Myrac Aldehyde

Endpoint summary

Description of key information

- Eye irritation: Not irritating (OECD TG 492, GLP, Rel. 1)

- Skin irritation: Irritating (OECD TG 439, GLP, Rel. 1)

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:

07 August 2017 - 14 August 2017

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:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

20 July 2012

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

other: epidermal keratinocytes

Cell source:

other: SkinEthic Laboratories, Lyon, France.

Source strain:

other: Not applicable

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM, 0.38 cm^2
- Tissue batch number(s): 17-EKIN-032
- Pre-incubation: On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for approximately 22 hours at 37°C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.

- Before the start of the test, the substance was checked for possible colour interference and possible direct MTT reduction.

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 36.6-37.5 °C
- Humidity: 74-90%, containing 5.0 ± 0.5% CO2 in air

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1 with phosphate buffered saline
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL in PBS
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

CELL VIABILITY MEASUREMENT:
After incubation in MTT, epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 μL isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 69.5 hours. The amount of extracted formazan was determined spectrophotometrically. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test item was classified according to remaining cell viability following exposure of the test item.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: one experiment with two OD measurements for each replicate.

DECISION CRITERIA (see table 1 in 'any other information on materials and methods')
- A test substance is considered irritant in the skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- A test substance is considered non-irritant in the in vitro skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

ACCEPTABILITY CRITERIA:
- The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤18.
- The mean relative tissue viability of the positive control should be ≤ 50% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
- The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Test material: 25 μL
Positive control: 25 μL, re-spread after 7 minutes contact time
Negative control: 25 μL

Duration of treatment / exposure:

15 minutes ± 0.5

Duration of post-treatment incubation (if applicable):

42 hours and 3 hours with MTT

Number of replicates:

3 tissues for the treatment group, the positive control group and the negative control group each.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of 3 replicates

Value:

13

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Mean tissue viability: 4.8%

Remarks on result:

other: SD: 1.0%

Other effects / acceptance of results:

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

DEMONSTRATION OF TECHNICAL PROFICIENCY: the results of the positive control (mean tissue viability of 4.8% with a standard deviation of 2.3%) showed that the test system functioned properly.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 of the three tissues of the negative control were within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability was 1.3.
- Acceptance criteria met for positive control: yes, the mean relative tissue viability of the positive control was 4.8% relative to the
negative control and the Standard Deviation value (SD) of the % viability was 2.3.
- Acceptance criteria met for variability between replicate measurements: yes, the SD calculated from individual % tissue viabilities of the three identically treated replicates was <3%

Mean OD570 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item:

Item	OD570 of tissues	Mean OD570 of triplicate tissues	± SD of OD570	Relative mean viability (%)
Negative Control Item	1.222	1.234	0.016	100
	1.227
	1.252
Positive Control Item	0.032	0.059	0.028	4.8
	0.087
	0.059
Test Item	0.162	0.156	0.013	13
	0.165
	0.142

SD = Standard deviation

*The mean viability of the negative control tissues is set at 100 %

Interpretation of results:

other: Skin irritant.

Remarks:

according to Regulation (EC) No. 1272/2008 and its amendments.

Conclusions:

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the substance compared to the negative control tissues was 13%. Since the mean relative tissue viability for the test substance was below 50% it is considered to be irritant.

Executive summary:

The possible skin irritation potential of the substance was tested in vitro using a human skin model through topical application for 15 minutes. The study procedures described in this report were according to OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 25 μL undiluted test substance. After a 42-hour incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. The positive control had a mean cell viability of 4.8% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 3%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 13%. Since the mean relative tissue viability for the substance was below 50% after 15 minutes treatment the substance is considered to be irritant.

Therefore, the substance has to be classified as a skin irritant according to CLP regulation (EC) n° 1272/2008.

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:

19 May - 02 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline 492 without any deviation

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:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

23 October 2015

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. 23712] were received on 31 May 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: 1 hour and 54 minutes 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 water or isopropanol was checked by adding 50 µL of the test item to 1 mL of distilled water or 2 mL of isopropanol, respectively.


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 21 hours and 05 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 a 1-hour and 54-minute 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 05 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 measured in triplicate samples 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:

75.62

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 with colourless deposit of test item in the bottom of the well was observed after 3 hours of incubation between 36.5°C and 38.2°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 with colourless deposit of test item in the bottom of the well was obtained in water after 1 hour of incubation between 36.5°C and 38.2°C, 5% CO2. A colourless solution with colourless deposit of test item in the bottom of the well was obtained in isopropanol after 3 hours of incubation at room temperature. No significant colouration appeared, therefore the test item was considered to be not interfering with the MTT assay.

MAIN TEST
- MTT assay results: The mean percent tissue viability of the RhCE replicates treated with the test substance was 75.62%, versus 34.89% 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	1.261	1.214	1.223	99.30	100.00	1.39
		1.180
		1.203
	2	1.274	1.231		100.70
		1.215
		1.205
Positive control	1	0.471	0.462	0.427	37.79	34.89	5.81
		0.461
		0.455
	2	0.393	0.391		31.98
		0.389
		0.392
Test item PH-16/0229	1	1.041	1.033	0.925	84.50	75.62	17.75
		1.011
		1.049
	2	0.842	0.816		66.75
		0.794
		0.813

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

OD: optical density

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions adopted and in accordance with the Regulation EC No. 1272/2008, the test item does not require classification for eye irritation or serious eye damage. Also, the test item does not require classification for eye irritation or serious eye damage according to UN GHS.
No hazard statement and no signal word are required.

Executive summary:

An in vitro eye irritation test using the Reconstructed human Cornea-like Epithelium (RhCE) (EpiOcular™ tissue) model was performed according to the OECD Guideline 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 1-hour and 54-minute 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 percent tissue viability of the RhCE replicates treated with the test substance was 75.62%, versus 34.89% in the positive control (Methyl acetate).

Under the experimental conditions adopted and in accordance with the Regulation EC No. 1272/2008 (CLP), the test item does not require classification for eye irritation or serious eye damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Eye irritation:

An in vitro eye irritation test using the Reconstructed human Cornea-like Epithelium (RhCE) (EpiOcular™ tissue) model was performed according to the OECD Guideline 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 1-hour and 54-minute 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 percent tissue viability of the RhCE replicates treated with the test substance was 75.62%, versus 34.89% in the positive control (Methyl acetate).

Under the experimental conditions adopted and in accordance with the Regulation EC No. 1272/2008 (CLP), the test item does not require classification for eye irritation or serious eye damage.

Dermal irritation:

The possible skin irritation potential of the substance was tested in vitro using a human skin model through topical application for 15 minutes. The study procedures described in this report were according to OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 25 μL undiluted test substance. After a 42-hour incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. The positive control had a mean cell viability of 4.8% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 3%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 13%. Since the mean relative tissue viability for the substance was below 50% after 15 minutes treatment the substance is considered to be irritant.

Therefore, the substance has to be classified as a skin irritant according to CLP regulation (EC) n° 1272/2008.

Justification for classification or non-classification

In a GLP study conducted according to OECD 492 guideline, exposure of epithelia to the registered substance led to percent tissue viability higher than 60% therefore under experimental conditions adopted and in accordance with CLP Regulation (EC) n° 1272/200 and the GHS, the registered substance does not require classification as eye irritant or serious eye damage.

In a GP study conducted according to OECD 439 guideline, topical application of the registered substance led to relative mean tissue viability of 13% after 15 minutes. Therefore, in accordance with CLP Regulation (EC) n° 1272/2008 and the GHS, the registered substance has to be classified as skin irritant.