N-[3-(dimethylamino)propyl]dodecanamide N-oxide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 - 09 Jun 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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:

adopted in 2017

Deviations:

yes

Remarks:

eyes immersed in physiological saline instead of HBSS

Test guideline 2

Qualifier:

equivalent or similar 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:

adopted in 2020

Deviations:

yes

Remarks:

eyes immersed in physiological saline instead of HBSS

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N-[3-(dimethylamino)propyl]dodecanamide N-oxide

EC Number:

263-218-7

EC Name:

N-[3-(dimethylamino)propyl]dodecanamide N-oxide

Cas Number:

61792-31-2

Molecular formula:

C17H36N2O2

IUPAC Name:

N-[3-(dimethylamino)propyl]dodecanamide N-oxide

Specific details on test material used for the study:

Freeze-dried test material was used to ensure that the purity was sufficiently high.

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Vitelco, 's Hertogenbosch, the Netherlands
- Storage, temperature and transport conditions of ocular tissue: Freshly isolated bovine eyes of young cattle were collected from the slaughterhouse. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing: The eyes were tested on the day of arrival in the laboratory.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 316.4 - 364.2 mg

NEGATIVE CONTROL
- Amount applied: 750 µL

POSITIVE CONTROL
- Amount applied: 750 µL
- Concentration: 20% (w/v)

Duration of treatment / exposure:

240 ± 10 min

Number of animals or in vitro replicates:

Three corneas were used for each treatment group.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, the Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of Duratec Analysentechnik GmbH (Hockenheim, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1 °C. The corneas were incubated for the minimum of 1 h at 32 ± 1 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS
Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, Duratec GmbH). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.

NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group.

TREATMENT METHOD: Open chamber method
The corneas were mounted in a corneal holder with the endothelial side against the O-ring of the posterior chamber. The anterior chamber was then positioned on top of the cornea and tightened with screws. Starting with the posterior chamber, both chambers of the corneal holder were then filled with cMEM medium and the corneas were incubated for minimum 1 h at 32 ± 1 °C. After the equilibration period an initial illuminescence reading the test item, the positive or the negative control was performed. The test item or control substances were applied on the corneas and the holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 min at 32 ± 1 °C. Possible pH effects of the test item on the corneas were recorded.

POST-INCUBATION PERIOD: No

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation, the solutions and the test item were removed and the epithelium was washed at least three times with MEM with phenol red (Eagle’s Minimum Essential Medium Life Technologies).

- POST-EXPOSURE INCUBATION: No

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed. Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer (OP-KIT).
- Corneal permeability: The passage of sodium fluorescein dye was measured with the aid of a microtiter plate reader (TECAN Infinite® M200 Pro Plate Reader). The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as Category 1.
Test substance with an IVIS ≤ 3 was regarded as No Category.
Test substance with an IVIS > 3; ≤ 55: no prediction can be made.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean value of 3 corneas

Value:

71

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Technical proficiency of the test system was demonstrated with 13 substances according to OECD 437.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
After treatment with the negative control (physiological saline) the calculated IVIS ranged from -0.5 to 0.7, and was therefore within the standard deviations of the current historical mean of the negative control (IVIS: -3.40 to 6.30) (please refer to Tables 1 and 2 under "Any other information on results incl. tables").
- Acceptance criteria met for positive control:
After treatment with the positive control (20% (w/v) imidazole) the calculated IVIS ranged from 125 to 168, and was therefore within the standard deviations of the current historical mean of the positive control (IVIS: 92 - 251) (please refer to Tables 1 and 2 under "Any other information on results incl. tables").

Any other information on results incl. tables

Table 1: Results of the BCOP test

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score^1, 2
Negative control	-0.1	0.007	0.0
Positive control	129	1.487	151
Test item	0.8	4.695	71

¹    Calculated using the negative control corrected mean opacity and mean permeability values for the positive control and test item.

²    In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Table 2: Historical control data generated in the testing laboratory from Jan 2017 - Jan 2020

	Negative control			Positive control
	Opacity	Permeability	In vitro Irritancy Score	In vitro Irritancy Score
Range	-3.50 – 6.20	-0.011 - 0.081	-3.40 – 6.30	92 – 251
Mean	1.06	0.013	1.27	149
SD	1.80	0.013	1.86	30
n	165	165	165	168

SD = Standard deviation

n = Number of observations

Applicant's summary and conclusion

Interpretation of results:

other: corrosive according to Regulation (EC) No. 1272/2008.

Conclusions:

Under the conditions of the BCOP assay, the test substance showed corrosive properties towards eyes. Application of the test substance to bovine corneas resulted in a calculated mean IVIS of 71 after 4 h of treatment.