Seventy‐two chemicals were tested for their mutagenic potential in the L5178Y tk+/− mouse lymphoma cell forward mutation assay, using procedures based upon those described by Clive and Spector (Mutat Res 44:269‐278, 1975) and Clive et al. (Mutat Res 59:61‐108, 1979). Cultures were exposed to the chemicals for 4 hr, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT), 3 μg/ml. The chemicals were tested at least twice. Significant responses were obtained with allyl isothiocyanate, p‐benzoquinone dioxime, benzyl acetate, 2‐biphenylamine HCl, bis(2‐chloro‐1‐methylethyl)ether, cadmium chloride, chlordane, chlorobenzene, chlorobenzilate, 2‐chloroethanol, chlorothalonil, cytarabine‐HCl, p,p′‐DDE, diazinon, 2,6‐dichloro‐p‐phenylenediamine, N,N‐diethylthiourea, diglycidylresorcinol ether, 2,4‐dimethoxy aniline‐HCl, disperse yellow 3, endosulfan, 1,2‐epoxyhexa‐decane, ethyl acrylate, ethyl benzene, ethylene thiourea, F D and C yellow Number 6, furan, heptachlor, isophorone, mercuric chloride, 4,4′‐methylenedianiline 2 HCl, methyl viologen, nickel sulfate‐6H2O, 4,4′‐oxydianiline, pentachloroethane, piperonyl butoxide, propyl gallate, quinoline, rotenone, 2,4,5,6‐tetrachloro‐4‐nitro‐anisole, 1,1,1,2‐tetrachloroethane, trichlorfon, 2,4,6‐trichlorophenol, 2,4,5‐trimetho‐xybenzaldehyde, 1,1,3‐trimethyl‐2‐thiourea, 1‐vinyl‐3‐cyclopetene dioxide, vinyl toluene, and ziram. Apart from 2‐biphenylamine‐HCl, 2‐chloroethanol, disperse yellow 3, ethylene thiourea, FD and C yellow number 6, phenol, and 1,1,2‐tetrachloroethane, rat liver S9 mix was not a requirement for these compounds. Chemicals not identified as mutagens were acid red, 11‐aminoundecanoic acid, boric acid, 5‐chloro‐o‐toluidine, coumaphos, cyclohexanone, decabromodiphenyl oxide, di(2‐ethylhexyl)adipate, ferric chloride, fluometuron, melamine, monuron, phenesterin, phthalimide, reserpine, sodium dodecyl sulfate, 4,4‐sulfonyldianiline, tetrachloroethylene, and zearalenone. The assay was incapable of providing a clear indication of whether some chemicals were mutagens; these were benzyl alcohol, 1,4‐dichlorobenzene, phenol, succinic acid‐2,2‐dimethyl hydrazide, and toluene.
Eighteen chemicals were tested for their mutagenic potential in the L5178Y tk+/- mouse lymphoma cell forward mutation assay by the use of procedures based upon those described by Clive and Spector [Mutat Res 44:269-278, 1975] and Clive et al [Mutat Res 59:61-108, 1979]. Cultures were exposed to the chemicals for 4 hr, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT), 3 micrograms/ml. The chemicals were tested at least twice. Significant responses were obtained with benzofuran, benzyl chloride, bromodichloromethane, butylated hydroxytoluene, chlorendic acid, o-chlorobenzalmalonitrile, 1,2,3,4-diepoxybutane, dimethyl formamide, dimethyl hydrogen phosphite, furfural, glutaraldehyde, hydroquinone, 8-hydroxyquinoline, and resorcinol. Apart from bromodichloromethane, butylated hydroxytoluene and dimethyl hydrogen phosphite, rat liver S9 mix was not a requirement for the activity of any of these compounds. Chemicals not identified as mutagens were water, tert-butyl alcohol, pyridine, and witch hazel.
A new protocol for testing vapors and gases in the L5178Y mouse lymphoma assay is presented. Four chemicals, propylene, 1,2-propylene oxide, 1,3-butadiene, and vinylidene chloride, were tested for their mutagenic potential. Cultures were exposed to the chemicals, which were delivered as vapors or gases, for 4 hr, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT), 3 microgram/ml. Each chemical was tested at least twice. Significant responses were obtained with 1,2-propylene oxide and vinylidene chloride, but neither cytotoxicity nor mutagenicity was induced by 1,3-butadiene; propylene could not be classified as either mutagenic or non-mutagenic in the assay. Rat liver S9 mix was not a requirement for the mutagenic activity of 1,2-propylene oxide, whereas the liver preparation markedly enhanced both the cytotoxicity and mutagenicity of vinylidene chloride.
Nine substances were tested for their mutagenic potential in the L5178Y tk+/tk- mouse lymphoma cell forward mutation assay, by means of procedures based upon those described by Clive and Spector (Mutat Res 44:269-278, 1975) and Clive et al (Mutat Res 59:61-108, 1979). Cultures were exposed to the chemicals for 4 hr, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT), 3 micrograms/ml. The coded chemicals were tested at least twice. Significant responses were obtained with calcium chromate, 3-(chloromethyl)pyridine, 1,2-epoxybutane, monochloroacetic acid, dicyclohexylthiourea, 2,4-diaminophenol hydrochloride, and pentachloroanisole. Apart from pentachloroanisole, rat liver S9 mix was not a requirement for the clearly mutagenic activity of any of these compounds. Compounds not identified as mutagens were 3-amino-1,2,4-triazole and sucrose.
The purpose of this work was to define, in this laboratory, the conditions of most general utility for the metabolic activation of chemicals to mutagens in the mouse lymphoma L5178Y cell tk+tk- ----tk-tk- assay. The approach used was to optimize the generation of non-toxic concentrations of NADPH from NADP glucose-6-phosphate (G6P) and G6P dehydrogenase, then use that system to examine the effects of increasing concentrations of Aroclor-1254-induced Fischer 344 male rat liver post-mitochondrial supernatant fluid (S9) upon the mutagenicity of chemicals. The promutagens were 2-acetylaminofluorene, cyclophosphamide, dimethylnitrosamine, methanol, 3-methylcholanthrene and procarbazine. It was determined that: (i) cell population growth was reduced at NADPH concentrations of greater than or equal to 1 mM; (ii) to ensure a maximum conversion of NADP to NADPH the G6P/NADP ratio should be two or greater; (iii) excess G6P (2.5 mM) is not harmful to the cells; (iv) toxicity due to S9 was observed at 12.5 mg whole liver equivalents (WLE)/ml (i.e. 50 microliters of a 25% liver S9 preparation per ml); (v) concentrations of S9 between 2.5 and 7.5 mg WLE/ml appeared to be adequate for the activation of the six promutagens to demonstrably mutagenic products.
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