1-methoxy-4-[3-methyl-4-(2-phenylethoxy)but-3-en-1-yl]benzene

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28-01-2021 to 08-02-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP with minor deviations. All relevant validity criteria were considered to be met.

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:

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

Deviations:

yes

Remarks:

Minor deviation in environmental conditions (humidity minimum 68%) ; 25 µL test item, negative control, positive controls were applied, respectively rather than 10 µL. These protocol deviations were not considered to affect the reliability of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Minor deviation in environmental conditions (humidity minimum 68%) ; 25 µL test item, negative control, positive controls were applied, respectively rather than 10 µL. These protocol deviations were not considered to affect the reliability of the study.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1-methoxy-4-[3-methyl-4-(2-phenylethoxy)but-3-en-1-yl]benzene

EC Number:

954-543-1

Cas Number:

2489703-47-9

Molecular formula:

C20H24O2

IUPAC Name:

1-methoxy-4-[3-methyl-4-(2-phenylethoxy)but-3-en-1-yl]benzene

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: In refrigerator (2-8°C) protected from light container flushed with nitrogen
- Other: colourless liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN TM Small Model
- Tissue batch number(s): Lot no.: 21-EKIN-005
- Production date: Not reported.
- Shipping date: Not reported. Although the CoA for the tissues QA is provided in the full study report.
- Delivery date: 27-01-2021 ; day prior to test initiation (ca. 24 hours)
- Date of initiation of testing: ca. 28-01-2021

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.0 °C, 5 ± 0.5% CO2 (actual: 35.6 – 37.0°C)
- Temperature of post-treatment incubation (if applicable): 37 ± 1.0 °C, 5 ± 0.5% CO2; for 42 hours. For further information see below.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After the exposure period of 15 ± 0.5 minutes at room temperature, 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. Subsequently the skin tissues were incubated for 42 hours at 37°C. For the cell viability measurements: cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labelled microtubes and extracted with 500 μL isopropanol. Tubes were stored refrigerated and protected from light for approximately 68 hours. The amount of extracted formazan was determined spectrophotometrically.
- Observable damage in the tissue due to washing: Not applicable.
- Modifications to validated SOP: Not applicable.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 3.0 mg/mL MTT concentrated diluted (x10) to 0.3 mg/mL MTT solution prepared in assay medium
- Incubation time: For the cell viability measurements: cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labelled microtubes and extracted with 500 μL isopropanol. Tubes were stored refrigerated and protected from light for approximately 72 hours. The amount of extracted formazan was determined spectrophotometrically.
- Spectrophotometer: M200 Pro Plate Reader
- Wavelength: 570 nm
- Filter: Without reference filter
- Filter bandwidth: No reported.
- Linear OD range of spectrophotometer: Not reported.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Within NC and PC historic control ranges presented within the report.
- Barrier function: See above.
- Morphology: See above.
- Contamination: Not applicable.
- Reproducibility: The relative mean tissue viability for the positive control treated tissues was 6.6% relative to the negative control following the 15-minute exposure/42-hour incubation period. With standard deviation of 5.3%. The positive control acceptance criterion was therefore satisfied. The negative control triplicate treated tissues mean OD570 was 1.239 and the standard deviation of viability was 7.4%. The acceptance criterion was therefore satisfied. The test item triplicate treated tissues viability standard deviation was 9.7%. The mean OD570 for both the negative and positive controls were within the historical control ranges.
- Other: As an additional quality control step, the results of the assay are compared to the historical ranges for negative and positive controls generated by the testing laboratory to confirm the functioning of the test system.

NUMBER OF REPLICATE TISSUES: Three (3), triplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: In the pretest for colour interference: the solution containing the test item was colourless. It was therefore unnecessary to run colour correction tissues. In the pre-test for direct MTT reduction: The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT. It was unnecessary to run killed tissues.
- Procedure used to prepare the killed tissues (if applicable): Not applicable.
- N. of replicates : Not applicable.
- Method of calculation used: Not applicable.
- Other: In the assessment of Colour Interference with the MTT endpoint, the solution containing the test item did not become coloured. This was taken to indicate the test item did not have the potential to cause colour interference.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One (n=1)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating to skin if e.g. the viability after 15-minutes exposure / 42-hours incubation is less than or equal to 50%,.
- The test substance is considered to be non-irritating to skin if e.g. the viability after 15-minutes exposure / 42-hours incubation is greater than 50%.
- Cut off points in accordance with OECD TG 439 and the GHS and CLP Classification systems.
- Skin irritation is expressed as the remaining cell viability after exposure to the test substance at exposure times 15-minutes / 42-hours incubation. Where necessary, direct MTT reduction, colour interference modifications were completed.

OTHER:
EpiSkin RHE Small Model (Lot no.: 21-EKIN-005). The test consists of topical application of the test item -one on the skin tissue for 15 minutes. After exposure the skin tissue is thoroughly rinsed to remove the test item and transferred to fresh medium. After a 42 hour incubation period, determination of the cytotoxic (irritancy) effect is performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment. The EPISKIN Small Model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum. Pre-test checks for Direct MTT reduction and Colour Interference were completed.

Preincubation:
On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for 22.5 hours at 37°C. Maintenance medium and Assay medium were supplied by a recognised supplier (documented in the full study report).

Application of test item and rinsing:
Test item: Tissues were treated with 25 µL test item for 15 minutes exposure period. Negative control: 25 µL PBS (negative control) was similarly applied. Positive control: 25 µL SDS (5% w/v) was respread after 7 minutes contact time to maintain distribution of the SDS - for the remainder of the contact period. This was not deemed necessary in the negative control or test item definitive test group. Minor deviation : 25 µL test item, negative control, positive controls were applied (ca. 65 µL/cm2), respectively rather than 10 µL (ca. 26 µL/cm2). These protocol deviations were not considered to affect the reliability of the study. After the exposure period of 15 ± 0.5 minutes at room temperature, 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. Subsequently the skin tissues were incubated for 42 hours at 37°C. All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 68 – 93%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.6 - 37.0°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on testing laboratory historical data these deviations are not considered significant.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µL
- Concentration (if solution): undiluted
- Other: Minor deviation : 25 µL test item, negative control, positive controls were applied (ca. 65 µL/cm2), respectively rather than 10 µL (ca. 26 µL/cm2). These protocol deviations were not considered to affect the reliability of the study

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL
- Concentration (if solution): Not applicable. (Phosphate buffered saline, pre-diluted)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL
- Concentration (if solution): 5% SDS (pre-diluted)

Duration of treatment / exposure:

15 ± 0.5 minutes at room temperature, after which the tissues were washed with phosphate buffered saline to remove residual test item.

Duration of post-treatment incubation (if applicable):

Subsequently the skin tissues were incubated for 42 hours at 37°C.

Number of replicates:

Triplicate; treatment and concurrent negative control and positive control groups

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean (n=3)

Value:

106

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Basis: mean. Time point: 15 minute exposure. Remarks: n=3; SD = 9.4% ; Score in terms of percentage of negative control.

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None reported.
- Direct-MTT reduction: Screened prior to test. Not applicable.
- Colour interference with MTT: Screened prior to test. Not applicable.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory previously demonstrated technical proficiency of the validated method with proficiency test items (information in the public domain). Additionally, concurrent positive control and negative controls were within the current historic control range (HCD) (documented in the full study report), each meeting the validity criteria respectively.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Acceptance criteria met for variability between replicate measurements: Yes.
- Range of historical values if different from the ones specified in the test guideline: Historic Control Data are presented in the full study report. All values for the control groups were within the ranges of the current Historic Control Data. Historic Control Data (n=147): the mean OD of the positive control was 0.108 ; range 0.026 to 0.549. In this same period the mean OD of the negative control was 1.020 ; range 0.507 – 1.426.

Any other information on results incl. tables

Table 1. Mean absorption and tissue viability in the in vitro skin irritation test with the test item

Item	OD570 of tissues	Mean OD570 of triplicate tissues	± SD of OD570	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	1.134	1.239	0.092	100*	7.4
	1.295
	1.289
Positive Control Item	0.033	0.082	0.066	6.6	5.3
	0.056
	0.157
Test Item	1.268	1.313	0.120	106	9.7
	1.221
	1.449

OD = Optical Density

SD = Standard deviation

* = The mean viability of the negative control tissues is set at 100%.

Values are corrected for background absorption (0.042). Isopropanol was used to measure background adsorption.

Negative control: Phosphate buffered saline (PBS)

Positive control: 5% (aq.) Sodium dodecyl sulphate (SDS)

 

The relative mean tissue viability for the positive control treated tissues was 6.6% relative to the negative control following the 15-minute exposure/42-hour incubation period. With standard deviation of 5.3%. The positive control acceptance criterion was therefore satisfied. The negative control triplicate treated tissues mean OD570 was 1.239 and the standard deviation of viability was 7.4%. The acceptance criterion was therefore satisfied. The test item triplicate treated tissues viability standard deviation was 9.7%. The mean OD570 for both the negative and positive controls were within the historical control ranges. All assay acceptance criteria were met.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the test item is not considered to be irritating to the skin.

Executive summary:

The study was performed to OECD TG 439 and EU Method B.46 to assess the skin irritation potential of the test item in accordance with GLP using a human three dimensional epidermal model (EPISKIN Small Model). The test was performed on a total of 3 tissues per test item together with negative and positive controls. 25 μL of the undiluted test item was added (with a pipette) into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 μL 5% SDS (positive control), respectively. The positive control was re-spread after 7 minutes contact time. Use of 25 µL test item, negative control and positive controls (ca. 65 µL/cm2), respectively rather than 10 µL (ca. 26 µL/cm2) protocol deviation was not considered to affect the reliability of the study. After the exposure period of 15 ± 0.5 minutes at room temperature, 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. Subsequently the skin tissues were incubated for 42 hours at 37°C. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. The positive control had a mean cell viability of 6.6% after 15 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 relative mean tissue viability obtained after 15 minutes treatment with the test item compared to the negative control tissues was 106%. The standard deviation value of the percentage viability of three tissues treated identically was ≤ 9.7%, indicating that the test system functioned properly. Since the mean relative tissue viability for the test item was above 50% after 15 minutes treatment it is not considered to be irritant. Under the conditions of this study the test item is not irritant to the skin.