Phosphoric acid, mono- and di-C16-20-(even numbered, branched and linear)-alkyl esters

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 September 05, 2016 to September 09, 2016

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

Test material

Test material information

Constituent 1

Reference substance name:

Phosphoric acid, mono- and di- (C16-20 - (even numbered, branched and linear)-alkyl esters

IUPAC Name:

Phosphoric acid, mono- and di- (C16-20 - (even numbered, branched and linear)-alkyl esters

Constituent 2

Reference substance name:

Isooctadecan-1-ol

EC Number:

248-470-8

EC Name:

Isooctadecan-1-ol

Cas Number:

27458-93-1

Molecular formula:

C18H38O

IUPAC Name:

Isooctadecan-1-ol

Test material form:

liquid

Specific details on test material used for the study:

- Name of test material (as cited in study report): Phosphoric acid, mono- and bis (C16-20 branched and linear alkyl) esters
- Batch: CH 200529/001
- Composition: UVCB
- Appearance: Lightly yellow liquid

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:

Skin tissue was moistened with 5 µl of Milli-Q water and at least 10 mg of the test substance was applied directly on top of the skin tissue for 15 ± 0.5 minutes. 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. Skin irritation was expressed as the remaining cell viability after exposure to the test substance.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 µL

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42h

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

cytotoxicity

Run / experiment:

Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT

Value:

99

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

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 99%. Since the mean relative tissue viability for the test substance was above 50%, it was considered to be non-irritant.
- The positive control had a mean cell viability of 5.3% 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 12% or less, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

other: CLP criteria not met

Remarks:

not classified

Conclusions:

Under the study conditions, the test substance was considered to be non-irritant to human skin in the in vitro skin irritation test.

Executive summary:

A study was conducted to determine the in vitro skin irritation potential of the test substance according to OECD Guideline 439 and EU Method B.46 (Reconstructed Human Epidermis Test Method), in compliance with GLP. Human three dimensional epidermal models (triplicates) were exposed to 25µL test substance for 15 min. After a 42 hour post-incubation period, a 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. Skin irritation was expressed as the remaining cell viability after exposure to the test substance. The positive control (25 µL 0.5% SDS) had a mean cell viability of 5.3% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control (25 µL PBS) tissues was within the laboratory historical control data range. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test substance compared to the negative control tissues was 99%. The standard deviation value of the percentage viability of three tissues treated identically was less than 16%, indicating that the test system functioned properly. Th experiment was considered valid. Since the mean relative tissue viability for the test substance was above 50% after 15 ± 0.5 minutes treatment, the test substance was considered to be non-irritant to human skin under the study conditions (Eurlings, 2016).