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Diss Factsheets

Toxicological information

Eye irritation

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Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 June 2016 - 17 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 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
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

Test animals / tissue source

other: epidermal keratinocytes
Details on test animals or tissues and environmental conditions:
EpiOcular™ kits and MTT-100 kits are purchased from MatTek Corporation (82105 Bratislava, Slovakia).

Test system

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
- Amount applied: 50 μL
Duration of treatment / exposure:
30 minutes
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
Duplicate tissues were treated with: test substance, positive control or negative control.
Details on study design:
RhCE tissue construct used, including batch number :
EpiOcular™ kits and MTT-100 kits are purchased from MatTek Corporation, batch 23714 (82105 Bratislava, Slovakia). The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) were cultured on specially prepared cell culture inserts..

Assessment of Direct Test Item Reduction by MTT:
Test items may have the ability to directly reduce MTT and to form a blue/purple reaction product which could have an impact on the quantitative MTT measurement. Therefore, it was necessary to assess this ability for the test item prior to conducting any assays with viable tissues. For this purpose approximately 50 μl of the test item were added to a 1 mL of a 1.0 mg/mL MTT solution (in DMEM) in a 6-well plate and the mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for three hours. A control (50 μL of deionised water in 1 mL of 1.0 mg/mL MTT solution) was run concurrently. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT. Since the MTT solution colour did not turn blue/purple, the test item is not presumed to be a MTT reducer. An additional test with freeze-killed tissues did not have to be performed.

Assessment of Coloured or Staining Materials:
Coloured test items or test items which become coloured after application to the tissues could interfere with the quantitative photometric MTT measurement if the colorant bound to the tissue and would be extracted together with MTT. Therefore, each test item had to be checked for its colorant properties.
Since the test item was colourless additional tests had to be performed to assess, if it will dye water or isopropanol. For this purpose each 50 μL of the test item were added to 1 mL of water and to 2 ml isopropanol in 6-well plates. The water mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for one hour, the isopropanol mixture 3 hours at room temperature Since the test item did not dye water or isopropanol, additional tests with viable tissues did not have to be performed.

Experimental Performance:
After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca++Mg++free-DPBS. The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH) for 30 minutes.
Test item exposure: After the 30 minute Ca++Mg++free-DPBS pre-treatment, the test and control items were tested by applying 50 μL topically on the EpiOcular™ tissues. The tissues were incubated at standard culture conditions for 30 minutes.
At the end of the 30 minutes treatment time, the test item was removed by extensively rinsing the tissues with Ca++Mg++-free DPBS (brought to room temperature). Three clean beakers containing a minimum of 100 mL each of Ca++Mg++-free DPBS were used per test item. Each test item utilized a different set of three beakers. The inserts containing the tissues were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps. The test or control items were decanted from the tissue surface onto a clean absorbent material and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker. The culture was then rinsed in the second and third beakers of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material by rotating the insert to approximately a 45° angle (open end down) and touching the upper lip to the absorbent material (to break the surface tension).
After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labelled 12-well plate for 12 minutes immersion incubation (post-soak) at room temperature. This incubation in Assay Medium was intended to remove any test item absorbed into the tissue.
At the end of the post-soak immersion, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the inserts were blotted on absorbent material, and transferred to the appropriate well of the pre-labelled 6-well plate containing 1 mL of warm Assay Medium. The tissues are incubated for approximately 120 minutes at standard culture conditions (post-treatment incubation).

MTT Assay:
After post-treatment incubation of 120 minutes the MTT assay was performed.
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues are placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions and rinsed 3 times with DPBS afterwards.
Each insert was removed from the 24-well plate after 180 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol was flowing into the insert on the tissue surface. The plates were sealed with a standard plate sealer, and were stored overnight at 2-8 °C in the dark and then shaken for 2-3 hours at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken.
The extract solution was mixed and two 200 μL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate.
The absorbance at 570 nm (OD570) of each well was measured with a plate reader reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro, version 4.7.1). No reference wavelength measurement was used.

Data Evaluation:
1) The mean OD value of the blank control wells (ODBlk) for each experiment were calculated.
2) The ODBlk from each OD value of the same experiment (blank corrected values) were subtracted.
3) The mean value of the two aliquots for each tissue (= corrected test item OD) were calculated.
4) The mean value of the two relating tissues for each control and test item (= corrected mean OD) were calculated. For further calculations only the corrected mean negative control OD value was needed.
5) The corrected OD value of the negative control corresponds to 100% viability.
Corrected negative control OD = Negative Control OD - ODBlk = 100% Viability

Calculations for Viability Tests:
1) The percent viability of each of the two relating tissues for each control and test item relative to the negative control (100% control) were calculated.
Viability [%]= 100 x corrected test item OD/corrected mean negative control OD
2) The difference of the viability between duplicate tissues was calculated. If the difference is >20% the test is considered as non-qualified.
3) The mean test item viability (TI viability) was calculated and the test item was classified according to the decision criteria.

Decision criteria:
If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labeled non-irritant.
If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labeled irritant.

Acceptability of the Assay:
The results are acceptable if:
1) The negative control OD is > 0.8 and < 2.5,
2) The mean relative viability of the positive control is below 50% of the negative control viability.
3) The difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items). This applies also to the killed controls (items and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

Results and discussion

In vitro

Irritation parameter:
other: % tissue viability
Negative 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 or isopropanol did not led to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not show blue colour.
Test item:
The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 98.6% (threshold for irritancy: ≤ 60%), consequently the test item was not irritant to eye.

- Acceptance criteria met for negative control: The negative control OD was > 0.8 and < 2.5 (2.118 and 2.013).
- Acceptance criteria met for positive control: The mean relative viability of the positive control was below 50% of the negative control viability (34.9%).
The difference of viability between the two relating tissues of a single item was < 20% (values between 5.1% and 17.7%) in the same run (for positive and negative control tissues and tissues of single test items).

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


Absolute value of the difference of the rel. absorbances (%)




viability (%)



viability (%)


Control Item








Positive Control Item








Test Item








OD = Optical Density

Applicant's summary and conclusion

Interpretation of results:
other: Not an eye irritant
according to Regulation (EC) No. 1272/2008 and its amendments.
Since the mean relative tissue viability for the substance was above 60% the substance is considered not to be an eye irritant.
Executive summary:

The possible eye irritation potential of the substance was tested in an in vitro test. Two type of tissues can be used either primary human epidermal keratinocytes or human immortalized corneal epithelial cells. The epidermal keratinocytes were used in the present test. The study procedures described in this report were according to OECD TG 492 guideline and GLP principles. Skin tissue was treated by topical application of 50 μL test substance for 30 minutes. After 120 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. Eye 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 34.9%. The relative mean tissue viability obtained after 30 minutes treatment with the substance compared to the negative control tissue was 98.6% presenting absence of eye irritation.