4'-methoxyacetophenone

Endpoint summary

Description of key information

Skin irritation/corrosion

The test substance was determined to be irritant to skin Category 2 (according to UN GHS and EU CLP regulation) in an in vitro Reconstructed Human Epidermis Test.

 

Eye irritation

The substance was considered to be irritant (UN GHS Category 2) as shown in a Human Cornea Model Test and a BCOP test.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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)

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

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

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

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.

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. 

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-05-05 to 2017-05-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

- Reconstructed human Cornea-like Epithelium

Vehicle:

water

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

- Controls: Each 50 μL were applied.

Duration of treatment / exposure:

- On duplicate tissues for 6 hours.

Duration of post- treatment incubation (in vitro):

- 18 hours

Number of animals or in vitro replicates:

- 2 replicates per test concentration and control

Details on study design:

- Details of the test procedure used: Procedure according to MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model
- RhCE tissue construct used, including batch number: EpiOcular™ kits from MatTek Corporation (82105 Bratislava, Slovakia), Lot No.: 23779
- Doses of test chemical and control substances used: 50 mg (test item), 50 μL (controls)
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: Incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH) for 6 hours
- Description of any modifications to the test procedure: There were no deviations from the guidelines and the study plan
- Indication of controls used for direct MTT-reducers and colouring test chemicals: Concurrent negative controls (50 μL of deionised water in 1 mL of 1.0 mg/mL MTT solution) and freeze-killed controls
- Number of tissue replicates used per test chemical and controls (positive control, negative control): 2
- Wavelength and used for quantifying MTT formazan: 570 nm (OD570), no reference wavelength measurement was used.
- Description of the method used to quantify MTT formazan: Plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro, version 4.7.1).
- Description of evaluation criteria used: Cell viability was measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide], present in cell mitochondria, into a blue formazan salt that was quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls was used to predict eye irritation potential. If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labeled non-irritant. If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labeled irritant. According to OECD guideline 492 a single test composed of at least two tissue replicates should be sufficient for a test chemical, when the result is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent tissue viability equal to 60±5%, a second test should be considered, as well as a third one in case of discordant results between the first two tests.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: Yes, data of 21 studies performed from July 2015 until end of March 2017 were evaluated.
- Complete supporting information for the specific RhCE tissue construct used: Yes.
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: Laboratory technical proficiency with the test system according to OECD 492 was demonstrated at Envigo CRS GmbH as documented in Envigo CRS project No. 1729900.
- Positive and negative control means and acceptance ranges based on historical data: Positive control; mean viability = 31.59%, rel. standard deviation = 11.56%; range of viabilities: 8.10% - 42.60% ; mean absorption = 0.48 (rel. standard deviation = 0.13); range of absorption: 0.22- 0.64. Negative control: Mean absorption = 1.50 (rel. standard deviation = 0.26), range of absorbance: 1.24 – 2.05.



- Acceptable variability between tissue replicates for positive and negative controls: According to guideline, the mean relative viability of the positive control should be below 50% of the negative control viability. This criterion was met in the definitive test.
- Acceptable variability between tissue replicates for the test chemical: According to guideline, the difference of viability between the two relating tissues of a single test item must be < 20% in the same run (for positive and negative control tissues and tissues of test items). This applies also to the freeze-killed tissues (items and negative control), which are calculated as percent values related to the viability of the relating negative control. This criterion was met in the definitive test.

Irritation parameter:

other: tissue viability [%]

Run / experiment:

1

Value:

<= 60

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Range of historical values if different from the ones specified in the test guideline:

Irritant / corrosive response data:

Not applicable.

Other effects:

Not applicable.

Table: Results after treatment for 6 hours with the test item and the controls

Dose Group	Absorbance Well 1 (Tissue 1/2)	Ab-sorbance Well 2 (Tissue 1/2)	Mean Absor-bance* (Tissue 1/2)	Mean Absorbance Tissue 1 and 2 minus Mean Blank	Mean Absorbance of 2 Tissues	Rel. Ab-sorbance [%] Tissue 1 and 2*	Absolute Value of the Difference of the Rel. Absorbances [%] Tissue 1 and 2	Mean Rel. Absorbance [% of Negative Control]*	Corrected Mean Rel. Absorbance [%]**
Blank	0.038	0.039	0.038
Negative Control	1.544	1.516	1.530	1.492	1.462	102.0	4.1	100.0
	1.466	1.475	1.471	1.432		98.0
Positive Control	0.421	0.457	0.439	0.401	0.409	27.4	1.2	28.0
	0.454	0.459	0.456	0.418		28.6
Test item	0.067	0.067	0.067	0.028	0.029	1.9	0.1	2.0	1.7
	0.068	0.068	0.068	0.030		2.1
Blank	0.038	0.039	0.038
Nagative Control Freeze-killed	0.078	0.080	0.079	0.041	0.044	2.8	0.4	3.0
	0.085	0.085	0.085	0.047		3.2
Test Item Freeze-killed	0.088	0.089	0.088	0.050	0.048	3.4	0.3	3.3
	0.084	0.084	0.084	0.046		3.1

* Relative absorbance [rounded values]: )()(100/controlnegativentrolpositivecotestitemabsorbanceabsorbance×

** Corrected mean rel. absorbance [rounded value]:

Mean rel. absorbancetest item – (mean rel. absorbancefreeze-killed test item – mean rel. absorbancefreeze-killed negative control)

Interpretation of results:

other: Cat. 1 or 2

Conclusions:

The test item is regarded as eye irritant or serious eye damage (no distinction possible between Cat.1 and 2).

Executive summary:

To assess the eye irritation potential of the test item, an in vitro study was performed by means of the Human Cornea Model Test. The study was conducted under GLP-conditions and according to OECD Guideline 492 following the MatTek Corporation Protocol for the EpiOcular™ Eye Irritation Test (OCL-200-EIT). An additional test with freeze-killed tissues to determine a correction factor for calculating the true viability in the main experiment was carried out, since the test item directly reduced MTT in the MTT interference pre-experiment. About 50 mg of the test item and each 50μL of the negative control (deionised water) and positive control (methyl acetate), respectively, were applied to each of duplicate EpiOcular™tissue for 6 hours. Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 28.0%, thus the validity of the test system is ensured. All validity criteria were fulfilled. In result, the mean relative absorption value of the tissues corresponding to the cornea viability decreased to 2.0% compared with the value of the negative control even without taking the determined correction factor into consideration (threshold for irritancy: ≤ 60%). Taking the correction factor in account, the viability was 1.7%. Since the mean percent tissue viability after exposure and post-exposure incubation is less than or equal (≤) to 60%, it can be concluded that the test item is irritant to the eye (UN GHS Category 2 or Category 1).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2013-05-17 to 2013-05-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Origin of isolated bovine eyes of donor cattle: Slaughterhouse; Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany

Vehicle:

Hank's balanced salt solution

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

- Test material (suspended): 750 µL
- Negative Control: 750 µL
- Positive Control: 750 µL

Duration of treatment / exposure:

240 minutes

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

90 minutes

Number of animals or in vitro replicates:

Three replicates each for the test item, positive control and negative control.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- Freshly isolated bovine eyes were collected; excess tissue was removed; transported in transport medium
- Preparation of corneas immediately after delivery
- Examination for defects: Macroscopically, corneas showing e.g. pigmentation, opacity, scratches, vascularization were disregarded
- Corneas of undamaged eyes were removed with scalpel and rounded scissors
- Sclera were left for stability and handling

NUMBER OF REPLICATES
- Test item: 3
- Negative control: 3
- Positive control: 3

NEGATIVE CONTROL USED
- 0.9% sodium chloride solution

POSITIVE CONTROL USED
- 20% Imidazole (in 0.9% sodium chloride solution)

APPLICATION DOSE AND EXPOSURE TIME
- Dose: 750 µL each (test item, positive and negative control)
- Exposure timae: 240 minutes

TREATMENT METHOD:
- Open chamber

POST-INCUBATION PERIOD: No

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least three (or until substance was removed verified by visual inspection) using wash medium; final rinse using incubation medium (EMEM) to remove phenol red from the anterior chamber

- POST-EXPOSURE INCUBATION: Yes, incubation for 90 minutes at 32 ± 1 °C after wash steps and before permeability was determined.


METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Before and after treatment with opacitometer (BASF-OP2.0)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: According to Guideline (an IVIS > 55 indicates corrosive or severe irritant properties of the test item)

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

24.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Validity criteria met for negative control: Yes, IVIS = 0.2 (within the standard deviations of current historical data of mean of negative control, IVIS: -0.4 - 2.9)
- Validity criteria met for positive control: Yes, IVIS =116.2 (within the standard deviation of current historical data of mean of positive control, IVIS: 73.5 - 142.8)

The test is valid.

Other effects:

No

Table: Results of the Bovine Corneal Opacity and Permeability Test (BCOP)

				IVIS
		Opacity	Permeability	per conrea	per group (mean value)	Standard deviation
Negative control	0.9% sodium chloride solution	0.556	0.000	0.561	0.2	0.3
		-0.142	0.001	-0.122
		0.139	0.003	0.179
Positive control	Imidazole (20%)	88.924	2.184	121.683	116.2	4.8
		67.641	3.106	114.234
		70.031	2.847	112.735
Test material	p-Methoxy-acetophenone	22.117	0.007	22.225	24.1	2.2
		26.443	0.006	26.531
		23.466	0.006	23.549

Interpretation of results:

other: not GHS Cat. 1

Conclusions:

The test item did not show an ocular corrosive or severe irritant potential under the conditions chosen.

Executive summary:

To investigate the ocular corrosive and severe irritant potential of the test item, a Bovine Corneal Opacity and Permeability (BCOP) Test was conducted under GLP conditions, according to OECD Guideline 437 and the Commission Regulation (EU) No. 1152/2010. Intact corneas of freshly isolated bovine eyes were exposed in triplicates to the test item and a reference substance (20% Imidazole). A negative control (0.9% sodium chloride solution) with three replicates ran in parallel. After preparation and examination for defects, the corneas were mounted in a cornea holder. Both compartments of the holder were filled with pre-warmed (32 °C) EMEM incubation medium. The opacity of the corneas was measured with an opacitometer before and after treatment. A volume of 750 µL each of the test item, the reference material and the negative control was added to the anterior chamber of the the corneas. Subsequently, the corneas were incubated for 240 minutes at 32 ± 1 °C in horizontal position. Afterwards, the corneas were washed at least three times with wash medium to ensure removal of the test item, the reference item or the negative control from the epithelium. Finally, they were rinsed with incubation medium (EMEM) in order to remove phenol red from the anterior chamber.Then the compartments of the cornea holders were filled with fresh incubation medium (EMEM) and the opacity after treatment was determined. After opacity measurement, the incubation medium of the posterior chamber was renewed and in the anterior chamber 1 mL of a fluorescin solution (dissolved in 5mg/L DPBS) was placed. Further incubation in horizontal position for 90 minutes at 32 +- 1 °C followed. After that, the permeability of the corneas was measured by determination of the the amount of sodium fluorescin that crossed into the posterior chamber of the cornea holder. For this reason, 360 µL medium from the posterior chamber per replicate were transferred into a 96 -well plate and the sodium fluorescin was detected spectrophotometrically at 490 nm (OD490) using a microplate reader. The opacity value of each cornea was calculated individually by substracting the initial baseline opacity from the opacity after treatment. The average change of opacity of the negative control was substracted from the change of opacity of each cornea exposed to the test item or the reference substance. The mean corrected change of opacity of the treatment groups was calculated by averaging of the individual corrected opacity values of the treated corneas. To estimate the permeability of the corneas, the average cornea value of the negative control was substracted from the original permeability value of corneas exposed to the test item or reference substance. The mean corrected OD450 value of each treatment group was calculated from from the individual corrected permeability. In result, the calculated IVIS was 0.2 and 116.2 for the negative control and the positive control, respectively. Thus, the controls were within two standard deviations of the current historical mean of the negative (IVIS: -0.4 - 2.9) and positive control (IVIS:73.5 - 142.8). Therefore, all validity criteria of the test were fulfilled. The calculated IVIS for the test substance was 24.1. Thus, the test item dit not show ocular corrosive or severe irritant potential, since the threshold value of IVIS = 55 was not exceeded.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Skin irritation

To assess the irritation potential of of the test item, an in vitro study 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), positive controls with 5% SLS solution in deionised water (MatTek) as well as an additional test with freeze-killed tissues (only in experiment 1) were conducted simultaneously. The test item, the negative control (DPBS, 30 µL) and the positive control (5% SLS, 30 µL) were applied to each triplicate tissue. In the first experiment, the corrected mean relative absorbance value for the test item 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, the mean relative absorbance value of the test item 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 category 2 according to UN GHS and EU CLP regulation. The acceptance criteria of the guidlelines were met.

Eye irritation

The eye irritating potential of the test material was investigated under GLP conditions by means of the Human Cornea Model Test (2017) according to OECD guideline 492 and following the MatTek Corporation Protocol for the EpiOcular™ Eye Irritation Test (OCL-200-EIT). About 50 mg of the test item and each 50μL of the negative control (deionised water) and positive control (methyl acetate), respectively, were applied to each of duplicate EpiOcular™tissue for 6 hours. Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 28.0. Thus, the validity criteria of the test were fulfilled. Since the mean percent tissue viability after exposure to the test item and post-exposure incubation was less than or equal (≤) to 60%, it can be concluded that the test item is irritant to the eye (UN GHS Category 2 or Category 1).

To assess the ocular corrosive and severe irritant potential of the test item, a Bovine Corneal Opacity and Permeability (BCOP) Test (2013) was conducted under GLP conditions, according to OECD Guideline 437 and the Commission Regulation (EU) No. 1152/2010. Intact corneas of freshly isolated bovine eyes were exposed in triplicates to the test item and a reference substance (20% Imidazole). A negative control (0.9% sodium chloride solution) with three replicates ran in parallel. A volume of 750 µL each of the test item, the reference material and the negative control was added to the anterior chamber of the cornea holder. In result, the calculated IVIS was 0.2 and 116.2 for the negative control and the positive control, respectively. Thus, the controls were within two standard deviations of the current historical mean of the negative (IVIS: -0.4 - 2.9) and positive control (IVIS:73.5 - 142.8). Therefore, all validity criteria of the test were fulfilled. The calculated IVIS for the test substance was 24.1. Thus, the test item did not show ocular corrosive or severe irritant potential, since the threshold value of IVIS = 55 was not exceeded.

Conclusion

The Human Cornea Model Test (2017) determined that the test substance is irritant to the eye, but cannot resolve between UN GHS Category 2 or Category 1. Taking into account the results of the the Bovine Opacity and Permeability (BCOP) Test (2013), it can be differentiated between UN GHS Categories 1 and 2. Because the threshold value of IVIS = 55 was not exceeded, and thus, the test item did not show ocular corrosive or severe irritant potential, the test substance has to be classified as Category 2 (irritant to the eye).

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin and eye irritation/corrosion, the test item is classified to be irritating to skin Category 2 and eye irritating Category 2 according to Regulation (EC) No 1272/2008 (CLP), as amended for the fifteenth time in Regulation (EU) 2020/1182.