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Trouiller study - unsupported conclusions

Via E-Mail George C. Prendergast, Ph.D. Editor-in-Chief Cancer Research 615 Chestnut Street 17th Floor Philadelphia, PA 19106-4404 Genetic Instability In vivo in Mice”, B. Trouiller, et al., Cancer Research, 2009; 69:8784 The Titanium Dioxide Stewardship Council (TDSC)1 and the Titanium Dioxide Manufacturers Association (TDMA)2 submit these comments on the UCLA study published in the journal Cancer Research the week of November 15, 2009, titled: “Titanium Dioxide Nanoparticles Induce DNA Damage and Genetic Instability In vivo in Mice.” The TDSC appreciates the scholarship that went into the study, but wishes to note several critically important deficiencies with the study. Importantly, the study does not differentiate clearly between pigmentary TiO2 (0.25-2.5μm) and TiO2 nanoparticles (<0.10μm). The study states that “Titanium Dioxide (TiO2) nanoparticles are manufactured worldwide in large quantities for use in a wide range of applications….” The study neglects to note, however, that the production of nanoparticle TiO2 comprises less than 1% that of pigment grade TiO2. Importantly also, the end uses are different. Pigment brings opacity and whiteness to paints, plastics, papers, inks, food, and drugs. Nanoparticles of TiO2 are transparent and their property of UV absorbency is desirable in cosmetics and catalysts. In the study, TiO2 nanoparticles were given to mice in drinking water. Contrary to what the study implies, TiO2 nanoparticles are not used in any applications intended for human or animal consumption, such as the toothpaste and food colorant examples noted in the text. Therefore, ingestion is not a relevant route of exposure for nanoparticle TiO2. Nanoparticle TiO2 is intended 1 The members of TDSC include: DuPont, Millennium Inorganic Chemicals -- A Cristal Company, Huntsman Corporation, TRONOX LLC, and Kronos Worldwide, Inc. TDMA was formed in 1974 to promote the interests of the European TiO2 industry. TDMA operates under the governance of CEFIC. George C. Prendergast, Ph.D. March 3, 2010 Page 2 for applications that involve its incorporation into a substrate or matrix. An effective combination of engineering controls and personal protective equipment minimize worker exposure during manufacturing and incorporation. Once in a substrate, there is no or very little exposure to the TiO2 dust. Toxicologists from TDSC member company laboratories have reviewed the referenced paper and have expressed concerns about how these studies were designed and conducted. Concerns include the lack of internal particle controls, lack of use of time course evaluations, and, a key concern, the high doses used in the study. A serious concern was that only one type of nanoparticle TiO2, a highly active photocatalyst, was used to represent all types of nanoparticle TiO2. The TiO2 photocatalyst used consists largely of anatase, is not surface treated, and has a primary particle size of 20 nm. These properties are not representative of all commercial grades of nanoparticle TiO2. A more comprehensive technical response to the study, in preparation by expert resources from both the TDMA and the TDSC, will be forthcoming in the next 1-2 months. The TDSC is aware of the challenges to environment, health, and safety brought by certain applications of nanotechnology. Our member companies’ commitment to product stewardship reflects these challenges and accounts for them accordingly. Thank you for considering these comments. If you have any questions, please call me at (410) 229-4560 or e-mail at Curt.DeMille@cristalglobal.com. Curt DeMille Chair Titanium Dioxide Stewardship Council COMMENTS ON DNA DAMAGE CAUSED BY
NANOPARTICULATE TITANIUM DIOXIDE: TROUILLER ET AL
PAPER 2009

EXECUTIVE SUMMARY

A publication by Trouiller et al. (2009) in the journal Cancer Research reports that
nanoparticulate titanium dioxide (TiO2, Aeroxide P25, Evonik, primary particle size of 21
nm) administered to mice in the drinking water produces a uniformly positive response in
a number of genotoxicity assays. The technical design of the studies appears to be
nonstandard, and the results implicate TiO2 as the causative agent for DNA damage,
resulting from single and double stranded breaks and oxidative damage that is considered
secondary to inflammation and/or oxidative stress. The authors claim that “These results
represent the first comprehensive in vivo genotoxicity study of TiO2 nanoparticles” and
further that “These data suggest that we should be concerned about a potential risk of
cancer or genetic disorders especially for people occupationally exposed to high
concentrations of TiO2 nanoparticles.”. For a number of reasons, the current studies fall
short of providing adequate support for the claims made. In particular, statistically
significant effects were generally only reported or measured at the highest exposure level
tested. The magnitude of most reported effects were on the order of 2-fold or less, and
thus represented marginal responses. As a result of the non-conventional test methods
employed, as well as a lack of more conventional endpoints for comparison, any
interpretation of possible human health hazards from this work is questionable.
BACKGROUND INFORMATION

Findings from Trouiller et al. 2009:

Trouiller et al. (2009) administered nanoparticulate TiO2 as a suspension in the drinking
water to mice at concentrations such that the mice received approximate total dosages of
0, 50, 100, 250 or 500 mg/kg body weight (bwt) over 5 days (male mice) or 10 days
(pregnant mice, on days 8.5 to 18.5 post coitum, 500 mg/kg bwt). A number of assays
were described in this paper that measured DNA damage (breakage), oxidative DNA
damage, and inflammation. DNA strand breakage (both single-strand and double strand)
was measured in the Comet assay, the micronucleus assay, and the -H2AX
immunostaining assay. Oxidated DNA damage was assayed by measuring 8-hydroxy-2’-
deoxyguanosine (8-OHdG) levels in livers from mice after 5 days of dosing. In addition,
DNA deletion (a genetic instability endpoint) was measured as unpigmented retinal
pigment epithelium (RPE) in the eyes of the offspring of homozygous (pun/pun) mice.
Inflammatory response was measured as the presence of mRNA levels of inflammatory
cytokines in peripheral blood. The results suggest that nanoparticulate TiO2 administered
orally produced a genotoxic response, possibly secondary to inflammation and oxidative
stress. All assays for DNA damage (with the exception of the -H2AX immunostaining
assay) showed significant and positive effects only at the highest dose level tested. The
Trouiller et al., 2009/Summarized Comments -H2AX immunostaining assay, a marker for DNA double-strand breaks, proved to be
the most sensitive assay employed and indicated significant increases in -H2AX-positive
cells in the bone marrow of mice (5 days of treatment) at all treatment levels. At the
highest dose level, proinflammatory cytokines (but not anti-inflammatory) were increased
indicative of a possible direct effect on circulating effector cells with secondary effects in
peripheral organs.
Genotoxicity Studies with TiO2:

There is little definitive information available concerning the genotoxic potential of TiO2
in vivo. Micronucleus formation in bone marrow cells and peripheral blood lymphocytes
was enhanced following intraperitoneal injection of TiO2 (form unspecified) in mice, but
the effect was not dose-dependent over the range 200 to 1500 mg/kg bwt. (Shelby et al.,
1993; Shelby and Witt, 1995). In a companion chromosomal aberration test in mice, no
clastogenic response was recorded (Shelby and Witt, 1995). Enhanced mutagenesis in rat
alveolar cells, measured at the hypoxanthine-guanine phosphoribosyl transferase (HPRT)
locus, was observed after intratracheal administration of 100 mg/kg bwt of TiO2,
(anatase, median diameter = 0.18 m), a dose that elicited persistent lung inflammation
and is suggestive of an effect due to cytotoxicity (Driscoll et al., 1997). Enhanced
mutagenesis was not observed at a dose of 10 mg/kg bwt.
There is conflicting information from available studies concerning the in vitro genotoxic
potential of nanoparticulate TiO2. Nanoparticulate TiO2 has failed to display genotoxic
activity in a number of standard in vitro assays including: the Ames test using Salmonella
typhimurium
or Escherichia coli (Warheit et al., 2007; Theogaraj et al., 2007);
chromosomal aberrations in Chinese hamster ovary cells (Warheit et al., 2007); or
cytogenetic studies in cultured rat liver epithelial cells (Linnainmaa et al., 1997). In
apparent contrast, studies reported by Wang et al. (2007) indicate genotoxicity and
cytotoxicity of ultrafine TiO2 produced in cultured human lymphoblastoid cells. Rahman
et al. (2002) have reported that ultrafine TiO2 induces micronuclei and apoptosis in
Syrian hamster embryo fibroblasts.
Limitations of the Trouiller et al. 2009 Study:

The genotoxicity results reported by the authors appear to be indicative of genotoxicity,
and at most provide a basis for confirmatory follow-up studies using more appropriate
and robust standardized methodologies. Indeed, from an experimental design
perspective, the Trouiller et al. study unfortunately has several weaknesses that limit
interpretation of the study results. These include but are not limited to the following
issues:
1. The authors provided inadequate physicochemical characterization of the nanoscale TiO2 particle-types used in the study. In particular, more information regarding surface chemistry and reactivity, surface coatings and purity should have been included (Warheit, 2008); Trouiller et al., 2009/Summarized Comments 2. Exceedingly high but unconfirmed doses of TiO2 nanoparticles were used in the study. Given the small group sizes (5 animals/group), and the use of estimated delivered doses, it is possible that certain animals received significantly larger delivered doses, thus providing a possible explanation for some of the minimal effects noted; 3. No attempt was made to accurately measure or to document the biokinetics or dose of ingested nanoscale TiO2 particles to any target tissues, thus a correlation of response with tissue dose is not possible. Considering the research approach and claims made based on the data, this would have been a necessary inclusion; 4. Benchmark positive control test samples, as well as positive control and historical control data, should have been included. The lack of such information precludes intra-laboratory comparisons of results and calls into question any interpretation of minimally significant effects; 5. No justification was provided for utilizing mice of an age of 4-5 months; younger mice (i.e., 2-month old) are generally used in standardized genetic toxicity assays. This deviation from normal protocol corresponds to a lack of appreciation of the potential age-effects on the erythrocyte population. The reported micronuclei results did not include a measurement of the ratio between immature and mature erythrocytes, and it is not clear which cell population was used for the micronucleus analyses. Therefore it cannot be excluded that the observed positive micronucleus result was caused by an age-related accumulation of micronuclei in the target cell population rather than exposure to TiO2. The claim that is made by the authors that “These results represent the first comprehensive in vivo genotoxicity study of TiO2 nanoparticles” is not supported by the data. In particular, the studies described in Trouiller et al. (2009) differ significantly in design from more robust studies conducted under recognized guidelines, thus making an interpretation of the findings difficult. Although the weight of evidence from these studies suggests DNA damage has occurred, there is no indication from the findings whether such damage would have been repaired, caused lethality or induced heritable mutations. An additional major deficiency is the lack of measures of cytotoxicity. DNA damage can result indirectly from such cytotoxicity and it is thus possible that some of the reported effects were observed in the presence of a cytotoxic response. In the Comet assay, the changes reported were < 1.5-fold and were quite minimal in nature. Without comparisons to laboratory control data, such a small increase cannot be evaluated. A 2.1-fold increase in micronuclei in peripheral blood, reported at the highest dose level only, suffers from a similar deficiency. The significance of a 1.27-fold increase in the incidence of pink-eyed unstable locus mutations measured only at the highest dose level is also difficult to interpret. This assay is not widely used and the biological relevance of such a minimal effect is difficult to evaluate. The biological relevance of any of the marginally positive findings can be argued. We have reason to suggest, based on the known photoreactivity of TiO2, with possible implications for such endpoints as oxidative DNA damage (8-OHdG) and the Comet assay, that perhaps some of the effects recorded Trouiller et al., 2009/Summarized Comments in the current studies were artifacts of the methods employed and were not a direct
consequence of the in vivo exposures.
The findings in the -H2AX assay were of more significance. The data showed dose-
related increases of up to 30% at the highest dose tested and with significant (p < 0.001)
increases produced at all dose levels. The -H2AX assay has proven useful for the
detection of DNA double-strand breakage and repair following treatment with ionizing
radiation or with a number of DNA damaging chemicals (Löbrich et al., 2010).
However, -H2AX foci can be produced at lesions other than double-strand breaks,
making the assay an indirect rather than direct measure of double-strand breaks. In the
current paper, no corroborating assay, such as the Comet assay, was performed on bone
marrow samples. In fact, the Comet assay performed on peripheral blood showed only a
minimal response and only at the highest dose level tested. The changes noted in the -
H2AX assay are most likely of biological significance, but without information to
indicate such a response would lead to a viable and permanent genetic alteration, it is not
possible to ascribe a potential health hazard based on this assay.
The proposal by Trouiller et al. (2009) that the measured genotoxicity of nanoparticulate
TiO2 may result from inflammation and oxidative stress is supported by evidence
presented. Increases in pro-inflammatory cytokines are in line with an induced oxidative
stress from TiO2 exposure. It is also reasonable to postulate that such oxidative stress
could lead to the other genotoxic results presented including single- and double-stranded
DNA breaks, deletions and oxidized DNA. However, levels of 8-hydroxy-2’-
deoxyguanosine (8-OHdG), indicative of an oxidative DNA lesion, were only increased
1.5-fold at the highest administered dose. With no data from other dose levels (dose-
response) and without concurrent cytotoxicity measures, the biological significance of
this small increase cannot be adequately assessed. The current study is further limited by
a lack of a single assay performed in all affected tissues, thus impeding any continuity of
interpretation.
In summary, the Trouiller et al. genotoxicity results with nanoscale TiO2 particulates
appear to be intriguing as a preliminary finding. However, the data would be more
compelling if the authors had generated results using a standardized in vivo genotoxicity
assay, along with adequate material characterization, appropriate assessments of doses to
target tissues, dose response and time course experimental designs, and corresponding
benchmark positive control test substances. Accordingly, it seems clear that standardized
models and reproducibility of the results are necessary prerequisites for the establishment
of verifiable genotoxicity assay results.
Discussion and Proposed Study Protocols

In the current studies reported by Trouiller et al. (2009), a mutagenic response in mice
treated orally with nanoparticulate TiO2 was reported and suggested to be secondary to an
inflammatory response and oxidative stress. In the absence of measures of cytotoxicity,
and with predominantly minimal effects observed at only the highest dose level tested,
the relevance of these findings for the assessment of potential adverse health effects is
Trouiller et al., 2009/Summarized Comments questionable. Oral absorption of nanoparticulate TiO2 with systemic distribution is suggested from the results; however, the lack of tissue measurements of TiO2 as well as the lack of a consistent bioassay system applied to all potential target tissue does not allow a meaningful correlation of tissue response with dose to be made. The authors contend “These data suggest that we should be concerned about a potential risk of cancer or genetic disorders especially for people occupationally exposed to high concentrations of TiO2 nanoparticles.”, with the implication that this applies to the oral route of exposure. However, more conventional 2-year feeding studies in rats and mice with pigment-grade TiO2 at levels in feed up to 50,000 ppm (5%) were without effect (NTP, 1979). And although lung tumors in rats, under conditions of lung-overload, are produced after chronic inhalation exposure to either pigment-grade or nanoparticulate TiO2 (Lee et al., 1985; Heinrich et al., 1995), tumors in other organs are absent. In addition, epidemiological evidence from well-conducted investigations has shown no causal link between TiO2 exposure and the risk of cancer in humans (Chen and Fayerweather, 1988; Fryzek et al., 2003; Boffetta et al., 2001 and Boffetta et al., 2004). In order to further investigate the preliminary findings of genotoxicity of nanoscale TiO2 particles reported by Trouiller et al., the following testing strategy is proposed:  Prior to commencing the study, the physicochemical characteristics of the nanoparticle test substance should be rigorously characterized, in a manner as previously described (Warheit, 2008). Some of these characterization elements include – crystal structure, particle size distribution, purity, surface area, and shape, as well as additional particle surface characteristics such as reactivity, coatings, and agglomeration.  Following adequate material characterization of the TiO2 nanoparticle-type, a standardized in vivo micronucleus assay (OECD 474) which detects chromosomal breakage and disturbance of mitotic spindles) (rat or mouse species) should be implemented, concomitant with a Comet assay (DNA damage). It is suggested that oral gavage should be the route of administration/exposure.  Positive control groups should include cyclophosphamide, as traditionally used for these studies, concomitant with a metal nanoparticulate positive control particle such as Chromium (Cr VI) or a Nickel compound. The inclusion of cyclophosphamide demonstrates the reproducibility of the assay. Moreover, the addition of Ni or Cr provides an attempt to establish a metal nanoparticulate positive control sample for this assay. The results obtained from a study employing this proposed testing strategy (Warheit and Donner, 2010) may produce useful insights and would serve to confirm or challenge the findings of Trouiller and coworkers. Trouiller et al., 2009/Summarized Comments
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