Ethyl decanoate

Endpoint summary

Description of key information

Based on the available key studies performed to assess the potential irritation and corrosion effects of the registered substance, no effect was observed in the three in vitro studies. Hence, according to CLP criteria, ethyl decanoate was not classified for skin and eye irritation.

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:

From 24 November 2017 to 14 December 2017

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:

No analysis certificate of the episkin model was given.

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: From the sponsor, batch : 16090585
- Expiration date of the lot/batch: 19 september 2018
- Purity test date: 22 September 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions:at least 6 months from the manufacture date
- Solubility and stability of the test substance in the solvent/vehicle: not applicable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: not applicable, the test item was used pure
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

FORM AS APPLIED IN THE TEST (if different from that of starting material)

Test system:

artificial membrane barrier model

Remarks:

Reconstructed human epidermis

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not applicable

Source strain:

other: not applicable

Justification for test system used:

The present test is proposed as an alternative in vitro approach to assess the skin irritant potential of chemicals or cosmetic ingredient.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM
- Tissue batch number(s): lot 17-EKIN-047
- Production date: not specified
- Shipping date: not specified
- Delivery date: not specified
- Date of initiation of testing: 21 November 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C ± 1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: thoroughly rinsed with PBS
- Observable damage in the tissue due to washing: not specified
- Modifications to validated SOP:no modification

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/ml
- Incubation time: 3 hours
- Spectrophotometer: not specified
- Wavelength: 570nm
- Filter: not filter used
- Filter bandwidth: not applicable
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: yes
- Barrier function: yes
- Morphology: stratified differentiated epidermis derived from human keratinocytes
- Contamination: not specified
- Reproducibility:yes

NUMBER OF REPLICATE TISSUES: triplicates were used

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues (if applicable): Freezed-killed tissue
- N. of replicates : triplicates
- Method of calculation used: % viability = [OD mean of treated condition / OD mean of control condition] x 100

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2 independant tests :pretest with interference and MTT reduction test and main test

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 30 minutes exposure and 42 hours post incubation is less than 50%.
- The test substance is considered to be non-irritant to skin if the viability after 30 minutes exposure and 42 hours post incubation is greater than or equal to 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50µL/cm2 corresponding to 19 µL (0.38cm2)
- Concentration (if solution): pure

VEHICLE
Not applicable

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 19 µL
- Concentration (if solution): pure

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 19µL
- Concentration (if solution): 5%

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

triplicates were used

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main test - Negative control

Value:

100

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main test - Test item

Value:

100

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main test - Positive control

Value:

17

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: the 5% SDS was used as positive control and showed proficiency of the test system to detect cellular cytotoxicity

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: conform
- Acceptance criteria met for positive control: conform
- Acceptance criteria met for variability between replicate measurements: conform
- Range of historical values if different from the ones specified in the test guideline: Historical values of positive control : 14±4% ; Positive control values in the test : 17%. Conform

Table 1 :Results

Condtion	Optical Density Mean	Cellular viability
Negative control	1.522 ± 0.012	100
Positive control	0.257 ± 0.015	17
Test item	1.523 ± 0.016	100

 

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the result of this study, the test item when applied on the EpiSkin model for 15 minutes did not induce a decrease of cellular viability. Hence, according to the CLP criteria, the test item, caprate ethyl, was not classified for skin irritation.

Executive summary:

This GLP compliant study was performed in order to assess the potential skin irritation potential of the test item, caprate ethyl. This study was performed according to the OECD TG 439 method.

EpiSkinTM was used as test system in this study. 19 µL of caprate ethyl was applied on the EpiSkinTM for 15 minutes. Buffered saline solution (PBS) was used as negative control and 5% Sodium dodecyl sulfate (SDS) was used as positive control. Triplicates were used for each condition. The 15 minutes exposure period was followed by a 42 hours post incubation period. After this step, the viability of the EpiSkinTM model was measured by a MTT assay.

The postive control induceda decrease of cellular viability (17% of viability). The negative control did not induced a viability decrease. The viability of the test system was 100% when exposed to the test item.

The acceptance criteria of the assay was considered as validated.

Based on the result of this study, the test item whan applied on the EpiSkin model for 15 minutes did not induce a decrease of cellular viability. Hence, according to the CLP criteria, the test item, caprate ethyl, was not classified for skin irritation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 10 November 2017 to 6 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar 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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by the sponsor, batch no. 16090585
- Expiration date of the lot/batch: 19 September 2018
- Purity test date: 22 September 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions:at least 6 months from the manufacture date
- Solubility and stability of the test substance in the solvent/vehicle: not applicable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: not applicable
- Preliminary purification step (if any): not applicable
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Pure

Vehicle:

unchanged (no vehicle)

Controls:

yes

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30±2 µL (60 µL/cm2)
- Concentration (if solution): pure

VEHICLE
No vehicle was used

Duration of treatment / exposure:

30 ± 2 minutes

Duration of post- treatment incubation (in vitro):

30 ± 2 minutes

Number of animals or in vitro replicates:

Duplicates were used for each condition

Details on study design:

- Details of the test procedure used
Prior to the application of the test item, HCE tissues are transferred into 24-well plates filled with EPISKIN maintenance medium. 30µL (±2µL) of test material and/or controls are topically applied on the surface of HCE tissues (0.5cm2) Tissues treated with test item and with control substances tested concurrently to test item are incubated for 30 minutes (±2) at standard culture conditions.
After exposure to the test material to the test material, the treated tissues are rinsed with PBS and transferred into 24 well plates filled with maintenance medium. This rinsing step is followed by a 30 minutes(±2) post exposure immersion in maintenance medium at standard culture conditions, prior to performing the MT assay.
In order to evaluate the tissue viabilit, MTT assay was performed. The inserts are transferred to a 24 well plate containing 300 µL of MTT solution at 1mg/ml in maintenance medium. After 180 minutes, incubation in standard culture conditions, the precipitated blue formazan is then extracted from the tissues using isopropanol. Tissues tested with liquid test items are extracted from both the top and the bottom of the tissues, while tissues with colourd liquids are extracted from the bottom of the tissue only.

- RhCE tissue construct used, including batch number HCE SkinEthicTM ; HCE/S/5 lot 17-HCE-058

- Doses of test chemical and control substances used 30 µL of pure test chemical, positive and negative control were used.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
30 minutes exposure period at 37°C ; post exposure immersion 30 minutes at 37°C ; 180 minutes of MTT incubation period

- Justification for the use of a different negative control than ultrapure H2O (if applicable) not applicable. The PBS was required according to the guideline (OECD TG 49 2)

- Justification for the use of a different positive control than neat methyl acetate (if applicable) methyl acetate was used

- Description of any modifications to the test procedure not applicable

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable)
Direct MTT reduction controls : 30µL of the test material was added to 300 µL of 1 mg/mL MTT solution and the mixture is incubated for 3 hours at standard culture conditions.
Colour Interference : 10 µL of test material are mixed with 90 µL of distilled water. The solution is incubated for 30 minutes at room temperature

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable)
duplicates were used

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)
570 nm

- Description of the method used to quantify MTT formazan
The optical densities OD from individual replicate tissues are first corrected by substracting the blank mean OD value. The mean from corrected OD values (ODcorr) is calculated in each experimental group.
For normal condition, the percentage of tissue viability (%viab) is calculated from the mean ODcorr values relative to the mean viability of negative control (PBS treated epithelium)
%Viab [Treated] = ([Treated]ODcorr mean / [Negative Control]ODcorr mean) x100

- Description of the qualification of the HPLC/UPLC-spectrophotometry system (if applicable) not applicable

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
The eye irritant potential of the test item is predicted by the mean percent tissue viability (mean between 2 tissue replicates) normalised to the negative control which is set at 100%. The percentage tissue viability cut-off value distinguishing classified from non-classified test items according to UN-GHS is 60% (higher than 60% of viability : not classified ; lower than 60% of viability : no prediction can be made)

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria
not specified

- Complete supporting information for the specific RhCE tissue construct used
Only batch and RhCE tissue was given

- Reference to historical data of the RhCE tissue construct
not specified

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals
the methyl acetate showed positive responce and the capability of the test system to measure a decrease of cellular viability.

- Positive and negative control means and acceptance ranges based on historical data
The mean viability of tissues treated with the positive control (methyl acetate n=2) is =< 30%.
The mean OD of the negative control (NC n=2) is >= 1.4 with an upper acceptance limit of =<2.5

- Acceptable variability between tissue replicates for positive and negative controls
Conform
- Acceptable variability between tissue replicates for the test chemical
Conform

Irritation parameter:

in vitro irritation score

Remarks:

% tissue viability

Run / experiment:

Maint test - Negative Control

Value:

100

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Remarks:

% tissue viability

Run / experiment:

Main test - Positive Control

Value:

2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

in vitro irritation score

Remarks:

% tissue viability

Run / experiment:

Main test - Test Item

Value:

100

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: The methyl acetate was used as positive control and showed potency of the test system to measured tissue viability

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: conform
- Acceptance criteria met for positive control: conform
- Range of historical values if different from the ones specified in the test guideline: not applicable

Table 1 :Results

Condtion	Optical Density Mean	Cellular viability
Negativecontrol	1.759 ± 0.011	100
Positive control	0.041 ± 0.001	2
Test item	1.766 ± 0.017	100

 

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of this study, the test item ethyl caprate, did not induced decrease of tissue viability. Hence, according to the CLP criteria, the caprate was not classified for eye irritation.

Executive summary:

This GLP compliant in vitro study was performed in order to assess the potential eye irritation potential of the test item, ethyl caprate. This study was performed according to the OECD TG 492 method.

HCE SkinEthicTM model was used as test system in this study. 30 µL of ethyl caprate was applied on the EpiSkinTM for 30 minutes. Buffered saline solution (PBS) was used as negative control and methyl acetate was used as positive control. Duplicates were used for each condition. The 30 minutes exposure period was followed by a 30 minutes post incubation period. After this step, the viability of the HCE SkinEthicTM model was measured by a MTT assay.

The postive control induced a decrease of cellular viability (2% of viability). The negative control did not induced a viability decrease. The viability of the test system was 100% when exposed to the test item.

The acceptance criteria of the assay was considered as validated.

Based on the results of this study, the test item ethyl caprate, did not induced decrease of tissue viability. Hence, according to the CLP criteria, the caprate was not classified for eye irritation.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 14 September 2017 to 6 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

according to guideline

Guideline:

other: Official method for irritation evaluation by application on chorioallantoic membrane of the egg. Order of the 29 November 1996. Official diary of the French Republic (Journal Officiel de la Republique Française) of the 28 December 1996.

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
supplied by the sponsor, batch no. 16090585
- Expiration date of the lot/batch: 19 September 2018
- Purity test date: 22 September 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions:at least 6 months from the manufacture date
- Solubility and stability of the test substance in the solvent/vehicle: not applicable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: not applicable
- Preliminary purification step (if any): not applicable
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

FORM AS APPLIED IN THE TEST (if different from that of starting material)

Pure

Species:

chicken

Strain:

other: White Leghorn

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EGGS
- Source:Couvoir de Cerveloup, Vourey
- Number of animals: 12 eggs were used in this study (50-65g)
- Characteristics: Incubation time 10 days at 37.5°C and 45-60% relative humidity

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.3 mL
- Concentration (if solution): pure

VEHICLE
No vehicle was used

Duration of treatment / exposure:

20 seconds

Observation period (in vivo):

Observation of the chorioallantoic membrane was performed before application and after 5 minutes exposure period.

Number of animals or in vitro replicates:

Four eggs were used per condition except for the negative condition when 2 eggs were used

Details on study design:

SELECTION AND PREPARATION OF EGGS:
After 10 days of incubation, the egg shell is carefully scratched around the air space and then pared off. The inner egg membrane is carefully moistened with 0.9% NaCl solution and removed using tapered forceps to ensure accessibility to chorioallantoic membrane (CAM)

QUALITY CHECK OF THE ISOLATED CORNEAS
Eggs which were considered as defective are discarded from the study.

NUMBER OF REPLICATES
Four eggs were used per condition except for the negative condition when 2 eggs were used

NEGATIVE CONTROL USED
0.9% NaCl solution

POSITIVE CONTROL USED
3% SDS Sodium dodecyl Sulfate

APPLICATION DOSE AND EXPOSURE TIME
0.3mL of test chemical, negative or positive control was applied

TREATMENT METHOD: 0.3mL of the test item are added to the CAM and eliminated by irrigation with 5 mL of 0.9% saline solution after 20 seconds

POST-INCUBATION PERIOD: No

METHODS FOR MEASURED ENDPOINTS:
The choriallantoic membrane was observed through a magnifying glass before (to control the integrity) and over 5 minutes after application. The macroscopic examination consisted in monitoring appearance of the following phenomena:
-Hyperaemia : secondary vessel, non visible before test product addition, become visible due to increased blood flow. Visible secondary vessels expand and become redder. This phenomenon may occur in vessels with higher diameter.
-Haemorrhage : blood flows out the blood vessels. Various aspect mùay indicate haemorrhage: diffuse fog, "cauliflower" aspect, dotty aspect (blood flows out from different points of the membrane).
-Coagulation : Opacity and/or thrombosis (opacity : opalescent shadow on the membrane or direct opacity due to protein denaturation.) (Thrombosis: blood flow holds up)

SCORING SYSTEM: See table 1 below in "Any other information on materials and methods incl. tables" section

DECISION CRITERIA: See table 2 below in "Any other information on materials and methods incl. tables" section

Irritation parameter:

in vitro irritation score

Run / experiment:

Main test - Negative control

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

Main test - Positive control

Value:

12

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

Main test - Test item

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on the results of the study, the test item did not induced adverse effect when applied on the chorioallantoic membrane of eggs. According to the REACh regulation, no prediction can be made. Indeed, the HET-CAM assay can only classified a substance as Category 1 UN-GHS.

Executive summary:

The GLP compliant in vitro study was performed to assess the potential eye irritation potential of the caprate ethyl. This HET-CAM (Hen's Egg Test Chorioallantoic Membrane) was performed according to the natoinal standard Journal Officiel de la République Française of 29 November 1996.

White Leghorn eggs were used. They were incubated 10 days before preparation. When prepared, 0.3 mL of test item, positive or negative control was applied on the chorioallantoic membrane for 5 minutes. Evaluation was thereafter performed using magnifying glass for hyperaemia, haemorrhage and coagulation.

The test item did not induced adverse effect when applied on the chorioallantoic membrane of eggs. According to the REACh regulation, no prediction can be made. Indeed, the HET-CAM assay can only classified a substance as Category 1 UN-GHS.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

A GLP compliant study was performed in order to assess the potential skin irritation potential of the test item, ethyl decanoate (ethyl caprate).

This study was performed according to the OECD TG 439 method. EpiSkinTM was used as test system in this study. 19 µL of ethyl caprate was applied on the EpiSkinTM for 15 minutes. Buffered saline solution (PBS) was used as negative control and 5% Sodium dodecyl sulfate (SDS) was used as positive control. Triplicates were used for each condition. The 15 minutes exposure period was followed by a 42 hours post incubation period. After this step, the viability of the EpiSkinTM model was measured by a MTT assay.The postive control induceda decrease of cellular viability (17% of viability). The negative control did not induced a viability decrease. The viability of the test system was 100% when exposed to the test item. Hence the substance is considered as non irritant for the skin.

Two key studies are available to assess potential eye irritation of the test item:

- The first study was performed according to the OECD TG 492 method. HCE SkinEthicTM model was used as test system in this study. 30 µL of ethyl caprate was applied on the EpiSkinTM for 30 minutes. Buffered saline solution (PBS) was used as negative control and methyl acetate was used as positive control. Duplicates were used for each condition. The 30 minutes exposure period was followed by a 30 minutes post incubation period. After this step, the viability of the HCE SkinEthicTM model was measured by a MTT assay. The postive control induced a decrease of cellular viability (2% of viability). The negative control did not induced a viability decrease. The viability of the test system was 100% when exposed to the test item.

- As the second key study, a HET-CAM (Hen's Egg Test Chorioallantoic Membrane) was performed according to the natoinal standard Journal Officiel de la République Française of 29 November 1996. White Leghorn eggs were used. They were incubated 10 days before preparation. When prepared, 0.3 mL of test item, positive or negative control was applied on the chorioallantoic membrane for 5 minutes. Evaluation was thereafter performed using magnifying glass for hyperaemia, haemorrhage and coagulation. The test item did not induced adverse effect when applied on the chorioallantoic membrane of eggs.

Additionally, ethyl decanoate belongs to category groups ( Long chain fatty acids (C8 to C18) alcohol esters (ethanol, isopropanol, octanol, hexanol and 2-ethylhexanol)). The results from studies performed on ethyl decanoate are consitent with negative results from source substances studies. The very similar behavior for skin and eye effects reinforces the category approach.

Justification for classification or non-classification

Based on the available key studies performed to assess the potential irritation and corrosion effects of the registered substance, no effect was observed in the three in vitro studies. Hence, according to CLP criteria, ethyl decanoate was not classified for skin and eye irritation.