ethyl tetrahydrofuran-2-carboxylate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-02-20 to 2014-02-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study Deviations to the OECD 431 (2014) guideline without significant effects on the results: - test for MTT reduction was conducted at room temperature - 3 minute exposure time was not conducted at room temperature

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

adopted 2013-07-26

Deviations:

yes

Remarks:

please refer to the field "Rationale for reliability incl. deficiencies" above

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2013-04-11

Test material

Test material information

Constituent 1

Reference substance name:

ethyl tetrahydrofuran-2-carboxylate

EC Number:

807-745-8

Cas Number:

16874-34-3

Molecular formula:

C7H12O3

IUPAC Name:

ethyl tetrahydrofuran-2-carboxylate

Test material form:

other: liquid

Details on test material:

- Physical state: liquid, clear, colourless
- Storage condition of test material: at room temperature

Test animals

Details on test animals or test system and environmental conditions:

Not applicable - Since this is an in vitro study there is no information on test animals.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: Control (50µL dionised water) skin cultures were used.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μL of the undiluted test item

Duration of treatment / exposure:

3 minutes and 1 hour

Observation period:

not applicable

Number of animals:

Number of skin tissue replicates per dose:
Test item: duplicates
Negative control: duplicates
Positive control: duplicates

Details on study design:

CELL CULTURE:
- Epi-200 kits (Lot no.: 19620) and MTT-100 assays were purchased from MatTek Corporation (Bratislava, Slovakia).
- EpiDerm™ tissue (surface 0.6 cm²) consisted of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consisted of organised basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.

TEST FOR DIRECT MTT REDUCTION:
- approximately 50 μL of the test item were added to 1 mL of MTT solution and the mixture was incubated in the dark at room temperature for 60 minutes.
- untreated MTT medium was used as control.
- if the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.

TREATMENT:
- at least 1 hour before dosing, EpiDerm™ tissues were transferred under sterile conditions into 6-well plates containing pre-warmed assay medium.
- after the pre-incubation of the EpiDerm™ tissues was completed the medium in each well was replaced by 0.9 mL fresh assay medium.
- duplicates of EpiDerm™ tissues were exposed to the test item, positive control (8.0 N potassium hydroxide) or negative control (50 μL deionised water) for each of two different exposure periods: 3 minutes and 1 hour.
- plates containing the treated tissues were placed into an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2).
- at the end of the exposure period the tissues were removed from the plate and rinsed with DPBS to remove any residual test material.

MTT ASSAY:
- MTT concentrate was diluted with the MTT diluents (ratio 1:4)
- 300 µL MTT solution was added to each well and the cell culture inserts were transferred to the MTT-plates.
- after a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) the MTT solution was aspirated from the wells and the wells were rinsed with DPBS.
- inserts were transferred into new plates and they were immersed in extractant solution (isopropanol).
- formazan salt was extracted for approximately 21 hours.
- after the extraction period the inserts were pierced to allow the extract to run into the well from which the insert was taken, and the insert was discarded.
- 3 × 200 μL aliquots of the blue formazan solution were transferred from each tissue into a 96-well flat bottom microtiter plate.
- optical density (OD) was determined in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany) at 570 nm (OD570) without reference filter.
- mean values were calculated for each set of 3 wells per tissue insert.

INTERPRETATION OF RESULTS:
- mean OD value obtained for the duplicate tissues per test item were used to calculate the percent viability relative to the negative control, which was arbitrarily set at 100%.
- test item is considered to be corrosive to skin (EU CLP Cat.1):
(1) if the viability after 3 minutes exposure is less than 50%, or
(2) if the viability after 3 minutes exposure is greater or equal than 50% and the viability after 1 hour exposure is less than 15%.
- test item is considered to be non-corrosive to skin:
(1) if the viability after 3 minutes exposure is greater or equal than 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: relative viability(%)

Value:

77.4

Remarks on result:

other:

Remarks:

Basis: mean. Time point: after 3 minutes incubation. Reversibility: no data. (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: relative viability(%)

Value:

5.1

Remarks on result:

other:

Remarks:

Basis: mean. Time point: after 60 minutes incubation. Reversibility: no data. (migrated information)

In vivo

Irritant / corrosive response data:

After exposure to the test item the relative absorbance value decreased to 77.4% after 3 minutes exposure (threshold for corrosivity: 50%). But after the 1 hour exposure period the relative absorbance value was reduced to 5.1 (threshold for corrosivity: 15%). Therefore, the test item was considered to be corrosive.

Any other information on results incl. tables

HISTORICAL DATA

Table 1: 3 minutes treatment

Positive Control		Negative Control
Mean viability	22.04%	Mean optical density	1.653
Standard deviation	6.58%	Standard deviation	0.305
Range of viabilities	0.00% – 33.07%	Range of optical densities	0.987 – 2.740

Table 2: 60 minutes treatment

Positive Control		Negative Control
Mean viability	9.96%	Mean optical density	1.664
Standard deviation	3.82%	Standard deviation	0.292
Range of viabilities	0.01% – 16.93%	Range of optical densities	1.110 – 2.241

Data of 159 studies performed from October 2004 until July 2013

RESULTS

Table 3: Results after treatment with the test item and the controls

Dose group	Exposure interval	Absorbance 570 nm tissue 1*	Absorbance 570 nm tissue 2*	Mean absorbance of 2 tissues	Rel. absorbance [% of negative control]**
Negative control	3 minutes	1.524	1.525	1.525	100.0
Positive control	3 minutes	0.182	0.273	0.228	14.9
Test item	3 minutes	1.224	1.138	1.181	77.4
Negative control	1 hour	1.596	1.646	1.621	100.0
Positive control	1 hour	0.095	0.070	0.083	5.1
Test item	1 hour	0.080	0.087	0 .083	5.1

* mean of three replicate wells after blank correction

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

- optical evaluation of the MTT-reducing capacity of the test item after an 1 hour incubation with MTT-reagent did not show blue colour and thereby was not considered to be an MTT reducer.

- after exposure to the negative control the absorbance values met the required acceptability criterion of mean OD570 ≥ 0.8 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 (14.9%) and for the 1 hour exposure period (5.1%) thus the validity of the test system and the specific batch of tissue models could be confirmed.

Applicant's summary and conclusion

Interpretation of results:

Category 1B (corrosive)

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item is corrosive to the skin.
According to the Directive 67/548/EEC and its subsequent amendments, the test substance is corrosive to the skin.
According to the Regulation (EC) No 1272/2008 and subsequent regulations, the test item is corrosive to the skin.

Executive summary:

In this in vitro study the test substance was tested for its skin corrosive potential according to the OECD guideline 437 (2013) by the means of the human skin model test using EpiDerm™. Duplicates of EpiDerm™ tissues were exposed to either the undiluted test item (50 µL), the positive control (8.0 N potassium hydroxide; 50 µL) or the negative control (deionised water; 50 µL) for each of two different exposure periods: 3 minutes and 1 hour (incubation temperature: 37 ± 1 °C). Afterwards the test and the control items were rinsed off the tissues, and a 3 hour incubation period (37 ± 1 °C) with MTT solution followed. MTT solution was then aspirated from the wells and the wells were rinsed with DPBS. Next, the formazan salt was extracted for about 21 hours at room temperature followed by the measurement of the optical density (OD570).

After exposure to the test item the relative absorbance value decreased to 77.4% after 3 minutes exposure (threshold for corrosivity: <50%). But after the 1 hour exposure period the relative absorbance value was reduced to 5.1 (threshold for corrosivity: <15%). Therefore, the test item was considered to be corrosive. The controls confirmed the validity of the study.