Aroclor-induced Rat Liver S9 Research Articles

Comprehensive interpretation of in vitro micronucleus test results for 292 chemicals: from hazard identification to risk assessment application

Risk assessments are increasingly reliant on information from in vitro assays. The in vitro micronucleus test (MNvit) is a genotoxicity test that detects chromosomal abnormalities, including chromosome breakage (clastogenicity) and/or whole chromosome loss (aneugenicity). In this study, MNvit datasets for 292 chemicals, generated by the US EPA’s ToxCast program, were evaluated using a decision tree-based pipeline for hazard identification. Chemicals were tested with 19 concentrations (n = 1) up to 200 µM, in the presence and absence of Aroclor 1254-induced rat liver S9. To identify clastogenic chemicals, %MN values at each concentration were compared to a distribution of batch-specific solvent controls; this was followed by cytotoxicity assessment and benchmark concentration (BMC) analyses. The approach classified 157 substances as positives, 25 as negatives, and 110 as inconclusive. Using the approach described in Bryce et al. (Environ Mol Mutagen 52:280–286, 2011), we identified 15 (5%) aneugens. IVIVE (in vitro to in vivo extrapolation) was employed to convert BMCs into administered equivalent doses (AEDs). Where possible, AEDs were compared to points of departure (PODs) for traditional genotoxicity endpoints; AEDs were generally lower than PODs based on in vivo endpoints. To facilitate interpretation of in vitro MN assay concentration–response data for risk assessment, exposure estimates were utilized to calculate bioactivity exposure ratio (BER) values. BERs for 50 clastogens and two aneugens had AEDs that approached exposure estimates (i.e., BER < 100); these chemicals might be considered priorities for additional testing. This work provides a framework for the use of high-throughput in vitro genotoxicity testing for priority setting and chemical risk assessment.

Mutagenic Activity of N-Nitrosodiethylamine in Cell Lines Expressing Human CYP2E1-Adequacy of Dimethylsulfoxide as Solvent.

Human CYP2E1 metabolizes many xenobiotics of low-molecular weight, thereby activating various promutagens/procarcinogens. In toxicological studies in vitro, dimethylsulfoxide (DMSO) is a common vehicle for organic compounds. However, it was observed to potently inhibit CYP2E1 activity. We were interested in whether it affects CYP2E1-dependent mutagenic responses. In this study, N-nitrosodiethylamine (NDEA), which is soluble in both water and DMSO, was used as a model promutagen. It induced Hprt gene mutations and micronuclei in a Chinese hamster V79-derived cell line expressing both human CYP2E1 and sulfotransferase (SULT) 1A1 (V79-hCYP2E1-hSULT1A1) even at low-micromolar concentrations, but was inactive in parental V79 cells. Mutagenicity of NDEA was also observed in a recombinant V79-hCYP2E1 cell line that expresses human CYP2E1 at a lower level. NDEA induced micronuclei in human L-02 hepatocytes which expressed CYP2E1 even more weakly. DMSO did not modify NDEA-induced gene mutations or micronuclei, up to 0.2% (v:v, the highest noncytotoxic concentration) in V79-hCYP2E1-hSULT1A1 cells. In parental V79-Mz cells, NDEA induced micronuclei with Aroclor 1254-induced rat liver S9 mix, and this effect was unaffected by DMSO up to 0.2%. However, it inhibited the effect of NDEA in L-02 (by 44%) and V79-hCYP2E1 cells (by 70%) at 0.2%, with the effects of NDEA remaining statistically significant. No effect of DMSO was observed on CYP2E1 protein expression in V79-hCYP2E1-hSULT1A1 or its mRNA transcripts in each cell line. We conclude that DMSO may not significantly affect CYP2E1-dependent mutagenic effects, at concentrations up to 0.2% in cells with relatively high CYP2E1 expression. Environ. Mol. Mutagen. 60:214-226, 2019. © 2018 Wiley Periodicals, Inc.

Utility of Rat Liver S9 Fractions to Study Skin-Sensitizing Prohaptens in a Modified KeratinoSens Assay

Prohaptens are chemicals, which may cause skin sensitization after being converted into electrophilic molecules by skin enzymes. Aroclor-induced rat liver S9 fractions represent the metabolic activation system most commonly used in in vitro toxicology. This system contains much higher enzyme activities compared with those reported in skin, but it may still serve as a surrogate system to study the potential of chemicals to act as prohaptens. To test this concept, the luciferase induction in KeratinoSens reporter cells treated with chemicals in presence and absence of S9 fractions was measured. Suspected prohaptens such as methyl isoeugenol, eugenol, or trans-anethole gave no, or only weak, ge ne induction in absence of S9 fractions, and a significantly enhanced luciferase induction in presence of S9, proving their prohapten status. Direct-acting haptens like 2,4-dinitrochlorobenzene or cinnamic aldehyde gave a reduced response in presence of S9. We evaluated whether this metabolic activation assay might be implemented in a tiered screening strategy to counter-screen negatives in the KeratinoSens assay to enhance sensitivity. To this aim, all chemicals classified negative were retested with this activation step. Among the 77 chemicals found as correct-negatives, 73 were also negative in presence of metabolic activation, thus this counterscreen would reduce specificity only slightly. However, this comprehensive screening showed that only a small fraction of the known skin sensitizers need activation by the S9 system. Therefore, the KeratinoSens-S9 assay appears useful for the in vitro evaluation of specific classes of potential prohaptens and to mechanistically rationalize their prohapten status.

Inhibition of cooked food-induced mutagenesis by dietary constituents: Comparison of two natural isothiocyanates

Sulforaphane(1-isothiocyanato-(4 R)-(methylsulphinyl)butane), a major constituent of broccoli (Brassica oleracea, var. italica) and a structurally related natural aliphatic isothiocyanate, sulforaphen (4-isothiocyanato-(1 R)-(methylsulphinyl)-1-( E)-butene), found in radish (Raphanus sativus L., Cruciferae) were investigated for their antimutagenic potential against different classes of cooked food mutagens (heterocyclic amines) in the Ames assay using Salmonella typhimurium TA98 and TA100 strains in the presence of Aroclor 1254-induced rat liver S9. Results of the in vitro antimutagenicity studies in the TA100 strain strongly suggest that both isothiocyanates were potent inhibitors of the mutagenicity induced by all the tested mutagens. Sulforaphen, possessing unsaturation in the alkyl chain of its structure, was, however, found to be 1.3–1.5 times more active than sulforaphane. These studies strongly warrant further investigations of sulforaphen for its potential as a chemopreventive agent.

Metabolic fate of the Ah receptor ligand 6-formylindolo[3,2- b]carbazole

The physiological role of the aryl hydrocarbon receptor (AhR), a member of the basic helix–loop–helix PER-ARNT-SIM (PAS) transcription factor family is not known. We have suggested that the AhR is involved in light signaling through binding of photoproducts with high AhR affinity. This suggestion is based on (i) the high AhR affinity of the tryptophan photoproduct formylindolo[3,2- b]carbazole (FICZ), (ii) the induction of rapid and transient expression of AhR-regulated genes by FICZ and by extracts of UV-irradiated tryptophan as well as (iii) the fact that light induces the AhR-regulated cytochrome P450s CYP1A1, CYP1B1 and CYP2S1. The transient mRNA expression caused by light and tryptophan photoproducts suggests that the biotransformation enzymes induced by AhR activation take part in a metabolic degradation of the natural AhR ligand. This study aimed at identifying the involvement of phase I and phase II enzymes in the metabolic degradation of FICZ. A cytochrome P450-dependent metabolism of FICZ giving rise to preferentially mono- and di-hydroxylated derivatives has earlier been reported. In the present study, rat and human hepatic S9 mixes were employed together with specific enzyme inhibitors and cofactors. Compared to the Aroclor-induced rat liver S9, the non-induced rat liver S9 and the human liver S9 caused a more complex metabolite profile of FICZ. The CYP1A1 enzyme was confirmed to be the most important enzyme for the first step in the metabolism. CYP1A2 was found to have overlapping specificity with CYP1A1 being able to form the same major metabolites although with different kinetics. CYP1B1 turned out to be preferentially involved in the further metabolism of dihydroxylated metabolites. Microsomal epoxide hydrolase, and as yet not identified forms of sulphotransferases and glucuronosyltransferases were also found to take part in the metabolic degradation of FICZ. Thus, tryptophan photoproducts fit into a model in which the ligand-activated AhR signaling is autoregulated by the induced metabolic enzymes.

Mutagenicity of steviol and its oxidative derivatives in Salmonella typhimurium TM677.

Stevioside is natural non-caloric sweetner isolated from Stevia rebaudiana BERTONI, which has been used as a non-caloric sugar substitute in Japan. Pezzuto et al. demonstrated that steviol shows a dose-dependent positive response in forward mutation assay using Salmonella typhimurium TM677 in the presence of metabolic activation system (Aroclor induced rat liver S9 fraction). Our studies were carried out to identify the genuine mutagenic active substance from among the eight steviol derivatives. Steviol indicate almost similar levels of mutagenicity under the presence of S9 mixture, as reported by Pezzuto et al. 15-Oxo-steviol was found to be mutagenic at the one tenth the level of steviol itself under the presence of S9 mixture. Interestingly, specific mutagenicity of the lactone derivative under the presence of S9 mixture was ten times lower than that of the lactone derivative without the addition of S9 mixture.

Genotoxicity evaluation of tetramethylbenzenes

The three tetramethyl isomers of benzene (prenitene, 1,2,3,4-; izodurene, 1,2,3,5-; and durene, 1,2,4,5-tetramethylbenzene) were studied using in vitro mutagenicity and in vivo genotoxicity tests. Potency of mutate induction by these solvents was evaluated in Salmonella typhimurium cells with, and without S9-mix made from Aroclor 1254-induced rat liver S9. The potency of induction of micronuclei (MN) and sister chromatid exchanges (SCEs) by solvents was evaluated in bone marrow of mice. Izodurene displayed mutagenic potency in strains TA97a, TA98 and TA100 only in the absence of the S9-mix. In MN tests, all three tetramethylbenzenes demonstrated no clastogenic activity on the bone marrow cells. All the tested solvents were active as genotoxic compounds in the SCE tests, demonstrating a dose–response relationships.

Lack of genotoxic effects of sucrose acetate isobutyrate (SAIB)

Sucrose acetate isobutyrate (SAIB) was tested for potential genotoxic activity in four different in vitro assay systems. Two independent trials of a Salmonella reverse mutation assay (using strains TA98, TA100, TA1535, TA1537 and TA1538) showed no increases in revertant frequencies at doses up to 10,000μg/plate which was non-toxic but exceeded the solubility limit. Similarly, no mutagenic response was observed at doses up to 1000μg/ml at the HGPRT locus in cultured CHO cells; SAIB was toxic and its solubility limit was exceeded at 50μg/ml. No clastogenic activity was detected in cultured CHO cells at concentrations up to 2000μg/ml. All three preceding in vitro tests were conducted both in the presence and absence of Aroclor 1254-induced rat liverS-9 metabolic activation systems. An unscheduled DNA synthesis assay also was performed using rat primary hepatocyte cultures with doses up to 1000μg/ml, and no DNA repair was detectable. Thus, SAIB was stringently tested at doses exceeding the solubility limit in culture medium and causing toxicity to CHO cells without obtaining any evidence for genotoxic activity as a mutagen, clastogen, or DNA-damaging agent.

Thalidomide: lack of mutagenic activity across phyla and genetic endpoints

The human and rabbit teratogen thalidomide has been tested for mutagenicity in a wide range of assays, ranging from bacterial gene mutation assays conducted in vitro to in vivo cytogenetic assays conducted using rabbits, and including a variety of human-derived tissues. Thalidomide was not mutagenic to 6 strains of Salmonella when tested both in the presence and absence of Aroclor-induced rat liver S9 mix. This inactivity was confirmed in strains TA98 and TA100 using a 1-h pre-incubation assay protocol with the same S9 mix (10% S9), and additionally, in strain TA98 using 3 concentrations of S9 (4%, 10% and 30% S9 in S9 mix). Thalidomide was not clastogenic either to cultured human lymphocytes (whole blood cultures, minus S9 mix) or to Chinese hamster ovary (CHO) cells treated in vitro. Further, no cytotoxicity was observed in purified human lymphocytes when exposed to thalidomide up to the limit of its solubility in the medium in the presence and absence of liver S9 from Aroclor-induced pregnant rabbit. The CHO assays were conducted without metabolic activation and in the presence of a variety of sources of auxiliary metabolic activation (PB/ βNP-induced rat liver S9 mix, pooled male and female human liver S9 mix, uninduced and Aroclor-induced pregnant rabbit liver S9 mix and foetal rabbit S9 mix). Thalidomide did not induce micronuclei in isolated human lymphocytes (minus S9 mix) and it was non-mutagenic to mouse lymphoma L5178Y TK +/− cells when tested to the limits of its solubility in the culture medium (±S9 mix). No indication of recombinogenic or clastogenic activity was observed for thalidomide when tested in Drosophila. In addition, it failed to induce chromosome aberrations in grasshopper neuroblasts when tested in the presence and absence of Aroclor-induced rat liver S9 mix. Some unusual chromosome morphologies were observed in the grasshopper cytogenetic preparations indicating a potential of thalidomide to interact with chromosomal proteins. However, this potential was not evident in the human lymphocyte micronucleus assay, and thalidomide was apparently not reactive to the proteins of the mouse skin, as it gave negative results in a mouse local lymph node assay for skin sensitizing agents. Thalidomide was inactive in bone marrow micronucleus assays conducted using males and females from two strains of mice, and female New Zealand white rabbits. It is concluded that thalidomide is neither a mutagen nor an aneugen. This conclusion is discussed within the context of the results of earlier mutagenicity studies, the recent claim that thalidomide may be a heritable germ cell mutagen to humans, and the current interest in thalidomide for the treatment of immune system-related diseases.

Diaminotoluenes induce intrachromosomal recombination and free radicals in Saccharomyces cerevisiae

The carcinogenicity of aniline-based aromatic amines is poorly reflected by their activity in short-term mutagenicity assays such as the Salmonella typhimurium reverse mutation (Ames) assay. More information about the mechanism of action of such carcinogens is needed. Here we report the effects on DEL recombination in Saccharomyces cerevisiae of the carcinogen 2,4-diaminotoluene and its structural isomer 2,6-diaminotoluene, which is reported to be non-carcinogenic. Both compounds are detected as equally mutagenic in the Salmonella assay. In the absence of any external metabolizing system both compounds were recombinagenic in the DEL assay, with the carcinogen being a more potent inducer of deletions than the non-carcinogen. In the presence of Aroclor-induced rat liver S9, however, the carcinogen 2,4-diaminotoluene became a 2-fold more potent inducer of deletions, and the non-carcinogen 2,6-diaminotoluene was rendered less toxic and no induced recombination was observed. 2,4-Diaminotoluene is distinguished from its non-carcinogen analog in the DEL assay, therefore, on the basis of a preferential activation of the carcinogen in the presence of a rat liver microsomal metabolizing system. Free radical species are produced by several carcinogens and have been implicated in carcinogenesis. We further investigated whether exposure of yeast to either 2,4-diaminotoluene or 2,6-diaminotoluene resulted in a rise in intracellular free radical species. The effects of the free radical scavenger N-acetylcysteine on toxicity and recombination induced by the two compounds and intracellular oxidation of the free radical-sensitive reporter compound dichlorofluorescin diacetate were studied. Both 2,4- and 2,6-diaminotoluene produced free radical species in yeast, indicating that the reason for the differential activity of the compounds for induced deletions is not reflected in any difference in the production of free radical species.

Metabolic Activation and Inactivation of Metanil Yellow and Orange II in the Salmonella typhimurium his− Reversion Assay

Among a variety of experimental protocols used, the combined use of 0.5% dextrose in bottom agar and 1 μmol of flavin mononucleotide (FMN) in preincubation mixtures without fraction S9 mix resulted in the highest level of induced Salmonella typhimurium his− reversions with both dyes metanil yellow and orange II with strain TA100. Strain TA98 yielded notably lower levels of reversions under the same conditions. The presence of uninduced hamster liver S9 fraction resulted in a weak mutagenic response while Aroclor 1254-induced rat liver S9 fraction resulted in the complete absence of mutagenicity with both strains and with both dyes.

Optimization of the Ames/Salmonella Mutagenicity Assay for Use with Extracts of Aquatic Sediments

Non-mutagenic components interfered with the ability of the standard Ames/salmonella assay to detect mutagenicity in extracts of contaminated Great Lakes sediments. The use of gel permeation chromatography (GPC) to remove these macromolecules from methylene chloride extracts prior to Ames testing enhanced the likelihood of transfer of mutagenic components into dimethyl sulf oxide (the assay solvent). Therefore, to optimize the assay's sensitivity we pre-treated sediment extracts using GPC and increased metabolic activity through the use of a 30% S9 mix. Increasing the level of Aroclor 1254-induced rat liver S9, typically used to metabolically activate promutagens, had the additional beneficial effect of reducing the cytotoxicity of the extracts. As applied in this study, the Ames assay can serve as a sensitive test for screening the mutagenic potential of large numbers of uncharacterized sediment extracts.

Cytotoxicity and mutagenicity of polycyclic aromatic hydrocarbon o-quinones produced by dihydrodiol dehydrogenase

Eight polycyclic aromatic hydrocarbon (PAH) o-quinones that can be generated by dihydrodiol dehydrogenase (DD) were examined for their cytotoxicity in H-4-II-e (rat hepatoma) cells and for their mutagenicity in the Ames test. Seven of the PAH o-quinones were potent cytotoxins yielding IC 50 values for cell survival in the range 1–30 μM. PAH o-quinones were grouped into three classes based on their cytotoxicity profiles: group I contained o-quinones (e.g., naphthalene-1,2-dione and 7,12-dimethylbenz[ a]anthracene-3,4-dione) which reduced cell viability and cell survival; group II contained o-quinones (e.g., benz[ a]-anthracene-3,4-dione and 5-methylchrysene-1,2-dione) which reduced cell survival but had no effect on cell viability; and group III contained o-quinones (e.g., benzo[ a]pyrene-7,8-dione) which had a pronounced effect on cell viability but minimal effects on cell survival. Using hepatoma cell suspensions and rat liver subcellular fractions, it was found that o-quinones underwent preferential enzymatic one-electron redox-cycling and produced Superoxide anion radical (O 2 ∸) and/or o-semiquinone anion or alternant radicals. o-Quinones that reduced cell viability produced O 2 ∸ and caused the most total free radical formation, while those that reduced cell survival produced o-semiquinone anion or alternant radicals only. PAH o-quinones were also tested as direct-acting mutagens in Salmonella typhimurium tester strains TA97a, TA98, TA100, TA102 and TA104. They were found to be more mutagenic than the test mutagens used for each tester strain, and were predominantly frameshift mutagens. The presence of an activating system (Aroclor-induced rat liver S9 plus NADPH) did not increase the mutagenicity of o-quinones in tester strains that are sensitive to oxidative mutagens (TA102 and TA104). These data suggest that PAH o-quinones produced by DD are cytotoxic and mutagenic by different mechanisms. The mechanism of cytotoxicity involves the formation of reactive oxygen species and/or o-semiquinone anion or alternant radicals. The mechanism of mutagenicity is independent of free radical formation and is related to the ability of PAH o-quinones to intercalate and covalently modify DNA.

Mutagenicity and mutation spectra of 2-acetylaminofluorene at frameshift and base-substitution alleles in four DNA repair backgrounds of Salmonella

We used colony probe hybridization procedures to determine the mutations in ∼ 600 revertants of the −1 frameshift allele hisD3052 and ∼ 200 revertants of the base-substitution allele hisG46 of Salmonella typhimurium induced by 2-acetylaminofluorene (2-AAF) in the presence of Aroclor-induced rat liver S9. 2-AAF was primarily a frameshift mutagen, exhibiting 5 times more frameshift than base-substitution activity. The only frameshift mutation 2-AAF induced at the hisD3052 allele was a hotspot (−2) deletion within the sequence CGCGCGCG. The addition of the pKM101 plasmid had a small effect on the mutagenic potency of 2-AAF at this allele in a uvr + background and no effect on the mutation spectra in either a uvr + or uvr − background. The small amount of base-substitution activity exhibited by 2-AAF at the hisG46 allele required the presence of both the pKM101 plasmid and the uvrB mutation. The base substitutions were G · C → T · A transversions (86%) and G · C → A · T transitions (14%), and 85% of the substitutions were at the second position of the CCC target of the hisG46 allele; the remainder were at the first position. We propose that the hotspot frameshift may be initiated by N-acetyl-2-aminofluorene adducts located at the C(8) position of any of the guanines except the first one in the CGCGCGCG hotspot sequence. The mutation might then result from correct incorporation of cytosine opposite the adducted guanine, followed by a 2-base slippage according to our recently proposed correct-incorporation/slippage model. The hotspot mutation may also result from a 2-AAF-induced B- to Z-DNA transition at the repeating GpC site as well as by the action of enzymes involved in DNA metabolism, such as DNA resolvases or topoisomerases, on DNA structures that have been distorted by 2-AAF adducts. The small amount of 2-AAF-induced base-substitution activity may be due to mispairing of adenine opposite the minor aminofluorene adduct at the C(8) position of guanine.

Mutagenic activity of newly synthesized sulfa drugs to Salmonella typhimurium

The mutagenic activity of seven newly synthesized sulfa drugs was studied in Salmonella typhimurium, using forward mutation to 8-azaguanine (8-AG) resistance and reversion mutation assays (Ames test) both in the absence and presence of Aroclor induced rat liver S9. In forward mutation assays, N 1-methylsulfanilamide, N 4-acetyl- N 1-methylsulfanilamide and N 4-acetyl- N 1-diethylsulfanilamide were mutagenic to S. typhimurium TM677 both in the presence and absence of metabolic activation while N 4-acetylsulfanilamide, N 1-diethylsulfanilamide and 4-nitro- N-2-pyridinylbenzenesulfonamide [2-( p-nitrobenzenesulfonamido)pyridine] were mutagenic only in the presence of metabolic activation. But 2-( N 4-acetylsulfanilamido)pyridine was mutagenic in neither the presence nor the absence of metabolic activation. However, none of the seven compounds had any mutagenic effect on S. typhimurium TA98 or TA100 in the absence or presence of metabolic activation, by the Ames test preincubation method. The relationship between the structure of the compounds and their mutagenic activity is also discussed.

Species Variation in the Genotoxicity of Batracylin

Batracylin (8-aminoisoindolo[1,2-b]quinazolin-1 2(10H)-one), an experimental chemotherapeutic agent, is a heterocyclic aryl amine. The presence of the free amino group suggests that this compound may be a carcinogen. Since many carcinogens or their product interact with DNA, the genotoxicity of batracylin was evaluated. The mutagenicity of batracylin was tested in Salmonella typhimurium (TA 98, TA 100, TA 102) with and without Aroclor-induced rat liver S9. Batracylin induced histidine revertants in all three strains with a higher number of mutants formed in the presence of S9. The maximum mutant incidence as well as the lowest concentration inducing a positive response was observed with TA 98 which detects frameshift mutations. Genotoxicity was further assessed by the induction of DNA repair in hepatocytes isolated from F-344 rats and two mouse strains differing in N-acetyltransferase activity. In hepatocytes from male F-344 rats, batracylin elicited DNA repair at concentrations of 5 × 10 −8 to 10 −6 M. Maximum repair was observed in both strains of mice at 5 × 10 −5 M batracylin, a concentration that was toxic to rat hepatocytes. Cells isolated from the rapid acetylator strain, C57BL/6J, tended to have higher net grain counts than observed with the slow acetylator (A/J) strain, although the differences were not statistically significant. These results demonstrate that batracylin is genotoxic in bacteria and mammalian cells. The species variation in the genotoxicity of batracylin is consistent with in vivo toxicity studies.

Mutagenicity of N, N'-dimethylurea and methylamine hydrochloride in the Ames Salmonella/microsome test: Absence of mutagenic response

Methyl isocyanate (MIC) in aqueous solution forms methylamine (MA) and N, N'-dimethylurea (DMU). MA in buffered system further converts into its salt form, methylamine hydrochloride (MAH). Therefore, MAH and DMU were evaluated for their mutagenic activity in the in vitro Ames Salmonella/ microsome mutagenicity test. The liquid preincubation protocol was followed, using tester strains TA98, TA100 and TA104 of Salmonella typhimurium, in the presence of 0, 5, 15 and 30% Aroclor 1254-induced rat liver S9 mixture. DMU and MAH did not induce a mutagenic response in any of the tester strains, both in the presence and in the absence of S9 mixture. The results therefore confirm that MIC in its native form or as its unknown metabolites is responsible for the mutagenic activity reported earlier by us in the his − tester strains TA100 and TA104 of Salmonella typhimurium (Mutation Res., 204 (1988) 123–129) and not due to its hydrolysis products, MA or DMU.

The genotoxic potential of linear alkylbenzene mixtures in a short-term test battery

Alkylate 215 (A-215), Alkylate 225 (A-225), and Alkylate 230 (A-230) are mixtures of C 10C 14 linear alkylbenzenes used as intermediates for the manufacture of detergents. These products were evaluated for genotoxic activity in the Ames bacterial mutagenesis assay (strains TA98, 100, 1535, and 1537), the CHO HGPRT mammalian cell forward gene mutation assay, and the in vivo rat bone marrow chromosome assay. The Ames and CHO HGPRT assays were conducted both with and without the addition of Aroclor-induced rat liver S9. The maximum concentrations evaluated were 10 mg/plate (A-215) and 3 mg/plate (A-225 and A-230) for the Ames test, and 1.5 mg/ml (A-215 and A-225) and 2.0 mg/ml (A-230) for the CHO HGPRT assay. In each case, the highest concentrations produced evidence of either toxicity or insolubility. The highest dose in the bone marrow cytogenetics assay was 12,700 mg/kg, a level which produced significant weight loss. The results of all tests were negative, indicating a lack of genotoxic activity as measured by the battery of tests used.

Mutagenic activity of a series of synthetic and naturally occurring heterocyclic amines in Salmonella

26 synthetic and naturally occurring heterocyclic amines were tested in the Salmonella/microsome assay (Ames test) using tester strains TA98 and TA100 in the presence of an Aroclor-induced rat-liver S9 fraction. 9 of the compounds were protein-pyrolysis products which had previously been shown to be mutagenic. Mutagenic potencies similar to previously reported values were demonstrated for these compounds with the exception that Trp-P-1 was only mutagenic in strain TA98 in our study, although it had previously been reported to be weakly mutagenic in strain TA100. 17 structurally diverse heterocyclic amines were synthesized and tested for mutagenicity. The structural diversity of these synthetic heterocyclic amines will enhance the sensitivity of future quantitative structure-activity relationship (QSAR) studies by demonstrating the structural characteristics essential for mutagenicity. The results of this study provide a large data base for the mutagenicity of this important class of compounds.

Ozone is mutagenic in Salmonella.

Ozone is a highly reactive gas that has been tested for genotoxicity in a number of systems. Induced genetic damage resulting from ozone treatment may not be readily observed because of the high toxicity of the chemical and difficulties in generating and administering controlled concentrations. The mutagenicity of ozone was investigated in Salmonella typhimurium using a plate test protocol designed for reactive vapours and gases. Ozone, at two to three consecutive doses, induced weak, albeit statistically significant, mutagenic responses in tester strain TA102 with and without Aroclor-induced rat liver S9 (lowest effective mean concentration of 0.019 ppm; 35 min total exposure). However, dose-related responses were not always obtained. No mutagenicity was detected in strains TA98, TA100, or TA1535, with or without S9. In strain TA104, ozone induced a weak response only at a single dose with S9; this response was not reproducible. Mutagenicity was dependent on the ozone flow rate and total exposure time, with variations in the optimum dose-time regimen leading to toxicity or complete inactivity. The data show that ozone is a very weak bacterial mutagen and only when tested under narrowly prescribed, subtoxic dosing conditions.