Fatty acids, tall-oil, esters with ethylene glycol

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 - 11 Jun 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted Jun 2018

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2012

Deviations:

not specified

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, tall-oil, esters with ethylene glycol

EC Number:

269-130-5

EC Name:

Fatty acids, tall-oil, esters with ethylene glycol

Cas Number:

68187-85-9

Molecular formula:

C38H66O4 C38H64O4 C40H68O4 C40H70O4 C38H68O4

IUPAC Name:

2-[(9Z)-octadec-9-enoyloxy]ethyl (9Z)-octadec-9-enoate; 2-[(9Z)-octadec-9-enoyloxy]ethyl (9Z,12Z)-octadeca-9,12-dienoate; 2-[(9Z,12Z)-octadeca-9,12-dienoyloxy]ethyl (9Z,12Z)-octadeca-9,12-dienoate

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EPISKIN Small Model™

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model™
- Tissue batch number(s): 18-EKIN-023, 18-EKIN-015 and 18-EKIN-022

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: at room temperature
- Temperature of post-treatment incubation: at 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: The tissues were washed with phosphate buffered saline to remove residual test substance.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Barrier function: According to the Supplier`s Data Sheet, the IC50 value was determined to be 2.1 and 2.2 mg/mL for the tissues with the batch numbers of 18-EKIN-023 and 18-EKIN-015 (acceptance criteria: 1.5 ≤ IC50 ≤ 3.0), respectively. The result of the tissue with the batch number 18-EKIN-022 was invalid.

NUMBER OF REPLICATE TISSUES: 3 tissues were used per treatment, negative and positive control, respectively.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues : Since the test item reacted with the MTT medium, in addition three killed tissues treated with test item and three killed untreated tissues were used for the cytotoxicity evaluation with MTT (Batch numbers: 18-EKIN-015 and 18-EKIN-022).
- Procedure used to prepare the killed tissues: Living epidermis was transferred to 12 well plates and incubated with 2 mL Milli-Q for 48 ± 1 h. After incubation, killed epidermis was stored at ≤ -15°C. Killed tissues were thawed by placing them for 1 h at room temperature in 12 well plates on 2 mL maintenance medium. Further use of killed tissues was similar to living tissues.
- N. of replicates: 3
- Method of calculation used:
Nonspecific MTT reduction (NSMTT) was calculated. NSMTT is the difference between the mean OD of the untreated killed tissues (ODkt_u+MTT) and test item treated killed tissues (ODkt_t+MTT) expressed as percentage of the mean of the negative control tissues (ODlt_u+MTT).
%NSMTT = [(ODkt_t+MTT – ODkt_u+MTT)/ mean ODlt_u+MTT] * 100

True tissue viability is calculated as the difference between the living test item treated tissues incubated with MTT medium (ODlt_t+MTT) and the difference between ODkt_t+MTT and ODkt_u+MTT.
OD= ODlt_t+MTT – (ODkt_t+MTT-ODkt_u+MTT)
%Viability = [OD/ mean ODlt_u+MTT] * 100

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: Duplicate measurements each of 3 independent tissues

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant or corrosive (category 1 or 2) to skin if after 15 min exposure and 42 h of post incubation is less than or equal to 50%.
- The test substance is considered to be non-irritant (no category) to skin if after 15 min exposure and 42 h of post incubation is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 µL

NEGATIVE CONTROL
- Amount(s) applied: 25 µL

POSITIVE CONTROL
- Amount(s) applied: 25 µL
- Concentration: 5% (aq) sodium dodecyl sulfate in phosphate buffered saline

Duration of treatment / exposure:

15 ± 0.5 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

Duplicate measurements each of 3 independent tissues

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 ± 0.5 min exposure with the test item

Value:

69

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction and colour interference with MTT: The test item was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because a color change was observed by adding MTT-medium it was concluded that the test item did interact with the MTT endpoint. In addition to the normal procedure, three killed tissues treated with test item and three killed non-treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test item was 0.64% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test item treated viable tissues.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the OD values of the three independent tissues for the negative control were 1.3171, 1.2761, 1.0732, 1.0738, 1.0012 and 0.9437, and thus within the range of the istorical control data (0.422 - 1.547).
- Acceptance criteria met for positive control: Yes, the positive control reduced the cell viability at 3.6 % and fulfilled the acceptance criteria.
- Acceptance criteria met for variability between replicate measurements: Yes, differences of three tissue cell viabilities in the negative control substance, the positive control substance and the test substance groups were 15%, 14% and 0.8%, respectively, and thus ≤ 18%.

Any other information on results incl. tables

Table 1: Individual absorption values

	Tissue A	Tissue B	Tissue C
	(OD570)	(OD570)	(OD570)
Negative control measurement 1	13.171	10.732	10.012
Negative control measurement 2	12.761	10.738	0.9437
Test item on viable tissue OD570measurement 1	0.8058	0.9558	0.6488
Test item on viable tissue OD570measurement 2	0.7956	0.9267	0.6155
Test item on killed tissue OD570measurement 1	0.1190	0.1076	0.1505
Test item on killed tissue OD570measurement 2	0.1203	0.1197	0.1391
Non treated killed tissue OD570measurement 1	0.0993	0.1442	0.1296
Non treated killed tissue OD570measurement 2	0.0939	0.1317	0.1166
Positive control OD570measurement 1	0.0807	0.0874	0.0726
Positive control OD570measurement 2	0.0826	0.0924	0.0716

OD: Optical density

SD: Standard Deviation

1) The test item values were corrected fot the non-specific MTT reaction (0.64%)

Table 2: Mean tissue viability (%)

	Mean tissue viability (% of control)	Standard deviation (%)
Negative control	100	15
Test item	69	14
Positive control	3.6	0.8

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008