Phosphoric acid, hexadecyl phosphate esters, potassium salts with cetostearyl alcohol

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 16, 2013 to July 22, 2013

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Phosphoric acid, hexadecyl phosphate esters, potassium salts with cetostearyl alcohol

Molecular formula:

C16H34O4P1K1 (representative: mono- C16 PSE, K+) C16H34O1 (representative alcohol: C16 alcohol)

IUPAC Name:

Phosphoric acid, hexadecyl phosphate esters, potassium salts with cetostearyl alcohol

Test material form:

solid: granular

In vitro test system

Test system:

human skin model

Remarks:

human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM))

Cell type:

non-transformed keratinocytes

Justification for test system used:

The test is based on the experience that irritant chemicals show cytotoxic effects following short term exposure to the stratum corneum of the epidermis. The test was designed to predict and classify the skin irritation potential of a test substance by assessment of its effect on a three dimensional human epidermis model. In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

- Triplicates skin tissues were moistened with sterile distilled water and then treated with 10 mg test substance for 15 ± 0.5 minutes. Negative (PBS) and positive (SDS 5%) controls were included as well in the experiment. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment (optical density measurement). Skin irritation was expressed as the remaining cell viability after exposure to the test substance.
- Possible inflammatory mediator (IL-1a) was also determined.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 mg

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Expressed as the reduction of mitochondrial dehydrogenase activity

Value:

ca. 108

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100% relative mean viability

Positive controls validity:

valid

Remarks:

19.5% relative mean viability

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- Skin irritation was expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test substance compared to the negative control tissues was 108.0%. Since the mean relative tissue viability for the test substance was above 50%, it was considered to be non-irritant. It was unnecessary to perform IL-1a analysis as the results of the MTT test were unequivocal.
- The positive control had a mean cell viability of 19.5% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) 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 2.0% or less, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

other: not classified based on EU CLP criteria

Conclusions:

Under the study conditions, the test substance was determined to be non-irritating to the skin.

Executive summary:

An in vitro study was conducted to determine the skin irritation potential of the test substance, 'mono- and di- C16 PSE, K+ and C16-18-OH', using Reconstructed Human Epidermis Test Method – EpiskinTM, according to OECD Guideline 439 and EU Method B.46, in compliance with GLP. Triplicates skin tissues were moistened with sterile distilled water and then treated with 10 mg test substance for 15 ± 0.5 minutes. Positive (5% Sodium Dodecyl Sulphate) and negative (Phosphate Buffered Saline Dulbecco's) controls were included in the experiment as well. After a 42 h post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment (optical density measurement). Skin irritation was expressed as the remaining cell viability after exposure to the test substance. Possible inflammatory mediator (IL-1a) was also determined. The relative mean tissue viability obtained after 15 minutes treatment with the test substance compared to the negative control tissues was 108.0%. Since the mean relative tissue viability for the test substance was above 50%, it was considered to be non-irritant. It was unnecessary to perform IL-1a analysis as the results of the MTT test were unequivocal. The positive control had a mean cell viability of 19.5% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) 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 2.0% or less, indicating that the test system functioned properly. The study was considered to have met all the validity criteria. Under the study conditions, the test substance was determined to be non-irritating to the skin (Harlan, 2014).

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