[No public or meaningful name is available]

Endpoint summary

Description of key information

Keratinosens^TM(OECD 442D): negative, non-sensitizer

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

9 - 24 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

- Test concentrations:Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated in the dosed plates were as follows: 400, 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39 and 0.20 µg/mL.
- All concentrations of the test item were tested in triplicate.
- Positive control :Positive control stock solution (cinnamic aldehyde, 6.4 mM stock in DMSO):64, 32, 16, 8 and 4 µM.
- Negative control: vehicle: 1% DMSO in exposure medium

- Test System
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). KeratinoSens™ cells were supplied by Charles River, Den Bosch who previously obtained them from Givaudan, Switzerland. Stocks of cells were prepared according to the methods specified in the European Union Reference Laboratory for Alternatives to Animal Testing (EURL-ECVAM) protocols.

- Environmental conditions:
Unless otherwise stated, all incubations of cells were performed in a humidified incubator set to maintain a temperature of 37°C, and an atmosphere of 5% CO2 in air.

- Cell Thawing:
The cells were removed from liquid nitrogen storage and warmed in a ca 37ºC water bath until thawed. The cells were resuspended in KeratinoSens™ Medium 2 (10 mL) and collected by centrifugation at ca 125 g for 5 min. The cell pellet was resuspended in KeratinoSens™ Medium 2 (10 mL) and seeded into two tissue culture dishes, with the final volume of cells and media being 10 mL/dish.

-Culture maintenance:
Cells were maintained in KeratinoSens™ Medium 1. Cells were passaged when ca 80 90% confluent; if it was required to passage cells of lower or higher confluence, the passage ratio was adjusted accordingly. Cells were washed twice with Dulbecco’s phosphate buffered saline (DPBS) containing EDTA (0.05%, w/v; 5 mL), then Trypsin/EDTA (1 mL) was added and dishes were returned to the incubator. After cells had detached (ca 10 min), they were resuspended in KeratinoSens™ Medium 1 (10 mL) and reseeded into fresh dishes using an appropriate split ratio and fresh KeratinoSens™ Medium 1 to bring the total dish volume up to 10 mL.



EXPEIMENTAL DESIGN
- Test system setup
In the week prior to testing, cells were split and grown for 4 days in tissue culture dishes.

For the 2 experiments performed, KeratinoSens™ cells were at passage numbers p22 (run 1) and p17 (run 2).

The cells were washed twice with DPBS containing EDTA (0.05%, v/v; 5 mL), then Trypsin/EDTA (1 mL) was added and plates returned to the incubator. After cells had detached (ca 10 min), they were resuspended, counted and adjusted to a density of 80,000 cells/mL in KeratinoSens™ Medium 2.

The cells were then seeded into 96 well plates, 125 µL per well, except for one empty well (blank). Four parallel plates were prepared: three white 96 well plates and one transparent 96 well plate. Plates were then incubated for ca 24 h.

Following incubation and prior to dosing, the medium was removed and replaced with KeratinoSens™ Medium 3 (150 µL).

- Preparation of master plates
On each testing occasion, a stock solution of Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated was prepared on the day of use. Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated was dissolved in ultrapure water (filter sterilised though a 0.45 µm filter) and filter sterilised through a 0.2 µM filter at a concentration of ca 40 mg/mL. Actual concentrations prepared were 102.4% and 100.7% of target, in test run 1 and test run 2, respectively.

Positive control stock solution (cinnamic aldehyde, 6.4 mM stock in DMSO) was also prepared on the day of use, taking purity into account. Actual concentrations were 100.34% and 101.0% of target, in test run 1 and test run 2, respectively. DMSO was the vehicle control.

The 100x solvent plate was then prepared using these stocks, following a pre-defined plate plan. DMSO vehicle controls (6 wells) and cinnamic aldehyde positive controls were included on every plate. Serial dilutions of the test item in ultrapure water (12 concentrations) and positive control in DMSO (5 concentrations) stock solutions were then prepared in the 100x plate (1:1, v/v).

Into a fresh plate corresponding to the 100x plate layout, KeratinoSens™ Medium 3 (240 µL/well) was added to wells that would receive cinnamic aldehyde or DMSO from the 100x plate and, KeratinoSens™ Medium 3 (230 µL/well) was added to wells that would receive the test items from the 100x plate. Test item or controls (10 µL) were transferred from the 100x plate to the 4x plate followed by DMSO (10 µL) to the test item wells, according to the pre-defined plate plan.

Each independent repetition was performed on a different day, with fresh stock solutions of test items and independently harvested cells.

Aliquots (50 µL) of each dosing solution from the 4x plate were transferred to the pre prepared replicate assay plates (Section 8.3, three white plates and one clear plate) and mixed by gentle aspiration. The blank wells were not dosed. All plates were sealed with sealing tape to avoid evaporation of volatile compounds and to avoid cross contamination between wells. The plates were then incubated for 48 h +/- 2 h.


- Luciferase assay
Following 48 h +/- 2 h incubation, dosing solutions were aspirated from the white assay plates and discarded. The cells were then washed once with DPBS (300 µL). KeratinoSens™ Medium 3 (100 µL, ambient temperature) was then added to each well, including the blank well. ONE-Glo™ (Promega) reagent mix was allowed to thaw and equilibrate to room temperature. The ONE-Glo™ reagent mix (100 µL) was then added to each well. The plates were incubated at room temperature, protected from light, for 6 min, before analysis with a luminometer

- Cytotoxicity assessment
For the cell viability assay, the solutions in the clear plate were aspirated and replaced with MTT solution (0.59 mg/mL in KeratinoSens™ Medium 3, 227 µL per well, including the blank well) following the 48 h +/- 2 h incubation. The plate was sealed with sealing tape and then incubated for 4 h +/- 30 min. After 4 h +/- 30 min incubation, the MTT solution was removed and sodium dodecyl sulphate solution (SDS, 10%, w/v, 200 µL per well) was added. The plate was sealed with sealing tape and placed protected from light in the incubator. After overnight incubation to dissolve the cells, plates were placed on an orbital shaker for 10 min to homogenise the solutions, then the optical absorption at 600 nm was determined for each well.

ACCEPTABILITY CRITERIA
• The luciferase induction obtained with the positive control, cinnamic aldehyde, should be statistically significant above the threshold of 1.5 in at least one of the tested (non cytotoxic) concentrations.
• The EC1.5 value for the positive control should be within two standard deviations of the historical mean of the testing facility (2.86 to 26.68)
• The average luciferase induction in the three replicates for cinnamic aldehyde at 64 µM (highest dose tested) should be between 2 and 8. If the latter criterion is not fulfilled, tests may still be accepted if there is a clear dose response, with increasing luciferase induction at increasing concentrations of cinnamic aldehyde.
• The average coefficient of variation of the luminescence reading for the vehicle control, DMSO, should be <20% in each experiment (18 wells). If the variability is higher, and is due to a single value, this result can be rejected, otherwise, the results should be discarded and the test repeated.

INTERPRETATION
- Data analysis
The following parameters are calculated in the KeratinoSensTM test method:
• The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control
• The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained
• The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.
In case the luciferase activity induction is larger than 1.5 fold, statistical significance is shown by using a two-tailed Student’s t-test, comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05). ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data. The lowest concentration with > 1.5 fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction in cellular viability at the EC1.5 determining concentration.

- Skin Sensitising Prediction Model
A KeratinoSensTM prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered negative:
1. The luciferase induction is >1.5-fold and statistically significant compared to the vehicle control.
2. The EC1.5 value is <1000 µM (<200 µg/mL for test chemicals with no defined MW) in all 3 repetitions or in at least 2 repetitions.
3. At the lowest concentration with a gene induction above 1.5-fold (i.e. at the EC1.5 determining value), the cell viability is >70%.
4. There is an apparent overall dose-response for luciferase induction, which is similar between the repetitions.
For each test item, concordant data from 2 independent repetitions (performed on different days) is required to assign sensitising potential.

Positive control results:

• Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.15 and the EC1.5 116.78 µM.
• Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.45 and the EC1.5 117.59 µM.

Key result

Run / experiment:

other: 1

Parameter:

other: maximal average fold induction of luciferase activity (Imax)

Value:

1.41

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

Imax: 3.15

Key result

Run / experiment:

other: 2

Parameter:

other: maximal average fold induction of luciferase activity (Imax)

Value:

1.41

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

Imax: 2.45

Key result

Run / experiment:

other: 1

Parameter:

other: EC 1.5 (µM) (concentration for which induction of luciferase activity is above the 1.5 fold threshold)

Vehicle controls validity:

valid

Remarks:

NA

Positive controls validity:

valid

Remarks:

EC 1.5: 16.78

Remarks on result:

not determinable

Key result

Run / experiment:

other: 2

Parameter:

other: EC 1.5 (µM) (concentration for which induction of luciferase activity is above the 1.5 fold threshold)

Vehicle controls validity:

valid

Remarks:

NA

Positive controls validity:

valid

Remarks:

EC 1.5: 17.59

Remarks on result:

not determinable

Other effects / acceptance of results:

All acceptance criteria were met for each run.
Gene induction in the positive control was significant in at least one concentration for run 1 and run 2, with a p-value of 0.03 and <0.01, respectively. The EC1.5 values for the positive control were 16.78 and 17.59 for Run 1 and Run 2, respectively. This was within the two standard deviations of the historical mean (2.84-26.28). The average luciferase induction in the three replicates for cinnamic aldehyde at 64 µM (highest dose tested) for Run 1 and Run 2 was 3.15 and 2.45, respectively. This was within the acceptance criteria of 2-8. The average coefficient of variation of the luminescence reading for the vehicle control, DMSO, was 12.04% and 12.89%, for Run 1 and Run 2 respectively.

Cell Viability:
No tested concentrations of the test item or positive control (cinnamic aldehyde) caused a reduction in cell viability.

Table 1          Run 1 Gene Induction and Cell Viability Results

Test Item	IC30 (µM)	IC50 (µM)	EC1.5 (µM)	Imax	Dose Response for Luciferase	Significant at p<0.05	Result Accepted
Fructose-1,6-Diphosphate^A	N/A	N/A	N/A	1.41	N/A	N/A	Yes
Positive Control – Cinnamic Aldehyde	N/A	N/A	16.78	3.15	Yes	Yes	Yes

^AFructose-1,6-Diphosphate, raw: Glucose anaerobically fermented bySaccharomyces cerevisiae, concentrated, phosphorylated

Coefficient of variation of the luminescence reading for the vehicle control for this test was 12.04%. This run satisfied all acceptance criteria and therefore passed.

N/A: Not applicable – unable to calculate values as viability was over 70% and there was no luciferase induction

Table 1          Run 2 Gene Induction and Cell Viability Results

Test Item	IC30 (µM)	IC50 (µM)	EC1.5 (µM)	Imax	Dose Response for Luciferase	Significant at p<0.05	Result Accepted
Fructose-1,6-Diphosphate^A	N/A	N/A	N/A	1.41	N/A	N/A	Yes
Positive Control – Cinnamic Aldehyde	N/A	N/A	17.59	2.45	Yes	Yes	Yes

^AFructose-1,6-Diphosphate, raw: Glucose anaerobically fermented bySaccharomyces cerevisiae, concentrated, phosphorylated

Coefficient of variation of the luminescence reading for the vehicle control for this test was 12.89%. This run satisfied all acceptance criteria and therefore passed.

N/A: Not applicable – unable to calculate values as viability was over 70% and there was no luciferase induction.

Table 1          Summary of Data

Test Item	IC30(µM) Values		IC30(µM) Geometric Mean	EC1.5(µM) Geometric Mean	Imax Average	KeratinoSens^™ Classification
Fructose-1,6-Diphosphate^A	N/A	N/A	N/A	N/A	N/A	Non-Sensitiser
Positive Control – Cinnamic Aldehyde	N/A	N/A	N/A	17.18	2.80	Sensitiser

^AFructose-1,6-Diphosphate, raw: Glucose anaerobically fermented bySaccharomyces cerevisiae, concentrated, phosphorylated

N/A: Not applicable – unable to calculate values as viability was over 70% and there was no luciferase induction.

Interpretation of results:

other: Test item did not induce activation of the ARE-dependant pathway in keratinocytes

Remarks:

Study will be used for classificatin in combination with other studies (Weight of Evidence)

Conclusions:

In conclusion Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated (EC No. 944-754-7) did not induce activation of the ARE-dependant pathway in keratinocytes under experimental conditions described in this report and therefore can be considered a non-sensitiser.

Executive summary:

Chemical exposure to humans often occurs via the skin and compounds able to pass through the skin can cause disease such as sensitisation. A skin sensitiser is a substance that will lead to an allergic response following skin contact.

The KeratinoSens TM assay detects up-regulation of the Keap1/Nrf2 antioxidant response element (ARE) in KeratinoSens TM cells, a human keratinocyte cell line (HaCaT) stably transfected with a reporter construct containing an ARE coupled to luciferase. 

Following exposure to Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated (EC No. 944-754-7) the luciferase activity and viability of the KeratinoSens TM cells was measured. An increase in luciferase activity at non-cytotoxic concentrations indicates upregulation of the reporter gene through activation of the Keap1/Nrf2/ARE signalling pathway, indicating skin sensitising potential.

KeratinoSens TM cells were exposed to Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated (in filter sterilised ultrapure water) at 12 concentrations in 96 well plates for 48 h. Luciferase activity was then determined by addition of ONE-Glo TM reagent (Promega) followed by measurement of luminescence. Cell viability was determined using an MTT assay.

In two independent runs of the test Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated did not induce luciferase activity or cause cytotoxicity at any concentration (no EC_1.5value or IC_30/50). The maximum luciferase induction value (I_max) was 1.41-fold for Run 1 and Run 2. All acceptance criteria were met. Therefore Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated can be classified as a non-sensitiser according to the UN GHS classification system since negative results (<1.5-fold induction) were observed at all test concentrations.

In conclusion Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated (EC No. 944-754-7) did not induce activation of the ARE-dependant pathway in keratinocytes under experimental conditions described in this report and therefore can be considered a non-sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

QSAR DEREK: was not performed as Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented bySaccharomyces cerevisiae, concentrated, phosphorylated (EC 944-754-7) is a complex UVCB substance.

 

No DPRA assay (OECD 442C) was performed because the test item is a UVCB and no detailed information on identity of constituents / molecular weights is available to calculate a single apparent molecular weight considering the individual molecular weights of each component in the mixture. Therefore Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated (EC 944-754-7) is out of the applicability domain of the DPRA assay.

 

A valid Keratinosens assay was performed according to OECD 442D and GLP principles. The test item was suspended in ultrapure water at 40 mg/mL. The 100x solvent plate was then prepared using this stock, following a pre-defined plate plan. Final test concentrations were 0.20 – 400 µg/mL. No precipitate was observed at any dose level tested. Two independent experiments were performed.

The test item did not cause cytotoxicity. No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was1.41-fold for both experiment. The test item is classified as negative in the KeratinoSens^TMassay since negative results (<1.5-fold induction) were observed at test concentrations up to 200µg/mL. Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated (EC No. 944-754-7) is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

 

Known classification of the main carbohydrate D-fructose 1,6-bis (dihydrogen phosphate), CAS 488-69-7 (35% acc. to CoA): not classified according to ECHA C&L inventory (1 notifier) and it is not expected that this substance is skin sensitising.

 

Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated (EC 944-754-7) contains also 3% protein (according to CoA). These proteins are the remaining proteins in solution after filtration of the Saccharomyces cerevisiaec ells used for the fermentation of Glucose. According to the ECHA C&L inventory, neither lysates of Saccharomyces cerevisiae (CAS 94350-12-6) nor Saccharomyces cerevisiae extract (CAS 84604-16-0) are classified as skin sensitisers.

 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The KeratinoSens^TM assay gave a clear negative result (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes).

The AMES test (OECD 471) was negative. Acommon feature of genotoxic substances is that they can bind covalently to DNA and cause direct DNA damage, therefore the AMES test provides supporting information about the electrophilicity of the substance of interest and hence its likely sensitization ability. In this case, the test item did not induce increased mutation frequency and there is therefore no indication that covalent binding of the test item to DNA occurred.

The components of Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated (EC No. 944-754-7) that could be suspected of inducing a sensitisation response, the carbohydrates (mainly D-fructose 1,6-bis(dihydrogen phosphate) and proteins (derived from Saccharomyces cerevisiae) are not classified as skins sensitisers.

In conclusion, Fructose-1,6-Diphosphate, raw: Glucose anaerobically fermented by Saccharomyces cerevisiae, concentrated, phosphorylated (EC No. 944-754-7) can be considered a non-sensitiser and is therefore not classified for skin sensitisation.