Florian
Weighardt
FLORIAN WEIGHARDT 1 , VALÉRIE ZUANG 2 , SANDRA COECKE 2 , NINA HASIWA 3 , JAMES M. MCKIM 4 , MICHAELA NOLL 5
1. HPC today
2. ECVAM, IHCP, Joint Research Centre (EC)
3. CAAT-Europe, University of Konstanz (D)
4. CeeTox Inc. Kalamazoo, MI (USA)
5. AgenoLab GmbH & Co. KG, Stuttgart (D)
ABSTRACT: In 2013 a total ban of animal testing for cosmetics is foreseen to become operative in the European Union. Up to today it is unclear whether this deadline will be met due to the lack of validated alternatives to a series of essential toxicological tests. Top ranking experts in this innovative field will showcase and discuss the current state of the art during a scientific seminar organized by HPC today on April 18th 2012 at the In Cosmetics fair of Barcelona (ES).
PRESENTATION OF THE SEMINAR
The Registration, Evaluation, Authorization of Chemicals
(REACH) Regulation issued in 2006, the 7 th Amendment of 2003 of the European Cosmetics Directive and the new European
Regulation on cosmetic products issued in 2009 created an unprecedented need for alternatives to animal testing in
Europe.
On one side the REACH Regulation set the bases for a huge testing effort to overcome the lack of safety assessment data for all new chemicals and for approximately 30.000 already existing “old” compounds. The registration process has only recently begun, and the estimates on the real testing demands are still under discussion. However, there is little doubt that systemic toxicity testing will account for more than 95 percent of the testing costs and animal use of REACH. It is clear that testing capacities are challenged and that alternative approaches, especially for systemic toxicities are strongly required.
On the other the 7 th Amendment of the Cosmetics Directive has been stepwise prohibiting animal based testing for the safety assessment of cosmetic products or ingredients foreseeing a total ban in 2009 and in 2013. The new Regulation on Cosmetics
Products of 2009 further developed what provided by the 7 th
Amendment. The scope of these regulatory implementations was to protect and improve the welfare of animals used for experimental purposes by promoting the development and use of scientifically valid methods of alternative testing. The main objective of this Amendment to the European Cosmetics
Directive has been the complete ban of the testing on animals for cosmetic ingredients and products. The way this ban had to be implemented consisted in a phased series of EU testing and marketing bans. The ban on animal testing and sales would start immediately as soon as alternative non-animal tests were available. A complete testing ban was foreseen for 2009, six years after the directive came into force irrespective of the availability of animal-free methods, except for repeated-dose toxicological endpoints (i.e., toxicokinetics, repeated dose toxicity, skin sensitization, carcinogenicity, and reproductive toxicity) where the EU marketing ban has been delayed until
2013 for tests carried out outside the EU.
REACH and the Cosmetics Directive have been and are heavily triggering the development of alternative testing methods to reduce to a minimum the need for animal testing and, in the case of cosmetics, to fully substitute them. Testing methods cover fields like topical sensitation / irritation / corrosion / toxicity, substance absorbtion, systemic toxicity, mutagenesis, carcinogenesis, reproductive toxicity, teratogenesis, toxicokinetics, ecotoxicology and biosafety of biological compounds. Nevertheless, despite the huge efforts to develop alternatives to animal testing toxicokinetics, repeated dose toxicity, carcinogenicity, skin sensitisation, and reproductive toxicity, it is questionable whether the required tests will be all available until the 2013 deadline for the total ban. In the case alternative and validated methods would not be available on time the Directive foresees that the 2013 deadline could be further extended.
Within this context HPC today in cooperation with the organisers of the 2012 In Cosmetics fair to be held in Barcelona, decided to organize a scientific/technical seminar on the state of the art of alternatives to animal testing. The speeches will provide an overview on the regulatory context and the state of the art in this field describing what is available and the latest developments in the setup of the alternatives, including in-silico approaches to the more complex animal testing methodologies, which should be banned from 2013. Speakers from the European Centre for
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the Validation of Alternative Methods (ECVAM) belonging to the European Commission, of the Centre for Alternatives to
Animal Testing - Europe (CAAT-EU) and from the Companies
CeeTox and Ageno Lab will provide a complete overview of the actual state of the art and of future developments.
SPEAKERS AND ABSTRACTS
Current status of alternative methods in the field of cosmetics
By Valérie Zuang. Competence Group
Leader – Coordination of Validation
Projects on Alternative Methods - ECVAM
- Institute for Health and Consumer
Protection - DG Joint Research Centre –
European Commission.
There has been a continuous effort at
EU level to find alternative approaches which avoid testing on animals wherever possible. The “Three
Rs Principle” (replacement, reduction and refinement of animal use) is present in all relevant EU legislations.
The 7 th amendment to the EU Cosmetics Directive prohibits to market animal-tested cosmetics in Europe after 2013. The
Directive further foresees that the 2013 deadline could be extended in case alternative and validated methods would not be available in time. The European Commission regularly reports on progress and compliance with the deadlines as well as possible technical difficulties in complying with this ban.
In 2010 and in view of the 2013 marketing ban, ECVAM was asked to coordinate and chair a working process involving
39 scientists nominated by stakeholders. The selected experts were asked to analyse the status and prospects of alternative methods and to provide a scientifically sound estimate of the time necessary to achieve full replacement of animal testing in the fields of repeated-dose toxicity, toxicokinetics, reproductive toxicity, skin sensitisation and carcinogenicity. This presentation will provide information on the outcome of this review and some proposals on the way forward.
Biotransformation: still a bottleneck for in vitro kinetic and dynamic approaches
By Sandra Coecke. Head ECVAM - In-
House Validation and Training Laboratory
- ECVAM - Institute for Health and
Consumer Protection - DG Joint Research
Centre – European Commission.
Current European legislations for cosmetics and chemicals emphasise the need for the development of alternative methods and integrated nonanimal test strategies. Knowledge on absorption, distribution, metabolism and excretion of a substance is a prerequisite when assessing the safety of consumers, patients and the environment.
A lot of the information can be generated at tissue, cell or subcellular level by using in vitro and in silico test methods and use the information gained as input parameters for physiologicallybased toxicokinetic modelling (PBTK). Such computer modelling avoids unnecessary animal experimentation and improves the predictivity of in silico (QSAR) and in vitro tests by integrating the results of various in vitro and non-testing approaches.
Essential input parameters for such models rely on the availability of reliable and relevant human metabolic competent sources, modelling the process of xenobiotic biotransformation, already at the level of the absorption barriers. The metabolic stability of a molecule and the rate of its biotransformation can affect the potential for its bioaccumulation, and whether it is likely to become more or less toxic, without it necessarily being converted into a more reactive metabolite.
For the in vitro dynamics experiments, it is a prerequisite to know whether the cell or tissue is exposed to the parent compound and/or its metabolites. This information is required upfront and can be obtained from toxicokinetic alternative methods that identify the main metabolites and the clearance rates of the parent compound and/or its metabolites when human exposure conditions are applied.
Although such simple in vitro screening tools for assessing metabolic stability and metabolic routes have been used for a long time (e.g. pharmaceutical sector), for the quantitative extrapolation of such in vitro observations to the in vivo consequences a substantial amount of work needs still to be done. A very concrete guidance to test developers, toxicologists, safety assessors and regulators on which priorities to focus will be needed in order to make further progress with kinetic and dynamic models based on in silico and in vitro input parameters.
Systemic toxicity – A possible roadmap
By Nina Hasiwa, Scientific Officer, CAAT-
Europe, University of Konstanz; CEO,
AtaX-Advice – Alternatives to animal
Xperiments - Konstanz, Germany.
In October 2011 an expert workshop on a “Roadmap for Systemic Toxicity” took place, organized by CAAT-Europe, where five white papers: Reproductive Toxicity,
Carcinogenicity, Sensitization, Repeated
Dose Toxicity, and Toxicokinetics were presented and discussed.
These will be published in January 2012 in ALTEX and presented in Brussels in March 2012. The talk will summarize the outcome and give an overview on the proposed measures to further the idea of working towards testing strategies for systemic toxicity without the use of animals.
Understanding alternative methods for the evaluation of skin sensitization
By James M. McKim. Chief Scientific
Officier and Founder – CeeTox Inc.
A major concern for the cosmetic industry is product induced skin sensitization. It is there necessary to test chemicals used in cosmetics or the finished products for the potential to cause dermal sensitization.
Historically, this test has been done in animals using the mouse local lymph node assay (LLNA) or the guinea pig maximization test (GPMT). The 7 th amendment to the
Cosmetics Directive has called for a ban on the use of animals for standard safety testing of cosmetics and the ingredients used in cosmetics. The deadline for the skin sensitization test is scheduled to go into effect in 2013. This has prompted the development and validation of several new non-animal alternative methods. Many of these are being evaluated by
ECVAM and should provide good alternative methods for the industry. This presentation will focus on providing the attendee with information on the latest non-animal alternative methods, how they work, who can perform them, how to interpret the data obtained and their strengths and weaknesses.

primary cells. Detecting in real-time impact and fast effects of compounds are shown and investigations of results is without delay. Further information is obtained using Gene expression, analytic of specific proteins and immunehistological analysis. With the data from screening systems it is more effective to go further by using 3D reconstructed skin models for more detailed information like barrier function and histological results.
Integrated 2d and 3d skin test systems for in vitro -toxicology and compound testing of cosmetic, chemical and medical device
By Michaela Noll Head of Department of Cell Systems Tissue engineering
AgenoLab GmbH & Co. KG, Stuttgart,
Germany.
The effectiveness of in vitro testing methods as alternatives to animal testing is enhanced by using and integrating different suitable in vitro models and test strategies that allow the prediction of potentially toxic effects and compound testing of consumer products. To generate a high degree of information investigations could be performed with different systems on cellular or tissue-like level. This is achieved by the use of primary human skin cells, cell lines, reconstructed full thickness skin models and human ex vivo skin. Beside classical assays like cytotoxicity MTT Test, we operate as new in vitro tool the Real-Time Cell Monitoring by impedance measurement with xCELLigence Cell Analyzer Systems
(Roche). This label free cell monitoring provides real-time information regarding for example proliferation, viability, acute and chronic toxicity, adhesion and spreading, dose dependent compound effects and quality control of
REFERENCES AND NOTES
Basic reviews on the state of the art
- S. Adler et al., “Alternative (non-animal) methods for cosmetics testing: current status and future prospects, 2010", Arch Toxicol ., 85 , pp. 367-
485(2011).
http://ecvam.jrc.it/ft_doc/Alternatives_for_cosmetics_report.pdf
- A. David Basketter et al., “t4 Report: A Roadmap for the Development of Alternative (Non-Animal) Methods for Systemic Toxicity Testing”, ALTEX ,
29 , pp. 5-91 (2012).
http://altweb.jhsph.edu/news/2012/altex29_1.html
Further papers on the topic from HPC today
- F. Weighardt, “Alternative testing methods in Europe - Reducing animal testing needs in the era of REACH”, HPC today , 2 , pp. 6-9 (2009).
http://hpc-today.teknoscienze.com/pdf/Weighardt_AlternTesting_
HPC2-09.PDF
- F. Weighardt, “Rethinking the toolbox of toxicology. The Centre for
Alternative to Animal Testing –Europe (CAAT-EU)”, HPC today , 2 , pp. 36-
39 (2010).
http://hpc-today.teknoscienze.com/pdf/weighardt2_HPC2-10.PDF
- J.M. McKim, “Progress toward the development of novel alternative testing strategies - Assessing the safety of cosmetics without the use of animals”, HPC today , 3 , pp. 30-32 (2011).
http://hpc-today.teknoscienze.com/pdf/McKIM_hpc3_2011.PDF
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