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Detection of mutagens in the urine of rats following topical application of hair dyes
241
Mutation Research, 66 (1979) 241--245 © Elsevier/North-Holland Biomedical Press
DETECTION OF MUTAGENS IN THE URINE OF RATS FOLLOWING TOPICAL APPLICATION OF HAIR DYES
MARINEL M. AMMENHEUSER and MICHAEL E. WARREN Department of Biology, Lamar University, Beaumont, Texas 77710 (U.S.A.) (Received 28 June 1978) (Revision received 29 September 1978) (Accepted 10 October 1978)
Summary Mutagens were detected in the urine of rats following topical application of two commercial oxidative-type hair dye preparations. The test system used was induction of back mutation with the bacterial tester strain TA1538, a histidine-phenylenediamine, one of the constituents of the hair
Introduction Recent investigations have shown that some of the ingredients of commercially available hair dyes are mutagenic in Drosophila [5,8], in mouse lymp h o m a cells [14] and in bacterial test systems [1,10,13,16]. Two of these dye ingredients, 4-nitro~>-phenylenediamine (4NOPD) and 2-nitro-p-phenylenediamine (2NPPD), also effected immunosuppressive activity in cultures of human lymphocytes [18], produced morphological transformation in mouse CH3 prostate ceils [4] and induced chromosome damage in cultured Chinese hamster cells [11]. Most previous studies have involved direct testing of dye formulations or Abbreviations: 4NOPD, 4-nitro~-phenylenediamine; 2NPPD, 2-nitro-p-phenylenedlamine.
242
their major constituents. The present study was designed to imitate more closely the actual conditions of use of hair-dye preparations. Materials and methods Both 4NOPD and 2NPPD were tested for mutagenicity with the Salmonella histidine auxotroph system [2] using bacterial tester strains provided by Dr. B.N. Ames, University of California at Berkeley. In confirmation of previous findings [ 1,10,16 ] these c o m p o u n d s were mutagenic to strain TA1538 w i t h o u t the use of a metabolic activation system. The most active compound, 4NOPD, was tested further by injecting 5 mg i.p. into male Texas: Sprague-Dawley rats and then assaying their urine for mutagenicity with T A 1 5 3 8 using the m e t h o d of Durston and Ames [7]. Injection of a commercial dye formulation (Dye B) was also attempted, b u t a dilution of 0.5 ml of the d y e with 2 ml sterile water proved to be lethal to the test animals within 6--24 h. Percutaneous penetration in rats was determined by topical application of 120 mg of 4NOPD in acetone and in isopropanol followed by urine assays with TA1538. Each solution was applied to shortened hair on the back of the animal, allowed to remain on the hair for 20 min after application and then removed b y shampooing and thorough rinsing. Test animals were confined in individual metabolism cages designed for the separation of urine and feces, and urine specimens were collected before dye application for control tests and every 24 h after d y e application for 4 days. This same procedure was used for the application of two commercial oxidative-type hair-coloring products (Dyes A and B) which both contained the direct dyes 4NOPD and 2NPPD. A third p r o d u c t (Dye C), similar in composition to Dyes A and B b u t w i t h o u t 4NOPD or 2NPPD, was also tested. Results and discussion When 50 #g of 2NPPD was assayed with strain TA1538 using 0.1 ml of dimethylsulfoxide as a solvent, only 153 revertant colonies (less controls) were produced. There were 316 colonies with 100 pg of 2NPPD. In contrast, 4NOPD was mutagenic in this particular test system at much lower c o n c e n t r a t i o n s 227 revertants with 1 pg, 1184 with 5 pg, and 2256 with 10 pg. Comparison of these results with the results of the injection studies (Table 1), indicates that only 1--1.5% of the injected 4NOPD appears in the urine in a form that is directly mutagenic in the Salmonella test with strain TA1538. The one rat that survived 24 h following injection of a complete d y e formulation excreted urine that showed mutagenic activity in proportion to the quantity of 4NOPD (0.5 mg) contained in the product. Mutagenic activity was detected in the urine of rats (Table 1) following topical application of 4NOPD to their backs, demonstrating that this direct d y e can penetrate the skin. There was a 4-fold increase in mutagenic activity when the 4NOPD was applied in acetone rather than isopropanol, b u t the latter results should be more comparable to actual use conditions since isopropanol is one o f the base ingredients of hair-coloring products [6]. Data obtained using the complete d y e product are more relevant to the
243 TABLE 1 MUTAGENICITY OF URINE FROM RATS FOLLOWING INJECTION AND TOPICAL APPLICATION OF 4NOPD Treatment
Volume o f urine per plate (rnl)
Total volume of urine e x c r e t e d in 24 h (ml)
N u m b e r of colonies per plate less controls a
N u m b e r of colonies per ml of urine
5.0 m g 4 N O P D i.P. b 5.0 m g 4 N O P D i.P. b 0 . 5 m l D y e B i.p. c 1 2 0 m g 4 N O P D in 4 m l a c e t o n e d 4 ml acetone 1 2 0 m g 4 N O P D in 8 m l i s o p r o p a n o l d 8 ml isopropanol
0.3 0.3 0.3 0.3 0.3 0.3 0.3
11.8 25.0 11.6 8.2 10.0 9.7 14.9
282 188 34 145 0 36 0
940 627 113 483 0 120 0
-+ 6 1 . 5 + 30.4 -+ 5.7 +- 3.5 +
3.9
a R e s u l t s are t h e m e a n + s t a n d a r d d e v i a t i o n o f a t least 3 p l a t e s t e s t e d w i t h T A 1 5 3 8 . C o l o n y c o u n t s ( m e a n 29 +- 3) f r o m assays o f u r i n e s a m p l e s c o l l e c t e d p r i o r to t r e a t m e n t have b e e n s u b t r a c t e d . Plates a s s a y e d w i t h T A 1 5 3 8 w i t h o u t urine averaged 2 5 s p o n t a n e o u s c o l o n i e s . b 5 m g of 4 N O P D w a s s u s p e n d e d in 1 m l o f sterile 0.9% NaCI a n d 1 . 7 5 % g u m acacia s o l u t i o n . c 0.5 m l o f D y e B w a s d i l u t e d w i t h 2 m l o f sterile w a t e r . This d o s a g e c o n t a i n e d 0.5 m g o f 4 N O P D . d A c e t o n e or i s o p r o p a n o l and 4 N O P D w e r e m i x e d and applied t o p i c a l l y as d e s c r i b e d in M e t h o d s .
study of the possible mutagenicity of commercial hair dyes, but it is beyond the scope of this study to investigate the metabolism and potential mutagenicity of the approximately 25 different ingredients in an oxidative hair-coloring formulation. The Salmonella test was particularly useful in this context since mutagenicity could be detected without isolation of the particular compound (or compounds) responsible for the mutagenic response. The results obtained do suggest that the 4NOPD in the two commercial dyes tested could account for at least some of the mutagenicity of the urine excreted by the rats dyed with these products (Table 2). A number of other ingredients or their reaction products could also contribute urinary mutagens, but may not be detectable in this particular test s y s t e m - they may require further metabolic activation or may not be present in sufficient concentration. Maximal levels of mutagenic activity occurred with urine collected during the first 24 h following dye application. Although the level of mutagenic activity was low for all of the assays, a dose--response was observed when increasing volumes of mutagenic urine were tested. For example, when 30 ml of Dye A was applied topically, 0.3 ml of urine produced 25 revertant colonies (less 27 control colonies) and 0.5 ml of urine produced 47 revertants. Urine samples collected 2--4 days after the dye treatment did not show statistically significant levels of mutagenicity. Control urine collected prior to dye application was not mutagenic and there was no significant difference between control plates assayed with 0.3 ml, 0.5 ml or 0.7 ml of urine. Dye C, which did not contain 4NOPD or 2NPPD, was negative in this test system (Table 2). Animal studies do not duplicate exactly the conditions of use and the resulting response of human subjects to the application of hair-coloring products, but an effort has been made in this study to apply the dyes in a manner similar to the one recommended by the manufacturer of the product. The surface area
244 TABLE 2 MUTAGENICITY OF URINE FROM RATS FOLLOWING TOPICAL APPLICATION OF THREE COMMERCIAL HAIR DYES Treatment a
30 ml Dye A 30 ml Dye A + 30 20 ml Dye A + 20 10 ml Dye A + 10 30 ml Dye B 30mlDyeB+30mlH 30 ml Dye B + 30 30 ml Dye C + 30 30 ml Dye C + 30
ml H202 ml H202 ml H202 202 ~c ml H202 ml H202 ~ c ml H202
Volume of urine p e r plate (ml)
Total volume of urine e x c r e t e d in 24 h (ml)
Number of colonies per plate less controls b
Number of colonies per ml of urine
0.5 0.5 0.5 0.5 0.5 0.7 0.7 0.5 0.7
10.6 9.4 14.2 13.0 14.8 6.1 6.5 4.4 7.2
47 31 24 6 20 32 34 1 0
94 62 48 12 40 46 49 2 0
+ 4.5 +- 6 . 0 +- 2 . 8 +- 3 . 5 -+ 3 . 5 -+ 2.6 -+ 2.1 -+ 2 . 8
a D y e A c o n t a i n e d 2 . 5 m g 4 N O P D p e r m l a n d D y e B c o n t a i n e d 1 m g 4 N O P D p e r ml. T h e r e w a s n o 4 N O P D i n D y e C. W h e r e i n d i c a t e d , e q u a l v o l u m e s o f h y d r o g e n p e r o x i d e d e v e l o p e r l o t i o n w e r e a d d e d t o t h e d y e f o r m u l a t i o n s in a c c o r d a n c e w i t h t h e m a n u f a c t u r e r ' s i n s t r u c t i o n s . b S t r a i n T A 1 6 3 8 w a s u s e d for all assays. R e s u l t s are t h e m e a n -+ standard d e v i a t i o n o f c o u n t s f r o m at l e a s t 3 plates. C o l o n y c o u n t s f r o m assays o f urine s a m p l e s c o l l e c t e d prior t o d y e a p p l i c a t i o n ( m e a n 2 7 _+ 4 . 3 ) have b e e n s u b t r a c t e d . Plates a s s a y e d w i t h T A 1 5 3 8 w i t h o u t urine averaged 2 5 s p o n t a n e o u s c o l o n i e s . e T w o t e s t s w e r e d u p l i c a t e d using f e m a l e rats. All o t h e r t e s t s w e r e p e r f o r m e d o n m a l e T e x a s : S p r a q u e D a w l e y rats w e i g h i n g an average o f a b o u t 4 5 0 g.
used on the rats was smaller than the human scalp in proportion to the volume of dye applied, and the application time was slightly longer (about 30 min), but after treatment the hair was shampooed and thoroughly rinsed in accordance with the manufacturer's instructions. Extra precautions were also taken to prevent oral ingestion o f the dye formulation, since humans are not likely to swallow dye during use. Another factor to be considered is the relative permeability of various skin surfaces. A comparative study of skin permeability [3] indicated that the skin on the back of a rat was about twice as permeable as the skin on the human forearm. However, investigation of the relative absorption of a substance from various regions of the human b o d y indicated that the scalp was 3.5 times more permeable than the forearm [9]. Thus it can be concluded that the skin on the back o f a rat is at least similar in permeability to the skin of the human scalp. Differences in the metabolism of absorbed substances also must be considered. For this reason it is hoped that further investigations will include studies on human volunteers. Because of the relatively large volume of urine excreted by human subjects, a newly developed system for concentrating mutagens from urine [20] should be a useful addition to future research. The present investigation indicates that mutagens a r e excreted in the urine following topical application of commercial hair
245
demiological findings that suggest a possible link between breast cancer and the long term use of hair-coloring agents [ 17 ], and the studies being conducted by the U.S. National Institute for Occupational Safety and Health [19] which also suggest an excess of cancer of the genital organs among hairdressers and cosmetologists. References 1 A m e s , B.N., H . O . K a m m e n a n d E. Y a m a s a k i , H a i r d y e s a r e m u t a g e n i c : i d e n t i f i c a t i o n o f a v a r i e t y o f m u t a g e n i c i n g r e d i e n t s , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 7 2 ( 1 9 7 5 ) 2 4 2 3 - - 2 4 2 7 . 2 A m e s , B.N., J . M c C a n n a n d E. Y a m a s a k i , M e t h o d s f o r d e t e c t i n g c a r c i n o g e n s a n d m u t a g e n s w i t h t h e S a l m o n e l l a / m a m m a l i a n - m i c r o s o m e m u t a g e n i c i t y t e s t , M u t a t i o n Res., 31 ( 1 9 7 5 ) 3 4 7 - - 3 6 4 . 3 B a r t e k , J., J . A . L a B u d d e a n d H . I . M a i b a c h , S k i n p e r m e a b i l i t y in vivo: c o m p a r i s o n in r a t , r a b b i t , pig a n d m a n , J . Invest. D e r m a t o l . , 5 8 ( 1 9 7 2 ) 1 1 4 - - 1 2 3 . 4 Benedict, W.F., Morphological transformation and chromosome aberrations produced by two hair dye components, Nature (London), 260 (1976) 368--369. 5 Blljieven, W . G . H . , M u t a g e n i c i t y o f f o u r h a i r d y e s in Drosophila melanogaster, M u t a t i o n Res., 4 8 (1977) 181--186. 6 C o r b e t t , J . F . , H a i r d y e s , i n : K . V e n k a t a r a m a n ( E d . ) T h e C h e m i s t r y o f S y n t h e t i c D y e s , Vol. 5, A c a d e m i c Press, N e w Y o r k , 1 9 7 1 , p p . 4 7 5 - - 5 3 4 . 7 D u r s t o n , W.E., a n d B . N . A m e s , A s i m p l e m e t h o d f o r t h e d e t e c t i o n o f m u t a g e n s in u r i n e : s t u d i e s w i t h t h e c a r c i n o g e n 2 - a c e t y l a m i n o f l u o r e n e , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 71 ( 1 9 7 4 ) 7 3 7 - - 7 4 1 . 8 F a h m y , M . J . , a n d O . G . F a h m y , M u t a g e n i c i t y o f h a i r d y e c o m p o n e n t s relative t o t h e c a r c i n o g e n b e n z i d i n e in Drosophila melanogaster, M u t a t i o n R e s . , 6 5 ( 1 9 7 7 ) 3 1 - - 3 8 . 9 F e l d m a n n , R . J . , a n d H . I . M a i b a c h , R e g i o n a l v a r i a t i o n in p e r c u t a n e o u s p e n e t r a t i o n o f 1 4 C ' c o r t i s o l in man, J. Invest. Dermatol., 48 (1967) 181--183. 10 Garner, R.C., and C.A. Nutman, Testing of some azo dyes and their reduction products for mutag e n i c i t y u s i n g Salmonella typhirnurium T A 1 5 3 8 , M u t a t i o n R e s . , 4 4 ( 1 9 7 7 ) 9 - - 1 9 . 11 K i r k l a n d , D J . , a n d S. V e n i t t , C y t o t o x i c i t y o f h a i r c o l o u r a n t c o n s t i t u e n t s : c h r o m o s o m e d a m a g e i n d u c e d b y t w o n i t r o p h e n y l e n e d i a m i n e s in c u l t u r e d C h i n e s e h a m s t e r cells, M u t a t i o n R e s . , 4 0 ( 1 9 7 6 ) 47--56. 1 2 M c C a n n , J . , E. C h o i , E. Y a m a s a k i a n d B . N . A m e s , D e t e c t i o n o f c a r c i n o g e n s as m u t a g e n s in t h e SaN m o n e l l a / m i c r o s o m e t e s t : a s s a y o f 3 0 0 c h e m i c a l s , P r o c . N a t l . A c a d . Sei. ( U . S , A . ) , 7 3 ( 1 9 7 5 ) 5 1 3 5 - 5139. 13 Mohn, G.R., and F.J. de Serres, On the mutagenic activity of some hair dyes, Mutation Res., 38 (1976) 116--117. 1 4 P a l m e r , K . A . , A . D e n u n z i o a n d S. G r e e n , T h e m u t a g e n i c a s s a y o f s o m e h a i r d y e c o m p o n e n t s , u s i n g t h e t h y m i d i n e k i n a s e l o c u s o f L 5 1 7 8 Y m o u s e l y m p h o m a cells, J . E n v i r o n . P a t h o L T o x i c o l . , 1 ( 1 9 7 7 ) 87--91. 1 5 P u r c h a s e , I . F . H . , E. L o n g s t a f f , J . A s h b y , J . A . S t y l e s , D. A n d e r s o n , P . A . L e f e v e r a n d F . R . W e s t w o o d , E v a l u a t i o n o f six s h o r t t e r m t e s t s f o r d e t e c t i n g o r g a n i c c h e m i c a l c a r c i n o g e n s a n d r e c o m m e n d a t i o n s for their use, Nature (London), 264 (1976) 624--627. 1 6 S e a r l e , C . E . , D . G . H a r n d e n , S. V e n i t t a n d O . H . B . G y d e , C a r c i n o g e n i c i t y a n d m u t a g e n i c i t y t e s t s o f some hair colourants and constituents, Nature (London), 255 (1975) 506--507. 1 7 S h a f e r , N., a n d R . W . S h a r e r , P o t e n t i a l o f c a r c i n o g e n i c e f f e c t s o f h a i r d y e s , N . Y . S t a t e J . o f M e d . , 76 (1976) 394--396. 1 8 S m i t h , N.S., N . P . B i s h u n a n d D. Williams, D e p r e s s i o n o f l y m p h o c y t e t r a n s f o r m a t i o n b y t w o h a i r d y e constituents, Microbios Lett., 1 (1976) 205--208. 1 9 U.S. D e p a r t m e n t o f H e a l t h , E d u c a t i o n , a n d W e l f a r e , 2 , 4 - D i a m i n o a n i s o l e in h a i r a n d f u r d y e s , N I O S H Current Intelligence Bulletin 78--111 (1978) 1--5. 2 0 Y a m a s a k i , E., a n d B . N . A m e s , C o n c e n t r a t i o n o f m u t a g e n s f r o m u r i n e b y a d s o r p t i o n w i t h t h e n o n p o l a r r e s i n X A D - 2 : c i g a r e t t e s m o k e r s h a v e m u t a g e n i c u r i n e , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 7 4 ( 1 9 7 7 ) 3555--3559.
Mutation Research, 66 (1979) 241--245 © Elsevier/North-Holland Biomedical Press
DETECTION OF MUTAGENS IN THE URINE OF RATS FOLLOWING TOPICAL APPLICATION OF HAIR DYES
MARINEL M. AMMENHEUSER and MICHAEL E. WARREN Department of Biology, Lamar University, Beaumont, Texas 77710 (U.S.A.) (Received 28 June 1978) (Revision received 29 September 1978) (Accepted 10 October 1978)
Summary Mutagens were detected in the urine of rats following topical application of two commercial oxidative-type hair dye preparations. The test system used was induction of back mutation with the bacterial tester strain TA1538, a histidine
Introduction Recent investigations have shown that some of the ingredients of commercially available hair dyes are mutagenic in Drosophila [5,8], in mouse lymp h o m a cells [14] and in bacterial test systems [1,10,13,16]. Two of these dye ingredients, 4-nitro~>-phenylenediamine (4NOPD) and 2-nitro-p-phenylenediamine (2NPPD), also effected immunosuppressive activity in cultures of human lymphocytes [18], produced morphological transformation in mouse CH3 prostate ceils [4] and induced chromosome damage in cultured Chinese hamster cells [11]. Most previous studies have involved direct testing of dye formulations or Abbreviations: 4NOPD, 4-nitro~-phenylenediamine; 2NPPD, 2-nitro-p-phenylenedlamine.
242
their major constituents. The present study was designed to imitate more closely the actual conditions of use of hair-dye preparations. Materials and methods Both 4NOPD and 2NPPD were tested for mutagenicity with the Salmonella histidine auxotroph system [2] using bacterial tester strains provided by Dr. B.N. Ames, University of California at Berkeley. In confirmation of previous findings [ 1,10,16 ] these c o m p o u n d s were mutagenic to strain TA1538 w i t h o u t the use of a metabolic activation system. The most active compound, 4NOPD, was tested further by injecting 5 mg i.p. into male Texas: Sprague-Dawley rats and then assaying their urine for mutagenicity with T A 1 5 3 8 using the m e t h o d of Durston and Ames [7]. Injection of a commercial dye formulation (Dye B) was also attempted, b u t a dilution of 0.5 ml of the d y e with 2 ml sterile water proved to be lethal to the test animals within 6--24 h. Percutaneous penetration in rats was determined by topical application of 120 mg of 4NOPD in acetone and in isopropanol followed by urine assays with TA1538. Each solution was applied to shortened hair on the back of the animal, allowed to remain on the hair for 20 min after application and then removed b y shampooing and thorough rinsing. Test animals were confined in individual metabolism cages designed for the separation of urine and feces, and urine specimens were collected before dye application for control tests and every 24 h after d y e application for 4 days. This same procedure was used for the application of two commercial oxidative-type hair-coloring products (Dyes A and B) which both contained the direct dyes 4NOPD and 2NPPD. A third p r o d u c t (Dye C), similar in composition to Dyes A and B b u t w i t h o u t 4NOPD or 2NPPD, was also tested. Results and discussion When 50 #g of 2NPPD was assayed with strain TA1538 using 0.1 ml of dimethylsulfoxide as a solvent, only 153 revertant colonies (less controls) were produced. There were 316 colonies with 100 pg of 2NPPD. In contrast, 4NOPD was mutagenic in this particular test system at much lower c o n c e n t r a t i o n s 227 revertants with 1 pg, 1184 with 5 pg, and 2256 with 10 pg. Comparison of these results with the results of the injection studies (Table 1), indicates that only 1--1.5% of the injected 4NOPD appears in the urine in a form that is directly mutagenic in the Salmonella test with strain TA1538. The one rat that survived 24 h following injection of a complete d y e formulation excreted urine that showed mutagenic activity in proportion to the quantity of 4NOPD (0.5 mg) contained in the product. Mutagenic activity was detected in the urine of rats (Table 1) following topical application of 4NOPD to their backs, demonstrating that this direct d y e can penetrate the skin. There was a 4-fold increase in mutagenic activity when the 4NOPD was applied in acetone rather than isopropanol, b u t the latter results should be more comparable to actual use conditions since isopropanol is one o f the base ingredients of hair-coloring products [6]. Data obtained using the complete d y e product are more relevant to the
243 TABLE 1 MUTAGENICITY OF URINE FROM RATS FOLLOWING INJECTION AND TOPICAL APPLICATION OF 4NOPD Treatment
Volume o f urine per plate (rnl)
Total volume of urine e x c r e t e d in 24 h (ml)
N u m b e r of colonies per plate less controls a
N u m b e r of colonies per ml of urine
5.0 m g 4 N O P D i.P. b 5.0 m g 4 N O P D i.P. b 0 . 5 m l D y e B i.p. c 1 2 0 m g 4 N O P D in 4 m l a c e t o n e d 4 ml acetone 1 2 0 m g 4 N O P D in 8 m l i s o p r o p a n o l d 8 ml isopropanol
0.3 0.3 0.3 0.3 0.3 0.3 0.3
11.8 25.0 11.6 8.2 10.0 9.7 14.9
282 188 34 145 0 36 0
940 627 113 483 0 120 0
-+ 6 1 . 5 + 30.4 -+ 5.7 +- 3.5 +
3.9
a R e s u l t s are t h e m e a n + s t a n d a r d d e v i a t i o n o f a t least 3 p l a t e s t e s t e d w i t h T A 1 5 3 8 . C o l o n y c o u n t s ( m e a n 29 +- 3) f r o m assays o f u r i n e s a m p l e s c o l l e c t e d p r i o r to t r e a t m e n t have b e e n s u b t r a c t e d . Plates a s s a y e d w i t h T A 1 5 3 8 w i t h o u t urine averaged 2 5 s p o n t a n e o u s c o l o n i e s . b 5 m g of 4 N O P D w a s s u s p e n d e d in 1 m l o f sterile 0.9% NaCI a n d 1 . 7 5 % g u m acacia s o l u t i o n . c 0.5 m l o f D y e B w a s d i l u t e d w i t h 2 m l o f sterile w a t e r . This d o s a g e c o n t a i n e d 0.5 m g o f 4 N O P D . d A c e t o n e or i s o p r o p a n o l and 4 N O P D w e r e m i x e d and applied t o p i c a l l y as d e s c r i b e d in M e t h o d s .
study of the possible mutagenicity of commercial hair dyes, but it is beyond the scope of this study to investigate the metabolism and potential mutagenicity of the approximately 25 different ingredients in an oxidative hair-coloring formulation. The Salmonella test was particularly useful in this context since mutagenicity could be detected without isolation of the particular compound (or compounds) responsible for the mutagenic response. The results obtained do suggest that the 4NOPD in the two commercial dyes tested could account for at least some of the mutagenicity of the urine excreted by the rats dyed with these products (Table 2). A number of other ingredients or their reaction products could also contribute urinary mutagens, but may not be detectable in this particular test s y s t e m - they may require further metabolic activation or may not be present in sufficient concentration. Maximal levels of mutagenic activity occurred with urine collected during the first 24 h following dye application. Although the level of mutagenic activity was low for all of the assays, a dose--response was observed when increasing volumes of mutagenic urine were tested. For example, when 30 ml of Dye A was applied topically, 0.3 ml of urine produced 25 revertant colonies (less 27 control colonies) and 0.5 ml of urine produced 47 revertants. Urine samples collected 2--4 days after the dye treatment did not show statistically significant levels of mutagenicity. Control urine collected prior to dye application was not mutagenic and there was no significant difference between control plates assayed with 0.3 ml, 0.5 ml or 0.7 ml of urine. Dye C, which did not contain 4NOPD or 2NPPD, was negative in this test system (Table 2). Animal studies do not duplicate exactly the conditions of use and the resulting response of human subjects to the application of hair-coloring products, but an effort has been made in this study to apply the dyes in a manner similar to the one recommended by the manufacturer of the product. The surface area
244 TABLE 2 MUTAGENICITY OF URINE FROM RATS FOLLOWING TOPICAL APPLICATION OF THREE COMMERCIAL HAIR DYES Treatment a
30 ml Dye A 30 ml Dye A + 30 20 ml Dye A + 20 10 ml Dye A + 10 30 ml Dye B 30mlDyeB+30mlH 30 ml Dye B + 30 30 ml Dye C + 30 30 ml Dye C + 30
ml H202 ml H202 ml H202 202 ~c ml H202 ml H202 ~ c ml H202
Volume of urine p e r plate (ml)
Total volume of urine e x c r e t e d in 24 h (ml)
Number of colonies per plate less controls b
Number of colonies per ml of urine
0.5 0.5 0.5 0.5 0.5 0.7 0.7 0.5 0.7
10.6 9.4 14.2 13.0 14.8 6.1 6.5 4.4 7.2
47 31 24 6 20 32 34 1 0
94 62 48 12 40 46 49 2 0
+ 4.5 +- 6 . 0 +- 2 . 8 +- 3 . 5 -+ 3 . 5 -+ 2.6 -+ 2.1 -+ 2 . 8
a D y e A c o n t a i n e d 2 . 5 m g 4 N O P D p e r m l a n d D y e B c o n t a i n e d 1 m g 4 N O P D p e r ml. T h e r e w a s n o 4 N O P D i n D y e C. W h e r e i n d i c a t e d , e q u a l v o l u m e s o f h y d r o g e n p e r o x i d e d e v e l o p e r l o t i o n w e r e a d d e d t o t h e d y e f o r m u l a t i o n s in a c c o r d a n c e w i t h t h e m a n u f a c t u r e r ' s i n s t r u c t i o n s . b S t r a i n T A 1 6 3 8 w a s u s e d for all assays. R e s u l t s are t h e m e a n -+ standard d e v i a t i o n o f c o u n t s f r o m at l e a s t 3 plates. C o l o n y c o u n t s f r o m assays o f urine s a m p l e s c o l l e c t e d prior t o d y e a p p l i c a t i o n ( m e a n 2 7 _+ 4 . 3 ) have b e e n s u b t r a c t e d . Plates a s s a y e d w i t h T A 1 5 3 8 w i t h o u t urine averaged 2 5 s p o n t a n e o u s c o l o n i e s . e T w o t e s t s w e r e d u p l i c a t e d using f e m a l e rats. All o t h e r t e s t s w e r e p e r f o r m e d o n m a l e T e x a s : S p r a q u e D a w l e y rats w e i g h i n g an average o f a b o u t 4 5 0 g.
used on the rats was smaller than the human scalp in proportion to the volume of dye applied, and the application time was slightly longer (about 30 min), but after treatment the hair was shampooed and thoroughly rinsed in accordance with the manufacturer's instructions. Extra precautions were also taken to prevent oral ingestion o f the dye formulation, since humans are not likely to swallow dye during use. Another factor to be considered is the relative permeability of various skin surfaces. A comparative study of skin permeability [3] indicated that the skin on the back of a rat was about twice as permeable as the skin on the human forearm. However, investigation of the relative absorption of a substance from various regions of the human b o d y indicated that the scalp was 3.5 times more permeable than the forearm [9]. Thus it can be concluded that the skin on the back o f a rat is at least similar in permeability to the skin of the human scalp. Differences in the metabolism of absorbed substances also must be considered. For this reason it is hoped that further investigations will include studies on human volunteers. Because of the relatively large volume of urine excreted by human subjects, a newly developed system for concentrating mutagens from urine [20] should be a useful addition to future research. The present investigation indicates that mutagens a r e excreted in the urine following topical application of commercial hair
245
demiological findings that suggest a possible link between breast cancer and the long term use of hair-coloring agents [ 17 ], and the studies being conducted by the U.S. National Institute for Occupational Safety and Health [19] which also suggest an excess of cancer of the genital organs among hairdressers and cosmetologists. References 1 A m e s , B.N., H . O . K a m m e n a n d E. Y a m a s a k i , H a i r d y e s a r e m u t a g e n i c : i d e n t i f i c a t i o n o f a v a r i e t y o f m u t a g e n i c i n g r e d i e n t s , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 7 2 ( 1 9 7 5 ) 2 4 2 3 - - 2 4 2 7 . 2 A m e s , B.N., J . M c C a n n a n d E. Y a m a s a k i , M e t h o d s f o r d e t e c t i n g c a r c i n o g e n s a n d m u t a g e n s w i t h t h e S a l m o n e l l a / m a m m a l i a n - m i c r o s o m e m u t a g e n i c i t y t e s t , M u t a t i o n Res., 31 ( 1 9 7 5 ) 3 4 7 - - 3 6 4 . 3 B a r t e k , J., J . A . L a B u d d e a n d H . I . M a i b a c h , S k i n p e r m e a b i l i t y in vivo: c o m p a r i s o n in r a t , r a b b i t , pig a n d m a n , J . Invest. D e r m a t o l . , 5 8 ( 1 9 7 2 ) 1 1 4 - - 1 2 3 . 4 Benedict, W.F., Morphological transformation and chromosome aberrations produced by two hair dye components, Nature (London), 260 (1976) 368--369. 5 Blljieven, W . G . H . , M u t a g e n i c i t y o f f o u r h a i r d y e s in Drosophila melanogaster, M u t a t i o n Res., 4 8 (1977) 181--186. 6 C o r b e t t , J . F . , H a i r d y e s , i n : K . V e n k a t a r a m a n ( E d . ) T h e C h e m i s t r y o f S y n t h e t i c D y e s , Vol. 5, A c a d e m i c Press, N e w Y o r k , 1 9 7 1 , p p . 4 7 5 - - 5 3 4 . 7 D u r s t o n , W.E., a n d B . N . A m e s , A s i m p l e m e t h o d f o r t h e d e t e c t i o n o f m u t a g e n s in u r i n e : s t u d i e s w i t h t h e c a r c i n o g e n 2 - a c e t y l a m i n o f l u o r e n e , P r o c . N a t l . A c a d . Sci. ( U . S . 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