Fatty acids, C16-18 and C18-unsatd., Me esters, epoxidized, reaction products with ethylene glycol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Principles of method if other than guideline:

CD 86 expression in dentritic cell line as marker for stimulation (Myeloid U937 Skin Sensitization Test, MUSST)

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Dentritic cell activation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C16-C18 and C18-unsatd., ME esters, epoxidized, reaction products with ethylen glycol

EC Number:

606-712-3

Cas Number:

211450-54-3

Molecular formula:

UVCB substance

IUPAC Name:

Fatty acids, C16-C18 and C18-unsatd., ME esters, epoxidized, reaction products with ethylen glycol

Test material form:

liquid: viscous

Details on test material:

Name of test material (as cited in study report): Sovermol 1102
Analytical purity: 100 %

Specific details on test material used for the study:

- Name of test substance: Sovermol 1102
- Test substance No.: 11/0565-1
- Batch identification: CE03250005
- Date of production: Nov 21, 2010

In vitro test system

Details on the study design:

MODEL
- U937 cell line (human dentritic-like cell line)
- Provider: German Resource Center for Biological Material (DSMZ, Germany, catalog no.: ACC 5).

CHOICE OF TEST CONCENTRATION
A pre-test (experimental conduct in accordance with GLP but without a GLP status) was performed. Cells were exposed to 9 concentrations of the test substance (0.5 μg/mL up to 2000 μg/mL) and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. The CV75 value (= estimated concentration that affords at least 75% cell viability) was determined by linear regression from the concentration response curve to be 56 μg/mL. In the main test, test substance was used at six final concentrations determined with regard to the CV75 value: CV75 x 2, CV75 x 1.5, CV75 , CV75/2, CV75/4, CV75/8. After 48 hour exposure U937 cells were stained with FITC labeled anti-human-CD 86 antibody and propidium iodide and the fluorescence intensity was analyzed using flow cytometry.

ANTIBODIES:
Antibodies: FITC Mouse anti-human CD86 (BD Pharmingen) IgG1 FITC CD86 (BD Pharmingen)

EXPOSURE DURATION:
48h

CONCENTRATIONS:
7 - 112 μg/mL (affording at least 70% viability)

VEHICLE:
Culture medium

CONTROLS
True negative control: lactic acid 200 μg/Ml
Positive control: Ethylene diamine (EDA – 70 μg/mL)

EVALUATION CRITERIA
A test substance was 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 at least two independent experiments.

ACCEPTANCE CRITERIA
If the acceptance criteria mentioned below were not met, repetition of the test was considered. A tested concentration is not to be further evaluated when relative viability is less than 70%. A study is considered acceptable if the positive and negative and vehicle control data lie within the range of the historical data. The cell viability of untreated cells must yield at least 90%. The expression marker CD86 of the vehicle control cells should lie between 8% – 20%.

Results and discussion

Positive control results:

Ethylene diamine gave the expected induction of CD86. For details, see 'any other information on results incl tables'

In vitro / in chemico

Results

Remarks on result:

other: see 'any other information on results incl. tables'

Other effects / acceptance of results:

OTHER EFFECTS:
- A solubility experiment was performed. No precipitation in medium occurred, the test substance was a homogeneous suspension in culture medium. The dilutions of the test substance were solutions.
- The substance was active in the MUSST assay.

ACCEPTANCE OF RESULTS:
The level of CD86 expression was not induced after 48 hours treatment with lactic acid (negative control) and was induced after 48 hours exposure to EDA. The level of expression was within the range of the historical negative and positive control data.

Any other information on results incl. tables

Table 1: Viablility assays

Concentration(mg/L)	%PI negative cells, replicate 1	%PI negative cells, replicate 2	%PI negative cells, replicate 3	relative viability
control	98.9	98.8	98.8	100
0.5	99.0	98.6	98.8	99.9
1	99.0	98.7	98.8	100.0
5	98.9	98.7	98.8	99.9
10	98.4	98.5	98.5	99.6
50	80.4	74.8	77.6	78.5
100	53.5	43.8	48.7	49.2
500	39.1	43.1	41.1	41.6
1000	81.7	85.4	83.5	84.5
2000	51.4	39.5	45.5	46.0

PI = propidium iodide

Table 2: CD86 Induction

	experiment 1		experiment 2		experiment 3
concentration (mg/L)	CD86 induction	rel. Viability (%)	CD86 induction	rel. Viability (%)	CD86 induction	rel. Viability (%)
VC	1.0	100.0	1.0	100.0	1.0	100.0
7.0	0.8	99.9	1.7	100.2	0.9	99.9
14.0	0.8	99.2	1.7	99.6	0.9	99.6
28.0	0.8	97.2	1.3	97.4	0.8	98.4
56.0	1.9	79.7	1.4	92.2	1.3	91.0
112.0	1.4	34.0	2.9	46.1	1.4	56.6

Applicant's summary and conclusion

Interpretation of results:

other: activation of dentritic cells in vitro