Ethyl hexanoate

Endpoint summary

Description of key information

In the key in vitro skin irritation study, the test item was found to be irritating to the skin in the in vitro human epidermis model assay (OECD 439).

In the key in vitro skin corrosion study, the test item was found to be non corrosive in the Human skin model test (OECD 431).

In the key in vivo eye irritation study, the test item was found to be not irritating to the eyes of rabbit (OECD 405).

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:

2015-06-12 to 2015-07-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

yes

Remarks:

without any impact on the outcome of the study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

without any impact on the outcome of the study

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SYSTEM
- Source: MatTek Corporation
- Cell culture: The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm Ø).
- Pre-incubation period: pre-incubation phase of the EpiDerm™ tissues started on the day of receipt.
- Treatment: EpiDerm tissues were treated with the test substance
- Amount/test concentration: 50 µL
- Duration of treatment: 3 minutes, 60 minutes
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure : room tempeature
- Temperature of post-treatment incubation (if applicable): 37+-1.5°C
CONTROL
- Negative Control: 50 µL deionised water was used as negative control per tissue;
- Positive Control: 50 µL 8.0 N potassium hydoxide (Sigma) was used a positive control per tissue
REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing (if done): Tissues washed with DPBS at least 20 times in order to remove any residual test material. Exess DPBS was removed by gently shaking the tissue inserts and blotting the lower surface with blotting paper.
SCORING SYSTEM:
Viability measured using MTT assay
mean tissue viability < 50% after 3 minutes exposure; corrosive: optional sub-category 1A
mean tissue viability = 50% after 3 minutes exposure AND < 15% after 60 minutes exposure; Corrosive: Optional Sub-category 1B and 1C
mean tissue viability = 50% after 3 minutes exposure AND = 15% after 60 minutes exposuref; Non-corrosive

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

undiluted

Duration of treatment / exposure:

3 minutes; 60 minutes

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

Duplicates

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

109

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes

Value:

108.6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: test item did not reduce MTT
- Colour interference with MTT:it did not change colour

Dose Group	Exposure Interval	Absorbance Well 1 (Tissue ½)	Absorbance Well 2 (Tissue ½)	Absorbance Well 3 (Tissue ½)	Mean Absorbance (Tissue ½)	Absorbance 570 nm Tissue 1*	Absorbance 570 nm Tissue 2*	Mean Absorbance of 2 Tissues	CV [%]	Rel Absorbance [% of Negative Control]**
Negative control	3 min	1.642 1.694	1.670 1.529	1.339 1.637	1.550 1.620	1.511	1.580	1.546	3.2	100.0
Positive control		0.442 0.381	0.422 0.421	0.395 0.352	0.420 0.384	0.380	0.345	0.363	6.9	23.5
Test item		1.772 1.651	1.744 1.659	1.875 1.644	1.797 1.651	1.757	1.612	1.685	6.1	109.0
Negative control	60 min	1.488 1.603	1.654 1.612	1.649 1.598	1.597 1.604	1.560	1.568	1.564	0.3	100.0
Positive control		0.203 0.184	0.207 0.181	0.185 0.201	0.198 0.189	0.162	0.152	0.157	4.3	10.0
Test item		1.800 1.631	1.775 1.692	1.799 1.714	1.791 1.679	1.754	1.642	1.698	4.7	108.6

* Mean of three replicates wells after blank correction

** relative absorbance [rounded values]: 100 * (absorbance test item / positive control) / (absorbance negative control)

Interpretation of results:

GHS criteria not met

Conclusions:

In the test study the skin corrosion potential of the test item was determined by means of the Human Skin Model Test (OECD 431). The relative absorbance value of the negative control the mean relative absorbance value
was 109.0% after 3 minutes exposure and 108.6% after 60 minutes exposure of the skin tissues to the test item. This value is above the threshold for corrosion of 50%. Therefore, the test item is not considered to possess corrosion potential.
Under the experimental conditions reported, the test substance is not corrosive to skin.

Executive summary:

This in vitro study was performed to assess the corrosive potential of the test substance by means of the Human Skin Model Test with EpiDerm™ tissues models. 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.

 

Independent duplicate tissues of EpiDerm™ were exposed to either the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively. The test item (50 µL) was dispensed directly onto duplicate EpiDermTM tissue surface, and spread to match the surface of the tissue. After exposure to the negative control the absorbance values met the required acceptability criterion of a mean OD570 =0.8 and =2.8 for both treatment intervals thereby confirming the acceptable quality of the tissues. Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period and for the 1 hour exposure period. The 1 hour exposure caused a decrease of the cell viability <15% of the negative control. The CV in the range 20 – 100% viability between the tissue replicates is =30%, thus the validity of the test system and the specific batch of the tissue models is confirmed. After exposure of the tissues to the test item the relative absorbance values were not reduced compared with the negative control value, neither after 3 minutes exposure, nor after 1 hour exposure (109.0% and 108.6%, respectively). Both values did not touch the threshold for corrosivity, which is defined to be <50% after the 3 minutes exposure and <15% after the 1 hour exposure. Therefore, the test item was not considered to be corrosive.

 

In conclusion, it can be stated that in this study and under the reported experimental conditions, the test item was non corrosive to skin according to EU CLP and UN GHS.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-01-22 to 2015-02-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

yes

Remarks:

deviations have no impact on the outcome of the study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

deviations have no impact on the outcome of the study

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

Details on test system
TEST SYSTEM
- Source: MatTek Corporation
- Cell culture: The EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture
inserts (MILLICELLs, 10 mm Ø).
- Pre-incubation period: pre-incubation phase of the EpiDerm™ tissues started on the day of receipt.
- Treatment: The test substance was added into the insert atop the corresponding EpiDerm™ triplicate tissues
- Amount/test concentration: 30µL
- Duration of treatment: 60 minutes
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 35 minutes at 37+-1.5°C and room temperature for 25 minutes
- Temperature of post-treatment incubation (if applicable): 37+-1°C
CONTROL
- Negative Control: 30 µL DPBS (Gibco) was used as negative control per tissue;
- Positive Control: 30 µL of a 5% SLS solution in deionised water (MatTek) was used a positive control per tissue, freshly prepared prior to the start of the experiment.
REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing (if done): Tissues washed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least three times
SCORING SYSTEM:
Viability measured using MTT assay
mean tissue viability < 50%; irritant (I), H315 (category 2)
mean tissue viability > 50%; non-irritant (NI)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Undiluted

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

24 hours

Number of replicates:

Triplicates

Irritation / corrosion parameter:

% tissue viability

Value:

30

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: mean relative absorbance

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: it did not reduce MTT
- Colour interference with MTT: it did not change colour

Dose Group	Exposure Interval	Absorbance Well 1 (Tissue ½)	Absorbance Well 2 (Tissue ½)	Absorbance Well 3 (Tissue ½)	Mean Absorbance (Tissue ½)	Absorbance 570 nm Tissue 1*	Absorbance 570 nm Tissue 2*	Mean Absorbance of 2 Tissues	CV [%]	Rel Absorbance [% of Negative Control]**
Negative control	60 min	1.791 1.760 1.762	1.787 1.751 1.764	1.778 1.733 1.665	1.785 1.748 1.730	1.746 1.709 1.691	1.715	101.8 99.6 98.6	1.6	100.0
Positive control		0.108 0.114 0.098	0.101 0.112 0.097	0.102 0.118 0.095	0.104 0.115 0.097	0.065 0.076 0.058	0.066	3.8 4.4 3.4	13.6	3.8
Test item		0.508 0.560 0.566	0.528 0.563 0.582	0.524 0.569 0.582	0.520 0.564 0.577	0.481 0.525 0.537	0.514	28.0 30.6 31.3	5.8	30.0

* Mean of three replicate wells after blank correction

** relative absorbance per tissue [rounded values]: 100 * (absorbance tissue) / (mean absorbance negative control)

*** relative absorbance per treatment group [rounded values]: 100 * (absorbance test item/positive control) / (mean absorbance negative control)

(mean absorbance )

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the experimental conditions reported, the test substance is 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 the test substance by means of the Human Skin Model Test. The test item did not prove to be a MTT reducer (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. Each 30 µL of the test item, the negative control (DPBS) or the positive control (5% SLS) were applied to triplicate tissue each.

 

After treatment with the negative control the absorbance values were well in the required range of the acceptability criterion of mean OD =0.8 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 the mean relative absorbance value decreased to 30.0% compared to the relative absorbance value of the negative control. This value is below the threshold for irritancy of =50%. Therefore, the test item is considered to possess an irritant potential.

 

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test substance is irritant to skin (Category 2).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999-09-06 to 1999-09-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

Feb. 1987

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

Jan. 1997

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

other: albino New Zealand White (SPF)

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Charles River, Deutschland GmbH, Stolzenseeweg 32-36, 88353 Kißlegg
- Weight at study initiation: 1.8 - 2.0 kg b.w.
- Housing: individual
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): free access to bottles with domestic quality drinking water acidified with hydrochloric acid to pH 2.5

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C ± 3°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): 10 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume): 0.1 mL
- Concentration (if solution): undiluted

Duration of treatment / exposure:

24 hours

Observation period (in vivo):

1, 24, 48, 72 hours

Number of animals or in vitro replicates:

4

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): rinsing with 20 mL 0.9% sodium chloride after Fluorescein was instilled
- Time after start of exposure: 24 hours

SCORING SYSTEM:
Cornea
Opacity degree of density (Area most dense was taken for reading): Scores 0 to 4, where 0 – No ulceration or opacity, and 4 – Complete corneal opacity, iris not discernable.
Area of cornea involved: Scores 1 to 4, where 1 – One quarter or less but not zero, and 4 – More than 3 quarters up to whole area.
Iris
Iris: Scores 0 to 2, where 0 – Normal, and 2 – No reaction to light, hemorrhage, gross destruction (any or all of these).
Conjunctiva
Redness (refers to palpebral and bulbar conjunctivae excluding cornea and iris): Scores 0 to 3, where 0 - Vessels normal, and 3 - Diffuse beef red.
Chemosis: Scores 0 to 4, where 0 - No swelling, and 4 - Swelling with lids more than half closed.
Discharge: Scores 0 to 3, where 0 - No discharge, and 3 - Discharge with moistening of the lids and hairs, and considerable area around the eye.

TOOL USED TO ASSESS SCORE: visual examination; with UV-light after instilling fluorescein and rinsing

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

other: mean of observations after 24, 48, 72 hours

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #1

Time point:

other: mean of observations after 24, 48, 72 hours

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

other: mean of observations after 24, 48, 72 hours

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

other: mean of observations after 24, 48, 72 hours

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

other: mean of observations after 24, 48, 72 hours

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #2

Time point:

other: mean of observations after 24, 48, 72 hours

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

other: mean of observations after 24, 48, 72 hours

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

other: mean of observations after 24, 48, 72 hours

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

other: mean of observations after 24, 48, 72 hours

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #3

Time point:

other: mean of observations after 24, 48, 72 hours

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

other: mean of observations after 24, 48, 72 hours

Score:

0.33

Max. score:

3

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

other: mean of observations after 24, 48, 72 hours

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #4

Time point:

other: mean of observations after 24, 48, 72 hours

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #4

Time point:

other: mean of observations after 24, 48, 72 hours

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

animal #4

Time point:

other: mean of observations after 24, 48, 72 hours

Score:

0.33

Max. score:

3

Irritation parameter:

chemosis score

Basis:

animal #4

Time point:

other: mean of observations after 24, 48, 72 hours

Score:

0

Max. score:

4

Interpretation of results:

GHS criteria not met

Conclusions:

The study reports mean scores for cornea opacity, iris lesion, redness of conjunctiva and oedema of conjunctiva (chemosis) as 0.0, 0.0, 0.2 and 0.0. Therefore from these results, the test substance is non-irritating to the eye.

Executive summary:

The eye irritant effect of the test substance was investigated according to the method recommended in the OECD Guideline No. 405, "Acute Eye lrritation/Corrosion", Feb. 1987, and EEC Guideline B.5 "Acute Toxicity (Eye Irritation)", Jan. 1997. Four female albino rabbits were exposed to 0.1 mL of the test article in the left eye. The eyes were examined and the changes were graded according to a numerical scale one hour, 24, 48 and 72 hours after dosing. Slight signs of irritation were observed on the treated eyes. The following mean values, based on the results from the 24, 48 and 72 hour readings, were calculated as 0.0, 0.0, 0.2 and 0.0 for cornea opacity, iris lesion, redness of conjunctiva and oedema of conjunctiva, respectively. According to the Regulation (EC) No 1272/2008 of 16 December 2008, the test substance does not meet CLP classification for eye irritation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In the supporting study the skin irritation potential of the test item was determined within an acute dermal toxicity study in 10 rabbits, showing moderate redness (10 of 10 animals), slight edema (2 of 10 animals) and moderate edema (7 of 10 animals).

Based on the available information this study was selected as supporting study as it is insufficiently documented and therefore less reliable. Data cannot be used for classification.

Justification for classification or non-classification

Based on the available data, the test substance is classified for skin irritation as Category 2, 'Causes skin irritation', H315, and does not meet CLP classification for eye irritation.