Sodium hydrogen substituted amino acid

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 June 2018 - 29 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Method B.40 bis In vitro skin corrosion: human skin model test (Annex to Regulation (EC) No 440/2008).

Version / remarks:

31 May 2008.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

d.d. 22 January 2018

Test material

Test material information

Constituent 1

Test material form:

liquid

Remarks:

aqueous solution

Details on test material:

Sodium hydrogen substituted amino acid, FC-C 13403
Apppearance: Clear Colorless solution

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): 28811
- Surface: 0.6 cm^2
- Pretreatment: The skin tissues were kept on agarose and stored in the refrigerator the day they were received. The next day, at least 1 hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 ml DMEM per well.

INTERFERENCE WITH THE MTT ENDPOINT:
- Test for color interference by the test item: 50 μL test item was added to 0.3 mL Milli-Q water and the mixture was incubated for approx. 1 hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue/purple color change was observed. A negative control (Milli-Q water) was tested concurrently.
- Test for reduction of MTT by the test item: 50 μL test item was added to 1mL MTT solution (1mg/mL) in phosphate buffered saline. The mixture was incubated for approx. 1 hour at 37.0 ± 1.0°C. At the end of the exposure time it was checked if a blue/purple color change was observed or a blue/purple precipitate was observed.

TEMPERATURE USED FOR TEST SYSTEM
All incubation, with the exception of the test item incubation for 3 minutes at room temperature, were carried out in the dark at 37.0 +/- 1.0C (actual range 36.6-37.2°C).

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: after exposure and after incubation tissues were washed with phosphate buffered saline.
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- Amount of MTT-medium: 300 μL
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: For each time point (3-minute and 1-hour): 2 for the test item, 2 for the negative control and 2 for the positive control

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: one experiment per exposure period (two in total) with 3 independent OD570 measurements per replicate.

ACCEPTABILITY CRITERIA
- The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
- The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
- In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.

DECISION CRITERIA (see table 1 in 'any other information on materials and methods')
A test item is considered corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.

A test item is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 μL
- The test item was applied directly on top of the skin tissues

NEGATIVE CONTROL
- Amount applied: 50 μL

POSITIVE CONTROL
- Amount applied: 50 μL (8N KOH)

Duration of treatment / exposure:

3 minute and 1 hour

Duration of post-treatment incubation (if applicable):

3 hours in MTT medium

Number of replicates:

2 tissues per test item per exposure time (4 tissues in total)

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean of 2 replicates

Run / experiment:

3-minute exposure

Value:

98

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

% tissue viability: 8.6

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean of 2 replicates

Run / experiment:

1-hour exposure period

Value:

67

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

% tissue viability: 10

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: the results of the positive control data were within the historical data range and thereby showing the test system functioned properly.

ACCEPTANCE OF RESULTS (see table 2 in 'any other information on results' for historical data):
- Acceptance criteria met for negative control: yes, the absolute mean OD570 of the two tissues of the negative control was within the laboratory historical control data range (i.e., 1.526 for the 3-minute exposure period and 1.743 for the 1-hour exposure period).
- Acceptance criteria met for positive control: yes, the mean relative tissue viability following 1-hour exposure to the positive control was <15 % (i.e., 10%).
- Acceptance criteria met for variability between replicate measurements: yes, the Coefficient of Variation (CV) between tissue replicates was ≤ 30% (i.e., <6%)

For individual OD measurements, see table 3 in 'any other information on results'.

Any other information on results incl. tables

Table 2 Historical Control Data

	Negative control		Positive control
	3-minute treatment (OD570)	1-hour treatment (OD570)	3-minute treatment (OD570)	1-hour treatment (OD570)
Range	1.258 – 2.615	1.371 – 2.371	0.0172 – 0.56	0.046 – 0.339
Mean	1.80	1.82	0.19	0.14
SD	0.26	0.22	0.09	0.05
n	111	110	106	103

SD = Standard deviation n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of November 2014 to November 2017.

Table 3 Individual OD Measurements at 570 nm

	3-minute application (OD570)        A            B		1-hour application (OD570)        A            B
Negative control OD570measurement 1 OD570measurement 2 OD570measurement 3	1.7656 1.5268 1.5540	1.6453 1.3875 1.5345	1.7194 1.7546 1.7556	1.8760 1.8096 1.8013
Test item OD570measurement 1 OD570measurement 2 OD570measurement 3	1.5934 1.5800 1.5035	1.5323 1.5549 1.5056	1.2957 1.2323 1.2006	1.1982 1.1669 1.1521
Positive control OD570measurement 1 OD570measurement 2 OD570measurement 3	0.1772 0.1827 0.1811	0.1680 0.1711 0.1696	0.2548 0.2258 0.2311	0.2089 0.2165 0.2108

OD = Optical density

Duplicate exposures are indicated by A and B.

Applicant's summary and conclusion

Interpretation of results:

other: Not corrosive

Conclusions:

In a in vitro skin corrosion test conducted according to OECD 431 guideline and GLP principles, Sodium hydrogen substituted amino acid solution was found to be not corrosive to the skin (mean tissue viability of 98% and 67% after a 3-minute and a 1-hour exposure period, respectively) under the experimental conditions described in this report.