4-methylpent-3-en-2-one

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Fully GLP Compliant

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Mesityl Oxide

IUPAC Name:

Mesityl Oxide

Constituent 2

Reference substance name:

4-methylpent-3-en-2-one

EC Number:

205-502-5

EC Name:

4-methylpent-3-en-2-one

Cas Number:

141-79-7

Molecular formula:

C6H10O

IUPAC Name:

4-methylpent-3-en-2-one

Details on test material:

- Name of test material (as cited in study report): Mesityl Oxide
- Physical state: liquid
- Analytical purity: 99.87%
- Purity test date: 16 April 2010
- Lot/batch No.: A1YB3N010101
- Expiration date of the lot/batch: 16 April 2012
- Storage condition of test material: Ambient condition

Test animals / tissue source

Species:

other: Bovine

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
Eye supply: Slaughter house
Age of animals : 6-12 months
Killing time: Approximately 8:30 to 11:00 in the morning (used in early afternoon).
Transport condition: HBSS 1x plus Penicillin/streptomicin maintained at approximately 4°C.
Approximate test starting time: 13:30

PREARATION OF CORNEAS

- Eyes were examined for the presence of any defects
- Each cornea with 2-3 mm of surrounding sclera was dissected from the eye using a scalpel, scissors and forceps and placed into a Petri dish containing HBSS.
- Each cornea was mounted into a pre-warmed testing chamber with the endothelial surface of the cornea placed in contact with the
O-ring of the posterior part of the chamber.
- The chamber was then filled with EMEM without phenol red maintained at 32 ± 1°C (posterior part of the chamber first to maintain convexity).

EQUILIBRATION OF CORNEAS
- incubation in a liquid bath at 32 ± 1°C at least 1 hour; the two chambers were drained (anterior first) and re-filled with EMEM without phenol maintained at 32 ± 1°C (posterior first).
Selection : Basal opacity of corneas was determined by means of an opacitometer
Corneas were distributed in treatment groups starting from those with minor opacity (maximum basal opacity score: 1).

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: 3 corneas for each treatment with the positive or negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 ml/cornea for 3 corneas
- Concentration (if solution): as supplied

POSITIVE CONTROL
Identity: Sodium hydroxide (NaOH)
Sigma Cat. No. : S5881
CAS : 1310-73-2
Batch no. : 00014227117
Purity: 98.7 %
- Amount(s) applied (volume or weight with unit): 0.75 ml/cornea for 3 corneas
- Concentration: 10% (w/v) solution in water (sterile water Bieffe Medital, batch 08K2901).

NEGATIVE CONTROL
Identity: Physiological saline (0.9% NaCl; Bieffemedital batch no. 07K1701)
- Amount(s) applied (volume or weight with unit): 0.75 ml/cornea for 3 corneas
- Concentration: As supplied

Duration of treatment / exposure:

Corneas were exposed in horizontal position for 10 minutes incubated in a liquid bath at 32 ± 1°C.

Number of animals or in vitro replicates:

3 corneas for treatment with the test item

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: After exposure, corneas (epythelial side) were rinsed thoroughly with EMEM with phenol red.
Finally, the anterior chamber was re-filled with EMEM without phenol red maintained at 32 ± 1°C.
- Time after start of exposure: 2 hours in a liquid bath at 32 ± 1°C.

TOOL USED TO ASSESS OPACITY: Opacitometer

PERMEABILITY
- On completion of the opacity measurements, EMEM was removed from both chambers (anterior first).
- The posterior chamber was re-filled with fresh EMEM without phenol red maintained at 32 ± 1°C.
- The anterior chamber (epythelial surface) was treated with 1 ml aliquot of sodium fluorescein solution 0.4% in EMEM without phenol red maintained at 32°C ± 1°C. The fluorescein solution used for treatment was checked for concentration (3.864 mg/ml, using a reference curve 0-20 µg/ml - 96.6% of the theoretical concentration).
- The corneas were horizontally incubated in a liquid bath at 32 ± 1°C for approximately 90 minutes.
- The medium was removed from the posterior chamber (endothelial surface) and the optical density measure.

TOOL USED TO ASSESS OPACITY: Spectrophotometer set at 490 nm

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: in vitro irritancy score (IVIS)

Basis:

other: Mesityl oxide

Time point:

other: 10 min

Score:

41.2

Results 2

Irritation parameter:

other: in vitro irritancy score (IVIS)

Basis:

other: positive control

Time point:

other: 10 min

Score:

165.4

Results 3

Irritation parameter:

other: in vitro irritancy score (IVIS)

Basis:

other: negative control

Time point:

other: 10 min

Score:

0.35

Irritant / corrosive response data:

The test item induced a slight increase of the corneal opacity with a mean value equal to 11.0.
At the macroscopic observation the three corneas showed slight opacity.

With reference to the permeability, the permeability of the cornea after treatment with fluorescein was similar to that of positive control (slightly minor) with a mean observed OD490 value equal to 2.0114, thus indicating a significant alteration of corneal barrier.

Negative control gave the expected results.
The positive control induced opacity of the whole cornea surface with a mean increase of the opacity value equal to 133.0. The corneal permeability was also increased with a mean observed OD490 value equal to 2.1587. One of the three values was excluded since the OD490 value, 2.384, was over the upper limit of detection of this experiment (OD490 = 2.360 for a 20 µg/ml fluorescein solution).

Any other information on results incl. tables

Mesityl oxide: ASSESSMENT OF OCULAR SEVERE IRRITATION/CORROSION BOVINE CORNEAL OPACITY/PERMEABILITY (BCOP) ASSAY

  

Treatment code		Opacity Value		Macroscopic observation		OD490
N1		0*		No macroscopic changes		0,002
N2		1		No macroscopic changes		0,002
N3		0		No macroscopic changes		0,006
	Mean	0,3			Mean	0,0033
	SD	0,6			SD	0,0023
	CV %	200,0			CV %	69,70
P1		140		Cornea heavily opaque		1,974
P2		126		Cornea heavily opaque		2,384°
P3		134		Cornea heavily opaque		2,350
	Mean	133,3			Mean	2,1620
	SD	7,0			SD	0,2659
	CV %	5,3			CV %	12,30
Corrected Mean		133,0		Corrected Mean		2,1587
(negative control)				(negative control)
A1		13		Cornea slightly opaque		2,275
A2		9		Cornea slightly opaque		1,909
A3		12		Cornea slightly opaque		1,860
	Mean	11,3			Mean	2,0147
	SD	2,1			SD	0,2268
	CV %	18,6			CV %	11,26
Corrected Mean		11,0		Corrected Mean		2,0114
(negative control)				(negative control)

N1-3: Negative control

P1-3 : Positive control

A1-3: Test item

 

*: value read was -1; it was assumed to be 0 for mean calculation.

°: this value was outside the spectrophotometer range, and thus it is excluded from mean calculation

Applicant's summary and conclusion

Interpretation of results:

irritating

Remarks:

Migrated information category 2 Criteria used for interpretation of results: other: REGULATION (EC) No 1272/2008

Conclusions:

The treatment with the test item induced a slight effect on corneal opacity and a strong effect on corneal permeability.
According to the criteria stated in the OECD guideline for this test, the test item is not defined as corrosive or severely irritant to the eye.
However, since the calculated IVIS value is near to the threshold of severe irritancy/corrosion, the test item might have an irritating potential in vivo.

Executive summary:

The potential of the test item Mesityl oxide to cause corrosion/severe irritation by using the Bovine corneal opacity and permeability (BCOP) Assay was examined in agreement with OECD guideline no. 437 (adopted 7 September 2009). The test item was tested without any further dilution (being a liquid) on the epithelial surface of three idoneous bovine corneas for an exposure period of 10 minutes and a recovery period of 2 hours. Positive and negative controls [a 10% (w/v) sodium hydroxide (NaOH) solution and physiological saline alone, respectively] were concurrently tested in the same number of replicates at the same treatment volume (0.75 ml). The mean opacity detected at the end of the test item recovery period was 11.0. After the determination of opacity the epithelial surface of every cornea was treated with a 0.4% solution of sodium fluorescein in EMEM for 90 minutes to investigate alteration in permeability. Mean OD₄₉₀value of the medium from the endothelial surface side was 2.0114 (similar to the positive control mean value of 2.1587). Positive and negative controls gave the expected results, indicating the good functioning of the test system. These results indicate that the test item induces slight changes on corneal opacity and has a strong effect on corneal permeability. The calculatedin vitroirritancy score (IVIS) for the test item is 41.2. According to the criteria stated in the OECD guideline for this test, the test item is not defined as corrosive or severely irritant to the eye. However, since the calculated IVIS value is near to the threshold of severe irritancy/corrosion, the test item might have an irritating potential in vivo.