Hexyl hexanoate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14th May 2018 - 18th May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted 29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test"

Version / remarks:

Official Journal of the European Union No. L142, 31 May 2008

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Hexyl hexanoate

EC Number:

228-952-4

EC Name:

Hexyl hexanoate

Cas Number:

6378-65-0

Molecular formula:

C12H24O2

IUPAC Name:

hexyl hexanoate

Test material form:

liquid

Specific details on test material used for the study:

Batch number 202462
Expiry date: 19 November 2019

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis

Vehicle:

unchanged (no vehicle)

Details on test system:

Test for the Interference of the Test Item with the MTT Endpoint
A test item may interfere with the MTT endpoint if it is colored and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed.

Test for Color Interference by the Test Item
Hexyl Caproate was checked for possible color interference before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the color interference, 50 µL of the test item or 50 µL Milli-Q water as a negative control were added to 0.3 mL Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple color change was observed.

Test for Reduction of MTT by the Test Item
Hexyl Caproate was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 50 µL of the test item or 50 µL Milli-Q water as a negative control were added to 1 mL MTT (Sigma, Zwijndrecht, The Netherlands) solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0ºC. At the end of the exposure time it was checked if a blue / purple color change or a blue / purple precipitate was observed.

Test System Set Up
Tissues
On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 mL DMEM.

Figure 1 - A Diagram of the Application (attached below)

DMEM (Dulbecco’s Modified Eagle’s Medium)
Supplemented DMEM, serum-free supplied by MatTek Corporation.
MTT medium
MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.
Environmental conditions
All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 43 - 84%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.3 - 37.2°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Test Item Preparation
No correction was made for the purity/composition of the test item.
The liquid test item was applied undiluted (50 µL) directly on top of the tissue.

Application/Treatment of the Test Item
The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 mL DMEM per well. The level of the DMEM was just beneath the tissue (see figure 1). The plates were incubated for approximately 2.5 hours at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM just before Hexyl Caproate was applied. The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to Hexyl Caproate and two for a 1-hour exposure. Fifty µL of the undiluted test item was added into the 6-well plates on top of the skin tissues.
For the negative and positive controls, 2 tissues were treated with 50 µL Milli-Q water (negative control) and 2 tissues were treated with 50 µL 8N KOH (positive control) for both the 3-minute and 1-hour time point.
 
After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 µL DMEM until 6 tissues (= one application time) were dosed and rinsed.

Cell Viability Measurement
The DMEM was replaced by 300 µL MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.


PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.]
- The test substance is considered to be non-corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.]
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439:

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The liquid test item was applied undiluted (50 µL) directly on top of the tissue.

Duration of treatment / exposure:

3 minutes for two tissues
1 hour for two other tissues

Duration of post-treatment incubation (if applicable):

The DMEM was replaced by 300 µL MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2.

Number of replicates:

2 replicates for each exposure time

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: tissue viability

Value:

94

Remarks on result:

other:

Remarks:

Basis: other: percentage of control. Time point: 3 minutes. Remarks: Negative control = 100%; Positive control = 14%. (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: tissue viability

Value:

112

Remarks on result:

other:

Remarks:

Basis: other: percentage of control. Time point: 1 hour. Remarks: Negative control = 100%; Positive control = 8%. (migrated information)

Other effects / acceptance of results:

Hexyl Caproate was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test item did not interfere with the MTT endpoint.
The mean absorption at 570 nm measured after treatment with Hexyl Caproate and controls are presented in Appendix 1, Table 1. The individual OD570 measurements are presented in Appendix 2.

Table 2 shows the mean tissue viability obtained after 3-minute and 1-hour treatments with Hexyl Caproate compared to the negative control tissues. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with Hexyl Caproate compared to the negative control tissues was 94% and 112% respectively. Because the mean relative tissue viability for Hexyl Caproate was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment Hexyl Caproate is considered to be not corrosive.

The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and the laboratory historical control data range (See Appendix 3). The mean relative tissue viability following the 1-hour exposure to the positive control was 7.6%.

In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was  20%, indicating that the test system functioned properly (Appendix 1, Table 3).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

An in vitro skin corrosion test was conducted according to OECD guideline 431 and GLP principles. It is concluded that this test is valid and that the test substance is not corrosive in the in vitro skin corrosion test.

Executive summary:

The objective of this study was to evaluate Hexyl Caproate for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of Hexyl Caproate was tested through topical application for 3 minutes and 1 hour.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch 202462 of Hexyl Caproate was a clear colourless liquid. Hexyl Caproate was applied undiluted (50 µL) was applied directly on top of the skin tissue. 

The positive control had a mean relative tissue viability of 7.6% after the 1-hour exposure. The absolute mean OD₅₇₀ (optical density at 570 nm) of the negative control tissues waswithin the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤20%,indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with Hexyl Caproate compared to the negative control tissues was 94% and 112%, respectively. Because the mean relative tissue viability for Hexyl Caproate was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment Hexyl Caproate is considered to be not corrosive.

In conclusion, Hexyl Caproate is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this study.