Ethyl enantate

Endpoint summary

Description of key information

The test item is not to be considered eye or skin irritant.

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:

10.11.2015 - 07.12.2015

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:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

other: keratinocytes

Cell source:

other: not specified

Source strain:

other: not applicable

Justification for test system used:

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: EpiDermTM tissues
- Tissue batch number(s): 23305 Kit A
- Shipping date: 01 December 2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C
- Temperature of post-treatment incubation (if applicable): room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 15

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Versamax® Molecular Devices, Softmax Pro, version 4.7.1
- Filter: 570 ± 1 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Positive control: 4.77 %
- Reproducibility: Data of 13 studies performed from July 2015 until end of October 2015

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT EXPERIMENTS TO DERIVE FINAL PREDICTION: 2
The experiment was performed twice, because in a first experiment the validity criteria of the assay were not met. The results of the first experiment are not used for evaluation and are not reported in this study.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the mean tissue viability is =< 50%
- The test substance is considered to be non-irritant to skin if the mean tissue viability > 50%

EVALUATION OF RESUTLS
- 100% tissue viability current test: Mean OD of 3 negative control tissues was calculated after blank correction.
- Viability test item or positive control: individual relative tissue viability is calculated according to: Relative viability (%) = [mean OD test item / mean OD positive control] * 100 - Mean viability test item or positive control: mean relative viability ± rel. standard deviation of 3 individual tissues is calculated

ACCEPTABILITY OF ASSAY
- Negative control: 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 SD of 3 identical replicates should be < 18%. OD values should not be below historically established boundaries.

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): 47 µL/cm2 of undiluted test item

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL

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

Duration of treatment / exposure:

60 minutes of which 35 minutes at 37 ± 1.5 °C

Duration of post-treatment incubation (if applicable):

after removal of the test item: 24 hours at 37 ± 1.5 °C

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

102.3

Negative controls validity:

valid

Remarks:

Mean Relative Absorbance: 100%

Positive controls validity:

valid

Remarks:

Mean Relative Absorbance: 4.9%

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 lead 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.

Details results:

Dose Group	Absorbance 570 nm Tissue 1*	Absorbance 570 nm Tissue 2*	Absorbance 570 nm Tissue 3*	Mean Absor- bance of 3 Tissues	Rel. Absor- bance [%] Tissue 1, 2 + 3**	Relative Standard Deviation [%]	Mean Rel. Absorbance [% of Negative Control]***
Negative control	1.705	1.552	1.747	1.668	102.2 / 93.1 / 104.7	6.1	100.0
Positive control	0.075	0.083	0.085	0.081	4.5 / 5.0 / 5.1	6.4	4.9
Test item	1.679	1.893	1.547	1.706	100.7 / 113.5 / 92.7	10.3	102.3

 

*Mean of 3 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 * mean absorbance tissue test item or positive control / mean absorbance negative control

Interpretation of results:

GHS criteria not met

Conclusions:

The mean relative absorbance value of the test item was 102.3%. The threshold for irritancy is ≤ 50% and therefore the test item is not considered to possess an irritant potential.

Executive summary:

In the current in vitro study the irritation potential of the test item was assessed in a Human Skin Model Test according to OECD TG 439 and GLP.

The experiment was performed twice, because in a first experiment the validity criteria of the assay were not met. The results of the first experiment are not used for evaluation and are not reported in this study.

In the pre-test phase the test item passed the MTT- and the Colour Interference tests.

In the main test 30 μL of the test item was applied to the tissue and spread to match the surface. The same volume was used for the negative control (DPBS) and the positive control (5% SLS). All were tested in triplicate. The treatment lasted for 60 minutes, whereafter the test item or control was washed off extensively. Further incubation of the tissues occured with MTT whereafter the amount of extracted colorant was determined photometrically at 570 nm.

The negative control absorbance values were well within the required acceptability criterion of mean OD≥0.8 and ≤ 2.8, showing the quality of the tissues. Treatment with the positive control induced a decrease in the relative absorbance to 4.9% ensuring the validity of the test system. The relative standard deviations between the % variabilities of the test item, the positive and negative controls in the main test were below 11.0% which is below the threshold of the OECD TG 439 and therefore the study is valid.

Compared to the relative absorbance value of the negative control the mean relative absorbance value of the test item was 102.3% after exposure of the skin tissues to the test item. The threshold for irritancy is ≤ 50% and therefore the test item is not considered to possess an irritant potential.

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:

30.08 - 03.09.1999

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 2.3 - 2.4 kg bw.
- Housing: individually in PPO cages (floor arca: 2576 cm2) with perforated floor.
- Diet: ad libitum; pelleted complete rabbit diet "Altromin 2123" from Altromin, D-32791 Lage, Lippe
- Water: ad libitum; domestic quality drinking water acidified with hydrochloric acid to pH 2.5 (to prevent microbial growth)
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 3
- Humidity (%): 55 +/- 15
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): cycle of 12 hours light and 12 hours darlaiess. Light was on from 06 to 18 h.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

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

Duration of treatment / exposure:

single administration, no rinsing

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 (if done): no

EXAMINATION
- after 1, 24, 48 and 72 hours
- after the first 24h reading, Fluorescein was instilled. The eye was subsequently rinsed with 20 ml 0.9% sodium chloride solution, and examined again using UV-light to detect possible corneal damage.

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:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

0.33

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Irritant / corrosive response data:

One hour alter application of the test article all animals showed some conjunctival vessels definitely injected.
After 24 hours some conjunctival vessels definitely injected were observed in animals No.1590, No.1591 and No. 1592. Animal No. 1589 was free of any signs of eye irritation. After 48 hours only animal No. 1592 showed some conjunctival vessels definitely injected. Animal No.1589, No.1590 and. No. 1591 were free of any signs of eye irritation. After 72 hours animal No. 1592 was free of any signs of eye irritation.

Individual scores

Animal/weight per kg	Cornea	Iris	Conjuctival redness	Chemosis
1589 / 2.3	0.0	0.0	0.0	0.0
1590 / 2.4	0.0	0.0	0.33	0.0
1591 / 2.4	0.0	0.0	0.33	0.0
1592 / 2.4	0.0	0.0	0.67	0.0
Mean	0.0	0.0	0.33	0.0

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is not eye irritant.

Executive summary:

In the current study the eye irritant effect of the test item was investigated according to the method recommended in the OECD Guideline 405 and the EEC Guideline B.5.

Four female albino rabbits were exposed to 0.1 ml of the test item in the left eye. The right eye remained untreated and served as control.

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 with 20 ml 0.9% sodium chloride solution 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.

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:

Animal	Cornea	Iris	Conjuctival redness	Chemosis
1	0.0	0.0	0.0	0.0
2	0.0	0.0	0.33	0.0
3	0.0	0.0	0.33	0.0
4	0.0	0.0	0.67	0.0
Mean	0.0	0.0	0.33	0.0

According to the CLP regulation the test item should not be classified as an eye irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion

There is one recent and well documented in vitro study available assessing the irritation potential of the test item in a Human Skin Model Test according to OECD TG 439 and GLP. The negative control absorbance values were well within the acceptability criterion, showing the quality of the tissues. The positive control induced a decrease in the relative absorbance to 4.9% ensuring the validity of the test system. The relative standard deviations were below the treshold, therefore the study was valid. Compared to the relative absorbance value of the negative control the mean relative absorbance value of the test item was 102.3%. Since the threshold for irritancy is ≤ 50% the test item is not considered to possess an irritant potential.

Eye irritation

For eye irritation, one well documented study is available in which the eye irritation and corrosion effects of the test item were assessed according to OECD Guideline 405 and the EEC Guideline B.5.

Four female albino rabbits were exposed to 0.1 ml of the test article in the left eye, while the other eye served as control. 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, however, the effects were fully reversible. The mean values, based on the results from the 24, 48 and 72 hour readings, were 0.00 for the Cornea, 0.00 for the Iris, 0.33 for Conjuctival redness and 0.00 for Chemosis. Hence, the substance was concluded to be not irritating to the eye.

Justification for classification or non-classification

Skin irritation/corrosion

For results coming from in vitro studies the threshold value for irritancy is ≤ 50%. The relative absorbance value of the test item was 102.3% and thus the test item is not considered to possess skin irritant potential.

Eye irritation

The CLP regulation states that a substance has irreversible effects on the eye (Category 1) if, when applied to the eye of an animal, a substance produces:

- 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

- 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 irritating to eyes (Category 2) if, when applied to the eye of an animal, a substance produces:

- at least in 2 of 3 tested animals, a positive response of: corneal opacity >= 1 and/or
 iritis >= 1, and/or
 conjunctival redness >= 2 and/or 
conjunctival oedema (chemosis) >= 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.

According to these criteria of the CLP regulation the test item should not be classified as a skin or eye irritant.