Bis-O-(benzylidene)-D-glucitol

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 01 March 2017. Experimental completion date: 06 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

yes

Remarks:

DPBS was used for rinsing According to the quality certificate of the test kit demonstrating its robustness, during treatment with 1% Triton X-100 the ET50 was determined as 4.77 hours ≤ ET50 ≤ 8.72 hours instead of 4.0 hours ≤ ET50 ≤ 8.7 hours

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

yes

Remarks:

DPBS was used for rinsing. According to the quality certificate of the test kit demonstrating its robustness, during treatment with 1% Triton X-100 the ET50 was determined as 4.77 hours ≤ ET50 ≤ 8.72 hours instead of 4.0 hours ≤ ET50 ≤ 8.7 hours

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: MatTek test protocol "In vitro EpiDerm Skin Corrosion Test (EPI-200-SCT)", 07 November 2014

Deviations:

yes

Remarks:

DPBS was used for rinsing. According to the quality certificate of the test kit demonstrating its robustness, during treatment with 1% Triton X-100 the ET50 was determined as 4.77 hours ≤ ET50 ≤ 8.72 hours instead of 4.0 hours ≤ ET50 ≤ 8.7 hours

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Bis-O-(benzylidene)-D-glucitol

EC Number:

251-136-4

EC Name:

Bis-O-(benzylidene)-D-glucitol

Cas Number:

32647-67-9

Molecular formula:

C20H22O6

IUPAC Name:

1-{2,6-diphenyl-hexahydro-[1,3]dioxino[5,4-d][1,3]dioxin-4-yl}ethane-1,2-diol

Test material form:

solid: particulate/powder

Details on test material:

Identification: Geniset D
CAS number: 32647-67-9
Appearance/physical state: white powder
Batch: 5434
Purity: 99.4%
Expiry date: 31 October 2018
Storage conditions: room temperature in the dark

Specific details on test material used for the study:

Identification: Geniset D
Batch: 5434
Purity: 99.4%
Appearance: White powder
Expiry Date: 31 October 2018
Storage Conditions: Room temperature

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

other: dionised water

Remarks:

(test item wetted with water)

Details on test system:

Test System
Epi-200 Kit Components Needed for the Assay
EpiDerm™ Kit Lot No.: 25803
1 Sealed 24-well plate Contains 24 inserts with EpiDerm™ tissues on agarose
2 24-well plates For MTT viability assay
4 6-well plates For storing inserts, or for topically applying test agents
1 bottle Serum-Free Assay Medium DMEM-based medium
1 bottle DPBS Rinse Solution For rinsing the inserts in MTT assay

MTT-100 Assay Kit Components
1 vial, 2 mL MTT concentrate
1 vial, 8 mL MTT diluent (supplemented DMEM) For diluting MTT concentrate prior to use in the MTT assay
1 bottle, 60 mL Extractant Solution (Isopropanol) For extraction of formazan crystals

MTT-Solution:
The MTT-solution was prepared freshly on day of use (resulting: 1 mg/mL).
For use in the pre-test (step 3): MTT from Sigma, Germany, DMEM from Gibco, Germany
For use in the main experiment: MTT concentrate from MatTek, MTT diluent from MatTek.

Cell Culture:
Epi-200 kits and MTT-100 assays were purchased from MatTek Corporation (Bratislava, Slovakia). The EpiDerm™ tissue consisted of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consisted of organised basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts (MILLICELL, 10 mm Ø).
EpiDerm™ tissues were shipped at 4 °C on medium-supplemented agarose gels in a 24-well plate and reached Envigo CRS GmbH on 04 April 2017. On day of receipt the pre-incubation phase of the EpiDerm™ tissues started.

Test for Direct MTT Reduction and Colour Interference:
A test item may interfere with the MTT endpoint if: a) it is coloured and/or b) able to directly reduce MTT. The MTT assay is affected only if the test item is present in the tissues when the MTT viability test is performed.
Some non-coloured test items may change into coloured test items in wet or aqueous conditions and thus stain tissues during the 60 min exposure. Therefore, before exposure, a functional check for this possibility should be performed (step 1).

Step 1
25 ± 2 mg of the test item were added to 0.3 ml of deionised water (transparent glass test-tube). The mixture was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) for 60 min. At the end of the exposure time, the mixture was shaken and the presence and intensity of the staining (if any) was evaluated. If the solution changed colour significantly, the test item is presumed to have the potential to stain the tissue. An additional test on viable tissues (without MTT addition) should be performed (step 2).
Since the test item did not dye water when mixed with it (milky suspension), step 2 did not have to be performed.

Step 3
All test items (including those already evaluated in step 1 and step 2) should be further evaluated for their potential to interfere with MTT. To test if an item directly reduces MTT, 25 ± 2 mg of the test item were added to 1 ml of a MTT/DMEM solution (1 mg/mL) and were incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 CO2) for 60 minutes. Untreated MTT/DMEM solution (1 mg/mL) medium was used as control. If the MTT/DMEM solution (1 mg/mL) turns blue/purple, the test item reduces MTT and an additional test on freeze-killed tissues (step 4) must be performed.
Since the test item did not prove to be a MTT reducer, an additional functional check did not have to be performed.

EXPERIMENTAL PERFORMANCE:
Pre-warming of EpiDerm™ Tissues:
At least 1 hour before dosing, EpiDerm™ tissues were removed from the refrigerator. Under sterile conditions using sterile forceps, the inserts were transferred into 6-well plates containing the pre-warmed assay medium. A 24-well plate was prepared as holding plate containing 300 μL assay medium. The holding plate was pre warmed in an incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) until use.

Treatment:
Duplicate EpiDermTM tissues were treated with the test item, positive control or negative control for the following exposure times:
• Test Item: 3 ± 0.5 minutes, 60 ± 5 minutes
• Negative Control: 3 ± 0.5 minutes, 60 ± 5 minutes
• Positive Control: 3 ± 0.5 minutes, 60 ± 5 minutes
After the pre-incubation of the EpiDermTM tissues was completed the DMEM-based medium in each well was replaced with 0.9 mL fresh assay medium. The 6-well plates for the 3 ± 0.5 minutes exposure periods stayed at room temperature in the sterile bench, the 6-well plates for the 60 ± 5 minutes exposure period were placed into an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2).
At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed with DPBS to remove any residual test material (20 times). Excess DPBS was removed by gently shaking the tissue insert and blotting the lower surface with blotting paper. The tissues were placed in the prepared holding plate until all tissues were rinsed.

MTT Assay:
Two 24-well plates were prepared prior to the end of the tissue pre-warming period. MTT solution (300 μL) was added to each well and the plates were kept in an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) until required.
Following rinsing, the tissues were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) the tissues were rinsed three times with DPBS and carefully dried with blotting paper. The inserts were transferred into new 24-well plates. The tissues were each immersed in 2 mL of extractant solution (isopropanol) pipetted in each well ensuring that the tissues were completely covered. The 24-well plates were sealed to minimise isopropanol evaporation. The formazan salt was extracted overnight for 17 hours without shaking at room temperature.
After the extraction period the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken, and the insert was discarded. 24-well plates were then placed on a shaker for 15 minutes until the solution was homogeneous in colour.
3 × 200 μL aliquots of the blue formazan solution were transferred from each tissue into a 96-well flat bottom microtiter plate. The OD was determined in a microplate reader (Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1)) at 570 nm (OD570) without reference filter. The mean values were calculated for each set of 3 wells per tissue.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 ± 2 mg (39.7 mg/cm2 according to guideline) of the test item were applied onto the surface of duplicate EpiDermTM tissue. The test item was wetted with 25 μL of deionised water.

50 μL of positive and negative control

Duration of treatment / exposure:

3 ± 0.5 minutes and 60 ± 5 minutes, for test substance, positive and negative control.

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

Duplicate

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

(test item)

Run / experiment:

3 minutes

Value:

99.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: test item non-corrosive to skin

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour

Value:

95.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: test item non-corrosive to skin

Other effects / acceptance of results:

The acceptance criteria are met:
• the mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time (range: 1.557 to 1.681)
• the mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% compared to the negative control (3.4%)
• the Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 30% (range: 2.1% to 13.7%)

Applicant's summary and conclusion

Interpretation of results:

other: non corrosive

Conclusions:

In conclusion, it can be stated that in this study and under the reported experimental conditions, Geniset D is non corrosive to skin according to EU CLP and UN GHS.

Executive summary:

This in vitro study was performed to assess the corrosive potential of Geniset D by means of the Human Skin Model Test with EpiDerm™ tissues models.

The test item did not reduce MTT (test for direct MTT reduction), and it did not dye deionised water, when mixed with it (test for colour interference). Consequently, additional tests with freeze-killed or viable tissues to determine correction factors for calculating the true viability in the main experiment were not necessary.

Independent duplicate tissues of EpiDerm™ were exposed to either the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively.

After exposure to the negative control the absorbance values met the required acceptability criterion of a mean OD570 ≥ 0.8 and ≤ 2.8 for both treatment intervals thereby confirming the acceptable quality of the tissues.

Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period and for the 1 hour exposure period. The 1 hour exposure caused a decrease of the cell viability < 15% of the negative control. The CV in the range 20 – 100% viability between the tissue replicates is ≤ 30%, thus the validity of the test system and the specific batch of the tissue models is confirmed.

After exposure of the tissues to the test item the relative absorbance value decreased to 99.1% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was reduced to 95.2%. Both values did not exceed the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test item is not considered to be corrosive.

In conclusion, it can be stated that in this study and under the reported experimental conditions, the test item Geniset D is non corrosive to skin according to EU CLP and UN GHS.