7 genotox-mutagen en

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Establishment of timetables for the phasing out of animal experiments for cosmetics

Genotoxicity/mutagenicity

Daniela Maurici2 , Marilyn Aardema1, Raffaella Corvi2, Marcus Kleber3, Cyrille Krul4; Christian Laurent5, Nicola Loprieno6, Markku Pasanen7, Stefan Pfuhler8, Barry Phillips9,

Enrico Sabbioni2, Tore Sanner10, Philippe Vanparys11,

1Procter and Gamble, USA; 2ECVAM-JRC, Italy; 3Cognis Deutschland GmbH & Co. KG, Germany; 4TNO Nutrition and Food Research, The Netherland; 5SCCNFP and EFSA, Bruxelles, Belgium; 6University of Pisa, Italy; 7National Agency for Medicines, Helsinki and Department of

Pharmacology and Toxicology, University of Oulu, Finland; 8Wella AG, Germany; 9Research Animals Department RSPCA, UK; 10Institute for Cancer Research, Norway; 11Johnson and

Johnson, Belgium.

General considerations

Mutagenicity refers to the induction of permanent transmissible changes in the structure of the genetic material of cells or organisms. These changes (mutations) may involve a single gene or a block of genes. Genotoxicity is a broader term that refers to the ability to interact with DNA and/or the cellular apparatus that regulates the fidelity of the genome, such as the spindle apparatus and topoisomerase enzymes. Genotoxicity and mutagenicity testing are an important part of the hazard assessment of chemicals for regulatory purposes. To assess genotoxicity and/or mutagenicity, different endpoints must be taken into considerations: beside point mutations induction, a compound can induce changes in chromosomal number (polyploidy or aneuploidy) or in chromosome structure (breaks, deletions, rearrangements). However, aneuploidy can arise as a result of both genotoxic and non-genotoxic events, since loss of chromosomes can be caused either by direct effects on the chromosome to produce an acentric fragment, or by interference with the site of attachment of the chromosome on the spindle. Due to the diversity of the endpoints, it is then clear that the potential genotoxicity and/or mutagenicity of a compound cannot be assessed by a single assay system. For this reason, the group of experts has attempted to suggest a strategy to better investigate the mutagenic and/or genotoxic potential of the cosmetic products taking into consideration the needs of the cosmetic industry.

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1. State of the art in the field of genotoxicity and mutagenicity tests in the view of the 7th Amendment

The cosmetic industry is committed to eliminate animal testing as soon as this is scientifically possible but is also committed to the highest safety standards for its products. It should not be forgotten that mutations and tumour induction are the most severe toxic effects since they are irreversible and very harmful to humans. The in vitro tests determine the potential of a compound to be mutagenic/genotoxic (= hazard identification). There is currently no single validated test that can provide information on all three end-points namely gene mutations, clastogenicity and aneuploidy. As a consequence, a battery of tests is needed to determine the genotoxic and mutagenic profile of a compound.

Although several in vitro tests are routinely used and accepted by regulatory authorities, they present crucial limitations which affect the usefulness of the assays to predict mutagenicity/genotoxicity potential of a substance in vivo in mammals and especially in humans. These limitations in general are: - lack of a “human like” metabolic capacity of the cell lines used - absence of toxicokinetics - oversensitivity compared to in vivo situations – low specificity - sometimes the use of cell lines that are not relevant to predict genotoxic endpoints at target organs - in repeated dosing, the target organ of mutagenicity/genotoxicity may be different than the area of application (hair, skin). Due to these limitations, no single in vitro test can fully replace an existing in vivo animal test yet. Therefore, a battery of tests is needed and/or it is necessary to optimise existing in vitro tests and /or develop new tests that focus on target cells.

The experts felt the necessity to first establish a strategy that would ultimately lead to partial animal replacement. This led to the identification of the testing gaps, namely new tests that need to be developed to lead to full replacement of animal testing.

The focus of this report is on dermally applied cosmetics since this is the largest category, though many of the same considerations would apply to cosmetic products applied via other routes (i.e. orally).

2. Proposed strategy

Strategy is divided in 4 stages: - Stage 1 characterizes the substance based on existing data and knowledge - Stage 2 is a basic in vitro test battery for hazard identification - Stage 3 is a follow up stage in in vitro model systems. This stage is reached if one or

more tests are positive in Stage 2 - Stage 4 is in vivo. This stage is reached if one or more tests in Stage 3 are positive

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Stage 1 It is important at this stage to collect information about the chemical characteristics of the compound and on its skin absorption using also analysis databases and applying computer–based approaches. If it can be proven that there is no dermal absorption and that the compound does not reach the basal cell layer of the skin, mutagenicity/genotoxicity testing is not required. Stage 2 This stage consists of an in vitro test battery for hazard identification. Battery of tests: - Ames test (B13-14/TG471) - Gene mutation test in mammalian cells (preferably mouse lymphoma test) - Micronucleus test and/or chromosomal aberration (preference for micronucleus as it detects not only clastogens but also aneugens more directly than in the metaphase assay) If UV-exposure is expected and the compound can be photoactivated, screening for photogenotoxicity is needed. Photomutagenicity in bacteria or mammalian cells testing is warranted for those chemicals that absorb light in the wavelength of 290 - 700 nm and are used as leave-on products All tests of the basic package should be performed. Although definitive proof of non-mutagenicity/non-genotoxicity is not possible, a compound could be operationally classified as a non-mutagen for human cells if all the tests of the basic battery gave valid negative results. With negative results in the basic battery, further testing may not be requested. Positive results in one or more of the tests trigger further testing to elucidate the mechanism of action in stage 3. Stage 3 At this point, the strategy is to focus on hazard identification in target cells in vitro. Stage 3 is supposed to act as an interme diate step which should, if it can be successfully validated, be able to eliminate "false positive" results from Stage 2. The battery of tests suggested here need to be developed and/or validated Skin cells are the site of the first contact for most cosmetics and are therefore considered to have a high level of exposure. The proposed tests are: - Comet assay in primary skin cells or models - Micronucleus test in primary skin cells or models (if chromosomal aberrations or micronuclei are induced in Stage 2) For photo-mutagenicity/genotoxicity, similar tests can be considered: - Photo-Comet assay on primary skin cells or models - Photo-micronucleus test on primary skin cells or models If the test(s) performed in stage 3 is negative, further testing should not be necessary. For non-dermal cosmetics, new tests such as primary cells or models would need to be developed and validated for the assessment of mutagenicity/genotoxicity.

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Stage 4 in vivo if necessary In vivo tests commonly used by cosmetics industries are: - classical in vivo micronucleus test (B12-TG 474) - unscheduled DNA synthesis (UDS) with mammalian liver cells (B39-TG 486) In vivo test that are occasionally used by cosmetics are: - in vivo Comet assay - bone marrow chromosome aberration test (B11-TG 475) - transgenic mutagenicity models (BigBlue, Mutamouse). These models may be appropriate for the determination of genotoxic or mutagenic effects (DNA strand breaks or gene mutations, respectively) in skin cells. Moreover, some testing strategies have been suggested by SCCNFP:

- SCCNFP Recommended strategy for testing hair dyes (SCCNFP/0720/03, 24-25 June 2003) - SCCNFP Notes of Guidance for the testing of cosmetics ingredients and their safety evaluation (SCCNFP/06903, 20 October 2003) - SCCNFP Mutagenicity/genotoxicity tests recommended for the safety testing of Cosmetics Ingredients to be included in the Annexes to Council Directive 76/768/EEC (SCCNFP/0755/03) References: - Anderson D. and Plewa MJ. (1998). The international “Comet assay workshop”, Mutagenesis 13,67-73. - Baker RS. et al. (1992). Tumorigenicity of cyclopenta[a]phenanthrene derivatives and micronucleus induction in mouse skin. Carcinogenesis, Mar; 13(3): 329-32, - Criswell KA. et al, (2003). Validation of a flow cytometric acridine orange micronuclei methodology in rats. Mutat Res., 528, 1-18 -Haesen S. et al. (1993). Induction of micronuclei and karyotype aberrations during in vivo mouse skin carcinogenesis. Carcinogenesis, Nov; 14(11): 2319-27. - He SI. and Baker RS. (1989). Initiating carcinogen, triethylenemelamine, induces micronuclei in skin target cells. Environ Mol Mutagen.;14(1):1-5. - Torous DK. et al, (2003). Comparative scoring of micronucleated reticulocytes in rat peripheral blood by flow cytometry and microscopy. Toxicol. Sci. 74 (2): 309-314

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3. Inventory of methods currently available

In vitro

# Annex V EC

#OECD TG Name of the test Endpoint

B 13-14 471 Bacterial Reverse Mutation test (Ames test)

Gene mutations in bacteria

B 10 473 Mammalian chromosome aberration test

Chromosome aberrations

B 17 476 Mammalian cell gene mutation test (mouse lymphoma test)

Gene mutations

B 19 479 Sister chromatid exchange assay in mammalian cells (SCE)

Mammalian DNA damage

B 15 480 Saccharomyces cerevisiae gene mutation assay

Gene mutations in yeast

B 16 481 Saccharomyces cerevisiae mitotic recombination assay

Recombination in yeast

B 18 482 Unscheduled DNA synthesis (UDS) in mammalian cells

Mammalian DNA damage in liver cells

In vivo

# Annex V EC

#OECD TG Name of the test Endpoint

B 12 474 Mammalian erythrocyte micronucleus test

Structural and numerical chromosome aberrations in somatic cells

B 11 475 Mammalian bone marrow chromosome aberration test

Chromosome aberrations

B 20 477 Sex-linked recessive lethal test in Drosophila Melanogaster

Gene mutations in germ line

B 22 478 Rodent dominant lethal test Chromosome aberrations and/or gene mutations in germinal tissue

B 23 483 Mammalian spermatogonial chromosome aberration test

Inheritable chromosome aberrations

B 24 484 Mouse spot test Mutagenicity in foetal cells B 25 485 Mouse heritable translocation assay Heritable chromosome

aberrations B 39 486 Unscheduled DNA synthesis (UDS)

test with mammalian liver cells Mammalian DNA damage in liver cells

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4. Inventory of the alternative methods currently available Mammalian chromosome aberration test B. 10/OECD TG # 473 Short description, scientific relevance and purpose The purpose of the in vitro chromosomal aberration test is to identify agents that cause structural chromosome aberrations in cultured mammalian cells. In addition, numerical chromosome changes such as polyploidy and duplication can be measured. Structural aberrations may be of two types, chromosome or chromatid. With the majority of chemical mutagens, induced aberrations are of the chromatid type, but chromosome-type aberrations also occur. The in vitro chromosome aberration test may employ cultures of established cell lines, cell strains or primary cell cultures. This test is used to screen for possible mammalian mutagens and carcinogens. Many compounds that are positive in this test are mammalian carcinogens; however, there is not a perfect correlation between this test and carcinogenicity. Developer of the method Evans HJ (1976) Known users Widely used by industry, CROs and academics Status of validation and/or standardization Worldwide accepted by regulatory authorities Field and limitations of applications See general limitations for in vitro tests Recommendations of use in the view of animal replacement As part of an in vitro test battery Ongoing development Ready to use References - Evans, H.J. (1976). Cytological Methods for Detecting Chemical Mutagens. Chemical mutagens, Principles and Methods for their Detection, Vol. 4, Hollaender, A. (ed) Plenum Press, New York and London, pp. 1-29. -Galloway, S.M., et al, (1978). Chromosome aberration and sister chromatic exchanges in Chinese hamster ovary cells: Evaluation of 108 chemicals. Environs. Molec. Mutagen 10 (suppl.10), 1-175. - Huang, Y., et al, (1983). Aphidicolin - induced endoreduplication in Chinese hamster cells. Cancer Res., 43, 1362-1364. - Locke-Huhle, C. (1983). Endoreduplication in Chinese hamster cells during alpha-radiation induced G2 arrest. Mutation Res., 119, 403-413. - Ishidate, M.Jr. and Sofuni, T. (1985). The in Vitro Chromosomal Aberration Test Using Chinese Hamster Lung (CHL) Fibroblast Cells in Culture. Progress in Mutat. Res, Vol. 5, Ashby, J. et al., (Eds) Elsevier Science Publishers, Amsterdam-New York-Oxford, 427-432.

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- Morita, T., et al, (1992). Clastogenicity of low pH to Various Cultured Mammalian Cells. Mutat. Res., 268, 297-305. - Richardson, et al, (1989). Analysis of Data from In Vitro Cytogenetic Assays. In: Statistical Evaluation of Mutagenicity Test Data. Kirkland, D.J., (ed) Cambridge University Press, Cambridge, pp. 141-154. - Scott, D., et al, (1991). Genotoxicity under Extreme Culture Conditions. A report from ICPEMC Task Group 9. Mutat. Res,. 257, 147-204. Bacterial reverse mutation test B 13-14/OECD TG# 471 Short description, scientific relevance and purpose The bacterial reverse mutation test uses amino-acid requiring strains of Salmonella typhimurium and Escherichia coli to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs. The principle of this bacterial reverse mutation test is that it detects mutations, which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesise an essential amino acid. The revertant bacteria are detected by their ability to grow in the absence of the aminoacid required by the parent test strain. The bacterial reverse mutation test is rapid, inexpensive and relatively easy to perform. The bacterial reverse mutation test is commonly employed as an initial screening for mutagenic activity. Developer Ames B. (1971) Known users Widely used by industry, CROs and academics Status of validation and standardisation Worldwide accepted by regulatory authorities Field and limitations of application See general limitations for in vitro tests. Recommendations of use in the view of animal replacement As part of an in vitro test battery Ongoing development Ready to use References - Ames, B.N., et al, (1975). Methods for Detecting Carcinogens and Mutagens with the Salmonella/Mammalian-Microsome Mutagenicity Test. Mutation Res., 31, 347-364. - Maron, D.M. and Ames, B.N. (1983). Revised Methods for the Salmonella Mutagenicity Test. Mutation Res., 113, 173-215.

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Saccaromyces Cerevisiae gene mutation assay B. 15/OECD TG # 480 Short description, scientific relevance and purpose A variety of haploid and diploid strains of the yeast Saccharomyces cerevisiae can be used to measure the production of gene mutations induced by chemical agents with and without metabolic activation. Forward mutation systems in haploid strains, such as the measurement of mutation from red, adenine-requiring mutants (ade-1, ade-2) to double adenine-requiring white mutants and selective systems such as the induction of resistance to canavnaine and cycloheximide, have been utilized. The most extensively validated reverse mutation system involves the use of the haploid strain XV 185-14C which carries the ochre nonsense mutations ade 2-1, arg 4-17, lys 1-1 and trp 5-48, which are reversible by base substitution mutagens that induce site specific mutations or ochre suppressor mutations. XV 185-14C also carries the his 1-7 marker, a missense mutation reverted mainly by second site mutations, and the marker hom 3-10 which is reverted by frameshift mutagens. In diploid strains of S. cerevisiae the only extensively used strain is D7 which is homozygous for ilv 1-92. Developer of the method Zimmermann F. (1975) Known users Was used by industry, CRO and academics. Rarely used at present Status of validation and standardisation Accepted by regulatory authorities Field and limitations of application See general limitations for in vitro tests Recommendations of use in the view of animal replacement Not used in a standard battery Ongoing development Ready to use References - Hannan MA, et al, (1978). Mutagenicity and recombinogenicity of daunomycin in Saccharomyces cerevisiae. Cancer Lett. Dec;5(6):319-24 - Mondon P, Shahin MM. (1992). Protective effect of two sunscreens against lethal and genotoxic effects of UVB in V79 Chinese hamster cells and Saccharomyces cerevisiae strains XV185-14C and D5. Mutat Res. May 16;279(2):121-8. - Sorenson WG, et al, (1981). Comparison of mutagenic and recombinogenic effects of some adenine analogues in Saccharomyces cerevisiae D7. Mutat Res. Jun;82(1):95-100.

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Saccaromyces Cerevisiae mitotic recombination assay B. 16/OECD TG # 481 Short description, scientific relevance and purpose Mitotic recombination in Saccharomyces cerevisiae can be detected between genes (or more generally between a gene and its centromere) and within genes. The former event is called mitotic crossing-over and generates reciprocal products whereas the latter event is most frequently non-reciprocal and is called gene conversion. Crossing-over is generally assayed by the production of recessive homozygous colonies or sectors produced in a heterozygous strain, whereas gene conversion is assayed by the production of prototrophic revertants produced in an auxotrophic heteroallelic strain carrying two different defective alleles of the same gene. The most commonly used strains for the detection of mitotic gene conversion are D4 (heteroallelic at ade 2 and trp 5) D7 (heteroallelic at trp 5) BZ34 (heteroallelic at arg 4) and JDl (heteroallelic at his 4 and trp 5). Mitotic crossing-over producing red and pink homozygous sectors can be assayed in D5 or in D7 (which also measures mitotic gene conversion and reverse mutation at ilv 1-92) both strains being heteroallelic for complementing alleles of ade 2. Known users Widely used by industry and academics Status of the validation or standardisation Worldwide accepted by regulatory authorities Field and limitations of application See general limitations for in vitro tests Recommendations of use in the view of animal replacement Not used in a standard battery Ongoing development Ready to use References - Zimmermann FK, Vig BK (1975). Mutagen specificity in the induction of mitotic crossing-over in Saccharomyces cerevisiae. Mol Gen Genet. Aug 27;139(3):255-68. - Zimmermann FK, et al, (1984). Testing of chemicals for genetic activity with Saccharomyces cerevisiae: a report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutat Res. May;133(3):199-244.

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Mammalian cell gene mutation test B. 17/OECD TG # 476 Short description, scientific relevance and purpose The in vitro mammalian cell gene mutation test can be used to detect gene mutations induced by chemical substances. Suitable cell lines include L5178Y mouse lymphoma cells, the CHO, CHO-AS52 and V79 lines of Chinese hamster cells, and TK6 human lymphoblastoid cells. In these cell lines the most commonly used genetic endpoints measure mutation at thymidine kinase (TK) and hypoxanthine-guanine phosphoribosyl transferase (HPRT), and a transgene of xanthine-guanine phosphoribosyl transferase (XPRT). The TK, HPRT and XPRT mutation tests detect different spectra of genetic events. The autosomal location of TK and XPRT may allow the detection of genetic events (e.g. large deletions) not detected at the HPRT locus on X-chromosomes. Developer of the method Chu E.H.Y. (for HPRT) and Clive D. (for TK) Known users Widely used by industry, CROs and academics Status of the validation and standardisation Accepted by regulatory authorities Field and limitations of application See general limitations for in vitro tests Recommendations of use in the view of animal replacement As a part of a standard battery Ongoing development Ready to use References - Aaron, C.S., et al, (1994). Mammalian Cell Gene Mutation Assays Working Group Report. Report of the International Workshop on Standardisation of Genotoxicity Test Procedures. Mutat. Res., 312, 235-239. - Aaron, C.S. and Stankowski, Jr.L.F. (1989). Comparison of the AS52/XPRT and the CHO/HPRT Assays: Evaluation of Six Drug Candidates. Mutation Res., 223, 121-128. - Chu, E.H.Y. and Malling, H.V. (1968). Mammalian Cell Genetics. II. Chemical Induction of Specific Locus Mutations in Chinese Hamster Cells In Vitro, Proc. Natl. Acad. Sci., USA, 61, 1306-1312. - Liber, H.L. and Thilly, W.G. (1982). Mutation Assay at the Thymidine Kinase Locus in Diploid Human Lymphoblasts. Mutat. Res., 94, 467-485. - Moore, M.M., et al, (1987). Banbury Report 28: Mammalian Cell Mutagenesis, Cold Spring Harbor Laboratory, New York. - Moore, M.M., et al, (1989). Differential Mutant Quantitation at the Mouse Lymphoma TK and CHO HGPRT Loci. Mutagenesis, 4, 394-403.

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- Scott, D., et al, (1991). Genotoxicity Under Extreme Culture Conditions. A report from ICPEMC Task Group 9. Mutat. Res., 257, 147-204. Unscheduled DNA synthesis (UDS) in mammalian cells B. 18/OECD TG # 482 Short description, scientific relevance and purpose The Unscheduled DNA Synthesis (UDS) test measures the DNA repair synthesis after excision and removal of a stretch of DNA containing the region of damage induced by chemical and physical agents. The test is based on the incorporation of tritium labelled thymidine (3H-TdR) into the DNA of mammalian cells which are not in the S phase of the cell cycle. The uptake of 3H-TdR may be determined by autoradiography or by liquid scintillation counting (LSC) of DNA from the treated cells. Mammalian cells in culture, unless primary rat hepatocytes are used, are treated with the test agent with and without an exogenous metabolic activation system. Developer of the method Williams G. (1976) Known users Widely used by industry, CROs and academics in the past Status of the validation and standardisation Accepted by regulatory authorities Field and limitations of application Used to resolve mechanisms of action. See general limitations for in vitro tests Recommendations of use in the view of animal replacement As a part of a standard battery Ongoing development Ready to use References - Casciano DA (2000). Development and utilization of primary hepatocyte culture systems to evaluate metabolism, DNA binding, and DNA repair of xenobiotics . Drug Metab Rev. Feb;32(1):1-13. - Williams, G.M (1976). Carcinogen-induced DNA repair in primary rat liver cell cultures: a possible screen for chemical carcinogens. Cancer Letters 1: 231-236 - Williams, G.M. (1977). Detection of chemical carcinogens by unscheduled DNA synthesis in rat liver primary cell cultures. Cancer Res. 37: 1845-1851

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Sister chromatid exchange assay in mammalian cells (SCE) B. 19/OECD TG # 479 Short description, scientific relevance and purpose The Sister Chromatid Exchange (SCE) assay is a short-term test for the detection of reciprocal exchanges of DNA between two sister chromatids of a duplicating chromosome. SCEs represent the interchange of DNA replication products at apparently homologous loci. The exchange process presumably involves DNA breakage and reunion, although little is known about its molecular basis. Detection of SCEs requires some means of differentially labelling sister chromatids and this can be achieved by incorporation of bromodeoxyuridine (BrdU) into chromosomal DNA for two cell cycles. Mammalian cells in vitro are exposed to the test chemical with and without a mammalian exogenous metabolic activation system, if appropriate, and cultured for two rounds of replication in BrdU-containing medium. After treatment with a spindle inhibitor (e.g. colchicine) to accumulate cells in a metaphase-like stage of mitosis (c-metaphase), cells are harvested and chromosome preparations are made. Known users Used by industry, CRO and academics in the past Status of the validation and standardisation Accepted by regulatory authorities Field and limitations of application See general limitations for in vitro tests Recommendations of use in the view of animal replacement Rarely used Ongoing development Ready to use References - Hagmar L, et al, (2001). The usefulness of cytogenetic biomarkers as intermediate endpoints in carcinogenesis. Int J Hyg Environ Health. Oct; 204(1):43-7. - Russo A. (2000). In vivo cytogenetics: mammalian germ cells. Mutat Res. Nov 20;455(1-2):167-89.

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In vitro mammalian micronucleus test Alternatives to in vivo micronuclei/ in vivo chromosome aberration test Short description, scientific relevance and purpose The purpose of the in vitro micronucleus assay is to identify agents that cause structural and numerical chromosome changes. The in vitro micronucleus test may employ cultures of established cell lines or primary cell cultures. Developer of the method Evans H. J. (1959) Known users Pharmaceutical, cosmetic industries, CROs and academics Status of validation and/or standardisation Inter-laboratory validation studies include: the Japanese collaborative studies, the European Pharmaceutical industry validation studies and the study coordinated by the French Society of Genetic Toxicology. Fields and limitations of application Micronuclei result from lesions/adducts at the level of DNA or chromosomes, or at the level of proteins directly or indirectly involved in chromosome segregation. Limitations: together with general limitations, apoptosis may interfere with the scoring of micronuclei giving rise to false positives Recommendations of use in the view of animal replacement As part of an in vitro battery Ongoing development A multicentre evaluation study, coordinated by the Institute Pasteur de Lille (France), is ongoing using a new transfected cell line which cannot go into apoptosis. The cell line is CTLL 2 stably transfected with the apoptosis inhibitor gene bcl2 Effort needed to complete validation of the method As many data are already available, the method could be validated by a weight of evidence approach. A draft of the in vitro micronucleus test guideline is expected to be submitted to the OECD in 2004. References - Aardema, MJ et al (2001). The In Vitro Micronucleus Assay Genetic Toxicology and Cancer Risk Assessment, Ed: W. N. Choy, Marcel Dekker, Basel. - Evans HJ et al. (1959). The relative biological efficiency of single doses of fast neutrons and gamma rays in Vicia faba roots and the effect of oxygen. Part II. Chromosome damage: the production of micronuclei. Intl. J. Rad. Biol. 1, 230-240.

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- Garriott M.L. et al. (2002). A protocol for the in vitro micronucleus test. I. Contributions to the development of a protocol suitable for regulatory submissions from an examination of 16 chemicals with different mechanisms of action and different levels of activity”. Mutat Res. 27; 517(1-2): 123-34. - Kirsch-Volders M. et al, (2003). Report from the in vitro micronucleus assay working group. Mutat. Res 540, 153-163. -Meintieres S. et al., (2001). Apoptosis can be a confusing factor in in vitro clastogenic assays. Mutagenesis., 16(3): 243-50. Erratum in: Mutagenesis, 16(5):453. -Meintieres S. et al, (2003). Using CTLL-2 and CTLL-2 bcl2 cells to avoid interference by apoptosis in the in vitro micronucleus test, Environ Mol Mutagen.;41(1):14-27. - Phelps JB et al. (2003). Relative percent cell survival and positive response in the in vitro micronucleus test. Mutat Res 537 115–116. - Phelps J.B. et al. (2002). A protocol for the in vitro micronucleus test. II. Contributions to the validation of a protocol suitable for regulatory submissions from an examination of 10 chemicals with different mechanisms of action and different levels of activity”. Mutat Res. 26; 521(1-2):103-12. -Wilhelm von der Hude et al (2000). In vitro micronucleus assay with Chinese hamster V79 cells results of a collaborative study with in situ exposure to 26 chemical substances. Mutat. Res, 468, 137-163. In vitro Comet assay Alternatives to in vivo test for DNA Damage Short description, scientific relevance and purpose The Comet assay is a method for measuring DNA strand breaks. DNA strand breaks may be introduced directly by genotoxic compounds or through the interaction with oxygen radicals or other reactive intermediates, or as a consequence of excision repair enzymes. It is highly sensitive and can detect DNA strand breaks in individual cells. The test can be conducted under neutral or alkaline conditions even if the test under alkaline conditions is more common and better standardized. The in vitro Comet assay may employ cultures of established cell lines, cell strains or primary cell cultures. The advantages of the Comet assay include:

- DNA strand breaks in individual cells are measured - only small number of cells is necessary - no proliferating cells are required - the assay can be performed on any cell line or tissue

Developer of the method Singh N.P. (1988) Known users Pharmaceutical and Cosmetic industries, CROs and academics

Status of validation and/or standardisation No ongoing validation studies

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Fields and limitations of application Used for screening purposes and to understand mechanisms of action. Used as a replacement for the in vivo UDS test and to look at genotoxicity in target cells. Limitations: no validation, no official guideline. However, for the in vivo comet assay, recommendations on acceptance criteria and on how to standardise protocols have been recently published (Tice RR et al, 2000). These recommendations and the standardised protocol may be useful also for the in vitro Comet assay.

Recommendations of use in the view of animal replacement The Comet assay could replace the in vivo UDS test. This may lead to reduction of animal use. If genotoxicity can be confirmed or ruled out in target tissues, this may lead to a replacement of further animal experiments.

Ongoing development No coordinated development is ongoing.

Efforts needed to complete validation of the method Efforts are needed to coordinate a formal validation or possibly a weight of evidence validation. References - Hartmann, A. et al. (2003). Recommendations for conducting the in vivo alkaline Comet assay. Mutagenesis Vol. 18, No. 1, 45-51. -Ostiling O. and Johanson K.J. (1987). Microelectrophoretic study of radiation-induced DNA damage in individual cells”, Biochem. Biophy. Res Commun:123, 291-298. - Singh NP et al, (1988): A Simple technique for quantification of low levels of damage in individual cells. Exp Cell Res 175,184-191. - Tice, R.R. et al. (2000). The single cell gel / comet assay: Guidelines for in vitro and in vivo genetic toxicology testing. Environm. Mol. Mutagen. 35, 206-221.

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In vitro micronucleus test in primary skin cells or models Alternatives to in vivo micronuclei/ in vivo chromosome aberration test Short description, scientific relevance and purpose Primary human keratinocytes Human keratinocytes obtained from foreskin have been shown to contain several isoenzymes of cytochrome P-450. They also continue to express biotransfomation activity in vitro. Primary human keratinocytes have been shown to be sensitive to micronucleus induction by some clastogens and low doses of UVB and UVA. On the other hand, a publication showed that colchicine did not induce micronuclei in human keratinocytes while micronucleus induction was found in other human cells. 3D skin models (three dimensional skin model) There are several 3D skin models which are commercially available. The HCE model (SkinEthic, Nice, France) consists of immortalized human corneal epithelial cells (HCE cell line) that are cultivated at the air-liquid interface in a chemically defined medium on a polycarbonate substrate and form an air-epithelial tissue, devoid of stratum corneum, resembling morphologically the corneal epithelium of the human eye. A possible endpoint measurement may be micronucleus induction. The SkinEthic HCE model has recently been taken up by a few industries to be evaluated for its potential as test model to detect clastogens/aneugens. Some companies considered the potential of the HCE model for photomutagenicity testing. The EpiDerm model (MatTec Corporation, Ashland, MA, USA) consists of normal, human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. This model is already in widespread use for testing of skin irritancy and dermal toxicity. Known users Cosmetic industries and CROs

Status of validation and/or standardisation The tests with primary keratinocytes as well as with 3D skin cultures are in a phase of research and development. The development of the in vitro micronucleus test on target cells is not yet coordinated. Fields and limitations of application Both tests must, at this point, be seen as an addition to a standard battery of tests and to be used for mechanistic purposes. Ongoing development No coordinated development is ongoing. Efforts needed to complete the validation of the method Efforts needed to complete the validation of the method are: (1) a protocol needs to be established that leads to reproducible results (2) metabolic capabilities of the primary keratinocytes as well as the keratinocytes growing in

3D cultures should be determined

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(3) the barrier function of the 3D models needs to be assessed to enable a comparison with the in vivo situation

(4) the in vitro micronucleus test on human keratinocytes or 3D skin models should be compared with data on in vivo micronucleus test on rodent keratinocytes to assess the predictivity of the test

References - Emri, G. et al. (2000). Low doses of UVB or UVA induce chromosomal aberrations in cultured human skin cells. J Invest Dermat, 115 (3): 435-440. - Heimann R. and R.C. Rice (1983). Polycyclic aromatic hydrocarbon toxicity and induction of metabolism in cultivated esophageal and epidermal keratinocytes. Cancer Res 43, 4856-4862. - Kukkelhoven, M.W.A.C. (1985). Covalent binding of benzo[a]pyrene metabolites to DNA of cultured human hair follicle keratinocytes, Arch. Toxicol., 57, 6-12. - Kuroki, T. et al.(1980). Metabolism of benzo[a]pyrene in human epidermal keratinocytes in culture, Carcinogenesis, 1, 559-565. - Kuroki, T. et al, (1987). Inter-individual variation of arylhydrocarbon-hydroxylase activity in cultured epidermal and dermal cells, Jpn. J. Cancer Res., 78, 45-53. - Lofti, C.F.P. and G.M. Machado-Santelli (1996). Comparative analysis of colchicines induced micronuclei in different cell types in vitro. Mutat Res 349, 77-832. - Nishikawa, T.et al, (1999). Study of a rat skin in vivo micronucleus test: data generated by mitomycin C and methyl methanesulfonate. Mutat Res, 444, 159-166. - Nishikawa, T. et al. (2002). Further evaluation of an in vivo micronucleus test on rat and mouse skin: results with five skin carcinogens. Mutat Res, 513, 93-102. - Van Pelt F.N.A.M. et al, (1990). Immunohistochemical detection of cytochrome P450 isoenzymes in cultured human epidermal cells. Jl Histochem and Cytochem 38, 1847-1851. - Van Pelt, F.N.A.M. et al, (1991) Micronucleus formation in cultured human keratinocytes following exposure to mitomycin C and cyclophosphamide. Mutat Res 252, 45-50. - Van Pelt, F.N.A.M., et al, (1991) Micronucleus formation in cultured human keratinocytes: Involvement of intercellular bioactivation. Toxic. in Vitro, Vol. 5, No. 5/6, pp. 515-518.

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In vitro Comet assay with primary skin cells or models Alternatives to in vivo test for DNA Damage Short description, scientific relevance and purpose Primary keratinocytes and cell lines In the Comet assay, any cell type can theoretically be used for genotoxicity testing. However, only a few publications were found using human keratinocytes as target cell. The Comet assay has been shown to be able to detect genotoxic damage by H2O2 in a human keratinocyte cell line H103 and by carbonyl stress and Photofrin in the human HaCaT keratinocyte cell line. Some companies are exploring the utility of the Comet assay on human keratinocytes. The assay is used more for mechanistic purposes, especially in the field of photogenotoxicity. 3D skin models (three dimensional skin model) There are several models commercially available. The SkinEthic HCE model (SkinEthic, Nice, France) consists of immortalized human corneal epithelial cells (HCE cell line) that are cultivated at the air-liquid interface in a chemically defined medium on a polycarbonate substrate and form an air-epithelial tissue, devoid of stratum corneum, resembling morphologically the corneal mucosa of the human eye. The EpiDerm model (MatTec Corporation, Ashland, MA, USA) consists of normal, human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. This model has already widespread use in the testing of skin irritancy and dermal toxicity. Known users Cosmetic industries and CROs

Status of validation and/or standardisation Not yet standardise. The in vitro Comet tests with primary keratinocytes as well as with the 3D skin cultures are in a phase of research and development. Fields and limitations of application Reconstituted skin models are recently taken into consideration to assess photogenotoxicity (Meunier JR et al, 2001). The Comet assay has already been successfully adapted for use with 3D buccal mucosa equivalents (Wolfreys A et al, 1999). Recommendations of use in the view of animal replacement The Comet assay on both models must, at this point, be seen as an addition to a standard battery of tests and for mechanistic purposes. Ongoing development The development of the in vitro Comet tests on target cells is not yet coordinated. Efforts needed to complete the validation of the method Efforts needed to complete the validation of the method are: (1) a protocol needs to be established that leads to reproducible results (2) metabolic capabilities of the primary keratinocytes as well as the keratinocytes growing into

3D cultures should be determined

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(3) the barrier function of the 3D models needs to be assessed to enable a comparison with the in vivo situation

(4) the in vitro micronucleus test on human keratinocytes or 3D skin models should be compared with data on in vivo micronucleus test on rodent keratinocytes to assess predictivity of the test

References - Meunier, J-R et al. (2001). Comet assay on Episkin® an in vitro reconstructed skin model: A new tool for the evaluation of (photo)genotoxic potential. Abastract Mutation Res. 483 (Suppl. 1) S168. - Roberts, M.J et al. (2003). DNA damage by carbonyl stress in human skin cells. Mutation Research 522, 45-46. - Thein, N. et al. (2000). A strong genotoxic effect in mouse skin of a single painting of coal tar in hairless mice and in Muta(TM)Mouse. Mutation Research, 468, 117-124. - Wolfreys, A. et al. (1999). Use of a 3D buccal mucosa tissue equivalent to assess DNA damage in the presence and absence of human saliva. Poster EEMS. - Woods, J.A. et al. (2004). The effect of Photofrin on DNA strand breaks and base oxication in HaCaT keratinocytes: A Comet assay study. Photochemistry and Photobiology, 79(1). - Yendle, J. E.et al. (1997). The genetic toxicity of time: importance of DNA-unwinding time to the outcome of single-cell gel electrophoresis assays. Mutation Research, 375, 125-136.

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In vitro toxicogenomics

Short description, scientific relevance and purpose Toxicogenomics is the application of genomics methods to address questions in the field of toxicology. Changes in gene/protein expression as a result of exposure to a toxic chemical or physical agent can be measured in virtually any tissue (in vitro or in vivo). The rapidly developing field of toxicogenomics is expected to have a large impact on both the fields of genetic toxicology and carcinogenicity as a result of increased understanding of these processes. Increased understanding of the biological pathways involved in genotoxicity and carcinogenicity will promote the development of better tools for assessing these endpoints. Initial studies suggest that patterns of induced gene expression changes may be characteristic of specific classes of toxic compounds and identification of these distinctive fingerprints can help classify agents with different mechanisms of action. This has the potential to reduce the amount of testing normally required to define a mechanism or mode of action. Known users Pharmaceutical industries and academics Status of the validation and standardisation Genomics methods are at the stage of research and development. Because the field of toxicogenomics is relatively new, most experimental results are not well enough established to be suitable for regulatory decision-making at this time. Laboratory techniques and test procedures may not be well validated. In addition, test systems may vary so that results may not be consistent or generalized across different platforms. Field and limitation of application They are useful mechanistic tools but the general consensus is they are not suitable at this time for regulatory decision-making. The findings from a specific study often cannot be extrapolated across species or to different study populations (e.g., various human subpopulations with different genetic backgrounds). Ongoing development A move to standardise assays is underway, and much more information should be available within the next several years. References - Aardema M and MacGregor JT (2002). Toxicology and genetic toxicology in the new era of “toxicogenomics”: impact of “omics” technologies. Mutat Res, 13-25. - Corton JC and Stauber AJ. (2000). Toward construction of a transcript profile database predictive of chemical toxicity. Toxicol Sci 58, 217-219. - Farr S. and Dunn RT. (1999). Concise review: gene expression applied to toxicology. Toxicol. Sci, 50, 1-9. - Holmes EW et al. (2001). Metabonomic characterisation of genetic variation in toxicological and metabolic responses using probabilistic neural networks. Chem Res Toxicol 14, 182-191. - Nuwayysir et al. (1999). Microarray and toxicology: the advent of toxicogenomics. Mol Carcinogenesis, 153-159. - Pennie WA (2000). Use of cDNA microarray to probe and understand the toxicological consequences of altered gene expression. Toxicol Lett 112/113, 473-477. - Rockett JC and Dix DJ. (1999) Application of DNA arrays to toxicology. Environ Health perspect. 107, 681-685.

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5. Recommendation for achieving reduction in animal use General suggestions: - when possible, animals from subchronic and/or chronic toxicity studies should be shared

to measure genotoxic effects. By applying flow cytometry or image analysis, blood from rodents could be evaluated for the presence of micronuclei (Criswell KA et al, 2003; Torous DK et al, 2003)

- each animal can serve as its own control and the kinetics can be followed in the same animal (sample can be taken over several times).

- multiple dosing can be performed on the same animal (micronuclei, comet assay) - no general need for both sexes - instead of two routine in vivo assays, select only one assay (considering genotoxic

endpoints) Specific suggestions: - in classical in vivo micronucleus test (B12- TG 474), a substantial decrease in the number of animals can be obtained by implementing flow cytometry analysis

In vivo tests to be removed from the list because they are not relevant for the purposes of the cosmetic industry: - rodent dominant lethal test , B22-TG 478 - mouse heritable translocation assay, B25-TG 485 - specific locus test - mouse spot test, B24-TG 484 - mammalian spermatagonial chromosome aberration test, B23-TG 483 - sex-linked recessive lethal test in Drosophila Melanogaster, B20-TG 477

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6. Final Comments Genotoxicity and mutagenicity testing are an important part of the hazard assessment of chemicals for regulatory purposes. General crucial limitations of in vitro tests are due to the absence of toxicokinetic characteristics and/or to the use of cell lines not relevant to predict genotoxicity at target organs. The current situation is that no single in vitro test can fully replace an existing in vivo animal test. The recommendations provided here are based on a step-wise approach. Stage1 characterises the substance based on existing data and knowledge including data on skin absorption. If systemic exposure of the compound in question cannot be ruled out, a battery of three in vitro tests for hazard identification has to be performed that should cover the endpoints gene mutation, clastogenicity and aneuploidy (stage 2). Stage 3 is a follow up stage in in vitro model systems on target cells (e.g. three dimensional skin models) that has to be performed in case of positive findings in stage 2. Stage 3 is supposed to act as an intermediate step which should, if it can be successfully validated, be able to eliminate "false positive" results from stage 2. If the test(s) performed in stage 3 is negative, further testing should not be necessary. Such tests are not available at the moment and much effort has to be undertaken to develop and validate those tests. Stage 2 of the strategy uses tests that are already adopted by regulatory authorities, whereas the time estimated for implementation and validation of the stage 3 tests is 8-10 years. However, if the outcome of stage 3 still shows a mutagenic or genotoxic potency of the compound tested or if the methods necessary to perform such an intermediate step cannot be successfully validated, confirmatory experiments in vivo will still be required (Stage 4). To overcome the limitations of in vitro testing and reach full animal replacement, model systems in the area of toxicokinetics and metabolism are required that can accurately predict or mirror the in vivo situation. Moreover, the emerging area of toxicogenomics could lead to a better understanding of the process of genotoxicity/mutagenicity which may help to develop the "right" in vitro models. Taking into account the state of the art in those areas, it seems highly unlikely that full replacement in the field of genotoxicity/mutagenicity can be accomplished within the next 12 years. This time estimation is based on the following rationale: (i) model systems in the area of toxicokinetics and metabolism that can accurately predict or mirror the in vivo situation are required and need to be developed, (ii) new in vitro tests on target cells for cosmetics need to be developed and validated against an extended database of reliable in vivo data on target cells which does not exist yet, (iii) various laboratories need to be mobilized to put research in these fields, and (iiii) laboratories and /or organizations need to be found (and founded) to coordinate these research programs. Regarding the reduction of animal use, the experts feel that the flexibility given in the currently used in vivo guideline approaches is not sufficiently utilised at the moment. Improvements in this field could instantly lead to a substantial reduction in the number of animals used within the cosmetic industry. In conclusion, the experts are of the opinion that a total replacement of animal testing in the field of mutagenicity/genotoxicity testing is not feasible within the next 12 years. A total replacement of the in vivo genotoxicity tests will depend, besides the development of in vitro tests on skin models, also on the progress in the fields of toxicokinetics and toxicogenomics.

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Current endpoints

addressed in animal test

Alternative tests available

In vitro Endpoints Purpose Area(s) of

application Validation

status Regulatory acceptance Comments

Estimated time to have the method validated (ESAC endorsement)*

Ames test (B13/14-TG471)

Gene point mutations

Partial replacement (tiered strategy and/or test battery)

Mutagenicity/ genotoxicity Adopted EC (Annex V),

OECD -------------- ------------------

S. Cerevisiae gene mutation (B15-TG 480)




OECD -------------- ------------------

Gene point mutations (B20/TG477; B22/TG478; B24/TG484)

Mammalian cell gene mutation test (B17-TG 476)




OECD -------------- ------------------

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Mitotic recombination in S. Cerevisiae (B16-TG 481)

DNA damage



OECD -------------- ------------------

Unscheduled DNA synthesis (USD) (B18-TG 482)

DNA damage



OECD --------------- --------------------

Sister chromatid exchange (SCE) (B19-TG 479)

DNA damage



OECD --------------

------------------

Alkaline Comet assay DNA damage


Genotoxicity

Optimised ----------------

Coordination for formal or weight of evidence validation

Formal validat: 4-6 years Weight of evidence validat: 2-3 years

DNA damage (B39/TG486)

Alkaline Comet in skin cells/model

DNA damage Full replacement Genotoxicity R&D ----------------

Development needs to be coordinated

8-10 years

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Chromosomal aberrations (B11/TG475; B22/TG478; B25/TG485; B23/TG483)

Mammalian chromosomal aberration assay (B10-TG 473)

Chromosomal aberrations



OECD -------------- ------------------

Micronucleus in cell lines

Aneugenes and clastogenes


Genotoxicity/ mutagenicity

Optimised OECD test guideline submitted

--------------

Formal valid: 3-4 years Weight of evidence validat: 1-2 years

Detects aneugenes and clastogenes (B12/TG474) Micronucleus in

target cells and skin models

Aneugenes and clastogenes

Full replacement

Genotoxicity/ mutagenicity R&D ---------------

Development needs to be coordinated

6-10 years

Mechanism based Toxicogenomics Various


Genotoxicity R&D ---------------

Further development and standard needed

10 +

* This table estimates the time needed to achieve ESAC endorsement for individual alternative tests assuming optimal conditions. It does not indicate the time needed to achieve full replacement of the animal test, nor does it include the time needed to achieve regulatory acceptance. “Optimal conditions” means that all necessary resources, for example technical, human, financial and coordination, are met at all times in the process and that the studies undertaken have successful outcomes.

7 genotox-mutagen en

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