Section 4

Health effects

Test Guideline No. 471

Bacterial Reverse Mutation Test

26 June 2020

OECD Guidelines for the

Testing of Chemicals
 OECD/OCDE 471
Adopted:
21 July 1997
Corrected
26 June 2020
(CAS RN parag.24)

OECD GUIDELINE FOR TESTING OF CHEMICALS

Bacterial Reverse Mutation Test

INTRODUCTION

1. 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 (1)(2)(3). 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
synthesize an essential amino acid. The revertant bacteria are detected 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 are involved in tumour
formation in humans and experimental animals. The bacterial reverse mutation test is rapid, inexpensive
and relatively easy to perform. Many of the test strains have several features that make them more
sensitive for the detection of mutations, including responsive DNA sequences at the reversion sites,
increased cell permeability to large molecules and elimination of DNA repair systems or enhancement of
error-prone DNA repair processes. The specificity of the test strains can provide some useful information
on the types of mutations that are induced by genotoxic agents. A very large data base of results for a
wide variety of structures is available for bacterial reverse mutation tests and well-established
methodologies have been developed for testing chemicals with different physico-chemical properties,
including volatile compounds.
3. Definitions used are set out in the Annex.

INITIAL CONSIDERATIONS

4. 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. Tests conducted
in vitro generally require the use of an exogenous source of metabolic activation. In vitro metabolic
activation systems cannot mimic entirely the mammalian in vivo conditions. The test therefore does not
provide direct information on the mutagenic and carcinogenic potency of a substance in mammals.

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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 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 these 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 sensitivity of the bacterial reverse mutation
test can lead to an overestimation of mutagenic activity.
6. 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 those which 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, 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 because they act through other, non-genotoxic mechanisms or mechanisms absent
in bacterial cells.

PRINCIPLE OF THE TEST METHOD

8. 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, these
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 reverse mutation test have been described.
Among those commonly used 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 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.

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DESCRIPTION OF THE METHOD

Preparations

Bacteria
11. 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 be used. 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 assay through the
determination of viable cell numbers by a plating experiment.
12. The recommended culture temperature is 37°C.
13. 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 be reliable and
reproducibly responsive between laboratories. These four S. typhimurium strains have GC base 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 by E.coli WP2 strains 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, 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 of E.coli [e.g. E.coli WP2 or E.coli WP2 (pKM101).]
14. 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 stock culture
preparation (histidine for S. typhimurium strains, and tryptophan for E. coli strains). Other phenotypic
characteristics should be similarly checked, namely: the presence or absence 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 presence of characteristic
mutations (i.e. rfa mutation in S. typhimurium through sensitivity to crystal violet, 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 within 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 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

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16. 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 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 used 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 cases 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).

Test substance/Preparation
17. 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 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 used, their inclusion should be supported by data indicating their compatibility. It is
recommended that wherever possible, the use of an aqueous solvent/vehicle be considered first. When
testing water-unstable substances, the organic solvents used 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 used are cytotoxicity and solubility in the final treatment mixture. It may be useful 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 presence of metabolic
activation systems. Insolubility should be assessed 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 used with
approximately half log (i.e. √10) intervals between test points for an initial experiment. Smaller intervals
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.

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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 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 following chemicals are examples
of suitable positive controls for assays with metabolic activation:

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]

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 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] TA1537, TA97 and TA97a
or ICR191 [CAS no. 17070-45-0]
(d) Cumene hydroperoxide [CAS no. 80-15-9] TA102
(e) Mitomycin C [CAS no. 50-07-7] WP2 uvrA and TA102
(f) N-Ethyl-N-nitro-N-nitrosoguanidine [CAS no. 4245-77-6] or N- WP2, WP2 uvrA and
Methyl-N-nitro-N-nitrosoguanidine [CAS no. 70-25-7] or 4- WP2 uvrA (pKM101)
nitroquinoline 1-oxide [CAS no. 56-57-5]
(g) Furylfuramide (AF-2) [CAS no. 3688-53-7] plasmid-containing strains

25. Other appropriate positive control reference substances may be used. The use of chemical class-
related positive control chemicals may be considered, when available.

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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, untreated controls should
also be used unless there are historical control data demonstrating that no deleterious or mutagenic effects
are induced by the chosen solvent.

PROCEDURE

Treatment with test substance

27. 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 108 viable cells) and 0.5
ml of sterile buffer are mixed with 2.0 ml of overlay agar. For the assay with metabolic activation, usually
0.5 ml of metabolic activation mixture containing an adequate amount of 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. The overlay 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 metabolic activation system
(0.5 ml) usually for 20 min. or more at 30°-37°C prior to mixing with the overlay agar and pouring onto the
surface of a minimal agar plate. Usually, 0.05 or 0.1 ml of test substance/test solution, 0.1 ml of bacteria,
and 0.5 ml of S9-mix or sterile buffer, are mixed with 2.0 ml 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 plate does not
necessarily invalidate the assay.
30. Gaseous or volatile substances should be tested by appropriate methods, such as in sealed
vessels (12)(14)(15)(16).

Incubation
31. 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.

DATA AND REPORTING

Treatment of results

32. 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 positive control 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/or solvent) 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

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to be confirmed on a case-by-case basis. In those cases where confirmation of negative results is not
considered necessary, justification should be provided. Modification of study parameters 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

35. 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 the number
of revertant colonies per plate in at least one strain with or without metabolic activation system (23).
Biological relevance of the results should be considered first. Statistical methods may be used as an aid
in evaluating the test results (24). However, statistical significance should not be the only determining
factor for a positive response.
36. A test substance for which the results do not meet the above criteria is considered non- mutagenic
in this test
37. 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. Results may 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 typhimurium and/or
Escherichia coli. Negative results indicate that under the test conditions, the test substance is not
mutagenic in the tested species.

Test report

39. The test report must include the following information:

Test substance:
- identification data and CAS no., if known;
- physical nature and purity;
- physicochemical properties relevant to the conduct of the study;
- stability of the test substance, if known.

Solvent/Vehicle:

- justification for choice of solvent/vehicle;

- solubility and stability of the test substance in solvent/vehicle, if known.

Strains:

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- strains used;
- number of cells per culture;
- strain characteristics.

Test conditions:

- amount of test substance per plate (mg/plate or µl/plate) with rationale for selection of dose and
number of plates per concentration;
- media used;
- type and composition of metabolic activation system, including acceptability criteria;
- treatment procedures.

Results:

- signs of toxicity;
- signs of precipitation;
- individual plate counts;
- the mean number of revertant colonies per plate and standard deviation;
- dose-response relationship, where possible;
- statistical analyses, if any;
- concurrent negative (solvent/vehicle) and positive control data, with ranges, means and standard
deviations;
- historical negative (solvent/vehicle) and positive control data, with e.g. ranges, means and
standard deviations.

Discussion of the results.

Conclusion.

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LITERATURE

(1) Ames, B.N., McCann, J. and Yamasaki, E. (1975). Methods for Detecting Carcinogens and
Mutagens with the Salmonella/Mammalian-Microsome Mutagenicity Test. Mutation Res., 31, 347-364.
(2) Maron, D.M. and Ames, B.N. (1983). Revised Methods for the Salmonella Mutagenicity Test.
Mutation Res., 113, 173-215.
(3) Gatehouse, D., Haworth, S., Cebula, T., Gocke, E., Kier, L., Matsushima, T., Melcion, C., Nohmi,
T., Venitt, S. and Zeiger, E. (1994). Recommendations for the Performance of Bacterial Mutation Assays.
Mutation Res., 312, 217-233.
(4) Kier, L.D., Brusick D.J., Auletta, A.E., Von Halle, E.S., Brown, M.M., Simmon, V.F., Dunkel, V.,
McCann, J., Mortelmans, K., Prival, M., Rao, T.K. and Ray V. (1986). The Salmonella
Typhimurium/Mammalian Microsomal Assay: A Report of the U.S. Environmental Protection Agency Gene-
tox Program. Mutation Res., 168, 69-240.
(5) Yahagi, T., Degawa, M., Seino, Y.Y., Matsushima, T., Nagao, M., Sugimura, T. and Hashimoto,
Y. (1975). Mutagenicity of Carcinogen Azo Dyes and their Derivatives. Cancer Letters, 1, 91-96.
(6) Matsushima, M., Sugimura, T., Nagao, M., Yahagi, T., Shirai, A., and Sawamura, M. (1980).
Factors Modulating Mutagenicity Microbial Tests. In: Short-term Test Systems for Detecting Carcinogens.
Ed. Norpoth K.H. and Garner, R.C., Springer, Berlin-Heidelberg-New York. pp. 273-285.
(7) Gatehouse, D.G., Rowland, I.R., Wilcox, P., Callender, R.D. and Foster, R. (1990). Bacterial
Mutation Assays. In: Basic Mutagenicity Tests: UKEMS Part 1 Revised. Ed. D.J. Kirkland Cambridge
University Press, pp. 13-61.
(8) Aeschbacher, H.U., Wolleb, U. and Porchet, L. (1987). Liquid Preincubation Mutagenicity Test for
Foods. J. Food Safety, 8, 167-177.
(9) Green, M. H. L., Muriel, W. J. and Bridges, B.A. (1976). Use of a simplified fluctuation test to detect
low levels of mutagens. Mutation Res., 38, 33-42.
(10) Hubbard, S.A., Green, M.H.L., Gatehouse, D., and J.W. Bridges (1984). The Fluctuation Test in
Bacteria. In: Handbook of Mutagenicity Test Procedures. 2nd Edition. Ed. Kilbey, B.J., Legator, M., Nichols,
W. and Ramel C., Elsevier, Amsterdam-New York-Oxford, pp. 141-161.
(11) Thompson, E.D. and Melampy, P.J. (1981). An Examination of the Quantitative Suspension Assay
for Mutagenesis with Strains of Salmonella typhimurium. Environmental Mutagenesis, 3, 453-465.
(12) Araki, A., Noguchi, T., Kato, F. and T. Matsushima (1994). Improved Method for Mutagenicity
Testing of Gaseous Compounds by Using a Gas Sampling Bag. Mutation Res., 307, 335-344.
(13) Prival, M.J., Bell, S.J., Mitchell, V.D., Reipert, M.D. and Vaughn, V.L. (1984). Mutagenicity of
Benzidine and Benzidine-Congener Dyes and Selected Monoazo Dyes in a Modified Salmonella Assay.
Mutation Res., 136, 33-47.
(14) Zeiger, E., Anderson, B. E., Haworth, S, Lawlor, T. and Mortelmans, K. (1992). Salmonella
Mutagenicity Tests. V. Results from the Testing of 311 Chemicals. Environ. Mol. Mutagen., 19, 2-141.
(15) Simmon, V., Kauhanen, K. and Tardiff, R.G. (1977). Mutagenic Activity of Chemicals Identified in
Drinking Water. In Progress in Genetic Toxicology, D. Scott, B. Bridges and F. Sobels (Eds.)., Elsevier,
Amsterdam, pp. 249-258.

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(16) Hughes, T.J., Simmons, D.M., Monteith, I.G. and Claxton, L.D. (1987). Vaporization Technique to
Measure Mutagenic Activity of Volatile Organic Chemicals in the Ames/Salmonella Assay. Environmental
Mutagenesis, 9, 421-441.
(17) Matsushima, T., Matsumoto, A., Shirai, M., Sawamura, M. and Sugimura, T. (1979). Mutagenicity
of the Naturally Occurring Carcinogen Cycasin and Synthetic Methylazoxy Methane Conjugates in
Salmonella typhimurium. Cancer Res., 39, 3780-3782.
(18) Tamura, G., Gold, C., Ferro-Luzzi, A. and Ames. B.N. (1980). Fecalase: A Model for Activation of
Dietary Glycosides to Mutagens by Intestinal Flora. Proc. Natl. Acad. Sci. USA, 77, 4961-4965.
(19) Wilcox, P., Naidoo, A., Wedd, D. J. and Gatehouse, D. G. (1990). Comparison of Salmonella
typhimurium TA 102 with Escherichia coli WP2 Tester strains. Mutagenesis, 5, 285-291.
(20) Matsushima, T., Sawamura, M., Hara, K. and Sugimura, T. (1976). A Safe Substitute for
Polychlorinated Biphenyls as an Inducer of Metabolic Activation Systems. In: "In vitro Metabolic Activation
in Mutagenesis Testing", Eds F.J. de Serres et al. Elsevier, North Holland, pp. 85-88.
(21) Elliott, B.M., Combes, R.D., Elcombe, C.R., Gatehouse, D.G., Gibson, G.G., Mackay, J.M. and
Wolf, R.C. (1992). Alternatives to Aroclor 1254-induced S9 in in vitro Genotoxicity Assays. Mutagenesis,
7, 175-177.
(22) Maron, D., Katzenellenbogen, J., and Ames, B.N. (1981). Compatibility of Organic Solvents with
the Salmonella/Microsome Test. Mutation Res., 88, 343-350.
(23) Claxton, L.D., Allen, J., Auletta, A., Mortelmans, K., Nestmann, E., and Zeiger, E., (1987). Guide
for the Salmonella typhimurium/Mammalian Microsome Tests for Bacterial Mutagenicity. Mutation Res.,
189, 83-91.
(24) Mahon, G.A.T., Green, M.H.L., Middleton, B., Mitchell, I., Robinson, W.D. and Tweats, D.J. (1989).
Analysis of Data from Microbial Colony Assays. In: UKEMS Sub-Committee on Guidelines for Mutagenicity
Testing Part II. Statistical Evaluation of Mutagenicity Test Data. Ed. Kirkland, D.J., Cambridge University
Press, pp. 28-65.

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ANNEX
DEFINITIONS

A reverse mutation test in either Salmonella typhimurium or Escherichia coli detects mutation in
an amino-acid requiring strain (histidine or tryptophan, respectively) to produce a strain
independent of an outside supply of amino-acid.

Base pair substitution mutagens are agents that cause a base change in DNA. In a reversion test
this change 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
the DNA, thus changing the reading frame in the RNA

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