Sodium 4-oxovalerate

Endpoint summary

Description of key information

H-CLAT: demonstration of sensitising potential

LuSens: no potential to activate the Nrf2 transcription factor

DPRA: no or minimal reactivity

Key value for chemical safety assessment

Skin sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The skin sensitisation potential of the substance was evaluated in a weight-of-evidence approach considering data from three in vitro studies on the substance, in which the three key events from the AOP (Key 1: peptide/protein binding; Key 2: keratinocyte response; Key 3: dendritic cell response) are evaluated.

The peptide binding potential of the substance was evaluated in an in chemico test according to the OECD Guideline 442C and the Protocol No 154 from ECVAM DB ALM. The principle is based on chemical reactivity of the test item with proteins. The interaction between the molecule and lysine or cysteine rich peptides is detected with the HPLC. The remaining concentration of peptides was measured after 24 hours of incubation with the test item at 25 °C. It is measured with the UV detector of the HPLC system, after gradient elution at 220 nm. The depletion rates of lysine and cysteine peptides were then calculated and used to evaluate the reactivity of the substance with proteins. The mean percentage of depletion of lysine and cysteine was 1.09 % and 1.35 % respectively while the mean depletion was 1.22 %. Based on the depletion rates and the prediction model, the substance reflects a no or minimal reactivity and thus gives a negative prediction for the DPRA.

The keratinocyre reposnse was evaluated by using the genetically modified keratinocyte cell-line “LuSens” according to the OECD Guideline 442D and EU Method B.60. The test employs the use of a reporter gene for luciferase placed under the control of the antioxidant response element (ARE) and hence monitors Nrf2 transcription factor activity. The measured endpoint is the up-regulation of luciferase activity after 48 h of incubation with the test substance at different concentrations. This up-regulation is an indicator for the activation of the Keap1/Nrf2/ARE signalling pathway. In order to conclude on the Nrf2 transcription factor activity of the test substance, two, independent and valid experiments were performed. The assay included 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 (2000 μM) was chosen based on the results obtained in the CRFT. A geometric series (factor 1.2) of eleven dilutions thereof was prepared. Precipitation of the test item was not visible in any of the experiments. DMEM (final concentration: 1 %) was used as solvent control and medium (500 ml DMEM and 5 ml FCS)as growth control (blank medium control). Lactic acid (5000 μM) was used as negative control and p-Phenylenediamine (80 μM) as positive control.

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

Under the experimental conditions of this study, the test item, was negative in the LuSens assay and is therefore considered not to have the potential to activate the Nrf2 transcription factor (no sensitizing potential).

The dendritic cell response of the substance was addressed in the human cell line activation test (h-CLAT) following the OECD Guideline 442E. THP-1 cell cultures were exposed to eight concentrations of the test item, ranging from 39.1 to 5000 μg/ml, for 24 hours. After exposure, the expression of two cell surface antigens, CD86 and CD54, was measured by flow cytometry method. Vehicle control (RPMI), negative control (LA) and positive controls (DNCB and NiSO4) run in parallel. The chemicals were tested in two independents experiments. RFI of CD54 was higher than 200 in two experiments at 5000 μg/ml concentration. Since the RFI of CD54 exceeded 200 % in two experiments the substance has a skin sensitisation potential.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the CLP Regulation (EC) No. 1272/2008:

Annex I: 3.4.2.2. Skin Sensitisers Annex I: 3.4.2.2.1. Hazard categories

Annex I: 3.4.2.2.1.1. Skin sensitisers shall be classified in Category 1 where data are not sufficient for sub-categorisation.

Annex I: 3.4.2.2.1.2. Where data are sufficient a refined evaluation according to section 3.4.2.2.1.3 allows the allocation of skin sensitisers into sub-category 1A, strong sensitisers, or sub-category 1B for other skin sensitisers.

Annex I: 3.4.2.2.1.3. Effects seen in either humans or animals will normally justify classification in a weight of evidence approach for skin sensitisers as described in section 3.4.2.2.2. Substances may be allocated to one of the two sub-categories 1A or 1B using a weight of evidence approach in accordance with the criteria given in Table 3.4.2 and on the basis of reliable and good quality evidence from human cases or epidemiological studies and/or observations from appropriate studies in experimental animals according to the guidance values provided in sections 3.4.2.2.2.1 and 3.4.2.2.3.2 for sub-category 1A and in sections 3.4.2.2.2.2 and 3.4.2.2.3.3 for sub-category 1B.

Based on the OECD442C and OECD442D results the substance does not have the potential to bind to peptides/protein and does not activate the dendritic cells, and thus does not satisfy the key event 1 and key event 2. On the contrary, data from the in-vitro OECD442E suggest that the substance has a skin sensitisation potential. According to the Guidance on the CLP criteria (ECHA, version 5.0, July 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 WoE 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 in a WoE assessment together with other data in order to assess skin sensitisation potency. According to Chapter R7a-Endpoint specific guidance (ECHA, version 16, July 2017) if information from test method(s) addressing one or two of the key events in column 1 already allows classification and risk assessment according to point 8.3, studies addressing the other key event(s) need not be conducted.

As per Bauch (2012) statistics revealed that the h-CLAT assay has a sensitivity of 75% or 72%, a specificity of 77% or 76%, and an overall accuracy of 76% or 74% when compared to human data or the LLNA, respectively. The in chemico method offered a sensitivity of 89% or 81%, a specificity of 82% or 76% and an overall accuracy of 86% or 79% when compared to human data or the LLNA, respectively. For the LuSens assay, the statistics estimated a sensitivity of 89% or 81%; a specificity of 77% or 71% and an overall accuracy of 84% or 77% when compared to the human data or LLNA, respectively. It seems that H-CLAT sensitivity is slightly lower than the one of the other two in vitro methods used.

Two out of three in vitro tests result negative; the QSAR Toolbox did not identify any alert for skin sensitisation.

Based on the above considerations, the substance is considered to be non-sensitiser and thus a classification for skin sensitisation as per the CLP Regulation (EC) No.1272/2008 is not attributed.

Bauch C, Kolle S.N, Ramirez T, Eltze T, Fabian E, Mehling A, Teubner W, van Ravenzwaay B, Landsiedel R. Putting the parts together: Combining in vitro methods to test for skin sensitizing potentials. Regulatory Toxicology and Pharmacology 63 (2012) 489–504