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.     

Genotoxicity of quinocetone,cyadox and olaquindox in vitro and in vivo

Quinocetone (QCT) and Cyadox (CYA) are important derivative of heterocyclic N-oxide quinoxaline (QdNO), used actively as antimicrobial feed additives in China. Here, we tested and compared the genotoxic potential of QCT and CYA with olaquindox (OLA) in Ames test, HGPRT gene mutation (HGM) test in V79 cells, unscheduled DNA synthesis (UDS) assay in human peripheral lymphocytes, chromosome aberration (CA) test, and micronucleus (MN) test in mice bone marrow. OLA was found genotoxic in all 5 assays. In Ames test, QCT produced His⁺ mutants at 6.9 μg/plate in Salmonella typhimurium TA 97, at 18.2 μg/plate in TA 100, TA 1535, TA 1537, and at 50 μg/plate in TA 98. CYA produced His⁺ mutants at 18.2 μg/plate in TA 97, TA 1535, and at 50 μg/plate in TA 98, TA 100 and TA 1537. QCT was found positive in HGM and UDS assay at concentrations ⩾10 μg/ml while negative results were reported in CA test and MN test. Collectively, we found that OLA was more genotoxic than QCT and CYA. Genotoxicity of QCT was found at higher concentration levels in Ames test, HGM and UDS assays while CYA showed weak mutagenic potential to bacterial cells in Ames test.     

Assessment of the Potential Genotoxicity of Perfluorodecanoic Acid and Chlorotrifluoroethylene Trimer and Tetramer Acids

Perfluoro-n-decanoic acid (PFDA) is a perfluorinated fatty acidthat produces hepatomegaly and increased peroxisomal ß-oxidationwhen administered to rodents. Chlorotrifluoroethylene (CTFE)trimer acid and CTFE tetramer acid are metabolites of the six-and eight-carbon oligomers of CTFE, respectively. They are structurallyrelated to PFDA, and CTFE tetramer acid has caused toxic effectsin rodents that are similar to those observed following PFDAadministration. Because of the correlation between peroxisomeproliferation and hepatocarcinogenesis, CTFE trimer acid, CTFEtetramer acid, and PFDA were evaluated in in vitro and in vivo/invitro bioassays to assess their potential genotoxic activity.The assays conducted were the Ames Salmonella/microsomal mutagenicityassay, the hypoxanthineguanine phosphoribosyltransferase (HGPRT)locus Chinese hamster ovary gene mutation assay, the sisterchromatid exchange (SCE) assay, chromosomal aberration assay,and an in vivo/in vitro unscheduled DNA synthesis (UDS) andS-phase DNA synthesis assay. All test articles were negativein the Ames assay, the HGPRT assay, and the SCE assay. In thechromosomal aberration assay CTFE trimer acid and CTFE tetrameracid were negative in cultures with and without S9 metabolicactivation. PFDA was also negative in the absence of metabolicactivation, but chromosmal aberrations were observed when PFDAwas incubated in the presence of S9 fraction. All test articleswere negative for inducing UDS but all induced S-phase replicativeDNA synthesis 16 hr after administration of the test articleto the test animals; only ULFE tetramer acid and PFDA inducedS-phase synthesis 48 hr after dosing: the usual timepoint examinedfor this response.     

Evaluation of the Genotoxic and Cytotoxic Potential of Mainstream Whole Smoke and Smoke Condensate from a Cigarette Containing a Novel Carbon Filter

A novel carbon filter has been developed which primarily reducesthe amount of certain vapor phase constituents of tobacco smokewith greater efficiency than the charcoal filters of cigarettescurrently in the market In vitro indicators of genotoxic andcytotoxic potential were used to compare the cigarette smokecondensate (particulate phase) or whole cigarette smoke (vaporphase and particulate phase) from cigarettes containing thenovel carbon filter with smoke condensate or whole smoke fromcommercial or prototype cigarettes not containing the novelcarbon filter. Ames bacterial mutagenicity, sister chromatidexchange (SCE) in Chinese hamster ovary (CHO) cells, and neutralred cytotoxicity assays in CHO cells were utilized to assessthe genotoxic and cytotoxic potential of the cigarette smokecondensates. SCE and neutral red cytotoxicity assays were utilizedto assess the genotoxic and cytotoxic potential of the wholesmoke. As expected, the novel carbon filter did not significantlyaffect the genotoxic or cytotoxic activity of the smoke condensate,although we did observe that the use of low-nitrogen tobaccoreduced the mutagenicity of the condensate in Salmonella typhimuriumstrain TA98. However, the whole smoke from cigarettes containingthe novel carbon filter demonstrated significant reductionsin genotoxic and cytotoxic potential compared to cigaretteswithout the novel carbon filter. The toxicity of the smoke wascorrelated (r = 0.7662 for cytotoxicity and r = 0.7562 for SCEinduction) to the aggregate mass of several vapor phase components(acetone, acetaldehyde, acrolein, acrylonitrile, 1,3-butadiene,ammonia, NO_x, HCN, benzene, isoprene, and formaldehyde) in thesmoke of the cigarettes utilized in this study. In conclusion,this novel carbon filter, which significantly reduced the amountof carbonyls and other volatiles in mainstream cigarette smoke,resulted in significant reductions in the genotoxic and cytotoxicactivity of the smoke as measured by these assays.     

Evaluation of Propanil and Its N-Oxidized Derivatives for Genotoxicity in the Salmonella typhimurium Reversion, Chinese Hamster 1 Ovary/Hypoxanthine Guanine Phosphoribosyl Transferase, and Rat Hepatocyte/DNA Repair Assays

Evaluation of Propanil and Its N-Oxidized Derivatives for Genotoxicityin the Salmonella 3 typhimurium Reversion, Chinese Hamster Ovary/HypoxanthineGuanine Phosphoribosyl Transferase, and Rat Hepatocyte/DNA RepairAssays.MCMILLAN, D. C, SHADDOCK, J. G., HEFLICH, R. H., CASCIANO,D. A. AND HINSON, J. A. 1988. Fundam. Appl. Toxicol. 11, 429—4439. Since the herbicide propanil (3,4-dichloropropionanilide)is an aromatic amide and many 7 other aromatic amides are genotoxicvia A'-hydroxy (7V-OH) metabolites, JV-oxidized derivativesof propanil and 3,4-dichloroaniline were synthesized and testedfor genotoxicity. Propanil, 3,4-dichloroaniline, and their N-OHderivatives were not mutagenic in the Salmonella typhimuriumreversion assay using tester strains TA97, TA98, TA100, andTA104, in both the presence and absence of exogenous metabolicactivation (S9). In addition, the test compounds were not muta3 genic in the Chinese hamster ovary/hypoxanthine guanine phosphoribosyltransferase (CHO/ HGPRT) assay, in both the presence and absenceof S9. 3,3',4,4'-Tetrachloroazobenzene (TCAB) and its azoxyderivative (TCAOB), which are synthetic contaminants and/ordegradation products of propanil, were also inactive in theS. typhimurium reversion and CHO/HGPRT assays (?S9). UnscheduledDNA synthesis (UDS) assays weie performed to determine if propanil derivatives were able to induce DNA damage in primary rathepatocytes. Although TCAB 3 was the only derivative testedwhich induced an elevation in DNA repair, the extent was notstatistically significant. Hepatocyte toxicity, as measuredby the release of lactate dehydrogenase 24 hr after exposure,was induced by all the test compounds in a concentration-dependentman. Incubations of [₁₄]N-OH-3,4-dichloroaniline with DNA invitro resulted in only a low level of binding that was not affectedby pH. This observation may partially explain the lack of mutagenicityobserved in genotoxicity assays with propanil derivatives.     

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.     

Toxicity of the Protein Kinase C Inhibitor Safingol Administered Alone and in Combination with Chemotherapeutic Agents

Safingol [(2S,3S)-2-amino-1,3-octadecanediol] potentiates thetoxicity of doxorubicin (DOX) and cisplatin (CIS) against tumorcells in vitro and in vivo. The present studies were conductedin rats and dogs to evaluate safingol toxicity when administerediv as a single agent and to evaluate safingol's ability to potentiatethe toxicity of established chemotherapeutic agents to normaltissues in vivo. In an escalating dose study, dogs were administeredsafingol iv at 5, 10, 20, 30, 40, and 75 mg/kg on Days 1 through6. Necropsies were performed on Day 7. Red urine was observedat 10 mg/kg and higher. Icterus was observed following 40 mg/kgwith additional signs of hypoactivity and anorexia occurringafter 75 mg/kg. Clinical and microscopic pathology revealedmarked hepatotoxicity, venous degeneration and necrosis at injectionsites, and evidence of intra-vascular hemolysis. Doses of 5,20, or 40 mg safingol/kg were utilized in single iv dose ratand dog studies. No evidence of adverse systemic toxicity wasseen up to 20 mg/kg in either species [for rats: C_max = 12,600(males) or 17,133 (females) ng/ml, AUC = 3853 (males) or 4365(females) ng x hr/ml; for dogs: C_max = 2533 ng/ml, AUC = 2851ng x hr/ml (no sex differences)]. Local effects of venous irritationor intravascular hemolysis were observed at all doses in ratsand at 20 and 40 mg/kg in dogs. A dose of 40 mg/kg [for rats:C_max = 31,233 (males) or 91,300 (females) ng/ml, AUC = 11,519(males) or 18,620 (females) ng x hr/ml; for dogs: C_max = 9033ng/ml, AUC = 11,094 ng x hr/ml (combined sex)] was associatedwith clinical pathologic and renal histomorphologic changesconsidered consequent to intravascular hemolysis in both species,lethality and testicular toxicity in rats, and clinical biochemicalchanges indicative of hepatobiliary injury in dogs. Studiesindicated that hemolysis occurred during infusion, was not causedby circulating levels of safingol, and was a function of doseconcentration and vein of delivery. Safingol at 10 or 20 mg/kgwas administered iv to rats 30–60 min prior to myelosuppressiveiv doses of DOX, CIS, or cyclophosphamide (CYP). Hematology,plus renal function and morphology for CIS-treated animals,was assessed 4 and 14 days later. Safingol did not potentiateDOX-, CIS-, or CYP-mediated leukopenia/thrombocytopenia. A minimalenhancement of CIS-mediated decrease in GFR and increase increatinine was observed at 20 mg safingol/kg. Dogs were administered20 mg safingol/kg iv followed 60 min later by 0.5 or 1.25 mgDOX/kg or 0.75 or 2.0 mg CIS/kg. A complete toxicologic assessment4 and 29 days postdose failed to show potentiation of DOX toxicityby safingol or vice versa. A renal lesion was inferred in dogsadministered 20 mg/kg safingol and 2 mg/kg CIS based on minimalto slight renal tubular regeneration observed 4 weeks post-treatment.There were no effects of safingol on the pharmacokinetic profilesof DOX or CIS or vice versa.     

An evaluation of the roles of mammalian cell mutation assays in the testing of chemical genotoxicity

The present status of the applicability of mammalian cell gene mutation assays in the safety evaluation of industrial chemicals is evaluated from the industrial and regulatory point of view, with emphasis being placed on the CHO/HGPRT and mouse lymphoma tk +/- assays. The CHO/HGPRT assay was concluded to be a highly specific assay, but it might be less sensitive to mutagens that mainly induced large deletions. The mouse lymphoma assay was concluded to be sensitive, but it might have a lower specificity due to experimental artifacts such as pH and osmolality changes. Mammalian gene mutation assays, when conducted within their limitations, are concluded to be valuable in safety evaluation, providing results complementary to the Ames test and cytogenetic assays.     

Nonclinical Toxicology Studies with Zidovudine: Reproductive Toxicity Studies in Rats and Rabbits

Zidovudine (ZDV) was evaluated for adverse effects on reproductionand fetal development in animal test species. Standard preclinicaltests for reproduction and fertility, developmental toxicity,and postnatal toxicity were conducted in CD (Sprague-Dawley)rats and a developmental toxicity study was conducted in NewZealand white rabbits. In an additional study, reproductiveoutcome was characterized in female rats given ZDV before, during,or after mating and drug levels in the plasma and milk of lactatingrats were determined. Finally, drug exposure data includingobserved peak plasma concentrations (C_max) and area under theconcentration-time curve (AUC) were evaluated for pregnant ratsand rabbits. In a reproduction/fertility study in CD rats, toxicityto the early rat embryo, manifested as an increase in earlyresorptions and a decrease in litter size, was noted followingdosage of the parental animals with 75 or 225 mg ZDV/kg bid.A dose of 25 mg/kg bid was a no-effect level in rats. At thetime of mating, male rats had been dosed for 85 days, and femaleshad been dosed for 26 days. To further evaluate the effectsof ZDV on reproduction, dosing of male rats was continued to149 days when they were mated a second time to virgin, untreatedfemales. All reproductive parameters were normal in the untreatedfemales from this second mating, indicating that the embryotoxiceffect of the drug was not likely mediated by a genotoxic orother effect in the male. A separate study in female CD ratsgiven 225 mg/kg bid for various periods pre- or postconceptionsuggests that the toxic effect of ZDV is primarily to the earlyrodent embryo. Early embryo death did not occur in rats or rabbitsin standard developmental (teratology) studies; however, pregnantNew Zealand white rabbits given 250 mg/kg bid during gestationDays 6–18 showed reduced weight gain, anemia, and an increasein late fetal deaths. No other evidence of developmental toxicitywas noted in either species, and ZDV was not teratogenic inrats or rabbits given up to 250 mg/kg bid during the periodof major organogenesis. At this dose, C_max, values in rats andrabbits were approximately 234 and 150 times higher, respectively,than the mean steady-state serum concentration in adults followingchronic oral administration of 250 mg every 4 hr. In both thereproduction/fertility study and a periand postnatal study inrats, liveborn offspring showed no adverse effects on survival,growth, or developmental measurements.     

Lack of Genotoxicity of the Cancer Chemopreventive Agent N-(4-Hydroxyphenyl)retinamide

Lack of Genotoxicity of the Cancer Chemopreventive Agent N-(4-Hydroxyphenyl)retinamide.PAULSON, J. D., OLDHAM, J. W., PRESTON, R. F., AND NEWMAN, D.(1985). Fundam. Appl. Toxicol. 5, 144–150. As part ofthe preclinical drug safety evaluation of the cancer chemopreventiveagent N(4-hydroxyphenyl)retinainide (HPR) in vitro and in vivotests were conducted to assess its genotoxic activity. Negativefindings from HPR testing were demonstrated in the Ames Salmonella/microosomalactivation test, the L5178Y mouse lymphoma assay, and a ratbone marrow cytogenetics study. These data imply that HPR lacksthe ability to induce point mutations or chromosomal aberrations,and is therefore not genotoxic. Limited testing of retinyl acetatein the Ames test, the L5178Y mouse lymphoma assay, and the primaryrat hepatocyte/DNA repair assay yielded consistently negativeresults. These findings and previously published results concerningretinoid genotoxicity are discussed.     

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.     

Absence of mutagenic activity in Salmonella and of clastogenic activity in CHO cells of Caramel Colours I, II, III and IV.

A total of 15 caramel colours were examined for genotoxic activity using the Salmonella typhimurium plate incorporation assay (Ames test). Five bacterial strains, TA1535, TA1537, TA1538, TA98 and TA100 were used in all the plate incorporation tests. Caramel colours can be divided into four classes, classification depending on the preparative method used. In this study, representatives of all four classes of caramel colour were tested for genotoxic potential in the Ames test, some of the caramel colours being tested both with and without a pre-incubation stage. None of the 15 caramel colours tested exhibited genotoxic potential in any of the five bacterial tester strains. The last two caramel colours tested, in the series of 15 [203-23-4 (Class II) and 311 (Class III)] were also assessed for clastogenic potential. For this test, cultures of CHO cells were exposed to the two caramel colours and metaphase preparations from these cultures examined for evidence of chromosomal aberrations. No evidence of chromosome damaging activity was observed.     

Reproductive and Developmental Toxicity Studies of a Linear Alkylbenzene Mixture in Rats

Alkylate 215, a mixture of linear decyl- to tridecylbenzenes,is an intermediate in the manufacture of detergent sulfonates.A two-generation reproduction study and a developmental toxicitystudy were conducted using single daily doses given by gastricintubation in a corn oil vehicle. In the reproduction study,groups of 30 rats/sex/group were given doses of 0, 5, 50, or500 mg/kg/day. F₀ animals received a 10-week premating treatmentperiod and were then mated to produce a single litter; F₁ adultswere selected from the F₁ litters. F₁ animals were dosed for11 weeks before mating to produce a single litter. Adults andweaned pups received a gross postmortem examination. Histopathologystudies were conducted on reproductive tissues, tissues withgross lesions, and the pituitary gland taken from each adultin the control and high dose groups. In the developmental toxicitystudy, groups of 24 mated female rats were given 0, 125, 500,or 2000 mg/kg/day on Days 6 through 15 of gestation. Dams wereterminated on gestation Day 20 and fetuses were examined forexternal, soft tissue, and skeletal defects. Results of thereproduction study were as follows. At 50 mg/kg/day, pup weightswere decreased at Day 7 in the F₁ litter. At 500 mg/kg/day,decreases were found in the F₁ females in premating and earlylactation weight gains; in both generations in premating weightgains in males and in weight gains during gestation in females;and in litter size, pup viability at birth, Day 0–4 sur vival, and pup weights on Days 14 and 21. The NOAEL for reproductiveeffects was 5 mg/kg/day. The developmental toxicity study foundeffects on several parameters. The only effect noted at 125mg/kg/day was a slight decrease in maternal weight gain. Maternalweight gains were depressed to a greater extent at 500 and 2000mg/kg/day. Ossification variations and delayed ossificationwere increased significantly at 2000 mg/kg/day and were abovecontrol levels at 500 mg/kg/day. The NOAEL for developmentaltoxicity was 125 mg/kg/day. Alkylate 215 did not have any unusualor selective reproductive or developmental toxicity.     

Genotoxic Properties of (E)-5-(2-Bromovinyl)-2'-deoxyuridine (BVDU)

(E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) is a 5-substituted2'-deoxyuridine antiviral compound that inhibits thymidylatesynthetase. The selectivity of BVDU for virus-infected cellshas been attributed to phosphorylation of BVDU by a virus-inducedthymidine kinase. Since the closely related compounds 5-bromo-2'-deoxyuridineand 5-iodo-2'-deoxyuridine are in vitro and in vivo mutagens,BVDU was tested for genotoxic activity in bacterial and mammaliancell mutation assays as well as in assays measuring DNA damage/repairand clastogenic activity. Mutation assays with BVDU at concentrationsranging from 10 to 5000 µg/plate using Salmonella typhimuriumstrains TA1535, TA1537, TA1538, TA98, and TA100 were negative,both with and without S9 activation. BVDU was also negativein the in vitro rat hepatocyte unscheduled DNA synthesis assayat concentrations of 750 and 1000 µg/ml. In contrast,BVDU was positive in the L5178Y TK^± mouse lymphoma mutationassay without S9 activation at five concentrations ranging from500 to 2000 µg/ml. A Chinese hamster ovary cell (CHO)/hypoxanthineguanine phosphoribosyl transferase gene mutation assay conductedwithout S9 over similar concentrations was negative. However,micronucleus induction by BVDU was detected with out S9 activationat concentrations between 500 and 1750 µg/ml using bothCHO and L5178Y cells. These results indicate that BVDU is apotential human clastogen.     

The genotoxic activity of glycerol in an in vitro test battery

Glycerol, a widely distributed constituent of food and an additive used in cigarette manufacture, has been tested for genotoxic potential in a battery of short-term genotoxicity assays. Glycerol was evaluated in the Ames Salmonella typhimurium mutagenesis assay (strains TA98, TA100, TA1535, TA1537 and TA1538), in the rat hepatocyte unscheduled DNA synthesis assay, in the Chinese hamster ovary (CHO) chromosome aberration assay, the CHO sister chromatid exchange assay and the CHO mammalian mutagenesis assay. All assays (except the rat hepatocyte unscheduled DNA synthesis assay) were conducted both with and without the addition of Aroclor-induced rat liver S-9. The results of all tests were negative, showing that neither glycerol nor its metabolites have genotoxic activity in the battery of tests used.     

Uranyl Nitrilotriacetate, a Stabilized Salt of Uranium, is Genotoxic in Nontransformed Human Colon Cells and in the Human Colon Adenoma Cell Line LT97

Previous uranium mining in the "Wismut" region in Germany enhancedenvironmental distribution of heavy metals and radionuclides.Carryover effects may now lead to contamination of locally producedfoods. Compounds of "Wismut" origin are probably genotoxic viatheir irradiating components (radon) or by interacting directlywith cellular macromolecules. To assess possible hazards, weinvestigated the genotoxic effects of uranyl nitrilotriacetate(U-NTA) in human colon tumor cells (HT29 clone 19A), adenomacells (LT97), and nontransformed primary colon cells. Theseare target cells of oral exposure to environmentally contaminatedfoods and represent different cellular stages during colorectalcarcinogenesis. Colon cells were incubated with U-NTA. Cellsurvival, cytotoxicity, cellular glutathione (GSH) levels, genotoxicity,and DNA repair capacity (comet assay), as well as gene- andchromosome-specific damage combination of comet assay and fluorescencein situ hybridization [FISH], 24-color FISH) were determined.U-NTA inhibited growth of HT29 clone 19A cells (75–2000µM,72 h) and increased GSH (125–2000µM, 24 h). U-NTAwas genotoxic (1000µM, 30 min) but did not inhibit therepair of DNA damage caused by hydrogen peroxide (H₂O₂), 4-hydroxynonenal,and 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]-pyridine.U-NTA was also genotoxic in LT97 cells and primary colon cells,where it additionally increased migration of TP53 into the comettail. In LT97 cells, 0.5–2mM U-NTA increased chromosomalaberrations in chromosomes 5, 12, and 17, which harbor the tumor-relatedgenes APC, KRAS, and TP53. It may be concluded that uraniumcompounds could increase alimentary genotoxic exposure in humansif they reach the food chain in sufficient amounts.     

Genotoxicity assessment of NIM-76 and its formulation (pessary) in an in vitro Ames Salmonella/microsome assay and in vivo mouse bone marrow micronucleus test

Abstract

The possible genotoxic potential of NIM-76, a volatile fraction obtained from neem oil, having promising contraceptive activity, as well as its formulation product, called pessary (7.5% NIM-76 in polyethylene glycol), were evaluated in the Ames assay and mouse bone marrow micronucleus (MN) assay. Genotoxicity of NIM-76 (0.1–1000?µg/plate) and pessary (0.1–10?000?µg/plate) were studied using the liquid preincubation protocol of the Ames assay both in the presence and absence of S9. Likewise, the ability of NIM-76 [1–1000?mg/kg body weight (b.w.)] and its formulation product (18.75–300?mg/kg b.w.) to induce clastogenic effects were studied in the female mouse bone marrow MN test by using a two-dose intraperitoneal treatment protocol. There was no increase in the number of revertant colonies resulting from NIM-76 or pessary at any of their doses over the respective negative control plates, either in the presence or absence of S9. Similarly, in the MN assay, neither of them showed any clastogenic activity because there was no significant increase in the frequency of micronucleated polychromatic erythrocytes, over the negative control group of animals. The use of this compound in humans is therefore not likely to have mutagenic effects and may be considered as safe with regard to genotoxic potential.     

Chinese hamster ovary cell assays for mutation and chromosome damage: data from non-carcinogens

In vitro genotoxicity tests are employed to screen chemicals for their capability to cause various DNA and chromosomal alterations, and the results are often used to predict their potential for carcinogenicity. However, there is controversy regarding the apparent low specificity of some in vitro genotoxicity assays, which result in a high false positive rate. Since we use and rely upon in vitro assays for risk assessment and prediction of carcinogenicity, this specificity issue is of serious concern to us. Hence, we selected ten compounds deemed non-carcinogens in the literature to test for the induction of gene mutation and chromosomal damage using the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) mutation assay performed concurrently with a CHO micronucleus assay. The chemical exposures for the two end-points were done simultaneously. The protocol for the two end-points was developed using the carcinogens N-methyl-N'-nitro-N-nitrosoguanidine, 3-methylcholanthrene, cyclophosphamide and 7,12-dimethylbenzanthracene. The non-carcinogens chosen were 4-nitro-o-phenylenediamine, p-phenylenediamine dihydrochloride, 3-nitropropionic acid, dichlorvos, 2-(chloromethyl)pyridine, N-(1-naphthyl)ethylenediamine 2HCl, O-anthranilic acid, 4-nitroanthranilic acid, anilazine and triphenyltin hydroxide. Each of these chemicals had been reported positive in the Ames test and/or the mouse lymphoma TK+/- mutation assay. In addition, eight of them were also reported positive in in vitro assays for chromosome aberrations and/or sister chromatid exchange (SCE). We found four of the ten chemicals negative for gene mutation and micronucleus induction without and with activation in the CHO/HGPRT mutation and CHO micronucleus assays. However, one of these four chemicals may be a potential carcinogen according to other carcinogenicity reviewers. Four other chemicals that induced only micronuclei were negative for gene mutation. Dichlorvos was positive for gene mutation and micronucleus induction without and with activation. This chemical has been shown recently to cause various tumors in rodents. One of the non-carcinogens was positive in the micronucleus test and equivocally positive in the mutation test. These results indicate that the CHO/HGPRT mutation assay may provide more relevant results than the CHO micronucleus assay, the mouse lymphoma mutation assay, or in vitro SCE and chromosome aberration assays when screening chemicals for potential carcinogenicity.     

Evaluation of ochratoxin A for mutagenicity in a battery of bacterial and mammalian cell assays

Ochratoxin A (OA), a nephrotoxic mycotoxin, was evaluated for genotoxic potential in a battery of in vitro and in vivo assays. OA was not mutagenic to Salmonella typhimurium, either with or without metabolic activation, in the plate incorporation (Ames) test at concentrations of 50-600 micrograms OA/plate or in the gradient plate assay at concentrations of 0.1-1000 micrograms OA/ml. No induction of unscheduled DNA synthesis was evident in primary cultures of rat hepatocytes exposed to concentrations of OA ranging from 0.000025 to 500 micrograms/ml. In the mouse lymphoma forward mutation assay, exposure of L5178Y TK+/- mouse lymphoma cells to OA did not increase the numbers of L5178Y TK-/- mutants. There was no significant difference between the numbers of sister-chromatid exchanges in cells from OA-treated Chinese hamsters and those in cells from the negative-control animals.     

A Comparison of the Acute Neuroactive Effects of Dichloromethane, 1,3-Dichloropropane, and 1,2-Dichlorobenzene on Rat Flash Evoked Potentials (FEPs)

Previous research showed that acute exposure to dichloromethane(DCM) produced a selective reduction in peak N₃₀ of flash evokedpotentials (FEPs) in rats. In contrast, acute exposures to p-xyleneor toluene selectively reduced FEP peak N₁₆₀. The present experimentscompared the effects of DCM (log P = 1.25; oil:water partitioncoefficient), 1,3-dichloropropane (DCP; log P = 2.00), and 1,2-dichlorobenzene(DCB; log P = 3.38) on FEPs recorded from adult Long-Evans rats.Before administration of test compounds, FEPs were recordedfor five daily sessions to develop FEP peak N₁₆₀. Test compoundswere dissolved in corn oil and administered ip at doses basedon proportions of their LD50 values. The doses were: DCM, 0,57.5, 115, 230, or 460 mg/kg; DCP, 0, 86, 172, 343, or 686 mg/kg;and DCB, 0, 53, 105, 210, or 420 mg/kg. Testing times afterdosing varied among compounds and were based on pilot studiesto measure both the times of peak effect and recovery. Eachsolvent produced significant changes in the latency and amplitudeof multiple components of the FEP waveforms. However, the predominanteffect of DCM was to reduce the amplitude of peak N₃₀ (ED50= 326.3 mg/kg), that of DCP was to reduce both peaks N₃₀ (ED50= 231.0 mg/kg) and N₁₆₀) (ED50 = 136.8 mg/kg), and that of DCBwas to reduce peak N₁₆₀) (ED50 = 151.6 mg/kg). There was noconsistent relationship between log P values and the potencyof the compounds to alter FEP peaks N₃₀ and N₁₆₀ The resultssuggest that organic solvents have multiple acute effects onthe function of the central nervous system, which are not predictablesolely by the compound's lipid solubility.