Ethyl enantate

Skin irritation / corrosion

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl enantate

EC Number:

203-382-9

EC Name:

Ethyl enantate

Cas Number:

106-30-9

Molecular formula:

C9H18O2

IUPAC Name:

ethyl heptanoate

In vitro test system

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

Results and discussion

In vitro

Results

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.