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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:
2017-04-28 to 2017-08-18
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: UN GHS
Version / remarks:
published 2003, 7h revision, 2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
1,3-dimethyl-3-phenylbutyl acetate
EC Number:
268-407-8
EC Name:
1,3-dimethyl-3-phenylbutyl acetate
Cas Number:
68083-58-9
Molecular formula:
C14H20O2
IUPAC Name:
1,3-dimethyl-3-phenylbutyl acetate
Test material form:
liquid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: human-derived epidermal keratinocytes cultured from a multilayered, hughly differentiated model of the human epidermis
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST SYSTEM
- Source: MatTek Corporation (Bratislava, Slovakia)
The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.

- Procedure used: The plastic bag containing the 24-well plate with epidermal tissues was opened under sterile conditions and the gauze was removed and the inserts were taken out under an airflow using forceps. Any remaining agarose was removed by gentle blotting on the sterile filter paper or gauze, and the tissues were placed in the empty, sterile 6-well plate. Prior to the exposure of the test item and of the controls the EpiDerm™ tissues were inspected for quality.
- Quality control for skin discs: The tissues were checked for air bubbles between agarose and insert (should be less than 30% of the total surface), liquid on top of the insert was removed with sterile cotton tips, if again moisture was observed on top of the inserts after the pre-incubation or in case of visible defects the respective skin models were discarded.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C,
- Temperature of post-treatment incubation (if applicable): 37 ± 1.5 °C,

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: The inserts were removed immediately from the 6-well plate and tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material and then 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: none reported
- Modifications to validated SOP: none reported

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: microplate reader (Versamax® Molecular Devices, Softmax Pro, version 4.7.1)
- Filter: 570 nm

NUMBER OF INDEPENDENT TESTING RUNS AND DECISION CRITERIA
- The test chemical is considered to be irritant to skin in accordance with UN GHS and EU CLP Category 2 if the tissue viability of three individual tissues after exposure and post-treatment incubation is less than or equal (≤) to 50%.
- The test substance is considered to be non-irritant to skin if the mean relative tissue viability of three individual tissues is reduced > 50% of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL (47 μL/cm2)
- Concentration (if solution): test item was applied undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL of DPBS was applied to triplicate tissue.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL of SLS was applied to triplicate tissue.
- Concentration (if solution): 5% SLS solution in deionised water
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
41 hours
Number of replicates:
Three replicates per test item, negative and positive controls were used.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
26.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
30.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
30.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
29.1
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 optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour of water. Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour. Therefore, the test item passed the MTT- and the colour interference pre-tests.

The is considered valid as all the acceptability criteria were met:

In 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 compared with the negative control to 3.3% 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 8% (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.

Any other information on results incl. tables

Table 1. Results

Dose group

Tissue No.

Absorbance 570 nm

Mean absorbance of 3 wells

Mean absorbance of 3 wells blank corrected

Mean absorbance of 3 tissues

Rel. absorbance [%] tissue 1, 2+3

Rel. standard deviation

Mean Rel. absorbance [%]

Well 1

Well 2

Well 3

Blank

 

0.038

0.038

0.037

0.037

0.000

 

 

 

 

Negative control

1

1.708

1.718

1.740

1.722

1.684

1.670

100.8

4.6

100.0

2

1.804

1.757

1.767

1.776

1.738

104.1

3

1.659

1.606

1.611

1.625

1.588

95.1

Positive control

1

0.090

0.095

0.094

0.093

0.055

0.055

3.3

0.6

3.3

2

0.093

0.093

0.091

0.092

0.055

3.3

3

0.092

0.093

0.093

0.093

0.055

3.3

Blank

 

0.037

0.037

0.036

0.037

0.000

 

 

 

 

Test item

1

0.489

0.473

0.477

0.480

0.443

0.485

26.5

7.5

29.1

2

0.557

0.537

0.535

0.546

0.506

30.3

3

0.555

0.540

0.536

0.544

0.507

30.4

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
The test item is considered as irritating to skin under test conditions as the mean relative absorbance value was reduced to 29.1% after exposure of the skin tissues compared to the relative absorbance value of the negative control.
Executive summary:

An in vitro study was performed to assess the irritation potential of test item by means of the Human Skin Model Test. The study was conducted according to OECD 439 and EU B.46 guidelines and is GLP-compliant.

Each three tissues of the human skin model EpiDerm were treated with the test item, the negative control (DPBS) or the positive control (5% SLS) for 60 minutes.

After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system. For the test item, the mean relative absorbance value decreased to 29.1% compared to the relative absorbance value of the negative control. This value is below the threshold for irritancy of ≤ 50%. Therefore, under the experimental conditions the test item is considered to be irritant to skin.

The study is reliable without restrictions (Klimisch 1) as it was conducted according to the standard methods and to GLP. It is therefore considered as relevant for key study for this endpoint.