Amines, C16-18 (even numbered)-alkyl , salts with phosphoric acid, mono- and di-C16-18 (even numbered) 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:

01 September 2020 - 07 September 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2012

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Amines, C16-18 (even numbered)-alkyl , salts with phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters

EC Number:

952-252-4

Molecular formula:

H4NO4P(C32H66) to H3NO4P(C54H111)

IUPAC Name:

Amines, C16-18 (even numbered)-alkyl , salts with phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters

Test material form:

solid

Details on test material:

Chemical registery number : 952-252-4
Chemical name : Amines, C16-18 (even numbered)-alkyl , salts with phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters

Specific details on test material used for the study:

Appearance: Beige solid
Storage conditions: At room temperature

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Adult donors

Details on animal used as source of test system:

EpiSkin TM Small/Human Epidermis
- Batch: 20-EKIN-036
- Cells are screened for potential biological contaminants (HIV1 and 2 antibodies, hepatitis C antibodies and hepatitis B antigen HBs, absence of bacteria, fungus and mycoplasma)
- Suggested expiration date: September 7, 2020

Justification for test system used:

Recommended test system in international guideline (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed.

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

NUMBER OF REPLICATE TISSUES: triplicate

NUMBER OF INDEPENDENT TEST SEQUENCES: One

ACCEPTABILITY CRITERIA
The in vitro skin irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the acceptance limits of OECD439 (lower acceptance limit =0.6 and upper acceptance limit =1.5) and the Standard Deviation value (SD) of the % viability should be =18.
b) The mean relative tissue viability of the positive control should be =40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be =18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be =18.
d) The %NSCliving should be = 30% relative to the negative control OD.

PREDICTION MODEL / DECISION CRITERIA
A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is = 50% of the mean viability of the negative controls.

A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 10.4 to 21.0 mg

NEGATIVE CONTROL
- Amount(s) applied: 25 µL PBS

POSITIVE CONTROL
- Amount(s) applied: 25 µL 5% SDS

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

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

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Relative mean tissue viability

Value:

121

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

COLOR INTERFERENCE
- Instead of MTT solution these tissues were incubated with assay medium. The non-specific color by the test item was 4.46% of the negative control tissues. The OD of the tissues incubated with assay medium was subtracted from the ODs of the test item treated tissues incubated with MTT medium.
The test item was checked for possible direct MTT reduction and color interference. The solutions did not turn blue / purple, nor a blue / purple precipitate was observed. The OD for the test item solution was >0.08. Therefore, it was concluded that the test item did interfere with the MTT endpoint.

RESULTS
- The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 121%. Since the mean relative tissue viability for the test item was above 50% the test item is considered to be non-irritant.

ACCEPTANCE OF RESULTS
- The positive control had a mean cell viability of 4% 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 = 6%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The in vitro skin irritation test was conducted according to OECD 439 guideline and in accordance with GLP principles. It is concluded that this test is valid and that the test substance is not irritant in the in vitro skin irritation test under the experimental conditions described in this report. Therefore,
the substance does not need to be classified according to GHS and CLP criteria.

Executive summary:

In an in vitro skin irritation test using a human skin model (EPISKIN Standard Model), the influence of Amines, C16-18 (even numbered)-alkyl , salts with phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters on the viability of human skin was tested.

At least 10 mg of the test item 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 is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.
The test item induced color interference in aqueous conditions. In addition to the normal procedure, three tissues were treated with test item. Instead of MTT solution these tissues were incubated with assay medium. The non-specific color by the test item was 4.46% of the negative control tissues. The OD of the tissue incubated with assay medium was subtracted from the ODs of the test item treated tissues incubated with MTT medium.
Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 121%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to be non-irritant.
The positive control had a mean cell viability of 4% 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 ≤ 6%, indicating that the test system functioned properly.

It is concluded that the test substance is not irritant in the in vitro skin irritation test under the experimental conditions in this report. Therefore, the substance does not need to be classified according to GHS and CLP criteria.