Assessment of genotoxicity of herbal medicinal products: Application of the “bracketing and matrixing” concept using the example of Valerianae radix (valerian root)
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An assessment of genotoxicity is a precondition for marketing authorization respectively registration of herbal medicinal products (HMPs), as well as for inclusion into the ‘Community list of herbal substances, preparations and combinations thereof for use in traditional herbal medicinal products’ established by the European Commission in accordance with Directive 2001/83/EC as amended, and based on proposals from the Committee on Herbal Medicinal Products (HMPC).
In the ‘Guideline on the assessment of genotoxicity of herbal substances/preparations’ (EMEA/HMPC/107079/2007) HMPC has described a stepwise approach for genotoxicity testing, according to which the Ames test is a sufficient base for the assessment of genotoxicity in case of an unequivocally negative result. For reducing efforts for testing of individual herbal substances/preparations, HMPC has also developed the ‘guideline on selection of test materials for genotoxicity testing for traditional herbal medicinal products/herbal medicinal products’ (EMEA/HMPC/67644/2009) with the aim to allow testing of a standard range of test materials which could be considered representative of the commonly used preparations from a specific herbal drug according to a ‘bracketing/matrixing’ approach.
The purpose of this paper is to provide data on the practical application of this bracketing and matrixing concept using the example of Valerianae radix, with the intention of facilitating its inclusion in the “Community list”. Five extraction solvents, representing the extremes of the polarity range and including also mid-range extraction solvents, were used, covering the entire spectrum of phytochemical constituents of Valerianae radix, thereby including polar and non-polar constituents. Extracts were tested in the Ames test according to all relevant guidelines. Results were unequivocally negative for all extracts. A review of the literature showed that this result is in accordance with the available data, thus demonstrating the lack of a genotoxic potential.
In conclusion the two guidelines on genotoxicity provide a practically applicable concept. Valerianae radix has no genotoxic potential, supporting its use in HMPs and its inclusion in the Community list.
- Herbal medicinal products;
- Valerian root;
- Bracketing and matrixing;
The frame for the regulation of HMPs in the European Union (EU) is given by directive 2001/83/EC as amended by directive 2004/24/EC. According to regulation (EC) No 726/2004, the committee for herbal medicinal products (HMPC) is responsible for preparing the view of the European Medicines Agency (EMA, formerly EMEA) on herbal medicines (Knöss and Chinou 2012). According to the guideline EMEA/HMPC/32116/2005, for many herbal substances and preparations, used in well-established or traditional herbal medicinal products (HMPs), a safety profile in accordance to modern standards is supported or at least partially substituted by their documented history of medicinal use. Thus, only if a safety concern is recognized or suspected, supporting non-clinical investigations may be needed (EMEA, 2008). In general, the combination of documented experience gained during long-standing use with bibliographic data is the main basis of the non-clinical assessment of traditional and well-established HMPs. Therefore, particular attention should be paid to effects that are difficult or even impossible to detect clinically. They include genotoxicity, carcinogenicity, and toxicity to reproduction. Especially the lack of data from genotoxicity testing may present a safety concern. Thus there is a need for the assessment of the genotoxic potential of herbal preparations. For many active substances data on genotoxicity are missing or data described in the literature are inadequate. If an adequate assessment cannot be made, further genotoxicity testing is required (EMEA, 2006b).
Guidelines on genotoxicity testing of pharmaceuticals, primarily directed to the assessment of new chemically defined active ingredients, have been established by ICH since the 1990s (ICH S2) and have been adopted as EMA guideline (EMA/CHMP/ICH/126642/2008). This guideline describes a battery approach of genotoxicity testing, in which pro- and eukaryotic test systems in in vitro and in vivo are employed ( EMEA, 2008). The basic requirement is to assess genotoxicity initially in a bacterial reverse mutation test, followed by tests in mammalian cells in vitro and a mandatory test in a mammalian model in vivo.
For HMPs, the HMPC has established a stepwise approach, in order to address both scientific aspects of genotoxicity testing and the special needs of HMPs within the current regulatory framework. This approach also includes the documented history of medicinal use, with the guideline EMEA/HMPC/32116/2005 setting the frame and the guideline EMEA/HMPC/107079/2007 giving detailed guidance. The basic assessment is a bacterial reverse mutation test, using a battery of different strains as well as metabolic activation. For technical information on how to perform this test (Ames-test; Salmonella typhimurium mutation assay) the OECD guideline no. 471 ( OECD, 1997a) is referred to. Only in cases of equivocal or positive results, which cannot be explained sufficiently or clearly attributed to specific constituents with a well-known safety profile (e.g. the ubiquitous quercetin or other flavonoids common also in food), additional in vitro tests, e.g. mouse lymphoma cell assay, and, if necessary, also in vivo studies are required.
Usually, European community herbal monographs established by the HMPC for well-established and/or traditional use cover a range of herbal preparations. In order to alleviate the manufacturers task of testing their own specific preparations, a distinct guidance was developed offering a strategy to reduce the number of test materials (EMEA, 2009). This approach suggests testing a representative range of preparations of an herbal drug rather than conducting individual tests as is otherwise required – the so-called ‘bracketing/matrixing’ approach (Wiesner and Knöss, 2010). The main objective of this guideline is to achieve consensus on a standard range of test materials which could be considered representative of the commonly used preparations of a herbal drug, with the intention to facilitate providing data necessary for allowing their entry to the Community list established by decision of the European Commission (2008/911EC).
This guideline mentions the option to extrapolate the results obtained with a specific preparation to closely related preparations. Only in case of extracts prepared with ethanol/water mixtures of substantially different concentrations, the demonstration of the phytochemical similarity of the test materials may be required (EMEA, 2009). This test design also assumes that the genotoxic potential of any intermediate preparation is represented by the results of the extremes tested.
Recently, the conduction of a joint project for testing genotoxicity of selected extracts using this “bracketing and matrixing” concept for more than 30 herbal drugs has been reported (Gaedcke et al., 2009 and Kelber et al., 2012). The present paper shows the results of the practical application of the “bracketing and matrixing” concept on a specific herbal drug, Valerianae radix.