7th Amendment Research Articles

Vaginal irritation testing-prospects of human organotypic vaginal tissue culture models.

Personal lubricants intended for local or systemic delivery via the vaginal route can induce vaginal irritation, damage the vaginal epithelial barrier which can enhance microbial entry, induce inflammation, and alter the microbiome of the vaginal ecosystem. Therefore, manufacturers of personal lubricants and medical devices are required to show biocompatibility and safety assessment data to support regulatory decision-making within a specified context of use. Furthermore, due to ethical concerns and the introduction of the 7th amendment of the European Council Directive which bans animal testing for cosmetic ingredients and products coupled with the Food and Drug Administration modernization Act 2.0 guidelines, there is a wave of drive to develop alternative test methods to predict human responses to chemical or formulation exposure. In this framework, there is a potential to use three-dimensional organotypic human vaginal-ectocervical tissue models as a screening tool to predict the vaginal irritation potential of personal lubricants and medicaments. To be physiologically relevant, the in vitro tissue models need to be reconstructed using primary epithelial cells of the specific organ or tissue and produce organ-like structure and functionality that recapitulate the in vivo-like responses. Through the years, progress has been made and vaginal tissue models are manufactured under controlled conditions with a specified performance criterion, which leads to a high level of reproducibility and reliability. The utility of vaginal tissue models has been accelerated in the last 20years with an expanded portfolio of applications ranging from toxicity, inflammation, infection to drug safety, and efficacy studies. This article provides an overview of the state of the art of diversified applications of reconstructed vaginal tissue models and highlights their utility as a tool to predict vaginal irritation potential of feminine care products.

Fiscal Federalism: An Analysis of Khyber Pakhtunkhwa's Share in the National Resources in the Post-18th Amendment Period

Decentralization of power and distribution of resources has remained a contentious issue between the Federal government and provincial governments in Pakistan. The 7th NFC award and 18th amendment to the constitution have further strengthened the federalist structure in Pakistan by adopting multiple criteria and ensuring provincial autonomy respectively. The Constitution of 1973 provided a well-structured mechanism for fair and just distribution of resources and inter-governmental harmony in the form of NFC and CCI and offered solutions to the contentious issue of distribution of power. Khyber Pakhtunkhwa is blessed with abundant natural resources in the form of water resources, crude oil production, natural gas and LPG and making extensive contributions to the energy sector of Pakistan. Although the constitution of Pakistan, in theory, is clear regarding revenue distribution the cleavages in its practical implementation in the context of the fiscal relationship between the federal government and Khyber Pakhtunkhwa are creating hurdles in their smooth relation.

A State-of-the-Art Review on the Alternatives to Animal Testing for the Safety Assessment of Cosmetics

Almost a decade after the stipulated deadline in the 7th amendment to the EU Cosmetics Directive, which bans the marketing of animal-tested cosmetics in the EU from 2013, animal experimentation for cosmetic-related purposes remains a topic of animated debate. Cosmetic industry continues to be scrutinised for the practice, despite its leading role in funding and adopting innovation in this field. This paper aims to provide a state-of-the-art review of the field on alternative testing methods, also known as New Approach Methodologies (NAMs), with the focus on assessing the safety of cosmetic ingredients and products. It starts with innovation drivers and global regulatory responses, followed by an extensive, endpoint-specific overview of accepted/prospective NAMs. The overview covers main developments in acute toxicity, skin corrosion/irritation, serious eye damage/irritation, skin sensitisation, repeated dose toxicity, reproductive toxicity/endocrine disruption, mutagenicity/genotoxicity, carcinogenicity, photo-induced toxicity, and toxicokinetics. Specific attention was paid to the emerging in silico methodology. This paper also provides a brief overview of the studies on public perception of animal testing in cosmetics. It concludes with a view that educating consumers and inviting them to take part in advocacy could be an effective tool to achieve policy changes, regulatory acceptance, and investment in innovation.

Alternative in vitro models used in the main safety tests of cosmetic products and new challenges.

Guided by ethical considerations and regulatory requirements such as the 7th Amendment to the European Cosmetics Directive N° 1223/2009, the cosmetic industry has developed and evaluated alternative test strategies such as in vitro assays, in silico approaches for toxicological endpoints and efficacy of cosmetic products and cosmetics ingredients. In consequence, the European Centre for the Validation of Alternative Methods (ECVAM) has proposed a list of validated cell-based in vitro models for predicting the safety and toxicity of cosmetic ingredients. These models have been demonstrated as valuable and effective tools to overcome the limitations of animal in vivo studies. For example, 3D human skin equivalent models are used to evaluate skin irritation potential; and excised human skin is used as the gold standard for the evaluation of dermal absorption. This review presents, in relation to the regulatory requirements, the main alternative in vitro models used in the safety tests of cosmetic products, focusing on skin sensitization, skin corrosion, skin irritation and skin absorption, with advantages and limitations of each model. Recent innovative 3D cell technologies such as Organ-on-a-Chip (OoC) models that can bring significant improvements for toxicology and efficacy testing are also presented. The development of OoC technology is promising for assessing the toxicity of substances contained in cosmetics, particularly for repeated dose toxicity, for which no alternative in vitro methods are currently available. Nevertheless, aside from the challenges, the technology needs to be validated and accepted by regulatory organizations as an effective method. Collaboration between researchers, regulatory organizations and industry would be required to achieve this validation.

Safety Testing of Cosmetic Products: Overview of Established Methods and New Approach Methodologies (NAMs)

Cosmetic products need to have a proven efficacy combined with a comprehensive toxicological assessment. Before the current Cosmetic regulation N°1223/2009, the 7th Amendment to the European Cosmetics Directive has banned animal testing for cosmetic products and for cosmetic ingredients in 2004 and 2009, respectively. An increasing number of alternatives to animal testing has been developed and validated for safety and efficacy testing of cosmetic products and cosmetic ingredients. For example, 2D cell culture models derived from human skin can be used to evaluate anti-inflammatory properties, or to predict skin sensitization potential; 3D human skin equivalent models are used to evaluate skin irritation potential; and excised human skin is used as the gold standard for the evaluation of dermal absorption. The aim of this manuscript is to give an overview of the main in vitro and ex vivo alternative models used in the safety testing of cosmetic products with a focus on regulatory requirements, genotoxicity potential, skin sensitization potential, skin and eye irritation, endocrine properties, and dermal absorption. Advantages and limitations of each model in safety testing of cosmetic products are discussed and novel technologies capable of addressing these limitations are presented.

The Symptoms of Distrust in the Fundamental Law of Hungary and in its 7th Amendment

The Symptoms of Distrust in the Fundamental Law of Hungary and in its 7th Amendment

The EpiOcular Eye Irritation Test (EIT) for hazard identification and labelling of eye irritating chemicals: protocol optimisation for solid materials and the results after extended shipment.

The 7th Amendment to the EU Cosmetics Directive and the EU REACH Regulation have reinforced the need for in vitro ocular test methods. Validated in vitro ocular toxicity tests that can predict the human response to chemicals, cosmetics and other consumer products are required for the safety assessment of materials that intentionally, or inadvertently, come into contact with the eye. The EpiOcular Eye Irritation Test (EIT), which uses the normal human cell-based EpiOcular™ tissue model, was developed to address this need. The EpiOcular-EIT is able to discriminate, with high sensitivity and accuracy, between ocular irritant/corrosive materials and those that require no labelling. Although the original EpiOcular-EIT protocol was successfully pre-validated in an international, multicentre study sponsored by COLIPA (the predecessor to Cosmetics Europe), data from two larger studies (the EURL ECVAM-COLIPA validation study and an independent in-house validation at BASF SE) resulted in a sensitivity for the protocol for solids that was below the acceptance criteria set by the Validation Management Group (VMG) for eye irritation, and indicated the need for improvement of the assay's sensitivity for solids. By increasing the exposure time for solid materials from 90 minutes to 6 hours, the optimised EpiOcular-EIT protocol achieved 100% sensitivity, 68.4% specificity and 84.6% accuracy, thereby meeting all the acceptance criteria set by the VMG. In addition, to satisfy the needs of Japan and the Pacific region, the EpiOcular-EIT method was evaluated for its performance after extended shipment and storage of the tissues (4-5 days), and it was confirmed that the assay performs with similar levels of sensitivity, specificity and reproducibility in these circumstances.

Evaluation of an optimized protocol using human peripheral blood monocyte derived dendritic cells for the in vitro detection of sensitizers: Results of a ring study in five laboratories

Allergic contact dermatitis is a delayed T-cell mediated allergic response associated with relevant social and economic impacts. Animal experiments (e.g. the local lymph node assay) are still supplying most of the data used to assess the sensitization potential of new chemicals. However, the 7th amendment to the EU Cosmetic Directive have introduced a testing ban for cosmetic ingredients after March 2013.We have developed and optimized a stable and reproducible in vitro protocol based on human peripheral blood monocyte derived dendritic cells to assess the sensitization potential of chemicals. To evaluate the transferability and the predictivity of this PBMDCs based test protocol, a ring study was organized with five laboratories using seven chemicals with a known sensitization potential (one none-sensitizer and six sensitizers, including one pro-hapten).The results indicated that this optimized test protocol could be successfully transferred to all participating laboratories and allowed a correct assessment of the sensitization potential of the tested set of chemicals. This should allow a wider acceptance of PBMDCs as a reliable test system for the detection of human skin sensitizers and the inclusion of this protocol in the toolbox of in vitro methods for the evaluation of the skin sensitization potential of chemicals.

Safety Evaluation of Cosmetic Ingredients: In Vitro Opportunities for the Identification of Contact Allergens

Irritant and allergic contact dermatitis are undesired side effects in the development of drugs and cosmetics as well as after contact with environmental or industrial chemicals. Over the last decades, a great deal of progress has been made in the development of alternative In vitro test to assess these issues. Driven by the 7th Amendment to the European Cosmetic Directive, the EU policy on chemicals (the registration, evaluation, authorization and restriction of chemicals (REACH) system), the update of the European legislation on the protection of animals used in research, and emerging visions and strategies for predicting toxicity, in vitro methods are likely to play a major role in the near future. On 12 December 2013, the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM, part of the European Commission Joint Research Centre) published its Recommendation on the Direct Peptide Reactivity Assay (DPRA) for skin sensitization, capable of distinguishing sensitizers from non-sensitizers. Other assays (i.e., KeratinoSens™ assay) will follow shortly. While a number of methods are at various stages of development and use, currently it is not possible to rank chemicals for their sensitizing potency, an issue that is important for a full safety assessment. It is expected that a predictive method to totally replace animal testing will be in the form of a test battery comprising molecular, cell-based, and/or computational methods, the so-called “Integrated Approaches to Testing and Assessment”. This review aims to discuss the state-of-the-art in the field of in vitro assessment of contact sensitizers.

Development and evaluation of an in vitro skin sensitization assay based on primary monocyte-derived dendritic cells (P6376)

Abstract Allergic contact dermatitis is a delayed T-cell mediated allergic response associated with relevant social and economic impacts. Until now, animal experiments (e.g. the local lymph node assay) are supplying most of the data used to assess the sensitization potential of new chemicals. However, becoming effective in March 2013, the 7th amendment to the EU Cosmetic Directive introduced an animal testing ban for the endpoint skin sensitisation for cosmetic ingredients. In vitro alternative methods are thus being actively developed. Although promising results have been obtained with cell lines, their reduced functionality and inherent genomic instability led us to reinvestigate the use of peripheral blood monocyte-derived dendritic cells (PBMDCs) for the establishment of a reliable in vitro sensitization test. We successfully solved the issues associated with the use of primary cells (optimized cytokine concentrations, incubation time, readout, pooled vs. single donors and cytoxicity) resulting in a stable and reproducible protocol. Here, we report on the adaption of the protocol to the 96-well format, allowing for a higher and cost-effective throughput of test substances. Comparative analyses of the effect of several skin sensitizers on CD86 expression display similar results for 24- and 96-well format. Upscaling of the PBMDCs advances its applicability for the reliable detection of human skin sensitizers.

Evaluation of the sensitizing potential of antibiotics in vitro using the human cell lines THP-1 and MUTZ-LC and primary monocyte‐derived dendritic cells

Since the 7th amendment to the EU cosmetics directive foresees a complete ban on animal testing, alternative in vitro methods have been established to evaluate the sensitizing potential of small molecular weight compounds. To find out whether these novel in vitro assays are also capable to predict the sensitizing potential of small molecular weight drugs, model compounds such as beta-lactams and sulfonamides – which are the most frequent cause of adverse drug reactions – were co-incubated with THP-1, MUTZ-LC, or primary monocyte‐derived dendritic cells for 48h and subsequent expression of selected marker genes (IL-8, IL-1β, CES1, NQO1, GCLM, PIR and TRIM16) was studied by real time PCR. Benzylpenicillin and phenoxymethylpenicillin were recognized as sensitizing compounds because they are capable to induce the mRNA expression of these genes in moDCs and, except for IL-8, in THP-1 cells but not in MUTZ-LC. Ampicillin stimulated the expression of some marker genes in moDCs and THP-1 cells. SMX did not affect the expression of these genes in THP-1, however, in moDCs, at least PIR was enhanced and there was an increase of the release of IL-8. These data reveal that novel in vitro DC based assays might play a role in the evaluation of the allergenic potential of novel drug compounds, but these systems seem to lack the ability to detect the sensitizing potential of prohaptens that require metabolic activation prior to sensitization and moDCs seem to be superior with regard to the sensitivity compared with THP-1 and MUTZ-3 cell lines.

Xenobiotic metabolism capacities of human skin in comparison with a 3D‐epidermis model and keratinocyte‐based cell culture as in vitro alternatives for chemical testing: phase II enzymes

The 7th Amendment to the EU Cosmetics Directive prohibits the use of animals in cosmetic testing for certain endpoints, such as genotoxicity. Therefore, skin in vitro models have to replace chemical testing in vivo. However, the metabolic competence neither of human skin nor of alternative in vitro models has so far been fully characterized, although skin is the first-pass organ for accidentally or purposely (cosmetics and pharmaceuticals) applied chemicals. Thus, there is an urgent need to understand the xenobiotic-metabolizing capacities of human skin and to compare these activities to models developed to replace animal testing. We have measured the activity of the phase II enzymes glutathione S-transferase, UDP-glucuronosyltransferase and N-acetyltransferase in ex vivo human skin, the 3D epidermal model EpiDerm 200 (EPI-200), immortalized keratinocyte-based cell lines (HaCaT and NCTC 2544) and primary normal human epidermal keratinocytes. We show that all three phase II enzymes are present and highly active in skin as compared to phase I. Human skin, therefore, represents a more detoxifying than activating organ. This work systematically compares the activities of three important phase II enzymes in four different in vitro models directly to human skin. We conclude from our studies that 3D epidermal models, like the EPI-200 employed here, are superior over monolayer cultures in mimicking human skin xenobiotic metabolism and thus better suited for dermatotoxicity testing.

Vaginal Irritation Models: The Current Status of Available Alternative and In Vitro Tests

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.

Alternative (non-animal) methods for cosmetics testing: current status and future prospects—2010

The 7th amendment to the EU Cosmetics Directive prohibits to put animal-tested cosmetics on the market in Europe after 2013. In that context, the European Commission invited stakeholder bodies (industry, non-governmental organisations, EU Member States, and the Commission's Scientific Committee on Consumer Safety) to identify scientific experts in five toxicological areas, i.e. toxicokinetics, repeated dose toxicity, carcinogenicity, skin sensitisation, and reproductive toxicity for which the Directive foresees that the 2013 deadline could be further extended in case alternative and validated methods would not be available in time. 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 summary, the experts confirmed that it will take at least another 7-9years for the replacement of the current in vivo animal tests used for the safety assessment of cosmetic ingredients for skin sensitisation. However, the experts were also of the opinion that alternative methods may be able to give hazard information, i.e. to differentiate between sensitisers and non-sensitisers, ahead of 2017. This would, however, not provide the complete picture of what is a safe exposure because the relative potency of a sensitiser would not be known. For toxicokinetics, the timeframe was 5-7years to develop the models still lacking to predict lung absorption and renal/biliary excretion, and even longer to integrate the methods to fully replace the animal toxicokinetic models. For the systemic toxicological endpoints of repeated dose toxicity, carcinogenicity and reproductive toxicity, the time horizon for full replacement could not be estimated.

Towards an integrated in vitro strategy for repeated dose toxicity testing

Springer-Verlag 2011 Currently, there is clear political incentive in the EU to minimize the number of animals involved in the chemical risk assessment process, particularly holding true for the cosmetics sector. Stringent requirements of full replacement of animals in safety testing are laid down in the EU Cosmetics Directive 76/768/EEC by the implementation of the 7th Amendment (2003/15/EC), setting a rigid time frame for the availability of valid 3R-alternative methods for the safety testing of cosmetic products and their ingredients (EU 2003). An important step forward to achieve this

EN

The 7th Amendment to the EU's Cosmetic Directive (now recast as Regulation 1223/2009) bans the testing of cosmetic ingredients and products on animals, effective 2009. An extension until 2013 was granted, for marketing purposes only, for three endpoints: repeated dose, toxicokinetics, and reproductive toxicity. If the European Commission determines that alternatives for these endpoints are not likely to be available, it can propose a further extension. To this end, the Commission has instructed experts to produce reports on the status of alternatives for the 2013 deadline. We criticized the draft reports on a number of issues. First, the experts fell into the "high fidelity fallacy trap," i.e. asserting that full replication of the in vivo response, as opposed to high predictivity, is required before an animal test can be considered useful for regulatory purposes. Second, the experts' reports were incomplete, omitting various methods and failing to provide data on the validity, reliability, and applicability of all the methods discussed, regardless of whether the methods were in vivo, in vitro, or in silico. In this paper we provide a summary of our criticisms and provide some of the missing data in an alternative proposal for replacement of animal tests by 2013. It is our belief that use of the Threshold of Toxicological Concern (TTC) will be a useful method to mitigate much animal testing. Alternative approaches for carcinogenicity and skin sensitization could be considered sufficient in the very near future, even though these tests are not listed under the 2013 extension. For repeated dose, toxicokinetics, and reproductive toxicity a combination of in vitro methods may be able to provide appropriate protection for consumers, especially when viewed in the context of the poor predictivity of the animal models they replace. We hope the revised report will incorporate these comments, since a more thorough and positive review is required if the elimination of animal testing for cosmetics in Europe and beyond is to be achieved.

In vitro detection of contact allergens: Development of an optimized protocol using human peripheral blood monocyte-derived dendritic cells

Allergic contact dermatitis is a delayed T-cell mediated allergic response associated with relevant social and economic impacts. Animal experiments (e.g. the local lymph node assay) are still supplying most of the data used to assess the sensitization potential of new chemicals. However, the 7th amendment to the EU Cosmetic Directive will introduce a testing ban for cosmetic ingredients after 2013. In vitro alternative methods are thus being actively developed. Although promising results have been obtained with cell lines, their reduced functionality and inherent genomic instability led us to reinvestigate the use of peripheral blood monocyte-derived dendritic cells (PBMDCs) for the establishment of a reliable in vitro sensitization test. To solve the issues associated with the use of primary cells, the culture and exposure conditions (cytokine concentrations, incubation time, readout, pooled vs. single donors and cytotoxicity) were re-assessed and optimized. Here we propose a stable and reproducible protocol based on PBMDCs. This should allow a wider acceptance of PBMDCs as a reliable test system for the detection of human skin sensitizers and the inclusion of this protocol in an integrated testing strategy.

A Frame Response to the European Commission Consultation on the Draft Report on Alternative (Non-animal) Methods for Cosmetics Testing: Current Status and Future Prospects — 2010

This response on behalf of FRAME to the European Commission's consultation on the five chapters of the Draft Report on Alternative (Non-animal) Methods for Cosmetics Testing: Current Status and Future Prospects--2010, is via a Comment in ATLA, rather than via the template supplied by the Commission. This is principally so that a number of general points about cosmetic ingredient testing can be made. It is concluded that the five draft chapters do not provide a credible basis for the Commission's forthcoming report to the European Parliament and the European Council on the five cosmetic ingredient safety issues for which the 7th Amendment to the Cosmetic Directive's ban on animal testing was postponed until 2013. This is mainly because there is insufficient focus in the draft chapters on the specific nature of cosmetic ingredients, their uses, their local effects and metabolism at their sites of application, and, in particular, on whether their possible absorption into the body would be likely to lead to their accumulation in target sites at levels approaching Thresholds of Toxicological Concern. Meanwhile, there continues to be uncertainty about how the provisions of the Cosmetics Directive should be applied, given the requirements of the REACH system and directives concerned with the safety of other chemicals and products.

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.

The Center for Alternatives to Animal Testing – Europe (CAAT-EU): a transatlantic bridge for the paradigm shift in toxicology

The Center for Alternatives to Animal Testing - Europe (CAAT-EU) was founded based collaboration between the Johns Hopkins Bloomberg School of Public Health and the University of Konstanz. CAAT-EU, housed at the University of Konstanz, will coordinate transatlantic activities to promote humane science in research and education, and participate, as partner or coordinator, in publicly and privately funded European projects. Thomas Hartung will serve as program liaison representing Johns Hopkins University and Marcel Leist as the University of Konstanz liaison. CAAT-EU aims to: 1) Set up transatlantic consortia for international research projects on alternative methods. 2) Establish a CAAT Europe faculty and advisory board composed of sponsor representatives and prominent academics from Europe . 3) Participate in the Transatlantic Think Tank for Toxicology (t4) devoted to conceptual work for the paradigm shift in toxicology. 4) Coordinate a series of information days in Europe on relevant developments in the US, similar to the 2009 series CAAT held in the US on EU issues (one on the 7th Amendment to the EU Cosmetics Directive and one on EU and US chemical regulation). 5) Support ALTEX as the official journal of CAAT and CAAT-EU. 6) Develop strategic projects with sponsors to promote humane science and new toxicology, especially with CAAT faculty members. 7) Develop a joint education program between Johns Hopkins and the University of Konstanz, such as e-courses and the existing Humane Science Certificate program developed by CAAT, a student exchange program, and collaboration with the International Graduate School "Cell-based Characterization of De- and Regeneration" in Konstanz.