Differences in the induction of SCEs between human whole blood cultures and purified lymphocyte cultures and the effect of an S9 mix

Studies for SCE induction are frequently performed on human blood cultures. Either whole blood cultures (WBC) or purified lymphocyte cultures (PLC) are employed. However, it has been shown that fundamental differences with respect to metabolic activity exist between these two systems. In order to further characterize the whole blood culture and the purified lymphocyte culture, differently acting substances were studied comparatively with and without an Aroclor-1254-induced S9 mix. Treatment with ethyl methanesulfonate (EMS), a direct mutagen, produced distinct SCE induction in both systems. Cyclophosphamide (CP) and benzo[a]pyrene (BP), two indirect mutagens, also led to a significant increase of SCEs both in WBC and PLC without S9 mix. Only with CP was this effect more pronounced after addition of S9 mix. Sodium selenite (Na2SeO3), which induced SCEs in WBC, did not show this effect in the PLC. After S9 mix was added to purified lymphocytes, an increase of SCEs by sodium selenite was observed as in WBC. H2O2, a radical former, led to SCE induction in purified lymphocytes but not in the whole blood culture. By adding S9 mix, a distinct reduction of the SCEs induced by H2O2 was established. These results show that human lymphocytes can metabolize indirect mutagens and that it should be kept in mind when using S9 mix that, besides mixed-function oxygenases, it also contains enzymes which influence the SCE-inducing effects of substances.     

Effect of tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate on induction of sister-chromatid exchanges in Chinese hamster V79 cells treated with mutagens

The effect of a tumor promoter, 12-O-tetradecanoyl-phorbol 13-acetate (TPA) alone and in combination with mitomycin C (MMC) or cyclophosphamide (CPP) on the induction of sister-chromatid exchanges (SCE) in Chinese hamster V79 cells was investigated. TPA alone at various doses and durations caused no increase of SCE frequency. MMC either at the dose of 0.03 or 0.003 μg/ml alone or in combination with TPA (2 μg/ml) all caused a significant increase of SCE frequencies. There was no difference in SCE frequencies between the cultures treated with MMC alone at 0.03 μg/ml and those treated with MMC plus TPA. However, cultures treated with MMC at 0.003 μg/ml plus TPA had significantly and consistently higher SCE frequencies than those treated with MMC alone at all durations. Treatment of CPP at 1 μg/ml activated by S9 mix caused significant increase of SCE frequencies. Surprisingly, the cultures treated with CPP, S9 mix plus TPA (2 μg/ml) caused a drastic reduction of SCE frequencies as compared to those treated with CPP and S9 mix only at all durations. These results indicate that TPA alone had no effect on SCE in V79 cells. TPA enhanced the SCE induction in V79 cells treated with MMC at a low dose, i.e. 0.003 μg/ml, but it inhibited SCE induction in cultures treated with the indirect mutagen CPP. Thus, TPA has no direct effect on genetic materials but it may indirectly alter the effects of a mutagen.     

In vitro genotoxic perspective of Tamiflu

The aim of this study was to investigate the genotoxic and/or cytotoxic effects of Tamiflu, commercial form of the oseltamivir antiviral and most frequently prescribed for the treatment of influenza infections, on cultured human peripheral lymphocytes by using sister chromatid exchange (SCE), chromosomal aberration (CA), and cytokinesis-blocked micronucleus (CBMN) assays. Cells were treated with 0.5, 1, 2 μg/mL oseltamivir, the Tamiflu capsule ingredient, for 24 or 48 h in the absence or presence of an exogenous metabolic activation system (S9 mix). The test chemical did not demonstrate any genotoxic effect dose-dependently but it showed a weak cytotoxicity on cells in this study. On the other hand, some concentrations of Tamiflu (2 μg/mL without S9 mix for 48 h and 1 μg/mL with S9 mix) induced SCE and also decreased significantly the proliferation index (PI) (48 h period) and the nuclear division index (NDI) (24 h period) (P < 0.05) in the absence of S9 mix. Considering the results, Tamiflu did not induce significant increases of CA or micronucleated cells in vitro in cultured peripheral blood lymphocytes under the treatment conditions used but weak SCE induction was observed. On the other hand, the weak cytotoxic effects observed disappeared in the cultures treated in presence of the S9 mix.     

Author index

A technique using human lymphocytes together with an Ames-type microsomal (S9) activation system for testig indirect chemical mutagens has been developed and examined. The cytotoxic drug cyclophosphamide (CP), which only displays mutagenic properties after metabolic activation, was used as a test chemical and mutagenicity assessed in terms of sister-chromatid exchange (SCE) induction. Direct exposure of lymphocytes to CP and S9 mix produced increases in the yield of SCE for CP concentrations down to 10^?6 M. Exposure times of 30, 60 and 120 min commencing at the beginning of cell culture or after 48 h gave different ranges of detection and response with later treatment being more effective for SCE induction. Variations in relative proportions of the S9 mix culture medium constituents affected the lymphocytes' tolerance of toxic factors and modifiec the mutagen's effect. CP pre-incubated with S9 mix for 1 h before adding to the lymphocyte cultures also produced increases in SCE levels but the method was complicated and did not reduce toxicity. Direct addition of CP and S9 mix to the lymphocytes without prior pre-incubation showed maximum sensitivity, minimum toxocity and was a simpler, more reliable technique.     

Hypersensitive character of Bloom syndrome B-lymphoblastoid cell lines usable for sensitive carcinogen detection

Various carcinogens were tested with regard to the induction of sister-chromatid exchanges (SCEs) and chromosome aberrations using 3 types of Bloom syndrome (BS) B-lymphoblastoid cell lines (LCLs) (type I with normal frequency of SCEs and normal karyotype; type II with high frequency of SCEs and normal karyotype; type III with high frequency of SCEs and abnormal karyotypes) in the presence and absence of S9 mix. Three types of BS B-LCLs and normal cells showed different responses to the various carcinogens in the level of SCE induction. BS type I cells had the same SCE response as normal cells to carcinogens. Some carcinogens that require metabolic activation (S9 mix) had little effect on type II cells without S9 mix but had high SCE levels with S9 mix. BS type III cells were highly susceptible to both direct and indirect carcinogens with respect to high SCE increase without S9 mix (ca. 140 SCEs/cell), though some carcinogens produced SCEs rated in the medium (ca. 120 SCEs/cell) range, and had a high rate (more than 10%) of centromere spreading (CS), in addition to quadriradials. Therefore BS type III is a unique cell line which can be used to detect carcinogens.     

Tea tannin components modify the induction of sister-chromatid exchanges and chromosome aberrations in mutagen-treated cultured mammalian cells and mice

The modifying effects of tannin components extracted from green tea and black tea on mutagen-induced SCEs and chromosome aberrations were studied. These tannin components did not affect spontaneous SCEs and chromosome aberrations in cultured Chinese hamster cells. The frequency of SCEs and chromosome aberrations induced by mitomycin C (MMC) or UV was enhanced by the posttreatment with tea tannin components. When cells were post-treated with tea tannin components in the presence of metabolic enzymes of rat liver (S9 mix), the modifying effects on the induction of SCEs and chromosome aberrations by mutagens were complicated. MMC- and UV-induced SCEs and chromosome aberrations were suppressed by the posttreatment with tea tannin components at low concentrations (less than or equal to 6.7 micrograms/ml) with S9 mix. At a high concentration of tea tannin components (20 micrograms/ml) with S9 mix, a co-mutagenic effect was observed. The modifying effects of tea tannin components were shown to occur in the G1 phase of the cell cycle. In cells from a patient with xeroderma pigmentosum (XP) and a normal human embryo, MMC-induced SCEs were suppressed by the posttreatment with tea tannin components in the presence of S9 mix, and enhanced in the absence of S9 mix. On the other hand, tea tannin components modified SCE frequencies in UV-irradiated normal human cells but not in UV-irradiated XP cells. Our results suggested that tea tannin components themselves inhibited DNA-excision repair and resulted in a co-mutagenic effect, while in the presence of S9 mix metabolites of tea tannin components promoted DNA-excision repair activity and resulted in an antimutagenic effect. MMC-induced chromosome aberrations in mouse bone marrow cells were suppressed by the pretreatment with green tea and black tea tannin mixture.     

A comparison of alternative distributions of postimplantation death in the dominant lethal assay

The action of beta-aminoethylisothiouronium bromide hydrobromide (AET) and sodium fluoride (NaF) on the clastogenic activity of Trenimon, cyclophosphamide, and bleomycin was tested on cultures of human peripheral lymphocytes with and without the addition of rat liver S9 mix. In addition, the influence of both anticlastogens on the SCE-inducing activity of Trenimon and cyclophosphamide was examined under the same conditions. In the absence of S9 mix both substances displayed the known anticlastogenic action when TR was the standard clastogen but acted coclastogenically in the experiments with BM. Under the influence of rat-liver S9 mix this action on TR-induced chromosome damage was decreased and only a slight anticlastogenic effect was observed in the experiments with activated cyclophosphamide. S9-activated BM lost some of its strong chromosome-damaging effect and AET proved clearly anticlastogenic under these test conditions. AET displayed a slight decreasing effect on SCE induced by TR, but had no effect on CP-induced SCE. No anti-SCE effect at all was found in the experiments with NaF. Detailed analyses revealed different actions of both anticlastogens on the different types of structural chromosome damage.     

Induction of sister-chromatid exchanges in human lymphocytes and Chinese hamster cells exposed to aflatoxin B1 and N-methyl-N-nitrosourea.

The effect of a 1-h exposure to aflatoxin B1 (AFB1) in inducing sister-chromatid exchange in Chinese hamster ovary (CHO) cells and human lymphocytes in the presence or absence of mixed function oxidase ("S9 mix") was compared. CHO cells were also exposed to a graded series of doses of N-methyl-N-nitrosourea, a powerful inducer of SCE whose action was independent of the presence or absence of S9 mix. CHO and human cells showed a close correlation in response to SCE induction by AFB1 and in both cell systems the additon of mixed function oxidases in the S9 mix resulted in a marked enhancement of action of AFB1.     

Genotoxic effects of fluoride evaluated by sister-chromatid exchange

The purpose of this investigation was to study the genotoxic potential of fluoride (in the form of sodium fluoride, NaF) using in vitro and in vivo sister-chromatid exchange (SCE) assays with Chinese hamster cells. The NaF concentrations used in cultures of Chinese hamster ovary (CHO) cells ranged from 0 to 6.3 mM, both with and without S9 activation. Fluoride analysis of the culture medium demonstrated that it contained little indigenous fluoride, and the concentration of added fluoride was not affected by the components of the medium or the S9 mix. The CHO cells cultured in 6.3 mM NaF almost vanished, and at the concentration of 5.3 mM NaF in cultures without S9 microsome, only M1 cells were observed. In in vivo studies, Chinese hamsters were intubated with NaF dosages of 0, 0.1, 1.0, 10, 60 and 130 mg/kg, and the bone marrow (CHBM) cells were examined for SCE frequencies. Bone fluoride data showed that the intubated NaF was effectively absorbed. Death occurred in 3 of the 8 animals given 130 mg NaF/kg. The results indicated that NaF, in dosages up to 5.3 mM in CHO cell cultures and 130 mg/kg in in vivo CHBM cells, did not significantly increase the SCE frequencies over those observed in the negative (distilled water) controls. However, examination of the cell cycle revealed an inhibitory effect of NaF on cell proliferation with doses of NaF at or greater than 1.0 mM in cultured CHO cells and at or greater than 60 mg NaF/kg in in vivo CHMB cells. The results of the present study indicated an inhibition of the cell cycle and death of the cells with increasing concentrations of fluoride but not effect of fluoride on SCE frequency in CHO and CHBM cells.     

Genotoxic effects of chemicals in the single cell gel (SCG) test with human blood cells in relation to the induction of sister-chromatid exchanges (SCE)

In a comparative study, henzo[a]pyrene (BaP), cyclophosphamide (CP), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and tetrachloroethylene (PER) were tested for their ability to induce genotoxic effects in the single cell gel (SCG) test and the sister-chromatid exchange (SCE) test with human blood cells. MNNG as well as S9 mix activated BaP- and CP-induced DNA effects in both tests in a dose-dependent manner. While the range of concentrations which induced DNA migration or SCE was the same for MNNG and for Bap, much higher CP concentrations were necessary for a positive response in the SCG test than in the SCE test. PER was tested in the absence and in the presence of S9 mix and neither induced DNA migration nor increased SCE frequencies. In these experiments, a clear cytotoxic effect of PER was observed. To investigate a possible influence of DNA repair on the effects in the SCG test, cells were treated for 2 h and further incubated for 1 h after removal of the test substance. This procedure caused a clear decrease in induced DNA migration in experiments with Bap and CP, whereas no reduction was found with MNNG. This modified protocol did not lead to the detection of DNA effects after treatment with PER. The results indicate that the SCG test responds to various DNA lesions and does not seem to be sensitive to non-genotoxic cell killing. Its sensitivity obviously depends on the type(s) of induced DNA lesions and the effects can be modified by DNA repair processes in a complex manner. For the detection of genotoxic properties of chemicals with the in vitro SCG test, a single evaluation at the end of the exposure period seems to be sufficient.     

Induction of sister chromatid exchanges and chromosome aberrations in cultured mammalian cells treated with aminophenylnorharman formed by norharman with aniline

Aminophenylnorharman (APNH) is a newly identified mutagenic heterocyclic amine formed by coupling of norharman with aniline in the presence of S9 mix. Furthermore, mutagenic amino-3'-methylphenylnorharman (AMPNH) and aminophenylharman (APH) have been identified from a reaction mixture of norharman and o-toluidine and that of harman and aniline, respectively, with S9 mix. Among these three heterocyclic amines, APNH shows most potent mutagenic activity towards Salmonella typhimurium TA98 and YG1024 with S9 mix. In the present study, the induction of sister chromatid exchanges (SCEs) by APNH was examined in Chinese hamster lung (CHL) cells in vitro, comparing it to those of AMPNH and APH. On incubation with rat S9 for 6h, followed by a recovery culture period of 18h, a dose-dependent effect was found at concentrations between 0.00125 and 0.01 microg/ml for APNH and between 0.3125 and 5 microg/ml for AMPNH and APH. The approximate chemical concentrations leading to a three-fold of control SCE levels calculated from slopes of the linear regressions of induced SCEs were 0.005 for APNH, 0.51 for AMPNH and 1.7 microg/ml for APH. Because of the very strong SCE-causing ability of APNH, we further explored its genotoxicity by examining the induction of chromosome aberrations in CHL cells. A dose-dependent effect was found for chromosome aberrations at concentrations between 0.00125 and 0.04 microg/ml of APNH. The aberrations observed were primarily chromatid exchanges (cte) and breaks (ctb). In conclusion, the potency of SCE induction and clastogenic activity induced by APNH is stronger than Actinomycin D, Mitomycin C (MMC) or 1,8-dinitropyrene which are considered to be the potent clastogens in the literature. Further studies are needed for elucidating mechanisms of the genotoxic actions of these compounds and for evaluating their potential hazards to human health.     

Induction of sister-chromatid exchanges in cultured human lymphocytes by Aldicarb, a carbamate pesticide

The aim of this work was to investigate the effects of Aldicarb on human lymphocytes in vitro in the presence of an exogenous metabolic activation system. This was done by means of an analysis of SCE and mitotic delay. CP was used to compare the chromosomal effects of Aldicarb with a known genotoxic agent. Our experiments showed that Aldicarb as well as CP induced a significant increase of SCE values in the absence of S9 mix. In vitro metabolic activation of both chemicals increased the SCE values. The addition of a metabolic system slightly decreased the successive mitotic progression of cells in culture.     

Cytogenetic effects of penicillamine

Penicillamine (PA), a drug used for the treatment of rheumatoid arthritis induces sister-chromatid exchanges (SCEs) and chromosome aberrations in cultivated mammalian cells. PA in concentrations from 400 micrograms/ml upward induced SCEs and proliferative delay in human blood cultures when added for the last 24 h of the culture period. In V79 Chinese hamster cells SCE induction was found after acute exposure to PA before the addition of BrdUrd and after chronic exposure during one cell cycle in the presence of BrdUrd. The effect of PA on SCE frequencies occurred both after treatment in complete medium and in serum-free medium and was not influenced by the application of an S9 mix. The simultaneous addition of peroxidase reduced the PA-induced SCEs whereas catalase did not show any effect. Chromosome analysis in the first mitosis after PA treatment revealed a significant increase in the incidence of chromosome aberrations and endoreduplication. The results are discussed with respect to the cause and the significance of the observed effects in connection with mutagenicity testing.     

Structures of mutagens produced by the co-mutagen norharman with o- and m-toluidine isomers

Norharman, abundantly present in cigarette smoke and cooked foods, is not mutagenic to Salmonella typhimurium strains. However, norharman shows mutagenicity to S. typhimurium TA98 and YG1024 in the presence of S9 mix when coexisting with aromatic amines, including aniline, o- and m-toluidines. We previously reported that the mutagenicity from norharman and aniline in the presence of S9 mix was due to the formation of a mutagenic compound, 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman). In the present study, we analyzed the mutagens produced by norharman with o- or m-toluidine in the presence of S9 mix. When norharman and o-toluidine were reacted at 37 degrees C for 20 min, two mutagenic compounds, which were mutagenic with and without S9 mix, respectively, were produced, and these were isolated by HPLC. The former mutagen was deduced to be 9-(4'-amino-3'-methylphenyl)-9H-pyrido[3,4-b]indole (amino-3'-methylphenylnorharman) on the basis of various spectral data, and this new heterocyclic amine was confirmed by its chemical synthesis. The latter mutagen was identified to be the hydroxyamino derivative. Amino-3'-methylphenylnorharman induced 41,000 revertants of TA98, and 698,000 revertants of YG1024 per microg with S9 mix. Formation of the same DNA adducts was observed in YG1024 when amino-3'-methylphenylnorharman or a mixture of norharman plus o-toluidine was incubated with S9 mix. These observations suggest that norharman reacts with o-toluidine in the presence of S9 mix to produce amino-3'-methylphenylnorharman, and this compound is metabolically activated to yield its hydroxyamino derivative. After activation by O-acetyltransferase, it might bind to DNA and exert mutagenicity in S. typhimurium TA98 and YG1024. When norharman and m-toluidine were reacted in the presence of S9 mix, 9-(4'-amino-2'-methylphenyl)-9H-pyrido[3,4-b]indole (amino-2'-methylphenylnorharman) was identified as a mutagen. Thus, the mutagenicity of norharman with m-toluidine may follow a mechanism similar to that with o-toluidine.     

Mutagenicity testing of seminal fluid: seminal fluid increases the mutagenicity of the precursor mutagen benzo[a]pyrene in the presence of S9 mix

Small amounts of seminal fluid strongly enhanced the mutagenicity of the precursor mutagen benzo[a]pyrene (BP) in the Salmonella/microsome test. This previously unreported effect was found only in the presence of S9 mix for metabolic activation. The increase far exceeded the additive effect expected from experiments where seminal fluid and BP were tested separately with S9 mix. Testing of the direct-acting mutagen 4-nitro-o-phenylene-diamine (NPD) together with seminal fluid resulted in a lower mutagenic activity than that of NPD alone. Seminal fluid had a bactericidal effect on the Salmonella bacteria, thus only volumes up to 40 microliter could be used per plate. The mutagenic effect of only seminal fluid and S9 mix was slightly increased over controls in a standard Ames test, but was equal to the spontaneous mutation rate with a preincubation test modified according to Kado and coworkers. There were no significant differences between seminal plasma from smokers and non-smokers in any experimental series. Seminal fluid concentrated 20-fold by extraction with the mutagen-removing adsorbant Mutasorb did not have any enhancing effect on the mutagenicity of BP, nor did it exhibit any mutagenic activity in itself with or without S9 mix.     

Cytogenetic studies on 1,1-dichloroethylene and its two isomers in mammalian cells in vitro and in vivo

Chromosomal aberration and sister-chromatid exchange (SCE) tests in vitro on 1,1-dichloroethylene (1,1-DCE), its two isomers, cis- and trans-1,2-DCE, and two possible metabolites of 1,1-DCE, chloroacetyl chloride and chloroacetic acid, were carried out using a Chinese hamster cell line, CHL. 1,1-DCE induced chromosomal aberrations in the presence of S9 mix prepared from the rat liver, but not in the absence of S9 mix. SCEs were also slightly induced by 1,1-DCE only in the presence of S9 mix. On the other hand, two isomers and two metabolites of 1,1-DCE induced neither chromosomal aberrations nor SCEs with and without S9 mix. 1,1-DCE, however, was negative even at a sublethal dose in the micronucleus test using mouse bone marrow, fetal liver and blood.     

Studies by means of the SCE assay in V79-E Chinese hamster cells on the mode of action of tri-substituted nitrosoureas

The genotoxic activity of 3,3-diethyl-1-methyl-1-nitrosourea ( DEMNU ), 1,3-dimethyl-3-phenyl-1-nitrosourea ( DMPNU ) and 1-chloroethyl-3-methyl-3-phenyl-1-nitrosourea ( CEMPNU ) was studied in the SCE assay in V79-E cells in vitro. These compounds are very stable in aqueous solutions, but are directly acting genotoxins . The SCE rates increase linearly with the length of the incubation period. This direct activity is presumably due to an intracellular catalytic decomposition. Whereas the SCE-inducing effect of DMPNU and CEMPNU is not influenced by addition of S9 mix, that of DEMNU is strongly potentiated by rat and Syrian hamster S9 mix. This DEMNU activation is an NADPH-dependent enzymatic reaction and is inducible by phenobarbital. The absence of a direct mutagenic effect of DEMNU in the Ames test, as reported by other authors, is probably caused by a striking insensitivity to tri-substituted nitrosoureas of the Salmonella assay. This assumption was substantiated by long-term application of very low DMPNU doses to V79-E. Long-term simultaneous treatment with DMPNU and bromodeoxyuridine (BUdR) significantly diminished the rate of SCE induction.     

Retinol (vitamin A) inhibition of dimethylnitrosamine (DMN) and diethylnitrosamine (DEN) induced sister-chromatid exchanges in V79 cells and mutations in Salmonella/microsome assays

When the Chinese hamster cell line V79 and the tester strain of Salmonella typhimurium TA100 were treated with the precarcinogens dimethylnitrosamine (DMN) or diethylnitrosamine (DEN) in the presence of S9 mix, a dose-dependent increase of sister-chromatid exchanges (SCE) in V79 cells and His+ revertants in TA100 resulted. DMN was a far more efficient SCE inducer than DEN, while DEN was a more efficient inducer of His+ revertants than DMN. Retinol (Rol) effectively inhibited DMN and DEN induced SCE in V79 cells and His+ revertants in TA100. Concurrent treatment of V79 cells with Rol at various doses and one dose of DMN or DEN in the presence of S9 mix caused a significant reduction of SCE as compared to SCE induced by DMN or DEN without Rol. Rol inhibition of DMN-induced SCE was dose-dependent. Rol was less efficient in inhibiting DEN-induced SCE, and no consistent dose-dependent inhibition was observed. At all doses, Rol significantly inhibited DMN and DEN induced mutation frequencies in TA100. At the highest dose of Rol (40 micrograms/plate), the inhibition of DMN and DEN induced His+ revertants reached about 90% and 60%, respectively. The possibility that Rol exerts its antimutagenic activities by inhibiting certain forms of the cytochrome P-450 isoenzymes required for activation of precarcinogens such as DMN and DEN is discussed.     

On the genotoxicity of the pesticides Endodan and Kilacar in 6 different test systems

Two pesticides, the fungicide Endodan (ethylene thiuram monosulphide) and the insecticide-acaricide Kilacar (bis(parachlorophenyl)cyclopropyl methanol), produced or used in the neighbouring countries of Bulgaria and Greece were investigated in a coordinated research programme for their genotoxic effects in a variety of test systems. This included the Ames test, Aspergillus nidulans for mitotic segregation, in vitro human lymphocyte cell cultures for SCE and chromosomal aberrations, in vivo bone marrow cells in hamsters and rats and the dominant lethal test in rats. The genotoxicity of Endodan was found to range from negative to slightly positive in different test systems. At concentrations of 7.5 and 12.0 micrograms/plate together with S9 mix it induced base-pair substitutions in the TA100 strain of Salmonella typhimurium at a rather low level. At a dose of 93 mg/kg b.w. it also caused chromosomal aberrations in acutely treated hamster bone marrow cells. A significant increase of SCE was also found in human lymphocyte cultures at a concentration of 20.0 micrograms/ml. Endodan was found to be negative in A. nidulans for somatic segregation, lymphocyte cultures for chromosomal aberrations and mitotic activity and in rats for dominant lethals and chromosomal aberrations. Kilacar was found to be a weak mutagen in the TA97 strain of S. typhimurium at concentrations of 2.5 and 5.0 micrograms/plate together with S9 mix. At concentrations of 1.0, 1.5 and 2 micrograms/ml Kilacar increased the number of mitotic segregants in A. nidulans by 160%, 220% and 156% respectively over the control. In Syrian hamster bone marrow cells after acute administration at concentrations of 0, 40, 80 and 160 mg/kg, the MI was 5.50, 4.30, 3.10 and 1.30 respectively, and an increase in chromosomal aberrations of about 300% over the control was observed with a concentration of 80 mg/kg. In human lymphocytes no significant changes were observed in either MI or SCE. In the dominant lethal test after chronic treatment of male rats at doses of 5.1, 10.2 and 102.0 mg/kg b.w. no significant mutagenic effect was found although a decrease was shown in the percentage of females with implants mated with treated males in the first week.     

Mutagenicity of rat-liver S9 to L5178Y mouse lymphoma cells

Rat-liver S9 preparations became highly mutagenic to cultured L5178Y mouse lymphoma cells when the exposure period was increased to 18-24 h or when S9 mix was preincubated in Fischer's medium at 37 degrees C for 19 h and then used to treat the cells for 4 h. Five different S9 preparations (from untreated and Aroclor 1254-treated Fischer 344 or Sprague-Dawley male rats) behaved similarly. S9 mix, which contained 1 mM NADP and 5 mM isocitrate as cofactors, was more mutagenic than S9 alone. Heat treatment of S9 did not destroy its mutagenic activity, but the addition of cofactors no longer stimulated an increase in mutagenicity, as observed with native S9. Treatment with cofactors was not mutagenic. These results implied the involvement of both energy-independent and NADPH-dependent enzymatic changes in S9 mix in producing mutagenic substances. The mutagenic treatments with S9 or S9 mix induced predominantly small TFT-resistant mutant colonies, which suggested that these treatments should be clastogenic to cultured mammalian cells. A warning was given that test chemicals evaluated as mutagenic only in the presence of S9 mix may instead be accelerating the decomposition of S9 mix into mutagens, and it may become necessary to experimentally distinguish between these two mechanisms before a chemical can be regarded as mutagenic.