Guidance

Annexe 3a. Detailed evaluation of in vitro papers with green and amber ratings

Published 11 October 2024

Note: some papers assessed several endpoints.

‘Green’ papers – micronucleus (MN) assay

Andreoli and others (2018)

Andreoli and others (2018) conducted a cytokinesis block micronucleus (CBMN) assay in peripheral blood mononuclear cells (PBMCs), using cytochalasin B (cytoB) and performed according to OECD TG487 with minor modifications. 1 x 10⁶ PBMC were suspended in medium with phytohaemaglutinin (PHA) to stimulate lymphocyte proliferation. Cells were treated using 2 different protocols. In protocol 1, cells were treated with PHA for 24 hours, then with TiO₂ (anatase, rutile or a mixture of both, at concentrations of 0, 10, 50, 100 or 200 micrograms per millilitre (µg/mL)) for 20 hours, after which cytoB was added for 28 hours (48-hour total treatment time). In protocol 2, cells were treated PHA for 43.5 hours and TiO₂ for 30 minutes, after which cytoB was added for 28 hours (28.5-hour total treatment time). Protocol 1 was considered the more robust methodology and hence more weight was put on these results.

The primary size of the commercial TiO₂ nanoparticles was 20 to 60 anometres (nm), 30 x- 100nm and 45 to 252nm for the anatase, rutile and mix, respectively, with a secondary size of 328nm (70 to 2130nm), 283nm (50 to 2,730nm) and 303nm (40 to 2,450nm), respectively, when dispersed by ultrasonication in MilliQ H2O, and 210nm (50 to 1,570nm), 226nm (50 to 3,340nm) and 328 (50 to 770nm), respectively, when dispersed in RPMI media without a surfactant. One thousand binucleated cells/concentration were analysed for micronucleated (MN) formation per experiment and 2 independent experiments were conducted (total of 2,000 binucleated cells/concentration). Hydrogen peroxide (500 micrometres (µM)) was used as a positive control. For cytotoxicity assessment, the cytokinesis block proliferation index (CBPI) was calculated, but no toxicity was observed after any treatment with TiO₂.

The MN frequency in the negative control (background) was 0.75 to 1% and the positive control was 5- to 8-fold higher than the background.

Following treatment with TiO₂, no increase in MN formation was detected with either treatment protocol.

COM opinion

This study was considered robust and well conducted with minor deficiencies. A flow-based method was used which does not confirm cellular uptake. The highest dose (200 µg/mL) exceeded the concentration recommended in the OECD guidelines for testing nanomaterials but despite this, was still negative. Hydrogen peroxide was used as a positive control, which is not a recommended positive control according to OECD TG487; however, it induced an unequivocal positive response (5- to 8-fold increase over background). Despite these reservations, the study was given a RAG rating of green.

The study detected induction of 8-oxo-2’-deoxyguanosine (8-oxodG) by anatase and rutile TiO₂ and the mixture, indicating oxidative damage which did not translate into a genotoxic signal.

Overall, no increase in MN formation was detected and the study was considered negative. This study was cited in the European Food Safety Authority (EFSA) review (EFSA, 2021).

Demir and others (2015)

Demir and others (2015) conducted a CBMN assay in human embryonic kidney (HEK293) cells and mouse embryonic fibroblast (NIH/3T3) cells using cytoB. 5x10⁵ HEK293 or NIH/3T3 cells were treated with 10, 100 or 1000 µg/ml anatase TiO₂ nanoparticles for 48 hours. CytoB was added for the last 24 hours prior to harvesting.

Two sizes of anatase nanoparticles were tested. The primary sizes of these were 21 ± 2.8nm and 50 ± 12nm and the secondary sizes were 22.94 ± 0.3nm and 50.72 ± 0.4nm. For these measurements, TiO₂ nanoparticles were dispersed by ultrasonication in 0.05% bovine serum albumin (BSA). One thousand binucleated cells/concentration were scored for MN in duplicate (total of 2,000 binucleated cells/concentration). Mitomycin C (MMC; 0.3µM) was used as the positive control. For cytotoxicity assessment, CBPI was calculated.

The MN frequency in the negative control (background) was 0.55% in HEK293 cells and 0.6% in NIH/3T3 cells; the positive controls induced approximately 8- and 6-fold higher Mn levels than background in HEK293 cells and NIH/3T3 cells, respectively.

Following treatment with TiO₂, no genotoxicity or cytotoxicity was detected between 10 to 100 µg/mL in either cell line for both sizes of nanoparticles. MN formation was only increased at the highest TiO₂ concentration (1,000 µg/ml) in both cell lines.

COM opinion

This study showed robust methodology. HEK293 is a non-standard cell line and is not recommended by OECD, but background MN frequency was in line with that seen in OECD-recommended cells. The doubling time of the cells is 24 to 48/36 hours. No evidence of nuclear uptake was provided. The increased MN formation observed at the highest concentration (1,000 µg/mL) in both cell lines was not considered to be relevant for genotoxicity assessment, as this is an extremely high concentration that would not be considered appropriate for testing nanomaterials (OECD, 2022). This concentration was therefore discounted from any assessment of effect. Despite these reservations, the study was given a RAG rating of green.

A Comet assay ± formamidopyrimidine DNA glycosylase (Fpg) in the study gave no evidence of oxidative DNA damage or cell transformation between 10 to 100 µg/ml.

Overall, no increase in MN formation was detected at the relevant concentration range and the study was considered negative. This study was cited in the EFSA review (EFSA, 2021).

Di Bucchianico and others (2017)

Di Bucchianico and others (2017) carried out a CBMN assay in a human bronchial epithelial cell line (BEAS-2B) using cytoB according to OECD TG487. 6 x 104 cells were treated with 3 different TiO2 nanoparticles: uncoated anatase (NM-100; 50 to 150nm), coated (no further information given) anatase (NM-101; 5 to 8nm) and coated rutile (NM-103; 20 to 28nm) at doses of 1, 5 or 15 µg/ml for 20 hours, after which cytoB was added for 28 hours.

MN were also evaluated using flow cytometry without cytoB, in which cells were treated with1, 5, 15 or 30 µg/mL for 48 hours.

Nanoparticles were dispersed using ultrasonication in 0.05 % BSA. 2000 binucleated cells were analysed for MN formation. Mitomycin C (0.05 µg/mL) was used as a positive control. Cytotoxicity was assessed by calculating the reduction in replication index (RI) of the treated cells compared to the negative control. The number of apoptotic, necrotic and mitotic cells per 1,000 cells was also evaluated as a measure of cytotoxicity and cell proliferation.

Cytotoxicity was minimal. The MN frequency in the negative control (background) was approximately 1% (for both methods employed) and the positive control was 8- to 9-fold higher than background (for manual versus flow method, respectively).

Following treatment with TiO₂, uncoated and coated anatase (NM-100 and NM-101, respectively) were both negative for MN formation in both methods, apart from 1 µg/mL NM-101 in the flow cytometry method which showed a small increase in MN formation. Low doses of NM-103 also showed a small increase in MN formation in both methods (2-fold increase at 1 µg/mL (manual) and 1.5-fold increase (flow method).

COM opinion

This study was well conducted and showed robust methodology. BEAS-2B is a non-standard cell line and is not recommended by OECD, but background MN frequency was in line with that seen in OECD-recommended cells. The doubling time of the cells is 26 hours. The Litron kit method normally requires 10,000 cells to be scored, whereas in this study only 2,000 nuclei were scored. Flow-based measures are not reliable to show cellular uptake. The number of replicates was unclear, although figures did contain error bars. Despite these reservations, the study was given a RAG rating of green.

There were indications of oxidative damage as the Comet assay, conducted ±Fpg, was positive after 3 hours with both NM-100 and NM-103, while after 24 hours only NM-101 was positive.

Overall, the study was weakly positive (1.5 to 2-fold increase) for the formation of MN at lower doses, but only in rutile-treated cells. This study was cited in the EFSA review (EFSA, 2021).

Li and others (2017)

Li and others (2017) conducted an MN assay without cytoB in human lymphoblastoid TK6 cells using 2 methods. Test 1 used flow cytometry and test 2 used manual microscopy. As TiO₂ emits fluorescence which could interfere with flow cytometry, results from test 1 were not considered further. In test 2, 3 x 105 cells were exposed to synthesised anatase TiO₂ nanoparticles at concentrations of 100 and 200 µg/mL.

The primary size of TiO₂ nanoparticles was 8.9 to 15.3nm and the secondary size was 860 to 892nm. The samples were prepared by probe sonication in sterilised water. A total of 2,000 binucleated cells were analysed for MN formation. MMC was used as the positive control (0.01 µg/mL) . Cytotoxicity was assessed by relative increase in cell counts (RICC) and relative population doubling (RPD).

In the manual assay (test 2), RICC ranged from 58 to 73% and RPD from 54 to 77% at 200 and 100 µg/mL, respectively. The MN frequency in the negative control was 1.85% and the positive control was 2.5-fold higher than background.

Following treatment with TiO₂, a significant increase in frequency of MN was detected (1.5- and 2-fold increase at 100 and 200 µg/ml, respectively).

COM opinion

The manual analysis method in test 2 was well conducted. However, only data for the lowest dose (100 µg/mL) was considered, as the highest dose (200 µg/mL) exceeded the concentration recommended in the OECD guidelines for testing nanomaterials and was therefore discounted. Despite these reservations, the study was given a RAG rating of green. No information was available from this study to aid in the understanding of any mechanism of action. No cellular uptake was reported.

Overall, test 2 indicated a 1.5-fold increase in MN formation which is not considered to be biologically relevant, hence the study was considered negative. A 2-fold increase was observed at the highest dose, which was discounted due to it being higher than OECD guidelines. This study was cited in the EFSA review (EFSA, 2021).

Unal and others (2021)

Unal and others (2021) conducted a CBMN assay with cytoB and a chromosomal aberration (CA) assay (see below) in human peripheral blood lymphocytes (HPBLs) isolated from whole blood from 3 non-smoking females. The cells (number not given) were exposed to TiO₂ nanopowder ((CAS 13463-67-7; rutile/anatase) at concentrations of 20, 40, 60, 80 or 100 µg/mL for 48 hours and cytoB was added after 44 hours.

The primary size of TiO₂ nanopowder was 10 to 360nm but secondary size was not stated. Stock nanoparticle suspensions were ultrasonicated in distilled water, diluted to final concentrations and then sonicated again. Three thousand binucleated cells were analysed for MN. Mitomycin C (0.2 µg/ml) was used as a positive control. Cytotoxicity was measured by CBPI (called Nuclear Division Index (NDI)).

There was no change in the NDI. The MN frequency in the negative control was 0.13% and the positive control was 20-fold higher than the background.

Following treatment with TiO₂, no significant increases in MN formation were observed at any dose. There was no evidence of uptake of nanoparticles.

COM opinion

This study was regarded to be good quality, although some of the study details are unclear, namely whether PHA was used to induce cell division (but as the positive control shows a 20-fold increase over background, it is assumed that it was). There is no evidence of uptake. Despite these reservations, the study was given a RAG rating of green.

Further experiments in this study included a Comet assay that showed increased DNA damage only at the highest concentration, which did not result result in fixed genetic damage (MN).

Overall, no increase in MN formation was detected and the study was considered negative. This study was identified during the recent literature search.

‘Green’ papers – hprt assay

Kazimirova and others (2020)

Kazimirova and others (2020) conducted a hprt forward mutation test in V79 cells according to OECD TG476. 1 x 106 cells/dish were exposed to an anatase/rutile TiO₂ nanoparticles mix at concentrations of 3, 5 or 75 µg/cm2 for 24 hours. After 24 hours, cells were washed and reseeded for 8 days, after which they were replated with 6-TG to detect mutation frequency.

The primary size of TiO₂ nanoparticles was 21nm (15 to 60nm) and secondary size was 228 ± 3.2nm as measured using dispersion procedure (DP) 1, and 184 ± 3.5nm using DP2. For DP1, the samples were prepared by ultrasonication in PBS and 10% FBS, and in DP2 samples were suspended in culture medium with HEPES buffer without FBS, and sonicated. Methyl methanesulphonate (MMS; 0.1 mM) was used as a positive control. Cytotoxicity was measured by determining plating efficiency (PE).

There were no substantial effects of exposure time (24, 48 and 72 hours), on cytotoxicity, although a tendency for a lower Relative Growth Activity at longer exposure was observed. No significant difference in PE values were seen. The hprt mutation frequency in the negative control was approximately 2 x 105 and the positive control was approximately 6-fold higher than background.

Following treatment with TiO₂, there was no evidence of increased hprt mutation frequency in cells treated with TiO₂ in spite of evidence of uptake of nanoparticles by cells.

COM opinion

This was considered to be a well-conducted assay using robust methodology and the study was given a RAG rating of green.

Overall, no evidence of increased mutation was seen and it was considered negative. This study was cited in the EFSA review (EFSA, 2021).

‘Green’ papers – CA assay

Unal and others (2021)

Unal and others (2021) conducted a CBMN assay with cytoB (see next paragraph) and a CA assay in HPBLs isolated from whole blood from 3 non-smoking females. The cells (number not given) were exposed to TiO₂ nanopowder (no further information given) at concentrations of 20, 40, 60, 80 or 100 µg/mL for 24 and 48 hours, with colchicine added 2 hours before harvest.

Three hundred metaphases per treatment were analysed. Mitomycin C (0.2 µg/mL) was used as a positive control. Cytotoxicity was measured by Mitotic Index (MI) in the CA assay.

At 24 hours the MI was significantly lower at 60 and 80 µg/ml (reduced by 20% at both concentrations compared to a negative control) but was still within the range of control values. There was no evidence of cytotoxicity at 48 hours. The CA in the negative control at both 24 and 48 hours was 1.3%, excluding gaps, and the positive control was 17- and 13-fold higher than the background, respectively.

Following treatment with TiO₂, a significant increase in CA was observed at all doses at 24 hours (3.6- to 5.6-fold increase over background), At 48 hours, similar results were obtained, but were significant only at 20, 80 and 100 µg/mL (3.6-, 2.9- and 2.9-fold increase). There was no evidence of uptake of nanoparticles.

COM opinion

This study was regarded to be good quality, although some of the study details are unclear, namely whether PHA was used to induce cell division (but as the positive control shows a 20-fold increase over background, it is assumed that it was). No dose response was observed as CA frequency decreased with increasing TiO₂ concentration. There was no evidence of uptake. Despite these reservations, the study was given a RAG rating of green.

Further experiments in this study included a Comet assay, which showed increased DNA damage only at the highest concentration.

Overall, the study was considered positive as there was a significant increase in CA at all doses at 24 hours, although there was no positive dose response as CA frequency decreased with increasing TiO₂ concentration. At 48 hours similar results were obtained but were significant only at 20, 80 and 100 µg/mL. This study was identified during the recent literature search.

‘Amber’ papers – MN assay

Kazimirova and others (2019)

Kazimirova and others (2019) conducted a CBMN assay in TK6 lympoblastoid cells and lymphocytes from human volunteers.

2 x 105 TK6 cells were treated with 3, 15 or 75 µg/cm² anatase/rutile TiO₂ nanoparticle mix for 4 or 24 hours. CytoB was added for the last 24 hours prior to harvesting, resulting in total treatment times of 28 or 48 hours.

Fourteen volunteers also participated in the study (ages 40 to 50 years old, 8 women and 6 men) but were not considered further.

The primary size of the anatase/rutile nanoparticle mix was 21nm (15 to 60nm) with a bimodal distribution of 102 ± 15nm and 285 ± 67nm when dispersed by ultrasonication in PBS and FBS.

2,000 binucleated cells per concentration were analysed for MN formation in 2 independent experiments. Micronuclei in 1,000 mononucleated cells per concentration were also analysed. Mitomycin C (0.3µM) was used as the positive control. For cytotoxicity assessment, CBPI was calculated.

The number of MN in the negative control (background) was 1.5 after 28 hours (4 hour treatment) and 4 after 48 hours (24 hour treatment), corresponding to frequencies of 0.075% and 0.2 %, respectively, and the positive control induced a level approximately 24-fold higher than the background.

Following treatment with TiO₂, no cytotoxicity and no increases in MN frequency were detected.

COM opinion

This study showed acceptable methodology. However, only the 24 hour treatment time was considered appropriate as the 4 hour treatment time followed by the addition of cytoB for 24 hours was considered a co-treatment. Data showed large variability (even when expressed as SEM of duplicates) which suggests a lack of reproducibility, and TK6 cells had high background MN levels at 48 hours. Due to these observations, the study was given a RAG rating of amber.

Overall, no increase in MN formation was detected and the study was considered negative. This study was cited in the EFSA review (EFSA, 2021).

Kurzawa-Zegota and others (2017)

Kurzawa-Zegota and others (2017) conducted a CBMN assay with cytoB in HPBLs. The blood lymphocytes were collected from 3 different cohorts of patients: group 1, healthy patients (n=20); group 2, polyposis coli patients (n=19); group 3, colon cancer patients (n=20). For the purpose of this review, only data from healthy volunteers is considered. 400 µL whole blood was incubated with PHA for 24 hours, then exposed to anatase titanium nanoparticles at concentrations of 10, 40 or 80 µg/mL for 20 hours, after which time cytoB was added (at 44 hours). Cells were harvested at 72 hours.

The primary size of TiO₂ nanoparticles was <25nm and the secondary size increased with time and dose and varied with the media used, ranging from 104.2nm in water to 1,303nm in RPMI. The samples were prepared by ultrasonication in water, PBS or RPMI 1,640 (although it is unclear which preparation was used for cell treatment). Two thousand binucleated cells were analysed for MN formation. Cytotoxicity was determined by CBPI. Mitomycin C (0.4µM) was used as a positive control.

No cytotoxicity was observed in any cohort. The frequency of MN in the negative control for group 1 was 0.36. The positive control was 11-fold higher than the negative control. No uptake was reported.

Following treatment with TiO₂, group 1 had significantly increased MN formation at the highest dose (6-fold increase over background).

COM opinion

The approach used in the study appears to be relevant. However, there was insufficient information on the population used, it was unclear how the nanoparticles were suspended for the MN assay, and there was no direct evidence of uptake. Due to these observations, the study was given a RAG rating of amber.

There was, additionally, a significant increase in DNA strand breaks detected by Comet assay in group 1 and dose-related increases in group 2 and especially in group 3. In a MN-FISH assay, there was a dose-related increase particularly for MN without centromeres in all groups.

Overall, this study indicated that TiO₂ significantly increased MN formation in a range of human lymphocytes and was considered positive. This study was cited in the EFSA review (EFSA, 2021).

Osman and others (2018)

Osman and others (2018) conducted a CBMN assay with cytoB in HPBLs from healthy volunteers and from a group of volunteers with respiratory disease. For the purpose of this review, only data from healthy volunteers is considered. The cells (number not given) were exposed to PHA for 24 hours and then treated with anatase nanoparticles at a concentration of 5 and 10 µg/mL for 20 hours. CytoB was added at 44 hours and cells were harvested at 72 hours.

The primary size of TiO₂ nanoparticles was 40 to 70nm but the secondary size was not determined. The samples were prepared by suspension in dimethyl sulfoxide (DMSO) filtration through a 0.2µm syringe filter and then sonicated. One thousand binucleated cells were analysed for MN formation. Mitomycin C (0.4µM) was used as a positive control. Cytotoxicity was determined by NDI.

No cytotoxicity was observed. The MN frequency in the negative control was 0.2% and the positive control showed a 15-fold increase over background in the healthy group.

No significant increase in MN formation was seen in the healthy controls when exposed to either of the TiO₂ doses. No cellular uptake data was stated.

COM opinion

This study focussed on the comparison of DNA damage seen in HPBLs from healthy controls and in those from patients with lung disease following exposure of the cells to TiO₂. Only data from healthy individuals were considered in this report. The experimental data and details of the TiO₂ nanoparticles used were limited, only 1000 binucleated cells were counted, only two concentrations of TiO₂ were examined, and there was no analysis of uptake. Due to these observations, the study was given a RAG rating of amber.

Overall, no evidence of genotoxicity was seen in healthy volunteers and the study was considered negative. This study was cited in the EFSA review (EFSA, 2021).

Prasad and others (2013)

Prasad and others (2013) conducted a CBMN assay with cytoB in BEAS-2B cells. 5 x 104 cells/cm² were treated with TiO₂ in the different media at doses of 10, 20, 50 or 100 µg/ml for 24 hours, after which cytoB was added for 18 hours.

TiO₂ nanoparticles (86% anatase and 14% rutile; primary particle size 27.5nm (range 14.2 to 64.6nm) were prepared in 3 different media that have been previously used in TiO₂ genotoxicity studies: keratinocyte growth media (KGM) supplemented with 0.1% BSA (KB); a medium that mimics BAL by containing phosphate buffered saline (PBS) supplemented with 0.6% BSA and 0.001% surfactant (referred to as dispersion medium (DM)); or KGM media supplemented with 10% foetal bovine media (KF). The nanoparticles were probe sonicated for dispersal. One thousand binucleated cells were analysed for MN formation in 2 independent experiments (total of 2,000 binucleated cells/concentration). MMS (100µM) was used as a positive control. Cytotoxicity was measured by CBPI.

The MN frequency in the negative control was 2 to 2.2% and the positive control showed a 3.5-fold increase over background. There did not appear to be cytotoxicity at any tested concentration.

Following treatment with TiO₂, only TiO₂ prepared in KF medium gave a dose-related significant increase in the formation of MN. This medium was considered by the authors to be that which facilitated the lowest amount of particle agglomeration, the greatest amount of nanoparticle cellular interaction, and the highest population of cells accumulating in S phase.

COM opinion

The study design and conduct were considered reasonable. However, BEAS-2B is a non-standard cell line and is not recommended by OECD. The doubling time of the cells is 26 hours. There was a high background frequency of MN (>2%), which was considered to be at the upper limit of acceptability. Only TiO₂ prepared in KF media gave a dose-related increase in MN formation. While this was statistically significant, the level of induction was very low, increasing from approximately 2% (background) to 2.8% at 20 µg/mL, 3% at 50 µg/ml and 3.8% at 100 µg/mL (maximum 1.7-fold increase). The low incidence of MN may be due to the exposure duration being less than one cell cycle, rather than 1.5 to 2 cycles as recommended by OECD. There was no firm evidence of cellular uptake, although the method used was not appropriate for uptake analysis. Due to these observations, the study was given a RAG rating of amber.

A Comet assay was also conducted and this was only significantly positive for DNA damage in KB media at the highest dose, although the increase over background was also very low despite being statistically significant (2% (background) increasing to approximately 3.5% at 100 µg/ml).

Overall, due to the level of MN induction being low (<2-fold), the study was considered to be negative. This study was cited in the EFSA review (EFSA, 2021).

Stoccoro and others, (2016 and 2017)

CBMN formation, with cytoB, was studied in mouse BALB/3T3 cells (Stoccoro and others, 2016) and human A549 cells (Stoccoro and others, 2017). TiO₂ nanoparticles (84% anatase and 16% brookite crystal phase) were treated in different ways: pristine (uncoated), silicate coated, or sodium citrate coated. Aeroxide P25 (a fine-particulate pure TiO₂) was used as a benchmark material.

7.5 x 105 BALB/3T3 cells were treated with anatase at concentrations of 10, 20 or 40 µg/cm² (32, 64, 128 µg/mL) for 48 hours. CytoB was added after 44 hours and the cells were harvested after 72 hours.

The primary sizes of the TiO₂ nanoparticles were 83.5, 57.5 or 155.6nm (uncoated, citrate coated or silicate coated, respectively) and secondary sizes were 1608, 68.3 or 563.2nm (uncoated, citrated or silicate coated, respectively) (Stoccoro and others, 2016).

7.5 x 104 A549 cells were treated with anatase at concentrations of 10, 20, 40 µg/cm² (32, 64, 128 µg/mL) for 48 hours. CytoB was added after 44 hours and cells were harvested after 72 hours.

The primary sizes of the TiO₂ nanoparticles were 1608, 91.3 and 563.2nm (uncoated, citrated and silicate coated, respectively) (Stoccoro and others, 2017).

A549 cells were dispersed in complete cell culture medium while citrate and 0.05% BSA were used for dispersion of BALB/3T3 cells. A total of 1,000 binucleated cells were analysed for MN formation in each assay. Mitomycin C (0.1 µg/ml) was used as a positive control. Cell cytotoxicity was assessed by CBPI. TEM was used to evaluate cytoplasmic uptake.

Cytotoxicity was <20% in BALB/3T3 cells and A549 cells, apart from citrate-coated nanoparticles which reduced cytotoxicity to approximately 50%. The MN frequency in the negative control was 1% in both cell lines; the positive control was 60-fold higher than background in A549 cells and 5-fold higher in BALB/3T3 cells.

Following treatment with TiO₂, in BALB/3T3 cells an increase in the formation of MN was only observed in citrate-coated TiO₂ nanoparticles at the highest dose (3% compared to 1% negative control), while the assay was negative for uncoated and silica-coated nanoparticles. In A549 cells, however, formation of MN was increased with all treated nanoparticles (except for the lowest dose of silicate-coated TiO₂). A dose-dependent increase in MN (2%, 3% and 4%) was observed with 32, 64 and128 µg/mL, respectively, compared with 1% for the negative control.

COM opinion

These combined studies were considered to be robust. However, BALB/3T3 cells and A549 cells are non-standard cell lines and are not recommended by OECD, but background MN frequencies were in line with that seen in OECD-recommended cells. The doubling time of BALB/3T3 cells is 18 hours and A549 cells is >22 hours. Only 1,000 binucleated cells were counted and the fold-increase with the positive control in A549 cells was high. Due to these observations, the study was given a RAG rating of amber.

A Comet assay with Fpg in BALB/3T3 cells suggested reactive oxygen species (ROS) involvement. The Comet assay with Endo III and Fpg in A549 cells also showed oxidative DNA damage. Fluorescence in situ hybridization (FISH) analysis suggested possible aneuploidy in MN with TiO₂ (but very weak) and all nanoparticles increased DNA methylation.

In BALB/3T3 cells, positive results for MN formation were only detected in citrate-coated nanoparticles. In A549 cells, increased formation of MN was seen with all treated nanoparticles and was considered positive. Overall, these studies showed increased formation of MN and were considered positive. These studies were cited in the EFSA review (EFSA, 2021).

Vales and others (2015)

Vales and others (2015) conducted a 4-week chronic CBMN assay with cytoB in BEAS-2B cells. 5.5 x 105 cells were exposed to NM-102 (anatase) at concentrations of 1, 10 or 20 µg/mL for up to 3 weeks. TiO₂ was added every 4 days and cells were subcultured weekly. For MN analysis, cells were harvested after 48 hours, 1 or 3 weeks following cytoB co-treatment (no further details were available).

The primary size of TiO₂ nanoparticles was 21.7nm and secondary size was 575.9nm. The samples were prepared by pre-wetting in ethanol, dispersed in 0.05% BSA and sonicated. A total of 2,000 binucleated cells were analysed for MN. Mitomycin C (150 ng/ml) was used as a positive control. Transmission electron microscopy (TEM) was used to detect cellular uptake into the vacuoles and the nuclear surface. Cytotoxicity was detected by CBPI.

The MN frequency in the negative control was 1.7% at 48 hours and 1 week, and 1.2% at 3 weeks; the positive control was 5-fold higher than background at 48 hours and 3 weeks, and 3-fold higher after 1 week.

Following treatment with TiO₂, there was no increase in MN formation at 48 hours, 1 week or 3 weeks.

COM opinion

This is a good quality study which was reasonably well-conducted. BEAS-2B is a non-standard cell line and is not recommended by OECD, but background MN frequency was in line with that seen in OECD-recommended cells. The doubling time of these cells is 26 hours. As well as the standard short-term exposure, non-standard long-term exposures (1 and 3 weeks) were used. Due to these observations, the study was given a RAG rating of amber.

Further studies reported in the paper (intracellular ROS measurement) showed no induction of ROS.

Overall, the results showed no evidence of a dose response, and only small, inconsistent increases, that were not biologically relevant, were observed; hence the study was considered negative. This study was identified during the recent literature search.

‘Amber’ papers – hprt assay

Vital and others (2022)

Vital and others (2022) conducted a hprt assay with cytoB in V79 cells according to OECD TG476. 3 x 105 cells were exposed to NM-100 (anatase) at concentrations of 1, 3, 10, 30, and 75 µg/cm³ for 24 hours.

The primary size was 110nm and the secondary size was 256.7 to 341.3nm in culture media at 0 hours and 218.0 to 260.6nm at 24 hours. The samples were prepared using the Nanogenotox dispersion protocol (no further details provided) in which a stock suspension of 2.56 mg/ml NM-100 was sonicated. MMS (0.1 mM) was used as a positive control. Cytotoxicity was assessed using plating efficiency.

In the hprt assay, cytotoxicity was 30% at the highest concentration. The hprt mutation frequency in the negative control was 9.59 x 10^-6 and the positive control was 4-fold higher than the background.

Following treatment with TiO₂, no evidence of uptake was reported. Significantly increased numbers of mutations were detected at low doses (1, 3, 10 µg/cm³), but increases were not significant at higher doses.

COM opinion

The study was of good design. However, the plating efficiency was 40% at the lowest concentration and 30% at the highest concentration. No uptake data was reported. Due to these observations, the study was given a RAG rating of amber.

Overall, there were some significant increases in hprt mutations at the lower doses but not the higher doses (lack of dose-response relationship) but overall, the study was considered positive. This study was identified during the recent literature search.

Summary

Following the screening of papers cited in the EFSA opinion (EFSA, 2021) as described in the methodology section, and a further assessment of newer literature (2021 to 2023; Annexe 1), 15 papers, reporting 16 assays, were identified to be of sufficient quality to warrant further assessment.

Regarding the in vitro genotoxicity of TiO₂, the studies considered to be of sufficient quality included 3 genotoxicity assays, namely CBMN (green = 5; amber = 8), hprt (green = 1; amber = 1) and CA (green = 1; amber = 0), all of which are recognised by the OECD and other international regulatory bodies. Several of the papers also outlined non-regulatory experiments on the role of oxidative stress and DNA interactions which may aid insight into mechanisms of action.

An overall summary of the data is presented in Table 4 and results of each study are summarised in tables 5 to 9.

Of the 6 green studies deemed to have used robust methodology only one reported an increase in MN frequency, in BEAS-2B cells following treatment with coated rutile TiO₂ (Di Bucchianico and others, 2017). The same study also reported negative results with coated and uncoated anatase. All other ‘green’ studies showed no increase in MN frequency following TiO₂ treatment in various cell lines or HPBLs. Kazimirova and others (2020) also reported no increase in hprt mutations in V79 cells following treatment with an anatase/rutile mixture, although Unal and others (2021) reported an increase in CA in HPBLs following treatment with TiO₂ nanopowder.

From the ‘amber’ papers, an increase in MN frequency was reported in HPBLs following treatment with anatase TiO2 (Kurzawa-Zegota and others, 2017), and in BALB/3T3 cells following treatment with a citrate coated anatase/brookite mix (Stoccoro and others, 2016) and in A549 cells with uncoated, citrate coated and silica coated anatase/brookite mix (Stoccoro and others, 2017). All other ‘amber’ studies showed no increase in MN frequency following TiO₂ treatment in various cell lines or HPBLs. Vital and others (2022) showed an increase in hprt mutations in V79 cells following anatase treatment.

Table 5. Summary of the ‘green’ MN results

Test material	Primary size	Concentration	Cell type	OECD recommended cells	Result	Reference
Anatase Rutile Mixture	20 to 60nm 30 x 100nm 45 to 252nm	10 to 200 µg/mL (200 µg/mL data excluded from interpretation [note 1])	PBMCs	Yes	Neg	Andreoli and others (2018)
Anatase	21 or 50nm	10 to 100 µg/mL (1,000 µg/mL data excluded from interpretation [note 1])	HEK293 NIH/3T3	No	Neg	Demir and others (2015)
Uncoated anatase (NM-100) Coated anatase (NM-101) Coated rutile (NM-103)	50 to 150nm 5 to 8nm 20 to 28nm	1 to 15 µg/mL	BEAS-2B	No	Neg (uncoated anatase) Neg (coated anatase) Pos (coated rutile) (parallel Comet assay assessment +/-fpg was suggestive of oxidative damage)	Di Bucchianico and others (2017)
Anatase	8.9 to 15.3nm	100 µg/mL (200 µg/mL data excluded from interpretation [note 1])	TK6	Yes	Neg	Li and others (2017)
Rutile/anatase nanopowder	10 to 360nm	20 to 100 µg/mL	HPBLs	Yes	Neg	Unal and others (2021)

Note 1: higher doses excluded from interpretation as they exceeded those recommended in OECD test guidelines.

Table 6. Summary of the ‘green’ hprt results

Test material	Primary size	Concentration	Cell type	OECD recommended cells	Result	Reference
Anatase/rutile mix	21nm	3 to 75 µg/cm2	V79	Yes	Neg	Kazimirova and others (2020)

Table 7. Summary of the ‘green’ CA results

Test material	Primary size	Concentration	Cell type	OECD recommended cells	Result	Reference
Rutile/anatase nanopowder	10 to 360nm	20 to 100 µg/mL	HPBLs	Yes	Pos	Unal and others (2021)

Table 8. Summary of the ‘amber’ MN results

Test material	Primary size	Concentration	Cell type	OECD recommended cells	Result	Reference
Anatase/rutile mix	21nm (15 to 60nm)	3, 15 or 75 µg/cm2	TK6	No	Neg	Kazimirova and others (2019)
Anatase	<25nm	10 to 80 µg/mL	HPBLs	Yes	Pos	Kurzawa-Zegota and others (2017)
Anatase	40 to 70nm	10 to 100 µg/mL	HPBLs	Yes	Neg	Osman and others (2018)
86% anatase, 14 % rutile	27.5nm	10 to 100 µg/mL	BEAS-2B	No	Neg	Prasad and others (2013)
84% anatase, 16% brookite Pristine (uncoated) Silicate coated Sodium citrate coated	83.5nm 57.5nm 155.6nm	32 to 128 µg/mL	BALB/3T3	No	Neg (uncoated) Pos (citrate coated) (Comet assay with Fpg suggested ROS involvement) Neg (silicate coated)	Stoccoro and others (2016)
84% anatase, 16% brookite Pristine (uncoated) Silicate coated Sodium citrate coated	83.5nm 57.5nm 155.6nm	32 to 128 µg/mL	A549	Yes, but not extensively validated	Pos (uncoated) Pos (citrate coated) (Comet with Endo III and Fpg in showed oxidative DNA damage. (FISH analysis suggested possible aneuploidy in MN with TiO2 (but very weak) and all nanoparticles increased DNA methylation) Pos (silicate coated)	Stoccoro and others, (2017)
NM-102 (anatase)	21.7nm	1 to 20 µg/mL	BEAS-2B	No	Neg	Vales and others (2015)
NM-102 (anatase) NM-103 (rutile, coated with hydrophobic Al) NM-105 (81.5% anatase and 18.5% rutile mix)	22 to 30nm	0.14 to 14 µg/mL	Caco-2 H29-MTX-E12	Yes, but not extensively validated	Neg	Vieira and others (2022)

Table 9. Summary of the ‘amber’ hprt results

Test material	Primary size	Concentration	Cell type	OECD recommended cells	Result	Reference
NM-100 (anatase)	110nm	1 to 75 µg/cm3	V79	Yes	Pos	Vital and others (2022)

Abbreviations

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

References

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*[RPMI is Roswell Park Memorial Institute 1640 Medium.RPMI is Roswell Park Memorial Institute 1640 Medium. *[SD]: Sprague-Dawley *[SOD]: superoxide dismutase *[TEM]: transmission electron microscopy *[TK]: toxicokinetic *[UF]: ultrafine