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Toxicological information

Eye irritation

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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: GLP and guideline study

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 Guideline for Testing of Chemicals No. 437, September 07, 2009 (“Bovine Corneal Opacity and Permeability Test, Method for Identifying Ocular Corrosives and Severe Irritants”)
GLP compliance:
yes (incl. QA statement)
Remarks:
testing lab.

Test material

Constituent 1
Chemical structure
Reference substance name:
Isopulegol
EC Number:
201-940-6
EC Name:
Isopulegol
Cas Number:
89-79-2
Molecular formula:
C10H18O
IUPAC Name:
isopulegol
Details on test material:
- Test substance : Cyclohexanol, 5-methyl-2-(1-methylethenyl)-, (1R,2S,5R)-
- Name of test material (as cited in study report): L-Isopulegol
- Physical state: liquid, colorless clear
- Analytical purity: 99.4 corr.area-%
- Lot/batch No.: Muster 7 CH
- Expiration date of the lot/batch: 01 Jun 2012
- Storage condition of test material: room temperature, under N2
- Other: pH-value approx. 5 (undiluted test substance)

Test animals / tissue source

Species:
other: isolated bovine corneas
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
The test system (target tissue) is the isolated bovine cornea. Bovine eyes are obtained as a by-product of freshly slaughtered cattle (age of the animals: minimum 12 months, maximum 60 months).

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: not applicable
Amount / concentration applied:
singe application of 750 µl the undiluted liquid test substance
Duration of treatment / exposure:
10 minutes followed by a 2-h post-incubation period
Observation period (in vivo):
not applicable
Number of animals or in vitro replicates:
not applicable
Details on study design:
Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in specially designed corneal holders that consists of anterior and posterior chambers and were equilibrated in a vertical position at about 32°C for at least 1 hour. Initial corneal opacity readings were taken for each cornea with an opacitometer (Kit BASF-OP2.0, BASF Ludwigshafen, self-construction) to exclude damaged corneas. Corneas with opacity values close to the median value of all corneas were selected as negative control. The remaining corneas were then distributed into treatment and positive control groups.
750 μL of the undiluted liquid test substance, highly deionized water (negative control, NC) or 1% (w/v) solution of sodium hydroxide in highly de-ionized water (positive control, PC) was applied into the anterior chamber using a pipette. Each treatment group consisted of 3 corneas.
The corneas were incubated in a horizontal position at about 32°C for approximately 10 minutes. The test substance, NC and PC were then removed from the anterior chamber using a syringe and the epithelium was washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red). Both chambers were then refilled with fresh Eagle’s MEM (without phenol red). The corneas were incubated for further 2 hours at about 32°C.
Before measurement, each cornea was observed visually and observations were recorded. Final corneal opacity readings were taken for each cornea with an opacitometer.
For determination of permeability the medium in the anterior chamber was replaced by 1 mL sodium fluorescein solution (4 mg/mL) and incubated for 90 ± 5 min in a horizontal position at about 32°C. The amount of sodium fluorescein that permeated through the corneas into the posterior chamber was measured spectrophotometrically.

Evaluation criteria:
IVIS (In Vitro Irritancy Score) >55 -> risk of serious damage to the eyes;
IVIS <=55 -> no risk of serious damage to the eyes

• IVIS per treatment group = mean opacity value + 15 * mean permeability OD value

Acceptance criteria:
A study is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean.
The negative control responses should result in opacity and permeability values that are less 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. If the prediction is not clearly identified in all corneas, the test will be repeated.

Results and discussion

Any other information on results incl. tables

IVIS (per treatment group)/SD:       27.1 / 3.1 (test substance)

IVIS (per treatment group)/SD:       144.7 / 11.3 (PC)

IVIS (per treatment group)/SD:       0 / 0.6 (NC)

Applicant's summary and conclusion

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