[No public or meaningful name is available]

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August - September 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Method Summary No. 164: EpiOcular™ Eye Irritation Test

Version / remarks:

22 July 2015

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2-(3a,4,5,6,7,7a-hexahydro-1H-4,7-methanoinden-5-yloxy)ethyl acrylate and 2-(3a,4,5,6,7,7a-hexahydro-1H-4,7-methanoinden-6-yloxy)ethyl acrylate

Molecular formula:

C15 H20 O3

IUPAC Name:

Reaction mass of 2-(3a,4,5,6,7,7a-hexahydro-1H-4,7-methanoinden-5-yloxy)ethyl acrylate and 2-(3a,4,5,6,7,7a-hexahydro-1H-4,7-methanoinden-6-yloxy)ethyl acrylate

Test material form:

liquid

Details on test material:

- Name: 2-Propenoic acid,2-[[3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-inden-5(or 6)-yl]oxy]ethyl ester
- CAS No.: 68169-12-0 (outside EU)
- EC Name: Reaction mass of 2-(3a,4,5,6,7,7a-hexahydro-1H-4,7-methanoinden-5-yloxy)ethyl acrylate and 2-(3a,4,5,6,7,7ahexahydro-1H-4,7-methanoinden-6-yloxy)ethyl acrylate
- EC Number: 951-956-9
- Batch No.: OX8J0799
- Molecular Weight: 248.32 g/mol
- Physical State: liquid
- Colour: clear
- Active Components: 98.52% active components (different isomers), 1.48% 2-Cyclopenta-1,3-dienyloxyethan-1-ol
- Purity: 98.52%
- Expiry Date: 17 September 2023
- Storage Conditions: room temperature
- Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Test animals / tissue source

Species:

human

Strain:

other: normal epidermal keratinocytes

Details on test animals or tissues and environmental conditions:

The test was carried out with the EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek). The model consists of normal, human-derived epidermal keratinocytes which have
been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable
layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

- Negative Control 50 μL Aqua dest.
- Positive Control 50 μL methyl acetate
- Test Item 50 μL (undiluted)

Duration of treatment / exposure:

30 min

Duration of post- treatment incubation (in vitro):

- Post-treatment incubation 12 min (RT) + 120 min (37°C)
- MTT incubation 3 h

Number of animals or in vitro replicates:

duplicate

Details on study design:

The test item was applied topically to the EpiOcular tissue. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid (MTT) after a 30 min exposure period and 120 min post-treatment period and compared to those of the concurrent negative controls.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Remarks:

Mean Relative Tissue Viability [%]

Run / experiment:

test item

Value:

80.17

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100.0

Positive controls validity:

valid

Remarks:

36.0

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The controls confirmed the validity of the study.
The mean absolute OD570 of the two negative control tissues was > 0.8 and < 2.8 (2.058).
The mean relative tissue viability (% negative control) of the positive control was < 50% (36.0%).
The maximum inter tissue difference of replicate tissues of all dose groups was < 20% (7.9%).

Any other information on results incl. tables

Ocular irritation potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with Aqua dest.

The mixture of 50 μL test item per 1 mL MTT medium showed reduction of MTT as compared to the solvent. The mixture turned blue/purple. Since the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value killed tissue controls were performed for quantitative correction of results.

NSMTT [%] = [(ODKT - ODKU)/ODNC] * 100 = -0.356%

Difference of NSMTT of the two duplicate tissues must be < 20%, otherwise not accepted.

NSMTT1 [%] = [(meanODKT1 - ODKU)/ODNC] * 100 = [(0.030 – 0.035)/2.010] = -0.25%

NSMTT2 [%] = [meanODKT2 - ODKU)/ODNC] * 100 = [(0.026 – 0.035)/2.010] = -0.45%

NSMTT1 - NSMTT2 = ± 0.2%

NSMTT was ≤ 60% (-0.4% relative to the negative control of living epidermis and could therefore be used for determination of the killed control corrected viability (KCCV) according to the following formula:

KCCV [%] = 80.17% – (-0.356%) = ~ 80.52%

The mixture of 50 μL test item per 1 mL Aqua dest. and per 2 mL isopropanol showed no colouring as compared to the solvent. Therefore, NSCliving equalled 0%.

The test item showed non-specific reduction of MTT but no relevant colouring potential after mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false negative results were necessary.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 60% (80.52% NSMTT- corrected).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed no irritant effects. The test item is classified as “non-irritant“ in accordance with UN GHS “No Category”

Executive summary:

In the present study the eye irritating potential of 2-Propenoic acid,2-[[3a,4,5,6,7,7a-hexahydro-4,7 -methano-1H-inden-5(or 6)-yl]oxy]ethyl ester; EC Name: Reaction mass of 2-(3a,4,5,6,7,7ahexahydro-1H-4,7-methanoinden-5-yloxy)ethyl acrylate and 2-(3a,4,5,6,7,7a-hexahydro-1H-4,7 -methanoinden-6-yloxy)ethyl acrylate was analysed. Since irritant substances are cytotoxic to the corneal epithelium after a short time exposure the cytotoxic effects of the test item on EpiOcular, a reconstituted three-dimensional human corneal epithelium model, were determined. Hereby, the test item was applied topically to the EpiOcular tissue. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid (MTT) after a 30 min exposure period and 120 min post-treatment period and compared to those of the concurrent negative controls.

The mixture of 50 μL test item per 1 mL MTT medium showed reduction of MTT as compared to the solvent. The mixture turned blue/purple. Since the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value killed tissue controls were performed for quantitative correction of results.

NSMTT [%] = [(ODKT - ODKU)/ODNC] * 100 = -0.356%

Difference of NSMTT of the two duplicate tissues must be < 20%, otherwise not accepted.

NSMTT1 [%] = [(meanODKT1 - ODKU)/ODNC] * 100 = [(0.030 – 0.035)/2.010] = -0.25%

NSMTT2 [%] = [meanODKT2 - ODKU)/ODNC] * 100 = [(0.026 – 0.035)/2.010] = -0.45%

NSMTT1 - NSMTT2 = ± 0.2%

NSMTT was ≤ 60% (-0.4% relative to the negative control of living epidermis and could therefore be used for determination of the killed control corrected viability (KCCV) according to the following formula:

KCCV [%] = 80.17% – (-0.356%) = ~ 80.52%

The mixture of 50 μL test item per 1 mL Aqua dest. and per 2 mL isopropanol showed no colouring as compared to the solvent. Therefore, NSCliving equalled 0%.

The test item showed non-specific reduction of MTT but no relevant colouring potential after mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false negative results were necessary.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 60% (80.52% NSMTT-corrected).

Conclusion

In this study under the given conditions the test item showed no irritant effects. The test item is classified as “non-irritant“ in accordance with UN GHS “No Category”