Carnosine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Apr - 5 May 2017

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 28 Jul 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No 440/2008 of 30 May 2008, 1st ATP 2009

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:

Carnosine

EC Number:

206-169-9

EC Name:

Carnosine

Cas Number:

305-84-0

Molecular formula:

C9H14N4O3

IUPAC Name:

(2S)-2-(3-aminopropanoylamino)-3-(1H-imidazol-4-yl) propanoic acid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm™ (EPI-200-SIT)

Source strain:

other: 00267

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200-SIT) (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number(s): 25810
- Shipping date: 3 May 2017
- Delivery date: 3 May 2017
- Date of initiation of testing: 3 May 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C for 35 min in the incubator; thereafter at room temperature for 25 min in a sterile bench
- Temperature of post-treatment incubation: Tissues were incubated for 24.7 hours at 37 ± 1.5 °C. After change of medium, tissues were again incubated for another 17.5 hours at 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Inserts were removed from the 6-well plate and tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After 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.

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 v.4.7.1)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm tissue was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 1.556 ± 0.088 (acceptance criteria: 1.0 - 3.0).
- 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 5.23 h (acceptance criteria: 4.77-8.72 h).
- Contamination: The cells used to produce the EpiDerm tissue were screened for the presence of viruses, bacteria, yeast and other fungi.
- Reproducibility: Absorbance of positive and negative control were within the range of historical controls.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Since the test substance did not directly reduce MTT in a pre-test, an additional test with freeze-killed tissues was not performed.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
- single experiment

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 1 hour exposure is ≤ 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 mg ± 2 mg (~ 39 mg/cm2) of the test item, wetted with 25 μL DPBS

NEGATIVE CONTROL
- Amount applied: 30 μL

POSITIVE CONTROL
- Amount applied: 30 μL
- Concentration: 5%

Duration of treatment / exposure:

35 min at 37 ± 1.5 °C and 25 min at RT

Duration of post-treatment incubation (if applicable):

42.2 h

Number of replicates:

triplicates

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min

Value:

102.9

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: The test substance did not interfere with the MTT assay (no reducing capacity).
- Colour interference with MTT: The test substance did not change colour when mixed with deionised water. Also its intrinsic colour was not intensive.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for 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 assuring the quality of the tissues.
- Acceptance criteria met for positive control: Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 4.5% thus ensuring the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: The relative standard deviations between the % variability values 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.
- Range of historical values if different from the ones specified in the test guideline: The relative standard deviations of historical positive controls were with 20.12% higher than standard deviations ≤ 18% in the test guideline. In the study, the relative standard deviations of the positive control were 5.1% and therefore ≤ 18%.

Any other information on results incl. tables

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

Test group	Mean absorbance at 570 nm*			Rel. absorbance (%)**			SD (%)	Rel. absorbance (% of negative control)***
	Tissue 1	Tissue 2	Tissue 3	Tissue 1	Tissue 2	Tissue 3
Negative control	1.504	1.372	1.266	108.9	99.4	91.7	8.6	100.0
Positive control	0.062	0.059	0.065	4.5	4.3	4.7	5.1	4.5
Test substance	1.281	1.401	1.578	92.8	101.5	114.3	10.8	102.9

* Mean of three replicate wells after blank correction (blank = 0.037)

** Relative absorbance per tissue (rounded values): 100 × (absorbance tissue) / (mean absorbance negative control)

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

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 reconstructed human epidermis test the test substance does not possess any skin irritating potential.

Executive summary:

The skin irritation potential of the test substance was determined by an in vitro skin irritation test using a reconstructed human skin model according to OECD Guideline 439 and in compliance with GLP (2016). After treatment with the test substance for 60 min the tissue viability was 102.9% compared to the negative control (threshold for irritancy ≤ 50%). Therefore, the test substance is not considered to be irritating to the skin.