Nα-acetyl-DL-tryptophan

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-05-25 - 2016-05-27

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 28 July 2015

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Skinethic skin irritation test -42bis Standard operating procedure (SOP) 2009

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz

Test material

Test material information

Constituent 1

Reference substance name:

Nα-acetyl-DL-tryptophan

EC Number:

201-739-3

EC Name:

Nα-acetyl-DL-tryptophan

Cas Number:

87-32-1

Molecular formula:

C13H14N2O3

IUPAC Name:

N-acetyltryptophan

Test material form:

solid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: SkinEthic™ RHE-model

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Remarks:

No vehicle used; Test item was applied neat to the tissues

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model (Episkin/Skin Ethic Laboratories, Lyon, France)
- Tissue batch number: 16-RHE-052
- Expiration Date: May 30, 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 42 ± 1 min at room temperature; thereafter at 37 °C for 42 ± 1 h

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were gently rinsed with DPBS in order to remove any residual test material. Excess DPBS was removed by gently shaking the tissue inserts and blotting the bottom of the tissues inserts with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h ± 5 min
- Spectrophotometer: microplate reader (ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the RHE model was assessed by undertaking a 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 6.9 h.
- Other: Absence of significant abnormalities after histological observations (HES stained vertical paraffin)

NUMBER OF REPLICATE TISSUES: The test item as well as the positive and negative control were tested in batch-triplicates. Therefore, a total number of nine tissues was used in this study.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test substance did not directly reduce MTT.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability is less than 50%
- The test substance is considered to be non-irritant to skin the viability is greater than or equal to 50%

ACCEPTABILITY CRITERA
The negative control OD values for the RHE-model have to be in the range of ≥ 0.8 and ≤ 3.0.
The negative control data meet the acceptance criteria if the mean OD value is higher or equal than a historically established boundary at 570 nm. The boundary is two standard deviations below the current historical mean (1.417). The standard deviation value is considered valid if ≤ 18% of the group mean value.
The positive control data meet the acceptance criteria if the mean viability value, expressed as % of the negative control, is lower than or equal to a historically established boundary. The boundary is three standard deviations above the current historical mean (3.26%).
The standard deviation between the three tissue replicates in each group shall be ≤ 18%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 16 mg per tissue

NEGATIVE CONTROL
- Amount applied: 16 µL per tissue (Dulbecco`s Phosphate-Buffered Saline)

POSITIVE CONTROL
- Amount applied: 16 µL per tissue
- Concentration: 5% aqueous solution of sodium dodecyl sulfate in deionised water

Duration of treatment / exposure:

42 min (± 1 minute)

Duration of post-treatment incubation (if applicable):

42 hours (± 1 hour)

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment 1 / Run 1

Value:

96

Vehicle controls validity:

not applicable

Remarks:

The test item was applied neat to the tissues

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:
The negative control OD values were 1.857, 1.830 and 1.822 and, thus, in the range of ≥ 0.8 and ≤ 3.0.

Acceptability of the Positive and Negative Control Data:
After treatment with the negative control (DPBS-buffer) the mean OD was 1.836 (standard deviation: 0.99%) and, thus, higher than the historically established threshold of 1.417.
After treatment with the positive control (5% aqueous solution of sodium dodecyl sulfate) the mean viability value was 1.25% (standard deviation: 2.51%) and, thus, lower than the historically established threshold of 3.26%.
The negative and positive control are acceptable as the difference compared with the historical data is minimal.

Variability of the Data:
The standard deviation between the three tissues replicates treated with the test item was 0.33% and, thus, ≤18%. The standard deviations between the three tissue replicates of the negative control and the positive control were 0.99% and 2.51%, respectively, and, thus, ≤18%.

The study met all acceptance criteria.

Any other information on results incl. tables

Table 1: Summary of Results

Group	Time / [min]	Mean OD	Mean Relative viability / [%]
Negative Control	42	1.836	100.00
Positive Control	42	0.023	1.25
Test Material	42	1.763	96.00

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008

Conclusions:

Under the conditions of the conducted test, the test substance did not show irritating properties towards reconstructed human epidermis tissue.