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Diss Factsheets

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:
30 Oct 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
adopted in 2009
Deviations:
no
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 2013
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
Test material form:
other: powder

Test animals / tissue source

Species:
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

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
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
Duration of post- treatment incubation (in vitro):
90 min at 32 ± 1°C
Number of animals or in vitro replicates:
3 corneas each for the test item, negative and positive controls
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
- 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

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

QUALITY CHECK OF THE ISOLATED CORNEAS
Corneas were visually checked for defects and any defective corneas were discarded. Corneas with an initial opacity reading above 7 units were discarded.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
0.9% NaCl

POSITIVE CONTROL USED
Imidazole (20% in 0.9 NaCl)

APPLICATION DOSE AND EXPOSURE TIME
Corneas were exposed to 750 µL of test substance and control substances for 4 h ± 5 min.

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes, 90 min at 32 ± 1°C

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The test substance was removed and the epithelium washed at least three times with MEM containing phenol red. Once the medium was macroscopically free of test substance, the cornea was finally rinsed with complete RPMI 1640 phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer (MC2, Clermont, France).
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490)
- Others (e.g, pertinent visual observations, histopathology): (please specify)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: Test substance with an IVIS ≥ 55.1 was regarded as severe irritant/corrosive and labelled Category 1 according to the decision criteria given in OECD 437.

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
Other effects / acceptance of results:
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 an 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: non-irritant to the eye according to Regulation (EC) No 1272/2008
Conclusions:
CLP: not classified
Under the conditions of the Bovine Corneal Opacity and Permeability (BCOP) Test the test item does not possess eye iritant properties.