(E,Z)-2,6-dimethylocta-2,4,6-triene

Endpoint summary

Description of key information

In accordance with Regulation EC No. 1272/2008 and with the results of a study conducted according to Guideline OECD 439, the test substance has to be classified in Category 2 “Irritating to skin”. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” are required.

In accordance with Regulation EC No. 1272/2008 and with the results of a study conducted according to Guideline OECD 431, the test substance is not predicted as causing skin corrosion (Category 1).

 

In accordance with Regulation EC No. 1272/2008 and with the results of a study conducted according to Guideline OECD 492, the test substance was identified as not requiring classification and labelling according to UN GHS Category 2 or Category 1.

 

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:

key study

Study period:

01-15 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline 439 with a minor deviation: standard deviation between negative control tissues replicates was greater than the maximum acceptable variability. Considering the results obtained, this deviation was considered as without impact on the conclusion of the study.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 28 July 2015

Deviations:

yes

Remarks:

standard deviation between negative control tissues replicates was greater than the maximum acceptable variability

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated 23 July 2009

Deviations:

yes

Remarks:

standard deviation between negative control tissues replicates was greater than the maximum acceptable variability

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

23 October 2015

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: foreskin

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- 0.50 cm² reconstructed epidermis (Episkin SA, RHE/S/17 Batch No. 16-RHE-130) were received on 13 December 2016.
- On the same day, the insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The inserts were placed in 6 wells culture plate which had been previously filled with 1 mL of growth medium (Episkin SA, batch No. 16 MPE 133) during 3 hours and 25 minutes. Then just before treatment, the inserts were placed in 24 wells culture plate which had been previously filled with 1mL of maintenance medium (Episkin SA, batch No. 16 MA 077).

TREATMENT
- The test item was applied, as supplied, at the dose of 16 µL, to the epidermal surface of 3 living skin models during 42 minutes at room temperature. To ensure a good contact with the epidermis, during all the treatment period, all liquid items were recovered with a nylon mesh provided by Episkin SA.
- In the same experimental conditions, a positive control (5% SDS), and a negative control (DPBS – PAN BIOTECH GmbH - Batch No. 9510916) were carried out. The 5% SDS solution was prepared by weighing 0.5 g of SDS (SIGMA Batch No. STBF1623V) in a 10 mL volumetric flask (QS 10 mL of distilled water). Then, the preparation was magnetically stirred, just before the treatment. To ensure a good contact with the epidermis, during all the treatment period, all liquid items were recovered with a nylon mesh provided by Episkin SA.


REMOVAL OF TEST MATERIAL AND CONTROLS
- 42 minutes after the test item application, the human epidermis were washed with 25 x 1 mL of DPBS (PAN BIOTECH GmbH, Batch No. 9510916). The rinsed tissues were checked for any coloration and noted to be whitish, comparable to negative control tissues. They were incubated for a 41-hour and 8-minute post-treatment incubation period in fresh medium at 37°C, 5% CO2. Then, the epidermis were put in contact with the MTT solution.


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- The cell viability was quantified by the measurement of the cell succinate dehydrogenase activity. This enzyme was responsible for the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS No. 298-93-1)] reduction into blue formazan crystal.
- The skin samples were placed in 300 µL of a MTT solution at 1.0 mg/mL for 3 hours at 37°C, 5% CO2. The precipitated blue formazan product was then extracted using isopropanol during 2 hours under gentle agitation in the dark, and the concentration of formazan was measured by determining the Optical Density (OD) at 570 nm, just after dilution of the extracts (1:2 in isopropanol).
- The OD of MTT extract was measured in triplicate. The measured OD was proportional to the number of living cells. The measurement of OD was performed using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.

PREDICTION MODEL / DECISION CRITERIA
The OD values obtained for each test sample were used to calculate a percentage of viability relative to the negative control, which was arbitrarily set at 100%. The cut-off values for the prediction of irritation associated with the RHE models were as follows:
- The test item is considered to be non-irritant to skin if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is >50%.
- The test item is considered to be irritant to skin if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is ≤50%. In accordance with Regulation EC No. 1272/2008, the test item has to be classified in Category 2 “Irritant”. The corresponding hazard statement is “H315: Causes skin irritation” with the signal word “Warning”.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 µL
- Concentration (if solution): Undiluted

Duration of treatment / exposure:

42 minutes at room temperature

Duration of post-treatment incubation (if applicable):

41-hour and 8-minute post-treatment incubation period in fresh medium at 37°C, 5% CO2

Number of replicates:

3 replicates of living skin models

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

main test (duration of exposure: 42 minutes)

Value:

4.4

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:

PRELIMINARY TESTS
- Test for direct interaction with MTT:
A yellow solution was observed after 3 hours of incubation between 37.8°C and 36.9°C, 5% CO2. Therefore, there was no direct interaction between the test item and MTT.
- Test for the detection of the colouring potential of the test item:
A colourless solution was obtained after 2 hours and 47 minutes of incubation at ambient temperature with gentle shaking. The mean of the corrected OD (blank subtracted) was 0.000 which is less than 0.08 (value corresponding to approximately twice the OD of the extracting solvent). Therefore the test item was considered to be not interfering with the MTT assay and there was no need to add non-specific coloration controls to the study.

MTT VIABILITY ASSAY RESULTS
The mean percent viability of the treated tissues was 4.4%, versus 1.5% in the positive control (5% Sodium Dodecyl Sulfate).

ACCEPTANCE OF RESULTS:
- The standard deviation of the negative control group was 26.0%, instead of ≤ 18% as initially scheduled. Considering the results obtained, this deviation is considered as without impact on the conclusion of the study.

Table 7.3.1/1: Main test - Individual and mean OD values and tissue viabilities for the test item, the negative and positive controls

 

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	Standard deviation (SD)
Negative control	1	2.015	1.964	1.574	124.8	100.0	26.0
		1.908
		1.969
	2	1.513	1.610		102.3
		1.632
		1.685
	3	1.063	1.147		72.9
		1.190
		1.188
Positive control	1	0.027	0.027	0.023	1.7	1.5	0.5
		0.028
		0.027
	2	0.015	0.015		1.0
		0.015
		0.015
	3	0.027	0.028		1.8
		0.029
		0.028
Test item	1	0.055	0.055	0.069	3.5	4.4	1.0
		0.055
		0.055
	2	0.086	0.085		5.4
		0.086
		0.084
	3	0.067	0.067		4.3
		0.067
		0.066

#: mean of 3 values (triplicate of the same extract)

OD: optical density; measured after a 1:2 dilution of the formazan extracts in isopropanol.

 

Acceptability criteria: SD ≤ 18%.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In accordance with Regulation EC No 1272/2008, the test substance has to be classified in Category 2 “Irritating to skin”. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” are required.
Considering the results obtained, an in vitro test of skin corrosivity has to be conducted to know if the test item has to be classified in Category 1 “Corrosive” or Category 2 “Irritant”.

Executive summary:

An in vitro skin irritation test using the Reconstructed human Epidermis (SkinEthic RHE® model) was performed according to Guideline OECD 439 and in compliance with GLP to predict the acute skin irritation potential of the test substance.

The test substance was applied as supplied, at the dose of 16 µL, to 3 living Reconstructed Human epidermis during 42 minutes, followed by a rinse with 25 mL of DPBS and a 41-hour and 8-minute post-incubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.

In the preliminary tests, the test substance was found not to have direct MTT reducing properties or colouring potential. In the main test, the mean percent viability of the treated tissues was 4.4%, versus 1.5% in the positive control (5% Sodium Dodecyl Sulfate).

Therefore, in accordance with Regulation EC No 1272/2008, the test substance has to be classified in Category 2 “Irritating to skin”. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” are required.

Considering the results obtained, an in vitro test of skin corrosivity has to be conducted to know if the test item has to be classified in Category 1 “Corrosive” or Category 2 “Irritant”.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September - October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU method B.40bis of the Council Regulation No. 440/2008.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

other: reconstituted epidermis (epiCS, Cell Systems)

Cell type:

other: epiCS, Cell Systems

Vehicle:

unchanged (no vehicle)

Details on test system:

HUMAN SKIN MODEL
The 0.6 cm² reconstituted epidermis (epiCS, Cell Systems) were received on 03 October 2017. The insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The insert was placed in a 6 wells culture plate which had been previously filled with 1 mL of culture medium . The culture dishes were incubated at 37±2°C, 5% CO2 during 21 hours and 15 minutes before treatment. Just before the treatment, the culture medium was replaced by a new culture medium .

TREATMENT
The test item was applied as supplied at the dose of 50 µL to the epidermal surface of the 2 living human skin models during 3 minutes at room temperature and during 1 hour at 37°C ± 1°C, 5% ± 1% CO2.
In the same experimental conditions, a positive control (8N KOH) and a negative control (distilled water) were carried out.

REMOVAL OF TEST MATERIAL AND CONTROLS3 minutes and 1 hour after the test item application the human epidermis were washed 20 times with 1 mL of DPBS (DPBS – Dutscher, Batch No. 1310817). The rinsed tissues were checked for any coloration and noted to be whitish, comparable coloration to that of the negative control tissues.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
The cell viability was quantified by measurement of the cell succinate dehydrogenase activity. This enzyme was responsible for the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS No. 298-93-1)] reduction into blue formazan crystal. The skin sample was placed in MTT solution of 1 mg/mL concentration for 2 hours and 55 minutes between 36.5°C and 38.0°C, 5% CO2. The precipitated blue formazan product was then extracted using isopropanol during 2 hours under agitation in the dark, and the concentration of formazan was measured by determining the Optical Density (OD) at 570 nm, just after dilution of the extraction in isopropanol (1:3).
The absorbance was measured in triplicate of MTT extract.
The measured absorbances were proportional to the number of living cells.

The measurement of OD was performed using the Elx800 absorbance microplate reader (controlled and calibrated every year if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.
The linearity range of optical density measured is validated for an optical density between 0 and 2.0.

VIABILITY CALCULATION:
- The results were expressed as a viability percentage compared with the negative control: viability % = (mean OD test item / mean OD negative control) * 100
- Data from individual replicate tissues (OD values and calculated percent tissue viability data for the test item and controls), mean percent tissue viability and standard deviation for each individual test item and control were reported in Table 7.3.1/1.

PREDICTION MODEL / DECISION CRITERIA:

The OD values obtained for each test sample are used to calculate a percentage of viability relative to the negative control, which is arbitrarily set at 100%. The cut-off values for the prediction of corrosion associated with the epiCS® models are as follows:

Viability measured after exposure time points (t=3 and 60 minutes)

STEP 1 (corrosive or not corrosive)
< 50% after 3 min exposure ==> Corrosive: Corresponding hazard statement “H314: Causes severe skin burns and eye damage” and signal word “Danger”
≥ 50% after 3 min exposure AND < 15% after 60 min exposure ==> Corrosive: Corresponding hazard statement “H314: Causes severe skin burns and eye damage” and signal word “Danger”
≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure ==> Non-corrosive

STEP 2 (subcategories for items identified as corrosive in step 1)
< 15% after 3 min exposure ==> Optional Sub-category 1A*
≥ 15% after 3 min exposure ==> A combination of optional Sub-categories 1B-and-1C

* According to the data generated in view of assessing the usefulness of the RhE test methods for supporting subcategorisation, it was shown that around 33% of the Sub-category 1A results of the epiCS® test methods may actually constitute Sub-category 1B or Sub-category 1C substances/mixtures (i.e. over-classifications).
It must be noted that a limitation of this Test Guideline is that it does not allow discriminating between skin corrosive sub-categories 1B and 1C.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): Undiluted

Duration of treatment / exposure:

during 3 minutes at room temperature and during 1 hour at 37°C ± 1°C, 5% ± 1% CO2.

Duration of post-treatment incubation (if applicable):

The skin sample was placed in MTT solution of 1 mg/mL concentration for 2 hours and 55 minutes between 36.5°C and 38.0°C, 5% CO2.

Number of replicates:

2 living human skin models

Irritation / corrosion parameter:

% tissue viability

Value:

93.86

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: at 3 minutes

Irritation / corrosion parameter:

% tissue viability

Value:

93.25

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: at 60 minutes

Other effects / acceptance of results:

MTT VIABILITY ASSAY RESULTS
- The mean percent viability of the treated tissues was 100%, versus 0.62% in the positive control .

Table 7.3.1/1: Main test - Individual and mean OD values and tissue viabilities for the test item, the negative and positive controls

INDIVIDUAL AND AVERAGE VALUES AFTER 3 MINUTES EXPOSURE

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	Standard deviation (SD)
Negative control	1	1.288 1.250 1.197	1.245	1.246	99.96	100.00	0.1
	2	1.274 1.236 1.227	1.246		100.04
Positive control	1	0.433 0.423 0.427	0.428	0.720	34.36	57.77	46.8
	2	1.069 0.967 0.997	1.011		81.17
Test item	1	1.150 1.131 1.159	1.147	1.169	92.09	93.86	3.5
	2	1.244 1.170 1.159	1.191		95.62

INDIVIDUAL AND AVERAGE VALUES AFTER 1 HOUR EXPOSURE

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	Standard deviation (SD)
Negative control	1	1.443 1.113 1.137	1.231	1.282	96.06	100.00	7.9
	2	1.341 1.314 1.340	1.332		130.94
Positive control	1	0.002 0.003 0.004	0.003	0.008	0.23	0.62	0.8
	2	0.014 0.013 0.013	0.013		1.01
Test item	1	1.170 1.089 1.087	1.115	1.195	87.01	93.25	12.5
	2	1.385 1.221 1.220	1.275		99.49

  Note #: mean of 3 values OD: optical density

Acceptability criteria:The mean OD of negative control tissues for the treatment of 3 minutes and for the treatment of 1 hour were respectively 1.246 and 1.282 instead of≥ 0.3 and ≤ 0.9as initially scheduled.

 

Considering the results obtained and the fact that these values remain in the range of our historical negative control data (see appendices 2 & 3), this minor deviation is considered as without impact on the conclusion of the study

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In accordance with Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that test item (E,Z)-2,6-DIMETHYLOCTA-2,4,6-TRIENE does not have to be classified in Category 1 “Corrosive”.
Hazard statement “H314: Causes severe skin burns and eye damage” with signal word “Danger” are not required.

Executive summary:

An in vitro skin corrosion test was performed according to OECD Guideline 431 and in compliance with GLP.

The test item was applied as supplied at the dose of 50 µL to 2 living Human skin model surfaces (epiCS^®, CellSystems^®) during 3 minutes and 1 hour. The application was followed by a rinse with 20 mL of DPBS. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.

 

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item and treated with the positive control item (potassium hydroxide 8N) were respectively 93.86% and 93.25% versus 57.77% and 0.62%.

 

In accordance with Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item does not have to be classified in Category 1 “Corrosive”. Hazard statement “H314: Causes severe skin burns and eye damage” with signal word “Danger” are not required.

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:

01-09 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline 492 with a minor deviation: difference of viability between negative control tissues replicates was more than the maximum acceptable variability. Considering the results obtained, this deviation was considered as without impact on the conclusion of the study.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other 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:

adopted 28 July 2015

Deviations:

yes

Remarks:

difference of viability between negative control tissues replicates was more than the maximum acceptable variability

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

2017

Species:

human

Details on test animals or tissues and environmental conditions:

- Description of the cell system used: 0.60 cm² Reconstructed human Cornea-like Epithelia [EpiOcular(TM) OCL-212-ver2.0, supplied by MatTek Corporation, batch No. 23755] were received on 07 December 2016.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): Undiluted

Duration of treatment / exposure:

30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions)

Duration of post- treatment incubation (in vitro):

- Post-exposure immersion period: 12 minutes at room temperature
- Post-exposure incubation period: 2 hours at standard culture conditions

Number of animals or in vitro replicates:

Test item, negative and positive controls were applied on duplicate tissues.

Details on study design:

PRELIMINARY TESTS
- Test for direct interaction with MTT:
The direct interaction of MTT with the test item was checked by adding 50 µL of the test item to 1 mL of MTT solution at 1 mg/mL.
- Test for the detection of the colouring potential of the test item:
The colouration potential of the test item in isopropanol was checked by adding 50 µL of the test item to 2 mL of isopropanol.


MAIN TEST
- Pre-incubation of the tissues:
On the day of receipt, the 12 tissues in their 24-well shipping container were equilibrated at room temperature for 15 minutes. Then the inserts (filter + epithelium) were gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. They were placed in 6 wells culture plate which had been previously filled with 1 mL of 37°C pre-warmed assay medium and incubated during 19 hours and 58 minutes at standard culture conditions.
After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca2+Mg2+Free-DPBS. The tissues were incubated at standard culture conditions for 30 minutes.
- Treatment and post-treatment incubation of the tissues:
The test item was applied, as supplied, at the dose of 50 µL, to the entire surface of 2 living RhCE tissue replicates during 30 minutes at standard culture conditions. In the same experimental conditions, a positive control (Methyl acetate), and a negative control (distilled water) were used. The controls were applied, as supplied, at the dose of 50 µL, to the surface of 2 viable RhCE tissue replicates during 30 minutes at standard culture conditions.
After the treatment, the test item and control substances were carefully washed from the RhCE tissues by extensive rinsing with Ca2+Mg2+Free-DPBS. The rinsed tissues were checked for any colouration and noted for comparable colour with the negative control. This rinsing step was followed by a 12-minute post-exposure immersion period at room temperature in 5 mL of fresh medium to remove any test item absorbed into the tissue. The RhCE constructs were then incubated for 2 hours post-exposure incubation at standard culture conditions in 1 mL of fresh medium at 37°C, 5% CO2.
- MTT viability assay:
Following the exposure to the test item, viability measurements are performed immediately after the post-exposure incubation period of the rinsed tissues in fresh medium. This period allows both for recovery from weak cytotoxic effects and for appearance of clear cytotoxic effects.
The RhCE tissue viability was measured by enzymatic conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1] by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured by Optical Density (OD) after extraction from tissues. The measured OD was proportional to the number of living cells.
The RhCE constructs were placed in 300 µL of a MTT solution at 1.0 mg/mL for 3 hours at standard culture conditions. The precipitated blue formazan product was then extracted from the tissues by placing each insert in 2 mL of isopropanol during 17 hours and 10 minutes at 6±3°C in the dark. The concentration of formazan was measured by determining the OD at 570 nm, just after dilution of the extractions in isopropanol (1:2). The OD measurement of formazan extracts at 570 nm was performed using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.

Irritation parameter:

other: % mean viability of the tissues

Run / experiment:

Main test

Value:

85.49

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:

PRELIMINARY TESTS
- Test for direct interaction with MTT:
A yellow solution was observed after 3 hours of incubation between 37.8°C and 36.9°C, 5% CO2. Therefore, there is no direct interaction between the test item and MTT.
- Test for the detection of the colouring potential of the test item:
A colourless solution was obtained in isopropanol after 2 hours and 47 minutes of incubation at ambient temperature with gentle shaking. The mean of the corrected OD (blank subtracted) was 0.001 which is less than 0.08 (value corresponding to approximately twice the OD of the extracting solvent). Therefore the test item was considered to be not interfering with the MTT assay.

MAIN TEST
- MTT assay results: The mean corrected percent tissue viability of the RhCE replicates treated with the test substance was 85.49%, versus 37.65% in the positive control (Methyl acetate). Refer Tables 7.3.2/1 for more details.

Table 7.3.2/1: Main test - Individual and mean corrected OD values and tissue viabilities for the test item, the negative and positive controls

 

	Tissue	OD	Mean OD/disc (#)	Mean OD/product	Viability %	Mean viability %	Difference of viability %
Negative control	1	0.911	0.906	0.776	116.83	100.00	33.66
		0.892
		0.917
	2	0.617	0.645		83.17
		0.659
		0.660
Positive control	1	0.308	0.313	0.292	40.36	37.65	5.42
		0.313
		0.319
	2	0.269	0.271		34.95
		0.273
		0.271
Test item	1	0.687	0.685	0.663	88.33	85.49	5.67
		0.683
		0.685
	2	0.616	0.641		82.66
		0.674
		0.635

#: mean of 3 values (triplicate of the same extract)

OD: optical density

 

Note

- The difference of viability between the two replicates of the negative control group was 33.66% instead of ≤20%. Considering the results obtained, this deviation was considered as without impact on the conclusion of the study.

Interpretation of results:

GHS criteria not met

Conclusions:

In accordance with Regulation EC No 1272/2008 and GHS Regulation, the test substance was identified as not requiring classification for eye irritation or serious eye damage. No hazard statement and no signal word were required.

Executive summary:

An in vitro eye irritation test using the Reconstructed human Cornea-like Epithelium (RhCE) (EpiOcular™ tissue) model was performed according to Guideline OECD 492 and in compliance with GLP to predict the acute eye irritation potential of the test substance.

The test substance was applied, as supplied, at the dose of 50 µL to two DPBS pre-treated RhCE (EpiOcular™ tissue model) during 30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with DPBS at room temperature, a 12-minute post-exposure immersion period at room temperature and a 2-hour post-exposure incubation at standard culture conditions. The tissue viability was measured by performing a MTT assay.

In the preliminary tests, the test substance was found not to have direct MTT reducing properties or colouring potential. In the main test, the mean corrected percent tissue viability of the RhCE replicates treated with the test substance was 85.49%, versus 37.65% in the positive control (Methyl acetate).

Therefore, in accordance with Regulation EC No 1272/2008 and CLP Regulation, the test substance was identified as not requiring classification for eye irritation or serious eye damage. No hazard statement and no signal word were required.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

An in vitro skin irritation test using the Reconstructed human Epidermis (SkinEthic RHE® model) was performed according to Guideline OECD 439 and in compliance with GLP to predict the acute skin irritation potential of the test substance.

The test substance was found not to have direct MTT reducing properties or colouring potential. The mean corrected percent viability of the treated tissues was 4.4%, versus 1.5% in the positive control (5% Sodium Dodecyl Sulfate). The mean percent tissue viabilities obtained with the positive control and negative controls were within the range of historical data and therefore validate the experiment.

Skin corrosion

An in vitro skin corrosion test was performed according to OECD Guideline 431 and in compliance with GLP to predict the acute skin irritation potential of the test substance.

The test substance was found not to have direct MTT reducing properties or colouring potential. The mean corrected percent viability of the treated tissues was 93.86% at 3 minutes and 93.25% at 1 hour, versus 57.77% and 0.62% in the positive control .

Eye irritation

An in vitro eye irritation test using the Reconstructed human Cornea-like Epithelium (RhCE) (EpiOcular™ tissue) model was performed according to Guideline OECD 492 and in compliance with GLP to predict the acute eye irritation potential of the test substance.The mean percent tissue viability of the RhCE replicates treated with the test substance was 85.49%, versus 37.65% in the positive control (Methyl acetate).

Justification for classification or non-classification

Based on the results of two studies conducted according to Guidelines OECD 439 and OECD 431, the registered substance is classified in category 2 (H315) for skin irritation according to Regulation EC No 1272/2008 (CLP) and GHS.

Based on the results of a study conducted according to Guideline OECD 492, the registered substance should not be classified for eye irritation according to Regulation EC No 1272/2008 (CLP) and GHS.