ethyl tetrahydrofuran-2-carboxylate

Endpoint summary

Description of key information

Skin corrosion: corrosive (OECD 431; GLP compliant)
Eye irritation: serious eye damage (worst case classification, based on corrosivity and Figure R.7.2-3 of Guidance on information requirements and CSA, Chapter R.7a, 2014)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

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

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

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.

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%.

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)

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)

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.

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.

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin corrosion:

An in vitro skin irritation study according to OECD439 (key_in vitro skin irritation_2014_RL1) is considered to be reliable without restrictionsand the results indicate that the test item is irritant to skin. Hence, an in vitro skin corrosion study according to OECD 431 (key_in vitro skin corrosion_2014_RL1) was performed to prove if the substance has corrosive properties. The study performance is considered to be reliable without restrictions. The results indicate that the test item is corrosive to skin.

Eye irritation

Two in vitro studies, BCOP and Human cornea model test, were conducted with the test item. Following the BCOP test no prediction could be made regarding the serious eye damaging properties of the test item. The Human cornea model test indicated that the test item has severe/extreme eye irritating potential. There was no internationally accepted guideline available yet for the latter test.

Based on these studies and according to the testing strategy for eye irritation potential in section R.7.2-3 (ECHA, 2014: Guidance on information requirements and CSA, Chapter R.7a) and section 3.3.2.6 (Guidance on the Application of the CLP Criteria Version 4.0 – November 2013), when the substance is classified as Skin Corr.1, the risk of severe damage to eyes is considered implicit, so the substance is classified for serious eye damage.

Justification for selection of skin irritation / corrosion endpoint:
Two in vitro studies, one on corrosion and one on irritation, were conducted with the test item. The test for corrosion showed that the test substance is corrosive to the skin. Hence, the test item is classified as corrosive to skin. The study for irritation, which indicated the test item as irritating to the skin, is used as supporting information.

Justification for selection of eye irritation endpoint:
Two in vitro studies, BCOP and Human cornea model test, were conducted with the test item. Following the BCOP test no prediction could be made regarding the serious eye damaging properties of the test item. The Human cornea model test indicated that the test item has severe/extreme eye irritating potential. There was no internationally accepted guideline available yet for the latter test. Based on these studies and according to the testing strategy for eye irritation potential in Figure R.7.2-3 (ECHA, 2014: Guidance on information requirements and CSA, Chapter R.7a) and section 3.3.2.6 (Guidance on the Application of the CLP Criteria Version 4.0 – November 2013), when the substance is classified as Skin Corr.1, the risk of severe damage to eyes is considered implicit, so the substance is classified for serious eye damage.

Effects on skin irritation/corrosion: corrosive

Effects on eye irritation: corrosive

Justification for classification or non-classification

Skin irritation/corrosion:

The test item does possess a skin corrosive potential and does require classification as corrosive to skin according to Directive 67/548/EEC and its subsequent amendments (C; R34 Causes burns) and Regulation (EC) No 1272/2008 and its subsequent regulations (Skin Corr. Cat. 1B; H314: Causes severe skin burns and eye damage).

Eye irritation:

Based on the classification of skin corrosiveness the test item does require classification as risk of serious eye damage according to

Directive 67/548/EEC and its subsequent amendments (Xi; R41: Risk of serious damage to eyes) and Regulation (EC) No 1272/2008 and its subsequent regulations (Eye Damage Cat. 1; H318 : Causes serious eye damage).