Rosetal A

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 October 2020 - 30 October 2020

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

[2,2-bis[(3,7-dimethyl-2,6-octadienyl)oxy]ethyl]benzene

EC Number:

266-805-6

EC Name:

[2,2-bis[(3,7-dimethyl-2,6-octadienyl)oxy]ethyl]benzene

Cas Number:

67634-02-0

Molecular formula:

C28H42O2

IUPAC Name:

(2,2‐bis(((E)‐3,7‐dimethylocta‐2,6‐dien‐1‐yl)oxy)ethyl)benzene

Test material form:

liquid

Specific details on test material used for the study:

No correction was made for the purity/composition of the test item.
The test item was stored at room temperature in a container flushed with nitrogen.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

EpiDerm Skin Model:
- All cells used to produce Epiderm™ are purchased or derived from tissue obtained by MatTek Corporation from accredited institutions.
- Cells are screened for potential biological contaminants (HIV-1, Hepatitis B, Hepatitis C, bacteria, yeast and fungi)

Justification for test system used:

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

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Tissue lot number: 33746, kit G
- Surface: 0.6 cm²
- The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment at 37.0 ± 1.0°C (actual range 36.6 - 37.3°C).

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2 replicates per exposure duration, two negative controls per exposure duration and two positive controls per exposure duration

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Since the test item was found to possibly interact with the MTT measurement by color interference in aqueous conditions, two additional tissues were treated with test item for 3 minutes and two additional tissues for 1-hour. Instead of MTT solution these tissues were incubated with DMEM.

ACCEPTABILITY CRITERIA:
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
c) In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.
d) The %NSC should be ≤ 30% relative to the negative control OD.

INTERPRETATION
A test item is considered corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.

A test item is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

See Table 1 (Any other information on methods inc. tables)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The liquid test item was applied undiluted (50 μL)

Duration of treatment / exposure:

3 minutes or 1 hour

Number of replicates:

2 replicates per exposure duration

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute exposure

Value:

101

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour exposure

Value:

94

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

MTT INTERFERENCE
- In addition to the normal 3-minute and 1-hour procedure, two tissues were treated with test item. Instead of MTT solution these tissues were incubated with DMEM. The color interference by the test item was 0.08% and 0.16% of the negative control tissues after 3 minutes and 1 hour respectively. The test item values were corrected for color interference using the relevant value.

RESULTS
- Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 101% and 94%, respectively. Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment the test item is considered to be not corrosive.

ACCEPTANCE OF RESULTS
- The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range.
- The mean relative tissue viability following the 1-hour exposure to the positive control was 11%.
- In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 15%, indicating that the test system functioned properly

Any other information on results incl. tables

Table 2. Mean Absorption in the in vitro Skin Corrosion Test with Rosetal A BHT

	3-minute application					1-hour application
	A (OD570)	B (OD570)	Mean (OD570)		SD	A (OD570)	B (OD570)	Mean (OD570)		SD
Negative control	1.692	1.830	1.761	±	0.098	1.802	1.837	1.820	±	0.025
Test item ^(1)	1.805	1.756	1.780	±	0.035	1.856	1.578	1.717	±	0.197
Positive control	0.216	0.140	0.178	±	0.054	0.149	0.245	0.197	±	0.068

SD = Standard deviation

Duplicate exposures are indicated by A and B.

⁽¹⁾ The test item values are corrected for color interference (0.08% and 0.16% after 3 min and 1 hr exposure, respectively).

In table 1 the values are corrected for background absorption (0.0436). Isopropanol was used to measure the background absorption.

 

Table 3. Mean Tissue Viability in the in vitro Skin Corrosion Test with Rosetal A BHT

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
Test item	101	94
Positive control	10	11

 

Table 4. Coefficient of Variation between Tissue Replicates

	3 minute	1 hour
Negative control	7.6	1.9
Test item	2.7	15
Positive control	35	39

CV (%) = 100 - [(lowest OD₅₇₀/highest OD₅₇₀) x 100%]

Applicant's summary and conclusion

Interpretation of results:

other: Not skin corrosive in accordance with EU CLP (EC no 1272/2008 and its amendments)

Conclusions:

The substance does not cause skin corrosion.

Executive summary:

In an in vitro skin corrosion test using a human skin model (EpiDerm Skin Model) performed according to OECD TG 431 and GLP principles, the influence of the test substance on the viability of human skin was tested. Fifty μL test substance was applied directly on top of 0.6 cm2 cultured skin. The positive control had a mean relative tissue viability of 11% after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 15%, indicating that the test system functioned properly.

The test item showed color interference in aqueous conditions. The OD of the treated tissues without MTT assay was subtracted from the ODs of the test item treated viable tissues with MTT assay.

The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 101% and 94%, respectively. Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive to skin.