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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 22 September 2016 and 26 September 2016.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 431 using the EPISKINTM Reconstructed Human Epidermis Model and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
(2015)
Deviations:
yes
Remarks:
Please see "Principles of method if other than guideline" below for further details.
Principles of method if other than guideline:
An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using freeze killed tissues was performed. However, the results obtained showed that negligible interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the freeze killed tissues for quantitative correction of results or for reporting purposes. This deviation was considered not to affect the purpose or integrity of the study.
GLP compliance:
yes (incl. QA statement)

Test material

1
Chemical structure
Reference substance name:
1-(2,4-dimethylcyclohex-3-en-1-yl)propan-1-ol
EC Number:
944-553-4
Cas Number:
1632042-40-0
Molecular formula:
C11H20O
IUPAC Name:
1-(2,4-dimethylcyclohex-3-en-1-yl)propan-1-ol
Test material form:
solid
Remarks:
White

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: epithelial
Cell source:
other: EpiDerm™ Reconstructed Human Epidermis Model Kit
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Human Skin Model
- Tissue batch number(s): 23356
- Delivery date: 22 September 2016
- Date of initiation of testing: 22 September 2016

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:Rinsing was achieved by filling and emptying each tissue under 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: 1.0 mg/mL
- Incubation time: Overnight.

NUMBER OF REPLICATE TISSUES: Two pre-labeled 6-well plates for both the 3 Minute and 60 Minute exposure periods.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: The determination of skin corrosion potential was performed in parallel on viable and freeze- killed tissues.
- Procedure used to prepare the killed tissues (if applicable): Freeze-killed tissues were prepared by placing untreated EPIDERMTM tissues in an empty 12 well plate and storing in a freezer (-14 to -30 °C) for a minimum of 24 hours. Before use each tissue was thawed by placing in 0.9 mL of assay medium for approximately 1 hour at room temperature.
- N. of replicates : In addition to the normal test procedure, the MTT reducing test item was applied to two freeze killed tissues per exposure period. In addition, two freeze killed tissues per exposure period remained untreated.
- Method of calculation used: A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
25 mg of test item was added to 300 µL of sterile water. The solution was incubated in the dark at 37 oC, 5% CO2 in air for 60 minutes. A visual assessment of the color was then made.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
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): 25 mg, used as supplied and added to second two tissues.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50µl sterile distilled water, used as supplied and added to first two tissues.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50µl 8.0N Potassium Hydroxide, used as supplied and added to final two tissues.
Duration of treatment / exposure:
3 minutes and 60 minutes
Duration of post-treatment incubation (if applicable):
3 hours for MTT incubation
Number of replicates:
3 minute exposure:
Test material x 2
Negative control x 2
Positive control x 2

60 minute exposure:
Test material x 2
Negative control x 2
Positive control x 2

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes exposure period
Value:
99.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: The test item was considered to be non-corrosive to the skin.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minute exposure period
Value:
112.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: The test item was considered to be non-corrosive to the skin.
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using freeze-killed tissues was performed during the determination of skin corrosion potential. However, the results obtained showed no degree of interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results or for reporting purposes.
- Colour interference with MTT: The solution containing the test item did not become colored. This was taken to indicate the test item did not have the potential to cause color interference.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD562 for the negative control treated tissues was 2.014 for the 3 Minute exposure period and 1.889 for the 60 Minute exposure period. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 4.4% relative to the negative control following the 60 Minute exposure period. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:
other: The test was considered to be non-corrosive to the skin
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
In a skin corrosion study using the EpiDermTM HUman Skin Model, performed according to OECD 431, the test item was considered to be non-corrosive to the skin.
Executive summary:

The skin corrosivity of the test substance was determined according to OECD Guideline 431 using the EPISKIN™ Reconstructed Human Epidermis Model. The relative mean viability of 3 and 60 minutes were 99.2% and 112.6% respecitvely in relation to the control. Therefore the substance is not corrosive to skin, according to EU CLP criteria.