In vivo genotoxicity testing strategies: Report from the 8th International Workshop on Genotoxicity Testing (IWGT).

The in vivo working group (WG) considered three topics: acceptable maximum doses for negative erythrocyte micronucleus (MN) tests, validation status of MN assays in non-hematopoietic tissues, and nuisance factors in the comet assay. The WG reached agreement on many issues, including: negative erythrocyte MN studies should be acceptable if dosing is conducted to Organisation for Economic Co-operation and Development (OECD) test guideline (TG) 474 recommendations and if sufficient bone marrow exposure is demonstrated; consensus on the evidence required to demonstrate "sufficient" exposure was not reached. The liver MN test using six-week-old rats is sufficiently validated to develop an OECD TG, but the impact of animal age warrants additional study. Ki-67 is a reliable marker for cellular proliferation in hepatocytes. The gastrointestinal tract MN test is useful for detecting poorly absorbed or rapidly degraded aneugens, and for genotoxic metabolites formed in the colon. Although current validation data are insufficient to support the development of an OECD TG, the methodologies are sufficient to consider as an appendix to OECD TG474. Comparison of comet assay results to laboratory historical control data (HCD) should not be used in data evaluation, unless the HCD distribution is demonstrated to be stable and the predominant source of HCD variation is due to animal, not study, factors. No universally acceptable negative control limit for any tissue was identified. Methodological differences in comet studies can result in variable data interpretations; more data are required before best practice recommendations can be made. Hedgehogs alone are unreliable indicators of cytotoxicity and additional investigations into cytotoxicity markers are required.

The IARC Monographs: Updated Procedures for Modern and Transparent Evidence Synthesis in Cancer Hazard Identification.

The Monographs produced by the International Agency for Research on Cancer (IARC) apply rigorous procedures for the scientific review and evaluation of carcinogenic hazards by independent experts. The Preamble to the IARC Monographs, which outlines these procedures, was updated in 2019, following recommendations of a 2018 expert advisory group. This article presents the key features of the updated Preamble, a major milestone that will enable IARC to take advantage of recent scientific and procedural advances made during the 12 years since the last Preamble amendments. The updated Preamble formalizes important developments already being pioneered in the Monographs program. These developments were taken forward in a clarified and strengthened process for identifying, reviewing, evaluating, and integrating evidence to identify causes of human cancer. The advancements adopted include the strengthening of systematic review methodologies; greater emphasis on mechanistic evidence, based on key characteristics of carcinogens; greater consideration of quality and informativeness in the critical evaluation of epidemiological studies, including their exposure assessment methods; improved harmonization of evaluation criteria for the different evidence streams; and a single-step process of integrating evidence on cancer in humans, cancer in experimental animals, and mechanisms for reaching overall evaluations. In all, the updated Preamble underpins a stronger and more transparent method for the identification of carcinogenic hazards, the essential first step in cancer prevention.

The presence of arginine may be a source of false positive results in the Ames test.

An increase in the number of revertant colonies in the Ames test is generally taken as a strong indication of mutagenic activity of a test compound. However, irrelevant positive findings may constitute a major problem in regulatory drug testing. In this study, mixtures containing only amino acids such as glycine, lysine, arginine and isoleucine, routinely used as peptide preservatives in polypeptide pharmaceutical products, were investigated for mutagenesis in the Ames Salmonella typhimurium test. The results demonstrated that in the presence of metabolic activation, all the solutions containing arginine induced an increase in the number of revertant colonies in strains TA98, TA100 and TA1535 compared with the solvent control. More specifically, for strain TA98, all arginine doses tested, i.e. from 0.4 to 8 mg/plate induced a statistically significant increase in the number of revertants. This increase was biologically significant from 1.2 to 8 mg/plate. For strain TA100, the five highest test doses, i.e., from 1.2 to 8 mg/plate, induced statistically and biologically significant increases in the number of revertants. A statistically significant increase in colony number was also observed in strain TA1535, but only at the maximal test dose of 8 mg/plate arginine. These increases were observed with arginine from two different sources, suggesting that the observed effect would not be due to the presence of potential impurities in the type of arginine used. Our findings show that a functional metabolic activation system was required to induce an increase in the number of colonies. The presence of vitamin C inhibited the arginine-induced increase in the number of revertant colonies in S. typhimurium strain TA98, suggesting a potential involvement of oxidative stress.

A comparison of transgenic rodent mutation and in vivo comet assay responses for 91 chemicals.

A database of 91 chemicals with published data from both transgenic rodent mutation (TGR) and rodent comet assays has been compiled. The objective was to compare the sensitivity of the two assays for detecting genotoxicity. Critical aspects of study design and results were tabulated for each dataset. There were fewer datasets from rats than mice, particularly for the TGR assay, and therefore, results from both species were combined for further analysis. TGR and comet responses were compared in liver and bone marrow (the most commonly studied tissues), and in stomach and colon evaluated either separately or in combination with other GI tract segments. Overall positive, negative, or equivocal test results were assessed for each chemical across the tissues examined in the TGR and comet assays using two approaches: 1) overall calls based on weight of evidence (WoE) and expert judgement, and 2) curation of the data based on a priori acceptability criteria prior to deriving final tissue specific calls. Since the database contains a high prevalence of positive results, overall agreement between the assays was determined using statistics adjusted for prevalence (using AC1 and PABAK). These coefficients showed fair or moderate to good agreement for liver and the GI tract (predominantly stomach and colon data) using WoE, reduced agreement for stomach and colon evaluated separately using data curation, and poor or no agreement for bone marrow using both the WoE and data curation approaches. Confidence in these results is higher for liver than for the other tissues, for which there were less data. Our analysis finds that comet and TGR generally identify the same compounds (mainly potent mutagens) as genotoxic in liver, stomach and colon, but not in bone marrow. However, the current database content precluded drawing assay concordance conclusions for weak mutagens and non-DNA reactive chemicals.

The microbial PhIP metabolite 7-hydroxy-5-methyl-3-phenyl-6,7,8,9-tetrahydropyrido[3',2':4,5]imidazo[1,2-a]pyrimidin-5-ium chloride (PhIP-M1) induces DNA damage, apoptosis and cell cycle arrest towards Caco-2 cells.

7-Hydroxy-5-methyl-3-phenyl-6,7,8,9-tetrahydropyrido[3',2':4,5]imidazo[1,2-a]pyrimidin-5-ium chloride (PhIP-M1) is a newly identified intestinal microbial metabolite from the food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Although the mutagenic potential of the endogenous N-hydroxy PhIP derivate has been reported, the risks associated with PhIP-M1 have not yet been explored. In this work, the cytotoxic and genotoxic effects originating from PhIP-M1 were assessed in the epithelial intestinal Caco-2 cell line. PhIP-M1 significantly decreased in a time- and dose-dependent manner mitochondrial dehydrogenase activity and protein synthesis, with IC50 values of, respectively, 180+/-39.4 and 173+/-20.3 microM after 24h, and 33.8+/-3.5 and 37.3+/-10.9 microM after 72 h. Apoptosis within the concentration ranges of cytotoxicity was confirmed by morphological examination, DAPI nuclear staining and annexin V staining. PhIP-M1 provoked cell cycle arrest, characterized by a significant increase in the number of nucleoids in the G2/M phase. A dose-dependent increase in DNA damage, as quantified by the alkaline comet assay, was observed after 3h in the 50-200 microM range. Because these PhIP-M1-induced genomic and cellular events may contribute to the carcinogenicity of PhIP, the potency of the colon microbiota to bioactivate PhIP must be included in future risk assessments.

Mutagenic potency in Salmonella typhimurium of organic extracts of soil samples originating from urban, suburban, agricultural, forest and natural areas.

The purpose of the present work was to assess the mutagenic potency of soil samples presumably not contaminated by industrial wastes and discharges. A set of 51 soil samples was collected from areas considered as not contaminated by a known industrial activity: 11 urban samples (collected in cities), 15 suburban samples (collected in villages), 7 agricultural samples, and 18 forest or natural samples. Each soil sample was collected at the surface (0-5cm deep), dried, sieved (2mm), homogenized before organic extraction (dichloromethane/acetone 1/1 (v/v), 37 degrees C, 4h, soil/solvent ratio 1/2, m/v), solvent exchange to DMSO and sterilizing filtration. The micro-method adaptation of the standard bacterial mutagenicity test on Salmonella typhimurium strain TA98 was performed with and without a metabolic activation system (rat-liver homogenate S9), and thus detected the effect of pro-mutagens and direct mutagens, respectively. The use of a pre-incubation method increased the sensitivity of the assay. The results obtained showed a wide range of effect levels, from no effect to clear mutagenicity. In particular, the extract of all 11 urban soil samples demonstrated mutagenic activity, while the extracts of 10 of the 15 suburban samples showed mutagenicity. On the other hand, the extract of only one of the 7 agricultural samples studied induced mutations, and none of the 18 natural or forest-soil samples investigated produced mutagenic extracts. These findings seem to indicate the crucial influence of the diffuse pollution originating from different human activities on the mutagenic potency of urban soil samples. These findings make it possible to classify the soils according to their mutagenic potency. No clear correlation was found between the mutagenicity detected in soil extracts and the measured polycyclic aromatic hydrocarbon (PAH) content of the soils investigated.

Influence of extraction parameters on the mutagenicity of soil samples.

The aim of this study was to investigate the influence of four extraction parameters (type of solvent, temperature, duration of extraction, and soil mass/solvent volume ratio) on the mutagenicity of soil extracts. Four urban soil samples were submitted to the micro-method adaptation of the Ames test on Salmonella typhimurium according to the following sequence: identification of the most sensitive strain (TA98 or TA100), the best solvent(s), the optimum extraction temperature and extraction time, and finally the optimal soil/solvent ratio. Extraction was thus performed using eight different solvents (distilled water, dichloromethane, acetonitrile, acetone, cyclohexane, methanol, hexane, or ethanol), two temperatures (room temperature or 37 degrees C), two durations (4 or 24 h), and two soil mass/solvent volume ratios (1:2 or 1:10). The results show that strain TA98 was more sensitive than strain TA100, and the observed mutagenicity was expressed as number of TA98 revertants per mg of soil equivalent. No mutagenicity was induced by the distilled water extracts, whereas most of the organic solvent extracts induced a significant mutagenic response. A dichloromethane/acetone mixture appeared to be the best compromise for extraction of mutagens from the urban soils tested. Moreover, the present study showed that a higher mutagenic activity was generally obtained with a temperature of 37 degrees C (compared to room temperature), with an extraction time of 24 h (compared to 4 h), and with a soil mass/solvent volume ratio of 1:10 (compared to 1:2).

In vitro and in vivo chromosomal aberrations induced by megazol.

With the re-emergence of Human African Trypanosomiasis (HAT) on the one hand, which are increasingly resistant to current therapies, and the stage-dependent effectiveness or even the prohibitive cost of these therapies on the other hand, megazol, a 5-nitroimidazole thiadiazole highly active against various trypanosomal species, was assessed for its genotoxic potential. Very little information has become available until now. Two batches of megazol were provided by two different suppliers: Far-Manguinhos, a part of the Fiocruz foundation, under the Brazilian Minister of Health, and Delphia, a French company. These two batches, obtained by different synthetic routes, were studied by means of the in vitro micronucleus assay on L5178Y mouse lymphoma cells, in its microscale version. Both batches of magazol displayed a strong genotoxic activity in this screening assay. A second batch from Delphia was then investigated by use of two tests, i.e. the in vitro metaphase analysis with human lymphocytes and the in vivo micronucleus test in rat bone-marrow. Megazol was shown to be a potent inducer of in vitro and in vivo chromosomal aberrations. Although megazol is a potent trypanocidal agent and is orally bio-available, its toxicity dictates that it should not be developed further for the treatment of HAT and Chagas disease. All development work has therefore been discontinued.

Study of serum interaction with a cationic nanoparticle: Implications for in vitro endocytosis, cytotoxicity and genotoxicity.

We used well-characterized and positively charged nanoparticles (NP(+)) to investigate the importance of cell culture conditions, specifically the presence of serum and proteins, on NP(+) physicochemical characteristics, and the consequences for their endocytosis and genotoxicity in bronchial epithelial cells (16HBE14o-). NP(+) surface charge was significantly reduced, proportionally to NP(+)/serum and NP(+)/BSA ratios, while NP(+) size was not modified. Microscopy studies showed high endocytosis of NP(+) in 16HBE14o-, and serum/proteins impaired this internalization in a dose-dependent manner. Toxicity studies showed no cytotoxicity, even for very high doses of NP(+). No genotoxicity was observed with classic comet assay while primary oxidative DNA damage was observed when using the lesion-specific repair enzyme, formamidopyrimidine DNA-glycosylase (FPG). The micronucleus test showed NP(+) genotoxicity only for very high doses that cannot be attained in vivo. The low toxicity of these NP(+) might be explained by their high exocytosis from 16HBE14o- cells. Our results confirm the importance of serum and proteins on nanoparticles endocytosis and genotoxicity.

Formation of conjugate adducts in the reactions of malonaldehyde-acetaldehyde and malonaldehyde-formaldehyde with guanosine.

The reactions of guanosine with malonaldehyde in buffered aqueous solutions in the presence of acetaldehyde or formaldehyde were studied. The reaction mixtures were analyzed by RP-HPLC. Two adducts were formed in the reaction of malonaldehyde and acetaldehyde and one in the reaction of malonaldehyde and formaldehyde. The products were isolated and purified by preparative C-18 chromatography and structurally characterized by UV absorbance, 1H NMR, and 13C NMR spectroscopy and mass spectrometry. The adducts formed in the reaction of malonaldehyde and acetaldehyde were identified as 7-(2,2-diformyl-1-methylethyl)-3-(beta-D-ribofuranosyl)pyrimido[1,2-a]-purin-10(3H)-one (M2AA-Guo I) and 2-(3,5-diformyl-4-methyl-1,4-dihydro-1-pyridyl)-9-(beta-D-ribofuranosyl)-purin-6(9H)-one (M2AA-Guo II). In the reaction of malonaldehyde and formaldehyde, the major product was identified as 7-formyl-3-(beta-D-ribofuranosyl)pyrimido[1,2-a]purin-10(8H)-one (M1FA-Guo). The highest yields of M2AA-Guo I and M2AA-Guo II, 7 and 2 mol %, respectively, were obtained in the reaction performed at pH 7.4 and 37 degrees C for 6 days, while M1FA-Guo was produced at a yield of 0.3 mol % after 3 days of reaction at pH 7.4 and 37 degrees C. The products are formed by reactions of malonaldehyde-acetaldehyde and malonaldehyde-formaldehyde condensation products with guanosine and are analogous to the previously identified condensation products formed with adenosine, cytidine, and proteins.

Identification of conjugate adducts formed in the reactions of malonaldehyde-acetaldehyde and malonaldehyde-formaldehyde with cytidine.

Malonaldehyde was reacted with cytidine in buffered aqueous solutions in the presence of acetaldehyde or formaldehyde. The reaction mixtures were analyzed by HPLC, and the products were isolated by preparative C18 chromatography and structurally characterized by UV absorbance, fluorescence emission, (1)H and (13)C NMR spectroscopy, and mass spectrometry. The major adducts formed in the reaction of malonaldehyde and acetaldehyde were identified as 7-(beta-D-ribofuranosyl)-4-methyl-6-oxo-6,7-dihydro-4H-pyrimido[1,6-a]pyrimidine-3-carbaldehyde (M(1)AA-Cyd) and 1-(beta-D-ribofuranosyl)-4-(3,5-diformyl-4-methyl-1,4-dihydro-1-pyridyl)pyrimidine (M(2)AA-Cyd). In the reaction of malonaldehyde and formaldehyde, the major product was identified as 7-(beta-D-ribofuranosyl)-6-oxo-6,7-dihydro-4H-pyrimido[1,6-a]pyrimidine-3-carbaldehyde (M(1)FA-Cyd). The highest yields of M(1)AA-Cyd and M(2)AA-Cyd, 3.2 and 0.5 mol %, respectively, were obtained in the reaction performed at pH 4.6 and 37 degrees C for 8 days, while M(1)FA-Cyd was produced at a yield of 0.3 mol % after 3 days of reaction at pH 4.0 and 37 degrees C. The products consist of units derived from malonaldehyde and acetaldehyde (M(1)AA-Cyd and M(2)AA-Cyd) or from malonaldehyde and formaldehyde (M(1)FA-Cyd), and are thus further examples of nucleoside modifications containing structural elements derived from aldehyde condensation reactions. Trace amounts of the adducts may be formed at physiological conditions and may be involved in the mutagenicity of the studied aldehydes.