2-[[(butylamino)carbonyl]oxy]ethyl acrylate

Endpoint summary

Description of key information

skin corrosion (OECD 431): not corrosiv
skin irritation (OECD 439): not irritating
eye irritation (OECD 437): not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 - 16 Oct 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

only the 4-hour exposure period was assessed

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Gyógyszerészeti és Egészségügyi Minőség- és Szervezetfejlesztési Intézet (National Institute for Quality- and Organizational Development in Healthcare and Medicines), Budapest, Hungary

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

SOURCE
- Source: SKINETHIC Laboratories, Lyon, France
- Age: adult

Justification for test system used:

The EPISKIN model has been validated for corrosivity testing in an international trial; it is considered to be suitable for this study (Statement on the scientific validity of the EPISKINTM Test (an in vitro test for skin corrosivity); ECVAM JRC Environment Institute, European Commission; Ispra; 03 April 1998).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model (EpiSkinTMSM), EPISKIN SNC Lyon, France
- Tissue batch number(s): 14-EKIN-039

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1 step
- Observable damage in the tissue due to washing: no; care was taken to avoid the damage of epidermis

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h
- Spectrophotometer: yes
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if mean tissue viability is < 35% after 4 h exposure.
- The test substance is considered to be non-corrosive to skin if mean tissue viability ≥ 35% after 4 h exposure.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL

NEGATIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration (if solution): 9 g/L saline

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration (if solution):

Duration of treatment / exposure:

4 h at RT

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Value:

121

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: No colour change was observed after 3 h of incubation during the check-test for possible direct MTT reduction with test item. The test item did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.
- Colour interference with MTT: The intrinsic colour of the test item was found to be colourless in small amount (such as treatment volume) and showed no ability to become coloured in contact with water during the check-test to detect the colouring potential of test item, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: Please refer to table 2.

Table 3. MTT assay after 4 h exposure.

	Negative control		Positive control		Test item
Tissue sample	1	2	1	2	1	2
OD570	0.774	0.716	0.048	0.013	0.942	0.864
OD570(mean values of replicates)	0.745		0.031		0.903
Viability (%)	100		4		121

Interpretation of results:

other: CLP/GHS criteria not met; no classification required according to Regulation (EC) No. 1272/2008

Conclusions:

CLP: not classified

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 - 6 Feb 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)

Version / remarks:

adopted in 2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Gyógyszerészeti és Egészségügyi Minőség- és Szervezetfejlesztési Intézet (National Institute for Quality- and Organizational Development in Healthcare and Medicines), Budapest, Hungary

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

SOURCE
- Source: SKINETHIC Laboratories, Lyon, France
- Age: adult

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no- skin irritating test substances (Statement on the scientific validity of the EPISKINTM Test (an in vitro test for skin corrosivity); ECVAM JRC Environment Institute, European Commission; Ispra; 03 April 1998).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model (EpiSkinTMSM), EPISKIN SNC Lyon, France
- Tissue batch number(s): 15-EKIN-005

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1 step
- Observable damage in the tissue due to washing: no; care was taken to avoid the damage of epidermis

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 15 ± 0.5 min
- Spectrophotometer: yes
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
The test substance is considered to be
irritant to skin (Category 2), if the mean relative viability after 15 minutes exposure and
42 hours post incubation is less or equal (≤) to 50% of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 10 µL

NEGATIVE CONTROL
- Amount(s) applied: 10 µL

POSITIVE CONTROL
- Amount(s) applied: 10 µL
- Concentration (if solution): 5%

Duration of treatment / exposure:

15 ± 0.5 min

Duration of post-treatment incubation (if applicable):

42 ± 1 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

72

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: No colour change was observed after 3 h of incubation during the check-test for possible direct MTT reduction with test item.
- Colour interference with MTT: The intrinsic colour of the test item was found to be colourless in small amount (such as treatment volume) and showed no ability to become coloured in contact with water during the check-test to detect the colouring potential of test item, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: Please refer to table 2.

Table 3. MTT assay after 15 min exposure.

	Negative control			Positive control			Test item
Tissue sample	1	2	3	1	2	3	1	2	3
OD570	0.854	0.862	0.835	0.212	0.110	0.192	0.596	0.528	0.724
OD570 (mean values of replicates)	0.850			0.171			0.616
Viability (%)	100			20			72

Interpretation of results:

other: CLP/GHS criteria not met; no classification required according to Regulation (EC) No. 1272/2008

Conclusions:

CLP: not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Jan 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Species:

other: cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST METHOD
The bovine corneal opacity and permeability (BCOP) test method is designed to measure the opacity of the cornea by quantifying the ability of light to pass through it.The permeability, as a result of the irritation potential of the test item, is determined using sodium fluorescein solution. The comparison of the opacity before and after the exposure to the test item and the determination of the permeability after the treatment provide an indication of the damaging effect of the test item. For this purpose the induction of opacity and increased permeability in an isolated bovine cornea after application of the test item was measured. The results of both criteria were combined. The resulting in vitro irritation factor (IVIS) was compared with the classification according to the UN GHS System.

IDENTIFICATION OF THE SOURCE OF THE EYES, STORAGE AND TRANSPORT CONDITIONS
- Source: Schlachthof Bensheim, Bensheim, Germany
- Donor animals: at least 9 month old
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes. The cornea were directly used in the BCOP on the same day.
- Transport medium and temperature conditions: Hank's Buffered Salt Solution (HBSS) supplemented with penicillin/streptomycin at ambient temperature.

PREPARATION OF THE EYES (BEFORE EXPOSURE)
- Eyes free of defects (scratches, neovascularisation): yes
- Dissection of the eyes and treatment: The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. Each cornea was mounted in a specially designed cornea holder.
- Description of the cornea holder: The cornea holder consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively.
- Test medium used in the cornea holder: The incubation medium consisted of MEM, supplemented with 1.1 g/500 mL sodium bicarbonate, 5 mL/500 mL L-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin. Immediately before starting the test, MEM was supplemented with 1% fetal calf serum.
- Equilibration time: 1 h at 32 ± 1 °C
- Quality check of the equilibrated corneas: At the end of the equilibration period, the basal opacity was determined (t0). Each cornea with a value of the basal opacity >7 was discarded.

DETERMINATION OF THE INITIAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer.
- Specification of the device: OP_KiT opacitometer, Electro Design, France

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.75 mL

POSITIVE SUBSTANCE
- Substance: 2-ethoxyethanol
- Amount(s) applied in the test: 0.75 mL

NEGATIVE CONTROL
- 0.9% NaCl (w/v) solution in deionised water (saline)

Duration of treatment / exposure:

10 min at 32±1 °C

Observation period (in vivo):

Not applicable

Number of animals or in vitro replicates:

3

Details on study design:

TEST CONDITIONS
- Short description of the method used: The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on the top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. The anterior compartment received the test item or the controls on the surface of the corneae. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath for 10 minutes.

POST-EXPOSURE TREATMENT
- Removal of the test substance: The test item was rinsed off from the application side with saline.

DETERMINATION OF THE FINAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer.
- Time of determination: After the test item was rinsed off from the application side, the corneae were incubated for further 2 hours in a vertical position, followed by a second opacity reading (t130).
- Specification of the device: OP_KiT opacitometer, Electro Design, France

DETERMINATION OF THE CORNEAL PERMEABILITY:
- Method: After the final opacity measurement was performed, the incubation medium was removed from the anterior compartment and replaced by sodium fluorescein solution. Corneae were incubated again in horizontal position for 90 ± 10 min in a water-bath at 32 ± 1 °C. The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured with a spectrophotometer as optical density at 490 nm (OD490).
- Amount and concentration of the dye: 1 mL sodium fluorescein solution (0.4% (w/v); dissolved in HBSS)
- Incubation time: 90 min at 32 ± 1 °C
- Treatment for measuring: Complete medium from the posterior compartment was removed, well mixed, transferred into a 96 well plate and OD490 was determined.
- Specification of the spectrophotometer: Versamax Molecular Devices

Irritation parameter:

cornea opacity score

Run / experiment:

mean out of 3

Value:

-0.33

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein leakage

Run / experiment:

number 1

Value:

0.009

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein leakage

Run / experiment:

number 2

Value:

0.004

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein leakage

Run / experiment:

number 3

Value:

0.004

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

mean out of 3

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes; With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae could be observed.
- Acceptance criteria met for positive control: yes; The positive control (2-ethoxyethanol) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damaging.

Table 1. Historical Data

	Positive control	Negative control
Mean IVIS	71.85	1.07
SD	17.57	0.71
Range of IVIS	33.59 – 153.20	0.00 – 2.84

Values of 274 studies with liquid test items performed from February 2007 until December 2014.

Interpretation of results:

other: CLP/GHS criteria not met; no classification required according to Regulation (EC) No. 1272/2008

Conclusions:

CLP: not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin

The skin corrosion and irritation potential of the test substance was determined by an in vitro skin corrosion and irritation test using a human skin model.

The skin corrosion potential of the test substance was determined by an in vitro skin corrosion test using a human skin model according to OECD Guideline 431 and in compliance with GLP (Ágh, 2014). A volume of 50 µL test substance was applied evenly to the epidermal surface of human skin tissues (EpiSkinTM) with a tissue size of 0.38 cm² for 4 h. Additional exposure times of 3 min and 1 hour were not examined. After the incubation period, the cytotoxic effect was assessed. Cell viability was measured by dehydrogenase conversion of MTT, present in cell mitochondria, into a blue formazan salt, that is quantitatively measured after extraction from tissues. Saline was used as negative control, glacial acetic acid was used as positive control. The relative mean tissue viability obtained after 4 h treatment with the test substance compared to the negative control tissues was 121%. Since the mean relative tissue viability for the test substance was above 50% after 4 h treatment the test substance is considered to be non-corrosive. The optical density of the negative control was well within the required acceptability criterion of 0.6 ≤ mean OD ≤ 1.5. The positive control glacial acetic acid revealed a mean cell viability of 4% (required ≤ 20%) after 4 h exposure and thus ensuring the validity of the test system. Variation within replicates was acceptable. Based on the results, the test substance was not corrosive to the skin under the conditions of the test.

The skin irritation potential of the test substance was determined by an in vitro skin irritation test using a human skin model according to OECD Guideline 439 and in compliance with GLP (Ágh, 2015). A volume of 10 µL test substance was applied evenly to the epidermal surface of human skin tissues (EpiSkinTM) with a tissue size of 0.38 cm² for 15 min. After a 42 ± 1 h post-incubation period, the cytotoxic (irritancy) effect was assessed. Cell viability was measured by dehydrogenase conversion of MTT, present in cell mitochondria, into a blue formazan salt, that is quantitatively measured after extraction from tissues. 1x PBS was used as negative control, 5% aq. SDS solution was used as positive control. The relative mean tissue viability obtained after 15 min treatment with the test substance compared to the negative control tissues was 72%. Since the mean relative tissue viability for the test substance was above 50% after 15 min treatment the test substance is considered to be non-irritant. The optical density of the negative control was well within the required acceptability criterion of 0.6 ≤ mean OD ≤ 1.5. The positive control 5% sodium dodecyl sulphate solution revealed a mean cell viability of 20% (required ≤ 40%) after 15 min exposure and thus ensuring the validity of the test system. Variation within replicates was acceptable. Based on the results, the test substance was not irritating to the skin under the conditions of the test.

In conclusion, the test substance is non-corrosive in the in vitro skin corrosion test and non-irritant in the in vitro skin irritation test under the experimental conditions used.

 

Eye

The eye irritation potential of the test substance was determined in a bovine corneal opacity and permeability test (BCOP test) according to OECD Guideline 437 and in compliance with GLP (Roth, 2015). After a first opacity measurement of the fresh bovine corneae, the neat test substance was applied directly to the epithelial surface of three cattle corneas in an incubation chamber in horizontal position for 10 min at 32 ± 1 °C. After the incubation phase the test substance was rinsed from the corneae. Further, the corneae were incubated for another 120 min at 32 ± 1 °C in a vertical position, while the anterior chamber contained incubation medium as well. Afterwards, opacity was measured a second time. In addition, the permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 ± 10 min at 32 ± 1 °C. The results of the opacity and permeability measurement of the test substance were used to calculate an in vitro irritation score (IVIS) of 0.00. With the negative control neither an increase of opacity nor permeability of the corneae could be served. The mean IVIS of the negative control was 1.15. The mean in vitro irritation score of the positive control (2-ethoxyethanol) was 67.84. All three values of the negative and positive controls were within the range of historical data of the test facility. Therefore, the test system was acceptable. Based on the results, the test substance was not irritating to the eye under the conditions of the test.

Justification for classification or non-classification

The available data on skin and eye irritation do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.