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

Endpoint summary

Description of key information

The test item did not induce significant or irreversible damage to the skin and eye  in an In vitro Skin Irritation Test according to OECD 439 and in an Bovine Corneal Opacity and Permeability Assay according to OECD 437 and is considered to be "not irritating" to the skin and eye (reference 7.3.1-1 and 7.3.2-1).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

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

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 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

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

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-06-20 to 2020-08-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Concentration: 20% (w/v) suspension in a 0.9% sodium chloride solution

VEHICLE
- Concentration: 0.9% sodium chloride solution
- Lot/batch no.: 18163013

Duration of treatment / exposure:

240 minutes at 32 ± 1°C

Number of animals or in vitro replicates:

3 replicates per test item/ positive control/ negative control

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Freshly isolated bovine eyes of cattle were collected from the slaughterhouse. Excess tissue was removed from the eyes. The eyes were kept and transported in transport medium cooled on ice.

The corneas were prepared immediately after delivery of the eyes to the laboratory. The corneas were carefully removed from the eyes using scalpel and rounded scissors. A rim of about 2 to 3 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the study were collected in incubation medium (pre-warmed at 32 ± 1°C) and the corneal diameter of each cornea was measured and recorded. Each cornea was mounted in a cornea holder (CiToxLAB, Veszprem, Hungary) with the endothelial side against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring without stretching the cornea. Afterwards, the anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex form.

QUALITY CHECK OF THE ISOLATED CORNEAS
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded.

NUMBER OF REPLICATES
3 replicates per test item/ positive control/ negative control

NEGATIVE CONTROL USED
0.9% sodium chloride solution

POSITIVE CONTROL USED
20% (w/v) imidazole in 0.9% sodium chloride solution

APPLICATION DOSE AND EXPOSURE TIME
- Test item: 20% (w/v) in 0.9% sodium chloride solution
- Positive control: 20% (w/v) in 0.9% sodium chloride solution
- Negative/vehicle control: 0.9% sodium chloride solution
- Exposure time: 240 minutes

TREATMENT METHOD:
- negative and positive control: closed chamber
- test item: open chamber

POST-INCUBATION PERIOD: No

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Three times with wash medium

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity value of each individual cornea was corrected for background opacity by subtracting the initial baseline opacity reading from the post treatment opacity reading. In addition, the opacity values of both the treatment and positive control groups were corrected for the mean negative control opacity values. From the individual corrected opacity values, a mean corrected opacity value was calculated for each group.

- Corneal permeability: For each cornea either treated with the positive control or the test item, an individual corrected OD490 value was calculated by subtracting the average negative control permeability value from each individual permeability reading. From the individual corrected permeability values, a mean corrected permeability value was calculated for each group.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: decision criteria as indicated in the test guideline was used

Irritation parameter:

in vitro irritation score

Run / experiment:

mean of three corneas

Value:

0.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Table 1: Opacity, permeability and in vitro irritancy scores calculated after treatment with the test item        

		Opacity	Permeability	IVIS
				per cornea	per group (mean value)	Standard deviation
Negative control	0.9% sodium chloride solution	-0.2	0.012	-0.020	0.2	0.6
		-0.5	0.012	-0.320
		0.7	0.015	0.925
Positive control	Imidazole (20%)	74.2	2.639	113.785	103.8	8.8
		73.9	1.556	97.240
		70.8	1.966	100.290
Test item	Art. 137119	2.0	-0.003	1.955	0.8	1.1
		0.9	-0.008	0.780
		-0.1	-0.008	-0.220

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present study, the test item did not show an eye hazard potential. The test item is not requiring classification for eye irritation or serious eye damage (UN GHS: No Category).

Executive summary:

After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 0.2 (study acceptance criteria range: -1,4 - 3.0). Treatment with the positive control (20% Imidazole) revealed an IVIS of 103.8 (study acceptance criteria range: 83.0 - 131.8). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment with the test item was 0.8 and, thus, lower than 3, i.e. according to OECD 437 the test item is not requiring classification for eye irritation or serious eye damage (UN GHS: No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation

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.

 

Eye irritation

After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 0.2 (study acceptance criteria range: -1,4 - 3.0). Treatment with the positive control (20% Imidazole) revealed an IVIS of 103.8 (study acceptance criteria range: 83.0 - 131.8). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment with the test item was 0.8 and, thus, lower than 3, i.e. according to OECD 437 the test item is not requiring classification for eye irritation or serious eye damage (UN GHS: No Category).

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for irritation/corrosion under Regulation (EC) No 1272/2008, as amended for fifteenth time in Regulation (EU) No 2020/217.