Butanal, reaction products with N2,N2'-1,6-hexanediylbis[N4,N6-dibutyl-N2,N4,N6-tris(2,2,6,6-tetramethyl-4-piperidinyl)-1,3,5-triazine-2,4,6-triamine and hydrogen peroxide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation

Remarks:

other: in-vitro method

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable in vitro testing method in pre-validation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Principles of method if other than guideline:

The myeloid U937 skin sensitization test (MUSST) is a dendritic cell activation test to predict skin sensitizing potential. The test is performed using the human pro-monocytic cell line U937 as surrogate for dendritic cells. As readout, the change in the expression of the cell membrane marker CD 86 measured by flow cytometry after 48 hours of test substance exposure is determined. A test substance is predicted to activate dendritic cells when CD86 cell surface expression exceeds the threshold in relation to vehicle control in at least two independent experiments.

GLP compliance:

no

Type of study:

other: Dendritic Cell Line Activation Assay

Test material

Test material information

Constituent 1

Reference substance name:

GSID 3056-1

IUPAC Name:

GSID 3056-1

Test material form:

solid - liquid: suspension

Details on test material:

- Name of test material (as cited in study report): GSID 3056-1
- Physical state: solid, brownish
- Analytical purity: 60.8 area-% (HPLC; analytical report 11L00523)
- Lot/batch No.: 1470 VB04
- Storage condition of test material: room temperature
- Stability under storage conditions over the study period: guaranteed by the sponsor (sponsor holds this responsibility)

In vivo test system

Test animals

Species:

human

Strain:

other: pro-monocytic cell line U937

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

other: not applicable

Vehicle:

other: ethanol and medium

Induction 2

Concentration / amount:

To solubilize the test item in the vehicle (ethanol, final conc. 0.25%), the test item was heated to 90°C.

Challenge

Challenge 1

Route:

other: not applicable

Vehicle:

other: ethanol and medium

Challenge 2

Concentration / amount:

To solubilize the test item in the vehicle (ethanol, final conc. 0.25%), the test item was heated to 90°C.

No. of animals per dose:

not applicable

Details on study design:

PRETEST FOR DOSE SELECTION AND CYTOTOXICITY
The test item was incubated for 48 h as suspension in ethanol and medium at 43.8 µg/mL. Precipitation occurred at this dose level, which was therefore identified as the highest measurable concentration. Regarding cytotoxicity (propidium iodide staining), the highest measurable concentration of 43.8 µg/mL was also found to be the estimated concentration that affords 75% cell viability (CV75) under the chosen exposure conditions with the test item, on U937 cells. Thus, 43.8 µg/mL was retained as the highest test concentration.

TEST CONCENTRATIONS
43.80, 21.90, 10.95, 5.48 and 2.74 µg/mL

TESTING
The pro-monocytic cell line U937 cells were incubated for 48 hours with the different concentrations of the the test item mentioned above. Thereafter, the cells were stained with FITC labeled anti-human-CD 86 antibody and propidium iodide, and the fluorescence intensity was analyzed using flow cytometry.
Propidium iodide staining served for evaluation of the cytotoxicity of the test item.
FITC labeled anti-human-CD 86 antibody was used for evaluation of the surface marker expression as indicator for dendritic cell activating potential.

CONTROLS
The strong sensitizer ethylene diamine (EDA, 70 µg/mL) was used as positive and lactic acid (LA, 200 µg/mL) as non-sensitizing negative control. CD 86 expression was examined after 48 hour of treatment, and the values were compared to historical data.

EVALUATION CRITERIA
A test substance is predicted to have a dendritic cell activating potential when the marker expression exceeded the threshold of 1.2 with respect to vehicle treated cells (VC) at any tested sufficiently non-cytotoxic (cell viability > 70%) concentration in two experiments.

Results and discussion

Positive control results:

70 µg/mL ethylene diamine induced CD86 2.7 fold in the first and 2.0 fold n the second experiment. Viability was only slightly reduced by treatment wit ethylene diamine.

In vivo (non-LLNA)

Results

Reading:

other: CD 86 expression induced in U937 cells after 48 h following treatment

Group:

test chemical

Dose level:

2.74 to 10.95 µg/mL

Clinical observations:

after 48 hours of exposure to the test item, CD 86 expression was induced in U937 cells at concentration between 2.74 and 10.95 µg/mL, affording at least 70% viability. It was concluded that the test item induces dendritic cell activation.

Remarks on result:

other: see Remark

Remarks:

Reading: other: CD 86 expression induced in U937 cells after 48 h following treatment. Group: test group. Dose level: 2.74 to 10.95 µg/mL. Clinical observations: after 48 hours of exposure to the test item, CD 86 expression was induced in U937 cells at concentration between 2.74 and 10.95 µg/mL, affording at least 70% viability. It was concluded that the test item induces dendritic cell activation..

Any other information on results incl. tables

Table1: Results of main experiment

Concentration [µg/mL]	1st experiment		2nd experiment
	CD86 induction	rel. viability	CD86 induction	rel. viability
vehicle control	1.00	100.0	1.00	100.0
2.74	0.94	99.9	1.22	100.0
5.48	1.03	99.9	1.57	99.9
10.95	1.40	99.8	1.29	99.9
21.90	1.04	99.5	0.60	99.7
43.80*	0.18	86.1	0.18	96.3
negative control	0.9	99.9	1.0	100.1
positive control	2.7	93.3	2.0	93.3

Applicant's summary and conclusion

Interpretation of results:

other: not applicable

Conclusions:

After 48 hours of exposure to the test item GSID 3056-1, CD 86 expression was induced in U937 cells at concentrations between 2.74 and 10.95 µg/mL affording at least 70% viability. From this was concluded that GSID 3056-1 induces dendritic cell activation and may be assumed to be sensitizing.