1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl 4-chloro-4-deoxy-α-D-galactose

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 December 2017 - 01 May 2018

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

Test guideline 1

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:

2013

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl 4-chloro-4-deoxy-α-D-galactose

EC Number:

259-952-2

EC Name:

1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl 4-chloro-4-deoxy-α-D-galactose

Cas Number:

56038-13-2

Molecular formula:

C12H19Cl3O8

IUPAC Name:

1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl 4-chloro-4-deoxy-α-D-galactose

Test material form:

solid: crystalline

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: MC16B92011
- Expiration date of the lot/batch: 31 Jan 2018
- Purity test date: 01 Feb 2018; 100.1%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15-25 degrees Celsius, protected from light

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: 20% w/v solution in 0.9% sodium chloride solution

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Local abattoir
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were removed after slaughter, completely immersed in Hank's Balanced Salt Solution (containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL) and transported on same day to testing facility. Upon arrival at test facility, corneas were excised from the eyes and loaded onto holders. Both chambers of each holder were filled with Minimal Essential Medium (MEM) and incubated at 32+/- 1 degrees Celsius for at least 1 hour.
- Time interval prior to initiating testing: Within 24 hours after receipt at test facility
- indication of any existing defects or lesions in ocular tissue samples: On arrival at the test facility the eyes were carefully examined for defects including increased opacity, scratches and neovascularisation. Only corneas free from such defects were used.; opacity was measured using opacitometer to look for scratches or increased neovascularization
- Indication of any antibiotics used: Yes; 100 IU/mL penicillin and 100 ug/mL streptomycin

Test system

Vehicle:

other: 0.9% sodium chloride solution

Remarks:

Test substance was dissolved in sodium chloride but study indicated that this was not a vehicle control but a negative control.

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:

4 hours

Duration of post- treatment incubation (in vitro):

1 hour 25 minutes

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Corneas were excised from the eyes and loaded onto specifically designed holders. Both chambers of each holder (posterior and anterior) were filled with warmed Minimal Essential Medium (MEM). Holders were incubated at 32 +/- 1 degress Celsius for at least 1 hour. After incubation, the media was removed from both chambers and fresh media was added.
QUALITY CHECK OF THE ISOLATED CORNEAS
The opacity of each cornea was measured using an opacitometer. Any corneas found to have scratches, increased neovascularization or an opacity of > 7 opacity units when examined prior to treatment were discarded.
NUMBER OF REPLICATES
3
NEGATIVE CONTROL USED
0.9% sodium chloride solution
SOLVENT CONTROL USED (if applicable)
None
POSITIVE CONTROL USED
20% w/v Imidazole in 0.9% sodium chloride solution
APPLICATION DOSE AND EXPOSURE TIME
750 µL of test material was applied to each of 3 corneas followed by 4 hour incubation at 32 degrees Celsius
TREATMENT METHOD: open chamber
POST-INCUBATION PERIOD: Yes. If YES please specify duration
1 hour and 25 minutes
REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Each cornea was washed with media containing phenol red (as a pH indicator) until the indicator showed no pH effect occurring. The corneas were then washed once in media without phenol red so that corneal opacity could be measured.
- POST-EXPOSURE INCUBATION:
After washing to remove test substance, each cornea was then incubated in vertical position in media and sodium fluroescein for 1 hour and 25 minutes.
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer
- Corneal permeability: Passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490)
- Others (e.g, pertinent visual observations, histopathology): Visual, but no histopathology was conducted

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IIVS = mean opacity value + (15 X mean permeabiliity value)
DECISION CRITERIA:
-Serious eye damage if IVIS greater than equal to 55
-Not classified for eye irritation if IVIS is equal to or less than 3
-If IVIS is greater than 3 and less than or equal to 55, no prediction can be made.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean

Value:

-0.003

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

0

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

-0.04

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Yes
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values if different from the ones specified in the test guideline: OK

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

To determine whether the test item causes serious eye damage or does not require classifying for eye irritation a GLP study according to OECD 437 was carried out. Under the conditions of this assay, the test substance produced an IVIS score of -0.04 and does not require classification for eye irritation.