Task Force Nanoscience
Titanium Dioxide Project
Development
Luigi Manzo
S. Maugeri Medical Centre Department of Internal Medicine
University of Pavia
3rd National N.I.C. Conference, Milano, 2-3 December 2009.
Safety of Manufactured Nanomaterials.
Key Uncertainties.
Health and environmental impacts
Adequacy of existing testing methods.
Regulatory framework
REACH Regulation and Safety Assessment of
Nanomaterials. A Roadmap Using Titanium Dioxide a
Model Agent.
Federchimica RSI Committee NIC Programme
Task Force Nanoscience REACH Centre Pavia University
.
REACH Regulation and Safety Assessment of
Nanomaterials. A Roadmap Using Titanium Dioxide a
Model Agent.
Aims
To review and evaluate existing toxicological, ecotoxicological
and biokinetic data on TiO2 according to legal requirements
(REACH Regulation).
To test the workability of the organisational set up of REACH
using TiO2 as a model nanomaterial.
To evaluate the adequacy of current testing methodologies and testing needs for hazard estimation, as required by REACH.
Number of PubMed Listed Publications,
2005-2008.
REACH Regulation and Safety Assessment of
Nanomaterials. A Roadmap Using Titanium Dioxide
as a Model Agent.
• Collection of all the relevant recorded knowledge (using
pre-defined inclusion/exclusion criteria)
• EBT data evaluation (using an ex-ante quality instrument)
• Data input (IUCLID-5)
• Data gap analysis, identification of research needs
Evidence-Based Safety Evaluation of
Nanosize Titanium Dioxide.
Limited data Humans Isolated studies Aquatic, terrestrial organisms (Ecotox) “Outcomes” research Laboratory animals “Outcomes” research Isolated (cell culture)
systems
Level of evidence
Bioreceptor
Cellular Uptake of Rutile, Anatase and Coated
TiO
2NPs.
Z. Pan et al, Small 2009..
Flow cytometry data showing different
particle uptake of cells incubated with 0.4 mg mL-1 rutile, anatase, and coated TiO2
nanoparticles for 2 days. The phase
contrast images of cells with attached particles were taken prior to the flow cytometry analysis.
control rutile anatase coated
control (median: 2.37) rutile TiO2 (median: 10.46) anatase TiO2 (median: 36.52) coated TiO2 (median: 3.85) 120 100 80 60 40 20 0 100 101 102 103 104 FL4-H C ounts
Comparative Biokinetics of Fine (FTiO
2) and Ultrafine
(UFTiO
2) Titanium Dioxide after Intratracheal
Instillation
Rats exposed to an equal surface area dose (0.52 mg/rat or 10.7
mg/rat for UFTiO2 and FTiO2, respectively).
Changes in TiO2 levels from 7 to 42 days post-exposure:
TiO2 remaining in the lung
UFTiO2: 51% decrease
FTiO2: 17% decrease
TiO2 accumulation in the tracheo-bronchial and thymic lymph nodes
UFTiO2: 246% increase
FTiO2: 134% increase
Tox Sci, 2009
“Lack of Dermal Penetration following Topical
Application of Coated and Uncoated Nano- and
Micron-Sized Titanium Dioxide to Intact and Dermoabraded
Skin of Mice”.
N.V. Gopee, C. Cozart, P. Siitonen, C.S. Smith, N.J.
Walker, P.C. Howard
US FDA Natl Center for Toxicological Research Jefferson AR, NIEHS Research Triangle Park NC
contral 10 nm 25 nm Degussa P2,5 60 nm normal size contral 10 nm 25 nm Degussa P2,5 60 nm normal size
skin sub.muscles heart liver
spleen lung kidney brain
conce ntrati on of T i (µg / g) conce ntrati on of T i (µg / g) A B 80 60 40 20 0 35 30 25 20 15 10 5 0 Titanium contents in tissue of hairless mice after dermal exposure
to different sized TiO2
nanoparticles for 60 days.
(A) Skin, subcutaneous muscle, heart, liver.
(B) spleen, lung, kidney, brain.
Proposed Mechanisms of “Primary” and
“Secondary” NP-Induced Genotoxicity.
In vitro genotoxicity testing allows for the identification of primary genotoxicity of nanoparticles, which may result from either direct (e.g. physical interaction between nanoparticles and genomic DNA) or indirect pathways (e.g. formation of ROS by nanoparticle-activated target cells). Secondary genotoxicity implies a pathway of genetic damage resulting from oxidative DNA attack by ROS, generated from activated phagocytes (neutrophils, macrophages) during particle-elicited inflammation.
Findings from Toxicity Assays Applied to
Nanoscale TiO
2• Pulmonary Bioassay: low toxicity
• Acute Oral Toxicity Test: low toxicity
• Skin Irritation Test: not a skin irritant
• Eye Irritation: minor ocular conjunctival
redness
• Skin Sensitization – LLNA: not a sensitizer
• Genotoxicity Tests – Ames: negative
Chromosomal Ab Study: negative
• Aquatic Battery - Rainbow Trout: low hazard
• Daphnia: low hazard
• Algae: medium concern
Updated (2009) Summary of Findings from
Recent Studies on Nanoscale TiO
2• Pulmonary Bioassay: high/medium concern
• Acute Oral Toxicity Test: low toxicity
• Skin Irritation Test: not a skin irritant
• Eye Irritation: minor ocular conjunctival
redness
• Skin Sensitization – LLNA: not a sensitizer
• Genotoxicity Tests: negative or positivenegative
• Aquatic Battery - Rainbow Trout: low hazard ?
• Daphnia: lowlow hazard ?hazard
Study limitations:
• Lack of material characterisation
• Unreplicable studies
• Unrealistic doses/concentrations
• Lack of comparative evaluation (no positive control) • Several eperiments “investigator-motivated”
Nano TiO2 data often constrasting with SDS information
Systematic Review of Nano TiO
2
Guidance for Initial Safety Assessment
.
• Minimal set of toxicological assays
• Biological fate of the test agent
• Realistic dosages and routes of exposure
• Case-by-case approach in study design
Safety Assessment of Nanomaterials. Initial
In Vitro Screening.
• Cytotoxicity (functional endpoints)
• ROS generation, oxidative stress
• Pro-inflammatory response
• Biocompatibility, blood contact properties
• Genotoxicity
Proposed Tiered Research Approach to Toxicity
Testing for Nanomaterials.
• Physico-chemical characterisation
• Preliminary in silico evaluation (SAR modelling, read-across, computational data gap filling, etc)
• In vitro (cell/tissue cultures) • In vitro ex vivo
• Limited, justifiable in vivo testing
Future Perspectives for Preclinical Testing
of Nanotechnology Derived Products.
• Study of biomarkers (most in vitro or ex-vivo)
that may be useful in identifying potential
risks to humans*
• New technologies to help identify early
toxicity and mode-of-action (omics, imaging)
.
Current understanding
• TiO2 prepared in a particular nanoform may be more
hazardous than in other physico-chemical forms. However this is not necessarily the case.
• There is no good evidence that unique hazardous properties
can arise from exposure to TiO2 in the nanoform.
• Our current knowledge is insufficent to conduct toxicological studies by alternative methods (cell cultures, read across, etc). However, considerable progress in recent years
• A case-by-case (mechanistic) approach should be used in study design
Titanium Dioxide Project
Federchimica
Dania Della Giovanna
