Octanoic acid, 2-phenoxyethyl ester

Endpoint summary

Description of key information

Skin corrosion in vitro (OECD 431): not corrosive

Skin irritation in vitro (OECD 439): not irritant

Eye irritation in vitro (OECD 437): not irritant

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 - 28 Nov 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

adopted 2004

Deviations:

yes

Remarks:

interference with MTT not determined

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

adopted 2016

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: EU Method B.40 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

adopted May 2008

Deviations:

not specified

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Source strain:

other: EpiDerm™; reconstructed three-dimensional human epidermis (EPI-200)

Vehicle:

unchanged (no vehicle)

Details on test system:

HUMAN SKIN MODEL TEST
- Model used: EpiDerm™ (Epi-200)
- Tissue batch number(s): 11208 kit H

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.0 ± 1.0 °C (actual range 35.9 - 37.2 °C)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Number of washing steps: 1

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 1 h
- Spectrophotometer: Multiskan Spectrum (Thermo Labsystems)
- Wavelength: 540 nm

REDUCTION OF MTT BY THE TEST SUBSTANCE
To assess the ability of the test substance to reduce MTT, approximately 100 µL of the test substance was added to a 24-well plate filled with 1 mL MTT medium. The mixture was incubated for approximately 1 h at room temperature in the dark. A negative control (sterile Milli-Q water) was tested concurrently.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
Negative control: 3 min treatment (range: 1.153 - 2.003; mean: 1.61; SD: 0.22; n = 36); 1 h treatment (range: 1.298 - 2.007; mean: 1.60; SD: 0.19; n = 36)
Positive control: 3 min treatment (range: 0.075 - 0.156; mean: 0.11; SD: 0.02; n = 36); 1 h treatment (range: 0.059 - 0.178; mean: 0.09; SD: 0.02; n = 36)

NUMBER OF REPLICATE TISSUES: duplicates per incubation time and for controls

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 min exposure is less than 50%, or if the viability after 3 min exposure is greater than or equal to 50% and the viability after 1 h exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 min exposure is greater than or equal to 50% and the viability after 1 h exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 8N

Duration of treatment / exposure:

3 min and 1 h

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min application with the test substance

Value:

93

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 h application with the test substance

Value:

113

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The relative mean tissue viability obtained after the 3-min and 1-h treatments with the test substance was 93% and 113%, respectively, compared with the negative control tissues.

In Table 1 the raw data are presented. Table 2 shows the mean tissue viability obtained after 3-min and 1-h treatments compared to the negative control tissues. Skin corrosion is expressed as the remaining cell viability after exposure to the test substance.

Table 1: Mean absorption at 540 nm

	A	B	Mean	±SD	A	B	Mean	± SD
Negative control (water)	1.613	1.815	1.714	0.143	1.558	1.443	1.501	0.082
Positive control (8 N KOH)	0.082	0.091	0.086	0.007	0.064	0.075	0.070	0.008
Test substance	1.619	1.556	1.587	0.045	1.758	1.697	1.697	0.087

Values are corrected for background absorption.

SD: standard deviation

A and B: duplicate cultures

Table 2: Mean tissue viability

	Viability [% of control]
	3 min	1 h
Negative control (water)	100	100
Positive control (8 N KOH)	5	5
Test substance	93	113

The absolute mean OD540 (optical density at 540 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following 3-min exposure to the positive control was 5%. The maximum inter-tissue variability in viability between two tissues treated identically was less than 15% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 8%. It was therefore concluded that the test system functioned properly.

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation(EC) No. 1272/2008

Conclusions:

Under the conditions of the RHE test method the test substance did not show corrosive properties.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 - 12 Jan 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted in 2015

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: L'Oréal Standard Operating Procedure, ECVAM Skin Irritation Validation Study, Validation of the Episkin Skin Irritation Test (42 h) assay for the prediction of acute skin irritation of chemicals

Version / remarks:

Jan 2005

Deviations:

not specified

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Source strain:

other: EpiSkin-Standard Model™

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiSkin-Standard Model
- Model used: EpiSkin-SM™
- Tissue batch number(s): 09-EKIN-001

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37.0 ± 1.0 °C (actual range: 36.5 – 37.4 °C)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Number of washing steps: 1

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Multiskan Spectrum (Thermo Labsystems)
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the relative mean tissue viability of three individual tissues after 15 min of exposure to the test substance and 42 h of post incubation is ≤ 50% of the mean viability of the negative controls.
- The test substance is considered to be non-irritant to skin if the relative mean tissue viaiblity of three individual tissues after 15 min of exposure to the test substance and 42 h of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 10 µL

NEGATIVE CONTROL
- Amount(s) applied: 10 µL

POSITIVE CONTROL
- Amount(s) applied: 10 µL
- Concentration: 5%

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

92

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The relative mean tissue viability obtained after 15 min treatment with the test substance compared to the negative control tissues was 92%. Since the mean relative tissue viability for the test substance was above 50% it is considered to be non-irritant.
The positive control had a mean cell viability after 15 min exposure of 3%. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was equal to or greater than 0.6. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly.

The mean absorption at 570 nm measured after treatment with the test substance and controls are presented in Table 1.

Table 2 shows the mean tissue viability obtained after 15 min treatment with the test substance compared with the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test substance.

Table 1: Mean absorption at 570 nm

	A	B	C	Mean	± SD
Negative control (PBS)	0.7690	0.7650	0.7680	0.7670	0.0020
Positive control (5% SDS)	0.0230	0.0310	0.0250	0.0260	0.0040
Test substance	0.6830	0.7160	0.7200	0.7060	0.0200

The values are corrected for background absorption.

PBS: Phosphate buffered saline

SDS: Sodium dodecyl sulphate

SD: Standard deviation

Table 2: Mean tissue viability

	Viability [% of control]
Negative control (PBS)	100
Positive control (5% SDS)	3
Test substance	92

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation(EC) No. 1272/2008

Conclusions:

Under the conditions of the RHE test method the test substance did not show irritant properties.

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:

11 Dec 2008

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:

adopted in 2013

Deviations:

yes

Remarks:

the study was conducted prior to the adoption of the test guideline, according to the draft version (dated 14 Aug 2008)

GLP compliance:

yes

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Slaughterhouse (Vitelco, 's-Hertogenbosch, the Netherlands)
- Storage, temperature and transport conditions of ocular tissue: Eyes were collected and transported in physiological saline in a suitable container

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 750 µL

CONTROLS
- Negative control: 750 µL
- Positive controls: 750 µL
- Concentration of positive control solution: Alkylbenzyldimethylammonium chloride 10% (w/v) in physiological saline and 99.9% ethanol

Duration of treatment / exposure:

10 ± 1 min

Duration of post- treatment incubation (in vitro):

120 ± 10 min

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Corneas were isolated and cultured in cMEM supplemented with 1% (v/v) foetal bovine serum and 1% (v/v) L-glutamine at 32 ± 1 °C for a minimum of 1 h prior to the start of the experiment. Opacity determinations were performed on each of the corneas using an opacitometer. Corneas that had an initial opacity reading higher than 3 were not used. Three corneas were selected at random for each treatment group.

QUALITY CHECK OF THE ISOLATED CORNEAS
Eyes were carefully examined for defects by holding the eyes submersed in physiological saline; those exhibiting unaccetable defects (opacity, scratches, pigmentation, neovascularisation) were discarded

TREATMENT METHOD
Each cornea was mounted in a cornea holder with the endothelial side against the O-ring of the posterior part of the holder. Afterwards, the anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. For equilibration, the corneas in the holder were incubated in at 32 ± 1 °C for at least one hour.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 3

METHODS FOR MEASURED ENDPOINTS
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM
In Vitro Irritancy Score (IVIS)
In vitro score range In vitro classification
0 - 3 Non irritant
3.1 - 25 Mild irritant
25.1 - 55 Moderate irritant
55.1 - 80 Severe irritant
> 80 Very severe irritant

DECISION CRITERIA
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

The positive control in vitro irritancy score should be reasonably within the laboratory historical positive control data range.
- The uncorrected negative control in vitro irritancy score is less than 3.1

Irritation parameter:

in vitro irritation score

Run / experiment:

mean out of 3 eyes

Value:

0.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

IVIS score of 157.9 for Alkylbenzyldimethylammonium chloride and 68.3 for ethanol, respectively

Irritation parameter:

other: Opacity

Run / experiment:

mean out of 3 eyes

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Mean opacity of 86 for Alkylbenzyldimethylammonium chloride and 24 for ethanol, respectively

Irritation parameter:

other: Permeability

Run / experiment:

mean out of 3 eyes

Value:

0.009

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Mean permeability of 4792 for Alkylbenzyldimethylammonium chloride and 2950 for ethanol, respectively

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The individual in vitro irritancy scores for the negative controls was 0 for all three corneas.
- Acceptance criteria met for positive control: The individual positive control in vitro irritancy scores ranged from 145 to 165 for benzalkonium chloride and ranged from 60 to 83 for ethanol and were within the range of historical control data. The corneas treated with the positive control substances were opaque after the 10 minutes of treatment. Thus, the test system functioned properly.
- Range of historical values if different from the ones specified in the test guideline:
- Negative control: Range = -1.0 - 2.2; Mean = 0.3; SD = 0.6; n = 41
- Positive control: Range = 113.8 - 202.7; Mean = 163.4; SD = 22.1; n = 42

Table 1: Individual results

	Eye No.	corrected final opacity	corrected final OD 450	in vitro irritancy score
				individual	mean
negative control	1	0	0.003	0	0
	2	0	0	0
	18	0	-0.002	0
positive control A	4	88	3.78	144.7	157.9
	5	89	5.07	165.1
	6	81	5.526	163.9
positive control B	16	26	3.816	83.2	68.3
	17	23	2.49	60.4
	3	23	2.544	61.2
test substance	13	0	0.006	0.1	0.1
	14	0	0.023	0.3
	15	0	-0.002	0

A Benzalkonium chloride

B Ethanol

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation(EC) No. 1272/2008

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation / corrosion

The skin corrosive potential of 2-phenoxyethyl octanoate was determined in an in vitro skin corrosion test using the EpiDerm™ model according to OECD guideline 431 (Notox, 2009). A concurrent negative and positive control was included. The skin corrosion is expressed as the cell viability after exposure to 2-phenoxyethyl octanoate. The relative mean tissue viability determined for the positive and negative control fell within the historical control data range and the respective controls were shown to be valid. The relative mean tissue viability obtained after 3 min and 1 h treatment with 2-phenoxyethyl octanoate compared with the negative control tissue was 93% and 113%, respectively. Under the conditions of this study, 2-phenoxyethyl octanoate is considered to be not corrosive to the skin.

The skin irritancy potential of 2-phenoxyethyl octanoate was determined in an in vitro skin corrosion test using the EpiSkin-Standard Model™ according to an ECVAM Skin Irritation Validation Study protocol. The protocol was published as OECD guideline 439 in 2015 (Notox, 2009). The skin irritation is expressed as the cell viability after exposure to 2-phenoxyethyl octanoate. A concurrent negative and positive control was included and fell within the historical control data range, and the respective controls were shown to be valid. The relative mean tissue viability obtained after 15 min exposure with 2-phenoxyethyl compared to the negative control tissue was 92%. Therefore, 2-phenoxyethyl octanoate is considered to be not irritant to the skin.

Eye irritation

The eye irritancy potential of 2-phenoxyethyl octanoate was determined in vitro using the Bovine Opacity and Permeability Test (BCOP test), according to the OECD guideline 437 draft version (Notox, 2009). The corneas treated with 2-phenoxyethyl octanoate showed opacity values of 0 and permeability values ranging from -0.002 to 0.023, the IVIS scores were 0.0-0.3. A negative and two positive controls were included; the IVIS scores for the controls fell within the cut-off values according to the current OECD guideline 437 and were considered to be valid. Based on the results of the BCOP test, 2-phenoxyethyl octanoate is considered to be not irritant to the eye.

Justification for classification or non-classification

The available data on skin and eye irritation and corrosion with 2-phenoxyethyl octanoate do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.