4-O-α-D-glucopyranosyl-D-glucitol

Endpoint summary

Description of key information

Skin irritation: not irritating to skin according to the results of the key in vitro skin irritation study with the target substance Maltitol (Reliability 1 key study; GLP compliant; OECD 439 test guideline) (CitoxLab Hungary, 2018).

Eye irritation: not irritating to eyes according to the results of the key ex vivo eye irritation / corrosion study with the target substance Maltitol

(Reliability 1 key study; GLP compliant; OECD 438 test guideline (CitoxLab Hungary, 2018).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 14 August 2018 to 05 November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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: not applicable
- 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 system:

human skin model

Source species:

human

Cell type:

other: three-dimensional human epidermis model (EPISKINTM (SM))

Cell source:

other: adult donors

Source strain:

other: not applicable

Details on animal used as source of test system:

Not applicable (EPISKINTM (SM) kits).

Justification for test system used:

The EPISKINTM (SM) model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Remarks:

prior to test item application, distilled water was applied to the epidermal surface in order to improve further contact between test item and epidermis.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN TM (SM) model
- Tissue batch number(s): 18-EKIN-036
- Production date: 04 September 2018
- Shipping date: not specified
- Delivery date: not specified
- Date of initiation of testing: 05 September 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: at room temperature (23.4-25.3°C)
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: volume not specified, once (After the 15 minutes incubation time, the EPISKINTM (SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
- Observable damage in the tissue due to washing: not specified
- Modifications to validated SOP: none specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL (MTT working solution)
- Incubation time: 3 hours at 37°C
- Spectrophotometer: not specified
- Wavelength: 570 nm
- Filter: not specified
- Filter bandwidth: not specified
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.
EPISKINTM (SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma.
The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EPISKINTM (SM) test kits used in the present study) and were documented in an appendix to the study report.
NUMBER OF REPLICATE TISSUES: three replicates were used for the test item

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE : not applicable (the test material did not interact with MTT).

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
-The test item may be considered to be non-irritant to skin in accordance with UN GHS (No Category), if the mean relative viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours post incubation is more than (˃) to 50% of the mean viability of the negative controls.
-The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean relative viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours post incubation is less or equal (≤) to 50% of the mean viability of the negative controls.
-In case the test chemical is found to be non-corrosive, and shows tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%, the test chemical is considered to be irritant to skin in accordance with UN GHS Category 2.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: not applicable (not different from TG 439 recommendations).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg
- Concentration (if solution): not applicable

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): not applicable

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): 5% (w/v)

Duration of treatment / exposure:

15 minutes (± 0.5 min) at room temperature (23.4-25.3°C).

Duration of post-treatment incubation (if applicable):

42 hours + 3 hours (MTT test)

Number of replicates:

3 replicates were used for the test item.
3 negative controls and 3 positive controls were also run in the assay.
Furthermore, as the test item was coloured, 2 additional test item-treated living tissues were used for the non-specific OD evaluation.

Irritation / corrosion parameter:

% tissue viability

Remarks:

relative viability compared to the negative control

Run / experiment:

1 (mean three samples)

Value:

93.8

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: none specified.
- Direct-MTT reduction: no colour change (yellow colour) was observed after three hours of incubation of the test item in MTT working solution, the test material did not interact with MTT.
- Colour interference with MTT: The mean optical density (measured at 570 nm) of tissues was 0.011, Non Specific Colour % was calculated as 1.3%

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS: All thee parameters below met the acceptability criteria, therefore the study was considered to be valid.
- Acceptance criteria met for negative control: As no colour change (yellow colour) was observed after three hours of incubation of the test item in MTT working solution, the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary. The false estimation of viability can be excluded.
- Acceptance criteria met for positive control: As the test item was coloured (white), two additional test item-treated living tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of tissues was 0.011, Non Specific Colour % was calculated as 1.3%. This value was below 5%, therefore additional data calculation was not necessary (mean blank value was 0.048).
- Acceptance criteria met for variability between replicate measurements: The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 3.1%.
- Range of historical values if different from the ones specified in the test guideline:

Negative control (PBS) (number of cases 251)
Mean optical density (OD): 0.788 ± 0.129 (SD) (Min. OD: 0.573 and Max. OD: 1.362)

Positive control (5% (w/v) SDS solution) (number of cases 246):
Mean optical density (OD): 0.065 ± 0.041 (SD) (Min. OD: 0.019 and Max. OD: 0.354)

Table 1 : Optical Density (OD) and the calculated relative viability % of the samples

Substance	Optical Density (OD)			Viability (% RV)	Standard Deviation
		Measured	Blank corrected
Negative control (Phosphate buffered saline)	1	0.834	0.786	97.0	2.7
	2	0.866	0.818	100.9
	3	0.876	0.828	102.1
	mean	-	0.811	100.0
Positive Control (5% (w/v) SDS solution)	1	0.072	0.025	3.0	1.0
	2	0.076	0.029	3.5
	3	0.088	0.040	4.9
	mean	-	0.031	3.8
Test item (Maltitol)	1	0.807	0.760	93.7	3.1
	2	0.833	0.786	96.9
	3	0.783	0.736	90.7
	mean	-	0.760	93.8

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this in vitro skin irritation study, the test item Maltitol, is not irritant to skin. The test material is not classified as irritating to the skin 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 skin irritation potential of the test item Maltitol in vitro using the reconstructed human epidermis model EPISKINTM (SM). This test was performed according to the OECD Test Guideline No. 439.

Material and methods

Disks of EPISKINTM (SM) (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2, in a >95% humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2, in a >95% humidified atmosphere, protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). Two additional disks were used to provide an estimate of color contribution (NSCliving) from the test item. The test item did not react with MTT and therefore the use of additional controls was not necessary. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

Results

Following exposure with Maltitol, the mean cell viability was 93.8% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

Conclusion

In conclusion, under the experimental conditions of this in vitro skin irritation study, the test item Maltitol, is not irritant to skin. The test material is not classified as irritating to the skin 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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

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)

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

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).

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.

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

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

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

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

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

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

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

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".

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

 

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin irritation:

A reliable key in vitro skin irritation study is available on the target substance Maltitol (Reliability 1; OECD 439 test guideline; CitoxLab Hungary, 2018). This test was performed to assess the skin irritation potential of Maltitol using the reconstructed human epidermis model EPISKIN. Disks of were treated with the test item and incubated for 15 minutes at room temperature followed by a cell viability test (MTT). Positive and negative controls fulfilled the required acceptance criteria and the study was considered to be valid. The results showed that following exposure with Maltitol, the mean cell viability was 93.8% compared to the negative control. This was above the threshold of 50%, therefore Maltitol was considered as being non-irritant to skin.

In vivo supporting peer reviewed local irritation data (Reliability 4; CIR, 2008) are also available on the target substance tested diluted at approximately 70% (Unpublished reports). The primary dermal irritation of Maltitol diluted at 69.09% was evaluated in 8 rabbits using the abraded and non-abraded procedures. After 24 and 72h of exposure, skin reactions were evaluated according to the Draize scoring method. The Primary Irritation Index (PII) was 0.1 (i.e., none to weak irritant under the test conditions). In a cumulative skin irritation study, Maltitol diluted at 69.09% was evaluated in guinea pigs. The substance was applied once daily for 3 days. The skin reactions were evaluated 24h following each application. It was concluded that Maltitol at 69.09% was a non- to weak irritant under the test conditions. Human data were also peer reviewed (Unpublished reports). Human primary skin irritation tests were performed with Maltitol up to 69.09% in 24 h closed patch tests (53.2, 64.5 and 69.09% concentrations were tested). The test material was applied to an adhesive patch and placed on the intact forearm of healthy female volunteers for 24h. The plaster was removed and skin responses were scored. No positive skin reaction was observed in any volunteer for all the tested doses at 24h after application of the test material. Therefore, it was concluded that under the experimental conditions of the tests, Maltitol up to 69.09% did not have a skin irritation potential.

Additionally, reliable in vitro data (Reliability 1; OECD 431 and 439 test guidelines; Seibersdorf Labor GmbH, 2010) are available on the analogue substance Syrups, wheat, hydrolyzed starch (No CAS / EC 931-687-3). In the skin corrosion study, the test substance was applied to the human skin model EpiDerm for 3 minutes and 60 minutes followed by a cell viability test. Positive and negative controls were within the required acceptance criteria. The relative absorbance values for the test substance were 97.8% after 3mn exposure and 98.2% after 1h exposure, which were above the threshold for corrosion (50%). Therefore, Syrups, wheat, hydrolyzed starch was considered as a non-corrosive in the in vitro Human Skin Model Test. In the skin irritation study, the test substance was applied to the human skin model EpiDerm for 60 mn followed by a cell viability test. Positive and negative controls were within the required acceptance criteria. The mean relative absorbance value for the test item was 96.9%, which was above the threshold for irritation (50%). Therefore, Syrups, wheat, hydrolyzed starch was considered as a non-irritant in the in vitro Human Skin Model Test and is not classified for skin irritation /corrosion.

Based on the results of the key in vitro skin irritation study (supported by the in vivo peer reviewed data with diluted Maltitol and the similar in vitro results obtained with the structural analogue Syrups, wheat, hydrolyzed starch), the registered substance Maltitol is not irritating to skin (not classified for skin irritation / corrosion).

Eye irritation:

A reliable key ex vivo eye irritation study is available on the target substance Maltitol (Reliability 1; OECD 438 test guideline; CitoxLab Hungary, 2018). This test was performed to assess the eye irritation potential of the test item Maltitol using isolated chicken’s eyes. In the study, three test item treated eyes were examined in each of the two experiments (as the test item was solid, negative results were confirmed by a second experiment according to the recommendations of the OECD No. 438 guideline), furthermore three positive control treated eyes and one negative control treated eye were examined in each experiment. The negative and positive control results were within the historical control data range. The results obtained in both experiments showed no corneal swelling during the four-hour observation period on all test item treated eyes, no significant corneal opacity change and no significant fluorescein retention change on test item treated eyes. No other corneal effect was observed in both experiments. Therefore, it was concluded that Maltitol is not irritant to eyes.

In vivo supporting peer reviewed eye irritation data (Reliability 4; CIR, 2008) are also available on the target substance tested diluted at approximately 70% (Unpublished report). The test material (at 69.09%) was instilled into one eye of each rabbit animal without irrigation. The other eye remained untreated and served as control. The eye reactions were evaluated according to the Draize scoring method. The eye irritation index of the test sample was 2.0 at 4h following instillation of test sample. It was therefore concluded that Maltitol at 69.09% is a not irritant to rabbit eyes under the test conditions.

Additionally, reliable in vitro and in vivo data (Reliability 1; OECD 437 and 405 test guidelines; Seibersdorf Labor GmbH, 2010) are available on the analogue substance Syrups, wheat, hydrolyzed starch (No CAS / EC 931-687-3). In the ex vivo study, bovine corneas were exposed to the test substance at a concentration of 20% in deionised water for 4h. An in vitro irritancy score (IVIS) was calculated. Both the negative and positive control results were within the historical control data range. A mean IVIS of 1.1 was reported for the test substance. According to the experimental conditions of this study and based on the latest updated version of the OECD 437 guideline, the results showed that the analogue substance Syrups, wheat, hydrolyzed starch is not irritant to eyes at 20%. In the in vivo eye irritation study, the analogue substance Syrups, wheat, hydrolyzed starch was instilled undiluted into the conjunctival sac of the left eye of each of 3 rabbits. Animals were exposed to the test substance for 24h and observed over a 72h period. No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred. No effects on the corneas, irises and conjunctivae were observed at any of the observation time points (i.e., 24, 48 and 72h). Based on the results of this study, it was concluded that the analogue substance Syrups, wheat, hydrolyzed starch was not irritant to rabbit eyes.

Based on the results of the key ex vivo eye irritation study performed with the target substance (supported by the in vivo peer reviewed data with diluted Maltitol and the similar ex vivo and in vivo results obtained with the structural analogue Syrups, wheat, hydrolyzed starch), the registered substance Maltitol is not irritating to eyes (not classified for eye irritation).

Justification for classification or non-classification

Skin irritation: According to the negative results of the in vitro skin irritation key study (OECD 439 test guideline, CitoxLab Hungary, 2018), the substance Maltitol is not classified for skin irritation according to the CLP criteria.

Eye irritation: According to the negative results of the ex vivo key study (OECD 438 test guideline, CitoxLab Hungary, 2018), the substance Maltitol is not classified for eye irritation according to the CLP criteria.