Guidance

Statement on the COM assessment of in vitro and in vivo genotoxicity of titanium dioxide

Published 11 October 2024

Introduction

Titanium dioxide has been the subject of multiple safety evaluations including 3 evaluations by the European Food Safety Authority (EFSA) in 2016, 2019 and 2021. In their most recent Opinion (2021), EFSA considered that some findings regarding immunotoxicity, inflammation and neurotoxicity with respect to TiO₂ nanoparticles, which are present in food grade TiO₂, may be indicative of adverse effects. On the basis of the currently available evidence and the uncertainties, in particular a concern regarding genotoxicity which could not be resolved, the EFSA Panel concluded that E171 could no longer be considered as safe for use as a food additive. Following this, in 2021 the COT published an interim position on titanium dioxide (COT, 2021) capturing the outcomes of discussions and outlining the next steps.

The Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment (COM) has undertaken a review of the mutagenicity TiO₂ as a food additive.

Screening and evaluation of papers

The in vitro and in vivo studies referenced in the EFSA opinion (EFSA, 2021) were collated. An additional literature search was carried out to identify papers published between 2021 to 2023 (see Annex 1 for search methodology). All papers were screened against a series of criteria to assess the characteristics of the nanomaterial used in the study and the generic study design (tier 1); and the generic experimental details of the genotoxicity study including adherence to Organisation for Economic Co-operation and Development (OECD) test guidelines (tier 2). These criteria were assessed by a sub-group of the COM. Finally, the experimental details of the study were thoroughly evaluated using expert judgement (tier 3). Annexes 2A and 2B gives a detailed explanation of the screening criteria for in vitro and in vivo papers, respectively. Annexes 3A and 3B describes in detail the evaluation of papers screen as green or amber for in vitro and in vivo papers, respectively.

COM opinion

After reviewing the in vitro genotoxicity studies performed to date on TiO₂, we note the following points:

1. There were 5 in vitro studies of the highest quality (labelled ‘green’ here) that used TiO₂ nanoparticles of different sizes and forms in the micronucleus assay. All 4 ‘green’ studies that used anatase TiO₂ nanoparticles reported negative results for the MN endpoint. Of the 2 green studies that used rutile TiO₂ nanoparticles, one was negative and the other was weakly positive for MN induction in a non-standard cell line but only at the 2 lowest doses used (1 and 5 mg/ml) (Di Bucchianico and others, 2017). Two green studies used TiO₂ nanoparticles of mixed anatase/rutile form and both were negative for MN induction.

2. There were 2 green studies that both used anatase/rutile TiO₂ nanoparticles in either the hprt gene mutation assay or CA assay. The TiO₂ nanoparticles were negative in the hprt assay. In the CA assay, the TiO₂ nanoparticles were positive, but the CA frequency decreased with increasing TiO₂ concentration, and despite the significant induction of CA, this study was negative with the micronucleus assay.

3. There were 8 amber studies (that is, ones that contained some suboptimal aspects) that used TiO₂ nanoparticles of different sizes and forms in the micronucleus assay. Four studies used anatase TiO₂ nanoparticles and 3 of these were negative for micronuclei induction. The one positive study reported a dose-dependent increase in micronuclei induction in lymphocytes from healthy individuals. All 3 studies that used nanoparticles of mixed anatase/rutile TiO₂ were negative for micronuclei induction. Two studies that used anatase/brookite TiO₂ nanoparticles reported positive results for micronuclei induction.

4. The one amber study on hprt mutations was positive at low anatase TiO₂ nanoparticle doses but not at higher doses (Vital and others, 2022).

5. Some ‘green’ studies included other assays (for example, Comet assay) to provide mechanistic information but results were inconsistent, showing either no increase (Demir and others, 2015), or an increase in oxidative DNA damage (Di Bucchianico and others, 2017) but only at the highest dose (Unal and others, 2021). Andreoli and others, 2018 and Stoccoro and others, 2017 showed ROS involvement.

After reviewing the in vivo genotoxicity studies performed to date on TiO₂, the committee note the following points:

1. The highest quality in vivo studies labelled here as ‘green’ (n=2), both show negative results for the micronucleus endpoint (Donner and others, 2016; Sadiq and others, 2012). There were no ‘green’ studies for other endpoints.

2. Only Donner and others, (2016) used pigment grade TiO₂ (including micro-sized anatase that was most similar to E171) and therefore was most relevant to the concern for human health in this case. This study showed no micronucleus induction.

3. The Donner and others (2016) paper also used a physiologically relevant oral route, which is most appropriate for the assessment of dietary exposure of food grade TiO₂.The authors acknowledge that absorption from the gastrointestinal (GI) tract is low, meaning poor bone marrow exposure. This is important for risk assessment purposes where the oral bioavailability of E171 in humans is very low (≤0.0013% - refer to COT opinion COT/2024/05).

4. The Sadiq and others (2012) study, that used an intravenous (iv) route (a route that is most likely to achieve bone marrow exposure), also showed a negative micronucleus response and confirmed bone marrow exposure to titanium.

5. The studies labelled as ‘amber’ (that is, contained some suboptimal aspects) showed a mixture of positive (4 out of 9) and negative (5 out of 9) results for the genotoxicity endpoints studied.

6. The positive studies included chromosomal and DNA damage endpoints and were all associated with cytotoxicity and/or indirect mechanisms of genotoxicity, such as oxidative damage and inflammation. There was no evidence of gene mutations, however no definitive conclusion can be made due to the deficiencies in the study designs and limited number of available studies.

7. The route of administration of nano-sized TiO₂ in these ‘amber’ studies was often not via the most relevant oral route (only 2 out of 9 studies) when considering the use of E171 as a food grade material. The less relevant endotracheal route was employed in 3 out of 9 studies and the i.v. route and i.p. route were employed in 3 out of 9 and 1 out of 9 studies, respectively. Often the dosing regimens employed in these studies were suboptimal and did not follow the recommendations of the OECD test guidelines, which also makes interpretation difficult.

8. All these ‘amber’ studies used a nano-sized TiO₂ material which is less relevant to the E171 material.

The COM opinion is that there is little evidence that TiO₂ nanoparticles are genotoxic in vitro, with the limited number of positive studies all reporting no dose-response effects with significant effects being observed at the lowest doses used. There is also a lack of replication of study outcomes using the same nanoparticle in different labs.

There is little evidence in the literature to suggest that there is a health concern related to in vivo genotoxicity induction by TiO₂, particularly via the oral route and especially the micro sized TiO₂ fraction (most studies used the nano-sized material).

Currently a definitive assessment of the safety of food grade E171 is difficult when there are no high-quality OECD-compliant studies that adequately incorporate the study design considerations and characterisation of the nanoparticulate fraction present in E171. The studies identified in this report are not representative of E171, where the fraction of nanoparticulate is less than 50% and according to the recent Guidance on the implementation of the Commission Recommendation 2022/C 229/01 on the definition of nanomaterial, E171 would not fall under the definition of an NM, hence we need GLP studies with E171 to definitively assess the hazard.

Abbreviations

Abbreviation	Meaning
ANS Panel	EFSA Panel on Food Additives and Nutrient Sources added to Food
BEAS-2B	bronchial epithelial cell line
BSA	bovine serum albumin
CBMN	cytokinesis block micronuclei
CBPI	cytokinesis block proliferation index
CP	cyclophosphamide
DMEM	Dulbecco’s Modified Eagle Medium
EFSA	European Food Safety Authority
EMS	ethyl methanesulphonate
FBC	fluidized bed crystallization
FISH	fluorescence in situ hybridization
Fpg	formamidopyrimidine DNA glycosylase
HEK	human embryonic kidney
HPBL	human peripheral blood lymphocytes
Hprt	hypoxanthine phosphoribosyl transferase
LDH	lactate dehydrogenase
MI	Mitotic Index
MMC	mitomycin C
MMS	methyl methanesulphonate
MN	micronuclei
MTT	3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide
NDI	Nuclear Division Index
OECD	Organisation for Economic Co-operation and Development
8-oxodG	8-oxo-2’-deoxyguanosine
PBMC	peripheral blood mononuclear cells
PBS	phosphate-buffered saline
PFL	water filtration media
PHA	phytohaemoglutinin A
RI	replication index
RICC	relative increase in cell counts
RNBR	relative nuclei to bead ratio
ROS	reactive oxygen species
RPD	relative population doubling
RPMI / RPMI 1640	Roswell Park Memorial Institute 1640 Medium
SCE	sister chromatid exchange
SEM	standard error of the mean
TEM	transmission electron microscopy
6-TG	6-thioguanine
TiO2	titanium dioxide (E171)
VIN	vinblastine

References

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