Safrole, eugenol and methyleugenol induce intrachromosomal recombination in yeast

Deletion of an integrated plasmid, a specific type of intrachromosomal recombination, was evaluated for inducibility with the phenylpropenes safrole, eugenol and methyleugenol in the yeast Saccharomyces cerevisiae. These phenylpropenes are found in food products, spices, pharmaceuticals and clove cigarettes. Safrole and eugenol are known carcinogens in animals and methyleugenol is a suspected carcinogen. These phenylpropenes are not detectable by the Ames assay and most other short-term tests used currently in predictive carcinogenesis. Like safrole, which has been shown to be nonmutagenic with the Ames assay, eugenol and methyleugenol were found to be nonmutagenic with the Ames assay. In contrast, with the yeast assays which screen for intra- and inter-chromosomal recombination in logarithmic phase cultures, all 3 compounds gave a positive dose-related response. These results demonstrate further that the yeast system can be modified easily to detect various genetic endpoints and that it deserves serious consideration as a test system for predictive carcinogenesis.     

Genotoxic effects of eugenol, isoeugenol and safrole in the wing spot test of Drosophila melanogaster

In the present study, the phenolic compounds eugenol, isoeugenol and safrole were investigated for genotoxicity in the wing spot test of Drosophila melanogaster. The Drosophila wing somatic mutation and recombination test (SMART) provides a rapid means to evaluate agents able to induce gene mutations and chromosome aberrations, as well as rearrangements related to mitotic recombination. We applied the SMART in its standard version with normal bioactivation and in its variant with increased cytochrome P450-dependent biotransformation capacity. Eugenol and safrole produced a positive recombinagenic response only in the improved assay, which was related to a high CYP450-dependent activation capacity. This suggests, as previously reported, the involvement of this family of enzymes in the activation of eugenol and safrole rather than in its detoxification. On the contrary, isoeugenol was clearly non-genotoxic at the same millimolar concentrations as used for eugenol in both the crosses. The responsiveness of SMART assays to recombinagenic compounds, as well as the reactive metabolites from eugenol and safrole were considered responsible for the genotoxicity observed.     

Floral scent production in Clarkia breweri (Onagraceae). II. Localization and developmental modulation of the enzyme S-adenosyl-L-methionine:(iso)eugenol O-methyltransferase and phenylpropanoid emission.

We have previously shown (R.A. Raguso, E. Pichersky [1995] Plant Syst Evol 194: 55-67) that the strong, sweet fragrance of Clarkia breweri (Onagraceae), an annual plant native to California, consists of 8 to 12 volatile compounds, including 4 phenylpropanoids. Although some C. breweri plants emit all 4 phenylpropanoids (eugenol, isoeugenol, methyleugenol, and isomethyleugenol), other C. breweri plants do not emit the latter 2 compounds. Here we report that petal tissue was responsible for the bulk of the phenylpropanoid emission. The activity of S-adenosyl-L-methionine: (iso)eugenol O-methyltransferase (IEMT), a novel enzyme that catalyzes the methylation of the para-4'-hydroxyl of both eugenol and (iso)eugenol to methyleugenol and isomethyleugenol, respectively, was also highest in petal tissue. IEMT activity was absent from floral tissues of plants not emitting (iso)methyleugenol. A C. breweri cDNA clone encoding IEMT was isolated, and its sequence was shown to have 70% identity to S-adenosyl-L-methionine:caffeic acid O-methyltransferase. The protein encoded by this cDNA can use eugenol and isoeugenol as substrates, but not caffeic acid. Steady-state IEMT mRNA levels were positively correlated with levels of IEMT activity in the tissues, and no IEMT mRNA was observed in flowers that do not emit (iso)methyleugenol. Overall, the data show that the floral emission of (iso)methyleugenol is controlled at the site of emission, that a positive correlation exists between volatile emission and IEMT activity, and that control of the level of IEMT activity is exerted at a pretranslational step.     

A study of the potential genotoxicity of methapyrilene and related antihistamines using the hepatocyte/DNA repair assay

Methapyrilene and four related antihistamines were evaluated for their ability to cause DNA repair measured autoradiographically as unscheduled DNA synthesis (UDS) in primary cultures of Fischer-344 rat hepatocytes. Methapyrilene failed to induce UDS at all doses tested while pyrilamine and tripelennamine induced a concentration-dependent increase in DNA repair. Doxylamine and thenyldiamine, previously untested in this system, induced a weak response at the highest non-toxic doses tested. Methapyrilene was clearly cytotoxic at doses of 100 microM and higher, as judged by morphology, and precursor incorporation into RNA and protein. Precursor incorporation into RNA was irreversibly inhibited 90% and 55% at 1000 microM and 100 microM methapyrilene, respectively, while precursor incorporation into protein was inhibited 80% and 60%. These data verify the genotoxicity of pyrilamine and tripelennamine and the failure of methapyrilene to elicit DNA repair, and suggest that doxylamine and thenyldiamine may be weak DNA-damaging agents.     

The induction of DNA-strand breaks and unscheduled DNA synthesis in F-344 rat hepatocytes following in vivo administration of caprolactam or benzoin

Benzoin (ZOIN) and caprolactam (CAP) were administered by gavage to Fischer 344 rats at a dose of 750 mg/kg and the hepatocytes isolated 12, 24 or 48 h after treatment. The isolated hepatocytes were subsequently examined for the induction of DNA-strand breaks (SB) and unscheduled DNA synthesis (UDS). Neither ZOIN nor CAP induced SB or UDS in hepatocytes, however ZOIN did induce an increase in the fraction of cells in S-phase 24 h after treatment. These results correlate well with the observed lack of carcinogenicity of these compounds.     

Further evidence that eugenol does not bind to DNA in vivo

The naturally-occurring alkenylbenzene, eugenol, was examined for its ability to form DNA adducts in the livers of mice that had been treated with up to 10 mg of the compound. No adducts were detected by 32P-postlabelling with a limit of detection of 1 adduct in 10(9) nucleotides. Under these conditions adducts were readily detected in liver DNA from the structurally-related hepatocarcinogen safrole.     

Cryopreservation and long-term storage of primary rat hepatocytes: Effects on substrate-specific cytochrome P450-dependent activities and unscheduled DNA synthesis

The effects of cryopreservation and long-term storage on substrate-specific cytochrome P45O-dependent activities and unscheduled DNA synthesis were studied in freshly isolated and cryopreserved hepatocytes derived from adult male Fischer 344 and Sprague-Dawley rats. Primary rat hepatocytes were isolated via an in situ collagenase perfusion technique, cryopreserved at –196°C, and thawed at 5 weeks and 104 and 156 weeks post-freezing. In Fischer 344 and Sprague-Dawley rats, cryopreserved hepatocytes were equivalent or similar to freshly isolated hepatocytes in substrate-specific activities for 7-ethoxyresorufin-0-deethylase and dimethylnitrosamine-N-demethylase and unscheduled DNA synthesis responses. No significant differences in activities toward 7-ethoxyresorufin-0-deethylase and dimethylnitrosamine-N-demethylase, the substrate-specific activities for cytochromes P4501A1 and P4501A2 and cytochrome P4502E1, respectively, were observed between freshly isolated and cryopreserved hepatocytes. Similar unscheduled DNA synthesis responses, a measure of DNA damage and repair, were observed after exposure to the genotoxic carcinogens 2-acetylaminofluorene, 7,12-dimethyEbenz[a]anthracene, and dimethylnitrosamine; although some decreases were also observed in Fischer 344 hepatocytes after 104 weeks and Sprague-Dawley hepatocytes after 156 weeks in the highest concentrations tested. These results suggest that cryopreserved hepatocytes, stored for extended periods of time in liquid nitrogen, are metabolically equivalent to freshly isolated hepatocytes in their ability to activate precarcinogens.Abbreviations 2-AAF 2-acetylaminofluorene - DDH₂O distilled deionized water - DMBA 7,12-dimethyIbenz[a]anthracene - DMN dimethylnitrosamine - DMNA dimethylnitrosamine-N-demethylase - DMSO dimethyl sulfoxide - EROD 7-ethoxyresorufin-O-deethylase - F344 Fischer 344 - FBS fetal bovine serum - %IR percentage of cells in repair - LN₂ liquid nitrogen - LSD least significant difference - CG cytoplasmic grains - NNG net nuclear grains - SD Sprague-Dawley - UDS unscheduled DNA synthesis - WE Williams' Medium E     

Determination of methyleugenol in rodent plasma by high-performance liquid chromatography

A method has been developed and validated for the analysis of methyleugenol, a volatile flavoring agent, in rodent plasma at concentrations from 0.050 to 10.0 μg/ml. The method involves the addition of acetonitrile to the plasma, removal of the protein precipitate, and analysis of the supernatant by reversed-phase (C₁₈) HPLC using an acetonitrile-water mixture with UV detection. The precision, accuracy, sensitivity, and specificity of the method were assessed. The stability of methyleugenol in plasma, during freeze-thaw cycles, refrigerated at the extract stage, and during the analysis was evaluated. The recovery of methyleugenol from plasma was also determined. This method was found to be acceptable for plasma concentrations in toxicokinetic studies of methyleugenol in rodents. Over 400 samples from toxicokinetic studies have been successfully analyzed to date. Kinetic data from a preliminary single administration intravenous and oral study in rats is also presented.     

Methyleugenol reduces cerebral ischemic injury by suppression of oxidative injury and inflammation

Abstract

The present study tested the cytoprotective effect of methyleugenol in an in vivo ischemia model (i.e. middle cerebral artery occlusion (MCAO) for 1.5 h and subsequent reperfusion for 24 h) and further investigated its mechanism of action in in vitro cerebral ischemic models. When applied shortly after reperfusion, methyleugenol largely reduced cerebral ischemic injury. Methyleugenol decreased the caspase-3 activation and death of cultured cerebral cortical neurons caused by oxygen-glucose deprivation (OGD) for 1 h and subsequent re-oxygenation for 24 h. Methyleugenol markedly reduced superoxide generation in the ischemic brain and decreased the intracellular oxidative stress caused by OGD/re-oxygenation. It was found that methyleugenol elevated the activities of superoxide dismutase and catalase. Further, methyleugenol inhibited the production of nitric oxide and decreased the protein expression of inducible nitric oxide synthase. Methyleugenol down-regulated the production of pro-inflammatory cytokines in the ischemic brain as well as in immunostimulated mixed glial cells. The results indicate that methyleugenol could be useful for the treatment of ischemia/inflammation-related diseases.     

Inhibition of xenobiotic-induced genotoxicity in cultured precision-cut human and rat liver slices

In this study precision-cut liver slices have been used to evaluate the effects of the flavone tangeretin, the flavonoid glycoside naringin and the flavanone naringenin (the aglycone derived from naringin) on xenobiotic-induced genotoxicity. Liver slices were cultured for 24 h in medium containing [3H]thymidine and the test compounds and then processed for autoradiographic determination of unscheduled DNA synthesis (UDS). The cooked food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) markedly induced UDS in cultured human liver slices and both 2-acetylaminofluorene (2-AAF) and aflatoxin B1 (AFB1) induced UDS in cultured rat liver slices. Tangeretin (20 and 50 microM) was found to be a potent inhibitor of 5 and 50 microM PhIP-induced UDS in human liver slices, whereas 20 and 50 microM naringenin was ineffective and naringin only inhibited genotoxicity at a concentration of 1000 microM. In rat liver slices 50 microM tangeretin inhibited 10 and 50 microM 2-AAF-induced UDS, whereas 50 microM naringenin and 100 and 1000 microM naringin were ineffective. None of the three flavonoids examined inhibited 5 microM AFB1-induced UDS in rat liver slices. The inhibition of PhIP- and 2-AAF-induced UDS by tangeretin is probably attributable to the inhibition of the human and rat cytochrome P-450 isoforms which are responsible for the bioactivation of these two genotoxins. Although flavonoids can modulate xenobiotic-induced genotoxicity in human and rat liver slices, any protective effect is dependent on the particular combination of genotoxin and flavonoid examined. These results demonstrate that cultured precision-cut liver slices may be utilised as an in vitro model system to examine the modulation of xenobiotic-induced genotoxicity by flavonoids and other dietary components.     

Influence of age,sex and strain on the in vitro induction of unscheduled dna synthesis in rat hepatocyte primary cultures

The activity of chemical-induced unscheduled DNA synthesis was evaluated in hepatocyte primary cultures from Fischer 344 and Sprague-Dawley rats over a period of two years. In this two-year study hepatocytes from both sexes and strains were prepared from animals 2, 8, 14, 20 and 25 months of age and UDS was measured by autoradiography following treatment with N-methyl-AP-vitro-N-nitrosoguanidine and 2-acetylaminofluorine. A dose-related positive response occurred for both compounds throughout the study in hepatocytes from male and female Fischer rats and male Sprague-Dawley rats. The magnitude of the response was greatest in hepatocytes from male Fischer rats and a markedly lower response in unscheduled DNA synthesis occurred in all cultures prepared from animals of both strains and sexes at 20 and 25 months of age. Hepatocytes from female Sprague-Dawley rats showed a low level of unscheduled DNA synthesis with N-methylN-vitro-N-nitrosoguanidine throughout the study. The most striking finding was the absence of a UDS response to 2-acetylaminofuorene by hepatocytes from Sprague-Dawley females at the 8, 14, 20 or 25 month periods. The results indicate an age-related decrease in chemical-induced unscheduled DNA synthesis activity among rats.Abbreviations 2AAF 2-acetylaminofluorine[deDMSO] - dimethylsulfoxide ³H-TdR, meth yl-3H-thymidine - MNNG N-methyl-N-vitro-N-nitrosoguanidine - UDS unscheduled DNA synthesis     

Strain differences in in vitro rat hepatocyte unscheduled DNA synthesis (UDS): effect of UV is independent of strain while increased sensitivity is apparent using Fischer-344 instead of Sprague-Dawley rats

The unscheduled DNA synthesis (UDS) assay measures DNA repair following in vitro treatment of rat primary hepatocytes. This report compares the UDS response of primary hepatocytes from 2 widely used rat strains, the Fischer-344 (F344) and Sprague-Dawley (SD) strains. Ultraviolet (UV) light and 5 known genotoxic chemicals were evaluated in each strain in parallel experiments. The chemicals tested were 2-acetylaminofluorene (2-AAF), 4-aminobiphenyl (4-AB), benzidine, dimethylnitrosamine (DMN) and N-propyl-N'-nitro-N-nitrosoguanidine (PNNG). Four of these compounds (2-AAF, 4-AB, benzidine and DMN) require metabolic activation. Benzidine and PNNG were both negative using SD rat hepatocytes, but were weakly positive using F344 rat hepatocytes. In the first of 2 experiments, 4-AB was inconclusive in SD hepatocytes, but strongly positive in F344 cells. In the second experiment, 4-AB was positive in hepatocytes from both strains. 2-AAF was more strongly positive in F344 cells than in SD cells. DMN and UV light induced positive dose responses with little or no differences between strains. It is concluded that hepatocytes from F344 rats may be more sensitive, qualitatively and quantitatively, than hepatocytes from SD rats as indicators of UDS. This difference is not due to intrinsic differences in DNA repair mechanisms but is probably due to differences in drug-metabolizing enzymes between these strains. Thus, for routine screening, F344 rats are preferable for measurement of the in vitro UDS-inducing potential of compounds.     

Lethal injury by diquat redox cycling in an isolated hepatocyte model

Hepatocyte isolated by collagenase perfusion of livers of male Fischer-344 rats, and treated with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (50 microM for 30 min at 37 degrees C) to inhibit glutathione reductase, were significantly more vulnerable to cytotoxicity of the bipyridyl herbicide diquat than similarly treated cells of Sprague-Dawley rats. Without compromise of cell defenses by BCNU, diquat was not cytotoxic to hepatocytes from either strain. Microsomal enzyme induction with phenobarbital (80 mg/kg ip for 3 days before hepatocyte isolation) did not potentiate killing of Fischer hepatocytes by diquat. Specific activities of NADPH-cytochrome P-450 reductase in isolated Fischer and Sprague-Dawley rat liver microsomes utilizing 1 mM diquat as acceptor were 0.085 +/- 0.017 and 0.076 +/- 0.028 mumol/mg.min (mean +/- SEM, N = 5), respectively, indicating the capacity for very active redox cycling of diquat by this route in both strains. The serine protease inhibitor, phenylmethylsulfonyl fluoride (100 microM), had no effect on diquat cytotoxicity, but both leupeptin (100 micrograms/ml) and antipain (50 or 100 microM) were able to delay, through not completely prevent, diquat-induced cell death. The phospholipase inhibitors, chlorpromazine (50 or 100 microM) and dibucaine (50 or 100 microM), similarly delayed but did not prevent cell death. Diquat increased the rate of hepatocyte phospholipid hydrolysis, measured as release into the suspending medium of [14C]arachidonic acid previously incorporated into hepatocyte lipids, but although chlorpromazine decreased phospholipid hydrolysis to the control rate, only partial protection against diquat cytotoxicity was seen. These data suggest that activation of phospholipase A2 and proteases by elevation of cytosolic free Ca2+ cannot account entirely for the loss of cell viability observed in the presence of cytotoxic concentrations of diquat.     

Investigation of the genotoxic effects of 2-amino-9H-pyrido[2,3-b]indole in different organs of rodents and in human derived cells

Aim of the present study was the investigation of the genotoxicity of amino-alpha-carboline (AalphaC) in human derived cells and of its organ-specific effects in laboratory rodents. This heterocyclic amine (HA) is contained in fried meat and fish in higher concentrations than most other cooked food mutagens. In the present experiments, AalphaC caused dose-dependent induction of micronuclei in the human derived hepatoma cell line HepG2 at concentrations > or =50 microM. In contrast, no significant effects were seen in Hep3B, another human hepatoma cell line, which may be explained by the concurrent lower activity of sulfotransferase (SULT), an enzyme playing a key role in the activation of AalphaC. A positive result was also obtained in the single cell gel electrophoresis (SCGE) assay in peripheral human lymphocytes, but the effect was only significant at the highest concentration (1000 microM). In Fischer F344 rats and ICR mice, the liver was the main target organ for the formation of DNA adducts (at > or =50 mg/kg bw), and in lungs and colon substantially lower levels were detected. Identical organ specificity as in the DNA adduct measurements was seen in SCGE assays with rats, whereas in mice the most pronounced induction of DNA migration was observed in the colon. Comparison of our results with data from earlier experiments indicate that the genotoxic potency of AalphaC is equal to that of other HAs, which are contained in human foods in much smaller amounts. Therefore, our findings can be taken as an indication that the human health risk caused by exposure to AalphaC is higher than that of other HAs that are formed during the cooking of meat and fish.     

Effects of anethole and structural analogues on the contractility of rat isolated aorta: Involvement of voltage-dependent Ca2+-channels

Anethole is a naturally occurring aromatic oxidant, present in a variety of medicinal plant extracts, which is commonly used by the food and beverage industry. Despite its widespread occurrence and commercial use, there is currently little information regarding effects of this compound on the vasculature. Therefore the actions of anethole on the contractility of rat isolated aorta were compared with those of eugenol, and their respective isomeric forms, estragole and isoeugenol. In aortic rings precontracted with phenylephrine (PE; 1 microM), anethole (10(-6) M-10(-4) M) induced contraction in preparations possessing an intact endothelium, but not in endothelium-denuded tissues. At higher concentrations (10(-3) M-10(-2) M), anethole-induced concentration-dependent and complete relaxation of all precontracted preparations, irrespective of whether the endothelium was intact or not, an action shared by eugenol, estragole and isoeugenol. The contractile and relaxant effects of anethole in PE-precontracted preparations were not altered by L-NAME (10 microM) or indomethacin (10 microM), indicating that neither nitric oxide nor prostaglandins were involved in these actions. The mixed profile of effects was not confined to PE-mediated contraction, since similar responses were obtained to anethole when tissues were precontracted with 25 mM KCl. Anethole and estragole (10(-6)-10(-4) M), but not eugenol or isoeugenol, increased the basal tonus of endothelium-denuded aortic rings, an action that was abolished by VDCC blockers nifedipine (1 microM) and diltiazem (1 microM), or by withdrawal of extracellular Ca(2+). Our data suggest complex effects of anethole on isolated blood vessels, inducing contraction at lower doses, mediated via opening of voltage-dependent Ca(2+)-channels, and relaxant effects at higher concentrations that are shared by structural analogues.     

Activity of germ-cell mutagens and nonmutagens in the rat spermatocyte UDS assay

The ability of 13 chemicals of known germ-cell mutagenicity to induce unscheduled DNA synthesis (UDS) in rat spermatocytes was examined. At selected times following i.p. injection of test compounds, spermatocytes were isolated from Fischer 344 rats by enzymatic digestion of the seminiferous tubules and cultured for 24 h in the presence of [3H]thymidine. 7 compounds, methyl methanesulfonate, triethylenemelamine, cyclophosphamide, methylnitrosourea, ethylnitrosourea, procarbazine, and dibromochloropropane produced positive UDS responses in spermatocytes. These chemicals are also positive for specific locus mutations, heritable translocations, or dominant lethal mutations when administered to male rodents. Mitomycin C, which produces DNA interstrand crosslinks and induces heritable mutations and translocations in male germ cells, failed to stimulate UDS in rat spermatocytes. Germ-cell nonmutagens N-methyl-N'-nitro-N-nitrosoguanidine, dimethylnitrosamine, 4-nitroquinoline 1-oxide, and ethylene dibromide were negative in the rat spermatocyte UDS assay. Correlation of these results with those of other assays for heritable mutations in germ cells indicates that the in vivo/in vitro spermatocyte DNA repair assay is useful in predicting the mutagenic potential of chemicals in male germ cells.     

Genotoxicity and cell proliferative activity of omeprazole in rat stomach mucosa.

Induction of unscheduled DNA synthesis (UDS) as a marker of genotoxicity and induction of ornithine decarboxylase (ODC) activity as a marker of cell proliferative activity by omeprazole were determined in the glandular stomach mucosa of male F344 rats after oral administration. Commercial enteric-coated omeprazole (Losec) at doses of 30 and 100 mg/kg body weight induced a dose-dependent increase in UDS but not replicative DNA synthesis in the pyloric mucosa of rat stomach 4 h after its administration. Dose-dependent significant induction of ODC activity was observed in fundic and pyloric mucosa with a maximum 8 h after administration of omeprazole at doses of 37.5-100 mg/kg body weight. These results show that omeprazole has genotoxicity and cell proliferative activity in the rat glandular stomach mucosa.     

Induction of unscheduled DNA synthesis in primary rat hepatocytes by benzidine-congener-derived azo dyes in the in vitro and in vivo/in vitro assays

The genotoxicity of the benzidine-congener-derived azo dyes. Direct Blue 1 ( DB1 ), Direct Blue 14 ( DB14 ), Direct Brown 95 ( DB95 ), and Direct Red 46 ( DR46 ) was studied in the in vitro and in vivo/in vitro unscheduled DNA synthesis (UDS) assays in primary rat hepatocytes to determine if in vivo metabolism of these compounds was required for induction of UDS. Hepatocytes were isolated, cultured, and treated with the azo dyes and [3H]thymidine (in vitro assay); alternatively, in the in vivo/in vitro assay, rats were intubated with the azo dyes, the hepatocytes isolated at 17 h after dosing and incubated in a medium containing [3H]thymidine. UDS was quantified by an autoradiographic method. None of the azo dyes induced UDS in the in vitro assay. However, DR46 did induce marginal, but significant UDS in 1 experiment (1.2 net grains at 500 micrograms/ml media). No significant UDS was observed when DR46 was tested in a subsequent in vitro assay. In the in vivo/in vitro assay, DB95 (100 mg/kg), DB14 (125 mg/kg), and DR46 (100 mg/kg) induced significant UDS (12, 2.1, and 3.5 net grains, respectively). None of the azo dyes tested was mutagenic in the Salmonella/microsome assay in the presence and absence of rat liver enzymes. Therefore, in vivo reduction of azo dyes, presumably by the gut microflora, is a requirement for the genotoxicity of these azo dyes in the primary rat hepatocyte UDS assay.     

Assessment of genotoxic potential of ethylenediamine: in vitro and in vivo studies

Ethylenediamine (EDA) was evaluated for potential genotoxic activity using a battery in vitro and in vivo mammalian tests. The tests employed were the Chinese hamster ovary (CHO) gene mutation assay, the sister-chromatid exchange (SCE) test with CHO cells, unscheduled DNA synthesis (UDS) assays with primary rat hepatocytes and a dominant lethal study with Fischer 344 rats. EDA did not produce a positive, dose-related, mutagenic effect in either the CHO mutation assay or in the SCE test when evaluated both with and without the addition of a rat-liver S9 activation system. With hepatocytes, no positive effects of EDA upon UDS values were noted in 2 separate studies using either a scintillation counting procedure or an autoradiographic method to determine UDS activity. In a dominant lethal study, male rats fed for 23 weeks with dietary levels of EDA X 2HCl of 0, 0.05, 0.15 or 0.50 g/kg/day, and mated with 1 virgin female/week for 3 consecutive weeks, showed no dose-related or statistically significant effects upon fertility, total number of implantations/female, or the number of living and dead implants per female; marked effects upon the incidence of dominant lethal mutations were noted in the positive control group injected intraperitoneally with one dose of 0.25 mg/kg triethylenemelamine. We conclude that EDA was not genotoxic in the in vitro and in vivo mammalian test systems employed.