[No public or meaningful name is available]

Endpoint summary

Description of key information

Skin Irritation/corrosion:

-         Episkin test for skin irritation (OECD 439): cell viability: 87%, non-irritant

-         Episkin test for skin corrosion (OECD 431): cell viability: 112%, non-corrosive

 

Eye Irritation:

-         BCOP test (OECD 437): IVIS score 11.4, no prediction can be made

-         Epiocular^TM(OECD 492): 14% cell viability, Cat. 1/Cat. 2

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:

12-13 July 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)

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008, Annex Part B, B.40Bis: “In Vitro Skin Corrosion: Human Skin Model Test”,

Version / remarks:

Official Journal of the European Union No.
L142, dated May 31st, 2008.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ECVAM Database Service on Alternative Methods to Animal Experimentation, INVITTOX Protocol No. 118; “EPISKINTM Skin Corrosivity Test”

Version / remarks:

updated December 2011 / February 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

EpiSkin Small Model (three-dimensional human epidermis model) manufactured by EPISKIN SNC Lyon, France

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EPISKIN model has been validated for corrosivity testing in an international trial; it is considered to be suitable for this study (STATEMENT ON THE SCIENTIFIC VALIDITY OF THE EPISKINTM TEST (AN IN VITRO TEST FOR SKIN CORROSIVITY); ECVAM JRC Environment Institute, European Commission; Ispra; 03 April 1998).

Vehicle:

unchanged (no vehicle)

Remarks:

100 µL NaCl solution (9 g/L saline) was added topically (to each unit) to ensure good contact with the epidermis

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model
- Supplier: SKINETHIC Laboratories 4, rue Alexander Fleming, 69366 Lyon Cedex 07 - France
- Tissue batch number(s): 17-EKIN-028
- Expiry date: 17 July 2017
- Date of initiation of testing: 12 July 2017

PRE-INCUBATION
The “maintenance medium” was pre-warmed to 37°C. The appropriate number of an assay plate wells were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37°C in an incubator with 5 % CO2, >= 95% humidified atmosphere.

APPLICATION / EXPOSURE
- Test Item: An amount of 20 mg test item was applied evenly to the epidermal surface of the two test skin units/exposure times respectively. Subsequently, 100 µL NaCl solution (9 g/L saline) was added topically (to each unit) to ensure good contact with the epidermis. The test item was spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary.
- Positive and negative control: A volume of 50 µL positive control (glacial acetic acid) or negative control (NaCl 9 g/L) was applied on the skin surface by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary.
- Additional controls for MTT direct interacting chemicals: In addition to the normal procedure, 2 killed treated tissues and 2 killed negative control tissues were used for the MTT evaluation in one run.
- Additional controls for dyes and chemicals able to colour the tissue: In addition to the normal procedure, two additional test item treated tissues were used for the non-specific OD evaluation.
- Additional controls for non-specific colour in killed tissues: In addition to the normal procedure, two killed treated tissues were used for avoiding a possible double correction for colour interference.
- Exposure: The plates with the treated epidermis units were incubated for the exposure time of 4 hours at room temperature (24.2-24.7°C).

NUMBER OF REPLICATE TISSUES FOR TEST ITEMS AND CONTROLS
Duplicates

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 25 ml PBS 1x Solution, once. The rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT TEST:
After the exposure of test item was terminated by rinsing with PBS, the EpiSkinTMSM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours (± 5 min) at 37°C in an incubator with 5 % CO2 protected from light, >=95% humidified atmosphere.

FORMAZAN EXTRACTION:
A disk of epidermis was cut from the unit (this involves the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 µL acidified isopropanol (one tube corresponding to one well of the tissue culture plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all epidermis material with the acidified isopropanol then incubated overnight at room temperature protected from light for formazan extraction. At the end of the incubation period, each tube was additionally mixed using a vortex mixer to help extraction.

CELL VIAVILITY MEASUREMENTS:
Following the formazan extraction, 2×200 µL sample from each tube was placed into the wells of a 96-well plate (labelled appropriately) and read the OD (Absorbance / Optical Density) of the samples in a 96-well plate spectrophotometer (Thermo Scientific; Multiscan FC) at 570 nm (±10nm; Read out range: 0-3.5 Abs, Linearity range: 0.2136 – 3.1752) using acidified isopropanol solution as the blank (6×200 µL).

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

PREDICTION MODEL / DECISION CRITERIA:
- The test substance is considered to be corrosive to skin (SubCat.1A) if mean tissue viability is < 35 % after 3 min exposure
- The test substance is considered to be corrosive to skin (subCat.1B or 1C) if Mean tissue viability is = 35 % after 3 min exposure and < 35 % after 1 hour exposure OR Mean tissue viability is = 35 % after 1 hour exposure and < 35 % after 4 hours exposure
- The test substance is considered to be non-corrosive to skin if mean tissue viability is = 35 % after 4 hours exposure

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 20 mg

VEHICLE
- no, but 100 µL NaCl solution (9 g/L saline) was added topically (to each unit) to ensure good contact with the epidermis.

NEGATIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration (if solution): NaCl 9 g/L

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL

Duration of treatment / exposure:

4 hours

Duration of post-treatment incubation (if applicable):

no post-treatment incubation

Number of replicates:

2 replicates of test item and 2 replicatesfor each control (negative, positive, additional controls for MTT direct interacting chemicals, additional controls for dyes and chemicals able to colour the tissue and additional controls for non-specific colour in killed tissues)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

112

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

difference of viability between the two tissue replicates: 8.1%

Positive controls validity:

valid

Remarks:

1% viability; the difference of viability between the two tissue replicates: 1.9%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: During the check-method for possible direct MTT reduction, colour change was observed after three hours of incubation. The test item interacted with the MTT, therefore additional controls and data calculations were necessary.
The non-specific MTT reduction (NSMTT) was determined to be 5.635 %. As the NSMTT were below 50 % the true MTT metabolic conversion in all occasions and the correction of viability percentages were undertaken.
- Colour interference with MTT: As the test item has an intrinsic colour (caramel), two additional test item-treated tissues were used for the non-specific OD evaluation. Mean OD (measured at 570 nm) of these tissues was determined as 0.005. The Non Specific Colour % (NSC %) was calculated as 0.70 % (below 5 %). Therefore additional data calculation was not necessary. A false estimation of viability can be precluded.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, OD value 0.769, difference of viability between the two tissue replicates: 8.1% (The mean OD value of the three negative control tissues should be between 0.6 and 1.5 and the standard deviation value (SD) of the % viability should be < or = 30)
- Acceptance criteria met for positive control: yes, 1 % viability, difference of viability between the two tissue replicates: 1.9% (The acceptable mean percentage viability range for positive controls is 0-20% and the standard deviation value (SD) of the % viability should be < or = 30.)
- Acceptance criteria met for variability between replicate measurements: yes, difference of viability between the two tissue replicates test item:4.9% (should be < or = 30)

Table 1: OD values and cell viability percentages of the positive and negative control

Controls	Optical Density (OD)		Viability (%)	Delta%
Negative Control: NaCl (9 g/L saline)	1	0.800	104	8.1
	2	0.738	96
	mean	0.769	100
Positive Control: Glacial acetic acid	1	0.004	1	1.9
	2	0.018	2
	mean	0.011	1

 

Table 2: OD values and viability percentages of the test item (including corrected values):

Test Item	Optical Density (OD)		TODTT	Viability (%)	Relative Viability (%)	Delta%
Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated	1	0.888	0.845	116	110	4.9
	2	0.926	0.882	120	115
	mean	0.907	0.864	118	112
	standard deviation (SD)			3.44	3.44

 

Table 3: OD values of additional controls for MTT-interacting test item:

Additional controls	Optical Density (OD)
Negative control killed tissues: NaCl (9 g/L saline)	No. 1	0.065
	No. 2	0.111
	mean	0.088
Test item treated killed tissues: Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated	No. 1	0.168
	No. 2	0.095
	mean	0.132

 

Table 4: OD values and NSC % of additional control:

Additional colour control	Optical Density (OD)		Non Specific Colour % (NSCliving %)	Delta %
Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated	No. 1	0.007	0.70	0.4
	No. 2	0.004
	mean	0.005

 

Remark: delta %: The difference of viability between the two relating tissues

              TOD_TT: true MTT metabolic conversion

Interpretation of results:

GHS criteria not met

Conclusions:

The results obtained from this in vitro skin corrosion test, using the EPISKIN model, indicated that Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated (EC No.: 944-754-7) has no skin corrosion potential under the utilised testing conditions. In conclusion, the test item Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated can be classified as Non-corrosive.

Executive summary:

EpiSkinTM SM test of the test item Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated (EC No.: 944-754-7) has been performed to predict its corrosion potential by measurement of its cytotoxic effect, as reflected in the MTT assay according to the OECD Test Guideline No. 431, 29 July 2016.

Disks of EPISKIN (two units / incubation time) were treated with test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. 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 and protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically.

NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls respectively.

The test item has an intrinsic colour (caramel), therefore two additional test item treated tissues were used for the non-specific OD evaluation.

The test item is a MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement.

The test item is a MTT-reducer and has an intrinsic colour (caramel). To avoid a possible double correction [TODTT (MTT and NSC)] for colour interference, a third control for non-specific colour in killed tissues (NSCkilled) was performed. Two killed treated tissues were used to avoid a possible double correction for colour interference.

For each treated tissue viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal 35 % of the negative control.

The test item did not show significantly reduced cell viability (corrected value) in comparison to the negative control after 4 hours of exposure. The average test item treated tissue relative viability was 112 % at 4 hours of exposure. The test item treated tissue viability was above 35 % of the mean negative control value after 4 hours of exposure.

Positive and negative controls showed the expected cell viability values within acceptable limits.

All assay acceptance criteria were met, the experiment was considered to be valid.

The results obtained from this in vitro skin corrosion test, using the EPISKIN model, indicated that the test item reveals no skin corrosion potential under the utilised testing conditions. In conclusion, the test item Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated (EC No.: 944-754-7) can be classified as Non-corrosive.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 August - 01 September 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)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 April 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Version 1.8 (February 2009)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

EpiSkin Small Model (three-dimensional human epidermis model) manufactured by EPISKIN SNC Lyon, France

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EpiSkin Model has been validated for irritation testing in an international trial. The EpiSkin method is a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404) for the purposes of distinguishing between skin irritating and no-skin irritating test substances.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model
- Supplier: SKINETHIC Laboratories 4, rue Alexander Fleming, 69366 Lyon Cedex 07 - France
- Tissue batch number(s): 17-EKIN-035
- Expiry date: 04 September 2017
- Date of initiation of testing: 30 August 2017

PRE-INCUBATION
The “maintenance medium” was pre-warmed to 37°C. The appropriate number of an assay plate wells were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37°C in an incubator with 5 % CO2, >= 95% humidified atmosphere.

EXPOSURE
- Test Item: The epidermal surface was first moistened with 5 µL deionised water in order to improve further contact between powder and epidermis. Subsequently, 10 mg of the test item was applied evenly to the epidermal surface. The test item was spread gently with a curved flat spatula in order to cover evenly all the skin surface if necessary.
- Positive and negative control: A volume of 10 µL positive control (SDS 5 % aq.) or negative control (1x PBS) was applied on the skin surface by using a suitable pipette. Chemicals were gently spread with the pipette tip in order to cover evenly all the epidermal surface if necessary.
- Additional controls for MTT direct interacting chemicals: 3 killed treated tissues and 3 killed negative control tissues were used for the MTT evaluation in one run.
- Additional controls for dyes and chemicals able to colour the tissue: two additional test item treated tissues were used for the non-specific OD evaluation.
- Additional controls for non-specific colour in killed tissues: two killed treated tissues were used for avoiding a possible double correction for colour interference.
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature.

NUMBER OF REPLICATE TISSUES FOR TEST ITEMS AND CONTROLS
Triplicates

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 25 mL PBS 1x solution, once. rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source
- Observable damage in the tissue due to washing: none

POST-INCUBATION
After rinsing the units were placed into the plate wells with fresh pre-warmed “maintenance medium” (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5 % CO2, >=95% humidified atmosphere.

MTT TEST
After the 42 hours incubation the EpiSkinTMSM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours (± 5 min) at 37°C in an incubator with 5 % CO2 protected from light, >=95% humidified atmosphere.

FORMAZAN EXTRACTION
A disk of epidermis was cut from the unit using a biopsy punch, placed into a tube of 500 µL acidified isopropanol (0.04N HCl), mixed by using a vortex mixer and incubated for 4 hours at room temperature protected from light with gentle agitation (~150 rpm). At the middle and at the end of the incubation period, each tube was additionally mixed using a vortex mixer to help extraction.

CELL VIAVILITY MEASUREMENTS
Following the formazan extraction, 2×200 µL sample from each tube was placed into the wells of a 96-well plate (labelled appropriately) and read the Absorbance / Optical Density of the samples in a 96-well plate spectrophotometer at 570 nm using acidified isopropanol solution as the blank (6×200 µL).

PREDICTION MODEL / DECISION CRITERIA:
The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal to 50% of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 10 mg

NEGATIVE CONTROL
- Amount(s) applied:10 µL
- Concentration: 1x PBS (Phosphate Buffered Saline)

POSITIVE CONTROL
- Amount(s) applied:10 µL
- Concentration: Sodium Dodecyl Sulphate (SDS) 5% aq. solution

Duration of treatment / exposure:

15 minutes (± 0.5 min)

Duration of post-treatment incubation (if applicable):

42 hours (± 1h)

Number of replicates:

Three replicates were used for the test item and controls, respectively.
In addition, three killed treated tissues and three killed negative control tissues were used for the MTT evaluation.
Furthermore, two additional test item-treated tissues were used for the non-specific OD evaluation and two killed treated tissues were used to avoid a possible double correction for colour interference.

Irritation / corrosion parameter:

% tissue viability

Value:

87

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: mean viability value of three tissues

Other effects / acceptance of results:

OTHER EFFECTS:
- Possible direct MTT reduction with test item
During the check-method for possible direct MTT reduction, colour change was observed after three hours of incubation. The test item interacted with the MTT, therefore additional controls and data calculations were necessary.
The non-specific MTT reduction (NSMTT) was determined (0%)*, the correction of viability percentage was not necessary.
*: The calculated NSMTT was -2.517%. However, for the calculation of non-specific MTT reduction, small negative numbers are counted as zero, because the reason of the small negative number is a slight difference between the used killed epidermis (biological variability).

- Colouring potential of test item
As the test item has an intrinsic colour (caramel), two additional test item-treated tissues were used for the non-specific OD evaluation. Mean OD (measured at 570 nm) of these tissues was determined as 0.017. The Non Specific Colour % (NSC %) was calculated as 1.6 % (below 5 %). Therefore additional data calculation was not necessary. A false estimation of viability can be precluded.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Mean OD value 1.035 and standard deviation value (SD) for the % viability 7.18 (The mean OD value of the three negative control tissues should be between 0.6 and 1.5 and the standard deviation value (SD) of the % viability should be = or < 18)
- Acceptance criteria met for positive control: 25% mean viability range standard deviation value (SD) for the % viability 2.19 (The acceptable mean percentage viability range for positive controls is 0-40% and the standard deviation value (SD) of the % viability should be = or < 18.)
- For test chemicals, the standard deviation value (SD) of the % viability should be = or < 18 : 3.35 % SD

Cell viability

The results of the optical density (OD) measured at 570 nm of each replicate and the calculated % viability of the cells is presented below:

Negative Control (1xPBS):

Replicate	Optical Density (OD)	Viability (%)
1	0.996	96
2	0.989	96
3	1.121	108
Mean	1.035	100
Standard Deviation (SD)		7.18

Positive Control (SDS 5% aq.):

Replicate	Optical Density (OD)	Viability (%)
1	0.288	28
2	0.247	24
3	0.251	24
Mean	0.262	25
Standard Deviation (SD)		2.19

Test item: Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated

Replicate	Optical Density (OD)	Viability (%)
1	0.886	86
2	0.945	91
3	0.884	85
Mean	0.905	87
Standard Deviation (SD)		3.35

OD values of additional controls for MTT-interacting test item:

Additional controls	Replicate	Optical Density (OD)
Negative control killed tissues: 1x PBS	1 2 3 mean	0.110 0.046 0.059 0.071
Test item treated killed tissues:	1 2 3 mean	0.050 0.047 0.040 0.045

OD values and NSC % of additional control:

Additional colour control	Replicate	Optical Density (OD)	Non Specific Colour % (NSC %)
(test item treated tissues without MTT incubation)	1 2 mean	0.015 0.018 0.017	1.6

Interpretation of results:

GHS criteria not met

Conclusions:

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilized testing conditions. The test item Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated (EC No. 944-754-7) is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category / CLP not classified).

Executive summary:

EpiSkinTM SM test of Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated (EC No. 944-754-7) has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439, 28 July 2015.

Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37°C for 42 hours in an incubator with 5 % CO2, >=95% humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light, >=95% humidified atmosphere. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

The test item has an intrinsic colour (caramel), therefore two additional test item treated tissues were used for the non-specific OD evaluation.

The test item acted directly on MTT (MTT-reducer), therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement.

SDS (5% aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control.

The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (<=) to 50% of the negative control.

In this in vitro skin irritation test using the EPISKIN model, the test item Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated did not show significantly reduced cell viability in comparison to the negative control (mean value: 87 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected cell viability values within acceptable limits. Positive and negative control values were within the corresponding historical control data ranges.

All assay acceptance criteria were met, the experiment was considered to be valid.

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilized testing conditions. The test item Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated (EC No. 944-754-7) is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category / CLP not classified).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28 August - 20 September 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)

Version / remarks:

2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: ABP, Perth, PH1 3XB
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Cow eyes were collected from freshly slaughtered cattle, and placed into containers containing Hank’s balanced salt solution (HBSS). Transport to test facility was on the same day as slaughter. Cold packs were used to keep the contents cool.
- Time interval prior to initiating testing: Corneas were prepared and used for testing on the day of collection
- indication of any existing defects or lesions in ocular tissue samples: Upon receipt, eyes were rinsed with HBSS and examined for defects (scratches, opacity or neovascularisation) prior to use.Any eyes showing defects were rejected from further use.
- Indication of any antibiotics used: Not specified

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 750 µL
- Concentration Test Item: 20.38 %, w/v in physiological saline
NEGATIVE CONTROL: Physiological saline
- Amount(s) applied: 750 µL
- Concentration: Sodium Chloride 0.9%, w/v
- Batch no.: 16B29T2C
POSITIVE CONTROL : Imidazole
- Amount(s) applied (volume or weight with unit): 750 µl
- Concentration (if solution): 20.36%, w/v in physiological saline
- Batch no.: SLBS6105

Duration of treatment / exposure:

4 h ± 10 min

Duration of post- treatment incubation (in vitro):

no post-treatment incubation

Number of animals or in vitro replicates:

Three Corneas per test item and four corneas for controls

Details on study design:

SELECTION AND PREPARATION OF CORNEAS:
Upon receipt, eyes were rinsed with HBSS and examined for defects (scratches, opacity or neovascularisation) prior to use. Any eyes showing defects were rejected from further use. Corneas from undamaged eyes were dissected, leaving a sclera margin of ca 3 mm. Collected corneas were placed epithelial side down in HBSS at ambient temperature until use. Prior to mounting corneas the width of the cornea was measured using a ruler.

QUALITY CHECK OF THE ISOLATED CORNEAS
Corneas were then mounted, epithelial side forward, into pre-warmed corneal holders. Once mounted, both chambers of the cornea holders were filled with pre-warmed EMEM (Eagles Minimum Essential Medium) without phenol red. The posterior chamber was filled first to encourage the cornea to return to its original curvature, and care was taken to avoid the introduction of bubbles into the medium. Holders were then allowed to equilibrate for >1 h in an incubator.
Unless otherwise stated, all incubations of corneas were conducted in an incubator set to maintain 32°C (actual temperatures have been recorded in the raw data). At the end of the equilibration period, the medium in both chambers was replaced with fresh, EMEM without phenol red. Baseline opacity readings were then taken using an Opacitometer (Duratec Analysentechnik GmbH) comprising a Testo 545 luminometer with a photo diode and xenon halogen light source in a custom enclosure. Damaged corneas (opacity >7 opacity units) were rejected from further use.

TREATMENT METHOD: closed chamber. Prior to dosing, cornea holders were tipped forward to avoid contact between the dosing solution and the corneal epithelium. Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated (20.38%, w/v in physiological saline; 750 µL) was applied to the surface of three corneas using syringe. The negative control, physiological saline (750 µL) and the positive control, imidazole (20.36%, w/v in physiological saline; 750 µL), were also applied to the surface of three corneas each, using a syringe. The anterior chambers were then sealed with tape. To begin exposure, the corneal holders were tilted to a horizontal position, taking care to ensure that the entire epithelial surface was covered with the dosing solution. The dosed units were then returned to the incubator for 4 h ± 10 min.

REMOVAL OF TEST SUBSTANCE
EMEM with and without phenol red were pre-warmed prior to use. Following the exposure incubation, the test items and control solutions were removed from the anterior chambers through the holes using a vacuum pump. Corneas were then rinsed three times with EMEM with phenol red (ca 5 mL per rinse), removing the EMEM each time, then rinsed once with EMEM without phenol red (ca 5 mL), which was also removed. Finally, both chambers were refilled with EMEM without phenol red, and any bubbles introduced into the medium were removed.

POST-INCUBATION PERIOD: no

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacity readings were then taken using an Opacitometer (Duratec Analysentechnik GmbH) comprising a Testo 545 luminometer with a photo diode and xenon halogen light source in a custom enclosure. Gross damage or other changes were observed by visual assessment.
- Corneal permeability: Following the measurement of opacity, the EMEM in both chambers of the cornea holder wasremoved. Posterior chambers were refilled with EMEM without phenol red and sealed. Sodium fluorescein solution (5 mg/mL in EMEM without phenol red; ca 1 mL) was added to the anterior chambers. Cornea holders were rotated to the horizontal position, and incubated for 90 min ± 5 min in an incubator set to maintain a temperature of 32°C. At the end of the permeability test, EMEM from the posterior chambers was collected. These samples were stored overnight in a refrigerator set to maintain a temperature of 4°C.
Following overnight storage, triplicate aliquots from each sample (380 µL) were transferred into a 96 well plate, and analysed with a Multiskan Go plate reader at 490 nm. Three blank wells containing EMEM without phenol red (380 µL) were analysed on the same plate, along with two aliquots of a quality control solution (sodium fluoresceinsodium in EMEM without phenol red, 5 µg/mL, 380 µL).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIS = Opacity change + (15 x A490 Value)

DECISION CRITERIA:
In accordance with OECD Test Guideline No. 437, irritancy was assigned on the basis of in vitro irritancy scores (IVIS), calculated from the opacity and permeability value for each cornea, as follows:

IVIS<=3: not classified according to GHS/CLP
3IVIS>55: Classified as Eye Damaging Category 1 according to GHS/CLP

Irritation parameter:

in vitro irritation score

Run / experiment:

Single experiment

Value:

11.39

Vehicle controls validity:

valid

Remarks:

IVIS: 0.00

Positive controls validity:

valid

Remarks:

IVIS: 113.41

Remarks on result:

not determinable

Remarks:

No Prediction can be made

Other effects / acceptance of results:

OTHER EFFECTS:
- Prior to baseline opacity checks cornea diameter was measured and the mean diameter for the 3 test item treated corneas was 2.3 cm. Negative and positive control corneas had a mean diameter of 2.2 cm and 2.4 cm, respectively.
- Visible damage on test system: not reported

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, mean post treatment opacity 4.94 (historical values range 3.83 +/- 1.82) / mean permeability corrected for blank of 0.032 (historical values range 0.045 +/- 0.043). The classification achieved was “No Category”.
- Acceptance criteria met for positive control: yes, mean IVIS 113.41 (historical range: Mean +/- 2SD =66.29 to 169.36). The classification achieved was “Category 1”.
- The BCOP results were similar for all corneas and are presented in full in Table 1 (see Any other information on results incl. tables section).

Table 1                         BCOP Results for Controls (n=4) and Test Item (n=3)

Treatment	Opacity Post Dose (Opacity Units, Uncorrected)	Corrected* Opacity Change (Opacity Units)	Permeability (A490), Corrected for Blank and Dilution Only	Permeability (A490), Corrected*	IVIS	IVIS (Mean)	UN GHS Category**
Physiological saline (negative control)	3.98 4.81 4.15 6.83	-0.41 -0.59 -0.10 1.11	0.037 0.045 0.015 0.030	0.005 0.013 -0.016 -0.001	-0.34 -0.40 -0.35 1.08	0.00	No Category
Imidazole (positive control)	72.10 61.92 69.49 54.94	66.30 57.29 64.94 51.57	3.248 3.292 2.798 5.023	3.216 3.260 2.766 4.991	114.54 106.19 106.44 126.45	113.41	Category 1
Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated.	16.34 17.71 18.27	11.72 10.75 12.55	0.003 0.004 0.032	-0.029 -0.028 0.000	11.28 10.33 12.56	11.39	No prediction can be made

* Corrected for Negative Control

**Classifications from OECD Guidelines for the Testing of Chemicals No. 437 (2013)

Interpretation of results:

other: no prediction could be made

Conclusions:

In conclusion, no prediction could be made for Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated (EC No.: 944-754-7) according to the UN GHS classifications and the CLP classification system using the OECD 437 BCOP test.

Executive summary:

In this study the irritation potential of Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated (EC No.: 944-754-7) was evaluated using the OECD 437 Bovine Corneal Opacity and Permeability (BCOP) assay.

Corneas were dissected from freshly obtained cow eyes, the cornea diameter was measured and they were mounted into corneal holders. Following a pre-dose equilibration period, and measurement of baseline opacity (using an opacitometer), the epithelial sides of the corneas were treated as follows:

Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated (20.38%, w/v in physiological saline; ca 750 µL) was applied to three corneas. Physiological saline (ca 750 µL) or imidazole (20.36%, w/v in physiological saline, ca 750 µL) as negative and positive controls, respectively, were applied to four corneas each. Following exposure for 4 h ± 10 min, the test or control items were rinsed off.

The opacity of each cornea was then determined, followed by measurement of permeability by assessment of the passage of sodium fluorescein through the cornea. Irritancy was assigned on the basis of in vitro irritancy scores (IVIS). The results are summarised as follows:

Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented Mean IVIS Score : 11.39

In conclusion, no prediction could be made for Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated (EC No.: 944-754-7) according to the UN GHS classifications and the CLP classification system.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

04 - 05 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) SOP; for the prediction of acute ocular irritation of chemicals. For use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model.

Version / remarks:

10 Feb. 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: The EpiOcular™ human cell construct (MatTek Corporation)

Details on test animals or tissues and environmental conditions:

DETAILS ON TEST SYSTEM:
- RhCE tissue construct used: EpiOcular™ (OCL-200-EIT)
- Supplier: MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, 821 05 Bratislava, Slovakia
-Lot No.: 27007
- Expiry date: 05 October 2017
- Storage: The EpiOcular™ (OCL-200-EIT) units were stored at refrigerator (2-8°C) until the preparation of tissues for treatment is started. The assay media supplied with the kits were stored at refrigerator (2-8°C).

JUSTIFICATION FOR SELECTION OF THE TEST SYSTEM
The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. From this validation study and its independent peer review it was concluded that the EpiOcular™ EIT is able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose. The EpiOcular™ EIT is recommended to identify chemicals that do not require classification for eye irritation or serious eye damage according to UN GHS (UN GHS No Category) without further testing, however, a drawback of this test is the inability to distinguish between Category 1 (corrosive to eye) and Category 2 (eye irritant). Thus, in case of a positive result further testing is required.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 mg

Duration of treatment / exposure:

6 hours (± 15 min)

Duration of post- treatment incubation (in vitro):

25 ± 2 minutes immersion incubation (Post-Soak)
18 hours ± 15 minutes (Post-treatment Incubation)

Number of animals or in vitro replicates:

Two

Details on study design:

DETAILS ON THE TEST PROCEDURE USED:

- Preparation of EpiOcular™ Tissues for Treatment
After the test kit arrival, the tissues were equilibrated to room temperature for about 15 minutes. The Assay Medium was pre-warmed to 37±1°C. The appropriate number of an assay plate wells (6-well plates) was filled with the pre-warmed medium (1 mL per well). The insert was transferred aseptically into the 6-well plates and pre-incubated at 37°C in an incubator with 5±1% CO2, 90±10% humidified atmosphere for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37°C and the EpiOcular™ tissues was incubated at standard culture conditions overnight (16 - 24 hours).

- Application
Two replicates were used for the test item and control(s) respectively.
Pre-Treatment: After the overnight incubation, the tissues were pre-wetted with approximately 20 µL of Ca++Mg++Free-DPBS. If the Ca++Mg++Free-DPBS did not spread across the tissues, the plate was tapped to assure that the entire tissue surface was wetted. The tissues were incubated at standard culture conditions for 30 ± 2 minutes.

Test Item: Approximately 50 mg test item was applied (each test item treated insert) topically onto the EpiOcular™ tissues. To avoid that test item is spilled into the medium under the tissue inserts, the tissue insert was removed from the medium, placed onto a sterile surface (e.g. the lid of a microtiter plate) and dosed by pouring the solid test article onto the tissue surface so that the surface of the tissue was completely covered by the test item. The insert was gently shaken from side to side to ensure that tissue was completely covered by the test item. After this procedure the tissue was then returned to its medium.

Positive and Negative Control: A volume of 50 µL positive control (methyl acetate) or negative control (sterile deionized water) was applied on the tissues surface by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the EpiOcular™ tissues surface if necessary.

Additional controls for MTT direct interacting chemicals
In addition to the normal procedure, 2 killed treated tissues and 2 killed negative control tissues were used for the MTT evaluation in one run.
Additional controls for dyes and chemicals able to colour the tissue:
In addition to the normal procedure, two additional test item treated tissues were used for the non-specific OD evaluation.
Additional controls for non-specific colour in killed tissues:
In addition to the normal procedure, two killed treated tissues were used for avoiding a possible double correction for colour interference.

- Exposure
The plates with the treated tissue units were incubated for the exposure time of 6 hours (± 15 min) at standard culture conditions (37°C in an incubator with 5 % CO2, 90±10% humidified atmosphere).

- Rinsing
After the incubation time the EpiOcular™ units were removed and rinsed thoroughly with Ca++Mg++ Free-DPBS as following way: Three clean beakers (glass or plastic with minimal 150 mL capacity), containing a minimum of 100 mL each of Ca++Mg++ Free-DPBS were used per test item. Each test item utilizes a different set of three beakers. The inserts containing the tissue was lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. Use of curved forceps facilitates handling and decanting. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps (taking care of not to damage the tissues by the forceps). The test or control items were decanted from the tissue surface onto a clean absorbent material (paper towel, gauze, etc.) and the tissues was dipped into the first beaker of DPBS and was swirled in a circular motion in the liquid for approximately 2 seconds and after was lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container.
This process was performed two additional times in the first beaker. The tissue was rinsed in the second and third beakers of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material. Decanting was most efficiently performed by rotating the insert to approximately a 45° angle and touching the upper lip to the absorbent material (to break the surface tension).

-Post-Soak and Post-incubation
After rinsing, the tissues were transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 25 ± 2 minute immersion incubation (Post-Soak) at room temperature. This incubation in Assay Medium was intended to remove any test item absorbed into the tissue.
At the end of the Post-Soak immersion, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the insert was blotted on absorbent material, and afterwards transferred to the appropriate well of the pre-labeled 6-well plate containing 1 mL of warm (37°C) Assay Medium. The tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation).

- MTT Test After Post-incubation
After the post-incubation the EpiOcular™ units were transferred into the MTT ready to use solution filled 24-well plate (300 µL of 1 mg/mL MTT per well) and then incubated for 3 hours (± 10 min) at 37±1°C in an incubator with 5±1 % CO2 protected from light, 90±10% humidified atmosphere.

- Formazan Extraction
Each insert was removed from the 24-well plate after 3 hours ± 10 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labeled 6-well plate containing 2 mL of isopropanol in each designated well so that isopropanol was flowing into the insert on the tissue surface. The plates were sealed with parafilm (between the plate cover and upper edge of the wells) and were either stored overnight at 2-8°C in the dark.
To extract the MTT, the plates were placed on an orbital plate shaker and shaken (150 rpm) for approximately 2 hours at room temperature. At the end of the extraction period, the tissue was not pierced. The corresponding negative, positive, and colorant controls were treated identically.

- Cell Viability Measurements
Following the formazan extraction, 200 µL sample(s) from each tube (2×200 µL) was placed into the wells of a 96-well plate (labelled appropriately) and read Absorbance / Optical Density of the samples in a 96-well plate spectrophotometer at the wavelength of 570 nm (±10nm; Read out range: 0-3.5 Abs, Linearity range: 0.2136 – 3.1752) using isopropanol solutions as the blank (8×200 µL).

OTHER INFORMATION ON MATERIALS AND METHODS
-Check-method for Possible Direct MTT Reduction by the Test Item
Approximately 50 mg test item was added to 1 mL MTT 1 mg/mL solution in a 6-well plate or tube and the mixture is incubated in the dark at 37 ±1°C, 5±1% CO2, 90±10% humidified atmosphere. The mixture is incubated for approximately three hours and then any colour change observed:
- Test item which do not interact with MTT: yellow
- Test item interacting with MTT: blue or purple
If the MTT solution’s colour becomes blue or purple, the test item interacts with the MTT. It is then necessary to evaluate the part of optical density (OD) due to the non-specific reduction of the MTT (i.e. by using killed tissues).

Additional controls for direct MTT interacting chemicals (MTT reducers):
In addition to the normal procedure, 2 killed treated tissues and 2 killed negative control tissues were used for the MTT evaluation in one run (untreated killed tissues may exhibit little residual NADH and dehydrogenase associated activity). The batch of killed tissues was different than the batches of the living tissues (batch no. of killed EpiOcular™ tissues: 27005). The same treatment steps are followed for these tissues as for the living tissues.

Freeze killed tissues for MTT reducers:
– Placing untreated EpiOcular™ constructs (in a 24 well plate) in the –20°C freezer overnight.
– Thawing to room temperature.
– Refreezing (–20°C).
– Once frozen, the tissue may be stored indefinitely in the freezer.
– Before use, the killed tissues are de-frozen at room temperature.
– Further use of killed tissues is similar to living tissues.


- Check-method to detect the colouring potential of test Item

Prior to treatment, the test item was evaluated for its intrinsic colour or ability to become coloured in contact with water and/or isopropanol. Blue, dark purple and black test articles may be directly tested on colorant controls without further tests because it is obvious that they can interfere with the blue MTT product.

Approximately 50 mg test item was added to 1mL of water in a tube and the mixture was incubated in the dark at 37±1 °C, 5±1% CO2, 90±10% humidified atmosphere for one hour and then colour checked (unaided eye assessment).
Approximately 50 mg test item was added to 2 mL isopropanol, the same amount as used for MTT extraction, and was incubated in tubes. The tube was placed on an orbital plate shaker and shaken (150 rpm) for approximately 2 hours at room temperature and then colour checked (unaided eye assessment).

The test item has an intrinsic colour (caramel). So Non Specific Colour % (NSC %) was determined in order to evaluate the ability of test item to stain the epidermis by using additional control tissues.

Additional controls for dyes and chemicals able to colour the tissue:
In addition to the normal procedure, two additional test item-treated tissues were used for the non-specific OD evaluation. These tissues followed the same treatment steps as the other tissues except for the MTT step: MTT incubation was replaced by incubation with fresh assay medium. OD reading was made following the same conditions as for the other tissues.


Additional controls for non-specific colour in killed tissues:
The test item is a MTT-reducer and has an intrinsic colour (caramel). To avoid a possible double correction [TODTT (MTT and NSC)] for colour interference, a third control for non-specific colour in killed tissues (NSCkilled) was performed. However, the NSCliving % was 0.79 % (below 5 %), so the (NSCkilled) was not determined and used during the calculation of true MTT metabolic conversion.

In addition to the normal procedure, two killed treated tissues were used to avoid a possible double correction for colour interference. In this additional control, the test item was applied on two killed tissue replicates, which undergo the entire testing procedure but were incubated with medium instead of MTT solution during the MTT incubation step.



- Assay acceptance criteria
- The mean OD value of the two negative control tissues should be between 0.8 and 2.5.
- The acceptable percentage viability for positive control (mean of two tissues) is:
30 minute exposure: below 50% of control viability (liquids)
6 hours exposure: below 50% of control viability (solids)
- The difference of viability between the two relating tissues of a single chemical is < 20% in the same run (for positive and negative control tissues and tissues of single chemicals). This applies also to the killed controls (single chemicals and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

Irritation parameter:

other: Mean Tissue Viability (% of negative control)

Value:

14

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Possible direct MTT reduction with test item:
During the check-method for possible direct MTT reduction, colour change was observed after three hours of incubation. The test item interacted with the MTT, therefore additional controls and data calculations were necessary.
The non-specific MTT reduction (NSMTT) was determined to be 0.220 %. As the NSMTT were below 60 % the true MTT metabolic conversion in all occasions and the correction of viability percentages were undertaken.

- Colouring potential of test item:
The test item showed no ability to become coloured in contact with water and isopropanol. However, as the test item has an intrinsic colour (caramel), two additional test item-treated tissues were used for the non-specific OD evaluation. Mean OD (measured at 570 nm) of these tissues was determined as 0.011. The Non Specific Colour % (NSC %) was calculated as 0.79 % (below 5 %). Therefore additional data calculation was not necessary. A false estimation of viability can be precluded.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: mean OD value 1.344
- Acceptance criteria met for positive control: 38 % viability at 6 hours exposure
- Difference of viability between the two tissue replicates: 1.4 % - 7.3%

Cell Viability

The results of the optical density (OD) measured at 570 nm of each replicate and the calculated % viability of the cells is presented below:

Negative Control: Sterile deionized water

Replicate	Optical Density (OD)	Viability (%)	Delta %
1	1.393	104	7.3
2	1.295	96	7.3
Mean	1.344	100

Positive Control: Methyl acetate

Replicate	Optical Density (OD)	Viability (%)	Delta %
1	0.469	35	6.2
2	0.552	41	6.2
Mean	0.510	38

OD values and viability percentages of the test item Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated (including corrected values):

Replicate	Optical Density (OD)	TODTT	Viability (%)	RelativeViability (%)	Delta %
1	0.186	0.183	14	14	0.99
2	0.205	0.202	15	15	0.99
Mean	0.196	0.193	15	14
Standard deviation (SD)			0.99	0.99

OD values of additional controls for MTT-interacting test item:

Negative Control killed tissues: Sterile deionized water

Replicate	Optical Density (OD)
1	0.034
2	0.036
Mean	0.034

Test item treated killed tissues:

Replicate	Optical Density (OD)
1	0.040
2	0.037
Mean	0.038

OD values and NSC % of additional control: Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated

Replicate	Optical Density (OD)	Non Specific Colour %(NSCliving %)	Delta %
1	0.011	0.79	0.0
2	0.011	0.79	0.0
Mean	0.011	0.79

Remark: Delta %: The difference of viability between the two relating tissues

Interpretation of results:

other: Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1) Test method (OECD 492) cannot resolve between UN GHS Categories 1 and 2. Further testing is required.

Conclusions:

The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, with the test item Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated (EC No. 944-754-7) indicated that the test item is Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1). However, this test method (OECD 492) cannot resolve between UN GHS Categories 1 and 2. Therefore further testing with other test methods will be required to decide on its final classification.

Executive summary:

The purpose of this study was to determine the acute ocular irritation potential of the test item Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated (EC No.944-754-7) on three-dimensional RhCE tissue in the EpiOcular™ model in vitro.
Before treatment the tissues were pre-wetted with approximately 20 µL of Ca⁺⁺Mg⁺⁺Free-DPBS and were incubated at standard culture conditions for 30 ± 2 minutes. Disks of EpiOcular™ (two units) were treated with (50 mg/units) test item and incubated for 6 hours (± 15 min) at standard culture conditions (37±1°C in an incubator with 5±1% CO₂, 90±10% humidified atmosphere).
Exposure of test material was terminated by rinsing with Ca⁺⁺Mg⁺⁺Free-DPBS solution. After rinsing, the tissues were incubated for a 25 ± 2 minutes immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion test item treated tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation). Fresh Assay Medium was used during the Post-Soak and Post-incubation. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37±1°C in an incubator with 5±1% CO₂protected from light, 90±10% humidified atmosphere. The precipitated formazan was then extracted using isopropanol and quantified spectrophotometrically. Sterile deionized water and methyl acetate treated tissues were used as negative and positive controls, respectively. The Disks of EpiOcular™ (two units / control) were treated with positive and negative control and incubated for 6 hours ± 15 minutes. The test item has an intrinsic colour (caramel), therefore two additional test item treated tissues were used for the non-specific OD evaluation.

The test item acted directly on MTT (MTT-reducer), therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement. The test item is a MTT-reducer and has an intrinsic colour (caramel). To avoid a possible double correction [TODTT (MTT and NSC)] for colour interference, a third control for non-specific colour in killed tissues (NSC_killed) was performed. Two killed treated tissues were used to avoid a possible double correction for colour interference.

The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean percent tissue viability after exposure and post-exposure incubation is less than or equal to 60% compared to the negative control. Depending on the regulatory framework in member countries, the test item is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%.

The test item Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated showed significantly reduced cell viability in comparison to the negative control (mean relative viability: 14 %). All obtained test item viability results were below 60% when compared to the viability values obtained from the negative control. Positive and negative controls showed the expected cell viability values within acceptable limits.The experiment was considered to be valid. 

The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, with the test item Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated (EC No. 944-754-7) indicated that the test item is Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1). However, this test method (OECD 492) cannot resolve between UN GHS Categories 1 and 2. Therefore further testing with other test methods will be required to decide on its final classification.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

A valid EpiSkinTM SM test with Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated was performed under GLP to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439, 28 July 2015. EPISKIN discs were exposed in triplicate to the test item for 15 min at room temperature. The EPISKIN discs treated with the test item did not show significantly reduced cell viability (87% viability compared to the negative controls). Therefore,Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category / CLP not classified). The study is rated as key study and as Klimisch 1 "reliable without restriction".

Skin Corrosion

A valid EpiSkinTM SM test with Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated was performed under GLP to predict its corrosion potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 431, 29 July 2016. EPISKIN discs were exposed in triplicate to the test item for 4 h at room temperature. The EPISKIN discs treated with the test item did not show significantly reduced cell viability (112% viability compared to the negative controls). Therefore Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated is considered to be non-corrosive to skin and is not classified (UN GHS No Category / CLP not classified). The study is rated as key study and as Klimisch 1 "reliable without restriction".

Eye irritation

OECD 492:

A valid study was performed under GLP to determine the acute ocular irritation potential of the test item Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated on three-dimensional RhCE tissue in the EpiOcular™ model in vitro, according to OECD guideline Nr. 492, 28 July 2015. Disks of EpiOcular™ (two units) were treated with (50 µL/units) test item and incubated for 6 hours (± 15 min) at standard culture conditions (37±1°C) in an incubator with 5±1 % CO2, 90±10% humidified atmosphere). The test item showed significantly reduced cell viability: mean relative viability: 14 %, i.e. <= 60%, in comparison to the negative control. The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, with the test item Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated indicated that the test item is Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1). However, this test method (OECD 492) cannot resolve between UN GHS Categories 1 and 2. Therefore an additional study was performed.

The study is used in a weight of evidence approach and is rated as Klimisch 1 "reliable without restriction".

OECD 437:

A valid assessment of ocular irritation ofFructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylatedIn Vitro Using the Bovine Corneal Opacity and Permeability Assay was performed under GLP according the OECD guideline Nr, 437, 26 July 2013.

Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated(750 µL), was applied to three corneas. Following exposure for 4 h 10 min, the test item or control items were rinsed off. The in vitro irritancy score (IVIS) calculated for this substance was 11.4.
If the IVIS score is <= 3, the substance does not require classification for eye damage according to the UN GHS classifications and the CLP classification system. If the IVIS score is >= 55, the substance has to be classified as corrosive to the eye (Category 1).
In conclusion, the BCOP assay categorised Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated (EC no. 944-754-7) as ‘No prediction can be made’ according to the UN GHS classifications and the CLP classification system.

The study is used in a weight of evidence approach and is rated as Klimisch 1 "reliable without restriction".

Justification for classification or non-classification

For Skin Irritation:

According to the results obtained with the EpiSkinTM SM test for irritation potential (OECD 439) and the EpiSkinTM SM test for corrosion potential (OECD 431)Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated (EC no. 944-754-7)is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category /CLP not classified).

For Eye Irritation:

The Epiocular test (OECD 492) indicated thatFructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated(EC no. 944-754-7)is Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1). However, this test cannot distinguish between C1 and C2. To distinguish between the two categories, a BCOP test (OECD 437) was performed. Using this test, C1 could be excluded. Therefore, the substance has to be classified as Irritant to the eyes (Category 2) according to the CLP Regulation (EC) No 1272/2008.