L-cystine dihydrochloride

Endpoint summary

Description of key information

Skin:

Based on the results of an in vitro skin irritation assay according to OECD 439, the test substance is considered to possess skin irritating potential (UN GHS: Category 1 or 2) (reference 7.3.1 -1).

Based on the results of an in vitro skin corrosion assay according to OECD 431, the test substance is considered to be corrosive to the skin (UN GHS: Category 1B or 1C) (reference 7.3.1 -2).

Eye:

Based on the results of an in vitro eye irriation assay according to OECD 437, the test substance is not considered to have an eye damaging potential UN GHS Category 1. No prediction can be made for eye damaging potential UN GHS Category 2 or no classification for eye damaging potential (reference 7.3.2 -1).

Based on the results of an in vitro eye irritation assay according to OECD 492, the test substance is considered to possess eye damaging potential (UN GHS Category 1 or 2) (reference 7.3.2 -2).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

26 September 2016 - 05 December 2016

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

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 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:

SkinEthic™ RHE-model RHE/S/17
- Batch no.: 16-RHE-114
- Expiration date: 07 November 2016

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: rinsed with minimum 25 mL DPBS; excess DPBS removed by shaking the tissue inserts and blotting the bottom of the tissue inserts with blotting paper
- Observable damage in the tissue due to washing: no data
- Modifications to validated SOP: none

DYE BINDING METHOD
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: microplate reader ELx800, BioTek Instruments GmbH
- Wavelength: 570 nm

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

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin category 2 if the viability is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 16 mg ± 2 mg per tissue

NEGATIVE CONTROL
- Volume applied: 16 µL ± 2 µL per tissue

POSITIVE CONTROL
- Volume applied: 16 µL ± 2 µL per tissue (5 % solution in deionized water)

Duration of treatment / exposure:

42 minutes (± 1 minute)

Duration of post-treatment incubation (if applicable):

42 hours (± 1 hour)

Number of replicates:

The test item as well as the positive and negative control were tested in batch-triplicates.

Irritation / corrosion parameter:

% tissue viability

Remarks:

replicate 1

Run / experiment:

1

Value:

28.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

replicate 2

Run / experiment:

1

Value:

29.27

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

replicate 3

Run / experiment:

1

Value:

30.77

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

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	1.925	98.52%	1.978	101.22%	1.959	100.26%	1.954	100.00%	1.37%
Positive Control	0.029	1.48%	0.028	1.45%	0.030	1.52%	0.029	1.48%	2.30%
Test item	0.557	28.50%	0.572	29.27%	0.601	30.77%	0.577	29.51%	3.91%

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the conditions of the present study, the test item is considered to possess an irritant potential to skin (UN GHS: Category 2).

Executive summary:

The objective of the present study was to investigate the potential of the test item to induce skin irritation in an in vitro human skin model.

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, 10µL 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 (DPBS-buffer) the mean OD was 1.954 (study acceptance criterion: > 1.431). Treatment with the positive control (5% aqueous solution of sodium dodecyl sulfate) revealed a mean viability value of 1.48% (study acceptance criterion: < 3.14%). Thus, the acceptance criteria were met.

Following treatment with the test item, the tissue viability was 29.51% and, thus, lower than 50%, i.e.according to OECD 439 the test item is considered as irritant to skin (UN GHS: Category 2).

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05 December 2016 - 06 February 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: B.40.bis. In vitro skin corrosion (Human skin model test)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: INVITTOX Protocol SkinEthicTM 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 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:

SkinEthic™ RHE-model RHE/S/17
- Batch no.: 16-RHE-130
- Expiration date: 19 December 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: ambient temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing step: using minimum volume of 20 mL DPBS, 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 data
- Modifications to validated SOP: none

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

NUMBER OF REPLICATE TISSUES: 2

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

PREDICTION MODEL / DECISION CRITERIA
- 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%.
- 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%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 20 ± 3 mg per tissue

NEGATIVE CONTROL
- Volume applied: 40 ± 3 µL per tissue

POSITIVE CONTROL
- Volume applied: 40 ± 3 µL per tissue (5 % solution in deionized water)

Duration of treatment / exposure:

3 minutes or 1 hour

Number of replicates:

The test item as well as the negative control were tested with two replicate tissues per time point (3 min and 1 hour exposure). The positive control was tested with two replicate tissues only for 1 hour.

Irritation / corrosion parameter:

% tissue viability

Remarks:

replicate 1 / 3 minutes

Run / experiment:

1

Value:

59.56

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

replicate 1 / 1 hour

Run / experiment:

1

Value:

3.68

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: positive indication of corrosion

Irritation / corrosion parameter:

% tissue viability

Remarks:

replicate 2 / 3 minutes

Run / experiment:

1

Value:

59.59

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

replicate 2 / 1 hour

Run / experiment:

1

Value:

4.63

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: positive indication of corrosion

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		Mean		CV
		OD	viability	OD	viability	OD	viability	viability
Negative Control	3 min	2.019	99.02%	2.059	100.98%	2.039	100.00%	1.39%
	1 hour	1.675	99.30%	1.699	100.70%	1.687	100.00%	0.99%
Positive Control	1 hour	0.009	0.51%	0.011	0.66%	0.010	0.59%	17.90%
Test item	3 min	1.214	59.56%	1.215	59.59%	1.215	59.57%	0.03%
	1 hour	0.062	3.68%	0.078	4.63%	0.070	4.16%	16.13%

Interpretation of results:

other: combination of optional sub-categories 1B and 1C

Conclusions:

The test item is considered to possess a corrosive potential to skin.

Executive summary:

The objective of the present study was to investigate the potential of the test item to induce skin corrosion in an in vitro human skin model.

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 skincorrosion 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, 8M) 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.

Following treatment with the test item, the tissue viability was ≥ 50% after 3 minutes exposure (mean viability: 59.57%) and < 15% after 1 hour exposure (mean viability: 4.16%), i.e.according to OECD 431 the test item is considered as corrosive to skin. Following step 2 of the prediction model, the test item is classified as a combination of the optional sub-categories 1B and 1C.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05 January 2017 - 01 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

Justification of the test method and considerations regarding applicability:
The reconstructed human cornea-like epithelium (RhCE) model is an accepted in vitro method to replace animal testing. The human eye EpiOcular™-model closely mimics the biochemical and physiological properties of the human eye, i.e. the cornea.

Characterisation of the test system:
- Designation: EpiOcular™ Tissue (OCL-200, OCL-212)
- Lot No.: 23759
- Keratinocyte strain: 4F1188
- Supplier: MatTek In Vitro Life Science Laboratories

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 mg per tissue

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

25 minutes at room temperature, 18 hours at 37 ± 1.5 °C

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used:

Preparation:
On day of receipt, the tissues were equilibrated in their 24-well shipping container to room temperature for about 15 minutes. Afterwards the tissues were removed from the shipping container using sterile forceps and transferred to 6-well plates containing 1 mL pre-warmed (37°C) assay medium. Any agarose adhering to the inserts was removed by gentle blotting on gauze or paper towel. Afterwards, the tissues were incubated at 37°C and 5% CO2 overnight (about 16.7 hours) without medium exchange.

Pre-Treatment:
After the overnight incubation, the tissues were pre-wetted with 20 µL DPBS and incubated at 37°C and 5% CO2 for 30 minutes (± 2 minutes).

Exposure and Post-Treatment:
After the 30 minute DPBS pre-treatment, the solid test item, the negative and the positive control were tested by applying 50 mg (test item) or µL (controls) topically on the EpiOcular™ tissues. The tissues were placed back into the culture medium after dosing and incubated at 37°C and 5% CO2 for 6 h.
At the end of the 6 hours treatment time, the positive control, negative control and the test item were removed by extensively rinsing the tissues with pre-warmed (room temperature) DPBS. Three clean beakers, containing a minimum of 100 mL each of DPBS were used per group. The inserts containing the tissue were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the two tissues per group were rinsed simultaneously by holding the replicate inserts together by their collars using forceps. The test item or control articles were decanted from the tissue surface onto a clean absorbent material and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed at least two additional times in the first beaker. The culture was then rinsed in the second and third beakers of DPBS at least three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material.
After rinsing, the tissues were immediately transferred in 5 mL of pre-warmed (room temperature) assay medium in a 12-well plate for 25 minutes at room temperature.
After the 25 minutes incubation, each insert was removed from the assay medium, the medium was decanted off the tissue, and the insert were blotted on absorbent material and transferred in 6-well plates filled with 1 mL of pre-warmed (37°C) assay medium for 18 h at 37°C and 5% CO2.

- RhCE tissue construct used, including batch number:

Designation: EpiOcular™ Tissue (OCL-200, OCL-212)
Lot No.: 23759
Keratinocyte strain: 4F1188
Supplier: MatTek In Vitro Life Science Laboratories

- Doses of test chemical and control substances used: 50 mg (test item), 50 µL (controls)
- Duration and temperature of exposure, post-exposure: exposure: 6 hours at 37 °C; post-exposure: 25 min at room temperature + 18 h at 37 °C

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: No. 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 and the test item has no colorant properties.

- Number of tissue replicates used per test chemical and controls: 2

- Wavelength used for quantifying MTT formazan: 570 nm

- Description of the method used to quantify MTT formazan:
After the post-treatment incubation period, the treated tissues were transferred in a 24-well plate filled with 300 µL MTT solution (1.0 mg/mL MTT). Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes (± 10 minutes) at 37°C and 5% CO2.
The inserts were removed from the 24-well plate after 180 minutes (± 10 minutes). The bottom of the inserts was blotted on absorbent material, and then transferred to a 6-well plate containing 2 mL isopropanol so that no isopropanol was flowing into the inserts. The plate was sealed with a standard plate sealer. To extract the MTT, the plates was placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. The corresponding negative and positive controls were treated identically.
The extract solution was mixed and 2 x 200 µL were transferred into a 96-well plate. The OD was read using a spectrophotometer at 570 nm wavelength. A functional test of the microplate reader was performed using a filter test plate.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
If the test item-treated tissue viability is >60.0% relative to negative control-treated tissue viability, the test item is labeled non-irritant (UN GHS No Category). If the test item-treated tissue viability is ≤ 60.0% relative to negative control-treated tissue viability, the test item is labeled irritant (UN GHS Category 1 or Category 2).

- Acceptance Criteria:
The results are acceptable if:

1. The negative control OD >0.8 and <2.5,

2. The mean relative viability of the positive control is:
a) 30 minute exposure: below 50% of control viability
b) 6 hour exposure: below 50% of control viability

3. Acceptable variability between tissue replicates: < 20 %

Irritation parameter:

other: tissue viability %

Remarks:

tissue 1

Run / experiment:

1

Value:

3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

other: tissue viability %

Remarks:

tissue 2

Run / experiment:

1

Value:

2.1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Table 1 Tissue viability

Treatment group	Absorbance well1 (tissue 1/2)	Absorbance well 2 (tissue 1/2)	Mean absorbance (tissue 1/2)	Mean absorbance minus mean blank (tissue 1/2)	Mean tissue absorbance*	Relative absorbance [%] (tissue 1/2)**	Difference of rel. absorbances [%]	Viability [% of negative control]**
Blank	0.039	0.039	0.039	0.000
Negative control	1.216	1.232	1.224	1.185	1.291	91.9	16.3	100.0
	1.415	1.453	1.434	1.396		108.1
Positive control	0.114	0.118	0.116	0.078	0.104	6.0	4.2	8.1
	0.169	0.171	0.170	0.131		10.2
Test item	0.089	0.066	0.077	0.039	0.033	3.0	0.9	2.6
	0.067	0.065	0.066	0.027		2.1

*       Mean of two replicate wells after blank correction

**     Relative absorbance (rounded values) =  100 x (absorbance treated group / absorbance negative control)

Interpretation of results:

other: Category 1 (irreversible effects on the eye) or Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Under the conditions of the present study, the test item did show an eye hazard potential. The test item is identified as potentially requiring classification and labelling according to UN GHS (Category 1 or Category 2).

Executive summary:

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

The white to colourless test item did not prove to be an MTT reducer in the MTT pre-test, and it did not prove to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed or viable tissues did not have to be performed.

Each 50 mg of the test item, were applied to each of duplicate tissue for 6 hours. Each 50 µL of the negative control (deionised water) and of the positive control (methyl acetate) were also applied to duplicate tissues each.

After treatment with the negative control the absorbance values (between 1.216 and 1.453) were well within the required acceptability criterion of mean OD > 0.8 and < 2.5 thus showing the quality of the tissues.

Treatment with the positive control induced a decrease below 50% compared with the negative control value in the relative absorbance (8.1%) thus ensuring the validity of the test system.

The difference of viability between the two relating tissues was < 20% in the same run (values between 0.9% to 16.3%) for test item tissues, positive and negative control tissues).

Irritating effects were observed following incubation with the test item. Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues decreased below 60% (2.6%).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

08 March 2016 - 22 April 2016

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany
- Age at study initiation: 17 - 33 months
- Corneal diameter: 24 - 26 mm

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.

SOURCE OF COLLECTED EYES
- Source: Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany
- Age at study initiation: 17 - 33 months
- Corneal diameter: 24 - 26 mm
- Storage, temperature and transport conditions of ocular tissue: cooled on ice
- Time interval prior to initiating testing: The corneas were prepared immediately after delivery of the eyes to the laboratory.
- Indication of any existing defects or lesions in ocular tissue samples: Eyes presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Volume applied: 750 µL
- Concentration: 20%

VEHICLE
- Volume applied: 750 µL

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

Three corneas were used per group (negative control, positive control and test item group).

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
All eyes were carefully examined macroscopically for defects. A rim of about 2 to 3 mm of tissue (sclera) was left for stability and handling of the isolated cornea.

QUALITY CHECK OF THE ISOLATED CORNEAS
Corneas presenting defects such as vascularization, pigmentation, opacity or scratches were discarded.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: 0.9% sodium chloride solution

POSITIVE CONTROL USED: Imidazole

APPLICATION DOSE AND EXPOSURE TIME: 750 µL of a 20% solution were applied for 240 minutes

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3 times with washing medium

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: baseline opacity was determined with a calibrated opacitometer, the light transmission through the corneas, given as lux value, was recorded in a table and thereafter converted into an opacity value (baseline opacity values).
- Corneal permeability: corneas were incubated again in an incubator in a horizontal position at 32 ± 1°C for 90 minutes, amount of fluorescein that crossed the cornea was measured spectrophotometrically 490 nm

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula (referring to OECD Guideline 437) was used to determine the In Vitro Irritancy Score (IVIS) of the negative control:
IVIS = mean opacity value + (15 x mean permeability OD490 value)
The following formula was used to determine the In Vitro Irritancy Score (IVIS) of the positive control and the test item:
IVIS = corrected opacity value + (15 x corrected permeability OD490 value)
- The In Vitro Irritancy Score (IVIS) was calculated for each individual treatment and positive control cornea. The mean In Vitro Irritancy Score (IVIS) value of each treated group was calculated from the individual In Vitro Irritancy Score (IVIS) values.

DECISION CRITERIA:
IVIS ≤ 3 No Category (according to GHS)
IVIS > 3; ≤ 55 No prediction can be made
IVIS > 55 Serious eye damage, Category 1 (according to GHS)

ACCEPTANCE CRITERIA:
A test is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean(IVIS positive control: 76.5 - 136.8).
The negative control responses should result in an IVIS that falls within three standard deviations of the current historical mean (IVIS negative control: -1.4 - 3.5).
A single test run with three corneas should be sufficient for a test item when the resulting classification is unequivocal. In cases of the following borderline results in the first testing run, a second test run should be considered.
- 2 of the 3 corneas give discordant predictions from the mean of all 3 corneas or
- 1 of the 3 corneas give discordant predictions from the mean of all 3 corneas, and the discordant result is >10 IVIS units from the cut-off threshold of 55.

Irritation parameter:

in vitro irritation score

Remarks:

replicate 1

Run / experiment:

1

Value:

41.445

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Remarks:

replicate 2

Run / experiment:

1

Value:

45.768

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Remarks:

replicate 3

Run / experiment:

1

Value:

55.232

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no
No observations (e.g. tissue peeling, residual test chemical, non-uniform opacity patterns) were seen in a visually inspection of the corneas after treatment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
The resulting classification of the test item in this study is unequivocal and no borderline results were obtained. Therefore, a single testing run composed of three corneas per group was sufficient.

Table 1 Opacity, permeability and IVIS results

		Opacity	Permeability	IVIS
				per cornea	per group (mean value)	Standard deviation
Negative control	0.9% sodium chloride solution	3.330	0.009	3.470	1.1	2.1
		0.204	0.006	0.299
		-0.536	0.006	-0.441
Positive control	Imidazole (20%)	63.371	2.149	95.606	111.9	14.6
		70.988	3.008	116.103
		74.970	3.260	123.870
Test item		41.465	-0.001	41.445	47.5	7.1
		45.808	-0.003	45.768
		55.247	-0.001	55.232

Interpretation of results:

other: not Category 1 (irreversible effects on the eye) based on GHS

Conclusions:

Based on the results of the present study, the test item is not requiring classification for eye damaging potential (UN GHS Category 1). No prediction can be made if the test item is requiring classification for eye irritation (UN GHS Category 2) or if the test item is not requiring classification (no UN GHS Category).

Executive summary:

This in vitro study was performed to evaluate the eye hazard potential of the test item by means of the BCOP (Bovine Corneal Opacity and Permeability Assay).

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 the test item, the positive, and the negative control were rinsed from the corneas 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 (0.9% sodium chloride solution) the calculated IVIS was 1.1 (study acceptance criteria range: -1.4 - 3.5). Treatment with the positive control (20% Imidazole) revealed an IVIS of 111.9 (study acceptance criteria range: 76.5 - 136.8). Therefore, the study fulfilled the validity criteria.

The IVIS obtained after treatment was 47.5 and, thus, higher than 3 and lower than 55, the test item is not requiring classification for eye damaging potential (UN GHS Category 1). No prediction can be made if the test item is requiring classification for eye irritation (UN GHS Category 2) or if the test item is not requiring classification (no UN GHS Category).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

OECD 439

A study was performed to investigate the potential of the test item to induce skin irritation in an in vitro human skin model.

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.

After treatment with the negative control (DPBS-buffer) the mean OD was 1.954 (study acceptance criterion: > 1.431). Treatment with the positive control (5% aqueous solution of sodium dodecyl sulfate) revealed a mean viability value of 1.48% (study acceptance criterion: <3.14%). Thus, the acceptance criteria were met.

Following treatment with the test item, the tissue viability was 29.51% and, thus, lower than 50%, i.e. according to OECD 439 the test item is considered as irritant to skin (UN GHS: Category 1 or 2).

OECD 431

A study was performed to investigate the potential of the test item to induce skin corrosion in an in vitro human skin model.

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, 8M) 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.

Following treatment with the test item, the tissue viability was ≥ 50% after 3 minutes exposure (mean viability: 59.57%) and < 15% after 1 hour exposure (mean viability: 4.16%), i.e.according to OECD 431 the test item is considered as corrosive to skin. Following step 2 of the prediction model, the test item is classified as a combination of the optional sub-categories 1B and 1C.

Conclusion: Since one in vitro test alone can not predict reliably the skin irritating/corrosive potential of the test item, an integrated test strategy, consisting of the tests according to OECD Guideline 439 (skin irritation, reference 7.3.1-1) and OECD Guideline 431 (skin corrosion, reference 7.3.1-2) was performed. According to the results of the performed tests (skin irritating, skin corrosive) the test item is considered to be skin corrosive. As no discrimination between Cat 1B and C can be made as precautionary approach the substance is classified as UN GHS: Category 1B.

Eye irritiation

OECD 437

An in vitro study was performed to evaluate the eye hazard potential of the test item by means of the BCOP (Bovine Corneal Opacity and Permeability Assay).

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 the test item, the positive, and the negative control were rinsed from the corneas 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 (0.9% sodium chloride solution) the calculated IVIS was 1.1 (study acceptance criteria range: -1.4 - 3.5). Treatment with the positive control (20% Imidazole) revealed an IVIS of 111.9 (study acceptance criteria range: 76.5 - 136.8). Therefore, the study fulfilled the validity criteria.

The IVIS obtained after treatment was 47.5 and, thus, higher than 3 and lower than 55, i.e. according to UN GHS classification no prediction can be made regarding the eye hazard potential of the test item.

OECD 492

An in vitro study was performed to assess the eye irritation potential of the test item by means of the Human Cornea Model Test.

The white to colourless test item did not prove to be an MTT reducer in the MTT pre-test, and it did not prove to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed or viable tissues did not have to be performed.

Each 50 mg of the test item, were applied to each of duplicate tissue for 6 hours. Each 50 µL of the negative control (deionised water) and of the positive control (methyl acetate) were also applied to duplicate tissues each.

After treatment with the negative control the absorbance values (between 1.216 and 1.453) were well within the required acceptability criterion of mean OD > 0.8 and < 2.5 thus showing the quality of the tissues.

Treatment with the positive control induced a decrease below 50% compared with the negative control value in the relative absorbance (8.1%) thus ensuring the validity of the test system.

The difference of viability between the two relating tissues was < 20% in the same run (values between 0.9% to 16.3%) for test item tissues, positive and negative control tissues).

Irritating effects were observed following incubation with the test item. Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues decreased below 60% (2.6%).

Conclusion:

Since one in vitro test alone can not predict reliably the eye irritating/damaging potential of the test item, an integrated testing strategy, consisting of the tests according to OECD Guideline 437 (eye damage, reference 7.3.2 -1) and OECD Guideline 492 (eye irritation, reference 7.3.2 -2) was performed. According to the results of the performed tests the test item is considered to be eye irritating (UN GHS Category 2, H319).

Justification for classification or non-classification

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

The available experimental data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on this data, the substance is considered to be classified for skin corrosion (UN GHS: Category 1B, H314) and eye irritation (UN GHS: Category 2, H319) under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EC) No 2017/776.