Hexanoic acid

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Dec 2011 - 24 Jan 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline Study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 437: Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants (adopted 7 Sept 2009)

GLP compliance:

yes (incl. QA statement)

Remarks:

BAYERISCHES LANDESAMT FÜR GESUNDHEIT UND LEBENSMITTELSICHERHEIT, LANDESINSTITUT FÜR ARBEITSSCHUTZ UND PRODUKTSICHERHEIT, München, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Hexanoic acid

EC Number:

205-550-7

EC Name:

Hexanoic acid

Cas Number:

142-62-1

Molecular formula:

C6H12O2

IUPAC Name:

hexanoic acid

Test animals / tissue source

Species:

other: cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

IDENTIFICATION OF THE SOURCE OF THE EYES, STORAGE AND TRANSPORT CONDITIONS
- Source: Attenberger Fleisch GmbH & Co. KG, Munich, Germany
- Date of eye collection: 14 Dec 2011
- Transport medium and temperature conditions: Hanks´ Balanced Salt Solution (HBSS) containing penicillin/streptomycin on ice

PREPARATION OF THE EYES (BEFORE EXPOSURE)
- Eyes free of defects (scratches, neovascularisation): yes
- Dissection of the eyes and treatment: Corneas were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders.
- Type of cornea holder used: MC2, Clermont, France
- Description of the cornea holder: the cornea holders consist of an anterior and a posterior compartment, which interface with the epithelial and endothelial sides of the cornea.
- Test medium and temperature conditions used in the cornea holder: RPMI 1640 without phenol red supplemented with 1% [v/v] fetal bovine serum and 2 mM L-glutamine
- Equilibration time: 1 h at 32 ± 1 °C in a water bath
- Quality check of the equilibrated corneas: initial opacity measurement; corneas with an initial opacity above 7 in the opacitometer were discarded.

DETERMINATION OF THE INITIAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer.
- Specification of the device: MC2, Clermont, France

Test system

Vehicle:

other: sesame oil

Controls:

other: 3 eyes each for the two negative controls (sesame oil and physiological saline 0.9% NaCl); 3 eyes for the positive control (70% ethanol)

Amount / concentration applied:

- Amount(s) applied in the test: 750 µL
- Concentration (if solution): 50%

Duration of treatment / exposure:

10 min at 32 ± 1 °C

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

3 eyes for the test item

Details on study design:

TEST CONDITIONS
- Short description of the method used: closed-chamber method.
The test substance or control substances were introduced into the anterior.

POST-EXPOSURE TREATMENT
- Removal of the test substance: Example: The test substance was removed after 10 min incubation and the epithelium washed at least three times. for the corneas treated with sesame oil as vehicle, the chambers were opened and the corneas were rinsed with 100 mL MEM.
- Medium for washing the corneas: MEM containing phenol red
- Medium for final rinsing: Example: RPMI 1640 without phenol red

DETERMINATION OF THE FINAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer.
- Time of determination: After refilling fresh RPMI 1640 without phenol red into the anterior chamber, the final opacity was measured after 2 hours incubation at 32 ± 1 °C
- Specification of the device: MC2, Clermont, France

DETERMINATION OF THE CORNEAL PERMEABILITY:
- Method: Sodium fluorescein solution was added to the anterior chamber of the cornea holder while the posterior chamber was filled with fresh medium. The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured via UV/VIS spectrophotometry at 490 nm recorded as optical density (OD490).
- Amount and concentration of the dye: 1 mL sodium fluorescein solution (4 mg/mL)
- Incubation time: 90 min at 32 ± 1 °C

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Remarks:

mean of all 3 eyes

Run / experiment:

10 min exposure with the test substance

Value:

143.61

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

62.51

Results 2

Irritation parameter:

other: opacity

Remarks:

mean of all 3 eyes

Run / experiment:

10 min exposure with the test substance

Value:

114

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

52.67

Results 3

Irritation parameter:

other: permeability

Remarks:

mean of all 3 eyes

Run / experiment:

10 min exposure with the test substance

Value:

1.974

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

0.656

Other effects / acceptance of results:

For hexanoic acid at a concenration of 50% (v/v) an in vitro irritation score of 143.61 was calculated and therefore the test substance is considered as severe eye irritant.

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 (0.9% NaCl)	4	5	1	2.33	-	-
	4	9	5
	4	5	1
Negative control (sesame oil)	4	7	3	2.33	-	-
	4	5	1
	5	8	3
Test substance	3	124	121	-	118.67	114.00
	3	112	109		106.67
	3	122	119		116.67
Positive control	5	56	51	-	48.67	52.67
	5	58	53		50.67
	5	66	61		58.67

 

 

Table 2: Permeability values (optical density (OD) at 490 nm)

Parameter	OD490 change	Mean OD490 change of NC	Corrected OD490 change	Mean OD490 value
Negative control (0.9% NaCl)	0.003	0.004	-	-
	0.009
	0.001
Negative control (sesame oil)	0.000	0.004	-	-
	0.003
	0.008
Test substance	1.842	1.977	1.838	1.974
	2.086		2.082
	2.004		2.00
Positive control	0.400	0.661	0.396	0.656
	0.770		0.766
	0.812		0.808

 

 

Table 3: In Vitro Irritancy Score (IVIS) values

	IVIS	Mean IVIS
Negative control (0.9% NaCl)	1.05	2.40
	5.14
	1.02
Negative control (sesame oil)	3.00	2.39
	1.05
	3.12
Test substance	146.24	143.61
	137.90
	146.67
Positive control	54.61	62.51
	62.16
	70.79

 

 

Applicant's summary and conclusion

Interpretation of results:

corrosive

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

CLP: Cat. 1, H318