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Development of a base set of toxicity tests using ultrafine TiO2particles as a component of nanoparticle risk management

David B. Warheit, Robert A. Hoke, Carol Finlay, E. Maria Donner,Kenneth L. Reed, Christie M. Sayes

DuPont Haskell Laboratory for Health and Environmental Sciences, Newark, DE, USA

Received 16 March 2007; received in revised form 24 April 2007; accepted 24 April 2007

Available online 27 April 2007

Abstract

Thedevelopment of a risk management systemfor nanoscaleor ultrafine particle-types requires a base setof hazarddata.Assessing

riskis a functionof hazard and exposure data.Previously, we have suggestedparalleltracks as a strategy forconducting nanoparticle

research. On the one hand, mechanistic studies on representative nanoparticles could be supported by governmental agencies.

Alternatively, with regard to commercial nanoparticles, the environmental, health and safety (EHS) framework would include a

minimum base set of toxicity studies which should be supported by the companies that are developing nano-based products. The

minimum base set could include the following criteria: substantial particle characterization, pulmonary toxicity studies, acute dermal

toxicity and sensitization studies, acute oral and ocular toxicity studies, along with screening type genotoxicity, and aquatic toxicity

studies.

We report here the toxicity results of a base set of hazard tests on a set of newly developed, well-characterized, ultrafine TiO 2

(uf-TiO2) particle-types.In vivo pulmonary toxicity studies in rats demonstrated low inflammatory potential and lung tissue toxicity.Acute dermal irritation studies in rabbits and local lymph node assay results in mice indicated that uf-TiO2 was not a skin irritant or

dermal sensitizer. Acute oral toxicity studies demonstrated very low toxicity and uf-TiO2 produced short-term and reversible ocular

conjunctival redness in rabbits. Genotoxicity tests demonstrated that uf-TiO2 was negative in both the bacterial reverse mutation

test and in an in vitro mammalian chromosome aberration test with Chinese hamster ovary cells. The results of aquatic toxicity

screening studies demonstrated that uf-TiO2 exhibited low concern for aquatic hazard in unaerated, 48 h, static acute tests using the

water flea, Daphnia magna; exhibited low concern for aquatic hazard in unaerated, 96 h, static acute tests using the rainbow trout,

Oncorhynchus mykiss; and exhibited medium concern in a 72 h acute test using the green algae Pseudokirchneriella subcapitata.

To summarize the findings, the results of most of the studies demonstrated low hazard potential in mammals or aquatic species

following acute exposures to the ultrafine TiO2 particle-types tested in this program.

2007 Elsevier Ireland Ltd. All rights reserved.

Keywords: Ultrafine particles; Nanoparticles; Nanomaterials; Nanoparticle toxicity testing; Minimum base set; Acute toxicity; Subchronic toxicity;

Pulmonary toxicity; Dermal toxicity; Skin sensitization; Oral toxicity; Ocular toxicity; Genotoxicity; Aquatic toxicity

Corresponding author at: DuPont Haskell Laboratory, 1090 Elkton

Road, PO Box 50, Newark, DE 19714-0050, USA.

Tel.: +1 302 366 5322; fax: +1 302 366 5207.

E-mail address: [email protected](D.B. Warheit).

1. Introduction

The intent of a nanoparticle risk management frame-

work is to implementa systematic process foridentifying

environmental health and safety (EHS) risks related to

exposures to newly developed engineered nanomaterials.

0378-4274/$ see front matter 2007 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.toxlet.2007.04.008
mailto:[email protected]://localhost/var/www/apps/conversion/tmp/scratch_6/dx.doi.org/10.1016/j.toxlet.2007.04.008http://localhost/var/www/apps/conversion/tmp/scratch_6/dx.doi.org/10.1016/j.toxlet.2007.04.008mailto:[email protected]

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The development and forthcoming commercialization

of many different engineered nanomaterial-types for

uses in a variety of industrial, chemical, manufactur-

ing, consumer, medical and diagnostic applications will

present a challenge for companies and regulators to

ensure the safety of products for workers and ultimately,

consumers. Moreover, the development of effectiveand safe products containing nanomaterials should be

a fundamental component of the product stewardship

process. This is an integral component of the broad

engagement process with stakeholders on EHS issues,

contributing to the public awareness and confidence

in products developed through nanoscale science and

engineering.

An EHS framework is being developed to character-

ize the potential risks related to exposures to nanoscale

or ultrafine particle-types. Of course, determination

of risk is a product of both exposure and hazardassessments. However, in many cases the exposure

potential cannot be ascertained, due in large part,

to the current limitations of technology to measure

nanoscale particle exposures in the workplace. Nonethe-

less, the risk management framework could include

a minimum base set of toxicity (hazard) screening

studies which provide a fundamental characterization

of the potential hazards of the particle-types being

investigated for human health and ecological effects.

This base set process is modeled, in part, on the

preparation of the SIDS (screening information datasets) used by OECD (Organisation for Economic

Co-operation and Development) and US EPA (United

States Environmental Protection Agency) for the inves-

tigation of HPV (High Production Volume) chemicals

(http://www.epa.gov/chemrtk/pubs/general/sidsappb.htm

http://www.epa.gov/chemrtk/pubs/general/sidsappb.pdf

http://www.oecd.org/dataoecd/13/14/36045229.pdf).

This base set serves as a reference point for the type of

screening information that should be addressed as a new

product is being developed. The generation of a base set

of information is important to sufficiently characterize

the EHS impacts of nanoscale or ultrafine materials andto build the foundational data that is necessary to develop

a risk assessment framework for nanomaterials.

The minimum base set is an evolving concept

designed to characterize the hazards associated with

exposures to nanomaterials, both in mammalian species

as well as in ecological environments. Justification for

these particular tests rests on the following criteria: (1)

potential routes of exposures (i.e., pulmonary, dermal,

oral and/or ocular); (2) screening for potential carcino-genic effects (mutation and chromosomal aberration

assays); and (3) screening for potential toxic aquatic

effects (exposures to rainbow trout, Daphnia, and algae).

The base hazard set of tests is not meant to provide for

an exhaustive assessment of toxicity, but is designed

to facilitate a reasonable balance between an adequate

toxicity characterization and a practical strategy for the

development of new nanomaterials. Thus, the goal is

to make the base set sufficiently robust in order to

guide adequate risk-evaluation processes, in a manner

commensurate with existing regulatory and voluntarystandards.

This manuscript briefly describes the methodology

and results of ten different toxicity studies conducted

with newly developed ultrafine TiO2 particle-types. Prior

to the commencement of the studies, substantial physic-

ochemical characterization of the test materials was

carried out. Subsequently, hazard studies were imple-

mented which included the following tests (see Table 1):

a doseresponse pulmonary bioassay study in rats with

postexposure periods lasting through 3 months; a der-

mal irritation test in rabbits; a skin sensitization study inmice; an acute oral toxicity study in rats; and an eye irri-

tation study in rabbits; two in vitro genotoxicity studies,

i.e. a bacterial reverse mutation test and chromosomal

aberration study; as well as three aquatic toxicity screen-

ing studies with rainbow trout, daphnia, and green algae.

Thus, it is proposed that these studies form the basis for

a minimum base set of hazard tests as a component of a

nanoparticle risk management system.

2. Methods

The following tests with newly developed ultrafine TiO2particle-types were conducted as components of the base set

of hazardassessment tests identified in Table 1. A briefdescrip-

Table 1

Base set hazard tests

Nanomaterial physiochemical characterization Mammalian hazard tests Genotoxicity tests Aquatic screening battery

Size and size distribution Pulmonary bioassay Bacterial reverse mutation Rainbow trout

Crystal structure Skin irritation Chromosomal aberration Daphnia

Chemical composition Skin sensitization Green algae

Surface reactivity Acute oral toxicity

Eye irritation
http://www.epa.gov/chemrtk/pubs/general/sidsappb.htmhttp://www.epa.gov/chemrtk/pubs/general/sidsappb.pdfhttp://www.oecd.org/dataoecd/13/14/36045229.pdfhttp://www.oecd.org/dataoecd/13/14/36045229.pdfhttp://www.epa.gov/chemrtk/pubs/general/sidsappb.pdfhttp://www.epa.gov/chemrtk/pubs/general/sidsappb.htm

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tion of each of the tests is provided in the methodology section

below.

2.1. Animals

All procedures using animals were reviewed and approved

by the Institutional Animal Care and Use Committee. TheDuPont Haskell Laboratory animal program is fully accred-

ited by the Association for Assessment and Accreditation of

Laboratory Animal Care (AAALAC).

2.2. Particle-types and physicochemical characterization

(see Fig. 1, Table 2)

An ILSI Research Foundation/Risk Science Institute Nano-

material Toxicity Screening Working Group has previously

developed the elements of a screening strategy for the haz-

ard identification of engineered nanomaterials (Oberdorster et

al., 2005). Fundamental to the conclusions of this report is thatadequate characterization of nanomaterials is required. This

represents one of the key aspects of toxicity screening strate-

gies. For the pulmonary bioassay studies conducted in rats, the

physicochemical characteristics of the ultrafine TiO2 particle-

types (identified as uf-A and uf-B) were measured in the dry

state (representing the supplied material) as well as in aque-

ous suspension, including water and phosphate-buffered saline

(PBS) solution vehicle (representing the administered material

exposed to the test subject).

An ultrafine particle is defined as a particle of average pri-

mary size of roughly 100 nm. Because both the uf-A, uf-B and

uf-C particulates have median particle sizes of

140 nm, withfractions of the size distribution that fall below 100 nm, they

are termed ultrafine. Often ultrafine particles have different

uses or have enhanced properties when compared to their bulk

counterparts. For certain uses they can be more effective than

their bulk counterparts because of their smaller size and higher

surface area.

For the pulmonary bioassay studies, two different ultrafine

TiO2 particles in the rutile crystal phase, designated as uf-A and

uf-B, were obtained from the DuPont Company. DuPont uf-A

is composed of a titanium dioxide core with an alumina sur-

face coating (98% titanium dioxide and 2% alumina) and

possesses a median particle size of 136 nm in water (aqueous

suspension buffered in 0.1%tetrasodium pyrophosphate) usingdynamic light scattering and an average BET surface area of

18.2 m2/g. DuPont uf-B is composed of a titanium dioxide core

with a silica and alumina surface coating (88 wt% titanium

dioxide, 7 wt% amorphous silica and5 wt% alumina,) and

possesses a median particle size of149.4 nm in water (aque-

ous suspension buffered in 0.1% tetrasodium pyrophosphate)

using dynamic light scattering andan average BETsurface area

of 35.7 m2/g (Brunauer et al., 1938) (see Fig. 1, Table 2).

Controls for the pulmonary bioassay study included fine-

sized TiO2 particle-types in the rutile form obtained from the

DuPont Company; P25 ultrafine TiO2 particles, consisting of

an 80:20 ratio of anatase:rutile TiO2 particles, purchased from

Fig. 1. Characterization of ultrafine and fine titanium dioxide (TiO2)

particles used in the pulmonary bioassay study. High resolution scan-

ning electron micrographs (HR-SEM) of ultrafine samples A and B

(labeled a and b) and fine sample (c). Micrographs depict variations in

surface treatment and particle size within each of the ultrafine and fine

TiO2 particle-types.

Degussa Corporation; and Min-U-Sil -quartz particles pur-

chased from US Silica Company. Fine-TiO2 is composed of a

titanium dioxide core with an alumina surface coating (99%

titanium dioxide and 1% alumina) and possesses a median

particle size of380 nm in water (aqueoussuspension buffered

in 0.1% tetrasodium pyrophosphate) using DLS and an average

BET surface area of 5.8 m2/g. All TiO2 rutile particle samples

underwent a neutralization process during production, which

neutralizes acidic chloride groups on the particle surface. P25

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Table 2

Characterization of ultrafine titanium dioxide (TiO2) particles used in the pulmonary (uf-A, uf-B); skin, ocular, oral, genetic, and aquatic toxicity

studies (uf-C)

Sample Surface area (m2/g) Crystalline phase Chemical reactivity delta ba Particle size and distribution (nm) pH in water

in watera (%) in PBS (%)

uf-A 18.2 100% rutile 10.1 136.0 35 1990 25 5.64uf-B 35.7 100% rutile 1.2 149.4 50 2669 25 7.14

uf-C 38.5 79% rutile; 21% anatase 0.9 140.0 44 4.80

Table of physical properties of the DuPont ultrafine TiO2 samples used in the study, including specific surface area, crystallinity, chemical reactivity,

particle size and distribution, and pH.a Indicates aqueous solution of 0.1% tetrasodium pyrophosphate.

ultrafine TiO2 particles, consist of 80% in the anatase crystal

phase and 20% in the rutile crystal phase and are composed

of 100 wt% titanium dioxide with a median particle size of

129.4 nm in water (aqueous solution buffered in 0.1% tetra-

sodium pyrophosphate) using dynamic light scattering and anaverage BET surface area of 53.0 m2/g.

Quartz particles (crystalline silica (-quartz), Min-U-Sil

5) with reported size range of 0.22m were obtained from

US Silica Company (Berkeley Springs, WV). Min-U-Sil 5 is

composed of100 wt% silicon dioxide in the -quartz crystal

phase, with a median particle size of480 nm in water (aque-

ous solution buffered in 0.1% tetrasodium pyrophosphate) and

an average surface area of 5.2 m2/g.

Each of the TiO2 samples were characterized to identify

its crystallinity and surface area in its dry native state and its

size, size distribution, pH, and chemical reactivity in water and

buffered solutions. X-ray fluorescence was used to measurepurity/composition. X-ray diffraction (XRD) (Philips XPERT

automated powder diffractometer, Model 3040) was used to

determine crystal structure, and crystallite size (Otwinowski

and Minor, 1997). XRD patterns do provide information on the

primary crystallite size, but do not, however, accurately repre-

sent the particle size of the dispersed aggregates. Therefore,

sizing data was obtained using two separate methods: dynamic

light scattering (DLS) andBET surface area analysis (Brunauer

et al., 1938). DLS measurements (Malvern Zetasizer Nano-

S, model Zen1600) were taken on each particle suspension

in water (aqueous suspension buffered in 0.1% tetrasodium

pyrophosphate) and phosphate-buffered saline (PBS) solution.

BET (Micromeritics ASAP 2405), to measure specific surfacearea, was taken on each particle sample in its dry state under

nitrogen.

For the remaining, non-pulmonary toxicity studies (i.e.,

dermal, oral, ocular, genotoxicity and aquatic studies), a next

generational form of ultrafine TiO2 particle-type (termed uf-

C) was used which has a similar particle size distribution to

uf-A and uf-B (Table 2). Ultrafine-C TiO2 had a crystalline

phase determination of 79% rutile and 21% anatase. X-ray flu-

orescence determined a composition of approximately 90 wt%

TiO2, 7% alumina, and 1% amorphous silica. Using dynamic

light scattering, the median particle size was 140 nm in water

(aqueous solution buffered in 0.1% tetrasodium pyrophos-

phate). The BET surface area was 38.5 m2/g. Similar to the

100% rutile TiO2 particle-types, uf-C TiO2 particle samples

underwent a neutralization process during production.

3. Toxicity studies

3.1. Pulmonary toxicity study in rats with

ultrafine-TiO2 particles

The aim ofthisstudy was to assess lung toxicity inrats

of newly developed, well-characterized, ultrafine-TiO2particles and compare them to TiO2 samples in two dif-

ferent size ranges and surface modifications. Groups of

male rats were intratracheally instilled with doses of 1 or

5 mg/kg of either two ultrafine rutile TiO2 particle-types(uf-A or uf-B); rutile fine-sized TiO2 particles; 80:20

anatase:rutile ultrafine-TiO2 particles; or -quartz parti-

cles (positive control). Phosphate-buffered saline (PBS)

solution instilled rats served as vehicle controls. Follow-

ing exposures, the lungs of PBS and particle-exposed

rats were evaluated for bronchoalveolar lavage (BAL)

fluid inflammatory markers, cell proliferation, and by

histopathology at post-instillation time points of 24 h, 1

week, 1 month, and 3 months.

The fundamental features of this pulmonary bioassay

are doseresponse evaluations and time-course assess-

ments to determine the sustainability of any observedeffect. Thus, the major endpoints of this study were the

following: (1) time course and dose/response intensity

of pulmonary inflammation and cytotoxicity; (2) air-

way and lung parenchymal cell proliferation; and (3)

histopathological evaluation of lung tissue (see Table 3

for the experimental protocol).

For the bronchoalveolar lavage studies, groups of

rats (5 rats/group/dose/time point) were intratracheally

instilled with single doses of either 1 or 5 mg/kg

ultrafine-TiO2 particle-type A (uf-A); ultrafine-TiO2

particle-type B (uf-B); fine-TiO2 particles; 80% anatase:

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Table 3

Protocol for ultrafine-TiO2 particle pulmonary bioassay study

20% rutile ultrafine TiO2 particle-types; or quartz (crys-

talline silica) particles (see Table 3). The intratracheal

instillation method of exposure can be a reliable qual-

itative screen for assessing the pulmonary toxicity ofinhaled particles (Warheit et al., 2005). All particle sam-

ples were prepared in a volume of phosphate-buffered

saline (PBS) solution and subjected to sonication for

15 min at 60 Hz. Groups of PBS-instilled rats served

as controls. The lungs of PBS and particle-exposed rats

were evaluated by BAL fluid analyses at 24 h, 1 week, 1

month and 3 months postexposure (pe).

For the lung tissue studies, additional groups of ani-

mals (4 rats/group/high dose/time period) were instilled

with the particle-types listed above plus the vehicle con-

trol, i.e., PBS. These studies and corresponding groups

of rats were dedicated to lung tissue analyses but only thehigh dose groups (5 mg/kg) and PBS controls were uti-

lized in the morphology studies. These studies consisted

of cell proliferation assessments and histopathological

evaluations of the lower respiratory tract. Similar to the

BAL fluid studies, the intratracheal instillation exposure

period was followed by 24 h, 1-week, 1-month, and 3-

month recovery periods (see Warheit et al., 1991, 1997,

2007 for more details).

For statistical analyses, each of the experimental val-

ues was compared to their corresponding sham control

values for each time point. A one-way analysis of vari-ance (ANOVA) and Bartletts test were calculated for

each sampling time. When the F-test from ANOVA was

significant, theDunnetts test wasused to compare means

from the control group to each of the groups exposed

to particulates. Significance versus PBS controls was

judged at the 0.05 probability level.

3.2. Acute dermal irritation study in rabbits

The acute dermal irritation tests were conducted

according to US EPA and OECD 404 guidelines

(USEPA, 1998; OECD, 2002). Ultrafine TiO2-C (uf-C)

particles were applied as a single 0.5 g dermal dose to

the shaved intact skin of three male New Zealand White

rabbits. The test substance, moistened with 0.25 mL ofdeionized water, was applied to a 6 cm2 area of skin. The

application area was covered with a 2-ply gauze square

which was held in place with non-irritating tape and cov-

ered with porous tape for a semi-occlusive dressing. The

rabbits were exposed to the test substance for 4 h after

which the test substance was removed. Test sites were

evaluated by Draize score for signs of dermal irritation

approximately 60 min, and 24, 48, and 72 h after test

substance removal. The rabbit that was initially treated

was also examined immediately after test substance

removal.

3.3. Dermal sensitization test: local lymph node

assay (LLNA) in mice

For the dermal sensitization component of the base

set study, responses in mice using the local lymph node

assay (LLNA) were utilized (OECD 429 Guideline).

The objective of this study was to evaluate the potential

of uf-C TiO2 particles to produce a dermal sensiti-

zation response in mice using the local lymph node

assay (LLNA). Five groups of five female CBA/JHsd

mice were dosed for 3 consecutive days with 0% (vehi-cle control), 5%, 25%, 50%, or 100% ultrafine TiO2particle-types on both ears. N,N-Dimethyl formamide

was used as the diluting vehicle. One group of five

female mice was dosed for 3 consecutive days with

25% hexylcinnamaldehyde (HCA) in 4:1 acetone:olive

oil (AOO) as a positive control and one group of five

female mice was dosed for 3 consecutive days with

AOO as a positive control vehicle. On test day 5 of

the assay, mice received 3H-Thymidine by tail vein

injection and were sacrificed approximately 5 h later.

The cell proliferation in the draining auricular lymph

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nodes of the ears from the test substance groups was

then evaluated and compared to the vehicle control

group.

A stimulation index (SI) was derived for each exper-

imental group by dividing the mean disintegrations per

minute (dpm) of each experimental group by the mean

dpm of the vehicle control group. The decision pro-cess in regard to a positive response includes an SI of

greater than or equal to 3.0 together with considera-

tion of doseresponse and, where appropriate, statistical

significance. Significance was judged at p < 0.05 except

for dpm data that were judged at p < 0.01. Lymph

node dpm data were log transformed to obtain nor-

mality or homogenous variances. When possible, an

EC3 value for the stimulation index data was derived

from linear interpolation of points on the doseresponse

curve immediately above and below the threefold

threshold.

3.4. Acute oral toxicity study in ratsup and down

procedure

The acute oral toxicity test-up and down procedure

was conducted according to US EPA and OECD 425

guidelines (USEPA, 2002; OECD, 2001). A single dose

of uf-C TiO2 particles suspended in deionized water

was administered by oral gavage to one fasted female

rat each at a dose of 175, 550, or 1750 mg/kg and

to three fasted female rats at a dose of 5000 mg/kg.The rats were dosed one at a time at a minimum of

48 h intervals. The rats were observed for mortality,

body weight effects, and clinical signs for 14 days

after dosing. All rats were necropsied to detect grossly

observable evidence of organ and tissue damage or

dysfunction.

3.5. Acute ocular irritation study in rabbits

The acute eye irritation tests were conducted accord-

ingtoUSEPAandOECD405guidelines(USEPA, 1998;

OECD, 2002). Uf-C TiO2 particles were evaluated foracute eye irritation potential in three young adult New

Zealand White rabbits. The study was conducted after

confirming that the compound was not a severe irritant

or corrosive to the skin.

Approximately 57 mg of test substance was admin-

istered to one eye of each animal. The eyes remained

unwashed following treatment. One rabbit was initially

treated. Since no severe irritation or corrosion was

observed, two additional rabbits were treated to complete

the test. The conjunctiva, iris, and cornea of each treated

eye were evaluated and scored according to a numer-

ical scale approximately 1, 24, 48, and 72 h following

administration of the test substance.

4. Genotoxicity test methods

4.1. Bacterial reverse mutation test

Uf-C TiO2 particles were evaluated for mutagenicity

in the Bacterial Reverse Mutation (Ames) Test using the

plate incorporation method. Salmonella typhimurium

strains TA98, TA100, TA1535, and TA1537 and

Escherichia coli strain WP2uvrA were tested in the

absence and presence of an exogenous metabolic activa-

tion system (Aroclor-induced rat liver S9). Sterile water

was chosen as the dosing vehicle based on the solu-

bility of the test substance and compatibility with the

target cells. The test substance formed a homogeneous

suspension at 50 mg/mL, the highest concentration thatwas tested in the study. A plating aliquot of 100L was

used. This dose was achieved using a concentration of

50 mg/mL and a 100L plating aliquot. The dose levels

in the study were 100, 333, 1000, 3333, and 5000g

per plate. Appropriate positive controls were included in

the study. The study was conducted according to the US

EPA and OECD 471 testing guidelines (USEPA, 1998;

OECD, 1998).

Milli-Q water was chosen as the dosingvehicle forthe

Ames test based on compatibility with the target cells.

The test substance formed a homogeneous suspension at50 mg/mL, the highest concentration that was tested in

the study. A plating aliquot of 100g/mL was used. The

dose levels in the study were 100, 333, 1000, 3333, and

5000g per plate.

4.2. In vitro mammalian chromosome aberration

test in Chinese hamster ovary cells

Uf-C TiO2 particles were tested for their ability

to induce structural chromosome aberrations in Chi-

nese hamster ovary (CHO) cells in the absence and

presence of an exogenous metabolic activation system(Aroclor-induced rat liver S9). The test substance was

prepared in Milli-Q water as this vehicle was deter-

mined to be the solvent of choice based on the solubility

of the test substance and compatibility with the tar-

get cells. The test substance formed a white suspension

in the vehicle at approximately 50 mg/mL, the highest

stock concentration prepared on the study. Osmolal-

ity, pH, and test substance precipitation were taken

into account for dose level setting, in addition to cell

count and mitotic activity. Cytogenetic evaluations of

structural aberrations were conducted in 200 cells in

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metaphase at 750, 1250, and2500g/mLforthe4hnon-

activated test condition; at 62.5, 125, and 250g/mL,

for the 4 h activated test condition, and at 25, 50, and

100g/mL for the 20 h non-activated test condition.

Numerical aberrations were recorded as well. Appro-

priate positive controls were included in the test. The

study was conducted according to the US EPA andOECD 473 testing guidelines (USEPA, 1998; OECD,

1998).

5. Aquatic toxicity test methods

5.1. Static, acute, 96 h toxicity screening tests with

Oncorhynchus mykiss (rainbow trout)

Theacute toxicity of fine anduf-C TiO2 particle-types

to the rainbow trout, O. mykiss was determined in unaer-

ated, 96 h static tests according to OECD 203 testingguidelines (OECD, 1992).

The study was conducted with four concentrations

each of fine-sized rutile TiO2 particles and ultrafine TiO2particles and a dilution water control at a mean tem-

perature of 12.2 C (range of 12.112.3 C) and 12.2 C

(range of 12.112.5 C), respectively. One test cham-

ber was used per test substance concentration for each

test substance with five test organisms in each chamber.

Based on visual observations, the dilution water con-

trols, 0.1 and 1.0 mg/L test concentrations were clear

and colorless with no precipitate at test start. The 10 and100 mg/L test concentrations were cloudy with a slight

amount of suspended substance present at test start. All

water quality parameters were within acceptable limits

during the exposure.


Daphnia magna

The acute toxicity of fine or uf-C TiO2 particles to the

water flea,D. magna (less than 24 h old) was determined

in unaerated, 48 h static tests according to OECD 202

testing guidelines (OECD, 2004).The study was conducted with four concentrations

each of fine or ultrafine TiO2 particles and a dilution

water control at a mean temperature of 20.2 C (range

of 20.120.3 C) and 20.1 C (range of 20.020.2 C),

respectively. One test chamber was used per test sub-

stance concentration for each test substance with 10

test organisms in each chamber. Based on visual obser-

vations, the dilution water controls, 0.1, and 1.0 mg/L

test concentrations were clear and colorless with no

precipitate at test start. The 10 and 100 mg/L test

concentrations were cloudy (white in color) and had

suspended substance present at test start. All water qual-

ity parameters were within acceptable limits during the

exposure.

5.3. Static, acute, 72 h growth inhibition toxicity

screening test to the green algae,

Pseudokirchneriella subcapitata

The acute toxicity of fine or uf-C TiO2 particles to

the green algae, P. subcapitata, was determined in a

72 h, static toxicity test according to OECD 201 testing

guidelines (OECD, 1984).

The study was conducted with a synthetic algal-

assay procedure (AAP) nutrient medium blank control

and five concentrations of fine or ultrafine TiO2 parti-

cles at a mean lighting intensity of 8938 lux (range of

82009500 lux), and a dilution water control at a mean

temperature of 23.8

C (range of 23.723.8

C) and ashaking speed of 100 rpm. Two replicates were used

per blank control and test concentration each with an

initial cell count (density) of 10,000 cells/mL. Based

on visual observations the 100 mg/L test concentration

solutions were very cloudy with suspended substance

present at test start. The 10 mg/L test concentration solu-

tions were slightly cloudy with suspended substance

present at test start. The blank control and remaining

test concentration solutions were clear and colorless

with no visible precipitate at test start. All environmen-

tal parameters were within acceptable limits during theexposure.

6. Results of the various assays (see Table 4)

6.1. Physicochemical characterization of particles

The particle size distribution (PSD) results for the

ultrafine TiO2 particle-types were highly agglomerated

following dispersion in the phosphate-buffered saline

solution, the vehicle utilized for the intratracheal instil-

lation exposures, although the uf-A sample showed a

slightly smaller average particle size (see Table 2). Thehigh degree of agglomeration in general arises from

the proximity of the buffer solution pH (near-neutral)

to isoelectric point of the samples. The relatively high

ionic strength of the buffer solution is also a factor

that promotes agglomeration. As described in an ear-

lier section, the uf-A and uf-B samples were utilized

for the pulmonary bioassay studies, and uf-C sam-

ples were used in the remaining studies. This is not

surprising since the development of materials for com-

mercialization can be an evolving process and one

developmental particulate candidate could demonstrate

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Table 4

Base set teststoxicity results

Test Result

Acute and short-term tests

Pulmonary bioassay Low toxicity

Acute oral toxicity test Low toxicity

Skin irritation test Not a skin irritantSkin sensitization Not a sensitizer

Eye irritation test Minor ocular conjunctival

redness

Genetic toxicity tests

Bacterial reverse mutation test Negative

Chromosomal aberration study Negative

Aquatic toxicity battery

Rainbow trout Low concern

Daphnia Low concern

Algae Medium concern

greater efficacy than another for a given commercial

application.

6.2. Pulmonary toxicity study in rats with

ultrafine-TiO2 particles

The ranking of lung inflammation/cytotoxicity/cell

proliferation and histopathological responses (in

descending order) was quartz > 80:20 anatase:rutile

uf TiO2 > fine-sized TiO2 = uf-A = uf-B. Exposures

to quartz and to a lesser degree, 80:20 anatase:rutile

TiO2 particles produced pulmonary inflammation,

cytotoxicity and adverse lung tissue effects. In contrast,

exposures to fine-TiO2 particles or to rutile uf-A/uf-B

TiO2 particle-types produced transient inflammation

and no adverse histopathological or cell proliferative

effects in the lungs of exposed rats at any postexposure

time period. It was concluded that differences in

responses to 80:20 anatase:rutile TiO2 particles versus

the rutile uf-A and uf-B TiO2 particle-types could be

related to crystal structure, inherent pH of the particles,

or surface chemical reactivity. Finally, the results

demonstrate that exposures to ultrafine-TiO2 particle-types can produce differential pulmonary effects, based

upon their composition, crystal structure and/or surface

reactivity.

6.3. Acute dermal irritation study in rabbits

The rabbits exhibited no dermal irritation during the

study. No clinical signs of toxicity were observed, and

no body weight loss occurred.

Under the conditions of this study, uf-C TiO2 particles

were not considered to be skin irritants.

6.4. Dermal sensitization test: local lymph node

assay (LLNA) in mice

No statistically significant differences in mean body

weights and body weight gains compared to the vehicle

control group were observed at any test concentration.

No clinical signs of toxicity were observed in the study.Statistically significant increases in cell proliferation

measurements compared to the vehicle control group

were observed at the 50% and 100% test concentrations.

Stimulation indices (SIs) of less than 3.0 were observed

at all test concentrations of uf-C TiO2 particle-types.

Therefore, the EC3 value (the estimated concentration

required to induce a threshold positive response, i.e.,

SI = 3) for the test substance under the conditions of this

study was not calculable. A 25% concentration of the

positive control, HCA, produced a dermal sensitization

response in mice. Therefore, the LLNA test system wasvalid for this study with ultrafine TiO2 particles. Under

the conditions of this study, uf-C TiO2 particles did not

produce a dermal sensitization response in mice.

6.5. Acute oral toxicity study in ratsup and down

procedure

No mortality occurred on the study. The single

rat dosed at 1750 mg/kg and the three rats dosed at

5000 mg/kg exhibited grey colored feces during the

study. No biologically important body weight losses

occurred. No gross lesions were present in the rats at

necropsy.

Based on this study, the oral LD50 for uf-C TiO2particles was greater than 5000 mg/kg for female rats.

6.6. Acute ocular irritation study in rabbits

The test substance produced conjunctival redness

(score 1 or 2) in the treated eye of all three rabbits. Flu-

orescein stain examinations did not reveal any corneal

injury. The treated eyes of the rabbits were normal by 24

or 48 h after instillation of the test substance. No clinicalsigns were observed, and no body weight loss occurred.

Based on this study, uf-C TiO2 particles produced

conjunctival redness in the treated rabbit eye which was

reversible.

7. Genotoxicity results

7.1. Bacterial reverse mutation test

No positive mutagenic responses or compound-

related toxicity were observed at any dose level with

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any tested strain (Salmonella typhimurium tester strains

TA98, TA1000, TA 1535, TA 1537, or Escherichia coli

strain WP2uvrA) when tested either with or without an

S9 metabolic activation system (Arocolor-induced rat

liver S9). Compound precipitate was observed at the top

three or four dose levels.

Uf-C TiO2 particles showed no evidence of muta-genicity in this study.

7.2. In vitro mammalian chromosome aberration

test in Chinese hamster ovary cells

In the preliminary toxicity assay, the highest concen-

tration tested was 5000g/mL, the OECD 473 guideline

limit dose for this test system. The osmolality or pH of

the highest test substance concentration in medium was

not significantly different from the vehicle control. Sub-

stantial toxicity (at least a 50% reduction in cell growthrelative to the solvent control) was not observed at any

concentration level in any test condition. Based on the

observedtest substanceprecipitation (generally regarded

as an overload dose) in the preliminary toxicity assay,

the highest concentration initially chosen for the chro-

mosome aberration assay was 2500g/mL, for all three

test conditions.

However, for the 4 h activated and the 20 h

non-activated test conditions, substantial test substance-

related inhibition of the mitotic activity was observed at

>750g/mL in the 4 h activated test condition, and allconcentration levels in the 20 h non-activated test condi-

tion. Therefore, cytogenetic evaluations were conducted

at 750, 1250, and 2500g/mL for the 4 h non-activated

test condition, at 62.5, 125, and 250g/mL, for the 4 h

activated test condition, and at 25, 50, and100g/mL for

the 20 h non-activated test condition. The percentage of

cells with structural or numerical aberrations in the test

substance-treated groups was not significantly increased

above that of the vehicle control at any concentration

(p < 0.05, Fishers exact test).

Uf-C TiO2 particles did not induce structural or

numerical chromosome aberrations in this study.

8. Aquatic toxicity screening results


O. mykiss (rainbow trout)

Exposure of rainbow trout to a dilution water control

and nominal fine TiO2 and uf-C TiO2 particle concentra-

tions of 0.1, 1.0, 10, and 100 mg/L resulted in 0, 0, 0, 10,

and 10% or 0, 0, 0, 0, and 0% immobility, respectively,

at the end of 96 h.

The 96 h LC50 for both types of TiO2 particles was

>100 mg/L based on nominal test concentrations. The

results demonstrated that fine or uf-C TiO2 particles each

exhibited low concern (Smrchek et al., 1993) for aquatic

hazard in unaerated, 96 h, static acute tests.


D. magna (daphnids)

Exposure of daphnids to thedilutionwatercontrol and

nominal fine or ultrafine TiO2 particle concentrations of

0.1, 1.0, 10, and 100 mg/L resulted in 0, 0, 0, 10, and

10% or 0, 0, 0, 10, and 0% immobility, respectively, at

the end of 48 h. The D. magna 48h EC50 values for fine

and ultrafine TiO2 particles, based on nominal concen-

trations were >100 mg/L.The results demonstrated that

fine and uf-C TiO2 particles exhibited low concern for

aquatic hazard in unaerated, 48 h, static acute tests.

8.3. Static, acute, 72 h growth inhibition toxicity

screening test to the green algae, P. subcapitata

Exposure of algae to nominal concentrations of 0.01,

0.1, 1, 10 and 100 mg/L fine TiO2 particles resulted in

2, 3, 2, 31, and 97% inhibition, respectively, based

on healthy cell count compared to the blank control at

the end of 72 h; percent inhibition of growth rate was

0, 1, 0, 8, and 66%, respectively. Exposure of algae to

nominal concentrations of 0.01, 0.1, 1, 10, and 100 mg/L

uf-C TiO2 particles resulted in 19, 6, 11, 15, and

94% inhibition, respectively, based on healthy cell count

compared to the blank control at the end of 72 h; per-

cent inhibition of growth rate was 3, 1, 2, 3, and

54%, respectively. Healthy cell counts increased in the

blank controls by at least a factor of 16 in 72 h, thereby

satisfying the appropriate test acceptance criteria.

The algae 72h EC50 values (95% fiducial limits)

based on inhibition of growth and healthy cell counts

were 16 (1222) mg/L for fine TiO2 particles and 21

(1626) mg/L for uf-C TiO2 particles. The 72 h EC50

values (95% fiducial limits) for growth rate based onnominal concentrations and healthy cell counts were 61

(5272) mg/L for fine-TiO2 particles and 87 (8391)

mg/L for uf-C TiO2 particles.

These results demonstrated that fine and uf-C TiO2particles exhibited medium concern under TSCA in a

72 h acute test (Smrchek et al., 1993).

9. Discussion

Ten different toxicity assays were carried out to eval-

uate the hazard potential of the ultrafine TiO2 (uf-TiO2)

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particle-types, uf-A, uf-B, or uf-C. To summarize the

hazard evaluation of uf-TiO2 particle-types, most of the

tests demonstrated low hazard potential in mammals or

aquatic species following acute exposures to ultrafine

TiO2 particles.

The hazard identification base set is an evolving

concept developed to characterize the inherent hazardsrelated to nanomaterial exposures, both in mammalian

species, as a representation of human health effects, and

in ecological environments using aquatic species. In the

absence of accurate exposure determinations for nano-

material aerosol exposures, information gained from

these hazard assessment tests provides a basis for mak-

ing reasonable and responsible decisions and for taking

action in limiting occupational and consumer exposures.

The intratracheal instillation method of exposure can

be a reliable qualitative screen for assessing the pul-

monary toxicity of inhaled particles. This comparisonwas recently made with different formulations of tita-

nium dioxide particle-types. The results demonstrated

that the intratracheal instillation-derived, pulmonary

bioassay studies represent an effective preliminary

screening tool for inhalation studies with the identical

particle-types used in this study (Warheit et al., 2005).

The justification for the base set hazard tests rests

on the following criteria: (1) potential routes of expo-

sures related to human health effects (i.e., pulmonary,

dermal, oral and/or ocular); (2) early screening for

potential carcinogenic effects (i.e., utilizing well estab-lished mutational and chromosomal aberrationscreening

assays); and (3) assessments of potential toxic effects

in representative aquatic organisms (i.e., exposures of

rainbow trout, Daphnia, and algae to nanomaterials).

It is important to note that the base hazard set of

tests described herein does not account for an exhaus-

tive evaluation or full toxicological profile for a given

nanomaterial, but rather serves to provide a reasonable

balance between an adequate toxicity characterization

anda practical strategy for the development of new nano-

materials. Thus, the aim of this strategy is to develop

hazard information sufficiently robust to guide adequaterisk-evaluation processes, commensurate with existing

regulatory and voluntary safety standards.

With regard to the development of testing strate-

gies to gauge the toxicity of nanomaterials, two notable

efforts have recently been reported. To this end, an

International Life Sciences Research Foundation/Risk

Sciences Institute working group developed the elements

of a screening strategy for assessing hazard profiles of

nanomaterials. According to this working group, the

three key elements of the toxicity screening strategy

were (1) physicochemical characteristics; (2) in vitro

assays (cellular and non-cellular) and (3) in vivo toxicity

assays.

The physicochemical properties that were considered

to be important for assessing toxic effects of nano-

materials included particle size and size distribution,

agglomeration state, shape, crystal structure, chemical

composition, surface area, surface chemistry, surfacecharge and porosity. Suggested in vitro assays were

proposed to focus upon specific biological and mech-

anistic pathways in ways that are not feasible in in vivo

tests. Tier I in vivo assays were proposed for pulmonary,

oral, skin and injection exposures. Tier I assessments

included markers of inflammation, oxidant stress, and

cell proliferation in portal-of-entry and selected remote

organs and tissues. More intensive Tier 2 evaluations

for pulmonary exposures were suggested for deposition,

translocation, toxicokinetic and biopersistence studies,

as well as effects of multiple exposures. Additional Tier2 studies could include potential effects on the repro-

ductive system, placenta, and fetus; alternative animal

models; and mechanistic studies (Oberdorster et al.,

2005).

More recently, the European Center for Ecotoxicol-

ogy and Toxicology of Chemicals (ECETOC) sponsored

a workshop to develop testing strategies to evaluate the

safety of nanomaterials. It brought together scientific

and clinical experts from industry, academia, govern-

ment agencies, research institutes, and nongovernmental

organizations. The fundamental questions addressed bythe workshop participants were the following: What can

we do today? What do we need tomorrow? (ECETOC,

2006).

Some of the summary conclusions of the workshop

included the following:

For assessment of nanoparticle physicochemical char-

acterization, the working definition of nanoparticles

was particle size

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studiesboth of which are genotoxicity screening

assays. The remaining studies in the battery of tests are

in vivo studies, using mammalian and aquatic organisms.

Clearly the development of validated in vitro screens to

substitute for in vivo tests would be beneficialin terms

of costs, timing, and reductions in animal use. Unfortu-

nately, none of the in vivo tests currently utilized in thebase set can be accurately replaced by in vitro assays.

We have made efforts to improve the predictability ofin

vitro assays to assess the in vivo pulmonary toxicity of

inhaled nanomaterials. The results obtained from a pre-

viously reported study utilizing in vitro methodologies

were not representative ofin vivo pulmonary inflamma-

tory or cytotoxic effects for the various particle-types

tested and thus were not well correlated. It was con-

cluded that in vitro cellular systems will need to be

further developed, standardized and validated in order

to provide useful screening data on the relative tox-icity of fine and nanoscale particulates (Sayes et al.,

2007).

In addition to the hazard identification base set

methodology described herein, bridging information can

provide additional useful information on a nanoparticle-

type. In this regard, when a material has few specific

hazard data, one way to inform decisions about it is to

bridge it to a material that has robust hazard data. The

two materials may then be entered into a toxicological

study, with the well-characterized material serving as

a reference particle for the material of interest. Thisbridging strategy has been utilized in a recent publica-

tion and is reviewed in the pulmonary bioassay results

herein, wherein toxicity assessments for new rutile-type

ultrafine TiO2 particle-types were conducted (Warheit et

al., 2007).

To summarize the findings reported herein, a hazard

identification base set methodology has been developed

which incorporates tests for assessing the toxicity of

ultrafine particle-types or nanomaterials in mammalian

and aquatic speciesrelated, in part, to potential routes

of exposure. When considering all of the test results, the

findings of most of the toxicity studies with the ultrafineTiO2 particle-types tested herein (i.e., uf-A, uf-B, and uf-

C) demonstrated low hazard potential in mammals and

aquatic species following short-term exposures.

Acknowledgments

This study was supported by DuPont Titanium

Technologies. We thank Drs. Brian Coleman, Ger-

ald Kennedy, Jr., Scott Loveless, Gary Whiting, and

Scott Frerichs for helpful comments on this manuscript.

The following individuals provided invaluable technical

assistance in the conduct of the various studies, Denise

Hoban, ElizabethWilkinson, Carolyn Lloyd, Lisa Lewis,

John Barr, Don Hildabrandt, Susan Munley, Lynn Ford,

Dr. John OConnor, Jeffrey Turner, Sr., Terry Lee Slo-

man, and Dr. Steven R. Frame.

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