4-O-α-D-glucopyranosyl-D-glucitol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 14 August 2018 to 18 October 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-O-α-D-glucopyranosyl-D-glucitol

EC Number:

209-567-0

EC Name:

4-O-α-D-glucopyranosyl-D-glucitol

Cas Number:

585-88-6

Molecular formula:

C12H24O11

IUPAC Name:

4-O-α-D-glucopyranosyl-D-glucitol

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by the sponsor; Batch/Lot number: ELEL7
- Expiration date of the lot/batch: 07 October 2022

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: not applicable
- Specific activity: not applicable
- Locations of the label: not applicable
- Expiration date of radiochemical substance: not applicable

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: controlled room temperature
- Stability under test conditions: not applicable
- Solubility and stability of the test substance in the solvent/vehicle: The solubility of the test item in physiological saline was tested prior to the Experiment I (30 mg test material in 1 mL physiological saline). The test item dissolved in physiological saline.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: none
- Final preparation of a solid: none

FORM AS APPLIED IN THE TEST (if different from that of starting material): tested as supplied

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): not applicable

OTHER SPECIFICS: none

Test animals / tissue source

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. (Address: 9600 Sárvár, Rábasömjéni út. 129., Hungary)
- Number of animals: not specified
- Characteristics of donor animals (e.g. age, sex, weight): from chickens (approximately 7 weeks old) which are used for human consumption.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): after collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box).
- Time interval prior to initiating testing: the heads were received at the testing laboratory and processed within 2 hours of collection in each experiment.
- indication of any existing defects or lesions in ocular tissue samples: after removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.
- Indication of any antibiotics used: not specified (chicken heads were collected after slaughter in a commercial abattoir from chickens which are used for human consumption).

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg
- Concentration (if solution): not applicable

VEHICLE
- Not applicable

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

Not applicable

Number of animals or in vitro replicates:

For both experiments (I and II): One eye was treated with negative control, three eyes with the test item and another three eyes with positive control.

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.
The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained

EQUILIBRATION AND BASELINE RECORDINGS
- The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoid too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity. The appropriate number of eyes was selected and after being placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.
- At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5% within the -45 min and the zero time. No changes in thickness (0.0%) were observed in the eyes in each experiment. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

NUMBER OF REPLICATES
One eye was treated with negative control, three eyes with the test item and another three eyes with positive control (experiments I and II).
The test item Maltitol showed no significant corneal effect in the first experiment. As the test item was solid, the negative results were confirmed by a second experiment according to the recommendations of the OECD No. 438 guideline.

NEGATIVE CONTROL USED
Physiological saline (Salsol solution, 0.9% (w/v) NaCl)

SOLVENT CONTROL USED (if applicable)
not applicable

POSITIVE CONTROL USED
Imidazole

APPLICATION DOSE AND EXPOSURE TIME
- After the zero reference measurements, the eye in its retainer was taken out of the chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position, while the test material was applied onto the centre of the cornea. In each experiment, 30 mg of the test item was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test item, taking care not to damage or touch the cornea.
In each experiment a negative control eye was treated with 30 μL of physiological saline; positive control eyes were treated with 30 mg powdered imidazole.
- The time of application was noted, the exposure period from the end of the application on the the cornea surface was 10 seconds.

OBSERVATION PERIOD
- The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: after an exposure period of 10 seconds from the end of the application, the cornea surface was rinsed thoroughly with 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material if possible. Additional gentle rinsing with at least 20 mL saline was performed at each time point when the positive control material remaining on the cornea was observed in each experiment. The positive control material treated eyes were rinsed additional gentle rinsing with five times with 3x20 mL physiological saline in each experiment.
- Indicate any deviation from test procedure in the Guideline

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal thickness and corneal opacity were measured at all time points.
- Damage to epithelium based on fluorescein retention: Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse.
- Swelling: measured with Haag-Streit BP 900® slit-lamp microscope (Depth-measuring device no. I; Slit-width setting: 9.5).
- Macroscopic morphological damage to the surface: morphological effects include “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test substance to the cornea. These findings can vary in severity and may occur simultaneously. The classification of these findings is subjective according to the interpretation of the investigator.
- Others (e.g, histopathology): at the end of the procedure, the corneas from the eyes were carefully removed from the eyes and placed individually into labelled containers of preservative fluid (10% (v/v) neutral buffered formalin) for potential histopathology and stored at room temperature.

SCORING SYSTEM:
- Mean corneal swelling (%)
- Mean maximum opacity score
- Mean fluorescein retention score at 30 minutes post-treatment

DECISION CRITERIA:
The conclusion on eye irritancy was based on the OECD No 438 guideline quantitative assessments. The mean maximum corneal swelling up to 240 min, the mean maximum corneal opacity and the mean fluorescein retention ICE classes are used for CLP and GHS classification.
See Tables 1-3 in the "any other information on materials and methods incl.tables" below.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Remarks:

mean maxiumum

Run / experiment:

I

Value:

0.33

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

fluorescein retention score

Remarks:

mean

Run / experiment:

I

Value:

0.33

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

percent corneal swelling

Remarks:

mean maxiumum at up to 75 min

Run / experiment:

I

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation parameter:

percent corneal swelling

Remarks:

mean maxiumum at up to 240 min

Run / experiment:

I

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 5

Irritation parameter:

cornea opacity score

Remarks:

mean maximum

Run / experiment:

II

Value:

0.33

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 6

Irritation parameter:

fluorescein retention score

Remarks:

mean

Run / experiment:

II

Value:

0.17

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 7

Irritation parameter:

percent corneal swelling

Remarks:

mean maxiumum at up to 75 min

Run / experiment:

II

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 8

Irritation parameter:

percent corneal swelling

Remarks:

mean maxiumum at up to 240 min

Run / experiment:

II

Value:

1.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: the positive control material was stuck on all cornea surfaces after the post-treatment rinse, the cornea surfaces were not cleared at 240 minutes after the post-treatment rinse in each experiment. No other morphological effect was observed in the study.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Imidazole, used as positive control, showed positive response to the ICE test as expected.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control Physiological saline was not classified for eye irritation or serious eye damage as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS).
- Acceptance criteria met for positive control: The positive control (Imidazole) was classified as inducing serious eye damage (Category 1) as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS).
- Range of historical values if different from the ones specified in the test guideline: The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical control data range in each experiment. The number of eyes used for each treatment group was adequate. These experiments were considered to be valid.

All the results are detailed in the section "any other information on results incl. tables".

Any other information on results incl. tables

1. TEST ITEM RESULTS

The test item Maltitol showed no significant corneal effect in the first experiment. As the test item was solid, the negative results were confirmed by a second experiment according to the recommendations of the OECD No. 438 guideline. The second experiment confirmed the negative results

Experiment I
Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.0%	I
Mean maximum corneal swelling at up to 240 min	0.0%	I
Mean maximum corneal opacity	0.33	I
Mean fluorescein retention	0.33	I
Other Observations	None
Overall ICE Class	3xI

 

Experiment II
Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.0%	I
Mean maximum corneal swelling at up to 240 min	1.1%	I
Mean maximum corneal opacity	0.33	I
Mean fluorescein retention	0.17	I
Other Observations	None
Overall ICE Class	3xI

 

2. POSITIVE CONTROL

Experiment I
Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	9.0%	II
Mean maximum corneal swelling at up to 240 min	24.3%	III
Mean maximum corneal opacity	4.00	IV
Mean fluorescein retention	3.00	IV
Other Observations	Imidazole was stuck on all cornea surfaces after the post-treatment rinse (3/3).The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.
Overall ICE Class	1xIII 2xIV

 

Experiment II
Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	11.2%	II
Mean maximum corneal swelling at up to 240 min	26.6%	III
Mean maximum corneal opacity	4.00	IV
Mean fluorescein retention	3.00	IV
Other Observations	Imidazole was stuck on all cornea surfaces after the post-treatment rinse (3/3).The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.
Overall ICE Class	1xIII 2xIV

 

3. NEGATIVE CONTROL

Experiment I
Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.0%	I
Mean maximum corneal swelling at up to 240 min	0.0%	I
Mean maximum corneal opacity	0.00	I
Mean fluorescein retention	0.00	I
Other Observations	None
Overall ICE Class	3xI

 

Experiment II
Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.0%	I
Mean maximum corneal swelling at up to 240 min	0.0%	I
Mean maximum corneal opacity	0.00	I
Mean fluorescein retention	0.00	I
Other Observations	None
Overall ICE Class	3xI

 

4. Historical Control data (updated on 23 January 2018)

Negative Control: Physiological Saline

 

Observation	Min. Value	Max. Value
Maximum corneal swelling at up to 75 min	-3.2 %	3.4 %
Maximum corneal swelling at up to 240 min	-4.8 %	3.4 %
Maximum corneal opacity change	0.00	0.50
Fluorescein retention	0.00	0.50
Number of studies	358

 Positive Control: Imidazole

 

Observation	Min. Value	Max. Value
Maximum corneal swelling at up to 75 min	-6.6 %	25.0 %
Maximum corneal swelling at up to 240 min	-15.9 %	36.7 %
Maximum corneal opacity change	3.50	4.00
Fluorescein retention	2.00	3.00
Number of studies	157

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test item Maltitol showed no significant corneal effect in the first experiment. As the test item was solid, the negative results were confirmed by a second experiment according to the recommendations of the OECD No. 438 guideline. The second experiment confirmed the negative results. Therefore, Maltitol is not irritant to eyes and is not classified for eye irritation or serious eye damage according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) criteria.

Executive summary:

This GLP compliant study was performed to assess the eye irritation potential of the test item Maltitol ex vivo using isolated chicken’s eyes. This test was performed according to the OECD Test Guideline No. 438 (25 June 2018).

Material and methods

In each experiment after the zero reference measurements, the eye was held in horizontal position and 30 mg test item was applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl solution). In the study, three test item treated eyes were examined in each experiment, furthermore three positive control treated eyes and one negative control treated eye were examined in each experiment.

Results

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in each experiment. The number of eyes used for each treatment group was adequate. Thus, the experiments were considered to be valid.

Experiment I: No corneal swelling was observed during the four-hour observation period on all test item treated eyes. No significant corneal opacity change (severity 0.0 on one test item treated eye or severity 0.5 on two test item treated eyes) was noted on test item treated eyes. No significant fluorescein retention change (severity 0.0 on one test item treated eye or severity 0.5 on two test item treated eyes) was noted on test item treated eyes. No other corneal effect was observed.

Experiment II: No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on all test item treated eyes. No significant corneal opacity change (severity 0.0 on one test item treated eye or severity 0.5 on two test item treated eyes) was noted on test item treated eyes. No significant fluorescein retention change was observed (severity 0.5 on one test item treated eye or severity 0.0 on two test item treated eyes) on test item treated eyes. No other corneal effect was observed.

Conclusion

Under the experimental conditions of this study, the test item Maltitol showed no significant corneal effect in the first experiment. As the test item was solid, the negative results were confirmed by a second experiment according to the recommendations of the OECD No. 438 guideline. The second experiment confirmed the negative results. Therefore, Maltitol is not irritant to eyes and is not classified for eye irritation or serious eye damage according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) criteria.