Mutagenicity of Textile Dyes Mendel Friedman," Martin J. Diamond, and James T. MacGregor Western Regioiial Research Center, Science and Education Administration, U.S. Department of Agriculture, Berkeley, California 947 10
Nine of twenty-eight textile dyes tested for mutagenicity in an Ames Salmonella typhimurium bacterial test have been found to be mutagenic. These findings suggest that further testing is appropriate to establish possible mutagenic hazards associated with dve Droduction and use. The safety of dyeing and other finishing treatments for wool, cotton, and other natural and synthetic fibers needs careful study before the safety of large-scale use can be assured (1-3). Although dyeing is an ancient art developed and extensively practiced in both the Old World and the New (4-7), the synthesis and use of synthetic dyes is only about a hundred
years old. Fiber-reactive dyes form a major class of synthetic dyes developed in the last 20 years ( 4 ) .Because these dyes have reactive side chains that can combine covalently with fibers during dyeing, exposure of living cells to fiber-reactive dyes could result in alkylation of amino and other functional groups in nucleic acids. They may, therefore, be mutagenic. Consequently, we have evaluated 28 dye formulations of 5 subclasses of fiber-reactive dyes as well as 3 non-fiber-reactive acid dyes for mutagenicity in an Ames Salmonella typhimurium test (8,9). Quantitative top agar assays were carried out as described by Ames (8) by using an Aroclor 1254-induced rat liver metabolizing system. The spot tests were carried out
Table 1. Mutagenicity of Fiber-Reactive Dyes and an Acid Dye in Salmonella fyphimurium Strains TA100, TA98, and TA1537 Quantitative Plate Tests of Fiber-Reactive Dyes revertantsiua dve added a TAlOO TA99 no S-9 no S-9 with S-9 with S-9
compd tsmsted
no S-9
TA1537 with S-9
*
bromoacrylamide dyes 1. Lanasol Blue 3R ((2.1. Reactive Blue 50; Lot 965G-Ciba) 2. lntracron Blue 3R (Lot 301612-3-2-lntracolor) 3. lntracron Blue 3G (Lot 32061 1K-15-lntracolor) 4. Lanasol Red G (C.I. Reactive Red 37; Lot 15767-3Ciba) 5. Lanasol Red 6G (C.I. Reactive Red 84) 6. Lanasol Scarlet 2R (C.I. Reactive Red 78; Lot 964GCiba) 7. Lanasol Yellow 4G (C.I. Reactive Yellow 39; Lot 960GCiba) vinyl sulfone dye 8. Remalan Brilliant Blue-B (C.I. Reactive Blue 36; Lot MO 5941Hoechst)
compd tested
9. Acid Green 58 10. control (Nle2SO)( f l std dev) 11. positive controls: aflatoxin 13, (1.0 p g spot test) 9-aminoacridine (100 pg spot test)
442/40
181/1000
473/200
223/5000
28/1000
468/40
185/1000
465/200
339/5000
52/40
28/5000
755/200
245/ 1000
261/200
278/5000
125/200
157/5000
1165/5000
1003/5000
53/5000
52/5000
1/50OOc
0/5000
343/5000
230/5000
8815000
5315000
6/5000 '
2/5000
60/5000
93/5000
315000 '
0/5000
8511000
118/5000
2715000
12/5000 '
0/5000
0/50OOc
175/1000
19/1000'
0/5000 '
0/5000
0/5000
3/50OOc
8/5000 '
0/5000 '
1/ 1000'
Spot Test of a Non-Fiber-Reactive Acid Dye revertant density surrounding dye spot, revertants/cm* TAlOO TA98 TA1537 no S-9 with S-9 no S-9 with S-9 no S-9 with S-9
27.6 2.6f0.26
19.8 2.4f0.19
0.64 0.45f0.06
17.9
0.94c 0.80f0.13
0.17' 0.33 0.12f0.04 0.14f0.05
18.0
>50.0
Revertants/plate after subtracting the spontaneous control value. The quantity of dye per plate is given in the denominator. Thus, the first number in the second column denotes that 40 p g dye induced 442 revertants. Spontaneous control value ( f l std dev) for the five experiments from which the data are derived, without and with S-9 metabolizing system, respectively, were as follows: TA100, 170 f 26; 164 f 22; TA98, 43 f 24; 41 f 17; TA1537, 9.2 f 3.6; 10.6 f 3.6. The least significant differences ( 79) at a confidence level of 0.001 were as follows: TAIOO/(no S-9), 56; TAlOO/(with S-9). 62; TA98/(no S-9), 25; TA98/(with S-9), 34; TA1537/(no S-9), IO;TA1537/(with S-9), 15. All tabular values exceed these least significant differences except those marked with a superscript c. Positive controls were carried out with each test as follows: TA98, 0.5 figlpiate of aflatoxin BI; TA100. 1.0 fig/piate of aflatoxin 6,; TA1537, 100 Fg/piate of 9-aminoacridine. The revertants per plate! ranged from 950 to 1575 for the aflatoxin B, and from 2400 to 3200 for 9-aminoacridine. Color index names, lot numbers, and manufacturers in parentheses when available.
This article not subject to U.S.Copyright. Published 1980 American Chemical Society
Volume 14, Number 9, September 1980
1145
by placing 1.0 mg of the test compound a t the center of the plate. A statistical treatment of the data is given in footnote a , Table I. The reactive sites of these dyes comprise bromoacrylamide (e.g., Lanasol, Intracron), chloroacrylamide (e.g., Lanasyn), vinyl sulfone (e.g., Remalan, Remazol, Levafix), chlorotriazine (e.g., Cibacron), and dichlorotriazine (e.g., Procion) side chains. Dyes were tested by using spot tests (8).Those exhibiting mutagenicity, bacterial growth inhibition, or questionable increases in bacterial revertant frequencies were retested by the quantitative overlay technique. Of the 28 products tested, 7 of 11bromoacrylamide dyes, 1of 4 vinyl sulfone dyes, and 1 of 3 acid dyes were mutagenic. No mutagenicity was observed among 4 dichlorotriazine, 1 chlorotriazine, 1chloroacrylamide, or 4 vinyl sulfone dyes that were spot tested. Table I illustrates representative data from all dyes that gave positive findings. The complete experimental results, including dose-response data, will be published elsewhere. The activities observed were usually less than those reported for better-known mutagens and carcinogens similarly tested (9).Intracron Blue 3G, Lanasol Blue 3R, and Intracron Blue 3R (C.I. Reactive Blue 50) (Table I), however, were approximately as mutagenic as the well-known mutagens and carcinogens nitrogen mustard and uracil mustard (9). Since we made no attempt to isolate and purify the active dye from commercial formulations, we do not know whether the dyes themselves or additives in the dye products are in fact responsible for the mutagenicity. The mutagenic components in the commercial samples obviously must be identified to evaluate the significance of exposure to the active ingredients. In one case (Acid Green 58), the test shows that a component of the dye formulation other than the dye itself may be responsible for the mutagenicity. Since the bacteria are fixed on the agar plate and the dyes are not, and since the ring of revertants on the petri plate was found outside the dye spot, the activity is evidently due not to the dye but to a more rapidly diffusing constituent(s) in the formulation. Bacterial growth on the plate was not inhibited. T o determine whether residual mutagenic activity remains after dyeing, we tested the spent dye after test-dyeing a wool fabric with Intracron Blue 3G, one of the most mutagenic dye samples listed in Table I. Wool fabric (50 g) was added to 2 L of a solution consisting of 1 g of dye, 5 g of Na$304,5 cm3 of acetic acid, and 0.5 cm3 of Albegal B (a dispersing agent). One hundred cm3 of this solution was removed for a control, and, after the remaining sample was boiled for 30 min, both the original and the remaining spent dye liquor were evaporated to dryness in vacuo under sterile conditions. An identical experiment in which dye was omitted was carried out as a control. No mutagenicity was observed in strains TA100, TA98, or TA1537, either with or without an in vitro metabolic activation system, when water or dimethyl sulfoxide extracts of the residue obtained after dyeing were tested. The activity expected from a sample of the original solid dye was confirmed in a sample of the dyebath removed before dyeing. The conditions of the mutagenicity test were such that 2% of the activity originally present would have been detected in strain TAlOO in the sample taken after dyeing. Thus, a t least 98% of the initial mutagenicity in the dyebath disappeared from the dyebath liquor during dyeing. Spectral measurement of the spent dyebath solution indicated that 97% of the dye is taken up by wool under these conditions.
1146
Environmental Science & Technology
Thus, though no generalizations should be made from data obtained with one dye, and though we believe that the spent dye liquors, freshly dyed fabrics, and finished fabrics should be further tested, the present results suggest that in one case where nearly all of the dye is absorbed by the wool not only do additional mutagens appear not to be formed during dyeing, but most, if not all, of the mutagenic constituent(s) originally present either decomposed or was taken up by the wool fabric during dyeing. Although our findings are a t present limited, they raise significant questions about possible mutagenic hazards associated with dye production and use. About one-third of the dyes tested contained measurable mutagenic constituents. Consequently, exposure t g these dyes probably should be minimized until any actual hazards from their use can be accurately defined. Exhaustion of dyes (4,10-16) and flameproofing (17) and mothproofing (18) agents in the dyeing process are often less complete than in the one case we tested. If the dye or mutagenic impurities were to escape hydrolysis or decomposition during dyeing and remain in the spent dye liquor effluents, they could present a mutagenic hazard. More likely, unbound dye in the fabric or in the effluent is in the hydrolyzed form and may have been inactivated. Similarly, dye bound to the fabric is no longer in its original reactive form. Nevertheless, possible mutagenicity associated with the final dyed textile product to be worn by the consumer deserves further study. Finally, epidemiological studies of dye-exposed workers may also help define the importance of this problem. Acknowledgment
It is a pleasure to thank Dorris P. Frederick and John F. Ash for excellent technical assistance. L i t e r a t u r e Cited (1) Kay, K. Text. Chem. Color. 1977,9, 275. (2) Kay, K. Text. Chern. Color. 1978,10, 47. (3) Tesoro, G. C., presented a t Symposium on “Recent Advances in Textile Dyeing and Finishing,” American Chemical Society Meeting, Miami, FL, September 1978, Abstracts CELL 30. (4) Peters, R. H. “Textile Chemistry”; Elsevier: Amsterdam, 1975; p p 582-648. (5) Beech, F. W. “Fiber-Reactive Dyes”; SAF International: New York, 1970. (6) Elliott, R. L. J. SOC.Dyers Colour. 1976,92, 303-5 and references cited there. (7) Kashwigi, K. M. Bull. Chern. SOC. Jpn. 1976,49, 1236-9. (8) Ames, B. N.; McCann, J.; Yamasaki, E. Mutat. Res. 1975, 31,
-_.
.?47--fiA
(9) McCann, J.; Choi, E.; Yamasaki, E.; Ames, B. N. Proc. Natl. Acad. Sci. U.S.A. 1975, 72, 5135-9. (10) Baumparte. U. Melliand Textilber. 1962.43. 1297-303. (11) Buhler, A.; Hurter, R.; Mausezahl, D.; Petipierre, J. C.-Proc.Int. Wool Text. Res. Coni., 5th, 1975 1976,5, 263-72. (12) Lewis, D. M. Wool Scz. Reu. 1974,49, 13-31. (13) Mausezahl, D. Textilueredlung 1970,5, 839-45. (14) Mosimann, W. Text. Chem. Color. 1969.1. 182-9. (15) Rouette, H. K.; Wilshire, J. F. K.; Yamase, I:; Zollinger, H. Text. Res. J . 1971,41, 518-25. (16) Venkataraman. K. “The Chemistrv of Svnthetic Dves”: “ , Academic Press: New York, 1972; Vol. VI: (17) Friedman, M. In “Flame-Retardant Polvmeric Materials”: Lewin, M., Atlas, S. M., Pierce, E. M., Eds.; Plenum Press: New York, 1977; Vol. 2, 229-84. (18) Friedman, M.; Ash. J. F.:. Brv. “ . R. E.: Simonaitis. R. A. J . Apric. Food Chem. 1979,27,’331-6. (19) Snedecor, G. W.; Cochran, W. G. “Statistical Methods”; 6th ed.; The Iowa State University Press: Ames, IA, 1967. Y
Received for review November 26,1979. Accepted M a y 30,1980.
