[4-[α-[4-(dimethylamino)phenyl]benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vitro

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non validate in vitro method Read across from a similar substance which has the same main component and with a different counter ion that doesn't influence the characteristics related to the specific end-point

Qualifier:

no guideline available

Principles of method if other than guideline:

NCTC2544/IL-18 method; pre-validation on going.
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 and to favour Th-1 type immune response by enhancing the secretion of pro-inflammatory mediators such as TNF-α, IL-8 and IFN-γ, (Okamura et al., 1995; Cumberbatch et al., 2001; Antonopoulos et al., 2008). IL-18 production in the human keratinocyte cell line NCTC 2544 has been identified as a potentially useful endpoint for identification and discrimination of contact versus respiratory allergens and/or irritants (Corsini et al., 2009; Galbiati et al., 2011). 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.

GLP compliance:

yes

Type of study:

other: NCTC2544/IL-18

Justification for non-LLNA method:

Existing study

Positive control results:

SI: 2.07

Remarks on result:

other: See report below

The CV80 (0.25 μg/ml) was used as the highest concentration tested for both salts. Cells were treated with increasing concentrations 0.031-0.25 μg/ml) or with PPD (60 μg/ml) as positive control for 24 h. 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
PPD60 μg/ml	10900±1780	2.07

According to the prediction model of this 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.

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. Based on the results obtained with the two different assays, both Malachite Green salts resulted positive; this study confirms the similar biological activity of Malachite Green salts.

Executive summary:

In vitro assessment of the allergenic potential of Malachite Green Chloride and Malachite Green Oxalate has been performed according to NCTC2544/IL-18 method (pre-validation on going). The CV80 (0.25 μg/ml) was used as the highest concentration tested for both salts. Cells were treated with increasing concentrations 0.031-0.25 μg/ml) or with PPD (60 μg/ml) as positive control for 24 h. Intracellular IL-18 was evaluated by ELISA, results were normalized for the cellular protein.

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

Reference for method:

1) Antonopoulos, C., Cumberbatch, M., Mee, J.B., Dearman, R.J., Wei, X., Liew, F.Y., Kimber, I., Groves, R.W., 2008. IL-18 is a key proximal mediator of contact hypersensitivity and allergen-induced Langerhans cell migration in murine epidermis. J. Leukoc.Biol. 83, 361-367.

2) Corsini, E., Mitjans, M., Galbiati, V., Lucchi, L., Galli, C.L., Marinovich, M. 2009. Use of IL-18 production in a human keratinocyte cell line to discriminate contact sensitizers from irritants and low molecular weight respiratory allergens. Toxicol In Vitro. 23, 789-796.

3) Cumberbatch, M., Dearman, R.J., antopoulos, C., Groves, R.W., Kimber, I., 2001. Interleukin-18 induces Langerhans cell migration by a tumor necrosis factor-α and IL-1β-dependent mechanism. Immunology 102, 323-330.

4) Galbiati, V., Mitjans, M., Lucchi, L., Viviani, B., Galli, C.L., Marinovich, M., Corsini, E. 2011. Further development of the NCTC 2544 IL-18 assay to identify in vitro contact allergens. Toxicol In Vitro. 25, 724-732.

5) Okamura, H., Tsutsui, H., Komatsu, T., Yutsudo, M., Hakura, A., Tanimoto, L., Torigoe∥, K., Okura, T., Nukada, T., Hattori, K., Akita, K., Namba, M., Tanabe, F., Konishi, K., Fukuda, S., and Kurimoto, M. (1995). Cloning of a new cytokine that induces IFN-γ production by T cells. Nature, 378: 88-91.

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), results show not clearly if MG could be classified as sensitising. 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."

Since the existing studies on sensitization properties on MG are not enough conclusive and many similar substances have positive results in this aspect, it was decided to perform further in vitro studies on MG.

Reach&Colors (2011) in vitro studies 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 both positive and those tests confirm the similar biological activity of MG salts.

MG salts should be considered as skin sensitizers.

Migrated from Short description of key information:

Sensitizing

Justification for selection of skin sensitisation endpoint:

]

Respiratory sensitisation

Endpoint conclusion

Additional information:

Skin sensitization: in vitro studies were conducted according to NCTC2544/IL-18 andTHP-1 activation methods, pre-validation on going, on MG Oxalate and Chloride salts and the two different assays showed that MG salts resulted both positive, confirming moreover the similar biological activity of MG salts. MG salts should be considered as contact sensitizers.

Respiratory sensitization: no data available

Justification for classification or non-classification

Results on in vitro test on human skin identified Malachite Green as a skin sensitizer. This result is supported, according to the application of CLP criteria, by the comparison with similar substances. Nevertheless data available do not allow to define the sub-category 1A or 1B.

According to CLP regulation (EC1272/2008) Malachite Green Acetate is classified as Skin Sens 1, H317 (May cause an allergic skin reaction).