Julia Scheel

A tiered approach to the use of alternatives to animal testing for the safety assessment of cosmetics: eye irritation.

Evaluation of the skin irritancy and corrosivity potential of an ingredient is a necessity in the safety assessment of cosmetic ingredients. To date, there are two formally validated alternatives to the rabbit Draize test for skin corrosivity in place, namely the rat skin transcutaneous electrical resistance (TER) assay and the Human Skin Model Test using EpiSkin, EpiDerm and SkinEthic reconstructed human epidermal equivalents. For skin irritation, EpiSkin, EpiDerm and SkinEthic are validated as stand-alone test replacements for the rabbit Draize test. Data from these tests are rarely considered in isolation and are evaluated in combination with other factors to establish the overall irritating or corrosive potential of an ingredient. In light of the deadlines established in the Cosmetics Directive for cessation of animal testing for cosmetic ingredients, a COLIPA scientific meeting was held in Brussels on 30th January, 2008 to review the use of alternative approaches and to set up a decision tree approach for their integration into tiered testing strategies for hazard and safety assessment of cosmetic ingredients and their use in products. In conclusion, the safety assessments for skin irritation/corrosion of new chemicals for use in cosmetics can be confidently accomplished using exclusively alternative methods.

Use of human in vitro skin models for accurate and ethical risk assessment: metabolic considerations.

Several human skin models employing primary cells and immortalized cell lines used as monocultures or combined to produce reconstituted 3D skin constructs have been developed. Furthermore, these models have been included in European genotoxicity and sensitization/irritation assay validation projects. In order to help interpret data, Cosmetics Europe (formerly COLIPA) facilitated research projects that measured a variety of defined phase I and II enzyme activities and created a complete proteomic profile of xenobiotic metabolizing enzymes (XMEs) in native human skin and compared them with data obtained from a number of in vitro models of human skin. Here, we have summarized our findings on the current knowledge of the metabolic capacity of native human skin and in vitro models and made an overall assessment of the metabolic capacity from gene expression, proteomic expression, and substrate metabolism data. The known low expression and function of phase I enzymes in native whole skin were reflected in the in vitro models. Some XMEs in whole skin were not detected in in vitro models and vice versa, and some major hepatic XMEs such as cytochrome P450-monooxygenases were absent or measured only at very low levels in the skin. Conversely, despite varying mRNA and protein levels of phase II enzymes, functional activity of glutathione S-transferases, N-acetyltransferase 1, and UDP-glucuronosyltransferases were all readily measurable in whole skin and in vitro skin models at activity levels similar to those measured in the liver. These projects have enabled a better understanding of the contribution of XMEs to toxicity endpoints.

A tiered approach to the use of alternatives to animal testing for the safety assessment of cosmetics: genotoxicity. A COLIPA analysis.

For the assessment of genotoxic effects of cosmetic ingredients, a number of well-established and regulatory accepted in vitro assays are in place. A caveat to the use of these assays is their relatively low specificity and high rate of false or misleading positive results. Due to the 7th amendment to the EU Cosmetics Directive ban on in vivo genotoxicity testing for cosmetics that was enacted March 2009, it is no longer possible to conduct follow-up in vivo genotoxicity tests for cosmetic ingredients positive in in vitro genotoxicity tests to further assess the relevance of the in vitro findings. COLIPA, the European Cosmetics Association, has initiated a research programme to improve existing and develop new in vitro methods. A COLIPA workshop was held in Brussels in April 2008 to analyse the best possible use of available methods and approaches to enable a sound assessment of the genotoxic hazard of cosmetic ingredients. Common approaches of cosmetic companies are described, with recommendations for evaluating in vitro genotoxins using non-animal approaches. A weight of evidence approach was employed to set up a decision-tree for the integration of alternative methods into tiered testing strategies.

Cross-Sector Review of Drivers and Available 3Rs Approaches for Acute Systemic Toxicity Testing

Acute systemic toxicity studies are carried out in many sectors in which synthetic chemicals are manufactured or used and are among the most criticized of all toxicology tests on both scientific and ethical grounds. A review of the drivers for acute toxicity testing within the pharmaceutical industry led to a paradigm shift whereby in vivo acute toxicity data are no longer routinely required in advance of human clinical trials. Based on this experience, the following review was undertaken to identify (1) regulatory and scientific drivers for acute toxicity testing in other industrial sectors, (2) activities aimed at replacing, reducing, or refining the use of animals, and (3) recommendations for future work in this area.

Eye irritation potential: Usefulness of the HET-CAM under the Globally Harmonized System of Classification and Labeling of Chemicals (GHS)

Extensive research has been conducted over the past decades to develop alternatives to the rabbit eye irritation test (Draize test) used in a regulatory context to assess eye irritation potentials. Although no single in vitro test has emerged as being completely acceptable for full replacement, various tests are considered to be suitable and are regularly used to assess certain aspects. Amongst these, the Hens Egg Test Chorioallantoic Membrane (HET-CAM) has gained regulatory acceptance in various countries to classify severe eye irritants. In this retrospective study, historical eye irritation data (in vivo and in vitro) from 137 samples (approx. 75% non-irritants; 25% (severe) irritants) tested both in the HET-CAM and Draize eye test was compared with regard to the predicted eye irritation classes under the GHS and the traditional EU classification system (DSD).The overall concordance was in the range of 80-90%. A high specificity (96-98%, depending on the classification system and the chosen discrimination) but rather low sensitivity (48-65%) was observed. The study indicates that HET-CAM results are useful as part of weight-of-evidence assessments or in tiered approaches to assess eye irritation potentials rather than as stand-alone classification method.

Report from the EPAA workshop: In vitro ADME in safety testing used by EPAA industry sectors

There are now numerous in vitro and in silico ADME alternatives to in vivo assays but how do different industries incorporate them into their decision tree approaches for risk assessment, bearing in mind that the chemicals tested are intended for widely varying purposes? The extent of the use of animal tests is mainly driven by regulations or by the lack of a suitable in vitro model. Therefore, what considerations are needed for alternative models and how can they be improved so that they can be used as part of the risk assessment process? To address these issues, the European Partnership for Alternative Approaches to Animal Testing (EPAA) working group on prioritization, promotion and implementation of the 3Rs research held a workshop in November, 2008 in Duesseldorf, Germany. Participants included different industry sectors such as pharmaceuticals, cosmetics, industrial- and agro-chemicals. This report describes the outcome of the discussions and recommendations (a) to reduce the number of animals used for determining the ADME properties of chemicals and (b) for considerations and actions regarding in vitro and in silico assays. These included: standardisation and promotion of in vitro assays so that they may become accepted by regulators; increased availability of industry in vivo kinetic data for a central database to increase the power of in silico predictions; expansion of the applicability domains of in vitro and in silico tools (which are not necessarily more applicable or even exclusive to one particular sector) and continued collaborations between regulators, academia and industry. A recommended immediate course of action was to establish an expert panel of users, developers and regulators to define the testing scope of models for different chemical classes. It was agreed by all participants that improvement and harmonization of alternative approaches is needed for all sectors and this will most effectively be achieved by stakeholders from different sectors sharing data.

Risk assessment of amorphous silicon dioxide nanoparticles in a glass cleaner formulation

Abstract Since nanomaterials are a heterogeneous group of substances used in various applications, risk assessment needs to be done on a case-by-case basis. Here the authors assess the risk (hazard and exposure) of a glass cleaner with synthetic amorphous silicon dioxide (SAS) nanoparticles during production and consumer use (spray application). As the colloidal material used is similar to previously investigated SAS, the hazard profile was considered to be comparable. Overall, SAS has a low toxicity. Worker exposure was analysed to be well controlled. The particle size distribution indicated that the aerosol droplets were in a size range not expected to reach the alveoli. Predictive modelling was used to approximate external exposure concentrations. Consumer and environmental exposure were estimated conservatively and were not of concern. It was concluded based on the available weight-of-evidence that the production and application of the glass cleaner is safe for humans and the environment under intended use conditions.

Optimised testing strategies for skin sensitization--the LLNA and beyond.

As toxicology in the 21st century progresses towards a future which aims at avoiding the use of in vivo testing, the endpoint of skin sensitisation can now be found in the front line. Accordingly, it was appropriate for several industry sectors to meet and review what has been learned from the currently most widely used in vivo method, the local lymph node assay (LLNA), and to consider the status of progress as we attempt to move beyond that test. No toxicology test is perfect, an experience brought into focus by issues of false positives and, to a lesser extent, false negatives in the LLNA. Use of weight of evidence arguments for classification and labelling, as well as for risk assessment was emphasised and it was also noted that a sufficient body of evidence now exists for conduct of methods other than the LLNA for carefully defined chemical classes. In terms of in vitro alternatives, progress towards methods which will deliver mainly hazard identification is being made, with some entering the final stages of validation, whereby (Q)SAR tools still need improvement to be used on a large scale in practise. As various other challenges also remain, e.g. testing lipophilic substances, as well as the development of non-animal methods which deliver reliable information on potency for risk assessment, these will remain a topic for continuing research and development.

Integrated risk assessment of a hydroxyapatite–protein-composite for use in oral care products: A weight-of-evidence case study

Risk assessment of cosmetic ingredients represents a regulatory standard requirement in Europe and other regions. An integrated approach was designed to assess the safety of HPC, a particulate composite of hydroxyapatite and protein (gelatin) for use in oral care products, employing a weight-of-evidence assessment and considering specific physico-chemical properties and exposure conditions. An initial evaluation of the constituents suggested that their chemical nature does not represent a particular health hazard per se. Hydroxyapatite is the main component of teeth and bones in mammals; gelatin is used in food and assumed to be safe once a BSE/TSE risk has been excluded. In vitro screening tests were chosen to further evaluate the biocompatibility: Hens egg test-chorioallantoic membrane (HET-CAM) to assess irritating effects towards mucous membranes; MTT cytotoxicity test with 3T3 fibroblasts; human corneal epithelial models to investigate inflammatory mediators and cytotoxicity; macrophage assays to measure cytotoxicity, inflammatory mediators and oxidative stress. Together with results from clinical studies, exposure estimates and analyses of kinetic properties, the presented information provides sound evidence to support the safe use of HPC. This is an example of a risk assessment for cosmetic use of small particles without the need for additional animal studies.

Classification and labeling of industrial products with extreme pH by making use of in vitro methods for the assessment of skin and eye irritation and corrosion in a weight of evidence approach

Classification and labeling of products with extreme pH values (≤ 2 or ≥ 11.5) is addressed in chemicals legislation. Following determination of pH and alkaline/acid reserve, additional in vitro tests are needed, especially to substantiate results less than corrosive. However, only limited experience with the practical application of in vitro methods to determine appropriate classifications for pH extreme products is available so far. Expert judgment and weight of evidence are given major roles under the globally harmonized system of classification and labeling of chemicals (GHS) and should be performed on a sound data basis. We have used a tiered testing strategy to assess 20 industrial products (cleaning and metal pretreatment) regarding their corrosive and irritating properties towards human skin models in vitro in the EpiDerm skin corrosion and/or skin irritation test. Nine dilutions of individual compounds were additionally tested. Non-corrosive samples were tested in the Hens egg test chorioallantoic membrane (HET-CAM). We demonstrate how data is combined in a weight of evidence expert judgment, and give examples of classification decisions. To our knowledge this is the first comprehensive analysis of industrial products with extreme pH values to determine irritating and corrosive properties by making use of in vitro methods in a weight of evidence approach.