Cyclopropanecarboxylic acid, 2-[1-(3,3-dimethylcyclohexyl)ethoxy]-2-methylpropyl ester

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12-06-2008 to 13-06-2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

equivalent or similar 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:

MatTek Corporation EpiOcular™ Eye Irritation Test (OCL-200-EIT)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: September 2006 ; signature: January 2007

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

460-490-0

EC Name:

-

Cas Number:

477218-42-1

Molecular formula:

C18H32O3

IUPAC Name:

2-[(1S)-1-[(1R)-3,3-dimethylcyclohexyl]ethoxy]-2-methylpropyl cyclopropanecarboxylate; 2-[(1S)-1-[(1S)-3,3-dimethylcyclohexyl]ethoxy]-2-methylpropyl cyclopropanecarboxylate

Test material form:

liquid

Details on test material:

Physical state: Liquid
- Storage condition of test material: In the refrigerator at + 2 to + 8 °C, protected from light

Test animals / tissue source

Species:

other: human-derived epidermal keratinocyte tissues

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: The test method is indicated as a validated test method recommended by OECD and/or recognised internationally for the sequential testing strategy for the assessment of ocular irritation.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 10231 Kit F).

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 of the test item
- Concentration (if solution): undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): Not applicable.
- Concentration (if solution): Not applicable.
- Lot/batch no. (if required): Not applicable.
- Purity: Not applicable.

Duration of treatment / exposure:

Negative control: 60 minutes at 37 ± 1°C
Positive control: 15 and 45 minutes at 37 ± 1°C
Test item: 3, 30 and 60 minutes at 37 ± 1°C

Duration of post- treatment incubation (in vitro):

10 to 20 min.

Number of animals or in vitro replicates:

Two tissues were used for each treatment group (test item, negative control and positive control).

Details on study design:

- Details of the test procedure used: See below.
- RhCE tissue construct used, including batch number: EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 10231 Kit F).
- Doses of test chemical and control substances used: 100 µL test item / 100 µL deionised water (negative control) / 100 µL 0.3% Triton X-100 (positive control)
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): After the incubation, the tissues were pre-wetted with 300 µL of assay medium. The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2) until use. After the pre-treatment, the test and control items were tested by applying 100 µL topically on the EpiOcular tissues. The tissues were incubated at standard culture conditions for 60 minutes (negative control), 15 and 45 minutes (positive control) and 3, 30 and 60 minutes (test item). At the end of the treatment time, the test item was removed by extensively rinsing the tissues with PBS (brought to room temperature). After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in pre-labelled 6-well plates for a 10-20 minute immersion incubation (post-soak) at room temperature. This incubation in Assay Medium was intended to remove any test item absorbed into the tissue. At the end of the post-soak immersion, each insert was removed from the Assay Medium, the medium was aspirated off the tissue, and the wells were triply rinsed with PBS. The inserts were immersed into extractant solution (2 mL isopropanol) for formazan salt extraction, sealed, and incubated for 18 hours without agitation at room temperature. After the extraction period, the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken. Afterwards the insert was discarded. The well plates were placed on a shaker for 15 minutes until the solution was homogeneous in colour. 3 x 200 µL aliquots of the formazan blue solution were transferred to a well plate and OD was read in a microplate reader at 570 nm without reference filter. Mean values (n=3) were calculated.
- 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 specified.
- Description of any modifications to the test procedure: Not specified.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): The test item did not directly-reduce MTT or was colour inference in pre-checks conducted prior to the exposure.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): Duplicate.
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm
- Description of the method used to quantify MTT formazan: Versamax® Molecular Devices, 85737 Ismaning, Germany – spectrophotometrically.
- Description of the qualification of the HPLC/UPLC-spectrophotometry system (if applicable): Not applicable.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: Not specified.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: Not specified.
- Complete supporting information for the specific RhCE tissue construct used: Attached to the full study report.
- Reference to historical data of the RhCE tissue construct: Full historic control data is attached to the full study report.
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: The test assay is validated at the test laboratory and certification of technical proficiency from the model supplier is attached to the full study report.
- Positive and negative control means and acceptance ranges based on historical data: See above.
- Acceptable variability between tissue replicates for positive and negative controls: Yes, < 6.2 % for positive control, negative control and test item replicates.
- Acceptable variability between tissue replicates for the test chemical: Yes.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: Mean tissue viability (% of negative control)

Remarks:

n=2

Run / experiment:

mean - test item (60 min)

Value:

114

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

other: Mean tissue viability (% of negative control)

Remarks:

n=2

Run / experiment:

mean - positive control (45 min)

Value:

22.2

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:

The ET50-value of the positive control was calculated to be 14.6 min. An ET50-value could not be calculated for the test item because viability was not reduced, where:
ET50 = a - ((a - b)(c - 50)) / (c - d)
where:
a = max. measured time in min with the% of negative control > 50%
b = min. measured time in min with the% of negative control < 50%
c = relative absorbance at time a in %
d = relative absorbance at time b in %

The lower the ET 50 value, the higher the eye irritant/cytotoxic potential of the test item. For classification purposes, an ET50-value greater than 60 is a non/minimal irritant; a value of 31-60 denotes a mild irritant; 3-30 denotes a moderate irritant and <3 denotes a severe/extreme irritant.

Based on the inability to calculate an ET50, the test item is not predicted to be an eye irritant.

OTHER EFFECTS:
- Visible damage on test system: None.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The test assay is validated at the test laboratory and certification of technical proficiency from the model supplier is attached to the full study report.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Range of historical values if different from the ones specified in the test guideline: See 'details on study design' for further information.

Any other information on results incl. tables

Table 1.0 : Mean absorption and Mean Tissue Viability in the EPIOCULAR Test

Dose Group	Treatment Interval	Mean Absorbance of 2 Tissues	Rel. Absorbance (% of Negative Control)
Negative Control	60 min	1.7315	100.0
Positive Control	15 min	0.8454	48.8
	45 min	0.3840	22.2
Test Item	3 min	1.7749	102.5
	30 min	2.0066	115.9
	60 min	1.9739	114.0

Acceptability of the Assay

a) The absolute mean OD570 of the two tissues of the negative control should reasonably be > 0.8 and < 2.5.

b) The mean relative tissue viability of the positive control should be < 50% relative to the negative control.

c) The difference between the % tissue viabilities of the two identically treated replicates should be < 20%.

All assay acceptability criteria were met.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the test item is not considered to be an eye irritant.

Executive summary:

The study was performed to assess the eye irritancy potential of the test item using the EpiOcular EIT model under OECD TG 492 guideline in accordance with GLP. The test item was found to not directly reduce MTT or colour interfere in pre-test checks. The test item was topically applied for 3, 30 and 60 minutes. After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. The tissues were transferred to fresh medium and incubated under standard culture conditions, prior to determination of the cytotoxic (irritancy) effect. The relative mean tissue viability obtained after treatment with the test item compared to the negative control tissues was 114% (60 min). The positive control had a mean cell viability of 22.2% after 45 minutes treatment. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptability criteria. The difference between the percentages of viability of two tissues treated identically was less than 20%, indicating that the test system functioned properly. Under the conditions of this study, since the test item relative mean viability was > 60% the test item is not predicted to be eye irritant.