70% and the fold induction of luciferase was 1.5x relative to the solvent controls. The goal of the study was to measure the activity of 3 known sensitizers (gluteraldehyde (GA) [strong], dimethyl maleate (DM) [moderate] and cinnamic aldehyde (CA) [moderate] spiked into four different botanical ingredients (each with a different excipient solvent systems). The “spiked” botanical ingredients were used as the test article as no sensitizing botanical ingredient was available. Activity of the spiked sample was measured relative to the EC1.5 of the neat sensitizer as a function of sensitizer concentration and extract composition. Three independent trials were performed on each test material. No appreciable cytotoxicity was observed with any of the samples. The recovery of the GA spike required at least a ~3 fold increase in concentration relative to the chemical alone and botanical ingredient #3 reduced the activity below detection. The DM and CA showed activity at about the same effective concentrations as the neat chemical although the DM showed reduced activity in botanical ingredient #3 as well. These data suggest that the KeratinoSens assay has the potential to identify electrophile allergens within a botanical ingredient matrix.
Source: Presented at the 2013 Society of Toxicology Annual Meeting
Authors: Michael Bauman, Kimberly Norman, Greg Mun, Hans Raabe
The Bovine Corneal opacity and Permeability (BCOP) assay can be used for predicting mild, moderate, and severe ocular irritation through quantitative assessment of the changes in opacity and permeability of the bovine cornea. In addition, histological evaluation of the corneas can be performed to assess the depth of damage. The BCOP assay with histology was used to determine the ocular irritation potential of prototype cleaning products with antimicrobial claims according to the guidance provided by the EPA-Office of Pesticide Program (OPP). Several prototype cleaners with similar formulation were evaluated along with a reference material. The results of the BCOP assay showed noticeable differences among the products. The in vitro score, determined by changes in opacity and permeability, of the corneas treated with products ranged from ~15 to 80. These scores indicate mild, moderate, and severe irritation according to the guideline provided in the EPAOPP document. In addition, the histological evaluation of the corneas showed differences in the depth of damage between moderate and severe category products, confirming the in vitro score.
The assay distinguished ocular irritation potential among similar prototypes demonstrating its effectiveness during product development. Additionally, the results demonstrate the utility of the BCOP assay with histology as a stand-alone assay for eye irritancy evaluation in the EPAOPP program.
Source: Presented at the 2013 Society of Toxicology Annual Meeting
Authors: Ramez Labib, Enrico Gilberti, Stephen Gettings, Hans Raabe
Phototoxicity is an acute toxic response after exposure to a phototoxicant and either UV radiation or visible light (UV/VIS). Phototoxicity from substances applied topically typically occurs at the site of photo-irradiation. Phototoxicity is the result of direct cellular damage caused by a non-immunological inflammatory response. Clinically, phototoxicity resembles an exaggerated sunburn (erythema, increased skin temperature, pruritis and edema). Phototoxicity reactions have been reported for both synthetic substances and those which occur naturally (e.g., botanical extracts). Although symptoms generally subside quickly, the potential for substances used in topical products to cause phototoxicity is clearly of concern for manufacturers of cosmetics, personal care and other consumer products. Historically, the potential to cause phototoxicity from substances applied topically was evaluated by utilizing various animal models. However in 1997 the 3T3 Neutral Red Uptake Phototoxicity Test (3T3 NRU PT) was validated by ECVAM’s Scientific Advisory Committee as an in vitro method for evaluating the phototoxic potential of chemicals shown to absorb in the UV/VIS range. To illustrate the utility of the 3T3 NRU PT as a useful screening tool in the safety evaluation of potential cosmetic ingredients, the results of the evaluation of 42 botanical extracts and 25 synthetic chemicals found to absorb in the UV/VIS range are reported. Most substances evaluated were found not to be phototoxic in vitro; however, 9 substances were identified as potentially/probably phototoxic in the 3T3 NRU PT and were eliminated from further consideration for use as cosmetic ingredients. Several substances found to be non-phototoxic in the 3T3 NRU PT were formulated with other ingredients in a prototype cosmetic formulation and subject to clinical testing. No manifestations of phototoxicity were observed in any of the test subjects in the prototype formulation containing any of the substances identified as non-phototoxic in vitro.
Source: Presented at the 2013 Society of Toxicology Annual Meeting
Authors: Jackie E. Bader; Gertrude-Emilia Costin; Allison Hilberer; Greg Mun; Jennifer R. Nash; Kimberly Norman; Nathan Wilt; Hans Raabe
The Bovine Corneal Opacity and Permeability (BCOP) assay is an ex vivo test used to evaluate the ocular irritation of a broad range of chemicals. In the regulatory classification and labeling arena, BCOP can be used to identify severe and corrosive eye irritants according to the OECD Test Guideline (TG) 437. However, BCOP has historically under-predicted certain anionic surfactants, when tested according to the standard liquid protocol. TG 437 specifies that liquid surfactants may be tested as 10% aqueous dilutions for 10 minutes (although alternate dilutions and exposure times may be conducted with scientific rationale), and the relevant guidance document (GD) No. 160 suggests that solid and concentrated liquid surfactants may be diluted to 10% for testing. However, GD No. 160 further directs that surfactant-based formulations are usually tested neat, but could be diluted with justification, imparting some confusion in identifying the most appropriate test methods. Since neither the basis for selecting the appropriate surfactant test methods, nor the justification for modifications are clearly presented in TG 437 or GD No. 160, we present on the testing of sodium lauryl sulfate (SLS) in the BCOP assay, using standard and modified dilutions and exposures, to elucidate the impact of these variables on eye irritation prediction. For example, in vitro scores of 20.7, 28.4, and 28.3 were obtained when testing SLS at concentrations of 50, 20, and 10% for 10 minutes, showing that irritation responses were not fully concentrationdependent, but demonstrated optimally at intermediate doses. When tested using modified exposure times, SLS showed time-related responses, with improvements in irritation predictions at the 20 and 30 minute exposures. Histopathology was performed to assess the surfactant-induced corneal changes. Based upon these results, a framework for testing surfactants, and surfactant-based formulations is proposed.
Source: Presented at the 2013 Society of Toxicology Annual Meeting
Authors: T. Abo; A. Hilberer; A. Heppenheimer; M. Watanabe; K. Ooshima; D. Cameron; A. Kirst; Y. Nukada; H. Sakaguchi; N. Nishiyama
STE test is an in vitro eye irritation test using cell viability as an end point in SIRC cells following just a 5 minute treatment, and the good correspondence has been confirmed between the STE irritation categories (non irritant [NI] and irritant [I]) and GHS categories (NC and category 1 [Cat. 1]/category 2 [Cat. 2]). Generally, cytotoxicity tests using cultured cells have an advantage of being simple, a quick procedure, and a low evaluation cost. The STE test has the advantages not only easy-to-use but also evaluable the eye irritation potential of water insoluble substances by using mineral oil as test vehicle. The STE test is planned for peer review in 2013 and may be accepted as an OECD test guideline for classifying ocular irritation. In this study, the technical transferability and inter-laboratory reproducibility of the STE test were evaluated in 3 contract research laboratories as a naive laboratory.
Source: Presented at the 2013 Society of Toxicology Annual Meeting
Authors: Cameron, David; Costin, Gertrude-Emilia; Kaufman, Lewis; Avalos, Javier; Downey; Martha Elena; Billhimer; Ward Loren; Gilpin, Sarah; Wilt, Nathan; Simion F. Anthony
Behentrimonium chloride (BTC) is a straight-chain alkyltrimonium chloride compound commonly used as an antistatic, hair conditioning, emulsifier, or preservative agent in personal care products. Although the European Union restricted the use of alkyltrimonium chlorides and bromides as preservatives to ≤0.1%, these compounds have been safely used at ≤5% in hundreds of cosmetic products for other uses than as a preservative. In vitro, clinical, and controlled consumer usage tests in barrier-impaired individuals were conducted to determine if whole body, leave-on skin care products containing 1-5% BTC cause dermal irritation or any other skin reaction with use. BTC-containing formulations were predicted to be non-irritants by the EpiDerm®* skin irritation test and the bovine corneal opacity and permeability (BCOP)/chorioallantoic membrane vascular assay (CAMVA) ocular irritation test battery. No evidence of allergic contact dermatitis or cumulative dermal irritation was noted under the exaggerated conditions of confirmatory human occlusive patch tests. No clinically assessed or self-reported adverse reactions were noted in adults or children with atopic, eczematous, and/or xerotic skin during two-week and four-week monitored home usage studies. These results were validated by post-marketing data for five body lotions, which showed only 0.69 undesirable effects (skin irritation) reported per million shipped consumer units during 2006-2011. No serious undesirable effects were reported during in-market use of the products. Therefore, if formulated in appropriate conditions at 1-5%, BTC will not likely cause dermal irritation or delayed contact sensitization when used in a whole-body, leave-on product.
European Union regulators announced today that the ban on the import and sale of cosmetics containing ingredients tested on animals has gone into effect. This ban follows the EU ban on animal testing of finished cosmetic products that went into effect in 2004. Today's ban removes extensions to the deadline allowing continued use of animal testing for those endpoints for which a suitable in vitro alternative has not been found. Through this action, the EU has also pledged to put forth a greater effort to push other parts of the world, like China, to accept alternatives. To read more about this ban, please click on the link above.
The Drug Controller General of India (DCGI) is said to have called for the fast-tracking of the deletion of two animal-tests from the regulatory safety standards governing cosmetics.Products ranging from eye-liners and lipsticks to shampoos and face-washes will be affected by this change. Companies wanting to test cosmetic products or ingredients for specific effects will have to submit a non-animal testing proposal to the DCGI for approval. It is expected that the Indian cosmetic standard IS4011 will be amended to reflect the changes, as directed by DCGI. To read the full article, please follow the link above.
US scientists have created a tiny device that simulates a working human lung and could reduce the need for testing pharmaceutical compounds in animals. The “lung-on-a-chip” contains hollow channels lined with living human cells that mimic the interface between the air sacks in the lung and the blood vessels beneath, allowing scientists to study the body’s response to lung infections and diseases. The chip’s inventors at the Wyss Institute for Biologically Inspired Engineering at Harvard University have received an annual international prize for the device’s potential to gradually reduce the use of animals in drug testing. Please click on the link above to read more at The Engineer.
Read about the activities and planned future work of the Evidence-based Toxicology Collaboration in there newsletter (linked above). The newsletter includes information on recent publications, a satellite meeting during the SOT meeting, and a summary of their 2012 meeting titled "Evidence-based Toxicology: Opportunities and Challenges".
Content includes information on the Short Time Exposure Assay (and our upcoming webinar on the same topic), a synopsis on our recent activities in China and Brazil, details concerning the LUSH prize for training, our MOU with the EPAA, and much more.
Queen Mary, University of London, has created a professorial chair in animal replacement science. The position is funded by the Dr Hadwen Trust, a charity that funds investigation into ways of replacing animals in research. According to the trust, the chair will "see the UK spearhead a collaborative global search for more ethical, human-relevant alternatives to animal testing". The professor will be based at Queen Mary's Blizard Institute, where researchers already are already working on developing in vitro models using human cells and tissue. Read more by following the link above.
A new animal- and cell-free method has been developed to measure the toxic activity of Botulinum neurotoxin (BoNT), e.g. Botox. The invention will avoid the use of mouse LD50 tests for toxicity testing, e.g. in BoNT-containing pharmaceutical products such as Botox.The method uses functionalized liposomes to determine the toxin’s activity under physiological conditions. Read more by following the link above.
In a development that could lead to faster and more effective toxicity tests for airborne chemicals, scientists from Rice University and the Rice spinoff company Nano3D Biosciences have used magnetic levitation to grow some of the most realistic lung tissue ever produced in a laboratory. The research is part of an international trend in biomedical engineering to create laboratory techniques for growing tissues that are virtually identical to those found in people's bodies. In the new study, researchers combined four types of cells to replicate tissue from the wall of the bronchiole deep inside the lung. The research is available online and scheduled to appear in a future issue of the journal Tissue Engineering Part C: Methods. Read more by following the link above.
Via Frame -
Amendments to the Animals (Scientific Procedures) Act 1986 came into force on January 1. The amended version of ASPA has been introduced to align UK legislation with European Directive 2010/63/EU. The Home Office has produced guides for researchers to help them with the changes.The ‘quick start’ guide provides advice on what the revised ASPA covers and guidance to holders of establishment licences, project licences and personal licences and new licence applicants. It also provides guidance on severity classification, humane killing and the accommodation and care of animals. More detailed draft guidance, covering more topics, will be published later in January for consultation.
A transitional guide sets out details of changes that researchers must make immediately in order to comply with the new regulations. It also details changes that will happen automatically.The new law requires re-authorisation for some activities and some types of animal, and the transitional guide sets out details of those amendments. For example, all cephalopods (octopus, squid, cuttlefish and nautilus) are now protected. Under the old regulations only Octopus vulgaris was included. The new rules also increase control of breeding of some frog species and zebra fish. Increased use of fish in regulatory testing accounted for a significant rise in the number of animals used for toxicology (safety testing) in the last Home Office statistics on the use of animals in laboratories in the UK.
Please follow these links to view the Quick Start and Transitional guides on the Home Office Website.
The next IVTIP meeting titled '2013: State of the art on alternatives from an industrial point of view: ready for regulation?' will be held in Southampton (UK) on May 14-16, 2013 hosted by British American Tobacco at their GR&D facility. Authors wishing to submit an abstract should send it to ivtip@planet.nl (1 page, title in bold, authors with affiliations, presenting author to be underlined, 350 words maximum, Arial 10, please indicate “oral” or “poster” presentation). Deadline for submission is February 1, 2013. Please consider the meeting program on the IVTIP website (www.ivtip.org) to make sure your paper falls within the scope of this event. Authors will be informed about the acceptance of their abstracts by February 20. All accepted abstracts will be included in the IVTIP Abstract Book and distributed to all participants. Please note that all presenters will be required to register for the conference.
Exhibition spaces available
A 2010 law passed by Israel took effect January 1, 2013 banning animal testing on cosmetic products imported into the country. Much like the European Cosmetics Directive, this law governs cosmetics, toiletries and detergents and includes a ban on the marketing of cosmetics that have been tested on animals in foreign countries as well. For more information on this law, please visit the website above.
This issue contains information on the election of board of directors and a summary of the first annual meeting.
The European Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) is inviting public comments on its latest recommendation concerning the 3T3 Neutral Red Uptake cytotoxicity test method. The deadline for receipt of these comments is January 31, 2013. The assay was recently validated by EURL ECVAM for its possible use to support the identification of substances not requiring classification for acute oral toxicity. The present recommendation outlines the assay's principle, its performance characteristics (based on available information and, mainly, evidence generated during validation) and the assay's limitations and makes recommendations for its implementation within an Integrated Testing Strategy (ITS) based primarily on non-animal approaches including structure-activity relationships and biokinetic modelling. The recommendation is based on the opinion of the ECVAM EURL Scientific Advisory Committee (ESAC) which peer-reviewed the validation study (the ESAC opinion is annexed to the recommendation document). Please visit the ECVAM website for downloadable pdfs of the relevant documents and instructions on how to submit your comments.
Mathematical modelling has the potential to solve biological questions, provide new insights which benefit science and medicine, and reduce reliance on animal models. Submit a problem to the NC3Rs Maths Study Group for the opportunity to work with mathematicians in addressing your research questions.
If you are interested in participating in the Study Group please submit a proposal describing your biological problem by 14 January 2013. Proposals should be no longer than two A4 pages and should include:
A brief background to the problem
Details of the problem
Any available data for informing possible mathematical models
Questions you would like to see answered
The potential impact on animal use
Any relevant references
Proposals for problems for the Study Group to consider at the workshop should be submitted to the NC3Rs at events@nc3rs.org.uk.
The U.S. Environmental Protection Agency Office of Pesticide Programs (U.S. EPA/OPP) and Health Canada’s Pest Management Regulatory Agency (PMRA) have cooperated on the development of a guidance document on the use of Quantitative Structure Activity Relationships [(Q)SAR] for pesticide risk assessors. This document has been developed under a North American Free Trade Agreement Technical Working Group (NAFTA TWG) project, 21st Century Toxicology: Integrated Approaches to Testing and Assessment.
The NAFTA TWG (Q)SAR Guidance Document provides an introduction to (Q)SAR and a flexible framework to assist with problem formulation for (Q)SAR, assessing the adequacy of predictions, and incorporating predictions into weight of evidence based assessments of pesticides. While the (Q)SAR Guidance Document was not designed to be a step-by-step manual, it provides useful guidance to evaluators who are reviewing predictions included in pesticide submissions or who are using (Q)SAR to help identify additional data requirements for pesticides, metabolites or degradates. Moreover, it also supplements, but does not replace, existing (Q)SAR guidance documents from the Organisation for Economic Cooperation and Development (OECD), the European Chemicals Bureau and other agencies.The NAFTA TWG (Q)SAR Guidance Document is currently available for download at the link above.
The Hamner Institutes for Health Sciences launched a partnership designed to advance a systems biology approach to the study of toxicity testing in pre-competitive research phases. Hamner said the partnership will develop human cell-based assays that map and model key cell signaling pathways in order to evaluate dose response, using toxicity pathway case studies with the goal of speeding implementation of recommendations from the National Research Council report “Toxicity Testing in the 21st Century: A Vision and a Strategy.” Partners sponsoring the research include Agilent Technologies, Illumina, Dow Chemical, Dow Corning, ExxonMobil, Unilever, and CropLife America, while the Long-Range Research Initiative of the American Chemistry Council supported earlier stages. Please click the link above to read the full article.
Earlier this month prospective EU Health Commissioner Tonio Borg expressed his support for the current March 2013 deadlines on animal testing put in place by the European Cosmetics Directive. "The ECEAE agrees with Mr. Borg's comments that industry would not find alternative means of testing if the marketing ban is not implemented as 'necessity is the mother of invention", it says in a statement. Likewise, HSI's senior EU policy adviser, Emily McIvor, agreed. "Tonio Borg's stated aim of overseeing full implementation of the 11 March 2013 ban represents a huge step forward..." Read more about ECEAE and HSI backing of Tonio Borg.
In contrast, Cosmetics Europe has urged caution over Tonio Borg's comments saying it may 'jeopardize' progress and undermine EU leadership in global animal welfare. Organization president Fabio Franchina comments, "[Borg's] sweeping statements threaten to remove that impetus and undermine the EU's hard-won leadership in driving the development of alternatives to animal testing. Furthermore, it is completely incompatible with the EU's existing Innovation 2020 strategy." Read more about Cosmetic Europe's concerns with Borg's remarks.
Marking its commitment to international cooperation, the European Partnership for Alternative Approaches to Animal Testing (EPAA) Co‐chairs signed a Memorandum of Understanding with the Institute for In Vitro Sciences (IIVS) President Rodger Curren during their 8th annual meeting on November 16th. EPAA and IIVS have agreed to establish a strategic partnership dedicated to the international dissemination of alternative techniques for safety evaluation. EPAA will provide sponsorship of up to €100,000 over the next two years to IIVS to support training activities in key regions, including China and Brazil. R. Curren stated that “EPAA’s efforts to promote international cooperation on 3Rs will be greatly complemented by our joint activities and solid international network.” To read more about the MOU and the EPAA annual meeting, please click on the link above. You can also view a signed copy of the MOU between EPAA and IIVS.
The European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) is renewing membership of its Scientific Advisory Committee (ESAC). ESAC is a key contributor to EURL ECVAM's workflow, notably through providing published ESAC opinions summarizing peer review advice on validation studies. ESAC opinions form the basis for issuing formal ECVAM Recommendations, summarizing the validity of alternative methods, their limitations and appropriate use.
EURL ECVAM is now launching an open call for applications for membership in the ESAC. Interested scientists with relevant competences are invited to apply. A Selection Committee composed of EURL ECVAM staff and external scientists will assess all applications for eligibility and will shortlist suitable candidates using defined selection criteria. The Selection Committee may also draw on the reserve lists created after the 2009 call for applications. Experts who are currently on the reserve list of the 2009 call do not need to reapply, unless their competence profile has changed considerably. All relevant details pursuant to the call can be found on ECVAM's website The deadline for applications is 30th November 2012.
IIVS staff recently returned from a training trip to China funded by a grant through PETA. Last week students and staff of the Beijing Technical and Business University attended a training session with IIVS that included education on the Bovine Corneal Opacity and Permeability Test. The students and staff were very enthusiastic to be part of the training. More information on the training session and the acceptance of non-animal test methods in China can be found in the article above.
The OECD QSAR toolbox is software that can be used to group chemicals into categories to help fill some of the gaps in the toxicity or ecotoxicity testing data needed to perform hazard assessment on chemicals. This tool could be a valuable addition to a tiered testing strategy that could also include follow-on in vitro testing. Version 3.0 of the software can be downloaded from the website link above.
The Organisation for Economic Co-operation and Development (OECD) has officially adopted two test guidelines for identification of substances with the potential to cause eye injury. One of these (OECD TG 405), an updated test guideline for the traditional rabbit eye test, incorporates specific procedures to avoid or minimize animal pain and distress when it necessary to use animals to identify substances with the potential to cause eye injuries. The other test guideline (OECD TG 460)provides a new method to identify substances that may cause serious eye injuries without using animals. Both guidelines, along with all of the current OECD test guidelines for the testing of chemicals) can be viewed on the OECD website by clicking on the link above.
Contents include: 3Rs Prize, David Sainsbury Fellowship Scheme, Project and Pilot Study grants and CRACK IT Challenges registration deadline. Please click on the link above to view the newsletter on the NC3R website.
Content includes information on the EPA's in vitro testing pilot program for ocular irritation testing on antimicrobial cleaning products (and our upcoming webinar on the same topic), a synopsis on our educational activities, information on a practical methods workshop for delegates from Russia, a dermal absorption workshop, and much more.
The U.S. Consumer Product Safety Commission (CPSC) has added a page to its website titled "Recommended Procedures Regarding the CPSC's Policy on Animal Testing." The page summarizes the CPSC policy on animal testing and their suggestions for the use of existing information and scientifically validated alternatives to animal testing in hazard assessment. The page also lists acceptable alternative methods for acute toxicity testing, ocular irritation testing, dermal irritation testing, and skin sensitization testing, and provides links to CPSC votes and approvals regarding animal testing policy. Please contact us to discuss strategies for implementing the CPSC's recommendations within your in vitro testing program. Click on the link above to view the page on the CPSC website.
The FDA has published a report following its most recent International Co-operation on Cosmetics Regulation meeting (ICCR-5), in which it outlines the applicability of animal testing alternatives in the four ICCR jurisdictions (Europe, Canada, Japan and the United States). The report describes the current regulatory climate in each region and it discusses a number of currently available OECD validated non-animal testing methods, such as for skin irritation testing and phototoxicity testing. It also outlines the use of tiered-testing approaches to safety assessment and the necessity of this type of testing paradigm as the focus is shifted to the use of non-animal testing methods. Click on the link above to read the report on the FDA website.
Congratulations to Dr. Horst Spielmann for receiving this year's Björn Ekwall Memorial Award. Dr. Spielmann has significantly contributed to the field of in vitro toxicology by developing non-animal tests aimed at reducing the number of animals used in experimentation such as the 3T3 phototoxicity assay and the embryonic stem cell test (EST) for determining embryotoxicity. Dr. Spielmann collaborated with Dr. Björn Ekwall over the course of several years. The Björn Ekwall Memorial Award will be awarded at the ESTIV 2012 meeting where Dr. Spielmann will deliver the Björn Ekwall Memorial Lecture titled Today Björn Ekwall would endorse the concept "Toxicology in the 21st Century".
The majority of cleaning products in the US do not undergo a pre-market registration process. However, once an “anti-microbial” claim is made for the product, it is regulated by the US Environmental Protection Agency’s Office of Pesticide Programs (US EPA OPP) and animal testing for hazard identification is required before the product can be sold. Click on the link above to view the full article.
This international workshop features presentations by representatives from around the globe who will speak on validation studies performed on the HET-CAM assay and their experiences in working with a number of materials in the assay. Registration is open to the public, but limited to 40 participants. More information, including links to the web page of the International Workshop on the HET-CAM Assay can be found by following the link above.
The Lush Training Prize aims to reward individuals and organizations involved in training researchers in non-animal methods. The training prize recognizes that many scientists involved in chemicals testing are not aware of the range of non-animal methods available or trained in using them. Establishing training programs around the world makes a huge difference to the field. The prize wishes therefore to reward individuals, teams, or organizations who have excelled in this field. The three groups on the short list are:
Institute of In Vitro Sciences
InterNICHE
The International QSAR Foundation
The winner will be announced October 15.
The paper chosen for the Editor's Highlight in the October 2012, Vol. 129, No. 2 issue of Toxicological Sciences is "Using Novel In Vitro NociOcular Assay Based on TRPV1 Channel Activation for Prediction of Eye Sting Potential of Baby Shampoos" by Anna Forsby, Kimberly G. Norman, Johanna EL Andaloussi-Lilja, Jessica Lundqvist, Vincent Walczak, Rodger Curren, Katharine Martin, and Neena K. Tierney.
Editor, Michael L. Cunningham notes that "Replacing animal testing for pain induction by chemicals is a goal of animal welfare research. Forsby et al. make a significant advancement in this field by the development of the NociOccular test for baby bath and shampoo formulations. This assay is a recombinate neuronal in vitro model of activation of the Transient Receptor Potential Vanilloid type 1 channel, a well characterized pain-inducing receptor. This research opens the way for the development of future assays to predict pain induction without the use of animals."
Source: Toxicol Sci. 2012 Oct;129(2):325-31
Authors: A. Forsby, K. Norman, J. EL Andaloussi-Lilja, J. Lundqvist, V. Walczak, R. Curren, K. Martin, and N. Tierney
The transient receptor potential vanilloid type 1 (TRPV1) channel is one of the most well-characterized pain-inducing receptors. The purpose of this study was to predict human eye stinging of 19 baby bath and shampoo formulations by studying TRPV1 activity, as measured by increase in intracellular free Ca2+. The NociOcular test, a novel recombinant neuronal in vitro model with high expression of functional TRPV1 channels, was used to test formulations containing a variety of surfactants, preservatives, and fragrances. TRPV1-specific Ca2+ influx was abolished when the TRPV1 channel antagonist capsazepine was applied to the cells prior to shampoo samples. The positive control, an adult shampoo that contains cocamide monoethanolamine (CMEA), a known stinging ingredient, was the most active sample tested in the NociOcular test. The negative control, a marketed baby shampoo, was negative in the NociOcular and human tests. Seven of the formulations induced stinging in the human test, and of those six were positive in the NociOcular test. Twelve formulations were classified as nonstinging in the human test, and of those ten were negative in the NociOcular test. There was no correlation between the clinical stinging results for the baby formulations and the data generated from other in vitro eye irritation assays (cytosensor microphysiometer, neutral red uptake, EpiOcular, transepithelial permeability). Our data support that the TRPV1 channel is a principal mediator of eye-stinging sensation induced by baby bath and shampoo formulations and that the NociOcular test may be a valuable in vitro tool to predict human eye–stinging sensation.
The National Center for the Replacement, Refinement, and Reduction of Animals in Research's (NC3Rs) latest newsletter is available on the web. The 47th issue includes information on the 3Rs prize, a call for fellowships, information on current vacancies, the CRACK IT 2012 Challenges launch, and two new CRACK IT Solutions. Please click on the link above for access to this newsletter.
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From the ESTIV2012 Organizing Committee - This is a friendly reminder that the Registration Deadline (15th September) for ESTIV2012 is fast approaching. This year the congress will take place October 16-19 in Lisbon, and will be co-hosted by AP Tox. ESTIV2012 will focus on the new developments in toxicity testing and the mechanism of action of chemicals and nanomaterials with a special emphasis on the following themes:
Dermal toxicity
Ocular toxicity
Dermal sensitisation
Innate immune responses in toxicity
Carcinogenicity testing
Reproductive & developmental testing
Systemic toxicity
Computational toxicity & toxicokinetics
Additional information and program is available online
Abstracts for the EUSAAT Annual Congress 2012 are now available to download from the FRAME website. The Congress (5 - 8 September) will focus on new approaches and methodologies — for example, disease models, non-animal tools for basic biomedical research, ‘-omics’ techniques and advanced 3D models. More details can be found on the FRAME website by following the link above. There is also a link to a pdf document of all of the conference abstracts on this page. The abstracts will also appear in a future issue of FRAME's scientific journal ATLA (Alternatives to Laboratory Animals)
The European Union Reference Laboratory for Alternatives to Animal Testing (EURL-ECVAM), the Institute for Health and Consumer Protection, has published two calls for grantholder positions to support EURL-ECVAM in the assessment and validation of alternative methods:
Deadline for the applications is 7 October 2012.
Subscribers to FRAME's Alternatives to Laboratory Animals (ATLA) journal may download the contents. Non-subscribers may purchase the entire issue. In addition, there are a number of free articles for review including: Editorial: The International Transport of Laboratory Animals: No Excuse for Biased Extremism of Any Kind, The Forthcoming Launch of Perspectives in Laboratory Animal Science (PiLAS), and the announcement of the Dorothy Hegarty Award Winners.
Dr. Rodger Curren, President of IIVS, stated that transparency, data sharing and active communication between scientists, industry and regulators is the best way to ensure the intelligent application of science to regulatory policy during a meeting of the American Chemical Society (ACS) yesterday in Philadelphia. Dr Curren described how such an approach was key to the successful use and acceptance of new in vitro ocular models for hazard testing of anti-microbial cleaning products. He said this approach could be modelled for use in the safety assessment of other consumer products, thus supporting the 3Rs approach and avoiding new animal experimentation. To read more from speakers at the meeting, please follow the link above.
People for the Ethical Treatment of Animals (PETA) and the Physicians Committee for Responsible Medicine (PCRM) have submitted public comments on the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM)'s draft 5 year plan. They have listed specific areas in which ICCVAM is not meeting its Congressional mandate to facilitate the acceptance of nonanimal testing methods government-wide. Please follow the link above to view a synopsis of their findings.
The 17th European Congress on Alternatives to Animal Testing (LINZ2012) and the 14th Annual Congress of the European Society for Alternatives to Animal Testing (EUSAAT 2012) are being held September 5-8 at the University of Linz, Austria. Early bird registration ends on Monday, August 13th. The preliminary program is available for review online. Please follow the link above to register for the event.
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 issue is now available at the link above.
The U.S. National Institutes of Health (NIH) has awarded 17 grants aimed at creating 3-D chips with living cells and tissues that accurately model the structure and function of human organs such as the lung, liver and heart. The grants were announced in a July 24th NIH news release (available on the NIH website by clicking on the link above). Once developed, these tissue chips will be tested with compounds known to be safe or toxic in humans. Data from these tests will help identify the most reliable drug safety signals, ultimately advancing research to help predict the safety of potential drugs in a faster, more cost-effective way. The initiative marks the first interagency collaboration launched by the NIH's recently created National Center for Advancing Translational Sciences.
More than 30 percent of promising medications have failed in clinical trials because they are determined to be toxic despite promising preclinical studies in animal models. Tissue chips, which are a newer human cell-based approach, may enable scientists to predict more accurately how effective a therapeutic candidate would be in clinical studies. Tissue chips merge techniques from the computer industry with modern tissue engineering by combining miniature models of living organ tissues on a transparent microchip. Ranging in size from a quarter to a house key, the chips are lined with living cells and contain features designed to replicate the complex biological functions of specific organs.
NIH's newly funded "Tissue Chip for Drug Screening" initiative is the result of collaborations that focus the resources and ingenuity of the NIH, the Defense Advanced Research Projects Agency, and the U.S. Food and Drug Administration. NIH's Common Fund and the National Institute of Neurological Disorders and Stroke led the trans-NIH efforts to establish the program. The NIH plans to commit up to $70 million to the program over five years.
Tissue chips are an example of innovative tools and methodologies that can be used to identify whether substances are likely to be safe or toxic to humans. In its draft 2013-2017 Five-Year Plan, the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) identifies "Promoting the Application and Translation of Innovative Science and Technology" as one of its core strategies to support the development of predictive alternative test methods. Innovative testing approaches such as tissue chips have the potential to more accurately and efficiently identify substances that may present human health hazards, while reducing and ultimately replacing animal use for this purpose.
In an article on AltTox, Nicholas Ball of The Dow Chemical Company describes the connection between REACH testing requirements and the obligation to utilize animal tests only as a last resort. Without simply adopting an unnecessarily conservative assessment of hazards, registrants must make the best use of the various options to reduce the need for new animal studies, and regulatory authorities must take into consideration the animal welfare aspects when reviewing dossiers and deciding on the need for additional studies. All of this must at the same time provide sufficient comfort that the hazards of substances are being identified and addressed as necessary so that human and environmental safety are not adversely impacted. Please follow the link above to AltTox to read the article in its entirety.
An article on Scientist Live discusses the progress made toward replacing animal test methods with alternatives at a recent meeting. SEURAT-1 is the first phase of a longer term research initiative aiming at Safety Evaluation Ultimately Replacing Animal Testing. Although there is an initial focus on chemicals found in cosmetics and personal care products, the methodology and tools being developed by SEURAT-1 scientists are intended for application in a variety of fields. Some of the latest results of SEURAT-1were presented at the Euroscience Open Forum (ESOF) in Dublin on 14 July. The new SEURAT-1 report on progress and related international efforts was launched on this occasion. Please follow the link above to read the entire article and learn more about what was presented in Dublin.
With China poised to accept its first ever non-animal test method for cosmetics by late summer, Dr. Brian Jones of IIVS says don't be surprised by how quickly the authorities accept and implement more. In an article in Cosmetics Design's new series "Voice of the Industry", the Director of Education and Outreach Programs discusses IIVS's role in helping to shape China's attitude to non-animal testing alternatives. Please follow the link above to view the entire article on the cosmetics design website.
The National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) is planning to convene an independent scientific peer review panel to assess the validation status of in vitro tests and integrated non-animal testing strategies proposed for identifying eye injury hazard potential of chemicals and products. The panel will be convened in collaboration with the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM).
NICEATM requests nominations of scientific experts who can be considered for the panel. NICEATM also requests data from substances tested in in vitro tests for identifying eye injury hazard potential. Of particular interest are data generated in the short time exposure (STE) and isolated rabbit eye (IRE) tests and data from approaches using two or more in vitro tests. However, NICEATM also requests data from other in vitro tests, as well as corresponding in vivo data from any ethical human or animal studies or accidental human exposures. For more information about the NICEATM-ICCVAM evaluation of the STE, IRE, and other eye safety test methods and strategies; submission of data; and nominations of scientific experts, please visit the NICEATM-ICCVAM website linked above.
Italy may soon become the European country with the most restrictive legislation on animal experimentation, if a draft law due for discussion in the Senate July 11th is approved in its current form. The Italian government says that it considers the draft “a good balance point”, but scientists fear that the restrictions would hamper biomedical research and damage the country's pharmaceutical industry. The draft being discussed in Italy contains several prohibitions that are not in the parent EU legislation, and that were not part of Italy’s regulations before the directive was adopted. It bans the breeding of primates, cats and dogs for laboratory use, and requires the government to impose “sufficiently cautious” norms for the authorized use of transgenic animals. It also bans experimentation on “anthropomorphic apes”, cats and dogs, unless the tests are mandatory for new drugs to be approved, or are aimed at “improving human health”. Finally, it prohibits all experiments without anaesthesia that cause pain to an animal. For more information, please follow the link above to the article.
Abstracts are being accepted now through July 31st for poster presentations during the first annual meeting of the American Society for Cellular and Computational Toxicology. The meeting will be held Sept. 21 at the Lister Hill Auditorium, National Library of Medicine, NIH in Bethesda, Md. and will feature a plenary lecture by Dr. Melvin Andersen, Associate Director of The Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, and ASCCT Board Member. Abstracts describing original research or policy analyses related to the development or implementation of in vitro and/or in silico toxicological testing methods are being accepted now until July 31. E-mail abstracts of 250 words or less as Microsoft Word documents using an Ariel 10 font to the ASCCT Secretary, Kristie Sullivan. Please include title, all authors (underline presenting author's name) and affiliations following the format of the sample Abstract here. Include email contact information for presenting author. A select number of abstracts will be chosen for oral presentations. Authors will be notified by Aug. 10. For more information on the society or their first annual meeting, please visit the link above
The European Union Reference Laboratory for alternatives to animal testing (EURL ECVAM) has received a Good Laboratory Practice accreditation from the Organization for Economic Co-operation and Development (OECD) for its work in developing alternative methods and approaches. Formally established last year due to the increasing need for new methods to be proposed for validation in the EU, the lab is active in testing topical, systemic and strategic toxicity activities. The OECD, an intergovernmental organization representing 29 industrialized countries in North America, Europe and the Pacific, provide GLP certification to laboratories meeting its guidelines of uniformity, consistency, reliability, reproducibility, quality, and integrity of non-clinical safety tests for chemicals from physio-chemical properties to kinetic and dynamic toxicity. Please click on the link above for more information.
The National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) in collaboration with the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) has developed a draft updated NICEATMICCVAM Five-Year Plan. This document will provide strategic direction for NICEATM and ICCVAM during 2013 to 2017. It outlines how, consistent with ICCVAM's statutory duties and purposes, NICEATM and ICCVAM will contribute to the transformation of safety testing by fostering and promoting the incorporation of scientific advances and innovative technologies into new improved test methods and integrated testing and decision strategies. As announced in the Federal Register, NICEATM and the National Institute of Environmental Health Sciences (NIEHS) request public comments on the draft 2013-2017 Five-Year Plan, which is available on the NICEATMICCVAM website at the link above. Comments are requested by August 13, 2012 and may be submitted online.
Content includes the announcement of our next webinar on skin irritation testing strategies using 3D tissue constructs, an article on the conduct of validation studies, a special workshop in Japan and much more.
Content includes the announcement of our next webinar on skin irritation testing strategies using 3D tissue constructs, an article on the conduct of validation studies, a special workshop in Japan and much more.
Registration for the First Annual Meeting of the American Society for Cellular and Computational Toxicology (ASCCT) is now open! Join your fellow ASCCT members for plenary and member lectures, poster presentations, the election of members to the board of directors, and a social cocktail event at the first annual scientific and business meeting.Abstracts describing original research or policy analyses related to the development or implementation of in vitro and/or in silico toxicological testing methods are being accepted now until July 15. Please visit the ASCCT website for details on registering for the meeting, submitting an abstract, and becoming an ASCCT member.
“Has this method been validated?” “Can you help validate our test?”
At IIVS, we are often asked these and similar questions for a variety of reasons. Sometimes clients would like to know whether a certain in vitro protocol has been validated (is it reproducible and reliable) and will it meet their testing goals. Sometimes they inquire if it is validated for regulatory use. Other times they would like assistance in validating a protocol to screen their unique product candidates. We are also frequently called upon by industry consortia and methods developers to participate in or lead a formal multi-laboratory validation of methods for potential regulatory use. What is assay validation, and what are the goals of a validation? Validation is a planned, detailed, fully documented process for the experimental and statistical evaluation of a proposed method, including the associated prediction model, within the constraints of its intended purpose. Simply put: Does the method do what it is intended to do? Please read the attached article for additional information.
Dr. Brian Jones of IIVS has written an article published at AltTox concerning the current state of cosmetic testing regulations in China. The article discusses the rapid progression of cosmetic regulations from their beginnings in 1990 to the current moves toward acceptance of alternative methods in place of animal testing. To read about the Chinese regulations and the need for sustained effort to assist them in moving towards acceptance of non-animal testing methods, please read the article above.
The Proceedings of the Eighth World Congress on Alternatives and Animal Use in the Life Sciences have now been published and can be accessed on the ALTEX website above. The organizers hope that it will serve as a lasting reminder of the excellent presentations and discussions that took place in Montréal last August. They hope that you will find the papers contained in these Proceedings to be inspiring, and that you will use them to stimulate the further implementation of the Three Rs in your own work. Finally, we look forward to building on the work presented and to meeting each of you again in Prague in 2014 for WC9.
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 439) or the Skin Corrosion Test (OECD TG 431). 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.
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.
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.
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.
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.
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.
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 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.
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!
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!
Source: Presented at the 7th World Congress on Alternatives and Animal Use in the Life Sciences
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.
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!
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.
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
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.
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.
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.
IIVS progress on optimization of Alternative Tests, ICCVAM Expert Panel Reviews BRD Addenda, Standards for In Vitro Tests, and more.
IIVS news on
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 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: 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.
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.
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.
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: 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.
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.
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: 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: 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: 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.
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 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: 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.
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.
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.
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.
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.
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.
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.
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.
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: 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: 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: Eurotox 2012
Authors: Anna Forsby, Kimberly Norman, Johanna EL Andaloussi-Lilja, Jessica Lundqvist, Beata Wojcik, Vincent Walczak, Rodger Curren, Katharine Martin and Neena Tierney
The Transient Receptor Potential Vanilloid type 1 (TRPV1) receptor is one of the most well characterized pain-inducing receptors. The purpose of this study was to predict human eye stinging of 19 baby bath and shampoo formulations by studying TRPV1 activity.
The NociOcular test, a novel recombinant neuronal in vitro model with high expression of functional TRPV1 channels was used to test shampoo formulations containing surfactants, preservatives, and fragrances (sodium laureth sulfate, cocoamidopropylbetaine, cocoglucoside, sodium benzoate, quaternium-15, etc.). The increase in intracellular free Ca2+ was analysed by fluorescence during exposure. TRPV1-specific Ca2+ influx was abolished when the TRPV1 channel antagonist capsazepine was applied to the cells prior to shampoo samples.
The positive control, i.e. adult shampoo, was the most active sample tested in the NociOcular test and also induced the worst stinging sensation . The negative control, i.e. marketed baby shampoo, was negative in both tests. Seven of the formulations induced stinging in the human test, and of those six were positive in the NociOcular test. Twelve of the formulations were classified as non-stinging in the human test, and of those ten were negative in the NociOcular test. None of the established in vitro tests for eye irritation were able to correctly predict the human stinging sensation of the baby products. Our data support that the TRPV1 channel is a principle mediator of eye stinging sensation induced by baby bath and shampoo formulations and that the NociOcular test may be a valuable in vitro tool to predict human eye stinging sensation.
For more information on this project, please contact Dr. Kimberly Norman of IIVS at knorman@iivs.org.
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.
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.
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.
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: 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: 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: 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: 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.
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.
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.
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.
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.
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.
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: 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: 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: 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: 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 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: 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: IIVS announcement
Authors: IIVS
Join us at the 2005 Practical Methods in In Vitro Toxicology Workshop in June.
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: 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.
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.
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.
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.
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 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.
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.
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.
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.
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: 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.
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.; 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.
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: 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: 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.
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
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: 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 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.
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 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.
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: 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: 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: 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: 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: 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.
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: 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: 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.