2,3-dihydro-2,2,6-trimethylbenzaldehyde

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 30, 2015 to July 21, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in 2015 at recognised contract research organisation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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:

2,3-dihydro-2,2,6-trimethylbenzaldehyde

EC Number:

204-133-7

EC Name:

2,3-dihydro-2,2,6-trimethylbenzaldehyde

Cas Number:

116-26-7

Molecular formula:

C10H14O

IUPAC Name:

2,6,6-trimethylcyclohexa-1,3-diene-1-carbaldehyde

Test material form:

other: liquid

Details on test material:

Batch N°SC00013340
Purity 82.7%
Expiry date 20.10.2015

In vitro test system

Test system:

human skin model

Remarks:

EPISKIN-SM, three-dimensional human epidermis model

Justification for test system used:

The EPISKIN-SM model has been validated for irritation testing in an international validation study [9] and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

EPISKIN-SM (Manufacturer: SkinEthic, France, Batch No.:15-EKIN-015, Expiry Date: 20 April 2015) is a three-dimensional human epidermis model. Adult humanderived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994) [7]. Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.
For killed epidermis, living epidermis units (Manufacturer: SkinEthic, France, Batch No.: 14-EKIN-044, Expiry Date: 24 November 2014) were placed in a 12 well plate with 2 mL of distilled water, then incubated at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere for approximately 48 hours. At the end of the incubation the water was discarded and the dead epidermis units were frozen on 21 November 2014. Before use, the killed tissues were thawed at room temperature (at least 30 minutes in 2 mL of Assay Medium). Further use of killed tissues was similar to living tissues.

Control samples:

yes, concurrent negative control

yes, concurrent no treatment

yes, concurrent positive control

Amount/concentration applied:

10 µL of test item were applied evenly to the epidermal surface. If necessary, the test item was spread gently on the skin surface with a pipette tip without damaging the epidermis. The amount was sufficient to cover the epidermal surface.

Duration of treatment / exposure:

42 hours

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

1.1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

According to EU classification, the irritancy potential of test substances is predicted for distinguishing between skin irritating (R38) and no-label (non-skin irritating) test substances OECD TG 404, OECD 439 & Method B.4 of Annex V to Directive 67/548/EEC [1,4,12,13]. The irritation potential of test substances can be also classified according to the United Nations Globally Harmonized System of Classification and Labelling of Chemicals [10]. In the present study, the irritancy potential of test substances is predicted by the mean tissue viability of tissues exposed to the test item. The test item is considered to be irritant to skin (Category 2, H315), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control. The prediction model (PM) is described below:
- If Mean tissue viability % is ≤ 50%: Irritant (I) with EU (67/548/EEC); Category 2 with UN GHS / CLP
- If Mean tissue viability % is > 50%: Non Irritant (NI) with EU (67/548/EEC); No Category with UN GHS / CLP

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In conclusion, in this in vitro EPISKIN model test with SAFRANAL, the results indicate that the test item is irritant to skin.

Executive summary:

An in vitro skin irritation test of SAFRANAL test item was performed in a reconstructed human epidermis model. EPISKIN-SM is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (detailed in 3.6. section). The irritation potential of the test item was evaluated according to the OECD No. 439 guideline.

Disks of EPISKIN (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). An additional disk was used to provide an estimate of colour contribution from the test item. Furthermore, three additional test item treated and three negative control treated killed epidermis units were used to determine the MTT interacting potential of the test item. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

Following exposure with SAFRANAL, the mean cell viability was 1.1% compared to the negative control (after adjustment for colour and non-specific MTT reduction). This is below the threshold of 50%, therefore the test item was considered as being irritant to skin. Note that this test protocol can not identify corrosives but usually only corrosive substances cause effects of this magnitude. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN model test with SAFRANAL, the results indicate that the test item is irritant to skin.