The EFSA Scientific Committee addressed in this document the peculiarities related to the genotoxicity assessment of chemical mixtures. The EFSA Scientific Committee suggests that first a mixture should be chemically characterised as far as possible. Although the characterisation of mixtures is relevant also for other toxicity aspects, it is particularly significant for the assessment of genotoxicity. If a mixture contains one or more chemical substances that are individually assessed to be genotoxic in vivo via a relevant route of administration, the mixture raises concern for genotoxicity. If a fully chemically defined mixture does not contain genotoxic chemical substances, the mixture is of no concern with respect to genotoxicity. If a mixture contains a fraction of chemical substances that have not been chemically identified, experimental testing of the unidentified fraction should be considered as the first option or, if this is not feasible, testing of the whole mixture should be undertaken. If testing of these fraction(s) or of the whole mixture in an adequately performed set of in vitro assays provides clearly negative results, the mixture does not raise concern for genotoxicity. If in vitro testing provides one or more positive results, an in vivo follow‐up study should be considered. For negative results in the in vivo follow‐up test(s), the possible limitations of in vivo testing should be weighed in an uncertainty analysis before reaching a conclusion of no concern with respect to genotoxicity. For positive results in the in vivo follow‐up test(s), it can be concluded that the mixture does raise a concern about genotoxicity.

The present opinion deals with an updated safety assessment of the food additive titanium dioxide (E 171) based on new relevant scientific evidence considered by the Panel to be reliable, including data obtained with TiO 2 nanoparticles ( NP s) and data from an extended one‐generation reproductive toxicity ( EOGRT ) study. Less than 50% of constituent particles by number in E 171 have a minimum external dimension < 100 nm. In addition, the Panel noted that constituent particles < 30 nm amounted to less than 1% of particles by number. The Panel therefore considered that studies with TiO 2 NP s < 30 nm were of limited relevance to the safety assessment of E 171. The Panel concluded that although gastrointestinal absorption of TiO 2 particles is low, they may accumulate in the body. Studies on general and organ toxicity did not indicate adverse effects with either E 171 up to a dose of 1,000 mg/kg body weight (bw) per day or with TiO 2 NP s (> 30 nm) up to the highest dose tested of 100 mg/kg bw per day. No effects on reproductive and developmental toxicity were observed up to a dose of 1,000 mg E 171/kg bw per day, the highest dose tested in the EOGRT study. However, observations of potential immunotoxicity and inflammation with E 171 and potential neurotoxicity with TiO 2 NP s, together with the potential induction of aberrant crypt foci with E 171, may indicate adverse effects. With respect to genotoxicity, the Panel concluded that TiO 2 particles have the potential to induce DNA strand breaks and chromosomal damage, but not gene mutations. No clear correlation was observed between the physico‐chemical properties of TiO 2 particles and the outcome of either in vitro or in vivo genotoxicity assays. A concern for genotoxicity of TiO 2 particles that may be present in E 171 could therefore not be ruled out. Several modes of action for the genotoxicity may operate in parallel and the relative contributions of different molecular mechanisms elicited by TiO 2 particles are not known. There was uncertainty as to whether a threshold mode of action could be assumed. In addition, a cut‐off value for TiO 2 particle size with respect to genotoxicity could not be identified. No appropriately designed study was available to investigate the potential carcinogenic effects of TiO 2 NP s. Based on all the evidence available, a concern for genotoxicity could not be ruled out, and given the many uncertainties, the Panel concluded that E 171 can no longer be considered as safe when used as a food additive.

The European Commission requested EFSA to provide advice on the following: (1) the suitability of the unscheduled DNA synthesis (UDS) in vivo assay to follow-up positive results in in vitro gene mutation tests; (2) the adequacy to demonstrate target tissue exposure in in vivo studies, particularly in the mammalian erythrocyte micronucleus test; (3) the use of data in a weight-of-evidence approach to conclude on the genotoxic potential of substances and the consequent setting of health-based guidance values. The Scientific Committee concluded that the first question should be addressed in both a retrospective and a prospective way: for future assessments, it is recommended no longer performing the UDS test. For re-assessments, if the outcome of the UDS is negative, the reliability and significance of results should be carefully evaluated in a weight-of-evidence approach, before deciding whether more sensitive tests such as transgenic assay or in vivo comet assay would be needed to complete the assessment. Regarding the second question, the Scientific Committee concluded that it should be addressed in lines of evidence of bone marrow exposure: toxicity to the bone marrow in itself provides sufficient evidence to allow concluding on the validity of a negative outcome of a study. All other lines of evidence of target tissue exposure should be assessed within a weight-of-evidence approach. Regarding the third question, the Scientific Committee concluded that any available data that may assist in reducing the uncertainty in the assessment of the genotoxic potential of a substance should be taken into consideration. If the overall evaluation leaves no concerns for genotoxicity, health-based guidance values may be established. However, if concerns for genotoxicity remain, establishing health-based guidance values is not considered appropriate.

The main dose-limiting side effect of cancer treatment with platinum compounds is peripheral neurotoxicity. To investigate the intracellular mechanisms of platinum drugs neurotoxicity we have studied the effects of cisplatin and oxaliplatin on the human neuroblastoma cell line SH-SY5Y. Both platinum compounds are toxic causing cellular death by inducing apoptosis but oxaliplatin is less neurotoxic than cisplatin. The study of the proteins involved in the intracellular transduction pathways that may cause apoptotic death, revealed a very similar pattern of changes after exposure to cisplatin or oxaliplatin. In particular, as demonstrated by densitometric analysis, after exposure to both platinum compounds the total amount of the anti-apoptotic protein Bcl-2 was significantly reduced. Conversely, the amount of the pro-apoptotic protein p53 significantly increased. Caspases 3 and 7 were activated, but their activation was a late event, indicating a secondary role in the apoptotic process. Among the mitogen activated protein kinases, only the p38 protein was activated (phosphorylated) early enough to have a possible role in inducing apoptosis, possibly through p53 stabilization. The results of the present study and the data of the literature demonstrate that the ways in which cisplatin and oxaliplatin are neurotoxic are very similar and include not only DNA damage, but also the modulation of specific molecules involved in regulating the cellular equilibrium between apoptotic death and the cell cycle.

The EFSA Scientific Committee was asked to provide guidance on the most appropriate in vivo tests to follow up on positive in vitro results for aneugenicity, and on the approach to risk assessment for substances that are aneugenic but not clastogenic nor causing gene mutations. The Scientific Committee confirmed that the preferred approach is to perform an in vivo mammalian erythrocyte micronucleus test with a relevant route of administration. If this is positive, it demonstrates that the substance is aneugenic in vivo . A negative result with evidence that the bone marrow is exposed to the test substance supports a conclusion that aneugenic activity is not expressed in vivo . If there is no evidence of exposure to the bone marrow, a negative result is viewed as inconclusive and further studies are required. The liver micronucleus assay, even though not yet fully validated, can provide supporting information for substances that are aneugenic following metabolic activation. The gastrointestinal micronucleus test, conversely, to be further developed, may help to assess aneugenic potential at the initial site of contact for substances that are aneugenic in vitro without metabolic activation. Based on the evidence in relation to mechanisms of aneugenicity, the Scientific Committee concluded that, in principle, health‐based guidance values can be established for substances that are aneugenic but not clastogenic nor causing gene mutations, provided that a comprehensive toxicological database is available. For situations in which the toxicological database is not sufficient to establish health‐based guidance values, some approaches to risk assessment are proposed. The Scientific Committee recommends further development of the gastrointestinal micronucleus test, and research to improve the understanding of aneugenicity to support risk assessment.

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