L-lysine, compound with S-(carboxymethyl)-L-cysteine (1:1)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 3 to 6, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

GLP compliance:

no

Remarks:

the available study was originally run not for REACH purposes; however, it is scientifically valid and well documented.

Test material

Test material information

Constituent 1

Reference substance name:

L-lysine, S-carboxymethyl-L-cysteine salt

IUPAC Name:

L-lysine, S-carboxymethyl-L-cysteine salt

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Cell type:

non-transformed keratinocytes

Details on test system:

A reconstructed artificial human skin model comprising normal human epidermal keratinocytes, growing as an integrated three-dimensional cell culture model, perfectly mimicking the human skin in vitro. The model exhibits normal barrier functions (presence of a differentiated stratum corneum). It was provided by SkinEthic.

The sample has been tested as that, undiluted. The positive control SLS has been dissolved at 5 % in water. Phosphate buffer alone has been used as negative control.
16 mg of the product have been applied on each epidermis unit in three replicas. The exposition has been carried out for 42‘ at room temperature. At the end of the exposure period the product was removed and the tissue was incubated at 37 °C, 5 % CO2 for 42 hours. At the end of the exposure the MTT assay was performed to evaluate the cell survival on the skin units.
The MTT assay is simple, accurate and yields reproducible results. The key component is (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) or MTT (Sigma M-2128). This product is of yellowish colour in solution. Mitochondrial dehydrogenases of viable cells cleave the tetrazolium ring, leading to the formation of purple crystals which are insoluble in aqueous solutions. The crystals are re-dissolved in acidified isopropanol and the resulting purple solution is measured spectrophotometrically. An increase or decrease in cell number results in a concomitant change in the amount of formazan formed, indicating the degree of cytotoxicity caused by the test material.
After 42-hour incubation of epidermis, the tissues is incubated for 3 h in 300 μl/well in a 24 well plate of 1mg/ml MTT solution at 37 °C. The solution is than removed and replaced with 1500 μl/well of isopropanol, with further 2 h incubation at room temperature under medium speed shaking.
The absorbance at 550 nm is measured with a microplate reader (Tecan modello Sunrise remote). The absorbance values are corrected by subtracting the reading of the blanks, with the diluent only.
The results are expressed in terms of viability:
% of cell viability = [OD(550 nm ) test product / mean OD(550 nm) negative control] × 100

Acceptance criteria of method
- for negative control (CN): the mean OD value of the 3 tissue has to be ≥ 1.2, the standard deviation has to be ≤ 18 %
- for positive control (CP): the viability mean (expressed as % of the NC) has to be < 40 %, the standard deviation has to be ≤ 18 %
- for the sample: the standard deviation has to be ≤ 18 %

Criteria for in vitro interpretation Classification
Mean tissue viability ≤ 50 % classified
Mean tissue viability > 50% not classified

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 16 mg, undiluted

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

average of 3 replicates

Value:

108.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Sample % mean cell viability (ds) after 42’ treatment and 42 h incubation Comment
test substance 108.9 (±8.5) not irritant
SLS 5 % 1.5 (±0.6) irritant
negative control (CN) 100 (±8.7) not irritant

Applicant's summary and conclusion

Interpretation of results:

other: not irritant according to the CLP Regulation (EC 1272/2008)

Conclusions:

Not irritant to the skin.

Executive summary:

Method

OECD 439. In vitro assay using a reconstructed artificial human skin model of normal human epidermal keratinocytes. Test sample was applied on 3 epidermis units for 42 minutes at room T. Then, the substance was removed and each tissue was incubated for 42 h at 37 °C and 5 % CO2. After incubation, evaluation of survival was done using the MTT assay and measuring absorbance at 550 nm. Based on absorbance values, a % of cell viability was derived.

Phosphate buffer was used as negative control; 5 % solution in water of SLS was used as positive control.

Results

Negative and positive controls were both valid. The mean cell viability of test substance was 108.9 ± 8.5 %, thus above the threshold of 50 %. Accordingly, the substance was considered as not irritant to the skin.