Reaction mass of methyl 2-[[(E)-(2,4-dimethylcyclohex-3-en-1-ylidene)methyl]amino]benzoate and methyl 2-[[(Z)-(2,4-dimethylcyclohex-3-en-1-ylidene)methyl]amino]benzoate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 February 2016 - 28 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The standard information requirements for REACH Annex VII substances for serious eye damage/eye irritation can be satisfied by an assessment of the available in vivo data, physicochemical properties (i.e. acid or alkali reserve) or in vitro eye irritation studies. The EpiOcular™ Eye Irritation Test (OECD 492) is an accepted in vitro test method to detect severe eye irritation/damage (Category 1) and/or the absence of effects (not classified under CLP).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 2-[[(Z)-(2,4-dimethylcyclohex-3-en-1-ylidene)methyl]amino]benzoate

Molecular formula:

C17H21NO2

IUPAC Name:

Methyl 2-[[(Z)-(2,4-dimethylcyclohex-3-en-1-ylidene)methyl]amino]benzoate

Constituent 2

Reference substance name:

Methyl 2-[[(E)-(2,4-dimethylcyclohex-3-en-1-ylidene)methyl]amino]benzoate

Molecular formula:

C17H21NO2

IUPAC Name:

Methyl 2-[[(E)-(2,4-dimethylcyclohex-3-en-1-ylidene)methyl]amino]benzoate

Test animals / tissue source

Species:

human

Strain:

other: cornea

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: Eye irritation is defined in Section 3.3.1.1 of Annex I to the CLP regulation as “[…] the production of changes in the eye following the application of test substance to the anterior surface of the eye, which are fully reversible within 21 days of application." According to Annex VII of the REACH Regulation, if new test data are required these must be derived from in vitro methods only. The EpiOcular™ model (OECD 492), using Reconstructed human Cornea-like Epithelium (RhCE), is an accepted in vitro test method to identify chemicals not requiring classification and labelling for eye irritation or serious eye damage.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organised basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. In a pre-validation study, in vitro eye test using the human cornea model EpiOcular™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for eye irritancy potential.

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
- Concentration (if solution): neat

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

2

Details on study design:

METHOD:
- Details of the test procedure used: The test consists of a topical exposure of the neat test item to a human reconstructed cornea model followed by a cell viability test. Tissues were incubated with the test item or controls for 30 minutes at standard culture conditions. At the end of the 30 minutes treatment time, the test item or control was removed by extensively rinsing the tissues. After rinsing, the tissues immersed in Assay Medium at room temperature for 12 minutes to remove any test item absorbed into the tissue. The tissues were then incubated in Assay Medium for about 120 minutes at standard culture conditions. The MTT assay was performed after the post-treatment incubation. The percent reduction of cell viability in comparison of untreated negative controls is used to predict eye irritation potential.

ENVIRONMENTAL CONDITIONS
- RhCE tissue construct used, including batch number: EpiOcular ™ from MatTek Corporation, keratinocyte strain 4F1188, lot numbers 21597 and 23707
- Doses of test chemical and control substances used: 50 μL, neat
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: 30 minute exposure at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH), 12 minute immersion at room temperature, and 120 minute post-treatment incubation at standard culture conditions
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: Negative control (deionised water) for direct MTT-reduction test
- Number of tissue replicates used per test chemical and controls (positive control, negative control): 2
- Wavelength used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm, linearity range not reported
- Description of the method used to quantify MTT formazan: At the end of the post-treatment incubation, tissues were placed into a 24-well plate containing 0.3 mL of MTT solution. The plate was incubated for about 180 minutes at standard culture conditions. After the incubation time, tissues were rinsed and transferred into another plate containing 2.0 mL of isopropanol per well. The plates were sealed and stored overnight at 2-8 °C in the dark. To extract the MTT, the plates were placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken. The extract solution was mixed and two 200 μL aliquots were taken for measurement.

EVALUATION CRITERIA
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: The mean viability for the colorant control was calculated and this mean colorant control "viability" value (CC "viability") was subtracted from the relating mean viability of the same test item (TI viability) to determine the colorant control corrected viability (CC corrected viability). The test item was classified regarding the colorant control corrected viability according to the prediction model. If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labelled non-irritant. If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labelled irritant.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: Positive control (mean viability 32.0%, relative standard deviation of mean viability 12.8%, range of viabilities 6.90-40.4%, mean absorption 0.538, relative standard deviation of mean absorption 0.258, range of absorbance 0.107-0.849), negative control (mean absorption 1.65, relative standard deviation of mean absorption 0.299, range of absorbance 1.27-2.05); data of 11 studies performed from July 2015 until end of February 2016.
- Complete supporting information for the specific RhCE tissue construct used: Lot number 21597 (tissue viability 1.779 ± 0.054; barrier function 20.25 min; sterile tissue), lot number 23707 (tissue viability 1.918 ± 0.095; barrier function 15.12 min; sterile tissue)
- Reference to historical data of the RhCE tissue construct: Acceptance criteria of tissue functionality and quality (tissue viability (OD 540-570nm) 1.1-3.0; barrier function (ET-50) 12.2=37.5 min; no contamination)
- Positive and negative control means and acceptance ranges based on historical data: The negative control OD must be > 0.8 and < 2.5.
- Acceptable variability between tissue replicates for positive and negative controls: < 20% in the same run
- Acceptable variability between tissue replicates for the test chemical: < 20% in the same run

Results and discussion

In vitro

Results

Irritation parameter:

other: Percent viability

Run / experiment:

Mean

Value:

107

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:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD was 2.012 to 2.109
- Acceptance criteria met for positive control: The mean relative viability of the positive control 40.4% of the negative control viability
- Range of historical values if different from the ones specified in the test guideline: Historical negative control OD range 1.27-2.05, historical range of viabilities for positive control 6.90-40.4%

Any other information on results incl. tables

Table 1. Absorbance results

Treatment	Replicate	OD_570nm (well 1)	OD_570nm (well 2)	OD_570nm (mean)	OD_570nm (mean of 2 wells, blank corrected)	OD_570nm (mean of 2 tissues, blank corrected)	Relative absorbance (%)	Absolute value of the difference of the relative absorbances (%)	Mean relative absorbance (% of negative control)
Blank	-	0.037	0.036	0.037	0.000	-	-	-	-
Negative control	1	2.012	2.041	2.026	1.990	2.021	98.5	3.1	100.0
Negative control	2	2.068	2.109	2.089	2.052	(see above)	101.5	(see above)	(see above)
Positive control	1	0.846	0.925	0.885	0.849	0.816	42.0	3.2	40.4
Positive control	2	0.805	0.837	0.821	0.784	(see above)	38.8	(see above)	(see above)
Test item	1	2.097	2.052	2.124	2.088	2.166	103.3	7.8	107.2 (without colourant correction); 107.0 (with colourant correction)
Test item	2	2.213	2.350	2.282	2.245	(see above)	111.1	(see above)	(see above)
Additional test with viable tissues without MTT reduction
Blank	-	0.036	0.035	0.036	0.000	-	-	-	-
Negative control	1	0.039	0.040	0.039	0.004	-	100.0	-	-
Test item	1	0.041	0.040	0.040	0.005	-	123.4	-	-

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The EpiOcular™ method (OECD 492) has been validated extensively and is an accepted in vitro test method to detect eye corrosion/irritation (Category 1) and/or the absence of effects (not classified under CLP), under REACH Annex VII. The viability of test item exposed tissues was 107.0% (threshold for irritancy: ≤ 60%), therefore the test item is not considered to possess eye irritating potential. Conducted according to the aforementioned guidelines and GLP, the EpiOcular™ test passed all validity criteria and was considered to be reliable without restriction (Klimisch 1).

Executive summary:

Eye irritation of the test item was evaluated with the EpiOcular™ Human Cornea Model (OECD 492). Cell viability of a stratified squamous epithelium (cultured from normal, human-derived epidermal keratinocytes), similar to that found in the human cornea, was evaluated using the MTT assay, which measures the conversion of (3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide (MTT) into a blue formazan salt. A 50 µL aliquote of the test item was applied to the tissue for 30 minutes, alongside a negative and positive control. The viability value of the test item exposed tissues was 107.0% (threshold for irritancy: ≤ 60%), therefore the test item is not considered to possess eye irritating potential.

The test item passed the MTT- and the Colour Interference pre-tests. Conducted according to OECD Test Guideline 492 and GLP, the study is considered to be reliable without restriction (Klimisch 1).