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Description of key information

A weight of evidence approach was performed using an in vivo LLNA, a Closed epicutaneous test (CET), an HRIPT, and two in vitro assays carried out with a read-across substance, namely DPRA, and LuSens. Furthermore, a grouping approach of salicylates published by RIFM was also considered.

The LLNA gave positive results, the CET and the HRIPT were negative, the DPRA and the LuSens for the read-across substance gave also negative results.

 

Using this WoE approach, the test item is considered not to be sensitizing to skin.

Key value for chemical safety assessment

Skin sensitisation

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

A weight of evidence approach was performed using an in vivo LLNA, a Closed epicutaneous test (CET), an HRIPT, and two in vitro assays carried out with a read-across substance, namely direct peptide reactivity assay (DPRA), h-CLAT, and LuSens. Furthermore, a grouping approach of salicylates published by RIFM and CIR was also considered.

LLNA

The purpose of this study was to assess the skin sensitization potential of the test substance after application to the dorsum of each ear of female CBA/N mice. The assay was performed according to OECD TG 442B and in compliance to GLP.

Based on the result of the dose range finding study, the high dose for the main study was selected as 50%. Two additional low dose levels (5 and 0.5%) were tested. In addition, the positive and negative control groups were included in the main study.

No abnormal clinical signs or deaths were observed in any animal. In the test substance groups, the body weight and ear thickness were not significantly different when compared to the negative control group. The erythema score, ear weight and stimulation index were significantly increased when compared to the negative control group.

In the positive control group, the erythema score, ear thickness, ear weight and stimulation index were significantly increased when compared to the negative control group and the stimulation index (SI) was calculated to be 3.06.

Based on the results of this study, the test substance produced the following stimulation indices: SI 2.21, 3.39, and 8.67 at doses of 0.5, 5, and 50%, respectively. EC 1.6 value was <0.5%.

CET

A Closed Epicutaneous Test (CET) was performed on female guinea pigs to determine the sensitizing potential of the test substance in petrolatum. The test was not performed according to a guideline but resembles the OECD TG 406 and it was not performed according to GLP.

In the induction phase of the CET, the test material was applied occluded for 48 hours at the highest non-irritative concentration (30%) on the shaved nape and this procedure is repeated three times per week for two weeks. FoIlowing a two-week rest period, the test material was applied at the maximum non-irritative concentration (1%) under occlusion for 48 hours on the flanks for chaIlenge.

This resulted in no indication of sensitization (rate: 0/8).

KeratinoSens

The in vitro KeratinoSensTMassay according to OECD 442D and GLP was performed with a read-across substance and enables detection of the sensitising potential of a test item by quantifying the luciferase activity in the transgenic cell line KeratinoSensTM. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

In the present study the test item was dissolved in DMSO. The following concentration range was tested in the assay: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 pM. Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSensTMcell line in at least two independent experiment runs.

DPRA

The in chemico DPRA is quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine. The test was performed according to OECD TG 442C and in compliance to GLP.

Experiment 1

In the present study the test item was dissolved in acetonitrile based on the results of the pre-experiments. However, due to the observed precipitation in both experiments and phase separations in the lysine run prior to the HPLC analysis, a test item concentration of 100 mM as well as the full contact of peptide and test item is not guaranteed. According to the evaluation criteria in the guideline, no firm conclusion on the lack of reactivity should be drawn from a negative result, if a test chemical is tested in concentration < 100 mM. Therefore, no prediction could be made.

Experiment 2:

In the second study the test item was dissolved in methanol based on the results of the pre-experiments.

For the 100 mM stock solution of the test item turbidity was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis, no precipitation, turbidity or phase separation was observed for any of the samples. For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis, phase separation was observed for the samples of the test item (including co-elution control) and the co-elution of the positive control. Samples were not centrifuged prior to the HPLC analysis.

As phase separation was observed in the lysine experiment, sensitising potential of the test item was predicted from the mean peptide depletion of the cysteine peptide. The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptide. The mean depletion of the cysteine peptide was ≤ 13.89% (0.30%). Based on the prediction model 2 the test item can be considered as non-sensitiser.

H-Clat

The potential of test substance to induce the cell membrane markers CD86 and CD54 expression was evaluated in the Human Cell Line Activation Test (h-CLAT). For this purpose the test substance was incubated with human pro-monocytic cell line THP-1 for ca. 24 hours at 37°C and membrane markers expression measured by flow cytometry. In order to determine the concentrations suitable for the main experiment a pre-test was performed. Cells were exposed to 10 concentrations of the test substance and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. The CV75 value (=estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve.

In the main test after 24 hour exposure THP-1 cells were stained with FITC labelled anti-human-CD86/ anti-human-CD54 antibody and propidium iodide and the fluorescence intensity was analysed using flow cytometry. A total of 3 valid experiments were performed. The expression of the cell surface marker CD54 clearly exceeded the threshold at oneconcentration with acellviability ≥50%inatleasttwoindependentruns.

The positive control (DNCB) led to an upregulation of CD54 and CD86 in all three experiments. The threshold of 150% for CD86 (264% experiment 1; 336% experiment 2; 304% experiment 3) and 200% for CD54 (231% experiment 1; 288% experiment 2; 257% experiment 3) were clearly exceeded.

Based on a weight of evidence approach according to Bauch et al 2012 two negative results drive the prediction of a test substance to be a non-sensitizer.

HRIPT

A Human Repeat Insult Patch Test (HRIPT) was performed to determine by repetitive epidermal contact the potential of the test material to induce primary or cumulative irritation and/or allergic contact sensitization. The test was performed according to GCP standard ICH E6.

107/112 qualified subjects, male and female, completed the study (the reason for discontinuation were not related to the application of the test).

Approximately 0.2 ml of test material at a concentration of 20% in DEP/EtOH 3:1was applied to the upper back to a 1" x 1" absorbent pad portion of a clear, adhesive dressing which was applied to form a semi-occlusive patch.

Patches were applied 3 times per week for a total of 9 applications. The first scoring was 24 h after application and evaluation was before re-application. Approximately 2 weeks after final induction patch application, a challenge patch was applied to a virgin test site adjacent to the original induction patch site following the same procedure as described for induction. The patch was removed and the site scored at the clinic day 1 and day 3 post-application. Erythema was scored numerically according to the key.

Under the conditions of the study, the test material indicated no potential for dermal irritation or allergic sensitization.

Conflicting results were obtained from different studies. While the LLNA gave positive results without reaching an EC1.6 value, all other studies including CET, HRIPT and an in vitro battery from a read-across substance gave negative results. RIFM published a toxicological and dermatological safety assessment of more than 15 salicylates (Belsito et al., 2007). Here, all salicylates under review, except for methyl 4 -methylsalicylate, have been evaluated for the potential to induce sensitization in humans in either a maximization test or in a repeated insult patch test (HRIPT). Sensitization reactions were observed in 2 maximization studies conducted with 20% benzyl salicylate and one non-specific reaction with pentyl salicylate. The rest of evaluated salicylates showed no sensitization potential when tested at concentrations of 1.25–100%. Animal studies resulted in the same problem that was faced for the above mentioned studies. They gave mixed results. They had been subjected to testing, either in standard guinea pig models [open epicutaneous test (OET), Draize tests, closed epicutaneous tests (CET), optimization assays, cumulative contact enhancement tests (CCET), Freund’s complete adjuvant tests (FCAT), etc.] or in the mouse local lymph node assay (LLNA). While some salicylates with aromatic side chains have been shown to induce skin sensitization at low doses of 0.1%, standard guinea pig models have failed to verify this with even 250 fold higher concentrations. As also human data could not verify these results (apart from benzyl salicylate), RIFM concluded that salicylates in general have no or very limited skin sensitization potential (latter one is only true for at most salicylates with aromatic side chains). Alkyl- and aliphatic ring side-chain salicylates have been assessed as having no sensitization potential. A CIR review substantiated suspicion of having received a false positive result in the above mentioned LLNA. It was shown that different results were obtained for salicylic acid with the same applied dose but by only changing the solvent in the LLNA (CIR, 2003).

Based on the WoE approach and under consideration of the RIFM and the CIR review it was concluded that the test item is not classified as skin sensitizer.

Reference

D. Belsito, D. Bickers, M. Bruze, P. Calow, H. Greim, J.M. Hanifin, A.E. Rogers, J.H. Saurat, I.G. Sipes, H. Tagami. 2007. Food and Chemical Toxicology 45 (2007) S318–S361.

Cosmetic Ingredient Review, 2003. International Journal of Toxicology, 22(Suppl. 3):1-108, 2003

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the WoE approach it was concluded that the test item is not classified as skin sensitizer.