4-O-β-D-galactopyranosyl-D-glucitol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

yes

Remarks:

Characterization of test and control substances not documented according to GLP, however, the purity of the materials used was certified by a reputable Supplier.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4-O-β-D-galactopyranosyl-D-glucitol

EC Number:

209-566-5

EC Name:

4-O-β-D-galactopyranosyl-D-glucitol

Cas Number:

585-86-4

Molecular formula:

C12H24O11

IUPAC Name:

4-O-beta-D-galactopyranosyl-D-glucitol

Specific details on test material used for the study:

DETAILS OF TEST MATERIAL
- Purity:100%
- Physical Description: White powdery solid
- pH: 5.1
- Stability: Test substance was expected to be stable for the duration of testing.
- Expiration Date: June 29, 2024
- Test substance was stored at room temperature; Testing was carried out on the test substance as received.

Test animals / tissue source

Species:

other: In vitro EpiOcular™ tissues containing stratified human keratinocytes

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: Test was performed according to OECD Guideline 492 using EpiOcular™ Eye Irritation Test (EIT). The test system (tissue construct model) was developed by MatTek Corporation and the EpiOcular EIT was validated and approved by regulatory authorities for evaluation of eye irritation potential. The assay allows for hazard identification of irritant substances in accordance with UN GHS and for discrimination between irritants that fall within Category 2 and non-irritants but does not discriminate between non-mandatory subcategories of the UN GHS.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: commercially available RhCE tissue constructs

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 mg

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

2

Details on study design:

TISSUE CONDITIONING (DAY 0): Prior to performing the test and whenever assay medium was used, it was allowed to come to room temperature. On the day the tissue was used, the plate was allowed to sit at room temperature for at least 15 minutes. Each tissue was inspected closely for air bubbles beneath the tissue and any found encompassing> 50% of the tissue surface were not used. Under sterile conditions each tissue insert was transferred into a well in previously prepared 6-well plates containing 1 mL of pre-warmed assay medium. Any agarose gel adhering to the outer surface of the inserts was blotted on sterile absorbent material before transferring. The tissues were incubated at 37.1°C and 5% CO2 for approximately 60 minutes, followed by a media change and further incubation until the following day (approximately 20 hours).

CHEMICAL EXPOSURE (DAY 1): After conditioning, all tissues were pre-wetted with 20 µL of sterile DPBS applied to the surface of the insert and incubated for 28 minutes at 37.l-37.4°C and 5% CO2 prior to test or control substance application. Tissues were removed from the incubator just prior to exposure. The test substance was allowed to thaw prior to use. Using a pipette, 50 mg of undiluted test substance(s), 50 μL of negative control (NC; deionized water), or positive control (PC; methyl acetate) was applied to the surface of two tissues/substance. Once all tissues were treated, plates were incubated for 6 hours at 37.0°C and 5% CO2. After exposure, the tissues were removed from the incubator and thoroughly rinsed of test and control substances. For each set of 2 tissues, 3 vessels containing at least 100 mL of sterile DPBS were used. The contents were decanted onto absorbent material and the inserts were fully submerged into the
first beaker of DPBS. Inserts were swirled to rinse, filled, and the liquid was decanted back into the beaker a total of 3 times. This was repeated in fresh DPBS two additional times. After the final rinse, the remaining DPBS was decanted onto absorbent material. The tissues inserts treated with solid substance (test substance) were transferred to new 12-well plates containing assay medium/well and allowed to soak submerged for 25 minutes at ambient temperature. After the post-treatment soak, the tissue inserts were removed, decanted, the outside blotted, and transferred to pre-labelled 6-well plates containing 1 mL of assay media/well. Plates were then incubated for approximately 18 hours at 37.0° C and 5% CO2.

MTT VIABILITY TEST: A 1 mg/mL MTT medium solution was prepared by combining 1 mL of the MTT concentrate (thawed) with 4 mL of MIT diluent provided. 300 μl of MTT medium was pipetted into a sufficient number of wells of a pre-labelled 24-well plate. Inserts were removed from the 6-well plates, residual media was blotted as needed and then transferred into the appropriately labelled wells of the 24-well plate. The tissues were incubated in MTT medium (37.0°C, 5% CO2) for approximately 3 hours. The tissue inserts were removed from the MTT medium, the bottoms were blotted on absorbent material, and the inserts were transferred to 6-well plates containing 1 mL of isopropanol in each well. The plate was sealed to prevent evaporation. Samples were extracted at ambient temperature for at least two hours under gentle shaking. After the extraction period was complete, the inserts were removed and discarded, and 1 mL of isopropanol was added to the extractant solution in each well. The solution was mixed in each well and duplicate 200 μL aliquots of each were transferred into a 96-well plate. Isopropanol was used as blanks.

EVALUATION: Optical density (OD) was read in a 96-well plate spectrophotometer using a wavelength of 570 nm without using a reference filter. Blank corrected OD values were calculated by subtracting the OD of the blank wells from the OD of all other measured wells. The mean OD value of each pair of aliquots was then calculated for each tissue including the killed tissue controls. The mean collected OD for treated viable tissues was calculated. The resulting mean OD for the negative control treated tissue corresponds to 100% viability for the assay.
Viability[%]= corrected test article OD+ corrected mean negative control OD x 100

VALIDATION CRITERIA: The results of the study were acceptable if the following criteria were met:
I) The negative control OD is> 0.8 and< 2.5,
2) The mean relative viability of the positive control is < 50% of control viability
3) The difference of viability between the two relating tissues of a single chemical is <20%

STATISTICAL ANALYSIS: Statistical analysis was limited to the calculation of the mean and standard deviation of OD, as well as % viability of the tissue.

Results and discussion

In vitro

Results

Irritation parameter:

other: % tissue viability

Value:

94.8

Negative controls validity:

valid

Remarks:

100% viability

Positive controls validity:

valid

Remarks:

28.3% viability

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none reported

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes; average optical density was 2.020; viability was 100% with SD of 7.54
- Acceptance criteria met for positive control: yes; average optical density was 0.571; viability was 28.3 with SD of 7.5

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was predicted to be a non-irritant (mean viability was 94.8%)

Executive summary:

The in vitro EpiOcular eye irritation test (EIT) was performed using the EpiOcular tissue construct, which models the cornea epithelium with progressively stratified, but non-cornified cells. The assay measures destruction of the ocular tissue, one component predictive of ocular irritation. The reduction of the viability of tissues exposed to chemicals in comparison to negative control treated tissue was used to predict the ocular irritation potential.

 

Viable tissue constructs were randomly allocated to three groups of two matrices each; one test substance group, a positive control group, and a negative control group. Methyl acetate and deionized water were used as the positive and negative controls, respectively.

 

The EpiOcular tissue were conditioned to the assay media prior to exposure to test or control substances. Each substance was applied directly to the surface of duplicate tissue matrices. Treated tissues were exposed to the test or control substance for 30 minutes and rinsed thoroughly with DPBS. Rinsed tissues were incubated for approximately 2 hours, followed by MIT (3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltctrazolium bromide) exposure for 3 hours. The tissues were then extracted with isopropanol in a refrigerator overnight for approximately 16 hours. The resulting extraction solution from each tissue was sampled and optical density (OD) measured using a spectrophotometer. The relative viability of each treated tissue was calculated based on the mean OD compared to that of the negative control treated tissue.

 

Average % viability was 94.8, 100.0, and 28.3 for lactitol, negative control (deionized water) and positive control (methyl acetate), respectively. Under the conditions of this study, lactitol is not considered to be an ocular irritant.