183

TXL 01924

Mutagenic activity of some textile dyes in

different test systems

Barbara Przybojewska, Boguslaw Barariski, Ewa Spiechowicz and

Krystyna Sitarek

(Received 20 July 1987)

(Accepted 9 October 1987)

Ke.v r+v&; Anthraquinone dye; Aro dye; Mutagenicity; (Mice)

The Salmonella/microsome mammalian tact, the micronucleus and the dominant lethal tests on mice
\%ere uied to study mutagenic effects of three dycc Lvidely used in the textile industry. Direct Black 19:l,
Dit-ect Red 81 and Acid Blue 62 increased the frequency of micronucleated polychromatic erythrocytes
in bone marrolc of mice. Out of all dye5 tested, only Direct Black 19:l appeared to be an indirect
mutagen inducing reverse mutations in two strains of Sub??one/lu /_vphr/??uriu~ TA 1538 and TA 9X.
None of them produced dominant lethal mutations in germ cells of male mice.

INTRODUCTION

The early and efficient detection of environmental mutagens and carcinogens and
the evaluation of their genetic effects constitute a very important problem linked
with human health protection. The examined dyes: Acid Blue 62 (C.I.62045), Direct
Black 19:l and Direct Red (C.I.28160) are widely used in the textile and dye in-
dustries in Poland and a considerable number of workers are exposed to them.
Detection of their mutagenic and genotoxic activity seems to be a very important
problem, especially if we take into consideration the fact that Acid Blue 62

Address for correspondence: Barbara Przq bojewska, Department of Occupational Medicine, Institute
of Occupational Wledicine, 90.950 Cdi, Teresy 8, Poland.
Abbreviation: MMS, methylmethane sulfonate.

037%4274/88/$ 03.50 ~0 1988 ElseLier Science Publishers B.V. (Biomedical Division)

1x4

represents a group of anthraquinone compounds and that the two other dyes belong
to a larger group of azo compounds. Anthraquinone derivatives arc closely related
to anthracycline antitumor antibiotics of known mutagenicity and carcinogenicity
[l-3]. Some azo compounds are reported to possess carcinogenic properties [4,5]
and some show mutagenic activity in bacterial tests [6,7].
General agreement now appears to exist that a battery of tests is required for tox-
icological evaluations in a mass-screening programme both because of the
mutagenic specificity and the range of genetic damage that can be produced 181. .A
testing system is recom~leIided that permits analysis of gene and cl~rorn(~soI~lc muta-
tions in somatic and germ c&s [9]. In this study, the Sa~monella~microso~~~e mam-
malian test, the micronucleus test and the dominant lethal mutation test on mice
were used to examine mutagenic activity of these dyes

MATERIAI S AND METHODS

The studied compounds Acid Blue h,,? Direct Black 19: 1 and Direct Ked 81 were
obtained from the Dye Factory at %gicrL, Poland. Their chemical structure is
presented in Fig. 1. The content of impurities is as follows: Acid Blue 62 -.- 3.48%
Na2SOJ, 0.02% 01’~ ; Direct Black 19: i - 5.10% Cl, 16% Nar SOA; Direct Red -~
8.1401, Cl.
In the Salmo~~ella/mi~rosome rnam~~al~an assay, dyes were tested using strains
TA 1535, TA 1537, TA 1538, TA 98 and TA 100 according to the test procedure
 18.5

TABLE I

SENSITIVITY OF THE SALMONELLA TYPHIMURIUM MUTANTS USED TO CONTROL

MUTAGENS AND ACTIVITY OF S-9 MIX

Data are the mean &SD of results obtained on two plates for one dose in two separate experiments.

Strain S-9 mix Compounds Dose level His ’ Numbel- of

(wWatc) colonies/plate spontaneous
revertants/plate

TA 1535 Sodium azide 1.5 10805 172 19+ 5

TA 1537 9-Aminoa~rid~ne 10 144t 9 8* !
TA 1.238 ._ 4-Nitro-o-phcnylenediamine 10 1374* 82 16-i 4
+ 2-Aminofluorene 10 > 2000 21t 7
‘T.4 98 4-Nitro-o-phenylenediamine 10 1014+ 118 21+ 1
+ 2-Aminofluorene 10 > 2000 23* 4
TA 100 Sodium azide 1.5 1452+ 165 119t16
+ 2-~~minofluorene 10 > 2000 131517

described by Ames et al. [lo] with later modifications [l I]. The tester strains were
kindly provided by Professor B.N. Ames, University of California, Berkeley, CA,
U.S.A. Sensitivity of the used strains and activity of the S-9 mix fraction were
checked out using appropriate mutagens according to the criteria given by De Serres
and Shelby [12]. The results of this study are shown in Table I.
S-9 mix was prepared from liver homogenate of rats treated with Aroclor 1254.
It contained (per ml) 0.1 ml S-9 fraction plus adequate cofactors, as described by
Ames et al. [lo]. In the Salmon~lla/microsome assay all tested dyes were dissolved
in aqua dist. and tested on two plates for each dose in two separate experiments with
and without 0.5 ml of S-9 mix per plate; three plates in each experiment were used
for spontaneous mutation control assay. All dyes were used as aqueous solutions
in the highest concentration which could be prepared. The dyes were studied at four
or six dose levels. The highest doses of the dyes in the Salmonella/microsome test
were chosen after preliminary solubility study according to the method described in
other reports [13,14]. Acid Blue 62 and Direct Red 81 were insoluble at a dose level
of 2500 pg/plate, in contrast to Direct Black 19: 1, and therefore the highest concen-
trations of both dyes were 1000 &plate. All dyes at the highest doses produced a
slight decrease in the number of revertants suggesting some toxicity at these doses.
The plates were incubated for 48 h at 37°C and then the numbers of reverted
bacterial colonies were counted. An investigated compound was judged to have in-
duced a positive response when a dose-related increase in the number of revertants
was observed and the number of revertants exceeded the negative control values by
at least 2-foId in at least two successive concentrations of the test chemical.
In preliminary experiments of the micronucleus test and the dominant lethal
‘C, r? “, - c I -
+I +I tl fl *I Cl
 187

TABLE III

THE FREQUENCY OF MICRONUCLEI IN BONE MARROW POLYCHROMATIC ERYTHRO-

CYTES OF BALB/c MICE AND THE RATIO OF NORMOCHROMATIC TO POLYCHROMATIC
ERYTHROCYTES

Compound Total Number Number of polychromatic Ratio of normo-

dose of erythrocytes chromatic to
(mgjkg) mice polychromatic
Total With micronuclei
erythrocytes
(% i SD)

Negative control _ 12 16 700 0.42?0.12 1.03

Acid Blue 62 87.50 4 7 500 0.63 t0.02* 0.98

(C.1.62045) 175 4 I 550 0.73 t0.25* 0.94
350 4 4 330 I. IO +0.26* 1.26
700 3 3 750 2.09+0.18* 1.60*
1400 3 3 400 2.40r0.16* 1.64*

Direct Black 19:1 16.66 4 7 600 0.80?0.11* I .04

50 4 7 340 0.82 i 0.37* I .4x
150 4 7 270 0.94*0.11* I .23
450 4 6 300 1.36 i 0.23* 1.45*
1350 4 7 320 1.54*0.39* 1.57s

Direct Red 81 91.25 4 4600 0.99*0.18* 1.15

(C.1.28160) 182.50 4 4 100 I .58 ? 0.79* 1.12
375 4 4 500 2.10 k 0.24* 1.28
750 4 5 350 2.58 * 0.83* 1.81*
1500 3 3 650 2.22i1.41 1.74*

Mitomycin C 2.50 4 4 000 2.77 I 0.41* 1.62*

(positive control) 5.00 3 2 900 3.78 i 0.78” 1.68*

*Significantly different from negative control as determined by the Wilcoxon test (P<O.O5).

assay, the LDso of all studied dyes administered i.p. to male mice was determined
using the Thompson and Weil method [15]. Median lethal doses of Acid Blue 62,
Direct Black 19:l and Direct Red 81 were 983, 1000 and 1048 mg/kg body weight,
respectively. Total doses of all dyes in the micronucleus test were in the range of
1.7 - 8.9% to about 140% of the LDso, while in the dominant lethal test it was equal
to about 125% LDzo.
In the micronucleus test the examined substances were dissolved in 0.9% aqueous
solution of NaCl just before treatment and administered to male mice of the
BALB/c strain weighing 25-30 g by i.p. injection, each dose in two equal portions
separated by 24 h intervals. The negative control group of animals received only
0.9% NaCl used as solvent. Mitomycin C (Sigma, London, U.K.) dissolved in 0.9%
18X

TABLE IV

KESULSS OF THE DOMINANT LETHAL MUTATION TEST‘ IN MALE BAl.t)/c MICE &.I)-
MINISTERED MMS IN A S1NGL.E i.p. INJECTION AT DOSES OF 20 .4ND 50 q/kg

NaCl was ad~linist~red to the animals in the positive control gr-oup. Each group of‘
animals consisted of four male mice. Bone marrow smears were prepared 30 h after
the first injection according to the procedure described by Schmid [16] with the
modification that 1% solution of sodium citrate was used instead of foetaf calf
serum and slides were stained in Giemsa (Merck) diluted 1:lO in Sorensen buffer
(pH 6.X).
In the dominant lethal assay the investigated dyes were given to male mice as solu-
tions in 0.9% aqueous solution of NaCl for 5 consecutive days by i.p. injection at
daily doses equal to 25’70 of the LD. 50, The animals in the negative control groups
were given i.p_ 0.940 aqueous solution of NaCl and those in the positive control
group were admiilistered methyl-methane sulfonate (ARMS) in a single i.p. injection
at doses of 20 and 50 mglkg. Each group of animals consisted of 40 males, except
TABLE V

DOMINANT LETHAL MUTATION TEST IN MALE BALB/c MICE ADMINISTERED ACID

BLUE 62 i.p. FOR 5 DAYS BEFORE MATING, AT A DOSE EQUAL TO 25% OF THE LDsII

Compound Mating Number Fertile Implants Live Resorptions

intervals of matings (mean k SD) embryos per female
(days) females (@+I) per female

Negative control l- 4 40 75 10.3 k2.4 9.6k2.8 0.8t0.8

5- 8 40 65 10.6t2.4 9,5 tr 2.8 1.2f 1.5
9-12 40 98 9.7i2.1 9.3 i 2.3 0.5 f 0.7
13-16 40 74 10.5 * 2.0 9.8 f 2.6 0.8+ 1.4
17-20 40 95 10.7 + 2.0 lO.Ok2.3 0.6+0.X
21-24 40 93 10.55 1.9 9.7* 1.9 0.8 * 0.9
25-28 40 93 9.9k3.0 9.7k3.1 0.3 f 0.6
29-32 40 78 10.0t2.1 9.3 + 2.3 0.7+0.8
33-36 40 88 11.4k2.4 10.5 k 2.6 0.9* 1.1
37-40 40 80 11.2 f 2.0 10.8i2.1 0.5 i 0.7
41-44 40 78 9.9 + 2.4 9.3 + 2.6 0.6 i I .O
45-48 40 85 9.5 * 2.0 8.8 k 2.4 0.7-to.9

Acid Blue 62 l- 4 40 x5 10.9-t 1.9 9.9k2.5 I.Oi 1.6

5- 8 40 98 11.7 i 2.0 10.7 k 2.9 l.O* 1.7
9-12 40 95 10.1 i 1.8 9. I * 1.9 0.92 1.6
13-16 40 88 lO.Y+ I.7 10.3i 1.7 0.6+0.8
17-20 40 88 10.9+ 2.6 10.3 f 2.7 0.7 * 0.8
21-24 40 98 10.7* 1.5 lO.O+ I.4 0.7 * 0.8
25-28 40 95 11.6i2.2 I I .2 + 2.2 0.5 * 0.8
29-32 40 98 10.8k2.5 10.1 I 2.6 0.8* 1.3
33-36 40 85 10.2+ I.6 9.4i 1.9 0.8 i I .o
37-40 40 85 10.9k2.6 10.5 i 2.4 0.4 i 0.8
41-44 40 85 9.3 k3.4 x.9 f 3.4 0.4 -c 0.7
45-48 40 98 9.9 i 2.4 9.4k2.3 0.5 i 0.9

the MMS-treated group, which consisted of 30 mice. The test procedure followed
the standard protocol for the dominant lethal test prepared by Ehling et al. [17].

RESULTS AND DISCUSSION

The results of the Salmonella/microsome mammalian assay are presented in

Table II. Among the dyes examined by this test, Direct Black 19:l appeared to be
an indirect mutagen inducing frameshift reverse mutations in strain TA 1538 and
TA 98. The two other dyes - Acid Blue 62 and Direct Red 81 - did not induce his+
reversion mutations in any of the used bacterial strains. The lack of mutagenic ac-
tivity of Acid Blue 62 on Ames strains of Salmonella typhimurium coincides with
the observations of Venturini and Tamaro [18].
As shown in Table III, all studied dyes increased the frequency of polychromatic
DObllNANT LETHAL MUTATION TEST IN MA1.E BALB/c MICE .4DivIINIS’l‘ERE~D UIREC’I
BLAC’K 19:l i.p. FOR 5 DAYS BEFORE hlATING, AT A DOSE bQUA1. TO 25% 01. THE l.Di,,

IO.5 A 2.6
lO.hi 3.2
10.8 J 2.5
IO.4 i 1.5
ll.li I.9
IO.h:+ 2.1
10.7 + 2.2
10.2 i 1.?
I I .? i 2.2
10.3i2.6
9.x .! 2.1
IO.? ? 7.2

erythrocytes with nlicronLicie~. The increase was significant and dose-related.

relatively lower increase in percentage of micronucleated polychromatic cryt hro-
cytes induced by Direct Red 81 at the highest dose (1500 mgikg) as compared to that
produced by this dye at half the dose (750 mg/kg) was probably connected with
mitotic delay, caused by the toxic action of the compound on bone marrow cells.
Toxicity of Direct Red 81 was noticed as a significant increase in the ratio of nor-
mochromatic to polychromatic erythrocytes. Such an explanation is in agreement
with the findings presented by Schmid [12], who stated that an increase in the ratio
of normochromatic to polychromatic erythrocytes may be a measure of mitotic
delay. The experiment with mitomycin C confirmed the validity of the micronucleus
test for detecting the effect of a chemical mutagen. ~~itornycjn C at doses of 2.5 and
5.0 mg/kg body weight delnonstrated a significant increase in the percentage of’
TARLE VII

DOMINANT I,ETHAL MUTATION TEST iN MALE BALB/c MICE Al>hlfNtSTERED UlRECT

RED 81 i.p.. FOR 5 DAYS BEFORE MATING, Al‘ A DOSE EQUAL TO 25% OF THE Llk

Compound Mating Number Fertile Implant, I.i\e Rcsorptions

intervalr Of matings (mean i SD) embryos ,xX l‘cmalc
(days) femaler ( “b ) per- female

Kcgati\ c colltrol l- 4 40 75 10.3i2.4 Y.6 ir 2.x 0.8 t 0.x

5- 8 40 65 lO.6i 2.4 Y.5 i 2.8 I.‘? I.3
9-12 30 Y8 9.712.1 9.3 t 2.3 0.5 i 0.7
13-16 40 73 IO.5 i 2.0 Y.X-2.6 0.8 2 I .J
17-20 40 95 10.7 + 2.0 lO.O-_2.3 0.6 t 0.x
21-23 40 93 10.5i 1.9 9.72 1.9 0.x k 0.‘)
25-28 40 93 Y.9i 3.0 9.7k3.1 0.3 _”0.6
29-32 40 78 lO.Oi2.l 9.322.3 0.7 i 0.x
33-36 JO 88 Il.42 2.4 lO.5i2.6 O.Yi I.1
37-40 40 80 11.2i2.0 108~2.1 0.5 i 0.7
41-44 40 78 9.9 z 2.1 Y.3t2.h O.fl -t I .o
45-48 40 x5 9.5 i 2.0 8.Xi2.4 0.7 k 0.‘)

Direct Red 81 I- 4 -10 xx 10.3ri-I.2 Y.5 i I .4 1.0-c I.6

5-- 8 40 95 lO.?i 1.5 Y.8* 1.5 0.6 + 0.6
Y-12 30 75 IO.5t2.1 lO.Oi 2.3 0.5 i 0.6
13-16 40 95 IO.‘), 1.4 Y.9 i 2.0 1.o f 1.6
17 20 30 98 lO.hi I.7 9.9 * 2.1 0.7+ 1.o
21-23 40 75 10.1 it.4 Y.X i 2.7 0.4 Lt0.7
25 2X 40 90 io.5 t 2.3 9.7 i 2.4 0.8 .L0.8”
29-32 40 YO lO.Oi2.5 9.1 k2.3 0.7 t 0.Y
33-36 40 93 10.01 1 .6 9.3 i I .Y 0.7 iI .o
37-40 30 98 10.6i I .h lO.Oz I .8 0.6t 1.o
41-44 40 87 10.7 i- 2.0 10.3 + 2.0 0.3+ 0.6
45-4x 40 95 10.9+2.11’ 10.3 i2.3” 0.6i 0.8
“‘Dif’f‘eretrce statistically +yificant (PcO.05)

micronucleated poIychromatic erythrocytes as compared to the negative control

vaIue (Table III).
MMS, which was used as a positive mlltagen, in the dominant lethal mutation test
(Table IV) produced at both applied doses an increase of dead implants in females
mated with treated male mice, but none of the dyes tested induced such an effect
(Tables V, VI and VII). All dyes given i.p. to male mice at a dose equal or close
to 25010 of their LDso induced an increase in the number of micronucleated
polychromatic erythrocytes, while their i.p. administration at a similar daily dose
for 5 consecutive days did not increase the frequency of dominant lethal mutations.
The highest total doses of tested dyes used in the micronucleus assays amounted to
140% of their LDso, which corresponds to the total dose (125% of the LD50) applied
in the dominant lethal test. The difference in the results obtained in both tests used
192

on mice seems to suggest a higher sensitivity of the somatic cells as compared to the
germ cells in detecting mutagenic activity of dyes. This observation demands confir-
mation because we have no data on the amount of dye in bone marrow and germ
cells in exposed mice.
Application of a battery of tests makes it possible to detect the mutagenic proper-
ties of all tested dyes, although out of the short-term tests used in this study the
micronucleus test appeared to be the most sensitive one.

RI FEKENCtS

I Brown, J.P. and Brown, J.R. (1976) Mutagenesis by 9,10-antraquinone derivatives and related con-
pounds in Sul~~ionrllu fvphimurrutn. Mutat. Kes. 40, 203 224.
2 Shahin, h1.M. and van Bowel, R.C. (1978) Comparison of mutagenic induction A, Q in recursion
\yhtcrn of Succhurom_vce.s crrevisiue and Suln~onellu /yphimuriutrr. hl utat Re\. 53, 1 IO.
3 Shahin, M.M. and con Bowel, R.C. (1976) Genetic activity of the antimicrobial food additives AI,-2
and H-103 in SrrccharotqyceL’r cerrvisiue. Mutat. Res. 3X, 379- 380.
4 Poirier, L.A., Miller, J.A., Miller, L.C. and Saw, K. (1967) ,~-Benlylo4~-.l’arninobenrene: Its car-
cinogenic activity in the rat and iti t-eactionj \\ith proteins and nucleic acid\ and their constituent\
in vitro. Cancer Kc,. 27, IhOO-1613.
5 Preussman, K.H., Druckrey, t-l., Lankocic, S. and Hodenbers, A.V. (1969) Chemical structure and
carcinogenicity of alifatic hydraLo, ago and arosy compounds and triarinc\ potential in \i\o
alhylating apent\. .4nn. N.Y. Acad. Sci. 163, 697-716.
6 Venitt, S. and Bushell, C.T. (1976) Mutagemclty of the food colour brown FK and constituent\ itI
Sulmonellu !vphirnurium. Mutat, Rej. 40, 309 3 16.
7 Yahag, T., I.egawa. hl., Seine. !‘., Matu~hima, T., Nagao. .\I., Sugimura, 1. and Hathimoto, y.
(197’)) Mutagenicit) of carcinogenic BIO dye and theit- tleri\ati\,e\. Cancer Lctt. I, Yl -YX.
s De Set-rer, 1.J. (1979) Problem\ a\wciated with the application of short-tclm tc\ts for mutagenicit!
in mas\-screening program\. Envil-on. Mutagen. I, 203-208.
9 Bochco~, N.P., Sram, R.J., Kuleohov. N.P. and Zhurkov, V.S. (1976) System for the cvaluatioll (11
the ri,k from chemical mutayxs for man: basic principle\ and practical recommendations. Murar.
Rer. 38, 191 -202.
10 .Ames, B.N., &lcCann, J. and \r’ama,aki, E. (1975) ,Methods for detectins carcinogens and mutagen\
~\ilh the Salmonella,‘rnarnrnalian micl-owme mutagenicity test. \lutat. Re$. 31, 337 364.
11 Rlaron. D.hl. and Ame\, B.N. (lY83) Rcviwd mcthod5 for- the Salmonella mutagenicity ie\t. Murat.
Rch. 113. 173-215.
12 De Serres. t,..l. and Shelby. h1.D. (1979) Recommendations on data production and analyii, u\iny
the Salrnon~lla/microsorne mutagemcity assay. Mutat. Re\. 64. I59 165.
13 Brurick, D., Giss, H.E. and Hoorn, A.J.W. (1978) Screening Program for the Idcntificatlon 01
Potential Chemical k4utagens and Carcinogens. Protocol No. 400, Litton Biomedic Inc., U.S.:\.
The Netherlands.
11 (1978) Repel-t of b!‘orkshop on Bacterial 111vitro Mutagenicity Twit Sg\tem\, held at Neuherberp,
Munich. hlutat. Re,. 53, 369 378.
15 U’eil, C.S. (lY52) Tablea for convenient calculation of mcdlan cffecti\e close (LL)<,, or EL&II) and ill-
itruction\ in their uw. Biometric5 8, 249 263.
16 Schmid, \h’. (lY73) Chemical mutagen testing on in viva somatic mammalian cells. Agent5 Action\
3, 77-85.
17 Ehling, U.H. (1977) Dominant lethal mutations in male mice. Arch. Touicol. 38, 1-I I.
18 Venturini. S. and Tamaro. M. (197’)) Mutagenicity of antraquinone and a/o dye\ in Ame\’
Sol~~wnellu t~phtmurium test. Mutat. Re\. 68. 307 -312