Toxicological and analytical investigations of noni (Morinda citrifolia) fruit juice

Morinda citrifolia (noni) is known to contain genotoxic anthraquinones in the roots. Because of the widespread use of noni juice, the possible genotoxic risk was examined through a battery of short-term tests. Noni juice was also chemically analyzed for the possible presence of anthraquinones. Noni juice extract in the Salmonella microsome assay showed a slight mutagenic effect in strain TA1537, due to the presence of flavonoids. No mutagenicity was observed in the mammalian mutagenicity test with V79 Chinese hamster fibroblasts. Rats treated with a noni juice concentrate did not show DNA repair synthesis (UDS) in primary rat hepatocytes, nor could DNA adducts or DNA strand breaks be observed. HPLC analysis of noni juice for anthraquinones was negative, with a sensitivity of <1 ppm. In summary, chemical analysis and genotoxicity tests reveal that noni juice does not have a genotoxic potential and that genotoxic anthraquinones do not exist in noni juice.     

Genotoxicity of glycidamide in comparison to 3-N-nitroso-oxazolidin-2-one

Acrylamide (AA) is generated by thermal processing of foods, depending on processing conditions and precursor availability. AA is not genotoxic by itself but becomes activated to its genotoxic metabolite glycidamide (GA) via epoxidation, mediated primarily by cytochrome P450 2E1. In the Comet assay in V79 cells and in human lymphocytes, GA induced DNA damage down to 300 microM concentration (4 h). After post-treatment with the DNA repair enzyme formamidopyrimidine-DNA-glycosylase (FPG), DNA damage became already detectable at 10 microM (4 h). By comparison, the N-nitroso compound 3- N-nitroso-oxazolidin-2-one (NOZ-2) is a much stronger genotoxic agent, significantly inducing DNA damage already at 15 min (3 microM). Post-treatment with FPG in this case did not enhance response. GA induced DNA damage in V79 cells rather slowly, with first response detectable at 4 h. The hPRT forward mutation test encompasses 5 days of expression time during which also repair can take place. GA-induced hPRT mutations only became detectable at concentrations of 800 microM and above. This is 80-fold higher than the lowest significant response to GA in the Comet assay (10 microM with FPG). In contrast, NOZ-2 was as effective in the hPRT test as in the Comet assay (3 microM). These results demonstrate substantial differences in the genotoxic potency of GA and NOZ-2. Whereas NOZ-2 is a pontent genotoxic mutagen, GA in comparison shows only low genotoxic and mutagenic potential, presumably as a result, at least in part, of preferential N7-G alkylation.     

Protection against Trp-P-2 DNA adduct formation in C57bl6 mice by purpurin is accompanied by induction of cytochrome P450

Purpurin, an anthraquinone constituent from madder root, has previously been reported as antimutagenic in the Ames Salmonella bacterial mutagenicity assay and as antigenotoxic in Drosophila melanogaster, against a range of environmental carcinogens. Short-term dietary supplementation with purpurin inhibits the formation of hepatic DNA adducts in male C57bl6 mice after a single dose of the heterocyclic amine dietary carcinogen Trp-P-2 (30 mg/kg). Inhibition of adduct formation was dose-dependent. No DNA adducts were observed in animals treated only with purpurin. The decrease in adduct formation was accompanied by significant, dose-dependent inductions of hepatic cytochrome P450-dependent dealkylations of methoxy- (CYP1A2), ethoxy- (CYP1A1), and pentoxy- (CYP2B) resorufins, total cytochrome P450, and NADPH cytochrome P450 reductase. It is hypothesized that purpurin exhibits chemopreventive potential by inhibiting the cytochrome P450-dependent metabolism of heterocyclic amines to their genotoxic N-hydroxylamines.     

Genotoxic Effects of Aluminum on the Neotropical Fish Prochilodus lineatus

Applying an integrated approach using the Comet, micronucleus (MN), and random amplified polymorphic DNA (RAPD) assays, occurrence of erythrocytic nuclear abnormalities (ENAs) and the liver activity of antioxidants enzymes (catalase and glutathione-S-transferase (GST)) was carried out to evaluate the effects of acute (6, 24, and 96 h) and subchronic (15 days) exposures to aluminum on fish Prochilodus lineatus. The Comet assay showed that fish erythrocytes exhibited significantly higher DNA damage after 6 and 96 h of Al exposure. MN frequencies were very low and did not increase significantly after Al exposures, while ENAs frequency increased significantly after all exposure periods. RAPD profiles obtained with DNA from fish fins collected before the toxicity tests were compared to the profiles with DNA from gills and liver of the same fish sampled after Al exposures. Alterations in RAPD profiles, including appearance and disappearance of bands, after 6 h, 24 h, and 15 days of Al exposure were detected. Fish exposed to Al for 6 and 24 h also showed significant increases in GST and catalase activities. These results indicated that Al exposure was genotoxic to P. lineatus, inducing DNA damage in peripheral erythrocytes. The induction of antioxidant enzymes might be an indication that Al causes oxidative damage to DNA, while the very low frequency of MN suggests that Al does not produce clastogenic or aneugenic effects. Genotoxic effects after 15 days of Al exposure was revealed only by RAPD, showing that this assay represents a sensitive method to detect genotoxic damage, occasionally not detected by other genotoxic tests used in toxicological genetics studies.     

Brassica oleracea L. Var. costata DC and Pieris brassicae L. aqueous extracts reduce methyl methanesulfonate-induced DNA damage in V79 hamster lung fibroblasts

Brassica oleracea L. var. costata DC leaves and Pieris brassicae L. larvae aqueous extracts were assayed for their potential to prevent/induce DNA damage. None of them was mutagenic at the tested concentrations in the Ames test reversion assay using Salmonella His(+) TA98 strains, with and without metabolic activation. In the hypoxanthine-guanine phosphoribosyltransferase mutation assay using mammalian V79 fibroblast cell line, extracts at 500 μg/mL neither induced mutations nor protected against the mutagenicity caused by methyl methanesulfonate (MMS). In the comet assay, none of the extracts revealed to be genotoxic by itself, and both afforded protection, more pronounced for larvae extracts, against MMS-induced genotoxicity. As genotoxic/antigenotoxic effects of Brassica vegetables are commonly attributed to isothiocyanates, the extracts were screened for these compounds by headspace-solid-phase microextraction/gas chromatography-mass spectrometry. No sulfur compound was detected. These findings demonstrate that both extracts could be useful against damage caused by genotoxic compounds, the larvae extract being the most promising.     

Approach for cancer risk estimation of acrylamide in food on the basis of animal cancer tests and in vivo dosimetry

The question about the contribution from acrylamide (AA) in food to the cancer risk in the general population has not yet had a satisfactory answer. One point of discussion is whether AA constitutes a cancer risk through its genotoxic metabolite, glycidamide (GA), or whether other mechanism(s) could be operating. Using a relative cancer risk model, an improvement of the cancer risk estimate for dietary AA can be obtained by estimation of the genotoxic contribution to the risk. One cornerstone in this model is the in vivo dose of the causative genotoxic agent. This paper presents an evaluation, according to this model, of published AA cancer tests on the basis of in vivo doses of GA in rats exposed in the cancer tests. The present status regarding data with importance for an improved estimation of the contribution from GA to the cancer risk of AA, such as in vivo doses measured in humans, is discussed.     

Ecotoxicological Evaluation in an Effluent and Petrochemical Waste Disposal Area

Water and sediment were studied to assess the impact of wastes from an area used for a disposal area of treated petrochemical effluents in Rio Grande do Sul, Brazil. The study was performed using the Daphnia magna (Straus 1820) for chronical evaluations, mutagenesis in Salmonella/microsome assays, and micronuclei induction in cultures of V79 cell to assess genotoxicity. Six sites were defined for chronic and genotoxic tests by micronuclei induction with liquid and sediment samples. Long-term tests were planned in semi-static flow, with microcrustaceans 2 to 26 h old in the beginning of assays. The minimum level of reproduction required to maintain the species was not reached. There are delays for the beginning of the reproductive process. Survival was also affected in some samples. The reproductive responses were more sensitive on identifying environmental quality than the survival rate. The study of mutagenicity by Salmonella/microsome assay made it possible to define the seasonality of the components showing greater frequency in winter. The predominant event was the frameshift mutation in assays with the presence of metabolism. However, the cytotoxic activity, although present in all seasons, was less frequent in winter. The genotoxicity analysis in V79 cells exposed to liquid samples from the area also showed that cytotoxicity was the most frequent event. This may have interfered in the detection of a potential micronuclei induction. The results showed that, even after treatment, effluents disposed on the soil continue with active pollutants interfering in cladoceran’s quality of life, cellular physiology, and DNA integrity.     

Genotoxic potential of glyphosate formulations: mode-of-action investigations

A broad array of in vitro and in vivo assays has consistently demonstrated that glyphosate and glyphosate-containing herbicide formulations (GCHF) are not genotoxic. Occasionally, however, related and contradictory data are reported, including findings of mouse liver and kidney DNA adducts and damage following intraperitoneal (ip) injection. Mode-of-action investigations were therefore undertaken to determine the significance of these contradictory data while concurrently comparing results from ip and oral exposures. Exposure by ip injection indeed produced marked hepatic and renal toxicity, but oral administration did not. The results suggest that ip injection of GCHF may induce secondary effects mediated by local toxicity rather than genotoxicity. Furthermore, these results continue to support the conclusion that glyphosate and GCHF are not genotoxic under exposure conditions that are relevant to animals and humans.     

Genotoxicity study of reaction products of sorbic acid

Sorbic acid (E200) and its salts (potassium and calcium sorbate: E202 and E203) are allowed for use as preservatives in numerous processed foods. Sorbic acid has a conjugated system of double bonds which makes it susceptible to nucleophilic attack, sometimes giving mutagenic products. Under conditions typical of food processing (50-80 degrees C), we analyzed the cyclic derivatives resulting from a double addition reaction between sorbic acid and various amines. Mutagenesis studies, involving the Ames test and genotoxicity studies with HeLa cells and plasmid DNA, showed that none of the products studied presented either mutagenic or genotoxic activities.     

Genotoxicity of snow in the Montreal metropolitan area

Snow is capable of scavenging particle-bound, mutagenic organic pollutants from the atmosphere. Between storms snow can act as a mechanical filter for airborne particulates and dry atmospheric fallout can contribute to contamination of the snowpack. In urban areas snow cover can store contaminants and provide a sensitive, time integrated record of local air contamination. We examined the genotoxicity of snow collected from 14 sites in the Montréal metropolitan area. Snow contaminants were extracted using dichloromethane and the genotoxicity of the extracts measured using the SOS Chromotest. Only one extract elicited a positive response in the absence of a metabolic activation mixture. Sites which provided genotoxic samples are diverse in nature. Some sites are adjacent to highways and might be expected to receive genotoxic contaminants from internal combustion vehicles. Extracts of snow from regional dump sites were not genotoxic. Mean ambient carbon monoxide and nitrogen dioxide levels were significantly higher at sites which provided genotoxic snow samples. The mean ambient concentration of suspended particulates at the positive sites was not significantly different from the concentration at the negative sites. However, a significant linear relationship was identified between the SOS response inducing potency (SOSIP) of the genotoxic samples and the ambient level of suspended particulates. The results presented confirm the presence of genotoxic material in urban snow and demonstrate that the SOS Chromotest can be used to rapidly screen complex environmental extracts for genotoxicity. High ambient levels of carbon monoxide, nitrogen dioxide measured at positive sites, and the relationship between SOSIP and the concentration of airborne particulate matter, suggests that the putative genotoxicants are fuel combustion by-products. This conclusion, while supported by previous research on atmospheric, particle-bound mutagens, is still speculative.     

用彗星实验技术检测环境遗传毒性物质

彗星实验（COMET Assay）是近年发展起来的在单细胞水平上定量检测DNA损伤的灵敏方法。经过不断改进和完善，用该方法检验的基因损伤已成为鉴别遗传毒性物质的敏感标记物，在致癌作用机制、环境污染监测以及环境毒理和风险评价等研究中，均发挥了重要作用，国内已有越来越多的研究人员开始采用这项技术。本文对彗星实验技术的发展过程、在环境中的应用以及未来的发展趋势进行综述，以利科研人员更加准确地掌握该技术，合理解释有关数据，来推动其进一步的应用和发展。     

水稻DNA损伤修复同源基因纯合突变系的鉴定

在长期的进化过程中,生物逐步形成了应对各种DNA损伤的修复机制,以保护基因组的完整性,减轻或消除DNA损伤的影响。本研究通过同源搜索找到了与DNA损伤修复相关水稻同源基因,在水稻插入突变体库中搜索出插入突变系,并成功引进了其中的26份T1代突变材料。通过对插入位点的分子鉴定和进一步培育,获得了14份涉及8个水稻DNA损伤修复同源基因的T3代纯合插入突变系,并对其进行了初步的遗传分析和表型考察。这些纯合突变系涉及的水稻基因可能在DNA错配修复(Os02g0592300、Os09g0407600、Os10g0509000)和碱基切除修复(Os02g0465112、Os06g0643600、Os05g0567500、Os04g0673400、Os09g0420300)中发挥重要作用。与亲本相比,除1份材料外,其他纯合突变体系及其野生型姐妹系的结实率均有显著降低;某些纯合突变系的千粒重与亲本相比有显著下降。这些水稻DNA损伤修复基因突变系的育成为开展水稻DNA修复的遗传学和分子生物学研究以及培育抗逆水稻提供了珍贵的实验材料。     

低剂量超辐射敏感与诱导辐射抗性的研究进展

低剂量超辐射敏感(HRS)和诱导辐射抗性(IRR)是辐射生物学的两个重要现象,研究这两个现象发生的机理对于辐射保护、控制肿瘤发生、研制新的功能材料具有重要的意义。辐射产生的DNA双链断裂(DNA double-strand breaks,DSBs)是辐射诱导细胞失活的主要损伤形式。对DSBs不能进行修复或者不能进行正确的重新连接是引起细胞死亡的主要原因。与DNA合成前期(first gap phase,G1期)和DNA合成期(synthetic phase,S期)相比,HRS在DNA合成后期(second gap phase,G2期)的表现非常明显。HRS和IRR之间相互转换调控的机制可能是G2期细胞中的一部分存在一个激活过程的发生。DNA损伤修复机制在HRS/IRR过程中发挥了重要作用。在低于20cGy的剂量条件下,细胞产生效率较低的损伤效应,细胞死亡迅速增加,低剂量超辐射敏感现象(HRS)出现;在高于20cGy的条件下,辐射造成DNA双链断裂,这些DSB本身或者由辐射引起的DNA变化激活了ATM(ataxia telangiectasia mutated),G2期专有的检测点被激活,细胞周期在G2期停顿下来,激活的G2期专有的检测点促进对DNA进行修复,提高了细胞的存活率,此时,诱导辐射抗性(IRR)出现。尽管这些结果使人们对HRS/IRR有了一个比较清晰地了解,目前仍然存在着一些关键问题有待解决。例如,为什么有些细胞系表现HRS/IRR现象,而有些细胞系不表现HRS/IRR现象;细胞中是否存在针对单一剂量的突变的HRS/IRR等。这些问题可能是将来重要的研究方向。     

用~3H-TdR研究混合重金属对鲫鱼DNA合成的影响

应用3 H 胸腺嘧啶核苷 ( 3 H TdR)示踪法研究混合重金属对鲫鱼 (Carassiusaura tus)遗传毒性的影响。结果表明 ,最佳取样时间为注射3 H TdR后 6h。鱼体各组织对混合重金属的毒性反应呈现双向效应 ,即低浓度时 ,表现出损伤、修复作用 ;高浓度时 ,DNA合成受到抑制。鱼卵、鱼鳃对重金属最敏感 ,损伤浓度为 0 0 773mg L ;而大脑对混合重金属耐受性最强。DNA损伤修复作用和DNA合成作用都存在剂量效应 ,随着暴露时间的延长 ,毒性作用增大。鱼体各组织对重金属的毒性反应可由DNA损伤修复变成合成抑制。经过 72h暴露后 ,3 H TdR在肝脏、鱼鳃、鱼卵中掺入量约低于对照的 5 0 %。     

Genotoxicity in German Surface Waters - Results of a Collaborative Study

As surface waters are widely used for the preparation of drinking water, appropriate test systems are required for the monitoring of possible genotoxic contaminations. In the course of a BMBF-funded collaborative project several methods (Ames test, umu test, alkaline elution, DNA unwinding assay, Comet assay and unscheduled DNA-synthesis test) have been examined for their ability to measure genotoxicity particularly in natural surface waters. The project was subdivided into two parts: In the first part appropriate test versions were developed (sensitized Ames test, luminometric umu test, alkaline elution using clams, Comet assay with fish cells or aquatic plants), adapted to the test subject and validated regarding their sensitivity towards standard genotoxins. All test results were statistically evaluated. In the course of the second part both natural and concentrated samples of the rivers Rhine, Elbe, Mulde, Wupper and one drinking water resource (Wahnbachbarrage) were tested. In parallel all samples were chemically analyzed. Among the unconcentrated samples several statistically positive test results were obtained both for the rivers Elbe and Rhine especially with the Ames test and the Comet assay. Only one river (Wupper) showed significant genotoxicity in the umu test. In this case chemical analysis revealed concentrations of about 41 and 47 µg/l of fluoroquinolonic acid, a bacterial gyrase inhibitor which may be responsible for this effect. No genotoxicity could be found in the drinking water resource even after concentration. The water extracts clearly showed different background genotoxicity in the umu test corresponding to increased pollution along the Rhine (Karlsruhe < K ln < D sseldorf). The eucaryotic in vitro tests revealed comparable results with nonconcentrated water samples. As a conclusion of the study we propose a graduated testing battery consisting of a bacterial (umu or Ames test) and an eucaryotic test like the Comet assay or the alkaline elution assay followed by an additional eucaryotic test (UDS test or micronucleus test) in a decisive function. There is a need for further evaluation by effect-orientated chemical analysis which should be done in the case of positive genotoxicity results in at least two tests or persistent positive results in only one.     

Oxygreen process applied on nongerminated and germinated wheat: role of hydroxamic acids

Wheat samples were taken at different stages of germination characterized by their falling number (which is a relevant indicator of germination) from 400 to 60 s. Each batch was treated by the Oxygreen process, a treatment by ozone, in a closed sequential batch reactor. Leucotriene B4 (LTB 4) was induced by germination, but ozone treatment did not increase this effect. Extract obtained from these wheat batches was applied on human epithelial bronchial cells. Wheat extract from nongerminated wheat did not induce any DNA adduct. More the wheat germination gets underway, more DNA adducts are observed. In contrast, germination did not affect the cell viability. Ozonization of wheat exemplified genotoxic effects only if the wheat was germinated. The implication of hydroxamic acids is discussed. In conclusion, ozonization of wheat, of high milling quality, does not pose any problem.     

Acrylamide carcinogenicity

The induction of cancer by chemicals is a multiple-stage process. Acrylamide is carcinogenic to experimental mice and rats, causing tumors at multiple organ sites in both species when given in drinking water or by other means. In mice, acrylamide increased the incidence and multiplicity of lung tumors and skin tumors. In two bioassays in rats, acrylamide administered in drinking water consistently induced mesotheliomas of the testes, thyroid tumors, and mammary gland tumors. In addition, brain tumors appeared to be increased. In one of the rat bioassays, pituitary tumors, pheochromocytomas, uterine tumors, and pituitary tumors were noted. The conversion of acrylamide metabolically to the reactive, mutagenic, and genotoxic product, glycidamide, can occur in both rodent and humans. Glycidamide and frequently acrylamide have been positive for mutagenicity and DNA reactivity in a number of in vitro and in vivo assays. The effects of chronic exposure of glycidamide to rodents have not been reported. Epidemiologic studies of workers for possible health effects from exposures to acrylamide have not shown a consistent increase in cancer risk. Although an increase in the risk for pancreatic cancer (almost double) was seen in highly exposed workers, no exposure response relationship could be determined. The mode of action remains unclear for acrylamide-induced rodent carcinogenicity, but support for a genotoxic mechanism based on in vitro and in vivo DNA reactivity assays cannot be ruled out. In addition, the pattern of tumor formation in the rat following chronic exposure supports a genotoxic mode of action but also suggests a potential role of endocrine modification.     

耐辐射球菌中priA类似基因突变对DNA修复和DNA转化的影响

DNA损伤很易阻断复制叉的前进。损伤DNA的修复以及接下来停止复制叉的重启动过程对细胞生存极为重要。依赖于PriA的复制重启动机制是细菌复制重启动的主要途径。为了解priA类似基因Dr2606在耐辐射球菌中的作用,并检测Dr2606在DNA修复中的作用,本研究用卡那霉素抗性基因代替Dr2606阅读框,构建了Dr2606缺失突变株,并对突变株进行UV和丝裂霉素处理,测定了Dr2606突变株的转化效率。Dr2606的突变导致菌体生长缓慢,细胞生存率急剧下降。意外的是,耐辐射球菌的DNA修复能力没有削弱。但突变株的转化效率大大削弱。这说明在耐辐射球菌中priA类似基因Dr2606对停止复制叉的重启动过程并不是必需的;耐辐射球菌不依赖于原点的复制重启动过程可能与其他细菌不同。     

Differentiation between bioavailable and total hazard potential of sediment-induced DNA fragmentation as measured by the comet assay with Zebrafish embryos

Goals, Scope and Background  While water quality strongly improved over decades in the Rhine River, sediments still reflect elapsed contaminations of organic pollutants and heavy metals. In comparing genotoxic effects induced by both sediment extracts and whole sediments, a ratio of bioavailable toxicity and total extractable toxicity is obtained. Since contaminated sites whose contaminants are toxic and as well bioavailable present an elevated risk to the ecosystem, such ratios may be used as a warning signal to identify sites of primary concern. Methods  Accordingly, two different exposure scenarios were compared to reveal the genotoxic potential of 18 sediment samples derived from 9 sample sites along the River Rhine. For assessment of effects on genome integrity, DNA fragmentation was measured using the comet assay with primary cells isolated from zebrafish embryos previously exposed to either organic sediment extracts or freeze-dried sediments at sublethal concentrations. Additionally, chemical data were used to determine responsible pollutants and correlate them with biological effects. Results  Whereas 17 out of 18 sediment extracts caused significant DNA damage to the embryo cells, only 4 native sediments showed a genotoxic potential. Thus, under field-like exposure conditions, a major part of potentially genotoxic compounds seem to remain particle-bound and ineffective, as shown for whole sediment exposure. Conversely, the organic extracts seem to contain enriched concentrations even of hardly soluble substances. Hence, organic extracts may be used as a screening tool to address potentially polluted sites, even though the relevance of these results for the field situation may be questionable. Investigations on native sediments determined few sites with bioavailable and therefore ecologically most relevant genotoxic sediment compounds. Discussion  However, these results may underestimate the total hazard potential of sample sites with hardly bioavailable substances. Chemical data revealed a variety of anthropogenic pollutants, ranging from PAHs to heavy metals. Nevertheless, chemical data on the measured priority pollutants did not fully explain the pollution pattern of the bioassays but clearly determined substances of concern (e.g., HCB, heavy metals) in particular sample sites. Conclusions  There is a striking advantage in assessing the genotoxicity by means of different exposure scenarios that focus on either bioavailable or extractable fractions, as the combination of the results allows obtaining information on specific properties of the genotoxicants and their bioavailability. An additional correlation with chemical data should be required to identify priority pollutants, as long as the responsible contaminant is known a priori. As many studies revealed inherent failures of such a correlation, an effect-driven analysis of pollutants is recommended as a promising tool to identify even non-priority pollutants by means of their ecotoxicological effectiveness.     

Validation of Microplate Bioassays for the Assessment of Contaminated and Remediated Sites

Background, Goal and Scope  Bioassays are frequently used to investigate the water extractable ecotoxicological and genotoxicological potential of contaminated soil samples. A laboratory intercomparison study was performed for validation of miniaturised biological test systems for the assessment of contaminated and remediated sites. The successful performance of this study resulted in an optimisation of microplate assays with respect to the testing of chemicals and environmental samples. Methods  For this purpose, miniaturised bioassays were chosen, which, because of their stage of development, are suitable for routine application in the characterisation of the water extractable ecotoxicological and genotoxicological potential of soils. These ecotoxicological and genotoxicological assays were performed with contaminated soil samples by three institutions at the same time. Results and Discussion  The toxicological assessment of the contaminated and remediated soil samples using LID-values, as a rule, was highly uniform. Some minor deviations could, for the most part, be explained by the heterogeneity of the soil samples and, to a lesser extent, by methodical aspects. The difference in sensitivity towards contaminants of the two bacteria Vibrio fischeri and Pseudomonas putida was pointed out. In the algae test with Desmodesmus subspicatus, the influence of the highest sample concentrations on the growth controls became obvious. It was recommended to modify the experimental setup of the microtitration plate, i.e. to place growth controls located next to both the lowest and the highest dilution steps of the sample. The Ames-test did in some cases provide new information on the genotoxicity of the samples, but is not considered useful in a test battery for the evaluation of the genotoxic potential because of its great expense in time and work. Conclusions  The investigations in this laboratory-intercomparison study for the assessment of the water extractable toxic potential of soil samples show that different bioassays are needed, which, in contrast to chemical-analytical methods, can detect the complete effects of all present pollutants in contaminated and remediated soils and solid substrates path-specifically. Recommendations and Outlook  If the recommended modifications for the performance of the bacterial and algae growth inhibition assays on microplates are taken into consideration, these tests can substitute the tests performed on a macro scale. The usefulness of the umu-test and the NM2009-test for the investigation of the genotoxic potential has been proven. Although the tests performed on microplates require much lower sample amounts, it is recommended that sample amounts be eluted in accordance with current guidelines to ensure representativity of the sample. Further work should focus on toxicity identification studies in the future by combining chemical and toxicological analyses.