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EC number: 211-190-1 | CAS number: 633-03-4
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
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:
- 2 011
- 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
- Reference substance name:
- Similar Substance 03 and 02
- IUPAC Name:
- Similar Substance 03 and 02
Constituent 1
In vivo test system
Test animals
- Species:
- other: Human cells
- Strain:
- other: Skin keratinocyte
Study design: in vivo (non-LLNA)
Inductionopen allclose all
- Route:
- other: Cell culture
- Vehicle:
- DMSO
- 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.
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