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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
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)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
5-(2,3-dimethyltricyclo[2.2.1.02,6]hept-3-yl)-2-methylpent-2-en-1-ol, stereoisomer
EC Number:
204-102-8
EC Name:
5-(2,3-dimethyltricyclo[2.2.1.02,6]hept-3-yl)-2-methylpent-2-en-1-ol, stereoisomer
Cas Number:
115-71-9
Molecular formula:
C15H24O
IUPAC Name:
5-(2,3-dimethyltricyclo[2.2.1.0~2,6~]hept-3-yl)-2-methylpent-2-en-1-ol
Constituent 2
Chemical structure
Reference substance name:
[1S-[1α,2α(Z),4α]]-2-methyl-5-(2-methyl-3-methylenebicyclo[2.2.1]hept-2-yl)-2-penten-1-ol
EC Number:
201-027-2
EC Name:
[1S-[1α,2α(Z),4α]]-2-methyl-5-(2-methyl-3-methylenebicyclo[2.2.1]hept-2-yl)-2-penten-1-ol
Cas Number:
77-42-9
Molecular formula:
C15H24O
IUPAC Name:
2-methyl-5-(2-methyl-3-methylenebicyclo[2.2.1]hept-2-yl)pent-2-en-1-ol
Constituent 3
Chemical structure
Reference substance name:
(Z)-5-[(6R)-4,6-dimethyl-6-bicyclo[3.1.1]hept-3-enyl]-2-methylpent-2-en-1-ol
Cas Number:
176777-61-0
Molecular formula:
C15H24O
IUPAC Name:
(Z)-5-[(6R)-4,6-dimethyl-6-bicyclo[3.1.1]hept-3-enyl]-2-methylpent-2-en-1-ol
Constituent 4
Chemical structure
Reference substance name:
(E)-(R)-(+)-5-(2,3-dimethyltricyclo[2.2.1.0^2,6]hept-3-yl)-2-methyl-2-penten-1-ol
Cas Number:
14490-17-6
Molecular formula:
C15H24O
IUPAC Name:
(E)-(R)-(+)-5-(2,3-dimethyltricyclo[2.2.1.0^2,6]hept-3-yl)-2-methyl-2-penten-1-ol
Constituent 5
Chemical structure
Reference substance name:
2-​Hepten-​1-​ol, 2-​methyl-​6-​(4-​methylphenyl)​-​, (2Z)​-
Cas Number:
122442-36-8
Molecular formula:
C15H22O
IUPAC Name:
2-​Hepten-​1-​ol, 2-​methyl-​6-​(4-​methylphenyl)​-​, (2Z)​-
Test material form:
liquid
Specific details on test material used for the study:
Batch# APISO-130608SD/SA

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
The bovine eyes were received from Spear Products on 01 Aug 2013 and transported to MB Research in Hank's Balanced Salt Solution with Pennstrep in a refrigerated container.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Liquids were tested undiluted. Volume applied: 0.75 ml of each liquid (ethanol, MEM or liquid test article)
Duration of treatment / exposure:
10 (±1) minute exposure
Duration of post- treatment incubation (in vitro):
2 hours
Number of animals or in vitro replicates:
3 replicates for each treatment (ethanol, MEM or liquid test article)
Details on study design:
Test System
The bovine eyes were received from Spear Products on 01 Aug 2013 and transported to MB Research in Hank's Balanced Salt Solution with Pennstrep in a refrigerated container.
Pretest Procedure
Fresh assay solutions were prepared prior to use. MEM solution was prepared by stirring together one jar of MEM powder (sufficient to make one liter of solution), 2.2 g Sodium Bicarbonate, 0.292 g L-Glutamine, 10 ml of Fetal Bovine Serum (FBS) and brought to a final volume of 1000 mL with distilled water. The MEM solution was kept in a 32 °C (±1 °C) incubator for the duration of testing. Hanks Balanced Salt Solution (HBSS) was prepared by stirring together one jar of HBSS powder (sufficient to make one liter), 0.35 g Sodium Bicarbonate and brought to a final volume of 1000 mL with distilled water. HBSS was maintained at room temperature.
In addition, MEM solution with Phenol Red was prepared by stirring together 9.3 g MEM with Phenol Red (sufficient to make one liter), 2.2 g Sodium Bicarbonate, 0.292 g L-Glutamine, 10 mL Fetal bovine Serum and brought to a final volume of 1000 mL with distilled water. The MEM solution with Phenol Red was kept in a 32 °C (±1 °C) incubator for the duration of testing.
The eyes were examined prior to use on the day of dosing. Any eye with a cornea exhibiting evidence of vascularization, pigmentation, opacity or scratches was discarded. Corneas from eyes that were free of defects were dissected from the surrounding tissues. A 2 - 3 mm rim of sclera was left attached to each cornea. The corneas were then placed in a container of fresh HBSS.
The dissected corneas were mounted in specially designed holders that were separated into anterior and posterior chambers and filled separately. Each cornea was mounted allowing the epithelium of the cornea to project into the anterior chamber. The posterior chamber was filled with MEM solution ensuring contact with the endothelium. The anterior chamber was filled with MEM solution, ensuring contact with the epithelium. Each cornea was visually inspected again to ensure there were no defects.
The entire holder was incubated at 32 °C (±1 °C) and allowed to equilibrate for at least one hour but not longer than two hours.
A pre-exposure determination of opacity was made for each cornea by measuring each against the blank supplied with the opacitometer.
Following the equilibration, fresh pre-warmed MEM was added to both chambers and baseline opacity readings were taken for each cornea. Any corneas that showed macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or an opacity unit of greater than 7, was discarded. The mean opacity of all equilibrated corneas was calculated. Three corneas with opacity values close to the median value for all corneas were selected as negative (or solvent) control corneas. The remaining corneas were then distributed into treatment and positive control groups.
A pre-exposure determination of opacity was made for each cornea by measuring each against the blank supplied with the opacitometer.
Study Procedure
Liquids were tested undiluted. Following the pretest observations, the MEM solution was removed from the anterior chamber. A volume of 0.75 mL of the ethanol, MEM or liquid test article was applied to the epithelium of each of the three positive controls, three negative controls, or three test article-treated corneas in a manner, which ensured the entire cornea was covered.
All holders and corneas were placed in a horizontal position (anterior side up) in the 32 °C (±1 °C) incubator. After 10 (±1) minute, the test article, ethanol or MEM solution in the controls were removed from the epithelium of the cornea and anterior chamber of the holder by washing with MEM solution containing phenol red. The anterior and posterior chambers of the holders were refilled with fresh MEM solution. Opacity measurements were made following the 10-minute exposure and MEM solution refill.
All corneas were incubated at 32 °C (±1 °C) for an additional two hours at which time the MEM solution in the anterior and posterior chambers was removed and the holders refilled with fresh MEM solution. A measurement of opacity was taken with each treated cornea compared to the blank supplied with the OP-KIT. This is the reading that was used in the final in-vitro calculations.
Immediately following the 2-hour opacity measurement, the MEM solution was removed from the anterior chamber and replaced with 1.0 mL of 0.4% sodium fluorescein solution (in Dulbecco's Phosphate Buffered Saline) for liquid test articles and corresponding controls. Each holder was returned to the 32 °C (±1 °C) incubator in a horizontal position (anterior side up) ensuring contact of the fluorescein with the cornea.
After 90 minutes, the fluid from the posterior chamber was removed and the amount of dye, which passed through the cornea, was measured as the optical density at 490 nm by a plate reader or spectrophotometer. A 1:1000 dilution of the fluorescein was prepared and measured in the plate reader or spectrophotometer as a measure of consistency.
Analysis of Data
Individual corrected opacity scores were calculated by subtracting the pretest score from the two-hour score. Corrected mean opacity scores were calculated by averaging the individual two-hour corrected opacity scores for a given dose group and subtracting the mean opacity score for the negative control group. A corrected mean opacity score was not calculated for the negative control, rather only the mean of the individual two-hour corrected opacity scores (with no subtraction of mean opacity score for negative control).
Individual corrected optical densities were calculated by subtracting the mean optical density for the negative control group from the individual optical density values. Individual corrected optical densities were not calculated for the negative control group. Corrected mean optical densities were calculated by averaging the individual corrected optical density values for a given dose group. A corrected mean optical density was not calculated for the negative control, rather only the mean of the individual optical densities.
The In Vitro Irritancy Score (IVIS) for the test article and positive control were calculated by adding the corrected mean opacity score to fifteen times the corrected mean optical density as shown by the equation below:
in Vitro Score = Corrected Mean Opacity Score + 15(Corrected Mean Optical Density Score)
OECD Guideline #437 defines a substance, which produces an In Vitro score of ≥55.1 as a corrosive or severe irritant. IVIS < 55.1 is not a corrosive or severe irritant and additional testing should be conducted for classification and labelling purposes.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Run / experiment:
mean value, test item
Value:
3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
mean value, positive control (Ethanol)
Value:
21.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
mean value, negative control (MEM)
Value:
-1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
calculated value, test item
Value:
3.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
calculated value, positive control (Ethanol)
Value:
28.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
calculated score, negative control (MEM)
Value:
-0.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The corrected mean optical density (permeability) score for TEST ARTICLE was 0.033.
The corrected mean optical density (permeability) score for NEGATIVE CONTROL was 0.020
The corrected mean optical density (permeability) score for POSITIVE CONTROL was 0.486

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The corrected mean opacity score was 3. The corrected mean optical density (permeability) score was 0.033. The in vitro irritancy score (IVIS) was calculated as 3.5. TFS plantation-grown Indian sandalwood (santalum album) oil, Batch# APIS0-130608SD/SA is not corrosive or a severe irritant.
Executive summary:

Objective: To determine the potential for ocular irritation using an alternative to the Draize methodology. This protocol is based on the methodology described in the current OECD Guideline for the Testing of Chemicals #437.

Method Synopsis: Three bovine corneas per group were dosed with 0.75 ml of TFS plantation-grown Indian sandalwood (santalum album) oil, Batch# APISO-130608SD/SA, Minimal Essential Media (MEM) (negative control), or 100% Ethanol (positive control). Following a two-hour exposure for each group of dosed corneas, opacity measurements and sodium fluorescein permeability were determined.

Summary/Conclusion:

Test Article:The corrected mean opacity score was 3. The corrected mean optical density (permeability) score was 0.033. The in vitro irritancy score (IVIS) was calculated as 3.5. TFS plantation-grown Indian sandalwood (santalum album) oil, Batch# APIS0-130608SD/SA is not corrosive or a severe irritant.

Negative Control: The corrected mean opacity score was -1. The corrected mean optical density (permeability) score was 0.020. The IVIS was calculated as -0.70.

Positive Control: The corrected mean opacity score was 21.3. The corrected mean optical density (permeability) score was 0.486. The IVIS was calculated as 28.6.

All Controls were within normal limits.

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