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Beskæftigelsesudvalget 2019-20
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Offentligt

Titanium

dioxide

nanomaterials:

Scientific basis

for setting

a health-based

occupational

exposure limit

Anne Thoustrup Saber, Sarah Søs Poulsen, Niels Hadrup,

Karin Sørig Hougaard, Nicklas Raun Jacobsen and Ulla Vogel

TITANIUM DIOXIDE NANOMATERIALS:

SCIENTIFIC BASIS FOR SETTING A HEALTH-

BASED OCCUPATIONAL EXPOSURE LIMIT

Anne Thoustrup Saber

Sarah Søs Poulsen

Niels Hadrup

Karin Sørig Hougaard

Nicklas Raun Jacobsen

Ulla Vogel

The National Research Centre for the Working Environment, Copenhagen 2018

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NFA-report

Title

Titanium dioxide nanomaterials: Scientific basis for setting a health-based

occupational exposure limit

Anne Thoustrup Saber, Sarah Søs Poulsen, Niels Hadrup, Karin Sørig

Hougaard, Nicklas Raun Jacobsen and Ulla Vogel

The National Research Centre for the Working Environment (NFA)

September 2018

978-87-7904-351-0

nfa.dk

Authors

Institution

Publisher

Published

ISBN

Internet version

The National Research Centre for the Working Environment (NFA)

Lersø Parkallé 105

DK-2100 Copenhagen

Phone: +45 39165200

Fax: +45 39165201

e-mail: [email protected]

Website: nfa.dk

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OREWORD

In 2015, the Danish Working Environment Council made 22 recommendations to

promote safe handling of nanomaterials (NMs) in the working environment, which were

enforced by the Minister of Employment. One of these recommendations was ‘That the

Danish Working Environment Authority in cooperation with relevant scientific experts

assesses whether adequate scientific documentation can be provided to use the scientific

quality committee for an assessment of the scientific evidence to determine limit values

for specific NMs in the work environment.’ (https://www.amr.dk/nano.aspx).

On this background, The Danish Working Environment Authority asked NFAto review

the scientific evidence with the aim of clarifying the possibilities for suggesting

nanospecific occupational exposure limits (OELs) for three different NMs (titanium

dioxide (TiO

), carbon black and carbon nanotubes (CNT)).

The purpose of the present report is to suggest a health-based OEL for nanosized TiO

Elizabeth Bengtsen and Karen Bo Frydendall, NFA, are gratefully acknowledged for

assistance with literature search.

The working group wishes to thank Chief Toxicologist Poul Bo Larsen, DHI, Denmark,

for reviewing the report.

Copenhagen, August 2018

iii

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XECUTIVE SUMMARY

In this report, a working group at the National Research Centre for the Working

Environment reviews data relevant to assessing the hazard of TiO

nanomaterials (TiO

NMs), i.e. human studies (Chapter 2), toxicokinetics (Chapter 3), animal studies (Chapter

4), mechanisms of toxicity (Chapter 5), previous risk assessments of TiO

NMs (Chapter

6), scientific basis for setting an occupational exposure limit (OEL) (Capter 7) and finally

we summarize and suggest a health based OEL for TiO

NM (Chapter 8). The focus of

this report is only occupational exposure by inhalation.

The present working group evaluated the relevant literature on TiO

NM from both

epidemiological and animal inhalation studies. None of the identified epidemiological

studies provided information on the particle size range of the TiO

, thus making it

impossible to determine whether the exposures included TiO

NM. Therefore it was

decided to base the suggested health-based OEL on data from experimental animal

studies.

Pulmonary inflammation and carcinogenicity was observed in inhalation studies in rats.

The present working group regards inflammation and carcinogenicity as the critical

adverse effects and the subsequent risk assessments are conducted based on studies

reporting these effects. TiO

NM induced cardiovascular effects were identified in animal

studies. However, none of these studies were sub-chronic or chronic inhalation studies

and therefore not suitable for OEL derivation. However, the present working group

regards the acute phase response as a biomarker of cardiovascular effects. Due to the

close association between pulmonary inflammation and the acute phase response, the

present working group regards inflammation as a proxy for cardiovascular effects

mediated by the acute phase response.

The present working group found strong dose response relationships for neutrophil

influx as a marker of pulmonary inflammation. Neutrophil influx was related to

deposited surface area. The working group considers inflammation as a threshold effect.

The present working group found that the mechanism of action of the carcinogenic effect

has not been fully clarified. Secondary genotoxicity due to particle-induced

inflammation is an important and well documented mechanism of action for the

development of lung cancer. However, the available data did not allow ruling out that

TiO

NM could also induce cancer through a direct genotoxic mechanism. Therefore, the

present working group considers carcinogenicity as a non-threshold effect.

Consequently, the present working group decided to perform the risk assessment based

on both a threshold effect for inflammation and a non-threshold effect for cancer.

For an OEL based on threshold effects, the following traditional approach suggested by

REACH is utilized: 1) identification of critical effect, 2) identification of the no observed

adverse effect concentration (NOAEC), 3) calculation of OEL using assessment factors

adjusting for inter and intra species differences. For non-threshold effects, the present

working group uses two approaches. The first method uses the measured lung burden in

rats exposed by inhalation and the alveolar surface area of rats and humans to estimate

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the human equivalent lung burden. The second method, suggested by The European

Chemicals Agency (ECHA), uses air concentrations directly.

The working group considered that data from two rat inhalation studies as the best basis

for the risk assessment. The following studies were selected to be used for calculation of

the derived-no-effect level (DNEL) and dose-response for excess cancer risk,

respectively: A 13 week sub-chronic inhalation study in rats (0, 0.5, 2.0 and 10 mg/m

)

and a 2 year chronic cancer inhalation study in rats (0 and 10 mg/m

). The table below

shows a DNEL for pulmonary inflammation derived based on the sub-chronic inhalation

study of rats, and extra lung cancer risk at 1 in 1 000, 1 in 10 000 and 1 in 100 000 derived

using two different approaches.

Overview of DNEL based on a threshold based mechanism of action and exposure levels

resulting in extra cancer risk levels at 1:1000, 1:10 000 and 1: 100 000 based on a non-threshold

based mechanism of action.

Mechanism of

action

Threshold

based

Non-treshold

based

DNEL

Extra cancer

risk

1:1000

1:10 000

1:100 000

Suggestion of an OEL for TiO

Inflammation

Lung cancer

(method I)

(method II)

10 µg/m

4 µg/m

0.4 µg/m

0.04 µg/m

47 µg/m

4.7 µg/m

0.47 µg/m

Both studies used for the risk assessment used P25 TiO

NM (15-40 nm diameter, 80%

anatase/20% rutile). TiO

NMs differ regarding size and surface area but also coating,

shape, crystal structure etc. The present working group notes that there is limited

available data on the biological effects of different physico-chemical properties, but the

present working group concludes that the majority of available data support that the

surface area (and therefore the size) of TiO

is a critical driver of particle-induced

inflammation and the acute phase response in the lungs. In support of this notion, The

National Institute for Occupational Safety and Health (NIOSH) showed that the

deposited surface area of TiO

particles of different sizes (fine and ultrafine) and

different crystal structure (80% anatase/20% rutile and 99% rutile) can explain the

observed variation in TiO

particle-induced pulmonary inflammation and lung cancer in

rat inhalation studies. This stresses the importance of the surface area as a predictor for

the inflammatory and carcinogenic response.

The present working group regards cancer as the most critical effect. The DNEL

approach relies heavily on the assumption of a threshold effect on inflammation and

carcinogenicity. The present working group is of the opinion that there is still

uncertainly whether this is the case for TiO

NM–induced carcinogenicity.

Two different approaches were used for calculating excess lung cancer risk based on the

same chronic inhalation study in rats. In the first approach, lung burden was used to

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estimate the exposure levels. In the second approach, air concentrations were used

directly. Independently of the applied method for risk assessment, the resulting OEL

suggestions were all very low. These levels are all more than 100-fold lower than the

current Danish OEL for titanium of 6 mg/m

(measured as Ti, corresponding to 10 mg/m

TiO

The present working group recommends the risk assessment approach estimating the

excess lung cancer risk based on lung burden, since this approach takes the retained

pulmonary dose into account. Thus, the expected excess lung cancer risk in relation to

occupational exposure to TiO

NMs is 1:1 000 at 4 µg/m

, 1:10 000 at 0.4 µg/m

and 1:100

000 at 0.04 µg/m

TiO

NM.

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ANSK SAMMENFATNING

I denne rapport vurderer en arbejdsgruppe ved Det Nationale Forskningscenter for

Arbejdsmiljø data, der er relevante for at vurdere faren ved udsættelse for titanium

dioxid nanomaterialer (TiO

NM), dvs. humane studier (kapitel 2), toksikokinetik

(kapitel 3), dyreforsøg (kapitel 4), toksicitetsmekanismer (kapitel 5), tidligere

risikovurderinger af TiO

NM (kapitel 6), det videnskabelige grundlag for fastlæggelse af

en grænseværdi (kapitel 7) og endelig opsummeres og foreslås en helbredsbaseret

grænseværdi for TiO

NM i arbejdsmiljøet (kapitel 8). Fokus i denne rapport er alene på

erhvervsmæssig eksponering ved indånding.

Den nærværende arbejdsgruppe evaluerede den relevante litteratur om TiO

NM fra

både epidemiologiske undersøgelser og inhalationsforsøg med dyr. Ingen af de

identificerede epidemiologiske undersøgelser indeholdt oplysninger om TiO

partikelstørrelse, hvilket gør det umuligt at afgøre, om eksponeringerne omfattede TiO

NM. Derfor blev det besluttet at basere den foreslåede sundhedsbaserede grænseværdi i

arbejdsmiljøet på data fra studier på forsøgsdyr.

Der blev observeret lungeinflammation og lungekræft i inhalationsundersøgelser af

rotter. Den nærværende arbejdsgruppe anser inflammation og kræft som de vigtigste

skadelige effekter. Derfor baseres de efterfølgende risikovurderinger på undersøgelser,

der rapporterer om disse effekter. Der blev identificeret TiO

NM-inducerede

kardiovaskulære effekter i dyreforsøg. Ingen af disse undersøgelser var imidlertid

subkroniske eller kroniske inhalationsundersøgelser og derfor var de ikke egnede til

risikovurdering. Den nærværende arbejdsgruppe anser dog akutfaseresponset som en

biomarkør for kardiovaskulære effekter. På grund af den stærke sammenhæng mellem

lungeinflammation og akutfasesponset betragter den nærværende arbejdsgruppe

inflammation som en proxy for hjertekareffekter medieret af akutfaserespons.

Den nærværende arbejdsgruppe fandt stærk dosis-respons-sammenhæng for neutrofilt

influx som markør for lungeinflammation. Det samlede lungedeponerede specifikke

overfladeareal prædikterede neutrofilt influx. Arbejdsgruppen anser inflammation for at

være en tærskeleffekt.

Den nærværende arbejdsgruppe fandt, at virkningsmekanismen for den

kræftfremkaldende effekt ikke er blevet fuldstændigt afklaret. Sekundær genotoksicitet

forårsaget af partikelinduceret inflammation er en vigtig og veldokumenteret

virkningsmekanisme for udvikling af lungekræft. De tilgængelige data tillod dog ikke at

udelukke at TiO

NM også kunne inducere kræft gennem en direkte genotoksisk

mekanisme. Derfor anser den nærværende arbejdsgruppe kræft som ikke-tærskel effekt.

Det blev derfor besluttet at udføre risikovurderingen baseret på både en tærskeleffekt for

inflammation og en ikke-tærskeleffekt for kræft.

For en grænseværdi i arbejdsmiljøet baseret på tærskeleffekt anvendes følgende

traditionelle tilgang, som anbefalet af REACH: 1) identifikation af kritisk effekt, 2)

identifikation af NOAEC, og 3) beregning af grænseværdi ved anvendelse af

vurderingsfaktorer, der justerer for inter- og intraspecies forskelle. For ikke-

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tærskeleffekter anvender den nærværende arbejdsgruppe to metoder. Ved den første

metode anvendes den målte lungedeponerede dosis hos rotter til at estimere den

tilsvarende eksponering i arbejdsmiljøet. Ved den anden metode anvendes

luftkoncentrationer direkte.

Arbejdsgruppen fandt, at data fra to inhalationsundersøgelser i rotter var det bedste

grundlag for risikovurderingen. Følgende undersøgelser blev udvalgt til beregning af

henholdsvis DNEL og kræftrisiko: En 13-ugers subkronisk inhalationsundersøgelse af

rotter (0, 0,5, 2,0 og 10 mg/m

) og en 2-årig kronisk kræftinhalationsundersøgelse af

rotter (0 og 10 mg/m

). Tabellen nedenfor viser en DNEL for lungeinflammation beregnet

på basis af det subkroniske inhalationsstudie af rotter og ekstra lungekræftrisiko hos 1

ud af 1.000, 1 ud af 10.000 og 1 ud af 100.000 beregnet på to forskellige måder.

Oversigt over DNEL baseret på en tærskelbaseret virkningsmekanisme og eksponerings-

niveauer, der resulterer i ekstra kræftrisikoniveauer på 1: 1000, 1:10 000 og 1: 100 000 baseret på

en ikke-tærskelbaseret virkningsmekanisme.

Virkningsmekanisme

Tærskel-baseret

Ikke tærskel-baseret

DNEL

Ekstra

kræftrisiko

1:1 000

1:10 000

1:100 000

Forslag til grænseværdi for TiO

Inflammation

Lungekræft

(metode I)

(metode II)

10 µg/m

4 µg/m

0.4 µg/m

0.04 µg/m

47 µg/m

4.7 µg/m

0.47 µg/m

Begge undersøgelser, som blev anvendt til risikovurderingen, benyttede P25 TiO

(15-40 nm diameter, 80% anatase / 20% rutil). TiO

NM'er er forskellige med hensyn til

størrelse og overflade, men også coating, form, krystalstruktur mv. Den nærværende

arbejdsgruppe bemærker, at der er begrænsede tilgængelige data om de biologiske

effekter af forskellige fysisk-kemiske egenskaber, men arbejdsgruppen konkluderer, at

størstedelen af de tilgængelige data støtter, at overfladearealet (og derfor også størrelsen)

af TiO

er en prædiktor for partikelinduceret inflammation og akutfaserespons i

lungerne. NIOSH har vist, at partikeloverfladearealet af TiO

-partikler af forskellige

størrelser (fin og ultrafin) og forskellige krystalstrukturer (80% anatase/20% rutil og 99%

rutil) kan forklare den observerede variation i TiO

-partikelinduceret lungeinflammation

og lungekræft i rotteinhalationsundersøgelser. Dette understreger vigtigheden af

overfladearealet som en prædiktor for det inflammatoriske respons og

kræftfremkaldende effekt.

Den nærværende arbejdsgruppe betragter kræft som den vigtigste effekt.

DNEL-tilgangen er stærkt afhængig af antagelsen om en tærskeleffekt for inflammation

og kræft. Den nuværende arbejdsgruppe er af den opfattelse, at der stadig er usikkerhed

om, hvorvidt dette er tilfældet for TiO

NM induceret kræft.

Der blev anvendt to forskellige metoder til beregning af den overskydende risiko for

lungekræft baseret på den samme kroniske inhalationsundersøgelse. Ved den første

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metode blev den lungedeponerede dosis brugt til at estimere eksponeringsniveauerne.

Ved den anden metode blev luftkoncentrationerne anvendt direkte. Uafhængigt af den

anvendte metode til risikovurderingen, er de beregnede forslag til grænseværdier alle

meget lave. Disse niveauer er mere end 100 gange lavere end den nuværende danske

grænseværdi i arbejdsmiljøet for titanium på 6 mg/m

(målt som Ti svarende til 10

mg/m

TiO

Den nærværende arbejdsgruppe anbefaler metoden, hvor den overskydende risiko for

lungekræft baseres på lungedeponeret dosis, da denne tilgang tager højde for den

faktiske lungedeponering. Således er den forventede overskydende lungekræftrisiko i

forbindelse med erhvervsmæssig udsættelse for TiO

NM 1: 1 000 ved 4 µg/m

, 1:10 000

ved 0,4 µg/m

og 1: 100 000 ved 0,04 µg/m

TiO

NM.

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ONTENTS

Foreword ....................................................................................................................................... iii

Executive summary ...................................................................................................................... iv

Contents .......................................................................................................................................... x

Abbreviations ............................................................................................................................... 11

Introduction.................................................................................................................................. 13

Human studies............................................................................................................................. 15

Human exposure ..................................................................................................................... 15

Epidemiological studies ......................................................................................................... 15

Toxicokinetics .............................................................................................................................. 20

Animal studies ............................................................................................................................. 22

Rodent versus human response ............................................................................................ 22

Intratracheal instillation versus inhalation .......................................................................... 22

Selection of studies and endpoints ....................................................................................... 23

Pulmonary inflammation ....................................................................................................... 23

Genotoxicity and cancer ......................................................................................................... 25

Cardiovascular effects............................................................................................................. 28

Reproductive toxicity .............................................................................................................. 29

Mechanisms of toxicity ............................................................................................................... 31

Pulmonary inflammation, genotoxicity and cancer ........................................................... 31

Cardiovascular effects............................................................................................................. 32

Dose-response relationships .................................................................................................. 33

Particle characteristics............................................................................................................. 34

Previous risk assessments of TiO

............................................................................................. 35

IARC .......................................................................................................................................... 35

ENRHES.................................................................................................................................... 35

NEDO ........................................................................................................................................ 36

NIOSH....................................................................................................................................... 36

Scaffold project ........................................................................................................................ 37

ECHA’s Committee for Risk Assessment (RAC) ................................................................ 37

Scientific basis for setting an occupational exposure limit .................................................... 40

Endpoint: Inflammation ..................................................................................................... 40

Endpoint: Cancer ................................................................................................................. 41

Conclusion .................................................................................................................................... 45

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BBREVIATIONS

3-ClTyr

3-NOTyr

5-OHMeu

8-OHdG

8-OHG

Apo-A1

ApoE

BAL

BMD

BMDL

CNT

CRP

DNEL

ECHA

EBC

eNOS

HDL

Hs-CRP

IARC

ICAM-1

ICP-MS

INEL

LDL

LOAEC

MAD

MMAD

MWCNT

NIOSH

NOAEC

NFA

OEL

o-Tyr

RAC

REACH

REL

ROS

SAA

3-chlorotyrosine

3-nitrotyrosine

5-hydroxymethyl uracil

8-hydroxy-2-deoxyguanosine

8-hydroxyguanosine

Assessment factor

Apolipoprotein A1

Apolipoprotein E

Broncho alveolar lavage

Benchmark dose

Benchmark dose lower bound

Complement factor 3

Confidence interval

Carbon nanotube

C reactive protein

Derived-no-Effect Level

European Chemicals Agency

Exhaled breath condensate

Endothelial nitric oxide synthase

High density lipoprotein

High-sensitivity C reactive protein

The International Agency for Research on Cancer

Intercellular cell adhesion molecule-1

Inductively Coupled Plasma Mass Spectrometry

Interleukine

Human indicative no-effect levels

Intraperitoneal

Low-density lipoproteins

Lowest observed adverse effect concentration

Malondialdehyde

Mass median aerodynamic diameter

Multi-walled carbon nanotube

National Institute for Occupational Safety and Health

Nanomaterial

Nitrogen oxide

No observed adverse effect concentration

National Research Centre for the Working Environment

Occupational exposure limit

o-tyrosine

ECHA’s Committee for Risk Assessment

Registration, Evaluation, Authorization and Restriction of Chemicals

Recommended exposure limit

Reactive oxygen species

Relative risk

Serum amyloid A

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SCCS

SMR

SOD

SP-D

TiO

TNF

TWA

VCAM-1

Scientific Committee on Consumer Safety

Standardized mortality ratio

Superoxide dismutase

Surfactant protein D

Titanium dioxide

Tumour necrosis factor

Time-weighted average

Vascular cell adhesion molecule-1

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NTRODUCTION

Titanium dioxide (TiO

) is a white solid inorganic and poorly soluble compound. TiO

various particle sizes including nanosizes have been used for almost 100 years in a

diverse range of industrial and consumer products. TiO

is used as white pigment in e.g

paints and as food colorant. Traditionally, TiO

has been considered a low toxicity

particles (Oberdorster et al. 2005). For that reason TiO

has previously been used as a

negative control particle in many animal studies. However, this view was changed with

studies showing lung cancer in rats following chronic exposure to a high dose of fine

TiO

(Lee et al. 1985) and a lower dose of TiO

nanomaterial (TiO

NM)(Heinrich et al.

1995). At the same time, toxicological studies showed that smaller TiO

particles induced

more inflammation than larger TiO

particles (reviewed by (Stone et al. 2017)). This

observation was followed up by toxicological studies of other types of low solubility

particles showing that more inflammation was induced by smaller particles compared to

larger particles with the same chemistry (Oberdorster et al. 2005).

The EU has adopted the following definition of a NM “A natural, incidental or

manufactured material containing particles, in an unbound state or as an aggregate or as

an agglomerate and where, for 50 % or more of the particles in the number size

distribution, one or more external dimensions is in the size range 1 nm - 100

nm.”(European Commission 2017). TiO

NMs differ regarding size and surface area but

also coating, shape, crystal structure etc. (OECD Environment 2016;NIOSH 2011).

In 2006, the International Agency for Research on Carcinogenicity (IARC) classified TiO

as possibly carcinogenic to humans (group 2B). This classification was based on

sufficient evidence of carcinogenicity in experimental animals and insufficient evidence

in humans. IARC does not differentiate between nano- and fine particles in their

classification (IARC 2010).

In 2011, the National Institute for Occupational Safety and Health (NIOSH)

recommended that exposure limits for TiO

are set based on their size: NIOSH

recommends exposure limits (RELs) of 2.4 mg/m

for fine TiO

and 0.3 mg/m

for

ultrafine TiO

. These RELs are estimated to equal lung cancer risk below 1 in 1,000

during working lifetime. Due to the lack of epidemiological studies of TiO

, NIOSH

choses to base the RELs on chronic rat inhalation studies and extrapolation to human

risk (NIOSH 2011).

To our knowledge, there are no legally binding NM-specific occupational exposure

limits (OELs) for TiO

. The present Danish OEL for titanium is 6 mg/m

(as Ti,

corresponding to 10 mg/m

for TiO

), and is regulated by the Danish Working

Environment.

The aim of the present report is to investigate if the present knowledge allows for a

suggestion of a health-based nanospecific OEL for TiO

In this document we review

the relevant literature on the adverse effects of TiO

NM. The risk assessment

methodology of this report will follow the guidelines suggested by REACH (ECHA

2012). First, threshold or non-threshold effects are determined. Threshold effect assumes

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that the organism can withstand a certain dose before adverse effects occur, whereas

non-threshold effects assume that any exposure to the substance can result in adverse

effects. For an OEL based on threshold effects, the following traditional approach is

utilized: 1) identification of critical effect, 2) identification of the NOAEC, 3) calculation

of OEL using assessment factors adjusting for inter and intra species differences. For

non-threshold effects, the present working group will use two different approaches for

calculating excess lung cancer risk. In the first approach lung burden will be used to

estimate the exposure levels. In the second approach, air concentrations were used

directly. Conclusively, the calculated OELs will be compared and lastly, a recommended

OEL for TiO

NM exposure will be proposed.

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UMAN STUDIES

Human exposure

There are limited data on occupational exposure TiO

NM. A review of reported

exposure to different types of engineered NMs included 29 exposure scenarios of TiO

NM exposure. TiO

NM exposure occurred in research laboratories, industrial-scale

synthesis units, in a pilot-scale synthesis unit, in a laboratory-scale production unit, and

in a university research laboratory. The mass concentrations of respirable TiO

in the

workers’ breathing zone ranged from 10 to 150 µg/m

during bag filling. TiO

NM was

mainly detected as aggregated structures in a size range spanning from nanometer to

micrometer (Debia et al. 2016).

A recent exposure assessment, which was not included in the review by Debia et al., was

performed at a Chinese TiO

manufacturing plant. Mass concentrations were assessed in

two different worksites at the plant: In the packaging workshop, total dust

concentrations were 3.17 mg/m

of which 1.22 mg/m

was dust in the nanosize range. In

the milling workshop, total dust concentrations were 0.79 mg/m

of which 0.31 mg/m

was dust in nanosize range. ICP-MS analysis showed that a rather small part of the dust

was TiO

: TiO

content in total dust was 46.4 µg/m

at the packaging workshop and 39.4

µg/m

at the milling workshop. TiO

NM constituted 16.7 and 19.4 µg/m

, respectively

(Xu et al. 2016).

Epidemiological studies

A few epidemiological studies have been performed to evaluate the adverse effects of

inhalation exposure to TiO

in humans. None of the epidemiological studies included

information on the size range of the TiO

particles. Thus, it is not possible to determine

whether the exposures included TiO

NM. In total eight studies were identified of which

five are cohort studies (Chen and Fayerweather 1988;Fryzek et al. 2003;Boffetta et al.

2004;Ellis et al. 2010;Ellis et al. 2013) and three are case-control studies (Siemiatycki, 1991

(as referred by (IARC 2010) 2010 and (NIOSH 2011); (Boffetta et al. 2001),(Ramanakumar

et al. 2008).

Except for the newest studies by Ellis et al. (Ellis et al. 2010;Ellis et al. 2013), the studies

are included in the evaluations of TiO

performed by IARC (IARC 2010) and/or NIOSH

(NIOSH 2011). The present working group refers to these publications for a more

detailed description of these studies. The evaluation of TiO

performed in 2006 by IARC

concluded that there was inadequate evidence of carcinogenicity in humans (IARC

2010). The evaluation of TiO

by NIOSH in 2011 concludes that “these studies provide no

clear evidence of elevated risks of lung cancer mortality or morbidity among those

workers exposed to TiO

dust”(NIOSH 2011).

Among the studies included in NIOSH and/or IARC, Chen and Fayerweather (Chen and

Fayerweather 1988), Fryzek et al. (Fryzek et al. 2003) and Boffetta et al. (Boffetta et al.

2004) in addition to all cause and lung cancer also assessed death caused by

cardiovascular diseases.

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The number of deaths from ischemic heart disease and cerebrovascular disease was

numerically increased among employees with TiO

exposure (n = 1 576) as compared

with employees without TiO

exposure (n=901) at two TiO

producing plants (Dupont,

US). However, this increase was not statistically significant at p<0.10. When the TiO

exposed workers were compared with the US reference group, the number of deaths

from diseases of the circulatory system was slightly decreased (Chen and Fayerweather

1988).

In the cohort study by Fryzek et al. (Fryzek et al. 2003) of 4 241 TiO

exposed workers at

four US TiO

plants, no significantly increased standardized mortality ratio (SMR) was

found for heart disease or cerebrovascular disease or for any other specific cause of death

as compared to the general background population.

In the European cohort study by Boffetta et al. (Boffetta et al. 2004) of 15 017 workers

employed at factories producing TiO

, no statistically significant increase in number of

deaths from cerebrovascular disease was found as compared with the general

background population.

The study by Ellis et al. (Ellis et al. 2010) investigated the mortality among workers

employed for at least 6 months in three DuPont TiO

plants in the United States (n=5054).

The general US population was used as reference. The mortality from all causes, lung

and larynx cancer, non-malignant respiratory disease and all heart disease was

statistically significantly decreased compared to the general population. The number of

cancers belonging to the category “Other respiratory cancers” was increased 2.5 fold

(95% CI: 0.62-6.46) compared to the general population. However, this was based on

only 3 cases and was not statistically significant. The very low standardized mortality

rates are characteristic when occupational active persons are compared with the general

population due to the healthy worker effect.

A second study by Ellis et al. (Ellis et al. 2013), sponsored by E.I. du Pont de Nemours

and Company, investigated the mortality among 3607 workers employed in the same

three DuPont TiO

plants as in the previous study by Ellis et al. (Ellis et al. 2010). The

study cohort overlapped with the 5054 workers in the previous study. Compared to the

previous study, stricter inclusion criteria were applied: In addition to having been

employed for at least 6 months, the job held had to have potential TiO

exposure, and no

more than 25% or 5 years missing job history was accepted. The outcomes were death

from all causes, all cancer, lung cancer, non-malignant respiratory disease, and all heart

disease. The number of employees and the age of the study group differed vastly

between the three factories which were included in the study. The employees at the

Edgemoor plant contributed with 56% of the person-years for follow-up, and 85% of the

deceased. Employees at Edgemoor plant were on average born in 1935, whereas

employees were on average born in 1952 and 1958, respectively, on the two other plants.

Consequently, the observed associations were driven by the Edgemoor plant, which

contributed with the largest study group and the most cases. The study used two

different reference groups, the US population and a control group of other Dupont

workers who were not exposed to TiO

. The present working group is of the opinion that

the reference group of non-exposed Dupont workers is the most appropriate reference

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group, since it is most comparable to the TiO

exposed Dupont workers regarding health

worker effects, lifestyle factors and level and type of medical insurance, as compared to

the general population in the US. However, the working group notes that the Dupont

workers in the reference group may be exposed to other hazardous agents but no

information regarding this is provided in the publication. There were no statistically

significant differences in mortality for any of the studied outcomes for the TiO

exposed

workers compared to the US population. However, when the TiO

exposed workers

were compared to the reference group of other Dupont workers, an increased mortality

was observed for the following endpoints: all causes (SMR 1.23; 95% CI 1.15–1.32), all

malignant neoplasms (SMR 1.17; 95% CI 1.02–1.33), and lung cancer (SMR 1.35; 95% CI

1.07–1.33). The associations were driven by an increased mortality found at the

Edgemoor plant. Increased heart mortality was seen on the Edgemoor plant when this

plant was compared to “other workers” as reference group, but not for all three plants

combined. The risk estimates did not show clear dose-response relationship with

increasing cumulative dose. Risk estimates for all causes and non-malignant respiratory

disease increased marginally with increasing cumulative exposure using a 10 year lag,

and risk estimates for all cancers and non-malignant respiratory disease increased

marginally with increasing cumulative exposure using no lag. The observed difference in

the SMRs using an employed population versus a general population as a reference

group is typically associated with healthy worker effect. The studies by Ellis et al. (Ellis

et al. 2010;Ellis et al. 2013) have some severe study limitations including lack of

information on TiO

particle size, smoking history and a lack of a description of how the

reference groups were selected (including the number of persons in the reference

groups). Furthermore, there was no description of other possible occupational exposures

of the Dupont workers in the reference group.

While none of the above mentioned studies had information on particle size, the adverse

effects of TiO

in the nanosize have been specifically studied in two human

biomonitoring studies:

In one study, biomarkers of inflammation, oxidative damage of nucleic acids, proteins

and lipids were analyzed in the exhaled breath condensate (EBC) of a cohort of TiO

manufacturing workers (n=36). The controls were healthcare personnel and technical

staff who were not employed at the factory and did not handle dusts (n=45). In the TiO

workshops, the median TiO

mass concentrations varied between 0.40 and 0.60 mg/m

In the facility, the median particle number concentration was approximately 2 x 10

particles/cm

of which approximately 80% of the particles were less than 100 nm. In the

research workspace, the air concentration (0.16 mg/m

) and the particle number (1.32 *

particles/cm

) were lower. The results have been published in a series of articles and

documented inflammation (Pelclova et al. 2016b), oxidative damage of nucleic acids and

proteins (Pelclova et al. 2016a) and lipid oxidative damage (Pelclova et al. 2017) in the

EBC of the cohort of TiO

NM manufacturing workers. TiO

concentrations in the EBC

were statistically significantly increased in the production workers (~20 µg TiO

/L,

p<0.001)) compared to both research personnel (2.00 µg/L) and controls (1.12 µg/L). The

levels of oxidative stress markers (8-OHdG, 8-OHG, 5-OHMeu, o-Tyr, 3-ClTyr, 3-

chlorotyrosine and 3-NOTyr were higher in the production workers than the workers

from the research wing of the plant and unexposed controls (Pelclova et al. 2016a).

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Inflammation was evaluated by measuring leukotriens in EBC. All of the measured

leukotrienes were statistically significantly increased compared to the control group

(Pelclova et al. 2016b). Lipid oxidation measured as malondialdehyde, 4-hydroxy-trans-

hexenal, 4-hydroxy-trans-noneal, 8-isoProstaglandin

F2α and aldehydes C

-C

was

increased in TiO

exposed workers compared to controls and a significant dose-response

relationship was found between exposure to TiO

and markers of lipid oxidation in the

EBC (Pelclova et al. 2017).

In another recent cross-sectional study, cardiopulmonary effects were analysed in

exposed workers (n=83) and controls (n=85) in a TiO

NM manufacturing plant in China.

The exposure is described in detail in (Xu et al. 2016) and is described in the above

paragraph on exposure in the present report. In short, the highest dust concentration was

measured in the packaging area where the total mass concentration of particles was 3.17

mg/m

of which 1.22 mg/m

was nanoparticles (39% of total mass). Only a minor part of

the dust was TiO

(46.4 µg/m

) and even less was TiO

NM (16.7 µg/m

). A number of

assessed markers of inflammation (IL-8, IL-6, IL-1β,

TNF-α, and IL-10),

oxidative stress

(SOD and MDA), cardiovascular disease (VCAM-1, ICAM-1, low-density lipoproteins

(LDL) and total cholesterol) and lung damage (surfactant protein D (SP-D) and

pulmonary function) were associated with occupational exposure to TiO

NM. The acute

phase proteins serum amyloid A (SAA) and high-sensitivity C reactive protein (hs-CRP)

and the inflammatory markers IL-1β

and IL-10

were also measured but for these markers

no significant differences between the groups were observed. Among the measured

biomarkers, SP-D was the only marker showing dose dependency: SP-D decreased with

increasing working time (Zhao et al. 2018).

IARC and NIOSH (IARC 2010;NIOSH 2011), concluded that the included

epidemiological studies did not show increased risks of lung cancer among workers

occupationally exposed to TiO

. However, in a recent study of workers exposed to TiO

at three different plants in the USA, statistically significantly elevated SMRs were found

for all causes, all cancers, and lung cancers when non-TiO2 exposed workers at other

Dupont factories were used as reference group while no increase was found when the

US population was used as reference group (Ellis et al. 2013). Mortality of heart disease

associated with TiO

exposure has been assessed by Chen et al. (Chen and Fayerweather

1988) and Ellis et al. (Ellis et al. 2010;Ellis et al. 2013). When using the US population or

other workers as reference group neither study showed increased heart mortality among

TiO

workers.

The present working group is of the opinion that the reference group of non-exposed

Dupont workers is the most appropriate reference group as compared to the general US

population, and therefore concludes that increased risk of all-cause mortality, malignant

neoplasms, and lung cancer was found in the study by Ellis et al. (Ellis et al. 2013).

In relation to assessing the effects of TiO

NMs, a major limitation is that none of the

studies on lung cancer and heart disease provided information on particle size.

However, the literature search identified two biomonitoring studies with information of

particle size. In these studies, biomarkers of effect were associated with occupational

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exposure to TiO

NM. The biomarkers reflect a local biological response to TiO

NM in

the pulmonary region of the exposed workers and a systemic response in the blood.

The ability to detect the effect of exposure to occupational carcinogens is also determined

by the population-specific lung cancer incidence. In Denmark, the life time risk of getting

lung cancer (0-74 years) is 4.9% for men and 4.5% for women, respectively, according to

The National Board of Health. In the US, life time lung cancer risk is similar, 7% for men

and 6% for women (American Cancer Society 2018). The relative lung risk caused by

occupational exposure to a carcinogen, which causes lung cancer the different risk levels,

1%, 0.1% and 0.01% are given in table 1. As can be seen in the table, exposures that cause

1% excess lung cancer will give relative risks of 1.2. According to power calculation,

detection of 1% excess cancer incidence with 5% lung cancer incidence in the reference

group would require group sizes of 8 000 participants (with 80% chance of detecting the

effect at 5% significance level). On the other hand, occupational exposures that cause

0.1% excess lung cancers (1 of 1 000, which is the acceptance level in the US),

corresponds to a RR of 1.04, which requires group sizes of 750 000 persons if the

background cancer incidence is 5%.

Table 1. Relative risk of lung cancer for carcinogens that cause 1%, 0.1% or 0.01% excess lung

cancer risk in a population with the current Danish lung cancer incidence

Life time risk (0-74 years)

2011-2015 in Denmark

Excess lung cancer risk

level

1:100

2:1000

1:1 000

1:10 000

1:100 000

Men

4.9%

RR= (4.9+1)/ 4.9= 1.20

RR= (49+2)/49= 1.041

RR= (49+1)/49= 1.02

RR= (490+1)/490= 1.002

RR= (4900+1)/4900= 1.000 2

Women

4.5%

RR= (4.5+1)/4.5= 1.22

RR= (45+2)/45=1.044

RR= (45+1)/45=1.02

RR= (450+1)/450= 1.002

RR= (4500+1)/4500= 1.000 2

Thus, the epidemiological studies on TiO

and lung cancer risk have limited statistical

power to detect carcinogenic effects of TiO

exposure, unless the excess lung cancer risk

associated with TiO

exposure was very high.

As none of the above mentioned epidemiological studies provided information on the

size range of the TiO

particles, thus making it impossible to determine whether the

exposures included TiO

NM, and no information on dose-response relationship, the

present working group has decided to include and base the suggested OEL of

experimental animal studies.

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OXICOKINETICS

Exposure to TiO

NM may occur by one of three exposure routes: inhalation, ingestion,

or dermal. Of these, inhalation and to some degree dermal are the main exposure routes

in the occupational setting. Dermal exposure through healthy skin is most likely not a

risk following short term exposure. However, uptake may occur through damaged skin

or if exposure is chronic (Christensen et al. 2011). The focus of this report is exposure by

inhalation.

Inhalation of particles results in deposition in the respiratory tract (nasopharyngeal,

tracheobronchial and alveolar regions) (Oberdorster et al. 2005). The deposition pattern

of particles in the different parts of the respiratory tract is strongly dependent on size of

the aerosolized particle agglomerate. Inhaled NMs deposit in the entire respiratory tract.

However, a large fraction of the inhaled NMs deposit in the alveolar region. In contrast,

most of the larger particles (> 1-2

µm)

deposit in the upper airways (Oberdorster et al.

2005) (Shi et al. 2013;Koivisto et al. 2012). In a study, mice were exposed by inhalation

1h/day for 11 days to 42 mg/m

aerosolized powder of rutile TiO

with an average

crystallite size of 21 nm. The pulmonary deposition fraction was estimated to be 8.6%

based on the observed particle size distribution in the aerosol (Hougaard et al. 2010).

A 12 week inhalation study in rats showed that pulmonary clearance of 21 nm TiO

was slower (t

1/2

= 501 days) than 250 nm TiO

particles (t

1/2

= 174 days) (Ferin et al. 1992). .

The main mechanism for particle clearance in the alveoli following TiO

NM inhalation

was phagocytosis by macrophages(Shi et al. 2013;Koivisto et al. 2012). Smaller particles

are less efficiently phagocytized than larger particles: A rat inhalation study with 20 nm

TiO

particles demonstrated that nanoTiO

particles are not efficiently phagocytized by

macrophages (Geiser et al. 2008). This results in prolonged residence time for particles in

the lungs increasing the possibility for inflammatory reactions and translocation into

lung tissue or the circulation. Ferin et al. showed that nanosized TiO

translocate from

the lungs to the blood circulation to a greater extent than larger TiO

particles (Ferin et al.

1992). As reviewed by Geiser & Kreyling, human studies have shown that the

translocated nanoparticle mass fraction is less than 1 % of the dose delivered to the lungs

(Geiser and Kreyling 2010).

Only few studies have measured translocation of TiO

NM from the lung into the

circulatory system to systemic tissue. Available data suggest that the rate of NM

migration to the circulatory system is low. The rate of translocation is likely to depend

on size, shape and surface modifications (Geiser and Kreyling 2010).

In a very comprehensive study, Kreyling and co-workers studied the biokinetics of

radiolabeled 70 nm TiO

NM following intratracheal instillation (Kreyling et al. 2017b),

intravenous injection (Kreyling et al. 2017a) and oral application (Kreyling et al. 2017c) in

rats. For the intratracheal and the intravenous studies, biodistribution was assessed

quantitatively 1 h, 4 h, 24 h, 7 d and 28 d after exposure. The biodistribution following

oral application was assessed on the same time points except for the latest time point.

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The intratracheal instillation study showed that after 1 h about 4% of the initial

peripheral lung dose had translocated from the pulmonary region. The TiO

NM were

mainly retained in the carcass (4% after 1 h and 0.3% after 28 d). In the liver and kidney

the fractions of TiO

NM remained constant (0.03%) (Kreyling et al. 2017b). A

comparison of the biodistribution after IV-injection (Kreyling et al. 2017a), gavage

(Kreyling et al. 2017c) and intratracheal instillation (Kreyling et al. 2017b), showed that

gavage and intratracheal instillation resulted in a similar patterns of biodistribution.

However, the rate translocation to secondary organs was higher following pulmonary

exposure (ca. 4.3% of the pulmonary deposited dose after 1 h) compared to oral exposure

(0.6% of the administered dose passed the gastro-intestinal-barrier after one hour). The

biodistribution following intravenous injection was very different from the

biodistribution of following pulmonary and oral dosing.

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NIMAL STUDIES

Rodent versus human response

Inhalation studies in mice and rats are used to assess potential human hazard where

human exposure studies and epidemiological studies are not available.

There is very limited data available on effects following inhalation of TiO

NMs in

humans. Rats are the preferred animal model in particle toxicology and are more

sensitive than mice to particle-induced lung cancer and fibrosis.

Intratracheal instillation versus inhalation

Inhalation studies are the gold standard of toxicity testing, as this exposure route is the

closest surrogate to human exposure. However, the deposited pulmonary dose can be

difficult to monitor after inhalation due to differences in sizes of the aerosolized particle

agglomerates. This can result in differences in deposition (Schmid and Cassee 2017). In

addition, exposure by inhalation requires a substantial amount of material and

specialized inhalation facilities, and it poses an occupational health risk to the

technicians handling the NMs.

Pulmonary deposition by intratracheal instillation is used in screening studies (Bourdon

et al. 2012;Husain et al. 2013;Poulsen et al. 2015b;Saber et al. 2012b;Saber et al. 2012a) and

has been proposed as an alternative to inhalation exposure. This exposure method

ensures that the same dose is delivered to the lung for all NM exposures, demands less

material and is more user-friendly. Intratracheal installation has previously been shown

to give widespread distribution of particles throughout the lung (Mikkelsen et al. 2011).

A number of studies have compared the toxicological response following inhalation and

instillation of nanomaterials. Two studies have compared the global transcriptional

profiles as a means to investigate the pulmonary biological response after inhalation

compared to instilled or aspirated NMs. Inhalation and intratracheal instillation of a

surface modified TiO

NM resulted in similar transcriptional changes, with the acute

phase response and inflammation as the most important pulmonary responses to inhaled

and instilled TiO

(Halappanavar et al. 2011;Husain et al. 2013). Similarly, Kinaret et al

(Kinaret et al. 2017) compared the global transcriptomic profiles of lung tissue from mice

exposed to a straight and long multi-walled carbon nanotube (MWCNT) by inhalation or

aspiration. The authors concluded that the perturbed pathways were very overlapping,

suggesting that the transcriptiomic response to MWCNT exposure was very similar for

inhaled and pulmonary dosed MWCNTs.

Other studies compared levels of pulmonary inflammation, measured as neutrophil

influx, after exposure by inhalation or intratracheal instillation in rodents. Two studies

using MWCNT reported that both methods resulted in pulmonary inflammation, with

inhalation being more potent at inducing inflammation (Morimoto et al. 2012;Porter et al.

2013). Baisch et al. reported that instillation of a high dose of TiO

nanoparticles induced

greater inflammation compared to low dose rate delivery through inhalation, even

though the same pulmonary deposited dose were delivered. The authors concluded that

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intratracheal instillation is useful for quantitative ranking of nanoparticle hazards, but

not for quantitative risk assessment (Baisch et al. 2014).

Selection of studies and endpoints

In the present report inhalation studies will be prioritized. For the description of

toxicological endpoints and mechanism of toxicity, studies using pulmonary deposition

as intratracheal instillation will be included where no quality inhalation studies are

available. Dose-response assessments, however, are solely conducted based on sub-

chronic and chronic inhalation studies.

Endpoints were evaluated based on reported adverse effects of TiO

NM exposure in

reports and in the scientific literature. The assessment by NIOSH used cancer as the

endpoint (NIOSH 2011). However, other previous assessments have mainly focused on

inflammation as critical effect (Christensen et al. 2011;Stockmann-Juvela et al.

2014;Nakanishi and Gamo 2011). This report will therefore include both endpoints.

Cancer and cardiovascular disease have been identified as two of the main mortality

causing diseases in the world (World Health Organization 2018;Cancer Risks UK 2018).

Both diseases are potentially initiated by inflammation, as described in

Mechanism of

toxicity.

In conclusion, the critical endpoints were chosen based on literature review and

mechanistic understanding.

Pulmonary inflammation

In a sub-chronic inhalation study by Ferin et al., rats were exposed to about 23 mg /m

two different sized anatase TiO

particles (21 nm and 250 nm) for 6 h/day, 5 days/week

for 12 week. Pulmonary inflammation was assessed 4, 8, 12, 41 and 64 weeks after start

of exposure. The 21 nm TiO

NM induced more neutrophil influx than the 250 nm TiO

particles and the filtered air already after 4 weeks of exposure. The number of

neutrophils were almost reduced to control level after 52 weeks post-exposure (Ferin et

al. 1992).

In a sub-chronic inhalation study by Bermudez et al, female rats, mice and hamsters in

groups of 25 were exposed to 0, 0.5, 2.0 or 10 mg/m

TiO

NM(P25, average primary

particle size of 21 nm) for 6 hours/day, 5 days/week for 13 weeks (Bermudez et al. 2004).

The mean mass-median aerodynamic diameter of TiO

NM and agglomerates was 1.37

µm

in exposure chamber. Pulmonary endpoints (inflammation, cytotoxicity, lung cell

proliferation and histopathology) were assessed 0, 4, 13, 26 and 52 weeks (49 weeks for

TiO

NM exposed hamsters) after end of exposure.

To assess inflammation, the total number of broncho alveolar lavage (BAL) cells and the

number of macrophages, neutrophils, eosinophils and lymphocytes in the BAL cells was

determined. The neutrophil influx as percent of total BAL cells is shown in Table 2.

Compared to controls, the percentage of neutrophils was not significantly increased in

hamsters at any dose or time point after end of exposure. Immediately after end of

exposure rats had increased percentage of neutrophils at 2 mg/m

and above while this

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was not the case for exposure at 0.5 mg/m

. From 4 weeks after exposure and later time

points, both rats and mice had increased percentage of neutrophils at the highest dose

(10 mg/m

In rats exposed at the highest dose (10 mg/m

), progressive epithelial and

fibroproliferative changes were observed through 13 weeks post-exposure. Most of these

changes were reported to be regressing over time (13-52 weeks post-exposure).

Inhalation and intratracheal instillation studies have shown that when rats and mice

were exposed TiO

, the ultrafine TiO

induced a much stronger pulmonary inflammatory

response compared to the same mass of fine TiO

particles. The inflammatory response

correlated with the surface area of the deposited particles irrespectively of size. This dose

response relationship has been observed for a number of low-toxicity low – solubility

particles and it is generally accepted that the inflammatory response of low toxicity-low

solubility particles including TiO

is proportional to the surface area of the instilled

particles rather than the mass (reviewed in Oberdörster et al (Oberdorster et al. 2005)).

We consider the Bermudez study (Bermudez et al. 2004) as a key study for this hazard

assessment. It is the only of the identified studies that is a sub-chronic inhalation study

with dose-response relationship of TiO

NM. In that study, a NOAEC of 0.5 mg/m

was

identified for pulmonary influx of neutrophils in rats which was the most sensitive of the

tested species. In addition to this study we have identified a range of inhalation studies

with shorter exposure duration using TiO

NM particles (Ma-Hock et al. 2009;Noel et al.

2012;Rossi et al. 2010a;Rossi et al. 2010b;Baisch et al. 2014;Kwon et al. 2012;Lindberg et al.

2012). Overall they support that a NOAEC level for pulmonary influx of neutrophils is in

the range of 0.5-2 mg/m

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Table 2. Pulmonary influx (%) of neutrophils in animals exposed by inhalation to TiO

(Bermudez et al. 2004).

Postexposure Concentration

(weeks)

(mg/m

)

0,5

Rats

0.4 ±0.42

0.5 ± 0.35

6.50 ± 4.23*

64.80 ±5.35*

0.3 ± 0.27

0.2 ± 0.27

0.90 ± 0.96

43.30 ± 3.27*

0.20 ± 0.27

1.20 ± 1.15

1.70 ± 1.60

41.9 ±14.10*

0.50 ±0.35

0.30 ± 0.27

1.90 ± 0.82

20.8 ± 7.64*

0.80 ± 0.84

0.60 ± 0.42

0.70 ± 0.57

12.00 ± 4.51*

Mice

0.00 ± 0.00

0.20 ± 0.27

14.50 ± 5.73*

0.20 ± 0.27

0.10 ± 0.22

12.40 ± 7.90*

0.10 ± 0.22

0.40 ± 0.55

0.30 ± 0.45

13.90 ± 6.81*

0.10 ± 0.22

0.30 ± 0.27

0.10 ± 0.22

17.00 ± 5.95*

Hamsters

2.30 ± 1.15

1.30 ± 6.84

1.00 ± 0.79

10.20 ± 14.65

2.30 ± 2.49

5.20 ± 4.72

4.10 ± 4.60

3.70 ± 3.49

7.80 ±

2.60 ±

2.70 ±

4.70 ±

3.10 ±

3.60 ±

2.30 ±

8.24

1.71

0.89

1.25

1.68

2.70

1.47

1.96

0.10 ± 0.22

4.50 ± 2.50

0.00 ± 0.00

5.50 ± 6.28

0.40 ± 0.65

5.20 ± 3.19

12.30 ± 4.80*

4.50 ± 1.70

*Significantly different from control, p < 0.05; NOAECs and

the lowest observed adverse

effect concentrations (

LOAECs

)

for each species at different time-points after end of

exposure are indicated with green and red text, respectively. Table is generated based on

Tables S1-S3 in Bermudez et al (Bermudez et al. 2004).

Genotoxicity and cancer

Genotoxicity and cancer are well studied, possible adverse effects of exposure to TiO

NM. Genotoxicity often occurs relative rapidly after exposure, whereas cancer is a more

complex pathological endpoint that requires longer time to develop. In this report, we

therefore chose to differentiate between genotoxicity in shorter-term studies and cancer

in long-term studies.

Cancer

Three chronic cancer TiO

inhalation studies were identified. One of these studies used

TiO

NM (P25, 80%anatase/20% rutile) which was tested in female Wistar rats (Heinrich

et al. 1995;Heinrich et al. 1995), while the other two studies used both fine, rutile TiO

but tested in male/female Wistar rats (Muhle et al. 1991) and in male/female Sprague-

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Dawley rats (Lee et al. 1985), respectively. Details on the study set-ups are summarized

in Table 3. An increased cancer incidence was detected in rats exposed to 10 mg/m

TiO

NM (Heinrich et al. 1995), while exposure to fine TiO

only increased cancer

incidence in rats exposed to the highest tested concentration (250 mg/m

) (Lee et al.

1985). In rats exposed to 10 mg/m

of TiO

NM, slight to moderate interstitial fibrosis in

the lungs was observed in all animals after 2 years of exposure (Heinrich et al. 1995). The

present working group notes that dose response relationship for TiO2 NM-induced

cancer could not be established since only one dose level was tested. However, NIOSH

has evaluated the rat cancer data from inhalation studies of TiO

in different sizes

(ultrafine and fine) and concluded that they fit on the same dose-response curve when

dose is expressed as total particle surface area in the lungs (NIOSH 2011). The present

working group considers this sufficient evidence of dose response relationship.

In summary, in the only identified chronic inhalation study of rats exposed to TiO

NM,

cancer was induced at 10 mg/m

(LOAEC = 10 mg/m

). TiO

has been classified as

possibly carcinogenic by IARC based on sufficient evidence of carcinogenicity in

experimental animals (IARC 2010). When NIOSH evaluated rat cancer data from

inhalation studies of TiO

in different sizes (ultrafine and fine) they concluded that all

the data points fit on the same dose-response curve when dose was expressed as total

particle surface area in the lungs (NIOSH 2011).

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Table 3. Overview of chronic rat inhalation studies

Reference

Muhle et al.

(Muhle et al.

1991)

Lee et al. (Lee

et al. 1985)

(reclassification

of tumors in

Warheit and

Frame (Warheit

and Frame

2006))

Type of TiO

Fine, rutile

Exposure

2 year, 0, or 5

mg/m

Whole body

inhalation for 6

hour/day, 5

days/week for

up to 2 years,

to 0, 10, 50, or

250 mg/m

No follow up

time after end

of exposure.

Lung tumor increase compared to controls

5 mg/m

: No increase

Fine, rutile

10 mg/m

: No increase

50 mg/m

: No increase

250 mg/m

: Increased

For the 250 mg/m

Bronchioalveolar carcinomas in 12/77 male

rats and 13/74 female rats

Squamous cell carcinomas in 1/77 males and

13/74 females)

Controls:

Bronchioalveolar carcinomas in 2/79 male

rats and 0/77 female rats

No squamous cell carcinomas.

10 mg/m

: Increased

At 30 months:

13/100: Adenocarcinoma

3/100: Squamous cell carcinoma

4/100: Adenocarcinoma

20/100: keratinzing cystic squamous-cell

tumors

32/100: Total number with tumors

Controls:

Adenocarcinomas: 1/217

No other lung tumors were observed

Heinrich et al.

(Heinrich et al.

1995)

Ultrafine P25

(15-40 nm

primary

particle size, 0.8

µm MMAD, 48

/g specific

surface area,

80%

anatase/20%

rutile)

18 hour/day, 5

days/week for

up to 2 years to

0, or 10 mg/m

followed by 6

months

without TiO

exposure

Genotoxicity

The genotoxic potential has been tested in many

in vivo

studies by analysis of different

endpoints including DNA strand breaks, DNA adducts and micronuclei. Some studies

indicate that TiO

NMs are genotoxic, while other studies do not (reviewed by (Shi et al.

2013)). The different physico-chemical properties of the tested TiO

particles (specific

surface area, coating, anatase/rutile, form) may explain why some studies are negative

and others positive. The present working group concludes that no firm conclusions can

be reached regarding genotoxicity.

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Cardiovascular effects

Only few studies have investigated the cardiovascular effects of pulmonary TiO

exposure. Some studies have assessed promising biomarkers for cardiovascular disease.

Plaque progression and vascular dysfuction

The lipid profile of mice significantly differs from that of humans. Mice do not develop

atherosclerosis, because rapid clearance of hepatic LDL results in low and rather stabile

total serum cholesterol levels, even after increased cholesterol intake and synthesis.

Atherosclerotic changes are therefore mainly investigated in ApoE -/- mice, which are

deficient in apolipoprotein E (apoE), a glycoprotein associated with all lipoproteins

except LDL. ApoE -/- mice develop spontaneous atherosclerosis as early as 3–4 months

of age when fed normal chow (Nakashima et al. 1994). This makes them suitable for

investigating cardiovascular effects.

A few studies have reported TiO

NM-induced accelerated plaque progression:

Modest effect on plaque progression was detected in ApoE-/- mice intratracheally

instilled with 0.5 mg/kg bodyweight TiO

NM (21 nm) once a week for 4 weeks. No

effect on vasodilatory function was detected in ApoE-/- mice intratracheally instilled

with 0.5 mg/kg bodyweight of three types of TiO

(rutile 288 nm TiO

, anatase/rutile 12

nm TiO

, and rutile 21 nm TiO

) at 26 and 2 hours before measurement (Mikkelsen et al.

2011).

ApoE-/- mice were exposed by tracheal instillation of 0, 10, 50 and 100 µg 5-10 nm TiO

NM once a week for 6 weeks. Compared to vehicle controls, the high dose group had

increased levels of CRP, nitrogen oxide (NO), endothelial nitric oxide synthase (eNOS),

total and high density lipoprotein (HDL) cholesterol in serum. In addition, the medium

and high dose group had increased plaque area and increased ratio of the lipid-rich core

area to plaque area, respectively. At the highest dose, TiO

NM exposure induced

systemic inflammation (measured as increased level of Hs-CRP), endothelial dysfunction

(measured as reduced serum level of nitric oxide and eNOS) and changed lipid

metabolism (measured as increased total cholesterol and decreased HDL in the serum)

(Chen et al. 2013).

Microvascular dysfunction was observed in rats exposed by inhalation to TiO

NM. The

microvascular dysfunction was associated with increased oxidative stress and decreased

NO production (Nurkiewicz et al. 2009).

Thrombus formation

Systemic administration of a single dose (1 mg/kg) of anatase TiO

NM (38 nm, 320 m

/g)

but not rutile TiO

NM (67 nm, 60 m

/g) accelerated thrombus formation in the

microcirculation in mice (Haberl et al. 2015).

Activation of complement factor 3 (C3) may promote the atherosclerotic process because

C3 activation products (C3a and C3b) are involved in the atherothrombotic process and

they are associated with lipid components in the vessel wall. Activation of C3 in blood

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was detected in C57BL/6 mice exposed by intratracheal instillation

to 18 or 162 µg of

TiO

NM (rutile, 21 nm) compared with vehicle controls (Husain et al. 2015).

Acute phase response

The acute phase response is induced in humans in response to infection, infarction and

trauma, and it is defined by increases in acute phase response proteins with the most

predominant being CRP, SAA, and fibrinogen. During an acute phase response these

proteins can increase thousand fold (Gabay and Kushner 1999). Elevated plasma levels

of CRP and SAA have been reported as a risk factor for cardiovascular disease in

humans (Johnson et al. 2004;Lowe 2001;Mezaki et al. 2003;Ridker et al. 2000). In mice, the

SAA isoforms are the main acute phase response proteins, while CRP is only moderately

induced by inflammatory stimuli (Whitehead et al. 1990;Pepys and Hirschfield 2003).

SAA (SAA1-4) is a highly conserved family of apolipoproteins associated with HDL.

Several studies have reported changes in

Saa

expression levels after pulmonary exposure

to TiO

NM. Inhalation of TiO

NMs as well as intratracheal instillation of a single dose

of TiO

NM (21 nm) in female C57BL/6 mice strongly increased

Saa1, Saa2

and

Saa3

mRNA levels in lung tissue in a dose-dependent manner (Saber et al. 2013;Halappanavar

et al. 2011;Husain et al. 2013). Time-mated mice were exposed by inhalation 1h/day to 42

mg/m

TiO

NM on gestation days 8–18.

Saa3

mRNA expression levels were increased 5

days and 4 weeks after the end of exposure (Saber et al. 2013).

Reproductive toxicity

Time mated female rats were exposed to TiO

NM (P25, 21 nm in diameter) by inhalation

for 8 non-consecutive days (4-6 h/day for 7.8 days). The mass concentration was 10

mg/m

. The

calculated cumulative, deposited dose was 217 ± 1.0

µg. Chromatin

immunoprecipitation and DNA sequencing were performed in the offspring fetal hearts

at gestation day 20; and it was reported that the experiments provide initial evidence

that significant epigenetic and transcriptomic changes occur in the cardiac tissue of

maternally TiO

NM exposed progeny (Stapleton et al. 2018a). Two other studies used a

similar dosing regimen. One found that gestational exposure to the 21 nm TiO

particles

(median aerodynamic diameter 130-150 nm) disrupted progeny cardiac function and

bioenergetics (Hathaway et al. 2017). In the other study, the median aerodynamic

diameter of the inhaled 21 TiO

NM particles was 171 nm and the calculated daily

maternal deposition was 13.9 ± 0.5 µg. At 5 months of age a standard battery of several

locomotion, learning, and anxiety tests was applied for testing of male offspring from

four control and four exposed dams (n=11). TiO

NM was associated with significant

working memory impairments in the radial arm maze and deficits in the visual platform

test, possibly reflecting deficits in initial motivation in male F1 adults (Engler-Chiurazzi

et al. 2016). In a final study from this research group, virgin and late stage pregnant [GD

19] female rats were exposed to TiO

NM (21 nm in diameter) by inhalation for 5 h. The

mass concentration was 10 mg/m

(median aerodynamic diameter particle diameter

173.2±6.4 nm in the exposure atmosphere), leading to a calculated deposited dose of 42.2

± 1.9 µg TiO

. Assessment in live animals showed that inhalation of TiO

NM disturbed

vascular reactivity differentially in different stages of estrous in non-pregnant females. In

addition, increased inflammatory activity was observed in these animals. The level of

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inflammatory markers in blood was altered during estrus and late gestation. The authors

suggest that female fertility may be impaired by TiO

NM inhalation (Stapleton et al.

2018b).

Another research group exposed time-mated female mice to TiO

NM (UV Titan, 21 nm

in diameter) by inhalation for 1h/day on gestation days 8 to 18. The mass concentration

was 42 mg/m

and the major particle size-mode was ~100 nm. Several outcomes were

studied in the time-mated females and their offspring. TiO

NM exposure was associated

with lung inflammation in the time-mated females 5 and 27 days post-exposure. In the

adult offspring, TiO

NM exposure was associated with moderate neurobehavioral

alterations. Cognitive function was unaffected but the offspring tended to avoid the

central zone in the open field assay. In addition, exposed female offspring displayed

enhanced prepulse inhibition in the acoustic startle test (Hougaard et al. 2010). Levels of

DNA strand breaks were evaluated using the comet assay. No effects were observed on

this endpoint in BAL cells or liver of time-mated females 5 and 27 days post exposure,

nor in the livers of their offspring at postnatal day 2 and 22 (Jackson et al. 2013). At

maturity, female F1 offspring were mated with unexposed males. Expanded-simple-

tandem-repeat-loci germline mutation rates were determined in the F2-generation and

found not to differ between TiO

and control F2 offspring (Boisen et al. 2012). Also

testicles were collected from the mature F1 and F2 males. Daily sperm production was

not statistically significantly affected in the F1- or F2-generation males originating in

dam with TiO₂ exposure compared to sham exposed dams

(Kyjovska et al. 2013).

Overall, the above described developmental toxicity effects were observed after 8 days of

exposure for 4-6 h per day at 10 mg TiO

NM/m

, or following exposure to 42 mg TiO

NM/m

for 1 h per day for 11 days. The corresponding daily doses were 5-7.5 mg/m3 per

8-h workday. When taking into account the LOAEC/NOAEC observed on increased BAL

neutrophils in BAL in other studies at 2/0.5 mg/m

(E.g. (Bermudez et al. 2004)),

developmental toxicity is not deemed to be the critical effect in the current investigation.

This, however, may change if experiments with lower mass concentrations of TiO

are

undertaken in the future.

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ECHANISMS OF TOXICITY

Pulmonary inflammation, genotoxicity and cancer

Pulmonary exposure to TiO

NM has consistently shown dose-dependent pulmonary

inflammation (NIOSH 2011)and deposited surface area has been identified as an

important predictor of pulmonary inflammation (NIOSH 2011). The present working

group notes that there is limited available data on the biological effects of TiO

NM with

different physico-chemical properties, but concludes that the majority of available data

support that the surface area (and therefore the size) of TiO

is a critical driver of

particle-induced inflammation in the lungs. The present working group concludes that

inhalation of TiO

NM induces dose dependent pulmonary inflammation and that

neutrophil influx is predicted by the total surface area of deposited particles. The

working group considers inflammation as a threshold effect.

Shi et al. reviewed TiO

NM-induced genotoxicity

in vivo

and

in vitro

and concluded

that:” The possible mechanisms for TiO

NM-induced genotoxicity involve DNA

damage directly or indirectly via oxidative stress and/or inflammatory responses”(Shi et

al. 2013).

IARC has classified TiO

as possibly carcinogenic to humans (group 2B) based on

sufficient evidence of carcinogenicity in experimental animals and insufficient evidence

in humans. IARC does not differentiate between nano- and fine particles in their

classification (IARC 2010).

NIOSH concludes that the inflammatory response and the induction of lung tumors by

TiO

and other low-toxicity low-solubility particles correlates well with the total surface

area of pulmonary deposited particles (NIOSH 2011). NIOSH furthermore concludes

that “TiO

is not a direct-acting carcinogen, but acts through a secondary mechanism that

is not specific to TiO

but primarily related to particle size and surface area (NIOSH

2011).

EU’s Scientific Committee on Consumer Safety (SCCS) concluded similarly in a recent

report that “..an inflammatory process and indirect genotoxic effect by ROS production

seems to be the major mechanism to explain the effects induced by TiO

”. However,

SCCS also stated that “a genotoxic effect by direct interaction with DNA cannot be

excluded since TiO

was found in the cell nucleus in various in vitro and in vivo studies”

(Scientific Committee on Consumer Safety (SCCS) 2017).

The present working group found that the mechanism of action of the genotoxic and

carcinogenic effects have not been fully clarified (Shi et al. 2013). Secondary

genotoxicity due to particle-induced inflammation is an important and well documented

mechanism of action for the development of lung cancer. However, the available data

did not allow ruling out that TiO

NM could also induce cancer through a direct

genotoxic mechanism. Therefore, the present working group considers carcinogenicity as

a non-threshold effect.

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Consequently, the present working group decided to perform the risk assessment based

on both a threshold and a non-threshold mechanism of action.

Cardiovascular effects

NM exposure can lead to cardiovascular effects either: 1. Directly, by translocation of

NMs from the lung to the vascular system. 2. Indirectly, as a consequence of pulmonary

inflammation and acute phase response. 3. Alterations in autonomic nervous system

activity to elicit peripheral effects.

Atherosclerosis is a central cardiovascular effect, which is manifested as increased

plaque deposition or build-up in the arteries. It is initiated by a biological, chemical or

physical insult to the artery walls. Translocated NMs could induce this insult by

interacting directly with the endothelium. This leads to the expression of cell adhesion

molecules (selectins, VCAM-1 and ICAM-1) on the endothelial lining of the arteries,

which facilitates the activation, recruitment and migration of monocytes through the

endothelial monolayer (Hansson and Libby 2006;Cybulsky et al. 2001). Inside the intima

layer, the monocytes differentiate into macrophages and internalize fatty deposits

(mainly oxidized low density lipoprotein), transforming them into foam cells, which is a

major component of the atherosclerotic fatty streaks. The fatty streaks reduce the

elasticity of the artery walls and the foam cells promote a pro-inflammatory environment

by secretion of cytokines and ROS. In addition, foam cells also induce the recruitment of

smooth muscle cells to the intima. Added together, these changes lead to the formation

of plaques on the artery walls. A fibrous cap of collagen and vascular smooth muscle

cells protects the necrotic core and stabilizes the plaque (Libby 2002;Virmani et al. 2005).

Although initially asymptomatic, narrowing of the blood vessels can lead to other

cardiovascular diseases, such as coronary artery disease or stroke. In addition, blood

clots can be formed if the plaque ruptures. These may travel with the bloodstream and

obstruct the blood flow of smaller vessels.

Pulmonary exposure to NMs may also promote accelerated atherosclerosis indirectly

through an induced pulmonary acute phase response. Introduction of NMs to the lung

promotes neutrophil influx and release of pro-inflammatory cytokines, which leads to

increased production of SAA proteins. The SAAs are hydrophobic proteins that upon

secretion in their target organs are able to translocate to the blood. A statistically

significant correlation between Saa3 mRNA levels in the lung and SAA3 protein levels in

the blood have previously been reported (Poulsen et al. 2015a), indicating that SAA3

produced in the target organ translocate to systemic circulation. SAA circulating in the

blood becomes incorporated with HDL, thereby replacing Apolipoprotein A1 (Apo-A1)

as the major HDL-associated protein and forming HDL-SAA. The formation of HDL-

SAA has a double effect on plaque progression: 1.

HDL

is a major component of reverse

cholesterol transport, a multi-stepped process resulting in the movement of cholesterol

through the blood from peripheral tissues (including the artery walls) to the liver. The

formation of SAA-HDL impairs the HDL-mediated reverse cholesterol transport,

resulting in reduced cholesterol transport and an increased systemic total cholesterol

pool (Lindhorst et al. 1997;Steinmetz et al. 1989). 2. SAA and SAA-HDL have been

shown to directly stimulate the transformation of macrophages into foam cells and to

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stimulate uptake of oxidized LDL in the macrophages (Lee et al. 2013). In addition, SAA-

HDL has a lower capacity to promote cellular cholesterol efflux from macrophages than

native HDL (Artl et al. 2000). Pulmonary neutrophil influx has been shown to correlate

with pulmonary Saa3 mRNA levels, SAA3 levels in blood and with deposited surface

area of instilled particles (Saber et al. 2014), which links deposited particle surface area

with biomarkers of risk of developing cardiovascular disease.

In conclusion, the present working group is of the opinion that pulmonary exposure to

particles including TiO

NMs can lead to accelerated plaque progression directly,

through translocation, or indirectly, through an induced acute phase response. No single

physicochemical property has been identified as the driver of cardiovascular effects, but

TiO

NM surface area a likely important due to the close association with pulmonary

inflammation. As for inflammation, we consider cardiovascular effects as a threshold

effect. This is based on identified dose-response relationships between particle exposure

dose and induced acute phase response (Poulsen et al. 2015a;Saber et al. 2013), and the

close interplay between inflammation, acute phase response and plaque progression.

Dose-response relationships

Inflammation

Strong dose-response relationships have been observed following inhalation (Bermudez

et al. 2004) and intratracheal instillation of TiO

NM (Saber et al. 2012a) when dose is

expressed as mass. Inhalation and intratracheal instillation studies have shown that

when rats and mice were exposed TiO

, the TiO

NM induced a much stronger

pulmonary inflammatory response compared to the same mass of fine TiO

particles.

The inflammatory response correlated with the surface area of the deposited particles

irrespectively of size. This dose response relationship has been observed for a number of

low-toxicity, low-solubility particles and it is generally accepted that the inflammatory

response of low toxicity-low solubility particles including TiO

is proportional to the

surface area of the deposited particles rather than the mass (reviewed by Oberdörster et

al. (Oberdorster et al. 2005)).

Acute phase response

Strong dose-response relationship has been observed for pulmonary

Saa

mRNA

expression levels in mice intratracheally instilled with TiO

NM (Saber et al. 2013).

Saa

mRNA expression levels correlates with neutrophil influx and total deposited surface

area (Saber et al. 2013).

Cancer

As for other low-toxicity low–solubility particles, on a mass basis, the rat tumor response

following pulmonary exposure to ultrafine TiO

(Heinrich et al. 1995) is much greater

than for fine TiO

(Lee et al. 1985). However, the tumor response in rats exposed to fine

and ultrafine TiO

fit on the same dose-response curve when dose is expressed as particle

surface area (NIOSH 2011). This indicates that for the same mass dose of TiO

the

tumour response is higher for ultrafine than for fine particles. Based on this, dose-

dependency is assumed for TiO

NM-induced lung cancer.

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Particle characteristics

TiO

NMs may vary regarding size (and therefore also surface area), crystal form,

coating etc. These are all characteristics that could influence the toxicity. As described

above in the paragraph on dose-response relationship, the surface area of TiO

NM is the

best dose predictor for both the inflammatory response and for lung tumors.

TiO

exists in different naturally occurring polymorphs including rutile and anatase.

NIOSH concluded that the dose-response relationships for pulmonary inflammation and

lung tumors were not affected by different crystal structures: “The difference in TiO

crystal structure in these sub-chronic and chronic studies did not influence the dose-

response relationships for pulmonary inflammation and lung tumors [Bermudez et al.

2002, 2004; Lee et al. 1985; Heinrich et al. 1995]. That is, the particle surface area dose and

response relationships were consistent for the ultrafine (80% anatase, 20% rutile) and fine

(99% rutile) TiO

despite the differences in crystal structure.”(NIOSH 2011).

The present working group notes that there is limited available data on the biological

effects of different physico-chemical properties, but the present working group

concludes that the majority of available data support that the surface area (and therefore

also the size) of TiO

is a critical driver of particle-induced inflammation and the acute

phase response in the lungs.

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REVIOUS RISK ASSESSMENTS OF

During the last couple of years, researchers, producers and organizations have proposed

recommended exposure limits (RELs), indicative or derived no-effect-level (INEL/DNEL)

and occupational exposure levels for TiO

NM. These have been set based on pulmonary

inflammation or lung cancer. The previous recommendations of exposure limits are

presented below and an overview can be found in table 4.

IARC

In 2006, the International Agency for Research on Cancer (IARC) classified TiO

possibly carcinogenic to humans (group 2B). This classification was based on sufficient

evidence of carcinogenicity in experimental animals exposed by inhalation and

insufficient evidence in humans. IARC does not differentiate between nano- and fine

particles in their classification (IARC 2010). In Denmark, substances classified as group 1,

2A and 2B by IARC are considered carcinogenic.

ENRHES

One of the first suggestions of a limit value for TiO

NM was made within the EU

project ENRHES (Christensen et al. 2011). Due to limited data on TiO

NM, the authors

suggest an

indicative

no effect level instead of a

derived

no effect level. The derivation of

an INEL was made under the assumption of a threshold driven mechanism of TiO

toxicity: TiO

NM induced oxidative stress/inflammation which may result in other

effects such as e.g. cancer.

The INEL 17 µg/m

was derived based on the sub-chronic inhalation study of mice, rats

and hamsters by Bermudez et al. (Bermudez et al. 2004). Because rats were the most

sensitive of the tested species, the data from the rats are used for the risk assessment. A

NOAEC (NOAEC

Bermudez

) of 0.5 mg/m

was identified for pulmonary influx of

neutrophils immediately after end of exposure in rats exposed 6 hour/day, 5 days/week

for 13 weeks to P25 TiO

NM (21nm, 80% anatase/20% rutile).

The calculations of INEL follow the approach given by ECHA (ECHA 2012):

First, the NOAEC

Bermudez

is modified to correct for an 8 hour working day (in Bermudez

et al. (Bermudez et al. 2004) the rats were exposed 6 hour a day) and to correct for a

higher breathing rate in workers (10 m

/day) compared to 6.7 m

/day at rest:

NOAEC

Corrected

= NOAEC

Bermudez

*6 hour/8 hour * 6.7 m

/10 m

= 0.25 mg/m

Secondly, the corrected NOAEC is adjusted by a number of assessment factors (most of

these are default values suggested by ECHA (ECHA 2012). The following assessment

factors are used. To adjust for interspecies extrapolation, an assessment factor of 1.5 was

used (default factor is 2.5) because the observed toxic effects do not involve metabolism

and therefore there is no need for allometric scaling):

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Interspecies extrapolation (default factor is 2.5):

Intraspecies interpolation (default factor for workers):

Extrapolation from sub-chronic to chronic (default factor):

The overall assessment factor,

Total

= 1.5 * 5 * 2 = 15

This results in an INEL for chronic inhalation for pulmonary inflammation of:

INEL = NOAEC

Corrected

/AF

Total

= 0.25 mg/m

/ 15 = 0.017 mg/m

= 17 µg/m

1.5

NEDO

The NEDO project also used the sub-chronic inhalation study by Bermudez et al.

(Bermudez et al. 2004) as basis for calculation (Nakanishi and Gamo 2011). However, in

contrast to Christensen et al. (Christensen et al. 2011), Nakanishi and Gamo chose to use

a NOAEC (NOAEC

Bermudez

) of 2.0 mg/m

for pulmonary influx of neutrophils in rats.

From 4 weeks after end of exposure and the following time points the NOAEC is 2.0

mg/m

, while the NOAEC used by Christensen et al (Christensen et al. 2011) is the

NOAEC immediately after end of exposure (please see table 2 for details, paragraph on

subacute studies).

The corrected NOAEC for human exposure is calculated to be 1.8 mg/m

The assessment factor is: AF=3

This results in the following recommendation by Nakanishi and Gamo, 2011:

OEL = NOAEC

corrected

/AF= 1.8 mg/m

/3= 0.61 mg/m

NIOSH

NIOSH suggested the following recommended airborne exposure limits (as time-

weighted average (TWA) concentrations for up to 10 hr/day during a 40-hour week): 0.3

mg/m

for ultrafine (including engineered nanoscale) TiO

and 2.4 mg/m

for fine TiO

“These recommendations represent levels that over a working lifetime are estimated to

reduce risks of lung cancer to below 1 in 1,000. The recommendations are based on using

chronic inhalation studies in rats to predict lung tumor risks in humans.”(NIOSH 2011).

NIOSH also derived exposure concentrations that are designed to prevent pulmonary

inflammation. These are 0.004 mg/m

for ultrafine TiO

and 0.04 mg/m

for fine TiO

These were derived based on a benchmark dose analysis for pulmonary inflammation in

rats followed by an extrapolation of the rat benchmark doses to humans. The starting

points for the calculations were 0.9 mg/m

and 0.11 mg/m

for the fine and ultrafine TiO

respectively. Compared to the RELs accepting a risk of cancer below 1 out of 1,000 there

would be a zero excess risk of cancer development due to secondary toxicity at exposure

limits preventing pulmonary inflammation.

NIOSH showed that total deposited particle surface area of TiO

particles of different

sizes (fine and ultrafine) and different crystal structure (80% anatase/20% rutile and 99%

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rutile) can explain the observed variation in TiO

particle-induced pulmonary

inflammation and lung cancer in rat inhalation studies: “…when rats were exposed to

TiO

in sub-chronic inhalation studies, no difference in pulmonary inflammation

response to fine and ultrafine TiO

particles of different crystal structure (i.e., 99% rutile

vs. 80% anatase/20% rutile) was observed once dose was adjusted for particle surface

area [Bermudez et al. 2002, 2004]; nor was there a difference in the lung tumor response

in the chronic inhalation studies in rats at a given surface area dose of these fine and

ultrafine particles (i.e., 99% rutile vs. 80% anatase/20% rutile) [Lee et al. 1985; Heinrich et

al. 1995]. Therefore, NIOSH concludes that the scientific evidence supports surface area

as the critical metric for occupational inhalation exposure to TiO

.”(NIOSH 2011).

Scaffold project

Recently, a recommendation of a limit value for TiO

NM was made within the frames of

the EU project Scaffold and was published by Stockmann-Juvala et al.(Stockmann-Juvela

et al. 2014).The Scaffold project identified pulmonary inflammation as the critical health

effect for TiO

. Similar to Christensen et al. (Christensen et al. 2011) and the NEDO

project (Nakanishi and Gamo 2011), the project uses the sub-chronic inhalation study by

Bermudez et al. (Bermudez et al. 2004) as basis for the calculation. Similar to Christensen

et al. 2011, Stockmann-Juvala et al., 2014 uses a NOAEC (NOAEC

Bermudez

) of 0.5 mg/m

for

pulmonary influx of neutrophils in rats immediately after end of exposure is chosen as

starting point for the calculations.

First, the NOAEC

Bermudez

is modified to correct for an 8 hour working day (in Bermudez

et al. (Bermudez et al. 2004) the rats were exposed 6 hour a day) and to correct for a

higher breathing rate in workers (10 m

/day) compared to 6.7 m

/day at rest:

NOAEC

Corrected

= NOAEC

Bermudez

*6 hour/8 hour * 6.7 m

/10 m

= 0.25 mg/m

Secondly, the corrected NOAEC is adjusted by an assessment factor to take differences

between sensitivity between individuals into account:

= 2.5

This results in an OEL for nanoTiO

OEL = NOAEC

Corrected

/AF = 0.25 mg/m

/ 2.5 = 0.1 mg/m

= 100 µg/m

No data were identified for the determination of an OEL for dermal exposure.

ECHA’s Committee for Risk Assessment (RAC)

ECHA’s Committee for Risk Assessment (RAC) has concluded that the available

scientific evidence meets the criteria in the CLP Regulation to classify TiO

as a substance

suspected of causing cancer through the inhalation route (RAC, 2017).

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Summary of the evaluations

As shown in table 4, three of the recommendations use the same approach on the results

from the same sub-chronic rat inhalation study (Bermudez et al. 2004). However, due to

different choice of starting point and/or different assessment factors the derived

recommendations are in the range from 17 µg/m

– 610 µg/m

(Christensen et al.

2011;Stockmann-Juvela et al. 2014;Nakanishi and Gamo 2011). Based on a benchmark

dose approach NIOSH suggested the following recommended airborne exposure limits

(as time-weighted average (TWA) concentrations for up to 10 hr/day during a 40-hour

week): 0.3 mg/m

for ultrafine (including engineered nanoscale) TiO

and 2.4 mg/m

for

fine TiO

. “These recommendations represent levels that over a working lifetime are

estimated to reduce risks of lung cancer to below 1 in 1,000. The recommendations are

based on using chronic inhalation studies in rats to predict lung tumor risks in

humans.”(NIOSH 2011)

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Table 4. Overview of suggested OELs for TiO

NM by different organizations/researchers

Methodology for OEL development and reference/project

ENRHES

NEDO

NIOSH

Scaffold

(Christensen et

(NIOSH 2011)

(Stockmann-Juvela

(

Nakanishi

and

al. 2011)

et al. 2014)

Gamo 2011)

Critical effect

Pulmonary

Lung cancer

Pulmonary inflammation

Pulmonary

inflammation

Key study

(Bermudez et al.

(Lee et al. 1985;Muhle et

(Bermudez et al.

2004)

al. 1991;Heinrich et al.

2002;Bermudez et al.

2004)

1995)

2004;Cullen et al.

2002;Tran et al. 1999)

Risk determinant

NOAEC

BMDL

BMD

NOAEC

associated with 1/1000

Particle surface area per

excess risk of cancer

gram of lung tissue

associated with 4%

inflammatory response of

neutrophils

Risk level in

0.5 mg/m

2 mg/m

0.5 mg/m

rodents

Corrected starting

0.25 mg/m

0.29 mg/m

0.11 mg/m

0.25 mg/m

point

Uncertainty factors

Interspecies

Intraspecies

Sub-chronic

to chronic

Overall uncertainty

factor

Suggested OEL

1.5

0.017 mg/m

(17 µg/m

)

0.61 mg/m

(610 µg/m

)

0.3 mg/m

(300 µg/m

)

2.5

0.004 mg/m

(4 µg/m

)

2.5

0.1 mg/m

(100 µg/m

)

NIOSH calculated exposure limits based on both pulmonary inflammation and lung cancer. However, NIOSH’s final recommendation is based

on lung cancer rather than pulmonary inflammations. For transparency, both results are shown in the table.

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CIENTIFIC BASIS FOR SETTING AN OCCUPATIONAL

EXPOSURE LIMIT

Different methods exist for calculating OELs. The choice of method depends on the

mode of action of the substance, and can fundamentally be split up in two approaches:

Threshold effects or non-threshold effects. The threshold effect approach relies on the

assumption that the organism can withstand a certain dose before adverse effects occur,

whereas for non-threshold effects it is assumed that any exposure to the substance can

result in adverse effects. In this report, we will calculate proposed OELs based both on

threshold effects and non-threshold effects.

Endpoint: Inflammation

Pulmonary inflammation is a defense mechanism when particles or other types of

foreign material enter the lungs. Particle-induced pulmonary inflammation is considered

to be one of the key steps leading to lung cancer by a secondary genotoxic mechanism

(NIOSH 2011). Furthermore there is a close interplay between inflammation, the acute

phase response and cardiovascular plaque progression (Poulsen et al. 2015a;Saber et al.

2013). Thus, the derivation of a DNEL based on inflammation has been made under the

assumption of a threshold-driven mechanism of TiO

NM toxicity: TiO

NM induced

oxidative stress/inflammation which may result in other effects such as e.g. cancer and

cardiovascular disease.

Our approach for an OEL for TiO

NM follows the traditional approach for setting

health-based OELs: 1) identification of critical effect, 2) identification of the NOAEC, 3)

calculation of OEL using assessment factors adjusting for inter and intra species

differences).

In the current report we use the DNEL as recommended by ECHA as the OEL for

toxicological effects having thresholds (ECHA 2012).

The DNEL of 10 µg/m

is derived based on the sub-chronic inhalation study of mice, rats

and hamsters by Bermudez et al. (Bermudez et al. 2004). Rats were the most sensitive of

the tested species, and the data from the rats are used for the DNEL derivation. A

NOAEC (NOAEC

Bermudez

) of 0.5 mg/m

was identified for pulmonary influx of

neutrophils immediately after end of exposure in rats exposed 6 hour/day, 5 days/week

for 13 weeks to P25 TiO

NM (21nm, 80% anatase/20% rutile). Histopathological changes

in the lungs were dose and time dependent.

The study by Bermudez et al (Bermudez et al. 2004) is the only sub-chronic dose-

response inhalation study with TiO

NM identified. In addition to this study we have

identified a range of inhalation studies of shorter exposure time using TiO

particles

(Ma-Hock et al. 2009;Noel et al. 2012;Rossi et al. 2010a;Rossi et al. 2010b;Baisch et al.

2014;Lindberg et al. 2012). Overall they support that a NOAEC level is in the range of

0.5-2 mg/m

The calculations of the DNEL follow the approach as set out in the REACH guidance

(ECHA 2012):

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First, the NOAEC

Bermudez

is modified to correct for an 8 hour working day (in Bermudez

et al. (Bermudez et al. 2004)) the rats were exposed 6 hour a day) and to correct for a

higher breathing rate in workers at light work (10 m

/day) compared to 6.7 m

/day at

rest:

NOAEC

Corrected

= NOAEC

Bermudez

*6 hour/8 hour * 6.7 m

/10 m

= 0.25 mg/m

Secondly, the corrected NOAEC is adjusted by a number of assessment factors (most of

these are default values suggested by ECHA.

Inflammation is considered an acute response. Due to the accumulation of particles over

time, we have chosen to use the default assessment factor 2 to extrapolate from sub-

chronic to chronic exposure. The following default assessment factors are used:

Interspecies extrapolation:

Intraspecies interpolation (default factor for workers):

Extrapolation from sub-chronic to chronic:

The overall assessment factor (AF

Total

= 2.5 * 5 * 2 = 25

This results in a DNEL for chronic inhalation for pulmonary inflammation of:

DNEL = NOAEC

Corrected

/AF

Total

= 0.25 mg/m

/ 25 = 0.01 mg/m

= 10 µg/m

2.5

Endpoint: Cancer

The present working group has chosen not to use the epidemiological study by Ellis et

al. (Ellis et al. 2013) as basis for an OEL suggestion for several reasons, including the lack

of information on particle size, lack of dose-response relationship between lung cancer

incidence and cumulative TiO

dose, lack of description of the reference groups

including information on exposure, lack of information about the lung cancer incidence

in the reference groups.

Instead, the derivation of an OEL based on cancer has been made under the assumption

of a non-threshold driven mechanism of TiO

NM toxicity.

The OEL is derived based on the chronic inhalation study of mice and rats by Heinrich et

al. (Heinrich et al. 1995). Lung tumor rate in mice exposed to TiO

was not statistically

different from the lung tumor rate in mice exposed to filtered air. Therefore, as the most

sensitive of the tested species, data from the rats are used for the risk assessment.

The lowest effect level for lung cancer was observed in rats, where increased lung cancer

incidence was found at 10 mg/m

. Lung cancer incidence in TiO

exposed rats was 32%

(32/100), while the cancer incidence in control rats was 0.5% (1/217).

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Increased lung cancer incidence was observed in rats at 10 mg/m

. Both malignant and

non-malignant tumors were included in accordance with the REACH guideline stating

that: “malignant tumours as well as benign tumours that are suspected of possibly

progressing to malignant tumours are taken into account in obtaining the dose-

descriptor values” (ECHA, 2012).

Table 5. Cancer incidence and TiO

lung burden at different TiO

mass air concentrations in a

chronic inhalation study (Heinrich et al. 1995)

Total cancer

incidence

TiO

lung

burden

(mg/lung)

0 mg/m

1/217

10 mg/m

32/100

Observed excess cancer incidence at 10 mg/m

(32/100- 1/217)/(1-1/217)= 0.32 =32 %

Method I

The present working group has chosen to use the approach used by Kasai et al (Kasai et

al. 2016) and Erdely et al (Erdely et al. 2013), who use the measured lung burden in rats

exposed by inhalation and the alveolar surface area of rats and humans to estimate the

human equivalent lung burden:

At 10 mg/m

, the amount of pulmonary deposited TiO

after 2 years of inhalation was

determined to be 39 mg/rat lung (Heinrich et al. 1995).

Human lung burden equals:

Rat lung burden (39 mg) × Human alveolar surface area (102 m

) / rat alveolar surface

area (0.4 m

) = 9945 mg per human lung.

We assume using standard values that human ventilation is 20 L/min during light work

(1.2 m

/h), work related exposure for 8 h per day, 5 days per week, 45 working weeks per

year, over a working lifetime of 45 years. The deposition rate was not reported to in the

Heinrich study. For the calculation, we have used a deposition of 8.6% based on an

inhalation study by (Hougaard et al. 2010). In that study, mice were exposed by

inhalation 1h/day for 11 days to 42 mg/m

aerosolized powder of rutile TiO

with an

average crystallite size of 21 nm. The pulmonary deposition fraction was estimated to be

8.6% based on the observed particle size distribution in the aerosol.

A lung burden of 9945 mg in humans would require that workers are exposed:

Air concentration = 9945 mg/(8h/day x 5 day/week x 45 weeks/year x 45 years x 1.2 m

x 0.086) = 1.2 mg/m

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Thus, at an air concentration of 1.2 mg/m

during a 45 year work life, an excess lung

cancer incidence of 32% is expected.

Assuming a linear dose-response relationship, then 1% excess lung cancer is expected at

(1.2 mg/m

)/32 = 0.04 mg/m

(40 µg/m

The TiO

NM air concentrations resulting in different excess lung cancer incidences are

given in the table below.

Table 6. Calculated excess lung cancer incidences at different TiO

NM mass concentrations

based on method I.

Excess lung cancer

incidence

1:1 000

1: 10 000

1: 100 000

TiO

NM Air

concentration (µg/m

)

0.4

0.04

Method II

ECHA (ECHA 2012; SCHER/SCCP/SCENIHR 2009), calculated based on the two year

TiO

NM inhalation study in rats by (Heinrich et al. 1995) (Table 5):

Excess cancer risk:

Observed excess cancer incidence at 10 mg/m

(32/100- 1/217)/(1-1/217)= 0.32 =32 %

Correction of dose metric for humans during occupational exposure (8h/d):

10 mg/m

x (18 h/day)/(8 h/day) x (6.7 m

/10 m

) = 15 mg/m

Calculation of unit risk for cancer:

Risk level = exposure level x unit risk

0.32 = 15 000 µg/m

x unit risk

Unit risk = 2.1 x 10

-5

per µg/m

At a dose of 1 µg/m

, 2.1 x 10

-5

excess cancers are expected.

Calculation of dose levels corresponding to risk level of 10

-5

(and other risk levels)

-5

risk level = exposure level x unit risk (2.1 x 10

-5

per µg/m

)

Exposure level (10

-5

) = 0.47 µg/m

Thus, at 0.47 µg/m

, 1:100 000 excess lung cancer cases can be expected.

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Table 7. Calculated excess lung cancer incidence at different TiO

NM mass concentrations

based on method II.

Excess lung cancer

incidence

1:1,000

1: 10,000

1: 100,000

TiO

NM Air

concentration (µg/m

)

4.7

0.47

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ONCLUSION

The present working group evaluated the relevant literature on TiO

NM from both

epidemiological and animal inhalation studies. None of the identified epidemiological

studies provided information on the particle size range of the TiO

, thus making it

impossible to determine whether the exposures included TiO

NM. Therefore it was

decided to base the suggested health-based OEL on data from experimental animal

studies.

Pulmonary inflammation and carcinogenicity was observed in sub-chronic and chronic

inhalation studies in rats. The present working group regards inflammation and

carcinogenicity as the critical adverse effects and the subsequent risk assessments are

conducted based on studies reporting these effects. TiO

NM induced cardiovascular

effects were also identified in animal studies. But as none of these studies were sub-

chronic or chronic inhalation studies, they were not suitable for risk assessment.

However, the present working group regards the acute phase response as a biomarker of

cardiovascular effects. Due to the close association between pulmonary inflammation

and the acute phase response, the present working group regards inflammation as a

proxy for cardiovascular effects.

The present working group found strong dose response relationships for neutrophil

influx as a marker of pulmonary inflammation (Bermudez et al. 2004). Neutrophil influx

was predicted by deposited surface area. The working group considers inflammation as

a threshold effect.

The present working group found that the mechanism of action of the carcinogenic effect

has not been fully clarified. Secondary genotoxicity due to particle-induced

inflammation is an important and well documented mechanism of action for the

development of lung cancer. However, the available data did not allow ruling out that

TiO

NM could also induce cancer through a direct genotoxic mechanism. Therefore, the

present working group considers carcinogenicity as a non-threshold effect.

Consequently, the present working group decided to perform the risk assessment based

on both a threshold and a non-threshold mechanism of action.

The working group considered that data from two rat inhalation studies were the best

basis for risk assessment. The following studies were selected to be used for calculation

of DNEL and excess cancer risk, respectively: A 13 week sub-chronic inhalation study in

rats (0, 0.5, 2.0 and 10 mg/m

) (Bermudez et al. 2004) and a 2 year chronic cancer

inhalation study in rats (0 and 10 mg/m

) (Heinrich et al. 1995). Table 8 shows a DNEL

for pulmonary inflammation derived based on the sub-chronic inhalation study of rats as

the most sensitive of three tested species, and exess lung cancer risk at 1 in 1 000, 1 in 10

000 and 1 in 100 000 derived using two different approaches.

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Table 8. Overview of DNEL based on a threshold based mechanism of action and exposure

levels resulting in extra cancer risk levels at 1:1000, 1:10 000 and 1: 100 000 based on a non-

threshold based mechanism of action using two different approaches.

Threshold

based

Non-threshold

based

DNEL

Extra cancer

risk

1:1000

1:10 000

1:100 000

Suggestion of an OEL for TiO

Inflammation

Lung Cancer

Lung cancer

(method I)

(method II)

10 µg/m

4 µg/m

0.4 µg/m

0.04 µg/m

4.7

0.47 µg/m

Both of studies used for the risk assessment used P25 TiO

NM (15-40 nm diameter, 80%

anatase/20% rutile). TiO

NMs differ regarding size and surface area but also coating,

shape, crystal structure etc. The present working group notes that there is limited

available data on the biological effects of different physico-chemical properties, but the

present working group concludes that the majority of available data support that the

surface area (and therefore the size) of TiO

is a critical driver of particle-induced

inflammation and the acute phase response in the lungs.

The present working group also notes that NIOSH showed that particle surface area of

TiO

particles of different sizes (fine and ultrafine) and different crystal structure (80%

anatase/20% rutile and 99% rutile) can explain the observed variation in TiO

particle-

induced pulmonary inflammation and lung cancer in rat inhalation studies. This stresses

the importance of the surface area as a predictor for the inflammatory and carcinogenic

response.

The present working group regards cancer as the most critical effect.

The DNEL approach relies heavily on the assumption of a threshold effect on

inflammation and carcinogenicity. The present working group is of the opinion that

there is still uncertainly whether this is the case for TiO

–induced

carcinogenicity.

Two different approaches were used for calculating excess lung cancer risk based on the

same chronic inhalation study. In the first approach, lung burden in rats after two years

of exposure was used to estimate the exposure levels for occupational exposure. In the

second approach, air concentrations were used directly. Independently of the applied

method for risk assessment, the resulting exposure levels were all very low. These levels

are all more than 100-fold lower than the current Danish OEL for titanium of 6 mg/m

(measured as Ti, corresponding to 10 mg/m

for TiO

The present working group recommends the approach using the excess lung cancer risk

estimates based on lung burden, since this approach takes the actual retained pulmonary

dose into account. Thus, the expected excess lung cancer risk based on lung burden

approach is 1:1 000 at 4 µg/m

, 1:10 000 at 0.4 µg/m

and 1:100 000 at 0.04 µg/m

TiO

NM.

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