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Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
30 Oct 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP-Guideline study, tested with the source substance isomaltulose (Cas# 13718-94-0). According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
BAYERISCHES LANDESAMT FÜR GESUNDHEIT UND LEBENSMITTELSICHERHEIT, LANDESINSTITUT FÜR ARBEITSSCHUTZ UND PRODUKTSICHERHEIT, München, Germany

Test material

Constituent 1
Chemical structure
Reference substance name:
6-O-α-D-glucopyranosyl-D-fructose
EC Number:
237-282-1
EC Name:
6-O-α-D-glucopyranosyl-D-fructose
Cas Number:
13718-94-0
Molecular formula:
C12H22O11
IUPAC Name:
6-O-alpha-D-glucopyranosyl-D-fructose
Details on test material:
- Name of test material (as cited in study report): Isomaltulose
- Analytical purity: ≥ 98%
- Physical state: fine white powder
- Lot/batch No.: L121291500
- Expiration date of the lot/batch: 8 April 2014
- Storage condition of test material: at room temperature

Test animals / tissue source

Species:
other: cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
IDENTIFICATION OF THE SOURCE OF THE EYES, STORAGE AND TRANSPORT CONDITIONS
- Source: Attenberger Fleisch GmbH & Co. KG, Munich, Germany
- Date of eye collection: 30 Oct 2012
- Transport medium and temperature conditions: Hanks´ Balanced Salt Solution (HBSS) containing 5% penicillin/streptomycin on ice

PREPARATION OF THE EYES (BEFORE EXPOSURE)
- Eyes free of defects (scratches, neovascularisation): yes
- Dissection of the eyes and treatment: corneas were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders.
- Type of cornea holder used: MC2, Clermont, France
- Description of the cornea holder: the cornea holders consist of an anterior and a posterior compartment, which interface with the epithelial and endothelial side of the cornea.
- Test medium and temperature conditions used in the cornea holder: RPMI 1640 without phenol red supplemented with 1% [v/v] fetal bovine serum and 2 mM L-glutamine
- Equilibration time: 1 h at 32 ± 1 °C in a water bath
- Quality check of the equilibrated corneas: initial opacity measurement; corneas with an initial opacity above 7 in the opacitometer were discarded.

DETERMINATION OF THE INITIAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer.
- Specification of the device: MC2, Clermont, France

Test system

Vehicle:
physiological saline
Controls:
other: 3 corneas each for the negative (physiological saline, 0.9% NaCl) and the positive control (20% imidazole)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied in the test: 750 µL
- Concentration (if solution): 20%

VEHICLE
- Amount(s) applied in the test: 750 µL
- Lot/batch no. (if required): 111214

POSITIVE SUBSTANCE
- Substance: imidazole
- Concentration: 20%
- Solvent: physiol. saline
- Amount(s) applied in the test: 750 µL
- Lot/batch no.: 109K5306V

Duration of treatment / exposure:
4 h ± 5 min at 32 ± 1 °C
Observation period (in vivo):
not applicable
Number of animals or in vitro replicates:
3 corneas for the test item
Details on study design:
TEST CONDITIONS
- Short description of the method used: closed-chamber method.
The test substance or control substances were introduced into the anterior chamber.

POST-EXPOSURE TREATMENT
- Removal of the test substance: The test substance was removed after 4 h incubation and the epithelium washed at least three times with MEM.
- Medium for washing the corneas: MEM containing phenol red
- Medium for final rinsing: RPMI 1640 without phenol red

DETERMINATION OF THE FINAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer.
- Time of determination: After refilling the anterior chamber with fresh RPMI 1640, the final opacity was measured.
- Specification of the device: MC2, Clermont, France

DETERMINATION OF THE CORNEAL PERMEABILITY:
- Method: Sodium fluorescein solution was added to the anterior chamber of the cornea holder while the posterior chamber was filled with fresh medium. The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured via UV/VIS spectrophotometry at 490 nm recorded as optical density (OD490).
- Amount and concentration of the dye: 1 mL sodium fluorescein solution (5 mg/mL)
- Incubation time: 90 min at 32 ± 1 °C

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
0.75
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

In vivo

Resultsopen allclose all
Irritation parameter:
other: opacity
Basis:
other: mean (out of all 3 eyes)
Time point:
other: 4 h
Score:
1
Reversibility:
other: not applicable
Remarks on result:
other: negative control (0.9% NaCl)
Irritation parameter:
other: opacity
Basis:
other: mean (out of all 3 eyes)
Time point:
other: 4 h
Score:
166.67
Reversibility:
other: not applicable
Remarks on result:
other: positive control (20% imidazole)
Irritation parameter:
other: opacity
Basis:
other: mean (out of all 3 eyes)
Time point:
other: 4 h
Score:
1.33
Reversibility:
other: not applicable
Remarks on result:
other: test item (20% isomaltulose)
Irritation parameter:
other: permeability
Basis:
other: mean (out of all 3 eyes)
Time point:
other: 5 h 30 min
Score:
0.017
Reversibility:
other: not applicable
Remarks on result:
other: negative control (0.9% NaCl)
Irritation parameter:
other: permeability
Basis:
other: mean (out of all 3 eyes)
Time point:
other: 5 h 30 min
Score:
1.846
Reversibility:
other: not applicable
Remarks on result:
other: positive control (20% imidazole)
Irritation parameter:
other: permeability
Basis:
other: mean (out of all 3 eyes)
Time point:
other: 5 h 30 min
Score:
0.028
Reversibility:
other: not applicable
Remarks on result:
other: test item (20% isomaltulose)
Irritation parameter:
other: IVIS
Basis:
other: mean (out of all 3 eyes)
Score:
1.26
Reversibility:
other: not applicable
Remarks on result:
other: negative control (0.9% NaCl)
Irritation parameter:
other: IVIS
Basis:
other: mean (out of all 3 eyes)
Score:
193.35
Reversibility:
other: not applicable
Remarks on result:
other: positive control (20% imidazole)
Irritation parameter:
other: IVIS
Basis:
other: mean (out of all 3 eyes)
Score:
0.75
Reversibility:
other: not applicable
Remarks on result:
other: test item (20% isomaltulose)
Irritant / corrosive response data:
Isomaltulose at a concenration of 20% (v/v) reached an in vitro irritation score of 0.75. According to the evaluation criteria, the test substance is not considerd as severe eye irritant.
The in vitro irritation score obtained with the positve control fell within the two standard deviations of the current historical mean and therefore the assay is considered to be valid.

Any other information on results incl. tables

Table 1: Opacity values

Parameter

Initial opacity

Final opacity

Opacity change

Mean opacity change of NC

Corrected opacity change

Mean opacity value

Negative control

(0.9% NaCl)

2

4

2

1

-

-

3

3

0

3

4

1

Positive control

4

155

151

-

150

165.67

4

183

179

178

4

174

170

169

Test substance

4

5

1

-

0

0.33

2

3

1

0

1

3

2

1

 

 

Table 2: Permeability values (optical density (OD) at 490 nm)

Parameter

OD490 change

Mean OD490 change of NC

Corrected OD490 change

Mean OD490 value

Negative control

(0.9% NaCl)

0.015

0.017

 

 

0.012

0.024

Positive control

1.812

 

1.795

1.846

1.906

1.889

1.870

1.853

Test substance

0.014

 

-0.003

0.028

0.035

0.018

0.086

0.069

 

 Table 3: Historical Data

 

IVIS (positive control)

 

Mean value (MV)

 

203.04

Standard Deviation (SD)

 

27.63

MV – 2x SD

 

147.79

MV + 2x SD

 

258.29

Applicant's summary and conclusion

Interpretation of results:
other: not corrosive/severe irritant
Conclusions:
There is regulatory acceptance in the EU that a substance can be considered a severe eye irritant (Serious eye damage Category 1/R41) based on a positive result in the Bovine Corneal Opacity and Permeability (BCOP) test. Negative in vitro corrosivity responses are not conclusive with respect to non-classification or classification as irritant (Category 2/R36) and shall therefore be subject to further evaluation.

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