Evaluation of genotoxicity induced by repetitive administration of local anaesthetics: an experimental study in rats

(1)

REVISTA

BRASILEIRA

DE

ANESTESIOLOGIA

OfﬁcialPublicationoftheBrazilianSocietyofAnesthesiology

www.sba.com.br

SCIENTIFIC

ARTICLE

Evaluation

of

genotoxicity

induced

by

repetitive

administration

of

local

anaesthetics:

an

experimental

study

in

rats

Gisele

Alborghetti

Nai

a,∗

,

Mariliza

Casanova

de

Oliveira

b

,

Graziela

de

Oliveira

Tavares

c

,

Laís

Fabrício

Fonseca

Pereira

c

,

Nádia

Derli

Salvador

Lemes

Soares

d

,

Patrícia

Gatti

Silva

d

a_Department_of_Pathology,_Universidade_do_Oeste_Paulista,_Presidente_Prudente,_SP,_Brazil b_Universidade_do_Oeste_Paulista,_Presidente_Prudente,_SP,_Brazil

c_Faculty_of_Dentistry_of_Presidente_Prudente,_Universidade_do_Oeste_Paulista,_Presidente_Prudente,_SP,_Brazil d_Medical_College_of_Presidente_Prudente,_Universidade_do_Oeste_Paulista,_Presidente_Prudente,_SP,_Brazil

Received16May2013;accepted25July2013 Availableonline25October2013

KEYWORDS Anaesthesia; Genotoxicity; Mutagenicitytests; Micronucleustests; Prilocaine Abstract

Backgroundandobjective: Previousstudiesregardingtheeffectsofsome localanaesthetics havesuggested thattheseagents cancausegenetic damage.However, theyhavenot been tested for genotoxicity related to repetitive administration. The aim ofthis study was to evaluatethegenotoxicpotentialoflocalanaestheticsuponrepetitiveadministration.

Methods:80maleWistarratsweredividedinto:groupA---16ratsintraperitoneallyinjected withlidocainehydrochloride2%;groupB---16ratsIPinjectedwithmepivacaine2%;groupC---16 ratsintraperitoneallyinjectedwitharticaine4%;groupD---16ratsIPinjectedwithprilocaine3% (6.0mg/kg);groupE---8ratssubcutaneouslyinjectedwithasingledoseofcyclophosphamide; andgroupF---8ratsintraperitoneallyinjectedwithsaline.EightratsfromgroupsAtoDreceived asingledoseofanaestheticonDay1oftheexperiment;theremainingratsweredosedoncea dayfor5days.

Results:Themediannumberofmicronucleiinthelocalanaestheticsgroupsexposedfor1or 5daysrangedfrom0.00to1.00,inthecyclophosphamide-exposedgroupwas10.00,andthe negativecontrolgroupfor1and5dayswas1.00and0.00,respectively(p<0.0001).Asigniﬁcant differenceinthenumberofmicronucleiwasobservedbetweenthecyclophosphamidegroup andalllocalanaestheticgroups(p=0.0001),butnotbetweenthenegativecontrolgroupand thelocalanaestheticgroups(p>0.05).

Conclusion: Nogenotoxicityeffectwasobserveduponrepetitiveexposuretoanyofthelocal anaestheticsevaluated.

∗_{Corresponding}_author.

E-mails:patologia@unoeste.br,gica@muranet.com.br(G.A.Nai).

Este é um artigo Open Access sob a licença de CC BY-NC-ND

(2)

PALAVRAS-CHAVE Anestesia; Testesde mutagenicidade; Testespara micronúcleos; Prilocaína

Avaliac¸ãodagenotoxicidadeinduzidapelaadministrac¸ãorepetidadeanestésicos locais:umestudoexperimentalemratos

Resumo

Justificativaeobjetivos: Estudosanterioressobreosefeitosdealgunsanestésicoslocais sug-eriramqueessesagentespodemcausaralteraçõesgenéticas.Noentanto,essesagentesnãosão testadosparagenotoxicidaderelacionadaàadministraçãorepetida.Oobjetivodesteestudo foiavaliaropotencialgenotóxicodeanestésicoslocaisapósrepetidasadministrações.

Métodos: 80ratosWistarmachosforamalocadosem:grupoA---16ratosreceberaminjeçãopor viaintraperitoneal(IP)decloridratodelidocaínaa2%;grupoB---16ratosreceberaminjeção IPcommepivacaínaa2%;grupoC--- 16ratosreceberaminjeçãoIPdearticaínaa4%;grupoD ---16ratosreceberaminjeçãoIPdeprilocaínaa3%(6,0mgkg−1_);_grupo_E_---₈_ratos_receberam

injeçãosubcutâneaemdoseúnicadeciclofosfamida;grupoF---8ratosreceberaminjeçãoIP comsoluçãosalina.OitoratosdosgruposdeAaDreceberamumadoseúnicadeanestésicono Dia1daexperiência;osratosrestantesforamdosadosumavezpordiadurantecincodias.

Resultados: Amedianadonúmerodemicronúcleosnosgruposcomanestésicoslocaisexpostos porumoucincodiasvarioude0,00a1,00; nogrupoexpostoàciclofosfamidafoi de10,00 enogrupocontrolenegativonoprimeiro equintodiasfoide1,00e0,00, respectivamente (p<0,0001).Umadiferenc¸asigniﬁcativafoiobservadanonúmerodemicronúcleosentreogrupo ciclofosfamidaetodososgruposcomanestésicoslocais (p=0,0001),mas nãoentreogrupo controlenegativoeosgruposcomanestésicoslocais(p>0,05).

Conclusão:Nenhumefeitodegenotoxicidadefoiobservadoapósaexposic¸ãorepetidaa qual-querumdosanestésicoslocaisavaliados.

Introduction

The development of safe and effective local anaesthetic

agentshasbeenoneofthemostimportantadvancesin

den-talscienceoverthelastcentury.Thedentalagentscurrently

available areextremely safeand meet mostof the

crite-riafor an ideallocalanaesthetic.These localanaesthetic

agentsinduceminimaltissueirritationandhavealowrisk

ofinducingallergicreactions.1

Alocalanaestheticismostoftenusedindentistryto con-trolpainandisalsowidelyusedinotherﬁeldsofmedicine.

Amongthe variousformulations oflocalanaesthetics, the

most commonly used types are anaesthetic salts of

lido-caine,mepivacaineandprilocaine.2

The combined use of a vasoconstrictor and a local

anaestheticagent wasﬁrstreported in1901, when Braun

simultaneouslyadministeredadrenalineandcocaine.3_Due

to the vasodilation properties of most anaesthetic salts,

the duration of anaesthesia is not always suitable,

illus-tratingthenecessityofconcomitantadministrationwitha

vasoconstrictor.Someadvantages ofthe combined

admin-istration of vasoconstrictors and anaesthetics are the

slow absorption of the anaesthetic salt (which reduces

toxicity and increases the duration of anaesthesia), a

reductioninthequantityof anaestheticrequiredto

anes-thetise the patient and an increase in the effectiveness

of the anaesthetic.2 _The _most _common _{vasoconstrictors}

used in combination with local anaesthetics belong to

the group of sympathomimetic amines, which includes

adrenaline, noradrenaline, levonordefrin, phenylephrine

andfelypressin.2

Genotoxic agents negatively affect the integrity of a

cell’s genetic material and are deﬁned as any substance

or chemical that damages DNA. Although the ability of

a substance to damage DNA does not automatically

ren-der it as a health hazard, it does raise concerns that

the substance may be a potential mutagen and/or

carci-nogen.4

Somelocalanaestheticshavenotbeentestedfor

carcino-genicityorgenotoxicity.Prilocaineisalocalanaestheticthat

hasbeenunderreviewbytheNationalToxicologyProgram

(NTP,USA)sinceOctober2007.5

Themicronucleustestiswidelyusedtoevaluatethe

abil-ity of a substance tobreak chromosomes (referred toas

its clastogenicity) or affect the formation of the mitotic

metaphase plate and/or spindle, both of which can lead

to inequitable distribution of chromosomes during

cellu-lardivision.6_The _micronucleus_test_generates_results_with

strongstatistical support;therefore,itis widelyusedasa

screeningtooltodeterminethesafetyofmanysubstances

andtoclassifyagentsascarcinogenicornon-carcinogenic.7

Theeaseofimplementationofthemicronucleustesthasled

towidespreadadoptionworldwideasastandard

genotoxic-itytesttomonitorthesafetyofagentsforuseinthehuman population.8

Tothebestofourknowledge,therearenostudiesinthe

literatureaddressingthegenotoxicpotentialofthe repeti-tiveuseoflocalanaesthetics.Localanaestheticsarewidely

usedin dentistryandmedicine, andstudiesthat evaluate

the risk of repetitive exposure to these substances may

contribute to a better understanding of their potentially

toxiceffectsongeneticmaterialandtheirpotentialriskto

exposedpatients.

Theobjectiveofthisstudyistoinvestigatethegenotoxic potentialoftherepetitiveuseoflocalanaestheticsusingthe

micronucleustest.

(3)

Methods

ThisstudywasapprovedbytheEthicsCommitteeonAnimal

Use(Protocoln◦930/11).

For this study, 80 male Wistar rats weighing between

200 and 250g were administered local anaesthetics

at 12 weeks of age. The rats were divided into

groups of four in large rectangular boxes (measuring

49cm×34cm×16cm)suitable for theaccommodation of

uptoﬁveadultrats.Theratswereplacedina

temperature-and humidity-controlled vivarium equipped with a 12-h

light/dark cycle light. All animals were weighed prior

to anaesthetic administration to calculate the proper

dosage.

The animals were divided into 6 groups: group A

--- 16 rats intraperitoneally (IP) injected with lidocaine

hydrochlorideandphenylephrine(Novocol® _100,_SS_White,

Rio de Janeiro, Brazil) at a dose of 4.4mg/kg, group

B --- 16 rats IP injected with mepivacaine 2%

(mepiva-caine, DFL, Jacarepaguá, Brazil) at a dose of 4.4mg/kg,

group C --- 16 rats IP injected with epinephrine and

articaine 4% (Septanest® _1:100,000, _Septodont, _Brussels,

Belgium) at a dose of 7.0mg/kg, group D --- 16 rats IP

injected with prilocaine 3% and felypressin (Cytanest®_,

Astra, Sao Paulo, Brazil) at a dose of 6.0mg/kg, group

E --- 8 rats subcutaneously injected with a single dose

of cyclophosphamide (Genuxal®_, _Baxter _Oncology _GmbH,

Halle/Westfalen, Germany) (50mg/kg) on Day 1 of the

experiment (positivecontrolgroup),8 _and _group_F _---_eight

mice IP injected with 0.5mL of saline (negative control

group).Becauseapreviousreportdemonstratedthe

forma-tionof micronuclei in responsetocyclophosphamide at a

doseof50mg/kg,thisdosewasusedforthepositivecontrol group.8

EightratsfromgroupsAtoDreceivedonedoseof

anaes-theticonDay1oftheexperiment.The remaininganimals

in these groups received a daily dose of anaesthetic for

ﬁvedays.The ratsingroup Fwereadministeredsalinein

asimilarmanner.

Eight rats fromgroups Ato D, four rats fromgroup F,

and all of the rats in group E wereeuthanised 24h after

administrationof the anaesthetic.The remaining animals

of groups A, B,C, D and F were euthanised5 days later.

Euthanasiawasperformedwithsodiumpentobarbital

(Syn-tec,Cotia, SãoPaulo, Brazil)atadoseof100mg/kgbyIP

injection.

Bonemarrowsampleswerecollectedfromthefemurof

each rat at the time of sacriﬁce, and two sample slides

were preparedper animal.8 _The _slides _were_stained _with

Giemsastain(Dolles,SãoPaulo,Brazil).Twothousand

poly-chromatic erythrocytes (1000 perslide) were countedfor

each animal at a magniﬁcation of 400× using an

opti-calmicroscope todetermine thenumber of micronuclei.8

Micronucleiweredeﬁnedasstructureswithprobablehalos

surrounding the nuclear membrane and a volume of less

thanone-thirdthatofthediameteroftheassociatednuclei;

themicronucleistainingintensitywassimilartothe

inten-sity of the associated nuclei, and both structures were

observed inthe samefocalplane.9_The _slide _analysis_was

performed in a blinded manner by a single person (MCO)

andreviewedbyasecondperson(GAN);bothresultswere

concordant.

Statistical

analysis

Thevarianceinmicronucleifrequencydidnothaveanormal

distributionwhenanalysedbytheKolmogorov---Smirnovtest

(p=0.0001)norwerethevarianceshomogenous(p=0.004)

by Levene test analysis. We therefore chose to use the

nonparametric Kruskal---Wallis test followed by multiple

comparisonswiththeStudent---Newman---Keulstestto

deter-mine statistical signiﬁcance. All statistical tests were

performedatasigniﬁcancelevelof5%.

Results

Themediannumberofmicronucleiobservedforeachgroup

wasasfollows: for the lidocainegroup, the 1-and 5-day

exposureswere1.00and0.50,respectively;forthe

mepiva-cainegroup,the1-and5-dayexposureswereboth1.00;for

thearticainegroup,the1-and5-dayexposureswere1.00

and0.00,respectively;andfortheprilocainegroup,the

1-and5-dayexposureswereboth0.00.Inthegroupexposedto

cyclophosphamide(thepositivecontrol),themedian

num-berofmicronucleiwas10.00.Thenegativecontrolgroups

for days 1 and 5 had median numbers of micronuclei of

1.00and0.00,respectively(p<0.0001) (Figs.1and2and Table1).

The number of micronuclei in the positive control

group(cyclophosphamide)signiﬁcantlydifferedfromthose

observedfor allofthe localanaestheticsstudied,both in

thetypeofanaestheticadministeredandthetimeof

expo-sure(p=0.0001).However,nosigniﬁcant differenceinthe

numberofmicronucleiwasobservedbetweenthenegative

controlgroupandthelocalanaestheticgroups,regardlessof thetypeofanaestheticadministeredorthetimeofexposure (p>0.05)(Table1). 13 41 19 13 11 10 8 6 4 3 2 1 0 Count Group Lido 1 Lido 5 MEPI 1 MEPI 5 ARTI 1 ARTI 5 Prilo 1 Prilo 5 Cyclo Control 1 Control 5 49 73

Figure 1 Micronuclei counts per study group (median and interquartile intervals). LIDO, lidocaine; MEPI, mepivacaine; ARTI,articaine;PRILO,prilocaine;CONTROL,negativecontrol; CYCLE,cyclophosphamide(positivecontrol);1,exposurefor1 day;5, exposurefor 5days;,outlier;*,theoutlier ofthe outlier;thenumberingovertheoutliercorrespondstothe num-beringofanimals.

(4)

Figure 2 Examples of a polychromatic erythrocyte with a micronucleus(largearrow)andanormalpolychromatic erythro-cyte(narrowarrow);theanimalwasexposedtomepivacainefor 5days(Giemsastaining,1000×).

Withtheexception ofthecyclophosphamide group,all

groupswereequalformultiplestatisticalanalyses.However,

signiﬁcant differences in the frequencies of micronuclei

wereobservedbetweentheprilocaine5-dayexposuregroup

andthe following groups: lidocaine1-day exposure group

(p=0.0466), the mepivacaine 1- (p=0.0437) and 5-day

(p=0.0460)exposuregroups andthearticaine1-day

expo-suregroup(p=0.0364)(Table1).

Discussion

Genotoxicitytestsareimportantfortheevaluationof

cellu-lartoxicityandtheidentiﬁcation ofpotentialcarcinogens

and mutagens. Several techniques are employed to test

Table1 Medianandinterquartilerangesofthefrequency ofmicronucleiforeachgroup(n=80).

Group Median Interquartilerange Lidocaine---1day 1.00a 3.00

Lidocaine---5days 0.50a,c 2.00 Mepivacaine---1day 1.00a 1.00 Mepivacaine--- 5days 1.00a 2.00 Articaine---1day 1.00a 2.00 Articaine---5days 0.00a,c 1.00 Prilocaine---1day 0.00a,c 1.00 Prilocaine---5days 0.00c 1.00 Negativecontrol---1day 1.00a,c 2.00 Negativecontrol---5days 0.00a,c 4.00 Cyclophosphamidea _10.00d _3.00

a_Positive _control._Results _with_different_letters_differ

signiﬁ-cantly(p<0.05).

an agent’s genotoxic activity,including assaysthat

deter-mineDNA/proteincross-linkingcoefﬁcients,mitochondrial

enzymaticactivity,cellproliferation,repairofDNAbreaks,

mitotic index,thetypeofdamageincurred, chromosomal

aberrations, chromosomal non-disjunctions, and levels of

apoptosis and necrosis.8 _The _micronucleus _test _has _been

used extensivelyto test the genotoxicity of many

chemi-cals.Micronucleiareeasily viewedin erythrocytesamples

andarestronglyindicativeofchromosomalaberrations.6

The micronucleus test was ﬁrst reported in 1970 by

Boller and Schmid and was subsequently used by Heddle

in 1977.10 _The _micronucleus_is_an _additional_nucleus,

sep-arated from the main nuclear core of a cell during cell

divisionandcompriseswholechromosomesorchromosomal

fragmentsthatlagbehindtheotherchromosomesuponthe

completionofmitosis.Themicronucleusresultsfrom

spon-taneousorexperimentallyinducedstructuralchangesinthe

chromosome(s),or throughcellularfusion errors,andit is

thereforeexcludedfromthenewnucleusthatisre-formed

intelophase.11

The advantages of the micronucleus assay over other

tests used to diagnose diseases and monitor

environmen-tal contaminants include its simplistic analysis, its high

detection sensitivity and accuracy of chromosome losses

andnondisjunctionevents,itsabilitytomeasurethelength

andprogressionofnucleardivisionanditsabilitytodetect

repairandexcisionevents.8_These_advantages_prompted_us

tochoosethemicronucleustesttoevaluatethegenotoxic

effectsofrepetitiveadministrationoflocalanaestheticsin thisstudy.

Adisadvantageofusingvasoconstrictorsin conjunction

withlocalanaestheticsisapparentwithintravascular

injec-tions,duringwhich highconcentrationsandlarge volumes

canleadtointoxication.12_Thus,_in_some_patients,_it_is

unac-ceptabletousevasoconstrictors,particularlyinthosewith

diabetesorheartdiseaseorpregnantwomen.Inthesecases,

themostcommonlyusedanaestheticsaltintheabsenceof

avasoconstrictorismepivacaine.12 _Thus,_mepivacaine_was

usedin thisstudywithout a vasoconstrictor.Thereareno

knownreportsintheliteraturethatevaluatethegenotoxic

actionofvasoconstrictors.

Prilocainewaspreviouslyreportedtoexhibitgenotoxic

activity in somatic cells and to be capable of inducing

homologous recombination. However, lidocaine and

arti-caine (Septanest®₎ _were _reportedly _incapable _of _inducing

chromosomalmutationorrecombination.13_No_genotoxicity

wasassociatedwiththeuseof anyofthedrugs examined

in this study.These results are in partial agreementwith

theliterature, anddifferonly withthe previousreport of

prilocainegenotoxicity.13

Ithas been demonstrated thatthe percentage ofcells

withpolyploidyandendoreduplicationincreasesupon

expo-suretoprilocainehydrochlorideandprocainehydrochloride

both inthepresenceandabsenceofexogenousmetabolic

activation.14 _These _results _indicate _that _such _chemical

agentsarepotentiallygenotoxictomammaliancells.14

How-ever,thisstudydidnotshowgenotoxicactionofprilocaine,

possiblybecauseitwasusedattherecommendeddoseper

kilogramofweight.

Nuclear condensation and fragmentation of chromatin

werepreviouslyobservedincellstreatedwithprilocaine.15

(5)

treatment in a dose- and time-dependent manner, with

maximaleffectsobservedataconcentrationof5mMafter

12---48hofexposure.15_Together_with_this_study,_these_data

show that usingprilocaine at the recommendeddose per

kilogramofbodyweightcannotcausegeneticdamage.

Lidocaineandprilocainearemainlymetabolisedin the

liverandsubsequentlyhydrolysedbyamideesters,releasing

themonocyclicaromaticamines,2,6-dimethylaniline(DMA)

and2-methylalanine(MA),respectively.5_Other_anaesthetics

thatcontainafractionofDMAincludebupivacaine,

mepiva-caineandropivacaine.5

The main carcinogenic mechanism of aromaticamines

such as DMA and MA occurs when cytochrome P450

metabolises these compounds into derivatives of

N-hidroxila.5_DMA_and_MA_can_be_further_metabolised_by_their

conjugation to reactive metabolites. The DNA lesion has

been described for DMA, but not for MA. The formation

of DNAadductsis thoughttobea possible mechanismby

which these compounds exert some of their carcinogenic

effects.16

However,theInternationalAgencyforResearchon

Can-cer (IARC) reported no carcinogenic effects of DMA in

humans, although there is sufﬁcient evidence of its

car-cinogenicityinrats.5_This_may_explain_the_greater_numbers

ofmicronucleiobservedinthelidocainegroup(regardless

of exposure time) compared to the prilocaine group and

the signiﬁcant difference between the groups exposed to

lidocainefor 1day and prilocainefor 5days (p=0.0466).

Although the frequency of micronuclei in the

lidocaine-exposed group was not signiﬁcantly different from that

of the negative control group, the higher frequency of

micronuclei in the lidocaine-exposed group (compared to

theprilocainegroup)maybeassociatedwiththeeffectsof

DMA,ametabolicproductoflidocaine.

Therearenoliteraturereportsregardingthegenotoxic

ormutagenicpotentialofmepivacaine.Inthisstudy,

mepi-vacaine exhibited nogenotoxicity, regardless of exposure

time.However,exposureofmepivacainefor1and5dayswas

signiﬁcantlydifferentfromthegroupexposedtoprilocaine

for5days(p<0.05).Mepivacainealsocontainsafractionof

DMA,5_which_may_explain_the_higher_number_of_micronuclei

observedinthisgroup,althoughthefrequencyof

micronu-cleiwasnotsigniﬁcantlydifferentfromthenegativecontrol group.

Mutagenicity studies in vitro and in vivo revealed no

genotoxic potential of articaine (CAS 23964-58-1) until

the maximum tolerated dose was reached. In agreement

with the data in this study, another articaine

prepara-tion (Septanest® _SP; _4% _articaine _HCl _and _epinephrine _1:

100,000)17 _exhibited _no _genotoxic _effects _in _an _in _vivo

studyusingtherecommendeddoseperkilogramofweight.

Although articaine belongs to the amide group of local

anaesthetics(whichalsoincludes lidocaine,prilocaineand

mepivacaine),itismetabolisedbycholinesteraseinserum

plasma into articainic acid via hydrolysis. Articainic acid

is an inactive metabolite and is partially metabolised in

the kidney into articainicacid glucuronide, rather than a

potentiallygenotoxicaromaticamine.18_This_result_may

par-tially explain the absence of genotoxicity upon articaine

exposure.However,the1-dayarticaineexposuregroupwas

signiﬁcantly different from the 5-day prilocaine exposure

group(p=0.0364).This maybeduetothepresenceofan

outlier in the articainegroup, which was well above the

overallmedianfrequencyofmicronuclei.

Conclusion

Inthepresent study,therewasnoincreasedfrequency of

micronucleiupon exposure toany ofthe local

anaesthet-icstested(lidocaine,mepivacaine,articaineandprilocaine)

whenusedattherecommendeddoseperkilogramofbody

weight, with either a single exposure or upon repetitive

administration. However,other teststhat assess

genotox-icity and mutagenicity should be applied to deﬁnitively

determinethattherepetitiveuseoftheseanaestheticshas

nogenotoxicormutagenicactivity.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

References

1.MoorePA,HershEV.Localanesthetics:pharmacologyand tox-icity.DentClinNorthAm.2010;54:587---99.

2.MarianoRC,SantanaSI,CouraGS.Análisecomparativadoefeito anestésicodalidocaína2%edaprilocaína3%.RevistaBrasileira deCirurgiaeImplantodontia---BCI.2000;7:15---9.

3.Jastak JT, Yagiela JA. Vasoconstritors and local anesthe-sia: a review and rationale for use. J Am Dent Assoc. 1983;107:623---30.

4.Smith MT. The mechanism of benzene-induced leukemia: a hypothesisandspeculationsonthecausesofleukemia.Environ HealthPerspect.1996;104:1219---25.

5.Duan JD, Jeffrey AM, Williams GM. Assessment of the medicines lidocaine, prilocaine, and their metabolites, 2,6-dimethylanilineand2-methylaniline,forDNAadductformation inrattissues.DrugMetabDisposJ.2008;8:1470---5.

6.FloresM,YamaguchiUM.Micronucleustest:anevaluationfor genotoxicscreening.JHealthRes.2008;1:337---40.

7.FenechM,HollandN,ChangWP,etal.TheHUmanMicroNucleus Project---aninternationalcollaborativestudyontheuseofthe micronucleustechniqueformeasuringDNAdamageinhumans. MutatRes.1999;428:271---83.

8.MacGregor JT, Heddle JA, Hite M, et al. Guidelines for the conduct ofmicronucleus assays inmammalian bone marrow erythrocytes.MutatRes.1987;189:103---12.

9.TolbertPE, Shy CM,AllenJW. Micronucleiand othernuclear anomaliesinbuccalsmears:methodsdevelopment.MutatRes. 1992;271:69---77.

10.Evans HJ. Historical perspectives on the development of the in vitro micronucleus test: a personal view. Mutat Res. 1997;392:5---10.

11.Ramírez A, Saldanha PH. Análise crítica de grupos controle no teste de micronúcleo na mucosa bucal. Genet Mol Biol. 1998;21:140.

12.PerusseR,GouletJP,TurcotteJY.Contraindicationsto vasocon-strictorsindentistry: partI.OralSurgOralMedOralPathol. 1992;74:679---86.

13.SchneiderLE,doAmaralVS,DihlRR,etal.Assessmentof geno-toxicityoflidocaine,prilonestandseptanestinthedrosophila Wing-Spottest.FoodChemToxicol.2009;47:205---8.

14.HagiwaraM,WatanabeE,BarrettJC,etal.Assessmentof geno-toxicityof14chemicalagentsusedindentalpractice:ability toinducechromossomeaberrationsinSyrianhamsterembryo cells.MutatRes.2006;603:111---20.

(6)

15.NakamuraK,KidoH,MorimotoY,etal.Prilocaineinduces apo-ptosisinosteoblasticcells.CanJAnaesth.1999;46:476---82. 16.PrestonRJ,Williams GM.DNA-reactivecarcinogens: modeof

action and human cancerhazard. Crit Rev Toxicol.2005;35: 673---83.

17.Leuschner J, Leblanc D. Studies on the toxicological pro-ﬁleofthelocalanaestheticarticaine.Arzneimittelforschung. 1999;49:126---32.

18.SnoeckM.Articaine:areviewofitsuseforlocalandregional anesthesia.LocalRegAnesth.2012;5:23---33.

www.sba.com.br

Este é um artigo Open Access sob a licença de CC BY-NC-ND

2010;54:587---99.

2000;7:15---9.

Jastak

1996;104:1219--25.

2008;8:1470---5.

2008;1:337---40.

1999;428:271---83.

1987;189:103---12.

Tolbert

Evans

Ramírez

Perusse

2009;47:205---8.

2006;603:111---20.

1999;46:476---82.

2005;35:673---83.

Leuschner

2012;5:23---33.