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Beskæftigelsesudvalget 2019-20
BEU Alm.del Bilag 101
Offentligt

Carbon

nanotubes:

Scientific basis

for setting

a health-based

occupational

exposure limit

Sarah Søs Poulsen, Nicklas Raun Jacobsen, Niels Hadrup,

Karin Sørig Hougaard, Anne Thoustrup Saber and Ulla Vogel

CARBON NANOTUBES: SCIENTIFIC BASIS FOR

SETTING A HEALTH-BASED OCCUPATIONAL

EXPOSURE LIMIT

Sarah Søs Poulsen

Nicklas Raun Jacobsen

Niels Hadrup

Karin Sørig Hougaard

Anne Thoustrup Saber

Ulla Vogel

Det Nationale Forskningscenter for Arbejdsmiljø, København 2018

BEU, Alm.del - 2019-20 - Bilag 101: Orientering om NFA’s forslag til grænseværdier for fem kemiske stoffer, fra beskæftigelsesministeren

NFA-report

Title

Carbon nanotubes: Scientific basis for setting a health-based occupational

exposure limit

Sarah Søs Poulsen, Nicklas Raun Jacobsen, Niels Hadrup, Karin Sørig

Hougaard, Anne Thoustrup Saber and Ulla Vogel

The National Research Centre for the Working Environment (NFA)

September 2018

978-87-7904-350-3

nfa.dk

Authors

Institution

Publisher

Published

ISBN

Internet version

The National Research Centre for the Working Environment (NFA)

Lersø Parkallé 105

DK-2100 Copenhagen

Tlf.: +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 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 nanomaterials in the work environment.’ (https://www.amr.dk/nano.aspx).

On this background, The Danish Working Environment Authority asked the National

Research Centre for the Working Environment to review the scientific evidence with the

aim of clarifying the possibilities for suggesting nanospecific occupational exposure

limits for three different nanomaterials (titanium dioxide, carbon black and carbon

nanotubes).

The purpose of the present report is to suggest a health-based occupational exposure

limit for carbon nanotubes.

Elizabeth Bengtsen and Karen Bo Frydendall, National Research Centre for the Working

Environment, 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

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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 carbon nanotubes (CNTs),

i.e. human studies (Chapter 2), toxicokinetics (Chapter 3), animal studies (Chapter 4),

mechanisms of toxicity (Chapter 5), previous risk assessments of CNTs (Chapter 6),

scientific basis for setting an OEL (Chapter 7) and finally summarize and suggest a

health-based occupational exposure limit for CNTs (Chapter 8). The focus of this report

is only occupational exposure by inhalation.

Carbon nanotubes are a very diverse class of nanomaterials with large variation in

physico-chemical properties including diameter, length, specific surface area, level and

type of contaminations, and surface modifications. Variation in toxicity potential based

on different physico-chemical properties has been reported, but at the time of this report,

the relationship between physico-chemical properties of CNTs and their inhalation

toxicity is not fully clarified. Furthermore, most commercially available CNT

preparations are very heterogeneous. Therefore, the present working group considers

toxicity data from all types of CNTs in order to obtain a precautious approach and

derivation of an OEL value that protects against as many different types of CNTs as

possible.

Assessments of human occupational exposure to CNTs at settings such as laboratories

and production sites have reported personal breathing zone concentration levels ranging

from non-detectable to ca. 80 µg/m

. This demonstrates that human occupational

exposure does occur during handling of CNTs. However, at present almost no human

data on toxicity and epidemiological studies is available. The current working group

therefore used studies in mice and rats to assess potential human hazard. Inhalation

studies were prioritized and risk assessments were solely based on these. However, for

the description of toxicological endpoints and mechanism of toxicity, studies using

pulmonary deposition from intratracheal instillation exposure and pharyngeal aspiration

exposure were included when no quality inhalation studies were available.

Pulmonary inflammation, and inflammatory-related changes, was the most commonly

reported adverse effect of pulmonary exposure to CNTs. Four sub-chronic and one

chronic study inhalation study in rats were identified as suitable for identification if

relevant NOAECs/ LOAECs and determining a derived-no-effect level (DNEL) for

pulmonary inflammation. In general, these studies identified ”no observed adverse

effect concentrations”(NOAECs) ranging from 0.05 mg/m

to 1 mg/m

and ”lowest

observed adverse effect concentrations” (LOAECs) ranging from 0.25 mg/m

to 5 mg/m

The deposited surface area of the CNTs was identified as a predictor of pulmonary

inflammation (neutrophil influx in the broncho alveolar lavage fluid). As dose-

dependency was identified for inflammation and as it was possible to detect a NOAEC,

inflammation was considered a threshold effect.

The genotoxic and carcinogenic potential of CNTs were investigated in several studies.

CNT-induced genotoxicity was reported; however, although diameter thickness was

suggested as a driver of CNT-induced genotoxicity, no clear coupling to physico-

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chemical properties could be identified. In addition, no dose response relationship was

identified regarding the genotoxic properties. One chronic cancer study in rats was

identified as suitable for risk assessment. This 2 year inhalation study investigated

pulmonary pathological changes after exposure to the long and thick MWCNT type

called MWNT-7/XNRI-7, and the authors reported lung adenomas and carcinomas at the

middle and high dose (0.2 and 2 mg/m

, 6 h/day, 5 days/week for 104 weeks), whereas

0.02 mg/m

was found as a NOAEC. The same MWCNT type (MWNT-7/XNRI-7) had

previously been classified as possibly carcinogenic by IARC. Dose-response relationships

have been identified for MWCNT-induced carcinogenic effects in several independent

studies. The present working group found that the mechanism of action of CNT-induced

carcinogenic effect has not been fully clarified. CNTs have reported to induce ROS

generation similar to carbon black. CNTs may also induce genotoxicity through they

fibrous shape, both in regards to diameter thickness and length. In addition, secondary

genotoxicity due to CNT-induced inflammation has been recognized as an important

and well-documented mechanism of action for the development of lung cancer. Based on

the lack of dose-response relationship for genotoxicity and the unclear mode of action for

cancer, the current working group did not find sufficient evidence for a threshold

mechanism for CNT-induced carcinogenicity and decided to consider it as non-threshold

effect.

CNT-induced cardiovascular effects were reported in several animal studies. Both

primary changes, such as accelerated plaque progression, and changes related to/or

leading to cardiovascular effects, such as the acute phase response, were identified.

Dose-response relationships have only sparsely been reported for CNT-induced

increased plaque progression, whereas dose-response relationships have been

established between CNT exposure and increased levels of acute phase response

proteins. CNT-induced atherosclerotic effects have solely used pulmonary deposition as

exposure method, and thus, the studies cannot be used to establish OELs. Due to the

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

current working group regards inflammation as a proxy for cardiovascular effects.

Cardiovascular effects are considered a threshold effects that is regulated in parallel to

inflammation.

The present working group regards inflammation and carcinogenicity as the critical

adverse effects of CNT exposure by inhalation and the subsequent risk assessments are

conducted based on studies reporting these effects. Based on dose-response relationships

and mode of action for these effects, the current working group decided to perform the

risk assessment based on both a threshold and a non-threshold mechanism of action.

Four sub-chronic and one chronic inhalation study in rats were identified as suitable for

determining a DNEL for pulmonary inflammation. A conservation approach was

selected and the DNEL was calculated based on the study using the CNT with the largest

specific surface area and reporting the lowest NOAEC estimate. The suggested exposure

limit based on inflammation was 1 µg/m

For the non-threshold approach on carcinogenic effects, the 2 year inhalation study in

rats were identified as suitable and excess cancer risks at the levels of 1:1,000, 1:10,000

and 1 in 100,000 were calculated based this study using two approaches (please see the

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accompanying table). In the first approach, lung burden was used to estimate the

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

Independently of the applied method for risk assessment, the acceptable exposure levels

were all very low. These levels are all more than 5 magnitudes lower than the present

Danish occupational exposure limit for bulk carbon black of 3.5 mg/m

Suggestion for an OEL for CNTs

Lung cancer

Mechanism of action

Inflammation

(Method I)

(Method II)

Threshold based

DNEL

1 µg/m

Non-threshold based Excess cancer risk:

1:1,000

0.03 µg/m

0.043 µg/m

1:10,000

0.003 µg/m

0.0043 µg/m

1:100,000

0.0003 µg/m

0.00043 µg/m

Table showing overview of DNEL based on a threshold based mechanism of action

and exposure levels resulting in excess cancer risk levels at 1:1000, 1:10 000 and 1: 100

000 based on a non-threshold based mechanism of action.

The present working group regards cancer as the most critical adverse effect of CNT

inhalation and recommends the approach using the excess lung cancer risk estimates

based on lung burden, since this approach takes the retained pulmonary dose into

account. Thus, the expected excess lung cancer risk based on lung burden approach is

1:1,000 at 0.03 µg/m

, 1:10,000 at 0.003 µg/m

and 1:100,000 at 0.0003 µg/m

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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 eksponering for

kulstofnanorør (CNTer), dvs. humane studier (kapitel 2), toksikokinetik (kapitel 3),

dyreforsøg (kapitel 4), toksicitetsmekanismer (kapitel 5), tidligere risikovurderinger af

CNTer (kapitel 6), videnskabeligt grundlag for fastlæggelse af en grænseværdi i

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

grænseværdi for CNTer i arbejdsmiljøet (kapitel 8). Fokus i denne rapport er alene på

erhvervsmæssig eksponering ved indånding.

Kulstofnanorør er en meget forskelligartet gruppe af nanomaterialer med store

variationer i de fysisk-kemiske egenskaber. Disse inkluderer diameter, størrelse, længde,

specifikt overfladeareal, mængde og type af metalurenheder og overflademodifikationer.

Variationer i CNTers fysisk-kemiske egenskaber har vist sig at kunne påvirke det

toksiske potentiale, men på nuværende tidspunkt er sammenhænge mellem CNTers

fysisk-kemiske egenskaber og deres toksicitet efter indånding endnu ikke fuldstændigt

klarlagt. Derudover er de fleste kommercielt tilgængelige CNT præparationer meget

heterogene af natur. Derfor anser den nærværende arbejdsgruppe alle CNT typer som

farlige ved indånding og foreslår at regulere alle CNTer som én gruppe.

Eksponeringsmålinger på arbejdspladser, såsom laboratorier og

produktionsvirksomheder, har påvist CNT koncentrationer i den personlige

indåndingszone som spænder fra under detektionsgrænsen til ca. 80 µg/m

. Dette viser,

at der er erhvervsmæssig eksponering for CNT ved håndteringen af CNT på

arbejdspladser. Men på nuværende tidspunkt forefindes der stort set ingen data på

toksicitet i mennesker eller epidemiologiske studier. Den nærværende arbejdsgruppe har

derfor brugt studier i mus og rotter til at vurdere den potentielle menneskelige

helbredsrisiko. Subkroniske og kroniske inhalationsstudier blev prioriteret og

risikovurderingerne blev udelukkende foretaget på baggrund af disse. Dog blev studier,

der anvendte lungedeponering ved intratracheal instillation, inkluderet til beskrivelse af

toksikologiske endepunkter og toksisitetsmekanismer, når der ikke forelå

inhalationsstudier af tilstrækkelig kvalitet.

Lungeinflammation, og inflammationsrelaterede ændringer var de hyppigst

rapporterede helbredseffekter efter lungeeksponering af CNTer. Fire sub-kroniske og et

kronisk inhalationsstudie i rotter blev fundet egnede til at bestemme en ”derived-no-

effect level” (DNEL) for lungeinflammation. Disse studier rapporterede generelt ”no

observed adverse effect concentrations” (NOAECs)mellem 0.05 mg/m

og 1 mg/m

”lowest observed adverse effect concentrations” (LOAECs) mellem 0.25 mg/m

og 5

mg/m

. Det deponerede overfladeareal af CNTer blev identificeret som en prædiktor for

lungeinflammation (neutrofilt influx i lungeskyllevæsken). Der var dosisafhængighed

for CNT-induceret lungeinflammation, og da det var muligt at bestemme en NOAEC,

blev inflammation anset for at være en tærskeleffekt.

CNTers genotoksiske og kræftfremkaldende potentialer er blevet undersøgt i flere

studier. CNT-induceret genotoksicitet er blevet rapporteret, men selvom

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diametertykkelse er blevet forslået som prædiktor for CNT-induceret genotoksicitet, er

der endnu ikke fundet en klar sammenhæng mellem genotoksicitet og de fysisk-kemiske

egenskaber for CNTer. Derudover blev der ikke fundet dosis-respons sammenhæng.

Et kronisk kræftstudie i rotter blev identificeret som egnet til risikovurdering. I dette 2

års inhalationsstudie blev patologiske ændringer i lungen efter eksponering for den

lange og tykke CNT type kaldet MWNT-7/XNRI-7 undersøgt, og forfatterne

rapporterede lungekræft ved den midterste og højeste dosis (0,2 og 2 mg/m

, 6

timer/dag, 5 dage/uge i 104 uger). Denne type CNT (MWNT-7/XNRI-7) er blevet

klassificeret som muligvis kræftfremkaldende (2b) af IARC. Der blev identificeret

dosisafhængighed for CNT-induceret kræft i flere uafhængige studier. Den nærværende

arbejdsgruppe fandt ikke, at mekanismen for CNT-induceret kræft er fuldstændig

klarlagt. Studier har vist, at CNTer kan inducere reaktive oxygen-forbindelser på samme

måde som carbon black. De kan muligvis også inducere genotoksicitet via deres

fiberform, både i forhold til diameter og til længde. Derudover er det velkendt at

sekundær genotoksicitet, pga. CNT-induceret inflammation, er en mekanisme for

udviklingen af kræft. På baggrund af den manglende dosisafhængighed for

genotoksicitet og en uklar virkningsmekanisme for kræft, fandt den nærværende

arbejdsgruppe ikke tilstrækkelig bevis for at CNT-induceret kræft er en tærskeleffekt.

CNT-induceret kræft blev derfor anset som en ikke-tærskeleffekt.

Resultaterne fra flere dyrestudier viste CNT-inducerede effekter på hjerte-karsystemet.

Dette gjaldt både primære effekter, som åreforkalkning, og ændringer relateret til eller

førende til hjerte-kareffekter, så som akutfaseresponset. Dosisafhængighed er kun i

begrænset omfang beskrevet for CNT-induceret øget åreforkalkning, hvorimod

dosisafhængighed er veldokumenteret for sammenhængen mellem CNT eksponering og

akutfaseproteiner. Men da de studier, hvor resultaterne viser CNT-inducerede effekter

på hjerte-karsystemet, udelukkende har brugt lungedeponering som

eksponeringsmetode, kan de ikke bruges til risikovurdering af CNTer. På grund af den

tætte sammenhæng mellem inflammation, akutfaseresponset og åreforkalkning har den

nærværende arbejdsgruppe valgt at anse inflammation som en proxy for effekter på

hjerte-karsystemet. Hjerte-kareffekter blev derfor anset som en tærskeleffekt på lige fod

med inflammation.

Den nærværende arbejdsgruppe anser inflammation og carcinogenicitet som de vigtigste

negative helbredseffekter forårsaget af indånding af CNT, og de efterfølgende

risikovurderinger er baseret på studier, der rapporterer disse effekter. Baseret på viden

om dosis-respons sammenhæng og underliggende biologiske mekanismer har den

nærværende arbejdsgruppe besluttet både at foretage risikovurderinger baseret på en

tærskeleffekt og på en ikke-tærskeleffekt. Fire subkroniske og et kronisk

inhalationsstudie i rotter blev identificeret som velegnede til fastlæggelse af DNEL for

lungeinflammation. Den nærværende arbejdsgruppe valgte en konservativ tilgang, og

derfor blev DNEL beregnet med udgangspunkt i det studie, som anvendte den CNT, der

havde det største specifikke overfladeareal, og som rapporterede det laveste NOAEC.

Den foreslåede grænseværdi for kulstofnanorør baseret på inflammation er 1 µg/m

Den nærværende arbejdsgruppe valgte at anvende ikke-tærskeleffekt tilgangen til

beregning af grænseværdi baseret på kræftrisiko, og et 2-års inhalationsstudie i rotter

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blev identificeret som velegnet. De eksponeringsniveauer, som resulterer i overskydende

kræftrisiko hos 1:1.000, 1:10.000 og 1 ud af 100.000 udsatte, blev beregnet på baggrund af

dette studie på to forskellige måder og er vist i tabellen nedenfor. Ved den første

beregningsmetode bruges den lunge-deponerede dosis til at estimere det tilsvarende

eksponeringsniveau. Ved den anden beregningsmetode blev luftkoncentrationer

anvendt direkte. Uafhængigt af metodevalg er alle de beregnede grænseværdier for

kulstofnanorør meget lave. Grænseværdierne er alle mere end 5 000 gange lavere end

den nuværende danske grænseværdi for carbon black (som er 3,5 mg/m

Forslag til grænseværdi for kulstofnanorør

Lungekræft

Virkningsmekanisme

Inflammation

(Metode I)

(Metode II)

Tærskeleffekt-baseret DNEL

1 µg/m

Ikke-tærskeleffekt-

Overskydende

baseret

lungekræft:

1:1.000

0,03 µg/m

0,043 µg/m

1:10.000

0,003 µg/m

0,0043 µg/m

1:100.000

0,0003 µg/m

0,00043 µg/m

Tabellen viser en oversigt over DNEL baseret på tærskeleffekt som

virkningsmekanisme for lungeinflammation og de eksponeringsniveauer, som

resulterer i overskydende kræftrisiko hos 1:1.000, 1:10.000 og 1 ud af 100.000 baseret på

en ikke-tærskeleffekt-baseret biologisk virkningsmekanisme for lungekræft.

Den nærværende arbejdsgruppe anser kræft for at være den vigtigste helbredseffekt ved

indånding af kulstofnanorør og anbefaler ydermere at bruge beregningsmetode I fordi

denne beregningsmetode tager udgangspunkt i den faktiske lungedeponerede dosis.

Det estimeres derfor at 0.03 µg/m

kulstofnanorør vil forårsage 1:1.000 overskydende

lungekræfttilfælde ved indånding i arbejdsmiljøet, mens 0,003 µg/m

kulstofnanorør vil

forårsage 1:10.000 overskydende lungekræfttilfælde og 0,0003 µg/m

forventes at

forårsage 1:100,000 overskydende lungekræfttilfælde.

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ONTENTS

Foreword ........................................................................................................................................ 3

Executive summary ....................................................................................................................... 4

Dansk sammenfatning .................................................................................................................. 7

Contents ........................................................................................................................................ 10

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

Introduction.................................................................................................................................. 12

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

Exposure ................................................................................................................................... 15

Biomonitoring .......................................................................................................................... 18

Toxicokinetics .............................................................................................................................. 19

Animal studies ............................................................................................................................. 21

Rodent versus human response ............................................................................................ 21

Intratracheal instillation versus inhalation .......................................................................... 21

Selection of studies and endpoints ....................................................................................... 22

Pulmonary inflammation ....................................................................................................... 22

Genotoxicity and cancer ......................................................................................................... 26

Cardiovascular effects............................................................................................................. 29

Reprotoxicity ............................................................................................................................ 31

Mechanisms of toxicity ............................................................................................................... 32

Pulmonary inflammation ....................................................................................................... 32

Genotoxicity and cancer ......................................................................................................... 33

Cardiovascular effects............................................................................................................. 34

Dose-response relationships .................................................................................................. 35

Previous risk assessments of carbon nanotubes ..................................................................... 37

Aschberger et al. 2010 ............................................................................................................. 37

Pauluhn 2010 ............................................................................................................................ 37

ENRHES.................................................................................................................................... 38

NIOSH....................................................................................................................................... 38

IARC .......................................................................................................................................... 38

Scientific basis for an occupational exposure limit ................................................................. 41

Calculations of exposure limits based on cancer as non-threshold effect ....................... 41

Calculations of exposure limits based on inflammation as threshold effect .................. 44

Conclusion .................................................................................................................................... 46

References ..................................................................................................................................... 48

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BBREVIATIONS

ALP

BAL

BALF

BET

CVD

CNT

CRP

DNEL

DSP

DWCNT

ECHA

ENRHES

IARC

ICAM

INEL

HARN

HDL

HiPCO

LDH

LDL

LOAEC

MWCNT

NIOSH

NOAEC

OEL

OECD

REACH

REL

SAA

SWCNT

TEM

TWA

VCAM

Alkaline phosphatase

Broncho alveolar lavage

Broncho alveolar lavage fluid

Brunauer–Emmett–Teller

Cardiovascular disease

Carbon nanotube

C-reactive protein

Derived-no-Effect Level

Daily sperm production

Double-walled carbon nanotubes

Elemental carbon

European Chemicals Agency

Engineered Nanoparticles: Review of Health and Environmental Safety

Electron microscopy

Gastrointestinal

The International Agency for Research on Cancer

Intercellular Adhesion Molecule

Human indicative no-effect levels

Intraperitoneal

High aspect ratio nanomaterials

High-density lipoproteins

High-pressure carbon monoxide method

Lactate dehydrogenase

Low-density lipoproteins

Lowest observed adverse effect concentration

Multi-walled carbon nanotube

National Institute for Occupational Safety and Health

Nanomaterial

No observed adverse effect concentration

Occupational exposure limit

Organisation for Economic Co-operation and Development

Registration, Evaluation, Authorisation and Restriction of Chemicals

Recommended exposure limit

Serum amyloid A

Single-walled carbon nanotube

Transmission electron microscopy

Time-weighted average

Vascular cell adhesion molecule

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NTRODUCTION

Carbon nanotubes (CNTs) comprise a group of poorly soluble, highly variable,

cylindrical, hollow, fibrous nanomaterials. They are grouped according to their number

of side-walls and generally divided into three categories: Single-walled (SW), double-

walled (DW), or multi-walled (MW) CNTs. Their side-walls are made of carbon atoms,

mainly in sp

configuration, arranged in inter-connected hexagon similar to that of

graphene sheets. As the name implies, SWCNTs consist of one rolled-up graphene layer,

whereas DW- and MWCNTs consist of two or more layers. The difference in wall

numbers affects the diameter and rigidity of the CNTs; whereas some are entangled,

others are fiber-like. Correspondingly, the diameters can vary from around 1 nm (most

SWCNTs) to up to 150 µm (some MWCNTs) (Jensen et al. 2015). Because of their long

lengths (up to several mm), CNTs are high aspect ratio (length:diameter) nanomaterials

(HARN). Long CNTs (over 15 µm in length) comply with the WHO fiber paradigm,

which states that fibers with long lengths, small diameters and high biopersistence

display increased toxicity, as they reach the alveoli region of the lungs, retain their

structure, and are difficult for the macrophages to phagocytize (Donaldson et al. 2010).

The physical appearance of CNTs can vary greatly from one type of CNT to another. The

rigidity of the CNT, and thus its level of fiber-like appearance, relies prominently on its

number of graphene walls. However, MWCNTs with few walls resembles SWCNT

physically more than fiber-like MWNCTs with several walls (Figure 1). It is therefore

difficult to address specific SW- or MWCNT effects. Instead of separating CNTs based on

wall numbers, the current working group has chosen to separating based on physico-

chemical properties as surface area, length, diameter, chemical composition etc. This

report therefore does not distinguish between SW- or MWCNT effects, and CNTs data

from both SWCNT and MWCNT were evaluated. Based on this, the current working

group has selected the most adequate data for OEL derivation.

Figure 1. Different CNTs. Left: Transmission electron microscope (TEM) image of

fiber-like MWCNTs (Poulsen et al. 2015b). Bar size: 1 µm. Middle: TEM image of

flexible MWCNTs (Poulsen et al. 2015b). Bar size: 1 µm. Right: TEM image of

SWCNTs (Balarak et al. 2016).

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CNTs can be synthesized though different processes and the method chosen affect the

level of impurities and structural defects. In general, most synthesis processes take place

in vacuum, on a catalyst (often a metal such as Fe, Ni or Co), with controlled process

gasses and controlled temperature (Ismail et al. 2018). The most commonly used method

for commercially available CNTs is chemical vapor deposition (CVD). In this process, the

type of CNT, its structure and its size can be controlled by the size and type of catalyst,

the choice of gas and the temperature. This means that physical dimensions are well

controlled although it may result in many structural defects on the CNTs, especially

MWCNTs. Another commonly used method for large-scale production is arc discharge,

which is a fairly easy and inexpensive method. The disadvantage to this method is that

carbon black, fullerenes and soot are generated as byproducts and that the raw CNTs

formed often contain a lot of metal catalyst residues. More precise, but also more

expensive, CNT synthesis processes, as laser ablation, also exist.

The most common structural defects introduced during the synthesis process are missing

carbon atoms or replacement of hexagons by pentagons or heptagons. Such defects may

increase the curvature of the CNTs due to elongation or compression of one side of the

CNT (Zhang and Li 2006). It is also possible that structural defects could render the

graphene sheets and thereby the CNTs more susceptible to biological degradation,

which would change their toxic potential. Equally, the presence of bioavailable metal

impurities from catalysts on the surface of the CNT could have influence their toxic

potential. Some metals, especially iron, are known to induce reactive oxygen species

(ROS) (Knaapen et al. 2004). ROS may induce damage to cellular components such as

DNA, lipids and proteins, thereby causing genotoxicity.

Pure, graphitized CNTs are very hydrophobic in nature. Functionalization of CNTs is

therefore an important tool for increasing their solubility in aqueous solutions or their

chemical binding in solid composites. This is also important for the potential use of

CNTs in biological applications. By changing their polarity, the dispersion and the

biological interactions in the lung milieu, as well as the fate of the CNTs, may change

dramatically. This could ultimately change the toxic potential of the CNTs. Several

studies have reported altered toxicity of functionalized CNTs compared to pristine CNTs

(Hamilton, Jr. et al. 2013;Jain et al. 2011;Poulsen et al. 2016;Sager et al. 2014;Sayes et al.

2006).

The overall structure and composition of CNTs facilitate excellent electric and thermal

conductivity, high tensile strength, and good chemical stability (Dresselhaus et al. 2004).

Due to these abilities, CNTs are desirable for use in a variety of products, including

composite materials, electronics, plastics and rubbers, coatings, insulation and in

biomedical applications (De Volder et al. 2013;Jensen et al. 2015). As an example, the

high-aspect ratio and high tensile strength of CNTs makes them ideal for low weight

materials, e.g. sports equipment and wind mill wings. Due to their many possible

applications, CNTs are already produced and utilized at commercial scale, and they are

available worldwide. The global market for CNT products is expected to grow from an

estimated USD 3.43 Billion in 2016 to USD 8.70 billion in 2022 (Markets and Markets

2017). Thus with increased production, the potential exposure risks for both workers and

consumers have also increased.

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To our knowledge, there are no legally enforced occupational exposure limits (OEL) for

CNTs. In Denmark, the approximate OEL is the current for bulk carbon black, which has

the same chemical composition as CNTs. The limit is 3.5 mg/m

and is regulated by the

Danish Working Environment Authority. The aim of the present report is to investigate

if the present knowledge allows for a suggestion of a health-based, OEL for CNTs. This

document will therefore review the relevant literature on the adverse effects of CNTs. As

suggested in the guidelines from REACH (ECHA 2012), the risk assessment

methodology in this report will be divided into threshold 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 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 (ECHA 2012): 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

current working group will use two approaches. The first method, used by Kasai et al.

2016 and Erdely et al. 2013, uses 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. The second method, suggested by (ECHA 2012;SCHER/SCCP/SCENIHR 2009),

uses air concentrations directly. Conclusively, the calculated OELs will be compared and

lastly, a recommended OEL for CNT exposure will be proposed.

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UMAN STUDIES

Exposure

CNTs are synthesized as powders and inhalation is therefore considered the main route

of exposure in humans, although dermal exposure is also possible. Exposure though

inhalation may occur during the entire CNT lifecycle: Manufacturing, storage,

transportation, product application, and end-of-life processes. However, in their final

applications CNTs rarely exist as free fibers. When bound to a surface or suspended in

either liquids or solids, the individual CNTs are surrounded and more or less bound in

the matrix. It has been reported that this diminishes their toxic potential substantially

(Saber et al. 2016). Therefore the greatest risk of human exposure is in the working

environment, especially during production and handling of large quantities of free

CNTs. The nano-sized properties of CNTs are important for scenarios involving human

exposure in the occupational settings:

•

More individual CNT per mass unit compared to larger fibers.

Larger surface area per mass unit compared to larger fibers.

More dusty and therefore stays in the air longer compared to larger fibers.

Measurement of personal exposure levels to CNTs in occupational settings has been

reported in several studies (Table 1). Whereas the earlier exposure assessment studies

measured total inhaled mass or total gravimetric mass in the personal breathing zone

(Han et al. 2008;Lee et al. 2010;Maynard et al. 2004;Methner et al. 2010), the more recent

studies measured elemental carbon (EC) concentrations in personal breathing zone

samples (Birch et al. 2011;Dahm et al. 2012;Dahm et al. 2015;Lee et al. 2015;Methner et al.

2012;Ono-Ogasawara et al. 2015;Shvedova et al. 2016;Takaya et al. 2012;Erdely et al.

2013;Hedmer et al. 2014), which is more specific for carbon-based particles, including

CNTs. As a possible consequence of this, the older studies tended to report higher

exposure levels compared to newer studies. In general, the newer occupational exposure

assessment studies found personal breathing zone concentration levels ranging from

non-detectable to ca. 80 µg/m

. The differences in personal breathing zone concentration

levels are primarily attributed to different worker exposure scenarios. These studies

demonstrate that human exposures to CNTs occur in occupational settings.

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Table 1. Measured personal occupational exposure levels of CNTs.

Type of samples collected

Estimated inhalable mass

Total gravimetric mass

Work place

Four facilities producing

SWCNTs

MWCNT research facility

Work process

HiPCO or laser ablation production

Handling of MWCNTs: Blending,

weighing, spraying, miling, etc..

Production and handling of MWCNTs

Personal breathing

zone mass

concentrations (μg/m

)

0.7 - 53

N.D. - 331.7

7.8 - 320.8

Reference

Maynard et al.

2004

Han et al. 2008

Lee et al. 2010

Two research institutes, two

labs and three industrial

facilities

Total carbon-inhalable size

Five research and

Handling of CNTs: Weighing, mixing,

fraction

development labs, and one

wet sawing, processing, drying, etc.

manufacturer

Elemental carbon-

A facility manufacturing

Carbon nanofiber handling

respirable size fraction

and processes vapor-grown

carbon nanofibers

Elemental carbon-

Five facilities producing

Production and handling of CNTs:

inhalable size fraction

CNTs and one developer of Weighing, mixing, sonicating, milling,

semiconductor

etc.

Elemental carbon-

Two facilities producing

Production and handling of CNTs and

inhalable size fraction

CNTs and two facilities

carbon nanofibers: Weighing,

producing carbon nanofibers

spraying, filtration, cleaning,

harvesting

Respirable elemental

Factory creating fabric from

Weaving

carbon mass

yarns covered with

concentrations

MWCNTs

Elemental carbon-

Eight facilities producing or

Production and handling of CNTs:

inhalable size fraction

using MWCNT

Weighing, mixing, sonicating, milling,

etc.

64-1094

Methner et al.

2010

Birch et al.

2011

Dahm et al.

2012

Methner et al.

2012

45 - 80

N.D. - 7.86

N.D. - 38

3.5 - 4.8

Takaya et al.

2012

Erdely et al.

2013

N.D - 79.6

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Elemental carbon-

respirable size fraction

Inhalable elemental carbon

mass concentrations

Respirable elemental

carbon mass

concentrations

Respirable elemental

carbon concentrations

Small-scale producer of

MWCNTs by arc discharge

A MWCNT manufacturing

company

Primary and secondary

manufacturers of CNT or

carbon nanofibers

A MWCNT manufacturing

facility

Production and handling of

MWCNTs: Cleaving, sieving,

cleaning, harvesting, grinding, etc.

Manufacturing of MWCNTs

<0.08 - 7.4

Hedmer et al.

2014

Lee et al. 2015

Dahm et al.

2015

Shvedova et al.

2016

5.5 - 9.3

0.02 - 2.94

Production and handling of CNTs:

Weighing, mixing, sonicating, milling,

etc.

Production and handling of

MWCNTs:

Harvesting,

disintegration, packaging, laboratory

handling, etc.

0.54 - 6.11

N.D: Not detected, i.e. below detection limit.

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Biomonitoring

Although carbon nanotubes have been known since the mid 50’s, they did not receive

much attention from the scientific community as a whole until Sumio Iijima described

MWCNTs in his Science article from 1991 (Iijima 1991). CNTs are therefore a relatively

new material, and large-scale productions have only started within the last decade.

Consequently, studies reporting toxicological effects after both occupational and non-

occupational human exposure are very scarce. At present date, only one study is

available in the literature.

Shvedova and colleagues investigated biomarkers in induced sputum and blood of

workers exposed to MWCNTs in a manufacturing facility in Tambov, Russia (Nanotech

Center Ltd.) (Shvedova et al. 2016;Fatkhutdinova et al. 2016). They recruited 8 workers

exposed to MWCNTs (as having direct contact with MWCNT aerosol for at least 6

months) and 7 non-exposed controls from the same facility. Further, exposure

assessment of the personal breathing zone at different tasks in the facility was also

conducted. The 8-h, TWA elemental carbon concentrations in respirable size fractions

were measured at different workstations and were in the range of 0.7–2.8 µg/m

(Fatkhutdinova et al. 2016). The control group, who did not handle MWCNTs, was not

exposed to MWCNTs. Exposed workers had more than 2-fold increased serum levels of

IL1B, TNF, IL4 and IL10 and increased sputum levels of Il1B, TNF, IL6, IL4, IL5, IL8 all

indicative of systemic inflammation. Acute phase response proteins C-reactive protein

(CRP) and serum amyloid A (SAA) were not assessed. No no-observed-adverse-effect

concentration (NOAEC) or lowest-observed-adverse-effect concentration (LOAEC) was

calculated, but the authors of the current report note that the exposure levels were

relatively low at 0.7–2.8 µg/m

, indicating that even this low level was sufficient to

induce markers for systemic inflammation in humans.

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OXICOKINETICS

The behavior and distribution of CNTs after exposure is of great importance for how and

where they affect the organism. The main entry way, which in the case of occupational

exposure is the lung and to some extent the skin, will undoubtedly receive the greatest

load and experience the greatest changes. However, extra-pulmonary alterations have

previously been reported after pulmonary exposure to CNTs (Poulsen et al.

2015a;Poulsen et al. 2017;Kim et al. 2015). Translocation of CNTs is a likely explanation

for these distal changes, although it is possible that secondary exposure through

inflammation plays a significant role. In order to understand potential target effects, it is

important to identify the tissues where CNTs accumulate.

Umeda and colleagues evaluated deposition pattern of MWCNTs MWNT-7 (D: 88±5 nm,

L: 5.0±4.5 µm) after inhalation exposure in F344 rats of both sexes (doses 0.2, 1 or 5

mg/m

MWCNT, 6 h/day, 5 days/week for 2 weeks)(Umeda et al. 2013). MWCNT

deposition was observed in the entire lung (bronchiolar space, alveolar space, alveolar

walls) and in the nasal cavity immediately after exposure. The MWCNTs were primarily

detected within alveolar macrophages with, a few free MWCNT fibers found in the

bronchi and alveolar space. The quantity of MWCNTs was higher in the rats exposed to

5 mg/m

MWCNTs compared to the lower doses. Deposited CNTs have been reported to

reach the sub-pleural region after pulmonary exposure in rodents (Ryman-Rasmussen et

al. 2009;Mercer et al. 2010). Inhaled MWCNTs (L:0.5–40 µm, D:10–50 nm, 1 or 30 mg/m

for 6 h) translocated rapidly to the sub-pleura region and were detected until the end of

the experiment 14 weeks post-exposure. The authors suggested that macrophages

facilitated the transport by engulfing the MWCNTs (Ryman-Rasmussen et al. 2009). In

concordance with this, Mercer et al. showed that 0.6% of the pulmonary deposited dose

had translocated to the sub-pleural region one day after pharyngeal aspiration of the

MWCNT MWNT-7 (L:3.9 µm, D: 49 nm)(Mercer et al. 2010). The translocation to the sub-

pleural region may suggest a similar mode of action as asbestos.

(Mercer et al. 2013) exposed male mice to MWCNTs MWNT-7 (aerodynamic diameter of

1.3 µm) by inhalation (5 mg/m

MWCNT aerosol for 5 hours/day for 12 days, 4

times/week for 3 weeks, estimated lung burden of 28.1 µg/lung). At 1 day and 336 days

after the exposure period they assessed the biodistribution of MWCNTs by darkfield

microscopy. They estimated that 7.3% of the lung burden measured at post-exposure day

1 was cleared from the lung at post-exposure day 336. The vast majority of this had

translocated to the lymph nodes, however 0.03% of the initial MWCNT lung burden was

found in the liver and lesser percentages was found in the kidney, heart, brain and

diaphragm.

Lung clearance and translocation to liver and spleen was assessed by (Czarny et al.

2014), who exposed female Balb/c mice to 20 µg

C skeleton-labelled MWCNTs by

pharyngeal aspiration. The MWCNTs had an average diameter of 41 nm, were 3.9 µm

long and appeared as straight single fibers in EM pictures. The limit of detection of the

radioactive labelling was determined to be in the order of 0.2 pg or 22 CNT fibers. Half

the dosed MWCNTs were cleared from the lung within 1 day. Of the remaining 10 µg,

10% was detected in the lung tissue 3 and 12 months post-exposure. At 12 months post-

exposure, 0.75% was found in liver and 0.20% was found in spleen. MWCNT

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translocation to liver and spleen was confirmed with EM. Thus, this study shows that

rigid MWCNTs was cleared from the lung and to some extent translocated to the liver

and spleen. 10% of the dosed MWCNTs remained in the lung and no further clearance

was detected the period between 3 and 12 months post-exposure. Based on the data

provided in the paper, lung clearance of the alveolar deposited dose is estimated to

occur with a half-life of 30 day.

In a two-year inhalation study, rats were exposed MWNT-7 at doses 0.02, 0.2 and 2

mg/m

(Kasai et al. 2016). The MWCNTs had diameters of 92.9-98.2 nm and mean lengths

of 5.8-5.9 um, and were mainly found as single fibers. Lung burden increased linearly

over time in a dose-dependent manner. Deposited dose was estimated to be 1.5-2.7% of

the inhaled dose. Deposited dose per lung differed between sexes, but no difference was

observed when normalized to lung weight. In addition to the lung, the MWCNTs were

observed as single or aggregated fibers in nasal cavity, larynx, trachea, lungs, lymph

nodes, spleen, liver, kidneys, olfactory bulb, and brain. In the kidney, olfactory bulb and

brain, the MWCNTs were only observed as single fibers.

Pauluhn performed a 13-week inhalation study in rats using Baytubes, which were 10

nm in diameter and 200-300 nm long (Pauluhn 2010b). The MWCNTs appeared curved

on EM pictures. The aerosol consisted mainly of CNT agglomerates. Pulmonary

clearance in the rats was estimated by assessing Co content in the lung. Half-lives for

clearance were 150-375 days. The highest clearance rate was observed for the highest air

concentration. The current working group considers the highest dose to give the best

estimate, as the Co content was close to the limit of detection.

Taken together, the data suggest that agglomerated CNTs are cleared away from the

lung slower than CNTs that are dispersed as single fibers. In agreement with this,

Pauluhn and Rosenbruch have shown that CNT clearance following inhalation exposure

in rats occurs faster with well-dispersed Baytubes CNTs compared to aggregates of the

same CNT (Pauluhn and Rosenbruch 2015). Half-lives for pulmonary clearance were

estimated to be 87 and 46 days for aggregated and dispersed CNTs, respectively.

Translocation can in principle occur either by translocation from lung to blood or by

secondary uptake via the GI tract following pulmonary clearance by mucociliary

transport. However, no uptake was detected following oral dosing of the radioactively

labelled MWCNTs suggesting that translocation occurred from lung to systemic

circulation (Czarny et al. 2014).

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NIMAL STUDIES

Rodent versus human response

As almost no human data on toxicity and epidemiological studies is available, inhalation

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

One human study reported MWCNT-induced systemic inflammation following

exposure to relatively low air concentrations of MWCNT (Shvedova et al.

2016;Fatkhutdinova et al. 2016). This finding is in overall agreement with strong

inflammatory potential of several different CNTs in sub-chronic inhalation studies in

rats (Pauluhn 2010b;Ma-Hock et al. 2009;Kasai et al. 2016;Kasai et al. 2015), although no

quantitative comparison of the responses has been performed.

Rats are the preferred animal model in particle toxicology and more sensitive than mice

to particle-induced lung cancer and fibrosis. However, rats do not express the acute

phase proteins serum amyloid a isoforms Saa1, Saa2 and Saa3, which are expressed by

humans and mice (Saa3 in mice only)(Cray et al. 2009). Serum amyloid is causally related

to plaque formation (Thompson et al. 2018). Since rats do not express SAA, a key acute

phase protein, they may be less well suited as model of human hazard assessment of

atherosclerotic effects. In this case, mice would be a more accurate model animal. Like

mice, humans also express SAA in lung tissue (Calero et al. 2014). Particle-induced acute

phase response in terms of increased SAA and CRP levels in blood was recently shown

in human volunteers following inhalation of ZnO nanoparticles (Monse et al. 2018).

Intratracheal instillation versus inhalation

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

closest surrogate to human exposure. For practical reasons, pulmonary deposition by

intratracheal instillation is widely used in screening studies (Bourdon et al. 2012b;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 nanomaterial 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),

also for MWCNT (Poulsen et al. 2016).

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 nanomaterials. Inhalation and intratracheal instillation

of a surface modified TiO

NP 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.

2017) compared the global transcriptomic profiles of lung tissue from mice exposed to a

straight and long 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.

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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. 2012b;Porter et

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

NPs induced

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

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

intratracheal instillation is useful for quantitative ranking of NP 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. Risk assessments, however, are solely conducted based on inhalation studies.

Hazard endpoints were evaluated based on reported adverse effects of CNT exposure in

reports and in the scientific literature. Previous assessments on CNTs have mainly

focused on inflammation as critical effect. However, these evaluations were conducted

prior to the pivotal, long term inhalation study in rats investigating the carcinogenic

potential of the long, thick MWCNT called MWNT-7 (Kasai et al. 2016) and the IARC

classification of MWNT-7 as possibly carcinogenic (2B) (Grosse et al. 2014). This report

will therefore include both endpoints. In addition, cancer and cardiovascular disease

have been identified as two of the main mortality causing diseases in the world, with a

combined estimate of approximately 26 million annual deaths worldwide (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, mechanistic understanding and general

importance in regards to worldwide mortality rates.

Pulmonary inflammation

Pulmonary inflammation, and inflammatory-related changes, is the most commonly

reported adverse effect of CNT exposure. However, the inflammogenic potential of

CNTs varies and is largely dependent on deposited dose and their physico-chemical

properties. The influx of neutrophilic cells into the BAL fluid is a commonly used and

reliable marker of pulmonary inflammation (Ma-Hock et al. 2009;Morimoto et al.

2012a;Erdely et al. 2009). Studies have shown that neutrophil influx correlates with

increases in both pro-inflammatory cytokine and acute phase response mRNA, and

protein levels (Bourdon et al. 2012b;Bourdon et al. 2012a;Bourdon et al. 2013;Husain et

al. 2013;Jackson et al. 2013;Poulsen et al. 2013;Poulsen et al. 2015b). In this report, the

current working group therefore chose pulmonary neutrophil influx in BAL fluid as the

marker of pulmonary inflammation.

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Due to the large number of studies reporting pulmonary inflammation after exposure to

CNTs, the current working group chose to highlight quality chronic and sub-chronic

inhalation studies (Table 2).

Chronic inhalation studies

One inhalation study was identified (Kasai et al. 2016). Here, rats were exposed to

MWCNTs MWNT-7 6h/day, 5 days/week for 2 years (D: 92.9-98.2 nm, L: 5.8-5.9 µm).

Statistically significantly increased neutrophil influx was observed at 2 mg/m

, but not at

0.2 or 0.02 mg/m

(Kasai et al. 2016). LOAEC for MWNT-7 in this study was therefore 2

mg/m

and NOAEC 0.2 mg/m

. The LOAEC and NOAEC levels for neutrophil influx

were lower in this chronic study, compared to the levels in the sub-chronic study of

5 mg/m

and 1 mg/m

, respectively, from the same authors (Kasai et al. 2015). This

indicates that the severity of pulmonary inflammation increases with exposure time and

thereby with increasing deposited dose.

Sub-chronic inhalation studies

Four different 13-week inhalation studies performed according to the OECD guidelines

have been published using 4 different MWCNTs (Ma-Hock et al. 2009;Pauluhn

2010b;Pothmann et al. 2015;Kasai et al. 2015). In all studies, rats were exposed 6h/day, 5

days/week for 13 weeks (Table 2).

Ma-Hock and colleagues exposed Wistar rats to aerosols of a short and thin MWCNT

type (D: 5-15 nm, L: 0.1-10 µm) at doses 0.1, 0.5, 2.5 mg/m

(Ma-Hock et al. 2009). The

authors reported no histopathological changes in any organ, except the lung, after the 13

weeks of exposure. Lung findings were dose-dependent and included increased lung

weights, pronounced multifocal granulomatous inflammation, diffuse histiocytic and

neutrophilic inflammation, and intra-alveolar lipoproteinosis at doses at 0.5 and 2.5

mg/m

. The investigators did not observe pulmonary fibrotic effects in the exposed rats.

At the lowest exposure level, 0.1 mg/m

, the authors reported minimal granulomatous-

type inflammation in the lungs and lung-associated lymph nodes, which were

considered sub-clinical and unlikely to be associated with functional effects. Based on

the observations reported by the authors, a NOAEC of 0.1 mg/m

and LOAEC of 0.5

mg/m

were established for this MWCNT type.

In the second sub-chronic inhalation study, Pauluhn exposed Wistar rats in nose-only

chambers to a short and thin MWCNT type (D: 10 nm, L: 0.2-0.3 µm) at doses 0.1, 0.4, 1.5,

and 6 mg/m

(Pauluhn 2010b). Pulmonary effects were examined up to 6 months post

exposure. Sustained pulmonary inflammation, in terms of increased neutrophil influx in

BAL fluid, was observed at doses 0.4 mg/m

and up. At 0.4 mg/m

and above, exposure-

related lesions in the upper and the lower respiratory tracts were revealed by

histopathology. Focally increased interstitial collagen staining, indicative of fibrosis, was

observed at doses 1.5, and 6 mg/m

, with borderline effects at dose 0.4 mg/m

. All

endpoints increased in intensity from exposure weeks 8 to 13, followed by a time-

dependent decrease in severity for all exposure groups. Based on the observations of the

study, a NOAEC of 0.1 mg/m

and LOAEC of 0.4 mg/m

were established for this

MWCNT type.

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Pothmann and colleagues exposed Wistar rats to a short and thin MWCNT type (D: 12.1

nm, L: 1.07 ± 1.1 µm) by nose-only exposure at doses 0.05, 0.25 and 5.0 mg/m

(Pothmann

et al. 2015). The animals were euthanized 1 or 90 days after the last exposure.

Significantly increased neutrophil levels were observed after exposure to the highest

dose (5.0 mg/m

) immediately after last exposure and at doses 0.25 and 5.0 mg/m

days after exposure. These increases were accompanied by increased levels of cytokines

IL-1β, IL-5, TNF-α, and IL-1

α.

Histopathological examination revealed focal/multifocal

collagen depositions after exposure to 5.0 mg/m

. Based on the observations of the study,

a NOAEC of 0.05 mg/m

and LOAEC of 0.25 mg/m

were established for this MWCNT

type.

In the last sub-chronic inhalation study, Kasai and colleagues exposed F344 rats by

whole-body inhalation to a long and thick MWCNT type (MWNT-7)(D: 94.1-98.0 nm, L:

5.53-6.19 µm) at doses 0, 0.2, 1 and 5 mg/m

(Kasai et al. 2015). In contrast to the previous

sub-chronic inhalation studies, the animals were exposed to aerosolized single fibers,

instead of agglomerates. The authors reported increased neutrophil influx in BAL fluid

at dose 1 mg/m

and granulomatous changes in the lung at dose 0.2 mg/m

and up.

Histopathological examination revealed focal fibrosis of the alveolar wall at dose 1

mg/m

and up. Based on the observations of this study, a NOAEC of 0.2 mg/m

and

LOAEC of 1 mg/m

for inflammation were established for this MWCNT type. However,

lactate dehydrogenase and alkaline phosphatase activities, and total protein levels were

all increased after exposure to 0.2 mg/m

MWCNTs.

Three of the four MWCNTs were thin (5-12 nm in diameter) and were aerosolized as

dense agglomerates. The BET surface area of these MWCNTs was approximately 10

times larger than the BET surface area of the fourth MWCNT, MWNT-7. The observed

NOAEC based on neutrophil influx was 0.05-0.1 mg/m

for the thin MWCNTs and 1

mg/m

for the thick MWNT-7, and thus appeared proportional to the BET.

Conclusion

In general, the chronic and sub-chronic inhalation studies identified NOAECs ranging

from 0.05 mg/m

to 1 mg/m

and LOAECs ranging from 0.25 mg/m

to 5 mg/m

(Table 2).

As dose-dependency was identified for inflammation and as it was possible to detect a

NOAEC, inflammation is considered a threshold effect.

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Table 2. Overview of sub-chronic and chronic inhalation studies in rats with inflammation as an endpoint

Sub-chronic studies

Ma-Hock et al. 2009

Pauluhn 2010b

Pothmann et al. 2015

Kasai et al. 2015

Rat strain

Male and female Wistar

Male and female rats,

Male and female

rats (N=10/group)

rats (N=6-10 per group)

RccHan©: WIST(SPF)

F344 rats

(N=10/group)

MWCNT

MWCNT Dimensions

Nanocyl NC 7000

D: 5-15 nm

L: 0.1-10 µm

BET: 250-300 m

Aerosolized as respirable

agglomerates

0.1, 0.5, 2.5 mg/m

6h/day, 5 days/week for

13 weeks

At the end of exposure

0.1 mg/m

0.5 mg/m

Baytubes

D: 10 nm

L: 0.2-0.3 µm

BET: 253 m

Aerosolized as respirable

agglomerates

0.1, 0.4, 1.5, 6 mg/m

6h/day, 5 days/week for

13 weeks

1 day, 28 days, 13 w, 26

0.1 mg/m

0.4 mg/m

0.1 mg/m

0.4 mg/m

(females not

assessed)

Graphistrength© C100

(NM-402)

D: 12.1 nm

L: 1.07 ± 1.1

μm

BET: 225.6 m

Aerosolized as respirable

agglomerates

0.05, 0.25 and 5.0 mg/m

6h/day, 5 days/week for

13 weeks

1 and 90 days

0.05 mg/m

0.25 mg/m

5 mg/m

MWNT-7

D: 94.1-98.0 nm

L: 5.53-6.19 µm

BET: 24-28 m

Aerosolized as single

fibers

0.2, 1, 5 mg/m

6h/day, 5 days/week

for 13 weeks

After overnight

fastening

1 mg/m

5 mg/m

0.2 mg/m

based on

LDH, ALP and total

protein in BALF

1 mg/m

Chronic study

Kasai et al. 2016

Male and female

F344/DuCrlCrlj rats

(N=50/group)

MWNT-7

D: 92.9-98.2 nm

L: 5.8-5.9 µm

BET: 24-28 m

Aerosolized as single

fibers

0, 0.02, 0.2, and 2

mg/m

6h/day, 5 days/week

for 104 weeks

At the end of

exposure

0.2 mg/m

2 mg/m

0.02 mg/m

0.2 mg/m

Air concentrations

(mg/m

)

Exposure setup

Post exposure time

points

NOAEC neutrophil

influx 1 day post-

exposure

LOAEC neutrophil

influx

1 day post-exposure

NOAEC fibrosis

1 day post-exposure

LOAEC fibrosis

1 day post-exposure

No fibrosis reported

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Genotoxicity and cancer

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

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

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

current working group therefore chose to differentiate between genotoxicity in shorter-

term studies and cancer in long-term studies.

Genotoxicity

The genotoxic potential, and thus the ability to induce cancerous changes, is likely

depended on the physico-chemical properties of the CNTs. Indeed, studies have

highlighted differences in CNTs’ ability to induce genotoxicity. Pulmonary exposure by

intratracheal instillation to 10 different MWCNTs (diameters 13-32 nm) in mice (doses: 6-

54 µg) increased DNA strand break levels in both BAL fluid and lung tissue for some of

the MWCNTs (Poulsen et al. 2016). No dose-response was observed. Using linear

regression analysis with independent physico-chemical parameters, the authors

identified diameter thickness as a possible predictor for DNA strand break levels in BAL

cells and lung tissue, even for these relatively thin MWCNTs. In concordance with this,

another study reported increased DNA strand breaks following pulmonary dosing of the

long and straight MWCNT MWNT-7 (1–200 µg/mouse by pharyngeal aspiration and

8.2–10.8 mg/m

for 4 days, 4 h/day by inhalation), but not a thin and entangled CNT

(diameter 8-15 nm) (Catalan et al. 2016). This could indicate that needle-like CNTs are

more genotoxic than thinner and often more entangled CNTs.

However, pulmonary exposure by intratracheal instillation of straight, thick and long

MWCNTs (D: 67 nm, L: 4 µm), and thin and short MWCNTs (D: 10 nm, L: 1.5 µm) in

mice both increased DNA strand break levels in lung tissue across doses (18-162 µg) and

time points (1-28 days) (Poulsen et al. 2015b). Similarly, two sub-chronic inhalation

studies in rats using thin CNTs (D: 10-15 nm and 12.1 nm, respectively) at comparable

doses reported opposing genotoxic potential. The first study showed increased levels of

DNA strand breaks in lung tissue from male and female rats following 28 days

inhalation (0.17-0.96 mg/m

, 6 h/day, 5 days per week) (Kim et al. 2014), whereas the

other reported no effect on DNA strand break levels, oxidative DNA damage or

micronuclei formation after 90 days inhalation (0.05-5.0 mg/m

, 6 h/day, 5 days per

week) (Pothmann et al. 2015). This indicates that other factors than diameter thickness

play a part in CNT-induced genotoxicity.

Cancer

The MWCNT MWNT-7 (D: 92.9-98.2 nm, L: 5.8-5.9 µm) was reported to cause peritoneal

mesotheliomas up to one year after non-pulmonary deposition in two studies (3-300 µg

in p53 heterozygous mice, or 0.5 or 5 mg MWCNT in rats twice with a 1-week interval,

respectively) (Takagi et al. 2012;Nagai et al. 2011). The same MWCNT also promoted

bronchioloalveolar adenoma and carcinoma in male mice after inhalation (5 mg/m

, 5

h/day, 5 days/week, 3 weeks) (Sargent et al. 2014). Based on these studies IARC classified

MWNT-7 as possibly carcinogenic (2B) (Grosse et al. 2014). In contrast, intraperitonal

injection of 2 or 20 mg short and thin (11 nm diameter, length ca 0.7 um) with or without

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structural defects in rats did not induce abdominal tumors up to 2 years after exposure,

whereas the positive control crocidolite asbestos (2 mg) did (Muller et al. 2009).

After publication of the IARC evaluation, more studies investigating the carcinogenic

potential of CNTs were published. (Rittinghausen et al. 2014) showed that IP injection of

4 different long (>7.9 um) and relatively thick (diameter: 37-85 um) MWCNTs at dose

levels of 1 and 5x10

fibers induced malignant mesotheliomas in a dose-dependent

manner for each of the studied MWCNTs. As the MWCNTs were dosed as 1 or 5x10

fibers, the dosed mass of the MWCNTs differed significantly between the 4 MWCNTs.

(Suzui et al. 2016) exposed rats to 1 mg NIKKISO MWCNTs by trans-tracheal

intrapulmonary spraying into the lung. MWCNTs were fractionized by size by filtering

through a 25 µm pore size sieve. The mean lengths of the MWCNT fractions were:

Unfiltered: 4.2 +/- 2.9 µm, flow-through: 2.6 +/- 1.6 µm, and retained > 2.6 µm. The

physico-chemical properties of NIKKISO were very similar to MWNT-7, except that the

flow-through fraction was shorter. The rats were exposed to the MWCNTs fractions 8

times during a 2 week period with a total dose of 1 mg/rat. The groups consisted of 12-15

rats and they were followed for 109 weeks post-exposure. Lung tumors were observed in

the combined three groups (37%) with no statistically significant differences between

groups. Mesotheliomas were statistically significantly increased in the combined group,

and were observed in the unfiltered and flow-through fractions although not statistically

significant. No mesotheliomas were observed in the retained fraction. NIKKISO were

carcinogenic even though the fibers were relatively short compared to MWNT-7, and no

difference in lung cancer incidence was observed across the different fractions. This

indicates that the length of MWCNTs is of less importance for their ability to promote

cancer.

A pivotal 2 year inhalation study investigated pulmonary pathological changes after

exposure to MWCNTs MWNT-7. F344 male and female rats (N=50 per exposure group)

were exposed to MWNT-7 for 6 h/day, 5 days/week for 104 weeks at concentrations of 0,

0.02, 0.2, and 2 mg/m

(Kasai et al. 2016). Scanning electron microscope of MWNT-7

demonstrated that most MWCNTs were aerosolized as single straight fibers with a mean

length of 5.8-5.9 µm.

The incidences of bronchiolo-alveolar carcinomas, total carcinomas (bronchiolo-alveolar

carcinomas, adenosquamous carcinoma, adenocarcinoma and squamous cell carcinoma),

and total carcinomas and/or adenomas were significantly increased in males exposed to

0.2 and 2 mg/m

MWNT-7 and females exposed to 2 mg/m

MWNT-7 compared with

their respective control groups (Table 3)(Kasai et al. 2016). The incidence of malignant

mesothelioma was not increased.

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Table 3. Total incidence of adenoma and/or carcinoma in the lungs of rats in the two

year inhalation study by (Kasai et al. 2016).

MWNT-7

0 mg/m

0.02 mg/m

0.2 mg/m

2 mg/m

concentration

Female rats

3/50

2/50

4/50

11/50*

Male Rats

2/50

13/50**

16/50**

*) p<0.05 **) p<0.01 by Fischer’s exact test

The total deposited MWCNT dose was calculated based on the number of fibers

observed and was concentration dependent. At 2 mg/m

, deposited dose was

approximately 1.8 mg/lung and 1.2 mg/lung for male and female rats, respectively, and

at 0.2 mg/m

it was approximately 0.15 and 0.12 mg/lung for male and female rats,

respectively. The number of fibers per gram body weight that induced lung carcinoma

was calculated to be 3.92 × 10

MWNT-7 fibers/gram body weight in males and 42.5 × 10

MWNT-7 fibers/gram body weight in females (Kasai et al. 2016).

In addition to the cancerous effects, exposure to MWNT-7 MWCNTs also induced

increased lung weight, pulmonary hyperplasia, granuloma formation and focal fibrosis,

and increased levels of total protein, lactate dehydrogenase, and alkaline phosphatase in

the BAL fluid at doses 0.2, and 2 mg/m

(Kasai et al. 2016). The dose of 2 mg/m

also

induced focal fibrosis in the pleura.

Conclusion

One specific thick and long carbon nanotube, MWNT-7/Mitsui-7/XNRI-7, has been

classified as possibly carcinogenic by IARC. After publication of the IARC evaluation,

the same MWCNT has been shown to induce lung adenomas and carcinomas by

inhalation in male (at 0.2 and 2 mg/m

) and female rats (at 2 mg/m

). Pulmonary dosing

of 1 mg/rat NIKKISO MWCNTs, which were shorter than MWNT-7, but otherwise with

similar physico-chemical properties, caused lung cancer. Other thick (>37 nm) and long

(>7.9 µm) have been shown to cause cancer following IP injection, whereas IP injection of

high doses (2 and 20 mg) of a thin (diameter 11 nm) and short (0.7 um) MWCNT did not

cause cancer in a 2-year study in rats. It is therefore very probable that other thick and

straight MWCNTs are equally carcinogenic by inhalation. No firm conclusions can be

reached regarding length because of the large heterogenicity of the tested samples.

Dose-response relationships have been identified for MWCNT-induced carcinogenic

effects in several independent studies (Kasai et al. 2016;Rittinghausen et al. 2014;Takagi

et al. 2012). However, in the study by (Rittinghausen et al. 2014) several different

MWCNTs were compared, and the carcinogenic potential could not be explained by

mass or by the number of CNT fibers. In contrast to the cancer studies, no consistent

dose-response relationship has been observed for CNT-induced DNA strand breaks in

the comet assay (Poulsen et al. 2016;Catalan et al. 2016;Poulsen et al. 2015b). Due to the

lack of dose-response relationship and the severity of cancer as a disease, the current

working group has therefore decided to consider genotoxicity and cancer as non-

threshold effects, as this is the most conservative approach.

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Cardiovascular effects

The term cardiovascular effects cover pathological changes in the entire cardiovascular

system. Atherosclerosis is a central cardiovascular disease, which is manifested as

increased plaque deposition or build-up in the arteries. In the later stages this can lead to

various other cardiovascular diseases, including coronary artery disease and stroke. For

CNT-induced cardiovascular effects, the present working group will focus on studies

using intratracheal instillation as pulmonary exposure route, as these adverse effects

primarily have been investigated in such studies. Differentiation between studies

reporting primary changes, such as accelerated plaque progression, and studies

reporting changes related to/or leading to cardiovascular effects, such as the acute phase

response, will be conducted.

Accelerated plaque progression

The lipid profile of mice differs significantly from that of humans. In mice, rapid

clearance of hepatic low-density lipoproteins (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 models

for investigating cardiovascular effects.

Several studies have reported CNT-induced accelerated plaque progression.

Intrapharyngeal instillation of 20 µg SWCNTs every other week for 8 weeks (total dose:

80 µg) in ApoE -/- mice fed either regular chow or high fat chow, revealed that SWCNT

induced accelerated plaque formation in mice fed on high fat chow (Li et al. 2007)

Similar increase was also observed in ApoE -/- mice exposed to SWCNT or DWCNT

once weekly for 10 weeks to 10 or 40 µg by pharyngeal aspiration (total dose 100 and 400

µg/mouse)(Suzuki et al. 2016). The authors reported accelerated plaque formation at the

high dose of 10 times 40 µg for both SWCNT and DWCNT. Two different short and thin

(D: 10-11 nm, L: 1.5 µm) MWCNT (NM-400 and NM-402) also induced accelerated

plaque formation in female ApoE -/- mice fed western diet after intratracheal instillation

once a week for 5 weeks to 25.6 µg/mouse (total dose 128 µg)(Cao et al. 2014).

In contrast, exposure to 4 or 40 µg of the thick and long MWCNT MWNT-7 (L: 3.86-5.7

µm, D: 49–74 nm) once a week for 10 weeks (total dose 40 or 400 µg) by intratracheal

instillation did not affect plaque formation in female ApoE -/- mice fed western diet

(Christophersen et al. 2016). The results indicate that accelerated plaque formation is

observed following pulmonary exposure to thin (SWCNT, DWCNT, NM-400 and NM-

402) but not thick (MWNT-7) MWCNT.

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 C-reactive protein (CRP), Serum amyloid A (SAA), and fibrinogen.

During an acute phase response these proteins can increase thousand fold (Gabay and

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Kushner 1999). Elevated plasma levels of CRP and SAA have been reported as a risk

factor for CVD in humans (Johnson et al. 2004;Lowe 2001;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 high density lipoproteins (HDL).

Several studies have reported changes in

Saa

expression or increased SAA protein levels

after pulmonary exposure to CNTs. Intratracheal instillation of both the thick MWCNT

MWNT-7 (D: 40–50 nm, L: 1– 4 µm) and two SWCNTs (D: 0.8–1.7nm, L: <1 µm) in female

C57BL/6 mice strongly increased

Saa3

mRNA levels in lung tissue (Saber et al. 2013). The

authors also observed elevated SAA3 protein levels in broncheoalveolar lavage fluid and

plasma after exposure to MWNT-7, whereas no changes in hepatic

Saa3

levels were

reported. Global gene expression analysis of pulmonary changes after exposure to a

small (L: 847 ±102 nm, D: 11 (6–17) nm) and a large MWCNT (L: 4048 ± 366 nm, D: 67

(24–138) nm) revealed that all

Saa

isoforms were among the most differentially regulated

genes 1, 3 and 28 days post-exposure (Poulsen et al. 2015b). Interestingly, both MWCNT

types also induced dose- and time-dependent increases in plasma protein SAA3 levels.

Increased SAA3 plasma protein levels have been reported in male mice exposed to a

pristine MWCNT (L: 15 ±5.0 µm and 13.5 ±1.50 nm) up to 1 year after intratracheal

exposure (Kim et al. 2015). This highlights the impact of pulmonary exposure to CNTs

on long-term vascular changes.

Changes in plasma SAA1/2 and SAA3 protein levels were investigated 1, 28 and 90 days

after pulmonary exposure to 14 commercial, well-characterized MWCNTs in female

C57BL/6J mice (Poulsen et al. 2017). In general, the majority of the MWCNTs induced

increases in both SAA1/2 and SAA3 plasma protein levels. No correlation was observed

between SAA1/2 and SAA3 plasma protein levels, which indicated that the changes are

controlled by different mechanisms. However, a very close correlation between

neutrophil influx and

Saa3

mRNA levels in lung tissue and SAA3 protein levels in blood

in mice have previously been reported (Poulsen et al. 2016;Saber et al. 2014), linking

inflammation and cardiovascular effects.

Conclusion

CNTs may promote atherosclerosis directly, by inducing accelerated plaque progression,

or indirectly, by inducing increased blood levels of acute phase response proteins. Dose-

response relationships have only sparsely been reported for CNT-induced increased

plaque progression, in part due to the low dynamic range of the assay, but also due to

study designs. In contrast, dose-response relationships have been established between

CNT exposure and increased levels of acute phase response proteins. However, the

studies reporting CNT-induced atherosclerotic effects have solely used pulmonary

deposition as exposure method, and thus, the studies cannot be used to establish OELs.

Due to the close interplay between inflammation, acute phase response and plaque

progression, inflammation as neutrophil influx can be used as a proxy for the acute

phase response. The current working group therefore considers cardiovascular effects as

a threshold effect that is regulated in parallel to inflammation.

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Reprotoxicity

A recent review summarized several studies of the potential for reproductive toxicity of

CNTs. Very few studies used the airway route of exposure (Ema et al. 2016). The review

concluded that there is potential for placental transfer of single- and multi-walled CNTs,

as reported in mouse studies following intravenous injection. Both SWCNTs and

MWCNTs were toxic to mouse fetuses when administered to the pregnant dam by

injection (intraperitoneal as well as intravenous). Oral gavage of MWCNTs induced no

developmental toxicity in mice and rats. In the following, only studies using airway

exposure to carbon nanotubes are reviewed. Sexually mature female mice received one

intratracheal instillation of 67 µg/mouse (~3.3 mg/kg) of MWCNTs (NM-400), one day

prior to cohabitation with a mature male. Pathological changes in lungs and liver were

observed in dams 6 weeks after instillation of the MWCNTs. The delivery of the first

litter was delayed by an average of 5 days in dams instilled with MWCNTs. No effects

were observed in gestation and litter parameters, offspring behavior in the open field or

startle test, or daily sperm production in the male offspring (DSP) (Hougaard et al. 2013).

To further characterize the delay in delivery of the first litter, estrous cycle regularity was

investigated by comparing vaginal smears before and after a single intratracheal

instillation of 67 µg of NM-400 MWCNT or vehicle. Reproductive function was analyzed

by measuring time to delivery of litters after instillation of 2, 18 or 67 µg of NM-400.

Exposure to MWCNT significantly prolonged the estrous cycle during which MWCNT

exposure took place compared to the pre-exposure estrous cycles. In contrast was the

estrous cycle immediately after the exposed cycle significantly shortened in MWCNT

exposed females. No consistent effects were seen on time to delivery of litter or other

gestational parameters. Litter parameters, such as litter size, sex ratio, implantations, and

implantation loss were not affected by exposure (Johansson et al. 2017).

MWCNTs suspended in 2% carboxymethyl cellulose were intratracheally instilled in

pregnant mice at 3, 4, or 5 mg/kg (Fujitani et al. 2012). At 4 and 5 mg/kg, exposed females

presented with reduced weight gain and significant increases in leucocyte counts in

peripheral blood. Fetal growth was significantly inhibited at 5 mg/kg. Data analysis

showed statistically significant increases in the number of litters with malformed fetuses,

at 4 and 5 mg/kg, owing mainly to increased incidences of fetuses with fusion of

vertebral bodies and arches. It should be noted that the vehicle used is unusual, and that

compared to control animals, the exposed females presented with 25 and 45% increases

in lung weight at 4 and 5 mg/kg, respectively. The finding warrants further

investigation, preferably exposing the pregnant animals by inhalation.

Conclusion

The findings in these studies indicate that CNTs may interfere with reproduction. The

applied route of instillation is intratracheal instillation in all three studies, and they can

therefore not be used as basis for derivation of occupational limit values.

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ECHANISMS OF TOXICITY

Pulmonary inflammation

Following pulmonary exposure, CNTs interact with the cellular membranes of resident

cells and proteins in the lung environment. When sensing CNTs through scavenger

receptors and/or toll-like receptors, macrophages and epithelial cells start secreting pro-

inflammatory cytokines. Alternatively, macrophages can initiate the inflammatory

response after sensing damage recognition-associated molecular patterns released from

damaged epithelial cells interacting with CNTs.

The surface area of the deposited CNTs is of great important for their level of interaction

with the resident pulmonary cell and thus their inflammogenic potential. In a study

investigating neutrophil influx after intratracheal instillation to 10 different MWCNTs at

three different time points (1, 28 and 92 days post-exposure), 83% of the observed

variation in neutrophil influx was predicted by BET surface area of the delivered dose

=0.69)(Poulsen et al. 2016). In multiple linear regression analysis with independent

physico-chemical properties, dose and BET surface area were the most consistent and

significant predictors of inflammation in terms of neutrophil influx)(Poulsen et al. 2016).

In agreement with this, particle-induced pulmonary inflammation was similarly

predicted by deposited surface area of low-toxicity-low solubility particles (Duffin et al.

2007;Schmid and Cassee 2017).

Identification of physico-chemical properties important for MWCNT-induced

inflammation, also revealed that MWCNT length and oxygen content (interpreted as

surface hydroxylation and carboxylation) affected the inflammatory potential of

MWCNTs (Poulsen et al. 2016). Length has previously been proposed as a determinant

of CNT-induced inflammation, albeit the strongest cases have been made after

intraperitoneal or pleural deposition (Murphy et al. 2011;Poland et al. 2008;Yamashita et

al. 2010). CNTs of a certain length (above 10 µm) can comply with the fiber paradigm

(Donaldson et al. 2011) resulting in frustrated phagocytosis due to the inability of

macrophages to completely engulf the long CNTs. This leads to lysosomal instability,

activation of the NALP inflammasome, and chronic stimulation of the cell, which results

in the macrophage releasing a range of pro-inflammatory molecules (Donaldson et al.

2011). Length could also be important for inflammogenicity of the vast types of CNTs

with lengths lesser than 10 µm. Kobler et al. reported that a thick, fiber-like MWCNT

appeared to escape vesicle enclosure more often compared to a smaller, entangled

MWCNT, which could lead to increased intracellular damage (Kobler et al. 2015).

However, the same two types of MWCNT induced comparable levels of neutrophil

influx up to 28 days after exposure by intratracheal instillation (Poulsen et al. 2015b). In

conclusion, the importance of length for CNT-induced inflammation is therefore largely

uncertain for CNTs with lengths below 10 µm.

A higher surface density of oxidized groups on the CNTs could increase their

hydrophilicity and dispersion in the lung. Supporting this, increased MWCNT oxygen

content (surface hydroxylation and carboxylation) was identified as protective of

inflammation 28 days after exposure to 10 different MWCNTs (Poulsen et al. 2016).

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Similar lowered or eliminated toxicity has previously been reported in the literature (Jain

et al. 2011;Sager et al. 2014). Hydrophilic CNTs may be cleared faster from the lung than

hydrophobic CNTs, as they more easily disperse in the lung environment. In addition,

the acidic treatment used to introduce the oxidized groups often introduces structural

defects in the CNTs. This renders the CNT more susceptible for enzymatic and oxidative

breakage, resulting in greater bio-degradability and thus more effective pulmonary

clearance (Liu et al. 2010).

In conclusion, CNTs are in general inflammogenic upon pulmonary exposure and their

surface area has been identified as important for the potency of the inflammatory

response. Inflammation is considered a threshold effect.

Genotoxicity and cancer

CNT-induced genotoxicity may occur by several mechanisms. These are briefly

described.

Surface-dependent ROS generation

Carbon-based nanomaterials such as carbon black are efficient generators of radicals

vitro

and in acellular assays (Jacobsen et al. 2008). SWCNT and MWCNTs also generate

ROS in acellular assays (Jackson et al. 2015;Jacobsen et al. 2008). ROS may result in

oxidation of the DNA, which can result in several types of DNA damage, including base

modifications, abasic sites, single-strand breaks, protein-DNA adducts, and DNA

crosslinks (Waris and Ahsan 2006). SWCNT-induced ROS is linked to generation of fpg-

sensitive sites

in vitro,

but did not affect the mutation frequency in the FE1 cell line

(Jacobsen et al. 2008).

Fiber-related DNA damage

Poulsen et al. (Poulsen et al. 2016) searched for physico-chemical predictors of DNA

strand break levels. Increasing MWCNT diameter was the most consistent and

significant predictor of DNA strand break levels in the comet assay. This supports a

mechanism related to the fibrous structure of thick MWCNTs. Similar to this, (Catalan et

al. 2016) compared genotoxicity of one entangled MWCNT and one straight MWCNT

(MWNT-7), and found that the straight MWCNT in contrast to the entangled MWCNT

induced DNA stand breaks in bronchoalveolar lavage cells and in lung tissue. The

straight MWCNT also induced micronuclei in alveolar type II cells, whereas this was not

assessed for the entangled MWCNT. The authors concluded that both primary and

secondary mechanisms may be involved in the observed genotoxicity of the straight

MWCNT.

The fiber paradigm as mechanism of CNT-induced carcinogenicity

Long CNTs have been proposed to induce gentoxicity according to the fiber paradigm

(Donaldson et al. 2011). Thus, macrophage interaction with long and straight MWCNTs

results in frustrated phagocytosis due to the inability of macrophages to completely

engulf the long CNTs. The slow clearance of insoluble MWCNT and frustrated

phagocytosis are the essential elements of the fiber paradigm (Donaldson et al. 2011).

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In conclusion, the mechanism of CNT-induced cancer has not been firmly established

yet. With respect to DNA damage there is no evidence indicating for a threshold mode of

action for CNTs. The current working group therefore assumes a non-threshold mode of

action for CNT-induced carcinogenicity.

Cardiovascular effects

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

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

inflammation. 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 CNTs 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 CNTs may also promote accelerated atherosclerosis indirectly

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

promotes neutrophil influx and release of proinflammatory 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

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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

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).

Identification of physico-chemical properties important for changes in plasma SAA

concentration indicated that increasing length predicts decreasing SAA1/2 plasma levels,

whereas metal content is important for SAA3 plasma levels (Poulsen et al. 2017). The

authors reported a significant correlation between plasma SAA3 levels and neutrophil

influx in the lung. Interestingly, pulmonary neutrophil influx has previously been shown

to correlate with deposited surface area of instilled MWCNT (Poulsen et al. 2016), which

links deposited CNT surface area with increased risk of developing cardiovascular

disease.

In conclusion, pulmonary exposure to CNTs can lead to accelerated plaque progression

directly, through translocation, or indirectly, through an induced acute phase response.

No single physico-chemical property has been identified as the driver of cardiovascular

effects, but CNT surface area a likely important due to the close connection to

pulmonary inflammation. As for inflammation, the current working group consider

cardiovascular effects as a threshold effect. This is based on identified dose-response

relationships between CNT 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. A very close correlation between neutrophil influx and

Saa3

mRNA levels in lung tissue and SAA3 protein levels in blood in mice were also

observed (Poulsen et al. 2016;Saber et al. 2014). Based on this, the current working group

suggests that inflammation in terms of neutrophil influx is used as biomarker of

cardiovascular risk.

Dose-response relationships

In general, dose response relationship is observed for health effects following pulmonary

exposure to CNTs.

Inflammation

Strong dose response relationships have consistently been observed for various markers

of pulmonary inflammation (Pauluhn 2010b;Ma-Hock et al. 2009;Pothmann et al.

2015;Kasai et al. 2015) and acute phase response (Poulsen et al. 2017;Saber et al.

2014;Saber et al. 2013;Poulsen et al. 2015b;Poulsen et al. 2015a). The working group

therefore considers inflammation as a threshold effect.

Cancer

Dose response relationship has been observed for carcinogenic effects of MWCNT in

several independent studies (Kasai et al. 2016;Rittinghausen et al. 2014;Takagi et al.

2012). The dose-response relationship was observed on mass-base for the individual

CNTs. However, in the study by (Rittinghausen et al. 2014) several different MWCNTs

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were compared, and differences in the carcinogenic potential between the MWCNTs

could not be explained by mass or by the number of CNT fibers.

Genotoxicity by comet assay

No consistent dose-response relationship has been observed for CNT-induced DNA

strand breaks in the comet assay (Poulsen et al. 2016;Catalan et al. 2016;Poulsen et al.

2015b). However, the lack of dose-response relationship may reflect that the level of

CNT-induced DNA strand breaks is low, leading to a low dynamic range of the comet

assay for this application. The mechanisms of CNT-induced genotoxicity and

carcinogenicity are not fully clarified. It is therefore not possible to conclude on a

threshold mechanism and a non-threshold approach will be used as the most

conservative approach.

Plaque progression

Dose-response relationships are rarely reported for CNT-induced increased plaque

progression. This is in part due to the low dynamic range of the assay, but also due to the

study designs, which often only include one dose. The exception is the study by Suzuki

and colleagues, which reported that their high doses of SWCNT and DWCNT induced

increased plaque progression after pharyngeal aspiration, whereas the low doses did not

(Suzuki et al. 2016). Alternatively, the acute phase response proteins could be used as

relevant biomarkers for plaque progression. Dose-response relationships are established

between CNT exposure and increased levels of these proteins. The current working

group considers cardiovascular effects as a threshold effect that should be regulated in

parallel to inflammation.

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REVIOUS RISK ASSESSMENTS OF CARBON

NANOTUBES

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

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

occupational exposure levels for CNTs. These have been set based on pulmonary

inflammation or lung cancer identified as the most critical effects of CNT exposure. The

previous recommendations of exposure limits are presented below and an overview can

be found in Table 4.

•

Aschberger et al. 2010, Critical Reviews in Toxicology (Aschberger et al. 2010).

Pauluhn 2010, Regulatory Toxicology and Pharmacology (Pauluhn 2010a).

ENRHES, Engineered Nanoparticles: Review of Health and Environmental

Safety, Final report, 2009 (ENRHES 2009).

NIOSH, 65, Occupational Exposure to Carbon Nanotubes and Nanofibers, 2013

(NIOSH 2013).

IARC, Monograph no. 111 (Grosse et al. 2014).

Ministry of Environment and Food of Denmark, Carbon nanotubes,

Environmental project No. 1805, 2015 (Jensen et al. 2015).

Aschberger et al. 2010

As part of a comprehensive review on carbon nanotube toxicity and exposure,

Aschberger and colleagues included an assessment of possible human exposure limits

(Aschberger et al. 2010). INELs were derived using the methodology suggested in the

REACH guidelines (chapter R.8 in ECHA, 2008), and were based on two sub-chronic

inhalation studies by (Pauluhn 2010b) and (Ma-Hock et al. 2009). In these studies a

NOAEC (Pauluhn 2010b) and a LOAEC (Ma-Hock et al. 2009) of 0.1 mg/m

were

identified for pulmonary inflammation after end of exposure in rats exposed 6 hour/day,

5 days/week for 13 weeks to two different MWCNTs (Baytubes and Nanocyl,

respectively). After applying default assessment factors, the authors estimated INELs of

2 µg/m

for Baytubes and 1 µg/m

for Nanocyl.

Pauluhn 2010

A study by Pauluhn from 2010 suggested an occupational exposure limit for Baytubes

MWCNTs (a thin, flexible MWCNT type) at 0.05 mg/m

(time-weighted average)

(Pauluhn 2010a). The author based his assessment on two previously published papers

by the same author, investigating toxicity and agglomeration of inhaled Baytubes

MWCNTs in rats (Pauluhn 2011;Pauluhn and Rosenbruch 2015). The author stated that,

in the case of the Baytubes MWCNTs, overload could be the trigger of the cascade of

events leading to lowered clearance and consequently increased MWCNT doses high

enough to trigger sustained pulmonary inflammation. The occupational exposure limit

in this study was calculated based on this mechanism. The author applied multiple

interspecies adjustments, but did not follow the REACH guidelines.

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ENRHES

Some of the first to establish derived-no-effect levels (DNEL) for CNTs was within the

frames of the EU project Engineered Nanoparticles: Review of Health and

Environmental Safety (ENRHES 2009). At the time of the report, no carcinogenicity

studies were identified for CNT exposure. The derivation of a DNEL was therefore made

under the assumption of a threshold driven mechanism of CNT toxicity. The calculated

DNELs of 33.5 µg/m

for pulmonary inflammation and 0.67 µg/m

for systemic immune

effects were derived based on a sub-acute study by (Mitchell et al. 2007). The calculations

of DNELs followed the methodology suggested in the REACH guidelines.

NIOSH

In 2010, the National Institute for Occupational Safety and Health (NIOSH) proposed a

recommended exposure limit (REL) of 7 µg/m

of CNTs in air as an eight-hour, time-

weighted average, respirable mass concentration. However, this value was re-adjusted to

be 1 µg carbon/m3 in April, 2013 (NIOSH 2013).This states: "The NIOSH REL is expected

to reduce the risk for pulmonary inflammation and fibrosis. However, because of some

residual risk at the REL and uncertainty concerning chronic health effects, including

whether some types of CNTs may be carcinogenic, continued efforts should be made to

reduce exposures as much as possible". The analysis conducted by NIOSH was focused

on the types of CNT included in published research studies, and it primarily based is

deriviation on the sub-chronic, rat inhalation studies (Ma-Hock et al. 2009;Pauluhn

2010b).

IARC

In 2014, the International Agency for Research on Carcinogenicity (IARC) classified the

MWCNT MWNT-7 as possibly carcinogenic to humans (group 2B). In Denmark,

substances classified as group 1, 2A and 2B by IARC are considered carcinogenic.

MWCNTs other than MWNT-7 were not classifiable as to their carcinogenicity to

humans (Group 3). SWCNTs were not classifiable as to their carcinogenicity to humans

(Group 3). These classifications were based on: 1. Inadequate evidence in humans for the

carcinogenicity of CNTs. 2. Sufficient evidence in experimental animals for the

carcinogenicity of MWNT-7 MWCNTs. 3. Limited evidence in experimental animals for

the carcinogenicity of two types of MWCNTs with dimensions similar to MWNT-7. 4.

Inadequate evidence in experimental animals for the carcinogenicity of MWCNT other

than MWNT-7. 5. Inadequate evidence in experimental animals for the carcinogenicity of

SWCNTs.

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Table 4. List of studies proposing occupational exposure limits based on LOAEC or NOAEC data in animal studies

Study

Aschberger et al. 2010

Pauluhn 2010a

NIOSH 2013

ENRHES 2009

Pulmonary

Systemic

Pulmonary

Critical effect

Pulmonary inflammation

inflammation and

inflammation

immune effects

inflammation

fibrotic events

Duration

Acute

Sub-chronic

Sub-acute

Ellinger-

Ziegelbauer

Pauluhn 2010b/

Mitchell et al.

Pauluhn 2010b

Key studies

and Pauluhn

Ma-Hock et al. 2009

2007

2009

MWCNT,

CNT type

Baytubes and

MWCNT, Baytubes

Baytubes and

Shenzhen

Baytubes

Nanocyl NC 7000

Nanotech

Risk determinant

LOAEC

NOAEC/LOAEC

NOAEC

NOAEC/LOAEC

NOAEC

LOAEC

Risk level in rodents

11 mg/m

0.1 mg/m

0.5 mg/m

0.3 mg/m

NOAEC

Corrected

5.5 mg/m

3 a

0.05 mg/m

3 a

0.05 mg/m

2.5 mg/m

0.15 mg/m

Corrected starting point

Default

-/2

assessment factors

Interspecies

2.5

Intraspecies

Uncertainty

Sub-chronic or

factors:

sub-acute to

chronic

Dose-response

issues:

LOAEC/NOAEC

extrapolation/

severity of effect

Overall uncertainty factor

Suggested OEL

37.5

150 µg/m

2 µg/m

/ 1 µg/m

50 µg/m

1 µg/m

33.5 µg/m

0.67 µg/m

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Table 4 description. a) Correction factors: 8 hour working hours and difference in

respiratory volume between animals in rest and workers at light activity. Corrected

L(N)OAEC

worker

for 8 working hours: (N(L)OAEC × 6 h/8 h). Correction for the

difference in respiratory volume between animals at rest and workers at light activity

(N(L)OAEC × 6.7 m

/10 m

). b) extrapolation from LOAEC to NOAEC. c) Interspecies

default factor: 2.5. Intraspecies default factor: 5 for workers/10 for public. Sub-chronic

to chronic default factor: 2. d) Altogether, overall assessment factors of 37.5 (3 × 2.5 × 5)

for acute and of 25 (2.5 × 5 × 2) and 50 (2 × 2.5 × 5 × 2), respectively, for chronic exposure

of workers. e) Sub-acute to chronic default factor: 6. f) Extrapolation from a LOAEC to

a NAEC (extrapolation factor of 3).

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CIENTIFIC BASIS FOR AN OCCUPATIONAL

EXPOSURE LIMIT

Different methods exist for calculating occupational exposure limits. 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 the non-threshold effects approach relies on the

assumption that any exposure to the substance can result in adverse effects. In this

report, the current working group will calculate proposed occupational exposure limits

based both on threshold effects or non-threshold effects.

Calculations of exposure limits based on cancer as

non-threshold effect

Carcinogenicity via genotoxic mechanisms is generally considered as a non-threshold

effect. This applies for CNT-induced carcinogenicity, as no evidence for threshold effects

are available for the mechanism of action for carbon nanotube-induced carcinogenicity.

Risk levels are calculated based on the 2 year inhalation study by (Kasai et al. 2016). In

this study, MWNT-7 MWCNTs were used. MWNT-7 are relatively thick and long

MWCNTs (D: 92.9-98.2 nm, L: 5.8-5.9 µm) and have very low levels of chemical

contaminations compared to other MWCNTs. It is therefore unlikely that the observed

effects are attributed to metal contaminations. The MWCNTs were aerosolized to

generate single fibers.

Lung burden was measured in lung tissue. Tissue was digested and fibers were counted

on SEM images of digested tissue. Time and air concentration-dependent accumulation

of MWCNTs were observed during the entire 2-year study. Lung deposition was 1.5-

2.7% (Kasai et al. 2016). Male rats were more sensitive compared to female rats, and thus

the proposed occupational exposure limit was calculated based on cancer incidents in

males. The authors reported statistically significantly increased lung cancer incidence in

male rats at 0.2 mg/m

. Both malignant and non-malignant tumors were included (Table

5).

Table 5. Cancer incidents and lung burden in male rats after exposure to different

doses of MWCNT in (Kasai et al. 2016).

0 mg/m

0.02 mg/m

0.2 mg/m

2 mg/m

Total cancer

2/50

13/50

16/50

incidences

MWCNT lung

152.4

1797.8

burden (µg/lung)

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Method I

The current working group has chosen to use the approach used by (Kasai et al. 2016)

and (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:

Observed cancer incidence at 0.2 mg/m

(13

0.2 mg

- 2

control

)/(50

control

- 2

control

) = 11/48 = 23%

Lung deposited dose in male rats at 0.2 mg/m

: 0.152 mg/lung.

The human equivalent dose is:

(Rat deposited dose) x (human alveolar surface area)/(rat alveolar surface area) =

0.152 mg x 102 m

/0.4 m

= 38.76 mg MWCNT per human lung.

The following standardized constants are assumed:

The standard value of human ventilation is 20 L/min during light work (1.2 m

/h).

An average work day is 8h per day.

An average work week contains 5 days.

In Denmark, an average employee work 45 weeks per year.

An average working life is 45 years.

The pulmonary deposition rate was reported to be 1.4-2.7% in the study Kasai and

colleagues (Kasai et al. 2016). However, in a 13 week inhalation study by Pauluhn, a

pulmonary deposition rate of 5.7 % was reported for aerosolized agglomerated

MWCNTs (Pauluhn 2010b). In order to take different kinds of MWCNTs as well as

different states of agglomeration into account, the current working group use the

pulmonary deposition rate of 5.7% as reported by (Pauluhn 2010b).

Using the values above, a lung burden of 38.8 mg in humans would require that workers

are exposed to:

Air concentration =

38.76 mg/(8h/day x 5 day/week x 45 weeks/year x 45 years x 1.2 m

/h x 0.057) =

0.00699 mg/m

Thus, at an air concentration of 7 µg/m

during a 45 year work life, an excess lung cancer

incidence of 23% is expected. Assuming a non-threshold linear dose-response

relationship, then 1% excess lung cancer is expected at:

(7 µg/m

)/23 = 0.304 µg/m

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Table 6. Calculated excess lung cancer incidence at different MWCNT mass and fiber

concentrations for 8 hour daily occupational exposure, Method I.

Excess lung cancer

MWCNT Air

Air concentration

incidence

concentration (µg/m

) MWNT-7 fibers/cm

1:1,000

0.03

0.3

1: 10,000

0.003

0.03

1: 100,000

0.0003

0.003

*) The calculations of fiber concentrations were based on the following: 1 µg MWNT-7

MWCNT corresponds to 9.03 x 10

MWNT-7 fibers (Kasai et al. 2016).

Only MWNT-7 has been shown to be carcinogenic by inhalation. One other type of

MWCNT did not induce cancer in a 2 year study (Muller et al. 2009). However, several

types of MWCNTs have been shown to be carcinogenic by other exposure routes

(Rittinghausen et al. 2014) or genotoxic (Poulsen et al. 2016;Catalan et al. 2016;Poulsen et

al. 2015b). Moreover, characterization of the physico-chemical properties of

commercially available CNTs has shown large deviations between empirical data and

data available from the supplier (Jackson et al. 2015). The present working group

therefore assume that all CNTs are carcinogenic.

Method II

Calculation based on approach suggested by ECHA (ECHA

2012;SCHER/SCCP/SCENIHR 2009), calculated based on the one year MWCNT

inhalation study in rats by (Kasai et al. 2016)(Table 5):

Excess cancer risk:

(13

0.2 mg

- 2

control

)/(50

control

- 2

control

) = 11/48 = 23%

Correction of dose metric into a human dose situation (8h/d):

0.2 mg/m

x (6h/day)/(8h/day) x (6.7 m

/10 m

) = 0.1 mg/m

or 100 µg/m

Calculation of unit risk for cancer:

Risk level = exposure level x unit risk

0.23 = 100 µg/m

x unit risk

Unit risk = 2.3 x 10

-3

per µg/m

At a dose of 1 µg/m

, 2.3 x 10

-3

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 (2.3 x 10

-3

per µg/m

)

Exposure level (10

-5

) = 4.3 x 10

-3

µg/m

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Table 7. Calculated excess lung cancer incidence at different MWCNT mass

concentrations for 8 hour daily occupational exposure, Method II.

Excess lung cancer

MWCNT Air

incidence

concentration (µg/m

)

1:1,000

0.043

1: 10,000

0.0043

1: 100,000

0.00043

Calculations of exposure limits based on

inflammation as threshold effect

Several studies have reported dose-dependent inflammatory effects as well as dose-

dependent acute phase response, a biomarker of risk of cardiovascular disease.

Inflammation, acute phase response and cardiovascular effects are therefore considered

threshold effects. However, at present only inflammogenicity has been investigated in

inhalation studies of sufficient quality for risk assessment. The current working group

therefore bases the risk assessment of threshold effects on the sub-chronic inhalation

studies investigating inflammogenicity. The choice of using the sub-chronic studies (Ma-

Hock et al. 2009;Pauluhn 2010b;Pothmann et al. 2015) , compared to the chronic one

(Kasai et al. 2016) is based on the CNT type being investigated (Table 2). In the sub-

chronic studies, the short, thin MWCNTs with large surface areas are used, whereas in

the chronic study it is the long, thick MWCNT type with small surface area. As described

Mechanism of toxicity,

CNT-induced inflammation has been shown to be predicted by

the specific surface area. In order to take the variation specific surface area into account

and as a precaution, the current working group has therefore decided to base the risk

assessment on the data from the sub-chronic studies.

NOAECs were noted for the highest air concentrations, which do not induce statistically

significantly increased levels of neutrophils (Table 2) (Ma-Hock et al. 2009;Pauluhn

2010a;Pauluhn 2010b;Pothmann et al. 2015). The lowest NOAEC for neutrophil influx

was reported by (Pothmann et al. 2015) to be 0.05 mg/m

. This is similar to the NOAECs

reported in other studies, albeit approximately half the concentration (Table 2). As a

precaution, the NOAEC of 0.05 mg/m

will be used for determining the proposed

occupational exposure limits for threshold effects.

Calculation

The DNEL for CNT-induced pulmonary inflammation is established based on the

methodology suggested in the REACH guidelines (ECHA 2012). Initially, a corrected

NOAEC is made that takes into account that rats were exposed for 6 h/day, whereas

humans are exposed for 8 h/day. In addition, exposed rats are at rest, whereas workers

are considered to do light activity. A total breathing volume of 10 m³ for an 8-hour shift

at light activity is therefore assumed, compared to 6.7 m

at rest.

Corrected NOAEC:

0.05 mg/m

x (6h/day)/(8h/day) x (6.7 m

/10 m

) = 0.0251 mg/m

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Next, assessment factors are applied. As no valid argumentation for using alternative

assessment factors could be found, the current working group uses the assessment

factors as proposed by ECHA (ECHA 2012):

Extrapolation from sub-chronic to chronic exposure: 2

Neutrophil influx is considered an acute response and this would normally not warrant

a duration factor from a sub-chronic study. However the accumulation of CNTs over

time would continue beyond sub-chronic exposure and thus the default assessment

factor of 2 to extrapolate from sub-chronic to chronic exposure were chosen.

Extrapolation from rats to humans: 2.5

Variation between workers: 5

Total assessment factor: 2 x 2.5 x 5 = 25

Suggested exposure limit based on inflammation:

0.0251 mg/m

/25 (total assessment factor) = 0.001 mg/m

= 1 µg/m

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ONCLUSION

The present working group evaluated the relevant literature on CNT toxicity from both

epidemiological and animal inhalation studies. However, as almost no human data on

toxicity and epidemiological studies is available, inhalation studies in mice and rats were

used to assess potential human hazard.

Carbon nanotubes are a very diverse class of nanomaterials with large variation in

physico-chemical properties including diameter, length, specific surface area, level and

type of contaminations, surface modifications. Therefore, as the relationship between

physico-chemical properties of CNTs and their inhalation toxicity is not fully clarified,

the present working group considers all types of CNTs a respiratory hazard and

proposes to regulate all CNTs as one group.

The present working group regards inflammation and carcinogenicity as the main

adverse effects of CNT exposure by inhalation and the subsequent risk assessments are

conducted based on studies reporting these effects. CNT-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

current 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 current working group regards inflammation as a

proxy for cardiovascular effects.

The present working group found that the mechanism of action of CNT-induced

carcinogenic effect has not been fully clarified. CNTs have reported to induce ROS

generation similar to carbon black. ROS may result in oxidation of the DNA, which can

result in several types of DNA damage, including base modifications, abasic sites, single-

strand breaks, protein-DNA adducts, and DNA crosslinks (Waris and Ahsan 2006).

CNTs may also induce genotoxicity through they fibrous shape, both in regards to

diameter thickness (Poulsen et al. 2016;Catalan et al. 2016) and length (Donaldson et al.

2011). In addition, secondary genotoxicity due to CNT-induced inflammation has been

recognized as an important and well-documented mechanism of action for the

development of lung cancer. Based on the above mentioned findings, the current

working group did not find sufficient evidence for a threshold mechanism for CNT-

induced carcinogenicity and decided to consider it as non-threshold effect.

In contrast to cancer, the mechanism of action for CNT-induced pulmonary

inflammation is more established and the present working group found strong dose

response relationships for various markers of pulmonary inflammation, including

neutrophil influx (Kasai et al. 2015;Ma-Hock et al. 2009;Pauluhn 2010b;Pothmann et al.

2015). Neutrophil influx was predicted by deposited surface area. The working group

considers inflammation as a threshold effect.

Based on the main adverse effects of pulmonary CNT exposure, inflammation and

cancer, the working group decided to perform the risk assessment based on both a

threshold and a non-threshold mechanism of action. Four sub-chronic and one chronic

BEU, Alm.del - 2019-20 - Bilag 101: Orientering om NFA’s forslag til grænseværdier for fem kemiske stoffer, fra beskæftigelsesministeren

study inhalation study in rats were identified as suitable for determining a DNEL for

pulmonary inflammation. A conservation approach was selected and the DNEL was

calculated based on the study using the CNT with the largest specific surface area and

reporting the lowest NOAEC estimate (Pothmann et al. 2015)(Table 8). For the non-

threshold approach on cancerous effects, a 2 year inhalation study in rats were identified

as suitable (Kasai et al. 2016) and excess cancer risks at the levels of 1:1,000, 1:10,000 and

1 in 100,000 were calculated using two approaches (Table 8).

Table 8. Overview of DNEL based on a threshold based mechanism of action and

exposure levels resulting in excess cancer risk levels at 1:1000, 1:10 000 and 1: 100 000

based on a non-threshold based mechanism of action

Suggestion for an OEL for CNTs

Lung cancer

Inflammation

(Method I)

DNEL

1 µg/m

Excess cancer risk:

1:1,000

0.03 µg/m

1:10,000

0.003 µg/m

1:100,000

0.0003 µg/m

Mechanism of action

Threshold based

Non-threshold based

Lung cancer

(Method II)

0.043 µg/m

0.0043 µg/m

0.00043 µg/m

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

approaches were used for calculating excess lung cancer risk based on the same chronic

inhalation study (Kasai et al. 2016). In the first approach, lung burden was used to

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

directly. Independently of the applied method for risk assessment, the acceptable

exposure levels were all very low. These levels are all more than 5 magnitudes lower

than the present Danish occupational exposure limit for bulk carbon black of 3.5 mg/m

The present working group recommends the approach using the excess lung cancer risk

estimates based on lung burden, since this approach takes the retained pulmonary dose

into account. Thus, the expected excess lung cancer risk based on lung burden approach

is 1:1,000 at 0.03 µg/m

, 1:10,000 at 0.003 µg/m

and 1:100,000 at 0.0003 µg/m

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