Titanium dioxide

Neurotoxicity

Description of key information

Long, T.C. et al. (2006, 2007): Investigation of intake and cellular changes in BV2 microglia, N27 neurons and primary culture of rat striatal cells after TiO2 exposure

Hougaard et al. (2010, 2011): Investigation of neurobehaviour, lung effects and fertility in mice after TiO2 inhalation exposure.

Younse et al. (2015): Investigation of neurobehaviour, histopathology and clinical chemistry parameter in rats after intraperitoneal TiO2 exposure.

Balvay, A. et al. (2013): Investigation of motorperformance and histopathology in mice after intracerebroventricular administration of TiO2.

Disdier, C. et al. (2017): Brain inflammation, blood brain barrier dysfunction and neuronal synaptophysin decrease after inhalation exposure to titanium dioxide nano-aerosol in aging rats

No conclusion can be drawn from the 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 section represent in vitro and in vivo experiments with investigations on neurotoxicity 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.

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

neurotoxicity: inhalation

Remarks:

developmental

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The reference shows significant methodological deficiencies: No. Of litters too low (13-14/dose; recommended 20 litters/dose); one concentration only; exposure during lactation missing; behavioural investigation was performed during light period (nocturnal animals); food consumption of dams not measured; data on clinical signs of dams not reported; clinical signs and detailed clinical observations of offspring missing; neuropathology of offspring missing; motor activity testing too short and missing for the pre-weaning period; Motor and sensory function measured only in adults ( 4 months old; recommeded 60 - 70 PND); learning and memory testing in adolescence missing; bw of weanlings missing Due to the above given arguments this reference is of no relevance for risk assessment purposes.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Time-mated mice (C57BL/6BomTac) were exposed by inhalation 1h/day to 42 mg/m³ aerosolized powder (1.7·106 n/cm³; peak-size: 97 nm) on gestation days 8-18. Endpoints included: maternal lung inflammation; gestational and litter parameters; offspring neurofunction and fertility.

FOLLOW-UP STUDY (Kyjovska et al. (2013))
The authors investigated the influence of maternal airway exposure to nanoparticulate titanium dioxide (TiO2, UV-Titan) on male reproductive function in the two following generations. Time-mated C57BL/6J mice were exposed by inhalation to UV-Titan. Body and testicle weight, sperm content per grams testicular parenchyma and daily sperm production (DSP) were assessed. The protocol for assessment of DSP was optimized for application in mice (C57BL/6J) and the influence of different parameters was studied.

GLP compliance:

not specified

Limit test:

no

Species:

mouse

Strain:

other: C57BL/6BomTac

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Europe, Ejby, Denmark
- Housing: grouped 5 or 6 in polypropylene cages with bedding and enrichment (removed during nursing). After exposure on gestational day 18, females were singly housed.
- Diet (ad libitum): Altromin 1324
- Water (ad libitum): tap water

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: dust

Vehicle:

clean air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: animals were placed separately in rooms of a "twelve-room-pie"; a cylindrical wire mesh cage (29 cm, height 9 cm) with radical partitions.
- System of generating particulates/aerosols: a microfeeder aerosolized powder particles through a dispersion nozzle at a pressure of 5 bar (Fraunhofer Institute für Toxicologie und Aerosolforschung, Hannover, Germany).
- Air flow rate: airflow in the exposure chamber was dynamic (20 L/min) with evenly distributed exposure atmosphere.
- Method of particle size determination: particle number and size distribution in the exposure atmosphere were monitored using a GRIMM Sequential
(Stepping) Mobility Particle Sizer (SMPS) system for sub-μm particles (12.8 to 486 nm; based on the rutile density of 4.25 g/cm3 [16]) and a GRIMM Dustmonitor (Model 1.106) for coarse particles (0.75 to > 15 μm). The SMPS consisted of a Long Electrostatic Classifier (Model No. 5.521) and a GRIMM Condensation Particle Counter (Model 5.400). The time resolution was 218 and 6 s for the SMPS and Dustmonitor data, respectively.

TEST ATMOSPHERE
- Brief description of analytical method used: mass-concentrations of total suspended dust was controlled periodically by filter sampling and adjusted to maintain a concentration of ~40 mg/m^3. Exposure air was sampled on pre-weighed Millipore Fluoropore filters (2.5 cm; pore size 0.45 μm) at an airflow of 2 L/min using Millipore cassettes, for 10 minutes. Filters were weighed immediately on a Sartorius Microscale (Type M3P 000V001). Final gravimetric data were obtained on acclimatized filters (50%RH and 20°C).
The particle number concentration in the exposure atmosphere was 1.70 ± 0.20·10^6/cm^3. The major particle size-mode was ~100 nm (geometric mean number diameter 97 nm), with a coarser size mode at ~4 μm. Smaller size modes were observed at ~20 nm and 1 μm. By number, 80% of the particles were between 40 and 200 nm and no particles were coarser than 12.5 μm detected. The mass-size distribution was strongly dominated by μm-size particles (geometric mean 3.2 μm) and 75% of the mass were represented by particles larger than 1.6 μm. The fraction of sub-100-nm-size particles amounted to 1% of the mass.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Please refer to the field "Details on exposure" above.

Duration of treatment / exposure:

Gestational day 8 to 18

Frequency of treatment:

1 hour/day

Remarks:

Doses / Concentrations:
42.4 ± 2.9 mg/m³ UV-Titan
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
40 mg UV-Titan/m³
Basis:
nominal conc.

No. of animals per sex per dose:

22 - 23 mice

Control animals:

yes, concurrent vehicle

Details on study design:

At 19 weeks of age, control and exposed offspring F1 were cross-mated to naïve CBA/J mice (12 weeks old; Charles River Wiga, Sulzfeld, Germany) and time-to-first-delivery of F2 litter, litter size, and gender ratio were recorded. A follow-up study was conducted with the F1 generation and F2 generation as described below.

FOLLOW-UP STUDY (Kyjovska et al. (2013))
At 19 weeks of age, control and exposed offspring F1 (males: 25; females: 25) were cross-mated to naïve CBA/J mice (12 weeks old; Charles River Wiga, Sulzfeld, Germany). One F1 male (C57BL/6J) per litter cohabited with one naïve CBA/J female mice to produce the F2 litters of the mixed (CBA/J)/(C57BL/6J) strain. Likewise, one F1 female C57BL/6J per litter cohabited with one naïve CBA/J male mouse to produce F2 litters of the mixed (C57BL/6J)/(CBA/J) strain.

Observations and clinical examinations performed and frequency:

MATERNAL EXAMINATION
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: females were observed for signs of toxicity and returned to cages less than 5 minutes after exposure.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: body weight was recorded before exposure on gestational days 9, 11, 14, and 18 as well as on postnatal day 1, 8, 11, 16, 19, and 22.

FOOD CONSUMPTION AND COMPOUND INTAKE: No data
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE: No data

OVARIES AND UTERINE CONTENT
- Gravid uterus weight: No data
- Number of corpora lutea: No data
- Number of uterine implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data

Neurobehavioural examinations performed and frequency:

PUP EXAMINATION:
- At weaning (postnatal day 22), one male and female per litter were randomly chosen for behavioural testing
Learning and memory was tested in the Morris water maze at age 11 and 15 weeks (males), and 12 and 16 weeks (females) as described in Hougaard et al. (2008)* with minor modifications. Activity was assessed for 3 minutes at 14 weeks of age in an open field using the dry water maze pool. Acoustic startle reaction (ASR) and prepulse inhibition (PPI) were tested at 4 months as described in Hougaard et al. (2005)* in two chambers (San Diego Instruments, San Diego, USA) with 70 dB(A) white background noise.

*References:
- Hougaard KS, Jensen KA, Nordly P, Taxvig C, Vogel U, Saber AT, Wallin H: Effects of prenatal exposure to diesel exhaust particles on postnatal development, behavior, genotoxicity and inflammation in mice. Part Fibre Toxicol 2008, 5(3):3.
- Hougaard KS, Andersen MB, Hansen AM, Hass U, Werge T, Lund SP: Effects of prenatal exposure to chronic mild stress and toluene in rats. Neurotoxicol Teratol 2005, 27:153-167.

Sacrifice and (histo)pathology:

MATERNAL EXAMINATION
- Sacrifice:
Non-pregnant time-mated females without implantations ("NP females") were euthanized on postnatal day 3 (i.e. 5 days post exposure) and subjected to bronchoalveolar lavage (BAL), as were dams with litters at postnatal days 24-25 ("P females"; 26-27 days post exposure). BAL cell composition and neutrophil influx was used to indicate lung inflammation. BAL was performed four times with 0.8 mL 0.9% sterile saline (Saber et al., 2005)*. The total number of cells and of dead cells in BAL samples was determined in cell suspension B by Nucleo-Counter. Differential counts of macrophages, neutrophils, lymphocytes, eosinophils, and epithelial cells were determined by counting 200 cells in cell supernatant fixed with 96% ethanol and stained with May-Grünwald-Giemsa stain. Total number of cells was calculated by combining data from differential cell counts with the total number of cells in BAL.
Approximately 25-75 mg tissue of the lung and liver were weighed and analysed for content of titanium (Ti). Maternal lung was included to determine remaining TiO2 and liver to assess systemic distribution in adults [Sadauskas et al., 2007, van Ravenzwaay et al., 2009)*. Tissues were digested and Ti content determined by quadrupole-
based inductively coupled plasma mass spectrometer (ICPMS 7500ce, Agilent Technologies, Tokyo, Japan) equipped with a collision/reaction cell (CRC). The limit of detection (LOD) for Ti in tissues, based on three times the standard deviation of repeated blank measurements, was estimated to be 0.2-5 mg/kg depending on sample intake and dilution.

*Reference
- Sadauskas E, Wallin H, Stoltenberg M, Vogel U, Doering P, Larsen A, Danscher G: Kupffer cells are central in the removal of nanoparticles from the organism. Part Fibre Toxicol 2007, 4(10):10.
- van Ravenzwaay B, Landsiedel R, Fabian E, Burkhardt S, Strauss V, Ma-Hock L: Comparing fate and effects of three particles of different surface properties: nano-TiO(2), pigmentary TiO(2) and quartz. Toxicol Lett 2009, 186:152-159.
- Saber AT, Bornholdt J, Dybdahl M, Sharma AK, Loft S, Vogel U, Wallin H: Tumor necrosis factor is not required for particle-induced genotoxicity and pulmonary inflammation. Arch Toxicol 2005, 79:177-182.

Statistics:

Gestational parameters were analysed by Mann-Whitney U-test, and time-to-first-delivery by log rank test (separately by gender). ANOVAs were applied to the remaining data when relevant with repeated measures in trials, days, or timebins. ANCOVA controlled for litter size in the analyses of weight gain during exposure, birth weights, and pre-weaning pup weights. Behavioral data were analysed by two-way ANOVA. Pairwise comparisons were performed by T-test or Mann Whitney U-test (p < 0.1). Analyses were performed in SYSTAT Software Package 9, MINITAB 14, and SAS 9.1.

FOLLOW-UP STUDY (Kyjovska et al. (2013))
Data were analysed by analysis of variance (ANOVA). Correlation analysis was used to determine Pearson correlation coefficient (variation between two halves
from one testicle, left and right testicle, correlation between sperm content and time-to-first F2 litter). All offspring in the F2 generation were a combination of two strains, i.e. C57BL/6J and CBA/J, but only the C57BL/6J allele had been exposed. The genetic background of the F2 generation therefore differed, depending on whether the F2 males were offspring of the prenatally exposed F1 males where the father was of the C57BL/6J strain ((CBA/J)/(C57BL/6J), i.e. paternal F2 males, PF2) or offspring of the prenatally exposed F1 females, where the mothers were of the C57BL/6J strain ((C57BL/6J)/(CBA/J), i.e. maternal F2 males, MF2). In analysis of the effects of nanoparticle exposure in the F2 generation, two ways ANOVA was therefore applied, with the factors of exposure (of the P generation) and gender (of the prenatally exposed F1 offspring). Time to delivery of first F2 litter in the CB study was assessed by log rank test. Data are given as mean
± SEM. P-values < 0.05 were considered statistically significant, 0.05 < P < 0.01 indicates a tendency to departure from the null-hypothesis. Statistical calculations were carried out using SYSTAT 9 (log rank test by SAS 9.2).

Description (incidence and severity):

Migrated information from 'Further observations for developmental neurotoxicity study'

Details on results (for developmental neurotoxicity):DETAILS ON EMBRYOTOXIC/TERATOGENIC EFFECTS
- values for titanium concentration in liver and milk were similar between control and exposed animals for all other samples.
- litter parameters were similar, apart from a slight decrease in pup viability in TiO2 litters (p = 0.083)
- no effects related to exposure were detected for offspring organ weights.
- in the Morris water maze, no change was observed in performance as a result of prenatal TiO2 exposure in either male or female offspring
- in the open field, ambulation differed by gender (p < 0.001) but not exposure, as females moved approximately 50% longer than males. Prenatally exposed
animals spent significantly less time than controls in the central zone of the field (p = 0.009), and visited the central zone less frequently (Exposure: p = 0.056; Gender: p = 0.003). Exposed males entered the central zone significantly less frequently than unexposed males (p = 0.021) and exposed females spent less time in the central zone than did unexposed females (p = 0.009).
- analysis of acoustic startle demonstrated that exposed male offspring startled less than control males and were less inhibited by prepulse, whereas the opposite pattern was apparent for female offspring. Statistical analysis substantiated a stronger prepulse inhibition in prenatally exposed females at the highest and lowest prepulse compared to control offspring (Figure 5B; p = 0.041 and p = 0.089, respectively). (migrated information)

Details on results:

DETAILS ON MATERNAL TOXIC EFFECTS
- lungs from exposed females contained 38 mg Ti/kg on day 5 after the exposure and 33 mg Ti/kg on days 26-27. No Ti was detected in unexposed female lungs (p = 0.0002). Values in the liver were similar between control and exposed animals for all other samples.
- similar numbers of control and exposed females delivered litters, and none of the time-mated females without litters displayed implantations.
- gestational parameters were similar
- only maternal lung weight showed overall statistical significant variation with exposure, in both absolute (p = 0.04) and relative (p = 0.05) measures. Pairwise comparisons showed both measures to be marginally increased in only in dams with litters (0.05

- more neutrophils were present in BAL in TiO2 exposed compared to unexposed females (p < 0.001), with significant exposure-pregnancy interaction (p = 0.02).
- BAL from exposed non-pregnant females contained 19 times more neutrophils in BAL than did unexposed non-pregnant females (5 days after exposure, p < 0.001).
- The exposed pregnant females displayed 3-fold more neutrophils compared to unexposed pregnant females (26-27 days after exposure, p = 0.02). Exposure resulted in overall change in macrophages (p = 0.002) and lymphocytes (p = 0.007) compared to unexposed pregnant females.
- fewer macrophages (p = 0.009) but more lymphocytes (p = 0.008) in exposed compared to unexposed nonpregnant females.
- no cell type showed significant change in exposed pregnant females compared to respective controls.
- a statistically significant increase in the total number of dead cells in BAL fluid (p = 0.03) was observed in BAL from the exposed pregnant females (p = 0.004) but not in BAL from exposed nonpregnant females. Total cell counts, total number of eosinophils, and epithelial cells in BAL were did not vary with exposure.

FOLLOW-UP STUDY (Kyjovska et al. (2013))
The influence of age on testicle size and sperm production was assessed for 56, 70, 210 and 350 days old mice:
- both absolute and relative testicle weights as well as SC/g testicular tissue and DSP increased significantly with age (0.001 < P < 0.002, ANOVA with age as factor).
- absolute testicle weights differed significantly from all other groups at 56 and 70 days of age (0.001 < P < 0.006), but were similar in the two oldest groups.
- for relative testes weights, animals aged 56 days displayed relatively smaller testes compared to all other groups (0.001 < P < 0.048).
- at 70 days of age, relative testes weights compared to that of the two oldest age groups.
- relative testes weights were higher in animals 350 days of age compared to 210 days old animals, probably due to lower body weights in the oldest animals.
- DSP differed statistically significantly between all studied groups (0.001 ≤ P).
- SC/g differed significantly between all groups (0.001 < P < 0.005), but between animals of 70 and 210 days of age.
Further findings:
- F1 male offspring of mothers exposed to nanosized TiO2 during gestation tended to display reduced sperm production compared to controls (SC/g:
P = 0.057; DSP: P = 0.125; Table 3). No differences were observed for the other assessed parameters.
- the time it took breeding couples, consisting of a prenatally TiO2 exposed F1 C57BL/6J male and a naïve CBA/J female, to deliver a first F2 litter was slightly (statistically non-significantly) prolonged compared controls (P = 0.136). In comparison, time-to-first F2 litter was similar in breeding couples of TiO2 exposed female F1 offspring compared to control female F1 offspring (18,19)*. These data for time-to-first F2 litter were used together with sperm counts in an overall regression analysis. The outcome indicated that overall, time-to-first F2 litter correlated negatively with sperm production (SC/g, P = 0.03, Pearson correlation coefficient 0.61), i.e. the lower the sperm count the longer the time-to-first F2 litter. Regression analysis was also performed separately for the two experimental groups. Also here the time-to-first litter increased with increased sperm counts, however non-significantly so, but suggests that sperm counts explain more of the variation for F1 TiO2-exposed offspring than in F1 control offspring.
- for testicles from the F2 offspring, two ways ANOVA revealed no influence of nanosized TiO2 exposure on male reproductive parameters at the age of 80 days in offspring of either genotype ((CBA/J)/(C57BL/6J) and (C57BL/6J)/(CBA/J)).
- irrespective of exposure, the recorded reproductive parameters of the F2 males differed highly significantly, depending on whether the father or the mother supplied the (C57BL/6J) allele.
- absolute and relative testicle weights, as well as SC/g and DSP were statistically significantly higher in F2 offspring derived from F1 males with a paternal C57BL/6J allele (PF2, (CBA/J)/(C57BL/6J)) compared to F2 offspring derived from F1 males with the paternal CBA/J allele (MF2, (C57BL/6J)/(CBA/J)) (P < 0.001 for all).

*References:
- Hougaard KS, Jackson P, Jensen KA, Sloth JJ, Loschner K, Larsen EH, et al. Effects of prenatal exposure to surface-coated nanosized titanium dioxide (UV-Titan). A study in mice. Particle and Fibre Toxicology 2010;7:16.
- Hougaard KS, Jackson P, Jensen KA, Sloth JJ, Loschner K, Larsen EH, et al. Correction: effects of prenatal exposure to surface-coated nanosized titanium
dioxide (UV-Titan). A study in mice. Particle and Fibre Toxicology 2011; 8(May (1)):14.

Based on:

test mat.

Basis for effect level:

other: According to the authors, inhalation of nano-sized coated TiO2 induced long-term lung inflammation in time-mated adult mice, and their gestationally exposed offspring displayed neurobehavioral alterations.

Remarks on result:

not measured/tested

Remarks:

Effect level not specified

Basis for effect level:

other: see 'Remark'

Remarks on result:

not measured/tested

Remarks:

Effect level not specified

Time-to-first-delivery of F2 litter was similar in control and exposed female offspring, but was extended in exposed male compared to control male offspring (32.9 ± 3.1 (SD) and 25.2 ± 16.8 (SD) days, respectively). However, this result did not reach statistical significance (p = 0.12). Litter size was similar in control and exposed F2 litters.

Conclusions:

According to the authors, inhalation of nano-sized coated TiO2 induced long-term lung inflammation in time-mated adult mice, and their gestationally exposed offspring displayed neurobehavioral alterations.

FOLLOW-UP STUDY
According to the authors, maternal particulate exposure did not affect daily sperm production statistically significantly in the F1 generation, although TiO2 tended to reduce sperm counts. They also stated that time-to-first F2 litter increased with decreasing sperm production and there was no effect on sperm production in the F2 generation originating after TiO2 exposure. Furthermore, the authors report statistically significant differences in sperm production between mouse strains.

The reference shows significant methodological deficiencies: No. Of litters too low (13-14/dose; recommended 20 litters/dose); one concentration only; exposure during lactation missing; behavioural investigation was performed during light period (nocturnal animals); food consumption of dams not measured; data on clinical signs of dams not reported; clinical signs and detailed clinical observations of offspring missing; neuropathology of offspring missing; motor activity testing too short and missing for the pre-weaning period; Motor and sensory function measured only in adults ( 4 months old; recommeded 60 - 70 PND); learning and memory testing in adolescence missing; bw of weanlings missing Due to the above given arguments this reference is of no relevance for risk assessment purposes.

Reference 2

Endpoint:

neurotoxicity, other

Remarks:

in vitro, cell culture

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

other: not rated according to Klimisch et al.

Rationale for reliability incl. deficiencies:

other:

Remarks:

The references contained in this summary entry represents in vitro experiments with investigations on neurotoxicity with very limited value for risk assessment purposes. The references do not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH andhazard 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.

Principles of method if other than guideline:

Long, T.C. et al. (2007):
Test substance: Titanium dioxide nanoparticles obtained from Degussa (TiO2 P25, anatase 70 %, rutile 30 %)
Test system: BV2 (immortalised mouse microglia cells), N27 (rat dopaminergic neurons), primary cultures derived from tissue plug of embryonic Sprague-Dawley rat brain striatum
Concentrations:
Assays: 2.5-120 ppm
Genomics: 20 ppm
Immunhistochemistry and morphometry: 5 ppm
Light and transmission electron microscopy: 20 ppm
Exposure conditions:
Assays: not documented precisely
Genomics: 3 h
Immunhistochemistry and morphometry: not specified
Light and transmission electron microscopy: 3h
Negative controls: untreated cells
Positive control: not documented precisely
Parameters investigated: intracellular ROS production, cell viability, caspase activity, cytotoxicity, RNA expression profiles, morphometry

Long, T.C. et al. (2006):
Test substance: Titanium dioxide nanoparticles obtained from Degussa (Degussa Aeroxide P25, anatase 70 %, rutile 30 %, N2-BET: 52.7±3.6 m²/g, zeta-potential (DMEM): -11.6 ± 1.2 mV, zeta-potential (HBSS): -9.25 ± 0.73 mV, pH(IEP): 6.4)
Test system: BV2 microglial cells
Concentrations:
Assays: 2.5 - 120 ppm
Transmission electron microscopy: 2.5 ppm
Exposure conditions:
Assays: 1h - 8 h
Transmission electron microscopy: 6 h, 18 h
Negative controls: untreated cells
Positive control: not specified
Parameters investigated: intracellular ROS production, cell viability, cytotoxicity, morphology

Mu, X. et al. (2020):
Test substance: Titanium dioxide nanoparticles (anatase) donated by Professor Yang Ping (Soochow University, China), purity: > 99.5%; average particle size: 8-9 nm (neurobasal medium); mean hydrdynamic diameter (neurobasal medium): 36.83 nm; zeta-potential (neurobasal medium): 44.5 mV
Test system: primary cultured hippocampal neurons isolated from 24-hour-old Sprague-Dawley rats after birth
Concentrations and exposure conditions:
Developmental assays: 0, 5, 15 and 30 μg/mL for 6h, 12h and 24h
Immunocytochemistry and Western blot: 0, 5, 15, or 30 μg/mL for 24 h
Nano-TiO2 Internalization assay: 0, 5, or 30 μg/mL for 6 h, 12 h and 24 h
Assays: 6 h, 12 h, 24 h
Negative controls: untreated cells
Positive controls: not specified
Parameters investigated: development of neuronal axons (length per cell), expression of axon development-related protein factors (Netrin-1, Sema3A, Neuropilin-1, Nogo-A, GAP-43, and microtubule-associated protein 2 (MAP2)) by ICC and Western blot, internalization assay with fluorescein isothiocyanate; determination of internalization of test item (preparation of fluorescein isothiocyanate-nano) in neurons
(FITC)-nano-TiO2

Remarks on result:

other:

Remarks:

Long, T.C. et al. (2007): Detection of ROS in BV2 microglial cells after TiO2 exposure (H2O2: >60 ppm TiO2; O2(-)-radical: > 100 ppm). Increase of caspase 3/7 activity after TiO2 exposure (> 40 ppm, measured at 6 h post-exposure). Reduced nuclear staining at > 100 ppm after 24 h and > 2.5 ppm after 48 h. Genomic pathways associated with cell cycling, inflammation, apoptosis and mitochondrial bioenergetics were affected after TiO2 exposure. Neuronal loss occured in primary cultures of rat brain striatum, whereas no effects were observed in N27 cells. BV2 and N27 cells internalised TiO2 aggregates in vacuoles.

Remarks on result:

other:

Remarks:

Long, T.C. et al. (2006): Detection of ROS in BV2 microglial cells after TiO2 exposure (H2O2: >10 ppm TiO2; O2(-)-radical: > 20 ppm). Viability of BV2 was not affected by TiO2 exposure. BV2 cells internalised TiO2 aggregates in vacuoles.

Remarks on result:

other:

Remarks:

Mu, X. et al. (2020): internalization assay: with increasing treatment concentration and time in culture, FITC-nano-TiO2 aggregate in primary cultured hippocampal neurons; most concentrated in the cytoplasm and cluster around the nuclei. ICC assay and Western blot assay: the axon length of neurons decreases significantly with increasing nano-TiO2 exposure concentration and time in culture. As the concentration of nano-TiO2 increased (5, 15, and 30 μg/mL), axon length was suppressed by 32.5%, 36.6%, and 53.8% at 6 hours; 49.4%, 53.8%, and 69.5% at 12 hours; and by 44.5%, 58.2%, and 63.6% at 24 hours, respectively, compared with controls. No significant difference in protein expression of microtubule-associated protein 2 (MAP 2); expression of semaphorin type 3A (Sema3A) significantly increased in the cultured primary neurons compared to controls; expression of GAP-43 and Netrin-1 significantly decreased in a dose-dependent manner. With the increase concentrations of nano-TiO2 (5, 15, and 30 μg/mL), the expression of MAP2 was not significantly affected, but alterations in the following proteins were observed: increases of 0.08-, 0.19-, and 0.29-fold for Sema3A; 0.06-, 0.24-, and 0.29-fold for Nogo-A; and decreases of 9.27%, 33.01%, and 44.47% for Netrin-1; 12.73%, 20.65%, and 33.33% for GAP-43 and 8.17%, 22.14%, 36.9% for Neuropilin-1, respectively.

Conclusions:

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 vitro experiments with investigations on neurotoxicity 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.

Long, T.C. et al. (2007):
The experimental study design does not allow to draw conclusions for the risk characterisation of TiO2 nanoparticles used in this study:
The cell culture models (BV2, N27), as well as the primary cultures (BrainBits: tissue plugs of embryonic Sprague-Dawley rat brain striatum) used in this study are not sufficiently validated for the use in risk assessment. Regarding the assays, results are questionable since there are experimental / methodical deficiencies (e.g. no data of calibration of the test system, no positive controls and an insufficient characterisation of test material).

Long, T.C. et al. (2006):
The experimental study design does not allow to draw conclusions for the risk characterisation of TiO2 nanoparticles used in this study:
The cell culture model (BV2) used in this study are not sufficiently validated for the use in risk assessment. Regarding the assays, results are questionable since there are experimental / methodical deficiencies (e.g. no data of calibration of the test system, no positive controls).

Mu, X. et al. (2020):
The experimental study does not allow to draw conclusions for the risk characterisation of TiO2 nanoparticles used in this study:
The cell culture model (hippocampal neurons of 24-hours old Sprague-Dawley rat brain) used in this study are not sufficiently validated for the use in risk assessment. Regarding the assays, the results are questionable since there are experimental / methodical deficiencies (e.g. no data calibration of the test system, no positive controls).

Reference 3

Endpoint:

neurotoxicity, other

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

other: not rated according to Klimisch et al.

Rationale for reliability incl. deficiencies:

other:

Remarks:

The references contained in this summary entry represents in vivo experiments with investigations on neurotoxicity with very limited value for risk assessment purposes. The references do not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH andhazard 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.

Principles of method if other than guideline:

Younes et al. (2015):
Test substance: TiO2 NPs were analyzed and provided by the Laboratory of Physics of Materials and Nanomaterials Applied at Environment (self-synthesised)
Doses/Concentrations: 20 mg/kg
No. of animals per concentration: 6 mice
Exposure duration: 20 days
Exposure frequency: every 2 days
Negative control: yes
Poistive control: not specified
Post exposure period: Observations: 1 and 14 days
Parameters investigated: Behaviour, blood biochemistry, haematology, histopathology

Balvay, A. et al. (2013):
Test substance: titanium dioxide nanoparticles (99 % purity, TiO2 P-25. spherical 22 nm, 85 % anatase, 15 % rutile) provided by EC-JRC in the framework of the OECD sponsorship program
Doses/Concentrations: 5 and 10 µg
No. of animals per concentration: 15
Exposure duration: not applicable (injection time: 10 min)
Exposure frequency: single application
Negative control: yes, vehicle control
Poistive control: not specified
Post exposure period: 8 weeks
Parameters investigated: element content analysis in brains, histopathology of brains, motor performances

Su, M. (2014):
Test substance: TiO2 NPs (self-synthesized) from Medical College of Soochow University, Suzhou, China; average particle size: 5-6 nm; surface area: 174.8 m²/g; mean hydrodynamic diameter: 208 to 330 nm (mainly 294 nm) and ζ potential: 9.28 mV of TiO2 NPs suspended in 0.5 % w/v hydroxymethylcellulose (HPMC) K4M solvent after 24 h (5 mg/L) incubation.
Doses/Concentrations: 0.25, 0.5 and 1 mg/kg bw
No. of animals per concentration: 25 female mice
Exposure duration: 9 consecutive months
Exposure frequency: daily
Negative control: yes, vehicle control (0.5 % w/v hydroxypropylmethylcellulose (HPMC))
Positive control: not specified
Post exposure period: not specified
Parameters investigated: weighing at start and end of study; morbidity and mortility; analysis of titanium content and histopathologic examination of hippocampi (N = 5 of each group); calculation of Hippocampal indices (coefficients of hippocampus, N = 10
each); determination of the gene expression level by real time PCR from isolated RNA of hippocampus and determination of protein levels of NGF, BDNF, TrkA, TrkB, CaMKIV, CREB1, CREB2, D1, D2, and TH in the hippocampi (N = 5 of each group) by ELISA.

Sheng, L. et al. (2014):
Test substance: TiO2 NPs (self-synthesized) from Medicall College of Soochow University, Suzhou, China; average particle size: 5-6 nm; surface area: 174.8 m²/g; mean hydrodynamic diameter: 208 to 330 nm (mainly 294 nm) and ζ potential: 9.28 mV of TiO2 NPs suspended in 0.5 % w/v hydroxymethylcellulose (HPMC) K4M solvent after 24 h (5 mg/L) incubation.
Doses/Concentrations: 1.25, 2.5 and 5 mg/kg bw
No. of animals per concentration: 40 female mice
Exposure duration: 6 consecutive months
Exposure frequency: daily
Negative control: vehicle control (0.5 % w/v hydroxypropylmethylcellulose (HPMC))
Post exposure period: not specified
Parameters investigated: clinical signs and mortality; weighing on start and end of study (regularly before behavioural experiments); Y-maze test of spatial recognition memory (N=10 in each group); hippocampi selected from all animals; hippocampi and blood were digested and analysed for titanium and iron content (N=5 in each group); histopathological examination of hippocampi (N=5 in each group); assay of gene expression by RT-PCR and protein expression ELISA of NR1, NR2A, NR2B, CaMKII, PSD-95, SynGAP, ERK1/2, Dexras1, CAPON, PAP7, DMT1, and nNOS in the hippocampi (N=5 in each group).

Species:

other: Younes et al. (2015): male rat (Wistar); Balvay, A. et al. (2013): C57Bl6 female mice; Su, M. et al. (2014) and Sheng, L. et al. (2014): female CD-1 (ICR) mice

Sex:

male/female

Route of administration:

other: Younes et al. (2015): intraperitoneal, Balvay, A. et al. (2013): intracerebroventicular; Su, M. et al. (2014) and Sheng, L. et al. (2014): intranasal inhalation

Dose descriptor:

NOAEL

Effect level:

< 20 other: mg / kg bw

Based on:

test mat.

Sex:

male

Remarks on result:

other:

Remarks:

Younes et al. 2015: The results from the elevated plus maze (effects on emotional behavior) showed that the TiO2-NP-injected group entered less frequently and spent less time in the open arms than control group. Moreover, subacute TiO2 NPs treatment increased significantly the anxious index (AI) (91.78±3.68 vs 77.13±6.12, p<0.05). TiO2 NPs treatment did not affect the body weight of the injected rats. The coefficient of the lung was significantly higher in TiO2-NP-treated rats and sacrificed after 14 days (S2) than the control group (8.04±0.33 vs 6.08±0.20, p<0.05). Regarding the index, the treatment decreased significantly the thymus index (1.33±0.24 for S1 vs 1.94± 0.25; p>0.05); this change seems to be reversible after 14 days. The concentration of hemoglobin, the hematocrit, and the percentage of red blood cells and white blood cells remained unchanged, but the platelet count increased significantly for treated rats and sacrificed after 1 day after the last injection (S1) than the control group. TiO2 NPs treatment increased significantly the aspartate aminotransferase/alanine aminotransferase ratio (AST/ALT ratio) (4.92±0.43 for S1 and 5.59±0.93 for S2 vs 2.98±0.22, p>0.05),and it also increased the LDH and glucose levels for treated rats sacrificed 14 days after the last injection (S2) (1138.15±126.94 vs 808.69±47.77, p<0.05 and 117.74±3.93 vs 153.69±8.89, p<0.05, respectively). In contrast, the serum levels of uric acid and creatinine remained unchanged compared to the control group. With intraperitoneal injection, the titanium may cause significant accumulation in the rat liver and lung (p<0.05) at different times (1 day and 14 days after the last injection). The titanium accumulation in the brain increased significantly for treated rats and sacrificed after 1 day after the last injection (S1) than the control group (2.05±0.12 vs 0.70±0.07, p<0.05). Histopathological examination did not show significant changes in morphology andpathological lesions in organs.

Sex:

female

Remarks on result:

other:

Remarks:

Balvay, A. et al. (2013): The motorperformance of mice of both dose groups (5 and 10 µg) detiorated significantly until week 8 in the accelarated rotarod test (4 to 40 rpm). In the rotarod test with fixed speed (20 rpm) the motorperformance of mice detiorated until week for and became null thereafter. Preliminary results of histopathological analysis indicated an activation of microglial cells. A causality of microglial activation and exposure could not be proven from the results.

Sex:

female

Remarks on result:

other:

Remarks:

Su, M. (2014): TiO2 NPs exposure resulted in (i) a significant reduction in body weight and (ii) the hippocampal indices, and (iii) an obvious increase in titanium accumulation (p < 0.05). No abnormal pathological changes in the hippocampus of controls but excessive proliferation of all glial cells and tissue necrosis in exposed mice (0.25, 0.5, or 1 mg per kg BW TiO2 NPs). (iv) A alteration of neurotrophins and related receptor expression in the TiO2 NPs-exposed hippocampus in mice; (v) NGF mRNA and its protein expression in the hippocampus were obviously decreased with an increasing dose of TiO2 NPs (P < 0.05). (vi) The mRNA and protein expression of TrkA also significantly decreased in mice exposed to TiO2 NPs (P < 0.05). (vii) BDNF mRNA and its protein expressions in the hippocampus were greatly reduced in exposed mice (P < 0.05). (viii) TrkB, were also remarkably inhibited in exposed mice (P < 0.05). (ix) A significant reductions in kinase expression of CaMKII and CaMKIV in the TiO2 NPs-exposed hippocampus of mice (P < 0.05); (x) marked reductions of gene and protein expression of the NGF-related transcriptional factors of CREB1 and CREB2 due to TiO2 NPs exposure (P < 0.05). (xi) Significant reductions of D1 and D2 expression in mice exposed to TiO2 NPs; (xii) lower expression of the TH gene and its protein in the hippocampus due to TiO2 NPs exposure.

Sex:

female

Remarks on result:

other:

Remarks:

Sheng, L. (2014): At Y-maze test (i) the percentage duration in the novel arm in control mice was significantly higher than that in the start and other arms, whereas the percentage duration in the novel arm in 1.25, 2.5, or 5 mg/kg BW TiO2 NP-exposed mice was significantly decreased as compared to the control mice throughout the experiment (P<0.05). (ii) The number of arm entries markedly decreased (P<0.05), with increased TiO2 NP dose. At ICP-MS analysation (iii) significant increases of titanium levels were observed, whereas iron levels were markedly reduced in the blood and hipocampus (P<0.01), with increased TiO2 NP dose. Titanium content in the control mice was negligent. (iv) Histopathological changes from hippocampal CA region with (significant oedema of glial cells, disperative replication of neuron cells, decreased size of cell volume, nuclear irregularity, and necrosis or abscission of neuron cells). The expression level of the actin3 gene (as endogenous control gene) was constant, with an expression ratio of almost one in all the samples. (vi) Changes in the expression levels of the 12 NMDA receptor subunit and/or postsynaptic signaling factor genes were evaluated and compared following exposure to TiO2 NPs for six consecutive months. (vii) Long-term exposure to TiO2 NPs resulted in a dose-dependent marked decrease in the mRNA and protein expression of NMDA receptor subunits, including NR1, NR2A and NR2B in the hippocampus, suggesting reductions of 16.67%, 64.71% and 75.49%; 15.63%, 67.8% and 78.07% for NR1; reductions of 34.52%, 67.85% and 75%; 41.18%, 71.67% and 78.39% for NR2A, reductions of 33.82%, 57.35% and 73.53%; 15.63%, 62.41% and 77.11% for NR2B, respectively. (viii)There were significant reductions in CaMKII, PSD-95, ERK1/2, Dexras1, CAPON, PAP7, and DMT1 expression, as the dose of TiO2 NPs increased; whereas there were marked increases of SynGAP and nNOS expression in the hippocampi (P<0.05).

Conclusions:

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 with investigations on neurotoxicity 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.

Younes et al. (2015):
The methodical setup is not adequately designed for risk assessment purposes of the test substance. The non-physiological route of administration via intraperitoneal injection is not guideline conform and not suitable to assess toxicity of the test substance after repeated administration. The test material is unsufficiently characterised (self-synthesised), only one dose was administered and age of the test animals were not specified.

Balvay, A. et al. (2013):
The methodical setup is not adequately designed for risk assessment purposes of the test substance. The non-physiological route of administration via intracerbroventricular injection is not guideline conform and not suitable to assess toxicity of the test substance after repeated administration. General health status of the animals is not documented. Furthermore, only preliminary data (1 to 3 animals per group) of the histopathological analysis were available since it was not completed until the date of publication.

Su, M. (2014):
Since the test item is poorly described and also the influence of the other simultaneously administered process chemicals is not addressed, this raises doubts as to whether (i) nano-sized TiO2 particles at all were used in this experiment, and (ii) whether the effects can really be attributed to titanium dioxide and (iii) the methodical setup is not in accordance with any guideline. A detailed evaluation of the statistics and given raw data with standard deviations showed that the standard deviations are always 5%. The non-physiological route of administration via intranasal inhalation is not guideline conform and not suitable to assess neurotoxicity. The experimental design is insufficiently documented (division of the test animals (individually or in groups) in the cages). Only one sex was used and the number of animals per group (n = 5 or 10 of 25) for some neurotoxicity tests on hippocampi is not sufficient to perform an appropriate statistical analysis. No functional observations or weekly body weights and the daily food consumption was not examined. Furthermore, there are no clinical signs, detailed clinical examinations and ophthalmonology. A positive control, historical control and individual data is missing. Overall, only selected parameters were investigated and the investigated endpoints are 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).

Sheng, L. (2014):
Since the test item is poorly described and also the influence of the other simultaneously administered process chemicals is not addressed, this raises doubts as to whether (i) nano-sized TiO2 particles at all were used in this experiment, and (ii) whether the effects can really be attributed to titanium dioxide and (iii) the methodical setup is not in accordance with any guideline. A detailed evaluation of the statistics and given raw data with standard deviations showed that the standard deviations are always 5% or 1 %. The non-physiological route of administration via intranasal inhalation is not guideline conform and not suitable to assess neurotoxicity. The experimental design is insufficiently documented (division of the test animals (individually or in groups) in the cages). Only one sex was used and the number of animals per group (n = 5 or 10 of 40) for neurotoxicity tests on hippocampi is not sufficient to perform an appropriate statistical analysis. No weekly body weights and the daily food consumption was not examined. The data of clinical signs and the mortality are missing.

Reference 4

Endpoint:

neurotoxicity: short-term inhalation

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

not specified

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

no guideline followed

Principles of method if other than guideline:

12 week and 19-month old male Fischer 344 rats were exposed by inhalation to 10 mg/m3 of TiO2 nano-aerosol (2 periods of 3hrs/day, 5 day/week, for 4 weeks). At the end of the 28-day inhalation period, animals were held for a recovery period of 3 or 28 days. Brains were collected for titanium analysis by ICP-MS. The blood brain barrier integrity was assessed by evaluating atenolol concentrations in brain and plasma and derivation of a partition coefficient. Additionally, the expression of the tight junction protein claudin-5 was evaluated in vessels of the midsagittal brain sections. In order to evaluate impact on neuronal activity, the expression of synaptophysin was analysed either by immunofluorescence and by transcriptional profiling via multiplexing approach. Cytokines/chemokines were assessed by multiplexing approach as well.

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material:
Manufacturer: Evonik, Distributor: Sigma Aldrich (Saint-Quentin Fallavier, France)

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Charles River Laboratories, France
- Age at study initiation:
12-13 weeks; 19 month
- Weight at study initiation:
300- 320g; 400- 425g
- Diet: ad libitum (A04 diet); aging rats were fed separately with A04 diet from 1 month of age to 7 months then with A05 diet adapted to long term studies
- Water: ad libitum

DETAILS OF FOOD AND WATER QUALITY:
A04 and A05 (safe diet)

ENVIRONMENTAL CONDITIONS
- Temperature: 22± 2 °C
- Humidity: 55± 10%
- Photoperiod (hrs dark / hrs light):
12/12

Route of administration:

inhalation: aerosol

Vehicle:

clean air

Remarks:

filtered air

Mass median aerodynamic diameter (MMAD):

ca. 905 nm

Remarks on MMAD:

count median aerodynamic diameter: 270 nm

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: combination of an aerosol generation system and inhalation tower for nose-only exposure
- Method of holding animals in test chamber: restraining tubes
- Method of conditioning air: integrated control of the exposure conditions (airflow, temperature and relative humidity) is managed and recorded using dedicated software.
- System of generating particulates/aerosols: nanostructured aerosols of TiO2 NPs were produced with a rotating brush generator (RBG) (RBG1000 PALAS)
- Temperature, humidity: 22 ± 2°C; 55 ± 10%
- Air flow rate: constant

TEST ATMOSPHERE
The aerosol monitoring and characterization were ensured by real time devices (condensation particle counter, electrical low pressure impactor, aerodynamic particle sizer, scanning mobility particle sizer spectrometer, optical light scattering dust monitor) and off-line analyses (gravimetric filter, particle size-distribution by cascade impactor, sampling for TEM observations).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The aerosol monitoring and characterization were ensured by real time devices (condensation particle counter, electrical low pressure impactor, aerodynamic particle sizer, scanning mobility particle sizer spectrometer, optical light scattering dust monitor) and off-line analyses (gravimetric filter, particle size-distribution by cascade impactor, sampling for TEM observations).

Duration of treatment / exposure:

28 days (20 effective exposure days)

Frequency of treatment:

6 hours/day (2 periods of 3 hrs/day), 5 days/week

Dose / conc.:

10.17 mg/m³ air (analytical)

Remarks:

SD ± 3.29 mg/m³ (adult rats)

Dose / conc.:

10.42 mg/m³ air (analytical)

Remarks:

SD ± 1.80 mg/m³ (elderly rats)

No. of animals per sex per dose:

not specified

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dosing schedule and dose selection corresponds to the 8 hours time-weighted average French Occupational Exposure Limit value for particles without significant toxicity. Inhalation of 10 mg/m³ of ultrafine (< 100 nm) titanium dioxide dusts was the lowest concentration associated with cancer in the respiratory tract in rats.
- Rationale for selecting satellite groups: recovery groups at different time points were included in order to decrease influences on the CNS level due to stress associated with experimental procedure
- Post-exposure recovery period in satellite groups:
adult rats: 0,3,28,90 and 180 days
elderly rats: 0,3,28 and 90 day

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: No data

OPHTHALMOSCOPIC EXAMINATION: No data

Specific biochemical examinations:

NEUROPATHY TARGET ESTERASE (NTE) ACTIVITY: No data

CHOLINESTERASE ACTIVITY: No data

Neurobehavioural examinations performed and frequency:

FUNCTIONAL OBSERVATIONAL BATTERY: No data

LOCOMOTOR ACTIVITY: No data

Sacrifice and (histo)pathology:

- Time point of sacrifice:3 and 28 days after the end of inhalation exposure period under isoflurane anesthesia
- Number of animals sacrificed:
- Parameters measured:
- Brain weight:
- Length and width of brain:
- Other:
- Procedures for perfusion:
- Number of animals perfused:
- Tissues evaluated: Parameters checked in table [No.?] were examined.
- Type of staining:
- Methodology of preparation of sections:
- Thickness:
- Embedding media:
- Number of sections:
- Number of animals evaluated from each sex and treatment group:

Other examinations:

Titanium content in brain:
Total brain tissues were weighed and thawed. Each sample was added with 8 mL of nitric acid + 2 mL of hydrofluoric acid and digested using a Microwave Assisted Reaction System (MARS) Express instrument. Microwave digestion program was 15 min; 150 °C; 1200 W then 15 min; 180 °C; 1200 W. After 20 min cooling, sample was rinsed 3 times using approximately 20 mL of 2% nitric acid solution in a polytetrafluoroethylene (PTFE) beaker then 2 mL of hydrogen peroxide was added. Beakers were heated at 180 °C until between 0.1 and 0.5 mL of solution remained. After cooling, the beakers were rinsed 3 times with 2% nitric acid solution before being stored for analysis.
Ti standard solutions for ICP-MS calibration were prepared at concentrations from 2 to 100 ng/L, by diluting a 1 g/L Ti standard stock solution (1.70363.0100, SCP Science) with 2% v/v HNO3 and 0.01% v/v Triton X-100. An internal standard solution 25 μg/L of Ge was prepared by diluting a 1000 mg/L stock solution (1.70320.0100, Merck) with 2% v/v HNO3. Ti analysis of acidified samples was carried out using a Varian 820-MS. Samples in 2% v/v HNO3 were analyzed with the Varian-820-MS. This mineralization method was optimized to obtain 96% recovery and a limit of quantification (LOQ) down to 13.7 ng/g in brain tissue and limit of detection of 4.1ng/g.

Neuroinflammation assessment:
Olfactory bulbs were dissected from the rest of the brain (cerebrum + cerebellum), homogenized separately in 10 volumes of TRIS lysis buffer 50 mM (Sigma) added with proteases inhibitor (Calbiochem) using homogenization for 60 sec in 15 mL TeenPrep Lysing Matrix D (MP Biomedicals). Homogenates were centrifuged for 5 min at 2000 g, supernatant was recovered for ultracentrifugation (30 min; 26184 g) and stored at −80 °C until assayed.
Multiple cytokines/chemokines (Il1β, IL6, EGF, MIP2, IFNγ, MCP1, IP10, VEGF, Fractalkine, RANTES, TNFα) were analysed in extracts from either the cerebral tissues (cerebrum + cerebellum), olfactory bulbs, or sera using a MILLIPLEX MAP kit (Merck Millipore, Billerica, US) and the Biorad Bioplex-200 analysis instrument (Bio-Rad laboratories, Hercules, US). Bioplex manager software was used to generate standard curves and calculate sample cytokines/chemokines concentrations. Assays were performed in duplicate for each brain extract. Results are expressed as mean of duplicate.

Blood brain barrier (BBB) permeability measurement:
4 hours before animal necropsy, rats were anesthetized with isoflurane and subcutaneously implanted with mini-osmotic pumps (Alzet model 2001D; DURECT Corp., Cupertino, California). Pumps were filled with atenolol dissolved in PEG200/DMSO (50/50) to deliver at 0.25 mg/kg/h. After 4 hrs, animals were anesthetized with isoflurane and euthanized. Plasma samples and brain were collected and weighed immediately after death. Atenolol concentration was quantified in the two compartments using tandem mass spectrometry coupled with liquid chromatography (LC/MSMS). BBB integrity was estimated by the partition coefficient (Kp) corresponding to the ratio of atenolol brain to plasma concentration (Cbrain and Cplasma).
Kp = Cbrain/Cplasma
For atenolol quantification, brains were mixed in ultrapure water (2 mL/g of tissue) using an Ultraturrax T65 system (IKA-Werke, Staufen, Germany). Extract suspensions (400 μL) were submitted to protein precipitation with 1 mL of methanol previously spiked with internal standard (atenolol-d7 4 μg/mL). After centrifugation (20000 g; 15 min; 4 °C) the supernatant was dried under nitrogen at 40 °C then resuspended in 1 mL 0.75 M NH4OH/methanol (80:20 v/v). Plasma (150 μL) was diluted with 150 μL of 0.75 M NH4OH/methanol (80:20 v/v) previously spiked with internal standard. Both brain and plasma extracts were submitted to solid-liquid extraction on isolute SLE + columns 1 or 6 mL (Biotage). The two eluates (3 mL of dichloromethane/isopropanol (70:30 v/v) then 3 mL of dichloromethane/isopropanol (70:30 v/v) + 0.2% formic acid) were pooled and evaporated to dryness. The dry extracts were resuspended in 200 μL of 5 mM ammonium acetate/methanol (95:5 v/v). Chromatography was performed using a Shimadzu HPLC system LC 20AD on a Kinetex C18 column (Phenomenex). The total run time was 5 min and the flow rate was 0.4 mL/min. Analyte (20 μL) was injected onto the column placed in an oven at 40 °C.
Detection was done by tandem mass spectrometry (Finnigan TSQ Quantum Discovery with Xcalibur and LC Quan softwares, Thermo) in positive electrospray mode. Tuning parameters were: capillary voltage 3 kV, source temperature 200 °C. The multiple reaction monitoring transitions for atenolol were m/z atenolol 267.18 > 145.1. Analyte was quantified by means of calibration curves using atenolol-d7 as internal standard. For plasma and brain extract assay, calibration ranges were from 1.0 to 200 ng/mL.

Immunofluorescence:
Midsagittal brain sections sampled at 28 days after the end of the inhalation period were embedded in OCT and cut on a cryostat (10 μm) were prepared for either claudin-5 and von Willebrand factor (vWF) double immunofluorescence, or either IL-1β or synaptophysin immunofluorescence. Brain sections were air-dried for 30 minutes and fixed in ice-cold acetone for 30 minutes and then rinsed in PBS. Sections were incubated with 3% BSA (60 min at RT), rinsed in PBS, and incubated with 150 μL per section of the appropriate primary antibody (claudin-5: 1:100, Invitrogen/Life Technologies, Carlbad, CA) and a FITC-tagged vWF (1:1000 dilution, Abcam) or IL-1β (1:1000, Abcam) or synaptophysin (1:1000, Abcam) alone, diluted in rinse wash buffer [1 part 5% blocking solution (0.5 mL Normal Rabbit Serum in 10 mL 3% w/v BSA) and 4 parts PBS] for 1 hr at RT and then rinsed 3 times with PBS. The slides were then incubated in 150 μL per section of the appropriate secondary antibody either Alexa Fluor 555 or Alexa Fluor 488 (1:1000 dilution, Vector Laboratories, Biovalley, Marne la Vallée, France) in the dark for 1 hr at RT. Slides were then rinsed 3 times in PBS and subsequently incubated with Hoescht nuclear stain (1 μL/mL; 150 μL/section) for one minute, rinsed again then cover-slipped with Aqueous Gel Mount (TBS, Fisher Scientific, Waltham, MA). Slides were imaged by fluorescent microscopy at 10x and 40x, using the appropriate excitation/emission filters, digitally recorded, and analyzed by image densitometry using Image J software (NIH). A minimum of 3 locations on each section (2 sections per slide), 3 slides and n = 3 per group were processed/analyzed. IL-1β and synaptophysin were quantified by total amount of fluorescence per unit area (consistent across regions/slides). Claudin-5 (double immunofluorescence, vessel specific) were measured by merging Alexa 488 (fluorescein isothiocyanate) and Alexa 555 (Cy3) signals into Red-Green-Blue (RGB) images. Colocalization was determined by quantifying total fluorescence of overlayed signals. Midbrain analysis included regions of brainstem, hippocampal formation, and cerebral cortex (somatosensory). Forebrain analysis included regions of the cerebral cortex (somatomotor) and cerebral nuclei/caudoputamen/striatum. Only vessels <50 μm were used for claudin-5 analysis.

Transcription profiling:
Rat brain capillaries and brain parenchyma were separated as described previously. Four to eight rats from each age group were used. Brains were extracted and stored in Hanks balanced salt solution (HBSS) supplemented with 1% (v/v) PSN on ice. The brains were cut sagittally into two halves and the cerebral cortices emptied of white matter. The meninges and the associated vessels were cleaned off by rolling on Whatman 3 mm chromatography paper. The homogenized tissue was pelleted by centrifugation (1500 rpm; 5 min). The pellet was digested in HBSS-1% PSN solution, 1 mg/mL collagenase/dispase, 10 U/μL DNase-I, and 1 μg/mL TLCK for 1 h at 37 °C. Digested tissue was then pelleted by centrifugation (1500 rpm; 5 min; 4 °C). The pellet was resuspended in 20% (w/v) BSA in HBSS-1% PSN solution and centrifuged (2800 rpm; 30 min). The resulting floating white matter corresponding to the brain parenchyma fraction. Centrifugation medium were removed carefully. The remaining parenchyma fraction was resuspended in centrifugation medium then half-dissolved in HBSS-1% PSN solution and pelleted by centrifugation (1500 rpm; 15 min). The brain parenchyma pellet was then washed several times before storage at −80 °C.

RNA was isolated from brain parenchyma fraction using the RNeasy Mini kit (Qiagen, France). The concentration and purity of the RNA samples were assessed spectrophotometrically at 260 and 280 nm using the NanoDrop ND-1000 instrument (NanoDrop Technologies, Wilmington, DE, USA). The A260/280 ratio ranged between 1.8 and 2. A sample of 0.5 μg of total RNA was converted to cDNA with random primers in a total of 10 μL using an RT2 first stand kit (Qiagen, France). The cDNA was diluted with DNA/RNAse-free distilled water to a volume of 110 μL. The quantitative expression of synaptophysin was determined using 0.4 μM cDNA for each primer set in the RT2 Pathway Focus profiler array from Qiagen. The RT2 Profiler array consists of a previously validated qRT-PCR primer set (1 μL) for synaptophysin. Thermocycling was carried out in a CFX96 real-time PCR detection system (Bio-Rad) using SYBR green fluorescence detection. The final reaction mixture contained 2 μL of diluted cDNA, 1 μL of one of the specific primer, 12.5 μL of distilled water and 9.5 μL of SYBR green master mix. The specific amplification conditions were 2 min at 50 °C, 10 min at 95 °C followed by 40 amplification cycles at 95 °C for
0.5 min, and 60 °C for 1 min to reinitialize the cycle again. The specificity of each reaction was also assessed by melting curve analysis to ensure the presence of only one product. Relative gene expression values were calculated as 2^−ΔCT, where ΔCT is the difference between the cycle theshold (CT) values for genes of interest and housekeeping
genes (hypoxanthine guanine phosphoryltransferase or Hprt).

Positive control:

not examined

Statistics:

Brain content Ti analysis:
Statistical analysis was performed using Stata 14 (StataCorp LP, TX,USA). After Box cox transformation on Ti concentration variable, statistical comparisons were accomplished using two-way ANOVA (the two determinants being treatment and time) followed by Bonferroni post-hoc test.

Blood brain barrier permeability measurement:
After homogeneity of variance confirmation by Hartley and Bartlett test, atenolol partition coefficients were analysed using one-way ANOVA between control and exposed groups followed by Tukey post hoc test (Prism 5.1 program, GraphPad Software, Inc, San Diego Ca).

Neuroinflammation assessment:
Cytokines and chemokines assays were analysed either using one-way ANOVA followed by Tukey post hoc test when Hartley and Bartlett test confirmed homogeneity of variances (Il1beta, IFNgamma, VEGF and IP10 for both aged groups; RANTES for aging group) or Kruskal-Wallis test followed by test by Dunn's post hoc (RANTES for young adult groups).

Immunofluorescence:
Statistical analysis was performed using one-way ANOVA between treatment groups followed by Holm-Sidak post hoc test (SigmaPlot SyStat Software Inc, San Joes, CA) and data expressed as mean ± SEM

Transcription profiling:
mRNA expression of synaptophysin were analysed using two-tailed Student's t-test after Hartley test confirmed homogeneity of variances.

All changes were considered statistically significant at p< 0.05

Clinical signs:

not specified

Dermal irritation (if dermal study):

not examined

Mortality:

not specified

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Neuroinflammation assessment:
- IL-1β was significantly upregulated 28 days after the end of the inhalation exposure in both age groups.
- VEGF (Vascular Endothelial Growth Factor) and fractalkine increased significantly at 28 days post-inhalation in both age groups.
- IFNγ, IP-10 (IFN-gamma-inducible protein 10) were also increased at 3 days and RANTES (Regulated on Activation, Normal T cell Expressed and Secreted) at 28 days after the end of the inhalation period in young adult group brains.

Blood brain barrier (BBB) permeability measurement:
- in the young adults group, cerebral atenolol concentrations and Kp between the control and exposed animals remained unchanged, suggesting a lack of BBB permeability modulation.
- in contrast, results from the aged rat brains showed that atenolol Kp remained unchanged at day 3, but significantly increased at day 28 post TiO2 nano-aerosol-exposure (0.2 ± 0.05 in exposed vs 0.2 ± 0.03 in controls).


Immunofluorescence:
- expression of claudin-5, one of the primary proteins expressed in Tight Junction intercellular connections between adjacent brain endothelial cells that contribute to BBB structural integrity, was down-regulated at 28 days after the end of the TiO2 nano-aerosol inhalation period in both the young and aged rat brains, compared to control animals.
- IL-1β expression in the cerebrum of TiO2 exposed animals (both young and aged) was increased compared to controls. An increase in IL-1β localization in the midbrain and forebrain in TiO2 exposed brains compared to the respective age-matched controls was observed
-a significant decrease of synaptophysin expression in the aging brains was observed between unexposed and exposed animals

Transcription profiling:
- a slight non-significant decrease in synaptophysin mRNA in the brain of young rats and a significant decrease in the brain of aged rats was observed after TiO2
nano-aerosol exposure.

Details on results:

Titanium content in brain:
- in both age groups, Ti analysis revealed lack of detectable translocation to the brain.
- this suggests that TiO2 nano-aerosol did not gain access in quantifiable amount to the CNS either across the BBB or through axonal translocation from the nasal mucosa.


Neuroinflammation assessment:
- increased in IL-1β expression in exposed aged rat brains compared within young rat brains, could be related with BBB disruption.
- an increase in BBB permeability in the aged group may be due in part to the increased expression of VEGFand/or Fractalkine
- IP-10 and RANTES up-regulation underscore the inflamed environment in the brain of exposed animals.
- up-regulation of cytokines/chemokines could mediate the increase in BBB permeability observed in exposed animals.

Blood brain barrier (BBB) permeability measurement:
- BBB permeability evaluation suggests that TiO2 nano-aerosol exposure results in exacerbation of BBB integrity loss that could be associated with age-related alterations. The decreased expression of claudin-5 also underscores age-related vulnerability and differences in response of the BBB to TiO2 nano-aerosol exposure

Neuronal activity:
- quantification of synaptophysin mRNA and protein expression revealed a decrease in the brains of aging exposed animals, suggesting a possible default in synaptic function.

Remarks on result:

not determinable because of methodological limitations

Remarks:

increased blood brain barrier permeability 28 days after the end of inhalation exposure in aging rats; increased cytokine levels in cerebral tissues: increased cytokine levels in cerebral tissues in young (IL1beta, fractalkine, VEGF, IFNgamma, RANTES and IP10) and aging rats (IL1beta, Fractalkine, VEGF, IFNgamma); decreased synaptophysin mRNA expression in brain parenchyma

Conclusions:

The study does 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 study design displays the following shortcomings: males only, only one dose level tested; clinical signs, heamatology, clinical chemistry, gross pathology and body weights were not determined; health status of rats was not assessed.

Additionally, an assignment, which and how many animals were used for which tests, was not possible. Some discrepancies were noted in reporting, i.e. it was reported that titanium content was analysed after a recovery period of 3 days or 28 days after the end of inhalation period. In the corresponding figure displaying the results graphically, 5 timepoints (0, 3, 28, 90 and 180 days) were displayed.

Effects observed in this study were: increased blood brain barrier permeability 28 days after the end of inhalation exposure in aging rats; increased cytokine levels in cerebral tissues: increased cytokine levels in cerebral tissues in young (IL1beta, fractalkine, VEGF, IFNgamma, RANTES and IP10) and aging rats (IL1beta, Fractalkine, VEGF, IFNgamma); decreased synaptophysin mRNA expression in brain parenchyma.

However, a causal relationship between titanium dioxide exposure and the effects seen in this study cannot be drawn, since titanium was not translocated to the brain. And presence or biopersistence of titanium in other organs relating to the described effects was not evaluated.

Endpoint conclusion

Endpoint conclusion:

no study available

Effect on neurotoxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Effect on neurotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Neurotoxicity

During a comprehensive literature search, four references were identified representing studies on neurotoxicity conducted with mice or rats receiving nano-form titanium dioxide via oral (gavage) or inhalation (nose only; whole body) administration. After a thorough reliability screening, these references were considered of limited relevance for hazard assessment purposes. The criteria for quality, reliability and adequacy of experimental data under REACH for hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X) are not fulfilled. These references are discussed briefly below, highlighting their findings and the reasons for their exclusion from health hazard assessment:

 

Disdier et al. (2017) exposed 12 week and 19-month old male Fischer 344 rats to 10 mg/m3 of TiO2 nano-aerosol via inhalation (2 periods of 3hrs/day, 5 day/week, for 4 weeks). At the end of the 28-day inhalation period, animals were held for a recovery period of 3 or 28 days. Brains were collected for titanium analysis by ICP-MS. The blood brain barrier integrity was assessed by evaluating atenolol concentrations in brain and plasma and derivation of a partition coefficient. Additionally, the expression of the tight junction protein claudin-5 was evaluated in vessels of the midsagittal brain sections. In order to evaluate impact on neuronal activity, the expression of synaptophysin was analysed either by immunofluorescence and by transcriptional profiling via multiplexing approach. Cytokines/chemokines were assessed by multiplexing approach as well.

The study design displays the following shortcomings: males only, only one concentration tested; clinical signs, haematology, clinical chemistry, gross pathology and body weights were not determined; health status of rats was not assessed.

Additionally, an assignment, which and how many animals were used for which tests, was not possible. Some discrepancies were noted in reporting, i.e. it was reported that titanium content was analysed after a recovery period of 3 days or 28 days after the end of inhalation period. In the corresponding figure displaying the results graphically, 5 timepoints (0, 3, 28, 90 and 180 days) were displayed.

Effects observed in this study were: increased blood brain barrier permeability 28 days after the end of inhalation exposure in aging rats; increased cytokine levels in cerebral tissues: increased cytokine levels in cerebral tissues in young (IL1beta, fractalkine, VEGF, IFNgamma, RANTES and IP10) and aging rats (IL1beta, Fractalkine, VEGF, IFNgamma); decreased synaptophysin mRNA expression in brain parenchyma

However, a causal relationship between titanium dioxide exposure and the effects seen in this study cannot be drawn, since titanium was not translocated to the brain. And presence or biopersistence of titanium in other organs relating to the described effects was not evaluated.

 

Hougaard et al. (2010, 2011) exposed time-mated mice (C57BL/6BomTac) to 42 mg/m³ aerosolized powder (1.7·106 n/cm³; peak-size: 97 nm) via inhalation for 1h/day on gestation days 8 - 18. The following parameters were investigated maternal lung inflammation; gestational and litter parameters; offspring neurofunction and fertility. In a follow-up study Kyjovska et al. (2013) investigated the influence of maternal airway exposure to nanoparticulate titanium dioxide (TiO2, UV-Titan) on male reproductive function in the two following generations. Time-mated C57BL/6J mice were exposed by inhalation to UV-Titan. Body and testicle weight, sperm content per grams testicular parenchyma and daily sperm production (DSP) were assessed. The protocol for assessment of DSP was optimized for application in mice (C57BL/6J) and the influence of different parameters was studied.

The results of the study by Hougaard et al. (2010, 2011) indicated that inhalation of nano-sized coated TiO2 induced long-term lung inflammation in time-mated adult mice, and their gestationally exposed offspring displayed neurobehavioral alterations. In addition, the results of the follow-up study showed that maternal particulate exposure did not affect daily sperm production statistically significantly in the F1 generation, although TiO2 tended to reduce sperm counts. They also stated that time-to-first F2 litter increased with decreasing sperm production and there was no effect on sperm production in the F2 generation originating after TiO2 exposure. Furthermore, statistically significant differences in sperm production between mouse strains was reported. The reference shows significant methodological deficiencies: only one very high dose was tested, exposure duration was too short, uterine content was not examined, food consumption was not monitored, skeletal alterations in pups was not examined. Due to the above given arguments this reference is of no relevance for risk assessment purposes.

 

Vasantharaja & Ramalingam (2018) investigated the toxicity of titanium dioxide nanoparticles on biochemical and histological changes in different regions of the brain (cerebrum, cerebellum, and medulla oblongata). Groups of 6 adult male Wistar rats received 50 and 100 mg/kg bw/day of titanium dioxide nanoparticles (33.95 nm) in 0.9 % saline solution via gavage for an exposure duration of 14 days. A vehicle control group was run concurrently. After exposure, the brain was removed and tissue samples of cerebrum, cerebellum and medulla oblongata were prepared for an membrane-bound enzymes assay (ATPases, alkaline phosphatase, 5’ nucleotidase and gamma-glutamyl transpeptidase). Furthermore, the samples were investigated for metal content (calcium, sodium, potassium, magnesium, iron, zinc, copper, and titanium). Lastly, the brain tissue samples were examined histopathologically. The results of a membrane-bound enzymes assay showed that compared to the control group, the enzymes ATPases, alkaline phosphatase, 5’ nucleotidase and gamma-glutamyl transpeptidase activities were statistically significant decreased in both titanium dioxide nanoparticles treated groups. Furthermore, the metal content analysis showed that calcium, sodium, iron, copper and titanium dioxide contents were statistically significant increased in both experimental groups compared to the control group, while the potassium, magnesium and zinc contents decreased compared to the control group. This reference had several reporting and experimental deficiencies, which do not allow an independent review about the exposure-effect correlation:

- number of animals was too low (n = 6) and males were only tested. The OECD guideline 424 foresees that normally 20 animals (10 males / 10 females)for each dose level and control group are investigated as well as both sexes. The reduction of animals causes a significant reduction of the statistical powder. Furthermore, there is no information on the effect of the test substance on females, which does not allow a conclusion on the effect in females.

- exposure duration was too short. The guideline foresees normally a exposure duration of at least 28 days. A shorter exposure duration needs to be justified. The justification is missing.

- the guideline foresees that at least three dose level and a control should be used. In this study, only two dose levels were tested. The use of two dose levels precludes the possibility to demonstrate any dose-related response and the determination of a No-Observed-Adverse Effect level (NOAEL).

 

- the guideline foresees the usage of a positive control to demonstrate the laboratory ability to carry out the study and the sensitivity of the procedures used. Apparently, no positive control was used in the study and, therefore, the sensitivity of the procedures cannot be assessed.

- clinical signs, mortality, detailed clinical examination, functional tests, body weight, and food consumption were not examined/recorded. These examinations are foreseen by the guideline. 

- incomplete histopathology was conducted. In the study, the cerebrum, cerebellum and medulla oblongata were only investigated.

- no individual data and historical data were provided, which would improve an evaluation of the data.

Based on the shortcomings given above, this publication was disregarded for the hazard and risk assessment.

 

Grissa et al. (2020) investigated the neurotoxicity of titanium dioxide nanoparticles (7 - 8 nm). Groups of 8 male Wistar rats received 50, 100 and 200 mg/kg bw of titanium dioxide nanoparticles in distilled water via gavage five times/week for an exposure duration of 8 weeks. A vehicle control group was run concurrently. After the last exposure, the brain was excised and weighed. The following investigations were carried out on the brain: titanium content in brain, oxidative antioxidant biomarkers, lipid peroxidation, nitric oxide levels, tumour necrosis factor-alpha (TNF-α) levels, histopathological changes, and examination for degenerated and apoptosis neurons. The results suggested that TiO2 nanoparticles can reach the brain and cross the brain blood barrier to been accumulated in the brain of rats causing cerebral oxidative stress damage, increasing nitric oxide levels and histopathological injury. At higher dose, the most cerebral injury by the highest accumulation of Ti was observed and by the remarkable increase of TNF-α besides to the most increase of degenerated and apoptosis neurons in the brain of exposed rats. TiO2 nanoparticles led to a neurotoxic damage accompanied by the increase of degenerated and apoptotic neurons in cerebral cortex. This reference had several reporting and experimental deficiencies, which do not allow an independent review about the exposure-effect correlation:

- test material was described insufficiently. The purity and the stability were not given.

- animal age and weight were not stated. According to the OECD guideline 424, dosing should normally begin as soon as possible after weaning, preferably not later than when the animals are six weeks, and, in any case, before the animals are nine weeks of age.

- number of animals was too low (n = 8) and males were tested only. The OECD guideline 424 foresees that normally 20 animals (10 males / 10 females) for each dose level and control group are investigated as well as both sexes. The reduction of animals causes a significant reduction of the statistical powder. Furthermore, there is no information on the effect of the test substance on females, which does not allow a conclusion on the effect in females.

- the guideline foresees the usage of a positive control to demonstrate the laboratory ability to carry out the study and the sensitivity of the procedures used. Apparently, no positive control was used in the study and, therefore, the sensitivity of the procedures cannot be assessed.

- a five-day dosing regime was used during the study. However, the OECD guideline foresees a seven day dosing regime and states that a shorter dosing regime needs to be justified. The justification is missing.

- clinical signs and mortality were observed according to the authors of the study, which is also foreseen by the guideline. However, data on these parameters were not presented in the publication, which makes it problematic to evaluate the systemic toxicity of the substance.

- detailed clinical examinations, functional tests, body weight, food consumption, and ophthalmological examination were not examined/recorded. These examinations are foreseen by the OECD guideline. Again, this makes it problematic to evaluate the systemic toxicity of the substance. Especially, due to the fact that functional tests were not conducted in the current study, it is impossible to concluded about the impact of the histopathological change observed in the brain.

- incomplete histopathology was conducted. In the study, the frontal lobes of the brain were only investigated.

- no individual data and historical data were provided, which would improve an evaluation of the data.

Based on the shortcomings given above, this publication was disregarded for the hazard and risk assessment.

 

 

Summary entry – Neurotoxicity

Another 6 references were identified during literature search, representing in vivo neurotoxicity investigations in rats and mice, following inhalation (intranasal), intraperitoneal, or intracerebroventicular of nano-sized titanium dioxide. Furthermore, an additional three references were identified representing in vitro neurotoxicity investigations in cell cultures (e.g. neurons, microglia). The study designs of the in vivo and in vitro studies are not in accordance with accepted guidelines and are therefore of limited relevance for chemicals hazard assessment. Further, the references usually lack significance due to, e.g., poor test item characterisation, missing dose response relationship, low number of animals used, preliminary data presented only, using non-physiological route, or cell culture model not validated. It is therefore concluded that all references do not fulfil the criteria for quality, reliability and adequacy of experimental data under REACH for hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The studies given below were therefore included in the IUCLID for information purposes only:

 

In vivo studies:

Younes et al. 2015: the methodical setup is not adequately designed for risk assessment purposes of the test substance. The non-physiological route of administration via intraperitoneal injection is not guideline conform and not suitable to assess toxicity of the test substance after repeated administration. The test material is insufficiently characterised (self-synthesised), only one dose was administered and age of the test animals were not specified.

 

Balvay, A. et al. (2013): the methodical setup is not adequately designed for risk assessment purposes of the test substance. The non-physiological route of administration via intracerbroventricular injection is not guideline conform and not suitable to assess toxicity of the test substance after repeated administration. General health status of the animals is not documented. Furthermore, only preliminary data (1 to 3 animals per group) of the histopathological analysis were available since it was not completed until the date of publication.

Su, M. (2014): since the test item is poorly described and also the influence of the other simultaneously administered process chemicals is not addressed, this raises doubts as to whether (i) nano-sized TiO2 particles at all were used in this experiment, and (ii) whether the effects can really be attributed to titanium dioxide and (iii) the methodical setup is not in accordance with any guideline. A detailed evaluation of the statistics and given raw data with standard deviations showed that the standard deviations are always 5%. The non-physiological route of administration via intranasal inhalation is not guideline conform and not suitable to assess neurotoxicity. The experimental design is insufficiently documented (division of the test animals (individually or in groups) in the cages). Only one sex was used and the number of animals per group (n = 5 or 10 of 25) for some neurotoxicity tests on hippocampi is not sufficient to perform an appropriate statistical analysis. No functional observations or weekly body weights and the daily food consumption was not examined. Furthermore, there are no clinical signs, detailed clinical examinations and ophthalmology. A positive control, historical control and individual data is missing. Overall, only selected parameters were investigated and the investigated endpoints are 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).

 

Sheng, L. (2014): since the test item is poorly described and also the influence of the other simultaneously administered process chemicals is not addressed, this raises doubts as to whether (i) nano-sized TiO2 particles at all were used in this experiment, and (ii) whether the effects can really be attributed to titanium dioxide and (iii) the methodical setup is not in accordance with any guideline. A detailed evaluation of the statistics and given raw data with standard deviations showed that the standard deviations are always 5% or 1 %. The non-physiological route of administration via intranasal inhalation is not guideline conform and not suitable to assess neurotoxicity. The experimental design is insufficiently documented (division of the test animals (individually or in groups) in the cages). Only one sex was used and the number of animals per group (n = 5 or 10 of 40) for neurotoxicity tests on hippocampi is not sufficient to perform an appropriate statistical analysis. No weekly body weights and the daily food consumption was not examined. The data of clinical signs and the mortality are missing.

 

Mohamed et al. (2019): the methodical setup is not adequately designed for risk assessment purposes of the test substance. The non-physiological route of administration via intraperitoneal injection is not guideline conform and not suitable to assess toxicity of the test substance after repeated administration. Since the test item is poorly described and also the influence of the other simultaneously administered process chemicals is not addressed, this raises doubts as to whether (i) nano-sized TiO2 particles at all were used in this experiment, and (ii) whether the effects can really be attributed to titanium dioxide. General health status of the animals is not documented. Further, only two doses were administered, which does not allow a dose-response related analysis. The concentration of the applied doses was not analysed and only male mice were used. Further, the methods are insufficiently described and individual data were not given. Overall, due to these limitations this study is considered unreliable.

 

Latif et al. (2019): the methodical setup is not adequately designed for risk assessment purposes of the test substance. The non-physiological route of administration via intraperitoneal injection is not guideline conform and not suitable to assess toxicity of the test substance after repeated administration. Since the test item is poorly described and also the influence of the other simultaneously administered process chemicals is not addressed, this raises doubts as to whether (i) nano-sized TiO2 particles at all were used in this experiment, and (ii) whether the effects can really be attributed to titanium dioxide. The dosing scheme of 28 days every second day is not justified, and the general health status of the animals is not documented. The number of animals per group (n=5) is too low for a valid statistical analysis. The concentration of the applied doses was not analysed, and only male rats were used. Further, the methods are insufficiently described, and individual data were not given. Overall, due to these limitations this study is considered unreliable.

 

In vitro studies:

Long, T.C. et al. (2007): the cell culture models (BV2, N27), as well as the primary cultures (BrainBits: tissue plugs of embryonic Sprague-Dawley rat brain striatum) used in this study are not sufficiently validated for the use in risk assessment. Regarding the assays, results are questionable since there are experimental / methodical deficiencies (e.g. no data of calibration of the test system, no positive controls and an insufficient characterisation of test material).

 

Long, T.C. et al. (2006): the cell culture model (BV2) used in this study are not sufficiently validated for the use in risk assessment. Regarding the assays, results are questionable since there are experimental / methodical deficiencies (e.g. no data of calibration of the test system, no positive controls).

 

Mu, X. et al. (2020): the cell culture model (hippocampal neurons of 24-hours old Sprague-Dawley rat brain) used in this study are not sufficiently validated for the use in risk assessment. Regarding the assays, the results are questionable since there are experimental / methodical deficiencies (e.g. no data calibration of the test system, no positive controls).

 

Conclusion

There is currently no relevant and reliable guideline compliant study available for the endpoint neurotoxicity. During comprehensive literature search, thirteen references were identified, which reported in vivo and in vitro studies investigating the potential of neurotoxicity of nano-form titanium dioxide. The study designs of these studies are not in accordance with accepted guidelines and are therefore of limited relevance for chemicals hazard assessment. Further, the references usually lack significance due to, e.g., poor test item characterisation, missing dose response relationship, low number of animals used, preliminary data presented only, using non-physiological route, or cell culture model not validated. It is therefore concluded that all references do not fulfil the criteria for quality, reliability and adequacy of experimental data under REACH for hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The studies were therefore included for information purposes only.

Justification for classification or non-classification

No conclusion can be drawn from the 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 section represent in vitro and in vivo experiments with investigations on neurotoxicity 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.