genotoxicity of three mouthwash products, cepacol®, periogard®, and plax®, in the drosophila...

Research Article

Genotoxicity of ThreeMouthwash Products, Cepacol1,Periogard1, and Plax1, in the Drosophila

Wing-Spot Test

Fa¤ bio Rodrigues, Maur|¤ cio Lehmann, Viviane Souza do Amaral,Maria Lu|¤ za Reguly, and Helo|¤ sa Helena Rodrigues de Andrade*

Laboratorio da Toxicidade Genetica – TOXIGEN, Universidade Luterana doBrasil – ULBRA/Canoas, Canoas, RS, Brazil

Antiseptic mouthwashes used in biofilm control arewidely available in the marketplace, despite incon-sistent data concerning their genetic and cellular tox-icity. In the present study, we investigated the geno-toxic potential of three antiseptics currently used forodontologic treatment, Cepacol1 (containing cetyl-pyridinium chloride), Periogard1 (chlorhexidinedigluconate), and Plax1 (triclosan). Genotoxicitywas evaluated using the Somatic Mutation andRecombination Test (SMART) in Drosophila mela-nogaster, employing flies having normal bioactiva-tion (the standard cross) and flies with increased cyto-

chrome P450-dependent biotransformation capacity(the high bioactivation cross). Periogard and Plaxproduced negative responses in both types of flies;however, Cepacol (75 and 100%) produced posi-tive responses in both the standard and high bioacti-vation assays, with the genotoxic responses mainlydue to the induction of mitotic recombination. Assaysperformed with ethanol and cetylpirydinium chlo-ride, two major ingredients of Cepacol, indicatedthat the genotoxity of the mouthwash is likely to bedue to ethanol. Environ. Mol. Mutagen. 48:644–649, 2007. VVC 2007Wiley-Liss, Inc.

Key words: SMART; cetylpyridinium chloride (CPC); chlorhexidine digluconate (CXD); triclosan (TCS)

INTRODUCTION

Antiseptic mouthwashes for personal oral hygiene are

widely used for their ability to inhibit dental plaque, a

complex biofilm formed in a series of discrete steps. Pla-

que begins with the accumulation of gram-positive strep-

tococci, followed by increasing deposits, which involve

gram-negative anaerobic bacteria [Xie et al., 2000; Lewis,

2001].

Three of the most extensively used mouthwash antisep-

tics are cetylpyridinium chloride (CPC), chlorhexidine

digluconate (CXD), and triclosan (TCS). Although these

agents have demonstrated the ability to inhibit the forma-

tion of biofilm, there is little publicly available informa-

tion on their genetic and cellular toxicity. A survey of the

effects of CPC and CPX in various experimental systems

indicates that CPC is cytotoxic [Burgalassi et al., 2001;

Chetoni et al., 2003] and that CXD can induce DNA dam-

age [Sakagami et al., 1988a,b; Eren et al., 2002; Ribeiro

et al., 2004]. Additionally, TCS was shown to induce so-

matic mutations in mice [Russel and Montgomery, 1980],

although a more recent report [Bargava and Leonard,

1996] failed to confirm this finding.

The present investigation was designed to evaluate

the genotoxic potential of three antiseptic mouthwash

products containing CPC, CXD, and TCS: Cepacol1 (0.05%

CPC), Periogard1 (0.12% CPX), and Plax1 (0.03% TCS).

The study employed the in vivo wing Somatic Mutation and

Recombination Test (SMART) in Drosophila melanogaster,which quantitatively measures both mutation and homolo-

gous recombination. There exists an extraordinary conserva-

tion of not only individual domains and proteins but also of

entire complexes and multi-step pathways between fly and

man [St. John and Xu, 1997]. This suggests that Drosophila

is a reasonable model for evaluating the potential of environ-

mental agents for inducing genetic damage in humans

[Bishop and Schiestl, 2003].

*Correspondence to: Heloısa H. R. de Andrade, Laboratorio da Toxicidade

Genetica – ULBRA, Predio 22, 48 andar, Sala 25, Av. Farroupilha, 8001,

92450-900, Canoas, RS, Brazil. E-mail: [email protected]

Grant sponsors: Conselho Nacional de Desenvolvimento Cientıfico e

Tecnologico (CNPq), Financiadora de Estudos e Projetos (FINEP), Fun-

dacao e Coordenacao de Aperfeicoamento de Pessoal de Nıvel Superior

(CAPES).

Received 4 August 2006; provisionally accepted 17 May 2007; and in

final form 1 June 2007

DOI 10.1002/em.20332

Published online 18 September 2007 in Wiley InterScience (www.interscience.

wiley.com).

VVC 2007Wiley-Liss, Inc.

Environmental andMolecular Mutagenesis 48:644^649 (2007)

MATERIALS ANDMETHODS

Chemicals

Cepacol [0.05% CPC (CAS no. 123-03-5)] was obtained from Aventis

Pharma (Sao Paulo, Brazil), while Periogard [0.12% CXD (CAS no. 55-

56-1)] and Plax [0.03% TCS (CAS no. 3380-34-5)] were purchased from

Colgate-Palmolive Company (Sao Paulo, Brazil). CPC was obtained

from Labsynth (Sao Paulo, Brazil). The molecular structures of the three

antiseptic substances are depicted in Figure 1. The test mouthwashes

were used at full strength (Cepacol [0.05% CPC], Periogard [0.12%

CXD] and Plax [0.03% TCS]) and/or in different dilutions, which were

prepared in distilled water just before use.

Wing Spot Test

The SMART assay detects different genetic endpoints by using crosses

of three different strains of Drosophila melanogaster which carry spe-

cific genetic markers (mwh and flr3) on the left arm of chromosome 3.

Parental flies used for the crosses were (i) flr3/In(3LR)TM3, ri pp sep

l(3)89Aa bx34ee Bds, (ii) ORR/ORR, flr3/In(3LR)TM3, ri pp sep l(3)89Aabx34e e Bds, and (iii) mwh/mwh.

Eggs derived from the standard (ST) cross (flr3/In(3LR)TM3, ri pp sep

l(3)89Aa bx34ee Bds virgin females crossed with mhw/mwh males) and

the high bioactivation (HB) cross (ORR/ORR, flr3/In(3LR)TM3, ri pp sepl(3)89Aa bx34e e Bds virgin females crossed with mwh/mwh males) were

collected for 8 hr on standard medium enriched with baker’s yeast.

Three days later, the larvae from both the crosses were transferred to

vials containing 1.5 g of dry Drosophila instant medium (Carolina Bio-

logical Supply, Burlington, NC) rehydrated with 5 ml of the test solu-

tions or distilled water. The larvae were allowed to feed on these media

until pupation [Andrade et al., 2004].

The flies generated by the ST and HB crosses were of two genotypes:

marker-heterozygous (mwhþ/flr3) and balancer-heterozygous (mwhþ/

TM3, Bds). The adults were collected and stored in 70% ethanol. Their

wings were mounted in Faure’s solution and inspected under 400 3magnification for the presence of mutant spots. The number of spots, as

well as type and size of the spots were analyzed. It is possible to distin-

guish three different categories of spots on marker-heterozygous wings

(mwh/flr3): (i) small single spots (1–2 cells in size), (ii) large single spots

(more than two cells), both expressing either the multiple wing hairs

(mwh) or the flare (flr3) phenotype, and (iii) twin spots, consisting of

both mwh and flr3 subclones. Only mwh single spots can be observed on

balancer heterozygous wings (mwh/TM3), as the inverted TM3 balancer

chromosome does not carry flr3 or any other suitable marker mutation.

While mutant clones in mwh/flr3 flies can be induced by somatic point

mutation, chromosome aberration, and/or mitotic recombination, this last

alteration is lethal in mwh/TM3 flies because of the presence of multiple

inversions in the TM3 balancer chromosome. Thus, by comparing spot

frequencies in these two genotypes, it was possible to quantify the

recombinagenic action of the test agents [Frei and Wurgler, 1996].

Statistical Analysis

The frequencies of each spot category per fly of the treated series

were compared to its concurrent negative control series (distilled water).

These statistical comparisons were performed using the Kastenbaum-

Bowman test for proportions, followed by the multiple-decision proce-

dure described by Frei and Wurgler (1988).

The recombinagenic activity of the test agents was calculated by com-

paring the standard frequency of clones per 105 cells obtained in the

mwh/flr3 and mwh/TM3 genotypes [Frei and Wurgler, 1996]. To ensure

an unbiased comparison, only mwh clones in mwh single spots and in

twin spots were used [Frei et al., 1992].

RESULTS

The genotoxicity of the three antiseptic mouthwashes was

judged by their responses in both the marker-heterozygous

(mwh/flr3) and balancer-heterozygous (mwh/TM3) genotypesin the ST and HB crosses (Tables I and II). The data obtained

in three individual experiments with each of the test agents

were pooled, since no statistical differences were found

between the individual trials. Each of the agents was tested

in a minimum of 400 flies from both the ST and the HB

crosses; i.e., a total of at least 800 flies were scored.

With few exceptions, neither Periogard nor Plax produced

a significant induction of any of the spot categories in either

the ST or HB version of the SMART; the exceptions were

an increase in small single spots produced by 12.5% Plax in

the HB cross and increases in large single spots produced

by 25 and 100% Periogard for the ST cross. Nevertheless,

neither Periogard nor Plax had a significant effect on total

spot frequencies, suggesting that the two agents do not act

as direct or indirect toxins (Tables I and II).

Cepacol, however, produced significant increases in the

frequencies of both small single spots and of total spots at

concentrations of 75 and 100% in both the ST and HB

crosses. In an attempt to estimate the recombinagenic

and/or mutagenic action of Cepacol, the TM3-balancer-

heterozygous flies exposed to this antiseptic were also an-

alyzed. No significant increases in the frequencies of spots

were observed in balancer-heterozygous flies treated with

the 75 and 100% concentrations of Cepacol, indicating

that this mouthwash was not mutagenic under these con-

ditions (Tables I and II). These results indicate that the

Fig. 1. Chemical structures of cetylpyridinium chloride (A), chlorhexi-

dine digluconate (B), and triclosan (C).

Environmental and Molecular Mutagenesis. DOI 10.1002/em

Genotoxicity of Mouthwash Products 645

genotoxicity of Cepacol in the SMART assay is restricted

to the agent’s ability to induce mitotic recombination.

Periogard and Plax contain, respectively, 9 and 6% of

ethanol, whereas Cepacol contains 16.8% of ethanol

(Eth). In attempting to determine whether ethanol or CPC

(0.05%) should be regarded as representing the genotoxic

hazard of Cepacol both drugs were tested individually at

the same concentrations present in the test concentrations

of the mouthwash. Treatment with CPC did not produce a

significant increase in any spot category in either ST or

HB cross flies (Table III). In contrast, 12.5 and 16.8%

Eth, which corresponds to the 75 and 100% doses of

Cepacol, produced significant increases in total spot fre-

quencies in ST cross flies, and 8.4, 12.5, and 16.8% Eth,

corresponding to the 50, 75, and 100% concentrations of

Cepacol, produced significant increases in total spots in

assays run with the HB cross (Table III).

DISCUSSION

The present study used the Drosophila SMART assay

to evaluate the genotoxic potential of three commercial

antiseptic mouthwashes: Cepacol (containing CPC), Perio-

gard (containing CXD), and Plax (containing TCS). The

results indicate that neither Periogard nor Plax is geno-

toxic in the assay (Tables I and II).

Although TCS was reported to be positive for somatic

mutation in the mouse spot test [Russel and Montgomery,

1980], acute, subacute/subchronic, and chronic treatments

with TCS were negative for mutagenicity, carcinogenicity,

and teratogenicity in male and female Sprague-Dawley rats

[Russel and Montgomery, 1980; Bhargava and Leonard,

1996]. Treatment with CDX induced primary DNA damage

both in rat peripheral blood and in oral mucosa cells as

assessed by the Comet assay, but did not induce micronuclei

[Ribeiro et al., 2004]. CDX also induced DNA damage in

human buccal epithelial cells and peripheral lymphocytes,

as detected by the Comet assay [Sakagami et al., 1988a,b;

Eren et al., 2002]. It is possible that the DNA damage

detected by Comet assay in these studies was an early event

that was repaired quickly, resulting in negative responses in

the micronucleus assay and our SMART assays.

In contrast to the negative results with Periogard and

Plax, Cepacol was positive in both the ST and HB crosses

TABLE I. Genotoxicity of Cepacol1, Plax1, and Periogard1 in the D. melanogaster Wing SpotTest using the Standard (ST) Cross

Genotypes

Dilutions

(%)

Number

of flies (N)

Spots per fly (number of spots)/statistical diagnosisa

Total mwh

clonesc (n)

Small single spots

(1–2 cells)b (m ¼ 2)

Large single spots

(>2 cells)b (m ¼ 5)

Twin spots

(m ¼ 5)

Total spots

(m ¼ 2)

CEPACOL1

mwh/flr3 0 40 0.88 (35) 0.15 (6) 0.03 (1) 1.05 (42) 38

12.5 40 0.83 (33)� 0.18 (7)i 0.05 (2)i 1.05 (42)� 40

25 40 1.08 (43)� 0.03 (1)� 0.03 (1)i 1.13 (45)� 45

50 40 0.88 (35)� 0.20 (8)i 0.08 (3)i 1.15 (46)� 46

75 40 1.68 (67)þ 0.15 (6)i 0.00 (0)i 1.83 (73)þ 73

100 40 1.60 (64)þ 0.18 (7)i 0.03 (1)i 1.80 (72)þ 72

mwh/TW3 0 40 0.90 (36) 0.03 (1) d 0.93 (37) 37

75 40 0.88 (35)� 0.00 (0)i 0.88 (35)� 35

100 40 0.85 (34)� 0.08 (3)i 0.93 (37)� 37

PLAX1

mwh/flr3 0 40 0.85 (34) 0.15 (6) 0.03 (1) 1.03 (41) 41

6.25 40 0.93 (37)� 0.20 (8)i 0.03 (1)i 1.15 (46)� 46

12.50 40 0.73 (29)� 0.13 (5)i 0.03 (1)i 0.88 (35)� 35

25 40 0.95 (38)� 0.08 (3)� 0.05 (2)i 1.08 (43)� 43

50 40 1.08 (43)� 0.10 (4)i 0.03 (1)i 1.20 (48)� 47

75 40 0.90 (36)� 0.35 (14)i 0.15 (6)i 1.40 (56)� 54

PERIOGARD1

mwh/flr3 0 40 0.93 (37) 0.05 (2) 0.05 (2) 1.03 (41) 41

12.5 40 0.93 (37)� 0.20 (8)i 0.00 (0)i 1.13 (45)� 44

25 40 0.78 (31)� 0.28 (11)þ 0.05 (2)i 1.10 (44)� 44

50 40 0.75 (30)� 0.08 (3)i 0.10 (4)i 0.93 (37)� 37

75 40 0.73 (29)� 0.08 (3)i 0.10 (4)i 0.90 (36)� 36

100 40 0.65 (26)� 0.33 (13)þ 0.10 (4)i 1.08 (43)� 43

ENU 0.05 mM 10 5.50 (55)þ 6.50 (65)þ 2.50 (25)þ 14.50 (145)þaStatistical diagnosis according to Frei and Wurgler (1988): þ, positive; �, negative; i, inconclusive; m, multiplication factor. Probability levels a ¼b ¼ 0.05.bIncluding rare flr3 spots.cConsidering mwh clones from mwh single spots and from twin spots.dOnly mwh single spots can be observed in mwh/TM3 heterozygotes as the balancer chromosome TM3 does not carry flr3 mutation.


646 Rodrigues et al.

in the SMART assay, with the positive responses being

mainly due to recombination (Tables I and II). Since

Cepacol contains 16.8% Eth and 0.05% CPC, both drugs

were tested individually at the same concentrations that

were present in the mouthwash (Table III). CPC was neg-

ative, and Eth positive in these assays. Consequently, the

induction of recombination that we observed for Cepacol

was probably due to the Eth contained in this mouthwash.

Published information relevant to the assessment of the

possible genotoxic potential of ethanol gave clear evi-

dence that it is not a bacterial or mammalian cell muta-

gen, although the in vitro assays carried out have gener-

ally not included exogenous metabolic activation. The

reported in vivo tests for chromosome aberration are all

negative and only few micronucleus tests have given posi-

tive results. The majority of SCE assays in cultured cells

have shown negative responses; however, these studies

did not use an exogenous metabolic activation system.

SCE can be induced in lymphocytes in vitro by acetalde-

hyde or ethanol treatment in the presence of alcohol dehy-

drogenase enzyme [Obe et al., 1986], which suggests that

acetaldehyde may be the agent responsible for SCE induc-

tion by ethanol in vitro and in animals.

Drosophila is well equipped to tolerate and utilize high

levels of ethanol encountered in its rotting-fruit niche,

which means that this metabolite is formed in significant

amounts in different tissues in vivo. Therefore, the genetic

effects observed in the present study—mitotic recombina-

tion increments in somatic cells of Drosophila mela-nogaster—might be due to an ethanol metabolite, presum-

ably acetaldehyde [Phillips and Jenkinson, 2001]. Acetal-

dehyde has been found to induce SCE and chromosome

aberrations in cultured mammalian cells. Although there

have been very few studies in intact mammals, the avail-

able evidence suggests that acetaldehyde produces similar

cytogenetic effects in vivo, which may be related to its

ability to form DNA–DNA and/or DNA–protein cross-

links. All in all, this might explain our data, as well as

the significant increases induced by ethanol in D. mela-nogaster X-chromosome nondisjunction, and sex-linked

recessive lethals [Rey et al., 1992; Phillips and Jenkinson,

2001; Montooth et al., 2006].

TABLE II. Genotoxicity of Cepacol1, Plax1, and Periogard1 in the D. melanogaster Wing SpotTest using the High Bioactivation (HB) Cross

Genotypes

Dilutions

(%)

Number

of flies (N)


Total mwh

clonesc (n)

Small single spots

(1–2 cells)b (m ¼ 5 )

Large single spots

(>2 cells)b (m ¼ 5)

Twin spots

(m ¼ 5)

Total spots

(m ¼ 2)

CEPACOL1

mwh/flr3 0 40 0.90 (36) 0.20 (8) 0.08 (3) 1.18 (47) 47

12.5 40 1.03 (41)� 0.08 (3)� 0.08 (3)i 1.18 (47)� 47

25 40 1.38 (55)þ 0.18 (7)i 0.00 (0)� 1.55 (62)� 62

50 40 1.00 (40)� 0.10 (4)� 0.13 (5)i 1.23 (49)� 47

75 40 1.60 (64)þ 0.18 (7)i 0.03 (1)i 1.80 (72)þ 72

100 40 1.65 (66)þ 0.25 (10)i 0.03 (1)i 1.93 (77)þ 77

mwh/TM3 0 40 1.00 (40) 0.03 (1) d 1.03 (41) 41

75 40 0.93 (37)� 0.15 (6)i 1.08 (43)� 43

100 40 1.33 (53)� 0.03 (1)i 1.35 (54)� 54

PLAX1

mwh/flr3 0 40 0.90 (36) 0.20 (8) 0.08 (3) 1.18 (47) 47

6.25 40 1.03 (41) 0.25 (10)i 0.10 (4)i 1.38 (55)� 54

12.5 40 1.38 (55)þ 0.03 (1)� 0.03 (1)i 1.43 (57)� 57

25 40 1.05 (42)� 0.33 (13)i 0.03 (1)i 1.40 (56)� 56

50 40 1.00 (40)� 0.28 (11)i 0.03 (1)i 1.30 (52)� 50

75 40 1.25 (50)i 0.20 (8)i 0.03 (1)i 1.48 (59)� 59

PERIOGRAD1

mwh/flr3 0 40 1.08 (43) 0.30 (12) 0.08 (3) 1.45 (58) 57

12.5 40 0.83 (33)� 0.15 (6)� 0.05 (2)� 1.03 (41)� 41

25 40 0.85 (34)� 0.25 (10)� 0.10 (4)� 1.20 (48)� 47

50 40 0.98 (39)� 0.20 (8)� 0.15 (6)� 1.33 (53)� 53

75 40 0.95 (38)� 0.30 (12)i 0.05 (2)i 1.30 (52)� 52

100 40 1.05 (42)� 0.38 (15)i 0.08 (3)i 1.50 (60)� 60

URE 20 mM 10 19.10 (191)þ 9.80 (98)þ 2.60 (26)þ 31.50 (315)þaStatistical diagnosis according to Frei and Wurgler (1998): þ, positive; �, negative; i, inconclusive; m, multiplication factor. Probability levels a ¼b ¼ 0.05.bIncluding rare flr3 spots.cConsidering mwh clones from mwh single spots and from twin spots.dOnly mwh single spots can be observed in mwh/TM3 heterozygotes as the balancer chromosome TM3 does not carry flr3 mutation.



In conclusion, the results of this study indicate that the

higher ethanol concentration present in Cepacol induces

mitotic recombination between homologous chromosomes

in the Drosophila SMART assay. Homologous mitotic

recombination can result in loss of heterozygosity or

genetic rearrangements, and these events are involved in

the genesis of numerous diseases, including cancer

[Bishop and Schiestl, 2003]. The evidence indicating that

the major effect of Cepacol is an increased frequency of

HR suggests that there may be a risk associated with the

use of this mouthwash, due to the high concentration of

ethanol present in its formulation. These results suggest

that it may be prudent to reevaluate currently available

odontology products by means of assays that are able to

detect a wide range of genetic lesions. In this context, the

SMART assay is one of the methodologies capable of

evaluating the induction of a range of genetic events

induced by complex mixtures like the mouthwashes ana-

lyzed in this study [Andrade et al., 2004].

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TABLE III. Genotoxicity of Ethanol and Cetylpyridinium Chloride in the D. melanogaster Wing Spot Test using both theStandard (ST) and High Bioactivation (HB) Crosses

Genotypes

Dilutions

(%)

Number

of flies (N)


Total mwhc

clones (n)

Small single spots

(1–2 cellsb) (m ¼ 2)

Large single spots

(>2 cells)b (m ¼ 5)

Twin spots

(m ¼ 5)

Total spots

(m ¼ 2)

Standard cross

Ethanol

mwh/flr3 0 40 0.55 (22) 0.35 (14) 0.05 (2) 0.95 (38) 38

2.1 40 0.95 (38)þ 0.15 (6)� 0.00 (0)i 1.10 (44)� 44

4.2 40 1.00 (40)þ 0.15 (6)� 0.00 (0)i 1.15 (46)� 46

8.4 40 0.60 (24)� 0.25 (10)� 0.20 (8)i 1.05 (42)� 42

12.5 40 1.55 (62)þ 0.20 (8)� 0.25 (10)þ 2.00 (80)þ 76

16.8 40 0.90 (36)þ 0.50 (20)i 0.05 (2)i 1.45 (58)þ 58

Cetylpyridinium chloride

mwh/flr3 0 40 0.85 (34) 0.10 (4) 0.10 (4) 1.05 (42) 42

12.5 40 1.05 (42)� 0.15 (6)i 0.05 (2)i 1.25 (50)� 50

25 40 0.85 (34)� 0.20 (8)i 0.00 (0)� 1.05 (42)� 42

50 40 0.95 (38)� 0.15 (6)i 0.10 (4)i 1.20 (48)� 48

75 40 1.10 (44)� 0.05 (2)i 0.00 (0)� 1.15 (46)� 46

100 40 1.15 (46)i 0.10 (4)i 0.03 (1)� 1.28 (51)� 51

High bioactivation cross

Ethanol

mwh/flr3 0 40 0.90 (36) 0.10 (4) 0.10 (4) 1.10 (44) 44

2.1 40 1.25 (50)i 0.00 (0)� 0.00 (0)� 1.25 (50)� 50

4.2 40 1.05 (42)� 0.15 (6)i 0.10 (4)i 1.30 (52)� 52

8.4 40 1.65 (66)þ 0.35 (14)þ 0.40 (16)þ 2.40 (96)þ 96

12.5 40 1.45 (58)þ 0.40 (16)þ 0.20 (8)i 2.05 (82)þ 82

16.8 40 1.75 (70)þ 0.30 (12)þ 0.15 (6)i 2.20 (88)þ 88

Cetylpyridinium chloride

mwh/flr3 0 40 0.95 (38) 0.10 (4) 0.10 (4) 1.15 (46) 46

12.5 40 1.10 (44)� 0.15 (6)i 0.05 (2)i 1.30 (52)� 52

25 40 0.90 (36)� 0.10 (4)i 0.10 (4)i 1.10 (44)� 44

50 40 1.25 (50)� 0.20 (8)i 0.00 (0)� 1.45 (58)� 58

75 40 1.28 (51)� 0.18 (7)i 0.00 (0)� 1.45 (58)� 58

100 40 1.15 (46)� 0.10 (4)i 0.03 (1)� 1.28 (51)� 51

aStatistical diagnosis according to Frei and Wurgler (1988): þ, positive; �, negative; i, inconclusive; m, multiplication factor. Probability levels a ¼b ¼ 0.05.bIncluding rare flr3 spots.cConsidering mwh clones from mwh single spots and from twin spots.


648 Rodrigues et al.

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Accepted by—R. Snyder



genotoxicity of three mouthwash products, cepacol®, periogard®, and plax®, in the drosophila...

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