Establishment of timetables for the phasing out of animal experiments for
cosmetics
Genotoxicity/mutagenicity
Daniela Maurici2 , Marilyn Aardema 1, Raffaella Corvi2, Marcus Kleber3, Cyrille Krul4;
Christian Laurent5, Nicola Loprieno 6, Markku Pasanen7, Stefan Pfuhler8, Barry Phillips9,
Enrico Sabbioni2, Tore Sanner10, Philippe Vanparys11,
1
Procter 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.
1
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
2
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 nonmutagenicity/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.
3
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
4
3. Inventory of methods currently available
In
vitro
In
vivo
# Annex V
EC
B 13-14
#OECD TG
Name of the test
Endpoint
471
Gene mutations in bacteria
B 10
473
B 17
476
B 19
479
B 15
480
B 16
481
B 18
482
Bacterial Reverse Mutation test
(Ames test)
Mammalian chromosome aberration
test
Mammalian cell gene mutation test
(mouse lymphoma test)
Sister chromatid exchange assay in
mammalian cells (SCE)
Saccharomyces cerevisiae gene
mutation assay
Saccharomyces cerevisiae mitotic
recombination assay
Unscheduled DNA synthesis (UDS)
in mammalian cells
# Annex V
EC
B 12
#OECD TG
Name of the test
Endpoint
474
Mammalian erythrocyte
micronucleus test
B 11
475
B 20
477
B 22
478
Mammalian bone marrow
chromosome aberration test
Sex-linked recessive lethal test in
Drosophila Melanogaster
Rodent dominant lethal test
Structural and numerical
chromosome aberrations in
somatic cells
Chromosome aberrations
B 23
483
B 24
B 25
484
485
B 39
486
Mammalian spermatogonial
chromosome aberration test
Mouse spot test
Mouse heritable translocation assay
Unscheduled DNA synthesis (UDS)
test with mammalian liver cells
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Chromosome aberrations
Gene mutations
Mammalian DNA damage
Gene mutations in yeast
Recombination in yeast
Mammalian DNA damage in
liver cells
Gene mutations in germ line
Chromosome aberrations
and/or gene mutations in
germinal tissue
Inheritable chromosome
aberrations
Mutagenicity in foetal cells
Heritable chromosome
aberrations
Mammalian DNA damage in
liver cells
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.
6
- 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.
9
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 Xchromosomes.
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
3
H-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(12):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(12):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
14
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.
15
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, humanderived 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
16
(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.
17
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, humanderived 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
18
(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.
19
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.
20
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
21
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.
22
Current
endpoints
addressed in
animal test
Gene point
mutations
(B20/TG477;
B22/TG478;
B24/TG484)
Alternative
tests available
In vitro
Endpoints
Purpose
Partial
replacement
Ames test
Gene point
(tiered
(B13/14mutations
strategy
TG471)
and/or test
battery)
Partial
replacement
S.
Cerevisiae
Gene point
(tiered
gene mutation
mutations
strategy
(B15-TG 480)
and/or test
battery)
Partial
Mammalian cell
replacement
point (tiered
gene mutation Gene
mutations
test
strategy
(B17-TG 476)
and/or test
battery)
Area(s) of
application
Validation
status
Regulatory
acceptance
Comments
Estimated time to
have the method
validated (ESAC
endorsement)*
Mutagenicity/
genotoxicity
Adopted
EC (Annex V),
OECD
--------------
------------------
Mutagenicity/
genotoxicity
Adopted
EC (Annex V),
OECD
--------------
------------------
Mutagenicity/
genotoxicity
Adopted
EC (Annex V),
OECD
--------------
------------------
23
Mitotic
recombination
in S. Cerevisiae
(B16-TG 481)
DNA damage
Unscheduled
DNA synthesis DNA damage
(USD)
(B18-TG 482)
DNA damage
(B39/TG486)
Sister chromatid
exchange (SCE)
(B19-TG 479)
DNA damage
Alkaline Comet
DNA damage
assay
Alkaline Comet
in skin
cells/model
DNA damage
Partial
replacement
(tiered
strategy
and/or test
battery)
Partial
replacement
(tiered
strategy
and/or test
battery)
Partial
replacement
(tiered
strategy
and/or test
battery)
Partial
replacement
(tiered
strategy
and/or test
battery)
Full
replacement
Mutagenicity/
genotoxicity
Adopted
EC (Annex V),
OECD
--------------
------------------
Mutagenicity/
genotoxicity
Adopted
EC (Annex V),
OECD
---------------
--------------------
Mutagenicity/
genotoxicity
Adopted
EC (Annex V),
OECD
--------------
------------------
Genotoxicity
Optimised
----------------
Genotoxicity
R&D
----------------
24
Coordination
for formal or
weight of
evidence
validation
Development
needs to be
coordinated
Formal validat:
4-6 years
Weight of
evidence validat:
2-3 years
8-10 years
Chromosomal
aberrations
(B11/TG475;
B22/TG478;
B25/TG485;
B23/TG483)
Detects
aneugenes
and
clastogenes
(B12/TG474)
Mechanism
based
Mammalian
chromosomal
aberration assay
(B10-TG 473)
Chromosomal
aberrations
Partial
replacement
(tiered
strategy
and/or test
battery)
Aneugenes
Micronucleus in
and
cell lines
clastogenes
Partial
replacement
(tiered
strategy
and/or test
battery)
Micronucleus in Aneugenes
target cells and and
skin models
clastogenes
Full
replacement
Toxicogenomics
Partial
replacement
(tiered
strategy
and/or test
battery)
Various
Mutagenicity/
genotoxicity
Genotoxicity/
mutagenicity
Genotoxicity/
mutagenicity
Genotoxicity
Adopted
Optimised
R&D
R&D
EC (Annex V),
OECD
--------------
------------------
OECD test
guideline
submitted
--------------
Formal valid:
3-4 years
Weight of
evidence validat:
1-2 years
---------------
Development
needs to be
coordinated
6-10 years
---------------
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.
25