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Toxicological information

Specific investigations: other studies

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Administrative data

specific investigations: other studies
surface reactivity in vitro
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
The aim of the study is to describe in vitro effects of rat alveolar macrophages (NR8383) of nanoscaled pigments. Corundum and quartz DQ12 particles served as negative and positive control.
The biologic effects of respirable particles can be determined with an in vitro test using cultured NR8383 rat alveolar macrophages (Wiemann et al., 2016). Four parameters are tested in the cell culture supernatant under serum-free conditions, namely H2O2, tumour necrosis factor alpha (TNFα), lactate dehydrogenase (LDH), and the lytic enzyme glucuronidase. These effects are relevant for the biological response of AM to particles and, if occuring also in vivo, for their clearance process of particles from the lung: Release of LDH reflects membrane damage and necrosis of AM. Glucuronidase is a representative of lytic enzymes released from (phago)lysosomes. These enzymes may damage surrounding tissue. It is released when macrophages are activated or upon destruction of the phagolysosomal membrane. H2O2 from AM may lead to oxidative damage of lipids, proteins and / or DNA putting at risk lung cells in the neighborhood of AM. Finally, TNFα is an important pro-inflammatory cytokine with a multitude of effects on various cell types. These include proliferation, apoptosis and triggering further immune responses. According to Wiemann et al. (2016), particles may be designated as "active particles" of at least 2 criteria are fulfilled at a certain concentration.
In the assay quartz DQ12 is a well accepted positive control as it elicits strong inflammation and acts progressively fibrogenic in the (rat) lung. In contrast, corundum particles alicit no such effects in the rat lung, even if a lung burden of 5 mg is administered, e.g. by intratracheal instillation. Conrundum is therefore used as a negative control. The remarkable difference between both types of mineral particles is reflected by in vitro testing with primary as well as cultured alveolar macrophages (NR8383) under serum-free conditions.
In case that particles settle completely such that a defined amount of particle mass is internalized by a known number of cells, experiments may provide information on a mean upper particle load tolerated. This may be helpful for planning in vivo experiments. Therefore, sedimentation and uptake of particles are routinely controlled by light microscopy.
GLP compliance:
Type of method:
in vitro
Endpoint addressed:
other: surface reactivity in vitro

Test material

Constituent 1
Chemical structure
Reference substance name:
Calcium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate
EC Number:
EC Name:
Calcium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate
Cas Number:
Molecular formula:
calcium 3-hydroxy-4-[(1E)-2-(4-methyl-2-sulfonatophenyl)diazen-1-yl]naphthalene-2-carboxylate
Test material form:
solid: nanoform
Details on test material:
- State of aggregation: solid
- Shape of particles: plate
- Surface area of particles: 58.9 m2/g
- particle size (TEM D50 nm): 8.4

Batch identification: 600819U1
CAS No.: 5281-04-9
Purity: 90.5 %
Expiry date: Feb 14, 2021
Specific details on test material used for the study:
Synonym: Irgalite Magenta SMA; Pigment Red 57:1
CAS No.: 5281-04-9
Purity: 90.5 %
Expiry date: Feb 14, 2021
Physical state/ appearance: solid / red
Additional test substance information
Mass-specific surface area (BET): 58.9 m2/g
TEM (min. Feret): 68.4 nm (D50)

Results and discussion

Details on results:
Pigment Red 57:1 was incompletely taken up by alveolar macrophages, possibly due to early cell damage. The cytotoxicity of Pigment Red 57:1 was dose-dependent and highly similar to Quartz DQ12 (i.e. significant upon 90 μg/mL), indicated by the release of LDH. The release of GLU (significant upon 45 μg/ml) also indicating an activation and/or release of the enzyme from phagolysosomes was even more pronounced than the response to quartz. However, unlike quartz DQ12, there was no induction of TNFα. Also in this case H2O2 production could not be measured due to strong optical interference.
Given the BET value of Pigment Red 57:1 and according to the active/passive classification of Wiemann et al. 2016, the substance was classified as to be active.

Applicant's summary and conclusion

Given the BET value of Pigment Red 57:1 and according to the active/passive classification of Wiemann et al 2016, the substance was classified as to be active