3-Pentanone, 1-hydroxy-2-methyl-

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:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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)

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-hydroxy-2-methylpentan-3-one

EC Number:

939-893-5

Cas Number:

27970-79-2

Molecular formula:

C6 H12 O2

IUPAC Name:

1-hydroxy-2-methylpentan-3-one

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
Test-substance No.: 15/0439-1
Batch identification: J 1812

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protect against humidity ; no direct
sunlight

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: freshly slaughtered cattle; Schlachthof Mannheim, Schlachthofstr. 21, 68165
Mannheim, Germany
- Age of the animals: minimum 12 months, maximum 60
months
- indication of any existing defects or lesions in ocular tissue samples: no

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL of the undiluted liquid test substance

Duration of treatment / exposure:

approximately 10 minutes

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to
3 mm rim of sclera. Isolated corneas were mounted in cornea holders that consists of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed Eagle’s MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour.
After the equilibration period the medium in both chambers was replaced with fresh prewarmed medium and initial corneal opacity readings were taken for each cornea with an
opacitometer. Any corneas that showed macroscopic tissue damage or an opacity value < 544 opacity units1 were discarded. The remaining corneas were then distributed into negative control, positive control and treatment groups.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
de-ionized water

POSITIVE CONTROL USED
100% ethanol, 100% dimethylformamide

APPLICATION DOSE AND EXPOSURE TIME
750 μL of the undiluted liquid test substance was applied into the anterior chamber
Exposure time: approximately 10 minutes

POST-INCUBATION PERIOD: yes. 2 hours

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: the epithelium was washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red).
- POST-EXPOSURE INCUBATION:
The corneas were incubated for further 2 hours at about 32 °C.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Before measurement, each cornea was observed visually and observations were recorded. Final corneal opacity readings were taken for each cornea with an opacitometer.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry. Three aliquots per cornea were transferred to a 96-well microtiter plate and the optical density (OD490) was determined.


SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
A study is considered acceptable if the PC gives an IVIS that falls within two standard
deviations of the current historic mean.
The NC responses should result in opacity and permeability values that are not higher than the established upper limits.
Since the IVIS per treatment group is determined from the mean of three single corneas, the
variability between the corneas treated per test substance should be acceptably low.
In cases of borderline results in the first testing run, a second testing run should be considered (but not necessarily required), as well as a third one in case of discordant mean IVIS results between the first two testing runs. In this context, a result in the first testing run is considered borderline if the predictions from the 3 corneas were non-concordant, such that:
- 2 of the 3 corneas gave discordant predictions from the mean of all 3 corneas, OR,
- 1 of the 3 corneas gave a discordant prediction from the mean of all 3 corneas, AND the
discordant result was >10 IVIS units from the cut-off threshold of 55.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

mean

Value:

39.7

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

other: permeability score

Run / experiment:

mean

Value:

0.514

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

47.4

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

See "attached background material"

In vivo

Irritant / corrosive response data:

See "attached background material"

Any other information on results incl. tables

See "attached background material"

Applicant's summary and conclusion

Conclusions:

The borderline result of the BCOP Test (mean IVIS 47.4) does not clearly allow for the evaluation of risk of serious eye damage. On basis of the results of this study a potential of the
test substance to bear a risk of serious eye damage cannot be excluded.

Executive summary:

The potential of Hydroxymethylpentanon to cause ocular irritation or serious damage to the eyes was assessed by a single topical application of 750 μL of the undiluted test substance to the epithelial surface of isolated bovine corneas. Three corneas were treated with the test substance for 10 minutes followed by a 2-hours postincubation period. In addition to the test substance a negative control (NC; de-ionized water) and two positive controls (PC1: 100% ethanol / PC2: 100% dimethylformamide) were applied to three corneas each.

Corneal opacity was measured quantitatively as the amount of light transmitted through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance.

Hydroxymethylpentanon showed an IVIS of 47.4 in the BCOP Test. Therefore, a potential of the test substance to bear a risk of serious eye damage cannot be excluded, the study has to be considered inconclusive.