Reaction mass of 1-O-α-D-glucopyranosyl-D-fructose and 6-O-α-D-glucopyranosyl-D-fructose and fructose and glucose and sucrose

Eye irritation

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 guideline

Test guideline 1

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

Test guideline 2

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

Test material information

Constituent 1

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

Results

Results 1

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)

Results 2

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)

Results 3

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)

Results 4

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)

Results 5

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)

Results 6

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)

Results 7

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)

Results 8

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)

Results 9

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.