Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

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:

equivalent or similar 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, C14-15 branched and linear alkyl esters, potassium salts

EC Number:

812-497-9

Cas Number:

1893414-79-3

Molecular formula:

n/a for an UVCB substance

IUPAC Name:

Phosphoric acid, C14-15 branched and linear alkyl esters, potassium salts

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

solid: crystalline

Specific details on test material used for the study:

Name: Phosphoric acid, C14-15 branched and linear alkyl esters, potassium salts
CAS No.: 1893414-79-3
Physical state: white solid at 20 °C
Batch No.: PU61810016
Re-certification date of batch: 09 March 2018
Purity: 100 % (UVCB, lyophilized solid, water content 0.85 % (w/w))
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: adult donors

Source strain:

not specified

Justification for test system used:

This test method is able to detect chemicals that cause skin irritation, i.e. produce reversible damage to the skin and allows for hazard identification in accordance with UN GHS “Category 2”. Depending on the regulatory framework it can also be used to identify non-classified chemicals.

Vehicle:

unchanged (no vehicle)

Details on test system:

In the present study the skin irritant potential of Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts was analysed. The EPISKIN-Standard Model (EPISKIN-SMTM), a reconstituted three-dimensional human epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404) to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as non-irritant.

Control samples:

yes, concurrent negative control

Amount/concentration applied:

10 µL (undiluted)
The test was performed on a total of 3 tissues per dose group.

Duration of treatment / exposure:

15 min of exposure

Duration of post-treatment incubation (if applicable):

42 h of post-incubation

Number of replicates:

The test was performed on a total of 3 tissues per dose group.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

92.6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

10 µL DPBS

Positive controls validity:

valid

Remarks:

10 µL 5% SDS solution

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

98.7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

10 µL DPBS

Positive controls validity:

valid

Remarks:

10 µL 5% SDS solution

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

105.1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

10 µL DPBS

Positive controls validity:

valid

Remarks:

10 µL 5% SDS solution

Remarks on result:

no indication of irritation

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

98.8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Acceptance Criteria
Value Cut off pass/fail
Mean OD570 nm Blank 0.042 < 0.1 pass
Mean Absolute OD570 nm NC 0.857 0.6 ≤ NK ≤1.5 pass
Mean Relative Viability [%] PC 13.0 ≤ 40% pass
Max. SD of % Viability [%] 3.9 – 11.4 ≤ 18% pass

Acceptance criteria of the OECD test guideline were fulfilled.

Validity controls as provided by the supplier (SkinEthic):

Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.

Morphology:
Histology scoring (HES stained vertical paraffin sections, n = 6):
Specification ≥ 19.5
Result: 21.8 ± 0.7, CV = 3.1%

Barrier function:
IC50 determination (SDS concentration, MTT test, n = 14):
Specification ≥ 1.5 mg/mL
Result: 2.4 mg/mL

Any other information on results incl. tables

Pre-experiment

The mixture of 10 µL test item per 2 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equaled 0%.

The mixtures of 10 µL of the test item per 90 µL aqua dest. and per 90 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSCliving equaled 0%

Experiment

The potential of the test item to induce skin irritation was analysed by using the three-dimensional human skin model EPISKIN-SM (SkinEthic) comprising a reconstructed epidermis with a functional stratum corneum. In the present study Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts was applied topically to the EPISKIN-SM tissue for 15 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay.

Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (98.8%) after 15 min treatment and 42 h post-incubation.

The controls confirmed the validity of the study. The mean OD570 of the six blank values was < 0.1. The mean absolute OD570 of the three negative control tissues was between 0.6 and 1.5. The mean relative tissue viability (% negative control) of the positive control was less than 40% (13.0%). The maximum standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (3.9% - 11.4%).

Table 1. Result of the Test Item Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts

Name	Negative Control	Positive control	Test item
Mean Relative Tissue Viability [%]	100.0	13.0**	98.9
SD Tissue Viability [%]*	3.9	11.4	6.3
CV [% Viability]	3.9	87.4	6.4

* Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%.

** Mean relative tissue viability of the three positive control tissues is ≤ 40 %.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed no irritant effects. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

Executive summary:

The potential of the test item to induce skin irritation was analysed by using the three-dimensional human skin model EPISKIN-SM (SkinEthic) comprising a reconstructed epidermis with a functional stratum corneum. In the present study Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts was applied topically to the EPISKIN-SM tissue for 15 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay. Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (98.8%) after 15 min treatment and 42 h post-incubation. The controls confirmed the validity of the study.