Micronucleus Analysis Research Articles

Induction of micronuclei and other nuclear abnormalities in mussels exposed to bisphenol A, diallyl phthalate and tetrabromodiphenyl ether-47

Analysis of micronuclei, nuclear buds, bi-polynucleated and fragmented-apoptotic cells was performed in gills of blue mussels exposed for 3 weeks to sublethal concentrations of bisphenol A, diallyl phthalate (for the both nominal concentration 50 ppb) and to tetrabromodiphenyl ether-47 (nominal concentration 5 ppb). Fourteen specimens from each treatment and control group were used for the analysis. Our results demonstrated a significant increase in micronuclei frequency after the treatment with bisphenol A ( P = 0.0243), diallyl phthalate ( P = 0.0005) and tetrabromodiphenyl ether-47 ( P < 0.0001; Mann–Whitney U-test). Induction of bi-nucleated ( P = 0.0028), fragmented-apoptotic ( P = 0.0004) cells and nuclear buds ( P = 0.0101) was found in mussels exposed to tetrabromodiphenyl ether-47 while treatment with diallyl phthalate increased the level of fragmented-apoptotic cells ( P = 0.0283). Bisphenol A was the only agent that resulted only in induction of micronuclei but not any other kind of nuclear injuries.

Mechanisms of formation of micronuclei in somatic cells of tailless amphibians normally and under the effect of N-nitroso-N-methylcarbamide

We studied the relationship between mitotic regimen and incidence of micronuclei in liver and bone marrow cells of tailless amphibians under the effect of genotoxic carcinogen N-nitroso-N-methylcarbamide. Micronuclei appeared in amphibian somatic cells normally and after mutagenic exposure as a result of abnormal mitosis and of "interphase chromatin diminution". This latter variant was most incident in lymphoid cells, presenting as "caudate nuclei", which should be taken into consideration when interpreting findings of micronucleus analysis.

Unstable chromosomal aberrations and micronuclei analyses in the biomonitoring of workers occupationally exposed to ionising radiation

The quantification of unstable chromosomal aberrations and micronuclei in peripheral blood lymphocytes is a method commonly used in biodosimetry by cytogenetic analysis, especially when physical dosimetry cannot be performed. In this context, the aim of this research was to compare these methods in the biomonitoring of health professionals occupationally exposed to ionising radiation. In parallel, the C-banding technique was applied to confirm the presence of unstable chromosomal aberrations (dicentrics and rings). For this, samples of peripheral blood from health professionals of three hospitals (Recife – Brazil) were collected and lymphocyte cultures were carried out based on classical cytogenetic techniques. The number of cells scored per subject was the same (1000) for each assay. Among the individuals, those who do not usually wear a lead apron had higher frequencies of unstable chromosomal aberrations and micronuclei than the ones who carefully observe the radioprotection rules.

In vivo evaluation of the mutagenic potential and phytochemical characterization of oleoresin from Copaifera duckei Dwyer

We characterized the chemical constituents of Copaifera duckei oleoresin and used dermal application to Wistar rats to evaluated its possible mutagenic and cytotoxic activities on peripheral blood reticulocytes and bone marrow cells. Chemical characterization of the oleoresin revealed the presence of sesquiterpene hydrocarbons, an unidentified neutral diterpene and diterpene acids. To evaluate mutagenicity evaluation the rats were treated with 10, 25 and 50% of the LD50 dose of the oleoresin for three consecutive days and peripheral blood collected after 0, 24, 48 and 72 h for micronucleus analysis. The rats were humanly sacrificed 24 hours after the last treatment and chromosome preparations made using standard techniques. At the three concentrations and the three time intervals tested we found that there were no statistically significant differences in either the mean number of micronucleated reticulocytes (MNRETs) or the number of chromosomal aberrations as to the negative control. However, at 25 and 50% of the LD50 dose of the oleoresin there was a significant decrease in the mitotic index (MI) as compared to the negative control. Under our experimental conditions, C. duckei V11 oleoresin produced no mutagenic effects on bone marrow cells or in peripheral reticulocytes as assessed by chromosome aberrations and the micronucleus test respectively, but showed cytotoxic activity at high doses.

Effects of 1-Week and 6-Week Exposure to GSM/DCS Radiofrequency Radiation on Micronucleus Formation in B6C3F1 Mice

The aim of this study was to examine the possible induction of micronuclei in erythrocytes of the peripheral blood and bone marrow and in keratinocytes and spleen lymphocytes of mice exposed to radiofrequency (RF) radiation for 2 h per day over periods of 1 and 6 weeks, respectively. The applied signal simulated the exposure from GSM900 and DCS1800 handsets, including the low-frequency amplitude-modulation components as they occur during speaking (GSM Basic), listening (DTX) and moving within the environment (handovers, power control). The carrier frequency was set to the center of the system's uplink band, i.e., 902 MHz for GSM and 1747 MHz for DCS. Uniform whole-body exposure was achieved by restraining the mice in tubes at fixed positions in the exposure setup. Mice were exposed to slot-averaged whole-body SARs of 33.2, 11.0, 3.7 and 0 mW/g during the 1-week study and 24.9, 8.3, 2.8 and 0 mW/g during the 6-week study. Exposure levels for the 1- and 6-week studies were determined in a pretest to confirm that no thermal effect was present that could influence the genotoxic end points. During both experiments and for both frequencies, no clinical abnormalities were detected in the animals. Cells of the bone marrow from the femur (1-week study), erythrocytes of the peripheral blood (6-week study), keratinocytes from the tail root, and lymphocytes from the spleen (both studies) were isolated on slides and stained for micronucleus analysis. Two thousand cells per animal were scored in erythrocyte and keratinocyte samples. In spleen lymphocytes, 1000 binucleated lymphocytes were scored for each animal. The RF-field exposure had no influence on the formation of red blood cells. After 1 week of exposure, the ratio of polychromatic to normochromatic erythrocytes was unchanged in the treated groups compared to the sham-exposed groups. Furthermore, the RF-field exposure of mice did not induce an increase in the number of micronuclei in erythrocytes of the bone marrow or peripheral blood, in keratinocytes, or in spleen lymphocytes compared to the sham-treated control.

Induction of micronuclei and nuclear abnormalities in Oreochromis niloticus following exposure to petroleum refinery and chromium processing plant effluents

The genotoxic effects of effluents from a petroleum refinery and a chromium processing plant were evaluated in Oreochromis niloticus (Pisces: Perciformes) using the micronucleus test. Fish were exposed to different concentrations (5, 10 and 20%, v/v) of the effluents for 3, 6 and 9 days. Micronucleus analyses were carried out on gill epithelial cells and peripheral blood erythrocytes. Nuclear abnormalities other than micronuclei, considered as genetic damage indicators, were also evaluated on erythrocytes. Cyclophosphamide at a single dose of 4 mg/L was used as a positive control. The results of this study showed that both effluents had genotoxic potential. On the other hand, the level of genetic damage induced by petroleum refinery effluent was considerably higher than that of chromium processing plant effluent. Our results further indicate that nuclear abnormalities other than micronuclei, such as blebbed and lobed nuclei, may also be used as indicators of genotoxic damage.

Assessment of the in vivo genotoxicity of vanadate: Analysis of micronuclei and DNA damage induced in mice by oral exposure

Vanadium compounds are able to interact with living cells exerting a variety of biological effects. The pentavalent form is the most stable and toxic form of the element. In systems in vitro pentavalent vanadium is an effective genotoxic agent, inducing DNA damage and chromosome malsegregation at low doses. On the other hand, no adequate in vivo data are available for the characterization of the genotoxic hazard following oral intake, the most relevant route of human exposure. In this study, the genotoxic effects produced by the oral intake of sodium ortho-vanadate (Na 3VO 4) were investigated. Male CD-1 mice were treated for 5 weeks with a range of concentrations of Na 3VO 4 in drinking water (0.75–1500 mg/l). Both micronuclei and primary DNA lesions as detected by comet assay were assessed in several tissues. Statistically significant increases of micronuclei in bone marrow were observed in mice receiving the two highest concentrations of Na 3VO 4 (750 and 1500 mg/l). A significant increase of comet tail length was observed in splenocytes of mice receiving Na 3VO 4 at 1500 mg/l, whereas no effect was observed in bone marrow and testis cells. No treatment-related effect on sperm chromatin structure or on testis cell population was observed. The determination of vanadium content in mouse tissues at the end of treatment highlighted a very low internal exposure, especially in soft tissues. Overall, the results obtained indicate that the genotoxic activity of pentavalent vanadium is expressed in vivo only following high dose exposure, possibly as a consequence of the poor bioavailability of the element.

Comprehensive measurement of chromosomal instability in cancer cells: combination of fluorescence in situ hybridization and cytokinesis‐block micronucleus assay

Most tumors show abnormal karyotypes involving either chromosome rearrangements and/or aneuploidies. The aim of our study is to measure the rate of both structural and numerical chromosome instability in two colorectal cancer cell lines: HCT116, and SW480 and its single subclones. To determine structural instability, we measured the nonclonal chromosome alterations of the last cell division by means of multicolor-fluorescence in situ hybridization (FISH). To quantify numerical instability, we used centromere-specific DNA probes to simultaneously detect chromosome loss and nondisjunctional events in binucleated cells obtained by cytokinesis-block micronucleus assay (CBMN). After clonal episodes, the structural chromosome instability rate increased significantly, confirming the large contribution of structural rearrangements to the heterogeneity of cancer cells. On the other hand, the aneuploidy rate was high and conserved in both the parental SW480 cell line and its subclones. The ability to differentiate chromosome loss and nondisjunction by the CBMN assay allowed us to conclude that no significant differences were detected among these events. Analysis of nucleoplasmic bridges, micronuclei, and nuclear blebs also demonstrated the differences among the structural instability rates of the parental cell line and its subclones. Overall, our results demonstrate the prevalence of structural over numerical chromosome instability in the subclones when comparing them with their parental cell line, confirming the contribution of ongoing chromosomal reorganizations in the generation of tumor cell heterogeneity.

Serotonin-Modulating Protein Content as a Biomarker of Environmental Pollution

This work concerns the ELISA-test determination of the serotonin-modulating anticonsolidation protein (SMAP) content under industrial and oil pollution. Exposure of goldfish Carassius auratus in fresh water containing oil at concentrations of 100 and 500 ppm for 5 days leads to significant increase of the content of SMAP in the livers. These studies are conducted in parallel with micronucleus analysis of erythrocytes. It is shown that although SMAP content changes at this test oil concentration, changes do not occur in micronucleus amounts. While studying the SMAP content in the livers of wild-living gobies Neogobius fluviatilis taken from a polluted zone of the Caspian Sea, decrease of SMAP content and increase of micronuclei amount are noticed. So, short-term exposure of fish to oil pollution leads to increase of the SMAP content, whereas long-term exposure results in decrease of its content. The possibility of application of the SMAP as a biomarker of environmental pollution is discussed.

Analysis of Micronuclei in the Transferrin-receptor Positive Reticulocytes from Peripheral Blood of Nasopharyngeal Cancer Patients Undergoing Radiotherapy by a Single-laser Flow Cytometer

The automated micronucleus test is now accepted as a simple, objective, and accurate method for evaluating potential mutagenic effects caused by physical, chemical or biotic factors. This paper describes a single-laser flow cytometry, based on an immunomagnetic isolation technique in combination with acridine orange staining, to detect frequencies of micronucleated transferrin-receptor positive reticulocytes from human peripheral blood. Using this flow cytometric system, we detected the frequencies of micronucleated transferrin-receptor positive reticulocytes from 10 nasopharyngeal cancer patients undergoing radiotherapy and the baseline of the frequencies of micronucleated transferrin-receptor positive reticulocytes from 7 healthy donors. The results showed that the mean frequency of micronucleated transferrin-receptor positive reticulocytes from healthy donors was 0.236% and that from nasopharyngeal cancer patients before radiotherapy was 0.297%. After radiotherapy it was significantly elevated. When the cumulative dose of radiotherapy was about 20Gy, it reached a maximum of 6.905%, and then, as the cumulative dose of radiotherapy continued to increase to 30Gy, 40Gy and 50Gy, the frequency decreased to 6.258%, 5.119% and 5.007% respectively. Our results indicated that the single-laser flow cytometric system was quick, reasonable and acceptable for detecting the frequency of micronucleated transferrin-receptor positive reticulocytes from human peripheral blood.

Micronucleus test in fish cells: A bioassay forin situ monitoring of genotoxic pollution in the marine environment

To evaluate the use of native fish species for assessing genotoxic pollution in the marine environment, micronucleus (MN) analysis was performed in peripheral blood erythrocytes and gill cells of the grey mullet (Mugil cephalus) from three sampling stations off the southeastern Mediterranean coast of Turkey. The frequencies of blebbed, notched, and lobed nuclei and binucleated cells also were evaluated in peripheral erythrocytes. The sampling sites were chosen on the basis of pollution levels; Karaduvar harbor, contaminated by different types of industrial effluents, and Mersin harbor, mainly contaminated by aromatic hydrocarbons, were selected as polluted areas. Erdemli harbor, a relatively unpolluted site, was used as the control area. Sampling was carried out at four different seasons. The frequencies of both micronuclei and other nuclear abnormalities (NAs) in mullets captured from polluted areas were significantly higher than those in mullets from the reference area. In general, gill cells had considerably higher MN frequencies than did erythrocytes, and genotoxic responses were higher in summer than in winter. The results of this study indicate that the MN test in fish is a suitable biomarker for in situ monitoring of genotoxic pollution in the marine environment. As demonstrated in this study, NAs other than micronuclei are also useful indices of chemical exposure and toxic responses. Therefore, measuring both micronuclei and NAs may increase the sensitivity of the test system.

Using fluorescence for improvement of the quantitative analysis of micronucleus in cell culture

The micronucleus test is of unquestionable importance, being widely used in studies with several purposes. Standardization of the technique has been proposed by several groups mainly when in vivo assays are performed. In cell cultures the staining method that predominates is the Feulgen's reaction, since it is specific for DNA. In this work, we evaluated the use of two fluorescent stains, SYTOX green ® and propidium iodide, in substitution for the Feulgen's reaction. Based on the results we concluded that the fluorescence microscopy allows the reliable detection of micronuclei, presenting the following advantages: the technique is easy and fast to perform, it avoids acid treatments; the fluorescence is long lasting; micronucleus counting becomes easier and more reliable by the use of RNAase treatment; the technique is more sensitive to detect the smallest micronuclei.

The Frequency of Micronuclei among Newborns from Kragujevac, Central Serbia, after NATO Bombing in the Spring of 1999

Kragujevac, as an important industrial and economic center of the region, is now placed on the top of the list of environmentally endangered cities in our country. The aim of this biomonitoring study was to evaluate cytogenetic damage in a sample of newborns from Kragujevac after contamination of the environment entailed by the intensive bombing of the industrial zone of this city in the spring of 1999. The frequency of micronuclei (MN) in peripheral blood lymphocytes in a total of 61 phenotypically healthy newborns was analyzed before and after the NATO bombing. Analysis of micronuclei has been performed using the cytokinesis-block technique (CB MN test). Average MN frequency in lymphocytes of newborns before the bombing (N = 25) was 5.77 ± 0.85/1000 analyzed cells. After the bombing (N = 36), the average frequency of MN increased by a factor of 1.4 (8.11 ± 0.85), compared to the control frequency before the bombing. Statistical difference (p < 0.05) was established by Student's t-test. Our data suggest that such changes in genetic material were a direct consequence of contamination of the living environment.

Micronuclei in lymphocytes of uranium miners of the former Wismut SDAG

We studied micronucleus frequencies in former German uranium miners of the Wismut SDAG (Sowjetisch-Deutsche Aktiengesellschaft). Various other groups were analyzed for comparison (individuals with lung tumors or lung fibrosis, controls). We had shown previously that micronucleus frequencies were not different among the various groups. Differences were observed, however, when centromere-positive and -negative micronuclei were distinguished. In the analyses presented here, we looked for the effects of smoking habits, alcohol consumption, vitamin uptake, chronic diseases, allergies, doing sports, γ-GT (gamma-glutamyltranspeptidase), lymphocyte numbers, CEA (carcinoembryonic antigen), X-ray diagnostics, computer tomographies, and scintigraphies. With the exception of more than one scintigraphy carried out during the last four months before micronucleus analysis, none of the factors mentioned above significantly affected micronucleus numbers. One result deserves specific attention: individuals with low percentages of binucleated lymphocytes after in vitro cytochalasin B exposure showed higher micronucleus frequencies than those individuals with high percentages of binucleated cells. The same result was obtained for various other populations that we monitored in the past.

In vitro antigenotoxic potential of acitretin in human lymphocytes treated with the antineoplastic alkylating agent ASE (NSC-71964)

Acitretin is widely used in the systemic treatment of severe forms of psoriasis and other skin disorders. ASE, namely 3β-hydroxy-13α-amino-13,17-seco-5α-androstan-17-oic-13,17-lactam- p-bis(2-chloro-ethyl)amino phenylacetate (AzaSteroidalEster, NSC-71964), is an alkylating agent with antineoplastic activity and mutagenic properties. The aim of this study was to investigate the possible genotoxic and/or antigenotoxic effects of acitretin in human lymphocyte cultures in vitro, using sister chromatid exchange (SCE) and cytokinesis-blocked micronucleus (CBMN) assays. Micronucleus (MN) analysis was achieved in combination with fluorescence in situ hybridization (FISH), using an α-satellite DNA pancentromeric probe. It was found that acitretin alone demonstrated no clastogenic or aneugenic activity. However, simultaneous incubation of lymphocyte cultures with ASE and acitretin resulted in a reduction of ASE-induced SCEs. For MN analysis lymphocytes were treated with ASE and acitretin at 21 and 41 h after culture initiation, corresponding to G 1 and G 2 phases, respectively, and lasted until cell harvest. Acitretin caused a decrease in ASE-induced MN when treatment of cells started at 41 h, but exerted no effect on them when treatment started at 21 h. These findings suggest that acitretin exerts antigenotoxic effects in human lymphocyte cultures, the expression of which may be related to the cycle phase of the cells upon onset and duration of the treatment, at least as far as MN frequency is concerned.

Absence of genotoxicity in human blood cells exposed to 50 Hz magnetic fields as assessed by comet assay, chromosome aberration, micronucleus, and sister chromatid exchange analyses.

In the past, epidemiological studies indicated a possible correlation between the exposure to ELF fields and cancer. Public concern over possible hazards associated with exposure to extremely low frequency magnetic fields (ELFMFs) stimulated an increased scientific research effort. More recent research and laboratory studies, however, have not been able to definitively confirm the correlation suggested by epidemiological studies. The aim of this study was to evaluate the effects of 50 Hz magnetic fields in human blood cells exposed in vitro, using several methodological approaches for the detection of genotoxicity. Whole blood samples obtained from five donors were exposed for 2 h to 50 Hz, 1 mT uniform magnetic field generated by a Helmholtz coil system. Comet assay, sister chromatid exchanges (SCE), chromosome aberrations (CA), and micronucleus (MN) tests were used to assess DNA damage, one hallmark of malignant cell transformation. The effects of a combined exposure with X-rays were also evaluated. Results obtained do not show any significant difference between ELFMFs exposed and unexposed samples. Moreover, no synergistic effect with ionizing radiation has been observed. A slight but significant decrease of cell proliferation was evident in ELFMFs treated samples and samples subjected to the combined exposure.

Cytogenetic biomonitoring of a floriculturist population in Italy: micronucleus analysis by fluorescence in situ hybridization (FISH) with an all-chromosome centromeric probe

Flower production in greenhouses associated with a heavy use of pesticides is very wide-spread in the western part of the Ligurian region (Italy). The formation of micronuclei in peripheral blood lymphocytes is a valuable cytogenetic biomarker in human populations occupationally exposed to genotoxic compounds. In the present study we investigated the micronucleus frequency in peripheral blood lymphocytes of 52 floriculturists and 24 control subjects by use of the cytokinesis-block methodology associated with fluorescence in situ hybridization with a pan-centromeric probe that allowed to distinguish centromere-positive (C+) and centromere-negative (C−) micronuclei. The comparison between floriculturists and controls did not reveal any statistically significant difference in micronucleus frequency, although an increase was observed with increasing pesticide use, number of genotoxic pesticides used and duration of exposure. An increase in C+ as well as in C− micronuclei and in the percentage of C+ micronuclei with respect to the total number of micronuclei was detected in floriculturists, suggesting a higher contribution of C+ micronuclei in the total number scored. The percentage C+ micronuclei was not related to the duration of exposure or to the number of genotoxic pesticides used, but a higher percentage (66.52% versus 63.78%) was observed in a subgroup of subjects using benzimidazolic compounds, compared with the floriculturist population exposed to a complex pesticide mixture not including benzimidazolics. These results suggest a potential human hazard associated with the exposure to this class of aneuploidy-inducing carcinogens.

Report from the in vitro micronucleus assay working group

At the Washington “2nd International Workshop on Genotoxicity Testing” (25–26 March 1999) current methodologies and data for the in vitro micronucleus test were reviewed. As a result, guidelines for the conduct of specific aspects of the protocol were developed. Agreement was achieved on the following topics: choice of cells, slide preparation, analysis of micronuclei, toxicity, use of cytochalasin-B, number of doses, and treatment/harvest times [Environ. Mol. Mutagen. 35 (2000) 167]. Because there were a number of important in vitro micronucleus validation studies in progress, it was not possible to design a definitive, internationally harmonized protocol at that time. These studies have now been completed and the data were reviewed at the Plymouth “3rd International Workshop on Genotoxicity Testing” (28–29 June 2002). Data from studies coordinated by the French Society of Genetic Toxicology, Japanese collaborative studies, European pharmaceutical industry validation studies, along with data from Lilly Research Laboratories were used to prepare conclusions on the main aspects of the in vitro micronucleus protocol. In this paper, the consensus agreements on the protocol for performing the in vitro micronucleus assay are presented. The major recommendations concern: 1. Demonstration of cell proliferation: both cell lines and lymphocytes can be used, but demonstration of cell proliferation in both control and treated cells is compulsory for the acceptance of the test. 2. Assessment of toxicity and dose range finding: assessment of toxicity should be performed by determining cell proliferation, e.g. increased cell counts (CC) or population doubling (PD) without cytochalasin-B, or e.g. cytokinesis-block proliferation index with cytochalasin-B; and by determining other markers for cytotoxicity (confluency, apoptosis, necrosis) which can provide valuable additional information. 3. Treatment schedules for cell lines and lymphocytes. 4. Choice of positive controls: without S9-mix both a clastogen (e.g. mitomycin C or bleomycin) and an aneugen (e.g. colchicine) should be included as positive controls and a clastogen that requires S9 for activity when S9-mix is used (e.g. dimethylnitrosamine, or cyclophosphamide in those cell types that cannot activate this agent directly). 5. Duplicate cultures and number of cells to be scored. 6. Repeat experiments: in lymphocytes, for each experiment blood from 2 different healthy young and non-smoking donors should be compared. In cell lines, the experiments need only to be repeated if the first one is negative. 7. Statistics: statistical significance should not be the sole factor for determining positive results. Biological meaning should serve as a guideline. Examples of statistical analyses are given.

Sister chromatid exchanges and micronuclei analysis in lymphocytes of men exposed to simazine through drinking water

In some cities of the autonomous community of Extremadura (south-west of Spain), levels of simazine from 10 to 30 ppm were detected in tap water. To analyse the possible effect of this herbicide, two biomarkers, sister chromatid exchanges (SCE) and micronuclei (MN), were used in peripheral blood lymphocytes from males exposed to simazine through drinking water. SCE and MN analysis failed to detect any statistically significant increase in the people exposed to simazine when compared with the controls. With respect to high frequency cells (HFC), a statistically significant difference was detected between exposed and control groups.

Mutagenic and cytotoxic effectiveness of diisopropyl xanthogen polysulphide in human lymphocyte cultures

The mutagenic and cytotoxic effectiveness of the new rubber vulcanisation accelerator diisopropyl xanthogen polysulphide (Robac AS 100) was tested in human lymphocyte cultures of four healthy probands. The concentrations of Robac AS 100 were 0.57, 5.7 and 57.0 μg/ml. Higher concentrations showed too high cytotoxicity to be evaluable. Without external activation, incubation time with Robac AS 100 was 21 h. In the presence of rat liver microsomes from aroclor-induced rats (2 mg microsomal protein/ml), incubation of the test compound was 2 h. Mutagenicity testing was performed by analysis of micronuclei (MN), structural chromosome aberrations (CAs) and sister chromatid exchanges (SCEs). The MN-rate was determined using the cytochalasin B (cyt B) block method. For evaluation of cytotoxicity, mitotic index (MI) and nuclear division index (NDI) were determined. The validity of the test methods was ascertained by positive controls: mitomycin C (MMC) and bleomycin (BLM) were used in experiments without exogenous activation and cyclophosphamide (CP) in experiments with exogenous activation. The presence of rat liver microsomes increased the mutagenic effect of Robac AS 100 in the SCE- and MN-test. But only the highest Robac AS 100-concentration (57.0 μg/ml) showed significantly increased mutagenic activity in all tests. However, cytotoxicity at this concentration was already substantial. Therefore, we consider the evidence for mutagenicity of Robac AS 100 as limited.