N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 July 2017 - 31 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2013

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Fatty Acids, C14 and C16 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Molecular formula:

C34H68N2O3

IUPAC Name:

Fatty Acids, C14 and C16 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Constituent 2

Reference substance name:

Fatty Acids, C15 and C16 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Molecular formula:

C35H70N2O3

IUPAC Name:

Fatty Acids, C15 and C16 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Constituent 3

Reference substance name:

Fatty Acids, C16 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Molecular formula:

C36H72N2O3

IUPAC Name:

Fatty Acids, C16 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Constituent 4

Reference substance name:

Fatty Acids, C15 and C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Molecular formula:

C37H74N2O3

IUPAC Name:

Fatty Acids, C15 and C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Constituent 5

Reference substance name:

Fatty Acids, C16 and C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Molecular formula:

C38H76N2O3

IUPAC Name:

Fatty Acids, C16 and C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Constituent 6

Reference substance name:

Fatty Acids, C17 and C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Molecular formula:

C39H78N2O3

IUPAC Name:

Fatty Acids, C17 and C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Constituent 7

Reference substance name:

Fatty Acids, C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Molecular formula:

C40H80N2O3

IUPAC Name:

Fatty Acids, C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Constituent 8

Reference substance name:

Stearic acid

EC Number:

200-313-4

EC Name:

Stearic acid

Cas Number:

57-11-4

Molecular formula:

C18H36O2

IUPAC Name:

stearic acid

Constituent 9

Reference substance name:

Palmitic acid

EC Number:

200-312-9

EC Name:

Palmitic acid

Cas Number:

57-10-3

Molecular formula:

C16H32O2

IUPAC Name:

palmitic acid

Constituent 10

Reference substance name:

Myristic acid

EC Number:

208-875-2

EC Name:

Myristic acid

Cas Number:

544-63-8

Molecular formula:

C14H28O2

IUPAC Name:

myristic acid

Constituent 11

Reference substance name:

2-(2-aminoethylamino)ethanol

EC Number:

203-867-5

EC Name:

2-(2-aminoethylamino)ethanol

Cas Number:

111-41-1

Molecular formula:

C4H12N2O

IUPAC Name:

2-(2-aminoethylamino)ethanol

Test material form:

solid

Details on test material:

Identification: N-(2-{[C16-18 (EVEN NUMBERED) ALKANOYL]AMINO}ETHYL)-N-(2-HYDROXYETHYL)[C16-18 (EVEN NUMBERED) ALKYLAMIDE
Storage Conditions: Room temperature

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature (in the dark*)
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: n/a
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no
- Preliminary purification step (if any): no
- Final dilution of a dissolved solid, stock liquid or gel: undiluted
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): n/a

OTHER SPECIFICS: no

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Freshly isolated bovine cornea obtained from AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany
- Number of animals: Not reported
- Characteristics of donor animals (e.g. age, sex, weight): At least 9-month-old donor cattle
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes were removed after slaughter, the isolated eyes were stored in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) in the cooled slaughter-house until transportation on the same morning to the laboratory using a Styrofoam box.
- Time interval prior to initiating testing: Same day
- indication of any existing defects or lesions in ocular tissue samples: No
- Indication of any antibiotics used: Penicillin/Streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) added to Styrofoam box during transportation.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL (of suspension)
- Concentration (if solution): The test item was tested as a 20% suspension (w/v) in saline

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a
- Concentration (if solution): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a

Duration of treatment / exposure:

240 minutes

Observation period (in vivo):

n/a

Duration of post- treatment incubation (in vitro):

90 minutes

Number of animals or in vitro replicates:

3

Details on study design:

APPLICATION DOSE AND EXPOSURE TIME

0.75 mL applied to each cornea and incubated at 32 ± 1 °C in the water-bath for 240 minutes.

TREATMENT METHOD

Closed chamber

POST-INCUBATION PERIOD

Following rinsing, the corneas were incubated (horizontally) for 240 minutes after which, corneal opacity was measured and then the anterior chamber emptied. For the permeability endpoint, 1 mL of sodium fluorescein (4 mg/mL solution) was added into the anterior chamber and the Corneae were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Each cornea was washed with media containing phenol red (as a pH indicator) until this indicator showed no pH effect occurring (and demonstrating that the test article had been removed successfully). The corneas were then washed once in media without phenol red
- POST-EXPOSURE INCUBATION: Following rinsing, the corneas were incubated (horizontally) for 2 hours after which, corneal opacity was measured and then the anterior chamber emptied. For the permeability endpoint, 1 mL of sodium fluorescein (4 mg/mL solution) was added into the anterior chamber and the corneas were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer
- Corneal permeability: Passage of sodium fluorescein dye measured with the aid of spectrophotometer (OD490)
- Others (e.g, pertinent visual observations, histopathology): (please specify) N/A

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: As described in OECD 437.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

ca. 0.48

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Table 1. Results after 240 Minutes Treatment Time

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Proposedin vitroIrritancy Score
		Mean		Mean
Negative Control	0	0.33	0.065	0.070	0.98	1.38	Not categorized
	0		0.068		1.02
	1		0.077		2.16
Positive Control	133.67*		0.006*		133.76	127.81	Category 1
	122.67*		0.011*		122.83
	126.67*		0.011*		126.83
Test Item	-0.33*		0.041*		0.28	0.48	Not categorized
	0.67*		0.033*		1.16
	-0.33*		0.022*		0.00

*corrected values

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

It was concluded that under the condition of this study, the test item, produced an IVIS score of 0.48 and therefore, the test item is not categorised according to UN GHS/EU CLP regulation.

Executive summary:

OECD 437 (2017) -The Bovine Corneal Opacity and Permeability (BCOP) test was conducted using  

N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

 in accordance with OECD Guideline 437 "Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage" (2013).

Undiluted test item of 0.75 mL volume was applied evenly to the surface of three corneas before being washed off with media solution after 10 minute test item contact time. A negative and positive control group, each containing 3 corneas, were also prepared using a similar concentration and volume of the test item .

Measurements for corneal opacity were made after 240 minutes incubation in the horizontal position with fresh media. Measurements for corneal permeability were made following 90  min incubation in the vertical position with sodium fluorescein.

Corneal opacity and corneal permeability media solutions were analysed by a spectrophotometer at 490 nanometers (OD490). The mean corrected opacity reading and permeability readings for the test item were 0.44 and 0.032, resulting in a mean In Vitro Irritation Score (IVIS) of 0.48.

It was concluded that under the condition of this study, the test item, produced an IVIS score of 0.48 and therefore, the test item is not categorised according to UN GHS/EU CLP regulation.