Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Not skin sesntitising

Key value for chemical safety assessment

Skin sensitisation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

No information on "skin sensitisation" is available for the Target Substance. However data on Similar Substance 02 has been taken into account for the assessment. More details about the similarity between the two substances are reported in section 13.

 

Initially the substance has been tested in an in vitro Skin Sensitisation test (human Cell Line Activation Test (h-CLAT)) according to the OECD guideline 442E. The study was conducted to investigate the potential of the test substance to activate monocytes and dendritic cells in the human monocytic leukemia cell line THP-1, by quantifying changes in the expression of cell surface markers (CD86 and CD54). The test article was dissolved in saline and a dose finding assay was conducted to determine a concentration showing 75 % THP-1 cell survival (CV75). The test article was dissolved at 500 mg/mL in saline, then eight stock solutions of each test article were prepared by 1.2-fold serial dilutions using saline. The stock solutions were then further diluted 50-fold into the culture medium (working solutions). Aliquots of 500 μL of each of the working solutions were mixed 1:1 with cell suspensions at 1 x 106 cells per well. Each plate was sealed using a plate sealer and then incubated at 37 ± 1 °C, 5 % (v/v) CO2 in air, in a humidified environment for 24 ± 0.5 hours. After the 24-hour incubation period, the cells were transferred into sample tubes, collected by centrifugation, washed with FACS buffer and then blocked with 600 μL of blocking solution (FACS buffer containing 0.01 % (w/v) globulin) at 4 °C for 15 minutes. After blocking, the cells were split into three aliquots of 180 μL into a 96-well plate, centrifuged and then stained with FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies at 4 °C for 30 minutes. The stained cells were washed with FACS buffer and re-suspended in FACS buffer and propidium iodide solution was added. The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.

No toxicity was noted in the dose finding assay at the maximum concentration, therefore the CV75 value for the test article was >5000 μg/mL. In the main study, the RFI for CD86 was <150 % in all 3 independent runs, the RFI for CD54 was < 200% in 2 out of 3 independent runs and cell viability was > 90 % in all 3 independent runs. All assay acceptance criteria were met. The test article was considered to be negative in the human Cell Line Activation Test. Subsequentally the substance was tested to investigate the potential to induce genes that are regulated by the antioxidant response element (ARE). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and nonsensitisers for the purpose of hazard classification and labelling. The test article was dissolved in dimethyl sulfoxide (DMSO) to the final stock concentration (40 mg/ mL). Serial dilutions were then made using DMSO to obtain 12 master concentrations of the test article (0.020, 0.039, 0.078, 0.156, 0.313, 0.63, 1.25, 2.5, 5, 10, 20 and 40 mg/mL). The master concentrations were then further diluted 25-fold into culture medium containing serum and finally used for treatment with a further 4-fold dilution factor so that the final concentrations ranged from 0.2 to 400 µg/mL. Aliquots of 50 µL of each of the final concentrationswere transferred to each of three luciferase plates and a single viability plate. Each plate was sealed using a plate sealer and then incubated at37 ± 1°C, 5 % (v/v) CO2in air, in a humidified environmentfor 48 ± 1 hours. After the 48-hour exposure period, the medium in the luciferase plates was replaced with fresh medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).The plate was sealed and incubated for 4 hours at 37 ± 1 °C, 5 % (v/v) CO2. The MTT medium was then removed and SDS (at 10 % w/v) added per well. The plate was sealed and placed into an incubator at 37 ± 1 °C, 5 % (v/v) CO2in air and left overnight. After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm using a SpectraMax M2e. After the 48-hour exposure period, the cells in the viability plate were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plates were then incubated for 20 minutes at 25 ± 2 °C, loaded into the luminescence plate reader and read. The results obtained under this test conditions are reported in the table below:

Criteria

Experiment 1

Experiment 2

Imax

1.03

1.27

Cell Viability

Not applicable

Not applicable

EC1.5

Not calculated

Not calculated

Dose Response

No

No

 

All acceptance criteria were met, with the exception that in Experiment 1, the EC1.5 for the positive control was above the range specified in the protocol at 16.90. However, luciferase induction values showed a clear dose response, therefore we consider that this was a valid experiment. As the Imax for the test article was below 1.5-fold in each experiment. The test article was considered to be negative in the ARE-Nrf2 Luciferase Test.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to the Guidance on the application of CLP criteria (ECHA, 2017): ''Validated in vitro/in chemico methods exist with the aim to identify a sensitising potential of a chemical. These include OECD TG442C (Peptide/protein binding), TG442D (keratinocyte response) and TG 442E (monocytic/dendritic cell response). The in vitro/in chemico tests are not regarded as stand alone tests and the result from such a test should be used together with other data in an overall assessment. Further, at present there is no agreed strategy on how to use in vitro/in chemico methods for direct estimation of sensitising potency, but data from such tests can be used together with other data in order to assess skin sensitisation potency. See also the Guidance on IR&CSA, especially Section R.7.3.4.1.''

The OECD guideline 442C was not applied as the test item is an UVCB substance.

Based on the results obtained in the two in vitro tests above described, the test substance has not been classified as skin sensitizer according to the CLP criteria (ECHA 2017).