An analysis of results from 305 compounds tested with the yeast RAD54-GFP genotoxicity assay (GreenScreen GC)-including relative predictivity of regulatory tests and rodent carcinogenesis and performance with autofluorescent and coloured compounds.

Data from 305 non-proprietary compounds tested using the yeast RAD54-GFP (Green Fluorescent Protein) assay, GreenScreen GC, are presented, together with a detailed comparison with results from in vitro and in vivo genotoxicity tests and rodent carcinogenesis. In addition, observations on reproducibility and the performance of the test with autofluorescent and coloured compounds are described. Like the Ames test, the GreenScreen assay is shown to exhibit high specificity (82%), meaning that compounds with positive results are very likely to be genotoxic carcinogens. This is in contrast to mammalian cell tests established for use in regulatory testing that provide disappointingly low specificity and the inevitable generation of confounding false positive data. The analysis confirmed the observations of earlier studies, showing that a combination of an Ames test (or surrogate) with the yeast test provides high specificity as well as high sensitivity in the identification of rodent carcinogens.

Report of the IWGT working group on strategies and interpretation of regulatory in vivo tests I. Increases in micronucleated bone marrow cells in rodents that do not indicate genotoxic hazards.

In vivo genotoxicity tests play a pivotal role in genotoxicity testing batteries. They are used both to determine if potential genotoxicity observed in vitro is realised in vivo and to detect any genotoxic carcinogens that are poorly detected in vitro. It is recognised that individual in vivo genotoxicity tests have limited sensitivity but good specificity. Thus, a positive result from the established in vivo assays is taken as strong evidence for genotoxic carcinogenicity of the compound tested. However, there is a growing body of evidence that compound-related disturbances in the physiology of the rodents used in these assays can result in increases in micronucleated cells in the bone marrow that are not related to the intrinsic genotoxicity of the compound under test. For rodent bone marrow or peripheral blood micronucleus tests, these disturbances include changes in core body temperature (hypothermia and hyperthermia) and increases in erythropoiesis following prior toxicity to erythroblasts or by direct stimulation of cell division in these cells. This paper reviews relevant data from the literature and also previously unpublished data obtained from a questionnaire devised by the IWGT working group. Regulatory implications of these findings are discussed and flow diagrams have been provided to aid in interpretation and decision-making when such changes in physiology are suspected.

Determination of genetic toxicity and potential carcinogenicity in vitro--challenges post the Seventh Amendment to the European Cosmetics Directive.

Genetic toxicology and its role in the detection of carcinogens is currently undergoing a period of reappraisal. There is an increasing interest in developing alternatives to animal testing and the three R's of reduction, refinement and replacement are the basis for EU and national animal protection laws the Seventh Amendment to the EU Cosmetics Directive will ban the marketing of cosmetic/personal care products that contain ingredients that have been tested in animal models. Thus in vivo tests such as the bone marrow micronucleus test, which has a key role in current testing strategies for genotoxicity, will not be available for this class of products. The attrition rate for new, valuable and safe chemicals tested in an in vitro-only testing battery, using the in vitro tests currently established for genotoxicity screening, will greatly increase once this legislation is in place. In addition there has been an explosion of knowledge concerning the cellular and molecular events leading to carcinogenesis. This knowledge has not yet been fully factored into screening chemicals for properties that are not directly linked to mutation induction. Thus there is a pressing need for new, more accurate approaches to determine genotoxicity and carcinogenicity. However, a considerable challenge is presented for these new approaches to be universally accepted and new tests sufficiently validated by March 2009 when the animal testing and marketing bans associated with the Seventh Amendment are due to come into force. This commentary brings together ideas and approaches from several international workshops and meetings to consider these issues.

Report of the IWGT working group on strategy/interpretation for regulatory in vivo tests II. Identification of in vivo-only positive compounds in the bone marrow micronucleus test.

A survey conducted as part of an International Workshop on Genotoxicity Testing (IWGT) has identified a number of compounds that appear to be more readily detected in vivo than in vitro. The reasons for this property varies from compound to compound and includes metabolic differences; the influence of gut flora; higher exposures in vivo compared to in vitro; effects on pharmacology, in particular folate depletion or receptor kinase inhibition. It is possible that at least some of these compounds are detectable in vitro if a specific in vitro test is chosen as part of the test battery, but the 'correct' choice of test may not always be obvious when testing a compound of unknown genotoxicity. It is noted that many of the compounds identified in this study interfere with cell cycle kinetics and this can result in either aneugenicity or chromosome breakage. A decision tree is outlined as a guide for the evaluation of compounds that appear to be genotoxic agents in vivo but not in vitro. The regulatory implications of these findings are discussed.

Testing strategies in mutagenicity and genetic toxicology: an appraisal of the guidelines of the European Scientific Committee for Cosmetics and Non-Food Products for the evaluation of hair dyes.

The European Scientific Committee on Cosmetics and Non-Food Products (SCCNFP) guideline for testing of hair dyes for genotoxic/mutagenic/carcinogenic potential has been reviewed. The battery of six in vitro tests recommended therein differs substantially from the batteries of two or three in vitro tests recommended in other guidelines. Our evaluation of the chemical types used in hair dyes and comparison with other guidelines for testing a wide range of chemical substances, lead to the conclusion that potential genotoxic activity may effectively be determined by the application of a limited number of well-validated test systems that are capable of detecting induced gene mutations and structural and numerical chromosomal changes. We conclude that highly effective screening for genotoxicity of hair dyes can be achieved by the use of three assays, namely the bacterial gene mutation assay, the mammalian cell gene mutation assay (mouse lymphoma tk assay preferred) and the in vitro micronucleus assay. These need to be combined with metabolic activation systems optimised for the individual chemical types. Recent published evidence [D. Kirkland, M. Aardema, L. Henderson, L. Müller, Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity, Mutat. Res. 584 (2005) 1-256] suggests that our recommended three tests will detect all known genotoxic carcinogens, and that increasing the number of in vitro assays further would merely reduce specificity (increase false positives). Of course there may be occasions when standard tests need to be modified to take account of special situations such as a specific pathway of biotransformation, but this should be considered as part of routine testing. It is clear that individual dyes and any other novel ingredients should be tested in this three-test battery. However, new products are formed on the scalp by reaction between the chemicals present in hair-dye formulations. Ideally, these should also be tested for genotoxicity, but at present such experiences are very limited. There is also the possibility that one component could mask the genotoxicity of another (e.g. by being more toxic), and so it is not practical at this time to recommend routine testing of complete hair-dye formulations as well. The most sensible approach would be to establish whether any reaction products within the hair-dye formulation penetrate the skin under normal conditions of use and test only those that penetrate at toxicologically relevant levels in the three-test in vitro battery. Recently published data [D. Kirkland, M. Aardema, L. Henderson, L. Müller, Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity, Mutat. Res. 584 (2005) 1-256] suggest the three-test battery will produce a significant number of false as well as real positives. Whilst we are aware of the desire to reduce animal experiments, determining the relevance of positive results in any of the three recommended in vitro assays will most likely have to be determined by use of in vivo assays. The bone marrow micronucleus test using routes of administration such as oral or intraperitoneal may be used where the objective is extended hazard identification. If negative results are obtained in this test, then a second in vivo test should be conducted. This could be an in vivo UDS in rat liver or a Comet assay in a relevant tissue. However, for hazard characterisation, tests using topical application with measurement of genotoxicity in the skin would be more appropriate. Such specific site-of-contact in vivo tests would minimise animal toxicity burden and invasiveness, and, especially for hair dyes, be more relevant to human routes of exposure, but there are not sufficient scientific data available to allow recommendations to be made. The generation of such data is encouraged.

An assessment of the utility of the yeast GreenScreen assay in pharmaceutical screening.

In this paper we describe an initial reproducibility study of 12 proprietary compounds followed by the assessment of 51 marketed pharmaceuticals and, lastly, a summary of the data so far from 2698 proprietary compounds from the Johnson & Johnson (J&J) compound library, in the yeast GreenScreen assay (GSA). In this assay, a reporter system in the yeast cells employs the DNA damage inducible promoter of the RAD54 gene, fused to the extremely stable green fluorescent protein (GFP). The assay proved to be very robust, the Excel templates provided by Gentronix with the assay interfaced well with in-house J&J systems with little adaptation, the assay was very rapid to perform and used very little compound. The results confirm previous work which suggests that the yeast GSA detects different classes of genotoxic compounds to the Ames assay and as a result can help screen out important genotoxic compounds at the pre-regulatory test phase that are missed by Ames-test-based screens alone. A combination of SAR evaluation of genotoxicity plus an Ames-test-based screen and the GSA provides a powerful pre-regulatory test battery to aid in the selection of successful drug candidates.

The GreenScreen genotoxicity assay: a screening validation programme.

A yeast (Saccharomyces cerevisiae) DNA repair reporter assay termed the GreenScreen assay (GSA) is described. This is a novel, cost-effective genotoxicity screen, developed to provide a pre-regulatory screening assay for use by the pharmaceutical industry and in other applications where significant numbers of compounds need to be tested. It provides a higher throughput and a lower compound consumption than existing eukaryotic genotoxicity assays and is sensitive to a broad spectrum of mutagens and, importantly, clastogens. We describe a simple, robust assay protocol and a validation study. The end-point of the test reflects the typically eukaryotic chromosomes and DNA metabolizing enzymes of yeast. The capacity for metabolic activation (MA) in yeast is limited compared with the mammalian liver or its extracts, but the assay does detect a subset of compounds that would require MA in existing genotoxicity tests. The GSA detects a different spectrum of compounds to bacterial genotoxicity assays and thus, together with an in silico structure-activity relationship (SAR) screen, and possibly a high throughput bacterial screen, would provide an effective preview of the regulatory battery of genotoxicity tests.

The preclinical toxicological evaluation of sumatriptan.

1 Sumatriptan is a potent and selective 5-HT1 receptor agonist marketed for the treatment of migraine by both oral and subcutaneous routes. An extensive toxicological programme employing high doses of sumatriptan was carried out in a range of animal species. The studies evaluated both the local and systemic tolerance to single and repeated dosing, effects on all stages of reproduction, as well as the genotoxic and oncogenic potential of sumatriptan. 2 The administration of relatively high single and repeated doses of sumatriptan was well tolerated by both rodents and dogs by the oral, subcutaneous and intravenous routes. Behavioural effects, suggestive of involvement of the central nervous system, were the most obvious result of such doses and were generally more pronounced in dogs than rodents. The reason for this may be related to the higher plasma concentrations of the drug achievable in dogs. Additional observations restricted to dogs, were transient, and included tachycardia, facial oedema and breaks in the continuity of secretion films on the corneal surface. A tendency for an increase in weight gain was seen for rats, while a slight decrease was usually seen for dogs. The only pathological changes related to treatment with high concentrations of sumatriptan consisted of local reactions at the site of subcutaneous administration. 3 Sumatriptan is an indole; the structures of this chemical class show varying propensities for nitrosation. However, appropriate testing with sumatriptan failed to identify any mutagenic nitroso compounds. 4 Sumatriptan was neither genotoxic nor oncogenic. 5 Reproductive studies demonstrated that sumatriptan was not teratogenic and had no effect on peri- and postnatal development. Some embryotoxicity was observed, but only at maternally toxic doses. A slight decrease in the success of insemination was also noted at high oral doses in rats. 6 Results of the toxicological programme performed in support of migraine therapy with sumatriptan provide good assurance of safety for subcutaneous and oral use.

Investigations into the induction of aneuploidy and polyploidy in mammalian cells by the anti-tussive agent noscapine hydrochloride.

Noscapine, a non-narcotic, centrally-acting anti-tussive drug induces polyploidy in Chinese hamster CHL cells; further studies were carried out to investigate whether similar effects could be induced in other rodent cells (Chinese hamster V79) and in human lymphocytes. In both cases, large increases in the frequency of polyploid cells were induced at test concentrations ranging from 15 to 120 micrograms/ml after 24 and 48 h continuous treatment in the absence of S9 mix. In addition, spindle damage was observed in V79 cells and human skin fibroblasts after 24 h treatment with test concentrations of 30 and 60 micrograms/ml. Furthermore, after treatment of human skin fibroblasts there was a marked increase in the proportion of cells containing chromosomes which had become dislocated from the spindle. Treatment of the mouse/human hybrid cell line R3-5 induced a significant increase in the number of 6-thioguanine resistant colonies and it was confirmed cytogenetically that these colonies had arisen due to loss of human chromosome 2. From these experiments it can be concluded that noscapine induces polyploidy in both rodent and human somatic cells, and that this could arise through a direct effect upon spindle structure and/or function. The aneugenic properties of noscapine are less certain and further work is required in this area. Exposure to the drug through its therapeutic use (15mg up to four times daily) could exceed, at least locally within the gastrointestinal (GI) tract, the concentration range shown to be active in these in vitro studies. An immediate topical hazard might exist within the buccal cavity and GI tract, but further confirmation of these in vitro results are required using suitable in vivo systems before definite conclusions can be made regarding any potential hazard associated with the administration of this drug.

Plasmid pGW16, a derivative of pKM101, increases post-UV DNA synthesis, but sensitises some strains of Escherichia coli to UV.

Plasmid pKM101, which carries muc genes that are analogous in function to chromosomal umu genes, protected Escherichia coli strains AB1157 uvrB+ umuC+, JC3890 uvrB umuC+, TK702 uvrB+ umuC and TK501 uvrB umuC against ultraviolet irradiation (UV). Plasmid pGW16, a derivative of pKM101 selected for its increased spontaneous mutator effect, also gave some protection to the UmuC-deficient strains, TK702 and TK501. However, it sensitised the wild-type strain AB1157 to low, but protected against high doses of UV, whilst sensitising strain JC3890 to all UV doses tested. Even though its UV-protecting effects varied, pGW16 was shown to increase both spontaneous and UV-induced mutation in all strains. Another derivative of pKM101, plasmid pGW12, was shown to have lost all spontaneous and UV-induced mutator effects and did not affect post-UV survival. Plasmids pKM101 and pGW16 increased post-UV DNA synthesis in strains AB1157 and TK702, whereas pGW12 had no effect. Similarly, the wild-type UV-protecting plasmids R46, R446b and R124 increased post-UV DNA synthesis in strain TK501, but the non-UV-protecting plasmids R1, RP4 and R6K had no effect. These results accord with the model for error-prone DNA repair that requires umu or muc gene products for chain elongation after base insertion opposite non-coding lesions. They also suggest that the UV-sensitizing effects of pGW16 on umu+ strains can be explained in terms of overactive DNA repair resulting in lethal, rather than repaired UV-induced lesions.

Uptake of tritiated thymidine by cells of the rat gastric mucosa after exposure to loxtidine or omeprazole.

The H2-antagonist loxtidine and the H+/K(+)-ATPase inhibitor omeprazole inhibit gastric acid secretion and both have been associated with the appearance of gastric tumours in rat cancer studies. Loxtidine is not genotoxic in a range of in vitro and in vivo assays. As false negative results can occur if the organotropic nature of the drug is not considered, both drugs were evaluated using an assay which estimates the uptake of tritiated thymidine by cells of the gastric mucosa (the target tissue) in comparison with the positive control, N-methyl-N-nitro-nitrosoguanidine (MNNG), which others have shown to induce genetic damage in the stomach mucosa of rats. Such uptake may be, in part, indicative of unscheduled DNA synthesis (UDS) resultant from genotoxic damage. Serum gastrin levels were also determined at various times after either loxtidine or omeprazole treatment. Increased uptake of tritiated thymidine was only obtained after omeprazole or MNNG treatment, when this was estimated scintillometrically. The nature of the formulation of omeprazole was critical. The uptake of tritiated thymidine was greatest when omeprazole was administered in vehicle which had been buffered to pH 9. These effects were unlikely to be due to the trophic effects of gastrin since serum gastrin levels were similar after either loxtidine or omeprazole treatment. Autoradiographic analysis of stomach sections was also carried out and revealed a 2- to 3-fold increase in the number of labelled cells within the fundic mucosa as compared to the control values after treatment with MNNG or Losec (enteric coated granules of omeprazole).(ABSTRACT TRUNCATED AT 250 WORDS)

Relevance of plasmid pKM101-mediated mutagenicity in bacteria to genotoxicity in mammalian cells.

Three structurally related compounds, 4-acetoxy-3-acetoxy-methyl-acetophenone (AAMAP), 1-[4'-hydroxy-3'-hydroxy-methylphenyl]-2-[benzyl-t-butylamino] ethanone hydrochloride (HHBEH) and 1-[4'-hydroxy-3'-hydroxymethyl-phenyl]-2-[benzyl-t-butylamino] ethanol (HHBE), gave positive dose-related mutagenic responses in the Ames test when Salmonella typhimurium strain TA100 was used as the test organism. Strain TA100 carries the hisG46 allele, which is revertable by base changes, together with plasmid pKM101, which encodes mucAB genes that are analogous to umuDC, the chromosomal SOS-repair genes of Escherichia coli K-12. None of the compounds was mutagenic in Ames strain TA1535, which is the plasmid-free derivative of strain TA100. Only AAMAP, and that at only the highest concentration tested, was mutagenic in strain TA98, which detects frameshift mutations and carries plasmid pKM101. No compound was significantly mutagenic in strain TA1538, which is the plasmid-free derivative of strain TA98. When the three compounds were tested for the induction of sister-chromatid exchanges (SCEs) in Chinese hamster cells, the two more potent mutagens, AAMAP and HHBEH were found to increase SCEs, whereas HHBE did not give a significant response at any concentration tested. Ames test data showing plasmid pK101-dependent mutagenesis are therefore, at least for these compounds, relevant indicators of eukaryotic genotoxicity.

Studies of error-prone DNA repair in Escherichia coli K-12 and Ames Salmonella typhimurium strains using a model alkylating agent.

4-Acetoxy-3-acetoxymethyl acetophenone (AAMAP) is mutagenic in Ames Salmonella typhimurium tester strains TA100 and TA98, which carry plasmid pKM101, but not in the isogenic plasmid-less strains TA1535 and TA1538. Similarly, no AAMAP-induced reversion of the his-4 allele is detectable in Escherichia coli K-12 umuC strains in the absence of the plasmid, even when the strains are treated with ethylene-diaminetetraacetate to increase permeability, or when the uvrB allele is introduced to increase error-prone DNA repair. AAMAP is, however, mutagenic in umuC+ strains or in umuC strains in which plasmid pKM101 has been introduced, suggesting that the plasmid-encoded MucAB or the chromosomally determined UmuDC proteins are required for mutagenesis. Mutation frequencies are higher in E. coli umuC (pKM101) strains, which resemble Ames tester strains of S. typhimurium, than in E. coli umuC+ or even umuC+ (pKM101) strains. Therefore, providing that the recommended pKM101-containing tester strains are used, the apparent absence of Umu-like protein activity in S. typhimurium may actually increase the sensitivity of the Ames test for the detection of mutagens that require error-prone DNA repair for activity.

Ondansetron: pre-clinical safety evaluation.

A programme of pre-clinical safety evaluation of ondansetron has been undertaken which involved a series of studies - single dose studies, repeat dose studies, reproduction studies, genotoxicity studies, oncogenicity studies, local irritancy studies, and a hypersensitivity study. Ondansetron was found to have a very good safety profile, and the only toxicity identified was associated with central nervous system activity when near lethal doses were administered. It was not genotoxic and had no reproductive or oncogenic potential.

Investigations into the genotoxic potential of loxtidine, a long-acting H2-receptor antagonist.

Loxtidine, a potent, non-competitive histamine H2-receptor antagonist was evaluated for genotoxic potential using a range of short-term mutagenicity assays. Unequivocally negative results were obtained in a Salmonella/plate incorporation assay and a liquid pre-incubation assay (using S. typhimurium strains TA1535, TA100, TA1537, TA1538 and TA98), a fluctuation assay [using Escherichia coli strains WP2, WP2 uvrA (R46) and 343/113 lys60 (R46)], a gene conversion assay (using Saccharomyces cerevisiae JD1) and a human peripheral lymphocyte cytogenetic assay. All of these in vitro tests were carried out in the presence and absence of rat liver S9 mix. In addition, the major metabolites of loxtidine in the rat were also negative in the same range of microbial mutagenicity assays. Loxtidine was inactive in the mouse micronucleus test after oral administration. The potential nitrosatability of loxtidine was investigated using an expanded version of the WHO Nitrosation Assay Procedure, and detectable quantities of mutagenic nitroso-species were not formed. The subsequent appearance of carcinoid tumours within the gastric fundus of rodents treated orally with loxtidine for most of their natural lifespan, led to additional assays being carried out on this compound to determine whether the tumorigenic effects were due to alternative mutagenic mechanisms. Negative results were obtained in an in vitro unscheduled DNA synthesis assay using primary rat hepatocytes, and an assay for spindle damaging agents using Muntjac skin fibroblasts. It can be concluded from these results that loxtidine is unlikely to be a genotoxic carcinogen. The increase in carcinoid tumour incidence observed in rats and mice after loxtidine treatment was probably related to the prolonged achlorhydria produced by this potent unsurmountable histamine H2-receptor antagonist.

The predictive value of batteries of short-term tests for carcinogens.

Carcinogenesis is a multistage process in which many factors may affect the outcome. No single short-term test, measuring a single endpoint, can predict chemically induced carcinogenesis with infallibility. Therefore it is necessary to use a battery of tests when screening new compounds. Ideally, tests to be used in this way should be validated rigorously. Test batteries should not be used in isolation but should be complemented by information from studies of metabolism, pharmacokinetics, etc. Such an approach is proving valuable for the detection of carcinogens already present in the environment and for screening new chemicals for latent hazards.