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Bacterial Reverse Mutation Test
The bacterial reverse mutation test uses amino-acid requiring strains of Salmonella typhimurium and Escherichia coli to detect point mutations, which involve substitution, addition ordeletion of one or a few DNA base pairs (1)(2)(3). The principle of this bacterial reverse mutationtest is that it detects mutations which revert mutations present in the test strains and restore thefunctional capability of the bacteria to synthesize an essential amino acid. The revertant bacteria aredetected by their ability to grow in the absence of the amino acid required by the parent test strain.
2. Point mutations are the cause of many human genetic diseases and there is substantial evidence that point mutations in oncogenes and tumour suppressor genes of somatic cells areinvolved in tumour formation in humans and experimental animals. The bacterial reverse mutationtest is rapid, inexpensive and relatively easy to perform. Many of the test strains have severalfeatures that make them more sensitive for the detection of mutations, including responsive DNAsequences at the reversion sites, increased cell permeability to large molecules and elimination ofDNA repair systems or enhancement of error-prone DNA repair processes. The specificity of the teststrains can provide some useful information on the types of mutations that are induced by genotoxicagents. A very large data base of results for a wide variety of structures is available for bacterialreverse mutation tests and well-established methodologies have been developed for testing chemicalswith different physico-chemical properties, including volatile compounds.
3. Definitions used are set out in the Annex.
The bacterial reverse mutation test utilises prokaryotic cells, which differ from mammalian cells in such factors as uptake, metabolism, chromosome structure and DNA repair processes. Testsconducted in vitro generally require the use of an exogenous source of metabolic activation. In vitrometabolic activation systems cannot mimic entirely the mammalian in vivo conditions. The testtherefore does not provide direct information on the mutagenic and carcinogenic potency of asubstance in mammals.
5. The bacterial reverse mutation test is commonly employed as an initial screen for genotoxic activity and, in particular, for point mutation-inducing activity. An extensive data base hasdemonstrated that many chemicals that are positive in this test also exhibit mutagenic activity inother tests. There are examples of mutagenic agents which are not detected by this test; reasons forthese shortcomings can be ascribed to the specific nature of the endpoint detected, differences inmetabolic activation, or differences in bioavailability. On the other hand, factors which enhance thesensitivity of the bacterial reverse mutation test can lead to an overestimation of mutagenic activity.
The bacterial reverse mutation test may not be appropriate for the evaluation of certain classes of chemicals, for example highly bactericidal compounds (e.g. certain antibiotics) and thosewhich are thought (or known) to interfere specifically with the mammalian cell replication system(e.g. some topoisomerase inhibitors and some nucleoside analogues). In such cases, mammalianmutation 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 because they act through other, non-genotoxic mechanisms or mechanisms
absent in bacterial cells.
Suspensions of bacterial cells are exposed to the test substance in the presence and in the absence of an exogenous metabolic activation system. In the plate incorporation method, thesesuspensions are mixed with an overlay agar and plated immediately onto minimal medium. In thepreincubation method, the treatment mixture is incubated and then mixed with an overlay agar beforeplating onto minimal medium. For both techniques, after two or three days of incubation, revertantcolonies are counted and compared to the number of spontaneous revertant colonies on solventcontrol plates.
9. Several procedures for performing the bacterial reverse mutation test have been described.
Among those commonly used are the plate incorporation method (1)(2)(3)(4), the preincubationmethod (2)(3)(5)(6)(7)(8), the fluctuation method (9)(10), and the suspension method (11).
Modifications for the testing of gases or vapours have been described (12).
10. The procedures described in this guideline pertain primarily to the plate incorporation and preincubation methods. 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. These compounds belong to chemical classes 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 recognised that certain classes of
mutagens are not always detected using standard procedures such as the plate incorporation method
or preincubation method. These should be regarded as "special cases" and it is strongly
recommended that alternative procedures should be used for their detection. The following "special
cases" could be identified (together with examples of procedures that could be used for their
detection): azo-dyes and diazo compounds (3)(5)(6)(13), gases and volatile chemicals
(12)(14)(15)(16), and glycosides (17)(18). A deviation from the standard procedure needs to be
scientifically justified.
Fresh cultures of bacteria should be grown up to the late exponential or early stationary phase of growth (approximately 109 cells per ml). Cultures in late stationary phase should not beused. It is essential that the cultures used 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 assaythrough the determination of viable cell numbers by a plating experiment.
The recommended culture temperature is 37°C.
At least five strains of bacteria should be used. These should include four strains of S. typhimurium (TA1535; TA1537 or TA97a or TA97; TA98; and TA100) that have been shown to bereliable and reproducibly responsive between laboratories. These four S. typhimurium strains haveGC base pairs at the primary reversion site and it is known that they may not detect certain oxidisingmutagens, cross-linking agents and hydrazines. Such substances may be detected by E.coli WP2strains or S. typhimurium TA102 (19) which have an AT base pair at the primary reversion site.
Therefore the recommended combination of strains is: 1. S. typhimurium TA1535, and2. S. typhimurium TA1537 or TA97 or TA97a, and3. S. typhimurium TA98, and4. S. typhimurium TA100, and5. 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 DNArepair-proficient strain of E.coli [e.g. E.coli WP2 or E.coli WP2 (pKM101).] Established procedures for stock culture preparation, marker verification and storage should be used. The amino-acid requirement for growth should be demonstrated for each frozen stockculture preparation (histidine for S. typhimurium strains, and tryptophan for E. coli strains). Otherphenotypic characteristics should be similarly checked, namely: the presence or absence of R-factorplasmids 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]; thepresence of characteristic mutations (i.e. rfa mutation in S. typhimurium through sensitivity to crystalviolet, and uvrA mutation in E. coli or uvrB mutation in S. typhimurium, through sensitivity to ultra-violet light) (2)(3). The strains should also yield spontaneous revertant colony plate counts withinthe frequency ranges expected from the laboratory's historical control data and preferably within therange reported in the literature.

An appropriate minimal agar (e.g. containing Vogel-Bonner minimal medium E and glucose) and an overlay agar containing histidine and biotin or tryptophan, to allow for a few cell
divisions, is used (1)(2)(9).
Metabolic activation

Bacteria should be exposed to the test substance both in the presence and absence of an appropriate metabolic activation system. The most commonly used system is a cofactor-supplemented post-mitochondrial fraction (S9) prepared from the livers of rodents treated withenzyme-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 used at concentrations in therange from 5 to 30% v/v in the S9-mix. The choice and condition of a metabolic activation systemmay depend upon the class of chemical being tested. In some cases it may be appropriate to utilizemore 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).
Test substance/Preparation

Solid test substances should be dissolved or suspended in appropriate solvents or vehicles and diluted if appropriate prior to treatment of the bacteria. Liquid test substances may be addeddirectly to the test systems and/or diluted prior to treatment. Fresh preparations should be employedunless stability data demonstrate the acceptability of storage.
est conditions


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 thanwell-known solvent/vehicles are used, their inclusion should be supported by data indicating theircompatibility. It is recommended that wherever possible, the use of an aqueous solvent/vehicle beconsidered first. When testing water-unstable substances, the organic solvents used should be free ofwater.
Exposure concentrations

Amongst the criteria to be taken into consideration when determining the highest amount of test substance to be used are cytotoxicity and solubility in the final treatment mixture. It may beuseful to determine toxicity and insolubility in a preliminary experiment. Cytotoxicity may bedetected by a reduction in the number of revertant colonies, a clearing or diminution of thebackground lawn, or the degree of survival of treated cultures. The cytotoxicity of a substance maybe altered in the presence of metabolic activation systems. Insolubility should be assessed asprecipitation in the final mixture under the actual test conditions and evident to the unaided eye. Therecommended maximum test concentration for soluble non-cytotoxic substances is 5 mg/plate or5 µl/plate. For non-cytotoxic substances that are not soluble at 5 mg/plate or 5 µl/plate, one or moreconcentrations tested should be insoluble in the final treatment mixture. Test substances that arecytotoxic 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 used with approximately half log (i.e. √10) intervals between test points for an initial experiment. Smallerintervals may be appropriate when a concentration-response 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.

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 thatdemonstrate the effective performance of each assay should be selected.
23. For assays employing a metabolic activation system, the positive control reference substance(s) should be selected on the basis of the type of bacteria strains used. The followingchemicals are examples of suitable positive controls for assays with metabolic activation: OECD/OCDE
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) Cyclophosphamide (monohydrate) [CAS no. 50-18-0 (CAS no. 6055-19-2)] 2-Aminoanthracene should not be used as the sole indicator of the efficacy of the S9-mix. If 2-aminoanthracene is used, each batch of S9 should also be characterised with a mutagen that requiresmetabolic activation by microsomal enzymes, e.g., benzo(a)pyrene, dimethylbenzanthracene.
For assays performed without metabolic activation system, examples of strain-specific Chemical and CAS No.
N-Ethyl-N-nitro-N-nitrosoguanidine [CAS no. 70-25-7] or 4-nitroquinoline 1-oxide [CAS no. 56-57-5] Other appropriate positive control reference substances may be used. The use 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 otherwise treated in the same way as the treatment groups, should be included. In addition, untreatedcontrols should also be used unless there are historical control data demonstrating that no deleteriousor mutagenic effects are induced by the chosen solvent.
Treatment with test substance
For the plate incorporation method (1)(2)(3)(4), without metabolic activation, usually 0.05 ml or 0.1 ml of the test solutions, 0.1 ml of fresh bacterial culture (containing approximately 108viable cells) and 0.5 ml of sterile buffer are mixed with 2.0 ml of overlay agar. For the assay withmetabolic activation, usually 0.5 ml of metabolic activation mixture containing an adequate amountof post-mitochondrial fraction (in the range from 5 to 30% v/v in the metabolic activation mixture)are mixed with the overlay agar (2.0 ml), together with the bacteria and test substance/test solution.
The contents of each tube are mixed and poured over the surface of a minimal agar plate. Theoverlay agar is allowed to solidify before incubation.
28. For the preincubation method (2)(3)(5)(6) the test substance/test solution is preincubated with the test strain (containing approximately 108 viable cells) and sterile buffer or the metabolicactivation system (0.5 ml) usually for 20 min. or more at 30°-37°C prior to mixing with the overlayagar and pouring onto the surface of a minimal agar plate. Usually, 0.05 or 0.1 ml of testsubstance/test solution, 0.1 ml of bacteria, and 0.5 ml of S9-mix or sterile buffer, are mixed with 2.0ml of overlay agar. Tubes should be aerated during pre-incubation by using a shaker.
29. For an adequate estimate of variation, triplicate plating should be used at each dose level.
The use of duplicate plating is acceptable when scientifically justified. The occasional loss of a platedoes not necessarily invalidate the assay.
30. Gaseous or volatile substances should be tested by appropriate methods, such as in sealed Incubation
All plates in a given assay should be incubated at 37°C for 48-72 hours. After the incubation period, the number of revertant colonies per plate is counted.
Treatment of results
Data should be presented as the number of revertant colonies per plate. The number of revertant colonies on both negative (solvent control, and untreated control if used) and positivecontrol plates should also be given.
33. Individual plate counts, the mean number of revertant colonies per plate and the standard deviation should be presented for the test substance and positive and negative (untreated and/orsolvent) controls.
34. There is no requirement for verification of a clear positive response. Equivocal results should be clarified by further testing preferably using a modification of experimental conditions.
Negative results need to be confirmed on a case-by-case basis. In those cases where confirmation ofnegative results is not considered necessary, justification should be provided. Modification of studyparameters to extend the range of conditions assessed should be considered in follow-up experiments.
Study parameters that might be modified include the concentration spacing, the method of treatment(plate incorporation or liquid preincubation), and metabolic activation conditions.
Evaluation and interpretation of results
There are several criteria for determining a positive result, such as a concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in thenumber of revertant colonies per plate in at least one strain with or without metabolic activationsystem (23). Biological relevance of the results should be considered first. Statistical methods maybe used as an aid in evaluating the test results (24). However, statistical significance should not bethe only determining factor for a positive response.
36. A test substance for which the results do not meet the above criteria is considered non- Although most experiments will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgement about the activity of the test substance. Resultsmay remain equivocal or questionable regardless of the number of times the experiment is repeated.
38. Positive results from the bacterial reverse mutation test indicate that a substance induces point mutations by base substitutions or frameshifts in the genome of either Salmonella typhimuriumand/or Escherichia coli. Negative results indicate that under the test conditions, the test substance isnot mutagenic in the tested species.
Test report
The test report must include the following information: identification data and CAS no., if known; physicochemical properties relevant to the conduct of the study; stability of the test substance, if known.
justification for choice of solvent/vehicle; solubility and stability of the test substance in solvent/vehicle, if known.
amount of test substance per plate (mg/plate or µg/plate) with rationale for selection ofdose and number of plates per concentration; type and composition of metabolic activation system, including acceptability criteria; OECD/OCDE
the mean number of revertant colonies per plate and standard deviation; dose-response relationship, where possible; concurrent negative (solvent/vehicle) and positive control data, with ranges, meansand standard deviations; historical negative (solvent/vehicle) and positive control data, with e.g. ranges, meansand standard deviations.
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A reverse mutation test in either Salmonella typhimurium or Escherichia coli detects mutation in anamino-acid requiring strain (histidine or tryptophan, respectively) to produce a strain independent of anoutside supply of amino-acid.
Base pair substitution mutagens are agents that cause a base change in DNA. In a reversion test thischange may occur at the site of the original mutation, or at a second site in the bacterial genome.
Frameshift mutagens are agents that cause the addition or deletion of one or more base pairs in theDNA, thus changing the reading frame in the RNA


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