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

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Malachite Green Oxalate and Chloride

IUPAC Name:

Malachite Green Oxalate and Chloride

Results and discussion

Positive control results:

SI: 2.07

In vitro / in chemico

Results

Remarks on result:

other: See report below

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.