Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-07-10 to 2018-10-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

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)
Version / remarks:
adopted in October 2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Absolute of Jasmine (Jasminum grandiflorum) obtained from Jasmine concrete by ethanol extraction
EC Number:
948-021-2
Molecular formula:
n.a.
IUPAC Name:
Absolute of Jasmine (Jasminum grandiflorum) obtained from Jasmine concrete by ethanol extraction
Test material form:
liquid
Details on test material:
- Batch: AH17.001
- Purity: 100%
- Physical state: liquid
- Colour: pale yellow-orange to orange reddish-brown
- Storage Conditions: ≤ 15 °C
- Expiry date: 30 June 2019
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was used as provided by the sponsor.

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES:
- Source: Isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany. On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice.

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
Duration of treatment / exposure:
10 min at 32 ± 1 °C
Duration of post- treatment incubation (in vitro):
2 h at 32 ± 1 °C
Number of animals or in vitro replicates:
3 corneas each for the test item, negative control and positive control
Details on study design:
SELECTION AND PREPARATION OF CORNEAS / QUALITY CHECK OF THE ISOLATED CORNEAS
The eyes were carefully examined for defects and any defective eyes were discarded. The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1 °C.

APPLICATION DOSE AND EXPOSURE TIME
750 μL of the test substance or the control substance was introduced into the anterior chamber. After 10 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red).

TREATMENT METHOD: closed chamber
After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI medium. An initial measurement was performed on each of the corneas using the opacitometer. Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas. The illuminance of each cornea was read and recorded. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay. The medium was removed from the anterior chamber and replaced with the test item or control. 750 µL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method). After 10 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an illuminance measurement was performed. Also, each cornea was observed visually and pertinent observations were recorded. After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 4 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIS = mean opacity value + (15 x mean permeability OD490 value)

DECISION CRITERIA:
- IVIS ≤ 3: UN GHS No Category
- IVIS > 3 to ≤ 55: No prediction can be made
- IVIS > 55: UN GHS Category 1

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean of three replicates
Value:
2.76
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid. The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control. For details on the results see box "Any other information on results incl. tables"

Any other information on results incl. tables

Table 1: In vitro Irritation Score (IVIS)

Cornea No. Test Item Corrected Opacity Value  Corrected OD490
Value 
IVIS
1 Negative Control
Physiological Saline
0.32 0.004

0.45

2 0.18 0.019
3 0.29 0.015
MV 0.26 0.013
1 Positive Control
100% Ethanol
23.35 1.249

46.49

2 26.52 1.572
3 28.13 1.276
MV 26.00 1.366
1 Test Item
Jasmine Absolute
3.45 -0.005

2.76

2 1.57 -0.001
3 3.39 -0.003
MV 2.80 -0.003

MV = mean value

Table 2: Opacity

Cornea No. Test Item Initial Opacity  Final Opacity Change of Opacity Value Corrected Opacity Value 
1 Negative Control
Physiological Saline
1.92 2.24 0.32  
2 1.96 2.13 0.18
3 1.96 2.24 0.29
MV 1.94 2.21 0.26
1 Positive Control
100% Ethanol
2.86 26.48 23.62 23.35
2 2.90 29.68 26.78 26.52
3 3.42 31.81 28.39 28.13
MV 3.06 29.32 26.26 26.00
1 Test Item
Jasmine absolute
-0.63 3.08 3.72 3.45
2 1.36 3.20 1.83 1.57
3 0.19 3.84 3.65 3.39
MV 0.31 3.37 3.07 2.80

MV = mean value

Table 3: Permeability

Cornea No. Test Item OD490 Corrected OD490
1 Negative Control
Physiological Saline
0.004  
2 0.019
3 0.015
MV 0.013
1 Positive Control
100% Ethanol
1.262 1.249
2 1.585 1.572
3 1.289 1.276
MV 1.379 1.366
1

Test Item
Jasmine absolute

0.008 -0.005
2 0.012 -0.001
3 0.010 -0.003
MV 0.010 -0.003

MV = mean value

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, based on the mean in vitro irritation score of 2.76 obtained in the bovine corneal opacity and permeability assay (OECD 437), the test substance Jasmine absolute is classified into UN GHS "No Category".
Executive summary:

The eye irritation potential of Jasmine absolute (100% purity) was investigated in the bovine corneal opacity and permeability assay (OECD 437). The undiluted test item was applied to the corneas. A mean in vitro irritation score of 2.76 was determined. The positive control induced the appropriate responses, indicating the validity of the assay. Based on the obtained mean in vitro irritation score, Jasmine absolute is classified into "No Category" according to the UN GHS classification criteria.