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Adopted:
21st July 1997 OECD GUIDELINE FOR TESTING OF CHEMICALS
Bacterial Rever Mutation Test
INTRODUCTION
1.The bacterial rever mutation test us 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 ba pairs (1)(2)(3). The principle of this bacterial rever mutation
test is that it detects mutations which revert mutations prent in the test strains and restore the functional capability of the bacteria to synthesize an esntial amino acid. The revertant bacteria are detected by their ability to grow in the abnce of the amino acid required by the parent test strain.
2.Point mutations are the cau of many human genetic dias and there is substantial evidence that point mutations in oncogenes and tumour suppressor genes of somatic cells are involved in tumour formation in humans and experimental animals. The bacterial rever mutation
test is rapid, inexpensive and relatively easy to perform. Many of the test strains have veral features that make them more nsitive for the detection of mutations, including responsive DNA quences at the reversion sites, incread cell permeability to large molecules and elimination of DNA repair systems or enhancement of error-prone DNA repair process. The specificity of the test strains can provide some uful information on the types of mutations that are induced by genotoxic agents. A very large data ba of results for a wide variety of structures is available for bacterial rever mutation tests and well-established methodologies have been developed for testing chemicals
with different physico-chemical properties, including volatile compounds.
3.Definitions ud are t out in the Annex.肤质检测
INITIAL CONSIDERATIONS
4.The bacterial rever mutation test utilis prokaryotic cells, which differ from mammalian cells in such factors as uptake, metabolism, chromosome structure and DNA repair process. Tests conducted in vitro generally require the u of an exogenous source of metabolic activation. In vitro metabolic activation systems cannot mimic entirely the mammalian in vivo conditions. The test there
fore does not provide direct information on the mutagenic and carcinogenic potency of a substance in mammals.
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5.The bacterial rever mutation test is commonly employed as an initial screen for genotoxic activity and, in particular, for point mutation-inducing activity. An extensive data ba has demonstrated that many chemicals that are positive in this test also exhibit mutagenic activity in other tests. There are examples of mutagenic agents which are not detected by this test; reasons for the shortcomings can be ascribed to the specific nature of the endpoint detected, differences in metabolic activation, or differences in bioavailability. On the other hand, factors which enhance the nsitivity of the bacterial rever mutation test can lead to an overestimation of mutagenic activity.
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6.The bacterial rever mutation test may not be appropriate for the evaluation of certain
class of chemicals, for example highly bactericidal compounds (e.g. certain antibiotics) and tho which are thought (or known) to interfere specifically with the mammalian cell replication system
(e.g. some topoisomera inhibitors and some nucleoside analogues). In such cas, mammalian
mutation tests may be more appropriate.
7.Although many compounds that are positive in this test are mammalian carcinogens, the
correlation is not absolute. It is dependent on chemical class and there are carcinogens that are not detected by this test becau they act through other, non-genotoxic mechanisms or mechanisms abnt in bacterial cells.
PRINCIPLE OF THE TEST METHOD
8.Suspensions of bacterial cells are expod to the test substance in the prence and in the
abnce of an exogenous metabolic activation system. In the plate incorporation method, the suspensions are mixed with an overlay agar and plated immediately onto minimal medium. In the preincubation method, the treatment mixture is incubated and then mixed with an overlay agar before plating onto minimal medium. For both techniques, after two or three days of incubation, revertant colonies are counted and compared to the number of spontaneous revertant colonies on solvent control plates.
9.Several procedures for performing the bacterial rever mutation test have been described.
Among tho commonly ud are the plate incorporation method (1)(2)(3)(4), the preincubation method (2)(3)(5)(6)(7)(8), the fluctuation method (9)(10), and the suspension method (11).
Modifications for the testing of gas or vapours have been described (12).
10.The procedures described in this guideline pertain primarily to the plate incorporation and
preincubation method s. Either of them is acceptable for conducting experiments both with and without metabolic activation. Some compounds may be detected more efficiently using the preincubation method. The compounds belong to chemical class that include short chain aliphatic nitrosamines, divalent metals, aldehydes, azo-dyes and diazo compounds, pyrollizidine alkaloids, allyl compounds and nitro compounds (3). It is also recognid that certain class of mutagens are not always detected using standard procedures such as the plate incorporation method or preincubation method. The should be regarded as "special cas" and it is strongly recommended that alternative procedures should be ud for their detection. The following "special cas" could be identified (together with examples of procedures that could be ud for their detection): azo-dyes and diazo compounds (3)(5)(6)(13), gas and volatile chemicals
(12)(14)(15)(16), and glycosides (17)(18). A deviation from the standard procedure needs to be
scientifically justified.
DESCRIPTION OF THE METHOD
Preparations
Bacteria
11.Fresh cultures of bacteria should be grown up to the late exponential or early stationary
pha of growth (approximately 109 cells per ml). Cultures in late stationary pha should not be ud. It is esntial that the cultures ud in the experiment contain a high titre of viable bacteria.
The titre may be demonstrated either from historical control data on growth curves, or in each assay through the determination of viable cell numbers by a plating experiment.
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12.The recommended culture temperature is 37°C.
13.At least five strains of bacteria should be ud. The should include four strains of S. typhimuriu
m (TA1535; TA1537 or TA97a or TA97; TA98; and TA100) that have been shown to be reliable and reproducibly responsive between laboratories. The four S. typhimurium strains have
GC ba pairs at the primary reversion site and it is known that they may not detect certain oxidising mutagens, cross-linking agents and hydrazines. Such substances may be detected li WP2 strains or S. typhimurium TA102 (19) which have an AT ba pair at the primary reversion site. Therefore the recommended combination of strains is:
1.S. typhimurium TA1535, and
2.S. typhimurium TA1537 or TA97 or TA97a, and
3.S. typhimurium TA98, and
4.S. typhimurium TA100, and
5. E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102.
In order to detect cross-linking mutagens it may be preferable to include TA102 or to add a DNA repair-proficient strain li [e.g. E.coli WP2 li WP2 (pKM101).]
14.Established procedures for stock culture preparation, marker verification and storage should
be ud. The amino-acid requirement for growth should be demonstrated for each frozen stock culture preparation (histidine for S. typhimurium strains, and tryptophan for E. coli strains). Other phenotypic characteristics should be similarly checked, namely: the prence or abnce of R-factor plasmids where appropriate [i.e. ampicillin resistance in strains TA98, TA100 and TA97a or TA97,
WP2 uvrA and WP2 uvrA (pKM101), and ampicillin + tetracycline resistance in strain TA102]; the prence of characteristic mutations (i.e. rfa mutation in S. typhimurium through nsitivity to crystal violet, and uvrA mutation in E. coli or uvrB mutation in S. typhimurium, through nsitivity to ultra-
violet light) (2)(3). The strains should also yield spontaneous revertant colony plate counts within世界品牌五百强
the frequency ranges expected from the laboratory's historical control data and preferably within the range reported in the literature.
Medium
15.An appropriate minimal agar (e.g. containing Vogel-Bonner minimal medium E and gluco) and an overlay agar containing histidine and biotin or tryptophan, to allow for a few cell divisions, is ud
(1)(2)(9).
Metabolic activation
16.Bacteria should be expod to the test substance both in the prence and abnce of an appropriate metabolic activation system. The most commonly ud system is a cofactor-supplemented post-mitochondrial fraction (S9) prepared from the livers of rodents treated with enzyme-inducing agents such as Aroclor 1254 (1)(2) or a combination of phenobarbitone and ß-naphthoflavone (18)(20)(21). The post-mitochondrial fraction is usually ud at concentrations in the range from 5 to 30% v/v in the S9-mix. The choice and condition of a metabolic activation system
may depend upon the class of chemical being tested. In some cas it may be appropriate to utilize
more than one concentration of post-mitochondrial fraction. For azo-dyes and diazo-compounds,
using a reductive metabolic activation system may be more appropriate (6)(13).
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Test substance/Preparation
17.Solid test substances should be dissolved or suspended in appropriate solvents or vehicles兄不友弟不恭
and diluted if appropriate prior to treatment of the bacteria. Liquid test substances may be added directly to the test systems and/or diluted prior to treatment. Fresh preparations should be employed unless stability data demonstrate the acceptability of storage.
Test conditions
Solvent/vehicle
18.The solvent/vehicle should not be suspected of chemical reaction with the test substance
and should be compatible with the survival of the bacteria and the S9 activity (22). If other than well-known solvent/vehicles are ud, their inclusion should be supported by data indicating their compatibility. It is recommended that wherever possible, the u of an aqueous solvent/vehicle be considered first. When testing water-unstable substances, the organic solvents ud should be free of water.
Exposure concentrations
19.Amongst the criteria to be taken into consideration when determining the highest amount of
test substance to be ud are cytotoxicity and solubility in the final treatment mixture. It may be uful to determine toxicity and insolubility in a preliminary experiment. Cytotoxicity may be detected by a reduction in the number of revertant colonies, a clearing or diminution of the background lawn, or the degree of survival of treated cultures. The cytotoxicity of a substance may be altered in the prence of metabolic activation systems. Insolubility should be assd as precipitation in the final mixture under the actual test conditions and evident to the unaided eye. The recommended maximum test concentration for soluble non-cytotoxic substances is 5 mg/plate or
5 µl/plate. For non-cytotoxic substances that are not soluble at 5 mg/plate or 5 µl/plate, one or more
concentrations tested should be insoluble in the final treatment mixture. Test substances that are cytotoxic already below 5 mg/plate or 5 µl/plate should be tested up to a cytotoxic concentration.
The precipitate should not interfere with the scoring.
20.At least five different analysable concentrations of the test substance should be ud with
approximately half log (i.e. √10) intervals between test points for an initial experiment. Smaller intervals may be appropriate when a concentration-respon is being investigated.
21.Testing above the concentration of 5 mg/plate or 5 µl/plate may be considered when
evaluating substances containing substantial amounts of potentially mutagenic impurities.
Controls
22.Concurrent strain-specific positive and negative (solvent or vehicle) controls, both with and
without metabolic activation, should be included in each assay. Positive control concentrations that demonstrate the effective performance of each assay should be lected.
23.For assays employing a metabolic activation system, the positive control reference
substance(s) should be lected on the basis of the type of bacteria strains ud. The following chemicals are examples of suitable positive controls for assays with metabolic activation:
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Chemical and CAS No.
9,10-Dimethylanthracene [CAS no. 781-43-1]
7,12-Dimethylbenzanthracene [CAS no. 57-97-6]
Congo Red [CAS no. 573-58-0] (for the reductive metabolic activation method)
Benzo(a)pyrene [CAS no. 50-32-8]
Cyclophosphamide (monohydrate) [CAS no. 50-18-0 (CAS no. 6055-19-2)]
2-Aminoanthracene [CAS no. 613-13-8]
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2-Aminoanthracene should not be ud as the sole indicator of the efficacy of the S9-mix. If 2-aminoanthracene is ud, each batch of S9 should also be characterid with a mutagen that requires metabolic activation by microsomal enzymes, e.g., benzo(a)pyrene, dimethylbenzanthracene.
24.For assays performed without metabolic activation system, examples of strain-specific positive controls are:
Chemical and CAS No.Strain
(a)Sodium azide [CAS no. 26628-22-8]TA1535 and TA100
(b)2-Nitrofluorene [CAS no. 607-57-8]TA98
(c)9-Aminoacridine [CAS no. 90-45-9]
or ICR191 [CAS no. 17070-45-0]
电子技术基础TA1537, TA97 and TA97a
(d)Cumene hydroperoxide [CAS no. 80-15-9]TA102
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(e)Mitomycin C [CAS no. 50-07-7]WP2 uvrA and TA102
(f)N-Ethyl-N-nitro-N-nitrosoguanidine [CAS no. 70-25-7] or
4-nitroquinoline 1-oxide [CAS no. 56-57-5]WP2, WP2 uvrA and WP2 uvrA (pKM101)
(g)Furylfuramide (AF-2) [CAS no. 3688-53-7]plasmid-containing strains
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25.Other appropriate positive control reference substances may be ud. The u of chemical class-related positive control chemicals may be considered, when available.
26.Negative controls, consisting of solvent or vehicle alone, without test substance, and otherwi treated in the same way as the treatment groups, should be included. In addition, untreated controls should also be ud unless there are historical control data demonstrating that no deleterious or mutagenic effects are induced by the chon solvent.
