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Skin irritation / corrosion

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skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 April 2016 - 06 June 2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This information is used for read across to Geranyl Isobutyrate MCS.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Citronellyl butyrate
EC Number:
EC Name:
Citronellyl butyrate
Cas Number:
Molecular formula:
3,7-dimethyloct-6-en-1-yl butyrate

In vitro test system

Test system:
human skin model
Source species:
Cell type:
other: epidermal keratinocytes
Cell source:
other: MatTek Corporation, 82105 Bratislava, Slovakia
Source strain:
other: Not applicable.
unchanged (no vehicle)
Details on test system:
- Model used: EpiDerm™, 0.6 cm^2
- Tissue batch number: 23339
- 30 μL of the test item was dispensed directly atop the EpiDerm™ tissue and spread to match the surface of the tissue for a complete treatment time of 60 minutes.

- Temperature used during treatment / exposure: 37 ± 1.5°C

Assessment of Direct Test Item Reduction of MTT:
MTT Salt Metabolism, Cell Viability Assay:
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt by mitochondrial succinate dehydrogenase in viable cells. One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of thecellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

Test for Direct MTT Reduction:
Prior to the start of the test, the test item’s colour interference potential had to be evaluated.
For this purpose 30 μL of the test item were mixed with 300 μL of deionised water. This mixture was incubated for 60 minutes at 37 ± 1.5 °C (5 ± 0.5% CO2). The test item was colourless and the colour of test item/water mixture did not change during the incubation period compared with the colour of the pure test item. Therefore, an additional test with viable tissues without MTT addition (step 2 according to study plan) was not necessary.
For correct interpretation of results it was necessary to assess the ability of the test item to directly reduce MTT. For this purpose approximately 30 μL of the test item were added to 1 mL of MTT solution and the mixture was incubated in the dark at 37 °C for 60 minutes.
Untreated MTT solution was used as control. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.
Since the colour did not turn blue/purple, the test item was not considered to be a MTT reducer. An additional test with freeze-killed tissues was not necessary.

The plastic bag containing the 24-well plate with epidermal tissues was opened under sterile conditions. Under an airflow using forceps, the gauze was removed and the inserts were taken out. Any remaining agarose that adheres to the outer sides of the inserts was removed by gentle blotting on the sterile filter paper or gauze, and the tissues were placed in the empty, sterile 6-well plate.
0.9 mL of the assay medium (20 – 25 °C) was pipetted into each well of sterile 6-well plates.
The inserts with the EpiDerm™ tissues were placed in the upper wells, and were preincubated for 60 minutes in the incubator (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH). Following, the inserts were transferred from upper wells into the lower wells of the 6-well plates, and, the pre-incubation was continued for further about 18 hours (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH).

After pre-incubation of EpiDerm™ tissues was completed, medium was replaced by 0.9 mL of fresh medium per well. The negative and positive control, the vehicle control, and the test item were added into the insert atop the corresponding EpiDerm™ triplicate tissues. The treatment time was 60 minutes in total. Within this period the 6-well plates were put into the incubator for 35 minutes at 37 ± 1.5 °C, 5 ± 0.5 % CO2. In the remaining period the plates were placed in a sterile bench at room temperature until the end of treatment.
After the end of the treatment interval 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. 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. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with sterile blotting paper. The tissues were carefully dried using sterile cotton tipped swap.
The tissues were then transferred into new 6-well plates with 0.9 mL of fresh assay medium in the upper row. The inserts were placed in the prepared holding plate. Tissues were incubated for approximately 24 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. After incubation the inserts were transferred into new 6-wells plates containing fresh medium. Thereafter tissues were incubated for another about 18 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. The complete incubation time was approximately 42 hours.

On the day of testing the MTT concentrate was diluted with the MTT diluent (1 mg/mL). The 24-well plates were prepared before the end of the tissue pre-warming period. A volume of 300 μL of the MTT solution was added to each well and the plates were kept in an incubator (37 ± 1 °C, 5 ± 0.5 % CO2) until further use.
After the 42-hours incubation period was completed for all tissues and exposure groups, culture inserts were transferred from the holding plates to the MTT-plates. After a 3-hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2), the MTT solution was aspirated from the wells, and the wells were rinsed three times with DPBS. Inserts were transferred onto new 24-well plates. The inserts were immersed into extractant solution by gently pipetting 2 mL extractant solution (isopropanol) in each insert. The level rose above the upper edge of the insert, thus tissues were completely covered from both sides. The 24-well plate was sealed to inhibit the isopropanol evaporation.
The formazan salt was extracted for about 69 hours without shaking in the refrigerator.
After the extraction period was completed, the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken. Afterwards the insert was discarded. The 24-well plates were placed on a shaker for 15 minutes until the solution was homogeneous in colour.
Per each tissue, 3 × 200 μL aliquots of the blue formazan solution were transferred into a 96- well flat bottom microtiter plate from the 15 minutes exposure. OD was read in a microplate reader (Versamax® Molecular Devices, Softmax Pro, version 4.7.1) with a 570 nm filter. Mean values were calculated from the 3 wells per tissue.

For the current test, a test item is considered irritant if the mean relative tissue viability of three individual tissues is reduced to ≤ 50% of the negative control.
For the current test, a test item is considered non-irritant if the mean relative tissue viability of three individual tissues is > 50% of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
- Amount applied: 30 μL
Duration of treatment / exposure:
60 minutes exposure period and 42 hours post-exposure incubation period.
Number of replicates:
A total of 9 tissues were used: Triplicate tissues were treated with: test substance, positive control or negative control.

Results and discussion

In vitro

Irritation / corrosion parameter:
% tissue viability
Vehicle controls validity:
Positive controls validity:
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Direct MTT Reduction and colour interference:
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.
Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.
Test Item:
The mean relative absorbance value of the test item, corresponding to the cell viability, did not decrease to (106.8%; threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin.

- Acceptance criteria met for negative control: 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.
- Acceptance criteria met for positive control: Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 4.3% thus ensuring the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: The relative standard deviations between the % variability values of the test item and negative controls in the main test were below 14% , thus ensuring the validity of the study. Since the blank corrected absorption values of the positive control were very low (values between 0.061 and 0.089) compared with the test item and negative control values, the acceptance criteria “% variability values in the main test were < 18%” was slightly exceeded (19%). Due to the clear positive result of the positive control, this circumstance does not affect the integrity of the study.

Any other information on results incl. tables

Mean OD570 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item:


OD570 of


Mean OD570

of triplicate


Relative SD (%)




viability (%)



viability (%)


Control Item










Positive Control Item










Test Item










OD = Optical Density

SD = Standard deviation

Applicant's summary and conclusion

Interpretation of results:
other: Not a skin irritant
according to EU CLP (EC No. 1272/2008 and its amendments).
Since the mean relative tissue viability for the substance was above 50% the substance is considered not to be a skin irritant.
Executive summary:

The possible skin irritation potential of the substance was tested in an in vitro test using a human skin model through topical application for 60 minutes. The study procedures described in this report were according to OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 30 μL test substance. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. The positive control had a mean cell viability of 4.3%. The relative mean tissue viability obtained after 60 minutes treatment with the substance compared to the negative control tissue was 106.8%. Since the mean relative tissue viability of the substance was above 50% after 60 minutes treatment, the substance is considered not to be a skin irritant.