Registration Dossier

Administrative data

Description of key information

The substance is not considered skin irritant and not considered eye irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02.08.2016-15.08.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
Instead of the MatTek EPI-200-SIT Kit, the MatTek EPI-200-PHO Test kit was used for the experimental performance. This Deviation had no influence on the outcome of the study (identical test kits with only the cell culture medium being different).
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: human-derived epidermal keratinocytes
Source strain:
other: not applicable
Vehicle:
unchanged (no vehicle)
Details on test system:
Details of the test procedure used
- EpiDerm™ tissue of human-derived epidermal keratinocytes was used (Epi-200- PHO Kit, Lot No.: 23349)
- Conditions of exposure: 37 ± 1.5 °C, 5 ± 0.5% CO2, 95%
- Washing: inserts gently rinsed with DPBS at least 15 times
- Number of tissue replicates used per test chemical and controls: 3
- MTT assay: incubation with 0.3 mL of MTT solution for 60 minutes at 37 ± 1.5 °C, 5 ± 0.5% CO2)
- Data evaluation: the following was calculated: The mean OD of the three negative control tissues was calculated after blank correction. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula: Relative viability(%) = (mean OD test item / positive control / mean OD negative control) x 100. For the test item and the positive control the mean relative viability ± rel. standard deviation of the three individual tissues was calculated
- Description of evaluation criteria: For the current test, an irritation potential of the test item of H315, GHS Cat 2 according to UN GHS (published 2003, last (6th) revision 2015) is recommended if the mean relative tissue viability of three individual tissues is reduced ≤ 50% of the negative control.
- Historical data positive control: Mean Viability: 4.64%; Rel. Standard Deviation: 11.2%; Range of
Viabilities: 4.00% - 5.90%; Mean Absorption: 0.0803; Rel. Standard Deviation: 12.6%; Range of Absorb
ance: 0.066-0.097
- Historical data negative control: Mean Absorption: 1.74; Rel. Standard Deviation: 8.68%; Range of
Absorbance: 1.48-1.98
- Acceptability of the Assay: the results are acceptable if (1) tissue viability is meeting the acceptance criterion, i.e. if the mean OD570 of the negative control tissues is ≥ 0.8 and ≤ 2.8 (negative control). If (2) the relative tissue viability of the positive control is ≤ 20% (positive control). If (3) the SD of 3 identical replicates is < 18%. OD values should not be below historically established boundaries.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
- Volume: 30 μL (47 μL/cm2 according to guideline)
Duration of treatment / exposure:
60 minutes
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Single test with three tissues
Value:
108.4
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.

Results of the pre-experiment:

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

Results after treatment with butyl butyrate and the controls:

Dose

Group

Exposure

Interval

Tissue

No.

Absorbance

570 nm

Well 1

Absorbance

570 nm

Well 2

Absorbance

570 nm

Well 3

Mean

Absorbance

of 3

Wells

Mean-

Absorba

nce

of three

wells

blank

corrected

Mean

Absorbance

of 3 tissues

after blank

correction

Rel.

Absorbance

[%]

Tissue

1, 2 + 3*

Relative

Standard

Deviation

[%]

Mean Rel.

Absorbance

[% of

Negative

Control]**

Blank

 

 

0.038

0.039

0.039

0.038

0.000

 

 

 

 

Negative

Control

60

min

Tissue 1

1.658

1.633

1.664

1.652

1.613

1.690

95.5

4.0

100.0

Tissue 2

1.792

1.783

1.755

1.777

1.738

102.9

Tissue 3

1.782

1.766

1.721

1.756

1.718

101.7

Positive

Control

60

min

Tissue 1

0.104

0.100

0.101

0.101

0.063

0.060

3.7

5.3

3.5

Tissue 2

0.094

0.096

0.096

0.095

0.057

3.4

Tissue 3

0.097

0.098

0.097

0.097

0.059

3.5

Test Item

60

min

Tissue 1

1.970

1.878

1.841

1.896

1.858

1.831

109.9

1.7

108.4

Tissue 2

1.889

1.844

1.773

1.835

1.797

106.3

Tissue 3

1.922

1.871

1.841

1.878

1.840

108.9

* relative absorbance per tissue [rounded values]: 100× (absorbancetissue)/( mean absorbancenegative control)

** relative absorbance per treatment group [rounded values]: 100× (mean absorbancetest item/positive control)/( mean absorbancenegative control)

The mean relative absorbance value of the test item, corresponding to the cell viability, was 108.4%. As the threshold for irritancy is a cell viability <= 50%, the test item was found to be not irritant to the skin.

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, butyl butyrate is not irritant to skin according to UN GHS and EU CLP regulation.
Executive summary:

This in vitro study was performed to assess the irritation potential of butyl butyrate by means of the Human Skin Model Test. The test was according to OECD 439 and GLP.

 

The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Also its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary.

 

Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative or the positive control for 60 minutes.

30 μL of the test item were applied to each tissue, and spread to match the surface of the tissue. 30 μL of either the negative control (DPBS) or the positive control (5% SLS) were applied to each tissue.

 

After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system.

 

After treatment with the test item butyl butyrate the mean relative absorbance value was 108.4% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%.
Therefore, the test item is not considered to
possess an irritant potential.

 

In conclusion, it can be stated that in this study and under the experimental conditions reported, butyl butyrate is not irritant to skin.

 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
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
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)
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).
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.
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.

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.

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation

For the skin irritation endpoint there is 1 study available.

 

In this in vitro key study the irritation potential of butyl butyrate was assessed by means of the Human Skin Model Test. The test was according to OECD 439 and GLP. The test item was used undiluted and the test was performed once in triplicate.

 

According to UN GHS (published 2003, last (7th) revision 2017) evaluation criteria, an irritation potential of the test item is observed if the mean relative tissue viability of three individual tissues is reduced to 50% of the negative control. After treatment with the test item butyl butyrate the mean relative absorbance value was 108.4% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of 50%.

 

Therefore, the test item should be classified as not skin irritating.

Eye Irritation

For the eye irritation endpoint there is 1 study available.

 

In this in vitro key study the eye irritation potential of the test item was assessed by the Human Cornea Model Test. The test was according to OECD 492 and GLP. The test item was used undiluted and the test was performed once in triplicate.

 

In the pre-tests, the test item did not reduce MTT and did not show interference with water or isopropanol.

 

50 μL of the test item, the negative control (deionised water) or the positive control (methyl acetate) was applied for 30 minutes.

 

The negative control was well within the required acceptability criterion showing the quality of the tissues, and the positive control induced a clear decrease ensuring the validity of the test system. 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%.

 

Therefore, the test item should be classified as not eye irritating.

Justification for classification or non-classification

Skin Irritation

In the in vitro key study with the Human Skin Model Test, the mean relative absorbance value was 108.4% compared to the relative absorbance value of the negative control, and based on UN GHS (published 2003, last (7th) revision 2017) criteria, the substance is classified as not irritating because this value is above the threshold for irritancy of 50%.

 

Eye Irritation

In the in vitro key study the relative mean absorption value corresponding to the viability of the tissues was 76.0%, which is above the cut off value of 60 %, indicating that the test item is not eye irritant.