OECD 471 bacteria reverse mutation test 9747101E

471

Adopted:

21st July 1997

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.

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.

1/11

471

OECD/OCDE

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.

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 10

9

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.

2/11

OECD/OCDE

471

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 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.

2.

3.

4.

5.

S. typhimurium TA1535, and

S. typhimurium TA1537 or TA97 or TA97a, and

S. typhimurium TA98, and

S. typhimurium TA100, and

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.g. WP2 or 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

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).

3/11

471

Test substance/Preparation

OECD/OCDE

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

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.

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:

4/11

OECD/OCDE

471

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.

(a)

(b)

(c)

(d)

(e)

(f)

(g)

Sodium azide [CAS no. 26628-22-8]

2-Nitrofluorene [CAS no. 607-57-8]

9-Aminoacridine [CAS no. 90-45-9]

or ICR191 [CAS no. 17070-45-0]

Cumene hydroperoxide [CAS no. 80-15-9]

Mitomycin C [CAS no. 50-07-7]

N-Ethyl-N-nitro-N-nitrosoguanidine [CAS no. 70-25-7] or

4-nitroquinoline 1-oxide [CAS no. 56-57-5]

Furylfuramide (AF-2) [CAS no. 3688-53-7]

Strain

TA1535 and TA100

TA98

TA1537, TA97 and TA97a

TA102

WP2 uvrA and TA102

WP2, WP2 uvrA and

WP2 uvrA (pKM101)

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.

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.

5/11

471

PROCEDURE

Treatment with test substance

OECD/OCDE

27. For the plate incorporation method (1)(2)(3)(4), without metabolic activation, usually 0.05

8

ml or 0.1 ml of the test solutions, 0.1 ml of fresh bacterial culture (containing approximately 10

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

8

with the test strain (containing approximately 10

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 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.

6/11

OECD/OCDE

Evaluation and interpretation of results

471

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

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:

-

-

Strains:

-

-

-

strains used;

number of cells per culture;

strain characteristics.

justification for choice of solvent/vehicle;

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

Test conditions:

-

-

-

-

amount of test substance per plate (mg/plate or µg/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.

7/11

471

Results:

-

-

-

-

-

-

-

-

OECD/OCDE

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, ranges, means

and standard deviations.

Discussion of the results.

Conclusion.

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.

Maron, D.M. and Ames, B.N. (1983). Revised Methods for the Salmonella Mutagenicity Test.

Mutation Res., 113, 173-215.

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.

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.

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.

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.

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.

Aeschbacher, H.U., Wolleb, U. and Porchet, L. (1987). Liquid Preincubation Mutagenicity Test

for Foods. J. Food Safety, 8, 167-177.

(2)

(3)

(4)

(5)

(6)

(7)

(8)

8/11

OECD/OCDE

(9)

(10)

471

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Maron, D., Katzenellenbogen, J., and Ames, B.N. (1981). Compatibility of Organic Solvents

with the Salmonella/Microsome Test. Mutation Res., 88, 343-350.

(11)

(12)

(13)

(14)

(15)

(16)

(17)

(18)

(19)

(20)

(21)

(22)

9/11

471

(23)

OECD/OCDE

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.

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.

(24)

10/11

OECD/OCDE

ANNEX

DEFINITIONS

471

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

11/11