Disodium L-cystinate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-07-20 to 2011-07-29

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study from supporting substance (structural analogue)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

April 13, 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Cystine

EC Number:

200-296-3

EC Name:

Cystine

Cas Number:

56-89-3

Molecular formula:

C6H12N2O4S2

IUPAC Name:

cystine

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Purity: >98.5%
- Storage condition of test material: at room temperature

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: normal, human-derived epidermal keratinocytes from EST-1000 kit from CellSystems Biotechnologievertrieb GmbH

Source strain:

other: human

Details on animal used as source of test system:

Not applicable

Justification for test system used:

The epidermis model (e.g. EST-1000™) is derived from human keratinocytes and consists of normal, human derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK, which are cultured on specially prepared cell culture inserts using serum free medium, attain levels of differentiation at the cutting edge of in vitro skin technology. Ultrastructurally, the skin models closely parallel human skin.

Vehicle:

not specified

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: normal, human-derived epidermal keratinocytes (NHEK) EST-1000™ (CellSystems Biotechnologievertrieb GmbH)
This skin model consists of normal human epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on specially prepared cell culture inserts (Millicells, 10 mm). It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C
- Temperature of post-treatment incubation (if applicable): Room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 3 rinses with PBS
-Volume and number of washing steps:
* Tissue was removed from 6-well plate after exposure period and gently rinsed using a wash bottle containing PBS to remove any residual test material
* Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper.
* The insert was placed in a prepared holding plate. All inserts were treated in the same manner.
* Then the inserts were transferred into a prepared 24-well "MTT assay plate" containing 300 ul prewarmed MTT solution.
- Observable damage in the tissue due to washing: Not reported
- Modifications to validated SOP: Not reported

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT Formazan salt (Sigma Alrdrich) dissolved in PBS
- Spectrophotometer: Optical density was read in a microplate reader (Versamax Molecular Devices)
- Wavelength: 570 nm (OD570)
- Filter: without reference filter

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if 1) the viability after 3 minutes exposure is less than 50% or 2) the viability after 3 minutes exposure is greater than or equal to 50% and the vibility after 1 hour exposure is less than 15%
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
- The mean OD value obtained for hte duplicate tissues per tset item were used to calculate the percent viability relative to the negative control, which was arbitratily set at 100%.
- The absolute OD570 of the negative control tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability met the acceptance criterion if the mean OD570 of the two tissues in both treatment intervals was ≥0.8. The assay met the acceptance criterion if mean relative tissue viability of the positive control was ≤30%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg of the test material were applied to the tissues and wetted with 25 ul deionised water and spread to cover the surface of the tissue.
- Concentration (if solution): not specified

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 ul per tissue

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 ul per tissue

Duration of treatment / exposure:

1) 3 mins
2) 1 hour

Duration of post-treatment incubation (if applicable):

3 hour incubation period in MTT-plates at 37 ± 1.5 °C, 5 ± 0.5% CO2

Number of replicates:

Experiment performed in duplicate (2 replicates)

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

ca. 1.015

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

No indication of corrosiveness

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour exposure

Value:

ca. 1.134

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

The test item is not corrosive to skin

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: not reported
- Direct-MTT reduction: No
- Colour interference with MTT: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After exposure to the negative control the absorbance values exceeded the required acceptability criterion of mean OD570 ≥ 0.8 for both treatment intervals thereby confirming the acceptable quality of the tissues.
- Acceptance criteria met for positive control: Exposure to the positive control decreased the relative absorbance as compared to the negative control to 27.3% for the 3 minutes exposure period and 0.5% for the 1 hour exposure period thus confirming the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: Not reported

Any other information on results incl. tables

Results after treatment with test item L-Cystine and with the positive and the negative control

Dose Group	Exposure Interval	Absorbance 570 nm Tissue 1*	Absorbance 570 nm Tissue 2*	Mean Absorbance of 2 Tissues	Rel. Absorbance [% of Negative Control]**
Negative Control	3 min	0.941	1.000	0.970	100.0
Positive Control	3 min	0.242	0.288	0.265	27.3
L-Cystine L-Cystine	3 min	0.997	1.033	1.015	104.6
Negative Control	1 hour	0.944	1.028	0.986	100.0
Positive Control	1 hour	0.004	0.005	0.004	0.5
L-Cystine	1 hour	1.201	1.067	1.134	115.0

* mean of three replicate wells after blank correction

** relative absorbance [rounded values]: 100 x (absorbance_{test item}) / (absorbance_{negative control})

The optical evaluation of the MTT-reducing capacity of the test item after one hour incubation with MTT-reagent did not show evidence of a blue colour and thereby was not considered to be an MTT reducer.

The test item L-Cystine is not considered to be corrosive to skin:

1) since the viability after 3 minutes exposure is greater than 50% and

2) the viability after 1 hour exposure is greater than 15%.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Test item L-Cystine is not considered to be corrosive to skin

Conclusions:

In an in vitro skin corrosion test (OECD 431) using the human skin model EST-1000™ the test item was tested as non-corrosive.

Executive summary:

The potential of L-Cystine to induce skin corrosion was analysed by using the epidermis model EST-1000™, derived from human keratinocytes and consists of normal, humanderived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK, which are cultured on specially prepared cell culture inserts using serum free medium, attain levels of differentiation at the cutting edge of in vitro skin technology. Ultrastructurally, the skin models closely parallel human skin. The test item was applied topically to the EST-1000™ tissue for 3 minutes and 1 hour followed by immediate determination of cytotoxic effects via MTT reduction assay.

Corrosivity potential of the test item was predicted from the relative absorbance values compared to the corresponding negative control tissues concurrently treated with deionised water. The positive control (8.0 N KOH) did induce the appropriate response. The controls confirmed the validity of the study. In this study under the given conditions the test item showed no corrosive effects. After exposure to the test item L-Cystine, the relative absorbance values did not decrease, neither after the 3 minutes exposure (104.6%; threshold for corrosivity: 50%), nor after the 1 hour exposure (115.0%; threshold for corrosivity: 15%). Therefore, the test item was not considered to be corrosive.