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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 - 17 Jun 2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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:
Jul 2015
GLP compliance:
yes (incl. QA statement)
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Constituent 1
Chemical structure
Reference substance name:
Citronellyl butyrate
EC Number:
EC Name:
Citronellyl butyrate
Cas Number:
Molecular formula:
citronellyl butyrate

Test animals / tissue source

other: EpiOcular™

Test system

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
- Amount applied: 50 µL

- Amount applied: 50 µL

- Amount applied: 50 µL
Duration of treatment / exposure:
30 min
Duration of post- treatment incubation (in vitro):
120 min
Number of animals or in vitro replicates:
in duplicates for each treatment and control group
Details on study design:
- RhCE tissue construct used, including batch number: EpiOcular™ tissue (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia), batch number: 23714
- Viability: The quality of the final product was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 1.836 ± 0.282 (acceptance criteria: 1.1 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 0.3% Triton X-100. The ET-50 value was determined to be 20.48 min (acceptance criteria: 12.2 - 37.5 min).
- Contamination: The cells used to produce the EpiOcular tissue were screened for the presence of viruses, bacteria, yeast and other fungi.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: 30 min exposure, 12 min post-exposure immersion and 120 min post-exposure incubation at 37 °C
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: The ability of the test substance to directly reduce MTT and to form a blue/purple reaction product was assessed in a pre-experiment. Since the MTT solution colour did not turn blue/purple, the test substance is not presumed to have reduced the MTT. An additional test with freeze-killed tissues did not have to be performed.
- Number of tissue replicates used per test chemical and controls: 2
- Description of the method used to quantify MTT formazan: The absorbance at 570 nm of each well was measured with a plate reader (Versamax Molecular Devices, Ismaning, Germany). No reference wavelength measurement was used.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: The test substance was considered to be not irritating to eye if the tissue viability after 30 min exposure, 12 min post-exposure immersion and 120 min post-exposure incubation is >60% relative to the negative control treated tissue.
- Acceptance criteria: The results are acceptable if the negative control OD is >0.8 and <2.5 and if the mean relative viability of the positive control is below 50% of the negative control viability.
- Reference to historical data of the RhCE tissue construct: Historical control data was used to assess the validity of the test.
- Acceptable variability between tissue replicates for the test chemical, positive and negative controls: The difference of viability between the two relating tissues of a single test substance is < 20% in the same run.

Results and discussion

In vitro

Irritation parameter:
other: % tissue viability
mean value of 2 tissues
Run / experiment:
30 min exposure
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Other effects / acceptance of results:
The optical pre-experiment (colour interference pre-experiment) to investigate the colour change potential of the test substance in water or isopropanol did not led to a change in colour. Optical evaluation of the MTT-reducing capacity of the test substance with MTT-reagent did not show blue colour.

- Acceptance criteria met for negative control: The negative control OD (2.118 and 2.013) was in the range of > 0.8 and < 2.5.
- Acceptance criteria met for positive control: Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 34.9%, thus the validity of the test system is ensured.

The difference of viability between the two relating tissues of a single substance is < 20% (values between 5.1% to 17.7%) in the same run (for positive and negative control tissues and tissues of single test substance).

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Under the conditions of the conducted test, the test substance did not exhibit irritating properties towards human-derived epidermal keratinocytes in the EpiOcular™ model.
Executive summary:

Irritating effects were not observed following incubation with CITRONELLYL BUTYRATE. Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues did not decrease below 60% (98.6%).