Genotoxicity hazard assessment of Caramel Colours III and IV.

Results from a battery of short-term tests in vitro and in vivo used to assess the genotoxicity of caramel colours are presented and discussed in relation to reports from the literature. No evidence of genotoxicity was found in the Salmonella plate incorporation test using five standard strains or in the Saccharomyces cerevisiae gene conversion assay using strain D4, either with or without S-9 for activation. A weak clastogenic effect for a sample of Caramel Colour III in CHO cells was abolished in the presence of S-9. Two samples of Caramel Colour IV were not clastogenic in CHO cells. Salmonella pre-incubation tests without S-9 also failed to reveal any mutagenic activity for any of the caramel colours tested. The Caramel Colour III sample that showed clastogenic activity in CHO cells in vitro did not induce micronuclei when evaluated in a mouse bone marrow assay. These results are in general agreement with reports in the literature regarding the genotoxicity of caramel colours, and support the conclusion that caramel colours do not pose a genotoxic hazard to humans.     

Genetic toxicology studies with glutaraldehyde.

Glutaraldehyde (GA; CAS no. 111-30-8) has a wide spectrum of industrial, scientific and biomedical applications, with a potential for human exposure particularly in its biocidal applications. The likelihood for genotoxic effects was investigated in vitro and in vivo. A Salmonella typhimurium reverse mutation assay showed no evidence for mutagenic activity with strains TA98, TA1535, TA1537 and TA1538, with or without metabolic activation. However, there was a weak mutagenic response (1.9-2.3-fold at the highest non-toxic concentration) with TA100 in the presence of metabolic activation. In a Chinese hamster ovary (CHO) forward gene mutation assay (HGPRT locus) there were no consistent, statistically significant, reproducible or dosage-related increases in the frequency of 6-thioguanine resistant cells. There were no reproducible or dosage-related increases in sister chromatid exchanges in an in vitro test in CHO cells. An in vitro cytogenetics study in CHO cells showed no evidence for an increase in chromosomal aberrations on treatment with GA, either in the presence or absence of metabolic activation. In vivo, a mouse peripheral blood micronucleus test showed no increase in micronucleated polychromatophils at sampling times of 30, 48 and 72 h after acute gavage dosing with GA at 40, 80 and 125 mg kg(-1) (corresponding to 25, 50 and 85% of the LD(50)). The absence of an in vivo clastogenic potential was confirmed by no increase in chromosomal aberrations in a rat bone marrow cytogenetics study with sampling at 12, 24 and 48 h after acute gavage dosing with GA (12.5, 30 or 60 mg kg(-1) with males, and 7.5, 20 or 40 mg kg(-1) with females). Thus, in this series of tests, GA produced genotoxic effects in vitro only in a bacterial reverse mutation assay with no evidence for in vivo genotoxicity.     

Studies on the acute toxicity primary irritancy and genotoxic potential of 1,3,5-triacryloylhexahydro-s-triazine (TAHT)

TAHT (1,3,5-triacryloylhexahydro-s-triazine), a reactive chemical coupling agent, was highly toxic following a single peroral dose of an aqueous suspension (10% w/v) to Wistar rats, or following application of TAHT in dichloromethane (DCM) solution (10% w/v) to covered skin of New Zealand rabbits. It was moderately toxic when applied dermally as an aqueous paste. Ocular contact with 25 mg of TAHT in a 5% aqueous suspension, or of 0.5 mg of TAHT in a 10% (w/v) solution in DCM, produced severe corneal damage, iritis and blepharo-conjunctivitis. A 30-min exposure of uncovered rabbit skin to 1 mg of TAHT in a 10% (w/v) aqueous suspension produced only slight skin irritation. However, 24-h exposures to TAHT on covered skin produced erythema, edema, ecchymoses, scabs, and death depending upon dosage and vehicle. In vitro genotoxicity studies revealed no positive effects upon gene mutations (HGPRT locus) or on sister chromatid exchanges (SCEs) of CHO cells exposed to TAHT with and without a rat-liver S9 metabolic activation system. TAHT did not increase the levels of [3H]thymidine incorporation in a test for unscheduled DNA synthesis with primary rat hepatocytes. In contrast, substantial increases in the number of chromosome breaks and rearrangements were observed in chromosome preparations used for the SCE analyses. The clastogenic activity of TAHT was confirmed in an in vitro chromosome aberration test with CHO cells. Treatment-related increases in chromosome breakage were observed at two independent sampling times and positive effects did not depend upon the presence or absence of a metabolic activation system. Clastogenic activity of TAHT was also demonstrated in vivo in a micronucleus test using mouse peripheral polychromatic erythrocytes. Significant, treatment-related increases in micronucleated polychromatic erythrocytes were obtained at two of three sampling times. The high degree of mammalian toxicity, severe eye irritancy and the in vitro and in vivo clastogenicity indicate that TAHT should be handled as a hazardous material using suitable caution and protective equipment.     

Application of simplified in vitro screening tests to detect genotoxicity of aristolochic acid

Aristolochic acid (AA), the active compound found in Aristolochia extracts, has been used as a traditional medicine. However, products containing AA were withdrawn from the market in the early 1980s because AA was found to be a potent carcinogen. Some genotoxicity studies of AA were conducted after the carcinogenicity of AA was reported. The purpose of this study was to check the ability of simplified, screening tests for genotoxicity to indicate the genotoxic activities of AA. Four commonly used in vitro genotoxicity endpoints were examined. In a bacterial mutation screening test, AA was mutagenic to tester strains TA98 and TA100 with and without rat liver S9. In the L5178Y mouse lymphoma cell gene mutation test, mutagenic activity was observed at 25 μg/ml with or without S9. A concentration-dependent increase in structural chromosome aberrations was observed in CHO cells, with significant increases at 50 μg/ml without S9 and at 25 μg/ml with S9. Significant increases in micronucleated binucleated cells were observed in CHO cells treated with AA at 25 μg/ml with or without S9. These results demonstrated that the genotoxicity of AA would have been easily detected if simple screening versions of in vitro genotoxicity assays had been used during early product development. It is suggested that simplified screening tests such as those used in this study would be a rapid and economical way of obtaining the preliminary genotoxicity profiles of new substances or products as an aid to decision-making for further development.     

Study of mutagenicities of phthalic acid and terephthalic acid using in vitro and in vivo genotoxicity tests

Genotoxicities of phthalic acid (PA) and terephthalic acid (TPA) were examined using three mutagenicity tests: Ames, chromosome aberration (CA), and micronucleus (MN). In the Ames test, these two agents did not produce any mutagenic responses in the absence or presence of S9 mix on the Salmonella typhimurium strains TA98, TA100, TA102, TA1535, or TA1537. The CA test also showed that PA and TPA exerted no significant cytogenetic effect on Chinese hamster ovary (CHO) cells. In the mouse MN test, no significant alteration in occurrence of micronucleated polychromatic erythrocytes was observed in ICR male mice ip administered any of these agents at doses of 0, 20, 100, 500, 2500 or 12,500 microM/kg. These results indicate that PA and TPA produced no mutagenic effects using these in vitro and in vivo mutagenic test systems.     

Genetic toxicity studies with genistein.

Genistein is a phytoestrogen that occurs naturally in the diet especially in soybeans and soy-based foods. Genistein and related phytoestrogens are of interest as chemopreventive agents for a variety of diseases and cancers based on epidemiologic evidence of reduced cancer rates in populations with a high intake of soy. Although soy and its constituents have been consumed at high levels in Asian populations without apparent adverse effects, concern has been raised of potential adverse effects due to estrogenic and other activities of the isoflavones. In these studies, genistein was evaluated for mutagenicity and clastogenicity in vitro in the S. typhimurium assay (Ames Test), the mouse lymphoma assay and in vivo in the micronucleus test in mice and rats. There was no evidence for a mutagenic effect in the in vitro S. typhimurium assay with and without metabolic activation (S9). In the in vitro mouse lymphoma assay, genistein increased resistant mutants with and without metabolic activation (S9), which were predominantly small colonies indicating that genistein acts as a clastogen. Three independent in vivo micronucleus tests were performed in Moro mice, RAIf rats and Wistar rats. MORO male and female mice were treated orally for 14 days at doses up to 20 mg/kg/day. RAIf and Wistar male and female rats were treated orally at doses up to 2000 mg/kg without an increase in micronuclei in treated mice or rats. It is concluded that genistein was not mutagenic in the S. typhimurium assay or mutagenic or clastogenic in vivo in the mouse and rat micronucleus test. In the mouse lymphoma assay, genistein induced an increase of predominantly small colonies indicating that genistein acts as a clastogen. This observation is in agreement with published data on the inhibitory action of genistein on topoisomerase II, which is known to lead to chromosomal damage with a threshold dose response.     

Mutagenicity of malachite green and leucomalachite green in in vitro tests.

The genotoxic potential of the fungicide malachite green (MG) and its reduced derivative leucomalachite green (LMG) was assessed in bacteria and mammalian cells using the standard Salmonella typhimurium/Ames and CHO/HGPRT tests. In vitro potential DNA damaging effects of MG and LMG were tested using the single-cell gel electrophoresis (Comet) assay on CHO cells. Malachite green was found to be extremely cytotoxic to bacteria and mammalian cells. It did not have any mutagenic activity, in any bacterial strains, in the presence or absence of metabolic activation for doses up to 10 microg per plate. In the CHO/HGPRT test, the mutagenic potential of MG could be evaluated only for very low concentrations ranging from 0.001 to 0.05 microg ml(-1) medium. When S9 fraction was added to the medium, the highest tested dose could be increased to 1 microg ml(-1). In these experimental conditions, MG did not increase the number of thioguanine-resistant mutants. Leucomalachite green was less toxic than MG to Salmonella typhimurium and did not have mutagenic activity in the Ames' test for doses up to 2000 microg per plate. It was also less cytotoxic than MG to CHO cells and was tested at doses ranging from 5 to 100 microg ml(-1). Overall results indicated that LMG was not mutagenic in the HGPRT test. In the Comet assay, MG induced DNA damage only at cytotoxic doses. Loss of cell viability was observed for doses of > or = 3 microg ml(-1), with parallel increase in DNA alterations as measured by the tail moment. After metabolic activation, however, DNA damage was observed at doses (15-20 microg ml(-1)) inducing only low cytotoxicity. In this case, the direct genotoxicity of MG metabolites could not be excluded. In the absence or presence of metabolic activation, LMG did not have any effect on cell viability or DNA damage for doses up to 500 microg ml(-1). This study indicates that LMG, which is the main residue found in fish tissues after treatment with MG, did not have any mutagenic or clastogenic effects in the experimental conditions used.     

In vitro and in vivo genetic toxicology studies with diethylene glycol monohexyl ether.

Diethylene glycol monohexyl ether (DEGHE; CAS no. 112-59-4), an industrial chemical, was investigated for the potential to produce genotoxic effects using three in vitro and two in vivo tests. No mutagenic activity occurred in either the absence or presence of metabolic activation with a Salmonella typhimurium reverse assay using strains TA98, TA100, TA1535, TA1537 and TA1538. In a Chinese hamster ovary (CHO) forward gene mutation test (HGPRT locus) there was an increase in the mutation frequencies, which were relatively small compared with the solvent control values, somewhat inconsistent between duplicate cultures and occurred particularly in the presence of metabolic activation. Linear regression analysis indicated a marginally significant trend for dosage versus mutation frequency, suggesting that DEGHE was weakly positive in this test. A sister chromatid exchange test in CHO cells showed no significant dosage-related effects in the presence or absence of metabolic activation. A peripheral blood micronucleus test in mice by dosing with an intraperitoneal injection of DEGHE did not show any potential for DEGHE to increase the incidence of micronucleated polychromatophilic erythrocytes. In a first femoral bone marrow chromosome aberration test in the rat by peroral dosing, DEGHE did not cause any increase in aberrations for 12-h and 24-h samples with males and females or with females at 48-h sampling. However, with males at 48 h the two lowest doses showed an increased number of aberrations, but not at the high doses. A repeat study in males with a larger number of doses and 24-h and 48-h samples did not replicate this finding. It is concluded that DEGHE may have limited weak mutagenic activity in vitro but is devoid of clastogenic potential.     

Genotoxic evaluation of the biocomponents of the cricket, Gryllus bimaculatus, using three mutagenicity tests

The mutagenic potential of the extracted components of Gryllus bimaculatus, a species of cricket, was evaluated using short-term genotoxicity tests including the Ames, chromosome aberration, and micronuclei tests. In a Salmonella typhimurium assay, G. bimaculatus extract did not produce any mutagenic response in the absence or presence of S9 mix with TA98, TA100, TA1535, and TA1537. Chromosome aberration testing showed that G. bimaculatus had no significant effect on Chinese hamster ovary (CHO) cells. In the mouse micronucleus test, no significant alteration in occurrence of micronucleated polychromatic erythrocytes was observed in ICR male mice intraperitoneally administered with G. bimaculatus extract at doses of 15, 150, or 1500 mg/kg. These results indicate that G. bimaculatus extract exerts no mutagenic effect in these in vitro and in vivo systems.     

Genetic toxicology profile of the new antineoplastic drug mitoxantrone in the mammalian test systems

As part of safety evaluation and drug development, 1,4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl) amino]-ethyl]amino]-9,10-anthracenedione dihydrochloride (mitoxantrone, NSC 301739, CL 232,315, Novantrone) was tested in the mammalian test systems to determine its mutagenic potential. Mitoxantrone produced significant clastogenic effect in bone marrow of rats treated for 5 days at greater than or equal to 0.5 mg/kg i.p. It produced apparent increases in DNA repair in the rat hepatocyte UDS (unscheduled DNA synthesis) test and increased SCEs (sister chromatid exchanges) in CHO cells and mutant frequencies in mouse lymphoma assay. In the cell transformation test using C3H/10T 1/2 cl 8 cells, mitoxantrone did not produce significant increases in type II or type III transformed foci. In the dominant lethal test in rats, mitoxantrone administered 2 mg/kg/d i.p. affected matings of treated males, however, total implantations as well as early deaths resulting from matings with surviving males were unaffected. These results show the potential of mitoxantrone to produce genetic activity in vitro and in the somatic cells in vivo but inability of the drug to cause morphological transformation in vitro or genotoxic effect in the germinal cells in vivo. The biological significance of findings such as above is uncertain. Examination of genetic end-points such as chromosomal assays in rodents on life time studies which are currently being completed will delineate the significance, if any, of these findings.     

Toxicological studies with dithranol and its 10-acyl analogues

The oral LD₅₀ values of an antipsoriatic drug, dithranol, were 1542 mg/kg in NMRI mice and 3216 mg/ kg in Wistar rats. Three 10-acyl analogues of dithranol (10-acetyl, 10-propionyl and 10-butyryl dithranol or butantrone) were more toxic both in mice and rats. They were mutagenic only in TA1537 of the five Salmonella typhimurium strains tested. None of them were mutagenic in two Escherichia coli strains. Butantrone was least toxic to test bacteria and had the lowest mutagenic activity on TA1537. In metaphase analysis of in vitro treated human lymphocytes, dithranol, 10-acetyl dithranol and 10-propionyl dithranol produced significant increases in the number of chromosome and chromatid gaps but without a clear dose-response relationship, and without inducing significant breaks. Butantrone did not cause significant increases in gaps or breaks. In the mouse micronucleus test, dithranol and butantrone caused no increases in micronucleated polychromatic or normochromatic erythrocytes, indicating lack of clastogenic activity in vivo at maximum tolerated doses. Hence, dithranol and its 10-acyl analogues have a weak mutagenic activity in vitro. The mutagenic activity of butantrone is lower than that of the other analogues and dithranol.     

Mutagenicity evaluation of glutaraldehyde in a battery of in vitro bacterial and mammalian test systems

Glutaraldehyde was evaluated for genotoxicity using a battery of four in vitro test systems: the Salmonella/microsome assay, the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyltransferase (CHO/HGPRT) gene mutation system, the sister-chromatid exchange test with Chinese hamster ovary cells, and measurements of unscheduled DNA synthesis in primary rat-hepatocyte cultures. No significant, dose-related increases in the various end-points were produced by glutaraldehyde in tests with or without the addition of a rat-liver metabolic activation system (S-9 mix) or with the cell-mediated activation of the hepatocyte test system. A range of concentrations which spanned cytotoxic to non-cytotoxic doses was evaluated in each test system and marked cytotoxicity was typically noted at micromolar concentrations. Within a range of biologically active concentrations, glutaraldehyde did not produce significant genotoxic effects with the assays and conditions used for these studies.     

Assessment of the ability of Imazaquin herbicide to induce chromosomal aberrations in vitro in cultured Chinese hamster ovary cells and micronuclei in vivo in mice.

The agricultural chemicals marketed to increase food production may not only combat pests and weeds but also present toxic properties and cause genetic damage to the fauna and flora. The Imazaquin herbicide (Scepter 70 DG-Cyanamid) has been widely used in soybean fields in Paraná (Brazil), but information on its genotoxicity is scarce. Thus, in vivo and in vitro studies were carried out to assess the possible clastogenic effect of this herbicide on eukaryote cells. In the in vitro studies, the Chinese hamster ovarian cell lines CHO-K1 (wild) and CHO xrs-5 (mutant) were treated at the three phases of the cell cycle (G1, S and G2) for chromosome aberration (CA) analysis. The in vivo assessment was carried out by the micronucleus test (MN) on Swiss mice (Mus musculus) bone marrow cells. The herbicide did not induce a significant increase in the CA frequency in any of the treatments. No statistically significant differences were observed in the MN frequencies among the groups treated with the herbicide and the negative control. From the test system used in this study, we can conclude that the Imazaquin herbicide did not act as a clastogenic agent either in vitro or in vivo.     

The genotoxicity of diaveridine and trimethoprim

We examined the genotoxicity of diaveridine and trimethoprim in the bacterial umu test, the bacterial reverse mutation test, the in vitro chromosome aberration test, the in vivo rodent bone marrow micronucleus test in two species, and the in vivo comet assay in five mouse organs. Both compounds were negative in the umu test (Salmonella typhimurium TA1535/pSK1002) and in the reverse mutation tests (S. typhimurium TA100, TA98, TA97, TA102, and Escherichia coli WP2 uvrA/pKM101) in the presence and absence of S9 mix. Diaveridine induced structural chromosome aberrations in cultured Chinese hamster CHL cells in the absence of a metabolic activation system, but not in the presence of a liver S9 fraction. No clastogenic activity in CHL cells was detected for trimethoprim. Bone marrow micronucleus tests in mice and rats conducted on diaveridine by single- and triple-oral dosing protocols were negative. The comet assay revealed that a single oral administration of diaveridine significantly induced DNA damage in liver, kidney, lung, and spleen cells, but not in bone marrow cells. The significant increase in migration values of DNA was reproducible with dose-response relationship. We suggest that the liver detoxifies the compound before it reaches the bone marrow, and that is why it is negative in the in vivo bone marrow micronucleus test. We concluded that diaveridine is genotoxic to mammalian cells in vitro and in vivo.     

Genotoxicity studies on selected organosilicon compounds: in vivo assays

Six organosilicon compounds which had been found to have clastogenic activity in an in vitro battery of genotoxicity assays were evaluated in rat bone marrow cytogenetic assays for assessing clastogenicity in an in vivo system. None of the six compounds produced significant increases in chromosome aberrations in the rodent assay. However, trimethylsilanol produced a single value at the high-dose level/48-hr sampling interval that was significantly elevated when compared to the low concurrent control value. Both an independent repeat of the bone marrow cytogenetic assay and performance of the rat dominant lethal test failed to substantiate the presence of any significant clastogenic activity. Organosilicon compounds involved in the synthesis and degradation of polydimethylsiloxanes were not genotoxic in the in vivo clastogenicity tests employed in these studies.     

Genotoxicity evaluation of lithium hypochlorite

Lithium hypochlorite (LiOCl), the pool and spa sanitizer/algicide, was evaluated for genotoxicity in a battery of studies designed to evaluate potential mutagenicity, DNA damage and chromosome aberrations. LiOCl was not mutagenic in the Ames test when tested in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, TA1538 or in the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) mutation assay in Chinese hamster ovary (CHO) cells without metabolic activation. LiOCl did not induce DNA damage in the unscheduled DNA synthesis assay using rat primary hepatocytes. Effects on metaphase chromosomes were evaluated in vitro in CHO cells at 12 and 18 h exposure without S9 and at 12 and 22 h following a 2 h exposure with S9. LiOCl induced a statistically significant increase in chromosome aberrations at the high dose only at both harvest times without S9 and at the late harvest time with S9. There were significant increases in chromosome aberrations at the low dose, low-mid and high doses, but not at the high mid-dose at the early harvest time with S9. However, LiOCl did not increase chromosome aberrations when tested orally in rats at maximally tolerated doses. Bone marrow cells, collected 6, 24 and 48 h after a single oral dose of LiOCl to rats (100, 500, 1000 mg/kg in males; 50, 250, 500 mg/kg in females) showed no increase in the incidence of aberrations. In general, the weight of the evidence indicates that LiOCl is not genotoxic.     

Genetic toxicology studies of an anti-AIDS drug

SC-48334 (N-butyldeoxynojirimycin) is an experimental anti-AIDS drug which is currently in clinical trials. This drug is an aminosugar derivative. Its biological properties have been previously published [1]. Since many antiviral agents which are nucleic acid analogs exhibit mutagenic and/or clastogenic properties, the genotoxic potential of SC-48334 was examined in the Ames Salmonella/microsome assay, the Chinese hamster ovary cell/hypoxanthine guanine phosphoribosyl transferase (CHO/HGPRT) assay and the mouse bone marrow micronucleus assay. No toxic or mutagenic effects were observed in either the bacterial or mammalian in vitro mutation assays. Likewise, no clastogenic activity was observed in the in vivo micronucleus assay. Therefore, the administration of this drug in humans is not likely to have mutagenic effects and would probably not have a carcinogenic effect.     

Mutagenicity and toxicity studies of p-phenylenediamine and its derivatives

The mutagenicity of p-phenylenediamine and its derivatives was tested using Ames Salmonella strains TA98 and TA100. p-Phenylenediamine was weakly mutagenic to TA98 with metabolic activation. 2-Nitro-p-phenylenediamine was directly mutagenic to both strains, while 2-methyl-p-phenylenediamine required S9 mix. All the test compounds induced a dose-related increase in chromosomal aberrations m Chinese hamster ovary (CHO) cells in the absence of the S9 mix. The mutagenicity and toxicity of these compounds did not correlate with their oxidation potentials, or any other tested physicochemical properties including the energy difference between the lowest unoccupied and the highest occupied molecular orbital, ionization potential, and dipole moment.     

Gene mutation assay of acrolein in the CHO/HGPRT test system

The mutagenic potential of acrolein has been studied with a wide range of in vitro and in vivo genetic toxicity assays. The data often have been conflicting, especially with the Ames assay. This study was undertaken to assess the mutagenic potential of acrolein using the CHO/HGPRT assay, both with and without metabolic activation. This assay system was chosen because it provides eukaryotic DNA as the target and is capable of detecting a range of mutational events. Because of its considerable toxicity, acrolein was tested over a very narrow dose range of 0.2-2 nl ml-1 without exogenous activation and 0.5-8 nl ml-1 with rat S-9 activation. Multiple assays were performed under both conditions. The results indicated that while acrolein was clearly very cytotoxic, it did not induce a significant mutagenic response in the presence or absence of metabolic activation.     

Genotoxicity evaluation of Isaria sinclairii (ISE) extract

The mutagenic potential Isaria sinclairii, a traditional Chinese medicine composed of the fruiting bodies of I. sinclairii and its parasitic host larva, was evaluated using short-term genotoxicity tests, namely, the Ames test, chromosome aberration (CA), and micronuclei (MN) tests. In a Salmonella typhimurium assay, I. sinclairii extract (ISE) did not produce any mutagenic response in the absence or presence of 59 mix with TA98, TA100, TA1535, and TA1537. In the chromosome aberration (CA) test, ISE induced no significant effect on Chinese hamster ovary (CHO) cells compared with control. In the MN test, no significant change in the occurrence of micronucleated polychromatic erythrocytes was observed in male ICR mice intraperitoneally administered ISE at doses of 15, 150, or 1500 mg/kg. These results indicate that ISE has no mutagenic potential in these in vitro and in vivo systems.