Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02.08.2016-11.08.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Butyl butyrate
EC Number:
203-656-8
EC Name:
Butyl butyrate
Cas Number:
109-21-7
Molecular formula:
C8H16O2
IUPAC Name:
butyl butanoate
Test material form:
liquid

Test animals / tissue source

Species:
human
Strain:
other:
Remarks:
not applicable
Details on test animals or tissues and environmental conditions:
- Source: MatTek Corporation (82105 Bratislava, Slovakia).
- EpiOcularTM tissue: normal, human-derived epidermal keratinocytes cultured to form a stratified squamous epithelium
- Surface: 0.6 cm
- Shipment: at 2 - 8 °C on medium-supplemented agarose gels
- Equilibration step: 15 minutes at room temperature
- Inspection: each tissue was inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used.
- Pre-incubation: standard culture conditions for 1 hour and after refreshing the Assay Medium at standard culture conditions overnight (17h in the experiment).

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
- Volume: 50 μL (83.3 μL/cm2)
- Concentration: undiluted
Duration of treatment / exposure:
30 minutes
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
2
Details on study design:
Details of the test procedure used
- RhCE tissue construct used, including batch number: EpiOcular Kit Lot No.: 23724
- Conditions of exposure: 37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH
- Washing: extensively rinsing the tissues with Ca++Mg++-free DPBS
- Number of tissue replicates used per test chemical and controls: 2
- Wavelength and band pass used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm (OD570), Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro, version 4.7.1. No reference wavelength measurement was used.
- MTT assay: incubation with 0.3 mL of MTT solution for 180 minutes at standard culture conditions, after incubation with MTT the inserts were incubated with isopropanol at 2-8°C overnight after which MTT was extracted for about 2 hours at room temperature
- Data evaluation: the following was calculated: mean of the blank control wells (ODBlk), ODBlk from each OD value of the same experiment (blank corrected values), mean of the two aliquots for each tissue (= corrected test item OD), mean of the two relating tissues for each control and test item (= corrected mean OD) (for further calculations only the corrected mean negative control OD value was needed), corrected OD value of the negative control corresponding to 100% viability (corrected negative control OD = Negative Control OD - ODBlk = 100% Viability)
- Description of evaluation criteria: If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is non-irritant and if the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labelled irritant. A single test composed of at least 2 tissue replicates should be sufficient for a test chemical when the result is unequivocal. In cases of borderline results, such as non- concordant replicate measurements and/or mean percent tissue viability equal to 60±5%, a second test should be considered, as well as a third one in case of discordant results between the first two tests (according to OECD 492).
- Historical data positive control: Mean Viability: 32.0%; Rel. Standard Deviation: 12.8%; Range of Viabilities: 6.90% - 40.4%; Mean Absorption: 0.538; Rel. Standard Deviation: 0.258; Range of Absorbance: 0.107- 0.849
- Historical data negative control: Mean Absorption: 1.65; Rel. Standard Deviation: 0.299; Range of Absorbance: 1.27 – 2.05
- 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

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Single test with two replicates
Value:
76
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The acceptance criteria were met.

Any other information on results incl. tables

Results of the pre-experiment:

The optical pre-experiment (colour interference pre-experiment) to investigate the test item's colour change potential in water or isopropanol did not lead to a change in colour.

Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not show blue color.

Results of the main experiment:

Results after treatment for 30 minutes

Dose Group

Absorbance Well 1 (Tissue 1/2)

Absorbance Well 2 (Tissue 1/2)

Mean Absorbance* (Tissue 1/2)

Mean Absorbance * Tissue 1 and 2

Mean Absorbance of 2 Tissues*

Rel. Absorbance [%] Tissue 1 and 2**

Absolute Value of the Difference of the Rel. Absorbances [%] Tissue 1 and 2

Rel. Absorbance [% of Negative Control]**

Blank

0.038

0.039

0.038

0.000

 

 

 

100.0

Negative Control

2.069

2.009

2.039

2.000

1.981

101.0

2.0

100.0

2.003

1.997

2.000

1.962

99.0

Positive Control

0.677

0.646

0.661

0.623

0.763

31.5

14.1

38.5

0.944

0.937

0.941

0.902

45.5

Test Item

1.531

1.533

1.532

1.494

1.505

75.4

1.2

76.0

1.553

1.557

1.555

1.517

76.6

* Mean of two replicate wells after blank correction


** Relative absorbance [rounded values]: 100 x (absorbance test item / positive control) / absorbance negative control

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 76.0% (threshold for irritancy: <= 60%).

Therefore, the substance was found to be not irritant to the eye.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item is not eye irritant.
Executive summary:

In the current study the eye irritation potential of the test item was assessed by means of the Human Cornea Model Test. The test was according to OECD 492 and GLP.

In the pre-tests, the test item did not reduce MTT and the intrinsic colour of the substance was not intensive and did not color water or isopropanol. Therefore, additional tests with freeze-killed or viable tissues did not have to be performed.

For this test, 50 μL of the test item, the negative control (deionised water) or the positive control (methyl acetate) was applied to tissue for 30 minutes and the test was performed in duplicate.

The absorbance values after treatment with the negative control were well within the required acceptability criterion showing the quality of the tissues, while treatment with the positive control induced a decrease below 50% compared to the negative control ensuring the validity of the test system.

The test was also valid as the difference in viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues).

Treatment with the test item did not indicate irritating effects. In the experiment the relative mean absorption value corresponding to the viability of the tissues was 76.0%. This result lies well above the borderline range of 60±5%.

In conclusion, in this study and under the experimental conditions reported, the test item is not eye irritant.