Esterification product of sunflower-oil fatty acids, with 1,4:3,6-dianhydro-D-glucitol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 6 January 2016 to 20 January 2016

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

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

1818326-42-9

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by the sponsor
- Expiration date of the lot/batch:August 2017
- Purity test date: August 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature and protected from light
- Stability under test conditions: not applicable, the test condition period was only 10 minutes
- Solubility and stability of the test substance in the solvent/vehicle: no vehicle was used
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test article was used as received
- Preliminary purification step (if any): not applicable
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: Not applicable

FORM AS APPLIED IN THE TEST (if different from that of starting material) Neat, as supplied

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Spear Products
- Number of animals: 9 corneas were used
- Characteristics of donor animals (e.g. age, sex, weight): not detailed
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): transported in HBSS medium with penicillin-streptomycin on the 7 January 2016 at the test facility
- Time interval prior to initiating testing: immediately after receipt
- indication of any existing defects or lesions in ocular tissue samples: no
- Indication of any antibiotics used: streptomycin and penicillin were used in medium

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): neat

VEHICLE
No vehicle was used

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

3 replicates per condition

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
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 32ºC (± 1º) and allowed to equilibrate for at least one hour but not longer than two hours.

QUALITY CHECK OF THE ISOLATED CORNEAS
A pre-exposure determination of opacity was made for each cornea by measuring each against the blank supplied with the opacitometer. Any cornea with a value greater than 7 units was discarded.

NUMBER OF REPLICATES
3 replicates were used per condition

NEGATIVE CONTROL USED
MEM Minimal Essential Medium

POSITIVE CONTROL USED
Ethanol

APPLICATION DOSE AND EXPOSURE TIME
750 µL for 10 minutes

TREATMENT METHOD: Close Chamber

POST-INCUBATION PERIOD: Yes 2 hours

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: he test article, ethanol or MEM solution was removed from the epithelium of the cornea and anterior chamber of the holder by washing with MEM solution containing phenol red. A final rinse was made with MEM without phenol red. The anterior and posterior chambers of the holders were refilled with fresh MEM solution.

- POST-EXPOSURE INCUBATION: All corneas were incubated at 32ºC (± 1º) 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.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: using OP-KIT
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- Others (e.g, pertinent visual observations, histopathology): (please specify)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
In Vitro Irritancy Score (IVIS) = Corrected Mean Opacity Score + (15 x Corrected Mean Optical Density Score)

DECISION CRITERIA: Criteria from OECD TG 437 was used

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Test Article - Calculated in vitro irritation score

Value:

1.03

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No damage

DEMONSTRATION OF TECHNICAL PROFICIENCY: The positive control showed technical proficiency to measure a positive result

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Conform
- Acceptance criteria met for positive control: Conform; The ethanol positive control In Vitro Irritancy Score was 22.56, which fell within the acceptance range of 20.43 – 34.27 (± 2 standard deviations of the historical mean).

Any other information on results incl. tables

Table 1 :Results

Cornea ID	Pretest Opacity Score	10-minute Opacity Score	2-Hour Opacity Score	Corrected Opacity Scores^1		OD490 nm (Permeability)	Corrected Optical Density^2
				10-minute	2-hour
10	0	1	2	1	2	0.018	0.001
11	0	2	1	2	1	0.015	-0.002
12	4	3	3	-1	-1	0.023	0.006
Corrected Mean Optical Density^3=							0.002
2-Hour Corrected Mean Opacity Score^4=							1.00

 

Table 2 Calculated In Vitro Irritation Scores

Test Article	Negative Control	Positive Control
HydraSynolIDL; IsosorbideDilinoleate
(INCI name proposed);	MEM	100% Ethanol
CAS# 1818326-42-9, Lot# CB 15026
1.00 + (15 x 0.002)	-0.33 + (15 x 0.017)	15.33 + (15 x 0.482)
1.00 + 0.03	-0.33 + 0.255	15.33 + 7.23
IVIS =   1.03	IVIS =  -0.07	IVIS =  22.56

 

Positive Control Historical Data
Mean IVIS	27.35
Standard Deviation	3.46
n	28

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of the study, the test item HydraSynol IDL; Isosorbide Dilinoleate induced an IVIS of 1.03. Hence, according to CLP criteria, no classification was required for the test substance for eye irritation.

Executive summary:

This GLP compliant In Vitro study was performed in order to determine the potential for ocular irritation of the test substance HydraSynol IDL; Isosorbide Dilinoleate according to OECD 437 method (BCOP Assay)

Three bovine corneas per group were dosed with 0.75 ml of HydraSynol IDL; Isosorbide Dilinoleate, Minimal Essential Medium as negative control and Ethanol as positive control condition. Following a 10-minute exposure for each group of dosed corneas, opacity measurements and sodium fluorescein permeability were determined.

2 hours of post incubation period was performed and measurements of opacity was taken with each treated cornea compared to the blank. After 90 minutes, the fluid from the posterior chamber was removed and the amount of dye, which passed through the cornea (permeability), was measured as the optical density at 490 nm by a spectrophotometer.

Calculated IVIS values are 1.0, -0.07 and 22.56 respectively for test substance, negative and positive control.

Under the experimental conditions of the study, the test item HydraSynol IDL; Isosorbide Dilinoleate induced an IVIS of 1.03. Hence, according to CLP criteria, no classification was required for the test substance for eye irritation.