[29H,31H-phthalocyaninato-N29,N30,N31,N32]cobalt

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

yes

Remarks:

The dilution strategy was different from the study plan. Given the slight solubility of the test item, the stock solution dilution was prepared 4X in treatment medium-4% DMSO instead of 100X. This has no impact on the study result

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

As Tier 1 approach and in order to avoid animal testing, no LLNA has been performed. Skin sensitisation has been assessed using appropriate in vitro assay.

Details on the study design:

125 µl of the cell suspension at 8.104 cells/ml (i.e. 104 cells per well) were distributed in three white plates for the induction measurement and two transparent plates to assess the cytotoxicity. The seeded plates were incubated 24 hours ± 1 hour at 37°C, 5% CO2.
In the 5 seeded plates, the medium was aspirated and replaced with 150 µl of treatment medium. Then the 4 X plate was replicated 5 times: 50 µl from the 4 X plate were placed in each of the three white plates and in the two transparent plates. The plates (1 X) were covered with an adhesive plastic foil to prevent evaporation and incubated for 48 hours ± 1 hour (37°C, 5% CO2).

After 48 hours, the medium was aspirated and each well was gently washed once with 200 µl of PBS. Then 100 µl of luciferase substrate (luciferine + ATP + lysing agent) were then added in each well. The plates were incubated at least 15 minutes at room temperature to ensure cells lysis.
The plates were placed in the luminometer then the luciferase activity was measured with an integration time of 2 seconds.

After 48 hours, the medium was aspirated and each well was gently washed once with 200 µl of PBS. Then, 225 µl of staining solution diluted at 0.6 mg/ml in treatment medium (from the 5mg/ml stock solution) were distributed in each well. The plates were covered with an adhesive plastic foil and incubated for 4 hours ± 30 minutes (37°C, 5% CO2).
After this contact time, the staining solution was eliminated and the cells were treated with 200 µl of 10% SDS one night in the dark (37°C, 5% CO2). After a 10 minutes homogenization, the absorbances were measured at 540 nm to assess cell viability

Cinnamaldehyde represents the positive control and culture medium represents the negatice control

Positive control results:

Positive control : Cinnamaldehyde
Geometric mean EC1.5 = 16.62
Mean Imax = 3.44

Run / experiment:

other: Assay 1

Parameter:

other: Imax

Remarks:

Mean value

Value:

2.54

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: Assay 1

Parameter:

other: Linear EC1.5 µM

Remarks:

Geometric mean value

Value:

160.98

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: Assay 1

Parameter:

other: EC1.5 Lin/Log (µM)

Remarks:

Geometric mean value

Value:

153.86

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Assay 2

Parameter:

other: Imax

Value:

1.5

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Other effects / acceptance of results:

For the 4 higher concentrations (from 2000 µM to 250 µM), the culture wells were stained by the test item.
The viability curves obtained are biphasic. From 0.98 µM to 125 µM, viability is decreasing, related to the concentrations and from 250 µM to 2000 µM viability is increasing. This is probably due to the blue staining of the well by the test item. The complementary assay on keratinocytes without gene insersion showed a viability curve with the same shape what seems to confirm the crossed reaction. IC70 was therefore calculated in the downward part of the curve.
In both repetitions, Imax are higher than 1.5 and the EC1.5 are lower than 1000 µM. The complementary assay did not showed any induction what indicates that inductions in the main test are not due to the crossed reaction. However at the EC1.5 concentration we can consider that the measured viability is overestimated. Taking into account the calculated IC70, at the EC1.5 concentration, viability should be lower than 70%.

Interpretation of results:

other: Based on a part of integrated approach, these results support a non-classification.

Conclusions:

Based on OECD 442D GLP compliant study, results are considered scientifically valid to be used as part of an integrated approach to support a non classification.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

Skin sensitisation

Based on OECD 442D GLP study, Cobalt Phthalocyanine did not show evidence of sensitising effect. However, theses results have to be considered as part of an integrated approach. It can be noted that OECD 442C assay is not applicable to metal compounds and results of assay OECD 442E depends of log Kow according the Guidance on information Requirements and chemical safety assessment - Chapter R.7a - Version 6.0 - 2017. Based on data of IUCLID (section 4.7), log Kow cannot be determined due to the low solubility of test item in water and octanol. Therefore, only OECD 442D test has been performed.

Besides, from data on 29H,31H-phthlalocyanine (EC number: 209 -378 -3, CAS number: 574 -93 -6) on ECHA website, no skin sensitisation properties is identified. For cobalt, a harmonised classification for skin sensitization has been established (refer to Annex VI of CLP Regulation) but due to the high molecular weight (571.47 g/mol) of test item and spatial conformation (cobalt is enclosed), no cobalt interaction is expected for this endpoint. Furthermore, according to C&L notifications from ECHA website, 47/49 notifications do not propose classification for skin sensitisation.

 

To conclude, in a weight of evidence approach and in order to avoid unnecessary testing on animals, considering all the elements listed above, it can be concluded that Cobalt Phthalocyanine does not have skin sensitising potential.