N-octylpyridin-4-amine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 15 February 2018, Experimental completion date 15 February 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

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N-octylpyridin-4-amine

EC Number:

265-019-0

EC Name:

N-octylpyridin-4-amine

Cas Number:

64690-19-3

Molecular formula:

C13H22N2

IUPAC Name:

N-octylpyridin-4-amine

Specific details on test material used for the study:

3.1 Test Item and Supporting Information
Information as provided by the Sponsor.
Identification: N-Octyl-4-pyridinamine
CAS Number: 64690-19-3
Batch: 80030735B
Purity: 99.6%
Physical state/Appearance: White solid
Expiry Date: 28 November 2018
Storage Conditions: 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 Bovine Eyes
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Preparation of Corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of Corneas and Opacity Reading
The medium from both chambers of each holder was replaced with fresh complete EMEM.
A pre treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.
Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Test Item Preparation and Analysis
For the purpose of this study the test item was prepared as a 20% w/v solution in sodium chloride 0.9% w/v.
The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration.

Reference Item Preparation
The negative control item, sodium chloride 0.9% w/v, was used as supplied.
The positive control item, Imidazole, was used as a 20% w/v solution in sodium chloride 0.9% w/v.

Duration of treatment / exposure:

240 minutes.

Duration of post- treatment incubation (in vitro):

90 minutes (permeability evaluation)

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

67.8

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

 Corneal Opacity and Permeability Measurement

Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in the table below.

  Corneal Epithelium Condition

The condition of each cornea is given in the table below.

The corneas treated with the test item were cloudy post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

 In VitroIrritancy Score

TheIn Vitroirritancy scores are summarized as follows:

Treatment	In VitroIrritancy Score
Test Item	67.8
Negative Control	1.1
Positive Control	109.8

 Criteria for an Acceptable Test

The positive controlIn VitroIrritancy Score was within the range of 65.1 to 123.3. The positive control acceptance criterion was therefore satisfied.

The negative control gave opacity of ≤2.4 and permeability ≤0.072. The negative control acceptance criteria were therefore satisfied.

 Conclusion

Category 1. UN GHS or EU CLP Causes serious eye damage.

Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity				Permeability (OD492)		In VitroIrritancy Score
		Pre-Treatment	Post-Treatment	Post-Treatment-Pre‑Treatment	Corrected Value		Corrected Value
Negative Control	2	2	3	1		0.013
	3	2	3	1		0.002
	6	4	5	1		0.006
				1.0*		0.007¨		1.1
Positive Control	7	3	78	75	74.0	1.960	1.953
	10	1	84	83	82.0	1.950	1.943
	11	2	73	71	70.0	3.005	2.998
					75.3·		2.298·	109.8
Test Item	12	3	68	65	64.0	0.812	0.805
	13	1	58	57	56.0	0.627	0.620
	14	2	53	51	50.0	0.813	0.806
					56.7·		0.744·	67.8

OD= Optical density           * = Mean of the post-treatment -pre‑treatment values             ¨= Mean permeability                     ·= Mean corrected value

Corneal Epithelium Condition Post Treatment

Treatment	Cornea Number	Observation Post Treatment
Negative Control	2	Clear
	3	Clear
	6	Clear
Positive Control	7	Cloudy
	10	Cloudy
	11	Cloudy
Test Item	12	Cloudy
	13	Cloudy
	14	Cloudy

 

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

The test item is classified as its in-vitro irritancy score is >55 (67.8)