Potential impurities in drug substances: Compound-specific toxicology limits for 20 synthetic reagents and by-products, and a class-specific toxicology limit for alkyl bromides.

This paper provides compound-specific toxicology limits for 20 widely used synthetic reagents and common by-products that are potential impurities in drug substances. In addition, a 15 µg/day class-specific limit was developed for monofunctional alkyl bromides, aligning this with the class-specific limit previously defined for monofunctional alkyl chlorides. Both the compound- and class-specific toxicology limits assume a lifetime chronic exposure for the general population (including sensitive subpopulations) by all routes of exposure for pharmaceuticals. Inhalation-specific toxicology limits were also derived for acrolein, formaldehyde, and methyl bromide because of their localized toxicity via that route. Mode of action was an important consideration for a compound-specific toxicology limit. Acceptable intake (AI) calculations for certain mutagenic carcinogens assumed a linear dose-response for tumor induction, and permissible daily exposure (PDE) determination assumed a non-linear dose-response. Several compounds evaluated have been previously incorrectly assumed to be mutagenic, or to be mutagenic carcinogens, but the evidence reported here for such compounds indicates a lack of mutagenicity, and a non-mutagenic mode of action for tumor induction. For non-mutagens with insufficient data to develop a toxicology limit, the ICH Q3A qualification thresholds are recommended. The compound- and class-specific toxicology limits described here may be adjusted for an individual drug substance based on treatment duration, dosing schedule, severity of the disease and therapeutic indication.

Arsenate (As V) in water: quantitative sensitivity relationships among biomarker, ecotoxicity and genotoxicity endpoints.

It is useful to test ecotoxicity and genotoxicity endpoints in the environmental impact assessment. Here, we compare and discuss ecotoxicity and genotoxicity effects in organisms in response to exposure to arsenate (As V) in solution. Eco(geno)toxicity responses in Aliivibrio fischeri, Lytechinus variegatus, Daphnia magna, Skeletonema costatum and Vicia faba were analyzed by assessing different endpoints: biomass growth, peroxidase activity, mitotic index, micronucleus frequency, and lethality in accordance with the international protocols. Quantitative sensitivity relationships (QSR) between these endpoints were established in order to rank endpoint sensitivity. The results for the QSR values based on the lowest observed effect concentration (LOEC) ratios varied from 2 (for ratio of root peroxidase activity to leaf peroxidase activity) to 2286 (for ratio of higher plant biomass growth to root peroxidase activity). The QSR values allowed the following sensitivity ranking to be established: higher plant enzymatic activity>daphnids≈echinoderms>bacteria≈algae>higher plant biomass growth. The LOEC values for the mitotic index and micronucleus frequency (LOEC=0.25mgAsL(-1)) were similar to the lowest LOEC values observed in aquatic organisms. This approach to the QSR of different endpoints could form the basis for monitoring and predicting early effects of pollutants before they give rise to significant changes in natural community structures.

Gene-TEQ--a standardized comparative assessment of effects in the comet assay using genotoxicity equivalents.

Existing methods for the comparison of genotoxic effects in the comet assay bear considerable disadvantages such as the problem to link information about concentration dependence and severity of effects. Moreover, given the lack of standardized protocols and the use of various standards, it may be extremely difficult to compare different studies. In order to provide a method for standardized comparative assessment of genotoxic effects, the concept of genotoxicity equivalents (Gene-TEQ) was developed. As potential reference compounds for genotoxic effects, three directly acting (N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl-methanesulfonate, and N-methyl-N-nitrosourea) and three indirectly acting (cyclophosphamide, dimethylnitrosamine, and 4-nitroquinoline-oxide) genotoxic substances were compared with respect to their cytotoxic (neutral red) and genotoxic (comet assay) concentration-response profiles in the permanent fish cell line RTL-W1. For further comparison, two sediment extracts from the upper Danube River were investigated as environmental samples. Based on the results of cytotoxicity and genotoxicity testing, MNNG was selected as the reference compound. At several exposure levels and durations, genotoxic effects of both the other pure substances and the environmental samples were calculated as percentages of the maximum MNNG effect and related to the absolute MNNG effect (EC values). Thus, genotoxicity equivalent factors (Gene-TEQs) relative to MNNG could be calculated. Gene-TEQs can easily be applied to pure substances, mixtures and field samples to provide information about their toxicity relative to the reference compound. Furthermore, the Gene-TEQ concept allows a direct comparison of environmental samples from different laboratories.

Genotoxic potential of arsenic at its reference dose.

Arsenic, a highly hazardous contaminant in our drinking water, accounts for various toxic effects (including cancer) in human. However, intake of arsenic @0.3 µg kg(-1)day(-1) through drinking water, containing arsenic at its guideline value or maximum contaminant limit (10 µg L(-1)), has been estimated to pose very little or no measurable risk to cancer in humans. The value also appears to be equal to the human reference dose (or index dose) of arsenic based on human skin toxicity data. The present work was a quantitative assessment of the genotoxic potential of arsenic in mice at doses equivalent to its human reference dose as well as its multiples. Significant increases in the frequencies of chromosome abnormalities in the bone marrow cells were registered over the control level upon exposure to all the doses of arsenic including its reference dose (or index dose). The assessment of arsenic genotoxicity in humans at low doses will therefore be highly instrumental in establishing a permissible limit of arsenic in drinking water.

In silico methods combined with expert knowledge rule out mutagenic potential of pharmaceutical impurities: an industry survey.

With the increasing emphasis on identification and low level control of potentially genotoxic impurities (GTIs), there has been increased use of structure-based assessments including application of computerized models. To date many publications have focused on the ability of computational models, either individually or in combination, to accurately predict the mutagenic effects of a chemical in the Ames assay. Typically, these investigations take large numbers of compounds and use in silico tools to predict their activity with no human interpretation being made. However, this does not reflect how these assessments are conducted in practice across the pharmaceutical industry. Current guidelines indicate that a structural assessment is sufficient to conclude that an impurity is non-mutagenic. To assess how confident we can be in identifying non-mutagenic structures, eight companies were surveyed for their success rate. The Negative Predictive Value (NPV) of the in silico approaches was 94%. When human interpretation of in silico model predictions was conducted, the NPV increased substantially to 99%. The survey illustrates the importance of expert interpretation of in silico predictions. The survey also suggests the use of multiple computational models is not a significant factor in the success of these approaches with respect to NPV.

Genotoxicity and oxidative stress biomarkers in Carassius gibelio as endpoints for toxicity testing of Ukrainian polluted river waters.

This study aimed to assess oxidative stress and genotoxicity biomarkers in Prussian carp Carassius gibelio laboratory-exposed to water from polluted Ukrainian rivers in order to evaluate their usefulness as endpoints in a short-term bioassay for toxicity testing of freshwaters. The micronucleus (MN) test and the frequency of cells with double nuclei (DN) in erythrocytes and gill cells were used as indicators of chromosome aberrations and abnormalities in cell divisions, respectively. Cellular antioxidant defenses i.e. antioxidant enzyme activities (catalase, Se-dependent glutathione peroxidase, total glutathione peroxidase and glutathione-S-transferase) and oxidative damage, i.e. lipid peroxidation (measured as thiobarbituric acid reactive substances) in the fish liver were used as biomarkers of oxidative stress. Exposure to the polluted river water samples for 96 h resulted in significantly increased MN and DN frequencies, limited increases in antioxidant enzyme activities and no changes in lipid peroxidation. Results suggest that MN and DN frequencies in C gibelio are useful endpoints in a short-term bioassay for genotoxicity testing of environmental water samples in contrast to the oxidative stress biomarkers applied that showed low potential for assessing sublethal effects after a 96 h exposure.

DNA damage in Gammarus fossarum sperm as a biomarker of genotoxic pressure: intrinsic variability and reference level.

In the perspective of a biomonitoring application for assessing genotoxicity of freshwater ecosystems, the Comet assay has recently been developed on spermatozoa in the amphipod Gammarus fossarum, in order to propose a sensitive and reliable genotoxicity biomarker in an ecologically relevant freshwater species. The appropriate use of a genotoxicity biomarker requires good knowledge of its basal level and its natural variability related to intrinsic biotic and environmental abiotic factors. We propose a procedure for which the lowest biomarker variability related to methodological and intrinsic biotic factors is obtained and a reference value of biomarker basal response taking into account its spatio-temporal changes has been defined. A strong impact of spermatogenesis status and exposure time on the response to genotoxicant pressure was observed. These reports led us to select a standard organism, i.e., the mature male gammarid in precopula. No effect of temperature and conductivity on baseline DNA damage was observed in the laboratory for the tested range (6-24 °C and 300/600 µS cm⁻¹). Similarly, no spatio-temporal change relative to season or the physico-chemical characteristics of the water was recorded during the field survey. On the basis of these results, a reference level with maximal threshold values has been proposed for the standard gammarid.

Follow-up actions from positive results of in vitro genetic toxicity testing.

Appropriate follow-up actions and decisions are needed when evaluating and interpreting clear positive results obtained in the in vitro assays used in the initial genotoxicity screening battery (i.e., the battery of tests generally required by regulatory authorities) to assist in overall risk-based decision making concerning the potential effects of human exposure to the agent under test. Over the past few years, the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Project Committee on the Relevance and Follow-up of Positive Results in In Vitro Genetic Toxicity (IVGT) Testing developed a decision process flow chart to be applied in case of clear positive results in vitro. It provides for a variety of different possibilities and allows flexibility in choosing follow-up action(s), depending on the results obtained in the initial battery of assays and available information. The intent of the Review Subgroup was not to provide a prescriptive testing strategy, but rather to reinforce the concept of weighing the totality of the evidence. The Review Subgroup of the IVGT committee highlighted the importance of properly analyzing the existing data, and considering potential confounding factors (e.g., possible interactions with the test systems, presence of impurities, irrelevant metabolism), and chemical modes of action when analyzing and interpreting positive results in the in vitro genotoxicity assays and determining appropriate follow-up testing. The Review Subgroup also examined the characteristics, strengths, and limitations of each of the existing in vitro and in vivo genotoxicity assays to determine their usefulness in any follow-up testing.

International amphibian micronucleus standardized procedure (ISO 21427-1) for in vivo evaluation of double-walled carbon nanotubes toxicity and genotoxicity in water.

Considering the important production of carbon nanotubes (CNTs), it is likely that some of them will contaminate the environment during each step of their life cycle. Nevertheless, there is little known about their potential ecotoxicity. Consequently, the impact of CNTs on the environment must be taken into consideration. This work evaluates the potential impact of well characterized double-walled carbon nanotubes (DWNTs) in the amphibian larvae Xenopus laevis under normalized laboratory conditions according to the International Standard micronucleus assay ISO 21427-1:2006 for 12 days of half-static exposure to 0.1-1-10 and 50 mg L(-1) of DWNTs in water. Two different endpoints were carried out: (i) toxicity (mortality and growth of larvae) and (ii) genotoxicity (induction of micronucleated erythrocytes). Moreover, intestine of larvae were analyzed using Raman spectroscopy. The DWNTs synthetized by catalytic chemical vapor deposition (CCVD) were used as produce (experiment I) and the addition of Gum Arabic (GA) was investigated to improve the stability of the aqueous suspensions (experiment II). The results show growth inhibition in larvae exposed to 10 and 50 mg L(-1) of DWNTs with or without GA. No genotoxicity was evidenced in erythrocytes of larvae exposed to DWNTs, except to 1 mg L(-1) of DWNTs with GA suggesting its potential effect in association with DWNTs at the first nonacutely toxic concentration. The Raman analysis confirmed the presence of DWNTs into the lumen of intestine but not in intestinal tissues and cells, nor in the circulating blood of exposed larvae.

Carcinogenicity categorization of chemicals-new aspects to be considered in a European perspective.

Existing systems of classification of carcinogens are a matter of discussion, world-wide. There is agreement that it should be distinguished between genotoxic and non-genotoxic chemicals. The risk assessment approach used for non-genotoxic chemicals is similar among different regulatory bodies: insertion of an uncertainty (safety) factor permits the derivation of permissible exposure levels at which no relevant human cancer risks are anticipated. For genotoxic carcinogens, case studies of chemicals point to a whole array of possibilities. Positive data of chromosomal effects only, in the absence of mutagenicity, may support the characterization of a compound that produces carcinogenic effects only at high, toxic doses. Non-DNA-reactive genotoxins, such as topoisomerase inhibitors or inhibitors of the spindle apparatus are considered in this respect. In such cases, arguments are in favour of the existence of "practical" thresholds. Taking existing concepts together, it is proposed to basically distinguish between "perfect" and "practical" thresholds. There is a wide consensus that for non-DNA-reactive genotoxins such as aneugens (aneuploidy, chromosome loss, non-disjunction) thresholds should be defined. It is being discussed as to whether the identification of possible threshold effects should also include other mechanisms of genotoxicity, in addition to aneugenic effects. Specific mechanisms of clastogenicity have been repeatedly addressed as also having thresholds, such as topoisomerase II poisons or mechanisms based on reactive oxygen. Oxidative stress as an important mechanism is triggered by exposure to exogenous factors such as ultraviolet (UV) and ionizing radiation, anoxia and hyperoxia, and by chemicals producing reactive oxygen species. The idea is receiving increased support that reactive oxygen species (ROS)-mediated processes of carcinogenesis have practical thresholds. Since reactive oxygen species are genotoxic in principle, questions arise whether chemicals that increase ROS production will superimpose to an endogenously produced background level of DNA lesions, related to mechanisms that may result in non-linear dose-effect relationships. The existence of "endogenous" DNA adducts has been generally accepted, and possible regulatory implications of the presence of endogenous carcinogens have been discussed. It is now becoming evident that a diversity of methods of carcinogenic risk extrapolation to low doses must be considered, dependent on the mode of action. Although there is an increasing international awareness of these developments, the system of classification of carcinogens of the European Union still remains static. This should be changed, as the philosophy of separation of a strictly sequential "hazard assessment" and "risk assessment" appears out-of-date.

[European Union legislation related to patulin]. / Nowe przepisy Unii Europejskiej w zakresie patuliny.

In the following review, European Union legislation concerning maximum level of patulin in foodstuffs was reported. Commission Regulation (EC) No 1425/2003 of 11 August 2003 amending Regulation (EC) No 466/2001, Commission Directive 2003/78/EC of 11 August 2003 laying down the sampling methods and the methods of analysis for the official control of the levels of patulin in foodstuffs and Commission Recommendation of 11 August 2003 on the prevention and reduction of patulin contamination in apple juice and apple juice ingredients in other beverages as regards patulin was reported.

Genotoxicity tests for new chemicals in Germany: routine in vitro test systems.

According to regulations in the European Union, new chemical substances must be notified before they can be introduced onto the market. One of the prerequisites for notification is that toxicological properties, including mutagenicity, are examined. In this paper, a report on routine in vitro mutagenicity testing is given for 776 new substances notified in Germany between 1982 and 1997. In general, the methodological quality of testing was in line with internationally accepted guidelines. Bacterial gene mutation tests (Bact) were conducted for nearly all of the substances, 13.4% were positive. Of the Bact-positive substances, 36 were also tested in the in vitro chromosomal aberration test (CAbvit) and the mammalian cell gene mutation test (MCGM). Twenty-six of these (72. 2%) were negative in both mammalian cell tests indicating that the genotoxic potentials of the substances are not relevant for man. Of all new substances, 333 were tested in CAbvit, here the percentage of positive findings was 25.2%. More than 80% of the in vitro clastogens were negative in the Bact. With respect to a sensitive detection of genotoxic potentials of substances, the combination 'Bact+CAbvit' is appropriate for basic testing. In our database CHL cells were more sensitive to clastogenic effects than other cell types. Only very few clastogens were identified as 'high toxicity clastogens'. MCGM tests were performed for 118 substances, quite often as follow-up in case of positive Bact tests. In total, 12.7% of the substances were positive in the MCGM. However, there was a clear difference in the frequencies of positive findings in HPRT tests (5.5%) and mouse lymphoma assays (MLA; 37.0%). None of the MCGM-positive substances was a 'unique positive', i.e., negative in Bact and CAbvit.

Research on the mechanisms of action of aneugenic chemicals and regulatory approaches for their control in the European Communities.

The European Communities have developed a wide range of regulatory instruments for the control of chemical products sold and used within its geographical area. An important part of the testing requirements for most chemicals within the European Communities is the preparation of an information package on the potential mutogen properties of each chemical. Currently, no test requirements specify a unique test for aneugenic activity, although current methods such as in vitro cytogenetic and bone marrow micronucleus assays provide some useful indirect information on aneugenic activity. During the past 15 years the European Communities supported a series of collaborative research projects that have investigated the mechanisms by which chemicals induce aneuploidy and developmental studies of test methods for the detection of aneugenic chemicals. These projects led to the development of in vitro methods for the detection and quantification of induced nondisjunction and chromosome loss and the measurement of aneuploidy in rodent bone marrow. The European Communities projects have demonstrated the aneugenic potential of a diverse range of chemicals and their potential role in inherited disease and tumour induction. However, regulatory guidelines have yet to be modified to take advantage of the methods developed for the detection and evaluation of aneugenic chemicals.

[Genetic-hygienic regulation of pesticide use taking into account their potential mutagenic danger]. / K voprosu o genetiko-gigienicheskoi reglamentatsii primeneniia pestitsidov s uchetom ikh potentsial'noi mutagennoi opasnosti.

The principle is described of genetic-hygienic reglamentation of pesticides-mutagens application based on the estimating of the degree of their genetic hazard using several parameters characterizing the peculiarities of their mutagenic effect. Substances may be classified into groups according to their mutagenic hazard. The results are given of the evaluation of the mutagenic hazards of some pesticides and recommendations are given for the genetic-hygienic reglamentation of their application taking into account their mutagenicity.

Test systems for assessing mutagenic potential of chemical substances.

The first report that certain chemicals have the potential of inducing heritable effects (mutation) appeared in the mid-1940's. Two decades passed before this and subsequent observations were translated into a concern that some of the chemicals to which we are exposed may constitute a hazard to man's genetic material and hence a threat to future generations of individuals. Such concern has led to the gradual evolution of the newest subdiscipline of toxicology, genetic toxicology. This subdiscipline is still young and faces many challenges in terms of developing sound toxicologic approaches for meeting specific evaluation needs in the interest of public health. This paper describes the current status of efforts in genetic toxicology and the types of steps which must be taken in order to begin to meet the needs and requirements of regulatory agencies.