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

Skin sensitisation

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

241-922-5

EC Name:

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

Cas Number:

18015-76-4

Molecular formula:

C23H25N2 x C2HO4

IUPAC Name:

4-{[4-(dimethylamino)phenyl](phenyl)methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium hydrogen oxalate

Constituent 2

Reference substance name:

Malachite Green Chloride

IUPAC Name:

Malachite Green Chloride

Results and discussion

Positive control results:

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

In vitro / in chemico

Results

Remarks on result:

other: results are reported in the other sections

Any other information on results incl. tables

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

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.

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.