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Description of key information

OECD 439 in vitro SkinEhic RHETM (Reconstructed Human Epidermis) Model

OECD Guideline 437 in vitro (Bovine Corneal Opacity and Permeability Test Method

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:
2017-11-2 to 2017-12-12
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)
GLP compliance:
yes
Specific details on test material used for the study:
Name of substance : MIRAMER SB
α-d-Glucopyranoside, β-d-fructofuranosyl,
benzoate
Supplier : Miwon Specialty Chemical Co.,Ltd
KTR code : TS-00832
Cas No. : 12738-64-6
Lot No. : 161228BO5
Purity : 100 %
Physical description : Flake (Solid)
Storage condition : Room temperature[(1 – 30 C°)]
Test system:
other: Reconstructed human epidermal (RHE) model
Source species:
human
Justification for test system used:
SkinEthic RHE tissue model is commonly used for in vitro skin irritation test,
Details on test system:
Quality assurance results for viability, barrier function and morphology were provided by manufacturer of skin (EPISKIN)
Culture condition:
Temperature 37 ± 1 °C
CO2 gas 5 ± 1 %
Humidity Under moist atmosphere
Incubator CO2 incubator (Thermo, Forma 3121)
The temperature and humidity during the test period were measured by COBEX recorder of a CO2 incubator.

Culture medium:
The culture medium used during the test period was provided by the manufacturer (EPISKIN)
Name Maintenance medium
Lot No. 17 SMM 022

Name Growth medium
Lot No. 17 SGM 033

SkinEthic RHE tissue model observation:
Before the beginning of incubation, each insert containing the RHA tissue was checked whether tissue surface were even and excess moisture were present and the seleted a tissue that was free of defects.

Tissue preparation:
Each insert containing the RHE tissue was removed from the agarose gel and transferred to the 6-well plate with 1 ml of growth medium. the plate was incubated at 37 °Cm 5% CO2 incubator for 2 hours.

Application of test substance:
18 ul or 10 ul water with 16 mg test substance was topically applied to the upper epithelial surface og the tissue with intervals of 1 minutes

Rinsing of test substances
According to the test substance application interval, treated RHE tissues was washed with PBS solution. The RHE tissue was washed 25 times (1 ml/time) to remove all residual test subtsnace and nylon mesh.

Post incubation:
Washed RHE tissue was transferred to the 6-well plate with 2 ml of groeth medium. The plate was incubated at 37 °C, 5% CO2 incubator for 42 hours.

Cell viabilty tests were performed on cells
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
16 mg 100% sucrose benzoate applied with an interval of 1 minute
Duration of treatment / exposure:
42 minutes
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
RHE model
Value:
72.5
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

The absorbance value of the negative control substance was 1.079. The result was within the acceptable range (0.8 ≤ OD ≥ 3.0) under the skin iiritaion test using SkinEhic RHE model.

The cell viability of the positive control group was 1.6 ± 0.2%

The cell viability of the test subtance group was 72.5 ± 4.5 %

Interpretation of results:
GHS criteria not met
Conclusions:
Sucrose benzoate is not a skin irritant
Executive summary:

This study was conducted to assess the toxic potential of skin irritation of MIRAMER SB in SkinEhic RHETM (Reconstructed Human Epidermis) Model. The test results were evaluated as follows.

The test was performed in three groups; negative control, positive control, and test substance group.

Direct dyeing and non-specific reduction of MTT were not observed on the Test substance - The absorbance value of the negative control group and the cell viability value of the positive control group were satisfied the acceptance criterion.

- The cell viability of positive control group (Group 2) was calculated as 1.6 ± 0.2 %, and for the test substance group (Group 3), test substance was calculated as 72.5 ± 4.5 %.

As a result of in vitro skin irritation test in MIRAMER SB in SkinEhic RHETM (Reconstructed Human Epidermis) Model, cell viability has exceeded the reference value of 50 %. Therefore It was considered to be a non-irritant substance corresponding to 'No category' based on the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).

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:
Nov 13th to Dec 1st 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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)
GLP compliance:
yes
Specific details on test material used for the study:
Name : MIRAMER SB
(α-d-Glucopyranoside, β-d-fructofuranosyl, benzoate)
Supplier : Miwon Specialty Chemical Co.,Ltd
KTR Code : TS-00832
Cas No. : 12738-64-6
Lot No. : 161228BO5
Purity : 100 %
Physical description : Flake (Solid)
Storage condition : Room temperature[(1 - 30)C°]

Species:
cattle
Details on test animals or tissues and environmental conditions:
Bovine eyes were collected by slaughterhouse on the day of slaughter and were transported in cooling condition. The information of bovine eyes was as the following:
Supplier : Hwajung-Food (Nonsan-si, Chungcheongnam-do, Korea)
Eyes excised date : 2017 - 11 - 16
Transfer condition : Cold (3.0 – 5.0) ℃
Number of arrived Eyes : 15 ea
Number of used cornea : 12 ea (3 ea/group)
Vehicle:
other: mineral oil
Controls:
yes, concurrent vehicle
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
20%
Duration of treatment / exposure:
The corneas were incubated in the presence of either the test substance, positive control, solvent control or negative control for 4 hours at (32 ± 1 C°) in an incubator.
Duration of post- treatment incubation (in vitro):
90 ± 5 minutes
Number of animals or in vitro replicates:
3 in each group (G1 : Negative control, G2 : Positive control, G3 : Solvent control, G4 : Test substance)
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The eyes, once they arrived at the laboratory, were grossly examined for damage (e.g., increased opacity, scratches, neovascularization and pigmentation) and those exhibiting defects were discarded in the laboratory. Corneas with a horizontal diameter ≥ 27.5 and ≤ 28.5 using vernier calipers (KG-EQM-429) was used in selected eyes.

Isolation of corneas : Selected eyes were stored in a dish containing 1X HBSS (containing penicillin/streptomycin solution) until all corneas were dissected. The cornea was dissected from eyes such that approximately 2 to 3 mm of sclera was present around the cornea. The isolated corneas were also re-stored in a dish containing 1X HBSS (containing penicillin/streptomycin solution) until all corneas were dissected.
The isolated corneas were mounted immediately in the corneal holders and then corneal holders were filled with cMEM (without phenol red). The holders were pre-incubated for 1 hour at (32 ± 1)℃ in an incubator

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
Name : Sodium Chloride 0.9% solution
Manufacturer : Sigma-Aldrich Corporation
Lot. No. : RNBD7305
Justification for selection : This negative control was recommended as guidelinesand used as negative control in BCOP assay widely.

SOLVENT CONTROL USED
Name : Mineral oil
Manufacturer : Sigma-Aldrich Corporation
Lot. No. : MKCB0402V
Justification for selection : Test substance had insolubility in water. Mineral oil had no adverse effects on the bovine cornea in solvent test. So mineral oil was used as solvent with the sponsor`s consent.

POSITIVE CONTROL USED
Name : Imidazole
Manufacturer : Sigma-Aldrich Corporation
Lot. No. : SLBH9399V
Justification for selection : This positive control was recommended as guidelines in case of non-surfactant solid test substances.

APPLICATION DOSE AND EXPOSURE TIME
The corneas were incubated in the presence of either the test substance, positive control, solvent control or negative control for 4 hours at (32 ± 1)C° in an incubator.

POST-INCUBATION PERIOD:


REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After 4 hours of exposure time, the negative control, solvent control, positive control and test substance were removed. The corneas were washed more than three times with cMEM (with phenol red). The corneas were then given a final rinse with cMEM (without phenol red) and replaced with cMEM (without phenol red) in anterior and posterior chamber of holder.
- POST-EXPOSURE INCUBATION: The corneas were incubated for 90 ± 5 minutes at (32 ± 1)C° in an incubator.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Final opacity unit was calculated according to the following formula: (I0/I-0.9894)/0.0251 where I0 is the illuminance(lux) through the reference holder, and I is the illuminance through the cornea holder
- Corneal permeability: At the end of the 90 ± 5 minutes incubation, the medium was collected from the posterior chamber of holder on a per cornea basis. Absorbance value of medium at 490 nm was measured using 96-well microtiter plate reader (KG-EQM-412). The medium volume in the 96 well plate was 300 uL/well and each media were filled in 3 wells in the 96 well plate. cMEM (without phenol red) was used as Blank.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
Irritation parameter:
cornea opacity score
Value:
<= 3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Interpretation of results:
GHS criteria not met
Conclusions:
Based on the observed results it was concluded, that MIRAMER SB was considered as a ‘UN GHS Category : No Category’ in the Bovine Corneal Opacity and PermeabilityTest (BCOP Test) under the test conditions chosen. The test subtsance Miramer SB (sucrose benzoate) is therefore not irritating to the eye.
Executive summary:

The purpose of this study was to evaluate the potential ocular irritancy/toxicity of a test substance MIRAMER SB as measured by the MIRAMER SB’s effect to induced opacity and permeability to fluorescein in an isolated bovine cornea. The test substance was administered to the test system as a 20 % solution (w/v in mineral oil) according to the OECD guideline. Mineral oil as solvent was selected by sponsor’s consent. The test results were as in the following: - Corneal opacity was measured quantitatively as the amount of light transmission through the cornea. The mean opacity value of test substance group was 0.8 ± 1.0 (opacity unit). The mean opacity value of negative control, solvent control and positive control were –0.3 ± 1.2, 0.5 ± 0.5 and 56.6 ± 11.9 respectively. The mean opacity value of the test substance relative to the solvent control group was 0.4 ± 1.2. - Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea by optical density. The mean OD490 value of test substance group was 0.013 ± 0.002. The mean OD490 value of negative control, solvent control and positive control were 0.005 ± 0.001, 0.011 ± 0.006 and 1.774 ± 0.428 respectively. The mean OD490 value of test substance relative to the solvent control group was 0.002 ± 0.002. - Both opacity and permeability were used to calculate an In Vitro Irritancy Score (IVIS) of the test substance relative to the negative control corneas. As a result of calculating the IVIS, test substance group was calculated as 1.0 ± 1.0. The IVIS of negative control, solvent control and positive control were –0.2 ± 1.2, 0.6 ± 0.6 and 83.2 ± 9.0 respectively. The IVIS of the test substance relative to the solvent control was 0.4 ± 1.0. The test result was considered acceptable that the IVIS of positive control falls within two standard deviations of the current historical mean.

Based on the observed results it was concluded, that MIRAMER SB was considered as a ‘UN GHS Category : No Category’ in the Bovine Corneal Opacity and PermeabilityTest (BCOP Test) under the test conditions chosen. The test subtsance Miramer SB (sucrose benzoate)is therefore not irritating to the eye.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Skin irritation:

As a result of in vitro skin irritation test in SkinEhic RHETM (Reconstructed Human Epidermis) Model a cell viability of

72.5 ± 4.5 % was obtained for sucrose benzoate which

exceeded the reference value for classification of 50 %. Therefore, sucrose benzoate is considered to be a non-irritant substance corresponding to 'No category' based on the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).

Eye irritation:

As a result of calculating the IVIS, test substance group was calculated as 1.0 ± 1.0. The IVIS of negative control, solvent control and positive control were –0.2 ± 1.2, 0.6 ± 0.6 and 83.2 ± 9.0 respectively. The IVIS of the test substance relative to the solvent control was 0.4 ± 1.0. This is below the cut-off value of 3 for classification as an eye irritant, thus no classification should apply for sucrose benzoate.