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Administrative data

Description of key information

not skin sensitiser

Key value for chemical safety assessment

Skin sensitisation

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

The in vitro 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 WoE assessment. Two in vitro tests were performed on Target Substance for Key Event 2-Keratinocyte response- and Key Event 3-Dendritic cell response as follows.

The first in vitro test was performed using the LuSens cell line. The LuSens test is an ARE (antioxidant response element) Reporter Gene Assay that was developed by the BASF SE (Ludwigshafen, Germany) and is based on the OECD 442D Guideline (KerationSens Assay). The assay was performed according to the BASF Protocol which differs in some points from the OECD Guideline No. 442D. The assay was performed in a cytotoxicity range finder test (CRFT) and two independent experiments (experiment I and II) with a treatment period of 48 h. The CRFT was performed to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the two experiments were determined.

In the experiments, the highest nominal applied concentration (25 µg/ml) was chosen with regard to the cytotoxic reaction in the CRFT. Furthermore, a geometric series (factor 1.2) of eleven dilutions was prepared. Precipitation of the test item was not visible in all experimental parts. DMSO (final concentration: 1 %) was used as solvent control and medium no. 3 as medium control. Furthermore, Lactic acid (5000 µM) was used as negative control and EGDMA (120 µM) as positive control.

No substantial and reproducible dose dependent increase in luciferase induction above 1.5 fold was observed in both experiments up to the maximal concentration of the test item.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item possesses no sensitizing potential in accordance to the BASF protocol.

The second in vitro study was performed according to the OECD Guideline No. 442E (2017). The test is based on the quantifying changes in the expression level of the two cell surface markers CD86 and CD54, which are associated with the process of activation of monocytes and dendritic cells. For this purpose the human monocytic leukaemia cell line THP-1 was used and treated with the test item for 24 h before evaluation.

In total a pre-test and three valid experiments (experiment I - III) with a treatment period of 24 hours were performed. For the experiments, the highest nominal applied concentration (1000 µg/mL) was chosen based on the results obtained in the pre-test. A geometric series (factor 1.2) of 7 dilutions was prepared. No precipitation of the test item was visible in the pre-test, but it was visible in all experiments at the four highest concentrations. As solvent control for the test item, RPMI 1640 was used in a final concentration of 1 % in culture medium.As positive control, 2,4-dinitrochlorobenzene (DNCB, CAS n. 97-00-7, ≥ 99% purity) was used.

In all experiments the RFI of CD86 was not ≥ 150 % with cell viability ≥ 50 %. The RFI of CD54 was not ≥ 200 % in experiment II, but was ≥ 200 % in experiment I and III with cell viability ≥ 50 %. Since the majority result of the three individual runs is positive, the test item is considered positive in the h-CLAT. Therefore the substance is considered to have the potential to activate dendritic cells.

In order to complete the overview of the profile and conclude on the presence or absence of skin sensitisation potential of the substance, data obtained for the Similar Substance 01 and Similar Substance 02 are taken into account. It is expected that the Target substance will present comparable skin sensitising profile. Justification for Read Across is given in Section 13 of IUCLID.

The potential of the test item to cause skin sensitisation reactions following topical application to the skin of CBA/JN mice, was assessed using the LLNA:BrdU-ELISA method, according to the OECD Guideline 442b (2010). Five concentrations [25 (maximum feasible concentration), 10, 5, 2.5 and 1 % w/w in acetone: olive oil 4:1 (v/v)] were tested in the preliminary phase, in order to identify a non toxic and minimally irritant concentration and avoid false positive results. Based on the results observed, in the main assay the test item was topically administered at the concentrations of 25, 10 and 5% (w/w), in acetone: olive oil 4:1 (v/v).

No mortality nor clinical signs were recorded in any animal. Changes in bodyweight observed during the study were within the expected range for this strain and age of animals.A slight increase in cell proliferation of draining lymph nodes was observed in the low dose group, with a Stimulation Index of 1.98. The other calculated indices (SI) were 0.95 and 1.28 respectively, in medium and high dose groups. No correlation with the doses nor statistical significance was observed. These results indicate that the test item may elicit a sensitisation response. However, due to the presence of an outlier in the low dose group, the absence of dose-response relationship and statistical significance, the observed reaction is not sufficient to indicate classification.

In order to evaluate the skin sensitisation potential of the test item, an in vivo maximization test on guinea pigs was performed according to the OECD guideline 406 (1981). A primary irritation experiment was performed in order to determine the test concentrations of the main study. In the main study ten animals (5 males, 5 females) were treated with the vehicle alone (petrolatum oil) and 20 animals (10 males, 10 females) were treated with the test substance. The intradermal induction was made by injection at 0.5 %; in the following epidermal induction 5 % of test item was used. After two weeks, the animals were challenged using doses of 3 %.

The substance resulted in a sensitisation rate of 0 % to the guinea pig after intradermal and epidermal induction.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Skin sensitisation

In the CLP Regulation (EC 1272/2008) a skin sensitizer is defined as a substance that will lead to an allergic response following skin contact. Validated in vitro/in chemico methods exist with the aim to identify a sensitising potential of a chemical. The subject of in vitro testing for skin sensitisation is discussed in the Guidance on IR&CSA, Section R.7.3.4. There are several validated test methods for the assessment of skin sensitisation potential in vitro and, for some of them, EU/OECD- adopted test guidelines are available. These test methods have been developed with the purpose of using several in chemico/in vitro methods together, as described in section 8.3.1 of Annex VII to the REACH Regulation. Annex VII to the REACH Regulation specifies that when new data need to be generated to fulfil the standard information requirement for skin sensitisation, as a first step in chemico/in vitro studies assessing three key events of skin sensitisation should be performed, unless data from fewer key events already allows classification and risk assessment, as specified in Annex VII, section 8.3, column 2. Indicators of potency such as the level of peptide depletion and concentration-responses can be obtained from the existing in chemico and in vitro tests, respectively. Data from the tests

(i) Direct Peptide Reactivity Assay (DPRA) for Key Event 1 Peptide/protein binding

(ii) ARE-Nrf2 Luciferase Test Method (KeratinoSensTM) for Key Event 2 Keratinocyte response

(iii) Human Cell Line Activation Test (h-CLAT) for Key Event 3 Monocytic /Dendritic cell response.

may be accepted to fulfil Annex VII requirement when used in combination with each other. These test methods were developed to address specific events of the skin sensitisation AOP (OECD, 2012). The AOP for skin sensitisation describes the current understanding of key events linked to skin sensitisation. As each of the test methods only addresses a specific key event of skin sensitisation, currently they should not be used in isolation to identify a skin sensitizer or a non-sensitizer.

In particular for the test substance:

(i) DPRA is not applicable to the testing of UVCB substances due to the defined molar ratio of the test substance and peptide.

(ii) no potential to activate the Keap1-Nrf2-ARE pathway under the conditions of the LuSens test

(iii) potential to activate dendritic cells under the conditions of the h-CLAT test.

The activation of dendritic cells as well of keratinocytes represents only two key events of the skin sensitisation AOP, information generated with these test methods considered alone may not be sufficient to conclude on the presence or absence of skin sensitisation potential of chemicals. Considering that the results obtained in theLuSens and h-Clat tests are contrasting and results from DPRA are not possible to be obtained, no conclusion can be drawn based on the data from the available in vitro studies. Due to the animal welfare, the evaluation of the skin sensitisation potential should be done byin vivotesting only as a last option. Consequently, other relevant complementary information derived from non testing methods is considered. In this way, the read-across approach from chemical analogues is considered. In particular data derived from two in vivo tests obtained for two analogue substances that cover all the functional groups of the target substance are taken into account.

Based on the results of skin sensitisation, no classification for skin sensitization is warranted under the CLP Regulation (EC 1272/2008).