Hexyl hexanoate

Endpoint summary

Description of key information

Studies conducted to recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

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

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

Specific details on test material used for the study:

Batch number 202462
Expiry date: 19 November 2019

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

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)

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).

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.

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:

15-May-2018 to 15-May-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

09 October 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

09 December 2010

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

The test article, a clear colourless liquid, was identified as Hexyl Caproate and was received as follows:

Test Article
Hexyl Caproate

CAS Number
6378-65-0

Storage
Room temperature,

Batch Number
202462

Expiration Date
19 November 2019

Purity
99.29%, assumed 100% for testing

A certificate of analysis for the test article was provided by the Sponsor and is presented in the Attachments.

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test System and Study Design

Specification
Corneas from bovine eyes were obtained from a local abattoir. The eyes were removed after slaughter, completely immersed in physiological saline in a suitably sized container and transported on the same day to the testing facility.

Assessment on Arrival
On arrival at the test facility the eyes were carefully examined for defects including increased opacity, scratches and neovascularisation. Only corneas free from such defects were used.

Excision and Preparation of Corneas
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32± 1°C. The corneas were incubated for the minimum of 1 hour at 32± 1°C.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The negative control substance was physiological saline, 750 µL, supplied by Eurovet Animal Health, Bladel, The Netherlands
The positive control substance was ethanol, 750 µL
The test article was administered without dilution.

Duration of treatment / exposure:

120 ± 10 minutes at 32±1°C.

Duration of post- treatment incubation (in vitro):

Corneas were incubated in a horizontal position for 10±1 minutes at 32±1°C

Number of animals or in vitro replicates:

3

Details on study design:

Preparation of Corneas:
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.

The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

Cornea Selection and Opacity Reading:
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

Treatment of Corneas and Opacity Measurements:
The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.

The opacity value (measured with the device OP-KIT) was calculated according to:
Opacity=[(I0/I)-0.9894]/(0.0251)

With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea.
The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea.

The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

Irritation parameter:

cornea opacity score

Run / experiment:

Mean value

Value:

ca. -0.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

opacity reading of 1.8

Positive controls validity:

valid

Remarks:

opacity reading of 16

Remarks on result:

no indication of irritation

Irritation parameter:

other:

Run / experiment:

Corneal Permeability, mean value

Value:

ca. 0.002

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

The mean group corrected optical density for the negative control was -0.016

Positive controls validity:

valid

Remarks:

The mean group corrected optical density for the positive control was 1.625

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

RESULTS:
Hexyl Caproate was tested neat.

Table 1 of Appendix 1 summarizes the opacity, permeability and in vitro irritancy scores of Hexyl Caproate and the controls. The opacity, permeability and in vitro scores of the individual corneas are shown in Table 2 - 5. The individual in vitro irritancy scores for the negative controls ranged from 0.8 to 2.7. The individual positive control in vitro irritancy scores ranged from 31 to 48 (Appendix 2, Table 5). The corneas treated with the negative and positive control items were translucent and
turbid, respectively, after the 10 minutes of treatment. The corneas treated with Hexyl Caproate showed opacity values ranging from -1.2 to 1.5 and permeability values ranging from 0.002 to 0.003. The corneas were translucent after the 10 minutes of treatment with Hexyl Caproate. No pH effect of the test item was observed on
the rinsing medium. Hence, the in vitro irritancy scores ranged from -1.2 to 1.5 after 10 minutes of treatment with Hexyl Caproate.

DISCUSSION:
The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 40 and within two standard deviations of the current historical positive control mean (Appendix 3). It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Hexyl Caproate did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.0 after 10 minutes of treatment.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, since Hexyl Caproate induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of Hexyl Caproate as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test).

This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of Hexyl Caproate was tested through topical application for 10 minutes.

The study procedures described in this report were based on the most recent OECD guideline.

Batch 202462 of Hexyl Caproate was a clear colourless liquid with a purity of 99.29%. The test item was applied as it is (750 µL) directly on top of the corneas.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical rangeindicating that the negative control did not induce irritancy on the corneas. The meanin vitroirritancy score of the positive control (Ethanol) was 40 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Hexyl Caproate did notinduce ocular irritation through both endpoints, resulting in a meanin vitroirritancy score of 0.0 after 10 minutes of treatment. In conclusion, since Hexyl Caproate induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance did not meet the criteria for classification as a skin or eye irritant specified by the Classification, Labelling, and Packaging regulation (1272/2008).