Bis-O-(benzylidene)-D-glucitol

Endpoint summary

Description of key information

In vitro Skin Corrosion Test:

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.

In vitro Skin Irritation Test: Human Skin Model Test

This in vitro study was performed to assess the irritation potential of Geniset D by means of the Human Skin Model Test according to OECD TG 439.

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

Three tissues of the human skin model EpiSkin™ were treated with the test item, the negative control (deionized water) or the positive control (5% SDS) for 15 minutes.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.6 till ≤ 1.5 thus showing the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control thus ensuring the validity of the test system.

After treatment with the test item the mean relative absorbance value slightly decreased to 98.2%. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the Geniset D is not irritant to skin according to UN GHS and EU CLP regulation.

Bovine Corneal Opacity and Permeability Assay (BCOP)

This in vitro study was performed to assess the corneal damage potential of Geniset D by means of the BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t₀), the 20% (w/v) suspension in saline (0.9% (w/v) NaCl in deionised water) of the test item Geniset D, the positive and the negative controls were applied to corneae fixed in an incubation chamber in horizontal position and incubated for 240 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae and opacity was measured again (t₂₄₀).

After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

With the negative control (physiological saline) neither an increase of opacity nor permeability of the corneae could be observed.

The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damage (CLP/EPA/GHS (Cat 1)).

Relative to the negative control, the test item Geniset D did not cause an increase of the corneal opacity or permeability. The calculated mean in vitro irritancy score was 0.82.

According to OECD 437 the test item is not categorized (GHS).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

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

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

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)

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

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

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

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%)

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.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 14 February 2017. Experimental completion date: 17 March 2017

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:

yes

Remarks:

There was the following deviation from study plan without impact on the outcome of the study: Concerning: Acceptability of the Assay The acceptance limit of the IC50 should be “between 1.5 and 3.0 mg/mL” instead of “between 1.0 and 3.0 mg/mL”.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

There was the following deviation from study plan without impact on the outcome of the study: Concerning: Acceptability of the Assay The acceptance limit of the IC50 should be “between 1.5 and 3.0 mg/mL” instead of “between 1.0 and 3.0 mg/mL”.

Qualifier:

according to guideline

Guideline:

other: UN GHS (published 2003, last (6th) revision 2015).

Deviations:

yes

Remarks:

There was the following deviation from study plan without impact on the outcome of the study: Concerning: Acceptability of the Assay The acceptance limit of the IC50 should be “between 1.5 and 3.0 mg/mL” instead of “between 1.0 and 3.0 mg/mL”.

GLP compliance:

yes (incl. QA statement)

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

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

other: wetted with deionised water

Details on test system:

EpiSkin™ Kit Components Needed for the Assay
EpiSkin™ Kit Lot No.: 17-EKIN-011
1 Sealed 12-well plate Contains 12 inserts with EpiSkin™ tissues on agarose
1 12-well plate For MTT viability assay
1 bottle Assay Medium Basic medium for use in MTT assays
1 bottle EpiSkin™ Maintenance Medium Basic medium for incubations

MTT-Solution:
The MTT-solution was prepared freshly on day of use with DMEM or assay medium (concentration: 0.3 mg/mL).

Cell Culture:
EpiSkin™ kits are purchased from SkinEthic Laboratories (69007 Lyon, France). The EpiSkin™ tissue consists of NHEK, which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized 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 EpiSkin™ tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts. EpiSkin™ tissues were shipped at ambient temperature on medium-supplemented agarose gels in a 12-well plate and reached Envigo CRS GmbH on 14 March 2017. On the day of receipt the pre-incubation phase of the EpiSkin™ tissues started.

Test for Direct MTT Reduction and Colour Interference:
Prior to the start of the test, the test item’s colour interference potential had to be evaluated. For this purpose the test item (10 ± 2 mg) was mixed with 90 μL of deionised water in a preexperiment. The test item/water mixture was gently shaken for 15 minutes at room temperature.
The colour of the test item/water mixture did not change during the incubation period compared with the colour of the pure test item. Therefore, the measurement of the OD of the
test item in water at 570 nm was not required and consequently not performed. There was not need to perform an additional test with viable tissues (without MTT addition) to determine a
correction factor for calculating the true viability in the main experiment.
For correct interpretation of results it is necessary to assess the ability of the test item to directly reduce MTT. To test for this ability 10 ± 2 mg of the test item was added to 2 mL of
MTT solution (0.3 mg/mL) and the mixture was incubated in the dark at 37 ± 1.5 °C (5 ± 0.5% CO2) for 3 hours. MTT solution was used as the control. Since the MTT solution colour did not turn blue/purple, the test item is not presumed to have reduced the MTT. Again, there was not need to perform an additional test with freeze-killed tissues to determine a correction factor for calculating the true viability in the main experiment.

EXPERIMENTAL PERFORMANCE:
Pre-warming of EpiSkin™ Tissues:
Under sterile conditions using sterile forceps, the inserts were transferred into 12-well plates containing the pre-warmed (37 ± 1.5 °C) maintenance medium. The EpiSkin™ tissues were incubated for about 23 hours.

Treatment:
The test item tissues were wetted with 5 μL of deionised water. The negative control, positive control and the test item were added into the insert atop the concerning EpiSkin™ triplicate
tissues for 15 minutes.
After the end of the treatment interval the inserts were immediately removed from the 12-well plate. The tissues were gently rinsed with PBS to remove any residual test material.
Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for approximately 42.5 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2.

IL-1 α Immunoassay:
Samples of all treatment groups were taken from the wells. The plates were shaken for approximately 15 minutes to homogenise the released mediators in the medium before
sampling. At least 1.6 mL medium from each well was taken and was stored in the freezer at –20 °C. Since the results derived from the MTT assay were not unclear or borderline, the IL-1 α concentration in the medium was not determined and the taken samples were discarded after report finalization.

MTT Assay:
A 12-well plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well. After the 42 ± 1 hour incubation period was completed for all tissues the cell culture inserts
were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was rinsed three times with PBS and the tissues
were plotted. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol containing 0.04 N HCl) into each vial. The tissue samples were completely covered by isopropanol. The formazan salt was extracted for about 3.5 hours at room temperature with gentle agitation.
Per tissue sample 2 × 200 μL aliquots of the formazan extract were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, version 4.7.1) with 570 nm filter. Mean values were calculated from the 2 wells per tissue sample.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

5 μL of deionised water were topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis. Each approximately 10 mg of
the test item were applied to the wetted tissues.

10 μL were applied to the tissues for both the positive and negative control

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

The tissues were incubated for approximately 42.5 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2.

Number of replicates:

triplicate

Irritation / corrosion parameter:

% tissue viability

Remarks:

(test item)

Run / experiment:

Mean of triplicate runs

Value:

98.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: not irritant to skin

Other effects / acceptance of results:

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not lead to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item after 3 hour incubation with MTT-reagent did not show blue colour.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.6 till ≤ 1.5 for the 15 minutes treatment interval
(values between 0.796 and 0.923) thus showing the quality of the tissues.
Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 3.9% thus ensuring the validity of the test system.
The rel. standard deviations between tissues of the same treatment group was below 9% (threshold of the "OECD TG 439 Guideline for the Testing of Chemicals: In vitro Skin
Irritation: Reconstructed Human Epidermis Test Method”: 18%), thus ensuring the validity of the study.

The data of the quality control (determined by SkinEthic Laboratories, 69007 Lyon, France) of the respective EpiSkin™ lot were met, (the acceptance limit of the
IC50 should be between 1.5 and 3.0 mg/mL after 18 hours treatment with SLS; result: 2.0 mg/mL).

Results after treatment with Geniset D and controls

Test Group	Absorbance 570 nm Tissue 1*	Absorbance 570 nm Tissue 2*	Absorbance 570 nm Tissue 3*	Mean Absorbance of 3 Tissues	Relative Absorbance [%] Tissue 1, 2 + 3**	Relative Standard Deviation [%]	Rel. Absorbance [% of Negative Control]***
Negative Control	0.792	0.76	0.888	0.813	97.4	8.2	100
					93.4
					109.2
Positive Control	0.032	0.034	0.029	0.032	3.9	7.6	3.9
					4.2
					3.6
Test Item	0.753	0.787	0.855	0.799	92.6	6.5	98.2
					96.8
					105.2

* Mean of two replicate wells after blank correction

** relative absorbance per tissue [rounded values]: ((100 x absorbance _tissue)/ mean absorbance _{negative control})

*** relative absorbance per treatment group [rounded values]:

((100 x mean absorbance _{test item})/ mean absorbance _{negative control})

Interpretation of results:

other: not irritant to skin

Conclusions:

After treatment with the test item the mean relative absorbance value was reduced to 98.2%.
This value is well above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, Geniset D is not irritant to skin according to UN GHS and EU CLP regulation.

Executive summary:

This in vitro study was performed to assess the irritation potential of Geniset D by means of the Human Skin Model Test according to OECD TG 439.

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

Three tissues of the human skin model EpiSkin™ were treated with the test item, the negative control (deionized water) or the positive control (5% SDS) for 15 minutes.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.6 till ≤ 1.5 thus showing the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control thus ensuring the validity of the test system.

After treatment with the test item the mean relative absorbance value slightly decreased to 98.2%. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the Geniset D is not irritant to skin according to UN GHS and EU CLP regulation.

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:

Experimental start date 17 February 2017. Experimental completion date 17 February 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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
Freshly isolated bovine eyes of at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were transported to the laboratory in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin). The corneae were isolated on the same day after delivery of the eyes.

Vehicle:

other: saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test item was tested as a 20% suspension (w/v) in saline. The test item absorbed the saline completely and formed a pulp, which was spread to match the surface of the corneae.
Due to the suspension’s consistency, it could not be pipetted exactly. Therefore, each approximately 0.75 ml were distributed to the cornea surface.

Volume of app. 0.75 mL of positive and negative control.

Duration of post- treatment incubation (in vitro):

The incubation time lasted 240 minutes.

Number of animals or in vitro replicates:

Triplicate

Details on study design:

Preparation of Corneae:
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was
carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. The corneae were
directly used in the BCOP test on the same day.

Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior
compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (Oring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea
and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex
position. Care was taken to assure no air bubbles were present within the compartments.

For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.
At the end of the incubation period, the basal opacity was determined (t0).

The basal opacity of all corneae was recorded. Each cornea with a value of the basal opacity > 7 was discarded. Sets of three corneae were used for treatment with the test item and the
negative and positive controls.

Exposure of the Corneae to the Test Groups:
The corneae were distributed as follows:
Groups Number of Corneae
1 Negative Control 3
2 Positive Control 3
3 Test Item 3
The anterior compartment received the test item suspension or negative or positive control at a volume of app. 0.75 mL each on the surface of the corneae. The corneae were incubated in
a horizontal position at 32 ± 1 °C in the water-bath.
The incubation time lasted 240 minutes.
Afterwards, the test item or control items, respectively, were rinsed off from the application side with saline, and fresh incubation medium was added into the anterior compartment and
opacity was measured (t240).
In the second step of the assay, permeability of the cornea was determined.

Opacity Measurement:
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer
(OP_KiT opacitometer (Electro Design, 63-Riom, France)) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed
in the photoreceptor compartment for treated cornea.
After exposure of the corneae to the test groups and after rinsing the opacity value was determined again (t240).

Permeability Determination:
Following to the opacity readings, the permeability was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the
incubation medium was removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 ± 10 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment were removed, well mixed and transferred into a 96 well plate.
The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490). The absorbance values were determined using the software SoftMax Pro
Enterprise (version 4.7.1).

IVIS Calculation:
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values.
Depending on the IVIS score obtained, the test item is classified into the following Category
according to OECD guideline 437:
IVIS In vitro Irritancy Score (GHS)
≤3 No Category
>3; ≤55 No prediction can be made
>55 Serious eye damage according to CLP/EPA/GHS (cat 1)

Irritation parameter:

in vitro irritation score

Remarks:

(test item)

Run / experiment:

mean of triplicate runs

Value:

0.82

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 1.22).

The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS = 105.46) corresponding to a
classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).

The test item Geniset D was tested as suspension. Relative to the negative control, the test item Geniset D did not cause an increase of the corneal opacity or permeability. The
calculated mean IVIS was 0.82 (threshold for serious eye damage: IVIS ≥ 55). According to OECD 437, the test item is not categorized.

Results after 240 minutes incubation time

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Proposed in vitro Irritancy Score
		Mean		Mean
Negative Control	0	0	0.89	0.081	1.34	1.22	No category
	0		0.083		1.25
	0		0.071		1.07
Positive Control	95.00*		0.077*		96.16	105.46	Category 1
	117.00*		0.059*		117.89
	102.00*		0.023*		102.35
Geniset D	0.00*		-0.005*		0.00**	0.82	No category
	1.00*		0.038*		1.57
	1.00*		-0.002*		0.97

*corrected values

**negative values are set to zero

Interpretation of results:

GHS criteria not met

Conclusions:

The test item Geniset D was tested as suspension. Relative to the negative control, the test item Geniset D did not cause an increase of the corneal opacity or permeability. The calculated mean IVIS was 0.82 (threshold for serious eye damage: IVIS ≥ 55). According to OECD 437, the test item is not categorized.

In conclusion, according to the current study and under the experimental conditions reported, Geniset D is not categorized (GHS).

Executive summary:

This in vitro study was performed to assess the corneal damage potential of Geniset D by means of the BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t₀), the 20% (w/v) suspension in saline (0.9% (w/v) NaCl in deionised water) of the test item Geniset D, the positive and the negative controls were applied to corneae fixed in an incubation chamber in horizontal position and incubated for 240 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae and opacity was measured again (t₂₄₀).

After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

With the negative control (physiological saline) neither an increase of opacity nor permeability of the corneae could be observed.

The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damage (CLP/EPA/GHS (Cat 1)).

Relative to the negative control, the test item Geniset D did not cause an increase of the corneal opacity or permeability. The calculated mean in vitro irritancy score was 0.82. According to OECD 437 the test item is not categorized (GHS).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of in-vitro skin corrosion and irritation studies, the substance is non-corrosive and is not an irritant to skin. The substance is therefore not classification according to EU CLP regulation as a corrosive or skin irritant.

Based on the result of an in-vitro eye (BCOP) study, the substance does not cause serious eye damage or irritation. The substance is therefore not classified according to EU CLP regulation as irritating to eyes.