Phenylacetaldehyde

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

6 - 28 Apr 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Phenylacetaldehyde

EC Number:

204-574-5

EC Name:

Phenylacetaldehyde

Cas Number:

122-78-1

Molecular formula:

C8H8O

IUPAC Name:

2-phenylacetaldehyde

Test animals / tissue source

Species:

human

Strain:

other: EpiOcular™

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 µL

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL

Duration of treatment / exposure:

30 min

Duration of post- treatment incubation (in vitro):

120 min

Number of animals or in vitro replicates:

in duplicates for each treatment and control group

Details on study design:

- RhCE tissue construct used, including batch number: EpiOcular™ tissue (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia), batch number: 23707
- Viability: The quality of the final product was assessed by undertaking an MTT cell viability test.
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 0.3% Triton X-100. The ET-50 value was determined to be 15.12 min.
- Contamination: The cells used to produce the EpiOcular tissue were screened for the presence of viruses, bacteria, yeast and other fungi.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: 30 min exposure, 12 min post-exposure immersion and 120 min post-exposure incubation at 37 °C
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: The ability of the test substance to directly reduce MTT and to form a blue/purple reaction product was assessed in a pre-experiment. Since the test substance proved to reduce MTT a functional check on killed controls was performed in the definitive assay to show that the test substance was not binding to the tissue and leading to a false MTT reduction signal.
- Number of tissue replicates used per test chemical and controls: 2
- Wavelength: 570 nm
- Filter: without reference filter
- Description of the method used to quantify MTT formazan: The optical density was determined in a microplate reader (Versamax, Molecular Devices) without reference filter.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: The test substance was considered to be not irritating to eye if the tissue viability after 30 min exposure, 12 min post-exposure immersion and 120 min post-exposure incubation is >60%.
- Acceptance criteria: The results are acceptable if the negative control OD is >0.8 and <2.5 and if the mean relative viability of the positive control is below 50% of the negative control viability.
- Reference to historical data of the RhCE tissue construct: Historical control data was used to assess the validity of the test.
- Acceptable variability between tissue replicates for the test chemical, positive and negative controls: The difference of viability between the two relating tissues of a single test substance is < 20% in the same run.

Results and discussion

In vitro

Results

Irritation parameter:

other: % tissue viability

Remarks:

mean value of 2 tissues

Run / experiment:

30 min exposure

Value:

47.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD (1.296 and 1.446) was in the range of > 1.0 and < 2.6.
- Acceptance criteria met for positive control: Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 32.5%, thus the validity of the test system is ensured.

The difference of viability between the two relating tissues of a single substance is < 20% (values between 1.1% to 7.3%) in the same run (for positive and negative control tissues and tissues of single test substance).

- Other observations: The test substance proved to be an MTT reducer in the MTT pre-test. It was not coloured and it did not dye water or isopropanol in the colour interference pre-test. Therefore, an additional test with freeze-killed tissues to gain a correction factor for evaluation of the main experiment was performed, but an additional test with a viable tissue was not necessary. Results did not have to be considered for correction of the mean relative absorbance of the test substance in the main experiment, since the viability resulted from the test substance exposed tissues was lower than the cut-off value of 60%, because any MTT reduction would not change the classification for the test substance.

Any other information on results incl. tables

Table 1. Results after 30 min incubation time

Test group	Absorbance*		Mean absorbance of 2 tissues*	Rel. absorbance (%)**		Absolute value of the difference of the rel. absorbance (%) Tissue 1 and 2	Rel. absorbance (% of negative control)**
	Tissue 1	Tissue 2		Tissue 1	Tissue 2
Blank	0.036	0.035	0.000	-	-	-	-
Negative control	1.289	1.388	1.338	96.3	103.7	7.3	100
Positive control	0.443	0.428	0.435	33.1	32.0	1.1	32.5
Test substance	0.619	0.646	0.633	46.3	48.2	2.0	47.3
Additional test with freeze killed tissues without MTT reduction***
Blank	0.036	0.035	0.000	-	-	-	-
Negative control	0.049	0.055	0.052	94.0	106.0	11.9	100.0
Test substance	0.717	0.706	0.712	1380.1	1359.3	20.8	1359.3

* Mean of two replicate wells after blank correction

** Relative absorbance (rounded values): 100 × (absorbance test substance/positive control) / (absorbance negative control)

*** Results did not have to be considered for correction of the mean relative absorbance of the test substance, since the viability resulted from the test substance exposed tissues was lower than the cut-off value of 50%, because any MTT reduction would not change the classification for the test substance.

Applicant's summary and conclusion

Interpretation of results:

other: Eye Irrit. Cat. 2 according to Regulation (EC) 1272/2008

Conclusions:

Under the conditions of the conducted test, the test substance possessed irritating properties towards human-derived epidermal keratinocytes in the EpiOcular™ model.