[(3aS*,4R*,5S*,7R*,7aS*)-4,7-methano-2,3,3a,4,5,6,7,7a-octahydro-1H-inden-5-yl]methyl acetate

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 to 18 February 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: 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; 29 June 2015

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

[(3aS*,4R*,5S*,7R*,7aS*)-4,7-methano-2,3,3a,4,5,6,7,7a-octahydro-1H-inden-5-yl]methyl acetate

Molecular formula:

C13H20O2

IUPAC Name:

[(3aS*,4R*,5S*,7R*,7aS*)-4,7-methano-2,3,3a,4,5,6,7,7a-octahydro-1H-inden-5-yl]methyl acetate

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Test item sourced from test sponsor, batch number: 10300010
- Expiration date of the lot/batch: January 2017
- Purity test date: Sum of Main Isomer: 96.30 Area %, Sum Isomers: 99.73 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Not reported

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: In a prevalidation study performed by Avon Products Inc. and MatTek Corporation, the 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 indicated this method as a sufficiently promising predictor for eye irritancy potential. The EpiOcular ™ Eye Irritation Test (EIT) was validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and cosmetics Europe between 2008 and 2013.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: 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 organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium.

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): 5 μL
- Concentration (if solution): Undiluted test item

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

3 hours

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used: 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 (June 2015) followed.
- RhCE tissue construct used, including batch number: EpiOcular™ kits and MTT-100 kits purchased. EpiOcular™ lot no. 21597
- Doses of test chemical and control substances used: 50 μL of undiluted test item, methyl acetate or deionised water.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): 30 minutes exposure at 37 ± 1.5 ºC; post-exposure immersion for 12 minutes at room temperature; post-exposure incubation for 120 minutes at 37 ± 1.5 ºC
- 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): 2 per test item, postive control or negative control.
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): Measured at 570 nm with a microplate reader without a reference filter. Absorbance values calculated with computer software.
- Description of the method used to quantify MTT formazan:
- 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: 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 viability is ≤ 60 % of treated negative control, the test item is labelled irritant.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria:
- Complete supporting information for the specific RhCE tissue construct used: Method validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and cosmetics Europe between 2008 and 2013.
- Reference to historical data of the RhCE tissue construct: Historic data from July 2015 to February 2016 show the postive control range of viabiltiies of 6.90 % - 40. 4 %. The range of absorbance for historic postive control data is 0.107 - 0.849 and for negative control data 1.27 - 2.05.
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: Historical report data reported.
- Positive and negative control means and acceptance ranges based on historical data: Positve control - mean viability: 32.0 %, range: 6.90 - 40.4 %, mean absorption: 0.538, range 0.107 - 0.849. Negative control - mean absorption: 1.65, range 1.27 - 2.05
- Acceptable variability between tissue replicates for positive and negative controls: < 20 %
- Acceptable variability between tissue replicates for the test chemical: < 20 %

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: Mean absorbance

Run / experiment:

2 Tissues

Value:

2.025

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

other: Relative absorbance (% of negative control)

Run / experiment:

Mean of 2 tissues

Value:

100.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

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

DEMONSTRATION OF TECHNICAL PROFICIENCY: Validity criteria met

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Negative control OD is > 1.0 and < 2.5 (1.99 and 2.053). The difference of viability between the two relating tissues of the postive control was 3.1 % (must be < 20%)
- Acceptance criteria met for positive control: Mean relative viability of the positive control is below 50% of the negative control viability (40.4%). The difference of viability between the two relating tissues of the postive control was 3.2 % (must be < 20%)
- Range of historical values if different from the ones specified in the test guideline: Not applicable

In vivo

Irritant / corrosive response data:

Irritating effects were not observed following 30 minutes incubation with the test item.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study, under the experimental conditions reported, the test item does not possess an eye irritating potential.

Executive summary:

The test item was assessed for eye irritation in a human cornea model test conducted following OECD 492, and UN GHS guidelines in undiluted form alongside a postive and negative control. The results of the test indicates that the test item does not possess an eye irritating potential according to the UN GHS regulations.

The study is a GLP compliant guideline experiment study available as an unpublished study report. The study has no restrictions and is fully adequate for assessment.