Disodium L-tyrosinate

Endpoint summary

Description of key information

Skin:

In an in vitro skin corrosion assay (RhE) according to OECD Guideline 431, the test item showed no skin corrosive potential (UN GHS: No Category) (reference 7.3.1-1).

In an in vitro skin irritation assay according to OECD Guideline 439 (RhE), the test substance did not show skin irritating properties (UN GHS: No Category) (reference 7.3.1 -2).

Eye:

In an ex vivo Bovine Corneal Opacity and Permeability Assay (BCOP) according to OECD Guideline 437, the test item showed serious eye damaging properties (UN GHS: Category 1, H318) (references 7.3.2-1).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05 December 2017 - 25 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Council Regulation (EC) No. 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 (REACH). B.40.bis. In vitro skin corrosion: human skin model test.

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: INVITTOX Protocol SkinEthic™ Skin Corrosivity Test

Version / remarks:

2012

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 reconstructed human epidermis model in vitro method is an accepted in vitro method to replace animal testing. The human skin RHE™ model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model RHE/S/17
- Tissue batch number: 16-RHE-130

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (3 min or 1 h)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: gently rinsing with a minimum volume of 20 mL DPBS using a pipette. Excess DPBS was removed by gently shaking the tissue inserts and blotting the bottom of the tissue inserts with blotting paper.
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h ± 15 min
- Spectrophotometer: microplate reader (ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany)
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The pre-test for direct MTT-reducing capacity of the test item did not result in blue color, i.e. the test item is not a direct MTT reducer.
In the pre-test medium coloration by the test item was observed, but no tissues were stained during the study. Therefore, no additional tissues for color control were treated according to the INVITTOX Protocol SkinEthic™ Skin Corrosivity Test (2012).

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50 %, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied : 20 ± 3 mg

NEGATIVE CONTROL
- Amount applied: 40 ± 3 µL

POSITIVE CONTROL
- Amount applied: 40 ± 3 µL

Duration of treatment / exposure:

3 min or 1 h

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

82.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 h

Value:

79.89

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

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

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Acceptability of the Test

Acceptability of the Negative Control: The negative control OD values were 2.019, 2.059, 1.675 and 1.699 and, thus, in the range of ≥ 0.8 and ≤ 3.0.

Acceptability of the Positive Control: After treatment with the positive control (potassium hydroxide, 8N) the mean viability value was 0.59 % after 1 hour exposure and, thus, lower than 15 %.

Test Substance Data Acceptance Criteria: The range between identically treated tissues was less than 30 % (27.10 % after 3 minutes exposure and 13.63 % after 1 hour exposure).

The study met all acceptance criteria

Table 1: Results

Group		Tissue 1		Tissue 2		Mean		CV
		OD	Viability (%)	OD	Viability (%)	OD	Viability (%)	Viability (%)
Negative Control	3 min	20.19	99.02	2.059	100.98	2.039	100.00	1.39
	1 h	1.675	99.30	1.699	100.70	1.687	100.00	0.99
Positive Control	1 h	0.009	0.51	0.011	0.66	0.010	0.59	17.90
Test Substance	3 min	1.481	72.66	1.883	92.35	1.682	82.50	16.88
	1 h	1.262	74.79	1.434	84.99	1.348	79.89	9.02

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vitro skin corrosion assay (RhE) according to OECD Guideline 431, the test item showed no skin corrosive potential.

Executive summary:

In an in vitro skin corrosion assay (RhE) according to OECD Guideline 431, the potential of the test item to induce skin corrosion in an in vitro human skin model was investigated.

The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin corrosion potential.

Duplicates of the human skin RHE-model were treated with the test item or the negative control for 3 minutes and additional 1 hour. Duplicates with the positive control were only treated for 1 hour. 40 ± 3 µL of either the negative control (deionised water) or the positive control (potassium hydroxide, 8N) were applied to the tissues. Before application of 20 ± 3 mg of the solid test item, 20 ± 2 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis.

After treatment with the negative control (deionised water) the mean OD per tissue replicate was 2.019 and 2.059 after 3 minutes exposure and 1.675 and 1.699 after 1 hour exposure (study acceptance criterion: ≥ 0.8 and ≤ 3.0). Treatment with the positive control (Potassium hydroxide, 8N) revealed a mean viability of 0.59% after 1 hour (study acceptance criterion: < 15%). Therefore, the study fulfilled the validity criteria.

Following treatment with the test item, the tissue viability was > 50% after 3 minutes exposure (mean viability: 82.50%) and > 15% after 1 hour exposure (mean viability: 79.89%), i.e. according to OECD 431 the test item is considered as non-corrosive to skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05 December 2017 - 15 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: SkinEthic Skin Irritation Test 42bis Standard Operating Procedure

Version / remarks:

2009

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 reconstructed human epidermis model in vitro method is an accepted in vitro method to replace animal testing. The human skin RHE™ model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e the epidermis, and has been validated by the ECVAM in 2008.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model RHE/S/17 (Episkin/SkinEthic Laboratories, Lyon, France)
- Tissue batch number: 18-RHE-001 and 18-RHE-009
- Date of initiation of testing: On day of receipt the pre-incubation phase of the tissues started

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: the test item, negative and positive control were removed immediately by gently rinsing with a minimum volume of 25 mL DPBS using a pipette. Excess DPBS was removed by gently shaking the tissue inserts and blotting the bottom of the tissue inserts with blotting paper.
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: ELx800 (BioTek Instruments GmbH, Bad Friedrichshall, Germany)
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test item is considered to be non-corrosive to skin if the tissue viability after exposure and post-treatment incubation is > 50%.
- The test item is considered to be irritant to the skin (UNGHS Category 2) if the viability is less than or equal to 50%.
- Since the in vitro skin irritation test according to OECD 439 cannot resolve between UN GHS Categories 1 and 2, further information on skin corrosion is required to decide on its final classification.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 16 ± 2 mg

NEGATIVE CONTROL
- Amount applied: 16 ± 0.5 µL

POSITIVE CONTROL
- Amount applied: 16 ± 0.5 µL

Duration of treatment / exposure:

42 ± 1 min

Duration of post-treatment incubation (if applicable):

42 ± 1 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1, Tissue 1

Value:

95.3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1, Tissue 2

Value:

89.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1, Tissue 3

Value:

85.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1, Mean

Value:

90

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

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


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Table 1: Optical Density and Tissue Viability

Group	Tissue 1		Tissue 2		Tissue 3		Mean		SD
	OD	viability	OD	viability	OD	viability	OD	viability	viability
Negative Control	1.890	103.8%	1.714	94.1%	1.858	102.0%	1.821	100.0%	5.2%
Positive Control	0.021	1.2%	0.020	1.1%	0.023	1.3%	0.021	1.2%	8.3%
Art. T1145	1.735*	95.3%	1.625*	89.2%	1.557*	85.5%	1.639	90.0%	5.4%

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vitro skin irritation assay according to OECD Guideline 439 (RhE), the test substance did not show skin irritating properties.

Executive summary:

In an in vitro skin irritation assay according to OECD Guideline 439 (RhE), the skin irritating properties of the test item were determined. The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential.

Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (DPBS-buffer) or the positive control (5 % aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Before application of 16 mg of the solid test item, 10 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis. All acceptability criteria after treatment with the negative control and the positive control were met.

All acceptability criteria after treatment with the negative control (DPBS-buffer) and the positive control (5% aqueous solution of sodium dodecyl sulfate) were met.

Following treatment with the test item, the tissue viability was 90.0% and, thus, higher than 50%, i.e. according to OECD 439 the test item is considered as non-irritant to skin (UN GHS: No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Additional information

Skin:

The objective was to assess the skin irritation and corrosion potential ofthe test substance. By using the currently available methods a single in vitro assay is not sufficient to cover the full range of skin irritating/corrosion potential. Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (according to OECD guideline 431) and Skin Irritation Test (according to OECD 439).

In an in vitro skin corrosion assay (RhE) according to OECD Guideline 431 (reference 7.3.1-1), the potential ofthe test item to induce skin corrosion in an in vitro human skin model was investigated. The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin corrosion potential. Duplicates of the human skin RHE-model were treated with the test item or the negative control for 3 minutes and additional 1 hour. Duplicates with the positive control were only treated for 1 hour. 40 ± 3 µL of either the negative control (deionised water) or the positive control (potassium hydroxide, 8N) were applied to the tissues. Before application of 20 ± 3 mg of the solid test item, 20 ± 2 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis. After treatment with the negative control (deionised water) the mean OD per tissue replicate was 2.019 and 2.059 after 3 minutes exposure and 1.675 and 1.699 after 1 hour exposure (study acceptance criterion: ≥ 0.8 and ≤ 3.0). Treatment with the positive control (Potassium hydroxide, 8N) revealed a mean viability of 0.59% after 1 hour (study acceptance criterion: < 15%). Therefore, the study fulfilled the validity criteria. Following treatment with the test item, the tissue viability was > 50% after 3 minutes exposure (mean viability: 82.50%) and > 15% after 1 hour exposure (mean viability: 79.89%), i.e. according to OECD 431 the test item is considered as non-corrosive to skin.

This result is extended by an in vitro skin irritation assay according to OECD Guideline 439 (RhE) (reference 7.3.1 -2). In this study, the skin irritating properties of the test item were determined. The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential.

Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (DPBS-buffer) or the positive control (5 % aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Before application of 16 mg of the solid test item, 10 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis. All acceptability criteria after treatment with the negative control (DPBS-buffer) and the positive control (5% aqueous solution of sodium dodecyl sulfate) were met.

Following treatment with the test item, the tissue viability was 90.0% and, thus, higher than 50%, i.e. according to OECD 439 the test item is considered as non-irritant to skin (UN GHS: No Category).

In conclusion, the test item did neither show skin corrosive nor skin irritating properties. According to the IATA strategy, the test item is considered as non-irritant to the skin.

Eye:

In an ex vivo Bovine Corneal Opacity and Permeability Assay (BCOP) according to OECD Guideline 437, the eye damaging potential of the test item was examined (references 7.3.2 -1). To determine the eye hazard potential the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item as a 20% (w/v) solution in a 0.9% sodium chloride solution. As negative control 0.9% sodium chloride solution and as positive control 20% (w/v) Imidazole was used. Three corneas were used per group (negative control, positive control or test item group). After a first opacity measurement of the untreated bovine corneas, 750 µL of the dissolved test item, positive or negative control were applied on the corneas and incubated for 240 minutes. After the incubation phase t the corneas were rinsed and the opacity was measured again. After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically. The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (IVIS).

After treatment with the negative control the calculated IVIS was 1.1 (study acceptance criteria range: -1.4 - 3.4). Treatment with the positive control revealed an IVIS of 102.3 (study acceptance criteria range: 79.9 - 134.6). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment with the test item was 78.1 and, thus, > 55, i.e. according to OECD 437 the test item is inducing serious eye damage (UN GHS: Category 1).

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

As a result the test item is considered not to be classified for skin irritation/corrosion under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.

In contrast, the test item should be considered as severely eye damaging (Cat. 1, H318) under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.