Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2017

Materials and methods

Test guideline
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 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

In vitro test system

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

Results and discussion

In vitro

Results
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.

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 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.

Applicant's summary and conclusion

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.