(2S)-2-amino-3-(4-{[bis(sodiooxy)phosphoryl]oxy}phenyl)propanoic acid

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-04-15 to 2020-05-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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)

Version / remarks:

2012-07-06

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2019-06-18

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(2S)-2-amino-3-(4-{[bis(sodiooxy)phosphoryl]oxy}phenyl)propanoic acid

EC Number:

860-352-3

Cas Number:

1610350-91-8

Molecular formula:

C9H10NO6PNa2

IUPAC Name:

(2S)-2-amino-3-(4-{[bis(sodiooxy)phosphoryl]oxy}phenyl)propanoic acid

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The in vitro skin irritation test using reconstructed human epidermis tissue and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™
- Tissue batch number: 30864

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 35 minutes at 37 ± 1.5°C and 5 ± 0.5% CO2, remaining 25 min at room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues washed with DPBS several times in order to remove any residual test material.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/mL
- Incubation time: 3 hours ± 5 minutes
- Spectrophotometer: Versamax® (Molecular Devices), using the software SoftMax Pro Enterprise (version 4.7.1)
- Wavelength: OD 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
Positive control (rel. viability): mean: 3.98%; standard deviation: 1.01% points; range of viabilities: 2.24-6.19%

Positive control (absorption) : mean: 0.07; standard deviation: 0.02; range of absorption: 0.03-0.11
Negative control (absorption): mean: 1.69; standard deviation: 0.19; range of absorption: 1.28-2

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Evaluation of colour interference: 30 µL of the test item mixed with 300 µL deionised water was incubated for 60 minutes at 37 ± 1.5 °C. Since the colour of the test item mixture did not change during the incubation period, additional testing with viable tissues was not required.
Evaluation of direct reduction of MTT: 30 µL of the test item added to MTT (1 mg/mL). Incubated in the dark at room temperature for 60 minutes. Since the colour did not turn blue/purple, the test item was not considered to be a MTT reducer.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 60 minutes exposure is greater than or equal to 50 %

- The test substance is considered to be non-corrosive to skin if the viability after 60 minutes exposure is greater than 50%

Control samples:

yes, concurrent negative control

yes, concurrent no treatment

Amount/concentration applied:

30 µL

Duration of treatment / exposure:

- 60 min (35 min in incubator at 37°C ± 1.5°C and 5 ± 0.5% CO2 and 25 min at room temperature)

Duration of post-treatment incubation (if applicable):

25.5 hours in fresh assay medium, then another 20 hours in assay medium

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean value of 3 replicates

Value:

90.86

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Table 1: Results after treatment with test item and controls

Treatment Group	Tissue No.	OD 570 nm			Mean OD of 3 Wells	Mean OD of 3 Wells blank corrected	Mean OD of 3 tissues	Rel. Viability [%] Tissue 1, 2 + 3	Standard Deviation	Mean Rel. Viability [%]
		Well 1	Well 2	Well 3
Blank		0.039	0.039	0.039	0.039
Negative Control	1	1.928	1.874	1.871	1.891	1.852	1.832	101.092	6.9	100.0
	2	1.757	1.727	1.722	1.735	1.696		92.589
	3	2.025	1.974	1.961	1.987	1.948		106.319
Positive Control	1	0.105	0.099	0.099	0.101	0.062	0.059	3.381	0.3	3.24
	2	0.102	0.100	0.102	0.101	0.062		3.399
	3	0.094	0.093	0.092	0.093	0.054		2.950
Test Item	1	1.736	1.698	1.689	1.708	1.669	1.664	91.088	0.6	90.86
	2	1.735	1.679	1.657	1.690	1.651		90.138
	3	1.730	1.706	1.702	1.712	1.673		91.344

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is non-irritant to skin according to UN GHS and EU CLP regulation.

Executive summary:

This in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test.

The test item did not prove to be a MTT reducer in the MTT interference pre-experiment. Also, its intrinsic color was not intensive and it did not change color when mixed with deionised water or isopropanol. Therefore, additional tests with freeze-killed tissues or viable tissues (without MTT addition) did not have to be performed.

Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative control (DPBS) or the positive control (5% SDS) for 60 minutes.

After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the viability as compared to the negative control for the 60 minutes treatment interval, thus assuring the validity of the test system.

After treatment with the test item the mean relative viability value was 90.86% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.