[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

no

Test guideline 2

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

Test guideline 3

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 material

Test material information

Constituent 1

Reference substance name:

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

Molecular formula:

Molecular formula of the eponymous compound: C6H14O12P2

IUPAC Name:

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

Test material form:

solid

Details on test material:

Batch No.: 18224600

In vitro test system

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)

Results and discussion

In vitro

Results

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)

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.