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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:
key study
Study period:
01 Aug 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

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 09 Oct 2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Test material

Constituent 1
Reference substance name:
Vinasses, residue of fermentation
EC Number:
932-215-9
Molecular formula:
Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance).
IUPAC Name:
Vinasses, residue of fermentation
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 28 Jun 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Slaughterhouse Moksel AG, Buchloe, Germany
- Storage, temperature and transport conditions of ocular tissue: The isolated eyes were transported on ice in Hank's Buffered Salt Solution (HBSS) containing penicillin/streptomycin.
- Time interval prior to initiating testing: On the test day, fresh eyes were collected from the slaughter house. The corneae were prepared immediately after delivery.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were carefully examined for visible defects and defective eyes were discarded.
- Indication of any antibiotics used: penicillin/streptomycin containing HBSS was used for transport.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 µL

NEGATIVE CONTROL:
- Amount applied: 750 µL
- Concentration: 0.9% NaCl solution in deionised water
- Lot no: 18095403

POSITIVE CONTROL:
- Amount applied: 750 µL
- Concentration: 100%
- Lot no.: K50117583
Duration of treatment / exposure:
10 ± 1 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:
triplicates for each treatment and control groups
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
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 Hanks’ balanced salt solution (HBSS). A specifically designed corneal holder was used to mount each cornea.

QUALITY CHECK OF THE ISOLATED CORNEAS
After an equilibration period, the initial opacity was determined. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay.

TREATMENT METHOD: Closed chamber method
The corneas were mounted in a corneal holder with the endothelial side against the O-ring of the posterior chamber. The anterior chamber was then positioned on top of the cornea and tightened with screws. Starting with the posterior chamber, both chambers of the corneal holder were then filled with complete RPMI 1640 medium and the corneas were incubated for 1 h at 32 ± 1 °C. After the equilibration period an initial illuminescence reading the test item, the positive or the negative control was introduced into the anterior chamber (closed chamber method). The corneas were incubated at 32 ± 1 °C and exposed to the appropriate test substance for 10 ± 1 min.

REMOVAL OF TEST SUBSTANCE
After the incubation either the test item or the control substance was removed and the epithelium was washed at least three times with minimum essential medium (MEM) (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI medium (without phenol red).

METHODS FOR MEASURED ENDPOINTS
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer (BASF-OP3.0, Duratec GmbH). The light was measured as illuminance (I = luminous flux per area, unit: lux).
- Corneal permeability: The passage of sodium fluorescein dye was measured with the aid of a spectrophotometer (Jenway 6405 UV/VIS) at 490 nm (OD 490).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA
Test substance with an IVIS > 55 was considered as severe irritant/corrosive and labelled Category 1 according to CLP/EPA/GHS.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3; ≤ 55: no prediction can be made.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Remarks:
mean value of 3 corneae
Run / experiment:
10 min exposure
Value:
1.31
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY:
Technical proficiency of the test system was demonstrated with 12 substances according to OECD 437.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.
- Acceptance criteria met for positive control: Yes, the positive control resulted in an IVIS value which fell within two standard deviations of the current historical mean.



Any other information on results incl. tables

Table 1: Results on opacity

Cornea No. Test Item Initial Opacity Final Opacity Change of Corrected Opacity Value Opacity Value
1 Negative 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  
4 Positive 2.86 26.48 23.62 23.35
5 2.9 29.68 26.78 26.52
6 3.42 31.81 28.39 28.13
MV 3.06 29.32 26.26 26
7 Test Item 1.85 2.79 0.94 0.68
8 1.85 2.1 0.25 -0.01
9 2.39 5.66 3.27 3.01
MV 2.03 3.51 1.49 1.22

MV= mean value

Table 2: Results on permeability

Cornea No. Test Item OD490 Corrected OD490 Value
1 Negative 0.004
2 0.019
3 0.015
MV 0.013
4 Positive 1.262 1.249
5 1.585 1.572
6 1.289 1.276
MV 1.379 1.366
7 Test Item 0.018 0.005
8 0.027 0.014
9 0.01 -0.003
MV 0.018 0.006

MV= mean value

Table 3: Results in vitro irritation score (IVIS)

Cornea No. Test Item Corrected Opacity Corrected
OD490 Value
IVIS
1 Negative 0.32 0.004  
2 0.18 0.019  
3 0.29 0.015  
MV 0.26 0.013 0.45
4 Positive 23.35 1.249  
5 26.52 1.572  
6 28.13 1.276  
MV 26 1.366 46.49
7 Test Item 0.68 0.005  
8 -0.01 0.014  
9 3.01 -0.003  
MV 1.22 0.006 1.31

MV= mean value

Table 4: Historical mean In Vitro Irritation Score of the positive control

  IVIS Positive Control - Ethanol 100 %
Mean Value (MV)  48.41
Standard Deviation (SD) 9.25
MV- 2xSD  29.91
MV+2xSD 66.91
Number of Replicates providing Historical Mean: 56

Positive controls are updated after every single experiment or at least every 3 months.

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) 1272/2008.
Conclusions:
CLP: not classified