Beskæftigelsesudvalget 2019-20
BEU Alm.del Bilag 101
Offentligt
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Carbon
black
nanomaterials:
Scientific basis
for setting
a health-based
occupational
exposure limit
Nicklas Raun Jacobsen, Niels Hadrup, Sarah Søs Poulsen, Karin Sørig Hougaard,
Anne Thoustrup Saber, Ulla Vogel
 BEU, Alm.del - 2019-20 - Bilag 101: Orientering om NFA’s forslag til grænseværdier for fem kemiske stoffer, fra beskæftigelsesministeren BEU, Alm.del - 2019-20 - Bilag 101: Orientering om NFA’s forslag til grænseværdier for fem kemiske stoffer, fra beskæftigelsesministeren
CARBON BLACK NANOMATERIALS:
SCIENTIFIC BASIS FOR SETTING A HEALTH
–BASED OCCUPATIONAL EXPOSURE LIMIT
Nicklas Raun Jacobsen
Niels Hadrup
Sarah Søs Poulsen
Karin Sørig Hougaard
Anne Thoustrup Saber
Ulla Vogel
The National Research Centre for the Working Environment, Copenhagen 2018
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NFA-report
Title
Carbon black: Scientific basis for setting a health-based occupational exposure
limit
Nicklas Raun Jacobsen, Niels Hadrup, Sarah Søs Poulsen, Karin Sørig
Hougaard, Anne Thoustrup Saber and Ulla Vogel
The National Research Centre for the Working Environment (NFA)
The National Research Centre for the Working Environment (NFA)
November 2018
978-87-7904-354-1
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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F
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 (NFA) 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 nanosized carbon black.
The working group wishes to thank Chief Toxicologist Poul Bo Larsen, DHI, Denmark,
for reviewing the report.
Copenhagen, November 2018
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E
XECUTIVE SUMMARY
Carbon black (CB) is the black dye of the world and millions of tonnes are used each
year. It is a solid inorganic and poorly soluble compound which differs between
products in size and surface area but also the levels of impurities such as polycyclic
aromatic hydrocarbons.
In this report, a working group at the NFA reviews scientific data relevant to assessing
the hazard of CB nanomaterials (CB NMs), i.e. human studies
,
toxicokinetics, animal
studies, mechanisms of toxicity, previous hazard and risk assessments of CB NMs,
scientific basis for setting an occupational exposure limit (OEL) and finally we
summarise and suggest a health-based OEL for CB NM. The focus of this report is only
occupational exposure by inhalation. The present working group evaluated the relevant
literature on CB NM from both epidemiological studies and pulmonary exposure in
animal studies. Cell culture studies were only used for the description and clarification
of mechanisms and modes of action.
Epidemiological data were inconclusive. Two European CB production cohorts show
evidence of excess cancer incidence; especially in the British cohort where a high
prevalence for all causes mortality and lung cancer was observed. This was mainly
driven by a large increase at two (out of five) facilities. Some indications of increased risk
were observed in a German cohort, but this was unrelated to years of
exposure/employment. In contrast, American CB employees demonstrated no excess
occurrence of cancer mortality. Actually, a decreased mortality was observed in spite of
some high estimated CB exposure levels. The latter result was explained by a strong
healthy worker effect. Also, smoking frequency over time and other work-related
exposures, in the general and in the CB worker populations may be a possible strong
confounder and was not controlled for in any of the studies. However, other cigarette
smoke induced diseases was not increased in UK cohort indicating that this was not the
cause for the observed excess lung cancer cases. None of the studies provided
information on the particle size range of the exposure. The present working group found
that the available epidemiological studies cannot be used for risk assessment of CB NM
and it was decided to base the suggested health-based OEL on data from experimental
animal studies.
Pulmonary inflammation and carcinogenicity was observed in inhalation studies in rats.
The present working group regards inflammation and carcinogenicity as the main
adverse effects and the subsequent hazard assessments are conducted based on sub-
chronic and chronic inhalation studies reporting these effects. The present working
group found a dose response relationship for neutrophil influx as a marker of
pulmonary inflammation. Neutrophil influx correlated with deposited surface area and
was inversely correlated with particle size. The working group considers inflammation
as a threshold effect.
The present working group concludes that there is substantial evidence for genotoxicity
of CB NM. The literature shows that CB NM can induce mutations, oxidative damage to
deoxyribonucleic acid (DNA) as well as DNA strand breaks in rats and mice. It is known
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that inflammation and associated cellular production of reactive oxygen species is
closely linked to genotoxicity. In addition, genotoxicity due to particle-induced
inflammation is an important and well- documented mechanism of action for the
development of lung cancer. However, the present working group found evidence for a
non-threshold mechanism-of-action for carcinogenicity. Therefore, the present working
group followed the European Chemicals Agency (ECHA) guidelines suggesting a
precautionary non-threshold approach. Consequently, the present working group
decided to perform the hazard assessment based on both a threshold mechanism for
inflammation and a non-threshold mechanism for cancer.
For an OEL based on threshold effects, the following traditional approach suggested by
Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) is
utilized: 1) identification of the critical effect, 2) identification of the no observed adverse
effect concentration (NOAEC), 3) calculation of the OEL using assessment factors
adjusting for inter and intra species differences and the duration of the study. For non-
threshold effects, the current working group uses two approaches. The first, Method I,
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 II, suggested by ECHA, uses air mass concentrations directly.
The working group considered that data from five rodent inhalation studies were the
best basis for the hazard assessment. The following studies were selected to be used for
calculation of derived no effect level (DNEL) and excess cancer risk, respectively: DNEL
studies were: A 12-month chronic inhalation study in rats (mass concentrations: 0, 2.5,
and 6.5 mg/m
3
), a 13-week sub-chronic inhalation study in mice, rats, and hamsters (0, 1,
7, and 50 mg/m
3
), and a 13-week sub-chronic inhalation study in rats (0, 1, 7, and 53
mg/m
3
). Cancer studies were: a 2-year chronic cancer inhalation study in rats (0 and 12
mg/m
3
) and a 2-year chronic cancer inhalation study in rats (0, 2.5 and, 6.5 mg/m
3
).
The table below shows the DNEL for pulmonary inflammation, and excess lung cancer
risk at 1 in 1 000, 1 in 10 000 and 1 in 100 000 derived using the above-mentioned two
different approaches. Independently of the applied method for hazard assessment, the
resulting OEL suggestions were all low compared to the current Danish OEL for CB of
3.5 mg/m
3
.
Overview of threshold-based DNEL and non-threshold-based exposure levels leading to excess
cancer risk using two different approaches.
Mechanism of
action
Threshold
DNEL
based
Non-threshold
Excess
based
cancer risk
1: 1 000
1: 10 000
1: 100 000
Suggestion of a health based OEL for CB NM
Inflammation
Lung
cancer Lung
cancer
(method I)
(method II)
20 µg/m
3 #
3 µg/m
3
0.3 µg/m
3
0.03 µg/m
3
45 µg/m
3
4.5 µg/m
3
0.45 µg/m
3
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#
Based on NOAEC values in 2 sub-chronic inhalation studies.
The present working group recommends the hazard assessment approach estimating the
excess lung cancer risk based on lung burden (Method I), since this approach takes the
retained pulmonary dose into account. Thus, the expected excess lung cancer risk in
relation to occupational exposure to CB NMs is 1: 1 000 at 3 µg/m
3
, 1: 10 000 at 0.3 µg/m
3
and 1: 100 000 at 0.03 µg/m
3
CB NM.
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DANSK SAMMENFATNING
Carbon black (CB) er verdens sorte farvestof og der bruges millioner af tons om året. CB
er et fast uorganisk materiale med lav opløselighed, og forskellige CB-produkter varierer
i størrelse, overfladeareal samt mængden af urenheder som fx polyaromatiske
hydrocarboner.
I denne rapport vurderer en arbejdsgruppe ved Det Nationale Forskningscenter for
Arbejdsmiljø (NFA) data, der er relevante for at vurdere faren ved udsættelse for CB-
nanomaterialer (CB NM), dvs. humane studier, toksikokinetik, dyreforsøg,
toksicitetsmekanismer, tidligere fare og risikovurderinger af CB NM samt det
videnskabelige grundlag for fastlæggelse af en grænseværdi. Endeligt opsummeres og
foreslås en helbredsbaseret grænseværdi for CB NM i arbejdsmiljøet. Fokus i denne
rapport er alene på erhvervsmæssig eksponering ved indånding. Den nærværende
arbejdsgruppe evaluerede den relevante litteratur om CB NM fra både epidemiologiske
undersøgelser og inhalationsforsøg med dyr. Celleforsøg er kun blevet evalueret hvor de
var nødvendige for at afklare og beskrive CB NMs virkningsmekanismer.
Der kunne ikke konkluderes endeligt på de epidemiologiske data omhandlende human
CB-eksponering. To europæiske kohorter med arbejdere fra CB produktionsfaciliteter
har vist, at arbejde med CB øger risikoen for at udvikle kræft. Specielt i en britisk kohorte
var der en høj prævalens af død generelt og død forårsaget af lungekræft. Dette blev
overvejende drevet af en stor effekt i arbejdere fra to ud af fem britiske
produktionsfaciliteter. Der var desuden indikationer på en øget risiko for lungekræft i en
tysk kohorte. Men dette var ikke korreleret til antallet af eksponeringsår. Der blev ikke
set øget forekomst af kræft i amerikanske CB-arbejdere. Faktisk blev der her set en
nedsat forekomst, og det på trods af, at de estimerede eksponeringsdoser var høje. Dette
blev forklaret som en stærk
healthy worker effect.
Evaluering af resultaterne kompliceres yderligere af, at det er svært at justere for rygning
samt andre eksponeringer i den generelle befolkning såvel som hos CB-arbejdere. Fx
korrigerede ingen studier for cigaretrygning. Det bemærkes dog, at andre sygdomme
forårsaget af cigaretrygning ikke var øget i den britiske kohorte, hvilket indikerer, at
dette ikke er baggrunden for det øgede antal dødsfald forårsaget af lungekræft. Ingen af
studierne beskrev størrelsesfordelingen eller renheden af den producerede CB.
Arbejdsgruppen fandt, at de tilgængelige epidemiologiske studier ikke kan bruges til
farevurdering, og det blev derfor besluttet at basere de foreslåede grænseværdier på
dyrestudier.
Der blev observeret lungeinflammation og lungekræft i inhalationsundersøgelser af
rotter. Arbejdsgruppen betragter inflammation og kræft som de vigtigste skadelige
effekter. Derfor baseres de efterfølgende farevurderinger på undersøgelser, der
rapporterer om disse effekter. Tydelig dosis-respons-sammenhæng for tilgang af
neutrofile celler til lungen som markør for lungeinflammation blev observeret. Det
doserede specifikke CB overfladeareal prædikterede tilgangen af neutrofile celler som
igen var omvendt korreleret til partikelstørrelse. Arbejdsgruppen anser inflammation for
at være en tærskeleffekt.
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Arbejdsgruppen konkluderer, at der er betydelig evidens for en DNA skadende effekt af
CB NM. Litteraturen viser, at CB NM kan forårsage mutationer, oksidative DNA-skader
samt DNA strengbrud i dyrestudier. DNA skadende effekter forårsaget af
partikelinduceret inflammation er en vigtig og veldokumenteret virkningsmekanisme
for udvikling af lungekræft. Også for CB NM er der betydelig evidens for denne
virkningsmekanisme. Med de tilgængelige data kan en direkte og kræftfremkaldende
mutagen mekanisme dog ikke afvises. Arbejdsgruppen fulgte derfor anbefalinger fra
ECHA som foreslår en tilgang baseret på forsigtighedsprincippet. Farevurderingen af CB
NM blev derfor udført både baseret på en tærskeleffekt for inflammation og en ikke-
tærskeleffekt for kræft. Arbejdsgruppen fandt evidens for en ikke-tærskel baseret
virkningsmekanisme for kræft. ECHA’s anbefalinger om forsigtighedsprincippet og
brugen af en ikke-tærskel-værdi blev derfor fulgt. Arbejdsgruppen foretog derfor en
farevurdering baseret på både en tærskel-mekanisme for inflammation og en ikke-
tærskel-mekanisme for kræft.
For en grænseværdi i arbejdsmiljøet baseret på tærskelværdier anvendes følgende
traditionelle tilgang, som anbefalet af REACH: 1) identifikation af kritisk effekt, 2)
identifikation af
no observed adverse effect concentration
(NOAEC), og 3) beregning af
grænseværdi ved anvendelse af vurderingsfaktorer, der justerer for inter- og
intraspecifikke forskelle. For effekter uden tærskelværdi anvender den nærværende
arbejdsgruppe to metoder. Ved den første, Metode I, anvendes den målte
lungedeponerede dosis hos rotter til at estimere den tilsvarende eksponering i
arbejdsmiljøet. Ved den anden, Metode II, anvendes de direkte luftkoncentrationer.
Arbejdsgruppen fandt, at data fra fem inhalationsundersøgelser i rotter var det bedste
grundlag for farevurderingen. Følgende undersøgelser blev udvalgt til beregning af
henholdsvis
derived no effect level
(DNEL) og kræftrisiko: For DNEL var der en 12
måneders kronisk inhalationsundersøgelse af rotter (massekoncentrationer: 0; 2,5; og 6,5
mg/m
3
), en 13-ugers subkronisk inhalationsundersøgelse af mus, rotter og hamstere (0; 1;
7; og 50 mg/m
3
), og en 13-ugers subkronisk inhalationsundersøgelse af rotter (0; 1; 7; og
53 mg/m
3
). Kræftstudier var: To 2-årige kroniske kræftinhalationsundersøgelser af rotter
(0 og 12 mg/m
3
, henholdsvis: 0; 2,5 og; 6,5 mg/m
3
).
Tabellen nedenfor viser den beregnede DNEL for lungeinflammation, og overskydende
lungekræftrisiko hos 1 ud af 1.000, 1 ud af 10.000 og 1 ud af 100.000 beregnet på to
forskellige måder. Uafhængigt af den anvendte metode til farevurderingen, er de
beregnede forslag til grænseværdier alle lave sammenlignet med den nuværende danske
grænseværdi på 3,5 mg/m
3
for CB i arbejdsmiljøet.
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Oversigt over tærskelbaseret DNEL og ikke-tærskelbaseret eksponeringsniveauer, der
resulterer i overskydende kræftrisikoniveauer. Beregnet på ved to forskellige metoder.
Virkningsmekanisme
Tærskel-baseret
Ikke tærskel-baseret
DNEL
Overskydende
kræftrisiko
1: 1 000
1: 10 000
1: 100 000
Forslag til grænseværdi for CB NM
Inflammation
Lungekræft
Lungekræft
(metode I)
1
(metode II)
2
¤
¤
20 µg/m
3 #
3 µg/m
3
0,3 µg/m
3
0,03 µg/m
3
45 µg/m
3
4,5 µg/m
3
0,45 µg/m
3
#
Baseret op NOAEC værdier fra to subkroniske inhalationsstudier.
Arbejdsgruppen anbefaler metoden, hvor den overskydende risiko for lungekræft
baseres på lungebyrde, da denne tilgang tager højde for den faktiske lungedeponerede
dosis. Således er den forventede overskydende lungekræftrisiko i forbindelse med
erhvervsmæssig udsættelse for CB NM 1: 1 000 ved 3 µg/m
3
, 1: 10 000 at 0,3 µg/m
3
and 1:
100 000 at 0,03 µg/m
3
CB NM.
Metode I er baseret på CB NM luftkoncentrationer som giver forhøjede antal af
lungekræft tilfælde. Udregnet med en deponeringsfraktion på 8,6 %.
1
Metode II er baseret på
unit-risk-metoden
som er beskrevet af det Europæiske
Kemikalieagentur
2
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C
ONTENTS
Foreword ....................................................................................................................................... iii
Executive summary ...................................................................................................................... iv
Dansk sammenfatning ................................................................................................................ vii
Contents .......................................................................................................................................... x
Abbreviations ............................................................................................................................... 11
Introduction.................................................................................................................................. 13
Human studies............................................................................................................................. 15
Toxicokinetics .............................................................................................................................. 28
Animal studies ............................................................................................................................. 31
Rodent versus human response ............................................................................................ 31
Intratracheal instillation versus inhalation .......................................................................... 31
Selection of studies and endpoints ....................................................................................... 32
Pulmonary inflammation ....................................................................................................... 32
Genotoxicity and cancer ......................................................................................................... 37
Cardiovascular effects............................................................................................................. 42
Reproductive toxicity .............................................................................................................. 46
Other toxicological endpoints................................................................................................ 48
Mechanisms of toxicity ............................................................................................................... 49
Pulmonary inflammation, genotoxicity and cancer ........................................................... 49
Non-threshold carcinogenic effect ........................................................................................ 51
Cardiovascular effects............................................................................................................. 54
Dose-response relationships .................................................................................................. 55
Particle characteristics/dose metrics ..................................................................................... 56
Previous hazard and risk assessments of CB .......................................................................... 58
International Agency for Research on Cancer..................................................................... 58
Scientific Committee on Consumer Safety ......................................................................... 58
Summary of the evaluations .................................................................................................. 59
Scientific basis for an occupational exposure limit ................................................................. 60
Endpoint: Inflammation ......................................................................................................... 60
Endpoint: Cancer ..................................................................................................................... 62
Conclusion .................................................................................................................................... 69
References ..................................................................................................................................... 72
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A
BBREVIATIONS
8-oxo-dGua
ApoE
-/-
BAL
BET
Bw
CB
CRP
DNA
DNEL
ECHA
H
HDL
Hprt
IARC
ICAM-1
LDL
LOAEC
LOAEL
mRNA
MWCNT
NFA
NIOSH
SRM
NM
NOAEC
NOAEL
OEL
PAH
REACH
RR
ROS
SAA
SCCS
SMR
TiO
2
UK
USA
VCAM-1
8-Oxo-2'-deoxyguanosine
Apolipoprotein E knockout mice
Broncho-alveolar lavage
Brunauer–Emmett–Teller
Body weight
Carbon black
C reactive protein
Deoxyribonucleic acid
Derived no effect level
European Chemicals Agency
Hour
High density lipoprotein
Hypoxanthine-guanine phosphoribosyltransferase
International Agency for Research on Cancer
Intercellular adhesion molecule-1
Low-density lipoproteins
Lowest observed adverse effect concentration
Lowest observed adverse effect level
Messenger ribonucleic acid
Multi-walled carbon nanotube
National Research Centre for the Working Environment
National Institute for Occupational Safety and Health
Standard reference material
Nanomaterial
No observed adverse effect concentration
No observed adverse effect level
Occupational exposure limit
Polyaromatic hydrocarbon
Registration, Evaluation, Authorisation and Restriction of Chemicals
Relative risk
Reactive oxygen species
Serum amyloid A
Scientific Committee on Consumer Safety
Standardised mortality ratio
Titanium dioxide
United Kingdom
United States of America
Vascular cell adhesion molecule 1
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Some carbon black nanomaterials will be frequently mentioned through this report as
they have been included in numerous studies. These are listed below, for easy access to
important physical and chemical parameters.
Product
Printex 90
Monarch 880
Elftex-12
Sterling V
α
Manufacturer
Degussa-Hüls, Germany
Cabot Corp., MA, USA
Cabot Corp., MA, USA
Cabot Corp., MA, USA
Size (diameter)
14 nm
16 nm
37 nm
70 nm
Surface area
337 m
2
/g
220 m
2
/g
43 m
2
/g
37 m
2
/g
PAH content
0.123 ppm
α
<0.1%
β
0.012 %
γ
329.7 ppm
α
Summarised content of; phenanthrene, anthracene, fluoranthene, pyrene, benzo(a)pyrene, and
benzo (ghi) perylene (Borm et al., 2005).
β
From Cabot Corp. technical data sheet; toluene extract
according to ISO 6209.
γ
The authors state that the extractable fraction of the CB was 68-times less
than that for their tested diesel exhaust soot.
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I
NTRODUCTION
Carbon black (CB), CAS number 1333-86-4, is a black solid inorganic and poorly soluble
compound. The fine black powder is produced by finely controlled incomplete
combustion of various carbonaceous (primarily petroleum) gases or liquid products. It is
produced by a variety of methods with furnace black being the far most common
product accounting for approximately 95% of the total produced CB (IARC, 2010). Other
methods yielding products such as thermal black, lampblack, acetylene black and
channel black are much less frequently used. CB products are typically fluffy powders of
very low density. It is aggregates or agglomerates of primary particles within a size
range of 10-100 nm; although larger and smaller products do exist. All products have a
very high surface area to mass ratio mainly between 30-1000 m
2
/g.
Global production exceeded 10 million tonnes in 2005 (IARC, 2010), and 13 million
tonnes in 2015 and are expected to reach 19.2 million tonnes in 2022 (Industry_Experts,
2012); thus, CB is a major industrial chemical, and the most used nanomaterial (NM) in
the world. Most CB products are almost pure elemental carbon with low amounts of
impurities of solvent-extractable organic compounds like polyaromatic hydrocarbons
(PAHs) (Borm et al., 2005; Jacobsen et al., 2008b). The level of such impurities varies
significantly with certain CB products, such as Sterling V, containing at least three orders
of magnitude more than e.g. CB Printex 90 (Borm et al., 2005). CB products at the high
end such as Sterling V approaches similar PAH-concentrations as found in e.g. diesel
exhaust particles (Jacobsen et al., 2008a; Wise and Watters, 2006).
The by far largest application for CB is as a reinforcing agent in rubber products. This
accounted for 93% of total CB use in 2013. Used as a reinforcement agent in automobile
tires accounted for 73%, whereas 20% were use in rubber hoses, rubber belts and similar.
Other use of CB includes printing inks, paints, cosmetics and plastic products and as
conductive fillers in batteries, which combined accounted for the remaining 7%
(Auchter, 2005; IHS_Markit, 2017). In general, rubber products contain the cheap furnace
blacks, whereas the more expensive high specialty CBs are used in the other products.
The high price for specialty CBs has, in spite of the smaller volume, propelled this class
to the forefront of CB research and development (IHS_Markit, 2017).
The International Agency for Research on Carcinogenicity (IARC) reclassified CB in 1996
to
possibly carcinogenic to humans
(group 2B). This classification was based on inadequate
evidence for carcinogenicity in humans, but sufficient evidence of carcinogenicity in
experimental animals of both CB and CB extracts. This IARC classification (volume 65)
was confirmed when CB was revisited in volume 93 (IARC, 2010).
To our knowledge, there exists no legally binding nano-specific occupational exposure
limit for CB NMs. The present Danish occupational exposure limit for CB is 3.5 mg/m
3
and is regulated by the Danish Working Environment Authority. CB has the annotation
K, meaning that the substance is regarded as carcinogenic (Arbejdstilsynet, 2007).
The aim of the present report is to investigate if the present knowledge allows for a
suggestion of a health-based nano-specific occupational exposure limit (OEL) for CB
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NM. In this document, we review the relevant literature on the adverse effects of CB. The
hazard assessment methodology of this report will follow the guidelines suggested by
Registration, Evaluation, Authorisation and Restriction of Chemicals (ECHA, 2012). First,
threshold and non-threshold effects are determined. Threshold effect assumes 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.
A part of the current work is to evaluate whether there is substantial evidence for the
involvement of a non-threshold mechanism in CB NM induced carcinogenicity. For an
OEL based on threshold effects, the following traditional approach is utilized: 1)
identification of the critical effect, 2) identification of the no observed adverse effect
concentration (NOAEC), 3) calculation of OEL using assessment factors adjusting for
inter and intra species differences. For non-threshold effects, the current working group
will use two different approaches for calculating excess lung cancer risk. In the first
approach lung burden will be used to estimate the exposure levels. In the second
approach, air concentrations were used directly. Conclusively, the calculated OELs will
be compared and lastly, a recommended OEL for CB NM exposure will be proposed.
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H
UMAN STUDIES
Several epidemiological studies have investigated the potential for adverse effects of
exposure to CB amongst workers at CB factories. The literature and previous evaluations
especially focus on 3 large cohort studies relating to CB facilities in the United Kingdom
(UK), Germany, and the United States of America (USA). These studies focus on causes
of mortality, with special emphasis on lung cancer.
A few smaller studies on CB workers have also been published. These include chest
radiographs amongst European CB workers; pulmonary function amongst Chinese and
American CB workers and dockyard workers occupationally exposed to CB while
unloading CB at the dock of Genova, Italy. These studies will be described towards the
end of this section.
Additionally, a range of other studies have been performed on people occupationally
exposed for CB in specific industries. These include workers in rubber/tire industry,
printing industry and battery manufacture (Bulbulyan et al., 1999; Greene et al., 1979;
Malker and Gemne, 1987; Oleru et al., 1983; Parent et al., 1996; Ramanakumar et al., 2008;
Straif et al., 2000, 1999; Szeszenia-Dabrowska et al., 1991). Although carbon black may
have been the dominating exposure in these industries, there are some indications that
exposure for e.g. asbestos and talc is confounders in these industries (IARC 2010).
Therefore, the present working group will not include these in the current evaluation.
IARC has previously evaluated the human carcinogenicity of CB and concluded
inadequate evidence
(IARC, 2010). IARC evaluated the available epidemiological studies
and considered the 3 large studies of CB workers in the United Kingdom, the USA, and
Germany to be the most informative when assessing cancer risk. The 2 European studies
indicated an excess risk. Although smoking is a possible confounder and smoking status
was unknown, it is unlikely to have explained the entire excess risk as there was no
excess of other diseases known to be associated with smoking. However, links between
increasing exposure and risk levels were equivocal for the UK study or not existing for
the German study with a rather crude exposure assessment. In contrast, the US study did
not suggest excess mortality for any reported cancer site but did not assess risk by level
of exposure. This has since been updated (Dell et al., 2015). There was no indication that
long-term employment for service workers had higher risks than short-term employed.
Tobacco smoking habits were not evaluated. IARC found isolated results for excess risks
for cancers of the urinary bladder, kidney, stomach and esophagus; but evaluated that
these are not sufficient to support an evaluation of human carcinogenicity (IARC, 2010).
The present working group agrees with the above-mentioned IARC review and
evaluation. Below is an update of the latest publication on epidemiological studies on CB
exposure. Older publications within the 3 large cohorts, already evaluated by IARC are
included for easier access and for a more complete story of these cohort studies.
UK
The cohort following male workers at 5 CB production plants (Merseyside,
West
Midlands,
South West England, Scotland and Wales) consisted at initiation of 1422 male
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process workers with at least 12 months of exposure between 1947 and 1974; with
follow-up until 1980 (Hodgson and Jones, 1985). Dust exposure was estimated based on
47 personal samples as well as background dust samples taken by the Factory
Inspectorate in 1976. Half of the samples had CB mass concentrations higher than the
time weight average OEL of 3.5 mg/m
3
; with highest levels observed for filter bag
replacement crew (79 mg/m
3
). The authors adjusted for regional variations in mortality
by assigning
regional specific male mortality rates to each factory. As two factories were
in areas, Merseyside and West Midlands Conurbation were mortality rates were
different compared to the region. These were adjusted for local mortality and age-
specific factors.
Increased risk of lung cancer (Standardised Mortality Ratio; SMR: 152)
were observed at all 5 plants. Notably this increase was not statistically significant. When
analysing the factories individually, the excess lung cancer deaths were statistically
significant at 2 plants both showing ~2-fold increased number of deaths compared to
what was expected for their individual region (12 cases were observed, and 5.8 cases
were expected). Adjustment for malignancies observed in the first 10 working years
(these are likely not related to exposure at work) did not change this (10 observed; 5.1
expected). The same 2 plants were also those with the lowest dust mass levels, although
the levels were still very high. A non-statistically significant increase in bladder cancer
(SMR: 250) deaths were also observed (5 plants combined). As the figures are too small,
an excess
risk cannot be concluded or excluded. The incomplete data makes the
interpretation difficult, and as mentioned by the authors do not allow for a negative
conclusion regarding CB exposure and lung and bladder cancer
(Hodgson and Jones,
1985).
The above study has since been followed up in 2001 and 2007, with certain adjustments
(Sorahan et al., 2001; Sorahan and Harrington, 2007). SMRs were calculated based on
both national and county-district specific mortality rates. The few workers not residing
in the districts around the factories were assigned to the most common district of the
factory at which they worked. Importantly, the longer follow-up time (up to an
additional 24 years) of now 1147 CB workers allowed for a more precise calculation of
risk estimates. Also, a more detailed retrospective exposure assessment was attempted.
This included literature data on job categories and the exposure levels of CB at the
factories (~15.000 measurements at 19 European factories between 1987-1995) (Gardiner
et al., 1996, 1993, 1992), but also additional visits to the factories, interviews and
additional collection of data. This enabled the generation of a job-exposure matrix with
individual estimates of exposure by year and by job category. Overall, the data showed
highly statistical significantly increased risk for death of lung cancer (2001; SMR: 173)
(2007; SMR: 146). The data was in both cases driven by the previously mentioned 2
factories (2001; SMR: 278 and 315) (2007; SMR: 219 and 259). Mortality from all causes,
when excluding lung cancer, was not significantly elevated (SMR: 106) (Sorahan et al.,
2001; Sorahan and Harrington, 2007). In 2001 the authors did not find evidence for
linking the excess lung cancer mortality to cumulative CB exposure
However, in 2007 the authors applied a ‘‘lugged analysis’’ to evaluate most recent 15
years of CB exposure. Notably the authors found that the risk of lung cancer mortality
appeared
linked to cumulative CB exposure in the most recent 15 years. This link was
made for all 5 factories as well as the 2 with the highest risks. This is an important
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finding because most epidemiological studies of occupational induced lung cancer focus
on distant past (Sorahan and Harrington, 2007). The authors conclude that it is highly
likely that occupational lung cancers, at least at 2 factories, were caused by CB or by CB
production associated exposures; and that if truly based on a causal relationship: “it
is
clear that current regulatory standards will only provide inadequate protection to workers at
some carbon black production facilities”
(Sorahan and Harrington, 2007). The authors argue
that smoking history and previous occupation involving exposure to lung carcinogens at
the 2 factories cannot explain the difference to the other 3 factories.
The 2 high risk plants
were also those with the lowest mass dust levels (although still very high). As the
authors also do, it is tempting to speculate in the importance of the size of the
manufactured CB
(Sorahan and Harrington, 2007)
but also in the content of extractable
organic material in the CB.
However, as interesting as this hypothesis is, it should be
emphasised that there was no
information concerning size and purity of the CB
produced at these plants.
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Table 1. Epidemiological studies of plants in the
UK
Reference
Location and exposure
Cohort
(Hodgson
5 CB plants in the UK.
1422 male workers
and Jones,
There is no direct
hired in the period
1985)
information on CB
of 1947 to 1975.
exposure in mg/m
3
Working at a
facility for more
than 1 year. Follow-
up period up to
1980.
Data on smoking
habits were not
available
(Sorahan et
5 CB plants in the UK
1147 male workers
al., 2001)
Exposure level was
hired in the period
between 0.5-30 mg/m
3
of 1950 to 1975 and
(1950s) with a decreasing working at a
facility for more
trend to 0.5-5 mg/m
3
(1980s) for administrative than 1 year.
(lowest) and cleaners
Follow-up period
(highest) respectively.
till 1996. Exposure
derived from a job-
exposure matrix
and records and
interviews
conducted at the 2
still functioning
factories.
Smoking history
unknown
(Sorahan and 5 CB plants in the UK
1147 male workers
Harrington,
Exposure as above.
hired in the period
2007)
of 1950 to 1975.
Working at a
facility for more
than 1 year.
Follow-up period
till 2004. Exposure
derived from a job-
exposure matrix
and records and
interviews
conducted at the 2
still functioning
factories.
Smoking history
unknown
Endpoints/Results
Excess lung cancer deaths
were observed at all 5 plants;
although only statistically
significant at 2 plants. The
same two with the lowest
mass dust levels.
Incompleteness of data made
interpretation of causes
complicated.
Mortality:
All cause (SMR: 113*), Lung
cancer (SMR: 173***).
Highly elevated at 2 factories.
No indication of risks
increasing length of
employment or estimated
exposure.
Mortality: All causes except
lung cancer (SMR: 106)
Lung cancer (SMR: 146**);
highly elevated at 2 of the
plants (SMR: 230***). CB (or
associated chemicals) had
effect on lung cancer with a
clear dose response at these 2
plants but not the other 3
plants.
The elevated lung cancer risk
was limited to those with
some employment in the most
recent 15 years.
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USA
The first publication on incidence of cancer amongst workers in the American CB
industry dates to 1950. In this cohort study, excess morbidity and mortality was
examined amongst 476 workers per year at one CB production facility in the years 1939-
1949 (Ingalls, 1950). The follow-up period was later extended to 1949-1956 (Ingalls and
Risquez-Iribarren, 1961), and both
these studies found that CB workers have no excess
risks of cancer mortality compared to other industrial groups or to National mortality
data or New York State cancer data.
The cohort was enlarged to encompass 4 CB production facilities with an average annual
number of employees of 1250. The workers were followed for 40 years between 1935 and
1974. No increased risks were found for all-cause-deaths, malignant neoplasms in the
respiratory or digestive systems, or mortality due to heart disease or ischemic heart
disease. Death rates were below expectations and sometimes significantly lowered. The
authors suggest that these findings are explained by a “healthy worker effect”
3
(Robertson and Ingalls, 1980). The exposure was considered equal for all workers and
evaluated as
person years of exposure;
i.e. there was no estimation of degree of exposure
(Robertson and Ingalls, 1980).
In 2006 Linda Dell and co-workers published a comprehensive industry wide cohort
study. It contained data from 18 CB production facilities and follow-up details of 5011
male and female workers hired for more than 1 year between the 1930s and 2004 (Dell et
al., 2006). Results were presented using race, gender, state and year-specific mortality
rates. No excess mortality caused by lung cancer was observed. The SMR was 89 (95%
confidence interval: 0.75–1.06) or 97 (95% confidence interval: 0.82–1.15); if including 11
deaths of unknown causes (15% of 76 cases totally). Mortality from all causes or ischemic
heart disease was 26% and 30% lower than expected. As no trends were observed
between cause of death and duration of employment, the authors suggest that there is no
increased mortality arising from employment in CB production (Dell et al., 2006). It
should be mentioned that mortality from all causes, excluding lung cancer, was
significantly reduced with an SMR of 72 (95% confidence interval: 68-76), suggesting that
all deaths were not traceable, or the application of an incorrect measure of person-years
working with CB production (IARC, 2009). Similar statistically significant reductions
were observed for all cause deaths (SMR: 74) and all cause cancer deaths (SMR: 83). At
least the data suggests a bias between the populations. A detailed exposure assessment
was not attempted. This might have provided risks in relation to (semi)-quantitative
estimates of CB exposure. Additionally, information on smoking or other possibly
confounding exposures was not available (Dell et al., 2006).
Recently an updated analysis of the above-mentioned cohort was published (Dell et al.,
2015). This study on the US cohort included a follow-up through 2011 of a full cohort of
6634 male and female workers CB workers and a smaller entry cohort of 3890 male
A healthy worker effect arises when workers must meet a certain health level in order
to function at the workplace. Workers of lower health will leave the job and thereby a
difference in health between the workers and the public will arise.
3
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hourly workers with an assumed larger potential for CB exposure. All workers were
employed for more than 1 year at one of 18 US CB production facilities. The paper
includes the new exposure metrics, and a job exposure matrix. The exposure matrix was
completed on the basis of more than 8000 measurements (primarily of total dust but also
of certain inhalable and respirable measurements) collected during 7 industry campaigns
between 1979 and 2007 (Kerr et al., 2002; Muranko et al., 2001; Smith and Musch, 1982).
Values were used for interpolation and backwards extrapolation before linkage to the
work history data (title and dates) for each cohort member. Mortality from lung cancer
was decreased for the full cohort but this was only borderline statistically significant for
the entry cohort (SMR 77 and 87, respectively). The authors concluded that their data did
not support an increased risk for lung cancer mortality for CB workers in this cohort, and
that the exposure (up to 43 mg CB/m
3
) experienced from the 1970s to the 1990s were not
associated with increased risk of lung cancer. Risk for mortality caused by non-
malignant respiratory diseases was not significantly altered for the 2 cohorts (SMRs: 88
and 109 for the full and entry cohort, respectively). Similarly, for chronic obstructive
pulmonary disease; stratified analyses, revealed no clear associations between excess of
lung cancer, non-malignant respiratory diseases or chronic obstructive pulmonary
disease and length of employment, time since the first employment, or time since
cessation of employment for any of the cohorts. Also “all-cause mortality”, “all cancer
mortality” and “all heart disease mortality” was decreased (SMRs: 78, 79 and 78,
respectively) in the full cohort. This was also statistically significant, albeit less
pronounced in the entry cohort (SMRs: 86, 87 and 84, respectively). The authors
acknowledge that their data set shows a strong healthy worker effect. Also, the smoking
frequency over time, in the general and CB working population is a possible strong bias.
The deficit in lung cancer deaths observed in general and in particular amongst certain
groups may suggest that workers were less likely to have been smokers (IARC, 2009).
Table 2. Epidemiological studies of plants in the USA
Reference
Location and exposure
Cohort
(Ingalls,
1 CB manufacturer with
476 workers per year
1950; Ingalls multiple units in
recruited in the
and
southwest USA. There is
period of 1940 to
Risquez-
no direct information on
1949 or 1940-1956
3
Iribarren,
CB exposure in mg/m
and working for
1961)
more than 12 months
at the facility.
Smoking habits were
unknown
(Dell et al.,
2006)
18 CB plants in the USA.
There is no direct
information on CB
exposure in mg/m
3
5011 male and
female CB workers
(worked at the plant
for more than 12
months) hired since
the 30s and followed
until end of 2003.
Age-, race-, sex-, and
calendar year-adjusted
SMRs were calculated
Endpoints/Results
The observed death rate was
0.21 per 1 000 per work year in
both tested periods (expected
was 0.49).
The result was that CB workers
face no excess risks for cancer
No increased risk for mortality
of lung, bladder, non-malignant
respiratory diseases.
Statistically reduced risk for all-
cause (SMR 74 and all-cancer
mortality (SMR 83).
No trends in risks were
observed with duration of
employment or time since first
hire for any cause of death incl.
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(Dell et al.,
2015)
18 CB plants in the USA.
Exposure level was
between 0-43 mg/m
3
(1980s) with a decreasing
trend to 0-12 mg/m
3
(2000s). The vast majority
of all measurements (1975-
2007) were in the range 0-5
mg/m
3
.
using state-specific
mortality rates
Smoking status and
history unknown
Full cohort: 6634
male and female CB
workers. A smaller
entry cohort of 3890
male hourly workers
with an assumed
larger CB exposure.
All with more than
12 months hire at one
of 18 US CB facilities.
Follow-up through
2011. A job-exposure
matrix was based on
measurement data
from 7 previous
industry campaigns.
Personal exposure
was estimated via
coupling to personal
work history.
State, race- and sex-
specific mortality
rates by age and
calendar interval
Smoking history
unknown
lung cancer.
Lung cancer mortality was stat.
significantly decreased in both
cohorts (SMR 77 & 87).
Similarly, “all-cause”, “all
cancer” and “all heart disease”
mortality was stat. significantly
decreased in the full cohort
(SMR 78, 79 and 78) and the
entry cohort (SMR 86, 87 and
84). No increased risk for
mortality caused by non-
malignant respiratory disease
and chronic obstructive
pulmonary disease.
The authors conclude: No
support for increased lung
cancer mortality for CB
workers. No associations with
total employment, time since
the first employment, or time
since cessation of employment
for any of the cohorts was
observed.
HR: 1.0 for 20-50 mg/m
3
*year;
HR: 1.3 for 50-100 mg/m
3
*year;
and HR: 1.4 for 100 or more
mg/m
3
*year compared with
referent (<20 mg/m
3
*year)
Germany
There are a range of studies on a cohort of CB workers from one production plant in
Germany followed in 1976 to 1998. The cohort consisted of 1528 male workers that
produced furnace black, lamp black, and gas black (Büchte et al., 2006; Morfeld et al.,
2016, 2006a, 2006b, Morfeld and McCunney, 2009, 2007; Wellmann et al., 2006). And one
study was likely in the same cohort (same number of deaths due to lung cancer, 50) in
1960 to 1998 (Vital status and causes of death were assessed for 1976 to 1998) (Wellmann
et al., 2006).
Concerning the study by Wellman and co-workers, the mortality of the cohort of 1535
male CB manufacturing plant workers was investigated. The workers were employed at
the plant for at least 1 year during the period of 1960 to 1998 (vital status and causes of
death assessed for the period of 1976 to 1998). Risk was calculated based on national
referent rates, and state referent rates. The SMR for all-cause-mortality (332 deaths) was
120 (95% confidence interval: 108 to 134), that of mortality from lung cancer (50 deaths)
was 218 (95% confidence interval: 161 to 287) using national rates as a reference.
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Comparisons to regional rates gave SMRs of 120 (95% confidence interval: 107 to 133)
and 183 (95% confidence interval: 136 to 241), respectively. There was no dose-response
relationship between lung cancer mortality and such indicators of occupational exposure
as years of employment and exposure to CB. The authors of the study concluded that:
“The mortality from lung cancer among German carbon black workers was increased. The high
lung cancer standardised mortality ratio cannot fully be explained by selection, smoking, or other
occupational risk factors, but the results also provide little evidence for an effect of carbon black
exposure.”
(Wellmann et al., 2006).
Buchte and co-workers performed a case–control study of lung cancer nested within the
cohort of 1528 subjects and the years were 1976–1998. Fifty lung cancer deaths were
analysed and showed no association to CB exposure (Büchte et al., 2006). Another study
was conducted on a sub-cohort comprising only subjects with information on their
smoking status. Moreover, an inception sub-cohort consisting of all study subjects from
the cohort who started working at the CB plant on or after January 1, 1960, was
identified to reduce the healthy worker selection biases. Combining the criteria, 4 study
groups were defined for analyses: The full cohort (cohort 1); The full cohort with
smoking information (cohort 2); The inception cohort (cohort 3); And the inception
cohort with smoking information (cohort 4). No positive association was found between
the 50 lung cancer deaths and CB exposure indices. However, these authors found that
certain models provided an indication that there was an increasing risk across duration
of work in the lamp black producing department. The authors of the study conclude that
their results do not suggest that CB exposure is a lung carcinogen. The lamp black results
may point at historical exposures to polycyclic aromatic hydrocarbons.” (Morfeld et al.,
2006b). A sensitivity analysis of the lung cancer SMRs was also applied to the data on
1522 of the German CB workers observed in the period of 1976 to 1998. Risks were
calculated based on national and regional mortality rates. Based on 47 lung cancer
deaths, the SMRs were 1.62, 1.72, and 2.08 (local, state, and national rates, respectively).
Adjustment for previous exposures and smoking provided additional correction factors
of 0.64 or 0.74. The authors of the study concluded: “Lung
cancer standardised mortality
ratios (95% confidence intervals) for the full cohort ranged from 1.20 (0.88 –1.59) to 2.08 (1.53–
2.77) in this sensitivity analysis. Thus, overall standardised mortality ratios are only weak
measures of causal associations and should be complemented by internal modelling of exposure
effects whenever possible
(Morfeld et al., 2006a).
In a follow-up to a British study of CB production workers (Sorahan and Harrington,
2007) in which the risk of lung cancer was reported to decline after cessation of
employment. The German cohort was re-evaluated by focusing on the first 15 years after
cessation of employment in terms of lung cancer SMR. This was analysed in the German
cohort of 1528 male workers and in an inception cohort consisting of 1271 males. In
contrast to the British study a rising trend in lung cancer SMR was observed (Morfeld
and McCunney, 2007). In a study of the same German cohort, a new analysis was
undertaken. The reason for this is that an analysis of a UK cohort had been published
(Sorahan and Harrington, 2007). In this publication the most recent 15 years of exposure
were assessed (‘‘lugging’’) to support the hypothesis that CB acts as a late stage lung
carcinogen. Thus, this was also tested in the German cohort of 1528 CB workers.
Negative coefficients were returned by all tested models. The authors of the study
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conclude that: “Despite
extensive searching, no exposure scenario suggested an adverse effect of
‘‘lugged’’ carbon black exposure on lung cancer mortality. Our analysis does not support the
hypothesis of carbon black being a late stage carcinogen.”
(Morfeld and McCunney, 2009).
SMR and Cox proportional hazards results from cohort studies of US, UK and German
CB production workers were combined. Mortality from all causes, heart disease,
ischemic heart disease and acute myocardial infarction were analysed. Full cohort meta-
SMRs (random effects) were 1.01 (95% confidence interval: 0.79–1.29) for heart disease;
1.02 (95% confidence interval: 0.80–1.30) for ischemic heart disease, and 1.08 (95%
confidence interval: 0.74–1.59) for acute myocardial infarction mortality. The authors of
the study conclude that “Our
results do not demonstrate that airborne CB exposure increases
all-cause or cardiac disease mortality”
(Morfeld et al., 2016).
Table 3. Epidemiological studies of plants in
Germany (and Germany, USA and UK combined)
Reference
Location and exposure
Cohort
Endpoints/Results
(Wellmann
A CB manufacturing plant in 1535 male CB
The authors of the study
et al., 2006)
Germany. There is no direct
workers, who had
conclude that: “the
results
information on CB exposure
worked
also provide little evidence for
in mg/m
3
, however, an expert for at least 1 year
an effect of carbon black
exposure”
on all-cause-
committee evaluated the
and were
exposure and established a
employed between mortality and lung cancer
mortality
job-exposure matrix and
1960 and 1998 at a
intensity of exposure was
single CB
assigned to each job title. The production plant.
highest score (20 units) was
Age- (five-year
assigned to jobs where
groups), sex-, and
carbon black was shovelled
calendar year-
into bags. This was
adjusted SMRs
performed up till the early
were calculated
1960s. A score of zero was
using national
assigned to the jobs with no
(West) Germany or
contact to CB
regional -specific
mortality rates
North-Rhine
Westphalia
(Büchte et
A CB production plant in
1528 CB workers,
No effect on lung cancer
al., 2006)
Germany.
1976 – 1998,
producing furnace
There is no direct
black, lamp black,
information on CB exposure
and gas black
in mg/m
3
. Applied an
adjusted job-exposure matrix
(Morfeld et
al., 2006b)
A CB production plant in
Germany. A few CB
measurements were
performed at the plant.
However, the authors did
not consider them valid for
the purpose of the article.
Thus, there is no direct
1528 CB workers,
1976 – 1998,
producing furnace
black, lamp black,
and gas black
No positive association was
found between the 50 lung
cancer deaths and CB
exposure indices. However,
these authors found that
certain models provided an
indication that there was an
increasing risk across
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(Morfeld et
al., 2006a)
information on CB exposure
in mg/m
3
. Also, certain
changes were done in the
job-exposure matrix
compared to Wellman et al.,
2006
A CB production plant in
Germany. There is no direct
information on CB exposure
in mg/m
3
duration of work in the lamp
black producing department
1528 CB workers,
1976 – 1998,
producing furnace
black, lamp black,
and gas black
(Morfeld
and
McCunney,
2007)
(Morfeld
and
McCunney,
2009)
A CB production plant in
Germany. There is no direct
information on CB exposure
in mg/m
3
A CB production plant in
Germany. There is no direct
information on CB exposure
in mg/m
3
1528 CB workers,
1976 – 1998,
producing furnace
black, lamp black,
and gas black
1528 CB workers,
1976 – 1998,
producing furnace
black, lamp black,
and gas black
(Morfeld et
al., 2016)
A CB production plant in
Germany. 18 CB plants in the
USA (also described above) 5
CB plants in the UK (also
described above).
Exposures for all 3 cohorts
were converted to 100
mg/m
3
-years (UK and US)
and unit/years (Germany).
The job-exposure matrix for
all 3 cohorts were used
SMR and Cox
proportional
hazards results
from cohort studies
of US, UK and
German CB
production
workers were
combined
Based on 47 lung cancer
deaths, the SMRs were 1.62,
1.72, and 2.08 (local, state,
and national rates,
respectively). Adjustment
for previous exposures and
smoking provided
additional correction factors
of 0.64 or 0.74
The cohort was re-evaluated
by focusing on the first 15
years after cessation of
work. A rising trend in lung
cancer SMR was observed
The most recent 15 years of
exposure was assessed by
so-called ‘‘lugging’’. No
exposure scenario suggested
an adverse effect of
‘‘lugged’’ CB exposure on
lung cancer mortality.
Full cohort meta- SMRs
(random effects) were 1.01
(95% confidence interval (CI)
0.79–1.29) for heart disease;
1.02 (95% CI 0.80–1.30) for
ischemic heart disease, and
1.08 (95% CI 0.74–1.59) for
acute myocardial infarction
mortality
Other studies
Employees (n=1755) at 22 North American plants were systematically administered
questionnaire and spirometry tests. In conclusion, the study shows a relationship
between exposure to CB and small reductions in the 1-second forced expiratory volume
(-2 mL/mg-year/m
3
total dust and -0.7 mL/mg-year/m
3
for the inhalable fraction) and
increased prevalence of chronic bronchitis for large exposures. Although the effect may
be limited, the authors conclude that workplace exposures to CB should be controlled to
the lowest practical levels (Harber et al., 2003).
Lung and bladder cancer was investigated in a group of 2286 longshoreman
occupationally exposed to CB dust at the dock of Genova, Italy. Workers exposed to high
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concentrations of CB (n=14) had a significantly increased frequency of bladder cancer
(standardised incidence ratios 204, 112–343). Gender and age-specific incidence rates for
the City of Genova were used to compute standardised incidence ratios. The authors
conclude that the increase in bladder cancer in longshoremen is probably related to high
CB exposure (Puntoni et al., 2001). In a follow-up study, cancer incidence was analysed
amongst 2101 longshoremen from the same dock. The workers CB dust exposure was
listed as low, moderate, and high. Incidence rates were calculated as mentioned above. A
positive CB exposure–response relationship was detected for bladder cancer (SIR 204,
95% CI 112–343; high CB exposure) (Puntoni et al., 2007).
Inhalation of CB NM (30 - 50 nm) and altered lung function and inflammation were
analysed in 81 CB-exposed male workers and 104 non-exposed male workers. The
exposure concentration was 14.9 mg/m
3
where 50.8% were less than 0.5 µm, and 99.6%
were less than 2.5 µm in aerodynamic diameter. A reduction of lung function parameters
including FEV1%, FEV/FVC, MMF%, and PEF% in CB workers was observed, and the
IL-1β, IL-6, IL-8, MIP-1beta, and TNF- alpha had 2.86-, 6.85-, 1.49-, 3.35-, and 4.87-folds
increase in serum of CB workers, respectively. The authors conclude that the data
strongly suggests that CB NM could be responsible for a reduction in lung function and
increased inflammation in the workers (Zhang et al., 2014).
As high levels of CB exposure had previously been associated with an increased
prevalence of chest radiograph abnormalities, the authors examined to what extent
current levels of exposure in the CB industry are associated with such effects.
Longitudinal analyses of workers in the European CB industry who provided three full-
size chest radiographs sequentially between 1987 and 1995. Data from 675 workers were
included. An association between cumulative CB exposure and new cases of chest
radiograph abnormalities and progression in small opacities was observed (majority
came from one factory). A large fraction of workers with abnormalities reversed to
normal; however, after adjusting for other confounders, this was not associated with
levels of exposure to CB dust. In conclusion, the results show that exposure to CB is
associated with increased risk of chest radiographic abnormalities, which may be
reversible after reduction or cessation of exposure (van Tongeren et al., 2002).
Power of the epidemiological studies
The ability to detect the effect of exposure to occupational carcinogens is also determined
by the population-specific lung cancer incidence. In Denmark, the life time risk of
developing lung cancer (0-74 years) is now 4.9% for men and 4.5% for women,
respectively, according to The National Board of Health. In the US, life time lung cancer
risk is similar, 7% for men and 6% for women (American Cancer Society 2018). The lung
cancer incidence has historically been much higher and is largely determined by the
smoking. The relative lung risk caused by occupational exposure to a carcinogen, which
causes lung cancer the different risk levels, 1%, 0.1% and 0.01% are given in Table 4. As
can be seen, exposures that cause 1% excess lung cancer will give relative risks of 1.2.
According to power calculation, detection of 1% excess cancer incidence with 5% lung
cancer incidence in the reference group would require group sizes of 8 000 participants
(with 80% chance of detecting the effect at 5% significance level). On the other hand,
occupational exposures that cause 0.1% excess lung cancers (1 of 1 000, which is the
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2130134_0028.png
acceptance level in the US), corresponds to a relative risk (RR) of 1.04, which requires
group sizes of 750 000 persons if the background cancer incidence is 5%.
Table 4. Relative risk of lung cancer for carcinogens that cause 1%, 0.1% or 0.01% excess lung
cancer risk in a population with the current Danish lung cancer incidence
Men
Women
Life time risk (0-74 years)
2011-2015 in Denmark
1
4.9 %
4.5 %
Excess lung cancer risk level
1: 100
RR= (4.9+1)/ 4.9= 1.20
RR= (4.5+1)/4.5= 1.22
2: 1 000
RR= (49+2)/49= 1.041
RR= (45+2)/45=1.044
1: 1 000
RR= (49+1)/49= 1.02
RR= (45+1)/45=1.02
1: 10 000
RR= (490+1)/490= 1.002
RR= (450+1)/450= 1.002
1: 100 000
RR= (4 900+1)/4 900= 1.000 2
RR= (4 500+1)/4 500= 1.000 2
RR: Relative risk
Thus, in spite of a small subsection of the cohorts being exposed to very high CB levels;
the epidemiological studies on CB and lung cancer risk have limited statistical power to
detect carcinogenic effects of CB exposure, unless the excess lung cancer risk associated
with CB exposure is very high.
Conclusion
The overall epidemiological evidence is not conclusive. The two European production
cohorts show evidence of excess cancer incidence. A very high prevalence for all causes
mortality (SMR 146) was observed in the British cohort. This was mainly driven by a
large increase at two facilities (SMR 230). In the German cohort similar trend on both all-
cause mortality and mortality from lung cancer was observed, but this was unrelated to
years of exposure/employment. Workers employed and exposed for many years should
have a higher occupational risk compared to workers recently employed. In contrast the
study of American CB employees demonstrated no excess occurrence of cancer mortality
when compared to the general population. A significantly decreased mortality was
observed in spite of some very high estimated exposure doses based on a job exposure
matrix. Concerning cardiac disease mortality, a study combining exposures from both
Germany, USA and UK showed no increased mortality associated with CB exposure
(Morfeld et al., 2016). When conducting epidemiological studies, it is a challenge to select
a control group with minimal bias; especially, when knowledge about the participants is
limited. Possible bias in all studies may come from the healthy worker effect, possible
misclassifications of exposures, previous occupational exposures and the unknown
smoking history. Smoking restrictions were implemented at American facilities in the
mid-90s, and this may have an impact on the lung cancer cases over time. In the UK
cohort there was no excess risk of other diseases known to be associated with smoking.
Also, high exposure exposures may have forced a healthy worker effect. None of the
epidemiological studies provided information regarding the particle size distribution of
the CB exposure and thus, did not allow to identify a possible CB NM exposure. CB
particles may likely have been larger in the earlier years leading to less deposition and
effect. The American insurance-based health plan generated through the employer may
give bias towards willingness or ability to seek medical aid and therefore, the general
population cannot be used as reference group for CB-exposed workers. The working
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group finds the difference between the European studies and especially the British and
the US cohort striking.
Thus, in conclusion, we cannot exclude a carcinogenic potential of CB NM based on the
present human epidemiological studies. Furthermore, the available epidemiological data
cannot be used for risk assessment.
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T
OXICOKINETICS
As CB is the black dye of the world and hence one of the most used chemicals (IARC,
2010), exposures may occur in many places via inhalation, dermal exposure or via
primary or secondary ingestion. Highest exposures and risks are expected to occur
through breathing of air in occupational settings. Dermal exposure occurs both in
occupational and consumer settings. However, dermal exposure levels are not expected
to be critical, and uptake through healthy undamaged skin are expected to be low or
non-existing. End-users of rubber, ink or paint products are not exposed to CB per se, as
it is bound within a product matrix (IARC 2010). Focus in this section will thus be on the
most critical exposure pathway; inhalation. Pulmonary deposition and systemic
biodistribution is of importance as it describes key sites for possible secondary effects
caused by translocation.
In general; the smaller the diameter of inhaled particles the deeper the pulmonary
penetration and deposition will be. Especially nanoparticles will deposit in the alveolar
ducts and alveolar sacs (ICRP, 1994; Jacobsen et al., 2009). Materials deposited from the
respiratory bronchioles to the larynx will be cleared by the ciliated epithelium (the
mucociliary escalator). The respiratory bronchioles are only partially ciliated, and the
alveolar sacs and ducts are not. Here the main mechanism for particle clearance is
macrophage phagocytosis.
The success of particle clearance relies on attracting the alveolar macrophages to the site
of particle deposition and particle uptake (phagocytosis). It has been shown that
nanoparticles are less efficiently phagocytized by macrophages than larger particles of
identical composition (Geiser et al., 2008; Kreyling et al., 2002; Oberdorster et al., 2005;
Oberdörster et al., 1992; Semmler et al., 2004). Twenty-four hours after inhalation of
various sized particles increased retention of particles within the lung is seen with
decreasing size of particles.
The poor phagocytosis of NMs could be due to a reduced response when phagocytes
and NM encounter. However, it could also be that the nanoparticles are too small to
initiate a cell generated substantial chemotactic signal or it could be caused by increased
adherence or uptake by epithelial cells. Actually, it has been suggested that the size
range of phagocytosis may be optimal in the lowest µm range (Tabata and Ikada, 1988).
Increased or long-term lung retention of nanoparticles may increase inflammation and
proximal as well as distal translocation to secondary target organs via the circulation.
Kinetics of CB particles are in general challenging to study as carbon is the second most
abundant element in the body. Thus only a few studies have performed quantitative
assessment of pulmonary retention of inhaled CB. In general, small variations over a
method of digesting dried tissues before measuring the optical density of the re-
suspended insoluble particles are used. The spectrophotometric results are compared to
a standard curve (Rudd and Strom, 1981).
Strom and co-workers exposed male rats for 20 h/d, 7 d/week for 1, 3, or 6 weeks to
filtered air or 7 mg/m
3
CB NM (Elftex-12, 37 nm, Cabot Corp., Boston, Mass.). After 1-, 3-,
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and 6-wk of exposures, the lung burdens were 1.1, 3.5, and 5.9 mg, respectively. One
year after the 1-, 3-, or 6-wk exposure, 8%, 46%, and 61% of the initial lung burden
remained in the lungs, respectively. Lymphatic translocation was determined 1-year post
exposure and showed lymph node burdens of 1%, 21%, and 27% of the initial lung
deposited material for the three exposure doses, respectively. A combined retention of
lung/lymph nodes was 9%, 67%, and 89% for the 1-, 3-, and 6-wk exposed animals
showing a clear decrease in lung clearance with increasing dose (Strom et al., 1989).
Rats were exposed 19 h/day, 5 days/week for 24 months to 12 mg/m
3
of CB NM (7.5
mg/m
3
for the first 4 months) (Printex 90, Degussa-Hüls, Germany). Following the
exposure 43.9 mg of CB NM material was retained in the lungs with determined half
times of 550 days and 6.7 mg in the lung associated lymph nodes (Creutzenberg et al.,
1990). The high half-times indicate a severely impaired or collapsed clearance function.
Another study focused on comparing three rodent species (female rats, mice and
hamsters) for particle retention kinetics. Four exposure concentrations were used; 0, 1, 7,
and 50 mg/m
3
high surface area CB (Printex 90, Degussa-Hüls, Germany; 300 m
2
/g) and
50 mg/m
3
low surface area CB NM (Sterling V, Cabot Corp., Boston, Mass.; 37
m
2
/g)(Elder et al., 2005). Exposure was 6 h/day, 5 days/week for 13 weeks. Increased dose
resulted in decreased clearance and low surface area CB NM was cleared faster than
high surface area CB NM. For low and middle dose, the retention half times were longest
for mice (133 and 343 days) followed by rats (64 and 115 days) and hamsters (42 and 53
days). Thus, these doses overwhelmed primarily the mouse lungs and to a lesser extent
the rat lungs leading to a very low and slow clearance. At the highest tested dose, the rat
lungs did show substantially longer retention half times compared to mice (322 days)
and hamsters (309 days). Normal retention half times has been suggested around 70 days
for rats and 55 days for mice (Kreyling, 1990; Oberdörster, 1995). Hamsters have the
most efficient clearance mechanisms also leading the least severe responses of the three
species.
One study has attempted to determine short term (24h) full body translocation of carbon
particle aggregates (25 nm) containing ~1% iridium (
192
Ir). Inhalation of pure
192
Ir
aggregates 20 nm or 80 nm was also performed (Kreyling et al., 2009). In general, most
material deposited in upper airways was rapidly cleared to the gastro intestinal tract to
the faeces. Here, as also shown before (Kreyling et al., 2002), absorption from the gastro
intestinal tract was negligible (Geraets et al., 2014). Twenty-four hours following the
inhalation 78 % of the retained dose was still in the lung; of this the 8 % was accessible by
lavage. Translocated carbon / iridium material agglomerates were detected; 0.2 % liver;
0.08 % heart; 0.06 % kidney; 0.05 % spleen; 0.05 % blood; 3% carcass (smooth tissue and
bone). Translocation was higher following inhalation of 20 nm
192
Ir aggregates and was
similar or slightly less following 80 nm
192
Ir aggregates (Kreyling et al., 2009).
A few short-term studies (generally 24h but one study up to 3 days) in humans using
carbon particles with attached
99m
technetium have also been performed (Brown et al.,
2002; Mills et al., 2006; Möller et al., 2008; P. Wiebert et al., 2006; Pernilla Wiebert et al.,
2006). All studies found that whether material size was 4-20 nm, 35 nm or 100 nm by far
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the most material remained in the lung and immediate surroundings and systemic
translocation was low and mainly consisted of free and not particle bound
99m
technetium.
Translocation of pulmonary deposited CB NM (Printex 90, Degussa-Hüls, Germany) to
the liver has also been documented using bright field and enhanced dark field
hyperspectral microscopy. In this study female mice were exposed by single
intratracheal instillation of 162 µg of CB NM suspension. CB NM was detected in the
liver but detection in other organs was not attempted (Modrzynska et al., 2018).
In summary; although little work has been performed on the kinetics of pulmonary
deposited CB NM, the data clearly shows low and slow pulmonary clearance. Three or 6
weeks of inhalation at 7 mg/m
3
resulted in a pulmonary and lymphatic retained dose of
68% and 89% 1 year after the inhalation ended (Strom et al., 1989). These results are from
a rat study but as shown by Elder and co-workers, mice actually have even slower
clearance rates compared to rats at this and lower inhalation concentrations (Elder et al.,
2005). It is the opinion by the present working group that there are no reasons to believe
that retention and systemic translocation and excretion of CB NM would be much
different for CB NM than for other low solubility and low toxic potency NMs. It has been
shown for several metals and metal oxides that smaller particles translocate from the
lung to the system to a greater extent than larger counterparts. This means higher
material accumulation in an increased number of organs for the smallest NM (Kreyling
et al., 2018, 2014, 2009; Sadauskas et al., 2009)(Balasubramanian et al., 2013; Ferin et al.,
1992; Kreyling et al., 2009, 2002; Oberdörster et al., 1994). The same conclusion has been
drawn in regards to translocation across placental barriers during rat pregnancy
(Semmler-Behnke et al., 2014). I.e. translocated particles may be found in several organs.
Knowledge from the general NM literature (radioactive gold, iridium or carbon
nanotubes) shows clear evidence that NM cross membranes and reach secondary target
organs where they accumulate. Although higher levels have been observed following
instillation with 1.4 and 2.8 nm gold (Schmid et al., 2017), evidence in general show that
only a smaller fraction of inhaled particles (<1 %) will translocate beyond the lung and
immediately associated tissue (lymph nodes and pleura) (Jacobsen et al., 2017; Kreyling
et al., 2002; Schmid et al., 2017). However, it is important to note that even if <1% escape
the lung and translocate into secondary organs such as liver, spleen, kidneys,
reproductive system and brain it may represent a very high number of NMs.
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A
NIMAL STUDIES
Rodent versus human response
Inhalation studies in mice and rats are used to assess potential human hazard where
human exposure studies and epidemiological studies are not available or inconclusive.
There is very limited data available on effects following controlled inhalation of CB NMs
in humans. Rats are the preferred animal model in particle toxicology and are more
sensitive than mice to particle-induced lung cancer and fibrosis (Kratchman 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 inhalation exposure. However, the deposited pulmonary
dose can be difficult to monitor precisely following inhalation of certain materials e.g. CB
NM. For these, inhalation models can be used taking into account differences in sizes of
the aerosolised particle agglomerates and deposition frequency making it possible to
estimate the delivered dose. In addition, exposure by inhalation requires a substantial
amount of material and specialised inhalation facilities, and it poses an occupational
health risk to the technicians handling the NMs.
Pulmonary deposition by intratracheal instillation is used in screening studies (Bourdon
et al. 2012;Husain et al. 2013;Poulsen et al. 2015b; Saber et al. 2012b; Saber et al. 2012a)
and has been proposed as an alternative to inhalation exposure. Intratracheal installation
has previously been shown to give widespread distribution of particles throughout the
lung (Mikkelsen et al., 2011; Poulsen et al., 2016). This exposure method ensures that the
same precise dose is delivered to the lung for all NM exposures, demands less material
and is more user-friendly.
Several studies have compared the toxicological response following inhalation and
instillation of NMs. Two studies have compared the global transcriptional profiles to
investigate the pulmonary biological response after inhalation compared to instilled or
aspirated NMs. Inhalation and intratracheal instillation of a surface modified titanium
dioxide (TiO
2
) NP resulted in similar transcriptional changes, with the acute phase
response and inflammation as the most important pulmonary responses to inhaled and
instilled TiO
2
(Halappanavar et al. 2011;Husain et al. 2013). Similarly, Kinaret
et al.,
(Kinaret et al. 2017) compared the global transcriptomic profiles of lung tissue from mice
exposed to a straight and long multiwalled carbon nanotubes (MWCNT) by inhalation or
aspiration. The authors concluded that the perturbed pathways were very overlapping,
suggesting that the transcriptomic response to MWCNT exposure was very similar for
inhaled and pulmonary dosed MWCNTs.
Other studies compared levels of pulmonary inflammation, measured as neutrophil
influx, after exposure by inhalation or intratracheal instillation in rodents. Two studies
using MWCNT reported that both methods resulted in pulmonary inflammation, with
inhalation being more potent at inducing inflammation (Morimoto et al. 2012;Porter et al.
2013). Baisch
et al.,
reported that instillation of a high dose of TiO
2
NPs induced greater
inflammation compared to low dose rate delivery through inhalation, even though the
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same pulmonary deposited dose was delivered. The authors concluded that intratracheal
instillation is useful for quantitative ranking of NP hazards, but not for quantitative
hazard assessment (Baisch et al. 2014).
Selection of studies and endpoints
In the present report inhalation studies will be prioritised. For the description of
toxicological endpoints and mechanism of toxicity, studies using intratracheal
instillation for pulmonary deposition, will be included to support the findings from the
inhalation studies. Hazard assessments, however, are solely conducted based on
subchronic and chronic inhalation studies. Endpoints were evaluated based on reported
adverse effects of CB NM exposure in reports and in the scientific literature. Cancer and
cardiovascular disease have been identified as two of the main mortality causing
diseases in the world (World Health Organization 2018;Cancer Risks UK 2018). Both
diseases are potentially initiated by inflammation, as described in the section
Mechanism
of toxicity.
The critical endpoints were chosen based on literature wide review.
Pulmonary inflammation
Concerning inflammation there are a long range of CB NM inhalation studies and
intratracheal instillation addressing this endpoint. The studies considered most relevant
for OEL derivation are described below; and chronic and subchronic inhalation studies
are also presented in Table 5.
In a chronic inhalation study by Mauderly and co-workers, male and female rats were
exposed to CB NM by inhalation. The dosage regimen was a mass concentration of 2.5 or
of 6.5 mg/m
3
for 16 h/day, 5 days/week for 12 or 24 months. The CB NM was Elftex-12
furnace black from Cabot Corp. (MA, USA) (Mauderly et al., 1994). This CB NM has
been reported elsewhere to have a diameter of 37 nm (SCCS, 2015). Neutrophils were
measured in bronchoalveolar lavage fluid (BAL) in lungs after 12 months of exposure.
The mass concentration of 2.5 mg/m
3
was determined to be the lowest observed adverse
effect concentration (LOAEC) in terms of increased neutrophil numbers in BAL.
Other endpoints investigated in the study were survival, additional BAL fluid endpoints,
organ weight, non-neoplastic lesions as well as neoplastic lesions. Concerning survival,
the median survival in days was for females: Control 696 days, low CB NM 707, high CB
NM 675 (statistically significantly decreased); and for males Control 639 days, Low CB
NM: 605 (statistically significantly decreased) and High CB NM 599 (statistically
significantly decreased). Concerning other BAL fluid endpoints, lactate dehydrogenase
and beta glucoronidase were increased at both dose levels. Concerning lung weight, the
weight was increased for all CB NM dosed groups. By the authors of the report it was
suggested that this reflected the inflammatory, proliferative and fibrotic lesions resulting
from the exposure. Notably the relative lung weights (lung weight/body weight) were
not increased. Concerning non-neoplastic lesions there are a range of described effects at
time points ranging from 3 months of exposure until 6 weeks after the end of the
experiments (Table 5). Notably, although clearly increased as evaluated by looking at the
absolute numbers given in the appendix of the graph, no statistical analysis was
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included. Because of this and because of the presence of neoplastic lesions in another
pivotal study (as described below) we have not focused on non-neoplastic changes here.
Concerning sub-chronic inhalation studies and neutrophil inflammation there are a
range of relevant studies. Of these we consider two to be the most important (Driscoll et
al., 1996; Elder et al., 2005). Elder and co-workers, investigated CB NM Printex 90 (14
nm) in three species, mice, rats and hamster. The mass concentrations were 1, 7 or 50
mg/m
3
. The duration was 6h/day 5 days /week for 13 weeks. The animals were evaluated
at 5 weeks, and 13 weeks (end of exposure), and after recovery periods of 3 or 11 months
post exposure. The NOAEC for neutrophil numbers in BAL was at the end of exposure 1
mg/m
3
for rats, mice and hamsters. Other endpoints investigated were body weight, lung
weight, as well as histopathology. Only minimal effects were seen on body weight. Only
high dose in hamsters showed decreased body weight at the end of exposure. The lung
weight was increased at high dose in all three animal species, and also at the mid dose in
mice at the end of exposure. Regarding histopathology, the density of alveolar type II
cells was increased at high dose at the end of exposure but only for rats and hamsters.
The percentage of cells in the S –phase was increased at high dose at the end of exposure
but only in rats (Elder et al., 2005).
Driscoll
et al.,
investigated CB NM Monarch 880 (16 nm) in rats. The inhalation mass
concentration was 1.1, 7.1 or 52.8 mg/m
3
and the duration was 6h/day, 5 days/week for
13 weeks. The NOAEC for increased neutrophil numbers in BAL was 1.1 mg/m
3
. Other
investigated endpoints included hypoxanthine-guanine phosphoribosyltransferase
(Hprt) gene mutation frequencies in alveolar epithelial cells, which were increased at 7
and 53 mg/m
3
at the end of exposure (Driscoll et al., 1996) suggesting that the used CB
NM is mutagenic by inhalation.
Two other inhalation studies in rats also measured pulmonary inflammation by
increased neutrophil numbers in BAL. These were done with 12 weeks of exposure and
resulted in LOAECs of 3.5 and 10 mg/m
3
(Henderson et al., 1992; Wolff et al., 1990). In
addition, there is a range of intratracheal instillation studies in mice and rats that also
reported pulmonary inflammation by increased neutrophil numbers in BAL. These were
done with CB NMs in the size range of 10 to 100 nm. These exposures resulted in lowest
observed adverse effect levels (LOAELs) in the range of 0.02 to 16 mg/kg body weight
(bw) and no observed adverse effect levels (NOAELs) in the range of 0.2 to 3.3 mg/kg bw
(Bend et al., 2007; Bengtson et al., 2017; Bourdon et al., 2012; Chang et al., 2005; Chen et
al., 2015; Cho et al., 2010; Danielsen et al., 2010; Götz et al., 2011; Hadrup et al., 2017;
Husain et al., 2015; Jacobsen et al., 2015, 2009, Kyjovska et al., 2015a, 2015b; Li et al., 1999;
Lu et al., 2009; Renwick, 2004; Roberts et al., 2015; Saber et al., 2012b, 2012a; Saber et al.,
2016; Sager and Castranova, 2009; Schinwald et al., 2012; Shvedova et al., 2005; Shwe et
al., 2005; Teeguarden et al., 2011; Yang et al., 1999).
In summary; inhalation of CB NM induced long lasting inflammation in rats, mice and
hamsters. Taking the data on increased neutrophil numbers in BAL from both the
chronic and the two subchronic studies into account (Driscoll et al., 1996; Elder et al.,
2005; Mauderly et al., 1994) we suggest that a NOAEC is placed at 1 mg/m
3
. This is based
on a LOAEC of 2.5 in a chronic study and NOAECs of 1 and 1.1 mg/m
3
in two
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subchronic studies. We acknowledge that the finding of a LOAEC of 2.5 mg/m
3
could
warrant a somewhat lower NOAEC level, but that also depends on a chosen assessment
factor for using a LOAEC instead of a NOAEC. If this factor for example is set to 3
(ECHA, 2012) the LOAEC in the chronic study would give a NOAEC also of
approximately 0.8 mg/m
3
.
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2130134_0037.png
Table 5. Overview of non-neoplastic endpoints in chronic subchronic CB inhalation studies used for evaluation of pulmonary inflammation hazard
levels in the current report
Reference
CB NM
Animal species / Exposure
Endpoints/Results
(Mauderly et
Elftex-12
Rats: Inhalation
BAL neutrophils after 12 months of exposure. Effects were
al., 1994)
furnace black,
for 16 h/day, 5 days/week for up to 2 years (12 months
observed at both doses. Also, lactate dehydrogenase and beta
37 nm
for inflammation measurements), to 0, 2.5, or 6.5
glucuronidase were increased in both doses.
mg/m
3
Survival (median) in days: Females: Control: 696 days, CB NM
2.5 mg/m
3
: 707 days, CB NM 6.5 mg/m
3
: 675 days (statistically
Regarding recovery time after exposure the following
significantly decreased). Males: Control 639 days, CB NM 2.5
was stated in Mauderly
et al.
(Mauderly et al., 1994):
mg/m
3
: 605 days (statistically significantly decreased), CB NM
“The
exposures were terminated at 24 months, and the
6.5 mg/m
3
: 599 days (statistically significantly decreased).
remaining rats were transferred to an animal housing room
where they were maintained in shoebox cages with
Lung weight: Increased for all CB NM dosed groups. No effect
hardwood-chip bedding (Murphy Forest Products,
on relative lung weight.
Montville, NJ) until mortality reached approximately 90%.
Non-neoplastic lesions there are a range of described effects (as
All remaining rats in blocks A and B were killed and
compared to control) at time points ranging from 3 months of
necropsies were performed between March 21 and 25, and
exposure until 6 weeks after the end of the experiments.
between April 10 and 12, 1991, or 41 to 45 and 40 to 42
Lesions described to be related to CB exposure consisted of
days after
alveolar macrophage hyperplasia and alveolar epithelial
the end of the 24-month exposures, respectively.”
hyperplasia. Notably, although clearly increased as evaluated
by looking at the absolute numbers given in the appendix of
the graph, no statistical analysis was included
(Elder et al.,
CB NM
Mice, rats and hamster: Inhalation of
BAL neutrophils 1-day post 13 week exposure:
2005)
Printex 90 (14
1, 7 or 50 mg/m
3
. The duration was 6h/day 5 days
Rats: Increased numbers at 7 and 50, but not at 1 mg/m
3
nm)
/week for 13 weeks.
Mice and hamster: The effects were similar to those observed
The animals were evaluated at 5 weeks, and 13 weeks
in rats.
(end of exposure), and after recovery periods of 3 or 11
months post exposure
Body weight: Decreased for hamsters at high dose (50 mg/m
3
).
No effects were observed at other doses and no effects were
observed in mice and rats. Lung weight: Increased for all
species at high dose (50 mg/m
3
), and for mice at the mid dose 7
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2130134_0038.png
(Driscoll et al.,
1996)
36
CB NM
Monarch 880
(16 nm)
Rats. Inhalation of 1.1, 7.1 or 52.8 mg/m
3
. The duration
was 6h/day, 5 days/week for 13 weeks
mg/m
3
). Histopathology, showed increased density of alveolar
type II cells at high dose (50 mg/m
3
) for rats and hamsters.
Cells in the S –phase was increased for rats at high dose (50
mg/m
3
)
BAL neutrophils increased at 7.1 and 52.8 but not 1.1 mg/m
3
Hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene
mutation frequencies in alveolar epithelial cells: Increased at
7.1 and 52.8 but not 1.1 mg/m
3
 BEU, Alm.del - 2019-20 - Bilag 101: Orientering om NFA’s forslag til grænseværdier for fem kemiske stoffer, fra beskæftigelsesministeren
Genotoxicity and cancer
Genotoxicity and cancer are well-studied endpoints and possible adverse effects of
exposure to CB NM. Genotoxicity are often studied shortly after exposure, whereas
cancer is a more complex pathological endpoint that requires longer time to develop. In
the below section, the working group has chosen to differentiate between genotoxicity in
shorter-term studies and cancer in long-term studies.
Cancer
IARC has classified CB as
possibly carcinogenic to humans
(group 2B). This classification
was based on inadequate evidence for carcinogenicity in humans, but sufficient evidence
of carcinogenicity in experimental animals (IARC, 2010).
There are some CB NM inhalation studies (Heinrich et al., 1995; Mauderly et al., 1994),
and intratracheal instillation studies (Davis 1975, Rat; Dasenbrock 1996, Rat; Pott 1994,
Rat;) having genotoxicity and cancer as main endpoints. The two pivotal chronic
inhalation carcinogenicity studies are by Heinrich and co-workers using CB NM Printex
90 (14 nm) (Heinrich et al., 1995) and by Mauderly and co-workers using Elftex-12
furnace black (37 nm)(Mauderly et al., 1994). These will be addressed in detail below.
The Heinrich study (CB NM Printex 90)
Female rats were exposed to CB NM Printex 90 by inhalation (18 h/day, 5 days/week for
up to 24 months). Printex 90 has a diameter of 14 nm, surface area 337 m
2
/g and is
specified as >99% pure CB (SCCS, 2015). The mass concentration was 7.2 mg/m
3
for the
first 4 months and then 12.2 mg/m
3
for the next 20 months. This amounts to an average
exposure of 11.6 mg/m
3
for 104 weeks. The rats were investigated for lung tumour
incidence. The tested dose resulted in a significantly increased (tumour) incidence.
Details of this study are presented in Table 6. Other endpoints such as mortality, body
weight, organ weight and BAL endpoints were also analysed. It was shown that the
lifetime of the rats was significantly shortened by CB NM exposure as compared to
controls. The body weight was significantly reduced from day 300 for CB NM exposed
rats as compared to controls. The lung weight was increased by CB NM exposure.
Concerning BAL endpoints, differential cell count, and the concentration of lactate
dehydrogenase, beta-glucuronidase, OH-proline and total protein in BAL showed CB
NM exposure related effects. The BAL cellularity was not detailed further than in the
article.
Female mice were exposed to CB NM Printex 90 by inhalation. The duration was 18
h/day, 5 days/week for up to 13.5 months. It is noted by the working group of the current
report that 13.5 months is a relatively short study of carcinogenicity where guidelines
suggest 24 months. The mass concentration was 7.2 mg/m
3
for the first 4 months and
then 12.2 mg/m
3
for the next 9.5 months. This amounts to an average exposure of 10.7
mg/m
3
. The mice showed no increase in lung tumour incidence. Details of this study are
presented in Table 6. Other endpoints included mortality, body weight and lung weight.
Increased mortality was described in the mice as being 20% for CB NM exposed animals
as compared to 10% in the control group. However, it was not stated whether this was
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statistically significant. The body weight was decreased and the lung weight was
increased for the CB NM exposed mice as compared to controls (Heinrich et al., 1995).
The Mauderly study (CB NM Elftex-12 furnace black)
Female and male rats inhaled CB NM Elftex-12 furnace black (Cabot Corp. Boston, MA,
USA) at mass concentrations of 2.5 or 6.5 mg/m
3
for 16 h/day, 5 days/week for 12 or 24
months. Elftex-12 has a diameter of 37 nm, a Brunauer–Emmett–Teller (BET) surface area
43 m
2
/g and extractable organic material about 0.04–0.29%. The size was not reported in
the study (Mauderly et al., 1994), but has been described elsewhere in the literature (e.g.
(SCCS, 2015)). Mauderly and co-workers reported a Mass Median Aerodynamic
Diameter of 101 nm as measured by small fraction parallel flow diffusion battery and a
Mass Median Aerodynamic Diameter of 2000 nm as measured by large fraction cascade
impactor (Mauderly et al., 1994). Neoplasms in lungs of female rats were found with
“statistical significance“. The lowest dose with a significant increased tumour incidence
was 2.5 mg/m
3
. There was no carcinogenic effect in male rats. Details of this study are
presented in Table 6. Inflammatory effects, also observed by Mauderly and co-workers
are described above in the section “Inflammation” (Mauderly et al., 1994). The same
results of Mauderly et al., which is a report, is also reported in the article (Nikula et al.,
1995).
Summary
In summary, Heinrich
et al.,
found significantly increased carcinogenicity at 11.6 mg CB
NM/m
3
in female rats whereas Mauderly
et al.,
found significantly increased
carcinogenicity at 2.5 mg CB NM/m
3
in female rats. This was the lowest tested dose in
both studies. Heinrich and co-workers additionally conducted a negative cancer study in
mice exposed for 10.7 mg/m
3
, however, this exposure was only for 13.5 months. It is also
noted that Mauderly
et al.
did not find an effect in male rats. CB has been classified as
possibly human carcinogen by IARC (group 2B) based on sufficient evidence of
carcinogenicity in experimental animals (IARC, 2010).
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2130134_0041.png
Table 6. Overview of chronic rat inhalation studies
Reference
CB NM
Animal species / Exposure
(Heinrich et al.,
Printex 90, 14
Rats (only females were investigated):
1995)
nm
18 h/day, 5 days/week for up to 2 years to 0, or 11.6
mg/m
3
(average exposure over the 2-year period)
followed by 6 months without CB NM exposure.
Mice (only females were investigated):
18 h/day, 5
days/week for up to 13.5 months (the animals were
kept at clean air for an additional 9.5 months) to 0, or
10.7 mg/m
3
(average exposure over the 13.5 month
period)
followed by 6 months without CB NM exposure
Lung tumour incidence
Rats:
11.6 mg/m
3
: Increased (p<0.001 by Fischer’s exact test
done by the authors of the current report)
Exposed: 39/100: Total number with tumours
20/100: Keratinizing cystic squamous-cell tumours
4/100: Squamous cell carcinoma
13/100: Adenoma
13/100: Adenocarcinoma
Controls: 1/217: Total number with tumours
1/217: Adenocarcinomas
Mice:
10.7 mg/m
3
: There was no carcinogenic effect
2.5 mg/m
3
:
Increased in females (p<0.01 by Fischer’s exact test
done by the authors of the current report).
Female rats:
8/107: Malignant or benign lung neoplasms
7/107: Malignant lung neoplasms
2/107: Adenoma
6/107: Adenocarcinoma
1/107: Mixed mesenchymal and epithelial type tumour
Male rats:
2/106: Malignant or benign lung neoplasms
1/106: Malignant lung neoplasms
1/106: Adenoma
1/106: Adenocarcinoma
(Mauderly et
al., 1994)
Elftex-12
furnace black,
37 nm
Rats (female and male): Inhalation
for 16 h/day, 5 days/week for up to 2 years, to 0, 2.5, or
6.5 mg/m
3
Regarding recovery time after exposure the following
was stated in Mauderly
et al.
(Mauderly et al., 1994):
“The
exposures were terminated at 24 months, and the
remaining rats were transferred to an animal housing room
where they were maintained in shoebox cages with
hardwood-chip bedding (Murphy Forest Products,
Montville, NJ) until mortality reached approximately 90%.
All remaining rats in blocks A and B were killed and
necropsies were performed between March 21 and 25, and
between April 10 and 12, 1991, or 41 to 45 and 40 to 42
days after the end of the 24-month exposures, respectively.”
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2130134_0042.png
40
6.5 mg/m
3
:
Increased in females (p<0.001 by Fischer’s exact test
done by the authors of the current report).
Female rats:
28/105: Malignant or benign lung neoplasms
21/105: Malignant lung neoplasms
13/105: Adenoma
20/105: Adenocarcinoma
1/105: Squamous cell carcinoma
1/105: Adenosquamous carcinoma
Male rats:
4/106: Malignant or benign lung neoplasms
4/106: Malignant lung neoplasms
1/106: Adenocarcinoma
2/106: Squamous cell carcinoma
1/106: Adenosquamous carcinoma
Controls:
Female rats:
0/105: Malignant or benign lung neoplasms
Male rats:
3/109: Malignant or benign lung neoplasms
2/109: Malignant lung neoplasms
1/109: Adenoma
1/109: Adenocarcinoma
1/109: Squamous cell carcinoma
 BEU, Alm.del - 2019-20 - Bilag 101: Orientering om NFA’s forslag til grænseværdier for fem kemiske stoffer, fra beskæftigelsesministeren
Genotoxicity
The genotoxic potential of CB NM exposure has been tested in several
in vivo
studies.
Therefore, we have chosen not to include
in vitro
or
ex vivo
exposures in this section.
Several of these are, however, included in the section “Mechanism of toxicity” where we
also discuss evidence for a non-threshold mechanism for genotoxicity and
carcinogenicity. The endpoints within this section include deoxyribonucleic acid (DNA)
strand breaks, DNA adducts and
Hprt
mutation. It is a general consensus that exposures
causing permanent changes to the DNA, e.g. mutations are also carcinogenic. The same
strong association has not yet been documented for DNA strand breaks measured by the
comet assay. Comet assay is a popular assay in the area of nanotoxicology (Karlsson,
2010), but based on the transient nature of the detected damage to DNA and the lack of a
clear link to carcinogenesis it will bear less weight as also discussed in a recent review
(Møller and Jacobsen, 2017). However, genotoxicity detected by the comet assay is
always a course for concern and should merit further studies clarifying the results.
Mutations have been detected in rat pulmonary alveolar epithelial cells following
inhalation exposure 6h/day, 5 days/week for 13 weeks to 7.1 and 52.8 mg/m
3
CB NM
(Monarch 880, 16 nm, 220 m
2
/g, Cabot Corp., MA, USA). The lung burdens were
estimated to 1826 and 7861 mg, respectively.
Hprt
mutation frequency was significantly
increased immediately after the 13 weeks of inhalation (7.1 and 52.8 mg/m
3
) but also
after a 3- and 8-month recovery period (52.8 mg/m
3
). For the high exposure dose the
increase in
hprt
mutation frequencies were 4.3-, 3.2-, and 2.7-fold greater than the air
control group, immediately after exposure and 3- and 8-months after exposure,
respectively. No adverse effects were detected following inhalation of 1.1 mg/m
3
(deposited dose 354 mg) (Driscoll et al., 1996). Instillation of a much smaller dose 0.2 mg
Printex 90/animal did not lead to any increase in mutant frequencies in the lungs of the
gpt
delta transgenic mouse model (Totsuka et al. 2009).
Rats inhaled CB NM Printex 90 (14 nm; 300 m
2
/g) or CB NM Sterling V (70 nm; 37 m2/g)
6h/day 5 days/week for 13 weeks. The mass concentration of CB NM Printex 90 was 1, 7
or 50 mg/m
3
whereas it was 50 mg/m
3
for CB NM Sterling V. Exposure to CB NM Printex
90 resulted in the formation of 8-Oxo-2'-deoxyguanosine (8-oxo-dGua) in lungs at the
highest dose immediately following the exposure period, and at both the high and
middle dose following a 44-week recovery period. No effect was observed at 1 mg/m
3
.
There was no effect of CB NM Sterling V, despite a retained particle surface area
comparable to the middle dose of Printex 90 (Gallagher 2003, Rat). Using very low
exposure doses (0.16 mg/m
3
for rats and 0.14 mg/m
3
for mice) for 4 h (rats) or 4 and 3x4 h
(mice) Wessels and co-workers did not detect oxidative DNA damage by the
formamidopyrimidine DNA glycosylase (FPG) modified comet assay in lung tissue of
pulmonary epithelial cells (Wessels et al., 2011).
Danielsen and co-workers exposed male rats orally to 0.64 mg/kg CB NM Printex 90. A
significant increase in 8-oxo-dGua was observed in liver, but not lung. No increase in
bulky PAH-DNA adducts were observed 24 h post exposure in lung or liver tissue
(Danielsen et al., 2010). Analysis for DNA adducts formed by CB NM exposure have
been examined in a few studies. Borm and co-workers tested Printex 90 (1, 7 and 50
mg/m
3
) and Sterling V (50 mg/m
3
) in a 13-week rat inhalation study (particles as
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mentioned in the above section). No PAH-DNA related adducts were observed in the
lung tissue compared to sham exposed rats (Borm et al., 2005). A lack of bioavailability
of surface PAHs was suggested due to tight adhesion. One study has reported a
significant increased level of DNA adducts in isolated rat type II alveolar cells when
compared to the filtered-air controls was reported (Bond et al. 1990). The rats were
exposed to 6.2 mg/m
3
(16 h/day, 5 days/week) for 12 weeks. The same material (CB NM
Elftex-12, Cabot Corp. Boston, MA, USA) tested negative for induction of DNA adducts
in another rat inhalation study (7 h/day, 5 days/week for 12 weeks at 10.0 mg/m
3
) (SCCS,
2015).
There is a range of inhalation and intratracheal instillation studies in which CB NM
induced DNA strand breaks using comet assay were examined. Mice inhaled CB NM
Printex 90 (aerosol agglomerate size was 65 nm) at a mass concentration of 20 mg/m
3
and
the total duration was 6 h distributed over 4 days. DNA strand breaks were detected in
BAL cells. Tissues were not analyzed (Saber et al., 2005). Pregnant mice inhaled CB NM
Printex 90 at a mass concentration of 42 mg/m
3
. The duration was 1 h on each of
gestation days 8 to 18; 11 h in total. This resulted in elevated DNA strand breaks damage
in the liver as measured by comet assay at 3 days of exposure. DNA damage was not
increased in BAL cells. Other tissues were not examined (Jackson et al., 2012a).
Concerning intratracheal instillation studies in mice and rats there are a range of studies
that report effects in the comet assay following CB NM Printex 90 exposure at doses in
the range of 0.02 for to 8.1 mg/kg bw (Bengtson et al., 2017; Bourdon et al., 2012; Hadrup
et al., 2017; Husain et al., 2015; Kyjovska et al., 2015a, 2015b; Saber et al., 2012a).
However, there are also studies in which no effect was observed using the same Printex
90 and
Lampblack 101
in the dose range of 0.6 to 8.1 mg/kg bw (Danielsen et al., 2010;
Jackson et al., 2012a; Saber et al., 2012b; Saber et al., 2012c; Saber et al., 2016, 2005).
In summary, the present working group concludes that there is clear evidence for
genotoxicity of CB NM. Above-mentioned
in vivo
studies show that CB NM can induce
mutations, oxidative damage to DNA as well as DNA strand breaks in rats and mice. It
is clear that inflammation is closely linked to genotoxicity via secondary cell driven
production of reactive oxygen species (ROS). Primary and secondary particle effects can
be challenging to separate within
in vivo
studies; however, the present working group do
find support for primary production of ROS could have some importance in the
genotoxicity of CB NM. This primary genotoxic effect does not include DNA bulky
adducts. Although a limited number of materials have been tested regarding adduct
formation, it is noteworthy that Sterling V, a dirty CB material containing high level of
PAHs, is amongst those. Therefore, this working group does not find sufficient evidence
for a direct acting mechanism via bulky PAH-DNA adduct formation.
Cardiovascular effects
The term cardiovascular effects cover all pathological changes in the entire circulatory
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
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this can lead to various other cardiovascular diseases, including coronary/ischemic heart
disease and stroke.
Only few studies have investigated the cardiovascular effects of pulmonary CB
NM exposure and some of these have investigated the acute phase response; a
promising early biomarker for cardiovascular disease. Below is a brief overview
of the scientific literature touching on the subject of CB NM-induced
cardiovascular effects.
Cardiac physiology
Concerning inhalation studies, mice were exposed to CB NM by inhalation of 0.55 mg/m
3
for 3 consecutive days (2 h/day). The CB NM aerosol was characterised by a count
median diameter of 0.7 µm and a mass median aerodynamic diameter of 1.0 µm. The
size and commercial name of the CB NM were not reported. CB NM exposure
demonstrated considerable (although some inconsistent) changes in the associations
between breathing and cardiovascular responses describing heart function (Hamade et
al., 2010). The relevance of the finding in this study in connection to the derivation of an
OEL is currently unclear and further investigation would be needed to clarify this. Rats
were exposed by inhalation to CB NM (86 nm). The particle exposure concentration was
1.3 × 10
5
, 6.2 × 10
5
, or 4.2 × 10
6
particles/cm
3
. Using a density of 1.7 g/mL this corresponds
to ~0.07, 0.3 and 2.4 mg/m
3
, respectively. The duration was 4 h/day, 5 days/week for 4
weeks. There was no effect on plasma coagulation, platelet aggregation, or on vasomotor
function (Kim et al., 2011).
Mice were exposed to ultrafine CB NM (Hiblack 41Y, 19 nm) by intra-tracheal instillation
(every 2 days for a total of 3 times) at 0.05, 0.15 and 0.6 mg/kg bw. At the two highest
doses heart rate variability indices were decreased. At the highest dose slight pulmonary
inflammation and myocardial injury were observed (Jia et al., 2012). Rats were exposed
to CB NM (N330, 28-36 nm; N990, 250-350 nm) by intratracheal instillation at 1, 3, or 10
mg/kg bw. No cardiac symptoms were detected by electrocardiographic endpoints
describing different measures of heart function and heart rate variability (Kim et al.,
2012).
Accelerated plaque progression and vascular dysfunction
The lipid profile of mice significantly differs from that of humans. Mice do not develop
atherosclerosis, because 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
apolipoprotein E knockout (ApoE
-/-
) mice, which are deficient in apolipoprotein E
(apoE), a glycoprotein associated with all lipoproteins except LDL. ApoE
-/-
mice develop
spontaneous atherosclerosis as early as 3–4 months of age when fed normal chow
(Nakashima et al. 1994). This makes them suitable for investigating cardiovascular
effects.
Regarding studies in knockout mice, ApoE
-/-
mice were exposed to CB NM Printex 90 (14
nm) by intratracheal instillation at 8.5 or 26 µg/mouse (~0.4 or ~1.3 mg/kg bw). CB NM
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exposure was not associated with promoted plaque progression in aorta or
brachiocephalic artery; and there was no effect on
Saa3
messenger ribonucleic acid
(mRNA) levels in lung. In contrast, plasma from CB NM exposed mice caused
vasoconstriction in aorta rings isolated from naïve mice (Christophersen et al., 2016).
ApoE
-/-
mice were exposed to CB NM (Printex 90, 14 nm) by intratracheal instillation.
Two consecutive CB NM doses each of 0.5 mg/kg bw caused decreased acetylcholine-
induced vasorelaxation in aorta segments. This was not observed following single doses
in the range of 0.05 to 2.7 mg/kg bw. CB NM exposure did not affect the progression of
atherosclerotic plaques in aged ApoE
-/-
mice. Moreover, CB NM did not affect vascular
cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1)
expression and did not affect the 3-nitrotyrosine levels in the vascular tissue of either
young or aged ApoE
-/-
mice (Vesterdal et al., 2010). Low density lipid receptor (LDLr)
-/-
Mice were exposed to CB NM by intratracheal instillation 1 mg/week for 10 weeks. This
was done in the presence or absence of 0.51% cholesterol diet. The CB NM had an
agglomerate size of 121 nm in diameter; no manufacturer name was given, and the total
dose given over 10 weeks was 500 mg/kg bw. CB exposure resulted in accelerated
development of atherosclerosis in mice receiving a high-cholesterol diet as compared to
control mice on cholesterol diet (Niwa et al., 2007).
Acute phase response
The acute phase response is an early defence system induced in e.g. humans in response
to e.g. infection, infarction, inflammation and trauma. 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 Kushner, 1999). Elevated plasma levels of CRP
and SAA are intimately linked to risk for cardiovascular disease in both epidemiological
(human) and animal studies (Johnson et al., 2004; Lowe, 2001; Mezaki et al., 2003; Ridker
et al., 2000). In mice, the four 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). In a recent review,
evidence for a close correlation between the deposited surface area of nanoparticles, the
generated pulmonary inflammation (neutrophil influx) and the resulting level of acute
phase response was presented (Saber et al., 2014). These results implied that even
smaller elevations of the inflammatory state have an influence on the risk for
cardiovascular disease.
Several studies have examined changes in
Saa
expression. Mice were exposed to CB NM
Printex 90 by inhalation. The mass concentration was 20 mg/m
3
and the duration was
1.5h/day for four consecutive days. There was no evidence of the induction of an acute
phase response in the livers of the mice. However, when considering this result the short
exposure time should be taken into account (Saber et al., 2009). Mice were exposed to CB
NM by intratracheal instillation of 0.67, 2, 6, or 162 µg Printex 90 CB NM (~0.04, 0.1, 0.3
and 8.1 mg/kg bw). There was only increased
Saa3
mRNA levels in lung at 8.1 mg/kg bw
(Kyjovska et al., 2015a). Mice were exposed to CB NM, Printex 90 by intratracheal
instillation at a dose of 162 µg/mouse (8.1 mg/kg bw). The
Saa3
mRNA level was
increased in lung at all three investigated time points 1, 3 and 28 days (Kyjovska et al.,
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2015b). Mice were exposed to CB NM at 18, 54 or 162 µg/mouse by intratracheal
instillation and humanely killed at 1, 3 or 28 days of recovery (~0.9, 2.7 and 8.1 mg/kg
bw). CB NM exposure resulted in increased expression of
Saa3
mRNA in lung tissue on
day 1 (all doses), 3 (all doses) and 28 (middle and high dose), but not in liver (Bourdon et
al., 2012).
Other cardiac endpoints
Mice were intratracheally instilled with 162 µg CB NM Printex 90 (~8 mg/kg bw) and
humanely killed at 1, 3 or 28 days post exposure. CB NM exposure was associated with
increased plasma levels of markers of endothelial inflammation (soluble-E-selectin,
soluble-ICAM-1, soluble-VCAM-1) and total PAI-1. CB NM exposure did not alter
cardiac gene expression as measured by gene array. It was concluded that CB NM
exerted adverse cardiovascular effects, in absence of changes in cardiac tissue gene
expression (Bourdon et al., 2013a). Mice were exposed to CB NM by oropharyngeal
aspiration at doses of 10 or 40 µg (~0.5 or ~2 mg/kg bw). The size of the CB NM ranged
from 71 to 96 nm as measured by dynamic light scattering (DLS). Twenty-four hours
after the instillation, the animals had their hearts perfused and excised. A 20 min period
of experimental cardiac ischemia was applied to the perfused hearts. There was no effect
of CB NM exposure on cardiac functional recovery, infarct size, and coronary flow rate
in the isolated perfused hearts (Tong et al., 2009). Rats were exposed to CB (N330, 28-36
nm; N990, 250-350 nm) by intratracheal instillation at 1, 3, or 10 mg/kg bw. N330 caused
accelerated platelet-dependent blood clotting at the highest dose. Both particles caused
prolonged activated partial thromboplastin time but only at the mid doses. No effect was
observed on prothrombin time (a measure of coagulability, the time it takes plasma to
clot after addition of tissue factor) (Kim et al., 2012).
In summary; one of the above studies on cardiovascular toxicity study finds effects at
0.55 mg CB NM/m
3
(2 h/day for 3 days). Considerable changes in heart function response
were observed. However, as the reported changes were not consistent, this study group
does not consider these effects to be suitable for OEL derivation. The other inhalation
study was performed using a mass concentration of 20 mg/m
3
, a much higher level
compared to studies on inflammation described above.
Concerning studies with intratracheal instillation, the above described studies reported
that doses having effect were in the range of 0.15 mg/kg bw to 500 mg CB NM/kg bw. A
NOAEL based on these intratracheal studies would be set to 0.05 mg/kg (bw). This
NOAEL could be hypothesised to translate to a mass concentration of ~0.7 mg/m
3
with a
deposition fraction of 50% and using this NOAEL as a NOAEL per day. However,
inhalation studies are deemed of higher importance than intratracheal instillation
studies. The NOAEC in inflammation studies (described above) was 1 mg/m
3
, a value
not far from a potential LOAEC of 0.55 mg/m
3
from the above described inhalation
study. Taken together, the present working group find that the data on cardiovascular
toxicity caused by CB NM are not strong enough to lower the suggested NOAEC of 1
mg/m
3
for pulmonary inflammation.
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Reproductive toxicity
Time mated female mice were exposed to CB NM Printex 90 either 1) by inhalation of 42
mg/m
3
for 1 h/day on gestation days 8-18; or 2) by intratracheal instillation to CB NM
Printex 90 on gestation days 7, 10, 15 and 18 at cumulative doses of 11, 54 and 268 µg CB
NM/mouse (equal to 0.55, 2.7 and 13.4 mg/kg bw). CB NM exposed mothers exhibited
persistent lung inflammation at 24-27 days after exposure for both administration routes,
albeit only at the highest intratracheal instillation dose. CB NM exposure did not affect
gestation or lactation. DNA strand breaks were increased in the liver of mothers and
their offspring following inhalation exposure only (Jackson et al., 2012a). Offspring from
the highest dose level of intratracheal instillation were subsequently tested in the open
field test. Female offspring showed an altered habituation pattern (Jackson et al., 2011).
Furthermore, DNA microarray was performed on tissues from male and female
offspring from the highest intratracheal instillation level. Liver was recovered on
postnatal day 2 and from dams 26-27 days after exposure. Maternal instillation exposure
to CB NM changed expression of several genes, significantly more in female compared
to male offspring, indicated that female offspring were more sensitive than male
offspring in regard to changes in liver mRNA levels. Affected pathways in female
offspring included: cellular signalling, inflammation, cell cycle regulation and lipid
metabolism. In males there were subtle changes in metabolism-related genes (Jackson et
al., 2012b). Finally, female and male F1-offspring were raised to maturity and
subsequently mated with unexposed animals. Testicles were collected from mature F1-
and F2-males. F2-offspring, whose fathers were prenatally exposed to CB NM Printex 90,
showed lowered sperm production (Kyjovska et al., 2013). The expanded simple tandem
repeat germline mutation rates were not different in CB NM-exposed F2 female
offspring as compared to controls (Boisen et al., 2013).
Pregnant mice were exposed to CB NM by intranasal instillation on gestational day 5
and 9 at a cumulative dose of 190 µg/kg bw. Splenocyte phenotypes as well as cytokine
mRNA levels were determined at day 1, 3, 5, and 14 days postpartum. In the CB NM
group there was a decrease in numbers of CD3
+
, CD4
+
and CD8
+
cells in the spleen from
1, 3 and 5-day-old offspring. In addition, CB NM exposure was associated with an
increased Interleukin-15 mRNA level in the spleen of new-born male offspring; and
increased Ccr7 and Ccl19 mRNA levels of female offspring spleen. The authors conclude
that exposure of pregnant mice to CB NM partially suppressed the development of the
offspring immune system (Shimizu et al., 2014). In another study by the same research
group, pregnant mice were exposed to CB NM using the same dosing regimen. CB NM
increased total thymocyte count and certain specific immunophenotypes (CD4
-
CD8
-
and
CD4
+
CD8
+
cells). In addition, an increase in total lymphocytes, and CD4
-
CD8
-
cells was
observed. The authors conclude that that maternal respiratory exposure to CB NM
during middle and late gestation may have allergic or inflammatory effects in male
offspring (El-Sayed et al., 2015). Several studies have used this exposure regimen and
investigated brains of the male offspring 6 to 12 weeks after birth. CB NM exposure was
generally associated with enlarged granules in perivascular macrophages and a decrease
in the number of periodic acid Schiff positively stained perivascular macrophages.
Furthermore, the expression level of glial fibrillary acidic protein was increased in
astrocytes, indicative of long-term activation of astrocytes and reactive astrogliosis. In an
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exposure-effect study, using cumulative doses of 5.8, 30 and 146 µg/kg body weight
(bw), 5.8 µg/kg (bw) could be identified as the NOAEL. The authors conclude that
maternal CB NM exposure is associated with a decrease in
normal
perivascular
macrophages, and the changes in expression of glial fibrillary acidic protein in astrocytes
are indicative of long-term activation of astrocytes and reactive astrogliosis in a wide
area of the CNS of the offspring. Overall, CB NM maternal exposure may increase the
risk for neurological changes in the offspring (Onoda et al., 2017b, 2017a, 2017c, 2014).
These latter outcomes have also been investigated following inhalation exposure. Time
mated mice were exposed for 45 min/day to 0, 4.6 or 37 mg/m
3
aerosolized Printex 90 on
gestation days 4–18, i.e. for a total of 15 days. No lung inflammation was observed in the
exposed females, when measured 11 or 28 days post-exposure. In the offspring, glial
fibrillary acidic protein expression levels were dose-dependently increased in astrocytes
in the cerebral cortex and hippocampus at six weeks of age, as also described above for
the instillation studies. Lysosomal granules were also enlarged in the brain perivascular
macrophages. Assessment of behaviour in the open field test at 90 days of age showed
dose dependent alterations, with prenatally exposed female offspring moving a longer
distance and males spending longer time in the central zone of the maze. Finally, the
number of parvalbumin-positive interneurons and the overall expression level of
parvalbumin were assessed at the highest dose level. Both were decreased in the motor
and prefrontal cortices at weaning and 120 days of age in prenatally exposed compared
to control offspring. The authors conclude that the effects are similar to those observed
after instillation exposure and furthermore that some of the observed effects resemble
those observed in mouse models of neurodevelopmental disorders (Umezawa et al.,
2018).
In summary, the present working group finds that the experimental studies indicate
possible adverse reproductive effects such as genotoxicity in liver and suppressed
development immune system in the offspring; to the least such an effect cannot be
excluded. It should, however, be underlined that the available data is far too limited and
does not yet include any pulmonary exposure guideline studies. Overall, within the
above studies on reproductive toxicity, effects were observed at exposure to 4.6 CB
NM/m
3
for 45 min /day. A NOAEL as low as 5.8 µg/kg (bw) was observed following
intranasal instillation exposure, and with a hypothetical deposition fraction of 50% this
could be translated to a mass concentration of 0.07 mg/m
3
. However, inhalation studies
are deemed of higher importance than intratracheal instillation studies. When using
intratracheal instillation a bolus dose is given. This results in a very high dose rate and
the importance of dose rate for inflammation and translocation to sites for reproductive
toxicity is unknown. The lowest observed LOAEC/LOAEL is following intratracheal
instillation. This taken together with the LOAECs and NOAECs observed for neutrophil
accumulation in BAL in the inhalation studies, described above, leads us to suggest that
reproductive toxicity is not the critical effect in the current report. This, however, may
change if experiments with reproductive endpoints with lower inhalation mass
concentrations of CB NM are undertaken in the future.
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Other toxicological endpoints
Following the overview of the literature, the present working group suggests that it is
highly likely that the critical endpoint of pulmonary exposure to CB NM is to be
identified among inhalation studies and the toxicity endpoints described above
(inflammation, genotoxicity and cancer, cardiovascular toxicity, reproductive toxicity).
However, to ascertain that no toxicological endpoints had been overlooked, a literature
search was done in the PUBMED database on the following: “Carbon black AND
sensitisation”, “Carbon black AND neurotoxicity AND inhalation”, “Carbon black AND
liver AND toxicity AND inhalation” and “Carbon black AND kidney AND toxicity AND
inhalation”. No relevant articles were identified following any of these searches.
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M
ECHANISMS OF TOXICITY
Pulmonary inflammation, genotoxicity and cancer
Toxicity of CB NM depends on the pulmonary deposition dose which is expected to
follow traditional fractional deposition patterns as described in the literature (Jacobsen et
al., 2009; Oberdörster et al., 2005). Pulmonary exposure to CB NM has consistently been
shown to cause dose-dependent pulmonary inflammation, with close correlation to
deposited surface area and inverse correlation to particle size (Bourdon et al., 2013b;
Elder et al., 2005; Saber et al., 2012b; Stoeger et al., 2007, 2006). One study has
demonstrated that this correlation is also valid using 6 finely tuned carbon materials all
within a narrow nano size range (primary particle size 10-50 nm and specific surface area
30-800 m
2
/g)(Stoeger et al., 2007, 2006).
Based on this, the present working group concludes that inhalation of CB NM induces
dose dependent pulmonary inflammation and that neutrophil influx is predicted by the
total surface area of deposited particles. In addition, the present working group
considers inflammation as a threshold effect.
The International Agency for Research on Carcinogenicity (IARC) has classified CB NM
as possibly carcinogenic to humans (group 2B) based on sufficient evidence of
carcinogenicity in experimental animals and
inadequate evidence
in humans for the
carcinogenicity of CB NM. In the evaluation, IARC does not distinguish between CB and
CB NM (IARC 2010). One pathway towards cancer suggested by IARC followed the
following line of events: Inhalation and deposition leading to inflammation with cell
injury and proliferation. This could directly cause mutations and carcinogenesis. A
second described pathway depended on increased levels of ROS leading to mutations
and carcinogenesis. The importance of inflammation in carcinogenesis of CB NM is
supported by the observation that 13 weeks of inhalation of CB NM only resulted in
mutations in the lung epithelium at doses that caused inflammation (7.1 and 52.8
mg/m
3
). A lower dose (1.1 mg/m
3
) did not result in inflammation or mutations (Driscoll
et al., 1996); this supports the idea of a threshold effect.
Several studies have shown that CB NM particles generate very high levels of ROS in a
concentration-dependent manner in both acellular and cellular tests (Folkmann et al.,
2009; Foucaud et al., 2007; Høgsberg et al., 2013; Jacobsen et al., 2008b; Koike and
Kobayashi, 2006; Saber et al., 2012b; Wilson et al., 2002). This opens for the possibility for
a primary and directly particle-driven imbalance in the oxidative stress defence. This
will be elaborated further below.
Secondary genotoxicity due to particle-induced inflammation and activated phagocytes
is another important and well-documented mechanism of action for the development of
lung cancer. It has previously been documented that exposure to CB NM may result in
damage to DNA as e.g. measured by the comet assay. Increased levels of DNA strand
breaks caused by CB NM exposure have been observed both
in vitro
and
in vivo
in BAL
cells and lung and liver tissue (Bourdon et al., 2012; Jackson et al., 2012a; Jacobsen et al.,
2009, 2007; Saber et al., 2005). The oxidatively damaged DNA lesions detected in CB NM
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Printex 90-exposed cultures encompass 8-oxo-dGua as well as ring-opened formamido-
pyrimidine lesions. These were detected in the formamidopyrimidine DNA glycosylase
(FPG) modified comet assay (Jacobsen et al., 2007). Increasing the exposure duration has
shown that long-term non-cytotoxic exposures to CB NM Printex 90 are associated with
a modest, but statistically significant increase in the
cII
and
lacZ
mutation frequency in
FE1-Muta
TM
Mouse cells (Jacobsen et al., 2007). The level of mutations was similar to that
observed following exposure to the standard reference material (SRM) 1650; a standard
reference diesel exhaust particle from a heavy-duty truck available from the National
institute for standards and technology (Jacobsen et al., 2008a).
The CB NM induced mutation spectrum was found to be in line with a fingerprint of
oxidative damage to DNA. E.g. the most frequent mutation was G:C→T:A transversions
that is likely caused by formation of 8-oxo-dGua mispairing with dA during replication.
It was suggested that the observed spectrum of CB NM Printex 90-induced mutations
was a direct consequence of oxidative damage to DNA, which in turn is a consequence
of the high ROS production (Jacobsen et al., 2008a).
Rats were exposed to saline or saline suspensions of 10 and 100 mg/kg bw of CB NM by
intratracheal instillation. Fifteen months after exposure, neutrophilic inflammation was
detected in all CB NM exposed rats. Additionally,
Hprt
mutation frequency was
increased in alveolar type II cells and epithelial hyperplasia was observed in the high
exposure group (Driscoll et al., 1997).
The cell mediated secondary genotoxicity was examined by exposing RLE-6TN cells to
macrophage or neutrophil enriched BAL cells from rats treated with 100 mg/kg bw of CB
NM. Both exposures (macrophages or neutrophils) increased
Hprt
mutation frequency;
however, the mutagenic activity appeared greatest for neutrophils. Addition of catalase
to the BAL cell exposures:RLE-6TN co-cultures inhibited the increase in
Hprt
mutation
frequency (Driscoll et al., 1997) suggesting a mechanism via oxidants and hydrogen
peroxide. Overall, the study suggests that exposures generating significant inflammation
are associated with increased level of mutations in epithelial cells.
However, inflammation alone does not always result in genotoxicity. Mice exposed for
one of 3 NM (TiO
2
, CeO
2
or CB NM; Printex 90) showed a strong and very similar
inflammatory response. All 3 materials translocated and were detected in the livers.
None of the materials caused increased genotoxicity at day 1. However, only the CB NM,
which was also the only NM to produce ROS, caused an increased level of DNA strand
breaks and this was observed after 1 month and 180 days (Modrzynska et al., 2018).
Particulates generated by combustion processes are often complex mixtures of organic
compounds and smaller levels of metals adhered to a carbon core. The same is true for
CB NM products, although the carbon content is high with normally only smaller traces
of other substances (Bingham and Cohrssen, 2012). It has been suggested that since CB
NM normally contains small amounts of PAHs (compared to soot) and desorption
occurs slowly and to a very low degree, the adverse health effects are associated with the
insoluble particle core (Borm et al., 2005; Jacobsen and Clausen, 2015).
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Non-threshold carcinogenic effect
In a recent evaluation of the genotoxicity of CB (Chaudhuri et al., 2018) the authors argue
that the apparent lack of DNA-PAH adducts following CB exposure strongly supports
the view of no direct interaction between DNA and CB. The general statement of no
PAH adducts is correct and is based on the lack of DNA-adduct formation in the lungs
of rats in several studies (Borm et al., 2005; Gallagher et al., 1994; Wolff et al., 1990). Bond
and co-workers did observe DNA adduct formation in rat alveolar type II cells, in a
similar inhalation experiment and using the same material as Wolff and co-workers
(Bond et al., 1990). Also, DNA-PAH adducts were analysed in A549 lung epithelial cells
following exposure to CB NM (Printex 90, Lampblack 101, N330 or Sterling V). Only
Sterling V, the CB NM with the highest PAH content, showed adduct spots using the
32
P
post-labelling detection method (Borm et al., 2005). However, the bioavailability of CB
surface PAH has been questioned several times. In general, removing such PAH requires
very harsh chemical treatments. E.g. Borm and co-workers did not observe leakage of
PAHs from CB particles when shaken in a range of saline/dipalmitoyl-phosphatidyl-
choline concentrations (24 h at 37°C in the dark) (Borm et al., 2005). Combined, the above
results could indicate no direct DNA interaction, but they could also indicate a low
bioavailability of PAH for the majority of CB NM. However, the present working group
see several evidences in favour of a non-threshold mechanism, thus supporting the latter
statement.
As detailed described above, CB NM has been shown to induce ROS in acellular and
cellular assays. The ROS induction is proportional to the specific surface area of the
carbon black particles (Saber et al., 2012b). ROS would be expected to cause oxidative
DNA damage (Møller et al., 2015) and DNA strand breaks, but not bulky DNA adducts.
It has been shown several times that CB NM can enter cell cytosol. E.g. transmission
electron microscopy of 16HBE cells exposed to CB NM (13 and 21 nm) for 24h showed
aggregates of NMs present either inside endosomes. About 80% of analysed cells
contained NM. Uptake was additionally supported by flow cytometry which showed a
dose dependent uptake of CB NM (Hussain et al., 2009). However, a central question is
whether CB NM enters the cell nuclei to exert a potential primary and direct genotoxic
effect. In this regard, it has been demonstrated
in vitro
that oxidised CB (127 nm in
hydrodynamic diameter) labelled with a fluorescent marker entered the nucleus of cells
in two cell lines; murine macrophage cells (RAW 264.7) and epidermal cervical
carcinoma calls (CaSki) (Amornwachirabodee et al., 2018).
CB NMs are great ROS producers. Hydrogen peroxide and hydroxyl radicals accounted
for < 20% of the ROS produced by Printex 90. Other CB-based black tattoo inks showed
similar results (Høgsberg et al., 2013). In this regard it is interesting that hydrogen
peroxide is a relatively stable ROS which are frequently used as positive control cell
exposures for the comet assay. Thus, a proportion of ROS produced outside of CB
exposed cells may move to the nucleus causing genotoxicity.
Two chronic cancer studies exist for inhalation of CB NM. In case of a non-threshold
mechanism the dose response curve for tumour induction should be linear and
extrapolate to the background tumour frequency. In Figure 1 the dose response curve of
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2130134_0054.png
the combined data from female rats in these two long-term chronic cancer studies are
presented (Heinrich et al., 1995; Mauderly et al., 1994). Both studies found increased
cancer incidence at their lowest tested CB NM concentration. The control groups (0
mg/m
3
) of the two studies overlap with a tumour frequency of 0.0% and 0.5%. We have
chosen to present the data as constrained; fixed through x=0 mg/m
3
and y= 0.25% (the
mean of the controls in the 2 studies) and without constrictions (free floating). The
computed regression lines have a very high coefficient of determination (R
2
); around 0.98
supporting the linear effect.
The regression lines are expressed as:
Constrained:
Not constrained:
y= 3.494x + 0.25
y= 3.462x + 0.5357
This means that the “free floating” linear regression suggests a tumour incidence about
0.5%. If we eliminate the background data (0 mg/m
3
) the regression line for the
remaining three points would be expressed as: y= 3.414x + 0.956 (R
2
=0.96).
C o n s t r a in e d
40
40
N o t c o n s t r a in e d
R a ts w ith tu m o u rs (% )
R a ts w ith tu m o u rs (% )
30
30
20
20
10
10
0
0
2
4
6
8
10
12
3
0
14
0
2
4
6
8
10
12
3
14
M a s s c o n c e n t r a tio n ( m g /m )
M a s s c o n c e n t r a tio n ( m g /m )
Figure 1. Frequency of female rats with tumours as a function of CB NM mass concentrations
in the chronic inhalation studies by (Heinrich et al., 1995; Mauderly et al., 1994). Notably the 2
values at 0 mg/m
3
overlap (0 and 0.5%). Left: The graph has been constrained and forced
through (0; 0.25). Right: Data represented without constrictions. Dotted lines represent 95%
confidence interval for the regression lines.
As mentioned previously it is challenging to separate primary and secondary
genotoxicity and to show if CB NM induces genotoxicity in the absence of inflammation.
Below we will show that a strong NM induced inflammation does not always lead to
genotoxicity and that CB genotoxicity has been observed without concomitant
inflammation.
As described above, mice were exposed to TiO
2
NM, CeO
2
NM and CB NM (Printex 90)
by a single intratracheal instillation of 162 µg/mouse. All three materials caused a very
similar and long lasting pulmonary inflammation. A strong inflammatory response was
observed following 1 day, much less but still very similar inflammation for all 3
materials following 1 month and a return to baseline was observed after 180 days; the
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last of the 3 tested time-points. DNA strand breaks were detected using comet assay in
liver, lung and BAL cells. None of the materials caused increased genotoxicity at day 1.
However, only the CB NM caused an increased level of DNA strand breaks and this was
observed after 1 month and 180 days. None of the materials gave any significant increase
in BAL cells or lung tissue. All 3 materials translocated and were detected in the livers
using enhanced dark-field hyperspectral microscopy; only the CB NM produced ROS
measured with the DCFH
2
assay. Therefore, the authors conclude that their findings
indicate that hepatic genotoxicity are caused by a direct genotoxic effect of translocated
CB NPs rather than being caused by inflammatory responses (Modrzynska et al., 2018).
Mice
were exposed to CB NM (Printex 90) to a single intratracheal instillation of 0.67, 2,
6, and 162 µg/mouse. Animals were examined following 1, 3, and 28 days post exposure.
The 3 low doses of CB NM induced a slight or no neutrophil influx one day after
exposure. DNA strand breaks in BAL cells, lung, and liver tissue were assessed and an
increase in genotoxicity were observed in BAL cells on day 1 (0.67 and 2 µg), on day 28
in lung tissue (2 µg) but not in liver tissue at any time point. The authors interpret the
genotoxicity as increased DNA damage and repair activity occurring in the absence of
substantial inflammation and therefore as being caused by primary particle genotoxicity
(Kyjovska et al., 2015a).
The cell line Muta™Mouse - FE1 was previously developed via spontaneous
immortalization of lung tissue from a male Muta™Mouse. A characterization of
development and the cell line that retain pulmonary
epithelial characteristics have
previously been published (White et al., 2003). Additional, results have shown
the origin
of the FE1 lung cell line as presenting a phenotype of both type I and type II alveolar and
have a similar Global transcriptional characterization and response as primary lung
epithelial cells were derived from mature male
Muta™Mouse
(Berndt-Weis et al., 2009).
The cell line was recently exposed for 3 levels of Mitsui-7 CNT (12.5, 25 and 100 µg/ml
corresponding to
3.9, 7.8 and 31.19 µg/cm
2
of Petri dish, respectively) for 24 h. A global
transcriptomic analysis showed that the cell line did not express inflammatory genes to
the extent of pulmonary tissue from exposed mice (Poulsen et al., 2013). This could be
interpreted as the
Muta™Mouse - FE1
is a cell line expressing low or no inflammation
upon NM exposure and then indicate that the genotoxicity previously observed using
the same cell line (Jacobsen et al., 2007; Jackson et al., 2015) was based on primary
genotoxicity caused by ROS production. In these cases, FE1 cells were exposed to CB
NM (Printex 90) for 75 µg/ml, 3 h and for 200 µg/ml, 24 h, respectively. In the latter
genotoxicity was only observed as tail length (TL) and not % tail DNA. No genotoxicity
was observed following CB NM (Printex 90) exposure up to 200 µg/ml, 24 h (Bengtson et
al., 2016).
In summary; for the mechanism of toxicity, the present working group notes that there is
limited available data on the biological effects of different physical - chemical properties
but concludes that most of the available data support that increased surface area (and
decreased size) is a critical driver of particle-induced inflammation, genotoxicity and
carcinogenicity in the lungs.
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Although a direct interaction between CB NM and DNA leading to genotoxic effect
cannot be ruled out, it seems that the major pathway for genotoxicity involves a primary
ROS production and a secondary cell mediated indirect genotoxicity.
The present working group does not find convincing evidence for a threshold
carcinogenic effect. On the contrary we find evidence pointing towards pathways of
genotoxicity involving both a primary ROS production and a secondary and indirect cell
mediated ROS production. A non-threshold effect is also supported with regards to
carcinogenicity. To the least, based on the above presented data, a non-threshold
mechanism of carcinogenicity cannot be excluded, and in such cases, we choose a
precautionary approach and recommend a linear extrapolation in the hazard assessment
of carcinogenicity. This precautionary approach is based on ECHA REACH R8 (ECHA,
2012) in which it is stated that:
“It is to be noted that the decision on a threshold and a non-
threshold mode of action may not always be easy to make, especially when, although a biological
threshold may be postulated, the data do not allow identification of it. If not clear, the assumption
of a non-threshold mode of action would be the prudent choice. For mutagens/carcinogens, it
should be stressed that the Carcinogens and Mutagens Directive (2004/37/EC) requires that
occupational exposures are avoided/minimised as far as technically feasible. As REACH does not
overrule the Carcinogens and Mutagens Directive, the approach to controlling workplace
exposure should therefore comply with this minimisation requirement.”
Consequently, the present working group decided to perform the hazard assessment
based on both a threshold effect for inflammation and a non-threshold mechanism of
action for carcinogenesis.
Cardiovascular effects
NM exposure can lead to cardiovascular effects either: 1. Directly, by translocation of
NMs from the lung to the vascular system. 2. Indirectly, as a consequence of pulmonary
inflammation and acute phase response. 3. Alterations in autonomic nervous system
activity to elicit peripheral effects.
Atherosclerosis is a central cardiovascular effect, which is manifested as increased
plaque deposition or build-up in the arteries. It is initiated by a biological, chemical or
physical insult to the artery walls. Translocated NMs could induce this insult by
interacting directly with the endothelium. This leads to the expression of cell adhesion
molecules (selectins, VCAM-1 and ICAM-1) on the endothelial lining of the arteries,
which facilitates the activation, recruitment and migration of monocytes through the
endothelial monolayer (Cybulsky et al., 2001; Hansson and Libby, 2006). Inside the
intima layer, the monocytes differentiate into macrophages and internalise fatty deposits
(mainly oxidised 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 stabilises the plaque (Libby, 2002; Virmani et al.,
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2005). Although initially asymptomatic, narrowing of the blood vessels can lead to other
cardiovascular diseases, such as coronary artery disease or stroke. In addition, blood
clots can be formed if the plaque ruptures. These may travel with the bloodstream and
obstruct the blood flow of smaller vessels.
Pulmonary exposure to NMs may also promote accelerated atherosclerosis indirectly
through an induced pulmonary acute phase response. Introduction of NMs to the lung
promotes neutrophil influx and release of pro-inflammatory cytokines, which leads to
increased production of SAA proteins. The SAAs are hydrophobic proteins that upon
secretion in their target organs are able to translocate to the blood. A statistically
significant correlation between Saa3 mRNA levels in the lung and SAA3 protein levels in
the blood have previously been reported (Poulsen et al., 2015), indicating that SAA3
produced in the target organ translocate to systemic circulation. SAA circulating in the
blood becomes incorporated with HDL, thereby replacing Apolipoprotein A1 (Apo-A1)
as the major HDL-associated protein and forming HDL-SAA. The formation of HDL-
SAA has a double effect on plaque progression: 1. HDL is a major component of reverse
cholesterol transport, a multi-stepped process resulting in the movement of cholesterol
through the blood from peripheral tissues (including the artery walls) to the liver. The
formation of SAA-HDL impairs the HDL-mediated reverse cholesterol transport,
resulting in reduced cholesterol transport and an increased systemic total cholesterol
pool (Lindhorst et al., 1997; Steinmetz et al., 1989). 2. SAA and SAA-HDL have been
shown to directly stimulate the transformation of macrophages into foam cells and to
stimulate uptake of oxidised LDL in the macrophages (Lee et al., 1985). In addition, SAA-
HDL has a lower capacity to promote cellular cholesterol efflux from macrophages than
native HDL (Artl et al., 2000). Pulmonary neutrophil influx has been shown to correlate
with pulmonary Saa3 mRNA levels, SAA3 levels in blood and with deposited surface
area of instilled particles (Saber et al., 2014), which links deposited particle surface area
with biomarkers of risk of developing cardiovascular disease.
In conclusion, the present working group is of the opinion that pulmonary exposure to
particles including CB NMs can lead to accelerated plaque progression directly, through
translocation, or indirectly, through an induced acute phase response. No single
physicochemical property has been identified as the driver of cardiovascular effects, but
CB NM surface area is likely important due to the close association with pulmonary
inflammation. As for inflammation, we consider cardiovascular effects as a threshold
effect. This is based on identified dose-response relationships between particle exposure
dose and induced acute phase response (Poulsen et al., 2015; Saber et al., 2013), and the
close interplay between inflammation, acute phase response and plaque progression.
Dose-response relationships
Inflammation
Strong dose-response relationships have been observed following inhalation (Driscoll et
al., 1996; Elder et al., 2005; Mauderly et al., 1994) when dose is expressed as mass.
Inhalation and intratracheal instillation studies have shown that when rats and mice
were exposed to CB NM the larger the BET surface area cause stronger pulmonary
inflammation. Pulmonary exposure to CB NM has consistently been shown to cause
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dose-dependent pulmonary inflammation, with close correlation to deposited surface
area and inverse correlation to particle size (Bourdon et al., 2013b; Elder et al., 2005;
Saber et al., 2012b; Stoeger et al., 2007, 2006). One study has demonstrated that this
correlation is also valid using 6 finely tuned carbon materials all within a narrow nano
size range (primary particle size 10-50 nm and specific surface area 30-800 m
2
/g)(Stoeger
et al., 2007, 2006). The dose response relationship has been observed for a number of
low-toxicity, low-solubility particles and it is generally accepted that the inflammatory
response of these materials including CB is proportional to the surface area of the
deposited particles rather than the mass as reviewed by (Oberdörster et al., 2005).
Cardiovascular toxicity
Dose-response relationship was observed for pulmonary
Saa
mRNA expression levels in
mice intratracheally instilled with CB NM (Kyjovska et al., 2015b)(Bourdon et al., 2012).
A weak dose response relationship was observed in hear rate variability indices
following intratracheal instillation of CB NM in mice (Jia et al., 2012). For the other
cardiovascular studies there was only effect at highest dose, only one dose investigated
of no effect at any of the investigated doses.
Cancer
The Heinrich study with CB NM Printex 90 inhalation in rats has only one dose and
therefore the results cannot form basis for an evaluation of dose response or not
(Heinrich et al., 1995). The Mauderly
et al.,
study was done with two mass concentrations
2.5 or 6.5 mg/m
3
(Mauderly et al., 1994). In females there was dose response as the
highest dose also exhibited increased number of rats with lung neoplasms and this
number was substantially higher than the increase observed at 2.5 mg/m
3
(Table 6).
Overview of chronic rat inhalation studies). Notably the Heinrich study was conducted
with CB NM Printex 90 which has a diameter of 14 nm and a BET surface area of 300
m
2
/g. This study gave rise to lower air concentrations resulting in different excess lung
cancer incidences as compared to a calculation based on Mauderly and co-workers and
CB NM Elftex-12 Furnace Black having a diameter of 37 nm and BET surface area of 43
m
2
/g (as described below) (Mauderly et al., 1994). This suggests that the smaller Printex
90 having a larger BET surface area induces cancer at a lower mass concentration as
compared to Elftex-12 Furnace Black. Suggesting that when taking these two studies
together, a dose response effect based on BET surface area is observed.
Particle characteristics/dose metrics
CB NMs may vary regarding size (and therefore also surface area), and in regard to the
levels of impurities. Of the described impurities PAHs are deemed to be of highest
concern.
In vitro
PAHs from CB NM have shown to become available for forming PAH-
DNA adducts. However, in the same publication it was stated that the
in vitro
conditions
showing this effect will not be encountered
in vivo,
and thus this mechanism is observed
to be highly unlikely
in vivo
(Borm et al., 2005). Therefore, the surface area of CB NM is
likely the best dose predictor for both the inflammatory response and for lung tumours.
The present working group notes that there is limited available data on the biological
effects of different physico-chemical properties, but the current working group
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concludes that the majority of available data support that the surface area (and therefore
also the size) of CB NM is a critical driver of particle-induced inflammation and the
acute phase response in the lungs.
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P
REVIOUS HAZARD AND RISK ASSESSMENTS OF
CB
During the last couple of years, IARC and the Scientific committee on consumer safety
(SCCS) have published evaluations of CB and highlights of these are presented below.
No evaluations of CB (NM) were found from: National Institute for Occupational Safety
and Health (NIOSH), New Energy and Industrial Technology Development
Organization (NEDO), Risk Assessment Committee (RAC), The Nordic Expert Group
(NEG) and The EU Scaffold project.
International Agency for Research on Cancer
In 2006, the International Agency for Research on Carcinogenicity (IARC) classified CB
as
possibly carcinogenic to humans
(group 2B). This classification was based on sufficient
evidence for carcinogenicity in rodent experiments.
“Three studies of female rats that
inhaled carbon black and three additional studies of female rats exposed intratracheally found
significant increases in the incidence of malignant lung tumours, providing sufficient evidence
that carbon black can cause cancer in animals. Solvent extracts of carbon black were used in one
study of rats in which skin tumours were observed after dermal application and several studies of
mice in which sarcomas were seen following subcutaneous injection, providing sufficient evidence
that carbon black extracts can cause cancer in animals.”
Additionally, IARC notes that there
was inadequate evidence to assess whether CB inhalation causes cancer in humans
(IARC, 2010).
In Denmark, substances classified as group 1, 2A and 2B by IARC are considered
carcinogenic by the Danish Working Environment Authority.
Scientific Committee on Consumer Safety
SCCS established a dossier evaluating the safety of CB NM (SCCS, 2015). The latest
recommendations regarding NMs were included. The main aim of the dossier was to
answer if CB in its nanoform is safe to use as colorant in cosmetics products. Generally,
the conclusion was that CB NM, with a size of 20 nm or larger, and a purity >97%, at a
concentration up to 10%, is considered to not pose any risk of adverse effects in humans
if applied to healthy, intact skin. The opinion paper specifies that the conclusion is for
intended use as a colorant in cosmetic products and does not apply to applications that
might lead to inhalation exposure.
As part of the opinion the SCCS evaluated the literature on toxicity and carcinogenicity
of CB. They conclude that “No
carcinogenic effect was observed after oral or dermal exposure.”
However, they note that studies are old and incomplete and, therefore, no conclusion
can be drawn. On the carcinogenicity following pulmonary exposure for CB the SCCS
concludes that their opinion is “that
carbon black can induce malignant tumours in female rats
after inhalation exposure or intratracheal instillations. The potency of carbon black particles with
diameter of 14 nm was higher than the potency of carbon black particle with diameter of 95 nm.
There is no empirical support for a dose threshold from the animal carcinogenicity studies”.
The
present working group has come to a similar conclusion.
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SCCS additionally notes that they find “that
the animal cancer data are relevant to humans
and that the use of nano carbon black in sprayable applications is not recommended”.
SCCS notes on inhalation toxicity that; “the
responses after inhalation of carbon black at 1 and
7 mg/m
3
among rats, mice and hamsters were similar in magnitude. The NOAEL for inhalation of
carbon black nanomaterials for rats, mice and hamster was 1 mg/m
3
.”
This NOAEL (NOAEC)
of 1 mg/m
3
is equal to the one the present working group is suggesting based on a
review of the available literature on inhalation of CB.
Summary of the evaluations
The IARC and SCCS opinions are in accordance with the finding in the current report.
SCCS suggests a NOAEL (NOAEC) of inhalation to be 1 mg/m
3
equal to the suggestions
of the present working group. In addition, both opinions agree on the genotoxic and
carcinogenic potential of CB. The present working group is also in line with the previous
hazard assessments regards to the genotoxicity and carcinogenicity of CB.
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S
CIENTIFIC BASIS FOR AN OCCUPATIONAL
EXPOSURE LIMIT
Different methods exist for calculating health-based OELs. The choice of method
depends on the mode of action of the substance and can fundamentally be split up in
two categories: Threshold effects or non-threshold effects. Threshold effect assumes 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.
In this report, we will calculate proposed occupational exposure limits based both on
threshold effects (inflammation) or non-threshold effects (carcinogenicity).
Endpoint: Inflammation
The derivation of a derived no effect level (DNEL) based on inflammation has been
made under the assumption of a threshold driven mechanism of CB NM toxicity: CB
NM induced oxidative stress/inflammation which may result in other effects such as e.g.
cardiovascular disease. The lung epithelium is covered by a thin layer of lung lining
fluid. This layer contains e.g. glutathione which gives it anti-oxidant capacity.
Our recommendation for an OEL for CB follows the traditional approach for setting
health-based OELs:
1)
2)
3)
Identification of critical effect
Identification of the NOAEC
Calculation of OEL using assessment factors adjusting for inter and intra
species differences
In the current report we use the DNEL as recommended by ECHA as the method for
calculating OEL for toxicological effects having a threshold [83].
Our calculation of a DNEL is based on pulmonary influx of neutrophils immediately
after end of exposure. Inflammation is a key effect linked to several adverse outcomes.
The calculation is based on a chronic inhalation study of rats (Elftex-12, 37 nm; 2.5 or 6.5
mg/m
3
for 16 h/day, 5 days/week for 12 or 24 months in rats) (Mauderly et al., 1994) and
two sub-chronic inhalation studies performed by Elder and co-workers (Printex 90, 14
nm; 1, 7 or 50 mg/m
3
, 6h/day 5 days/week for 13 weeks in mice, rats and hamster) and
Driscoll and co-workers (Monarch 880, 16 nm; 1.1, 7.1 or 52.8 mg/m
3
, 6h/day, 5
days/week for 13 weeks in rats). Mauderly and co-workers observed effect at the lowest
tested mass concentration; resulting in a LOAEC of 2.5 mg/m
3
(Mauderly et al., 1994). No
effect was observed at 1 and 1.1 mg/m
3
in the sub-chronic studies leading to a NOAEC of
1 mg/m
3
.
Overall these studies support that a NOAEC level could be set to 1 mg/m
3
.
The calculations of the DNEL follow the approach as set out in the REACH guidance
[83]:
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First, the NOAEC is modified to correct for an 8-hour working day with higher
breathing rate of 10 m
3
/day in workers compared to 6.7 m
3
/day at rest. Mauderly
et al.,
used a 16 h exposure whereas the sub-chronic studies used a 6 h exposure period. Below
we correct the NOAEC 6h at rest to NOAC 8h at higher breathing:
NOAEC
Corrected
= NOAEC
6h subchronic studies
* (6 hour/8 hour) * (6.7 m
3
/10 m
3
)
= 1 mg/m
3
* (6 hour/8 hour) * (6.7 m
3
/10 m
3
)
= 0.5025 mg/m
3
= 0.5 mg/m
3
NOAEC
Corrected
Secondly, the corrected NOAEC is adjusted by a number of assessment factors (most of
these are default values suggested by ECHA (2008). The following default assessment
factors are used:
Interspecies extrapolation:
Intraspecies interpolation (default factor for workers):
Extrapolation from sub-chronic to chronic:
The overall assessment factor AF
Total
= 2.5 * 5 * 2 =
2.5
5
2
25
This results in a DNEL for chronic inhalation for pulmonary inflammation of:
DNEL = NOAEC
Corrected
/AF
Total
= 0.5025 mg/m
3
/ 25 = 0.0201 mg/m
3
DNEL = 20 µg/m
3
Alternatively, as no NOAEC was observed, the LOAEC of 2.5 mg/m
3
observed at
12 months of exposure in the chronic study by (Mauderly et al., 1994) could also be used
for the calculation of a DNEL. In such a case an assessment factor in the range of 3 to 10
could be used to convert the LOAEC to a NOAEC as described by (ECHA, 2012). If we
used the least conservative safety factor of 3 as recommended by ECHA in the majority
of cases (ECHA, 2012), we would obtain a NOAEC of 0.83 mg/m
3
. In this case the
assessment factor for extrapolation from a subchronic study to a chronic (a factor of 2) is
omitted. Adjusting for the longer inhalation period per day (16 h/8 h) and the higher
breathing rate for workers (6.7 m
3
/10 m
3
) in the Mauderly study, we would obtain a
corrected NOAEC of 0.83 mg/m
3
. This is divided by a combined assessment factor of 12.5
(inter- and intra-species) and results in a DNEL of 90 µg/m
3
. This value is higher than the
20 µg/m
3
value obtained based on a NOAEC value in the subchronic studies. However, it
is noted that this calculation is based on the least conservative assessment factor for the
use of a LOAEC instead of a NOAEC.
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Endpoint: Cancer
Carcinogenicity is generally considered a non-threshold effect. The present working
group recommends and have applied the same for CB NM-induced carcinogenicity, as
the present working group finds evidence of non-threshold mechanism of CB-induced
cancer. This approach is supported by and based on ECHA REACH R8 (ECHA, 2012).
For comparison, however, towards the end of this section a calculation using a
carcinogenic threshold approach is given.
Risk levels are calculated based on two investigations; Heinrich
et al.,
who used CB NM
Printex 90 (14 nm) (Heinrich et al., 1995) and Mauderly
et al.,
who used CB NM Elftex-12
(37 nm) (Mauderly et al., 1994).
Heinrich et al., and CB NM Printex 90
The derivation of an OEL based on cancer has been made under the assumption of a
non-threshold driven mechanism of CB NM toxicity.
The OEL is derived based on the chronic inhalation study of female mice and rats by
Heinrich and co-workers (Heinrich et al., 1995). Lung tumour rate in mice exposed to CB
NM was not statistically different from the lung tumour rate in mice exposed to filtered
air. Therefore, as the most sensitive of the tested species, data from the rats are used for
the hazard assessment.
The lowest effect level for lung cancer was observed in rats, where increased lung cancer
incidence was found at 11.6 mg/m
3
; the only tested dose in the study. The rats inhaled 7.2
mg/m
3
for the first 4 months and 12.2 mg/m
3
for 20 months. Thus, the average exposure
was 11.6 mg/m
3
for 104 weeks. Lung cancer incidence in CB NM exposed rats was 39%
(39/100), while the cancer incidence in control rats was 0.5% (1/217). Both malignant and
non-malignant tumours were included in accordance with the REACH guideline stating
that:
“malignant tumours as well as benign tumours that are suspected of possibly progressing to
malignant tumours are taken into account in obtaining the dose-descriptor values”
(ECHA,
2012).
At 11.6 mg/m
3
, the amount of pulmonary deposited CB NM after 2 years of inhalation
was determined to be 43.9 mg/rat lung (Heinrich et al., 1995).
Table 7. Cancer incidence and lung burden in female rats after
Heinrich et al.
0 mg/m
3
11.6 mg/m
3
Total cancer incidences
1/217
39/100
#
CB NM lung burden (mg/lung)
43.9
#
Include benign keratinising cystic squamous-cell tumours
Method I
Observed excess cancer incidence at 11.6 mg/m
3
:
(39/100- 1/217) / (1-1/217) = 0.387 = 39 %
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The current working group has chosen to use the approach used by Kasai
et al.,
(Kasai et
al., 2016) and Erdely
et al.,
(Erdely et al., 2013), who use the measured lung burden in rats
exposed by inhalation and the alveolar surface area of rats and humans to estimate the
human equivalent lung burden. At 11.6 mg/m
3
, the amount of pulmonary deposited CB
NM after 2 years of inhalation was determined to be 43.9 mg/rat lung (Heinrich et al.,
1995).
Human lung burden equals:
Rat lung burden × Human alveolar surface area / rat alveolar surface area
43.9 mg × 102 m
2
/ 0.4 m
2
Human lung burden equals = 11 195 mg per human lung
4
.
We assume using standard values that human ventilation is 20 L/min during light work
(1.2 m
3
/h), work-related exposure for 8 h per day, 5 days per week, 45 working weeks
per year, over a working lifetime of 45 years. The deposition rate was not reported to in
the Heinrich study. For the calculation, we have used a deposition of 8.6% based on an
inhalation study with TiO
2
by (Hougaard et al., 2010). In that study, mice were exposed
by inhalation 1h/day for 11 days to 42 mg/m
3
aerosolized powder of rutile TiO
2
with an
average crystallite size of 21 nm. The pulmonary deposition fraction was estimated to be
8.6% based on the observed particle size distribution in the aerosol (Hougaard et al.,
2010). A more conservative approach would be to use a higher deposition fraction as
suggested by other studies. E.g. in a CB NM inhalation study of pregnant mice a
deposition fraction of 34.8 % was used (Jackson et al., 2012a). If we used this higher
deposition the suggested exposure limits would be reduced by approximately 4-fold.
A lung burden of 11195 mg in humans would require that workers are exposed, through
a full work life for:
Air concentration = 11195 mg / (8h/day x 5 day/week x 45 weeks/year x 45 years x 1.2
m
3
/h x 0.086) = 1.3 mg/m
3
.
Thus, at an air concentration of 1.3 mg/m
3
during a 45-year work life, an excess lung
cancer incidence of 39% is expected. If we assume a linear dose-response relationship,
then 1% excess lung cancer would be expected at: (1.3 mg/m
3
/39) = 0.03 mg/m
3
.
The CB NM air concentrations resulting in different excess lung cancer incidences for a
deposition fraction of 8.6% are given in the table below.
4
Human lung is defined as both lungs
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Table 8. Calculated excess lung cancer incidence at different CB
NM mass concentrations based on method I.
Excess lung cancer incidence
Deposition fraction: 8.6%
CB NM concentration
1: 1 000
3 µg/m
3
1: 10 000
0.3 µg/m
3
1: 100 000
0.03 µg/m
3
If using a more conservative approach based on a deposition fraction of 34.8 % as
described above the suggested exposure limits would be reduced by approximately 4-
fold. For example, at 1: 1 000 this would be 0.8 µg/m
3
.
Method II
ECHA (European Chemicals Agency (ECHA) 2012a;SCHER/SCCP/SCENIHR 2009),
calculated based on the two year CB NM inhalation study in rats by (Heinrich et al.,
1995) (Table 9). The calculations are based on results from female rats. No effects were
observed in male rats:
Excess cancer risk:
Observed excess cancer incidence at 11.6 mg/m
3
:
(39/100- 1/217) / (1-1/217) = 0.387 = 39 %
Correction of dose metric for humans during occupational exposure (8h/day):
11.6 mg/m
3
x (18 h/day) / (8 h/day) x (6.7 m
2
/10 m
2
) = 17.5 mg/m
3
Calculation of unit risk for cancer:
Risk level = exposure level x unit risk
0.39 = 17 500 µg/m
3
x unit risk
Unit risk = 2.2 x 10
-5
per µg/m
3
At a dose of 1 µg/m
3
, 2.2 x 10
-5
excess cancers are expected.
Calculation of dose levels corresponding to risk level of 10
-5
(1: 100 000), 10
-4
(1: 10 000)
and 10
-3
(1: 1 000).
10
-5
risk level = exposure level x unit risk (2.2 x 10
-5
per µg/m
3
)
Exposure level (10
-5
) = 0.45 µg/m
3
Thus, at 0.45 µg/m
3
, 1:100 000 excess lung cancer cases can be expected.
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Table 9. Calculated excess lung cancer incidence at different CB
NM mass concentrations based on method II.
Excess lung cancer incidence
CB NM Air concentration
1: 1 000
45 µg/m
3
1: 10 000
4.5 µg/m
3
1: 100 000
0.45 µg/m
3
Mauderly et al., and CB NM Elftex-12
The derivation of an OEL based on cancer has been made under the assumption of a
non-threshold driven mechanism of CB NM toxicity. The below OEL is derived based on
the chronic inhalation study of rats by Mauderly
et al.,
(Mauderly et al., 1994). The
calculations are based on results from female rats. No significant effects were observed
in male rats.
The lowest effect level for lung cancer was observed in rats, where increased lung cancer
incidence was found at 2.5 mg/m
3
. Lung cancer incidence in CB NM exposed rats was 8%
(8/107), while the cancer incidence in control rats was 0% (0/105). Both malignant and
non-malignant tumours were included in accordance with the REACH guideline stating
that:
“malignant tumours as well as benign tumours that are suspected of possibly progressing to
malignant tumours are taken into account in obtaining the dose-descriptor values”
(ECHA,
2012).
At 2.5 mg/m
3
, the amount of pulmonary deposited CB NM after 2 years of inhalation
was determined to be 21.0 mg/rat lung (Mauderly et al., 1994).
Table 10. Cancer incidence and lung burden in female rats
after Mauderly et al.
0 mg/m
3
2.5 mg/m
3
Total cancer incidences
0/105
8/107
#
CB
NM
lung
burden
21.0
(mg/lung)
#
Include malignant and benign neoplasms
Method I
Observed excess cancer incidence at 2.5 mg/m
3
:
(8/107- 0/105) / (1-0/105) = 0.075 = 8 %
The current working group has chosen to use the approach used by Kasai
et al.,
(Kasai et
al., 2016) and Erdely
et al.,
(Erdely et al., 2013), who use the measured lung burden in rats
exposed by inhalation and the alveolar surface area of rats and humans to estimate the
human equivalent lung burden. At 2.5 mg/m
3
, the amount of pulmonary deposited CB
NM after 2 years of inhalation was determined to be 21.0 mg/rat lung.
Human lung burden equals:
Rat lung burden × Human alveolar surface area / rat alveolar surface area
21.0 mg × 102 m
2
/ 0.4 m
2
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Human lung burden equals = 5 355 mg per human lung
5
.
We assume using standard values that human ventilation is 20 L/min during light work
(1.2 m
3
/h), work-related exposure for 8 h per day, 5 days per week, 45 working weeks
per year, over a working lifetime of 45 years. The deposition rate was not reported to in
the Heinrich study. For the calculation, we have used a deposition of 8.6% based on an
inhalation study with TiO
2
by (Hougaard et al., 2010). In that study, mice were exposed
by inhalation 1h/day for 11 days to 42 mg/m
3
aerosolised powder of rutile TiO
2
with an
average crystallite size of 21 nm. The pulmonary deposition fraction was estimated to be
8.6% based on the observed particle size distribution in the aerosol.
A lung burden of 5 355 mg in humans would require that workers are exposed through a
full work life for:
Air concentration = 5 355 mg / (8h/day x 5 day/week x 45 weeks/year x 45 years x 1.2
m
3
/h x 0.086) = 0.64 mg/m
3
.
Thus, at an air concentration of 0.64 mg/m
3
during a 45-year work life, an excess lung
cancer incidence of 8% is expected. If we assume a linear dose-response relationship,
then 1% excess lung cancer is expected at: (0.64 mg/m
3
/8) = 0.08 mg/m
3
.
The CB NM air concentrations resulting in different excess lung cancer incidences for a
deposition fraction of 8.6% are given in the table below.
Table 11. Calculated excess lung cancer incidence at different CB
NM mass concentrations based on method I.
Excess lung cancer incidence
Deposition fraction: 8.6%
CB NM concentration
1: 1 000
8 µg/m
3
1: 10 000
0.8 µg/m
3
1: 100 000
0.08 µg/m
3
If using a more conservative approach based on a deposition fraction of 34.8% as
described above the suggested exposure limits would be reduced by approximately 4-
fold. For example, at 1: 1 000 this would be 2 µg/m
3
.
Method II
ECHA (European Chemicals Agency (ECHA) 2012a;SCHER/SCCP/SCENIHR 2009),
calculated based on the two year CB NM inhalation study in rats by (Heinrich et al.,
1995) (Table 12):
Excess cancer risk:
Observed excess cancer incidence at 2.5 mg/m
3
:
(8/107- 0/105) / (1-0/105) = 0.075 = 8 %
5
Human lung is defined as both lungs
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Correction of dose metric for humans during occupational exposure (8h/day):
2.5 mg/m
3
x (18 h/day) / (8 h/day) x (6.7 m
2
/10 m
2
) = 3.8 mg/m
3
Calculation of unit risk for cancer:
Risk level = exposure level x unit risk
0.08 = 3800 µg/m
3
x unit risk
Unit risk =1.97 x 10
-5
per µg/m
3
At a dose of 1 µg/m
3
, 1.97 x 10
-5
excess cancers are expected.
Calculation of dose levels corresponding to risk level of 10
-5
(1: 100 000), 10
-4
(1: 10 000)
and 10
-3
(1: 1 000).
10
-5
risk level = exposure level x unit risk (1.97 x 10
-5
per µg/m
3
)
Exposure level (10
-5
) = 0.51 µg/m
3
Thus at 0.51 µg/m
3
, 1: 100 000 excess lung cancer cases can be expected.
Table 12. Calculated excess lung cancer incidence at different CB
NM mass concentrations based on method II.
Excess lung cancer incidence
CB NM Air concentration
1: 1 000
51 µg/m
3
1: 10 000
5.1 µg/m
3
1: 100 000
0.51 µg/m
3
Summary
The CB NM air concentrations resulting in different excess lung cancer incidences for a
deposition fraction of 8.6%, Heinrich and Mauderly data overview:
Table 13. Combined results based on Method I
Excess
lung
cancer
Based on Heinrich
incidence
CB NM concentration
1: 1 000
3 µg/m
3
1: 10 000
0.3 µg/m
3
1: 100 000
0.03 µg/m
3
Based on Mauderly
CB NM concentration
8 µg/m
3
0.8 µg/m
3
0.08 µg/m
3
Table 14. Combined results based on Method II
Excess
lung
cancer
Based on Heinrich
incidence
CB NM concentration
1: 1 000
45 µg/m
3
1: 10 000
4.5 µg/m
3
1: 100 000
0.45 µg/m
3
Based on Mauderly
CB NM concentration
51 µg/m
3
5.1 µg/m
3
0.51 µg/m
3
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The present working group recommends a hazard assessment of carcinogenicity of CB
NM based on a non-threshold mechanism as calculated above and thoroughly discussed
earlier in the report (see section Mechanisms of toxicity).
Non-threshold based excess cancer risk (1: 1 000) at 3 µg/m
3
- 45 µg/m
3
However, for reference an alternative calculation based on a threshold approach is
presented here. This calculation is based on the “potential NOAEC” of the Mauderly et
al. and Heinrich et al. studies. However as both studies showed effects at all tested doses
(2.5 and 6.5 mg/m
3
in Mauderly et al., 11.6 mg/m
3
in Heinrich et al.); this calculation is
based on the lowest “LOAEC”, 2.5 mg/m
3
and then the calculation is similar to the
calculation on a DNEL of inflammation using the Mauderly et al. study above and result
in a DNEL of 90 µg/m
3
. This is calculated using the lowest assessment factor 3 (selected
in the range of 3 to 10) and thus represents the least conservative assessment factor for
converting a LOAEC to a NOAEC. It is stressed that the calculation is against the current
presented evidence as well as against the ECHA guidelines (ECHA, 2012).
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C
ONCLUSION
The present working group evaluated the relevant literature on CB NM concerning
epidemiological and animal inhalation studies. None of the identified epidemiological
studies provided information on the particle size range or purity. Also, the
epidemiological data was inconclusive with results showing both large excess risks as
well as reduced risks from working at CB factories. A strong healthy worker effect is
expected in the latter case; but in general, the studies could not be corrected for smoking
frequency and other exposures. However, within the cohort showing the largest excess
risk for mortality caused by lung cancer, cigarette smoking is not expected to be a large
confounder, as other cigarette smoke induced diseases were not increased. The results of
epidemiological studies point in different directions, as to whether CB is carcinogenic or
not. Based on the available human epidemiological studies, we cannot use the available
epidemiological studies for risk assessment. Therefore, we decided to base the suggested
health based OEL on data from experimental animal inhalation studies.
Pulmonary inflammation and carcinogenicity were observed in sub-chronic and chronic
inhalation studies in rats. The present working group regards inflammation and
carcinogenicity as the main adverse effects and the subsequent hazard assessments are
conducted based on studies reporting these effects. CB NM induced cardiovascular and
reproductive 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 hazard
assessment. However, 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 dose response relationships for neutrophil influx as a
marker of pulmonary inflammation (Driscoll et al., 1996; Elder et al., 2005; Mauderly et
al., 1994). Neutrophil influx was predicted by deposited surface area. The working group
considers inflammation as a threshold effect.
The present working group concludes that there is substantial evidence for genotoxicity
of CB NM. CB NM can induce mutations, oxidative damage to DNA as well as DNA
strand breaks in rats and mice. It is clear that inflammation is closely linked to
genotoxicity via secondary cell driven production of ROS. Primary and secondary
particle effects can be challenging to separate within
in vivo
studies; however, the present
working group do find support for primary production of ROS could have some
importance in the genotoxicity of CB NM. Additionally, some evidence for a linear and
non-threshold relationship for CB NM-induced carcinogenesis are presented.
Consequently, the present working group decided to perform the hazard assessment
based on both a threshold (inflammation) and a non-threshold mechanism of action
(cancer).
The working group considered that data from five rodent inhalation studies were the
best basis for the hazard assessment. The following studies were selected to be used for
calculation of DNEL and excess cancer risk, respectively: DNEL studies were: A 12-
month chronic inhalation study in rats (0, 2.5, and 6.5 mg/m
3
) (Mauderly et al., 1994), a
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13-week sub-chronic inhalation study in mice, rats, and hamsters (0, 1, 7, and 50 mg/m
3
)
(Elder et al., 2005), and a 13-week sub-chronic inhalation study in rats (0, 1, 7, and 53
mg/m
3
) (Driscoll et al., 1996). Cancer studies were: a 2-year chronic cancer inhalation
study in rats (0 and 12 mg/m
3
) (Heinrich et al., 1995) and a 2-year chronic cancer
inhalation study in rats (0, 2.5 and, 6.5 mg/m
3
) (Mauderly et al., 1994). Table 15 shows a
DNEL for pulmonary inflammation derived based on the sub-chronic inhalation study of
rats, and the lowest evaluated excess lung cancer risk at 1 in 1 000, 1 in 10 000 and 1 in
100 000 derived using two different approaches. As a precautionary principle, the lowest
values are presented.
Table 15. Overview of threshold-based DNEL and non-threshold-based exposure levels
leading to excess cancer risk using two different approaches.
Suggestion of an OEL for CB NM
Mechanism
of
Inflammation
Lung cancer
Lung cancer
action
(method I)
(method II)
Threshold based
DNEL
20 µg/m
3 #
Non-threshold
based
Excess cancer
risk
1: 1 000
3 µg/m
3
1: 10 000
0.3 µg/m
3
1: 100 000
0.03 µg/m
3
#
Based on NOAEC values in 2 subchronic inhalation studies
45 µg/m
3
4.5 µg/m
3
0.45 µg/m
3
Studies used for the hazard assessment used either CB NM Printex 90 (14 nm) or CB NM
Elftex-12 Furnace Black (37 nm). CB NMs differ regarding size and surface area but also
in the levels of impurities such as PAHs. The present working group notes that there is
limited available data on the biological effects of different physico-chemical properties,
but the current working group concludes that the majority of available data support that
the surface area (and therefore also the size) of CB NM is a critical driver of particle-
induced inflammation and the acute phase response in the lungs (Stoeger et al., 2006).
Two different approaches were used for calculating excess lung cancer risk based on the
same two chronic inhalation studies. 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 hazard assessment, the resulting OEL
suggestions were all very low. These levels are all more than 100-fold lower than the
current Danish OEL for CB of 3.5 mg/m
3
.
CB NMs are similar to the soot particles in diesel engine exhaust although with less
adhered organics. The relative importance and general bioavailability of adhered
organics are questioned and remains to be elucidated. Both CB NM and diesel particles
consist mainly of an insoluble carbon core and both materials have shown similar results
when tested for e.g. mutagenicity and carcinogenicity. Long-term non-cytotoxic
in vitro
exposures to CB NM Printex 90 were associated with a statistically significant increase in
the
cII
and
lacZ
mutation frequency in FE1-Muta
TM
Mouse cells (Jacobsen et al., 2007). The
level of mutations was similar to that observed following exposure to SRM 1650 a
reference diesel exhaust particle from a heavy-duty truck (Jacobsen et al., 2008a). This
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similarity extends into a rat inhalation study which found no significant difference in
carcinogenic potency between inhalation of CB NM and diesel exhaust (Nikula et al.,
1995). Both materials have for long been registered as possibly or probably carcinogenic
to humans by IARC (CB, Group 2B). However, IARC recently re-evaluated diesel
exhaust to be a Group 1 carcinogen (previously Group 2A) (IARC, 2014). A recent
epidemiological meta-analysis points towards carcinogenicity at very low diesel
exposure levels (17 excess lung cancer deaths per 10 000 at life time occupational
exposures of 1 µg diesel exhaust/m
3
) (Vermeulen et al., 2014). Two hundred and 689
excess lung cancer deaths per 10 000 at a life time occupational exposure of 10 or 25 µg
diesel exhaust/m
3
, respectively. The recommended safe exposures are thus lower based
on the human meta-analysis data compared to the rat study. We see many similarities
between CB NM and soot particles and find a comparison of effects interesting.
The present working group recommends the hazard assessment approach estimating the
excess lung cancer risk based on lung burden, since this approach takes the retained
pulmonary dose into account. Thus, the expected excess lung cancer risk in relation to
occupational exposure to CB NMs is 1: 1 000 at 3 µg/m
3
, 1: 10 000 at 0.3 µg/m
3
and 1: 100
000 at 0.03 µg/m
3
CB NM.
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