1. The in vivo alkaline comet (single cell gel electrophoresis) assay (hereafter called simply the comet assay) is used for the detection of DNA strand breaks in cells or nuclei isolated from multiple tissues of animals, usually rodents, that have been exposed to potentially genotoxic material(s). The comet assay has been reviewed and recommendations have been published by various expert groups (1) (2) (3) (4) (5) (6) (7) (8) (9) (10). This Test Guideline is part of a series of Test Guidelines on genetic toxicology. A document presented as an Introduction to the Test Guidelines on genotoxicity (11) can also be referred to and provides succinct and useful guidance to users of these Test Guidelines.

Oecd guideline for testing of chemicals

TiO(2) nanoparticles (< 100 nm diameter) have been reported to cause oxidative stress related effects, including inflammation, cytotoxicity and genomic instability, either alone or in the presence of UVA irradiation in mammalian studies. Despite the fact that the aquatic environment is often the ultimate recipient of all contaminants there is a paucity of data pertaining to the potential detrimental effects of nanoparticles on aquatic organisms. Therefore, these investigations aimed to evaluate the potential cytotoxic and genotoxic effects of TiO(2) nanoparticles on goldfish skin cells (GFSk-S1), either alone or in combination with UVA. Whilst neutral red retention (NRR) assay (a measure of lysosomal membrane integrity) was used to evaluate cell viability, a modified Comet assay using bacterial lesion-specific repair endonucleases (Endo-III, Fpg) was employed to specifically target oxidative DNA damage. Additionally, electron spin resonance (ESR) studies with different spin traps were carried out for qualitative analysis of free radical generation. For cell viability, TiO(2) alone (0.1-1000 microg ml(-1)) had little effect whereas co-exposure with UVA (0.5-2.0 kJm(-2)) caused a significant dose-dependent decrease which was dependent on both the concentration of TiO(2) and the dose of UVA administered. For the Comet assay, doses of 1, 10 and 100 microg ml(-1) in the absence of UVA caused elevated levels of Fpg-sensitive sites, indicating the oxidation of purine DNA bases (i.e. guanine) by TiO(2). UVA irradiation of TiO(2)-treated cells caused further increases in DNA damage. ESR studies revealed that the observed toxic effects of nanoparticulate TiO(2) were most likely due to hydroxyl radical (OH) formation.

Hydroxyl radicals (*OH) are associated with titanium dioxide (TiO(2)) nanoparticle-induced cytotoxicity and oxidative DNA damage in fish cells.

Methods of in vitro toxicology.

Based on the current weight of evidence of all available data, the risk for humans from the use of nano-structured titanium dioxide (TiO2) or zinc oxide (ZnO) currently used in cosmetic preparations or sunscreens is considered negligible. There is a large body of information that when viewed in its entirety is considered as sufficient to demonstrate that these nano-structured ultraviolet (UV) filters, irrespective of various treatments (coatings) or crystalline structure, can be regarded as safe for use at concentrations up to 25% in cosmetic products to protect the skin from harmful effects of solar UV radiation. “Nano” TiO2 and ZnO formulated in topically applied sunscreen products exist as aggregates of primary particles ranging from 30–150 nm in size. These aggregates are bonded such that the force of sunscreen product application onto the skin would have no impact on their structure or result in the release of primary particles. Multiple studies have shown that under exaggerated test conditions neither nano-structured TiO2 nor ZnO penetrates beyond the stratum corneum of skin. Further, the distribution and persistence of these nano-structured metal oxides is the same compared to larger pigment-grade (i.e., >100 nm) particles, demonstrating equivalence in the recognition and elimination of such material from the body. Finally, the in vitro genotoxic and photogenotoxic profiles of these nano-structured metal oxides are of no consequence to human health. Whereas the most logical, straightforward conclusion based on data from internationally-recognized guideline studies and current 20+ year history of human use is that nano-structured TiO2 and ZnO are safe, there will continue to be questions as “nano” conjures images of technology gone awry. Despite this rather sober view, the public health benefits of sunscreens containing nano TiO2 and/or ZnO outweigh human safety concerns for these UV filters.

/pdf/human-safety-review-of-nano-titanium-dioxide-and-zinc-oxide-18kx5ohcoo.pdf

Human safety review of “nano” titanium dioxide and zinc oxide

http://fragrancematerialsafetyresource.elsevier.com/sites/default/files/AB-1-Ford-Criteria_0.pdf

R. Clothier

Papers

The International EU/COLIPA In Vitro Phototoxicity Validation Study : Results of Phase II (Blind Trial). Part 1 : The 3T3 NRU Phototoxicity Test