Ethyl octanoate

Endpoint summary

Description of key information

The test item is not irritant to the skin or to the eye.

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:

09.06.2016 - 20.06.2016

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)

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm TM tissues (Epi-200-SIT Kit)

Justification for test system used:

Dermal irritation is generally defined as "the production of reversible inflammatory changes in the skin". The potential for chemical induced skin irritation is usually determined in vivo in the Draize rabbit skin irritation test as described in OECD guideline 404. However, because systemic reactions play a minor role in modulating local skin toxicity potential of chemicals, skin irritation potential may be predicted by in vitro systems, provided they are sufficiently complex to mimic human skin barrier and cell reactivity. In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDermTM and EpiSkinTM and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200-SIT Kit
- Tissue batch number(s): 23341
- Delivery date: 14.06.2016
- Date of initiation of testing: 14.06.2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 35 minutes at 37 °C and 25 minutes at room temperature
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL
- Volume and number of washing steps: at least 15 times

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 42 hours
- Spectrophotometer: Versamax® Molecular Devices, Softmax Pro, version 4.7.1
- Filter: 570 nm

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The mean OD of 3 replicates is determined taking into account the blank control. The relative viability (%) is calculated as [(mean OD test item/positive control) / mean OD negative control] x 100. A substance is considered skin irritant category 2 according to UN GHS (published 2003, last (6th) revision 2015) if the mean relative tissue viability of three individual tissues is reduced ≤ 50% of the negative control.

ACCEPTANCE CRITERIA
- Negative control: The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD570 of the negative control tissues is ≥ 0.8 and ≤ 2.8.
- Positive control: An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is ≤ 20%.
- Standard deviation: The rel. SD of 3 identical replicates should be < 18%. OD values should not be below historically established boundaries.
- Historical data and the quality certificate of the supplier of the test kit demonstrated the robustness of the test system / test kit.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): undiluted

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5%

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

42.75 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of 3 replicates

Value:

99.9

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.
The acceptance criteria were met.

Results

 

Dose Group	Tissue No.	Absorbance 570 nm Well 1	Absorbance 570 nm Well 2	Absorbance 570 nm Well 3	Mean Absorbance of 3 Wells	Mean Absorbance of 3 wells blank corrected	Mean Absorbance of 3 tissues after blank correction *	Rel. Absorbance [%] Tissue 1, 2 + 3**	Relative Standard Deviation [%]	Mean Rel. Absorbance [% of Negative Control]***
Blank		0.037	0.038	0.038	0.038	0.000
Negative Control	1	1.870	1.865	1.837	1.857	1.820	1.889	96.3	3.3	100.0
	2	1.954	1.947	1.944	1.948	1.911		101.1
	3	1.997	1.966	1.963	1.975	1.938		102.6
Positive Control	1	0.123	0.127	0.127	0.126	0.088	0.086	4.7	6.5	4.5
	2	0.131	0.126	0.126	0.128	0.090		4.8
	3	0.118	0.117	0.117	0.117	0.079		4.2
Test Item	1	1.977	1.972	1.964	1.971	1.933	1.887	102.3	2.8	99.9
	2	1.873	1.853	1.871	1.866	1.828		96.8
	3	1.930	1.939	1.942	1.937	1.899		100.5

* Mean of three replicate wells after blank correction

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

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

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is not irritant to the skin.

Executive summary:

In this in vitro study the irritation potential of the test item was assessed by means of the Human Skin Model Test. The study was according to OECD 439 and GLP.

The test item did not reduce MTT (test for direct MTT reduction), and 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.

The main study consists of topical exposure of the test item to the human reconstructed epidermis model followed by a cell viability test. The cell viability is measured by dehydrogenase conversion of MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide], in cell mitochondria, into a blue formazan salt that is quantitatively measured (as optical density (OD)) after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict skin irritation potential and used for the purpose of classification as irritating or non-irritating. The test chemical is considered to be irritant to skin in accordance with UN GHS and EU CLP Category 2 if the tissue viability after exposure and post-treatment incubation is ≤ 50%.

Three tissues of the human skin model EpiDermTM were treated with the test item, the negative (DPBS) or the positive (5% SLS) control for 60 minutes.

Hereafter the skin tissues are washed and further incubated for about 42.75 hours, whereafter the tissues were treated with MTT for 3 hours following about 69.5 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD≥0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus showing the quality of the tissues.

Treatment with the positive control induced a sufficient decrease to 4.5% in the relative absorbance compared to the negative control for the 60 minutes treatement interval, thus ensuring the validity of the test system.

The relative standard deviations between the % variability values of the test item, the positive and negative controls in the main test were below 7% (threshold of the OECD TG: <18%), thus ensuring the validity of the study.

After treatment with the test item the mean relative absorbance value decreased to 99.9% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50% and therefore, the test item is not to be considered to possess irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is not irritant to the skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

11.09.2000 - 15.09.2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

other: SPF albino rabbits of the stock Chbb:HM(SPF) - Littlerussian

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 2.3 - 2.8 kg b.w.
- Housing: During a pre-period of at least one week and throughout the experiment the rabbits were caged individually in PPO cages (floor area: 2576 sq.cm) with perforated floor.
- Diet: pelleted complete rabbit diet "Altromin 2123", ad libitum
- Water: ad libitum, domestic quality drinking water acidified with hydrochloric acid to pH 2.5 to prevent microbial growth

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C ± 3°C
- Humidity (%): 55% + 15%
- Air changes (per hr): 10 times/hour
- Photoperiod (hrs dark / hrs light): Light was on from 6 a.m. to 6 p.m..

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

- Amount(s) applied (volume or weight with unit): 0.1 mL
- Concentration (if solution): undiluted

Duration of treatment / exposure:

24 hours

Observation period (in vivo):

1, 24, 48, and 72 hours

Number of animals or in vitro replicates:

4

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing : 20 ml 0.9% sodium chloride solution
- Time after start of exposure: 24 hours

SCORING SYSTEM: See 'Any other information on materials and methods'

TOOL USED TO ASSESS SCORE: fluorescein

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

other: 24, 48, 72 hours

Score:

0

Reversibility:

other: not applicable

Irritation parameter:

iris score

Basis:

mean

Time point:

other: 24, 48, 72 hours

Score:

0

Reversibility:

other: not applicable

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

other: 24, 48, 72 hours

Score:

0.3

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: 24, 48, 72 hours

Score:

0.3

Reversibility:

fully reversible within: 48 hours

Irritant / corrosive response data:

One hour after application of the test article animal No. 1884 showed a diffuse, crimson red conjunctiva with individual vessels not easily discernible, a swelling above normal and a discharge different from normal. In animal No. 1885 a diffuse, crimson red conjunctiva with individual vessels not easily discernible were observed as well as a swelling above normal. Animal No. 1886 showed some conjunctival vessels definitely injected, a swelling above normal and a discharge different from normal. In animal No. 1887 some conjunctival vessels definitely injected and a swelling above normal were observed.
24 hours after application of the test article animals No. 1884, No. 1885, No. 1886 and No. 1887 showed some conjunctival vessels definitely injected and a swelling above normal.
48 and 72 hours after application of the test article all four animals were free of any signs of eye irritation.

ASSESSMENT
According to the CLP Regulation (EC) No 1272/2008 the substance is considered to have irreversible effects on the eye (category 1) if, when applied to the eye of an animal, a substance produces:
1. at least in one animal effects on the cornea, iris or conjunctiva that are not expected to reverse or have not fully reversed within an observation period of normally 21 days; and/ or
2. at least in 2 of 3 tested animals, a positive response of: corneal opacity >= 3 and/or iritis > 1.5
calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material.

The substance is considered to be irritant to the eyes (category 2) if, when applied to the eye of an animal, a substance produces:
1. at least in 2 of 3 animals, a positive response of: corneal opacity >= 1 and/or iritis >= 1 and/or conjunctival redness >= 2 and/or conjunctival oedema (chemosis) >= 2
2. calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material, and which fully reverses within an observation period of 21 days.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is not eye irritating.

Executive summary:

In the current study the potential eye irritant effect of the test item was assessed according to the OECD 405 and EEC Guideline B.5 and in compliance to GLP. The day before testing both eyes of the animals were examined to ensure there were no defects or irritation.

In this test only one eye was treated while the other eye remains untreated and serves as control.

0.1 ml of the test article was placed in one eye of the rabbits by gently pulling the lower lid away from the eyeball to form a cup into which the test article was dropped. The lids were then gently held together for 1 second.

The eyes were examined and the grade of ocular reaction was recorded one hour and 24 hours later. After the first 24 hour reading Fluorescein was instilled. After rinsing the eyes were examined again using UV-light to detect possible corneal damage. The eyes were also examined 48 and 72 hours after the treatment.

The study was initiated with one rabbit. Since no marked eye irritation was seen in this animal another three rabbits were included in the study.

Therefore, in total 4 female albino rabbits were exposed to the test article. The eyes were examined and the changes were graded according to a numerical scale one hour, 24, 48 and 72 hours after dosing.

No or only 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: Cornea opacity 0.0; Iris lesion 0.0; Redness of conjunctiva 0.3 and Oedema of conjunctiva 0.3. All findings were reversible within 48h.

Based on these results, the test item shall not be classified as eye irritating.

According to the CLP regulation the test item shall not be classified as eye irritating

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

For this endpoint there are three studies available with relevant information to assess the skin irritant potential of the test item.

The first study is an in vitro key study according to OECD 439 using the Human Skin Model Test EpiDerm. The test item did not reduce MTT, change colour or had intensive intrinsic colour and so additional tests with freeze-killed or viable tissues were not necessary.

The human reconstructed epidermis model was exposed to the test item, the negative (DPBS) or the positive (5% SLS) control for 60 minutes. After washing and further incubation the tissues were treated with MTT and the colorant is extracted from the cells and photometrically measured at 570 nm.

The percent reduction of cell viability is calculated and compared to the negative control. The test chemical is considered to be irritant to skin Category 2 if the tissue viability is ≤ 50%.

The negative control absorbance values were well within the required acceptability criterion showing the quality of the tissues and the positive control induced a sufficient decrease in the relative absorbance ensuring the validity of the test system. The relative standard deviations were below the threshold of the OECD TG ensuring the validity of the study.

The test item had a mean relative absorbance value of 99.9% which is above the threshold for irritancy of ≤ 50% and therefore, the test item is not to be considered as skin irritant.

The other information that is available was obtained from two supporting studies performed on human volunteers. These studies are included in section 7.10.5 of IUCLID. In these studies the skin irritant potential of the test item was evaluated in human patch tests, performed as a pre-test of a human maximisation test. In these two studies either 5 or 25 healthy adult volunteers completed the experiment. A patch of ethyl octanoate at 2% in petrolatum was applied to normal sites on the volar forearms or backs for 48 hours under occlusion. The test item did not produce any skin irritant reactions and is considered not irritant to the skin.

The supporting human data confirms the in vitro test and in conclusion the test item is considered not irritant to the skin.

Eye irritation

For the eye irritation endpoint one in vivo study is available in which the potential of the test item to induce eye irritant effects in rabbits was assessed according to OECD TG 405 and in compliance to GLP. One eye was treated with the test item while the other eye remained untreated and served as control. The study was initiated with one rabbit and as no marked eye irritation was seen in this animal another three rabbits were included in the study. The eyes were examined and the changes were graded according to a numerical scale one hour, 24, 48 and 72 hours after dosing.

No or only 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: Cornea opacity 0.0; Iris lesion 0.0; Redness of conjunctiva 0.3 and oedema of conjunctiva 0.3. All effects were reversible within 48h.

Based on these results, the test item is not considered to be eye irritating.

Justification for classification or non-classification

Skin irritation

The criteria for skin corrosion and irritation are set out in the CLP Regulation (EC) No 1272/2008 in section 3.2. As indicated in paragraph 3.2.1.2. the results of a validated in vitro test and relevant human data can be used for classification purposes. In an in vitro study according to OECD 439 the substance was found to be not irritant to the skin and data available from human volunteer studies confirm this observation. Therefore, the test item should not be classified as skin irritant.

Eye irritation

In an in vivo test according to OECD 405 the test item was assessed for its eye irritant potential. Based on the results, it can be concluded that the test item shall not be classified as eye irritating.