(4E)-4-(3,3,4-trimethylcyclopentylidene)butanal

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-11-13 to 2020-11-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 492 and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

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:

18 June 2019

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(4E)-4-(3,3,4S-trimethylcyclopentylidene)butanal

Molecular formula:

C12 H20 O

IUPAC Name:

(4E)-4-(3,3,4S-trimethylcyclopentylidene)butanal

Constituent 2

Reference substance name:

(4E)-4-(3,3,4R-trimethylcyclopentylidene)butanal

Molecular formula:

C12 H20 O

IUPAC Name:

(4E)-4-(3,3,4R-trimethylcyclopentylidene)butanal

Test material form:

liquid

Details on test material:

Appearance: Very pale yellow liquid

Test animals / tissue source

Species:

human

Strain:

other: Human Keratinocyte

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: The EpiOcular™ EIT was validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. The EpiOcular™ EIT was endorsed as an in vitro test that can be used to identify those chemicals not requiring classification and labelling for eye irritation or serious eye damage in accordance with UN GHS (No Category).
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: EpiOcularTM Human Corneal Model (0.6 cm2 ) provided by MatTek In Vitro Life Science Laboratories, Bratislava – Slovakia. No biological contaminant detected.
- Cell line used, its source: Keratinocyte strain 4F1188.
- RhCE tissue used, including batch number: EpiOcular Tissue Lot Number: 30634.

Test system

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

Duration of treatment / exposure:

30 ±2 minutes at 37 °C, 5% CO2

Duration of post- treatment incubation (in vitro):

120 ±15 minutes at 37 °C, 5% CO2

Number of animals or in vitro replicates:

Duplicate cultures for test item, negative and positive controls

Details on study design:

- Details of the test procedure used: This method is based on the MatTek Corporation Protocol: 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; 02 October 2017.
- Volume of test chemical and control substances used: 50 µL
- Duration and temperature of exposure period: 30 ±2 minutes at 37 °C, 5% CO2
- Duration and temperature of post-exposure immersion period: 12 ±2 minutes at room temperature
- Duration and temperature of post-exposure incubation period: 120 ±15 minutes at 37 °C, 5% CO2
- Justification for the use of a different negative control than ultrapure H2O (if applicable): not applicable
- Justification for the use of a different positive control than neat methyl acetate (if applicable): not applicable
- Description of any modifications to the test procedure: none
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): not applicable
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): not applicable
- Description of the method used to quantify MTT formazan, if applicable: The optical density (OD) was measured at 570 nm (OD570) using a Labtech LT-4500 microplate reader. The upper limit of accuracy for measured absorbance of MTT Formazan at 570 nm (OD570), filter band pass 10 nm, was determined to be at an optical density of 2.6.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: The relative mean tissue viabilities were compared to the mean of the negative control (n=2) treated tissues. The relative mean tissue viabilities were calculated as follows: Relative mean tissue viability = mean OD570 of test item mean / OD570 of negative control x 100. Mean Tissue Viability >60% : No Category / Mean Tissue Viability ≤60% : No prediction can be made according to the EU CLP and the GHS.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: In addition to the acceptability criteria a comparison of laboratory historical data on negative and positive controls was made to verify the functioning of the test system.
- Complete supporting information for the specific RhCE tissue construct used: yes, Certificate of Analysis from MatTek is attached to the report.
- Reference to historical data of the RhCE tissue construct: Acceptance criteria were met for Tissue Viability, Barrier function and Sterility.
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: not included in the report, but available upon request to the lab

Results and discussion

In vitro

Results

Irritation parameter:

mean percent tissue viability 

Run / experiment:

Mean of duplicate cultures

Value:

73.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

36.7%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none

ACCEPTANCE OF RESULTS:
- Positive and negative control means and acceptance ranges based on historical data: All values for the control groups were within the ranges obtained by the testing laboratory.
- Acceptable variability between tissue replicates for positive and negative controls:
The relative mean tissue viability for the positive control treated tissues was 36.7% relative to the negative control treated tissues (below 50%). The positive control acceptance criterion was therefore satisfied.
The mean OD570 for the negative control treated tissues was 2.104 (> 0.8 and < 2.8). The negative control acceptance criterion was therefore satisfied.
- Acceptable variability between tissue replicates for the test chemical: The difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items). This applies also to the killed controls (test items and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

Any other information on results incl. tables

Table 7.3.2/1: Individual Scores and Mean Scores for Corneal Effects – Test Item

 

Item	OD570 of tissues (tvt)	OD570 Tissues corrected for[tkt-ukt]	Correct Mean OD570 of duplicate tissues	Individual tissue viability (%)	Relative mean viability (%)	Difference in viability (%)
Negative Control Item	2.209	-	2.104	105.0	100*	10.0
	1.999	-		95.0
Positive Control Item	0.737	-	0.771	35.0	36.7	3.3
	0.805	-		38.3
Test Item	1.540	1.498	1.539	71.2	73.2	3.9
	1.622	1.580		75.1

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions, the test item was classified as non-irritant according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Executive summary:

The purpose of this study was to identify chemicals not requiring classification and labelling for eye irritation or serious eye damage using the EpiOcular™ Eye Irritation Test (EIT) according to the OECD Test Guideline 492 Reconstructed human Cornea-like Epithelium (RhCE) test method.

Duplicate tissues were treated with the test item for an exposure period of 30 minutes. At the end of the exposure period each tissue was rinsed before incubating for 120minutes. The test item was found to directly reduce MTT and therefore additional non-viable, freeze-killed, tissues were incorporated into the testing for correction purposes. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. After MTT-loading each tissue was placed in 2 mL of isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 570 nm (OD570). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viability of the test item treated tissues, corrected for direct MTT reduction, was 73.2%.

The criteria required for acceptance of results in the test were satisfied.

Under the test conditions, the test item was classified as non-irritant according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.