Reaction mass of Benzenepropanal, 4-ethyl-α,α-dimethyl- and 3-(2-ethylphenyl)-2,2-dimethylpropanal

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 May 2016 to 30 May 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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

(2015)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2009)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

1

Reference substance name:

Reaction mass of Benzenepropanal, 4-​ethyl-​α,​α-​dimethyl- and 3-(2-ethylphenyl)-2,2-dimethylpropanal

EC Number:

916-329-6

Molecular formula:

C13H18O

IUPAC Name:

Reaction mass of Benzenepropanal, 4-​ethyl-​α,​α-​dimethyl- and 3-(2-ethylphenyl)-2,2-dimethylpropanal

Test material form:

liquid

Details on test material:

As described in 1.2 of dossier

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: EPISKIN™ in vitro Reconstructed Human Epidermis (RHE) Model Kit

Justification for test system used:

The EPISKIN™ model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes 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 a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum. Following a full validation study the EpiSkin™ reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items. The procedure followed is based on the recommended EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ in vitro Reconstructed Human Epidermis (RHE) Model Kit
- Supplier: SkinEthic Laboratories, Lyon, France
- EpiSkinTM Tissues (0.38cm2) lot number: 16-EKIN-021
- Delivery date: 24 May 2016
- Date of initiation of testing: 24 May 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 reader
- Wavelength: 562 nm

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA
- If the viability after 15 minutes exposure is greater than 50 % the test substance is not considered to be a skin irritant
- If the viability after 15 minutes exposure is less than or equal to 50% the test substance is considered to be a skin irritant

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 10 µL

NEGATIVE CONTROL
- Amount(s) applied: 10 µL

POSITIVE CONTROL
- Amount(s) applied: 10 µL
- Concentration: 5% w/v aqueous solution
- To ensure satifactory contact with positive control item the solution was spread over the entire surface of the epidermis using a pipette tip. After a 7 minute contact time the SDS solution was re-spread to maintain the distribution

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Exposure time: 15 minutes

Value:

9.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

other: Skin irritant category 2

Remarks:

in accordance with CLP (1272/2008 and its updates)

Conclusions:

Under the conditions of this study the test item was considered to be irritating to the skin, because the relative mean tissue viability was below 50% after 15 min exposure.

Executive summary:

The possible skin irritation potential of the test substance was tested in vitro using the EPISKIN™ reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The study procedures in the study were according to OECD TG 439 and GLP principles. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Triplicate tissues were treated with 10 µL test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. The optical density was measured at 562 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). The relative mean viability of the test item treated tissues was 9.8% after the 15-Minute exposure period and 42-hours post-exposure incubation period. Under the conditions of this study the test item was considered to be irritating to the skin, because the relative mean tissue viability was below 50% after 15 min exposure.