4'-methoxyacetophenone

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-05-05 to 2017-10-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

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:

according 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:

4'-methoxyacetophenone

EC Number:

202-815-9

EC Name:

4'-methoxyacetophenone

Cas Number:

100-06-1

Molecular formula:

C9H10O2

IUPAC Name:

1-(4-methoxyphenyl)ethanone

Specific details on test material used for the study:

Chemical Name: 4’-Methoxyacetophenone
CAS No.: 100-06-1
EC No.: 202-815-9
Batch: 10300067
Expiry Date: 30 May 2018
Appearance: Pale white to white crystals
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: The EpiDerm™ (82105 Bratislava, Slovakia) tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis.

Source strain:

other: not applicable; human-derived epidermal keratinocytes

Details on animal used as source of test system:

Not applicable

Justification for test system used:

Because systemic reactions play a minor role in modulating local skin toxicity potential of chemicals, skin irritation potential may be predicted by in vitro systems, provided they are sufficiently complex to mimic human skin barrier and cell reactivity. In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDerm™ and EpiSkin™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN DISC PREPARATION
- Procedure used: Epi-200 SIT kits and MTT-100 assay diluent purchased from MatTek Corporation (82105 Bratislava, Slovakia). On day of receipt the pre-incubation phase of the EpiDerm™ tissues started.
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C
- Temperature of post-treatment incubation: 37 ± 1.5 °C
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 3; after treatment, tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least three times. Afterwards the inserts were once again rinsed with sterile DPBS from the inside and the outside.
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No, there was no alteration from the MatTek test protocol.
DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Microplate reader (Versamax® Molecular Devices, Softmax Pro, version 4.7.1)
- Filter: Yes, 570 nm filter
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2 experiments; mean values were calculated from the 3 wells per tissue per experiment.
PREDICTION MODEL / DECISION CRITERIA
- For the current test, an irritation potential of a test item leading to H315 (according to regulation (EC) 1272/2008), and GHS category 2 according to UN GHS (published 2003, last (6th) revision 2015) is recommended if the mean relative tissue viability of three individual tissues is reduced ≤ 50% of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

other: freeze-killed control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 mg ± 2 mg (~ 39 mg/cm2 according to guideline) of the test item were applied to triplicate tissues, which were wetted with 25 µL DPBS prior to application.


CONTROLS
Concurrent controls were used for several studies performed simultaneously.
NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL

Duration of treatment / exposure:

The treatment time was 60 minutes in total.

Duration of post-treatment incubation (if applicable):

The complete incubation time was approximately 41 hours.

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

49.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

23.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY:

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

1st Experiment:
Treatment with the positive control induced a decrease in the relative absorbance compared with the negative control to 3.3% thus ensuring the validity of the test system. The relative standard deviations between the % variability values of the test item, the positive and negative controls in the main test were below 6% (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: < 18%), thus ensuring the validity of the study.

2nd Experiment:
In the second experiment no freeze-killed tissues were included since not effects on the interference with MTT were obtained in experiment one. In this experiment the negative control absorbance values were well within the required acceptability criterion of mean OD >= 0.8 and <= 2.8 for the 60 minutes treatment interval thus showing the quality of the tissues.

Treatment with the positive control induced a decrease in the relative absorbance compared with the negative control to 3.3% thus ensuring the validity of the test system. The relative standard deviations between the % variability values of the test item, the positive and negative controls in the main test were below 11% (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: < 18%), thus ensuring the validity of the study.

Any other information on results incl. tables

Table 1: Results after treatment with the test item and of the controls in the first experiment

Dose Group	Tissue No.	Absor-bance 570 nm Well 1	Absor-bance 570 nm Well 2	Absor-bance 570 nm Well 3	Mean Absor-bance of 3 Wells	Mean Absor-bance of three Wells blank corrected	Mean Absor-bance of 3 Tissues	Rel. Absor-bance [%] Tissue 1, 2 + 3*	Relative Standard Deviation [%]	Mean Rel. Absorbance [%]**	Corrected Mean Rel. Absorbance [%]***
Blank		0.038	0.038	0.037	0.037
Negative Control	1	1.708	1.718	1.740	1.722	1.684	1.670	100.8	4.6	100.0
	2	1.804	1.757	1.767	1.776	1.738		104.1
	3	1.659	1.606	1.611	1.625	1.588		95.1
Positive Control	1	0.090	0.095	0.094	0.093	0.055	0.055	3.3	0.6	3.3
	2	0.093	0.093	0.091	0.092	0.055		3.3
	3	0.092	0.093	0.093	0.093	0.055		3.3
Test Item	1	0.829	0.771	0.830	0.810	0.773	0.823	46.3	5.4	49.3	49.5
	2	0.902	0.893	0.889	0.895	0.858		51.4
	3	0.887	0.870	0.865	0.874	0.837		50.1
Negative Controlfreeze-killed	1	0.103	0.101	0.099	0.101	0.065	0.062	3.9	5.8	3.7
	2	0.096	0.097	0.095	0.096	0.060		3.6
Test Itemfreeze-killed	1	0.098	0.096	0.096	0.097	0.060	0.058	3.6	4.0	3.5
	2	0.094	0.094	0.092	0.093	0.057		3.4

*                      Relative viability [rounded values]

**                    Mean relative viability [rounded values]

***                  Corrected mean relative absorbance [rounded values]

                        

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 49.5% (threshold for irritancy: ≤ 50%), consequently the test item was (borderline) irritant to skin.

Table 2: Results after treatment with the test item and of the controls in the second experiment

Dose Group	Tissue No.	Absor-bance 570 nm Well 1	Absor-bance 570 nm Well 2	Absor-bance 570 nm Well 3	Mean Absor-bance of 3 Wells	Mean Absor-bance of three Wells blank corrected	Mean Absor-bance of 3 Tissues	Rel. Absor-bance [%] Tissue 1, 2 + 3*	Relative Standard Deviation [%]	Mean Rel. Absorbance [%]**
Blank		0.037	0.038	0.036	0.037
Negative Control	1	1.489	1.488	1.505	1.494	1.457	1.352	107.7	7.9	100.0
	2	1.386	1.402	1.395	1.394	1.357		100.4
	3	1.276	1.285	1.279	1.280	1.243		91.9
Positive Control	1	0.074	0.078	0.077	0.076	0.039	0.044	2.9	10.8	3.3
	2	0.081	0.082	0.081	0.081	0.044		3.3
	3	0.085	0.087	0.085	0.086	0.049		3.6
Test Item	1	0.333	0.329	0.333	0.332	0.295	0.316	21.8	8.6	23.4
	2	0.382	0.383	0.385	0.383	0.346		25.6
	3	0.344	0.343	0.342	0.343	0.306		22.6

*                      Relative viability [rounded values]

**                    Mean relative viability [rounded values]

 

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 23.4% (threshold for irritancy: ≤ 50%), consequently the test item was irritant to skin. 

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In this study and under the experimental conditions reported, the test item is irritant to skin according to UN GHS and EU CLP regulation.

Executive summary:

To assess the irritation potential of of the test item, an in vitrostudy was performed by means of the Human Skin Model Test (EpiDerm™) according to OECD Guideline 439, EU Method B.46 and UN GHS in compliance with GLP principles. Epi-200 SIT kits and MTT-100 assay diluent were purchased from MatTek Corporation (82105 Bratislava, Slovakia). An amount of 25 mg ± 2 mg (~ 39 mg/cm² according to guideline) of the test item were applied to tissues, which were wetted with 25 µL DPBS prior to application. Concurrent negative controls with DPBS (MatTek) and positive controls with 5% SLS solution in deionised water (MatTek) were conducted simultaneously. Since the test item passed the colour interference pre-test, but caused purple precipitate in the MTT interference pre-test, an additional test with freeze-killed tissues had to be performed. The test item, the negative control (DPBS, 30 µL) and the positive control (5% SLS, 30 µL) were applied to each triplicate tissue. The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 41 hours the tissues were treated with the MTT solution for 3 hours following 2.5 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm. After treatment with the test item in the first experiment, the corrected mean relative absorbance value decreased to 49.5% compared to the relative absorbance value of the negative control. This value is below the threshold for irritancy of ≤ 50% and is in the borderline range of 50 ± 5%. According to Guideline a second experiment was performed. In the second experiment no freeze-killed tissues were included since no effects on the interference with MTT were obtained in experiment one. After treatment with the test item, the mean relative absorbance value decreased to 23.4% compared to the relative absorbance value of the negative control. This value is far below the value of the threshold for irritancy. In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is irritant to skin according to UN GHS and EU CLP regulation. The acceptance criteria of the guidelines were met.