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EC number: 609-463-9 | CAS number: 376653-41-7
The objective of this study was to evaluate the potential of the test item to activate the Nrf2 transcription factor which is indicative of the potential of the test item to be a skin sensitizer. This test is a part of a tiered strategy for the evaluation of skin sensitisation potential. The data generated with this test under the present Test Guideline can be used to support the discrimination between skin sensitizers and non‑sensitizers in the context of a tiered approach to testing and assessment.
This in vitro test uses the KeratinoSens cell line, an immortalized and genetically modified Human adherent HaCaT keratinocyte cell line. The KeratinoSens cell line is stably transfected with a plasmid containing a firefly luciferase gene under the transcriptional control of the SV40 origin of replication promoter. This promoter is fused with an ARE sequence. Sensitizers with electrophilic properties provoke the dissociation of Keap-1 from the transcription factor Nrf2. The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase.
The KeratinoSens cells were first plated on 96-well plates and grown for 24 hours at 37°C. Then the medium was removed and the cells were exposed to the vehicle control or to different concentrations of test item and of positive controls. The treated plates were then incubated for 48 hours at 37°C. At the end of the treatment, cells were washed and luciferase production was measured by flash luminescence. In parallel, the cytotoxicity was measured by a MTT reduction test and was taken into consideration in the interpretation of the sensitisation results. Three independent runs were performed as part of this study.
All acceptance criteria were fulfilled for the positive and negative controls in each run; all runs were therefore considered to be valid.
This first run was performed using the following test item concentrations: 0.98, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µM in culture medium containing 1% DMSO.
At these tested concentrations:
. slight to strong precipitate was observed in test item-treated wells at concentrations ≥ 62.5 µM at the end of the 48-hour treatment period,
. no noteworthy decrease in cell viability was noted (i.e. cell viability > 70%), thus no IC30and IC50 was calculated,
. no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations. Moreover, the Imaxvalue was 1.48 (i.e. < positive threshold of 1.5).
The evaluation criteria for a negative response were met in this first run.
During the second run, same concentrations as those used in the first run were tested.
. precipitate was observed in test item-treated wells atconcentrations ≥ 125 µM at the end of the 48‑hour treatment period,
. statistically significant gene-fold inductions above the threshold of 1.5 were noted in comparison to the negative control at concentrations ≥ 15.63 µM (except at the highest concentration of 2000 µM) with an apparent dose response relationship,
. the Imaxvalue was 2.08 and the calculated EC1.5 was 11.39 µM.
The evaluation criteria for a positive response were therefore met in this second run.
However, the two runs performed were not concordant (i.e. first run negative and second run positive). Therefore, a third run was performed.
During the third run, same concentrations as those used in the first two runs were tested.
. gene-fold inductionsabove the threshold of 1.5 were noted in comparison to the negative control at concentrations ≥ 500 µM with an apparent dose response relationship. These gene-fold inductions were found statistically significant at the highest concentrations of 2000 µM,
. the Imaxvalue was 1.78.
In this third run, statistically non-significant inductions above 1.5-fold (i.e. inductions of 1.7 and 1.6 at concentrations of 500 and 1000 µM, respectively) were followed by a higher concentration with a statistically significant induction (i.e. induction of 1.8 at 2000 µM). Thus, the parameters used for the EC1.5 extrapolation were corrected manually using the formula described in the § Results analysis and was 311.73 µM.
The evaluation criteria for a positive response were therefore met in this third run.
No geometric mean IC30or IC50 was calculated since the cell viability was > 70% in all runs.
Since two of the three runs performed were considered as positive, the final outcome is therefore positive.
This positive result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of the integrated approach to testing and assessment used for skin sensitization evaluation, according to the OECD Guideline 442D.
Additionally, the Log P value of the test item is slightly > 5 (i.e. 5.66) and therefore between 5 and 7. However, this Log P value is not considered to be a limitation for the applicability of this test since the positive outcome obtained in two of the three runs performed guaranted the test system exposure to the test item.
Under the experimental conditions of this study, the test item was positive in the KeratinoSens assay and therefore was considered to activate the Nrf2 transcription factor and have the potential to be a skin sensitizer.
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