4'-methoxyacetophenone

Eye irritation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

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)

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

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

- Reconstructed human Cornea-like Epithelium

Test system

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.

Results and discussion

In vitro

Results

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:

In vivo

Irritant / corrosive response data:

Not applicable.

Other effects:

Not applicable.

Any other information on results incl. tables

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)

Applicant's summary and conclusion

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).