Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
other: read-across from similar substance
Adequacy of study:
key study
Study period:
2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Detailed justification of read across is attached in section 13. Source study has reliability 2.

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) plays 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 posseses 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 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:
no
Type of study:
other: THP-1 activation and NCTC2544/IL-18 assays

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

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:
positive indication of skin sensitisation

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 0.25 μg/ml  6987±634  1.33 
 Chl 0.125 μg/ml  6084±758  1.16 
 Chl 0.0625 μg/ml  5258±459  1.00 
 Chl 0.031 μg/ml  4791±294  0.91 
 Oss 0.25 μg/ml  7767±593  1.48 
 Oss 0.125 μg/ml  5943±209  1.13 
 Oss 0.0625 μg/ml  5700±465  1.08 
 Oss 0.031 μg/ml  4925±268  0.94 
 PPD 60 μg/ml  10900±1780  2.07 

Applicant's summary and conclusion

Interpretation of results:
other: skin sentising according to the CLP regulation (EC 1272/2008)
Conclusions:
According to the results, test substance is considered as a contact sensitiser.
Executive summary:

Method

Test substance was tested as oxalate and chloride salt.

Keratinocytes play a key role in all phases of skin sensitisation, and interleukin-18 (IL-18) plays a key proximal role in the induction of allergic contact sensitisation. 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 test substance at concentrations of 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.

Evidences of a skin sensitising potential are:

According to the prediction model method, a substance is considered as a sensitiser if:

- SI (stimulation index) exceeds the value of 1.2 with a dose-related increase in intracellular IL-18 content

- statistically significant release of IL-8 cells is observed in treated cells with respect to vehicle-treated cells

- SI exceeds 1.5 for CD86 expression.

Results

In cells treated by test substance in both salifications the following was found:

- SI threshold was reached at concentration between 0.125 - 0.25 µg/ml.

- statistically significant IL-8 release and CD86 expression (SI > 1.5)

Accordingly, test substance was considered as skin sensitiser.