Poly(oxy-1,2-ethanediyl), α-(carboxymethyl)-ω-hydroxy-, C16-18 (even numbered) and C18-unsatd. alkyl ethers

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2013-12-02 to 2014-01-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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:

26 July 2013

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:

20 July 2012

Deviations:

no

Principles of method if other than guideline:

Temporary deviations from the temperature and humidity in the incubator (time not exceeding 1 hour) are considered not to affect the study integrity based on laboratory historical data.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 004

Cas Number:

288259-40-5

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Dihydrogen oxide

Constituent 3

Reference substance name:

Alcohols, C12-16

EC Number:

272-490-6

EC Name:

Alcohols, C12-16

Cas Number:

68855-56-1

Molecular formula:

Mixture of homologues

IUPAC Name:

tetradecan-1-ol

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 30467967
- Expiration date of the lot/batch: 2015-02-28
- Purity: 93,5%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2-8°C) in the dark

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no correction for purity/composition
- Preliminary purification step (if any): no
- Final dilution of a dissolved solid, stock liquid or gel: The liquid test substance was applied undiluted (25 μI) directly on top of the tissue.

In vitro test system

Test system:

human skin model

Remarks:

EPISKIN Small Model™ (EPISKIN-SM™, 0.38 cm2, Batch no.: 14-EKIN-001

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model™
Three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Tissue batch number(s): 14-EKIN-001
- Production date: 2014-01-21
- Expiration date: 2014-01-27
- Date of initiation of testing: 2014-01-27

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 15 minutes at room temperature
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C (actual range 29.6 - 37.°C) for 42 h

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance.
- Observable damage in the tissue due to washing: not mentioned
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL in PBS
- Incubation time: 3 h at 37°C
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Morphology: Well-differentiated epidermis consisting of 3 basal layer, several spinous und granular layers and a thick stratum corneum
- Contamination: no


NUMBER OF REPLICATE TISSUES:
total of 3 tissues per test substance together with negative and positive controls

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is - The test substance is considered to be non-irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µL
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL
- Concentration (if solution): undiluted

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL
- Concentration (if solution): 5%

Duration of treatment / exposure:

15 minutes
The positive control was re-spread after 7 minutes contact time

Duration of post-treatment incubation (if applicable):

42 hours at 37°C

Number of replicates:

The test was performed on a total of 3 tissues per test substance together with negative and positive controls.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

test substance: mean of triplicates

Value:

87

Negative controls validity:

valid

Remarks:

set to 100%

Positive controls validity:

valid

Remarks:

27 % of control

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
The relative mean tissue viability obtained after 15 minutes treatment with the testsubstance compared to the negative control tissues was 87%.
Since the mean relative tissue viability for the testsubstance was above 50% the testsubstance is considered to be non-irritant.
The positive control had a mean cell viability after 15 minutes exposure of 27%.
The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.
The standard deviation value of the percentage viability of three tissues treated identically was 6 to 15%, indicating that the test system functioned properly.

- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline:
-negative control range (OD): 0.576 - 1.994 (mean 0.91 ± 0.17; n=171)
-positive control range (OD): 0.020 - 0.408 (mean 0.10 ± 0.07; n=170)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The testsubstance is not irritating in the in vitro skin irritation test under the experimental conditions described in this report.

Executive summary:

In a dermal irritation study performed in accordance with OECD Guideline 439 (In Vitro Skin Irritation) (26 July 2013) and EU method B.46 (In vitro skin irritation: reconstructed human epidermis model test) (20 July 2012), the test substance Polyoxyethylene(4.5) lauryl miristyl ether carboxylic acid (82%) was applied to the three-dimensional human epidermis model tissue (EPISKIN Small Model (EPISKIN-SM, 0.38 cm², Batch no.: 14-EKIN-001) for an exposure period of 15 minutes. The number of replicate tissues was three.

The test substance was applied undiluted in an amount of 25 µl directly on top of the skin tissue. After 15 minutes exposure at room temperature, the tissues were washed with phosphate buffered saline. Subsequently the tissue constructs were incubated for 42 h at 37°C. Cytotoxicity (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The positive (5% SDS) and negative (PBS) control gave responses that were within the acceptance criteria and as such demonstrated the validity of the study.

The relative mean tissue viability obtained after 15 minutes treatment with Polyoxyethylene(4.5) lauryl miristyl ether carboxylic acid compared to the negative control tissues was 87%. Since the mean relative tissue viability for the test substance was above 50%, Polyoxyethylene(4.5) lauryl miristyl ether carboxylic acid (82% a.i.) is identified to be not irritating.

 

Data generated by fully validated in-vitro methods can be used for REACH purposes provided that the information for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

Based on the result of the in-vitro skin irritation study, Polyoxyethylene(4.5) lauryl miristyl ether carboxylic acid as neat substance would be Not Classified (NC) for skin irritation.