[4-[4-(diethylamino)benzhydrylene]cyclohexa-2,5-dien-1-ylidene]diethylammonium hydrogen sulphate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation

Remarks:

other: in vitro

Type of information:

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

Adequacy of study:

weight of evidence

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

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 03 and 02

IUPAC Name:

Similar Substance 03 and 02

In vivo test system

Test animals

Species:

other: Human cells

Strain:

other: Skin keratinocyte

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

other: Cell culture

Vehicle:

DMSO

Induction 2

Concentration / amount:

0.25, 0.125, 0.0625, 0.031 µg/ml

Challenge

Concentration / amount:

0.25, 0.125, 0.0625, 0.031 µg/ml

Details on study design:

RANGE FINDING TESTS:Preliminary experiments were conducted to establish the dose range based on cell viability (CV75 for the THP-1 assay and CV80 for the NCTC2544/IL-18 assay, where CV represents the Cellular Viability at 75 or 80 % relative to vehicle treated cells). Cell viability was assessed by the MTT assay for the NCTC2544/IL-18 assay, accordingly to internal SOPs and previously published manuscripts (Mitjans et al., Toxicol In Vitro 22: 386-95, 2008; Corsini et al., Toxicol In Vitro 23: 789-96, 2009; Mitjans et al., Toxicol In Vitro 24:1803-9, 2010; Galbiati et al., Toxicol In Vitro 25: 724- 32, 2011).Both salts were cytotoxic in both cell lines at concentrations < 1 μg/ml.For the NCTC2544/IL-18 assay a CV80 of 0.25 μg/ml (0.68 μM for Malachite Green Chloride; 0.55 μM for Malachite Green Oxalate) was calculated for both compounds.EXPERIMENTAL MODEL AND PARAMETER EVALUATED - NCTC 2544 cell line (Istituto Zooprofilattico di Brescia, Italy) cells were seeded at the cell density of 2.5 x 105 cells/ml cultured in 96-well or 24-well plate (0.5-0.1 ml/well). Cells were treated in 0.1-0.5 ml of RPMI 1640 containing 2 mM L-glutamine, 0.1 mg/ml streptomycin, 100 IU/ml penicillin, supplemented with 10% heated-inactivated foetal calf serum (media) and cultured at 37C in 5% CO2. After overnight adherence, cells were treated for 24 h with Malachite at different concentrations, PPD (60 µg/ml) or DMSO as vehicle control. For IL-8 release 0.5 x 106 cells were seeded in 24-well plate. - For the assessment of IL-18 production in cells seeded in 24-well plate after incubation, culture medium was discarded, monolayer gently washed twice with 1 ml of PBS, and cells lysed in 0.25 ml of 0.5% Triton X-100 in PBS. Intracellular IL-18 content was assessed by specific sandwich ELISA commercially available (MBL, Nagoya, Japan). Plates were then frozen at -80°C. IL-8 release was assessed in cell-free supernatants by a specific sandwich ELISA, commercially available (ImmunoTools, Friesoythe, Germany), being 15.6 pg/ml the limit of detection.- Classification criteria: > 1.2 fold statistically significant increase in intracellular IL-18.- Replicate: each experiment was done 2-4 times with representative results shown. STATISTICAL ANALYSIS Each experiment was done 2-4 times with reprCellsesentative results shown. Results are expressed mean ± SD of 3-4 independent samples. Statistical analysis was performed with the Dunnett’s multiple comparison test, with **p<0.05 and **p<0.01 versus vehicle treated cells.

Positive control substance(s):

yes

Remarks:

p-phenylendiamine (PPD, CAS N° 16- 50-3, 60 μg/ml final concentration)

Results and discussion

Positive control results:

SI: 2.07

In vitro / in chemico

Results

Parameter:

other: skin sensitization

Value:

1.2

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, the tested substances should be considered as contact sensitizers. Based on the results obtained with the two different assays, both salts resulted positive; this study confirms the similar biological activity of the tested substances.

Executive summary:

In vitro assessment of the allergenic potential of the tested substances 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, the tested substances 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.