Methanaminium, N-[4-[[4-(dimethylamino)phenyl]phenylmethylene]-2,5-cyclohexadien-1-ylidene]-N-methyl-, ethanedioate

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The experiment is scientifically acceptable, nevertheless the in vitro test followed a not yet validated method (pre-validation on going). Read across from a similar substance which has the same main component and with a different counter ion that does not influence the characteristics related to the specific end-point.

Principles of method if other than guideline:

Keratinocytes play a key role in all phases of skin sensitization, and interleukin-18 (IL-18) has been shown to play a key proximal role in the induction of allergic contact sensitization. NCTC 2544 is a commercially available skin epithelial-like cell line originating from normal human skin, which posses a good expression of cytochrome P450-dependent enzymatic activities. THP-1 activation was used to assess the effects on dendritic cells activation (CD68 expression and IL-8 release). Cells were treated with increasing concentrations of both salts (0.25, 0.125, 0.0625, 0.031 µg/ml) or with PPD and LPS as positive controls for 24 h. CD86 expression was assessed by flow cytometry and IL-8 release by ELISA.

GLP compliance:

yes

Type of study:

other: THP-1 activation and NCTC2544/IL-18 assayes

Positive control results:

SI: 2.07
IL-8: ca 1600 (pg/ml); CD86: SI ca 2.0

Remarks on result:

other: results are reported in the other sections

Both salts were cytotoxic in both cell lines at concentrations ≤ 1 g/ml.

Results are expressed as mean ± SD of 3-4 independent samples.

According to the prediction model method, a substance is considered as a sensitizer when the SI exceeds the value of 1.2 with a dose-related increase in intracellular IL-18 content. Both salts reach this threshold at concentration between 0.125 - 0.25 µg/ml. In general the dose response of the two salts are similar.

THP-1 ACTIVATION ASSAY

Cells were treated with increasing concentrations of both salts or with LPS as positive control for 24 h. CD86 expression ws assessed by flow cytometry and IL-8 release by ELISA.

Both salts induce a prodes: but statistically significant IL-8 release; furthermore both salts induce CD86 expression (SI≥ 1.5).

NCTC2544/IL-18 ASSAY

The CV80 (0.25 μg/ml) was used as the highest concentration tested for both salts. Intracellular IL-18 was evaluated by ELISA, results were normalized for the cellular protein.

TREATMENT	Intracellular IL-18 (pg/mg)	SI
Control DMSO	5256±524
Chl 0.25μg/ml	6987±634	1.33
Chl 0.125μg/ml	6084±758	1.16
Chl 0.0625μg/ml	5258±459	1.00
Chl 0.031μg/ml	4791±294	0.91
Oss 0.25μg/ml	7767±593	1.48
Oss 0.125μg/ml	5943±209	1.13
Oss 0.0625μg/ml	5700±465	1.08
Oss 0.031μg/ml	4925±268	0.94
PPD 60 μg/ml	10900±1780	2.07

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: expert judgment

Conclusions:

Accordingly to the results, Malachite Green salts should be considered as contact sensitizers.

Executive summary:

Keratinocytes play a key role in all phases of skin sensitization, and interleukin-18 (IL-18) has been shown to play a key proximal role in the induction of allergic contact sensitization. NCTC 2544 is a commercially available skin epithelial-like cell line originating from normal human skin, which posses a good expression of cytochrome P450-dependent enzymatic activities. THP-1 activation was used to assess the effects on dendritic cells activation (CD68 expression and IL-8 release). Cells were treated with increasing concentrations of both salts (0.25, 0.125, 0.0625, 0.031 µg/ml) or with PPD and LPS as positive controls for 24 h. CD86 expression was assessed by flow cytometry and IL-8 release by ELISA.

According to the prediction model method, a substance is considered as a sensitizer when the SI exceeds the value of 1.2 with a dose-related increase in intracellular IL-18 content. Both salts reach this threshold at concentration between 0.125 - 0.25 µg/ml. In general the dose response of the two salts are similar. Furthermore, statistically significant release fo IL-8 vs vehicle treated cells and stimulation index ≥ 1.5 for CD86 expression are considered as positive results; both salts induce a prodes, but statistically significant IL-8 release and both salts induce CD86 expression (SI > 1.5), thuis they should be classified as senstitizers.

Conclusion

Malachite Green salts should be considered as contact sensitizers.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

No response in Clemmensen's study (1984) was elicited in the Guinea Pig maximization test on Malachite Green (MG).

Despite the fact that in the Bielicky's study (1969) patch test of MG resulted in same case positive and in others negative (6 positive cases on 9 patients tested). In the conclusions of that study, authors have reported this comment: "The simultaneous positive reactions after crystal violet (contained in gentian violet), brilliant green and MG in the majority of sensitive patients indicate the possibility of cross-sensitization. MG was not therapeutically used in our patients and this supports the opinion that there was no isolated sensitization to the individual dyes. It is not easy to decide which of the two remaining dyes was the primary sensitizer. If we support the contention that the reaction to the primary sensitizer is stronger, then for patients 8, 10, and 11 the brilliant green was the primary sensitizer and in patient 3 the crystal violet". Therefore, a clear conclusion on skin sensitising potential cannot be treated.

Since the existing studies on sensitization properties on MG are not satisfactorily conclusive and many similar substances are known skin sensitizer, it has been decided to perform further in vitro investigations on MG.

REACH&colours Kft (2011) in vitro experiments were conducted according to NCTC2544/IL-18 andTHP-1 activation methods, pre-validation on going, on MG oxalate and chloride salts. Based on the results obtained with the two different assays, MG salts resulted to be both capable to induce contact sensitizers.

Migrated from Short description of key information:

Skin sensitizing

Justification for selection of skin sensitisation endpoint:

The experiment is scientifically acceptable.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Results on in vitro test on human skin identified Malachite Green as a skin sensitizer; nevertheless data available do not allow to define the sub-category 1A or 1B.

According to CLP Regulation (EC 1272/2008), Malachite Green oxalate can be classified as Skin Sens 1, H317 (May cause an allergic skin reaction).