Cyclooctane

Endpoint summary

Description of key information

The test item is considered to possess an irritant potential to skin (reference 7.3.1-1).

The test item is not considered as corrosive to skin (reference 7.3.1-2).

The test item did not show a potential to cause eye irritation or damage to the eye (reference 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:

2021-08-31 to 2021-09-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EU method B.40bis IN VITRO SKIN CORROSION: RECONSTRUCTED HUMAN EPIDERMIS (RhE) TEST METHOD

Version / remarks:

2019-07-31

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2019-06-14

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

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 (Episkin/SkinEthic Laboratories, Lyon, France)
- Tissue batch number: 21-RHE-104
- Expiry date: 2021-09-06
- Date of initiation of testing: 2021-08-31

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: minimum volume of 20 mL DPBS, gentle rinsing
- Observable damage in the tissue due to washing: Not specified
- Modifications to validated SOP: No.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours (± 15 minutes)
- Spectrophotometer: ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany
- Wavelength: 570 nm
- Filter: Not specified
- Linear OD range of spectrophotometer: Not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
Please refer to “Any other information on materials”.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Pre-tests for MTT-reducing capacity and colorant properties of the test item were conducted. No such properties were detected and therefore, no additional controls were needed.

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 %.
- 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:

40 ± 3 µL per tissue

Duration of treatment / exposure:

test item and negative control: 3 min and 1 h
positive control: 1 h

Duration of post-treatment incubation (if applicable):

Not applicable

Number of replicates:

2 per substance and time point

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean 1 h exposure

Value:

90.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean 3 min exposure

Value:

102

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: Not specified.
- Direct-MTT reduction: No.
- Colour interference with MTT: No.

ACCEPTANCE OF RESULTS:
Please refer to “Any other information on results”.

The results of the reconstructed human epidermis model:

Group		Tissue 1		Tissue 2		Mean		CV
		OD	Viability	OD	Viability	OD	Viability	Viability
Negative Control	3 min	1.697	100.8 %	1.668	99.1 %	1.683	100.0 %	1.2 %
	1 hour	1.561	101.5 %	1.515	98.5 %	1.538	100.0 %	2.1 %
Positive Control	1 hour	0.009	0.6 %	0.008	0.5 %	0.009	0.6 %	16.7 %
Test item	3 min	1.653	98.2 %	1.779	105.7 %	1.716	102.0 %	5.2 %
	1 hour	1.362	88.6 %	1.413	91.9 %	1.388	90.3 %	2.5 %

 

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	1.515 to 1.697

 

Acceptance Criteria stated by Episkin/SkinEthic Laboratories:

	Acceptance Criterion	Result
Mean OD negative control	≥0.8 and ≤3.0	1.683 (3 min) 1.538 (1 hour)
Mean viability positive control	< 15 % after 1-hour exposure	0.6 %
Range between identically treated tissues with test item	< 30 %	7.6 % (3 min) 3.7 % (1 hour)

 

Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:

	Acceptance Criterion	Result
Mean OD negative control	≥1.605 (3 min) ≥1.408 (1 hour)	1.683 (3 min) 1.538 (1 hour)
Mean viability positive control	≤1.01 %	0.6 %

 

Negative Control, Positive Control and Test Substance Data Acceptance Criteria stated by the Testing Laboratory:

	Group	Acceptance Criterion	Result
Range between identically treated tissues	Negative control	< 30 %	1.7 % (3 min) 3.0 % (1 hour)
	Positive control	< 30 %	20.0 % (1 hour)
	Test substance	< 30 %	7.6 % (3 min) 3.7 % (1 hour)

The study met all acceptance criteria.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is not considered as corrosive to skin.

Executive summary:

The objective of the study conducted according to OECD TG 431 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 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 the liquid test item, the negative control (deionised water) or the positive control (potassium hydroxide, 8N) were applied to the tissues. After treatment with the positive control the mean viability value was 0.6 % and, thus, lower than the historically established threshold of 1.01 %. After treatment with the negative control the mean ODs were 1.683 (3 minutes exposure) and 1.538 (1 hour exposure) and, thus , higher than the historically established thresholds of 1.605 and 1.408, respectively. Thus, the acceptance criteria were met. Following treatment with the test item, the tissue viability was ≥50 % after 3 minutes exposure (mean viability: 102.0 %) and ≥15 % after 1 hour exposure (mean viability: 90.3 %). Therefore, under the conditions of the present study, the test item is not considered to possess a corrosive potential to skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2021-06-23 to 2021-06-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2019-07-31

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2020-06-26

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

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: 21-RHE-104
- Expiry date: 2021-06-28
- Date of initiation of testing: 2021-06-23

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 °C and 5 % CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: minimum volume of 25 mL DPBS, gentle rinsing
- Observable damage in the tissue due to washing: Not specified
- Modifications to validated SOP: No.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours (± 5 minutes)
- Spectrophotometer: ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany
- Wavelength: 570 nm
- Filter: Not specified
- Linear OD range of spectrophotometer: Not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
Please refer to “Any other information on materials”.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Pre-tests for MTT-reducing capacity and colorant properties of the test item were conducted. No such properties were detected and therefore, no additional controls were needed.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
A test item is considered as non-irritant to skin (UN GHS No Category) if the tissue viability after exposure and post-treatment incubation is > 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 ≤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:

16 µL ± 0.5 µL per tissue

Duration of treatment / exposure:

42 minutes (± 1 minute)

Duration of post-treatment incubation (if applicable):

42 hours (± 1 hour)

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

2

Vehicle controls validity:

not applicable

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: Not specified.
- Direct-MTT reduction: No.
- Colour interference with MTT: No.

ACCEPTANCE OF RESULTS:
Please refer to “Any other information on results”.

Results of the reconstructed human epidermis model:

Group	Tissue 1		Tissue 2		Tissue 3		Mean		SD
	OD	viability	OD	viability	OD	viability	OD	viability	viability
Negative Control	1.755	108.8 %	1.560	96.7 %	1.525	94.5 %	1.613	100.0 %	7.7 %
Positive Control	0.026	1.6 %	0.024	1.5 %	0.033	2.0 %	0.028	1.7 %	17.6 %
Test item	0.033	2.0 %	0.030	1.9 %	0.035	2.2 %	0.033	2.0 %	10.0 %

 

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	1.525 to 1.755

 

Acceptability of the Positive and Negative Control stated by Episkin/SkinEthic Laboratories:

	Acceptance Criterion	Result
Mean OD negative control	≥1.2	1.613
Mean viability positive control	<40 %	1.7 %
SD of group-mean value	≤ 18 %	17.6 % (positive control) 7.7 % (negative control)

 

Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:

	Acceptance Criterion	Result
Mean OD negative control	≥1.422	1.613
Mean viability positive control	≤2.70 %	1.7 %

 

Test Item Data Acceptance Criteria:

	Acceptance Criterion	Result
SD of group-mean value	≤18 %	10.0 %

The study met all acceptance criteria.

Interpretation of results:

other: The test item requires classification for skin irritation or corrosion. Further testing is required to establish the classification category.

Conclusions:

The test item is considered to possess an irritant potential to skin.

Executive summary:

The objective of the study conducted according to OECD TG 439 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 test item, the negative control (DPBS-buffer) or the positive control (5 % aqueous solution of sodium dodecyl sulfate) were applied to the tissues. All acceptability criteria after treatment with the negative control and the positive control were met. Following treatment with the test item, the tissue viability was 2.0 % and, thus, lower than 50 %. Under the conditions of the present study, the test item is considered to possess an irritant potential to skin (UN GHS: Category 2 or 1).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-06-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EU method B.69 (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:

2019-07-31

Deviations:

no

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:

2019-06-18

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

other: keratinocyte strain F1188

Details on test animals or tissues and environmental conditions:

- Justification of the test method (RhCE) 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.
- RhCE tissue used, including batch number: The EpiOcular™ Tissues (OCL-200, OCL-212) was obtained from MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia. Lot number 34914 and keratinocyte strain F1188 was used. For details on tissue quality please refer to “Any other information on materials”.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 µL per tissue

Duration of treatment / exposure:

30 ± 2 minutes at 37 °C and 5 % C02

Duration of post- treatment incubation (in vitro):

12 ± 2 minutes at room temperature and 120 ± 15 minutes at 37 °C and 5 % C02

Number of animals or in vitro replicates:

duplicates

Details on study design:

- Details of the test procedure used
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 (16-24 hours) without medium exchange. 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).
After the 30-minute DPBS pre-treatment, the test item, the negative and the positive control were tested by applying 50 µL 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 30 ± 2 minutes.
At the end of the 30 ± 2 minutes treatment time, the positive control, negative control and the test item were removed by extensively rinsing the tissues with pre-warmed (37 °C) 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 12 ± 2 minutes at room temperature. After the 12 ± 2 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 120 ± 15 minutes at 37 °C and 5 % CO2.
- Doses of test chemical and control substances used: 50 µL per tissue
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: Pre-tests for MTT-reducing capacity and colorant properties of the test item were conducted. No such properties were detected and therefore, no additional controls were needed.
- Wavelength used for quantifying MTT formazan, and linearity range of measuring device: Spectrophotometer: ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany; 570 nm wavelength
- 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 pre-labeled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol was flowing into the insert on the tissue surface. The plate was sealed with a standard plate sealer. To extract the MTT, the plate was placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. At the end of the extraction period, the tissues were pierced and the liquid within each insert was decanted into the well from which it was taken.
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: All formulas for the calculation of the relative viability were given by the supplier of the human in vitro eye model (MatTek In Vitro Life Science Laboratories). The mean OD (optical density) of the two negative control tissues was calculated. This value corresponds to 100 % tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability was calculated according to the following formula: Viability % = (test item OD/ mean negative control OD) x 100
The prediction model according to OECD 492 was used. 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.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: The negative control data meet the acceptance criteria if the mean OD value is higher or equal than a historically established boundary at 570 nm. The boundary is two standard deviations below the current historical mean. The positive control data meet the acceptance criteria if the mean viability value, expressed as % of the negative control, is lower than or equal to a historically established boundary. The boundary is two standard deviations above the current historical mean.
- Positive and negative control means and acceptance ranges based on historical data: Acceptance criterion for mean OD negative control: ≥1.088; Acceptance criterion for mean viability positive control: ≤57.9 %
- Acceptance Criteria according to OECD Guideline 492 were used. The results are acceptable if:
1. The negative control OD is >0.8 and <2.5
2. The mean relative viability of the positive control is:
a) 30-minute exposure (treatment of liquid test items): below 50 % of control viability
b) 6-hour exposure (treatment of solid test items): 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:

mean percent tissue viability 

Value:

85.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. Please refer to "Any other information on results".
- Acceptance criteria met for positive control: Yes. Please refer to "Any other information on results".

Results of RhCE model:

Group	Tissue 1		Tissue 2		Mean		SD	Difference between tissue replicates
	OD	Viability	OD	Viability	OD	Viability	Viability
Negative Control	1.999	97.9 %	2.084	102.1 %	2.042	100.0 %	2.97	4.2 %
Positive Control	0.307	15.0 %	0.323	15.8 %	0.315	15.4 %	0.57	0.8 %
Test item	1.701	83.3 %	1.802	88.2 %	1.752	85.8 %	3.46	4.9 %

 

Acceptance Criteria according to OECD Guideline 492:

	Acceptance Criterion	Result
Negative control OD	> 0.8 and < 2.5	1.999 and 2.084
Mean viability positive control	< 50 %	15.4 %
Difference of viability between the two relating tissues of a single chemical	<20 %	4.2 % (negative control) 0.8 % (positive control) 4.9 % (test item)

 

Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:

	Acceptance Criterion	Result
Mean OD negative control	≥1.088	2.042
Mean viability positive control	≤57.9 %	15.4 %

The study met all acceptance criteria.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item did not show an eye hazard potential.

Executive summary:

The objective of the present study conducted according to OECD TG 492 was 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 30 ± 2 minutes. 50 µL of either the test item, the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues. All acceptability criteria after treatment with the negative control and the positive control were met. Following treatment with the test item, the tissue viability was 85.8 % and, thus, higher than 60 %. Under the conditions of the present study, the test item did not show an eye hazard potential. The test item is identified as not requiring classification and labeling according to UN GHS (No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

A study according to OECD TG 439 was conducted to determine whether or not the test item requires no classification. Based on the result of the study a further in vitro study (according to OECD TG 431) was conducted and the study are used together in a weight of evidence approach. 

 

OECD 439 (reference 7.3.1-1)

The objective of the study conducted according to OECD TG 439 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 test item, the negative control (DPBS-buffer) or the positive control (5 % aqueous solution of sodium dodecyl sulfate) were applied to the tissues. All acceptability criteria after treatment with the negative control and the positive control were met. Following treatment with the test item, the tissue viability was 2.0 % and, thus, lower than 50 %. Under the conditions of the present study, the test item is considered to possess an irritant potential to skin (UN GHS: Category 2 or 1).

 

OECD 431 (reference 7.3.1-2)

The objective of the study conducted according to OECD TG 431 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 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 the liquid test item, the negative control (deionised water) or the positive control (potassium hydroxide, 8N) were applied to the tissues. After treatment with the positive control the mean viability value was 0.6 % and, thus, lower than the historically established threshold of 1.01 %. After treatment with the negative control the mean ODs were 1.683 (3 minutes exposure) and 1.538 (1 hour exposure) and, thus , higher than the historically established thresholds of 1.605 and 1.408, respectively. Thus, the acceptance criteria were met. Following treatment with the test item, the tissue viability was ≥50 % after 3 minutes exposure (mean viability: 102.0 %) and ≥15 % after 1 hour exposure (mean viability: 90.3 %). Therefore, under the conditions of the present study, the test item is not considered to possess a corrosive potential to skin.

 

Conclusion

The test item was determined to require classification for skin irritation (Category 2) or skin corrosion (Category 1) in a study conducted according to OECD TG 439. Furthermore, the test item was determined to be non-corrosive in a study according to OECD TG 431. Considering the results of both studies together in a weight of evidence approach, a potential to cause skin irritation is determined for the test item. Therefore, a classification for skin irritation (Category 2) is applied to the test item. 

 

Eye irritation

The objective of the present study conducted according to OECD TG 492 was 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 30 ± 2 minutes. 50 µL of either the test item, the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues. All acceptability criteria after treatment with the negative control and the positive control were met. Following treatment with the test item, the tissue viability was 85.8 % and, thus, higher than 60 %. Under the conditions of the present study, the test item did not show an eye hazard potential. The test item is identified as not requiring classification and labeling according to UN GHS (No Category).

Justification for classification or non-classification

Classification, Labelling, 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.

Based on available data on skin irritation/corrosion, the test item requires classification for causing skin irritation (Category 2, H315: Causes skin irritation) according to Regulation (EC) No 1272/2008 (CLP).

Based on available data on eye irritation/corrosion, the test item does not require classification according to Regulation (EC) No 1272/2008 (CLP).