methyl-bis(2-arylpropyl)dihydro-heteropolycycle

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-01-29 to 2019-01-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals No. 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:

25 June 2018

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2015

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: 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

Version / remarks:

29 June 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)

Test material

Test material information

Constituent 1

Test material form:

liquid

Test animals / tissue source

Species:

human

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:

1. Negative Control 50 µL Aqua dest.
2. Positive Control 50 µL methyl acetate
3. Test Item 50 µL (undiluted)
The test was performed on a total of 2 tissues per dose group.

Duration of treatment / exposure:

incubation: 30 +/- 2 min.

Duration of post- treatment incubation (in vitro):

- post soak incubation: 12 +/- 2 min.
- post treatment incubation: 120 +/- 15 min.
- MTT incubation: 3h +/- 15 min.

Number of animals or in vitro replicates:

2

Details on study design:

The potential of the test item to induce eye irritation was analysed by using the three-dimensional human corneal epithelium model EpiOcular, consisting of normal, human-derived epidermal keratinocytes mimicking characteristics of the corneal epithelium.
In the present study the test item was applied topically to the EpiOcular tissue for 30 min followed by 12 min post-soaking incubation after removal of the test item. After a 120 min post-treatment period cytotoxic effects were determined via MTT reduction assay.
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.

Results and discussion

In vitro

Results

Irritation parameter:

other: mean relative tissue viability

Run / experiment:

1

Value:

104.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: value in %

Other effects / acceptance of results:

Value Cut off pass/fail
Mean Absolute OD570 nm NK 1.624 0.8 < NK < 2.5 pass
Mean Relative Viability PC [%] 39.9 < 50% pass
Max. Difference of % Viability [%] 11.1 < 20% pass

Any other information on results incl. tables

The mixture of 50 µL test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%. 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, NSC_living equalled 0%. The test item showed no non-specific reduction of MTT and 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% (104.6%).

Result of the Test Item Methyl-bis(2 -arylpropyl)dihydro-heteropolycycle

Name	Negative Control		Positive Control		Test Item
Replicate Tissue	1	2	1	2	1	2
Absolute OD570	1.574	1.684	0.667	0.676	1.558	1.765
	1.569	1.669	0.663	0.689	1.660	1.806
Mean Absolute OD570	1.624****		0.674		1.697
OD570(Blank Corrected)	1.531	1.640	0.624	0.632	1.514	1.721
	1.525	1.626	0.619	0.646	1.617	1.762
Mean OD570of the Duplicates (Blank Corrected)	1.528	1.633	0.621	0.639	1.566	1.742
Total Mean OD570of the 2 Replicate Tissues (Blank Corrected)	1.580*		0.630		1.654
SD of Mean OD570of the Duplicates (Blank Corrected)	0.074		0.012		0.125
Relative Tissue Viability [%]	96.7	103.3	39.3	40.4	99.1	110.2
Relative Tissue Viability Difference [%]^***	6.7		1.1		11.1
Mean Relative Tissue Viability [%]	100.0		39.9**		104.6

^*              Corrected mean OD₅₇₀ of the negative control corresponds to 100% absolute tissue viability

^**             Mean relative tissue viability of the positive control is < 50%

^***            Relative tissue viability difference of replicate tissues is < 20%

^****        Mean absolute OD₅₇₀ of the negative control is ≥ 0.8 and ≤ 2.5       

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 the test item 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 TM, a reconstituted three-dimensional human corneal epithelium model, were determined. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 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 no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.

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, NSC_living equalled 0%.

The test item showed no non-specific reduction of MTT and 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% (104.6%).