A weight of evidence review of the genotoxicity of titanium dioxide (TiO2).

Titanium dioxide is a ubiquitous white material found in a diverse range of products from foods to sunscreens, as a pigment and thickener, amongst other uses. Titanium dioxide has been considered no longer safe for use in foods (nano and microparticles of E171) by the European Food Safety Authority (EFSA) due to concerns over genotoxicity. There are however, conflicting opinions regarding the safety of Titanium dioxide. In an attempt to clarify the situation, a comprehensive weight of evidence (WoE) assessment of the genotoxicity of titanium dioxide based on the available data was performed. A total of 192 datasets for endpoints and test systems considered the most relevant for identifying mutagenic and carcinogenic potential were reviewed and discussed for both reliability and relevance (by weight of evidence) and in the context of whether the physico-chemical properties of the particles had been characterised. The view of an independent panel of experts was that, of the 192 datasets identified, only 34 met the reliability and quality criteria for being most relevant in the evaluation of genotoxicity. Of these, 10 were positive (i.e. reported evidence that titanium dioxide was genotoxic), all of which were from studies of DNA strand breakage (comet assay) or chromosome damage (micronucleus or chromosome aberration assays). All the positive findings were associated with high cytotoxicity, oxidative stress, inflammation, apoptosis, necrosis, or combinations of these. Considering that DNA and chromosome breakage can be secondary to physiological stress, it is highly likely that the observed genotoxic effects of titanium dioxide, including those with nanoparticles, are secondary to physiological stress. Consistent with this finding, there were no positive results from the in vitro and in vivo gene mutation studies evaluated, although it should be noted that to definitively conclude a lack of mutagenicity, more robust in vitro and in vivo gene mutation studies would be useful. Existing evidence does not therefore support a direct DNA damaging mechanism for titanium dioxide (nano and other forms).

A tiered approach to investigate the inhalation toxicity of cobalt substances. Tier 4: Effects from a 28-day inhalation toxicity study with tricobalt tetraoxide in rats.

Lung cancer following inhalation in rodents is a major concern regarding exposure to cobalt substances. However, little information is available on adverse effects and toxicity following long-term inhalation exposure to poorly soluble cobalt substances with low bioavailability. Thus, the present study focused on pulmonary effects of the poorly soluble tricobalt tetraoxide (5, 20, 80 mg/m³) in a 28-day inhalation exposure study. Lung weights increased with increasing exposures. Bronchoalveolar lavage fluid analysis and histopathology revealed lung tissue inflammation at the mid-dose with increasing severity in the high-dose group and post-exposure persistency. Markers for cellular damage and cell proliferation were statistically significantly increased. No increase in 8-OH-dG lesions was observed, indicating an absence of oxidative DNA lesions. The primary effect of inhaled Co3O4 particles is inflammation of the respiratory tract strongly resembling responses of inhaled "inert dust" substances, with a NOAEC of 5 mg/m³ under the conditions of this test.

A tiered approach to investigate the inhalation toxicity of cobalt substances. Tier 3: Inflammatory response following acute inhalation exposure correlates with lower tier data.

In vitro studies have shown that cobalt substances predominantly induce pre-inflammatory biomarkers, resulting in a grouping of substances either predicted to cause inflammation following inhalation, or those with a different reactivity profile (poorly-reactive). There is a lack of data on whole-organ lung responses following inhalation of these substances, especially relating to the poorly-reactive group. It is of interest to generate tissue-specific histopathological correlation to better ascertain the predictive nature of the lower tier tests (i.e. tier 1 - bioelution, tiers 2a and b - in vitro markers and ToxTracker testing), in order to understand the type of effects caused by the poorly-reactive group and to gauge long-term effects. Eight cobalt substances were tested in vivo in a customized 4-h toxicity test; with animals sacrificed up to 14-days post-exposure. Histopathological severity scores were assigned based on inflammatory and pre-carcinogenic markers. A clear pattern emerged, with the reactive substances causing a persistent increase in one or more of the selected markers, and absence of these markers with poorly-reactive substances. Longer-term studies should be conducted to investigate the repeated dose effects of the poorly-reactive substances.

The underlying mode of action for lung tumors in a tiered approach to the assessment of inhaled cobalt compounds.

A mode of action (MOA) for cobalt substances based on the "International Programme on Chemical Safety Conceptual Framework for Evaluating a MOA for Chemical Carcinogenesis" is presented. The data recorded therein were generated in a tiered testing program described in the preceding papers of this special issue, as well as data from the public domain. The following parameters were included in the evaluation: solubility of cobalt substances in artificial lung fluids (bioelution), in vitro biomarkers for cytotoxicity, reactive oxygen species and hypoxia mimicry, inhalation toxicity following acute exposure and repeated dose inhalation effects. Two distinct groups of cobalt substances emerged: substances inducing all effects across the MOA form one group, associated with the adverse outcome of lung cancer in rodents upon chronic exposure. Another group of cobalt substances induces no or very limited effects in the in vitro and acute testing. Higher tier testing with a representative of this group, tricobalt tetraoxide, showed a response resembling rat lung overload following exposure to high concentrations of poorly soluble particles. Based on the fundamental differences in the lower tier toxicological profile, cobalt substances with an unknown hazard profile can be assigned to either group based on lower tier testing alone.

Bioelution, Bioavailability, and Toxicity of Cobalt Compounds Correlate.

Based on the wide use of cobalt substances in a range of important technologies, it has become important to predict the toxicological properties of new or lesser-studied substances as accurately as possible. We studied a group of 6 cobalt substances with inorganic ligands, which were tested for their bioaccessibility (surrogate measure of bioavailability) through in vitro bioelution in simulated gastric and intestinal fluids. Representatives of the group also underwent in vivo blood kinetics and mass balance tests, and both oral acute and repeated dose toxicity (RDT) testing. We were able to show a good correlation between high in vitro bioaccessibility with high in vivo bioavailability and subsequent high in vivo toxicity; consequently, low in vitro bioaccessibility correlated well with low in vivo bioavailability and low in vivo toxicity. In vitro bioelution in simulated gastric fluid was the most precise predictor of the difference in the oral RDT lowest observed adverse effect levels of 2 compounds representing the highly and poorly bioaccessible subset of substances. The 2 compounds cobalt dichloride hexahydrate and tricobalt tetraoxide differed by a factor of 440 in their in vitro bioaccessibility and by a factor of 310 in their RDT lowest observed adverse effect level. In summary, this set of studies shows that solubility, specifically in vitro bioelution in simulated gastric fluid, is a good, yet conservative, predictor of in vivo bioavailability and oral systemic toxicity of inorganic cobalt substances. Bioelution data are therefore an invaluable tool for grouping and read across of cobalt substances for hazard and risk assessment.

New studies on the in vitro genotoxicity of sodium molybdate and their impact on the overall assessment of the genotoxicity of molybdenum substances.

The potential of molybdenum substances to cause genotoxic effects has been studied previously. However, a review of existing in vitro data, including an assessment of relevance and reliability, has shown that inconsistent results have been observed in the past. To resolve the inconsistencies, new studies were performed with the highly soluble sodium molybdate dihydrate according to OECD test guidelines. In a bacterial reverse mutation assay sodium molybdate dihydrate did not induce reverse mutations in five strains of Salmonella typhimurium. No mutagenic or clastogenic effect was observed at the tk locus of L5178Y mouse lymphoma cells. In a micronucleus test in cultured human peripheral blood lymphocytes no clastogenic or aneugenic effects were seen. These results can be read across to other inorganic molybdenum substances, that all release the molybdate ion [MoO4]2- under physiological conditions as the only toxicologically relevant species. In summary, a weight of evidence assessment of all available in vitro data shows no evidence of genotoxicity of molybdenum substances.

Flow cytometry: interesting tool for studying binding behavior of DNA on inorganic layered double hydroxide (LDH).

BACKGROUND: A new method was established to characterize the binding kinetics of DNA toward layered double hydroxides (LDHs). The setup consisted of a newly developed sampling tube that allows the injection of analyte during the flow cytometric measurement. METHODS: Layered double hydroxides consist of cationic metal hydroxide layers and exchangeable interlayer anions. This negatively charged structure permits biomolecules such as DNA to adsorb, and a so-called DNA-LDH hybrid is formed. The hydroxide layers can be removed in acidic media and the DNA will be released. CERATOFIX (a registered trademark of Sud-Chemie AG NA that belongs to the family of LDHs, produced by Sud-Chemie AG). The chemical structure can be summarized as [Mg(2)Al(OH)(6)](CO(3))(0.5). The binding capacity and kinetic characteristics of different types of CERATOFIX NA for a model DNA was determined by flow cytometry. RESULTS: The static binding capacities of the different LDHs were determined after 1- and 16-h incubation with DNA solution, showing different binding patterns between the LDH materials. The binding kinetics were revealed by flow cytometric measurements in short-term and long-term kinetic experiments, showing that the majority of DNA adsorbs within the first 60 s. CONCLUSIONS: DNA removal from cell culture supernatants is one of the major concerns in downstream processing. Due to the anion exchange capabilities of LDHs it seemed a very interesting approach to use these materials for binding of DNA for elimination purposes.

The total synthesis of (-)-callystatin A.

Callystatin A is a prominent member of a class of natural products which display promising growth inhibition of cancer cells in their biological profile. The challenging structure and the interesting biological activity of (-)-callystatin A fueled our interest in the synthesis of this marine natural product. We achieved the total synthesis using a highly convergent approach joining four subunits together with a Wittig olefination, a selective Heck reaction and an aldol reaction as the pivotal steps. The aldol reaction as one of the final transformations during the synthesis opens fast access to a variety of structural analogues and circumvents tedious protecting group manipulations. Here we report an improved synthesis utilizing a modified vinyl iodide which shortens the synthesis by two steps. Additionally, first biological results will be reported.