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Mutagenic effects of hair colourants on bacteria and mammalian cells
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Institute of Cancer Research, Pollards Wood Research Station, Chalfont St. Giles (U.K.) Mutagenic effects of hair colourants on bacteria and mammalian cells Recent work (Searle, Harnden, Venitt and Gyde, 1975; Ames, Kammen, and Yamasaki, 1975) indicates that a high proportion of proprietary hair colourants contain constituents possessing mutagenic and possibly carcinogenic properties. We have n o w tested 30 hair colourants, available in the UK, for mutagenicity in the Ames Salmonella system. Twenty-four colourants were positive: of these, 6 were "colour-rinses", 8 were "semi-permanent colourants", and 10 permanent colourants used in conjunction with an oxidant. Three of the colourrinses were mutagenic in TA1535 (base-pair substitution) and 3 were mutagenic in T A 1 5 3 8 (frameshift). All the other colourants were mutagenic only in TA1538. The permanent colourants required metabolic activation for mutagenicity, whereas the semi-permanent colourants were mutagenic in the absence of metabolic activation. A range of individual hair colourant constituents were also tested and found to be mutagenic. 4-nitro-o-phenylenediamine and 2-nitro-p-phenylenediamine, 2 widely used colourant constituents, were c y t o t o x i c to a near-diploid line of Chinese hamster prostate cells (CHMP/E). Both c o m p o u n d s caused a dose-dependent decrease in colony-forming ability following a 5-day treatment at doses up to 50 #g/ml. During a 7-day exposure of these cells to 25 pg/ml of their c o m p o u n d , chromatid gaps and breaks appeared after 2--3 days, followed by an increasing frequency of exchanges, dicentrics and abnormal chromosomes at later times. These results emphasize the need for rigorous long-term toxicological testing of hair colourant constituents in view of the increasing use of these products by large numbers of the human population.
29 G.R. Mohn and F.J. de Serres, Environmental Mutagenesis Branch, National Institute of Environmental Health Sciences Research Triangle Park (U.S.A.) On the mutagenic activity of some hair dyes In two recent publications, [B. Ames et al., PNAS 72 (1975) 2423-2427 and C. Searle et al., Nature 255 (1975) 506-507], several ingredients of oxidativetype hair dye formulations were described as mutagenic in plate test techniques using strain Salmonella t y p h i m u r i u m TA 1538 as indicator of frameshift mutations. In our laboratory we were able to confirm those results and to extend them using spot test techniques -- to Escherichia coli 3 4 3 / 1 1 3 (induction ofarg ÷back mutations). However, in standard-type liquid tests, repeatedly shown previously to be at least as sensitive as spot tests, we obtained consistent negative results in a variety of indicator organisms -- including TA 1538. The purpose of the present study was thus to investigate the discrepancies
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between results in plate and in liquid tests. In order to keep the reaction mixtures as simple as possible, t w o chemicals which -- in partial confirmation of previous findings -- to n o t require microsome-mediated activation were selected, namely 4-nitro-o-phenylenediamine (4-NOPD) and 2,5-diaminoanisole (2,5DAA); the mutation system employed was detection of h/s* revertants (from his D30s2) in strain S. typhimurium TA 1538. The results can be summarized as follows: both c o m p o u n d s are mutagenic in the plate test, 4-NOPD being a b o u t ten times more active than 2,5-DAA. In liquid tests at 37 °C, using stationary and growing cells at incubation times up to 4 h, no increase in mutation frequency above the spontaneous level can be observed, even in the case of 2,5DAA, at concentrations which inactivate substantially the cell. With incubation times longer than 20 h, however, 4-NOPD treated bacterial suspensions show a more than 100-fold increase of mutation frequency without any inactiviation of colony forming ability; experiments with 2,5-DAA are in progress. The signification of these results for understanding of the mechanisms of hair dye mutagenicity as for routine mutagenicity testing procedures will be discussed.
30 C.E. Voogd, National Institute of Public Health, Bilthoven (Netherlands)
The mutagenic action of some nitrothiazoles and nitrothiophenes It is well-known, that some heterocyclic c o m p o u n d s with a nitro-group, e.g. nitrofurans and nitroimidazoles, possess a mutagenic action. As some nitrothiazoles were used for growth promoting purposes, we investigated the mutagenic properties of these c o m p o u n d s as they may be mutagenic. The fluctuation test was used to demonstrate an increase of the mutationfrequency. A KlebsieUa pneumoniae mutant requiring uracil and proline for growth was used as a testorganism. We found that 2-acetamido-5-nitrothiazole is a p o t e n t mutagenic compound. The mutagenic activity of N(5-nitrothiazole-2-yl) benzamide is somewhat less followed b y 2-bromo-5-nitrothiazole and 2-amino-5-nitrothiazole. Furthermore 2-amino-5-chlorothiazole and in a lesser degree 2-aminothiazole are also mutagenic. The mutagenic activity of 4-nitroisothiazole is equal to that of 2amino-5-nitrothiazole. Even w i t h o u t a nitrogen atom in the nucleus these compounds are mutagenic. So 2-nitrothiophene is more mutagenic than 3-nitrothiophene. With thiazole, isothiazole and thiophene no mutagenic action could be demonstrated. Although the presence of a nitro-group seems a prerequisite for a strong mutagenic action of these compounds, the relation between chemical structure and mutagenic activity of these substances remains unclear.
31 S. Igali and R.C. Von Borstel, "Frederic Joliot-Curie" National Research Institute of Radiobiology and Radiohygiene, Budapest (Hungary) and Dept. of Genetics, University of Alberta, E d m o n t o n (Canada)
Institute of Cancer Research, Pollards Wood Research Station, Chalfont St. Giles (U.K.) Mutagenic effects of hair colourants on bacteria and mammalian cells Recent work (Searle, Harnden, Venitt and Gyde, 1975; Ames, Kammen, and Yamasaki, 1975) indicates that a high proportion of proprietary hair colourants contain constituents possessing mutagenic and possibly carcinogenic properties. We have n o w tested 30 hair colourants, available in the UK, for mutagenicity in the Ames Salmonella system. Twenty-four colourants were positive: of these, 6 were "colour-rinses", 8 were "semi-permanent colourants", and 10 permanent colourants used in conjunction with an oxidant. Three of the colourrinses were mutagenic in TA1535 (base-pair substitution) and 3 were mutagenic in T A 1 5 3 8 (frameshift). All the other colourants were mutagenic only in TA1538. The permanent colourants required metabolic activation for mutagenicity, whereas the semi-permanent colourants were mutagenic in the absence of metabolic activation. A range of individual hair colourant constituents were also tested and found to be mutagenic. 4-nitro-o-phenylenediamine and 2-nitro-p-phenylenediamine, 2 widely used colourant constituents, were c y t o t o x i c to a near-diploid line of Chinese hamster prostate cells (CHMP/E). Both c o m p o u n d s caused a dose-dependent decrease in colony-forming ability following a 5-day treatment at doses up to 50 #g/ml. During a 7-day exposure of these cells to 25 pg/ml of their c o m p o u n d , chromatid gaps and breaks appeared after 2--3 days, followed by an increasing frequency of exchanges, dicentrics and abnormal chromosomes at later times. These results emphasize the need for rigorous long-term toxicological testing of hair colourant constituents in view of the increasing use of these products by large numbers of the human population.
29 G.R. Mohn and F.J. de Serres, Environmental Mutagenesis Branch, National Institute of Environmental Health Sciences Research Triangle Park (U.S.A.) On the mutagenic activity of some hair dyes In two recent publications, [B. Ames et al., PNAS 72 (1975) 2423-2427 and C. Searle et al., Nature 255 (1975) 506-507], several ingredients of oxidativetype hair dye formulations were described as mutagenic in plate test techniques using strain Salmonella t y p h i m u r i u m TA 1538 as indicator of frameshift mutations. In our laboratory we were able to confirm those results and to extend them using spot test techniques -- to Escherichia coli 3 4 3 / 1 1 3 (induction ofarg ÷back mutations). However, in standard-type liquid tests, repeatedly shown previously to be at least as sensitive as spot tests, we obtained consistent negative results in a variety of indicator organisms -- including TA 1538. The purpose of the present study was thus to investigate the discrepancies
117
between results in plate and in liquid tests. In order to keep the reaction mixtures as simple as possible, t w o chemicals which -- in partial confirmation of previous findings -- to n o t require microsome-mediated activation were selected, namely 4-nitro-o-phenylenediamine (4-NOPD) and 2,5-diaminoanisole (2,5DAA); the mutation system employed was detection of h/s* revertants (from his D30s2) in strain S. typhimurium TA 1538. The results can be summarized as follows: both c o m p o u n d s are mutagenic in the plate test, 4-NOPD being a b o u t ten times more active than 2,5-DAA. In liquid tests at 37 °C, using stationary and growing cells at incubation times up to 4 h, no increase in mutation frequency above the spontaneous level can be observed, even in the case of 2,5DAA, at concentrations which inactivate substantially the cell. With incubation times longer than 20 h, however, 4-NOPD treated bacterial suspensions show a more than 100-fold increase of mutation frequency without any inactiviation of colony forming ability; experiments with 2,5-DAA are in progress. The signification of these results for understanding of the mechanisms of hair dye mutagenicity as for routine mutagenicity testing procedures will be discussed.
30 C.E. Voogd, National Institute of Public Health, Bilthoven (Netherlands)
The mutagenic action of some nitrothiazoles and nitrothiophenes It is well-known, that some heterocyclic c o m p o u n d s with a nitro-group, e.g. nitrofurans and nitroimidazoles, possess a mutagenic action. As some nitrothiazoles were used for growth promoting purposes, we investigated the mutagenic properties of these c o m p o u n d s as they may be mutagenic. The fluctuation test was used to demonstrate an increase of the mutationfrequency. A KlebsieUa pneumoniae mutant requiring uracil and proline for growth was used as a testorganism. We found that 2-acetamido-5-nitrothiazole is a p o t e n t mutagenic compound. The mutagenic activity of N(5-nitrothiazole-2-yl) benzamide is somewhat less followed b y 2-bromo-5-nitrothiazole and 2-amino-5-nitrothiazole. Furthermore 2-amino-5-chlorothiazole and in a lesser degree 2-aminothiazole are also mutagenic. The mutagenic activity of 4-nitroisothiazole is equal to that of 2amino-5-nitrothiazole. Even w i t h o u t a nitrogen atom in the nucleus these compounds are mutagenic. So 2-nitrothiophene is more mutagenic than 3-nitrothiophene. With thiazole, isothiazole and thiophene no mutagenic action could be demonstrated. Although the presence of a nitro-group seems a prerequisite for a strong mutagenic action of these compounds, the relation between chemical structure and mutagenic activity of these substances remains unclear.
31 S. Igali and R.C. Von Borstel, "Frederic Joliot-Curie" National Research Institute of Radiobiology and Radiohygiene, Budapest (Hungary) and Dept. of Genetics, University of Alberta, E d m o n t o n (Canada)