Reaction mass of 3-methyl-1-octylpyridinium chloride and 4-methyl-1-octylpyridinium chloride

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (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:

Reaction mass of 3-methyl-1-octylpyridinium chloride and 4-methyl-1-octylpyridinium chloride

EC Number:

941-484-1

Molecular formula:

not possible

IUPAC Name:

Reaction mass of 3-methyl-1-octylpyridinium chloride and 4-methyl-1-octylpyridinium chloride

Test material form:

liquid: viscous

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir.
On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories.
Immediately after arrival of the eyes, cornea preparation was initiated. The eyes were carefully examined for defects and any defective eyes were discarded.
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1 °C in a water bath.
The calibration of the opacitometer was performed before each test and was documented in the raw data.
After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI. An initial opacity measurement was performed on each of the corneas using an opacitometer (MC2, Clermont, France). Three corneas with opacity readings approximately equivalent to the median opacity of all corneas were selected as negative-control corneas. The opacity of each cornea was read against an air-filled chamber and recorded. Corneas that have an initial opacity reading above 7 units were not dosed. The medium was removed from the anterior chamber and replaced with the test item or control.
Enough of the test substance to cover the whole cornea was introduced into the anterior chamber by removing the window-locking ring and glass window prior to treatment. 750 μL of the control substance was introduced into the anterior chamber (closed chamber method). After 10 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an opacity measurement was performed after 2 hours incubation at 32 ± 1 °C.
After the opacity measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. Sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: negative control and positive control

Amount / concentration applied:

Enough of the test substance to cover the whole cornea was introduced into the anterior chamber by removing the window-locking ring and glass window prior to treatment.
750 μL of the control substance was introduced into the anterior chamber (closed chamber method).

Duration of treatment / exposure:

10 minutes incubation at 32 ± 1 °C

Number of animals or in vitro replicates:

3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive control treated with ethanol 100%

Details on study design:

Enough of the test substance to cover the whole cornea was introduced into the anterior chamber by removing the window-locking ring and glass window
prior to treatment. 750 μL of the control substance was introduced into the anterior chamber (closedchamber method).

After 10 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an opacity measurement was performed after 2 hours incubation at 32 ± 1 °C.

After the opacity measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh
complete RPMI. 1 mL of a 4 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer.

Results and discussion

In vivo

Irritant / corrosive response data:

The following mean in vitro irritation score was calculated: 86.08
Therefore the test item is classified as corrosive / severe irritant to the eye (UN GHS Category 1)

Any other information on results incl. tables

Bovine Corneal supplier: Abattoir A. Moksel AG, Buchloe, Germany.

In Vitro Irritation Score:

Cornea No.	Test Item	Corrected Opacity	Corrected OD490 Value	IVIS
1	Negative Control	0.0	0.025	0.91
2		1.0	0.008
3		1.0	0.016
MV		0.67	0.016
4	Positive Control	60.33	2.046	80.10
5		46.33	2.036
6		42.33	2.005
MV		49.67	2.029
7	Test Item	52.33	2.096	86.08
8		42.33	2.098
9		69.33	2.090
MV		54.67	2.094

Opacity:

Cornea No.	Test Item	Initial Opacity	Final Opacity	Change of Opacity Value	Corrected Opacity Value
1	Negative Control	3	3	0
2		3	4	1
3		3	4	1
MV		3.00	3.67	0.67
4	Positive Control	4	65	61	60.33
5		4	51	47	46.33
6		4	47	41	42.33
MV		4.00	54.33	50.33	49.67
7	Test Item	1	54	53	52.33
8		4	47	43	42.33
9		2	72	70	69.33
MV		2.33	57.67	55.33	54.67

Permeability:

Cornea No.	Test Item	OD490	Corrected OD490 Value
1	Negative Control	0.025
2		0.008
3		0.016
MV		0:016
4	Positive Control	2.062	2.046
5		2.052	2.036
6		2.021	2.005
MV		2.045	2.029
7	Test Item	2.112	2.096
8		2.114	2.098
9		2.106	2.090
MV		2.111	2.094

Historical mean In Vitro Irritation Score of the positive control:

	IVIS Positive Control
Mean Value (MV)	75.72
Standard Deviation (SD)	12.24
MV- 2xSD	51.24
MV+2xSD	100.19

MV = Mean Value

Applicant's summary and conclusion

Interpretation of results:

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

Remarks:

Migrated information

Conclusions:

According to the evaluation criteria the test item Pyridine derivative 2 is classified as corrosive / severe irritant to the eye (UN GHS Category 1).