[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin 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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

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.

In vitro test system

Test system:

human skin model

Remarks:

EpiDerm

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

The test was carried out with the reconstituted three-dimensional human skin model EpiDerm (MatTek). This skin model consists of normal human epidermal keratinocytes (NHEK) which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts (Millicell). The EpiDerm epidermis model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible. It consists of organised basal, spinous and granular layers and a multi-layered stratum corneum analogous to patterns found in vivo.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

- Negative control 30 μL DPBS
- Positive control 30 μL 5% SDS solution
- Test Item Liquids: 30 μL (undiluted)

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

test item

Value:

92.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100.0

Positive controls validity:

valid

Remarks:

4.4

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 three negative control tissues was >= 0.8 and <= 2.8 (1.724). The mean relative tissue viability (% negative control) of the positive control was < 20% (4.4%). Standard deviation of viability of replicate tissues of all dose groups was <= 18% (0.5% - 17.6%).

Any other information on results incl. tables

The mixture of 30 μL test item per 1 mL MTT medium showed reduction of MTT compared to the solvent. The mixture turned blue/purple.

For quantitative correction of results, two killed tissues were treated with 30 μL of the test item (KT) and two killed tissues were left untreated as a control (KU), respectively. NSMTT was calculated relative to the negative control of living tissues (NC) according to the following formula:

NSMTT [%] = [(ODKT - ODKU)/ODNC] * 100 = -8.7%

Mean ODKT = 0.109

Mean ODKU = 0.241

Mean ODNC = 1.518

NSMTT was ≤ 30% (-8.7%) relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) of the test item treated living tissues TM was therefore corrected according to the following formula:

TODTT = ODTM – (ODKT – ODKU) = 1.685

The mixture of 30 μL of the test item per 300 μl aqua dest. and per 300 μL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equaled 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 > 50% (100.4%, NSMTT-corrected) after 60 min treatment and 42 h post-incubation.

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 relative mean tissue viability after 60 min of exposure and 42 h post-incubation was > 50%. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

Executive summary:

In the present study the skin irritant 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,4,5,6,7,7a-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 was analysed. The EpiDerm™-Standard Model (EPI-200™), a reconstituted three-dimensional human epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404) to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as non-irritant. 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 60 min exposure and 42 h post-incubation period and compared to those of the concurrent negative controls.

The mixture of 30 μL test item per 1 mL MTT medium showed reduction of MTT compared to the solvent. The mixture turned blue/purple.

For quantitative correction of results, two killed tissues were treated with 30 μL of the test item (KT) and two killed tissues were left untreated as a control (KU), respectively. NSMTT was calculated relative to the negative control of living tissues (NC).

NSMTT was ≤ 30% (-8.7%) relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) of the test item treated living tissues TM was therefore corrected according to the following formula: TODTT = ODTM – (ODKT – ODKU) = 1.685

The mixture of 30 μL of the test item per 300 μl aqua dest. and per 300 μL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equaled 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 > 50% (100.4%, NSMTT-corrected) after 60 min treatment and 42 h post-incubation.

Conclusion

In this study under the given conditions the test item showed no irritant effects. The relative mean tissue viability after 60 min of exposure and 42 h post-incubation was > 50%. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.