[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 September 2020 - 7 September 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

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:

Reaction products of 1,3-cyclohexanedimethanamine and 12-hydroxystearic acid

Molecular formula:

C44H86N2O4

IUPAC Name:

Reaction products of 1,3-cyclohexanedimethanamine and 12-hydroxystearic acid

Test material form:

solid: particulate/powder

Remarks:

white

Details on test material:

Chemical registery number : 951-139-7
Chemical name : Reaction products of 1,3-cyclohexanedimethanamine and 12-hydroxystearic acid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Adult donors

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize 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:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model(TM)
- Tissue batch number: 20-EKIN-036
- Surface: 0.38 cm^2
- Expiration date: 7 September 2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during exposure: room temperature
- Temperature of incubation: 36.3 - 37.3°C (actual range)
- Humidity: 81 - 95% (actual range)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: tissues were washed with phosphate buffered saline to remove residual test item (1 washing step)
- Observable damage in the tissue due to washing: no

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

NUMBER OF REPLICATE TISSUES: 3 tissues per test item together with negative and positive
controls

NUMBER OF INDEPENDENT TEST SEQUENCES: 1 single run

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
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.

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.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10.6 to 24.3 mg

NEGATIVE CONTROL
- Amount applied: 25 µL PBS

POSITIVE CONTROL
- Amount applied: 25 µL 5% SDS

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of 3 replicates

Value:

110

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

COLOR INTERFERENCE AND MTT
The test item was checked for color interference in aqueous conditions. Addition of the test item to Milli-Q and isopropanol resulted after subtraction of the blank in an OD of -0.0019 and -0.0004, respectively. Therefore, it was concluded that the test item did not induce color interference.
In addition, because no color change was observed in the presence of MTT it was concluded that the test item did not interact with the MTT endpoint.

RESULTS
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 110%. Since the mean relative tissue viability for the test item was above 50% the test item is considered to be non-irritant.

CONTROLS
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 4%. 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 <9%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vitro skin irritation test, performed according to OECD guideline 439 and under GLP principles, Reaction products of 1,3-cyclohexanedimethanamine and 12-hydroxystearic
acid (BA-1801) showed to be non-irritant (mean tissue viability of 110%). 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 Reaction products of 1,3-cyclohexanedimethanamine and 12-hydroxystearic acid (BA-1801) on the viability of human skin was tested. Skin tissue was moistened with 5 μL of Milli-Q water and 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.
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 110%. 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 <9%, indicating that the test system functioned properly.

Since the mean relative tissue viability after exposure to the test substance was above 50%, no classification is required for skin irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).