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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
not specified

Data source

Reference
Reference Type:
publication
Title:
A study of the uptake and biodistribution of nano-titanium dioxide using in vitro and in vivo models of oral intake
Author:
MacNicoll, A. et al.
Year:
2015
Bibliographic source:
J. Nanopart. Res. 17: 66.

Materials and methods

Objective of study:
absorption
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of 6 male Sprague-Dawley rats were administered a single dose of 4.6 mg/kg of either two different titanium dioxide nanoforms (40 nm and 40-50 nm) each or two micron-sized (120 nm and < 5 µm) titanium dioxide form each. During a post-administration period of 96 hours, rats were housed in metabolic cages for collection of urine and faeces. Furthermore, blood samples were taken during the post-administration period. After sacrifice (96 hours post treatment), tissue samples (liver, brain, heart, kidney, spleen and GI tract) were collected. The samples of urine, faeces, blood and tissues were analysed for titanium levels via ICP-MS.
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid: nanomaterial & solid: particulate
Details on test material:
The authors determined the particle size distribution and the agglomeration state of each material using a number of characterisation methods. These included transmission electron microscopy, scanning electron microscopy and CLS disc centrifugation for all the test materials. Each of the characterisation methods required a varying degree of dilution. These dilutions were prepared from a working stock dispersion of each material.

1) Name of test material (as cited in study report): NanoAmor 5430 MR
- Analytical purity (supplier information): 99.7 %
- Particle size (nominal; supplier information): ~ 15 nm
- Particle size (measured): 40 nm
- Crystal structure (supplier information): anatase

2) Name of test material (as cited in study report): Sigma cat # 637252
- Analytical purity :(supplier information) 99.5 %
- Particle size (nominal; supplier information): <100 nm
- Particle size (measured): 40 - 50 nm
- Crystal structure (supplier information): rutile

3) Name of test material (as cited in study report): NanoAmor 5430 MR
- Analytical purity (supplier information): 99.7 %
- Particle size (nominal; supplier information): ~ 15 nm
- Particle size (measured): 120 nm
- Crystal structure (supplier information): anatase

4) Name of test material (as cited in study report): Sigma cat # 224227
- Analytical purity (supplier information): 99.5 %
- Particle size (nominal; supplier information): < 5000 nm
- Particle size (measured): up to 5 µm
- Crystal structure (supplier information): rutile
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
All of the supplied ‘nanomaterials’ tested contained around ~80 % by weight of larger (typically between 200 and 500 nm) structures. Conversion of mass-based particle size distribution to particle number-based values still showed the majority of the particles to be the nanoscale.
High-powered sonication, followed by centrifugation, separated the nano-sized fraction by sedimenting larger-sized clusters. The nano-dispersions obtained by this method were stable in aqueous media over a few hours. The nanoscale fractions isolated as a supernatant from this procedure were either used straightaway as such, or freeze-dried and resuspended before use. Average nano-fraction obtained by this method was around 17 % (n = 3) of the original as measured by weighing after freeze-drying. Similar results were obtained by DLS method, although the hydrodynamic sizes measured by DLS are generally larger than those measured by other methods. The results of DLS measurements showed that between 10 and 17 % of the particles had a size in the nanoscale, whilst the remaining particles were in larger size range (600–700 nm). It was found that particle agglomeration had again taken place in freeze-dried nanofractions on resuspension in aqueous media. The materials and sample preparation methods were therefore adapted appropriately to obtain a uniform nano-fraction for use in in vivo experiment.

1) Name of test material (as cited in study report): NanoAmor 5430 MR (40 nm)
Dispersed in water and ovalbumin solution, sonicated, nano-fraction separated by centrifugation and used straightaway in experiments

2) Name of test material (as cited in study report): Sigma cat # 637252 (40 - 50 nm)
Dispersed in water and ovalbumin solution, sonicated, nano-fraction separated by centrifugation and used straightaway in experiments

3) Name of test material (as cited in study report): NanoAmor 5430 MR (120 nm)
Dispersed in water and ovalbumin solution. Nanofraction separated by centrifugation and freeze dried, which upon resuspension showed reagglomeration of particles to clusters of ~120 nm

4) Name of test material (as cited in study report): Sigma cat # 224227 (up to 5 µm)
Dispersed in water, sonicated and used straightaway in experiments as the non-nano control (< 5 µm)
Radiolabelling:
not specified

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
not specified
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Labs
- Age at study initiation: 8 weeks
- Weight at study initiation: 200 to 429 g (mean: 347.5 ± 46.6 g)
- Housing: a specially designed cage system was used to house individual rats to collect the faeces and urine samples during the experiment (McKenzie et al. 2010)*

*Reference:
- McKenzie J, Charlton A, Donarski J, MacNicoll A, Wilson J (2010) Peak fitting in 2D 1 H–13 C HSQC NMRspectra for metabolomic studies. Metabolomics 6(4):574–582.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: deionised water or 5 % ovalbumin
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Titanium dioxide materials were suspended in either 10 mg/mLdeionised water or 5 % ovalbumin solution.
The required amount of titanium dioxide dose was calculated for each rat and the rats were anaesthetised for test item administration.

Duration and frequency of treatment / exposure:
one administration only
Doses / concentrations
Dose / conc.:
4.6 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
6 male rats
Control animals:
yes, concurrent vehicle
Positive control:
not specified
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, and tissue samples (liver, brain, heart, kidney, spleen, large intestine and small intestine)

- Time and frequency of sampling: samples of blood, urine and faeces were collected immediately after oral gavage, and then at different time intervals over 4 days (blood: 0-2 , 24, 48, 72, and 96 hours; urine and faeces: 0 - 24, 24 - 48, 48 - 72, and 72 - 96 hours). Blood samples (0.3–0.4 mL) were collected and stored in lithium heparin tubes. Analysis of the sample showed no detectable titanium above background levels in the urine samples, which ruled out possible leaching of titanium dioxide from the faecal pellets into urine. After 96 hours postadministration of titanium dioxide, rats were killed and tissue samples of liver, brain, heart, kidney, spleen, GI tract (large and small intestine) were collected. The tissue samples were kept frozen and the urine, blood and faecal samples were kept in a cold room until analysed for titanium.
For titanium analysis, the samples were digested in acid/hydrogen peroxide and titanium dioxide quantified (as Ti ion) by inductively coupled plasma mass spectrometry (ICP-MS). For this, the TiO2 particles were solubilised by alkali or acid digestion, appropriately diluted, and measured using a ThermoFisher ‘Axiom’ ICP mass spectrometer tuned within 3,000–5,000 resolution to isolate the titanium signal from potential polyatomic interferences. Alkali digestion was used to solubilise TiO2 particles in aliquots of blood and urine and acid digestion was used for tissue samples. The digestion of faecal samples posed a particular challenge and the acid digestion was adapted to ensure complete digestion of TiO2 particles.


Statistics:
The measurement results were analysed in a linear mixed model for main effects only. Fixed Effects: nominal size + pre-treatment + vehicle, with rat, day and analytical run as crossed random effects. This model and method was chosen in response to the way that the experiment had been undertaken, rather than as a reflection of an underlying model for the effects that the various kinds of treatment might have. A value of p<0.05 was used for determining whether a main effect in the model was statistically significant.

Results and discussion

Preliminary studies:
A preliminary in vivo study was carried out to measure the background levels of titanium in rats before the main study. Three rats were used for this study, one of which was treated with the vehicle only to provide samples for determination of background exposure to titanium dioxide from diet and the environment, and the other two were orally administered with NanoAmor TiO2 (nominal particle size ~15 nm, measured size 40 nm) at 2 and 5 mg/kg bw. Samples (200 µL) of blood were collected at appropriate time intervals after titanium dioxide administration. Samples of urine and faeces were also collected and analysed for titanium content by ICP-MS.
Results:
The results of analysis of blood and urine indicated that very little titanium dioxide was absorbed within 24 hours at both 2 and 5 mg/kg bw treatment levels. However, the background levels of titanium were significantly above the limit of detection. The authors first thought that diet was the source of high background titanium levels, but an unexpected source was found to be the cardboard ‘‘toys’’ that were provided in the cages as ‘environmental enrichers’ which contained a considerable amount of titanium (~4.5 µg/g). As the rats with high background titanium levels were not suitable for this study, a fresh breeding nucleus of rats was established by feeding on a ‘titanium-free’ diet, and not providing them with the cardboard toys. The parental generation was maintained on this regime for 3–4 weeks before mating. Titanium levels in the tissues and faeces of the 50–60-day-old offspring of these rats were monitored to establish low background levels before use in the main in vivo study.
Main ADME resultsopen allclose all
Type:
absorption
Results:
The statistical analysis showed (with the exception of faecal excretion) that there no significant differences between any of the TiO2 treatments.
Results:
Tissue levels were very low (ng/g): liver (1.43-3.74), kidney (
Results:
Tissue titanium levels (in ng/g) of the control animals were in liver (2.46), in kidneys (

Toxicokinetic / pharmacokinetic studies

Details on absorption:
The results show that oral administration of TiO2 nano- (or larger) particles did not lead to translocation of titanium to blood in rat at any of the time intervals studied during the 96 hour post-treatment.
The result of statistical analysis showed that there were no significant effects between titanium dioxide treatments following oral exposure to titanium dioxide particles; and the variation in measurement results within each organ was consistent with that caused by analytical measurement variation.
The results show that oral administration of titanium dioxide nano- (or larger) particles did not lead to translocation of titanium to distribution to various organs in rat at any of the time intervals studied during the 96 h post-treatment. With the possible exception of one particle size, the mean concentration of titanium in the GI tract was not significantly affected by treatment.
Trace amounts of titanium were detected in the GI tract samples.
The results show that oral administration of titanium dioxide nano- (or larger) particles did not lead to translocation of titanium to urine in rat at any of the time intervals studied during the 96 hour post-treatment.
The orally administered titanium dioxide materials in all cases were found to be excreted in the faeces, although this happened at different elimination rates in different test animals.
The recoveries of titanium dioxide in faecal samples for the two Sigma materials (<100 and <5,000 nm) were much lower than the samples from the animals treated with other materials. Since titanium dioxide was not absorbed/translocated from the GI tract in any of the materials tested, this discrepancy indicates a possible analytical error due to incomplete solubilisation of the Sigma materials in faecal samples.
In all instances, the titanium dioxide materials were not completely cleared from the faeces at the end of the 96 hour sampling period.
Details on distribution in tissues:
not specified
Details on excretion:
not specified

Metabolite characterisation studies

Metabolites identified:
not specified

Bioaccessibility

Bioaccessibility testing results:
not specified

Applicant's summary and conclusion

Conclusions:
The following experimental result can be summarised here briefly:
- there was no detectable titanium in any of the urine samples. Interestingly, they observed a high dietary background in normal laboratory diet, which is why the main study was run with a low (0.44 ng/g) titanium diet. It was however later found that the source of the high background were cardboard “environment enricher” toys place in the rat cages (the abolishment of which led to low titanium background levels).
- the statistical analysis showed (with the exception of faecal excretion) that there no significant differences between any of the TiO2 treatments.
- in view of the dose given (5 mg/kg bw), the tissue levels were generally very low (in ng/g): liver (1.43-3.74), kidney (- the tissue titanium levels (in ng/g) of the control animals (n=6) were in liver (2.46), in kidneys (