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Genetic toxicity in vitro

Description of key information

Titanium dioxide has been tested in bacterial reverse mutation assays, in vitro gene mutation and clastogenicity tests as well as in vivo. All tests show a negative response, thus titanium dioxide does not require classification for mutagenic properties. Endpoint Conclusion: No adverse effect observed (negative).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Titanium dioxide has been tested in bacterial reverse mutation assays, in vitro gene mutation and clastogenicity tests as well as in vivo. All tests show a negative response, thus titanium dioxide does not require classification for mutagenic properties. Endpoint Conclusion: No adverse effect observed (negative).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Germ cell mutagenicity studies with pigment-grade titanium dioxide

Pigment size titanium dioxide was tested in various in vitro and in vivo systems for its ability to induce gene, chromosome or genome mutations. The systems used include bacterial reverse mutation tests, mammalian cell mutagenicity, chromosome aberration and micronuclei test as well as in vivo experiments in mice and rats for chromosome aberration or micronuclei formation in the bone marrow or peripheral blood. The tests were conducted with two crystalline forms of titanium dioxide, namely anatase and rutile or mixtures thereof.

 

In vitro genetic toxicity tests

Gene mutation in bacteria

Myhre, A. (2011) investigated the mutagenic potential of pigment size titanium dioxide (rutile phase; 100% pure) in the bacterial reverse mutation test according OECD TG 471 and under GLP. S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A were exposed using the plate incorporation method at doses of 333, 667, 1000, 3333 and 5000 µg/plate. No positive mutagenic responses were observed at any dose level or with any tester strain in either the absence or presence of S9 metabolic activation. No toxicity was observed at any dose level with any tester strain in either the absence or presence of S9. Test substance precipitation was observed at all dose levels (333 to 5000 µg/plate) with all tester strains both in the absence and presence of S9 activation. Appropriate positive controls demonstrated the sensitivity of the test system.

 

Callander, R.D. (1996) investigated the mutagenic potential of hydrophobically coated pigment grade titanium dioxide (rutile phase; 98.5% pure) in the bacterial reverse mutation test according OECD TG 471 and under GLP. S. typhimurium TA 1535, TA 1537, TA 98, TA 100, E. coli WP2 uvr A pKM101 and E. coli WP2 were exposed using the plate incorporation method at doses of 100, 200, 500, 1000, 2500 and 5000 µg/plate. In two separate experiments, hydrophobically coated pigment grade titanium dioxide did not induce any significant, reproducible increases of revertant colonies in any of the tester strains used, either in the presence or absence of S9 activation. Appropriate positive controls demonstrated the sensitivity of the test system.

 

In the DNA damage and repair test in Bacillus subtilis (recombination assay), Kanematsu, N. et al. (1980) investigated the DNA damaging potential of titanium dioxide (further details on the test item identity was not reported, pigment size only estimated by author) in B. subtilis strains H17 (rec+) and M45 (rec-). Titanium dioxide showed no genotoxic effects in the rec-assay applied in concentrations of 5-500 mM. Due to the poor reporting quality, this report is considered as supporting information only.

 

 

Summary - Gene mutation in bacteria

There was no evidence for mutagenic activity with titanium dioxide (rutile phase) in bacterial reverse mutation tests using the plate incorporation and pre-incubation method with and without metabolic activation employed at concentrations up to 5 mg/plate. The experiments were conducted with 4 different S. typhimurium and up to 2 E. coli strains. These findings are supported by a negative outcome in a DNA damage assay (rec-assay) in B. subtilis. No information on mutagenic activity in bacteria of the anatase phase is available.

 

 

In vitro mammalian cell gene mutation

Clay, P. (1997) investigated the mutagenic potential of hydrophobically coated pigment grade titanium dioxide (rutile phase; 98.5% pure) in the in vitro mammalian cell gene mutation test according OECD TG 476 and under GLP. Cultures of L5178Y TK+/- cells were treated in duplicate with doses of hydrophobically coated pigment grade titanium dioxide for 4 hours in the presence or absence of S9 mix at concentrations of 31, 63, 125, 250, or 500 µg/mL. In the first experiment, a concentration of 1000µg/mL resulted in the formation of heavy white precipitate which interfered with cell counting and plate scoring due to large amount of substance remaining in the medium after treatment period. The maximum concentration of 500 µg/mL was considered appropriate for testing, since no interference with the scoring was observed, albeit white precipitate was still observable. No significant increases in mutant frequency, compared to the control solvent cultures, were observed in cultures with hydrophobically coated pigment grade titanium dioxide in either the presence or absence of S9 mix. Negligible cytotoxicity was observed at the highest test concentration. Appropriate positive controls demonstrated to sensitivity of the test system.

 

Summary - In vitro mammalian cell gene mutation

There was no evidence for mutagenic activity of hydrophobically coated pigment grade titanium dioxide (rutile phase) in the mouse lymphoma assay, up to the maximum concentration limited by heavy precipitate. No information on the mutagenic potential in mammalian cells of uncoated rutile and anatase is available.

 

 

In vitro clastogenicity and aneugenicity

Glover, K.P. (2011) investigated the clastogenic potential of pigment size titanium dioxide (rutile phase; 100% pure) in the in vitro mammalian chromosome aberration test according to OECD 473 and under GLP. Chinese hamster ovary (CHO) cells were treated for 4 and 20 hours with metabolic activation and for 4 hours with S9 activation at concentrations of 25, 50, 75, 100 and 150 µg/mL in two separate experiments. The maximum concentration was limited by substantial toxicity (greater than a 50% reduction in cell growth compared with vehicle control) observed at 250 μg/mL for the 4-hour test conditions and at 100 μg/mL for the 20 hour test condition. Pigment size titanium dioxide did not induce structural or numerical chromosome aberrations in the in vitro mammalian chromosome aberration test in Chinese hamster ovary cells either with or without S9 metabolic activation. Appropriate positive controls demonstrated the sensitivity of the test system.

 

Fox, V. (1997) investigated the clastogenic effects of hydrophobically coated pigment grade titanium dioxide (rutile phase, 98.5% pure) in human peripheral blood lymphocytes. The study was conducted according to OECD 473 and under GLP. Duplicate cell cultures were exposed towards doses of 10 (5), 50, or 100 µg TiO2/mL for 3 hours with and without metabolic activation. The maximum concentration was limited by the reduction of the mitotic index by more than 50%. Hydrophobically coated pigment grade titanium dioxide did not induce structural or numerical chromosome aberrations in the in vitro mammalian chromosome aberration test in human lymphocytes either with or without S9 metabolic activation. Appropriate positive controls demonstrated the sensitivity of the test system.

 

Ivett, J.L. (1989) reported on the clastogenic potential of titanium dioxide (unknown crystalline phase, purity 98.5%) in a chromosome aberration assay in CHO cells. Cells were exposed at concentrations of 0, 15, 20 or 25 µg/mL for 2 or 8 hours, with and without metabolic activation respectively. The highest dose was limited by the formation of precipitate. 100-200 Colcemid treated cells were scored analysed for aberrations. Titanium dioxide did not induce biologically relevant chromosomal aberrations in CHO cells after 2- and 8-hours treatment with and without metabolic activation.

 

Miller, B.M., et al.(1995) investigated the micronucleus formation in CHO cells with titanium dioxide (no further details on crystalline phase or particle size were provided) with and without metabolic activation using pulse and continuous treatment. Cells were exposed to titanium dioxide concentrations of 0.025 to 10 µg/mL (without metabolic activation) and 0.25 to 10 µg/mL (with S9) were used. No increase on the MN frequency was observed up to the maximum concentration limited by precipitation of the test item.

 

Proquin, H. et al. (2017) investigated effects on chromosome aberration by titanium dioxide E171 in HCT116 cells via the CBMN. Cells of the human colon adenocarcinoma cell line were exposed to titanium dioxide E171 at concentrations of 5, 10, 50 and 100 µg/cm² for 24 h. Afterwards, cytokinesis was blocked, and subsequently, the cells were fixed and stained with the nuclei dye Hoechst. MN occurrence was determined in 1000 binucleated cells. Cytotoxicity was determined using Trypan blue viability test. Moreover, kinetochore co-localisation was investigated via immunostaining and fluorescence microscopy. The authors observed no significant cytotoxicity in HCT116 cells treated with titanium dioxide E171. However, the authors found a significant increase in the MN occurrence, in binucleated cells, at titanium dioxide E171 concentrations of 5, 10, 50 µg/cm². The highest concentration was not analysable due to interference with scoring. According to the authors, titanium dioxide E171 interacts with the centromere region of kinetochore poles during mitosis, based on observed co-localisation in immunostainings. However, based on major reporting deficiencies, no conclusion can be drawn on the results presented. The test item is not sufficiently characterised. Information on purity, impurity elements and surface modifications are not specified. The authors do not state on changes of pH or osmolality in test item-treated cell cultures. Moreover, the authors do not adequately report on precipitates. However, analyses of precipitation are crucial since particle accumulation on the cell surface is known to be potential confounder leading to false positives. Furthermore, type of negative control is not specified, positive control data is completely missing. Cytotoxicity is not measured in accordance with OECD TG 487, which recommends scoring at least 500 cells for the cytokinesis-block proliferation index (CBPI) or the replication index (RI). Historical control data is not adequately reported. Data on cell line is missing, since the authors do not state on cell doubling time, metabolic activation, and potential mycoplasma contamination. Evaluation and scoring criteria are not specified.

 

Di Bucchianico, S. et al. (2017) assessed effects on the number and structure of chromosomes induced by pigment size titanium dioxide (TiO2 NM100; crystalline phase: anatase; particle size distribution: 50-150 nm; surface area: 9.2 m²/g) via two different in vitro micronucleus test methods. In the first assay, BEAS-2B (human bronchial epithelial cell line) cells were treated with titanium dioxide at concentrations of 1, 5, and 15 µg/mL (0.2-3.2 µg/cm²) for 48 h. Cytokinesis-blocked cells were assessed for chromosomal aberrations via microscopic analysis. In the second experiment, cells of the same cell line were treated with titanium dioxide at concentrations of 1, 5, 15, and 30 µg/mL (0.7-3.9 µg/cm²) for 48 h. Flow cytometry was used to analyse the occurrence of chromosomal aberrations in cells which were not treated with a cytokinesis-blocking agent. The mitotic index was not significantly changed after treatment with either of the titanium dioxide nanoforms. None of the treatments with titanium dioxide resulted in a significantly increased MN formation. The reference is considered to be not reliable due to several deficiencies in reporting and methodology. The authors performed only a long-term test with 48-hour treatment, whereas a short-term test (3-6 hours) was not performed. A positive control for aneugenicity was not implemented in the assays. The highest concentrations tested did not reach 55 ± 5% cytotoxicity. Further, information on test material precipitation in the medium is not provided. Details on the cell line are missing, i.e. mycoplasma contamination, modal chromosome number and cell cycle times are not specified. Statements on marked changes of pH value or osmolality of test item-treated cell cultures are missing. Details on particle characterisation, e.g. type of test material coating, are missing.

 

Lu, P.J., et al. (1998) examined micronuclei formation of titanium dioxide in CHO cells using the cytokinesis block protocol with continuous treatment. The test item was described as titanium dioxide standard solution, no further details on crystal phase or particle size are provided. Cells were exposed with concentrations of 0, 1, 2, 5, 10 and 20 µM in DMSO for 18 hours. At least 1000 binucleated cells were scored per dose. The MN frequency was significantly and dose-dependently enhanced in the treated cells at the same dose range 2.5 to 3-fold. Relative cell survival was determined by a colony forming protocol. The method is insufficiently described and it is unclear how the cytotoxicity/growth inhibition was determined. The cytotoxicity tests were conducted up to 5 mM, whereas the MN experiments were conducted up to 20 mM, hence no information on the cytotoxicity for the maximum concentration is available. The maximum test item concentration of 20 mM titanium dioxide (molar mass: 79.9) corresponds to 1.6 mg/mL. As reported by other authors and depending on the cell line used, doses of 25-150 µg/mL cause significant cytotoxicity (mitotic index < 50 %), thus a ten- to sixty-fold higher concentrations showing no cytotoxic effects appear grossly implausible. Secondly, titanium dioxide shows a solubility of < 1 µg/L in a 24-hours transformation/dissolution test (according to OECD testing series 29 and under GLP). Consequently, the solubility was exceeded in the above described experiments by more than six orders of magnitude. Although the test guideline foresees testing at concentrations with (slightly) visible precipitation, one may safely assume that any mutagenic effect observed in the experiments can be attributed to an unspecific particle effect. Based on the reasons above, the results need to be treated with great caution. Consequently the study is considered not suitable for hazard and risk assessment purposes.

 

Türkez, H. et al. (2007) investigated the clastogenic potential of titanium dioxide in peripheral blood lymphocytes. The reference exhibits serious shortcomings, rendering it unsuitable for hazard assessment purposes: method is insufficiently described, cytotoxicity/growth inhibition was not measured, solubility exceeded in the experiments by more than three orders of magnitude. The test material was insufficiently characterised. Due to an absence of a clear designation, purity or impurity information, it remains unclear whether the test item was in fact of industrial origin and therefore of relevance for the hazard and risk assessment of titanium dioxide.

 

Riebe-Imre, M. et al.(1994) investigated the clastogenic potential of titanium dioxide in hamster airway epithelial cell line. TiO2 induced at very low concentrations a dose-dependent increase of the MN formation in M3E3/C3 cells (nearly 2-fold at the highest concentration of 2 µg/mL). However, the control culture already shows a MN frequency of 4%, being 10-times higher compared with other in vitro test systems. This raises doubts on the suitability of the test system for the in vitro MN test - no historical control data were provided. The test material was insufficiently characterised. Due to an absence of a clear designation, purity or impurity information, it remains unclear whether the test item was in fact of industrial origin and therefore of relevance for the hazard and risk assessment of titanium dioxide.

 

Andreoli, C. et al. (2018) evaluated two different pigment size titanium dioxide particle types (i: anatase, 50-270 nm; ii: rutile, 50-3000 nm) for their potential to induce clastogenicity or aneugenicity in peripheral blood mononuclear cells using the MN assay. The cells were exposed to the each of the two titanium dioxide particle types for 72 hours using two different treatment protocols. In the first assay (delayed co-treatment), the treatment was initiated 24 h after mitogen treatment and cytochalasin B was added 20 hours after treatment start. In the second assay (co-treatment), the test material exposure stated 43.5 hours after mitogen treatment and cytochalasin B was added 30 minutes after treatment and start. The cultures were treated with the different titanium dioxide particles at concentration levels of 50, 100, and 200 µg/mL. In parallel, cytotoxicity was evaluated using the cytokinesis-block proliferation index (CBPI). In general, the two titanium dioxide particle types tested did not induce statistically significant increases in the MN formation frequency, irrespective of the treatment protocol. The only exception was a statistically significantly increase (2.0-fold above negative control) observed for the titanium dioxide rutile type particles at a concentration of 50 µg/mL using the co-treatment protocol. However, this finding is considered to be a chance finding, since no such effect was observed at higher concentrations using the same protocol and not at any concentration in the delayed co-treatment assay. The CBPI was not statistically significantly decreased after titanium dioxide treatment under any condition tested. The publication shows several reporting and methodological deficiencies. The number of analysable test concentration levels is for some experiments lower than recommended (2 vs 3). In cultures treated with 200 µg/mL TiO2 anatase bulk particles (delayed co-treatment), test material aggregates precluded the scoring of micronuclei. The number of cells scored for chromosomal damage were partly lower than recommended in the current test guideline. Moreover, information on test material precipitation in the cultures is not provided. The authors performed only two different long-term tests with 72-h treatment, whereas a short-term test (3-6 h) was not performed. In the delayed treatment protocol, the mitogen stimulation period was shorter than recommended (24 vs. 44-48 h). The authors used only hydrogen peroxide as positive control substance, which is not recommended according to the test guideline followed. Essential details on cell cultures are missing, i.e. environmental conditions and normal cell cycle time. Justification for dose range is missing. Statements on confounding factors, such as marked pH and osmolality changes of the treated culture medium, are missing. The test material characterisation lacks details. Evaluation criteria are not specified. 

 

Summary - In vitro clastogenicity and aneugenicity

Titanium dioxide does not induce clastogenic or aneugenic mutations in mammalian cells up to concentrations limited by toxicity or precipitation. One study shows a dose-response effect for titanium dioxide after 18 hours exposure up to a 3-fold increase in the micronucleus frequency. However, the experimental procedure raises doubt whether the positive findings were caused by the test item itself. It is highly likely that the clastogenic events were caused by an unspecific particle effect due to the excessive exposure concentrations applied in this study. Consequently, it is concluded that titanium dioxide (rutile phase and unknown phase) does not induce chromosome or genome mutations in mammalian cells. No information on clastogenic or aneugenic activity in mammalian cells of the anatase phase is available.

 

Sister chromatid exchange (induced DNA damage)

Ivett, J.L. (1989) examined the DNA damaging effect of titanium dioxide (unknown crystalline phase, purity 98.5%) in a sister chromatid exchange assay in CHO cells. Titanium dioxide was added at concentrations of 0, 2.5, 8.3 and 25 µg/mL in duplicate cultures, with and without metabolic activation for 2 and 25 hours, respectively. For both testing conditions 10 µM bromodeoxyuridine (BrdU) was added 2 hours after dosing, Colcemid was added 2-2.5 hours prior harvest. 50 and 25 cells were scored per dose in the initial and the confirmatory experiment, respectively. Titanium dioxide did not induce sister chromatid exchanges up to the highest dose, limited by solubility. The test item is therefore considered non genotoxic in the sister chromatid exchange assay.

 

Lu, P.J., et al. (1998) examined sister chromatid exchange in CHO cells after titanium dioxide exposure. The test item was described as titanium dioxide standard solution, no further details on crystal phase or particle size are provided. Cells were exposed with concentrations of 0, 1, 2, 5 µM in DMSO for 24 hours. Colcemid was added to the cultures 2 hours prior harvest. 30 cells in metaphase were scored per dose. SCE frequencies were significantly increased in a dose-dependent manner. Relative cell survival was determined by a colony forming protocol. The method is insufficiently described and it is unclear how the cytotoxicity/growth inhibition was determined. The maximum test item concentration of 5 mM corresponds to 400 µg/mL. As reported by other authors and depending on the cell line used, doses of 25-150 µg/mL cause significant cytotoxicity (mitotic index < 50%), thus a three to sixteen-fold higher concentrations showing no cytotoxic effects appear grossly implausible. Secondly, titanium dioxide shows a solubility of < 1 µg/L in a 24 hours transformation/dissolution test (according to OECD testing series 29 and under GLP). Consequently, the solubility was exceeded in the above described experiments by more than five orders of magnitude. Although the test guidance foresees testing at concentrations with (slightly) visible precipitation, one may safely assume that any genotoxic effect observed in the experiments can be attributed to an unspecific particle effect. Based on the reasons above, the results need to be treated with great caution. Consequently the study is considered not suitable for hazard and risk assessment purposes.

 

Summary - In vitro sister chromatid exchange

Two studies on the in vitro SCE of titanium dioxide are available, one study showing no genotoxic effects during 2 and 25 hours exposure. The second study shows a dose-response effect for titanium dioxide after 24 hours exposure. However, the experimental procedure raises doubt whether the positive findings were caused by the test item itself. It is highly likely that the genotoxic effects were caused by an unspecific particle effect due to the excessive exposure concentrations applied in this study. Consequently, it is concluded that titanium dioxide (unknown crystalline phase) does not induce genotoxicity in the in vitro SCE assay.

 

In vitro DNA damage

The potential of pigment size titanium dioxide NM100 to induce early DNA damage was investigated in the alkaline comet assay by Norppa et al. (2013). The comet assay performed is part of the Nanogenotox testing programme (discussed in detail in a separate section below). Titanium dioxide NM- 100 showed a positive result in the 16HBE cell line (Norppa et al., 2013). However, the study is considered to be not reliable as discussed in detail below.

 

Proquin, H. et al. (2017) evaluated DNA damage induced by different titanium dioxide particles in Caco-2 cells using the Comet assay. Caco-2 cells were exposed to titanium dioxide E171, titanium dioxide nanoparticles (crystalline phase: 99.5% anatase; particle size distribution: 10-30 nm (SEM); surface area: 50-150 m²/g) or pigment size titanium dioxide particles (average particle size: 535 nm) at concentrations of 0.143 and/or 1.43 µg/cm² for 24 hours. Moreover, Caco-2 cells were treated with titanium dioxide particles in combination with azoxymethane, a known genotoxicant. Hydrogen peroxide was used as positive control. After treatment, 50 cells per slide and experiment were scored for median tail intensity. Experiments were performed in four biological replicates and in duplicate. Cytotoxicity was determined using Trypan blue viability test at titanium dioxide E171 concentrations of 0.143-143 µg/cm². The authors observed cytotoxicity for titanium dioxide E171, in Caco-2 cells, at concentrations ≥14.3 µg/cm². Titanium dioxide nanoparticles and pigment size titanium dioxide particles showed significant cytotoxicity at 143 µg/cm². Furthermore, the authors conclude that the titanium dioxide test materials had the capacity to induce single-strand DNA breaks under the conditions tested. The publication is considered not reliable based on the following major methodological and reporting deficiencies. For titanium dioxide nanoparticles and pigment size titanium dioxide particles was only one concentration tested, which allows no conclusion on dose-response relations ship. Titanium dioxide E171 was tested at only two different concentrations and there was no dose-response relationship observed. Moreover, historical control data are completely missing. The type of negative control used is unclear. Based on these circumstances the positive findings are not relatable and most probably chance findings. Moreover, it was shown that results from Comet assay show very high inter-laboratory variance due to differences in the proficiency of the different labs. Thus, it is inevitable to evidence proficiency to produce reliable results. However, this necessity is not given here. Notably, methodological information on the comet assay, e.g. lysis, electrophoreses, staining, are insufficient and impedes retracing the procedure. Furthermore, the test items are not sufficiently characterised. Information on purity, impurity elements and surface modifications are not specified. The authors do not state on changes of pH or osmolality in test item-treated cell cultures. Moreover, the authors do not adequately report on precipitates However, analyses of precipitation are crucial since particle accumulation on the cell surface is known to be potential confounder leading to false positives. Further, the cell line is insufficiently characterised due to a lack of information on normal cell proliferation time, metabolic activation, and potential mycoplasma contamination. Evaluation and scoring criteria are not specified. Information on hedgehog occurrence is not provided.

 

Andreoli, C. et al. (2018) investigated on DNA damage in human peripheral blood monocytes after exposure to two different pigment size titanium dioxide particle types (i: crystalline phase: anatase; ii: crystalline phase: rutile). In the first experiment, the cells were treated with the titanium dioxide particles at concentrations of 10, 50, 100, and 200 µg/mL for 24 hours. Afterwards, a conventional alkaline comet assay was performed, and the cells were scored for proportion of DNA in tail. In the second experiment, the cells were treated at the same titanium dioxide concentration levels for 6 or 24 hours. After the treatment, the DNA was extracted and the level of 8-oxodG was analysed using HPLC. Negative and positive control cultures were run concurrently. Cytotoxicity was evaluated using the trypan blue exclusion test. According to the authors, none of the titanium dioxide particle types tested showed increased cytotoxicity in the trypan blue exclusion test. In the conventional alkaline comet assay, both particle types induced a statistically significant increase in the proportion of DNA in tail only at the highest concentration level tested (200 µg/mL). The positive control substance, hydrogen peroxide, resulted in markedly and statistically significantly increased levels of DNA damage. Further, the titanium dioxide anatase particle-treated cultures showed statistically significant increases in the 8-oxodG levels starting at concentrations of 200 and 50 µg/mL, when treated for 6 and 24 hours, respectively. The treatment with titanium dioxide rutile particles induced statistically significantly increased 8-oxodG levels at concentrations levels of 100 and 200 µg/mL. The publication shows several reporting and methodological deficiencies. The number of analysed cells is too low the OECD test guideline for in vivo testing recommends scoring of at least 150 randomly selected cells. The authors performed in parallel an MNvit experiments using the same maximum concentration (200 µg/mL). In the MNvit, the authors reported that heavy precipitation precluded the scoring of MN frequencies in some cultures. In the comet assay, however, the authors provided not any information on test material precipitation, which potentially could have had interfered with the comet assay results. It is unclear whether the cells were kept during cell lysis and electrophoresis under dark conditions. Justification for the selection of concentration levels is missing. Statements on marked changes of pH value or osmolality of test material-treated cell cultures are missing. Details on particle characterisation, including surface treatment, manufacturer, and reactivity, are missing. Dye exclusion tests are in current standard test guidelines, considered to be not adequate to evaluate cytotoxicity. The results of the cytotoxicity are not tabulated. Scoring criteria are not specified. Evaluation criteria are not specified. Information on the occurrence of hedgehogs is not provided. The methodology followed to investigate on oxidative DNA damage is only poorly described.

 

Di Bucchianico, S. et al. (2017) evaluated DNA damage in BEAS-2B (human epithelial bronchial cell line) cells after treatment with titanium dioxide NM100 (crystalline phase: anatase; particle size distribution: 50-150 nm; surface area: 9.2 m²/g) using the comet assay. The cells were treated with the pigment size titanium dioxide particles at concentration levels of 1, 5, and 15 µg/mL for 3 and 24 hours. Cells treated with Ro 19-8022 photosensitiser (together with light irradiation) were used as positive controls. The assay was performed in three different versions. DNA strand breaks were assessed using the conventional alkaline comet assay. DNA oxidation was evaluated by performance of a modified alkaline comet assay. In this version, the treated cells were additionally incubated with FPG-enzyme, which detects purine oxidation products. Finally, the authors evaluated lab light as potential confounder of in vitro comet assays with titanium dioxide NM100. To this end, cells were exposed for several to normal laboratory light before alkaline treatment and electrophoresis. In each sample, a total of 50 cells were scored for the proportion of DNA in tail. All experiments were run in triplicates. Cell viability was determined using the Alamar Blue reagent. Additionally, cytotoxicity was evaluated by determination of apoptotic/necrotic cells via microscopy and flow cytometry. Moreover, mitotic index, cell cycle perturbations and cell survival were determined by flow cytometry. The authors concluded that titanium dioxide NM100 shows in general no or low cytotoxic effects at the concentration levels tested. Moreover, they concluded that the test item did not cause DNA strand breaks. However, titanium dioxide NM100 was concluded to show a weak genotoxic potential characterised by the induction of oxidative DNA damage. Further, they showed that laboratory light can interfere with the results of the in vitro comet assay when using titanium dioxide NM100. The results of the modified alkaline comet assay using FPG-enzyme are implausible and raise some doubts on the reliability of the assay. Treatment with TiO2 NM100 showed a significant induction of oxidative DNA damage after 3 h. However, after 24 h the increase in % DNA in tail was neither significant nor dose dependent. This observation is implausible since TiO2 particles are not metabolised and do not further dissolve. Thus, transient effects are not expected. Moreover, the publication shows some reporting and experimental deficiencies. Cytotoxicity was not measured after 24 h TiO2 nanoparticle treatment. Evaluation criteria are not specified. Information whether an exogenic metabolic activation system was used. Mycoplasma contamination, modal chromosome number and normal cell proliferation time are not specified. Moreover, the authors did not report on confounding factors, i.e. test material precipitation or pH and osmolality effects of the test material on the culture medium. Justification for dose range is missing. A trend test was not performed. Further, information on hedgehog occurrence is not provided.

 

Summary - In vitro DNA damage

Reliable references reporting on DNA damage in mammalian cells after exposure to pigment size titanium dioxide particles are not available. Thus, no conclusion on the DNA damaging potential in mammalian cells can be drawn based on the information given.

 

 

In vivo genetic toxicity tests

Donner, E.M. (2011) investigated the induction of micronuclei in the bone marrow of male and female ICR mice following a single oral administration of titanium dioxide (rutile phase, pigment grade, 100% pure). The test was conducted in accordance with OECD TG 474 and under GLP. Animals (10M/10F) received doses of 500, 1000 and 2000 mg/kg bw in deionised water and were sacrificed 24 and 48 hours post exposure. At least 2000 PCEs per animal were scored for the presence of micronuclei and the proportion of PCEs among 1000 total erythrocytes (PCE/NCE ration) was determined. The test item did not induce biologically relevant increases in micronucleated polychromatic erythrocytes in the bone marrow of mice when administered up to the limit dose of 2000mg/kg bw via oral route.

In a testing programme, a series of three different pigment grade titanium dioxide samples were tested for the induction of micronuclei in the peripheral blood of rats after single oral administration (Myhre, A., 2014a, Wessels, A., 2014a and 2014b). Animals received doses of 0, 500, 1000, 2000 mg/kg bw. All tests were conducted in accordance with OECD TG 474/EC B.12 and under GLP:

 In the study by Myhre, A. (2014a) pigment grade titanium dioxide (TiO2pg-1: anatase phase, 100% purity, d50: 120nm (primary particle size by TEM ECD), SA: 8.1 m²/g) was used as test item. The vehicle control, low, intermediate, and positive control groups contained 5 animals/sex, the high-dose group contained 7 animals/sex. Peripheral blood samples were taken via sublingual vein bleeding approx. 48 and 72 hours post exposure. A total of 20,000 RETs were analysed per blood sample via flow cytometry for the presence of micronuclei and toxicity as indicated by the frequency of immature erythrocytes among the total erythrocytes (PCE/NCE ratio). Additionally, titanium concentrations were determined in blood and liver at 48 or 72 hours. No statistically significant increases in the micronucleated RET frequency were observed in any evaluated test substance-treated group of male or female animals at either time point. There were no statistically significant decreases in the PCE/NCE ratio in any test-substance treated group. Under the conditions of this study, titanium dioxide did not induce formation of micronuclei in rat peripheral blood up to the limit dose of 2000 mg/kg bw. No discernible dose-dependent increases of titanium concentrations in the blood and liver of treated rats relative to control rats could be measured.

 In the studies by Wessels, A. (2014a and b) two different pigment grade titanium dioxide samples were used: (i) TiO2pg-2: rutile phase, 100% purity, d50: 165nm (primary particle size by TEM ECD), SA: 7.1 m²/g (ii) TiO2pg-3: rutile phase, rutile purity, d50: 132 nm (primary particle size by TEM ECD), SA: 17.1 m²/g. All dose groups contained 5 animals/sex. Peripheral blood samples were taken via tail vein approx. 48 and 72 hours post exposure. A total of 20,000 RETs were analysed per blood sample via flow cytometry for the presence of micronuclei and toxicity as indicated by the frequency of immature erythrocytes among the total erythrocytes (PCE/NCE ratio). No statistically significant increases in the micronucleated RET frequency were observed in any evaluated test substance-treated group of male or female animals at either time point. There were no statistically significant decreases in the PCE/NCE ratio in any test-substance treated group. Under the conditions of this study, titanium dioxide did not induce formation of micronuclei in rat peripheral blood up to the limit dose of 2000 mg/kg bw.

 

Saber, A.T. et al.(2012a) instilled intratracheally 54 µg of two different TiO2 nanoparticle types (NanoTiO2 and PhotocatTiO2) and one microscale TiO2 particle type (FineTiO2) in female mice. A comet assay was conducted with BAL fluid cells, which were extracted one day, three days, and 28 days post exposure. Comet tail lengths were calculated per treatment group. Cytotoxicity measurements (LDH and live/dead counts) were performed in a separate in vitro assay, using MutaTM Mouse lung epithelial cells and exposing them to 0 – 800 µg/ml of TiO2 particles. According to the authors, treatment with FineTiO2 and NanoTiO2 resulted in significantly increased DNA damage (1.3 fold, p < 0.05). No severe toxicity was observed in the in vitro cytotoxicity assay. The study shows major shortcomings in study design. The authors performed cytotoxicity measurements only in a separate in vitro assay and with different cells, which is not representative for the cytotoxicity in the in vivo system. Thus, any positive finding might be a cause of cytotoxicity and not direct DNA damage. Controls are partially missing or inadequate. The authors used an in vitro system (H2O2 exposed A549 cells) as positive control, which is inadequate to demonstrate proficiency. The OECD recommends usage of a group of at least three analysable animals treated with a positive control substance. Historical data are missing. BAL cell suspensions were frozen and thawed later for the comet assay analysis. However, the OECD recommends performing slide preparation as soon as possible (ideally within one hour), since freezing of tissues increases the variation between samples and is therefore not recommended for the comet assay. In addition, details on freezing procedure, storage time, and thawing are largely missing. Moreover, laboratory’s proficiency in preparing and analysing comets’ characteristics in comet assays is a prerequisite for reliable data. It was shown that there is huge variability in tail DNA intensities even within negative or positive controls (Tavares, A.M. et al. (2014); Nanogenotox Programme). One dose was testes, whereas the OECD recommends usage of at least three different doses, which does not allow a dose-response relationship analysis. Comet tail length was measured only 24 hours after treatment, exceeding the OECD recommended measurements foreseen after 2-6 h and 16-26 h after last treatment. Details on animal weights, clinical observations, housing, and test group randomization are missing. Evaluation criteria and scoring of comets are not sufficiently described. Results for two control group animals were excluded as outliers because the data points for these mice were more than two standard deviations from the rest of the data. However, the authors do not state on the experimental reason for these incidences. It is not stated whether these cases were due to the test animals per se or mistakes during the assay procedure. Further, data should not be excluded simply because being declared as outliers. The decision to remove the results from the data set on the basis of the standard deviation is arbitrarily and subjective. It would be more adequate to perform designated tests to determine objectively outliers (e.g. Grubbs test). Further, methods and evaluation criteria are insufficiently described. Statements on lysis, electrophoresis, and slide preparation are missing. The scoring system is not specified sufficiently and hedgehog quantity was not specified. The number of analysed comets is not specified. Comet tail lengths were used as endpoint for DNA damage analysis, although recommended by the OECD. Lung inflammation was concurrently observed for NanoTiO2, indicated by significantly increased neutrophil levels. Inflammation and subsequent issue damage may have potentially interfered with the comet assay and led to false positives. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment. 

 

Shelby, M.D., et al. (1995) investigated the clastogenic and aneugenic effect of titanium dioxide (no further details on crystal phase or particle size were provided) in male B6C3F1 mice in two parallel experiments following single intraperitoneal injection. In the micronucleus analysis, animals were dosed 0, 250, 500, 1000 mg/kg bw and 0, 500, 1000, 1500 mg/kg bw in the first and second experiment, respectively. The total dose was given in three portions over 24 hours. Animals were sacrificed 24 hours after the final injection, bone marrow and peripheral blood smears (two slides/tissue/mouse) were prepared and 2000 polychromatic erythrocytes (PCE) were scored per animal for frequency of micronucleated cells. Under the reported experimental conditions titanium dioxide did not induce significant elevated levels of micronuclei in the bone marrow cells of the mouse. Therefore titanium dioxide is considered to be non-clastogenic and non-aneugenic in this micronucleus assay. In the chromosome aberration analysis, animals were dosed 0, 625, 1250, 2500 mg/kg bw and sacrificed 17 or 36 hours post exposure. Animals received colchicine two hours prior sacrifice. Bone marrow cells were fixed, stained and 50 cells per animal were scored for presence of chromosomal aberration. Under the reported experimental conditions titanium dioxide did not induce chromosome aberrations in the bone marrow of mice. The intraperitoneal route of administration is non-physiological and therefore not considered relevant for risk assessment purposes of industrial chemicals, these test results are reported for information purposes. 

 

Saber, A.T. et al.(2012b) instilled intratracheally pure TiO2 nanoparticle suspension, pure carbon black suspension, or with suspended dusts of indoor paint with or without TiO2 nanoparticles in female mice. Instilled concentration of pure nanomaterials and dusts were 18, 54, and 162 µg and 54, 162, and 486 µg, respectively. Comet assays were conducted with BAL fluid and hepatic cells one day, three days, and 28 days post exposure. Comet tail lengths were normalized to positive controls, and calculated per treatment group. According to the authors, treatment with the highest TiO2 nanoparticle dose resulted in significantly increased DNA damage in hepatic cells one day post exposure. Carbon black induced DNA damage in BAL cells after one day at 18 and 162 µg/mouse, after three days at 162 µg/mouse, and after 28 days at all concentrations. DNA damage in hepatic cells was observed at all concentrations, but only one and 28 days post exposure. TiO2 nanoparticle-containing dust of paint induced no DNA damage. Dust of TiO2 free paint induced DNA damage only in hepatic cells at the highest concentration 28 days post exposure, which was interpreted as chance finding. The study shows major shortcomings in study design. The authors did not perform any cytotoxicity measurement. However, analysis of cytotoxicity is important, since cytotoxic conditions interfere with the outcomes of the comet assay and lead to artefactual positive findings. Controls are partially missing or inadequate. The authors used an in vitro system (H2O2 exposed A549 cells) as positive control, which is inadequate since it is not representative for the in vivo conditions. Historical data are missing. BAL cell suspensions were frozen and thawed later for the comet assay analysis. However, the OECD recommends performing slide preparation as soon as possible (ideally within one hour), since freezing of tissues increases the variation between samples and is therefore not recommended for the comet assay. In addition, details on freezing procedure, storage time, and thawing are largely missing. The lysing procedure varied significantly in time (1-3.5 h), which hampers the comparability of the results. Therefore, the authors normalised the treated sample to a concurrent in vitro positive control, which is an inadequate normalisation. Although the variability was admitted by the authors, it has not been taken into account in the interpretation of the results. Moreover, laboratory’s proficiency in preparing and analysing comets’ characteristics in comet assays is a prerequisite for reliable data. It was shown that there is huge variability in tail DNA intensities even within negative or positive controls (Tavares, A.M. et al. (2014); Nanogenotox Programme). One dose was testes, whereas the OECD recommends usage of at least three different doses, which does not allow a dose-response relationship analysis. Comet tail length was measured only 24 hours after treatment, exceeding the OECD recommended measurements foreseen after 2-6 h and 16-26 h after last treatment. Details on animal weights, clinical observations, housing, and test group randomization are missing. Evaluation criteria and scoring of comets are not sufficiently described. Further, methods and evaluation criteria are insufficiently described. Statements on lysis details, electrophoresis, and slide preparation are missing. The scoring system is not specified sufficiently and hedgehog quantity was not specified. The number of analysed comets is not specified. Comet tail lengths were used as endpoint for DNA damage analysis, however, it is recommended by the OECD to use %tail DNA Lung inflammation was concurrently observed for NanoTiO2, indicated by significantly increased neutrophil levels. Inflammation and subsequent issue damage may have potentially interfered with the comet assay and led to false positives. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment. 

 

Sycheva, L. et al. (2011) conducted a micronucleus assay in male mice after administration of titanium dioxide via gavage at doses of 40, 200, 1000 mg/kg bw/day for consecutive 7 days. The publication shows reporting and experimental deficiencies. The test material was insufficiently characterised. Due to an absence of a clear designation, purity or impurity information, it remains unclear whether the test item was in fact of industrial origin and therefore of relevance for the hazard and risk assessment of titanium dioxide. Some experimental procedures were insufficiently described, some procedures were not guideline compliant/deviate from established procedures e.g. lack of fixation, unclear staining procedure, unclear scoring for cytotoxicity. Experimental procedure for cell isolation from brain tissue and liver is missing. Unclear how the mitotic index was determined in forestomach and colon epithelial cells. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

 

Sycheva, L. et al. (2011) conducted a comet assay in male mice after administration of titanium dioxide via gavage at doses of 40, 200, 1000 mg/kg bw/day for consecutive 7 days. The publication shows reporting and experimental deficiencies. The test material was insufficiently characterised. Due to an absence of a clear designation, purity or impurity information, it remains unclear whether the test item was in fact of industrial origin and therefore of relevance for the hazard and risk assessment of titanium dioxide. Some experimental procedures were insufficiently described, some procedures were not guideline compliant/deviate from established procedures e.g. lack of fixation, unclear staining procedure, unclear scoring for cytotoxicity. Experimental procedure for cell isolation from brain tissue and liver is missing. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

 

The study by Bettini, S. et al. (2017) investigated on potential genotoxic effects of titanium dioxide E171 (pigment-scale, containing a nanoscale fraction). Male rats got daily intragastric gavages of titanium dioxide E171 suspensions for seven days at a concentration of 10 mg/kg bw/day. Afterwards, a comet assay was performed, and 100 Peyer's Patch (PP) cells were analysed regarding their % tail DNA. The cytotoxicity of titanium dioxide E171 was measured ex vivo in cells isolated from PP of untreated cells. According to the authors, treatment with titanium dioxide E171 suspension (without as well as with Fpg treatment) did not result in increased DNA damage in PP cells when compared to controls. A dose-dependent cytotoxic and anti-proliferative effect on the T cells was observed. The study is considered to be not reliable based on following deficiencies. The study shows deficiencies in design and reporting. E171 supplier is not specified. The number of analysed cells is too low (100; 150 are recommended by the OECD TG489: In Vivo Mammalian Alkaline Comet Assay, 2016). The authors tested only one dose (at least three are recommended by the OECD TG489). Positive controls and historical data are not specified. The exact time point of sampling and comet analysis is not specified. Moreover, the cytotoxicity measurements are described insufficiently. Information on occurrence and frequency of hedgehogs is not provided. Information on animal husbandry are largely missing. No details on environmental conditions, housing, diet supply, water supply, acclimation period, and test group randomization. The age of test animals is only specified as adult. Animal body weights are only specified for the study initiation. Methods are described insufficiently. Details on cell lysis, electrophoresis, and tissue and slide preparation are missing. Concentration of gavaged solution is not specified. Evaluation criteria description is insufficient.

 

The potential to induce DNA damage in splenic tissue of male rats after oral administration of titanium dioxide E171 was investigated by Hashem, M.M. et al. (2020). Ten male rats per group received daily gavages of 20 and 40 mg titanium dioxide E171/kg bw over a period of 90 days. Tissue sampled from the spleen was evaluated for DNA damage using the comet assay. The tail length, proportion of DNA in tail, and tail moment were analysed as indicator for the grade of DNA damage. According to the authors, nucleoids obtained from spleen tissues of male rats treated with titanium dioxide E171 showed a statistically significant and dose dependent increase in each DNA damage parameter evaluated, when compared to the vehicle control group. The publication shows several reporting and methodological deficiencies. Only two dose groups were included, which limits dose-response relationship evaluations. The comet assay methodology was only poorly described and restricted to a citation of a single reference. The number of nucleoids and animals evaluated is not specified. The authors stated only that the mean was based on ten replicates. Clinical signs were observed but their occurrence is not specified. The age of the test animals and group sizes in cages are not specified. The purity of the test material is not stated. Details on the sample preparation are missing. The positive findings observed in the comet assay were accompanied by increased toxicity at both doses, since the spleen showed cellular infiltration, haemorrhagic lesions, pyknotic nuclei, and even necrosis (high dose group). Based on the findings of pyknotic nuclei and necrosis it is not possible to differentiate whether genotoxicity, cytotoxicity, or both are causative for the increased DNA damage in treatment groups. Both dose groups showed significantly decreased terminal body weights and a depressed body weight gain. The comet assay results are not presented in a tabular format. Information on occurrence and frequency of hedgehogs is not specified. Evaluation and scoring criteria are missing. Historical control data is not included. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

 

Jensen, D.M. et al.(2019) evaluated the DNA damage in liver and lung tissue of lean Zucker rats after repeated oral exposure to titanium dioxide E171 using the comet assay. Groups of ten male rats received one gavage per week at doses of 50 and 500 mg titanium dioxide E171/kg bw over a period of ten weeks. Afterwards, the liver and lung were removed and homogenised. Subsequently, the authors performed both a conventional alkaline comet assay and a modified comet assay using FPG and hOGG1 glycosylase to specifically examine on oxidative DNA damage. For each sample, 100 randomly chosen nucleoids per slide were scored for DNA damage. Each sample was run and scored in duplicates. According to the authors, titanium dioxide E171 induced neither DNA strand breaks nor oxidative DNA damage under the conditions of the test. However, the authors did not demonstrate the exposure of the liver and lungs and the highest dose selected did not induce any toxicity. Thus, target organ exposure was not demonstrated, and selection of the doses is not compliant with the requirements set out in the current test guideline. Moreover, the number of dose groups is lower than recommended in the current test guideline (2 vs. 3). Instead of including a positive control group, the authors treated THP-1 cells with KBrO3 as positive control. Thus, an adequate positive control group is missing. The rats were exposed only one a week over a period of 10 weeks. However, according to the current test guideline, the exposure should be performed on a daily base. Moreover, the tissues were harvest only 24 hours after the last exposure, whereas an additional harvest time after 2-6 hours is recommended. The test material was insufficiently characterised. The DNA damage was not evaluated using the parameters recommended in the current test guideline. Instead, the authors used an arbitrary scoring system which is transformed into lesions per 10^6 bp using an investigator-specific calibration curve based on the induction of DNA strand breaks by radiation. This scoring system appears not adequate to evaluate the outcome of the comet assay. The proficiency to evaluate DNA damage using the comet assay was not demonstrated. Details on the comet assay, including temperature during lysis and electrophoresis, are missing. Information on the occurrence and frequency of hedgehogs is not specified. Evaluation and scoring criteria are missing. Historical control data is not included. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

 

The in vivo data for pigment grade titanium dioxide present a consistent pattern. In none of the relevant and reliable studies using physiological route of application, show an increase of chromosomal aberrations or micronuclei. The analysis of titanium concentration in liver and blood after single oral application up to the limit dose of 2000 mg/kg bw indicates a lack of absorption and systemic availability of titanium dioxide. It is therefore concluded that titanium dioxide does not cause clastogenic or aneugenic events in animals.

 

 

Germ cell mutagenicity studies with ultrafine titanium dioxide

 

A large number of references/publications dealing with the in vitro genetic toxicity potential of engineered nanomaterials were identified. All references were rated based on minimum criteria for genetic toxicity tests with engineered nanomaterials, as recommended in various recent reviews on the applicability of standard OECD test methods for poorly soluble nano particles (Greim 2010, Warheit 2010, Landsiedel 2009). References not fulfilling the minimum quality criteria are reported in summary entries for information purposes, presenting some essential details only.

The comprehensive NanoGenotox testing programme up to six different titanium dioxide nano materials were assessed for their mutagenicity and genotoxicity in a large number of in vitro and in vivo assays. The outcome of this programme is discussed below in a separate chapter. The results of that testing programme are reported in individual robust study summaries for all test system and test item combinations.

 

In vitro genetic toxicity tests

Gene mutation in bacteria

Landsiedel, R., et al. (2010) investigated the mutagenic potential of two coated titanium dioxide NANO2 samples (1: Titanium dioxide, aluminium hydroxide, dimethicone/methicone copolymer, purity of titanium dioxide core: ≥99% rutile phase, titanium dioxide -content: 79–89%; 2: Titanium dioxide, dimethoxydiphenylsilane/ triethoxycaprylylsilane crosspolymer, hydrated silica, aluminium hydroxide, purity of titanium dioxide core: ≥99% rutile phase, titanium dioxide -content: 69-73%) in the bacterial reverse mutation test according OECD 471 and under GLP. S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 were exposed with concentrations of 20, 100, 500, 1000, 2500 and 5000 µg per plate using the plate incorporation and plate incubation protocol. Both samples did not show mutagenic activity in the standard plate incorporation nor in the preincubation assay with and without exogenous metabolic activation tested up to 5,000 mg per plate.

 

Ford, L.S. (2006) investigated the mutagenic potential of ultrafine titanium dioxide (87% rutile, 13% anatase phase) in the bacterial reverse mutation test according to OECD TG 471 and under GLP. S. typhimurium TA 1535, TA 1537, TA 98 and TA 100; E. coli WP2 uvr A were exposed using the plate incorporation and preincubation method at doses of 100, 333, 1000, 3333, and 5000 µg/plate. Under the conditions of this study, ultrafine titanium dioxide showed no evidence of mutagenicity in the bacterial reverse mutation test either in the presence or absence of S9 activation. Appropriate positive controls demonstrated to sensitivity of the test system.

 

Butler, K.S. et al.(2014) evaluated the ability of TiO2 NPs (W740x, Degussa) to induce reverse mutation in histidine-requiring strains of Salmonella typhimurium and tryptophan-requiring strains of Escherichia coli. Salmonella typhimurium tester strains TA 98, TA 100, and TA 102 as well as E. coli tester strains WP2 and WP2 uvrA pKM101 were exposed to TiO2 NPs at eight different concentration levels ranging from (0.000056 to 56 µg/plate). After a 1-hour pre-incubation period, the bacterial cultures were incubated for 48 hours. Afterwards, the plates were scored for the number of revertant colonies. The experiment was performed in triplicates. Additionally, the authors assessed TiO2 NP association with Salmonella via flow cytometry and nanoparticle internalisation via TEM. Moreover, a bacterial DNA repair assay was performed. The bacterial strains treated with TiO2 NPs did not show increases in the revertant colony number, when compared to the vehicle control cultures. The publication shows several reporting and methodological deficiencies. The authors did not specify if the titre of viable cells was determined. Moreover, some Salmonella tester strains required by the OECD TG 471 (1997) were not included in the assay. Information on potential test material precipitation and cytotoxicity are not provided. The selection of the top dose is not consistent with criteria set out in the test guideline. The description of the methodology lacks some details. Historical control data is not included. Information on evaluation and scoring criteria are not provided. The results are not presented in a tabular format and individual culture results are not presented. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

 

 

Summary entry - Gene mutation in bacteria

The references contained in this summary entry represent gene mutation studies in bacteria with very limited value for risk assessment purposes. All references do not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The information contained therein were included for information purposes only.

The test material used in the references summarised in the summary entry was largely characterised insufficiently. As detailed in the introductory statement to chapter for human health hazard assessment, it is problematic to base the hazard and risk assessment of clearly characterised titanium dioxide on information generated with poorly characterised test item, with no market relevance. In accordance with ECHA guidance, all references that do not provide sufficient information on the substance identity were disregarded for hazard and risk assessment purposes (for further information please refer to the introductory statement).

 

Woodruff, R.S. et al.(2012): Reasonably well described publication with little reporting deficiencies. Test material insufficiently characterised.

Lopes, I. et al. (2012): Publication with reporting and experimental deficiencies. Test material insufficiently characterised. Unclear experimental procedure – dosing unclear. Number of strains used insufficient for a complete assessment.

Pan, X. et al. (2010): Publication with reporting and experimental deficiencies. Test item poorly characterised. No confirmatory experiment performed. Number of strains insufficient for a complete assessment. Dosing should have reached 5mg/plate.

Jomini, S. et al. (2012): Only three concentration levels were used. Not all required Salmonella tester strain were included. Both protocols used are deviating from the standard test guideline.

Casey, B. J. et al (2013): Publication with reporting and experimental deficiencies. Only two strains of S. typhimurium were used, the test system sensitivity was not demonstrated and no information on cytotoxicity were given.

 

 

Summary - Gene mutation in bacteria

There was no evidence for mutagenic activity with titanium dioxide in bacterial reverse mutation tests using the plate incorporation and pre-incubation method with and without metabolic activation employed at concentrations up to 5 mg/plate. Results were obtained using pure rutile phase or mixed rutile/anatase phase titanium dioxide particles. The experiments were conducted with 5 different S. typhimurium and 1 E. coli strain.

 

 

In vitro mammalian cell gene mutation

Kazimirova, A. et al. (2020) evaluated the ability of titanium dioxide NANO4 (Aeroxide P25) to induce gene mutations at the Hprt locus in Hamster lung fibroblast V79-4 cells. The test substance was dispersed according to different dispersion procedures resulting in different agglomerate sizes. All experiments were performed with each titanium dioxide nanoparticle dispersion type. The cell cultures were exposed to the two different titanium dioxide nanoparticle dispersion types at concentrations of 3, 15, and 75 µg/cm² for 24 hours. The cytotoxicity of the titanium dioxide nanoparticle suspensions was evaluated by determination of the plating efficiency and relative growth activity. According to the authors, the two different titanium dioxide nanoparticle dispersion types induced neither excessive cytotoxicity nor an increased gene mutation frequency under the conditions tested. The publication shows several reporting and methodological deficiencies. The selection of the top concentration (75 µg/cm²) is not consistent with the criteria set out in the OECD TG 476 (2016), since the test item was tested neither up to precipitating (at least not reported) nor cytotoxic (plating efficiency >75%) concentrations. Notably, the cytotoxicity was not evaluated using the relative survival index, rather than unadjusted plating efficiency. Further, data on cell counts at start and end of the treatment are missing. The number of concentration levels tested was lower than recommended by the current test guideline. Moreover, the numbers of cells cultured during expression and plated for mutant selection were lower than recommended. Apart from these deviations from the acceptability criteria of the test, the exposure period was deviating from the recommendation of the current test guideline (24 hours vs. 3 - 6 hours). The description of the cell line lacks details, since information on normal cell cycle time, mycoplasma infection state, modal chromosome number, and number of passages are missing. The results on cytotoxicity and gene mutation experiments are not presented in a tabular format and information on a potential dose-response relationship is not provided. Historical control data is not provided. The authors did not report on evaluation and scoring criteria.

 

Teubl, B.J. et al.(2015) investigated on the potential of titanium dioxide NM104 to induce gene mutations at the Hprt locus of V79 Chinese hamster lung fibroblasts. The cells were exposed to titanium dioxide NM104 for 4 hours at a concentration of 100 µg/mL. After appropriate periods of phenotype expression and mutant selection, the mutant frequency was calculated. Additionally, the cytotoxicity was evaluated in a parallel experiment using the colorimetric MTS assay. According to the authors, titanium dioxide NM104 did not induce an increased mutant frequency in V79, when compared to cell cultures treated with PBS. Moreover, the MTS assay did not reveal cytotoxic effects induced by titanium dioxide NM104. The publication shows several reporting and methodological deficiencies. Information on the test material preparation and the vehicle used are missing. Only a single concentration level was tested. The results are not shown. The cloning efficiency was not discussed. The authors performed an MTS assay in a separate assay in order to evaluated cytotoxicity of the test material. The assay was, however, performed in a different cell line. The description of the cell line is insufficient, since details on the source, passage number, and normal cell doubling time are missing. Information on the mutant frequencies observed in negative and positive control cultures are not provided. Moreover, information on potential test material precipitation in the culture medium is not provided. The authors did not state on the statistical tests performed. Historical control data are not included. Information on evaluation and scoring criteria are missing. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

 

Teubl, B.J. et al.(2015) investigated on the potential of titanium dioxide NM103 to induce gene mutations at the Hprt locus of V79 Chinese hamster lung fibroblasts. The cells were exposed to titanium dioxide NM103 for 4 hours at a concentration of 100 µg/mL. After appropriate periods of phenotype expression and mutant selection, the mutant frequency was calculated. Additionally, the cytotoxicity was evaluated in a parallel experiment using the colorimetric MTS assay. According to the authors, titanium dioxide NM103 did not induce an increased mutant frequency in V79, when compared to cell cultures treated with PBS. Moreover, the MTS assay did not reveal cytotoxic effects induced by titanium dioxide NM104. The publication shows several reporting and methodological deficiencies. Information on the test material preparation and the vehicle used are missing. Only a single concentration level was tested. The results are not shown. The cloning efficiency was not discussed. The authors performed an MTS assay in a separate assay in order to evaluated cytotoxicity of the test material. The assay was, however, performed in a different cell line. The description of the cell line is insufficient, since details on the source, passage number, and normal cell doubling time are missing. Information on the mutant frequencies observed in negative and positive control cultures are not provided. Moreover, information on potential test material precipitation in the culture medium is not provided. The authors did not state on the statistical tests performed. Historical control data are not included. Information on evaluation and scoring criteria are missing. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

 

 

Summary entry - In vitro mammalian cell gene mutation

The references contained in this summary entry represent gene mutation studies in mammalian cell lines with very limited value for risk assessment purposes. All references do not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The information contained therein were included for information purposes only.

The test material used in the references summarised in the summary entry was largely characterised insufficiently. As detailed in the introductory statement to chapter for human health hazard assessment, it is problematic to base the hazard and risk assessment of clearly characterised titanium dioxide on information generated with poorly characterised test item, with no market relevance. In accordance with ECHA guidance, all references that do not provide sufficient information on the substance identity were disregarded for hazard and risk assessment purposes (for further information please refer to the introductory statement).

 

Chen, Z. et al. (2014): Publication with reporting and experimental deficiencies. Only three concentration were used (OECD foresees four analysable concentrations). Vehicle/solvent control was missing. Exposure duration was too short (OECD foresees usually three to six hours). Cytotoxicity was not reported. Individual data was missing. Historical control data was missing. Justification of choice of vehicle was missing. Information on potential mycoplasma contamination was missing. Information on pH effects was missing.

 

Wang, J. et al. (2019): The CD59 mutation assay is not a standard genotoxicity assay and lacks validation. Only one concentration level was included. The type of negative control is unclear. The cell line used is insufficiently characterised.

 

 

Summary - In vitro mammalian cell gene mutation

Reliable publications reporting on gene mutation in mammalian cells after exposure to titanium dioxide nanoparticles are not available. Further studies on in vitro gene mutation in mammalian cells were conducted under the EU Nanogenotox Programme using four different nano forms of titanium dioxide. The results and conclusion are given below and in the respective study records.

 

 

In vitro mammalian chromosome aberration/micronucleus test

Riley, S. (1999) investigated the induction of chromosomal aberrations in CHO cells by trimethoxy caprylylsilane coated titanium dioxide NANO5 (crystal phase: 85% anatase, 15% rutile, 96.5% pure, 7.5% coating). The study was conducted according to OECD 473 and under GLP. Cells were exposed to, in experiment 1: 68.72, 209.7 and 800 µg/mL (-S9) and 167.8, 640, and 800 µg/mL (+S9); experiment 2: 167.8, 512 and 800 µg/mL (+S9). The metabolic activation in experiments 1 and 2 was derived from Aroclor 1254 and ß-naphthoflavone induced male rats, respectively. The highest concentration induced approx. 60% and 29% reduction in cell number in the absence and presence of metabolic activation, respectively. Treatment of cultures with titanium dioxide in the absence and presence of S9 resulted in frequencies of cells with structural aberrations that were similar to frequencies seen in concurrent solvent control cultures. Aberrant cell frequency in the majority of concurrent solvent control cultures and of treated cultures fell with the historical negative (normal) control range.

Mixed phase ultrafine titanium dioxide NANO6 (89.3% titanium dioxide (89.3% rutile, 10.7% anatase) 7.2% aluminium hydroxide 1.1% amorphous silica 1.8% trimethylolpropane 0.6% Citric acid) was tested by Donner, E.M. (2006) for the induction of structural and numerical chromosomal aberrations in CHO cells. In the main experiments the test item was applied at concentrations of:

125, 250, 750, 1250, and 2500 μg/mL (4h, -S9), three top doses used for CA analysis

62.5, 125, 250, 500, 700 μg/mL (4h, +S9), lowest three concentrations used for CA analysis

6.25, 12.5, 25, 50, 100 μg/mL (20h, -S9), three top doses used for CA analysis.

Cultures at 4 with and 20 hours without metabolic activation were limited by cytotoxicity. Under the conditions of this study, titanium dioxide did not induce structural or numerical chromosome aberrations in the in vitro mammalian chromosome aberration test in Chinese hamster ovary cells in either the non-activated or activated test conditions.

 

Landsiedel, R., et al. (2010) investigated the clastogenic and aneugenic potential of coated titanium dioxide NANO2 (Titanium dioxide, aluminium hydroxide, dimethicone/methicone copolymer, purity of titanium dioxide core: ≥99% rutile phase, titanium dioxide-content: 79–89%, surface area (BET): 100 m²/g) in the in vitro mammalian cell micronucleus test according to OECD TG 487 and under GLP. V79 cells were exposed for 4 and 24 hours to concentrations of 75, 150, and 300 µg/mL and 18.8, 37.5, and 75.0 µg/mL, respectively. Quadruplicate cultures were prepared and 2,000 cells were analysed for micronuclei for each test group. Toxicity indicated by strongly reduced quality of the cells was observed from 625 µg/mL onward after 4 h treatment and from 156.3 µg/mL onward after 24 h treatment. In addition, the occurrence of precipitation on the slides strongly influenced the toxicity assessment from 625 µg/mL onward. Ultrafine titanium dioxide did not increase the number of cells containing micronuclei up to the highest evaluable concentrations of the test substance, thus is considered not to be clastogenic or aneugenic substance under the experimental conditions of this study.

 

Jugan, M.L. et al. (2012) investigated the clastogenic and aneugenic potential of two ultrafine titanium dioxide samples (NANO6, A12: self-synthesized, 95% anatase, mean diameter: 12 nm, specific surface area: 92 m²/g; NANO4, A25: Aeroxide P25, 86% anatase, mean diameter: 24 nm, specific surface area: 46 m²/g) in the in vitro mammalian cell micronucleus test according to OECD TG 487. A549 cells were exposed for 24 hours to concentrations of 50, 100 and 200 µg/mL in triplicate, followed by 24 hours cytoB treatment. Cytotoxicity, measured by MTT and colony forming ability, was measured after 48 hours exposure, showing no excessive toxicity up to 100 µg/mL. Approximately 1500 nuclei were scored per slide, showing a decrease in the portion of binucleated cells compared with the negative control. The number of clastogenic and aneugenic events did not increase for any of the concentrations tested.

 

Di Bucchianico, S. et al. (2017) assessed effects on the number and structure of chromosomes induced by titanium dioxide NANO2 (TiO2 NM103; crystalline phase: rutile; particle size distribution: 20-28 nm; surface area: 51 m²/g) and NANO7 (TiO2 NM101; crystalline phase: anatase; particle size distribution: 5-8 nm; surface area: 316 m²/g) via two different in vitro micronucleus test methods. In the first assay, BEAS-2B (human bronchial epithelial cell line) cells were treated with both titanium dioxide nanoforms at concentrations of 1, 5, and 15 µg/mL (0.2-3.2 µg/cm²) for 48 h. Cytokinesis-blocked cells were assessed for chromosomal aberrations via microscopic analysis. In the second experiment, cells of the same cell line were treated with both titanium nanoforms at concentrations of 1, 5, 15, and 30 µg/mL (0.7-3.9 µg/cm²) for 48 h. Flow cytometry was used to analyse the occurrence of chromosomal aberrations in cells which were not treated with a cytokinesis-blocking agent. The mitotic index was not significantly changed after treatment with either of the titanium dioxide nanoforms. None of the treatments with titanium dioxide NANO7 resulted in a significantly increased MN formation, except for the treatment at the lowest concentration (1 µg/mL) in cells analysed via flow cytometry. Moreover, cells treated with titanium dioxide NANO2 at 1 and 5 µg/mL showed statistically significantly increased micronucleus frequencies. Noteworthy, the positive responses were found, for both nanoforms, only in low but not high dose groups. Moreover, the data, as indicated by the diagram, shows no dose-response. If at all, the data indicates a negatively correlated dose-response. Thus, the finding is at least questionable and seems to be more of an incidental nature. The reference is considered to be not reliable due to several deficiencies in reporting and methodology. The authors performed only a long-term test with 48 h treatment, whereas a short-term test (3-6 h) was not performed. A positive control for aneugenicity was not implemented in the assays. The highest concentrations tested did not reach 55±5% cytotoxicity. No statement on precipitation. Moreover, details on the cell line are missing, i.e. mycoplasma contamination, modal chromosome number and cell cycle times are not specified. Statements on marked changes of pH value or osmolality of test item-treated cell cultures are missing. Details on particle characterisation, e.g. type of test material coating, are missing. 

Armand, L. et al. (2016) investigated on potential effects on chromosomal aberration induced by titanium dioxide NANO4 (AEROXIDE P25; crystalline phase: 86% anatase, 14% rutile; average particle diameter (TEM): 24±6 nm; surface area: 46±1 m²/g (average)) using the cytokinesis-block micronucleus (CBMN) assay. A549 (human lung carcinoma cells) were treated with 1, 2.5, 5, 10, and 50 µg/mL TiO2-NPs for 1 day, 1 week, 2 weeks, 1 month or 2 months. The cell cultures were treated with cytochalasin B and 1000 binucleate cells per replicate were scored for occurrence of MN. Additionally, the authors investigated cytotoxicity via MTT, trypan blue staining, and propidium iodide staining. Cell proliferation was assessed by determination of the population doubling level (PDL) at each exposure concentration and at each cell passage. Titanium dioxide NANO4 showed no cytotoxicity after long-term exposure, as no overt toxicity was found in MTT assay, trypan blue staining and cell counting as well as propidium iodide staining and flow cytometry. However, the highest titanium dioxide NANO4 concentrations (10 and 50 µg/mL) and the longest exposure time significantly decreased cell proliferation, as indicated by the PDL, after each passage. The micronucleus frequency of titanium dioxide NANO4 treated cells was not statistically different from unexposed cells under all conditions tested. The reference is considered to be not reliable based on deficiencies in reporting and methodology. Details on cell culture are missing. Cell density at seeding is not stated. Justification for top dose is missing in absence of the determination of cytotoxicity by recommended parameters and missing information on precipitates. Moreover, the authors performed no short-term incubation (3-6 h) as recommended by the OECD.

 

Catalán, J. et al. (2012) investigated the clastogenic potential of ultrafine titanium dioxide (anatase phase, 99.7% pure, BET surface area: 222 m²/g) in human lymphocytes, in accordance with OECD TG 473. Cells were exposed at concentrations of 6.25, 12.5, 25, 50, 100, 150 and 300 µg/mL. The nanomaterials were ultrasonicated prior to exposure, to ensure maximum dispersion. Cells were exposed for 24, 48 and 72 hours without metabolic activation. The mitotic indices were not significantly affected by the nanomaterials at any exposure time. Two hundred metaphases were scored per dose and replicate (100 cells/ replicate culture), making a total of 400 cells for each experimental point. Authors conclude a dose-dependent increase at 100 and 300 μg/mL for all aberrations without gaps after 48 hours exposure. However, these isolated statistical positive finding were caused by the low aberration frequency in the control culture at that exposure time. A dose-response relationship has not been demonstrated at this experimental time, and no dose-dependent or statistically significant increase has been observed after 24 or 72 hours exposure. High background levels of %cells with aberrations in the control cultures over all experiments raises doubts on the suitability of the test system - %CA without gaps reached up to 3%, being 3fold above the expected value. It is therefore concluded that ultrafine titanium dioxide (anatase phase) does not induce chromosomal aberrations in human lymphocytes. Due to the use of an unsuitable test system, the reference is considered not reliable and therefore disregarded for the hazard assessment. 

 

The in vitro micronucleus experiment published by Kansara, K. (2015) exhibits significant shortcomings putting into question the results of this study. The exposure concentrations between 25 and 100 mg/L exceeded the solubility of titanium dioxide more than 4000 to 16,000 fold. Cytotoxicity was measured using the colorimetric MTT assay – any interference with the test item was not determined, thus underestimation of the cytotoxicity cannot be excluded. Further, the measurement of cell viability using the MTT assay is considered unsuitable when using PSLTs, CBPI is considered the more appropriate method. Cells were harvested and cleaned via centrifugation, which does not separate the test item from the cells, thus exposure duration becomes ill defined. In addition to these shortcomings, further absence of mycoplasma infection, cell culture conditions were not reported, cell proliferation/cytotoxicity (CBPI or RI from at least 500 cells) was not stated, test item insufficiently described. Historical control for the negative and positive control data are not reported. 

 

Browning, C.L. et al. (2014) examined the potential of titanium dioxide P25 nanoparticles to induce chromosomal aberrations in primary and immortalised human skin fibroblast cells (BJ cells). The cytotoxicity and genotoxicity testing showed no statistically significant effects at any concentration of 10, 50 and 100 µg/cm² compared to the negative control. The information does not allow an independent assessment due to major methodical and reporting deficiencies and is not in accordance with any guideline. The justification for dose selection is not given by the authors and the choice of the cell lines is not justified. Moreover the chosen cell model is inappropriate for the assessment of TiO2 nanoparticles effects to the skin. Furthermore, since the criteria for the chromosomal aberration analysis is not given and the number of metaphases counted was to low (in comparison with test guideline OECD 473 In vitro mammalian chromosome aberration assay), it is not possible to draw an independent conclusion on the effects of the test substance on the treated cells. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment. 

 

Stoccoro, A. et al. (2017) The aim of the study was to investigate potential chromosomal damage caused by treatment with different types of TiO2 nanoparticles and to reveal differences in the toxic properties of coated vs non-coated nanoparticles. Human alveolar type II-like epithelial A549 cells were treated with 10, 20, and 40 µg/cm² (32, 64, and 128 µg/mL) titanium dioxide nanoparticle suspensions for 48 hours. Afterwards, a micronucleus test was performed and 1000 binucleated A549 cell were scored for the occurrence of micronuclei, nuclear buds (NBUD), and nucleoplasmic bridges (NPB). Cytotoxic and cytostatic effects were measured by analysing clone formation efficiency (CFE), cytokinesis-block proliferation index (CBPI), replication index (RI), apoptotic cell ratio, and necrotic cell ratio. The study shows serious shortcomings in study design and reporting: Statements on the test material are partially missing. Purity of the test material is not stated. No statement on osmolality of the administered titanium dioxide nanoparticle suspension in cell culture. Details on the micronucleus test method are missing (slide preparation, cell scoring system, and DNA counterstaining). Essential details on cell culture are unclear or largely missing. The authors do not mention whether the test was conducted with or without exogenous metabolic activation of the cells. Cell harvest times after treatment are not specified. Evaluation criteria of the micronucleus test are unclear or missing. The authors provide no information on scorable or non-scorable cells. The results section shows some deficiencies in reporting. Historical data are missing. Individual culture data are not presented and the data are not summarised in tabular form. It is stated that TiO2 nanoparticle resulted in increased cytotoxicity and necrosis rates. Cytotoxicity could have had interfered with the test leading to false positives. Further, the authors analysed 1000 binucleated cells. However, the OECD recommends the analysis of at least 2000 binucleated cells. The authors do not state on dose-response relationship of the micronucleus occurrence after TiO2 treatment. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

 

Andreoli, C. et al. (2018) evaluated different titanium dioxide nanoparticles for their potential to induce clastogenicity or aneugenicity in peripheral blood mononuclear cells using the MN assay. The cells were exposed to titanium anatase nanoparticles (particle size: 15 nm), titanium dioxide rutile nanoparticles (particle size: 10x40 nm), and titanium dioxide anatase/rutile nanoparticles (25-70 nm) for 72 hours using to different treatment protocols. In the first assay (delayed co-treatment), the treatment was initiated 24 h after mitogen treatment and cytochalasin B was added 20 hours after treatment start. In the second assay (co-treatment), the test material exposure stated 43.5 hours after mitogen treatment and cytochalasin B was added 30 minutes after treatment and start. The cultures were treated with the different titanium dioxide particles at concentration levels of 50, 100, and 200 µg/mL. In parallel, cytotoxicity was evaluated using the cytokinesis-block proliferation index (CBPI). The titanium dioxide particle types tested were found to be negative in the MN assay irrespective of the treatment protocol used. Moreover, the CBPI was not significantly altered after the treatment with titanium dioxide under any condition tested. The reference is considered to be not reliable based on the following shortcomings. The test materials are insufficiently characterised. Information on test material precipitation in the cultures is not provided. The authors performed only two different long-term tests with 72-hour treatment, whereas a short-term test (3-6 hours) was not performed. In the delayed treatment protocol, the mitogen stimulation period was shorter than recommended (24 vs. 44-48 hours). The authors used only hydrogen peroxide as positive control substance, which is not recommended according to the test guideline followed. Essential details on cell cultures are missing, i.e. environmental conditions and normal cell cycle time. Justification for dose range is missing. Statements on confounding factors, such as marked pH and osmolality changes of the treated culture medium, are missing. Evaluation criteria are not specified. 

 

Brandão, F. et al. (2020) evaluated potential clastogenic and aneugenic effects in four different human cell lines after treatment with titanium dioxide NANO4 (Aeroxide P25; crystalline phase: 80% anatase, 20% rutile; particle size: 25 nm) using the MN test. The authors investigated on the MN formation in lung epithelial A549, human glioblastoma A172, hepatocellular carcinoma human cell line HepG2, and in human neuroblastoma SH-SY5Y cells. Each cell line was treated with titanium dioxide NANO4 at concentrations levels of 10, 50, 100, and 200 µg/mL for both 3 and 24 hours. After the treatment, the cells were cultured for an additional period of 24 hours in fresh medium, harvested, and subsequently, stained. The number of cells showing micronuclei was analysed by flow cytometry. Mitomycin C (A549, SH-SY5Y, and A172 cells) and benzo(a)pyrene (HepG2 cells) were used as positive control substances. The cytotoxicity of TiO2 NPs in A172 cells was evaluated using the colorimetric MTT assay. According to the authors, the A172 cells showed a statistically significantly decreased cell viability, when compared to vehicle control cultures, only after treatment with 200 µg/mL titanium dioxide NANO4 for 24 hours. However, the cell viability was still above 60%. For the other cell lines cytotoxicity was not evaluated. In the MN test, none of the cell lines evaluated showed a statistically significant increase in the MN formation frequency under the conditions tested. The positive control substances induced a statistically significantly increased MN formation rate. The description of the methodology is insufficient and lacks essential details. The number of cells analysed for MN occurrence is not specified. It remains unclear whether the authors used a cytokinesis block or not. Moreover, the cell proliferation time is not specified, and thus, it is unclear whether the cells have undergone cell division during or following treatment with the test item. Further, the cell density at seeding is not specified. Moreover, information on potential test item precipitation in the different media used is not provided. Cytotoxicity was not evaluated using the parameter recommended in the OECD TG 487 (2016) and was evaluated only for A172 using the MTT assay, which is not considered to adequate for cytotoxicity testing of PSLTs. The top concentration selected is not consistent with the criteria set out in the OECD TG 487, since the test material was not tested up to precipitating (at least not reported) or cytotoxic (according to the authors: > 60% cell viability for all cell lines) concentrations. The MN frequency observed for all vehicle control cultures was unexpectedly high (≥ 5%). An aneugenic positive control group was not included. The cells used are partly no standard cell lines used for genotoxicity testing and description of the cell lines lacks details, since information on the passage number, mycoplasma infection state, modal chromosome number, normal proliferation time, and karyotype stability are missing. The test concentration is specified in µg/mL which is not adequate for adherent cell lines. Evaluation and scoring criteria are not specified. Historical control data is not provided. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment. 

 

Liao, F. et al. (2019) investigated on the clastogenic and aneugenic potential of four different sizes of titanium dioxide anatase nanoparticles (T10: 10 ± 2.3 nm; T30: 30 ± 5.1 nm, T50: 50 ± 7.6 nm, and T100: 100 ± 14.3 nm) in human umbilical vein endothelial cells (HUVECs) using the cytokinesis-block micronucleus assay. The cells were exposed to the different titanium dioxide nanoparticles types at concentration levels of 1, 5, and 25 µg/mL for 24 hours. After the cytokinesis-block, the cells were harvested, stained, and scored for the presence of micronuclei via microscopy. The treatment with titanium dioxide T10, T30, and T50 resulted in statistically significantly increased micronucleus frequencies at all concentration levels tested. In contrast, T100 treatment resulted only at the highest concentration level (25 µg/mL) in a statistically significantly increased micronucleus occurrence. The publication shows severe deficiencies with regard to reporting and the methodology applied. The test material was insufficiently characterised, since information on the purity, manufacturer, coatings, surface reactivity, and morphology are missing. Details on the preparation of the test material are missing, especially information on the sonication procedure are missing. The cell line used in no standard in genotoxicity testing and not recommend by the current test guideline (OECD TG 487). Essential cell details, including the source, passage number, karyotype stability, and mycoplasma contamination, are not included. The methodology of the micronucleus assay is only poorly described. Potential cytotoxic effects of the test materials are neither examined nor discussed. Moreover, information on potential test material precipitation and effects on the pH and osmolality of the medium are not provided. Positive control cultures were not included. The type of negative control used is unclear. Furthermore, the authors provide no information on scoring and acceptability criteria. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

 

Kazimirova, Z. et al. (2019) evaluated the clastogenic and aneugenic potential of titanium dioxide NANO4 (Aeroxide TiO2 P25) by its effect on micronucleus formation in cultured human peripheral blood lymphocytes and in TK6 lymphoblastoid cells. The whole blood samples were obtained from eight male and six female donors and were not pooled for the analysis of the micronucleus formation rate. The samples were exposed to titanium dioxide NANO4 at concentrations levels of 3, 15, and 75 µg/cm² for 24 hours. Afterwards, the cultures were treated for 72 hours with phytohaemagglutinin. The last 28 hours of incubation, the cells were treated with cytochalasin B in order to block cytokinesis. The TK6 cells were exposed to the same concentration levels for 4 and 24 hours. Subsequently, the cells were treated with cytochalasin B for another 24 hours. The occurrence of micronuclei was scored in 2000 binucleated cells per concentration. The CPBI was calculated for cytotoxicity evaluations. According to the authors, human peripheral blood lymphocytes showed no statistically significant increase in the micronucleus frequency when results from the individual donors were combined. Notably, the individual analysis revealed statistically significantly increases for donor V1, V2, and V10 after titanium dioxide NANO4 treatment at concentrations levels of 75, 15, and 3 µg/cm², respectively. However, none of these responses was clearly concentration dependent and the responses showed in general high variability. In contrast, the positive control cultures showed a clear and statistically significant induction of the micronucleus formation. Cytotoxicity, as evaluated by the CBPI, was not evident at any concentration level tested. In TK6 lymphoblastoid cells, the titanium dioxide NANO4 treatment did neither induce a statistically significantly altered micronucleus frequency nor cytotoxicity. The publication shows several reporting and methodological deficiencies. The description of the micronucleus test is insufficient and lacks essential details. The scoring methodology is not described. The top concentration selected is not consistent with the criteria set out in the OECD TG 487, since the test material was not tested up to precipitating (at least not reported) or cytotoxic concentrations. The results are insufficiently described, since it is unclear whether the micronucleus frequencies are number of micronuclei per 100, 1000, or 2000 binucleated cells. If the results are presented in number of micronucleated cells per 100 binucleates, the control values were unexpectedly high (approx. 4% after prolonged treatment of TK6 cells; approx. 5% in lymphocytes in combined results and up to approx. 12% in lymphocytes after individual analyses). Further, the results appear to be highly variably in control and treatment cultures. The authors did not report on potential test item precipitation, pH effects, and effects on osmolality in the culture medium. The donors are older than recommended by the current test guideline (40-50 years vs. 18-35 years). Moreover, it is not stated why single donors are excluded from the analyses. The proliferation of the lymphocytes was stimulated after treatment with the test item, instead of 40-48 hours before treatment as recommended. Further, short-term exposure was not included in the MN assay using lymphocytes. Details on the TK6 cell line is missing, since information on the source, passage number, mycoplasma infection state, normal cell doubling time, modal number of chromosomes, and karyotype stability are not reported. The test concentrations are specified in µg/cm² which is not adequate for free-floating cells. An aneugenic positive control substance was not included in the assay. Evaluation criteria are not specified. Historical control is not provided. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

 

Stoccoro, A. et al. (2016) evaluated potential cytogenetic effects in Balb/3T3 mouse fibroblasts after treatment with TiO2 P25 NPs. The cell cultures were treated with TiO2 NPs at concentration levels of 10, 20, and 40 µg/cm² (corresponding to 32, 64, and 128 µg/mL) for 48 hours. Untreated cells served as negative control. Positive control cultures were treated with mitomycin C. After 44 h, the cells were treated with cytochalasin B in order to block cytokinesis. The experiment was performed in quadruplicates. A total of 1000 binucleated cells were scored for the occurrence of micronuclei. The cytokinesis-block proliferation index (CBPI) and replication index (RI) were determined in order to evaluate cytotoxicity. Additionally, the occurrence of nuclear buds and nucleoplasmic bridges were scored. Furthermore, a total of 500 cells was scored for the occurrence of apoptotic and necrotic cells. In a separate experiment, the cell viability was evaluated using the colony forming efficiency assay. Moreover, the cellular uptake of the test material was analysed using TEM. The TiO2 treated cell cultures showed a statistically significantly increased micronucleus frequency only at the lowest concentration level, (i.e., 10 µg/mL), when compared to untreated cell cultures. This effect is considered to be a chance finding based on the absence of this effect at higher TiO2 NP concentration levels. Similarly, the nuclear bud frequency was statistically significantly increased only after treatment with the lowest TiO2 NP concentration. The proportion of nucleoplasmic bridges was not statistically significantly altered at any TiO2 NP concentration tested. The CBPI and RI were statistically significantly decreased only in cell cultures exposed to 20 µg/cm², indicating a lack of marked cytostatic effects. In contrast, the apoptotic and necrotic indices were statistically significantly increased at all TiO2 NP concentration tested, indicating increased cytotoxicity. Furthermore, the colony forming efficiency assay indicated decreased cell viability under all conditions tested. According to the authors, the TiO2 NPs were found both inside and outside of the cells, when analysed via TEM. The publication shows several reporting and methodological deficiencies. The test material is only poorly characterised, since information on the source, manufacturer, and purity are missing. Moreover, information on the vehicle used and details on the test material preparation are missing. The description of the micronucleus test is insufficient, since information on slide preparation, cell scoring system, and DNA counterstain are missing. The number of cells scored for the occurrence of micronuclei is lower than recommended by the OECD test guideline (OECD TG 487, 2014) in force at that time (1000 vs. 2000 binucleated cells). The TiO2 NPs were tested for cytogenetic using only a long-term exposure scheme; however, according to the test guideline, short-term exposure should be performed and evaluated as well. Information on potential precipitation of the test material in the culture medium is missing. The number of cells analysed in order to determine the CBPI and RI is not specified. An aneugenic positive control group was not included. The cell line used is not a standard cell line used for genotoxicity testing and description of the cell lines lacks details, since information on the potential mycoplasma contamination, modal chromosome number, normal proliferation time, and karyotype stability are missing. Individual culture data are missing, and the results are not presented in a tabular format. Evaluation and scoring criteria are not specified. Historical control data is not provided. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

 

 

Summary entry - In vitro clastogenicity and aneugenicity

The references contained in this summary entry represent in vitro clastogenicity experiments in mammalian cells with very limited value for risk assessment purposes. All references do not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The information contained therein were included for information purposes only.

Shukla, S. et al. (2013): Publications with some reporting and relevant experimental deficiencies. Test material insufficiently characterised.

Guichard, Y. et al. (2012): Reference with reporting and experimental deficiencies.

Aufderheide, M.; Roller, M. (2000): Publication with reporting and experimental deficiencies. Test material insufficiently characterised.

Srivastava, R.K. et al. (2013): Publication with reporting and experimental deficiencies. Test material insufficiently characterised.

Magdolenova, Z. et al. (2012): Reasonably well described publication with little relevance for chemicals safety assessment. Test material insufficiently characterised.

Jaeger, A. et al. (2012): Publication with reporting and experimental deficiencies. Test material insufficiently characterised.

Shukla, R. K. et al. (2011): Reasonably well described publication. Test item poorly characterised.

Osman, I. et al. (2010): Reasonably well described publication Test material insufficiently characterised.

Di Virgilio, A.L. et al. (2010): Publication with reporting deficiencies. Test item poorly characterised.

Falck, G.C.M. et al. (2009): Reasonably well described publication with experimental deficiencies. Test item sufficiently characterised.

Demir, E. et al. (2015): Publication with some reporting and experimental deficiencies. Unclear culture and incubation conditions. Test item insufficeintly characterised. The cytotoxicity measured via inappropriate/imprecise method.

Gurr, J.-R. et al.(2005): It's unclear whether 4 or 10 µg/mL TiO2 was used for exposure (at least 3 concentrations should been evaluated). Moreover, the authors have not used any concurrent positive controls, which are needed to demonstrate the sensitivity of the assay to identify clastogens. For negative controls, historical data is not specified and the cytokinesis-block proliferation index (CBPI) or Replication Index (RI) from at least 500 cells was not reported (ref. to OECD TG 487).

Rahman, Q. et al. (2002): Cytotoxicity data and the relative increase in cell count (RICC) for demonstrating cell division were not reported. (ref. to OECD TG 487). The results of the apoptotic body formation can not be verified due to blurred microscopic images. Regarding to the DNA-fragmentation analysis, the authors observed a DNA-fragmentation after 24 and 48h exposure with 10 µg/mL TiO2. That cannot be confirmed, the agarose gel electrophoresis lanes are smeary, it cannot be determined if the gel was correctly prepared.

Kang, J. K. et al.(2008): Since the increase in micronucleus frequency was only observed in the high dose (50 and 100 µg/mL) it is probable that excessive cytotoxicity is a confounding factor for the result in micronucleus assay at the same concentrations. Furthermore, the results are questionable as the micronucleus assay was not validated (no positive controls) and the cytokinesis-block proliferation index (CBPI) or Replication Index (RI) from at least 500 cells was not reported (ref. to OECD TG 487).

Shi, Y. et al. (2009): The authors had not used any concurrent positive controls, which are needed to demonstrate the ability of the assay to identify clastogens. Analytical verification of target concentration is missing and the cytokinesis-block proliferation index (CBPI) or Replication Index (RI) from at least 500 cells was not reported (ref. to OECD TG 487).

Valdiglesias, V. et al. (2013): The study design is not in accordance with any guideline. The number of cells scored were not specified and the cytokinesis-block proliferation index (CBPI) or Replication Index (RI) from at least 500 cells was not reported (ref. to OECD TG 487). Moreover, the test substance is not sufficiently characterised.

Vales, G. et al. (2014): The study design is not in accordance with any guideline. There are two different exposure durations stated, it is unclear whether the cells were exposed for 24 or 48 hours to titanium dioxide nanoparticles. Furthermore, two uncommon exposure durations were chosen (e.g., 1 and 3 weeks).

Srivastava, R. K. (2011): The study design is not in accordance with any acceptable guideline. The number of cells scored were not specified and the cytokinesis-block proliferation index (CBPI) or Replication Index (RI) from at least 500 cells was not reported (ref. to OECD TG 487). Moreover, the test substance is not sufficiently characterised. No data is given on the correlation of cytotoxicity vs. micronucleus frequency and the dosimetry is unclear since the mg/cm² is not used as dose descriptor.

Prasad, R. Y. et al. (2014): The study design is not in accordance with any acceptable guideline. The experimental procedure is not described (e. g. number of / metaphases scored and evaluation criteria not specified) and the presentation of the results do not allow a conclusive evaluation. The micronucleus frequency of the control is already outside of historical control of other laboratories (0.1 - 1% vs. 0 - 1.5%).

Roszak, J. et al. (2013): The slight cytogenetic potential of the tested nanoparticles in primary human lymphocytes could not be confirmed, since the measured statistically significant increase in the micronucleus frequency at concentrations of 60, 100 and 250 µg/mL (0.3 ± 0.008, 0.5 ± 0.008, and 0.53 ± 2.1 %) in comparison to the vehicle control (0.15 ± 0.006) is in the historical control range of other laboratories (0.1 - 1.5 % / 0.1 - 1.0 %, of the 95th-percentile).

Uboldi, C., et al. (2016): The publication shows major reporting and methodological deficiencies. The test items are self-synthesised and are insufficiently characterised. Only one concentration was tested, thus dose-response relationship cannot be considered. The authors do not state on precipitates or changes in osmolality or pH value of test item-treated cell cultures. The method section contains only very limited information on the experimental procedure. Evaluation and scoring data are missing. Historical control data is not specified.

Zijno, A., et al. (2015): Positive control was not tested for 6 h exposure. Historical control data is missing. No information on metabolic activation of cells and only one condition was tested. Details of TiO2 NP suspension preparation and characterization are missing. Evaluation and scoring criteria are missing.

Li, Y., et al. (2017): Reporting is confusing, fold change and percent are used interchangeably. Positive results were only reported for the microscope-based cell counting and were, however, only slightly increased. Historical control data are missing. Purity, and impurity elements are not specified. Only two concentrations are used for the microscopic analyses, whereas OECD TG 487 recommends testing at least three concentrations. Statements on a change of pH value or osmolality as well as precipitates of the test item-treated cell culture are missing. Information on metabolic activation of the cells is not provided.

Reis, E.M., et al.(2016): Statement on precipitates is missing, which, however, could be a potential confounder of the assay leading to false-positives. Dosimetry is unclear since the mg/cm² is not used as dose descriptor. Statement on metabolic activation is missing. No trend test was performed. Historical control data is missing. Sonication procedure is insufficiently described. Particle characterisation was performed before sonication of test material. However, sonication procedure potentially influences particle sizes as wells aggregates and agglomerates.

 

 

Summary -In vitro clastogenicity and aneugenicity

Based on the reliable studies summarised above, ultrafine titanium dioxide does not induce clastogenic or aneugenic mutations in mammalian cells up to concentrations limited by toxicity or precipitation. Consequently, it is concluded that ultrafine titanium dioxide does not induce chromosome of genome mutations in mammalian cells.

 

 

In vitro DNA damage

Jugan, M.L. et al. (2012) investigated the DNA damaging potential of five ultrafine titanium dioxide samples via comet assay: TiO2-A12 (95% anatase, mean diameter: 12 nm, specific surface area: 92 m²/g, spherical shape), TiO2-R20 NPs (90% rutile, mean diameter: 21 nm, specific surface area: 73 m²/g, spherical shape), TiO2-A25 (AEROXIDE® P25, 86% anatase, mean diameter: 24 nm, specific surface area: 46 m²/g, spherical shape), TiO2-R68 (elongated shaped 100% rutile, mean diameter: L=68 nm D=9 nm, specific surface area: 118 m²/g), TiO2-A140 (100% anatase, mean diameter: 142 nm, specific surface area: 10 m²/g). Genotoxicity was assessed by using the alkaline comet assay after exposure for 4, 24 or 48 h to 100 µg/mL ultrafine titanium dioxide in A549 cells. Comets were scored on at least 50 cells per slide. Results are presented as median of % DNA in comet tail ± standard deviation. After 4 h of exposure, whatever the TiO2-NPs, a significant increase in the level of DNA breaks was observed. This increase in the level of breaks further increased after 24 h of exposure, still it was statistically significant only after exposure to TiO2-A12, -A25 and -R20, but not to TiO2-R68 and -A140. After 48 h of exposure, the frequency of breaks drastically decreased in exposed cells. It reached the basal level, i.e., the level of DNA breakage observed in unexposed cells, in all samples except in cells exposed to TiO2-A12, where a significant DNA fragmentation was still observed. In a parallel experiment, oxidative DNA damages by detection of 8-oxodGuo lesions were observed, showing a similar pattern as the comet assay. Authors hypothesized that the observed DNA damages were (i) caused by ROS being formed by an unknown mechanism, state however that a direct mechanism can be excluded. The DNA damages observed after 4 and 24 hours abated after 48 hours to a level comparable with the negative control. Consequently, the DNA damage observed after 4 and 24 hours is a reversible effect, which was not detectable after 48 hours via comet assay or via micronucleus test (see above). Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment. 

 

Petković, J. et al. (2011) investigated the DNA damaging effect of ultrafine titanium dioxide in human hepatoma cells (HepG2). Two different samples were used: TiO2-An: particle size <25 nm, 99.7% pure, specific surface area (BET): 129.3 m²/g, particle shape: spherical crystallites, XRD: Anatase type; TiO2-Ru: particle size ~10x40 nm, 99.5% pure, specific surface area (BET): 116.7 m²/g, particle shape: elongated crystallites, XRD: Rutile type. Cells were exposed at concentrations of 0, 1, 10, 100 and 250 µg/mL for 2, 4 and 24 hours. In each experiment positive controls (0.5 mM t-BOOH and 50 mM BaP) and a vehicle control (cell growth medium containing 10% PBS) were included. 50 randomly selected nuclei per experimental point were analysed. Three independent experiments were performed for each of the treatment conditions. The percent of tail DNA was used to measure the level of DNA damage. Cytotoxicity was assessed in a parallel experiment via MTT assay. The weak statistically significant findings for ultrafine anatase sample do neither show a time nor a dose dependency. The result on DNA damage for ultrafine anatase is therefore of questionable biological relevance. Ultrafine rutile did not show an increase of DNA damage. 

 

The publication by Kansara, K. et al. (2015) exhibits significant reporting deficiencies, unclear experimental conditions - culture concentrations were given as µg/mL, which is pointless for adherent growing cells - basic information on the cell lines were not reported, i.e. stability of modal chromosome number, cell doubling time and absence of mycoplasma infection - cell seeding conditions were not reported - information on the usage of metabolic activation was missing - cell proliferation/cytotoxicity (CBPI or RI from at least 500 cells) was not stated - information on pH missing - negative control was not clearly described (vehicle, medium, untreated) - historical control data for the negative and positive control is missing - individual data was not presented. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment. 

 

Stoccoro, A. et al. (2017) investigated DNA damage caused by treatment with different types of TiO2 nanoparticles and to reveal differences in the toxic properties of coated vs non-coated nanoparticles. A549 cells were treated with 10, 20, and 40 µg/cm² (32, 64, and 128 µg/ml) TiO2 NP suspensions for 48 h. Afterwards, cells remain untreated or were treated with either Fpg or Endo III to detect oxidized bases. A comet assay was performed and % tail DNA was determined. Colony forming efficiency (CFE) was determined as measure of cytotoxicity. Further, ratios of apoptotic and necrotic cells were determined. According to the authors, with TiO2 Aeroxide P25 showed the strongest capacity to induce DNA damage characterized by a dose-dependent and significant increase of % tail DNA at all concentrations tested. Pristine and silica coated TiO2 NPs showed a significantly increased DNA damage at higher concentrations. Treatment with silica-coated TiO2 NPs resulted in no significant induction of DNA damage. Concurrently, TiO2 showed a cytotoxic capacity characterized by a slight but significant decrease of the CFE at all concentrations (1.25, 2.5, 5, 10, 20, 40, and 80 µg/cm²) and exposure durations (24, 48, and 72 h), except for 1.25 and 10 µg/cm² after an exposure period of 48 h. The study has several shortcomings in study design and reporting. Statements on the test material are partially missing. Purity of the test material is not stated. No statement on osmolality and pH of the administered TiO2 nanoparticle suspension in cell culture. The methods section is described insufficiently. It was not specified whether an alkaline or neutral comet assay was performed. Harvest times after treatment are not specified, modal chromosome number, cell proliferation rate, and number of passages are not stated. Number of scored cells, hedgehog number is not specified and not historical control data were provided. The description of statistical analysis is insufficient. The authors do not specify whether the data sets were tested for normal distribution or homogeneity of variances. The authors state that TiO2 nanoparticle resulted in increased cytotoxicity and necrosis rates. Noteworthy, that cytotoxicity is a confounder of comet assay, leading to false positive findings. Number of analysed cells is too low the OECD test guideline for in vivo testing recommends scoring of at least 150 randomly selected cells. Further, the author used lesion-specific endonucleases to reveal oxidative DNA damages induced by TiO2 nanoparticles. However, it is reported that treatment with these exogenous enzymes increases the inter-sample-variability. Thus, positive findings provide only limited data for a hazard conclusion. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

 

El Yamani et al. (2017) tested the in vitro genotoxicity of four different nanomaterials by using a high throughput system, which should improve the output of comet assays for risk assessment purposes. A549 and TK6 cells were treated with TiO2 NM100 at 10 different concentrations for 3 or 24 h. An alkaline comet assay was performed with cells treated with or without Formamidopyrimidne-DNA Glycosylase (Fpg) enzyme, which detects oxidised bases. Afterwards, % tail DNA was determined. Cytotoxicity was determined using colony forming efficiency (CFE) assay and the colorimetric AlamarBlue assay. According to the authors, 3 h treatment with TiO2 NM100 suspensions resulted in a dose-dependent increase of DNA damage in both cell lines. The same was observable when using the lesion-specific Fpg endonuclease, indicating oxidation of purines. However, DNA damage was strongly reduced at 24 h of exposure. The DNA damaging capacity was generally higher for A549 cells than for TK6 cells. There was no significant cytotoxicity observable for A549 cells independent of the concentration and assay used. The study shows several shortcomings in study design and reporting. The test material is not characterised sufficiently. Purity and impurity elements of the test material are not stated. Information on comet assay evaluation criteria are unclear or missing. The authors do not state on scorable vs. non-scorable cells and hedgehog occurrence is not stated. Details on statistical analysis are missing. Performance of a trend test was not specified. There are no statements on the normality of homogeneity of variances of the analysed data. Historical data are missing. A vehicle control was not used in the assay. Cytotoxicity assays were only performed for the A549 cells but not for the TK6 cells. Measurement of cytotoxicity is essential to interpret the findings, since cytotoxicity is reported to be a confounder of comet assay potentially leading to false positives. Further, the author used lesion-specific endonucleases to reveal oxidative DNA damages induced by TiO2 nanoparticles. However, it is reported that treatment with these exogenous enzymes increases the inter-sample-variability. Thus, positive findings provide only limited data for a hazard conclusion. Only treatment with 140 µg/ml TiO2 NM100 resulted in a significantly increased DNA damage only in TK6 cells after 3 h of exposure. Such isolated finding at one concentration and one time point is likely to be a chance finding and should have been verified in a second experiment. The authors do not state on pH effects or effects on osmolality of the treatment suspension in cell culture. Results of CFE assay are shown as negative control-normalised values. The displayed results for TiO2 NM100 treated cells show significant cytotoxicity at the lowest concentration tested. However, the cytotoxicity decreases with increasing TiO2 NM100 concentrations, which is implausible. Number of analysed cells is too low, the OECD test guideline for in vivo testing recommends scoring of at least 150 randomly selected cells. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment. 

 

Haase, A. et al. (2017) determined the genotoxic capacities of 16 nanomaterials using an in vitro 3D human bronchial tissue model. The model was treated with a TiO2 NM-105 suspension for 60 h at a concentration of 50 µg/cm². An alkaline comet assay was performed and DNA damage measured by % tail DNA was determined. Cytotoxicity was determined by analysing the LDH activity and ATP vs total protein ratio. According to the authors, they were not able to detect increased DNA damage. Cytotoxic effects were observed in the ATP, but not in the LDH assay. However, the authors stated that the ATP showed strong variability and needs to be optimized.  

 

Armand, L. et al. (2016) assessed effects on DNA damage by titanium dioxide NANO4 (P25, Aeroxide) using the comet assay. The comet assay was performed either as conventional alkaline comet assay or as modified alkaline comet assay using formamidopyrimidine-DNA glycosylase (Fpg) to assesses oxidative DNA damage. A549 (human lung carcinoma cells) were treated with 1, 2.5, 5, 10 and 50 µg/mL titanium dioxide NANO4 for 1 day, 1 week, 2 weeks, 1 month or 2 months. Afterwards, 50 comets per slide were scored for % DNA in tail. Three slides were analysed per run, and four independent experiments were performed. Additionally, the authors investigated cytotoxicity via MTT, trypan blue staining, and propidium iodide staining. Cell proliferation was assessed by determination of the population doubling level (PDL) at each exposure concentration and at each cell passage. The authors concluded that titanium dioxide NANO4 showed no cytotoxicity after long-term exposure, as no overt toxicity was found in MTT assay, trypan blue staining and cell counting as well as propidium iodide staining and flow cytometry. However, they concluded that highest titanium dioxide NANO4 exposure concentrations and the longest exposure time significantly decreased cell proliferation, as indicated by counting the PDL after each passage. Moreover, they concluded that titanium dioxide NANO4 showed a genotoxic potential based in the effect on DNA damage in the alkaline comet assay. Since the damage is even more pronounced in the modified alkaline Comet assay, they concluded further, that DNA damage is also caused by oxidative stress. However, based on the results presented no conclusion on the DNA damaging potential of titanium dioxide can be drawn due to reporting and methodological deficiencies. The authors did not state on precipitates as well as pH or osmolality changes in cell cultures during titanium dioxide NANO4 exposure. They only stated, based on a selection of TEM images, that there were no major particle accumulations on the cell membrane. However, analyses of precipitation are crucial since particle accumulation on the cell surface is known to be potential confounder leading to false positives. This is especially important for long-term experiments as presented herein. No information whether the Comet assay was performed in absence of light. Titanium dioxide P25 is a photocatalytic particle which is known to cause secondary DNA damage. Historical control data is missing. Positive control was run, but the results were not presented. However, analysis and presentation of controls is crucial to show validity of the test system. Essential details on electrophoresis are missing. A trend test, in order to investigate on a potential concentration response relationship, was not performed. Moreover, laboratory’s proficiency in preparing and analysing comets’ characteristics in comet assays is a prerequisite for reliable data. It was shown that there is huge variability in tail DNA intensities even within negative or positive controls (Tavares, A.M. et al. (2014); Nanogenotox Programme).

 

Di Bucchianico, S. et al. (2017) evaluated DNA damage induced by titanium dioxide NANO2 (NM103) using the in vitro comet assay. The BEAS-2B (human epithelial bronchial cell line) cells were treated with titanium dioxide NANO2 at concentration levels of 1, 5, and 15 µg/mL for both 3 and 24 h. The assay was performed in three different versions. DNA strand breaks were assessed using the conventional alkaline comet assay. DNA oxidation was evaluated by performance of a modified alkaline comet assay. In this version, the treated cells were additionally incubated with FPG-enzyme, which detects purine oxidation products. Finally, the authors evaluated lab light as potential confounder of in vitro comet assays with titanium dioxide NANO2. To this end, cells were exposed for several to normal laboratory light before alkaline treatment and electrophoresis. In each sample, a total of 50 cells were scored for the proportion of DNA in tail. All experiments were run in triplicates. Cell viability was determined using the Alamar Blue reagent. Additionally, cytotoxicity was evaluated by determination of apoptotic/necrotic cells via microscopy and flow cytometry. Moreover, mitotic index, cell cycle perturbations and cell survival were determined by flow cytometry. The authors concluded that titanium dioxide NANO2 showed in general no or low cytotoxic effects at the doses tested. Moreover, they concluded that the test item did not cause DNA strand breaks. However, they concluded that titanium dioxide NANO2 showed a weak genotoxic potential characterised by the induction of oxidative DNA damage. Further, they showed that lab light can interfere with the results of the in vitro comet assay when using photocatalytic titanium dioxide nanoparticles. The results of the modified alkaline comet assay using FPG-enzyme are implausible and raise some doubts on the reliability of the assay. Treatment with titanium dioxide NANO2 showed a significant induction of oxidative DNA damage after 3 h. However, after 24 h the increase in % DNA in tail was neither significant nor concentration dependent. The same was observable for titanium dioxide NANO2 in the cell viability assay. This observation is implausible since titanium dioxide nanoparticles are not metabolised and do not further dissolve. Thus, transient effects are not expected. Moreover, the publication shows some reporting and experimental deficiencies. Cytotoxicity was not measured after 24-hour titanium dioxide treatment. Details on particle characterisation are missing. It is stated that titanium dioxide NM103 nanoparticles are coated, however, details on the surface modification are missing. Evaluation criteria are not specified. The authors provide no statement on a potential exogenic metabolic activation of the cell line. Moreover, information on mycoplasma contamination, modal chromosome number and cell cycle times are not specified. Statements on marked changes of pH value or osmolality of test item-treated cell cultures are missing. Information on precipitation of the test item is not given. A justification the selection of concentration is missing. The authors conducted no trend test to evaluate a potential concentration response relationship. Information on hedgehog occurrence and number is not specified.

 

Di Bucchianico, S. et al. (2017) evaluated DNA damage induced by titanium dioxide NANO7 (NM101) using the in vitro comet assay. BEAS-2B (human epithelial bronchial cell line) cells were treated with titanium dioxide NANO7 at concentration levels of 1, 5, and 15 µg/mL for both 3 and 24 h. Untreated cells and cells treated with Ro 19-8022 photosensitiser (together with light irradiation) were used as negative and positive controls, respectively. The assay was performed in three different versions. DNA strand breaks were assessed using the conventional alkaline comet assay. DNA oxidation was evaluated by performance of a modified alkaline comet assay. In this version, the treated cells were additionally incubated with FPG-enzyme, which detects purine oxidation products. Finally, the authors evaluated lab light as potential confounder of in vitro comet assays with titanium dioxide NANO2. To this end, cells were exposed for several to normal laboratory light before alkaline treatment and electrophoresis. In each sample, a total of 50 cells were scored for the proportion of DNA in tail. All experiments were run in triplicates. Cell viability was determined using the Alamar Blue reagent. Additionally, cytotoxicity was evaluated by determination of apoptotic/necrotic cells via microscopy and flow cytometry. Moreover, mitotic index, cell cycle perturbations and cell survival were determined by flow cytometry. The authors concluded that titanium dioxide NANO7 showed in general no or low cytotoxic effects at the doses tested. Moreover, they concluded that the test item did not cause DNA strand breaks. However, they concluded that the titanium dioxide nanoparticles showed a weak genotoxic potential characterised by the induction of oxidative DNA damage. Further, they showed that lab light can interfere with the results of the in vitro comet assay when using photocatalytic titanium dioxide nanoparticles. The publication shows some reporting and experimental deficiencies. Cytotoxicity was not measured after the 24-hour titanium dioxide nanoparticle treatment. Details on particle characterisation are missing. It is stated that titanium dioxide NM101 nanoparticles are coated, however, details on the surface modification are missing. Evaluation criteria are not specified. The authors provide no statement on a potential exogenic metabolic activation of the cell line. Moreover, information on mycoplasma contamination, modal chromosome number and cell cycle times are not specified. Statements on marked changes of pH value or osmolality of test item-treated cell cultures are missing. Information on precipitation of the test item is not given. A justification the selection of concentration is missing. The authors conducted no trend test to evaluate a potential concentration response relationship. Information on hedgehog occurrence and number is not specified.

 

Andreoli, C. et al. (2018) investigated on DNA damage in human peripheral blood monocytes after exposure to three different titanium dioxide nanoparticle types ((i) crystalline phase: anatase, particle size: 15 nm; (ii) crystalline phase: rutile, particle size: 10 x 40 nm; (iii) crystalline phase: rutile/anatase, particle size: 25 - 70 nm). In the first experiment, the cells of two different male donors were treated with titanium dioxide nanoparticles at concentrations of 10, 50, 100, and 200 µg/mL for 24 hours. Afterwards, a conventional alkaline comet assay was performed, and the cells were scored for the proportion of DNA in tail. In the second experiment, the cells were treated at the same concentration levels for 6 or 24 hours. After the treatment, the DNA was extracted and the level of 8-oxodG was analysed using HPLC. Cytotoxicity was evaluated using the trypan blue exclusion test. According to the authors, none of the titanium dioxide nanoparticle types tested showed increased cytotoxicity in the trypan blue exclusion test. In the conventional alkaline comet assay, titanium dioxide rutile nanoparticles induced a statistically significant increase in the proportion of DNA in tail starting at a concentration level of 100 µg/mL. The titanium dioxide anatase and rutile/anatase nanoparticles induced statistically significantly increased proportions of DNA in tail already at a concentration level of 50 µg/mL. Moreover, the treatment with each of the titanium dioxide nanoparticle types resulted in a shift of cell populations showing low DNA damage levels to populations showing high DNA damage levels. Further, all titanium dioxide nanoparticles types tested induced an increase in steady-state levels 8-oxodG DNA at concentration levels of 100 and 200 µg/mL under the conditions tested. The publication shows several reporting and methodological deficiencies. The number of analysed cells is too low the OECD test guideline for in vivo testing recommends scoring of at least 150 randomly selected cells. The authors performed a MNvit experiment in parallel using the same maximum concentration (200 µg/mL). In the MNvit, the authors reported that heavy precipitation precluded the scoring of MN frequencies in some cultures. In the comet assay, however, the authors provided not any information on test material precipitation, which potentially could have had interfered with the comet assay results. It is unclear whether the cells were kept during cell lysis and electrophoresis under dark conditions, which is critical especially for titanium dioxide anatase nanoparticles. Moreover, no information is provided on potential test material remnants during electrophoresis, which could have had potentially interfered with the assay. Justification for the selected concentration range is missing. Statements on marked changes of pH value or osmolality of test material-treated cell cultures are missing. Details on particle characterisation, including surface treatment, manufacturer, and reactivity, are missing. Dye exclusion tests are in current standard test guidelines, considered to be not adequate to evaluate cytotoxicity. The results of the cytotoxicity are not tabulated. Scoring criteria are not specified. Evaluation criteria are not specified. Information on the occurrence of hedgehogs is not provided. The methodology followed to investigate on oxidative DNA damage is only poorly described. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

 

Santonastaso, M. et al. (2019) evaluated the DNA damage in human sperm cells after in vitro treatment with titanium dioxide NANO4 (Aeroxide P25). The human sperm cell samples were obtained from donors aged between 25 and 39 years. In a conventional alkaline comet assay, the samples were treated with titanium dioxide nanoparticles at concentration levels of 1 and 10 µg/L for 15, 30, 45, or 90 minutes. The DNA damage was evaluated by determination and comparison of the proportion of DNA in tail. According to the authors, the sperm vitality was not statistically significantly changed after the titanium dioxide NANO4 treatment. However, the sperm motility was statistically significantly reduced after 45- and 90-min exposure at both titanium dioxide nanoparticle concentration levels tested. In the comet assay, the proportion of DNA in tail was statistically significantly increased after 30 min at both concentration levels tested, when compared to untreated control aliquots. The proportion of DNA in tail increased with increased exposure times and the strongest response was observed after 90 min exposure (≤ 3-fold increased above untreated control). In general, the number of samples used for each experiment, the number of replicates, and the number of independent experiments is unclear. Only two concentration levels were tested, and thus, concentration response relationship cannot be evaluated adequately. The comet assay methodology is only poorly described, since information on the temperature during lysis and electrophoresis, the number of samples used, and the number of nucleoids analysed is missing. Further, information on the presence of particles remnants during electrophoresis are not provided. Moreover, the authors did not report on light conditions during exposure, lysis, and electrophoreses. The exposure to light could have confounded the results due to the photocatalytic activity of the particles. The authors do not state on changes of pH or osmolality in test item-treated cell aliquots. Moreover, the authors do not report on precipitates. Evaluation and scoring criteria are not specified. Information on hedgehog occurrence is not provided. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

 

Liao, F. et al. (2019) performed a conventional alkaline comet assay in order to evaluate the DNA-damaging potential of four differently sized titanium dioxide anatase nanoparticles types (T10: 10 ± 2.3 nm; T30: 30 ± 5.1 nm, T50: 50 ± 7.6 nm, and T100: 100 ± 14.3 nm) in human umbilical vein endothelial cells (HUVECs). The cell cultures were treated with the titanium dioxide anatase nanoparticle types at concentrations of 1, 5, and 25 µg/mL for four hours. Afterwards, the comets were prepared and the OTM was determined via fluorescence microscopy. The assay was performed in three independent experiments using triplicate cultures. The titanium dioxide anatase nanoparticle types, T10, T30, and T50, induced statistically significantly increased OTMs at all concentration levels tested. Treatment with the largest particle type, T100, resulted only at 5 and 25 µg/mL in a statistically significantly increased OTM, when compared to the negative control cultures. The responses appeared to be dependent on the concentration level and the particle size. Based on the following shortcomings, the study is considered to be not reliable. The test material was insufficiently characterised, since information on the purity, manufacturer, coatings, surface reactivity, and morphology are missing. Further, details on the preparation of the test material are missing, especially information on the sonication procedure are missing. The cell line used in no standard in genotoxicity testing and not recommend by the current test guideline (OECD TG 487). Essential cell line details, including the source, passage number, and mycoplasma infection state, are not included. Details on the staining methodology applied are missing. Cytotoxicity was not evaluated and could have led to false positives. Moreover, information on test material precipitation and effects on the pH and osmolality of the medium are not provided. Moreover, no information is provided whether test material was present during electrophoresis, which could have had potentially interfered with the assay. A justification for the selected concentration range is missing. Cellular uptake of the test material was not evaluated. A positive control was not included. The type of negative control used is unclear. Information on scoring and acceptability criteria are not provided. Scoring was not performed using an automatic or semi-automated image-analysis system. The DNA damage was evaluated using the OTM, whereas proportion of DNA in tail is the recommended parameter according to the in vivo comet assay test guideline (OECD TG 489). Information on the occurrence and number of hedgehogs is not provided.

 

Chakrabarti, S. et al, (2019) evaluated titanium dioxide nanoparticles (particle size: 58.25 ± 8.11 nm) for their potential to induce DNA strand breaks in murine RAW 264.7 macrophage cells using the conventional comet assay. The cell cultures were exposed for 24 hours to titanium dioxide nanoparticle concentration levels of 10, 25, 50, 75, and 100 µg/mL. The nucleoids were scored for several DNA damage parameters, i.e. tail length, proportion of DNA in tail, tail moment, and olive moment. A total of 100 randomly chosen nucleoids were analysed per concentration. In a separate experiment the cell viability was assessed using the resazurin stain. Additionally, apoptosis and cell cycles were analysed using flow cytometry. All DNA damage parameters evaluated showed a concentration dependent increase after exposure to the titanium dioxide nanoparticles. Moreover, the tail length, proportion of DNA in tail, and tail moment were statistically significantly increased at all concentrations tested when compared to negative control cultures. The olive moment was statistically significantly increased at concentration levels of 25 µg/mL and above. Similarly, the treatment with titanium dioxide resulted in statistically significant and concentration related decrease in cell viability (decreased to 78.36%-46.62% of the viability of the control culture) as indicated by the resazurin assay. The flow cytometry experiments showed marked and statistically significant reduction in cell viability (reduced to 53.5%-25.3%) and arrest in G2/M cycle, when compared to negative control cultures. However, the test material was insufficiently characterised, since information on the purity, crystalline phase, manufacturer, coatings, and morphology are missing. Further, the authors provided no information on the vehicle used or any details on the preparation of the test material. The cell line used is not a standard cell line used for genotoxicity testing. Moreover, the description of the cell line is insufficient, since information on the passage number, mycoplasma contamination, and normal proliferation time are missing. The statistically significant increases in DNA damage parameters were associated with statistically significant and marked cytotoxicity (46.5 - 74.7% cytotoxicity), as indicated by flow cytometric analyses, at all concentration tested. Thus, it is unclear whether the effects on DNA damage parameters are due to cytotoxicity, genotoxicity, or a combination of both. Potential interference of the test material with the colorimetric cytotoxicity assay was not tested. Information on potential test material precipitation as well as pH and osmolality effects on the culture medium are not provided. Further, the authors did not state on potential particle remnants during electrophoresis. The number of cells analysed per concentration is lower than recommended in an equivalent in vivo test guideline (OECD TG 489; 100 cells per concentration vs. ≥ 150 cells per animal). The test concentration is specified in µg/mL which is not adequate for adherent cell lines. The type of negative control used is not specified. A positive control culture was not included. Evaluation and scoring criteria are not specified. Historical control data is not provided. Information on occurrence of hedgehogs in not provided. The authors did not investigate on the cellular uptake of the test material. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

 

Kazimirova, Z. et al. (2019) investigated on DNA damage in human peripheral blood mononuclear cells after treatment with titanium dioxide NANO4 (Aeroxide P25) using the alkaline comet assay. The test material was dispersed according to different protocols in order to get dispersions with low agglomeration (DP1) or high agglomeration (DP2) levels. The cell samples are obtained from eight male and six female donors and were not pooled for comet assay. The cells were exposed to each type of titanium dioxide nanoparticle suspension (DP1 and DP2) at concentrations levels of 3, 15, and 75 µg/cm² for 4 and 24 hours. After cell lysis cells were either left untreated or treated with formamidopyrimidine DNA glycosylase (FPG) to investigate on oxidative DNA damage. For each group duplicate slides were prepared and 100 nucleoids per slide were scored for the proportion of DNA in tail. According to the authors, the titanium dioxide nanoparticle suspension DP1 induced slight but statistically significant increases (≤ 2-fold) in the proportion of DNA in tail, when compared to untreated control cultures, at concentration levels of 75 and ≥ 15 µg/cm² after the 4- and 24-hour exposure, respectively. In contrast, exposure to the titanium dioxide suspension DP2 did not result in a statistically significant increase in the proportion of DNA in tail at any concentration tested and independent of the exposure duration. Moreover, none of the two titanium dioxide nanoparticle suspension types induced oxidative DNA damage as evidenced by the comet assay using the FPG enzyme. Individual analysis of the proportion of DNA in tail per donor revealed that 9/13 and 8/13 donors showed statistically significantly increased values after treatment with titanium dioxide nanoparticle DP1 suspension for 4 and 24 hours, respectively. Moreover, treatment with titanium dioxide suspension DP2 resulted in 8/13 and 5/13 donors a statistically significantly increased proportion of DNA in tail when treated for 4 and 24 hours, respectively. The publication shows several reporting and methodological deficiencies. The description of the comet assay methodology is insufficient and lacks essential details. Information on temperature, light conditions, and particle remnants during electrophoresis are not provided. Especially, the exposure to light could have confounded the results due to the photocatalytic activity of the particles. The scoring methodology is not described. The authors did not report on potential test item precipitation, pH effects, and effects on osmolality in culture medium. The donors are older than usually recommended by OECD genotoxicity test guidelines (40-50 years vs. 18-35 years). Moreover, it is not stated why single donors are excluded from the analyses. Cytotoxicity was not assessed in parallel. Potential concentration-response relationships were not analysed. However, the combined results showing a statistically significant increase after the 24-hour treatment with titanium dioxide nanoparticle suspension DP1 appeared to be not clearly concentration related. Further, most of the responses observed in the individual analysis showed no clear concentration response relationship. The recurrent observation that lower concentrations showed higher responses than the highest test concentration indicates high experimental variability. Moreover, the background response is highly variable and only single responses are above the highest background values observed. The results are not presented in a tabular format. Evaluation and scoring criteria are not specified. Historical control is not provided. No information on hedgehog occurrence. Particle uptake by the cells was neither examined nor discussed. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

 

Murugadoss, S. et al. (2020) performed a comparative study on the DNA damaging potential of two different titanium dioxide nanoparticle types ((i) NM10200a, crystalline phase: anatase, particle size: 117 nm; (ii) NM10202a, crystalline phase: anatase, particle size: 17 nm) each prepared in suspensions with different agglomeration states. Each of the different suspensions were tested in human bronchial epithelial cells (HBE), human monocytic cells (THP-1), and in Caco2 cells. The cell cultures were exposed to the four different titanium dioxide nanoparticle suspension types at concentration levels of 5, 25, 50, and 100 µg/mL for 24 hours. Afterwards, an alkaline comet assay was performed. Three independent experiments were performed in duplicates. A total of 50 nucleoids per experiment were scored for the proportion of DNA in tail. In two separate experiments, the cytotoxicity of the titanium dioxide nanoparticle suspension was examined using the colorimetric WST-1 assay and by determination of the cellular leakage of LDH using a kinetic assay. In the comet assay, the proportions of DNA in tail were statistically significantly increased in all cell lines exposed independent of the titanium dioxide nanoparticle size or agglomeration state. According to the authors, the response induced by the two different titanium dioxide nanoparticle suspension types were comparable under all conditions tested. Only in THP-1 cells, large agglomerates induced a stronger response when compared to the small agglomeration suspension containing the same titanium dioxide nanoparticle type. The four different titanium dioxide nanoparticle suspensions did not induce cytotoxicity as evaluated by means of WST-1 assay and the LDH leakage assay. The publication shows several reporting and methodological deficiencies. The test material was insufficiently characterised, since information on the purity and coatings are missing. The composition of the suspension medium is not described. The THP-1 and HBE cell lines are no standard cell lines regularly used for genotoxicity testing. The methodology is only poorly described and nearly all essential details on the comet assay are missing. The number of cells analysed per concentration is lower than recommended in an equivalent in vivo test guideline (OECD TG 489; 100 cells per experiment vs. ≥ 150 cells per animal). Potential interference of the test material with the colorimetric cytotoxicity assay was not tested. Information on potential test material precipitation as well as pH and osmolality effects on the culture medium are not provided. Further, the authors did not state on potential particle remnants during electrophoresis. The results from positive control cultures are neither discussed nor shown. Evaluation and scoring criteria are not specified. Historical control data is not provided. Information on occurrence of hedgehogs in not provided.

 

Stoccoro, A. et al. (2016) evaluated the DNA damaging capacity of TiO2 P25 in Balb/3T3 mouse fibroblasts using the modified alkaline comet assay. The cells cultures were exposed to 10, 20, and 40 µg TiO2 NP/cm² for 2, 24, 48, and 72 hours. Negative (untreated) and positive (hydrogen peroxide) control cultures were run concurrently. In the first assay, the cell cultures were investigated for primary DNA using and the nucleoids were not incubated with restriction enzymes. In the subsequent assay, the nucleoids were incubated either with endonuclease III or formamidopyrimidine glycosylase in order to investigate on oxidative DNA damage. Each assay was performed in three independent experiments. A total of 100 randomly selected nucleoids per sample were scored for the proportion of DNA in tail. In a separate experiment, the cell viability was evaluated using the colony forming efficiency assay. Moreover, the cellular uptake of the test material was analysed using TEM. According to the authors, cell cultures exposed to TiO2 NPs for 2 and 24 hours showed a statistically significantly increased proportion of DNA in tail at all concentration tested, when compared to untreated control cultures. However, no such effect was observed after prolonged exposure (48 and 72 hours) independent of the concentration level tested. This effect pattern appears, however, to be only limitedly plausible for a metal oxide and was not further investigated by the authors. Analysis of oxidised DNA lesions revealed a statistically significantly increased proportion of DNA in tail after the 2-hour exposure independent of the TiO2 NP concentration tested and after the 24-hour exposure at the lowest concentration level. The response appears to be negatively correlated with the concentration level, and thus, the nature of this effect remains unclear. Moreover, after incubation with endonuclease III, the proportion of DNA in tail was statistically significantly increased only after the 2-hour exposure period. Furthermore, the colony forming efficiency assay indicated decreased cell viability under all conditions tested. According to the authors, the TiO2 NPs were found both inside and outside of the cells, when analysed via TEM. The publication shows several reporting and methodological deficiencies. The test material is only poorly characterised, since information on the source, manufacturer, and purity are missing. Moreover, information on the vehicle used and details on the test material preparation are missing. The number of analysed cells is too low, since the equivalent OECD test guideline for in vivo testing recommends scoring of at least 150 randomly selected cells. The description of the methodology lacks essential details, since information on cell lysis, electrophoresis, neutralisation, and DNA stain are missing. Moreover, the light conditions during the experiment are not clearly stated, since the authors stated only that “experiments were performed for each treatment by avoiding direct light exposures of preparing slides”. Information on potential marked changes of osmolality of test material-treated cell cultures are not provided. Furthermore, the authors did not state on test material precipitation in the culture medium and potential particle remnants during electrophoresis. The evaluation of potential cytotoxic effects was restricted to a separate colony forming efficiency assay. The assay revealed that all concentration levels tested were cytotoxic at exposure periods longer than 2 hours. The results on DNA damage are partly implausible. Individual culture data are missing, and the results are not presented in a tabular format. Evaluation and scoring criteria are not specified. Historical control data is not provided. Information on the occurrence of hedgehogs is not included. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

 

Tomankova, K. et al. (2015) performed comet assays in order to evaluate the DNA damaging capacity of two TiO2 NP types (TiO2 and Nanorutil) in three different mammalian cell lines. The cell lines used were NIH3T3 mouse fibroblasts, SVK14 human keratinocytes, and BJ human foreskin fibroblasts. In a preceding experiment, the IC50 values of the two test materials in the different cell lines were determined using the MTT assay. In the subsequent comet assay, the cells were exposed to the test materials for six hours at the respective IC50 concentration, which ranged from 508.6 to 5659.8 mg/L. Afterwards, the cells were stained with SYBR Green and scored for the proportion of DNA in tail. Additionally, the authors investigated on ROS generation, cellular uptake, apoptosis induction, and mitochondrial membrane potential change. According to the authors, the test material, TiO2, when tested at the IC50 concentration, induced a statistically significantly increased proportion of DNA in tail, apoptosis rate, and ROS generation in all cell lines tested. Moreover, Raman spectroscopical analysis revealed cellular uptake of the test material in SVK14 cells. The treatment with Nanorutil resulted in a statistically significantly increased proportion of DNA in tail and ROS generation in all cell lines tested. Moreover, the apoptosis rate was increased in BJ and NIH3T3 cells after Nanorutil treatment at the IC50 concentration. Further, SVK14 cells showed uptake of Nanorutil. The mitochondrial membrane potential was not altered by TiO2 and Nanorutil independent of the cell lines tested. The publication shows several reporting and methodological deficiencies. The purity of the test material is not specified. Moreover, information on the vehicle used and details on the test material preparation are missing. Further, the concentration tested (508.6 to 5659.8 mg/L) exceeded by far the solubility of titanium dioxide (< 1 µg/L in a 24-hour transformation/dissolution test (according to OECD testing series 29 and under GLP)). The number of cells scored for DNA damage is not specified. The test was performed using only a single concentration level per test material and cell line. The comet assay was performed exclusively at IC50 concentrations. The methodology is only poorly described, since details on cell lysis, electrophoresis, neutralisation, and DNA staining are missing. Furthermore, the light conditions during the experiment are not specified, and thus, photocatalytic reactions leading to false positive cannot be excluded. Information on marked effects on the pH and osmolality of the culture medium by test material treatment are not provided. Moreover, the authors did not state on test material precipitation in the culture medium and potential particle remnants during electrophoresis. Individual culture data are missing, and the results are not presented in a tabular format. Evaluation and scoring criteria are not specified. Historical control data is not provided. Information on the occurrence of hedgehogs is not included. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

 

Danielsen, P.H. et al.(2020) evaluated the DNA damaging potential of four different anatase titanium dioxide nanoparticles in lung and liver cells of female C57BL/6jBomtac mice after a single intratracheal instillation. The titanium dioxide nanoparticles, TiO2 NM-1 (MKN-A015), TiO2 NM-2 (MKN-A100), TiO2 tubes (self-synthesised), and TiO2 cubes (self-synthesised), were administered once at total dose levels of 18, 54, and 162 µg per mice. Liver, Lung, and BALF cells were obtained after 1, 3, 28, and 90 days post-exposure. The DNA damaging potential was evaluated using the alkaline comet assay. The extent of DNA damage was evaluated by determination of the proportion of DNA in tail and tail length. A vehicle control group was run concurrently. The quartz (DQ12) was included as benchmark material. In addition, the authors investigated on inflammation via BALF analyses and lung histopathology (c.f. section 7.5.4). According to the authors, mice exposed to titanium dioxide nanoparticles via intratracheal instillation showed only a few significant increases in lung, liver, and BALF cells. These sporadic findings were considered to be chance finding due to a general lack of clear dose-dependencies. The publication shows several reporting and methodological deficiencies. The administration via intratracheal instillation is a non-physiological route of exposure and considered to be not relevant. The number of animals and nucleoids per animal evaluated for DNA damage is not specified. The sensitivity of the assay and the proficiency of the laboratory is not demonstrated, since a positive control group was not included and the results from the in vitro positive control were not shown. Moreover, the validity of the assay was not demonstrated, since historical negative control data are not provided. The results are restricted to obtained comet tail length, data on the proportion of DNA in tail are, however, missing. The authors did not provide evidence for the exposure of the liver. The characterisation of the test material lacks some details, e.g., purity and potential surface modifications. Evaluation and scoring criteria are missing. The test animals and the test animal housing conditions are not sufficiently described. Based on the above-mentioned shortcomings the reference is considered not reliable.

 

 

Summary entry - In vitro DNA damage

The references contained in this summary entry represent in vitro DNA damage experiments in mammalian cells (Comet assay) with very limited value for risk assessment purposes. All references do not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The information contained therein were included for information purposes only.

The test material used in the references summarised in the summary entry was largely characterised insufficiently. As detailed in the introductory statement to chapter for human health hazard assessment, it is problematic to base the hazard and risk assessment of clearly characterised titanium dioxide on information generated with poorly characterised test item, with no market relevance. In accordance with ECHA guidance, all references that do not provide sufficient information on the substance identity were disregarded for hazard and risk assessment purposes (for further information please refer to the introductory statement).

Protocols employed for the conduct of the in vitro comet assay do not meet minimal quality standards for conduct of the comet assay. Application of the comet assay to intact cells and tissues must carefully control for natural process that can produce DNA fragmentation and false positive assay outcomes. Cytotoxicity (Henderson et al., 2008; Fairbairn et al., 1996), apoptosis (Choucroun et al., 2001; Fairbairn et al., 1996), oxidative and temperature stress and terminal differentiation must all be carefully assessed for their impact upon assay outcomes. Further, the studies do not control for the presence of residual particles during processing and electrophoresis of the cells – presence of particles during these steps might also lead to false positive results. Information can also be obtained from detailed evaluation of the shape of Comet tails and the distribution of DNA within the tail (Lee et al., 2003). Little meaningful information is provided on any of these accepted response parameters. The studies summarised below controlled for none of these sources of artifactual false positives. Interpretation of the relevance of both positive and negative results from this test system cited above is therefore unclear and was not used for the current assessment of in vitro genotoxicity of nano and bulk size titanium dioxide.

 

Shukla, S. et al. (2013): Cytotoxicity measured in an independent experiment via MTT and neutral red uptake. No significant cytotoxicity observed in 6h exposure group. Authors conclude that TiO2 NP induce a significant (p < 0.05) concentration-dependent increase in oxidative DNA damage (10-80 mg/mL). TiO2 NPs induced a statistically significant (p < 0.05) increase in the number of micronucleated cells at 20 mg/ml. The positive findings were received in singulate cultures and were not confirmed in a secondary experiment. The positive finding in the MN was not dose-dependent and needs to be considered as incidental. Authors applied very high exposure concentrations.

Wan, R. et al. (2012): The authors conclude that nano TiO2 did not induce DNA damage. Experimental conditions insufficiently described. Authors focus on the effects of other NP with little information on effects caused by TiO2 NP. Details on test item characterisation were not reported.

Guichard, Y. et al. (2012): The authors conclude that No significant micronucleus formation was detected in SHE exposed to particles (5, 10, 50 µg/cm²) for 24 h. At the highest particle concentration (50 µg/cm²), all TiO2 particles except rutile nanoparticles caused increased DNA damage after 24 h of exposure. DNA damage observed at highest concentration was measured at signs of increased cytotoxicity. Conclusion on TiO2 causing direct DNA damage cannot be supported. Experimental procedures insufficiently described.

Bhattacharya, K. et al. (2009): Experimental procedure of the comet experiments insufficiently described. Authors conclude that TiO2-NP did not induce DNA-breakage measured by the Comet-assay in both human cell lines. Slight cytotoxicity was seen only at the highest dose in BEAS-2B, whereas cytotoxicity was seen in IMR-90 cells in doses, up to 40% at the highest dose.

Woodruff, R.S. et al.(2012): No treatment related increase in revertant colonies was observed compared with the control culture. Positive controls induced a significant increase in revertants. Cytotoxicity was caused in a dose dependent manner up to 55% viability at the highest dose. No DNA damage was observed at any concentration via comet assay (reported as %DNA in tail). Significant DNA damage was observed in the positive control culture.

Magdolenova, Z. et al. (2012): No DNA damage was observed at 20 and 40 µg dose. Unusual test design, which is likely not able to detect TiO2 NP induced DNA damage, since incubation duration was too short.

Shukla, R. K. et al. (2011): Cytotoxicity determined in a separate experiment via MTT and neutral red uptake. Cell viability >90% after 6h exposure. A statistically significant and dose dependent increase of DNA damage, expressed as tail moment was observed. Authors hypothesise that the genotoxicity might be caused by reactive oxygen species. Historical data were not presented for the cell line, thus findings are difficult to interpret.

Osman, I. et al. (2010): Cytotoxicity determined via MTT and NR uptake. Comet: cell viability between 70 and 85% (highest dose 65% viability). Authors conclude that TiO2 NP induce dose-dependent increase of DNA damage. No confirmatory experiments were performed. Selection of cell line not appropriately justified.

Hamzeh, M.; Sunahara, G. (2013): Publication with experimental deficiencies. The study design is not guideline compliant (e.g. only one exposure duration was chosen and only two concentrations have been tested). The number of counted cells were too low.

Hackenberg, S. et al. (2011): Publication with reporting deficiencies. Test material insufficiently characterised. Experimental procedure insufficiently described (lysis, unwinding, electrophoresis). Long exposure duration not justified. Number of replicates unknown.

Wang, S. et al. (2011): Publication with reporting and experimental deficiencies. Test material insufficiently characterised. Experimental procedure insufficiently described. Unclear why exposure duration of 60 days was chosen. Findings were not correlated with cytotoxicity.

Falck, G.C.M. et al. (2009): Reasonably well described publication with experimental deficiencies. Test item sufficiently characterised. Cytotoxicity was not correlated with %tail DNA formation. Cytotoxicity was expressed as total cell count 48h after exposure; scoring times do not match with the timing of genetic toxicity experiments. DNA damage as detected by comet especially in the high dose groups appears to be the cause of cytotoxicity. None of the positive MN findings highlighted by the authors were dose-dependent and could be regarded as incidental findings. Confirmatory experiments were not conducted.

Karlsson, H. et al. (2009): Publication with reporting and experimental deficiencies. Test item sufficiently characterised.

Demir, E. et al. (2015): Publication with some reporting and experimental deficiencies. Unclear culture and incubation conditions. Test item insufficiently characterised. The cytotoxicity measured via inappropriate/imprecise method.

Chen, Z. et al. (2014): The test item was not sufficiently characterised. The number of cells counted were too low to give a statistically significant prediction of the genotoxic potential of the test item. Moreover, the extreme standard deviation of the measured % tail DNA does not allow to draw a conclusion of the induced DNA damage.

Hackenberg, S. et al. (2017): The test item source is not reliable and the primary cell line is not sufficiently described. The experimental procedure is insufficiently described (lysis, unwinding, electrophoresis) and only one exposure duration was chosen.

Gurr, J.-M. et al.(2005): Occurring DNA-damage as a secondary effect is probable due to apoptosis. The positive control is not specified, only 1h of incubation time was applied (2 - 6h and 16 - 26h after single-exposure is recommended as sampling time by OECD for in vivo studies), at least the result of almost one concentration (3 are recommended by OECD TG 489) was reported and the number of evaluated cells is not specified.

Kang, J. K. et al.(2008): The observed DNA damage in the Comet assay is likely a secondary toxic effect due to cytotoxicity (up to 70%). Furthermore, there was no validation of the Comet assay performed, positive controls are not specified and only 60 cells per sample were scored (150 are recommended by OECD TG 489 to provide a adequate statistical power).

Shi, Y. et al. (2009): A validation of the Comet assay was not performed (no positive controls), only 100 cells per sample were scored (150 are recommended by OECD TG 489 toprovide a adequate statistical power) and analytical concentration verification is missing.

Prasad, R. et al. (2013): The study design is not in accordance with any guideline. In comparison with a suitable guideline, the acceptability criteria of the OECD test guideline 489 (in vivo mammalian alkaline comet assay) were not fulfilled since adequate number of cells (> 150 cells per slide) have not been analysed and the sampling time is not in accordance with the recommendation (2 - 6 and 16 - 26 hours after last treatment).

Ursini, C.L. et al. (2014): The study design is not in accordance with any guideline. In comparison with a suitable guideline, the acceptability criteria of the OECD test guideline 489 (in vivo mammalian alkaline comet assay) were not fulfilled since adequate number of cells (> 150 cells per slide) have not been analysed to reach a statistically significant level.

Valdiglesias, V. et al. (2013): The study design is not in accordance with any guideline. In comparison with a suitable guideline, the acceptability criteria of the OECD test guideline 489 (in vivo mammalian alkaline comet assay) were not fulfilled since adequate number of cells (> 150 cells per slide) have not been analysed and the sampling time is not in accordance with the recommendation (2 - 6 and 16 - 26 hours after last treatment). Moreover, the test substance is not sufficiently characterised.

Vales, G. et al. (2014): The study design is not in accordance with any guideline. In comparison with a suitable guideline, the acceptability criteria of the OECD test guideline 489 (in vivo mammalian alkaline comet assay) were not fulfilled since adequate number of cells (> 150 cells per slide) have not been analysed and the sampling time is not in accordance with the recommendation (2 - 6 and 16 - 26 hours after last treatment).

Petkovic, C. et al. (2011): The dosing in this study is unclear, since adherent growing cells were used and dosimetry is only given in mg/mL without volume per well (mg/cm² should be used as dose descriptor). Moreover the test substance is poorly characterised and confounding effects of TiO2 particles on MTT assay was not determined.

Botelho M.C. et al. (2014): The result cannot be verified, since dosimetry is unclear since adherent growing cells were used and dosimetry is only given in mg/mL without volume per well (mg/cm² should be used as dose descriptor) and residual TiO2 nanoparticle effects were not determined on the testing system. Moreover cell line properties seemed implausible as apoptosis of approximately 20 % could be observed in the untreated negative control cell line.

Demir, E. et al. (2013): The study design is not in accordance with any guideline. In comparison with a suitable guideline, the acceptability criteria of the OECD test guideline 489 (in vivo mammalian alkaline comet assay) were not fulfilled since adequate number of cells (> 150 cells per slide) have not been analysed and the sampling time is not in accordance with the recommendation (2 - 6 and 16 - 26 hours after last treatment). Moreover, the test substance is not sufficiently characterised.

Gerloff, K. et al. (2012): The study design is not in accordance with any guideline. In comparison with a suitable guideline, the acceptability criteria of the OECD test guideline 489 (in vivo mammalian alkaline comet assay) were not fulfilled, since only one concentration was tested, scoring criteria is not specified e.g. adequate number of cells which were counted have not been specified and the sampling time is not in accordance with the recommendation (2 - 6 and 16 - 26 hours after last treatment).

Ghosh, M. et al. (2013): The study design is not in accordance with any guideline. In comparison with a suitable guideline, the acceptability criteria of the OECD test guideline 489 (in vivo mammalian alkaline comet assay) were not fulfilled, since no positive control was used to determine the sensitivity of the Comet assay and the sampling time is not in accordance with the recommendation (2 - 6 and 16 - 26 hours after last treatment).

Hackenberg, S. (2010): The study design is not in accordance with any guideline. In comparison with a suitable guideline, the acceptability criteria of the OECD test guideline 489 (in vivo mammalian alkaline comet assay) were not fulfilled, since an unsuitable cell line was used, the test material was not sufficiently characterised, adequate number of cells (> 150 cells per slide) have not been analysed and the sampling time is not in accordance with the recommendation (2- 6 and 16 - 26 hours after last treatment).

Kermanizadeh, A. et al. (2013): The study design is not in accordance with any guideline. In comparison with a suitable guideline, the acceptability criteria of the OECD test guideline 489 (in vivo mammalian alkaline comet assay) were not fulfilled, since an adequate number of cells (> 150 cells per slide) have not been analysed and the sampling time is not in accordance with the recommendation (2- 6 and 16 - 26hours after last treatment). Number of replicates were not specified.

Kermanizadeh, A. et al. (2012): The study design is not in accordance with any guideline. In comparison with a suitable guideline, the acceptability criteria of the OECD test guideline 489 (in vivo mammalian alkaline comet assay) were not fulfilled, since an adequate number of cells (> 150 cells per slide) have not been analysed and the sampling time is not in accordance with the recommendation (2- 6 and 16 - 26hours after last treatment). Number of replicates were not specified.

Meena, R. et al. (2012): The study design is not in accordance with any guideline. In comparison with a suitable guideline, the acceptability criteria of the OECD test guideline 489 (in vivo mammalian alkaline comet assay) were not fulfilled, since an adequate number of cells (> 150 cells per slide) have not been analysed and the sampling time is not in accordance with the recommendation (2- 6 and 16 - 26hours after last treatment). Number of replicates were not specified, a positive control was not specified and the test material is not sufficiently characterised.

Tiano, L. et al. (2010): The cell lines were irridiated with genotoxic UV-rays while TiO2 nanoparticle exposure. It is not possible to draw a conclusion on the genotoxic potential of the test substance due tothe experimental setup. The negative control showed a cell viability decrease of approx. 70 % and a tail intensity in the range of 0 - 60%. The tail intensity of the treated cultures showed a tail intensity in the range of 0 - 100%. The study does not allow any meaningful evaluation of the test substance and conclusion on the effects of the test items. All test items were poorly characterised. Only one concentration was administered and the study does not comply with any guideline.

Magdalenova, Z. et al. (2012): The results of the Comet assay could not be verified, since a high cytotoxicity (decreased cell proliferation) was measured at the mid- and top concentrations in the adherent growing Cos-1 and EUE cells. According to the OECD test guideline 489 (in vivo mammalian alkaline comet assay) it is not recommended to conduct a Comet assay with such an extreme cytotoxicity (up to 5 % cell viability at the highest concentration).

Jugan, M.L. et al. (2012): The experimental procedure and results are very poorly described and no experimental data is given. The self-synthesised TiO2 nanparticles are not relevant for chemicals safety testing.

Prasad, R. Y. et al. (2014): The study does not comply with any acceptable guideline. The experimental procedure ( e.g. number of cells counted, justification dose selection, evaluation criteria) is not described and the presentation of the results do not allow a conclusive evaluation of the genotoxic potential of the test substance. Cytotoxicity data is missing.

Roszak, J. et al. (2013): The study design is not in accordance with any guideline. In comparison with a suitable guideline, the acceptability criteria of the OECD test guideline 489 (in vivo mammalian alkaline comet assay) were not fulfilled, since an adequate number of cells (> 150 cells per slide) have not been analysed. Authors observed a genotoxic effect after exposure to the highest concentrations of 100 and 250 µg/mL, which is likely a secondary toxic effect due to cytotoxicity.

Saquib, Q., et al. (2012): The study design is not in accordance with any guideline. In comparison with a suitable guideline, the acceptability criteria of the OECD test guideline 489 (in vivo mammalian alkaline comet assay) were not fulfilled, since an adequate number of cells (> 150 cells per slide) have not been analysed. Moreover, DNA damage was only observed at the highest concentration. For the highest concentration, however, was cytotoxicity not specified. Lower concentration showed significant cytotoxicity after 24 h exposure, thus effect on olive tail moment could have been a secondary toxic effect due to cytotoxicity. No trend was performed. The diagram presented, however, shows no dose-response relation at all. Statement on pH value, osmolality or precipitation of the test item-treated cell culture are missing.

Zijno, A., et al. (2015): Positive findings in the Comet assays performed showed no dose-response or time-response relationship, and thus, these findings are most probably chance findings. Moreover, the publication shows major reporting and experimental deficiencies. The number of scored comets is not specified. Historical control data is missing. Data on metabolic activation of cells is missing. Details of TiO2 NP suspension preparation and characterization are missing.

Elje, E. et al. (2020): The study design is not in accordance with any guideline. Concurrent and historical negative control data are missing. The test material is not a commercially available product.

 

References:

Choucroun, P., Gillet, D., Dorange, G., Sawicki, B. and Dewitte, J.D. (2001). Comet assay and early apoptosis. Mutat Res 478: 89-96.

Fairbairn, D.W., Walburger, D.K., Fairbairn, J.J. and O’Neill, KL. (1996). Key morphologic changes and DNA strand breaks in human lymphoid cells: discriminating apoptosis from necrosis. Scanning 18:407-416.

Henderson, L., Wolfreys, A., Fedyk, J., Bourner, C. and Windebank, S. (1998). The ability of the Comet assay to discriminate between genotoxins and cytotoxins. Mutagenesis 13:89-94.

Lee, M., Kwon, J. and Chung M-K (2003). Enhanced prediction of potential rodent carcinogenicity by utilizing Comet assay and apoptotic assay in combination. Mutat Res 541:9-19.

Barillet, S et al. (2009): The Comet assay used in this study is not validated, since there is no data of positive controls shown. Furthermore, DNA damage evaluation was not conducted after different sampling times (2 - 6h and 16 - 26h after single-exposure is recommended as sampling time by OECD for in vivo studies). At least, it is unclear why the most cytotoxic nanoparticles were used in the Comet assay, DNA damage could be a secondary effect induced by cytotoxicity. Furthermore, only 50 cells per sample were scored (150 are recommended by OECD TG 489 to provide an adequate statistical power).

 

 

Other DNA damage/repair assays:

Aufderheide, M.; Roller, M. (2000): Experimental procedure insufficiently described. No relevant increase in UDS in any of the cell line used. Slight increase in cell R3/1 but without any dose-response relationship.

Di Virgilio, A.L. et al. (2010): Cytotoxicity determined via MTT and NR uptake. SCE: conc. above 5µg/mL could not be scored due to cytotoxicity, significant induction of SCE in both doses, no dose response relationship. Cytotoxicity in SCE not verified by MTT and NR assays. Experimental procedures poorly described.

Wang, J. et al. (2019): A validated test guideline for the γ-H2AX assay is not available. Only one concentration level was tested. The human-hamster hybrid (AL) cell line is not a standard cell line regularly used for genotoxicity testing. The cell line is only poorly described.

 

 

Summary - In vitro DNA damage

Merely one reliable reference is available reporting on DNA damage in mammalian cells after exposure towards ultrafine titanium dioxide. The work by Petkovic shows an increase of DNA damage in mammalian cells after treatment with ultrafine anatase with no dose- or time-response relationship. The presence of oxidative DNA lesions indicates an indirect mode of action. Whether this was cause by ROS formed on the surface of the particles by light/UV-irradiation remains unclear. The lack of dose or time dependency also remains unexplained. Based on the given information it is concluded that ultrafine titanium dioxide does not cause significant and persistent DNA damage to an extent which is considered relevant for the human health hazard assessment.

 

In vivo genetic toxicity tests

In vivo clastogenicity and aneugenicity

In a testing programme, a series of three different ultrafine titanium dioxide samples were tested for the induction of micronuclei in the peripheral blood of rats after single oral administration (Myhre, A., 2014b and 2014c, Wessels, A., 2014c). Animals received doses of 0, 500, 1000, 2000 mg/kg bw. All tests were conducted in accordance with OECD TG 474/EC B.12 and under GLP:

In the study by Myhre, A. (2014b and 2014c) two ultrafine titanium dioxide samples (TiO2uf-1: 89.3% anatase phase, 10.7% rutile phase, D50(TEM ECD)=19 nm, SA: 50.4 m²/g; TiO2uf-3: rutile phase, D50(TEM ECD)=22 nm, SA: 58.8 m²/g) were used as test item. The vehicle control, low, intermediate, and positive control groups contained 5 animals/sex, the high-dose group contained 7 animals/sex. Peripheral blood samples were taken via sublingual vein bleeding approx. 48 and 72 hours post exposure. A total of 20,000 RETs were analysed per blood sample via flow cytometry for the presence of micronuclei and toxicity as indicated by the frequency of immature erythrocytes among the total erythrocytes (PCE/NCE ratio). Additionally, titanium concentrations were determined in blood and liver at 48 or 72 hours. One mid-dose animal was accidentally killed on TD3 prior to blood collection and was therefore not considered in the MN analysis (Myhre 2014c, TiO2uf-3). This has no impact on the outcome or validity of this study since it was a mid-dose animal and there is no indication of any test substance related effects in this or any other test substance treated group. No statistically significant increases in the micronucleated RET frequency were observed in any evaluated test substance-treated group of male or female animals at either time point. There were no statistically significant decreases in the PCE/NCE ratio in any test-substance treated group. Under the conditions of this study, titanium dioxide did not induce formation of micronuclei in rat peripheral blood up to the limit dose of 2000 mg/kg bw. No discernible dose-dependent increases of titanium concentrations in the blood and liver of treated rats relative to control rats could be measured.

In the studies by Wessels, A. (2014c) ultrafine titanium dioxide (TiO2uf-2) was used: anatase phase, D50(TEM ECD)=19 nm, SA: 82 m²/g. All dose groups contained 5 animals/sex. Peripheral blood samples were taken via tail vein approx. 48 and 72 hours post exposure. A total of 20,000 RETs were analysed per blood sample via flow cytometry for the presence of micronuclei and toxicity as indicated by the frequency of immature erythrocytes among the total erythrocytes (PCE/NCE ratio). No statistically significant and dose-dependent increases in the micronucleated RET frequency were observed in any evaluated test substance-treated group of male or female animals at either time point. Two isolated statistically significant increased were seen for males at 2000 mg/kg bw and for females at 500 mg/kg bw. Since these findings (i) do not show a dose response relationship (ii) are within the range of the historical negative control data, they are not considered biologically relevant. There were no statistically significant decreases in the PCE/NCE ratio in any test-substance treated group. Under the conditions of this study, titanium dioxide did not induce formation of micronuclei in rat peripheral blood up to the limit dose of 2000 mg/kg bw.

 

Sadiq, R. et al. (2012) investigated the induction of micronuclei in the peripheral blood of male B6C3F1 mice. Five animals per dose group were exposed via intravenous injection of three doses (0.5, 5, 50mg/kg/day) ultrafine titanium dioxide (anatase, primary particle size: 12 nm, aggregate size in medium approx. 170 nm) over three consecutive days, the maximum dose was limited by the feasibility to administer higher doses by that method. Concurrent positive and negative control groups were used. The frequency of micronucleated reticulocytes was determined by the acquisition of approximately 20,000 C71-positive reticulocytes for each animal. Bone marrow exposure was demonstrated by toxicity and elevated titanium levels in the bone marrow tissue, analysed via ICP-MS (fold increase over control ranged from 12.1 to 14.2). Titanium dioxide nanoparticles were not clastogenic or aneugenic in the micronucleus assay in mice at the dose levels showing target organ exposure. 

 

Sycheva et al. (2011) conducted a micronucleus assay in male mice after administration of titanium dioxide via gavage at doses of 40, 200, 1000 mg/kg bw/day for consecutive 7 days. The publication shows reporting and experimental deficiencies. The test material was insufficiently characterised. Due to an absence of a clear designation, purity or impurity information, it remains unclear whether the test item was in fact of industrial origin and therefore of relevance for the hazard and risk assessment of titanium dioxide. Some experimental procedures were insufficiently described, some procedures were not guideline compliant/deviate from established procedures e.g. lack of fixation, unclear staining procedure, unclear scoring for cytotoxicity. Experimental procedure for cell isolation from brain tissue and liver is missing. Unclear how the mitotic index was determined in forestomach and colon epithelial cells. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment. 

 

The micronucleus study by Dobrzyńska, M.M. et al. (2014) exhibits significant methodological deficiencies, intravenous administration is an unphysiological route of application and bears only very limited value for risk assessment purposes. The number of PCE analysed is too low, thus statistical robustness of the results is impaired. No positive control was used, thus sensitivity of the system was not demonstrated. Historical control data are not reported. No information on tissue toxicity was reported, thus it remains questionable whether positive findings were artefactual due to overt toxicity or a true genotoxic response. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment. 

 

In the study by Relier, C. et al. (2017) male Sprague-Dawley rats were intratracheally instilled three times, over a period of eight days, with a TiO2 nanoparticle suspension with concentrations of 0.17, 0.83, and 3.33 mg/mL which resulted in doses of 0.5, 2.5, and 10 mg/kg. A micronucleus assay was conducted with extractions from bone marrow before treatment, 2 hours and 35 days after treatment. 2000 PCE were scored for micronuclei frequency. Cytotoxicity was assessed by analysing the relative number of immature erythrocytes. According to the authors, the number of micronuclei was significantly increased after 35 days at all three doses, in a dose-independent manner. However, it was also stated that the observed changes were slight and might therefore be regarded as biologically irrelevant. The study shows inconsistencies in reporting, study design shortcomings and results, which are not biologically meaningful. The authors state that the rats were instilled three times with 100 µl TiO2 nanoparticle suspensions with a concentration of 0.17, 0.83, and 3.33 mg/mL allegedly resulting in total doses of 0.5, 0.25, and 10 mg/kg. Recalculating the (not reported) bodyweight of the animals, based on the given parameters (i) instillation volume, (ii) concentration of the test item in the vehicle and (iii) the resulting dose, would result in a total body weight of 102, 99.6 and 99.9g for the low, mid and high-dose animals. A typical weight of a 10 weeks old, male Sprague-Dawley rat (gender, species, and age used in this study) is about 300 g. It is therefore unclear whether the authors applied wrong doses due to a calculation error or whether the animals were of a different age (for 100g male Sprague-Dawley rats one would assume 4-5 week old animals). The authors state that they triggered hyperventilation for a better distribution of the test substance in the lungs. However, they make no statements on the induction process per se. They do not state on potential side-effects of this treatment on the results. This treatment further increases the artificiality of the exposure route and renders the study of little relevance for risk assessment purposes. Authors make no statements on the animal weights before or during the study. Furthermore, data on housing, clinical observations, environmental conditions, test group randomization, euthanasia, and food consumption are missing. The preparation of the bone marrow is insufficiently described. Evaluation criteria for the micronuclei assay are not described. Historical data are missing, the historical data for rat vehicle controls of other reputable laboratories show a range of 0.03-0.22% in micronucleation frequency. The MN frequencies measured over all doses and time points range between approx. 0.05 and 0.15% and therefore within the historical negative control range. Due to a lack of a dose response relationship and a MN frequency, which is within the historical control range, the significantly increased MN frequencies should have been considered as chance finding with no biological relevance. Based on the above-mentioned severe shortcomings, the reference is considered not reliable and was disregarded in the hazard assessment. 

 

Troullier, B. et al (2009) treated male C57Bl/6Jpun/pun mice for five days with P25 TiO2 nanoparticles suspended in drinking water at concentrations of 60, 120, 300, and 600 µg/ml (total dose after five days: 50, 100, 250, and 500 mg/kg). A micronuclei assay was conducted with peripheral blood taken before and after treatment. Approximately 2000 erythrocytes were scored for micronuclei occurrence. According to the authors, micronuclei frequency was significantly increased only at the highest TiO2 nanoparticle dose (500 mg/kg), when compared to untreated controls. The study shows serious reporting and experimental shortcomings. The actual dose received was calculated on an average 30g body weight and an average of 5 mL water intake. This is an unjustified simplification, since the bodyweight for 12-20-week-old animals differs significantly between e.g. male and female animals, also the average bodyweight of a 12-20 week old female mouse ranges between 21 and 24g. Such dose calculation. The author state that four to five months old mice were used. The current OECD test guideline recommends using young animals at an age of six to ten weeks. The authors do not justify selected species, strains, and dosing. The transgenic C57Bl/6Jpun/pun mouse is an uncommon strain for this assay, with no historical data available. Due to the very low solubility and the difference in density of a factor of 4, one has to assume that titanium dioxide settles on the bottom of the flask, leading to a higher total dose. The authors verified neither the actual dose received nor the homogeneity of the suspension before during and after the dosing of the animals using an appropriate analytical method. Authors provide test results a bar chart in which the MN results of the four dose groups were presented parallel to the control groups. From the different height of the control bars one has to assume that also four different control groups (with n=5) were used. This is a disproportionate use of animals and is not in-line with the ethical principles in toxicological testing practice. The MN frequency for untreated mice was given in the text as 4.3 ±0.93/2000 cells, whereas the bar chart reports approx. 4.3/1000 cells. In case the latter value it the true measured frequency, it would be above reported control data for mice, which is 2.5 ±0.46/1000 PCE (Zeiger et al. 2009). Cytotoxicity measurements were not performed, which does not allow a conclusive statement, whether a true test item specific effect was observed or an artefactual response due to increased cytotoxicity. Clinical observations were not reported. Time points of blood sampling and analysis, the proportion of immature erythrocytes to total erythrocytes is not specified. The authors conducted a student’s t-test to test for significant changes before and after treatment. However, the student’s t-test is not a suitable for paired data. Individual animal values and results from the lower doses (50, 100, 250 mg/kg) are not specified. Further, the authors state not on a potential dose-response relationship. Moreover, the author state that the highest TiO2 nanoparticle dose leads to an inflammatory response, measured via qRT-PCR of pro-inflammatory marker gene expression. Inflammatory responses could have interfered with the results and could have led to false positive findings in the genotoxicity assays. Based on the above-mentioned shortcomings, the reference is rated not reliable and disregarded for the hazard assessment. 

 

Kamel, A.F. and Saleh, A.S. (2019) evaluated the potential of titanium dioxide nanoparticles to induce clastogenicity and aneugenicity in bone marrow erythrocytes from male Swiss albino mice. The mice received daily intraperitoneal injections of titanium dioxide nanoparticle suspensions at doses of 37.5, 75.0, and 150 mg/kg bw for ten days. The occurrence of micronuclei was scored in 1000 polychromatic erythrocytes per animal. According to the authors, intraperitoneal injection of titanium dioxide nanoparticles induced statistically significantly increased micronucleus frequencies in all dose groups. Moreover, the effect showed a positive dose-response relationship. The ratio of polychromatic to normochromatic erythrocytes was slightly elevated in all exposure groups when compared to the vehicle control group. The publication shows severe deficiencies with regard to reporting and the methodology applied. The test material was administered via intraperitoneal injections, which is considered to be a non-physiological exposure route and is considered to be of limited value for the risk assessment purposes. The test material was insufficiently characterised, since information on purity, crystalline phase, manufacturer, size distribution, and surface modifications are missing. Moreover, the description of the test material is implausible, since the test material is specified to be a nanomaterial, whereas the nominal size is specified to be >100 nm. Result figures are at least partly manipulated and/or incorrectly assigned. Information on the number of animals scored for micronucleus occurrence is not provided. The number of immature erythrocytes scored for micronucleus is lower than recommended. The number of cells scored for the determination of the polychromatic to normochromatic erythrocytes ratio is not specified. Details on the administration of the test material are missing. Clinical observations were not included. Information on the body weights at start and termination of the treatment are not provided. Information on evaluation and scoring criteria are missing. Historical control data is not provided. Based on the above-mentioned shortcomings, the reference is rated not reliable and disregarded for the hazard assessment. 

 

The potential of titanium dioxide nanoparticles to induce chromosomal damage in bone marrow cells from male Swiss albino mice was evaluated by Kamel and Saleh (2019). Groups of six male mice received daily intraperitoneal injections of titanium dioxide nanoparticle suspensions at doses of 37.5, 75.0, and 150 mg/kg bw for ten days. The occurrence and type of chromosomal aberrations were scored in 50 arrested metaphases per animal. According to the authors, the treatment with titanium dioxide nanoparticles, via intraperitoneal injections, resulted in statistically significantly increased proportions of metaphases with structural chromosomal aberrations in Swiss albino mice. The effect was observed in all three dose groups and was considered to be dose related. The publication shows severe deficiencies with regard to reporting and the methodology applied. The test material was administered via intraperitoneal injections, which is considered to be a non-physiological exposure route, and thus, considered not relevant for risk assessment purposes. Furthermore, the test material was only poorly characterised. Moreover, the description of the test material is implausible, since the test material is specified to be a nanomaterial, whereas the nominal size is specified to be >100 nm. The number of metaphases scored for chromosomal aberration is lower than recommended. The mitotic index or other cytotoxicity parameters were not evaluated. Details on the administration of the test material are missing. Information on clinical observations and initial/terminal body weights were not included. A positive control group was not included. The authors did not report on the evaluation and scoring criteria. Historical control data is not provided.

 

Chakrabarti, S. et al. (2019) evaluated potential clastogenic and aneugenic events in bone marrow erythrocytes of Swiss albino mice after sub-chronic exposure to titanium dioxide nanoparticles (particle size: 58.25 ± 8.11 nm). Five mice per sex per group received oral titanium dioxide nanoparticle doses of 200 and 500 mg/kg bw over a period of 90 days. After the exposure period, 2000 polychromatic erythrocytes per animal were scored for the occurrence of micronuclei via light microscopy. Additionally, the ratio of immature erythrocytes among total erythrocytes was scored as indicator for bone marrow toxicity. Moreover, post-mortem investigations included clinical chemistry, haematological examinations, and histopathological examination of the liver and kidney. According to the authors, the treatment with 500 mg TiO2-NPs/kg bw resulted in a statistically significantly increased micronucleus frequency in polychromatic erythrocytes, when compared to vehicle control values. The ratio of immature erythrocytes to total erythrocytes was not altered after treatment with titanium dioxide nanoparticles. The biochemical analyses of the serum revealed hepatotoxicity. The findings were substantiated by the results of the histopathological examination of the liver. Moreover, the haematological analyses revealed statistically significantly decreased RBC, WBC, and platelet counts as well as a decreased haemoglobin level and a slightly increased proportion of basophils. The histopathological examination of the kidney revealed nephrotoxicity. Notably, gross necropsy revealed signs of bleeding in the cranial and peritoneal cavities in animals from the high dose group which died prematurely. Behavioural responses observed in mice of the high dose group were depression, light reduction in locomotion, aggressiveness, touched sensitivity, and respiratory moment. The publication shows several reporting and methodological deficiencies. The positive finding on chromosomal damage was only observed in presence of hepatotoxicity, nephrotoxicity, and altered haematological parameters. It is not clear whether premature deaths occurred in the high dose group. On the one hand the authors stated: “…signs of bleeding were observed in the cranial and peritoneal cavities of the dead animals treated with 500 mg/kg …”, but on the other hand the tables indicated no mortality. Moreover, the authors stated that each group contained ten animals, whereas the table indicates that only six animals were analysed. Notably, premature deaths could have evidenced that the MTD was exceeded, and thus, the acceptability criteria, set out in the OECD TG 474, might have been violated. The number of dose groups is lower than recommended in the current OECD TG 474 (2 vs. 3). Moreover, the number of analysed immature erythrocytes is lower than recommended in the current OECD TG 474 (2000 vs. 4000 per animal). Further, the number of cells scored for the determination of the immature to total erythrocyte ratio is not specified. The test material was insufficiently characterised, since information on the purity, crystalline phase, manufacturer, coatings, and morphology are missing. Details on animal housing are missing. The test animals were not kept under optimal environmental conditions (temperature: 25 ± 2°C; humidity: 70 ± 5%). Body weights before and during treatment are not specified. Evaluation and scoring criteria are missing. Historical control data is not included. Based on the above-mentioned shortcomings, the reference is rated not reliable and disregarded for the hazard assessment.

 

Potential clastogenic effects in bone marrow cells of Swiss albino mice after sub-chronic exposure to titanium dioxide nanoparticles (particle size: 58.25 ± 8.11 nm) were evaluated by Chakrabarti, S. et al. (2019). Groups of five mice per sex per group were gavaged with titanium dioxide suspension at doses of 200 and 500 mg/kg bw over a period of 90 days. All animals received a single dose of colchicine via intraperitoneal injection three hours before euthanasia. After euthanasia, the femur was removed, and bone marrow cells were obtained. The bone marrow cells were stained and evaluated for the mean proportion of chromosomal aberrations via microscopy. Additionally, post-mortem investigations included clinical chemistry, haematological examinations, and histopathological examination of the liver and kidney. According to the authors, the animals dosed with 500 mg TiO2-NPs/kg bw showed a statistically significantly increased (2.5-fold) mean proportion of chromosomal aberrations, when compared to the vehicle control group. Moreover, the mean total number of aberrant cells was 3.3-fold higher in high-dose animals as compared to the control value. Further, hepatotoxicity was evidenced by serum markers and histopathological examinations. The haematological analyses revealed statistically significantly decreased RBC, WBC, and platelet counts as well as a decreased haemoglobin level and a slightly increased proportion of basophils. The histopathological examination of the kidney revealed nephrotoxicity. Notably, gross necropsy revealed signs of bleeding in the cranial and peritoneal cavities in animals from the high dose group which died prematurely. Behavioural responses observed in mice of the high dose group were depression, light reduction in locomotion, aggressiveness, touched sensitivity, and respiratory moment. The publication shows several reporting and methodological deficiencies. The positive finding on chromosomal damage was only observed in presence of hepatotoxicity, nephrotoxicity, and altered haematological parameters. It is not clear whether premature deaths occurred in the high dose group. On the one hand the authors stated: “…signs of bleeding were observed in the cranial and peritoneal cavities of the dead animals treated with 500 mg/kg …”, but on the other hand the tables indicated no mortality. Moreover, the authors stated that each group contained ten animals, whereas the table indicates that only six animals were analysed. Notably, premature deaths could have evidenced that the MTD was exceeded, and thus, the acceptability criteria, set out in the OECD TG 475, might have been violated. The mitotic index of the bone marrow cells was not investigated. The number of dose groups is lower than recommended in the current OECD TG 475 (2 vs. 3). The test material was insufficiently characterised, since information on the purity, crystalline phase, manufacturer, coatings, and morphology are missing. The number of animals evaluated for chromosomal aberrations is not specified. Individual results are not specified. The types of chromosomal aberrations scored and used for calculation of the chromosomal aberration frequency are not specified. Details on animal housing are missing. The test animals were not kept under optimal environmental conditions (temperature: 25 ± 2°C; humidity: 70 ± 5%). Body weights before and during treatment are not specified. Evaluation and scoring criteria are missing. Historical control data is not included. Based on the above-mentioned shortcomings, the reference is rated not reliable and disregarded for the hazard assessment.

 

Kazimirova, A. et al. (2019) investigated on potential clastogenic and aneugenic effects of intravenously injected titanium dioxide NANO4 (Aeroxide P25) on bone marrow erythrocytes of female Wistar rats. Six to eight female rats received a single intravenous injection of titanium dioxide NANO4 at a dose of 0.59 mg/kg bw. The test animals were sacrificed after 1 day, 1 week, 2 weeks, and 4 weeks. A total of 2000 immature erythrocytes were scored for the occurrence of micronuclei. Additionally, the proportion of immature among total number of erythrocytes was scored in order to evaluate potential toxic effects of the test material. According to the authors, titanium dioxide NANO4 was found to be negative in the erythrocyte micronucleus test under the conditions tested. Moreover, the proportion of immature among total erythrocytes was not significantly altered. The publication shows several reporting and methodological deficiencies. The test material was intravenously injected, which is considered to be a non-physiological route of exposure with only limited relevance for risk assessment purposes. The concentration and volume of the administered suspension are not specified. Only one dose group was included, which precludes dose-response relationship evaluations. The number of immature erythrocytes scored for the occurrence of micronuclei was lower than recommended by the current OECD TG 474 (2000 vs. 4000 immature erythrocytes per animal). The dose selection is not in accordance with the current test guideline, since the test material was not tested up to the MTD. Exposure of the bone marrow was not demonstrated, since the ratio of immature to total erythrocytes was not significantly altered and plasma levels of the test substance were not analysed. Information on body weights and clinical signs are not provided. A positive control group was not included, and the proficiency of the laboratory was not demonstrated. Information on the evaluation and scoring criteria are not provided. Historical control data is not included. Based on the above-mentioned shortcomings, the reference is rated not reliable and disregarded for the hazard assessment.

 

 

Summary entry - In vivo clastogenicity and aneugenicity

The references contained in this summary entry represent in vivo clastogenicity/aneugenicity experiments with very limited value for risk assessment purposes. All references do not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The information contained therein were included for information purposes only.

The test material used in the references summarised in the summary entry was largely characterised insufficiently. As detailed in the introductory statement to chapter for human health hazard assessment, it is problematic to base the hazard and risk assessment of clearly characterised titanium dioxide on information generated with poorly characterised test item, with no market relevance. In accordance with ECHA guidance, all references that do not provide sufficient information on the substance identity were disregarded for hazard and risk assessment purposes (for further information please refer to the introductory statement).

Chen, Z. et al. (2014): Reference with reporting and experimental deficiencies: no positive control was used; negative control was not clearly described (vehicle, solvent, medium); number of cells analysed per animal was too low (1000 MNPCE, guideline foresees at least 2000 MNPCE), individual data was missing; historical control data was missing; evaluation criteria not stated.

Wang, Y. et al. (2014): Chinese article with abstract in English language only

Rad, J.S. (2013): The study does not comply with any guideline. The test material is insufficiently characterised, the dose regime is unclear and there are inconsistent information about the number of animals used. The evaluation criteria are unclear and there is no information about the methodical setup. The non-physiological route of intraperitoneal injection is not recommended by any guideline. It is not possible to give a judgement about the effects of the test substance, since the results are very poorly described.

Saghiri, Z. et al. (2012): The authors stated that exposure to the TiO2 nanoparticles induced a significant increase in MN frequency. In comparison with historical data of other laboratories the MN frequency is still in the control range of MN frequency. The vehicle control as stated in the results showed a MN frequency of 0% which is not plausible, the positive control is missing. The self-synthesised test material is insufficiently characterised and there are inconsistent information about the number of animals used. The evaluation criteria is unclear. The non-physiological route of intraperitoneal injection is not recommended by any guideline. It is not possible to give a judgement about the effects of the test substance, since the results are very poorly described and interpreted.

Song, M.F. et al. (2012): The study does not comply with any acceptable guideline. The administration procedure is not described (e.g. dosimetry and frequency of administration unclear) and the non-physiological route of intraperitoneal exposure is not appropriate for hazard assessment of a test substance. The single dose of 3 mg / mouse as stated by the authors does not allow a dose-response analysis of the test substance. Based on the poorly described administration procedure, it seems that an excessive i.p. dose of approximately 120 mg / kg BW (of an insoluble metal) was administered. The micronucleus frequency of all tested particles was nearly similar, the effect is rather a particle effect, which is not caused by the intrinsic properties of the test substance.

Xu, J. et al. (2013): Reference with reporting and experimental deficiencies: The method section in described insufficiently. Statements on animals total body weight and age are missing. Volume of administration is not specified and it is not clear whether the concentration of the stock solution was changed or different volumes administered to achieve the stated total doses. Historical data is not specified. Evaluation criteria are described insufficiently. Excessively high doses of TiO2 were administered (140-1387 mg/kg). The number of analysed polychromatic erythrocytes is too low (1000; 4000 are recommended by the OECD). Further, only four animals per sex and dose were analysed (at least five are recommended by the OECD). The bone marrow was sampled after 14 days, however the OECD recommends sampling latest 48 hours after treatment. Furthermore, individual mouse values are not specified. 

Rizk, M.Z. et al.(2017): The test material was administered via intraperitoneal injection. The method description lacks nearly all essential details. The test material is insufficiently described. Only three animals per group were examined. Only 100 cells per animal were scored for chromosomal aberrations. Information on body weights and food consumption are not provided. All positive findings were associated with marked hepatotoxicity.

 

 

Summary - In vivo clastogenicity and aneugenicity

With regard to clastogenicity and aneugenicity, the reliable in vivo data for ultrafine titanium dioxide present a consistent pattern. In none of the studies administering titanium dioxide via physiological route in rats or mice an increase of micronuclei was observed. The maximum doses/concentrations were limited by technical feasibility or the limit as foreseen in the guideline. It is therefore concluded that titanium dioxide does not cause local or systemic clastogenic or aneugenic events in animals.

 

In vivo DNA damage

Two comet assays investigated the DNA damage effect of nano-sized titanium dioxide following intratracheal instillation (Naya et al., 2012) or inhalation (Landsiedel et al., 2010).

In the study by Naya et al. young male Crl:CD(SD) rats were instilled with a single dose of 1 and 5mg/kg or with a repeated dose of 0.2 and 1 mg/kg once a week for 5 weeks with ultrafine titanium dioxide (primary particle size: 19 nm, anatase phase, 99.99% pure, surface area (BET): 316 m²/g). The histopathological findings 3 and 24 hours after single instillation showed a slight infiltration of alveolar macrophages and/or neutrophils at 5 and 1 and 5 mg/kg dose group, respectively. For the repeated treatment, a similar inflammatory response was observed 3 hours after final dosing only in the high-dose group animals. No DNA damage has been observed via alkaline comet assay in the lungs epithelial cells, after scoring 100 cells per rat. A positive control substance induced significant DNA damage after 3 hours, demonstrating the sensitivity of the test system. Consequently, ultrafine titanium dioxide does not induce DNA damage after single or repeated intratracheal instillation in rats. The test material was insufficiently characterised. Due to an absence of a clear designation, purity or impurity information, it remains unclear whether the test item was in fact of industrial origin and therefore of relevance for the hazard and risk assessment of titanium dioxide.

In the study by Landsiedel et al. young male Crl:Wi Han rats were exposed by inhalation to 10 mg/m³ of coated ultrafine titanium dioxide (Titanium dioxide, aluminium hydroxide, dimethicone/methicone copolymer, purity of titanium dioxide core: ≥99% rutile phase, titanium dioxide-content: 79–89%, surface area (BET): 100 m²/g). The alkaline comet assay was performed in alveolar lavage cells three weeks after exposure. Compared with the negative control no increase in the tail intensity, tail length or tail moment, nor in the hedgehogs formed upon inhalation of ultrafine titanium dioxide was observed. Consequently ultrafine titanium dioxide does not induce DNA damage after inhalation exposure in rats.

 

The work by Lindberg et al. investigated the induction of local or systemic genotoxic events in C57BL/6J mice after inhalation of self-synthesised ultrafine titanium dioxide (74% anatase, 26% brookite, agglomerate size: 10-60 nm, average primary particles size: 21 nm, surface area (BET): 61 m²/g) at 0.8, 7.2, and (the highest concentration allowing stable aerosol production) 28.5 mg/m³. DNA damage was assessed by the alkaline comet assay in lung epithelial alveolar type II and Clara cells sampled immediately following the exposure. The percentage of DNA in the comet tail from 100 cells per animal (two replicates, 50 cells each) was used as a measure of the amount of DNA damage. MN were analysed by acridine orange staining in blood PCEs collected 48 h after the last exposure. Blood samples were collected from the tails of the mice and whole blood smears were prepared. The frequency of micronucleated erythrocytes in 2000 PCEs and in 2000 normochromatic erythrocytes per mouse was scored. Also the percentage of PCEs was assessed (scored until the number of PCEs or NCEs reached 2000) to indicate possible bone marrow toxicity. The experiments were conducted using self-synthesised material by thermal decomposition of titanium tetraisopropoxide. The reaction mixture was directly attached to the inhalation chamber with no purification step in between. One has therefore to assume that animals were exposed towards a mixture of reaction products and not a pure titanium dioxide sample. A dose-dependent deposition of ultrafine titanium dioxide in lung tissue was seen. Although the highest exposure level produced a clear increase in neutrophils in BAL fluid, indicating an inflammatory effect, no significant effect on the level of DNA damage in lung epithelial cells or micronuclei in PCEs was observed, suggesting no genotoxic effects by the 5-day inhalation exposure to ultrafine titanium dioxide (anatase phase). Consequently, ultrafine titanium dioxide does not induce DNA damage after inhalation exposure in rats. Based on the unclear exposure conditions and the test item being a reaction mixture rather than a pure substance, the study is considered not relevant and was disregarded for hazard assessment. 

 

For the evaluation of the references by Saber et al. (2012a and b), please refer to the discussion of the in vivo studies with pigment-grade titanium dioxide presented above. 

 

Sycheva et al. (2011) conducted a comet assay in male mice after administration of titanium dioxide via gavage at doses of 40, 200, 1000 mg/kg bw/day for consecutive 7 days. The publication shows reporting and experimental deficiencies. The test material was insufficiently characterised. Due to an absence of a clear designation, purity or impurity information, it remains unclear whether the test item was in fact of industrial origin and therefore of relevance for the hazard and risk assessment of titanium dioxide. Some experimental procedures were insufficiently described, some procedures were not guideline compliant/deviate from established procedures e.g. lack of fixation, unclear staining procedure, unclear scoring for cytotoxicity. Experimental procedure for cell isolation from brain tissue and liver is missing. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment. 

 

In the study by Dobrzynska, M.M. et al. (2014) the genotoxic potential of nanosized titanium dioxide (anatase/rutile powder; 21 nm) was investigated using the alkaline comet assay. Male rats were given a single dose (5 mg/kg bw) of the test substance by intravenous administration and the animals were sacrificed 24 hours, 1 week and 4 weeks after administration. Two negative control groups (solvent and untreated controls) were run concurrently. Bone marrow cells were obtained for analysis. Comet tail moment and percentage of DNA in Comet tail were chosen as parameters. The exhibits significant methodological deficiencies. Intravenous administration is an unphysiological route of application and bears only very limited value for risk assessment purposes. Samples were collected 24 hours, 1 week and 4 weeks after the single injection. Only a single dose was tested, which precludes a dose-response relationship analysis. A positive control group was not included, and thus, the sensitivity of the system was not demonstrated. Historical control data are not reported. No information on tissue toxicity was reported, thus it remains questionable whether positive findings were artefactual due to overt toxicity or a true genotoxic response. The number of analysed cells was too low, individual results were not reported and acceptability and evaluation criteria not reported Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

 

Meena, R. et al. (2014) report the induction of adverse effect in testes after i.v. injection of TiO2 NP. The results of this study cannot be evaluated, due to severe reporting deficiencies. The methods section describes the dissection and preparation of the brain, whereas the results section presented findings in the testes. Furthermore, the experimental design exhibits shortcomings, such as unphysiological route of application, testes cell type not characterised, gross necropsy not reported or performed, clinical signs not reported, positive control missing, number of cells analysed too low, individual results were missing. Consequently, the reference is rated not reliable and therefore disregarded for the hazard assessment. 

 

In the study by Relier, C. et al. (2017) male Sprague-Dawley rats were intratracheally instilled three times, over a period of eight days, with a TiO2 nanoparticle suspension with concentrations of 0.17, 0.83, and 3.33 mg/mL which resulted in doses of 0.5, 2.5, and 10 mg/kg. Comet assays were conducted with lung, peripheral blood, and liver cells extracted two hours post exposure, and 35 days post exposure. The median of 150 tail DNA percentages was calculated per rat. Six rats per dose group were analysed. Hedgehog percentages were analysed as indicators for cytotoxicity. According to the authors, treatment with two highest doses of TiO2 nanoparticles induced significant DNA damages in liver cells at both post-exposure time points. In lung cells, a biologically meaningful and significant increase was only observed after 35 days in both doses. In blood cells, TiO2 nanoparticles did not induce DNA damage at biological relevant levels, at any dose and point in time. Exposure with TiO2 did not result in increased hedgehog levels. The study shows inconsistencies in reporting, study design shortcomings and results which are not biologically meaningful. The authors state that the rats were instilled three times with 100 µl TiO2 nanoparticle suspensions with a concentration of 0.17, 0.83, and 3.33 mg/mL allegedly resulting in total doses of 0.5, 0.25, and 10 mg/kg. Recalculating the (not reported) bodyweight of the animals, based on the given parameters (i) instillation volume, (ii) concentration of the test item in the vehicle and (iii) the resulting dose, would result in a total body weight of 102, 99.6 and 99.9g for the low, mid and high-dose animals. A typical weight of a 10 weeks old, male Sprague-Dawley rat (gender, species, and age used in this study) is about 300 g. It is therefore unclear whether the authors applied wrong doses due to a calculation error or whether the animals were of a different age (for 100g male Sprague-Dawley rats one would assume 4-5 week old animals). The authors state that they triggered hyperventilation for a better distribution of the test substance in the lungs. However, they make no statements on the induction process per se. They do not state on potential side-effects of this treatment on the results. This treatment further increases the artificiality of the exposure route and renders the study of little relevance for risk assessment purposes. Authors make no statements on the animal weights before or during the study. Furthermore, data on housing, clinical observations, environmental conditions, test group randomization, euthanasia, and food consumption are missing. The authors report a significant increase of DNA damage in lung cells, after 35 days, at the highest doses. However, the reported significantly increased mean tail intensities are still in the acceptable range of vehicle controls (OECD Series on Testing and Assessment No. 196). A lack of historical data precludes evaluation of the reported results for the other organs. Labs proficiency in preparing and analysing tail DNA intensities in comet assay is a prerequisite for reliable data. It was shown that there is huge variability in tail intensities even within negative or positive controls (Tavares, A.M. et al. (2014); Nanogenotox Programme). This variability in combination with lack of controls and historical data renders positive findings of this assay of little relevance for risk assessment purposes. Analysis of hedgehog percentages as measurement for cytotoxicity is questionable, since their aetiology and relation to cytotoxicity is not clarified so far. Only for the lung a second indicator, LDH level in BAL fluid, was used as measurement for cytotoxicity. The analysis of cytotoxicity is an important parameter, since cytotoxicity is known to be a potent confounder. Further, lung inflammation, indicated by significantly increased neutrophil levels, was observed concurrently at the highest dose. Inflammation could have potentially interfered with the comet assay and led to false positives in the lung test. Although the authors cite that Inflammatory cells have been shown to induce mutagenic effects after TiO2 inhalation exposure (Driscoll et al., 1997b), it was not taken into account in the overall conclusion.

 

Asare et al. (2015) administered male, wildtype and Ogg1(-/-) KO, mice via single intravenous TiO2 nanoparticle suspension injections at a dose of 2 mg/ml (final dose: 5 mg/kg). Extracted cells remained either untreated or were treated with Fpg enzyme. Comet assays were conducted with lung, liver, and testis cells extracted one and seven days post exposure. The median of 100 tail DNA percentages was calculated per mouse. Five to six mice per treatment were analysed. According to the authors, TiO2 nanoparticle-exclusive treatment does not result in a statistically significant increase of DNA damage after both post exposure periods. However, treatment of extracted cells with Fpg enzyme leads to a significant increase of DNA damage only in testis and after seven days post exposure. The study shows some study design shortcomings. The authors administered the test material intravenously into the tail vein. However, intravenous injection is an unphyisiological route of exposure and bears only very limited value for hazard assessment purposes. Furthermore, the study does not fulfil several requirements of the OECD test guideline 489, as follows: The authors analysed 100 comets, although a minimum of 150 are recommended. Only one does was used in the experiments, which precludes any conclusion on a dose-response relationship. The OECD guideline recommends a sampling period of two to six hours after the last treatment. Particularly in case of an intravenous administration, shorter sampling intervals should have been chosen due to a rapid systemic distribution. Animals were analysed one and seven days after the last treatment. Transient DNA lesion could have been lost during this period. Moreover, test material’s cytotoxicity and clinical observations of the test animals are not specified. Hedgehog occurrence is not specified. Furthermore, historical data is missing and the results of the positive control are not reported. Laboratory’s proficiency in preparing and analysing tail DNA intensities in comet assays is a prerequisite for reliable data. A huge variability in tail DNA intensities was shown even within negative or positive controls (Tavares, A.M. et al. (2014); Nanogenotox Programme). This variability in combination with lack of controls and historical data renders positive findings of this assay of little relevance for hazard assessment purposes. The test material was insufficiently characterised. Due to an absence of a clear designation, purity or impurity information, it remains unclear whether the test item was in fact of industrial origin and therefore of relevance for the hazard and risk assessment of titanium dioxide. Description of the methods is not sufficient, i.e. details on tissue preparation, and electrophoresis conditions are largely missing. Body weight, age of the wildtype mice, caging, acclimation and food consumption were not reported. Recalculating the animal weights on the basis of the given concentration of the suspension, total administered dose and the range of the administered volume, it is concluded that the body weight was ranging between 20-28 g (difference > 20%) per mice. However, the OECD recommends that the weight variation at study start should not exceed 20%. The authors show a significant increase of comet tail intensity for testis cells being treated with TiO2 nanoparticles and crude Fpg enzyme extraction. However, combined treatment bears only very limited value for risk assessment purposes. Based on the above-mentioned shortcomings the reference is rated not reliable and therefore disregarded for the hazard assessment. 

 

The information (follow-up study of Heinrich et al. 1995), contained in the reference by Gallagher et al. (1994) represents in vivo DNA damage experiments in mammalian cells (32P-Postlabeling) with very limited value for risk assessment purposes. The study design described in the reference for detecting genotoxicity is not in accordance with any harmonised protocol (e.g. only one concentration tested) and is consequently inappropriate for risk and hazard assessment purposes under REACH (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The information contained therein were included for information purposes only.

 

Boisen et al. (2012) exposed pregnant C57BL/6 mice by whole-body inhalation to TiO2 UV-Titan L181 nanoparticles (~42.4 mg UV-Titan/m³) or filtered clean air on gestation days (GD) 8–18. Female C57BL/6 F1 offspring were raised to maturity and mated with unexposed CBA males. The F2 descendants were collected and ESTR germline mutation rates in this generation were estimated from full pedigrees of F1 female mice (UV-Titan-exposed F2 offspring). ESTR mutation rates were examined. ESTR mutation rates of 0.029 (maternal allele) and 0.047 (paternal allele) in UV-Titan-exposed F2 offspring were not statistically different from those of F2 controls: 0.037 (maternal allele) and 0.061 (paternal allele). The generated information is not required for building an expert judgement and further assessment of the test substance under REACH (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X) for the following reasons:

(i) Titanium dioxide nanoparticles are not able to pass the blood stream via the blood-air barrier after inhalation. The major amount of particles will be eliminated through muco-ciliary escalation and subsequent swallowing. It is questionable if any nanoparticles could ever reach the target tissue, particularly because the placental barrier existed as a second effective barrier in exposed maternal animals.

(ii) The experimental conditions / housing conditions of animals are insufficiently described, important data e.g. age of animals at study initiation and housing conditions are not specified.

(iii) The study design is not in accordance with any guideline and not performed under GLP directive. Testing only one concentration is uncommon and an analytical verification of the concentration is recommended.

(iv) small effective sample size and the very large maternal dose used in the study hamper interpretation

(v) the study does not investigate a direct mutagenic effect on exposed animals but rather epigenetic effects transmitted from the parent to the descendants. No plausible mechanism has been presented by the authors for the alleged epigenetic effects.

 

Jackson et al. (2013) time-mated female mice (C57BL/6Bom-Tac) were exposed to filtered clean air or approx. 42 mg/m³ UV-Titan on gestation day (GD) 8–18 for 1 h/day by a whole-body exposure. DNA strand breaks were examined in bronchoalveolar lavage (BAL) and liver cells of the time-mated mice (5 and 26–27 days after inhalation exposure), and in livers of the offspring (post-natal days (PND) 2 and 22) using the alkaline Comet assay. Furthermore, hepatic gene expression in newborns using DNA microarrays was investigated. No statistically significant genotoxic effect was measured in maternal exposed mice and offspring compared to the control (filtered air). The newborn offspring, irrespective of nanoparticle exposure, had higher levels of DNA strand breaks compared with the offspring at weaning (p-value = 0.001). Changes in gene expression in the offspring were observed and suggested to be a secondary effect to maternal inflammation after exposure. The generated information concerning genotoxicity and toxicogenomics is not required for building an expert judgement and further assessment of the test substance under REACH (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X) for the following reasons:

(i) The study design is not in accordance with any guideline. The acceptability criteria of the OECD test guideline 489 (in vivo mammalian alkaline comet assay) were not fulfilled since there is only one concentration tested, adequate number of cells (> 150 cells per slide) have not been analysed, a positive control was not specified in the results and the sampling time is not in accordance with the recommendation (2 - 6 hours after last treatment).

(ii) A significant dose-response related effect could not be observed (due to lack of concentrations tested). 

 

Troullier et al (2009) treated male C57Bl/6Jpun/pun mice for five days with P25 TiO2 nanoparticles suspended in drinking water at concentrations of 60, 120, 300, and 600 µg/ml (total dose after five days: 50, 100, 250, and 500 mg/kg). An alkaline comet assay was conducted with peripheral blood taken before and after treatment. 150 to 200 comets per sample were randomly captured and scored for change in tail moment. According to the authors, tail moments of the comets were significantly increased after TiO2 nanoparticle treatment at the highest dose (500 mg/kg), when compared to untreated controls. Thus, the authors concluded that the test item is able to induce DNA strand breaks. The study shows serious reporting and experimental shortcomings. The actual dose received was calculated on an average 30g body weight and an average of 5 mL water intake. This is an unjustified simplification, since the bodyweight for 12-20 week old animals differs significantly between e.g. male and female animals, also the average bodyweight of a 12-20 week old female mouse ranges between 21 and 24g. Such dose calculation. The author state that four to five months old mice were used. The current OECD test guideline recommends using young animals at an age of six to ten weeks. The authors do not justify selected species, strains, and dosing. Laboratory’s proficiency in preparing and analysing tail DNA intensities in comet assays is a prerequisite for reliable data. It was shown that there is huge variability in tail DNA intensities even within negative or positive controls (Tavares, A.M. et al. (2014); Nanogenotox Programme). This variability in combination with lack of controls and historical data renders positive findings of this assay of little relevance for risk assessment purposes. The transgenic C57Bl/6Jpun/pun mouse is an uncommon strain for this assay, with no historical data available. Due to the very low solubility and the difference in density of a factor of 4, one has to assume that titanium dioxide settles on the bottom of the flask, leading to a higher total dose. The authors verified neither the actual dose received nor the homogeneity of the suspension before during and after the dosing of the animals using an appropriate analytical method. Historical data and positive controls are not specified. Cytotoxicity measurements were not performed, which does not allow a conclusive statement, whether a true test item specific effect was observed or an artefactual response due to increased cytotoxicity. Clinical observations were not reported. Description of methods is insufficient. Details on tissue preparation, electrophoreses, and slide preparation are missing. Moreover, evaluation criteria are not sufficiently described. Moreover, the author state that the highest TiO2 nanoparticle dose leads to an inflammatory response, measured via qRT-PCR of pro-inflammatory marker gene expression. Inflammatory responses could have interfered with the results and could have led to false positive findings in the genotoxicity assays. The authors conducted a student’s t-test to test for significant changes before and after treatment. However, the student’s t-test is not a suitable for paired data. Based on the above-mentioned shortcomings the reference is rated not reliable and thus considered unsuitable for risk assessment purposes.

 

The study by Bettini, S. et al. (2017) investigated on potential genotoxic effects of titanium dioxide NANO4 (Aeroxide P25). Male rats got daily intragastric gavages of titanium dioxide NANO4 suspension for seven days at a concentration of 10 mg/kg bw/day. Afterwards, a comet assay was performed, and 100 Peyer's Patch (PP) cells were analysed regarding their % tail DNA. The cytotoxicity of titanium dioxide NANO4 was measured ex vivo in cells isolated from PP of untreated cells. According to the authors, treatment with the titanium dioxide NANO4 suspension (without as well as with Fpg treatment) did not result in increased DNA damage in PP cells when compared to controls. A dose-dependent cytotoxic and anti-proliferative effect on the T cells was observed. The study shows deficiencies in design and reporting. The study shows deficiencies in design and reporting. The number of analysed cells is too low (100; 150 are recommended by the OECD*). The authors tested only one dose (at least three are recommended by the OECD*). Positive controls and historical data are not specified. The exact time point of sampling and comet analysis is not specified. Moreover, the cytotoxicity measurements are described insufficiently. Information on occurrence and frequency of hedgehogs is not provided. Information on animal husbandry are largely missing. No details on environmental conditions, housing, diet supply, water supply, acclimation period, and test group randomization. The age of test animals is only specified as adult. Animal body weights are only specified for the study initiation. Methods are described insufficiently. Details on cell lysis, electrophoresis, and tissue and slide preparation are missing. Concentration of gavaged solution is not specified. Evaluation criteria description is insufficient. *OECD TG489: In Vivo Mammalian Alkaline Comet Assay, 2016

 

Kazimirova, A. et al. (2019) evaluated the potential of titanium dioxide NANO4 (Aeroxide P25), administered via intravenous injection, to induce DNA damage in peripheral blood mononuclear cells of female Wistar rats. Six to eight female rats received a single intravenous injection of titanium dioxide NANO4 at a dose of 0.59 mg/kg bw. The test animals were sacrificed after 1 day, 1 week, 2 weeks, and 4 weeks. Peripheral mononuclear blood cells were obtained, and the DNA damage state was evaluated in a conventional and a modified alkaline comet assay using the formamidopyrimidine glycosylase. A total of 400 nucleoids were evaluated for the proportion of DNA in tail as indicator for the DNA damage state. According to the authors, the intravenous administration of titanium dioxide NANO4 resulted in a statistically significantly increased proportion of DNA in tail only in rat peripheral blood mononuclear cells obtained one day after the treatment. Samples from longer post-exposure periods were not statistically significantly different from samples obtained from untreated control rats. Moreover, the modified comet assay did not reveal significant oxidative damage in the exposure groups under all conditions tested. However, the interpretation of these results is precluded based on several shortcomings with regard to reporting and the methodology applied. The test material was intravenously injected, which is considered to be a non-physiological route of exposure with only limited relevance for risk assessment purposes. Moreover, the concentration and volume of the administered suspension are not specified. Only one dose group was included, which precludes dose-response relationship evaluations. The methodology is insufficiently described, since essential details on the comet assay are missing. Cytotoxicity was not investigated, and thus, a differentiation between real genotoxic effects and cytotoxic effects is not possible. Information on body weights and clinical signs are not provided. Information on the test animals are largely missing. The statistically significant effect on the proportion of DNA damage one day after the titanium dioxide nanoparticle treatment appeared to more due to a low negative control value rather than a markedly increased response due to the treatment with the test material. The proportion of DNA in tail are highly variable (approx. 2% after one week vs. 6% after 2 weeks). Results obtained from the conventional comet assay are depicted only in a diagram, while the results obtained from the modified comet assay are not shown at all. Untreated peripheral blood mononuclear cells exposed in vitro to hydrogen peroxide were used as positive control, instead of running a real positive control group in parallel. Information on the occurrence and frequencies of hedgehogs is not specified. Evaluation and scoring criteria are missing. Historical control data is not included. Based on the above-mentioned shortcomings, the reference is rated not reliable and disregarded for the hazard assessment.

 

In this study by Dobrzyńska, M. M. (2014) the genotoxic potential titanium dioxide NANO4 (NM105; crystalline phase: anatase/rutile powder; particle size: 21 nm) was investigated using the alkaline comet assay. Male rats were given a single dose (5 mg/kg bw) of titanium dioxide NANO4 by intravenous administration and the animals were sacrificed 24 hours, 1 week and 4 weeks after administration of the test substance. Bone marrow cells were obtained for analysis. Comet tail moment and percentage of DNA in comet tail were chosen as parameters. According to the authors, intravenous administration of titanium dioxide NANO4 did not induce statistically significant DNA damage under the conditions of the test. The study by Dobrzyńska (2014) exhibits significant methodological deficiencies. Intravenous administration is an unphysiological route of application and bears only very limited value for risk assessment purposes. Samples were collected 24 hours, 1 week and 4 weeks after the single injection (OECD foresees at both 2 - 6 and 16 - 26 hours after a single administration). Only a single dose was tested, which precludes a dose-response relationship analysis. A positive control group was not included, and thus, the sensitivity of the system was not demonstrated. Historical control data are not reported. No information on tissue toxicity was reported, thus it remains questionable whether positive findings were artefactual due to overt toxicity or a true genotoxic response. The number of analysed cells was too low, individual results were not reported and acceptability and evaluation criteria not reported Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

 

Saber, A.T. et al.(2018) administered different pristine titanium dioxide nanoparticles ((i) crystalline phase: rutile, particle size: 10.5 nm, surface area: 139.1 m²/g; (ii) crystalline phase: rutile, particle size: 38 nm, surface area: 28.2 m²/g) in suspension, suspended sanding dusts of paint containing titanium dioxide nanoparticles, and pristine carbon black in female mice via intratracheal instillation. Titanium dioxide nanoparticles and titanium dioxide sanding dusts were administered at doses of 18, 54, and 162 µg/animals and 54, 162, and 486 µg/animals. Alkaline comet assay was performed on liver samples, and lung BAL cell extracts on poste exposure days 1, 3, and 28. According to the authors, none of the test materials induced significant increases in DNA damage, when compared to controls. Comet results from exposed lung BAL extracts were disregarded due to great variability of results obtained. The study shows major shortcomings in reporting and study design. The authors did not perform any cytotoxicity measurement. Controls are partially missing or inadequate. The authors used an in vitro system (H2O2 exposed A549 cells) as positive control, which is inadequate since it is not representative for the in vivo conditions. Historical data are missing. Details on comet assay methodology, including cell lysis and electrophoresis, are missing. Moreover, the authors do not state on number of cells scored, hedgehog occurrence, scoreable vs. unscoreable cells, particles in comet head and potential interference of particles with DNA stain. BAL cell suspensions were frozen and thawed later for the comet assay analysis. However, the OECD recommends performing slide preparation as soon as possible (ideally within one hour), since freezing of tissues increases the variation between samples and is therefore not recommended for the comet assay. In addition, details on freezing procedure, storage time, and thawing are largely missing. The authors disregarded results from comet on lung BAL cells due to great variability of results obtained. Thus, it is questionable whether the methodology is under control. Laboratory’s proficiency in preparing and analysing comets’ characteristics in comet assays is a prerequisite for reliable data. It was shown that there is huge variability in tail DNA intensities even within negative or positive controls (Tavares, A.M. et al. (2014); Nanogenotox Programme). DNA damage, represented by %DNA in tail, was measured only 24 hours after treatment, exceeding the OECD recommended measurements foreseen after 2-6 h and 16-26 h after last treatment. Information on exposure are missing. Generation and administration of test suspension are either insufficiently described or completely missing. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

 

Chakrabarti, S. et al. (2019) evaluated the potential DNA damage in liver and kidney cells of Swiss albino mice after sub-chronic exposure to titanium dioxide nanoparticles (particle size: 58.25 ± 8.11 nm). Five mice per sex per group received oral titanium dioxide nanoparticle doses of 200 and 500 mg/kg bw over a period of 90 days. The liver and kidney tissue samples were evaluated for DNA damage using the alkaline comet assay. The resulting tail length, proportion of DNA in tail, tail moment, and the olive tail moment were analysed to evaluate the grade of DNA damage. Additionally, the cell cycle and apoptosis rate in kidney and liver cells were analysed using flow cytometry. Moreover, post-mortem investigations included clinical chemistry, haematological examinations, and histopathological examination of the liver and kidney. According to the authors, the high dose group showed statistically significantly increased DNA damage, independent of the parameters evaluated, in both kidney and liver cells, when compared to the vehicle control group. The effect was considered dose dependent. The low dose group showed no change at all in each DNA damage parameter evaluated. The cell viability was statistically significantly reduced in a dose dependent manner in both tissues and each dose group. Moreover, both titanium dioxide dose groups showed a statistically significantly decreased level of cells in G0/G1 and S cycles of mitosis, and the high dose group showed a statistically significant increase of cells in the G2/M cycle. Thus, cytotoxicity is indicated by both a decreased cell viability and a disrupted cell cyclicity in animals dosed with titanium dioxide nanoparticles. The publication shows several reporting and methodological deficiencies. DNA damage parameters statistically significantly increased were only found in presence of cytotoxicity as indicated by a statistically significantly decreased cell viability and disrupted cell cyclicity in both dose groups. Moreover, hepatotoxicity, nephrotoxicity, altered haematological parameters were observed in the high-dose group. It is not clear whether premature deaths occurred in the high dose group. On the one hand the authors stated: “…signs of bleeding were observed in the cranial and peritoneal cavities of the dead animals treated with 500 mg/kg …”, but on the other hand the tables indicated no mortality. Moreover, the authors stated that each group contained ten animals, whereas the table indicates that only six animals were analysed. Notably, premature deaths could have evidenced that the MTD was exceeded, and thus, the acceptability criteria set out in the OECD TG 489 might have been violated. Further, target tissue toxicity is known to effects on the response in the comet assay. The number of dose groups is lower than recommended in the current OECD TG 489 (2 vs. 3). The test material was insufficiently characterised. The methodology is only poorly described and nearly all essential details on the comet assay are missing. The number of animals and nucleoids per animal evaluated for DNA damage is not specified. Information provided in the main text and tables are partly conflicting. Details on animal housing are missing. The test animals were not kept under optimal environmental conditions (temperature: 25 ± 2°C; humidity: 70 ± 5%). Body weights before and during treatment are not specified. Information on hedgehogs is not given. Evaluation and scoring criteria are missing. Historical control data is not included. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

 

Murugadoss, S. et al. (2020) investigated on the DNA damage in female C57BL/6Jrj mice BAL cells after oropharyngeal aspiration of 17 or 117 nm titanium dioxide anatase nanoparticles, each prepared in suspensions containing predominantly small or large agglomerates. The mice were treated once with each of the titanium dioxide nanoparticle suspension types at three different dose level (approx. 2, 10, and 50 µg/mouse). Three days after the test material administration, the mice were sacrificed, and lungs cells were obtained by BAL. The DNA damage in BAL cells was examined via the alkaline comet assay. Untreated BAL cells exposed to hydrogen peroxide served as positive control. A total of 50 nucleoids per well was scored for DNA damage, i.e. % DNA in tail, in each of the three independent experiments performed. According to the authors, oropharyngeal aspiration of suspensions containing either predominantly small or large agglomerates of either 17 or 117 nm titanium dioxide nanoparticles induced no DNA damage in BAL cells, when compared to the vehicle control group. However, the publication shows several reporting and methodological deficiencies. The test material was administered via oropharyngeal aspiration, which is a non-physiological exposure route and only of limited relevance for risk assessment purposes. Furthermore, the dose selection was not performed according to the requirements of the OECD TG 489, since the highest dose tested should be the MTD. The test material was insufficiently characterised, since information on the purity and coatings are missing. Moreover, the composition of the suspension medium is not described. The methodology is only poorly described and nearly all essential details on the comet assay are missing. The dose levels tested are not explicitly specified. Untreated BAL cells exposed to hydrogen peroxide were used as positive control instead of a real positive control group. The results on DNA damage are not shown and only restricted to no/yes conclusions and results obtained from positive and negative control groups are not reported. Information on the occurrence and frequency of hedgehogs is not specified. Evaluation and scoring criteria are not reported. Historical control data is not included. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

 

Murugadoss, S. et al. (2020) investigated on the DNA damage in female C57BL/6Jrj mice BAL cells after oral administration of 17 or 117 nm titanium dioxide anatase nanoparticles, each prepared in suspensions containing predominantly small or large agglomerates. The mice received a single gavage with one of the four different titanium dioxide nanoparticle suspension types at doses of approximately 10, 50, and 250 µg/mouse. Three days after the test material administration, the mice were sacrificed, and the blood was collected. The DNA damage in blood cells was examined via the alkaline comet assay. A total of 50 nucleoids per well was scored for DNA damage, i.e. % DNA in tail, in each of the three independent experiments performed. According to the authors, the proportion of DNA in tail was statistically significantly increased only after the treatment with the small agglomerate suspension composed of 17 nm titanium dioxide nanoparticles and the large agglomerate suspension composed of 117 nm titanium dioxide nanoparticles. The effect was statistically significantly at all dose levels tested. However, none of the statistically significant responses appear to be dose related. The authors concluded that small agglomerate suspensions per se did not show a greater biological response when compared to suspensions composed of large agglomerates. However, the publication shows several reporting and methodological deficiencies. The test material was insufficiently characterised, since information on the purity and coatings are missing. Moreover, the type of blood cells analysed is not specified. Cytotoxicity was not investigated at all. Information on body weights and clinical signs are not provided. The composition of the suspension medium is not described. The methodology is only poorly described and nearly all essential details on the comet assay are missing. The dose levels tested are not explicitly specified. Information on the test animals are largely missing, since details on the weights before and during and after the treatment, group assignment, group sizes in cages, feed and water availability, and acclimation period are not provided. The statistically significant responses elicited by titanium dioxide 17 nm-SA and 117 nm-LA are apparently not dose related. The dose-response relationship, however, is not discussed. The effect on DNA damage was highly similar between animals treated with the two different 117 nm TiO2 NP suspension. However, only the response induced by the large agglomerate suspension was assigned to be statistically different, which seems implausible based on the data. Results obtained from positive and negative (only mean without SD) control groups are not shown for any of the parameters tested. Information on the occurrence and frequencies is not provided. Evaluation and scoring criteria are missing. Historical control data is not included. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

 

 

Summary entry - In vivo DNA damage

No conclusion can be drawn from the above publications due to lack of quality, reliability and adequacy of the experimental data for the fulfilment of data requirements under REACH.

The references contained in this summary entry represent in vivo experiments on genetic toxicity with very limited value for risk assessment purposes. All references do not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VIIX). The information contained therein were included for information purposes only.

The test material used in the references summarised in the summary entry was largely characterised insufficiently. As detailed in the introductory statement to chapter for human health hazard assessment, it is problematic to base the hazard and risk assessment of clearly characterised titanium dioxide on information generated with poorly characterised test item, with no market relevance. In accordance with ECHA guidance, all references that do not provide sufficient information on the substance identity were disregarded for hazard and risk assessment purposes (for further information please refer to the introductory statement).

 

Jin, C. et al. (2013): The methodical setup is not guideline compliant or adequately designed for risk assessment purposes of the test substance. The non-physiological route of administration via intranasal application is not guideline conform and not suitable to assess genetic toxicity. Moreover, the test substance is not sufficiently characterised, only one concentration was administered and the number of test animals were not specified.

 

Li, N. et al. (2009): The self-synthesised titanium dioxide nanoparticles were not sufficiently characterised and the influence of the other simultaneously administered process chemicals is not addressed. The non-physiological route of administration via intraperitoneal injection is not guideline conform and not suitable to assess genetic toxicity.

 

Carmona E.R. et al. (2015): The test material is not sufficiently characterised and an unsuitable test system was chosen (studies reporting DNA damage in bacteria, induction of SCE in mammalian cells, or tests in yeasts or drosophila are no longer recommended as part of regulatory testing by many agencies worldwide and there are no up-to-date OECD guidelines for their conduct). Interpretation of the relevance of both positive and negative results from such tests is therefore unclear, and so these studies are considered to have a minor contribution to the overall assessment of genotoxic potential. was used in this study for assessing the genotoxic potential of titanium dioxide nanoparticles. Moreover, no historical data is given by the authors and scoring criteria were inappropriate and do not allow a quantitative analysis. Microscopical analysis allows no conclusions on the effects of titanium dioxide on animals (necropsy poorly performed, scale missing, differences between treated and untreated animal difficult to interpret).

 

Relier, C. et al (2017). A significantly increased number of foci, belonging to category 3 (5-10 foci per cell), and mean foci per cell were only found in lung cells after 2 hours post-exposure at the highest dose. However, a significant cytotoxic response was observed at the same conditions (LDH level in BAL). The other tissues showed no significantly increased number of foci independent of dose. The methodical setup is not based on any established guideline. Information on housing, weights of animals, test group randomization, euthanasia, tissue preparation, and environmental conditions are missing. Species justification, and vehicle justification are missing. Data on evaluation criteria are not sufficient. Individual rat values are missing.

 

Reis, E.M., et al. (2016): The test material is not sufficiently characterised and an unsuitable test system was chosen (studies reporting DNA damage in bacteria, induction of SCE in mammalian cells, or tests in yeasts or drosophila are no longer recommended as part of regulatory testing by many agencies worldwide and there are no up-to-date OECD guidelines for their conduct). Interpretation of the relevance of both positive and negative results from such tests is therefore unclear, and so these studies are considered to have a minor contribution to the overall assessment of genotoxic potential. was used in this study for assessing the genotoxic potential of titanium dioxide nanoparticles. Moreover, no historical data is given by the authors and positive findings showed no dose-response relationship, which renders the findings presented probably to be chance findings.

 

Vasantharaja, D. et al. (2019): The methodology is only poorly described and lacks all essential details. The test substance is insufficiently characterised, since information on crystalline phase, manufacturer, coating, and particle size distribution are missing. Only two test groups are included. A positive control was not included. The positive findings were observed in presence of liver toxicity, indicated by statically significantly increased ALT, AST, and LDH values and histopathological alteration of the liver. Number of cells analysed is not specified. Test animals are insufficiently characterised.

 

Hadrup, N. et al. (2017): The methodology is insufficiently described and lacks essential details. Only one dose level was tested. The number of cells per animal scored for DNA damage is not specified. Moreover, the proportion of DNA in tail for each sample was normalised to negative A549 controls on each slide. Potential cytotoxic effects of the test material on BALF cells were not evaluated. The positive findings, observed 24 hours after the intratracheal instillations, are most likely induced by bolus effects.

 

Elnagar, A.M.B. et al.(2018): The methodology is poorly described, since all essential details are missing. The number of cells per animal scored for DNA damage is not specified. The exposure group sizes are not specified. Quantitative results are not shown. Only one dose level was tested precluding dose-response relationship evaluations.

 

 

Summary - In vivo DNA damage

Merely one in vivo DNA damage assay was considered reliable for hazard assessment purposes. The analysis of bronchi-alveolar lavage cells via alkaline comet assay showed no significant effect on the level of DNA damage after inhalation exposure towards 10 mg/m³ for five consecutive days. The maximum concentration was reaching the maximum tolerated dose. It is therefore concluded that titanium dioxide does not cause local or systemic clastogenic or aneugenic events in animals.

 

 

In vivo gene mutation assays

Suzuki, T. et al. (2020) performed a transgenic rodent assay in order to investigate on gene mutation in male C57BL/6J gpt delta mice after titanium dioxide NANO4 (Aeroxide P25) exposure. The mice were treated at titanium dioxide NANO4 doses of 2, 10, and 50 mg/kg bw via intravenous injection. Ninety days post-exposure, the male mice were sacrificed, and the middle liver lobes were obtained and the mutation frequencies at the gpt and Sci- loci were evaluated. According, to the authors, titanium dioxide NANO4 treatment did not result in a statistically significantly increased mutation frequency at both loci investigated. Notably, the titanium burden in the liver was statistically significantly increased and showed dose dependency. Thus, the authors concluded that that titanium dioxide NANO4 particles have no mutagenic effects on the liver, even though the particles remain in the liver long-term. The study shows deficiencies with regards to reporting and the methodology applied. The test material was administered via intravenous injections, which is considered as a non-physiological route of exposure. Furthermore, the test material is not sufficiently characterised and details on the test material preparation are missing. The methodology of the mutation assay is not described and the methods for enumeration of mutants are not specified. Moreover, the liver samples were obtained 90 days postexposure, the current OECD TG 488 foresees, however, sampling after 3 or 28 days postexposure. The number of packing reactions is not specified. No positive control group was included. Historical control data is not reported. Based on the above-mentioned shortcomings the reference is considered not reliable.

 

Summary entry - In vivo gene mutation assays

No conclusion can be drawn from the above publications due to lack of quality, reliability and adequacy of the experimental data for the fulfilment of data requirements under REACH.

The references contained in this summary entry represent in vivo experiments on genetic toxicity with very limited value for risk assessment purposes. All references do not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VIIX). The information contained therein were included for information purposes only.

The test material used in the references summarised in the summary entry was largely characterised insufficiently. As detailed in the introductory statement to chapter for human health hazard assessment, it is problematic to base the hazard and risk assessment of clearly characterised titanium dioxide on information generated with poorly characterised test item, with no market relevance. In accordance with ECHA guidance, all references that do not provide sufficient information on the substance identity were disregarded for hazard and risk assessment purposes (for further information please refer to the introductory statement).

 

Relier, C. et al (2017). The exposure to TiO2 nano-particles lead to no increased mutation rate in the PIG-a mutation assay. However, insufficient method description, a lack of details on evaluation criteria, absence of availability check of the test substance in the target organ, and missing values from the individual rats do not allow to draw a conclusion on the basis of the presented results. The methodical setup is not based on any established guideline. There is no established OECD-guideline for this type of assay. Information on housing, weights of animals, test group randomization, euthanasia, tissue preparation, and environmental conditions are missing. Species justification, and vehicle justification are missing. Data on evaluation criteria are not sufficient. Individual rat values are missing.

 

 

Summary - In vivo gene mutation assays

Reliable references reporting on gene mutation in vivo after exposure to titanium dioxide nanoparticles are not available. Thus, no conclusion on the potential to induce gene mutations in intact organisms can be drawn based on the information given.

 

 

Nanogenotox programme

In a comprehensive testing programme, a total of six different titanium dioxide nanomaterials were tested in several in vitro and in vivo test systems for their ability to induce (i) unspecific DNA damage, (ii) chromosome or genome mutation and (iii) gene mutation. An overview of the physicochemical parameters of the tested titanium dioxide samples is presented in the table below.

Table: Overview of the titanium dioxide samples used in the testing programme

Sample

Phase

Average XRD crystalline size

Average TEM particle size

Average BET & SAXS SSA

NM-100

anatase

56.7 - >100 nm

110 nm

9 m²/g

NM-101

anatase

7 nm

6 nm

316 m²/g

NM-102

anatase

21 nm

22 nm

78 m²/g

NM-103

rutile

23 nm

25 nm

51 m²/g

NM-104

rutile

23 nm

25 nm

56 m²/g

NM-105

anatase

rutile

23 nm

60 nm

24 nm

15 nm

46 m²/g

The testing programme was coordinated by the French Agency for Food, Environmental and Occupational Health & Safety (ANSES), involving 16 associated partners from 11 member states and 13 collaborating partners. The programme started in March 2010 for a period of 3 years, the final report was published in March 2013 (http://www.nanogenotox.eu/). An overview of all genetox tests executed within this programme is given in the table below.

Table: Overview of the in vitro genotoxicity assays conducted with titanium dioxide nano particles, samples NM-102 – NM-105.

Cell line

Assay

BEAS 2B

MN

Comet 3h

Comet 24h

16 HBE

MN

Comet 3h

Comet 24h

A549

MN

Comet 3h

Comet 24h

CACO-2

MN

Comet 3h

Comet 24h

Lymphocytes

MN

L5178Y

MLA

NHEK

MN

Comet 3h

Comet 24h

Skin 3D

Comet 3h

Comet 24h

 

Table: Overview of the in vitro genotoxicity assays conducted with titanium dioxide nano particles.

Species

Route

Assay

NM-101

NM-102

NM-103

NM-104

NM-105

Rat

Intratracheal

Comet

5 tissues

 

 

 

 

 

MN

1 tissue

 

 

 

 

 

Gavage

Comet

5 tissues

 

 

 

 

 

FpG Comet

2 tissues

 

 

 

 

 

MN

2 tissues

 

 

 

 

 

Intravenous

MN

1 tissue

 

 

 

 

 

Mice (LacZ)

Intravenous

gene mutation

1 tissue

 

 

 

 

 

Comet

2 tissues

 

 

 

 

 

MN

1 tissue

 

 

 

 

 

IUCLID study records were generated for each test item and test system combination, in order toreport the test results transparently. This resulted in 54 study records for the in vitro systems and 23 study records for in vivo systems.

Due to the large number of test results (some of the in vitro experiments were conducted as round robin test over a range of up to 12 different laboratories), the overall results are discussed here only in summary.

For the reliability rating according to Klimisch, all experiments were checked against the relevant OECD technical guidelines relevant at the time of study conduct. The in vivo comet assay study design was checked against the OECD test guideline 489 (26 September 2014), which is based on internationally agreed study designs suggested by various authors in the public domain (e.g. Kirkland 2008, Brendler-Schwaab 2005, Burlinson 2007 and 2012, Smith 2008, Hartmann 2003, McKelvey-Martin 1993, Tice 2000, Singh 1998, Rothfuss 2010). The comparison with OECD 489 is therefore appropriate, since the majority of these protocols existed at the time of study planning and conduct.

 

The reports exhibited a number of shortcomings in the description of the experimental procedures and in the presentation of the test results. Further, the overall reporting does not appears to be final, since e.g. the report on the in vivo micronucleus assay in colon of rats following gavage administration is still flagged as “to be amended”. The major shortcomings of the reporting and experimental design are given below:

 

General

        i.           

      ii.           

    iii.           

    iv.           

      v.           

 

In vitro experiments

        i.           

      ii.           

    iii.           

    iv.           

 

In vivo experiments

        i.           

      ii.           

    iii.           

    iv.           

      v.           

    vi.           

   vii.           

 

Such shortcomings in the reporting and the test design would normally result in a reliability rating of 3 or 4, i.e. rendering those studies not suitable for hazard assessment purposes of industrial chemicals. However, since it can be assumed that the experiments were conducted by professional labs with documented competence for the execution of those tests and that the two journal articles (published after project finalisation: Tavares, A.M. et al. (2014); Louro, H. (2014)) demonstrated that more details are available, than were provided in the Nanogenotox reports.

Consequently, all individual experiments conducted equivalent to accepted guidelines were rated with a reliability rating of 2, with the restriction that the individual studies cannot be used as key studies, but only as a part in a weight of evidence approach. Studies that conducted not following a guideline, such as in vitro comet assays, were rated as not reliable (RL3). The results of the individual experiments are presented and discussed below in detail.

 

Results

In vitro

Four titanium dioxide samples were tested in a micronucleus assay in six different cell lines. Authors concluded that the micronucleus assay was positive for four tested TiO2 samples (NM-102 – NM-105) in NHEK cells and also in the micronucleus assay in primary lymphocytes for NM-102, NM-103 and NM-104. However, the micronucleus assay was negative for all TiO2 samples in the other four remaining cell lines.

The comet assay was positive for all TiO2 in Caco-2 cells after the 24-hour treatment except for NM-104 (negative). NM- 100 showed a positive result in the 16HBE cell line as well as NM-102 (3-hour treatment) and NM- 105 (24 hour treatment). Four TiO2 samples (NM-102 - NM-105) showed a positive result at the 3- and 24-hours treatment time in the NHEK cell line. In addition, NM-102 showed a positive result in the BEAS 2B cell line at both treatment times. Lastly, NM-102 and NM-105 caused a positive result in the A549 cell line at the 3-hour treatment time.

In 3D human reconstructed full thickness skin models, all TiO2 nanomaterials (NM-102, NM-103, NM-104, and NM-105) investigated for DNA damage were negative in the comet assay. In contrast, the chemical positive control methylmethanesulfonate (MMS) consistently generated a significant increase in DNA damage. The highest dose studied by this protocol was 246 μg/cm² skin surface which showed no interference during the analysis. Transmission electron microscopic (TEM) analysis did not identify penetration of the stratum corneum of reconstructed human full thickness skin models even after 72-hour exposure with TiO2.

The L5178Y mouse lymphoma gene mutation assay was negative for all forms of TiO2 tested (NM-102 – NM-105).

 

In vivo

After intratracheal instillation in male Sprague-Dawley rats, none of the TiO2nanomaterials (NM-101 – NM-105) induced DNA damage in broncho alveolar lavage (BAL) cells in a comet assay. Two TiO2samples (NM-102 and -103) showed an insignificant increase with no dose dependency in the liver. None of the TiO2nanomaterials studied showed genotoxic effects in lung, spleen, and kidney.

Furthermore, intratracheal instillation with NM-101 – NM-105 in male Sprague-Dawley rats induced no increase in micronucleus frequency in a bone marrow micronucleus assay.

Following oral administration by gavage in male Wistar rats, some genotoxic effects were observed with the comet assay with TiO2in spleen, jejunum (NM-103), colon (NM-102 and -105), and bone marrow (NM-104). However, all TiO2 nanomaterials studied showed no genotoxic effects in liver, kidney, and lymphocytes samples. No mutagenicity in bone marrow was observed in a bone marrow micronucleus assay using the same type of rats and the same route of administration.

Micronucleus assays in rats were included using single and repeated (5 times) intravenous administrations with NM-103 and NM-104 in order to increase the micronucleus potency to reach systemic organs. No increase in micronuclei could be detected in bone marrow.

In a further series of testing C57Bl/6 pUR288 transgenic male mice were used. Negative results for bone marrow micronucleus assay after repeated intravenous (i.v.;2 times) exposure to NM-102 were obtained. The genotoxicity effect of NM-102 was also tested in a gene mutation assay (Mutation Lac Z assay) and a comet assay using the transgenic male mice and i.v. administration, which showed a negative effect in the liver and spleen.

 

Conclusion

 

The overall quality of the summary report and the individual deliverable reports of the Nanogenotox programme are insufficient to follow the experimental design and findings. In some cases the results section does not appear to match the findings reported in the results section. The material and methods section merely provides a brief description of the essential methods, which hinders an assessment of the whole study design by a peer-reviewer. A small sub-set of experiments were published after completion of the programme (Louro, H. et al. (2014); Tavares, A.M. et al. (2014)), which provides further information on the overall study design compared with the final report of the Nanogenotox Programme.

 

In vitro

The in vitro experiments returned predominantly negative results from the micronucleus tests. Merely one cell line (NHEK, which is also not commonly used in in vitro genotoxicity testing experiments) returned a dose-response relationship over all test items. However, the MNBN frequencies were within the historical data range of other cell lines and primary human lymphocytes seen by other labs (0-15 MNBN per 1000 BN cells in vehicle/negative control), which puts the biological relevance into question whether this dose-response relationship is a true substance- induced finding. Whether this particular cell line is more susceptible to a physical insult by PSPs leading to an increased frequency of MN formation, was not further discussed by the authors. Based on the overwhelmingly negative findings with the five remaining cell lines and primary human lymphocytes, the dose-response relationship finding with NHEK cells is considered as incidental finding with no biological relevance for the human health hazard assessment. Consequently, titanium dioxide is considered non-clastogenic and non-aneugenic in a large range of in vitro mammalian cell systems. The mouse lymphoma assay in L5178Y cells returned a clear negative result over a range of four titanium dioxide samples, indicating an absence of potential to induce gene mutations by nano-sized titanium dioxide.

 

The comet assays returned largely positive findings over a wide range of different cell systems (pulmonary, intestinal, and epidermal). It is unclear whether the differences among cell systems were caused by intrinsic differences of their susceptibility, or by experimental variations. A major concern is that a dose dependency of the comet findings could not be established in any of the experiments, which raises doubts on a substance intrinsic ability to induce DNA damage. Most disturbingly, a very large inter-laboratory difference among the comet results for selected nano materials was observed, highlighting the difficulty to obtain conclusive results with this assay. Based on the outcome of the in vitro comet assays with titanium dioxide, it is concluded that there is inconclusive evidence on a DNA damaging potential of titanium dioxide. This is based on an overall lack of a dose-response relationship and the large inter-laboratory differences in results.

In summary, there is no conclusive evidence of a genotoxic potential of five different titanium dioxide samples in a number of different in vitro tests systems, covering general DNA damage, gene, chromosome or genome mutations,

 

In vivo

Inconclusive positive in vivo findings in the come assay are restricted to isolated organs or tissues and are also route dependent. After oral administration by gavage genotoxic effects were observed in spleen and jejunum (MN-103), colon (NM-102 and NM-105) and bone marrow (NM-104) in a comet assay with rats. However, those findings were largely lacking a dose-response relationship or were even not statistically significant. Further, the authors highlighted that the involved laboratories showed pronounced differences in the evaluation of the comet assays, as expressed in the figure below.

Figure: Variability of comet assay responses in liver for negative (vehicle) and positive (MMS) controls among the different laboratories involved in the WP. Each point is the median of one animal.

 

This highlights the lack of repeatability of these experiments and the lack of reliability of this test. In addition, some labs lacked historical control data for some tissues (e.g. spleen). Such lack of historical control data aggravates the interpretation of positive findings, since the variability of the comet findings are not known. It is therefore unclear whether a significant increase of DNA damage may have been within a normal biological variation and thus caused by an incidentally low negative control. These shortcomings for the in vivo comet assay highlight that the criteria for acceptability of that test system (especially in organ systems or tissues not commonly investigated) are not yet established.

For the other assays in rats (comet assay (intratracheal administration), bone marrow micronucleus assay (intratracheal, intravenous, and oral administration), and transgenic mice (bone marrow micronucleus assay, comet assay, gene mutation assay), an overall negative outcome was investigated over a range of up to five different titanium dioxide samples (NM-101 – NM-105).

In summary, there is no conclusive evidence of a genotoxic potential of five different titanium dioxide samples in a number of different in vivo tests systems, covering general DNA damage, gene, chromosome or genome mutations.

 

 

Discussion

 

In conclusion, the Nanogenotox testing programme almost exclusively returned negative findings in genotoxicity tests with a long-lasting testing history and for which well-established guidelines and procedures are available. In cases where inconclusive positive findings were received, these were mostly isolated findings in a single cell line and could not be repeated in other cell lines or primary human lymphocytes, thus should be regarded as not biologically relevant. The overall negative findings in the in vivo systems corroborates the mostly negative in vitro test results. For test systems investigating the induction of unspecific DNA damage (Comets assay), the in vitro tests largely returned a positive outcome, which were however not biologically relevant or not statistically significant. Similarly, the positive in vivo findings of the comet assay showed serious shortcomings in an inter-laboratory comparison. Considering the overwhelmingly negative tests results in this testing programme, it is concluded that nano-sized titanium dioxide is non-mutagenic, non-clastogenic or non-aneugenic over a wide range of different test systems.

 

 

 

Overall conclusion

The genetic toxicity of pigment grade and ultrafine titanium dioxide was assessed in various in vitro and in vivo experiments, using different crystalline forms (anatase and rutile) as well as surface modified titanium dioxide. The surface modifications applied to titanium dioxide particles is aimed to increase the dispersibility in lipid suspensions. Consequently, negative results obtained with hydrophobic coated titanium dioxide are considered as worst case exposure conditions. In summary, there is a consistent pattern showing a complete lack of genotoxic effects over a wide range of test systems for all commercially relevant crystalline forms of titanium dioxide.

Justification for classification or non-classification

Genetic toxicity, in vivo

The in vivo data for pigment grade titanium dioxide present a consistent pattern. In rigorous GLP and guideline compliant studies as discussed above, combining chromosome aberration and micronucleus endpoints up to limit doses in mice, titanium dioxide did not show test item induced genetic toxicity.

 

Genetic toxicity, in vitro

None of the in vitro genotoxicity studies rated as reliable showed any effect in bacterial reverse mutation assays, in mammalian cell gene mutation tests (TK assay) or in mammalian cell chromosome aberration or micronucleus tests, thus supporting the negative findings in the in vivo tests as cited above.

The classification criteria acc. to regulation (EC) 1272/2008 as germ cell mutagen are not met, thus no classification applicable.