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Diss Factsheets

Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01.12.2016-23.01.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted July 28, 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes (incl. QA statement)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Mainzer Strasse 80, 65189 Wiesbaden, Germany

Test material

Constituent 1
Reference substance name:
2,6-Octadienal, 3,7-dimethyl-, acid-isomerized
EC Number:
291-768-8
EC Name:
2,6-Octadienal, 3,7-dimethyl-, acid-isomerized
Cas Number:
90480-35-6
IUPAC Name:
1-methyl-4-(propan-2-yl)benzene; ethanol

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: human-derived epidermal keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: human skin model EpiDerm™
- Tissue batch number: 23388
- Delivery date: 17 January 2017
- Date of initiation of testing: 17 January 2017 (preincubation phase)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C, 5 ± 0.5% CO2
- Temperature of post-treatment incubation: 37 ± 1.5 °C, 5 ± 0.5 % CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: Rinsed with DPBS at least 15 times, after the rinsing the inserts were submerged in DPBS at least three times. Afterwards the inserts were once again rinsed with sterile DPBS from the inside and the outside.
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: (1 mg/mL)
- Incubation time: 3 hours
- Spectrophotometer: Versamax® Molecular Devices, Softmax Pro, version 4.7.1
- Wavelength: 570 nm
- Filter: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Positive: 4.56%, range 2.76 - 6.77 %

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
the test item was considered to reduce MTT and an additional test with freeze-killed tissues was performed.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the mean tissue viability is ≤ 50 %.
- The test substance is considered to be non-irritant to skin if the mean tissue viability is > 50 %

Acceptability of the Assay
Criterion 1
Negative control: The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD570 of the negative control tissues is ≥ 0.8 and ≤ 2.8.

Criterion 2
Positive control: An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is ≤ 20%.

Criterion 3
Standard deviation: The SD of 3 identical replicates should be < 18%. OD values should not be below historically established boundaries.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
- Volume: 30 μL (47 μL/cm2 according to guideline) of undilted test item
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
approximately 43 hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of three tissues
Value:
>= 37.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The acceptance criteria were met.

Any other information on results incl. tables

Results after treatment with the test item and the controls

Dose Group

Tissue

No.

Absorbance

570 nm

Well 1

Absorbance

570 nm

Well 2

Absorbance

570 nm

Well 3

Mean

Absorbance

of 3

Wells

Mean-

Absorba

nce

of three

wells

blank

corrected

Mean

Absorbance

of 3 tissues

after blank

correction*

Rel.

Absorbance

[%]

Tissue

1, 2 + 3**

Relative

Standard

Deviation

[%]

Mean Rel.

Absorbance

[% of

Negative

Control]**

Blank

 

 

 

0.036

0.037

0.037

0.037

0.000

 

 

 

 

Negative

Control

1

1.757

1.682

1.699

1.713

1.676

1.533

109.3

8.1

100.0

2

1.540

1.486

1.463

1496

1.460

95.2

2

1.540

1.483

1.475

1.500

1.463

95.4

Positive

Control

1

0.077

0.075

0.076

0.076

0.039

0.042

2.6

8.2

2.7

2

0.078

0.077

0.076

0.077

0.040

2.6

2

0.082

0.082

0.083

0.082

0.046

3.0

Test Item

 

1

0.729

0.703

0.691

0.707

0.671

0.574

43.8

15.9

37.4

2

0.617

0.595

0.584

0.598

0.562

36.6

2

0.541

0.524

0.515

0.526

0.490

31.9

* Mean of three replicate wells after blank correction

** relative absorbance per tissue [rounded values]:100× (absorbancetissue)/( mean absorbancenegative control)

*** relative absorbance per treatment group [rounded values]:100× (meanabsorbancetest item/positive control)/( mean absorbancenegative control)

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is irritant to skin according to EU CLP regulation.
Executive summary:

This in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test. The test was according to OECD 439 and GLP.

The test item passed the colour interference pre-test, but an additional test with freeze-killed tissues had to be performed due to the test item’s MTT-reducing property determined in the MTT interference pre-test.

30 μL of either the test item, the negative control (DPBS) or the positive control (5% SLS) were applied to triplicate tissue each. The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for approximately 43 hours the tissues were treated with the MTT solution for 3 hours following about 68 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval thus showing the quality of the tissues.

Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 2.7% thus ensuring the validity of the test system. The relative standard deviations between the % variability values of the test item, the positive and negative controls in the main test were below 16% (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: < 18%), thus ensuring the validity of the study.

Compared to the relative absorbance value of the negative control the mean relative absorbance value was reduced to 37.4% after exposure of the skin tissues to the test item. Since this value is below the threshold for irritancy of ≤ 50%, a correction procedure using the correction factor determined in the additional test with freeze-killed tissues was not necessary.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is irritant to skin.