Acetylcysteine

Endpoint summary

Description of key information

Skin

In an in vitro skin irritation study the substance is not considered to possess an irritant potential to the skin (reference 7.3.1-1).

Eye

In an in vitro eye irritation study using the EpiOcular model, the eye hazard potential of the test item could not be predicted (reference 7.3.2 -1). In order to further evaluate the eye hazard potential of the substance the Bovine Corneal Opacity and Permeability Test according to OECD TG 437 was performed. No prediction regarding the eye hazard potential can be made (reference 7.3.2-2). However, based on the available data the substance is classified for eye irritation, Cat. 2 according to Regulation (EC) No 1272/2008 (CLP) and no further testing is necessary.

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:

March 21, 2018 - June 22, 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:

July 28, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ECVAM 2009, Performance Standards for In-Vitro Skin Irritation test Methods boased on Reconstructed Human Epidermis (RHE)

Qualifier:

according to guideline

Guideline:

other: SkinEthic Skin Irritation Test Standard Operating Procedure (SOP): Using the Reconstructed Human Epidermis (RHE) model, INVITTOX

Version / remarks:

Version 2.1, 2009

GLP compliance:

yes (incl. QA statement)

Remarks:

Merck KGaA, Non-Clinical Safety, Frankfurter Strasse 250, 64293 Darmstadt, Germany

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE-model RHE/S/17 obtained from Episkin/SkinEthic Laboratories, Lyon, France
- Tissue batch number(s): 18-RHE-041 (1st run) , 18-RHE-068 (2nd run)

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: gently rinsing with minimum volume of 25 mL DPBS using pipette
- 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 hours at 37°C
- Spectrophotometer: ELx800, BioTek Instruments GmbH
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD=1.3
- Barrier function: 4.6h
- Morphology: well differentiated epidermis consisting of basal, spinous, granular layers and a stratum corneum
- Contamination: no
- Reproducibility: yes

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2

PREDICTION MODEL
- The test substance is considered as non-irritant to skin if the viability after exposure and post-treatment incubation is greater than 50 %. A test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post-treatment incubation is equal or greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 16 mg per tissue

NEGATIVE CONTROL
- Amount(s) applied: 16 µL

POSITIVE CONTROL
- Amount(s) applied: 16 µL
- Concentration: 5% aqueous solution

Duration of treatment / exposure:

42 min

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1st run

Value:

82.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

3.6%

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2nd run

Value:

98.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

6.7%

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: none
- Colour interference with MTT: none

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: Results of the 1st Run

Group	Tissue 1		Tissue 2		Tissue 3		Mean		SD
	OD	viability	OD	viability	OD	viability	OD	viability	viability
Negative Control	2.034	104.1%	1.912	97.9%	1.913	98.0%	1.953	100.00	3.6%
Positive Control	0.031	1.6%	0.028	1.4%	0026	1.3%	0.028	1.4%	14.3%
Test item	1.661	85.0%	1.646	84.3%	1.537	787%	1.615	82.7%	4.2%

 

Table 2: Results of the 2nd Run

Group	Tissue 1		Tissue 2		Tissue 3		Mean		SD
	OD	viability	OD	viability	OD	viability	OD	viability	viability
Negative Control	1.794	107.7%	1.607	96.5%	1.594	95.7%	1.665	100.00	6.7%
Positive Control	0.025	1.5%	0.021	1.3%	0.021	1.3%	0.022	1.4%	7.1%
Test item	1.562	93.8%	1.581	95.0%	1.798	108.0%	1 647	98.9%	8.0%

Table 3: Acceptability of the Test (Run 1)

Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:
	Acceptance Criterion	Result
Negative control OD	≥ 0.8 and ≤ 3.0	1.912 to 2.034
Acceptability of the Positive and Negative Control stated by Episkin/SkinEthic Laboratories:
	Acceptance Criterion	Result
Mean OD negative control	≥1.2	1.953
Mean viability positive control	< 40%	1.4%
SD of group-mean value	≤18%	14.3% (positive control) 3.6% (negative control)
Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:
	Acceptance Criterion	Result
Mean OD negative control	≥1.453	1.953
Mean viability positive control	≤ 2.89%	1.4%
Test Item Data Acceptance Criteria:
	Acceptance Criterion	Result
SD of group-mean value	≤ 18%	4.2%

Table 4: Acceptability of the Test (Run 2)

Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:
	Acceptance Criterion	Result
Negative control OD	≥ 0.8 and ≤ 3.0	1.594 to 1.794
Acceptability of the Positive and Negative Control stated by Episkin/SkinEthic Laboratories:
	Acceptance Criterion	Result
Mean OD negative control	≥1.2	1.665
Mean viability positive control	< 40%	1.4%
SD of group-mean value	≤18%	7.1% (positive control) 6.7% (negative control)
Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:
	Acceptance Criterion	Result
Mean OD negative control	≥1.450	1.665
Mean viability positive control	≤ 2.87%	1.4%
Test Item Data Acceptance Criteria:
	Acceptance Criterion	Result
SD of group-mean value	≤ 18%	8.0%

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the study the substance is not considered to possess an irritant potential to skin (UN GHS: No Category).

Executive summary:

A study according OECD TG 439 was conducted to investigate the potential of the test item to induce skin irritation in an in vitro human skin model (SkinEthic RHE-model).

The substance 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. Because of non-concordant replicate results after treatment with the substance between the first (non-valid) run and the second run, a third run was performed. Only the two valid runs performed are reported as run 1 and run 2. 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 82.7% in run 1 and 98.9% in run 2 and thus, higher than 50% in both runs, i.e. according to OECD TG 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

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:

April 17, 2018 - April 19, 2018

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:

October 9, 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EpiOcular Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; For use with MatTek Corporation´s Reconstructed Human EpiOcular Model; MatTek Corporation

Version / remarks:

June 29, 2015

Qualifier:

according to guideline

Guideline:

other: DB-ALM Protocol No. 164: Ocular Irritation Assay for Chemicals using EpiOcular EIT

Version / remarks:

September 14, 2015

GLP compliance:

yes (incl. QA statement)

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 mg per tissue

Details on study design:

- Details of the test procedure used
The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) 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 eye irritation potential.

- RhCE tissue construct used, including batch number
EpiOcular Tissue (OCL-200, OCL-212), Lot No. 27033, MatTek In Vitro Life Science Laboratories

- Doses of test chemical and control substances used
Solid test item: 50 mg per tissue
Negative control: 50 µL per tissue
Positive control: 50 µL per tissue

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods
6 hours treatment at 37°C and 5% CO2, 25 min post-exposure immersion followed by 18 hours post-exposure incubation at 37°C and 5% CO2

- Number of tissue replicates used per test chemical and controls (positive control, negative control): 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.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
The test item is identified as not requiring classification and labeling according to UN GHS (No Category) if the mean percent tissue viability is more than 60%. In this case no further testing in other test methods is required. If the mean percent tissue viability is less than or equal 60%, no prediction can be made. In this case, further testing with other test methods will be required because RhCE test methods show a certain number of false positive results and cannot resolve between UN GHS Categories 1 and 2.

- Complete supporting information for the specific RhCE tissue construct used
Tissue viability: OD [540-570] 1.996
Barrier function: ET-50 = 27.45 min
Sterility: no contamination

- 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. The difference of viability between the two relating tissues of a single chemical is <20% in the same run (for positive and negative control tissues and tissues of single chemicals). This applies also to the killed controls (single chemicals and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

Irritation parameter:

other: % viability

Run / experiment:

1st Experiment

Value:

2.4

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

17.8%

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: non

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

The test item is not a direct MTT reducer and the test item has no colorant properties.

Interpretation of results:

other: no prediction can be made

Conclusions:

Under the conditions of the present study, the eye hazard potential of the test item cannot be predicted.

Executive summary:

A study according OECD TG 492 was conducted to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model.

The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) 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 eye irritation potential. Duplicates of the EpiOcular™-model were treated with the test item, the negative or the positive control for 6 hours (± 15 minutes). 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues.

After treatment with the negative control (sterile deionized water) the mean OD was 1.686 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 17.8% (study acceptance criterion: <50%). Thus, the acceptance criteria were met. Following treatment with the test item, the tissue viability was 2.4% and thus, lower than 60%. i.e. according to OECD TG 492, no prediction can be made regarding the eye hazard potential of the test.

 

 

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

May 23, 2018 - July 6, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

09 October 2017

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:

18 May 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Concentration: 20% (w/v)

VEHICLE
- Concentration: 0.9% sodium chloride

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

Three corneas were used per group

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. All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded. 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
For equilibration, the corneas in the holder were incubated in a vertical position at 32 ± 1°C for about one hour. At the end of the incubation period, the incubation medium was replaced by fresh pre-warmed (32 ± 1°C) incubation medium in both compartments. The 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). Any corneas that showed macroscopic tissue damage (e.g. scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded.

NUMBER OF REPLICATES : 3

SOLVENT CONTROL USED: yes

POSITIVE CONTROL USED : yes

APPLICATION DOSE AND EXPOSURE TIME
750 µL of the suspended test item (i.e. 150 mg/750 µL), positive or negative control were applied on the corneas.

TREATMENT METHOD: closed chamber method

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

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
Incubation medium was used as final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement. Fresh incubation medium was replaced in both compartments prior to reading the opacity value after treatment.
The post treatment 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 (post treatment opacity values).
In addition, each cornea was observed visually and pertinent observations were recorded (e.g. tissue peeling, residual test chemical, non-uniform opacity patterns).

- Corneal permeability:
An increased permeability of the cornea is indicative for an impairment of the integrity of the corneals' epithelial cell layers. To determine the permeability of the treated corneas, fresh incubation medium was added to the posterior compartments and 1 mL of a fluorescein solution was administered to the anterior compartments.
The corneas were incubated again in an incubator in a horizontal position at 32 ± 1°C for 90 minutes. The amount of fluorescein that crossed the cornea was measured spectrophotometrically in the medium from the posterior chamber. Therefore, 3 x 360 µL medium from each posterior chamber were well mixed, transferred into a 96-well plate, and read with a microplate reader (ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany) at 490 nm (OD490). A functional test of the microplate reader was performed using a filter test plate.

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:
The IVIS cut-off values for identifying test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given in the following:

IVIS: ≤ 3: No Category
IVIS: > 3; ≤ 55:No prediction can be made
IVIS: > 55: Category 1

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 run 1: 82.8 - 132.9, IVIS positive control run 2: 82.4- 132.8).
The negative control responses should result in an IVIS that falls within three standard deviations of the current historical mean (IVIS negative control run 1: -1.4 - 3.1, IVIS negative control run 2:-1.4-3.1).
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

Run / experiment:

1

Value:

52.1

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

52.9

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system:
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
In a first run, two of the three corneas treated with the test item gave discordant predictions from the mean of all three corneas. Due to this borderline a second run was performed according to OECD Guideline 437. The repeat testing run corroborated the prediction of the initial testing run. Therefore, a final decision was taken without further testing.

Run 1:

		Opacity	Permeability	IVIS
				per cornea	per group (mean value)	Standard deviation
Negative control	0.9% sodium chloride solution	0.5	0.004	0.560	0.7	0.4
		1.1	0.003	1.145
		0.4	0.002	0.430
Positive control	Imidazole (20%)	66.1	1.715	91.825	91.5	9.3
		51.6	2.026	81.990
		75.3	1.692	100.680
Test item	Art. 112422	56.9	0.000	56.900	52.1	6.9
		44.3	-0.005	44.225
		55.3	-0.002	55.270

 

Run 2:

		Opacity	Permeability	IVIS
				per cornea	per group (mean value)	Standard deviation
Negative control	0.9% sodium chloride solution	2.7	0.002	2.730	1.5	1.1
		0.8	0.001	0.815
		1.0	-0.001	0.985
Positive control	Imidazole (20%)	69.6	2.045	100.275	96.7	6.7
		66.4	2.302	100.930
		55.3	2.243	88.945
Test item	Art. 112422	49.0	0.001	49.015	52.9	3.7
		56.3	0.000	56.300
		53.3	0.001	53.315

 

Interpretation of results:

other: No prediction can be made

Conclusions:

Under the conditions of the present study, the eye hazard potential of the test item N-Acetyl-L-cysteine cannot be predicted.

Executive summary:

The objective of the present study was to examine the potential of the test item Art. 112422 (N-Acetyl-L-cysteine) to induce serious eye damage in the BCOP assay. The BCOP assay with isolated fresh bovine corneas is an accepted in vitro model for ocular hazard assessment.

To determine the eye hazard potential the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item Art. 112422 (N-Acetyl-L-cysteine) as a 20% (w/v) suspension 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 uL of the suspended 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).

In a first run, two of the three corneas treated with the test item gave discordant predictions from the mean of all three corneas. Due to this borderline a second run was performed according to OECD Guideline 437. The repeat testing run corroborated the prediction of the initial testing run. Therefore, a final decision was taken without further testing.

After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 0.7 in run 1 (study acceptance criteria range: -1.4-3.1) and 1.5 in run 2 (study acceptance criteria range: -1.4-3.1). Treatment with the positive control (20% Imidazole) revealed an IVIS of 91.5 in run 1 (study acceptance criteria range: 82.8 - 132.9) and 96.7 in run 2 (study acceptance criteria range: 82.4 - 132.8). Therefore, the study fulfilled the acceptance criteria.

The IVIS obtained after treatment with Art. 112422 (N-Acetyl-L-cysteine) was 52.1 in run 1 and 52.9 in run 2 and, thus higher than 3 and lower than 55, i.e. according to OECD 437 no prediction can be made regarding the eye hazard potential of the test item.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

A study according OECD TG 439 was conducted to investigate the potential of the test item to induce skin irritation in an in vitro human skin model (SkinEthic RHE-model).

The substance 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 min). 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 deionized water was spread to the epidermis surface to improve the contact between the test item and the epidermis. Because of non-concordant replicate results after treatment with the substance between the first (non-valid) run and the second run, a third run was performed. Only the two valid runs performed are reported as run 1 and run 2. 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 82.7% in run 1 and 98.9 in run 2 and thus, higher than 50% in both runs, i.e. according to OECD TG 439 the test item is considered as non-irritant to skin (UN GHS: No Category) (reference 7.3.1-1).

 

Eye Irritation

A study according OECD TG 492 was conducted to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model.

The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) 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 eye irritation potential. Duplicates of the EpiOcular™-model were treated with the test item, the negative or the positive control for 6 hours (± 15 minutes). 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues.

After treatment with the negative control (sterile deionized water) the mean OD was 1.686 (study acceptance criterion: > 0.8 and < 2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 17.8 % (study acceptance criterion: <50 %). Thus, the acceptance criteria were met. Following treatment with the test item, the tissue viability was 2.4 % and thus, lower than 60%. i.e. according to OECD TG 492 no prediction can be made regarding the eye hazard potential of the test (reference 7.3.2-1).

 

As no prediction can be made from the EpiOcular Test a second in vitro test was performed in a bottom-up approach as recommended by OECD (IATA for serious eye damage and eye irritation, 2017). The induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item Art. 112422 (N-Acetyl-L-cysteine) as a 20 % (w/v) suspension in a 0.9 % sodium chloride solution according to OECD TG 437. 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 suspended 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). In a first run, two of the three corneas treated with the test item gave discordant predictions from the mean of all three corneas. Due to this borderline a second run was performed according to OECD Guideline 437. The repeat testing run corroborated the prediction of the initial testing run. Therefore, a final decision was taken without further testing. After treatment with the negative control (0.9 % sodium chloride solution) the calculated IVIS was 0.7 in run 1 (study acceptance criteria range: -1.4-3.1) and 1.5 in run 2 (study acceptance criteria range: -1.4-3.1). Treatment with the positive control (20 % Imidazole) revealed an IVIS of 91.5 in run 1 (study acceptance criteria range: 82.8 - 132.9) and 96.7 in run 2 (study acceptance criteria range: 82.4 - 132.8). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment with Art. 112422 (N-Acetyl-L-cysteine) was 52.1 in run 1 and 52.9 in run 2 and, thus higher than 3 and lower than 55, i.e. according to OECD 437 no prediction can be made regarding the eye hazard potential of the test item (reference 7.3.2-2).

 

The test item was evaluated for eye irritation and serious eye effects in validated in vitro test methods using a Bottom-up approach (see Scott et al.: A proposed eye irritation testing strategy to reduce and replace in vivo studies using a Bottom-up and Top-Down approaches.) No prediction can be made based on both test systems therefore a weight of evidence approach is used to come to a conclusion. In the in vitro eye irritation tests adverse effects on cell viability and opacity of the cornea are observed but the substance is not identified as UN GHS Cat.1.

The IVIS in the Bovine Corneal Opacity and Permeability method (OECD TG 437) was about 52 in both runs and thus very close to the threshold of 55 for UN GHS Category 1. Furthermore, the mean cell viability obtained in the Eye Irritation Test (OECD TG 492) is 2.4 % and thus far below the threshold of 60 % which triggers “No Category”. In conclusion, the test item should be classified for eye irritation as UN GHS Cat.2. Therefore, no further testing is necessary.

Justification for classification or non-classification

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

The available experimental test data for the skin irritation potential are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based these available data, the test item is not classified as skin irritant according to Regulation (EC) No 1272/2008 (CLP), as amended for the 11th time in Regulation (EU) No 2018/669.

The WoE evaluation of the consistency, quality and relevance of all available data allows a decision on the eye irritation/serious eye damage potential of the test item. The test item is classified for eye irritation Cat.2 according to Regulation (EC) No 1272/2008 (CLP), as amended for the 11th time in Regulation (EU) No 2018/669. No further testing is necessary.