Phenylacetaldehyde

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Jan - 18 Feb 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 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted 27 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

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm™

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number: 23314
- Delivery date: 16 February 2016
- Date of initiation of testing: 16 February 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (3 minutes exposure), 37 ± 1.5 °C (60 min exposure)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were gently rinsed using a wash bottle containing DPBS to remove any residual test material (20 times).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: microplate reader (Versamax, Molecular Devices, SoftMax Pro Enterprise v.4.7.1)
- Wavelength: 570 nm
- Filter: without reference filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm™ tissue 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 1% Triton X-100. The ET-50 value was determined to be 6.55 h.
- Contamination: The cells used to produce the EpiDerm™ tissue were screened for the presence of viruses, bacteria, yeast and other fungi.

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues: freezing
- N. of replicates : 2
- Method of calculation used: Since the MTT reducing test substance extract was classified as corrosive by the skin irritation test (tissue viability <50 %), the correction procedures were not necessary.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: single experiment with 2 incubation periods

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is less than 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 µL

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
- Concentration: 8 N

Duration of treatment / exposure:

3 and 60 min

Number of replicates:

in duplicates for each treatment and control group

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 2 tissues

Run / experiment:

3 minutes exposure

Value:

39.3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 2 tissues

Run / experiment:

60 minutes exposure

Value:

10.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS
- Direct-MTT reduction: Optical evaluation of the MTT-reducing capacity of the test substance after 1 h incubation with MTT-reagent showed blue colour. An additional test with freeze-killed tissues had to be performed, but correction of the viability values was not necessary, since the test substance proved to be corrosive even without correction.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean negative control OD, both for the 3 and 60 min exposure period, was in the range of ≥ 0.8 and ≤ 2.8 for every exposure time.
- Acceptance criteria met for positive control: Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 min exposure period (23.0%) and for the 60 min exposure period (14.8%) thus confirming the validity of the test system and the specific batch of tissue models.
- Acceptance criteria met for variability between replicate measurements: The coefficient of variation in the range 20 – 100% viability between tissue replicates was < 14%.

Any other information on results incl. tables

Table 2. Results after treatment with the test substance and controls

Test group	Absorbance at 570 nm*		Mean absorbance of 2 tissues	Coefficient of variation (%)	Rel. absorbance (% of negative control)**
	Tissue 1	Tissue 2
3 minutes treatment
Negative control	1.559	1.359	1.459	13.7	100.0
Positive control	0.359	0.311	0.335	3.3	23.0
Test substance	0.559	0.589	0.574	2.0	39.3***
60 minutes treatment
Negative control	1.337	1.452	1.394	7.9	100.0
Positive control	0.236	0.176	0.153	4.1	14.8
Test substance	0.157	0.148	0.153	0.6	10.9***

* Mean of three replicate wells after blank correction

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

*** Since the MTT reducing test substance extract was classified as irritant by the skin irritation test (tissue viability <50% after 3 minutes exposure), the correction procedures were not necessary.

Applicant's summary and conclusion

Interpretation of results:

other: Skin Corr. Cat. 1B (H314)

Remarks:

according to Regulation (EC) 1272/2008

Conclusions:

Under the conditions of the reconstructed human epidermis test the test substance was corrosive to skin. According to the EpiSkinTM prediction model as described in OECD TG 431 a substance which causes cell viability >= 35% after 3 min exposure and < 35% after 60 min exposure, shall be regarded as a skin corrosive Category 1B / 1C. Thus, the test substance was classified as Skin Corr. Cat. 1B according to Regulation (EC) 1272/2008.