N-[3-(dimethylamino)propyl] amides C18-22 (even numbered)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2 Feb 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

26 July 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)

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Department of Health of the Government of the United Kingdom, UK

Test material

Test material information

Constituent 1

Reference substance name:

N-[3-(dimethylamino)propyl] amides C18-22 (even numbered)

EC Number:

946-742-7

Molecular formula:

not applicable, the substance is UVCB

IUPAC Name:

N-[3-(dimethylamino)propyl] amides C18-22 (even numbered)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, in the dark

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: local abattoir
- Characteristics of donor animals:12 - 60 months
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The isolated eyes were transported in Hanks' Balanced Salt Solution (HBSS, with streptomycin (100 μg/mL) / penicillin (100 IU/mL)), ice cooled.
- Time interval prior to initiating testing: The corneae were prepared immediately upon arrival.
- indication of any existing defects or lesions in ocular tissue samples: no
- Indication of any antibiotics used: streptomycin / penicillin

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 750 µL
- Concentration: 20% w/v in vehicle

VEHICLE
- Amount(s) applied: 750 µL
- Concentration: 0.9% w/v
- Lot/batch no.: 3011542

Duration of treatment / exposure:

4 h at 32 ± 1 °C

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

not applicable

Number of animals or in vitro replicates:

Number of eyes for the test item/ negative control/ positive control: 3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: A careful macroscopic examination was performed on all eyes to detect the presence of any defects. Only corneas free of damage were used. The cornea was removed from the eye leaving a rim of about 2 mm of sclera. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in HBSS until they were mounted in BCOP holders. A pre-treatment opacity reading was taken for each cornea.

QUALITY CHECK OF THE ISOLATED CORNEAS: Yes, the eyes were free of defects.

NUMBER OF REPLICATES: 3

SOLVENT CONTROL USED: Yes, sodium chloride (0.9% w/v)

POSITIVE CONTROL USED: Yes, imidazole (20% in sodium chloride (0.9% w/v))

APPLICATION DOSE AND EXPOSURE TIME: 750 µL test item (20%), 4 h

TREATMENT METHOD: Closed chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The cornea was rinsed three times with Eagle’s Minimum Essential Medium (EMEM) containing phenol red and one time with EMEM without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: A post-exposure opacity reading was taken.
- Corneal permeability: Passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (OD492) (Anthos 2001 microplate reader).
- Others (e.g, pertinent visual observations, histopathology): In addition each cornea was visually examined.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS), calculated as follows: IVIS = mean opacity value + (15 x mean permeability OD492 value). Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

DECISION CRITERIA: The decision criteria as indicated in the TG (OECD 437) was used:
IVIS ≤ 3: No category. Not requiring classification to UN GHS or EU CLP
3 < IVIS ≤ 55: No prediction of eye irritation can be made
IVIS > 55: Category 1: UN GHS or EU CLP Causes serious eye damage

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

negative control - mean out of all 3 eyes

Value:

0.3

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

positive control - mean out of all 3 eyes

Value:

68

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: value is corrected

Results 3

Irritation parameter:

cornea opacity score

Run / experiment:

test item - mean out of all 3 eyes

Value:

9.3

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: value is corrected

Results 4

Irritation parameter:

fluorescein leakage

Remarks:

permeability

Run / experiment:

negative control - mean out of all 3 eyes

Value:

0.02

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 5

Irritation parameter:

fluorescein leakage

Remarks:

permeability

Run / experiment:

positive control - mean out of all 3 eyes

Value:

1.35

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: value is corrected

Results 6

Irritation parameter:

fluorescein leakage

Remarks:

permeability

Run / experiment:

test item - mean out of all 3 eyes

Value:

0.258

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: value is corrected

Results 7

Irritation parameter:

in vitro irritation score

Run / experiment:

negative control - mean out of all 3 eyes

Value:

0.6

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 8

Irritation parameter:

in vitro irritation score

Run / experiment:

positive control - mean out of all 3 eyes

Value:

88.3

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 9

Irritation parameter:

in vitro irritation score

Run / experiment:

test item - mean out of all 3 eyes

Value:

13.2

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
Acceptance criteria:
The test was acceptable if the positive control produced an IVIS which fell within two standard deviations of the historical mean during 2015 (for this testing facility: 50.8 - 100.4).
The test was acceptable if the negative control produced an IVIS which is less than or equal to the upper limit for background opacity and permeability historical values for bovine corneas treated with the respective negative control. When testing solids, the negative control should show an opacity of ≤ 5.4 and a permeability of ≤ 0.070.

- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Any other information on results incl. tables

Table 1: Opacity values

Parameter	Initial opacity	Final opacity	Opacity change	Mean opacity change of NC	Corrected opacity change	Mean opacity value
Negative control (NC)	3	3	0	0.3	-	-
	3	3	0
	3	4	1
Test substance	6	13	7	-	6.7	9.3
	2	10	8		7.7
	2	16	14		13.7
Positive control	6	72	66	-	65.7	68.0
	2	75	73		72.7
	2	68	66		65.7

Table 2: Permeability values (optical density (OD) at 492 nm)

Parameter	OD492	Mean OD492 change of NC	Corrected OD492 change	Mean OD492 value
Negative control (NC)	0.023	0.020	-	-
	0.017
	0.020
Test substance	0.323	-	0.303	0.258
	0.162		0.142
	0.349		0.329
Positive control	1.663	-	1.643	1.35
	1.132		1.112
	1.315		1.295

Corneal epithelium condition post treatment:

While the negative control showed no effect on the corneal epithelium (clear), both the positive control and the test item led to a cloudy epithelium.

Applicant's summary and conclusion

Interpretation of results:

other: non-corrosive (Eye Irrit. 2 or not classified according to Regulation (EC) No 1272/2008)

Conclusions:

Under the conditions of the test, the test substance was shown to have no corrosive potential in the Bovine corneal opacity and permeability (BCOP) test prediction model. The result does not allow for the non-classification or classification as irritant of the test substance and therefore classification for Eye Irrit. 2 was chosen as worst case.