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Mutagens in rat urine after dermal application of 1,3-diaminobenzene
Mutation Research, 138 (1984) 137-143 Elsevier
137
MTR 00921
Mutagens in rat urine after dermal application of 1,3-diaminobenzene Steen Clemmensen and Henrik Rye Lam Institute of Toxicology, National Food Institute, 19, Markhoj Bygade, DK- 2860 Soborg (Denmark) (Received 23 January 1984) (Revision received 28 June 1984) (Accepted 5 July 1984)
Summary The mutagenicity of urine from rats treated topically on the skin with 1,3-diaminobenzene was studied by the Salmonella/mammalian-microsome assay. Urine samples were either passed directly through micropore filters or extracts were prepared using XAD-2 resin before testing in the frameshift strain TA98. Significant mutagenic activity was found only after metabolic activation with rat-liver microsomes. The activity was higher in extracts from rats treated with a mixture of hydrogen peroxide and 1,3-diaminobenzene than from rats which were exposed to 1,3-diaminobenzene only. After fractionation of the urine by HPLC it could be demonstrated that the mutagenic activity was not due to the parent amine but related to metabolites in two of the fractions. To a lesser extent these two partially purified fractions were also mutagenic without $9 activation even though it was not possible to demonstrate this effect in unfractionated urine extracts. A third fraction containing two metabolites did not exert demonstrable mutagenic activity. The implications for the assessment of hazard to man are discussed.
Arylamines are important precursors in the synthesis of dyes and plastics and are used as ingredients in commercial products. Many arylamines have shown mutagenic a n d / o r carcinogenic properties. One of these - - 1,3-diaminobenzene or meta-phenylenediamine (MPD) - - is used in the production of more than 100 dyestuffs and in many polymeric materials. However, this use leads to only minor human exposure because the processes mainly take place in dosed systems. A more pronounced exposure derives from the use as an ingredient in photographic developers and oxidative hair dyes (Fishbein, 1981). Positive responses to MPD have repeatedly been observed in bacterial mutagenicity tests (Ames et al., 1975a; Garner and Nutman, 1977; Shahin et al., 1980a; De Giovanni-Donelly, 1981). Point mutations have also been demonstrated in the L5178Y 0165-1218/84/$03.00 © 1984 Elsevier Science Publishers B.V.
mouse lymphoma test (Palmer et al., 1977). A mixture of hydrogen peroxide and M P D produced a larger response in the Salmonella test than MPD alone (Venitt and Searle, 1976). M P D was able to reach the mouse testis in amounts large enough to inhibit DNA synthesis (Seiler, 1977). A weakly positive result was found in a dominant lethal test in rats. However, this could not be confirmed (Sheu and Green, 1979; Burnett et al., 1977). With the exception of one study using subcutaneous injection (Saruta, 1962) all other carcinogenicity studies have been negative whether the route of administration was skin painting (Burnett et al., 1975; Holland et al., 1979) or oral administration (Weisburger et al., 1978). The available data have been evaluated by an international working group (IARC, 1978), which described the evidence as inadequate for the determination of carcinogenic
138 risk to humans. The study by Holland et al. (1979) was published later, but this was not definitive either, because the maximal dose that could be administered dermally to the mice, was small compared to doses known to give negative responses for carcinogenic arylamines like 4-aminobiphenyl (IARC, 1974) and 2,4-diaminoanisole (Sontag, 1981). It is difficult to evaluate skin painting studies with arylamines as no positive studies have been mentioned for those arylamines evaluated by I A R C (1982). Other approaches to gain information for hazard evaluation should be tried, when the available carcinogenicity studies prove inadequate. One approach would be to look for evidence of genetic toxicity in excreta from animals treated with MPD, In this study we administered M P D transderreally to rats in order to minimize interference from gastrointestinal metabolism as well as in partial simulation of the major route for human exposure. The Salmonella/mammalian-microsome assay on urine and urine extracts provided the biological end-point. Materials and methods
Chemicals 1,3-Diaminobenzene (MPD), CAS No. 108-45-2, 98% pure, was obtained from Merck-Schuchardt, F.R.G. Hydrogen peroxide, methanol and acetone from Ferak AG, F.R.G. The emulsion base consisted of: 22 g polyethyleneglycol 1000, 6.5 g cetyl palmitate, 6.5 g lauryl diethanolamide, 6.5 g 2-octyldecanol, 6 g isopropanol, 6 g 25% ammonia, 0.5 g ethylenediaminotetraacetic acid and 58.5 g distilled water, all cosmetic grade. Animals 6-week-old male Wistar rats (Mol : W I S T (SPF)) from Mrlleghrd Breeding Centre, Ejby, Denmark, were kept individually in metabolism cages with free access to water and IT-chow 101. R o o m climate was controlled at 2 2 + I ° C , relative humidity 60 + 10%, 8 air changes per hour and light from 9 p.m. to 9 a.m. Skin application An area of 8 cm x 8 cm on the back of the rats was clipped closely at least 3 h before application.
Only animals with intact skin were used for the experiments. The test material was prepared immediately before use by dissolving M P D in distilled water, and when specified by mixing 9 parts emulsion base with 11 parts of aqueous M P D a n d / o r hydrogen peroxide each to a final concentration of 4% (w/w). 1.5 ml test material (60 mg MPD) was applied evenly over an area of 4 cm × 4 cm and covered by 4-ply gauze, occlusive tape (Leucoflex ®, Beiersdorf, F.R.G.) and an elastic dressing. Application was terminated within 30 min after dissolving the MPD. The dressing was removed after 24 h and excess material carefully wiped off.
Preparation of urine extracts 24-h samples of urine were collected just before and for 2 days after application and immediately frozen at - 2 0 ° C until processed. Pooled urine from 4 rats was run through a 1 c m × 5 cm column of Amberlite XAD-2 (BDH Chemicals, Great Britain) at 2 - 3 m l / m i n , washed with 50 ml water to remove histidine. The adsorbed material was eluted with 50 ml methanol. The solvent was removed by evaporation under reduced pressure and solids dissolved in 0.5 ml sterile water per animal. Separation of metabolites 50-/~1 aliquots of urine extracts were injected into a Hewlett-Packard 1084 B liquid chromatograph (HPLC) equipped with a RP-18, 5-/xm column, using a flow rate of 1 m l / m i n , oven temperature 25 o C and a linear methanol:water gradient change from 10 to 46% over 24 min, thereafter increased to 100% methanol in 1 rain. Absorbance in the eluate was monitored at 280 nm. A 0.75-ml sample was passed through the H P L C by repeated injections of 50-/xl aliquots. From each injection 5 fractions were collected at preset time intervals: (1) 2.0-7.4 min, (2) 7.4-10.0 min, (3) 10.0-12.0 rain, (4) 14.0-19.0 min and (5) 20.0-23.0 min. The respective fractions from each time interval were pooled, evaporated under reduced pressure and dissolved in 2 ml distilled water before mutagenicity testing. Mutation assays Mutagenicity was determined in Salmonella
139 TABLE 1 REVERTANTS PER PLATE OF U R I N E FROM RATS F O L L O W I N G DERMAL APPLICATION OF MPD Treatment
TA98
TA100
- $9
+ $9
- $9
+ $9
Blank Water MPD
34+4 42+6 385:5
334- 3 484- 5 834-16
1265:59 2064-33 175+14
150±12 216+21 1764-13
Results are mean + S.D. of 5 plates. (a) 1.5 ml sterile water or (b) 1.5 ml 4% aqueous MPD was applied for 24 h under occlusion. 0.3 ml micropore-filtrated urine was added to each plate except blanks, where 0.3 ml phosphate buffer was used.
typhimurium strains TA98 (frameshift) and TA100 (base-pair substitution) obtained from Dr. Bruce Ames, Berkeley, CA. Microsomal preparations ($9) from Wistar rats treated with Arochlor 1254 (500 m g / k g ) was used for activation. Assays were performed according to Ames et al. (1975b) using quintuple determinations of 0.1 ml per plate. Results
Mutagenic activity in unprocessed urine An aqueous solution containing 60 mg MPD was applied topically to shaven dorsal skin of rats in order to provide a reservoir for absorption. The
site was occluded to increase skin permeability. Mutagenic activity could be detected when 0.3 ml micropore-filtered urine was tested in the Salmonella/mammalian-microsome assay (Table 1). The response was significant only in TA98 and only after metabolic activation. In comparison direct testing of 48 #g M P D (0.45 pmole) produced 294 revertants per plate - - a result similar to those previously reported by Venitt and Seade (1976). The investigation was therefore continued only in TA98.
Mutagenicity of urine extracts Urine extracts were prepared by adsorption
TABLE 2 M U T A G E N I C I T Y IN TA98 OF U R I N E EXTRACTS FROM RATS F O L L O W I N G TOPICAL APPLICATION OF MPD Treatment
Number of revertants per plate Without $9
$9 added
0-24 h
24-48 h
0-24 h
24-48 h
MPD in water MPD in emulsion base
40±4 36+4
53+3 46±3
122±6 >1000 56+ 4 a
> 1000 >1000 91+ 7 a
MPD ± H202 in water
44±4
68±4
MPD + H202 in emulsion base
41+4
46±3
>1000 63+ 4a >1000 420-4-10 b
>1000 556±17a >1000
H202
in emulsion base
Controls c
>1000 b
494-3
46±3
414- 3
444- 4
41-/-4
43±5
44± 4
414- 4
Results are mean + S.D. of 5 plates. Pooled urine from 4 animals treated with 1.5 ml test material ( + 4% MPD, ± 6% H202) were o n XAD-2 as described in Materials and methods. 0.1 ml urine extract per plate was applied. Blanks were in the r a n g e 30-40 revertants p e r p l a t e . a U r i n e e x t r a c t 25-fold diluted before t e s t i n g . b 50-fold diluted. c Mean of 3 untreated groups. processed
140 higher response was o b t a i n e d from extracts of urine from a n i m a l s treated with a n aqueous mixture of h y d r o g e n p e r o x i d e a n d M P D . T h e a b s o r p t i o n of a c o m p o u n d a n d t h e r e b y the m u t a g e n i c activity of the urine can be m o d i f i e d b y selection of vehicle as well as b y degree of occlusion. This was d e m o n s t r a t e d when an e m u l s i o n b a s e c o n t a i n i n g surface-active substances was used. I n this case the response was increased b y an o r d e r of m a g n i t u d e over that seen when either M P D alone or M P D a n d h y d r o g e n p e r o x i d e was a p p l i e d in a neutral aqueous solution (Table 2).
c h r o m a t o g r a p h y on X A D - 2 resin in o r d e r to increase the sensitivity of the assays. W i t h this p r o cedure no m u t a g e n i c activity was detected in extracts from urine collected before a p p l i c a t i o n n o r in urine extracts from rats treated with either water, aqueous h y d r o g e n peroxide, emulsion b a s e or h y d r o g e n p e r o x i d e c o n t a i n i n g emulsion base. W h e n aqueous M P D was a p p l i e d topically, m a x i m a l m u t a g e n i c response was found in urine collected b e t w e e n 24 a n d 48 h after a p p l i c a t i o n ( T a b l e 2) a n d only very little activity r e m a i n e d in urine collected later ( d a t a not shown). A m u c h
4
HPLC of urinary extracts
!
o
!
l
O
lO
0 ~0
Control
!
I
rain.
0
I
O
|
I0
t ~
[ rain.
M P O - t reeted
Fig. 1. HPLC chromatogram of urinary extracts (for details see Materials and Methods). Control urine was obtained 24 h before treating the rat topically with 1.5 ml 4% aqueous MPD. Peaks Nos. 3 and 4 are metabolites of MPD discussed in the text.
141
Mutagenic activity of fractions of urine extracts
TABLE 3
When urine extracts from treated rats were separated by HPLC two distinct peaks (Nos. 3 and 4 in Fig. 1) not present in urine from control animals were seen. Several other peaks that contained mainly endogenous materials were also present. Selected fractions (Nos. 1-6 in Table 3, see also Fig. 1) were collected and tested in TA98 with and without metabolic activation. HPLC of the concentrated fractions revealed that no hydrolysis or other changes had taken place during purification. Approximately half the total activity seen after metabolic activation was detected in fraction 4, which presumably contains only one metabolite in the peak with retention time 15 min. The remaining activity was present in the highly polar fraction 1 eluted between 2 and 7 rain, which contains a considerable amount of endogenous material. Both fractions also exhibited a significant though lower activity towards TA98 even without $9. This pattern could not be demonstrated with the crude urine extract itself. None of the other fractions showed any response. This includes fraction 3, which contains at least two metabolites in the peaks between 10 and 12 min. Only very small amounts of MPD, eluted between 7 and 8 rain, were present. The identity of the metabolites is presently under elucidation.
REVERTANTSPER PLATEIN TA98 OF FRACTIONATED URINE FROM RATSTOPICALLYTREATED WITH MPD
Discussion and conclusion Using the concentration procedure used in this study it was not possible to detect activity in a single day's urine in TA98 without metabolic activation, though it was easily detected when the urine was further purified and fractionated by HPLC. This demonstrates that the activity of even major metabolites can be overlooked unless the amount of substance available is comparable to that used for direct plate testing. If we assume that the molar absorption coefficient at 280 nm for M P D and the metabolites in fraction 4 are similar, we can calculate that the equivalent of 0.3/~mole of the fraction 4 metabolite produced more than 1000 revertants while 0.45 #mole of MPD produced 294. Direct testing of a mixture of MPD and hydrogen peroxide gave a larger number of revertants
Fraction No.
Retentiontime (rain)
- $9
+ $9
1 2 3 4 5
2.0- 7.4 7.4-10.0 10.0-12.0 14.0-19.0 20.0-23.0
260+ 89 31+ 6" 39+ 6 229+ 7 30+ 8
> t 000 33+ 8 30+15 >1000 32+ 6
Blank
35+_ 8
31+ 7
Results are mean+S.D, of 5 plates. Urine was collected 24 h after topical application of 4% aqueous MPD, treated with XAD-2, evaporated and fractionated by HPLC using a 5-#m RP-18 column and a methanol-water gradient. than the same amount of MPD alone (Venitt and Searle, 1976) and this suggests that compounds more mutagenic than MPD are formed. It follows from the large increase in mutagenic response in urine extracts from rats treated with such a mixture (Table 2) that some of these compounds must be absorbed through the skin. Though it is still unclear whether these compounds are further metabolized or not, this should be taken into account when the safety of oxidative hair dyes is considered. Recently it has been shown that post-excretion activation is needed before urinary metabolites of other arylamines such as benzidine, 2-naphthylamine, 4-aminobiphenyl and 2,4-diaminoanisole exhibit mutagenic activity in the Salmonella/microsome test, while their non-carcinogenic class-control pair members: 3,3',5,5'-tetramethylbenzidine, 2-aminobiphenyl and 1-aminonaphthalene are non-active under similar experimental conditions (Shahin et al., 1980b; Bos et al., 1980; Tanaka et al., 1980). These investigators also find the mutagenic activity of the urinary metabolites higher than that found by direct testing of the parent compounds. Contrary to the positive mutagenicity tests, results from long-term tests have been found negative within the limits of the experiments. But even when performed to the most current standards they have limited powers of resolution ( C h u e t al., 1981). Also the probability of a negative outcome is high when the substance in question is adminis-
142
tered at a low dose rate to few a n i m a l s over a relatively short period. W h e n the i n f o r m a t i o n is critically evaluated in this m a n n e r , m a n y contradictions can be explained a n d a better u n d e r s t a n d i n g o b t a i n e d as p o i n t e d out b y Purchase et al. (1981). T h e m a x i m u m tolerated dose was low in the oral study b y Weisburger et al. (1978) b u t as n o details are available, it is impossible to evaluate whether there was a n y trend in observed effects or not. The doses that can be m a d e available b y skin p a i n t i n g are generally low c o m p a r e d to those available b y oral a d m i n i s t r a t i o n , even if loss b y incomplete a b s o r p t i o n a n d b y d e s q u a m a t i o n is disregarded. But such studies do offer the possibility that local effects can be seen. If the mode of activation of the test substance is reasonably well established a n d k n o w n to be f u n c t i o n i n g in the skin, as in the case of p o l y a r o m a t i c hydrocarbons, we would find the n o n - a p p e a r a n c e of local tumors reassuring. If it is n o t - - a n d very little is k n o w n a b o u t arylamine m e t a b o l i s m i n the skin - - the significance of a negative o u t c o m e is less d e a r . It follows from this investigation that when living m a m m a l s are exposed to M P D through the skin, c o m p o u n d s that are more genotoxic than the p a r e n t a m i n e itself are formed, a n d that this effect is more p r o n o u n c e d when M P D is applied together with hydrogen peroxide. T h o u g h the identity of the metabolites is still u n d e r elucidation a n d their m o d e of activation needs to be related to possible m e c h a n i s m s of carcinogenicity, we believe, with regard to the i n a d e q u a c y of the available carcinogenicity studies that the potential hazard to m a n should be recognized and exposure to 1 , 3 - d i a m i n o b e n z e n e should be reduced as m u c h as possible. Acknowledgments T h e technical assistance of Vivian Jorgensen a n d Joan G l u v e r is gratefully acknowledged.
References Ames, B.N., H.O. Kammen and E. Yamasaki (1975a) Hair dyes are mutagenic: Identification of a variety of mutagenic ingredients, Proc. Natl. Acad. Sci. (U.S.A.), 72, 2423-2427.
Ames, B.N., J. McCann and E. Yamasaki (1975b) Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian-microsome mutagenicity test, Mutation Res., 31,347-364. Bos, R.P., R.M.E. Brouns, R. van Doorn, J.L.G. Theuws and P,Th. Henderson (1980) The appearance of mutagens in the urine of rats after the administration of benzidine and some other aromatic amines, Toxicology, 16, 113-122. Burnett, C., B. Lanman, R. Giovacchini, G. Wolcott, R. Scala and M. Keplinger (1975) Long-term toxicity studies on oxidation hair dyes, Food Cosmet. Toxicol., 13, 353-357. Burnett, G., R. Loehr and J. Corbett (1977) Dominant lethal mutagenicitystudy on hair dyes, J. Toxicol. Environ. Health, 2, 657-662. Chu, K.C., C. Cueto and J.M. Ward (1981) Factors in the evaluation of 200 NCI carcinogenicitybioassays,J. Toxicol. Environ. Health, 8, 251-280. De Giovanni-Donelly, R. (1981) The comparative response of Salmonella typhimurium strains TA1538, TA98 and TA100 to various hair-dye components, Mutation Res., 91, 21-25. Fishbein, L. (1981) Aromatic amines of major industrial importance: use and occurrence, IARC Sci. Publ., 10, 51-73. Garner, R.C., and C.A. Nutman (1977) Testing of some azodyes and their reduction products for mutagenicity using Salmonella typhimurium TA1538, Mutation Res., 44, 9-19. Holland, J.M., D.G. Gosslee and N.J. Williams (1979) Epidermal carcinogenicity of bis(2,3-epoxycyclopentyl)ether,2,2bis(p-glycidyloxyphenyl)propaneand m-phenylenediamine in male and female C3H and C57BL/6 mice, Cancer Res., 39, 1718-1725. IARC (1974) 4-Aminobiphenyl,in: IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 1, IARC, Lyon, pp. 74-79. IARC (1978) meta-Phenyldiamine, in: IARC Monographs of the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 16, IARC, Lyon, pp. 111-124. IARC (1982) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Chemicals, Industrial Processes and Industries Associated with Cancer in Humans, IARC, Lyon, Suppl. 4. Palmer, K.A., A. de Nunzio and S. Green (1977) The mutagenic assay of some hair dye components, using the thymidine kinase locus of L 5178 Y mouse lymphoma cells, J. Environ. Pathol. Toxicol., 1, 87-91. Purchase, I.F.H., D.B. Clayson, R. Preussmann and L. Tomatis (1981) Activity of 42 compounds in animal carcinogenicity studies, in: F.J. de Serres and J. Ashby (Eds.), Evaluation of Short-Term Tests for Carcinogens, Elsevier, Amsterdam, pp. 21-32. Saruta, N., S. Yamaguchi and T. Matsuoka (1962) Sarcoma produced by subdermal administration of m-phenylenediamine and m-phenylenediaminehydrochloride, Kyushu J. Med. Sci., 13, 175-180. Seiler, J.P. (1977) Inhibition of testicular DNA synthesis by chemical mutagens and carcinogens, Preliminary results in the validation of a novel short term test, Mutation Res., 46, 305-310.
143 Shahin, M.M., A. Bugaut and G. Kalopissis (1980a) Structure-activity relationship within a series of m-diaminobenzene derivatives, Mutation Res., 78, 25-31. Shahin, M.M., D. Rouers, A. Bugaut and G. Kalopissis (1980b) Structure-activity relationships within a series of 2,4-diaminoalkoxybenzene compounds, Mutation Res., 79, 289-306. Sheu, C.W., and S. Green (1979) Dominant lethal assay of some hair-dye components in random-bred male rats, Mutation Res., 68, 85-98. Sontag, J.M. (1981) Carcinogenicity of substituted benzenediamines (phenylendiamines) in rats and mice, J. Natl. Cancer Inst., 66, 591-602.
Tanaka, K.-I., S. Marui and T. Mii (1980) Mutagenicity of extracts of urine from rats treated with aromatic amines, Mutation Res., 79, 173-176. Venitt, S., and C.E. Searle (1976) Mutagenicity and possible carcinogenicity of hair colourants and constituents, IARC, Sci. Publ., 13, 263-272. Weisburger, E.K., A.B. Russfield, F. Homburger, J.H. Weisburger, E. Boger, C,G. van Dongen and K.C. Chu (1978) Testing of twenty-one environmental aromatic amines or derivatives for long-term toxicity or carcinogenicity, J. Environ. Pathol. Toxicol., 2, 325-356.
137
MTR 00921
Mutagens in rat urine after dermal application of 1,3-diaminobenzene Steen Clemmensen and Henrik Rye Lam Institute of Toxicology, National Food Institute, 19, Markhoj Bygade, DK- 2860 Soborg (Denmark) (Received 23 January 1984) (Revision received 28 June 1984) (Accepted 5 July 1984)
Summary The mutagenicity of urine from rats treated topically on the skin with 1,3-diaminobenzene was studied by the Salmonella/mammalian-microsome assay. Urine samples were either passed directly through micropore filters or extracts were prepared using XAD-2 resin before testing in the frameshift strain TA98. Significant mutagenic activity was found only after metabolic activation with rat-liver microsomes. The activity was higher in extracts from rats treated with a mixture of hydrogen peroxide and 1,3-diaminobenzene than from rats which were exposed to 1,3-diaminobenzene only. After fractionation of the urine by HPLC it could be demonstrated that the mutagenic activity was not due to the parent amine but related to metabolites in two of the fractions. To a lesser extent these two partially purified fractions were also mutagenic without $9 activation even though it was not possible to demonstrate this effect in unfractionated urine extracts. A third fraction containing two metabolites did not exert demonstrable mutagenic activity. The implications for the assessment of hazard to man are discussed.
Arylamines are important precursors in the synthesis of dyes and plastics and are used as ingredients in commercial products. Many arylamines have shown mutagenic a n d / o r carcinogenic properties. One of these - - 1,3-diaminobenzene or meta-phenylenediamine (MPD) - - is used in the production of more than 100 dyestuffs and in many polymeric materials. However, this use leads to only minor human exposure because the processes mainly take place in dosed systems. A more pronounced exposure derives from the use as an ingredient in photographic developers and oxidative hair dyes (Fishbein, 1981). Positive responses to MPD have repeatedly been observed in bacterial mutagenicity tests (Ames et al., 1975a; Garner and Nutman, 1977; Shahin et al., 1980a; De Giovanni-Donelly, 1981). Point mutations have also been demonstrated in the L5178Y 0165-1218/84/$03.00 © 1984 Elsevier Science Publishers B.V.
mouse lymphoma test (Palmer et al., 1977). A mixture of hydrogen peroxide and M P D produced a larger response in the Salmonella test than MPD alone (Venitt and Searle, 1976). M P D was able to reach the mouse testis in amounts large enough to inhibit DNA synthesis (Seiler, 1977). A weakly positive result was found in a dominant lethal test in rats. However, this could not be confirmed (Sheu and Green, 1979; Burnett et al., 1977). With the exception of one study using subcutaneous injection (Saruta, 1962) all other carcinogenicity studies have been negative whether the route of administration was skin painting (Burnett et al., 1975; Holland et al., 1979) or oral administration (Weisburger et al., 1978). The available data have been evaluated by an international working group (IARC, 1978), which described the evidence as inadequate for the determination of carcinogenic
138 risk to humans. The study by Holland et al. (1979) was published later, but this was not definitive either, because the maximal dose that could be administered dermally to the mice, was small compared to doses known to give negative responses for carcinogenic arylamines like 4-aminobiphenyl (IARC, 1974) and 2,4-diaminoanisole (Sontag, 1981). It is difficult to evaluate skin painting studies with arylamines as no positive studies have been mentioned for those arylamines evaluated by I A R C (1982). Other approaches to gain information for hazard evaluation should be tried, when the available carcinogenicity studies prove inadequate. One approach would be to look for evidence of genetic toxicity in excreta from animals treated with MPD, In this study we administered M P D transderreally to rats in order to minimize interference from gastrointestinal metabolism as well as in partial simulation of the major route for human exposure. The Salmonella/mammalian-microsome assay on urine and urine extracts provided the biological end-point. Materials and methods
Chemicals 1,3-Diaminobenzene (MPD), CAS No. 108-45-2, 98% pure, was obtained from Merck-Schuchardt, F.R.G. Hydrogen peroxide, methanol and acetone from Ferak AG, F.R.G. The emulsion base consisted of: 22 g polyethyleneglycol 1000, 6.5 g cetyl palmitate, 6.5 g lauryl diethanolamide, 6.5 g 2-octyldecanol, 6 g isopropanol, 6 g 25% ammonia, 0.5 g ethylenediaminotetraacetic acid and 58.5 g distilled water, all cosmetic grade. Animals 6-week-old male Wistar rats (Mol : W I S T (SPF)) from Mrlleghrd Breeding Centre, Ejby, Denmark, were kept individually in metabolism cages with free access to water and IT-chow 101. R o o m climate was controlled at 2 2 + I ° C , relative humidity 60 + 10%, 8 air changes per hour and light from 9 p.m. to 9 a.m. Skin application An area of 8 cm x 8 cm on the back of the rats was clipped closely at least 3 h before application.
Only animals with intact skin were used for the experiments. The test material was prepared immediately before use by dissolving M P D in distilled water, and when specified by mixing 9 parts emulsion base with 11 parts of aqueous M P D a n d / o r hydrogen peroxide each to a final concentration of 4% (w/w). 1.5 ml test material (60 mg MPD) was applied evenly over an area of 4 cm × 4 cm and covered by 4-ply gauze, occlusive tape (Leucoflex ®, Beiersdorf, F.R.G.) and an elastic dressing. Application was terminated within 30 min after dissolving the MPD. The dressing was removed after 24 h and excess material carefully wiped off.
Preparation of urine extracts 24-h samples of urine were collected just before and for 2 days after application and immediately frozen at - 2 0 ° C until processed. Pooled urine from 4 rats was run through a 1 c m × 5 cm column of Amberlite XAD-2 (BDH Chemicals, Great Britain) at 2 - 3 m l / m i n , washed with 50 ml water to remove histidine. The adsorbed material was eluted with 50 ml methanol. The solvent was removed by evaporation under reduced pressure and solids dissolved in 0.5 ml sterile water per animal. Separation of metabolites 50-/~1 aliquots of urine extracts were injected into a Hewlett-Packard 1084 B liquid chromatograph (HPLC) equipped with a RP-18, 5-/xm column, using a flow rate of 1 m l / m i n , oven temperature 25 o C and a linear methanol:water gradient change from 10 to 46% over 24 min, thereafter increased to 100% methanol in 1 rain. Absorbance in the eluate was monitored at 280 nm. A 0.75-ml sample was passed through the H P L C by repeated injections of 50-/xl aliquots. From each injection 5 fractions were collected at preset time intervals: (1) 2.0-7.4 min, (2) 7.4-10.0 min, (3) 10.0-12.0 rain, (4) 14.0-19.0 min and (5) 20.0-23.0 min. The respective fractions from each time interval were pooled, evaporated under reduced pressure and dissolved in 2 ml distilled water before mutagenicity testing. Mutation assays Mutagenicity was determined in Salmonella
139 TABLE 1 REVERTANTS PER PLATE OF U R I N E FROM RATS F O L L O W I N G DERMAL APPLICATION OF MPD Treatment
TA98
TA100
- $9
+ $9
- $9
+ $9
Blank Water MPD
34+4 42+6 385:5
334- 3 484- 5 834-16
1265:59 2064-33 175+14
150±12 216+21 1764-13
Results are mean + S.D. of 5 plates. (a) 1.5 ml sterile water or (b) 1.5 ml 4% aqueous MPD was applied for 24 h under occlusion. 0.3 ml micropore-filtrated urine was added to each plate except blanks, where 0.3 ml phosphate buffer was used.
typhimurium strains TA98 (frameshift) and TA100 (base-pair substitution) obtained from Dr. Bruce Ames, Berkeley, CA. Microsomal preparations ($9) from Wistar rats treated with Arochlor 1254 (500 m g / k g ) was used for activation. Assays were performed according to Ames et al. (1975b) using quintuple determinations of 0.1 ml per plate. Results
Mutagenic activity in unprocessed urine An aqueous solution containing 60 mg MPD was applied topically to shaven dorsal skin of rats in order to provide a reservoir for absorption. The
site was occluded to increase skin permeability. Mutagenic activity could be detected when 0.3 ml micropore-filtered urine was tested in the Salmonella/mammalian-microsome assay (Table 1). The response was significant only in TA98 and only after metabolic activation. In comparison direct testing of 48 #g M P D (0.45 pmole) produced 294 revertants per plate - - a result similar to those previously reported by Venitt and Seade (1976). The investigation was therefore continued only in TA98.
Mutagenicity of urine extracts Urine extracts were prepared by adsorption
TABLE 2 M U T A G E N I C I T Y IN TA98 OF U R I N E EXTRACTS FROM RATS F O L L O W I N G TOPICAL APPLICATION OF MPD Treatment
Number of revertants per plate Without $9
$9 added
0-24 h
24-48 h
0-24 h
24-48 h
MPD in water MPD in emulsion base
40±4 36+4
53+3 46±3
122±6 >1000 56+ 4 a
> 1000 >1000 91+ 7 a
MPD ± H202 in water
44±4
68±4
MPD + H202 in emulsion base
41+4
46±3
>1000 63+ 4a >1000 420-4-10 b
>1000 556±17a >1000
H202
in emulsion base
Controls c
>1000 b
494-3
46±3
414- 3
444- 4
41-/-4
43±5
44± 4
414- 4
Results are mean + S.D. of 5 plates. Pooled urine from 4 animals treated with 1.5 ml test material ( + 4% MPD, ± 6% H202) were o n XAD-2 as described in Materials and methods. 0.1 ml urine extract per plate was applied. Blanks were in the r a n g e 30-40 revertants p e r p l a t e . a U r i n e e x t r a c t 25-fold diluted before t e s t i n g . b 50-fold diluted. c Mean of 3 untreated groups. processed
140 higher response was o b t a i n e d from extracts of urine from a n i m a l s treated with a n aqueous mixture of h y d r o g e n p e r o x i d e a n d M P D . T h e a b s o r p t i o n of a c o m p o u n d a n d t h e r e b y the m u t a g e n i c activity of the urine can be m o d i f i e d b y selection of vehicle as well as b y degree of occlusion. This was d e m o n s t r a t e d when an e m u l s i o n b a s e c o n t a i n i n g surface-active substances was used. I n this case the response was increased b y an o r d e r of m a g n i t u d e over that seen when either M P D alone or M P D a n d h y d r o g e n p e r o x i d e was a p p l i e d in a neutral aqueous solution (Table 2).
c h r o m a t o g r a p h y on X A D - 2 resin in o r d e r to increase the sensitivity of the assays. W i t h this p r o cedure no m u t a g e n i c activity was detected in extracts from urine collected before a p p l i c a t i o n n o r in urine extracts from rats treated with either water, aqueous h y d r o g e n peroxide, emulsion b a s e or h y d r o g e n p e r o x i d e c o n t a i n i n g emulsion base. W h e n aqueous M P D was a p p l i e d topically, m a x i m a l m u t a g e n i c response was found in urine collected b e t w e e n 24 a n d 48 h after a p p l i c a t i o n ( T a b l e 2) a n d only very little activity r e m a i n e d in urine collected later ( d a t a not shown). A m u c h
4
HPLC of urinary extracts
!
o
!
l
O
lO
0 ~0
Control
!
I
rain.
0
I
O
|
I0
t ~
[ rain.
M P O - t reeted
Fig. 1. HPLC chromatogram of urinary extracts (for details see Materials and Methods). Control urine was obtained 24 h before treating the rat topically with 1.5 ml 4% aqueous MPD. Peaks Nos. 3 and 4 are metabolites of MPD discussed in the text.
141
Mutagenic activity of fractions of urine extracts
TABLE 3
When urine extracts from treated rats were separated by HPLC two distinct peaks (Nos. 3 and 4 in Fig. 1) not present in urine from control animals were seen. Several other peaks that contained mainly endogenous materials were also present. Selected fractions (Nos. 1-6 in Table 3, see also Fig. 1) were collected and tested in TA98 with and without metabolic activation. HPLC of the concentrated fractions revealed that no hydrolysis or other changes had taken place during purification. Approximately half the total activity seen after metabolic activation was detected in fraction 4, which presumably contains only one metabolite in the peak with retention time 15 min. The remaining activity was present in the highly polar fraction 1 eluted between 2 and 7 rain, which contains a considerable amount of endogenous material. Both fractions also exhibited a significant though lower activity towards TA98 even without $9. This pattern could not be demonstrated with the crude urine extract itself. None of the other fractions showed any response. This includes fraction 3, which contains at least two metabolites in the peaks between 10 and 12 min. Only very small amounts of MPD, eluted between 7 and 8 rain, were present. The identity of the metabolites is presently under elucidation.
REVERTANTSPER PLATEIN TA98 OF FRACTIONATED URINE FROM RATSTOPICALLYTREATED WITH MPD
Discussion and conclusion Using the concentration procedure used in this study it was not possible to detect activity in a single day's urine in TA98 without metabolic activation, though it was easily detected when the urine was further purified and fractionated by HPLC. This demonstrates that the activity of even major metabolites can be overlooked unless the amount of substance available is comparable to that used for direct plate testing. If we assume that the molar absorption coefficient at 280 nm for M P D and the metabolites in fraction 4 are similar, we can calculate that the equivalent of 0.3/~mole of the fraction 4 metabolite produced more than 1000 revertants while 0.45 #mole of MPD produced 294. Direct testing of a mixture of MPD and hydrogen peroxide gave a larger number of revertants
Fraction No.
Retentiontime (rain)
- $9
+ $9
1 2 3 4 5
2.0- 7.4 7.4-10.0 10.0-12.0 14.0-19.0 20.0-23.0
260+ 89 31+ 6" 39+ 6 229+ 7 30+ 8
> t 000 33+ 8 30+15 >1000 32+ 6
Blank
35+_ 8
31+ 7
Results are mean+S.D, of 5 plates. Urine was collected 24 h after topical application of 4% aqueous MPD, treated with XAD-2, evaporated and fractionated by HPLC using a 5-#m RP-18 column and a methanol-water gradient. than the same amount of MPD alone (Venitt and Searle, 1976) and this suggests that compounds more mutagenic than MPD are formed. It follows from the large increase in mutagenic response in urine extracts from rats treated with such a mixture (Table 2) that some of these compounds must be absorbed through the skin. Though it is still unclear whether these compounds are further metabolized or not, this should be taken into account when the safety of oxidative hair dyes is considered. Recently it has been shown that post-excretion activation is needed before urinary metabolites of other arylamines such as benzidine, 2-naphthylamine, 4-aminobiphenyl and 2,4-diaminoanisole exhibit mutagenic activity in the Salmonella/microsome test, while their non-carcinogenic class-control pair members: 3,3',5,5'-tetramethylbenzidine, 2-aminobiphenyl and 1-aminonaphthalene are non-active under similar experimental conditions (Shahin et al., 1980b; Bos et al., 1980; Tanaka et al., 1980). These investigators also find the mutagenic activity of the urinary metabolites higher than that found by direct testing of the parent compounds. Contrary to the positive mutagenicity tests, results from long-term tests have been found negative within the limits of the experiments. But even when performed to the most current standards they have limited powers of resolution ( C h u e t al., 1981). Also the probability of a negative outcome is high when the substance in question is adminis-
142
tered at a low dose rate to few a n i m a l s over a relatively short period. W h e n the i n f o r m a t i o n is critically evaluated in this m a n n e r , m a n y contradictions can be explained a n d a better u n d e r s t a n d i n g o b t a i n e d as p o i n t e d out b y Purchase et al. (1981). T h e m a x i m u m tolerated dose was low in the oral study b y Weisburger et al. (1978) b u t as n o details are available, it is impossible to evaluate whether there was a n y trend in observed effects or not. The doses that can be m a d e available b y skin p a i n t i n g are generally low c o m p a r e d to those available b y oral a d m i n i s t r a t i o n , even if loss b y incomplete a b s o r p t i o n a n d b y d e s q u a m a t i o n is disregarded. But such studies do offer the possibility that local effects can be seen. If the mode of activation of the test substance is reasonably well established a n d k n o w n to be f u n c t i o n i n g in the skin, as in the case of p o l y a r o m a t i c hydrocarbons, we would find the n o n - a p p e a r a n c e of local tumors reassuring. If it is n o t - - a n d very little is k n o w n a b o u t arylamine m e t a b o l i s m i n the skin - - the significance of a negative o u t c o m e is less d e a r . It follows from this investigation that when living m a m m a l s are exposed to M P D through the skin, c o m p o u n d s that are more genotoxic than the p a r e n t a m i n e itself are formed, a n d that this effect is more p r o n o u n c e d when M P D is applied together with hydrogen peroxide. T h o u g h the identity of the metabolites is still u n d e r elucidation a n d their m o d e of activation needs to be related to possible m e c h a n i s m s of carcinogenicity, we believe, with regard to the i n a d e q u a c y of the available carcinogenicity studies that the potential hazard to m a n should be recognized and exposure to 1 , 3 - d i a m i n o b e n z e n e should be reduced as m u c h as possible. Acknowledgments T h e technical assistance of Vivian Jorgensen a n d Joan G l u v e r is gratefully acknowledged.
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Tanaka, K.-I., S. Marui and T. Mii (1980) Mutagenicity of extracts of urine from rats treated with aromatic amines, Mutation Res., 79, 173-176. Venitt, S., and C.E. Searle (1976) Mutagenicity and possible carcinogenicity of hair colourants and constituents, IARC, Sci. Publ., 13, 263-272. Weisburger, E.K., A.B. Russfield, F. Homburger, J.H. Weisburger, E. Boger, C,G. van Dongen and K.C. Chu (1978) Testing of twenty-one environmental aromatic amines or derivatives for long-term toxicity or carcinogenicity, J. Environ. Pathol. Toxicol., 2, 325-356.