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EC number: 205-250-6
CAS number: 136-52-7
van den Brule & Lison (2016):
Table 1: Endotoxin level in cobalt
Absorbance (405 nm)
CoCl2 640 μM
CoCl2 64 μM
CoCl2 64μM + 0.5 U LPS/mL
In order to collect further evidence on the mode of action for
cobalt-induced carcinogenicity (see chapter “Summary and discussion of
carcinogenicity” or the endpoint summary in IUCLID section 7.7-
Carcinogenicity), further in vitro mechanistic studies were conducted.
The aim of these studies was to elucidate the role of genotoxicity,
oxidative stress and HIF1a activation, which are recognised key events
in cells and tissues after cobalt exposure.
In the ToxTracker assay reported by Derr and Brandsma (2021 and Hendriks
(2019) mouse embryonic stem (mES) reporter cell lines were exposed to 20
different cobalt substances. The ToxTracker assay is used to identify
the biological reactivity and potential carcinogenic properties of these
cobalt substances in a single test. The test system monitors activation
of specific cellular signalling pathways for detection of the biological
reactivity of the cobalt substances. ToxTracker consists of a panel of
six different mES GFP reporter cell lines representing four distinct
biological responses that are associated with carcinogenesis, i.e.
general cellular stress, DNA damage, oxidative stress and the unfolded
protein response. The results for the endpoints cytotoxicity,
genotoxicity, oxidative stress, p53 activation, protein damage and HIF1a
activation are summarised below.
Cytotoxicity: At the maximum tested concentrations in the absence of a
metabolising system, compounds CoOx, CoPro, Co(OH)2, CoBNeo, Co , CoCl2,
CoAcAc, Co3Et, CoCO3, CoNeo, CoBOct, and CoBProinduced significant
levels of cytotoxicity (>50%). Exposure to CoO caused approx. 40 %
cytotoxicity. In the presence of a metabolising system, there was no
increased cytotoxicity observed for any of the compounds. For CoRes,
CoLiO2, CoStea, CoNaph, and CoOOH, little cytotoxicity was observed and
the exposure concentration was limited by the solubility of the
substances in cell culture medium.The six ToxTracker reporter cell lines
showed a comparable cytotoxic response to the test compounds.
Genotoxicity: When tested in the absence or presence of a metabolising
system, none of the compounds showed an induction of the Bscl2-GFP
and/or Rtkn-GFP genotoxicity reporters. The Bscl2-GFP reporter is
associated with induction of promutagenic DNA lesions and DNA
replication inhibition. Induction of the Rtkn-GFP genotoxicity reporter
correlates with induction of DNA strand breaks.
Oxidative stress: CoOx, CoPro, Co(OH)2, Co2Et, CoBNeo, Co, CoCl2,CoAcAc,
CoRes, CoCO3, CoNeo, CoBOct, CoBPro, and CoO activated the Srxn1-GFP and
Blvrb-GFP oxidative stress reporters significantly, when tested in
absence or presence of a metabolising system. CoRes also activated the
Srxn1-GFP reporters, but only a weak induction (>1.5 fold) of Blvrb-GFP
which was not sufficient to reach the 2-fold threshold for a positive
ToxTracker response. CoLiO2, CoStea, CoNaph, and CoOOH did not activated
the Srxn1-GFP and/or Blvrb-GFB oxidative stress reporters in absence and
presence of S9. The activation of the oxidative stress reporter
correlated with the Co concentration in medium. CoS and Co3O4 did not
induce oxidative stress in ToxTracker. Induction of the Srxn1-GFP
reporter is associated with activation of the Nrf2 antioxidant response
and activation of the Blvrb-GFP reporter is associated with the Hmox1
P53 activation: Btg2-GFP was induced upon exposure to CoOx,
Co(OH)2,Co2Et, CoCO3, COBOct, and CoBPro only in the absence of S9. The
substances CoBNeo, Co, CoPro and CoAcAc showed a weak (>1.5 fold)
activation of the Btg2-GFP reporter in absence and/or presence of a
metabolising system, but induction levels did not reach the 2-fold
induction threshold for a positive ToxTracker result. Also, a weak
activation of Btg2-GFP (>1.5 fold) was observed for CoPro, CoNeo, and
CoO, but the induction levels did not reach the 2-fold threshold for a
positive ToxTracker response. The Btg2-GFP reporter is activated by p53
in response to various types of cellular stress such as protein damage,
oxidative stress, DNA damage or cytotoxicity. For CoOx, Co(OH)2, Co2Et
CoBNeo, Co and CoAcAc no activation of the Bscl2-GFP or Rtkn-GFP
reporters for genotoxicity was observed. Thus, the activation of the
Btg2-GFP marker does not appear to be related to DNA damage. As all six
substances activated both markers for oxidative stress as well as HIF1ɑ
target genes related to hypoxia, the activation of Btg2-GFP is likely to
be related to these processes.
Protein damage: The Ddit3-GFP reporter, associated with protein damage
and the unfolded protein response, was only weakly (>1.5 but < 2-fold)
activated by CoBNeo, Co, CoRes, CoCl2, CoNeo, CoBOct, Co(OH)2, and
CoBPro - the induction levels did not reach the 2-fold threshold for a
positive ToxTracker response.
Hypoxia assessment by qPCR analysis of various HIF1ɑ target genes:
Cellular hypoxia in the ToxTracker cells will activate the Hif1ɑ
transcription factor and induce expression of various target genes.
CoBNeo, Co, CoCl2 and CoAcAc all increased the expression of the HIF1ɑ
target genes Hmox1, Slc2a1 and Ddit4 by more than 2-fold. In addition,
Hmox1, BNIP3 and Ddit4 were all induced more than 2-fold upon exposure
to CoOX, CoPro, Co(OH)2, Co2Et, CoCo3, CoNeo, CoBOct, CoBPro and Coo.
CoRes activated HMOX, Slc2a1 and Ddit4 but not BNIP3. Slc2a1 activation
was also observed for CoPro, Co2Et, and CoCO3. CoAcAc also induced the
expression of Bnip3 and Eno1 and CoCl2 activated Bnip3, but not Eno1.
More than 2 fold increase in expression of Eno1 was only observed for
CoCl2, CoBOct, CoBPro, but not CoO. The substance that induced hypoxia
also activated the oxidative stress reporters in ToxTracker.
The study by van den Brule and Lison (2018) investigated the cytotoxic
activity and potential to stabilize the intracellular hypoxia-inducible
factor (HIF)-1a content in alveolar epithelial A549 of 18 different
Based on the experiments conducted in A549 cells with 18 different
cobalt substances, it appears that these can be sub-divided in 3 groups
on the basis of the four main endpoints investigated: (i) solubility in
the culture medium, (ii) solubility (bioaccessibility) in lysosomal
fluid, (iii) cytotoxicity to A549 cells, and (iv) the capacity to
stabilize HIF-1a in A549 cells.
Group 1a includes compounds (at least partly) soluble in the culture
medium. These compounds are cytotoxic, stabilize HIF-1a and can be
considered to exert a biological activity through Co ions. This group
comprises the substances: CoCl2, CoSO4, CoAcAc, CoNO3, CoPro.
Group 1b includes those compounds that remain as particulate in culture
medium, but are readily solubilized in lysosomal fluid. These compounds
are generally cytotoxic, stabilize HIF-1a, and can be considered to
exert a biological activity through Co ions. This group comprises the
substances: CoO, Co, CoCO3, CoOx, Co(OH)2, Co2Et, CoBPro, CoB2Et.
Group 2 includes compounds that remain as particulate in culture medium
and are poorly dissolved in lysosomal fluid. These compounds have a low
cytotoxicity, do not stabilize HIF1-a. They can be considered not to
exert a biological activity through Co ions. This group comprises the
substances: Co3O4, CoS, CoLiO2, CoOOH.
Both studies clearly demonstrate that there is a distinct different in
the biological responses in vitro after exposure towards different
cobalt substances. This information will be considered further in the
mode of action analysis and the read-across of the cobalt category
substances. Further details on the read-across approach for
inhalation is provided in the report attached to IUCLID section 13.2.
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