Reaction products of Sodium 1-amino-4-bromo-9,10-dioxoanthracene-2-sulphonate coupled with Aniline, subsequently coupled with p-(1,1-dimethylpropyl)phenol, subsequently coupled with Ethyl benzoylacetate, subsequently coupled with Fuming Sulfuric acid, sodium salts.

Endpoint summary

Description of key information

human Cell Line Activation Test (h-CLAT) - OECD 442E: negative

ARE-Nrf2 Luciferase Test - OECD 442D: positive

LLNA OECD 429: negative

Key value for chemical safety assessment

Skin sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The evaluation of the skin sensitising potential of test substance relies on a weight of evidence approach. In vitro studies, addressing key events II and III o skin sensitisation gave opposite results. The I key event could not be assessed as the current prediction model cannot be used with UVCB substances, such as test substance.

Consequently, an in vivo assay, i.e. LLNA, was run to clarify the skin sensitisation potential of test substance.

human Cell Line Activation Test (h-CLAT)

The skin sensitization potential of the substance was studied in in vitro skin sensitisation human cell line activation test, according to the OECD guideline 442E.

The extent of cytotoxicity induced on THP-1 cells by the test item was studied in two dose finding tests. CV75 could not be determined, since there was no test item concentration that resulted in 75 % cell viability compared to the solvent/vehicle control. Thus the highest soluble concentration (50 mg/ml) was used for setting the dose-range for measuring CD86 and CD54 expression in the main test. Eight doses were used in three independent runs between 500 –140 µg/ml.

The increase in CD86 marker expression (RFI) was not equal to or greater than 150 % at any tested dose (with > 50 % of cell viability) compared to the respective negative controls in any of the independent runs. Therefore, all three runs were negative for CD86 marker expression.

The increase in CD54 marker expression (RFI) was greater than 200 % compared to the respective negative controls at the highest tested concentration (500 µg/ml - with 89.4 % of cell viability) However the increase of CD54 expression in the second and third runs was not equal to or greater than 200 % at any tested dose (with > 50 % of cell viability) compared to the respective negative controls. Based on the majority result of the three individual runs (2 out of 3 were negative), prediction for CD54 marker expression was concluded as negative, as well.

Based on these results and the h-CLAT prediction model, the test item demonstrated a non-sensitizing potential under the experimental conditions of human Cell Line Activation Test.

ARE-Nrf2 Luciferase Test

The skin sensitization potential of test item was studied using the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method) accordin to OECD guideline 442D. In order to derive a prediction for the test item the results of three independent tests were used, since the results of the first two valid tests were not concordant and a third one was needed in order to derive a conclusion. However, the second test was needed to be repeated due to not fulfilling all validity criteria for the positive control for the first time. The luciferase activity induction obtained with the positive control, trans-cinnamaldehyde, was statistically significant above the threshold of 1.5-fold in all tests. For the test item, twelve doses ranging from 2000.00 μg/ml to 0.98 μM were used in the first two valid tests and then a narrower fold dilution was applied for the last test ranging from 1000.000 μg/ml to 11.561 μM. The test item induced cytotoxicity (viability below 70 %) in KeratinoSens™ cells compared to the solvent/vehicle in the first and second valid run, while no cytotoxicity was induced in the third run. IC30 and IC50 values were determined in the first run as 986 μM and 1321 μM respectively and in the second valid run as 916 μM and 1106 μM. Two out of three valid tests were concluded positive for luciferase gene induction, meaning that the induction values of the test item exceeded the 1.5-fold threshold, therefore EC1.5 values could be determined. EC1.5 values were 79 μM and 181 μM in the first and third run respectively. Moreover, a clear dose response could be observed also in three out of three tests. Based on these results and the KeratinoSens™ prediction model, the test item i concluded positive for skin sensitization potential under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

Local Lymp Node Assay (LLNA)

The skin sensitization potential of test substance was evaluated in vivo following dermal exposure in the LLNA according to OECD guideline 429.

Preliminary tests were performed to find an appropriate vehicle and the maximum applicable concentration.

DMF was selected and used as vehicle for the test item formulations. No significant adverse effects (systemic toxicity or irritation) were observed in the dose range finding test up to this maximum concentration. Accordingly, the test item was examined in the main test as 25 %, 10 %, 5 % and 2.5 % (w/v) formulations in DMF.

An appropriate positive control (a-hexylcinnamaldehyde, HCA), and furthermore two negative control groups dosed with the vehicles of the test and positive control groups, respectively, were employed.

The positive control item [25 % (w/v) HCA in acetone: olive oil 4:1 (v/v) mixture (AOO)] induced significant stimulation over the relevant control (SI = 12.1) thus confirming the validity of the assay.

No mortality or signs of systemic toxicity were observed during the main test. No significant, treatment related effect on the body weights was observed in any dose group. No signs of significant irritation or any other local effects were observed at the treatment site (ears) in any treatment group.

No significantly increased lymphoproliferation (indicated by an SI >= 3) compared to the relevant control (DMF) was noted for the test item at the applied test concentrations. The observed stimulation index values were 1.7, 1.5, 1.2 and 1.0 at test item concentrations of 25 %, 10 %, 5 % and 2.5 % (w/v), respectively. No significant dose-response relationship was observed. The lack of a significantly increased lymphoproliferation (indicated by an SI >= 3) up to the maximum attainable concentration of 25 % (w/v, based on solubility) as well as the lack of a significant dose-related response is considered as evidence that the substance is not a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), skin sensitiser means a substance that will lead to an allergic response following skin contact.

Available in vitro and in vivo data was taken into account to draw a conclusion on classification of test substance. Overall, available data is:

- positive result in an in vitro study according to OECD guideline 442D;

- negative result in an in vitro study according to OECD guideline 442E;

- negative result in an in vivo study according to OECD guideline 429.

On these bases, the substance is considered as devoid of a skin sensitisation potential and it is not classified within the CLP Regulation.