5-(hydroxymethyl)-2-furaldehyde

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

24 May 2019 - 31 July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

67-47-0

Molecular formula:

C6H6O3

impurity 1

Reference substance name:

Fructose

EC Number:

200-333-3

EC Name:

Fructose

Cas Number:

57-48-7

Molecular formula:

C6H12O6

IUPAC Name:

D-fructose

impurity 2

Reference substance name:

1-(furan-2-yl)-2-hydroxyethanone

Cas Number:

17678-19-2

Molecular formula:

C6H6O3

IUPAC Name:

1-(furan-2-yl)-2-hydroxyethanone

impurity 3

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

impurity 4

Reference substance name:

Sodium formate

EC Number:

205-488-0

EC Name:

Sodium formate

Cas Number:

141-53-7

Molecular formula:

CH2O2.Na

IUPAC Name:

sodium formate

impurity 5

Reference substance name:

Sodium nitrate

EC Number:

231-554-3

EC Name:

Sodium nitrate

Cas Number:

7631-99-4

Molecular formula:

HNO3.Na

IUPAC Name:

sodium nitrate

impurity 6

Reference substance name:

Glucose

EC Number:

200-075-1

EC Name:

Glucose

Cas Number:

50-99-7

Molecular formula:

C6H12O6

IUPAC Name:

D-glucose

impurity 7

Reference substance name:

Sodium acetate

EC Number:

204-823-8

EC Name:

Sodium acetate

Cas Number:

127-09-3

Molecular formula:

C2H4O2.Na

IUPAC Name:

sodium acetate

impurity 8

Reference substance name:

2-furaldehyde

EC Number:

202-627-7

EC Name:

2-furaldehyde

Cas Number:

98-01-1

Molecular formula:

C5H4O2

IUPAC Name:

2-furaldehyde

impurity 9

Reference substance name:

Sodium 4-oxovalerate

EC Number:

243-378-4

EC Name:

Sodium 4-oxovalerate

Cas Number:

19856-23-6

Molecular formula:

C5H8O3.Na

IUPAC Name:

sodium 4-oxopentanoate

impurity 10

Reference substance name:

Formaldehyde

EC Number:

200-001-8

EC Name:

Formaldehyde

Cas Number:

50-00-0

Molecular formula:

CH2O

IUPAC Name:

formaldehyde

Test material form:

solid: crystalline

Details on test material:

freeze-dried material
Batch: 1808A-RO-C-I1-FD-Chg#3
Appearance Crystal / brown
Storage: Refrigerator (2 - 8 °C)
Expiring date: 31 December 2019

Specific details on test material used for the study:

SOURCE OF BATCH MATERIAL
- Batch No.: 1808A-RO-C-I1-FD-Chg#3
- Storage Condition: 2-8°C

In chemico test system

Details on the study design:

SUMMARY
The reactivity of a test chemical and synthetic Cysteine (Ac-RFAACAA-OH) or Lysine (Ac-RFAAKAA-OH) containing peptides is evaluated by combining the test chemical with a solution of the peptide and monitoring the remaining concentration of the peptide following 24 hours of interaction time at room temperature. The peptide is a custom material containing phenylalanine to aid the detection and either Cysteine (“C”) or Lysine (“K”) as the reactive center.
Relative concentrations of the peptides following the 24 hour incubation are determined by high performance liquid chromatography with gradient elution and UV detection at 220 nm. Reaction samples, reference controls A, B and C, co-elution controls and positive controls are prepared and analyzed in triplicates in batches of up to 26 chemicals (including controls).


EXPERIMENTAL PROCEDURES

SYNTHETIC PEPTIDES
-Batch:
* Cys: 150219HS-MHe
* Lys: 020517HS_MHEW0119-2
- Purity:
* Cys: 92.33%
* Lys: 97.31

BUFFERS USED
- Phosphate buffer:
* pH 7.5 ± 0.05
* 18 (v/v) % 0.1 M sodium phosphate monobasic (of Sodium Phosphate Monobasic Monohydrate (NaH2PO4 · H2O) in purified water)
* 82 (v/v) % 0.1 M sodium phosphate dibasic (of Sodium Phosphate Dibasic Heptahydrate (Na2HPO4 · 7H2O) in purified water)
- Ammonium acetate buffer
* pH 10.2 ± 0.05
* 0.1 M Ammonium Acetate (CH3CO2NH4) in purified water

SOLUBILITY ASSESSMENT
- Ultrapure water was selected as the most suitable solvent for the test material

PREPARATION OF PEPTIDE STOCK SOLUTIONS
- CYSTEINE: Stock solution of 0.501 mg/mL (0.667 mM) in phosphate buffer
- LYSINE: Stock solution of 0.518 mg/mL (0.667 mM) in ammonium acetate buffer


PEPTIDE ASSAYS

PREPARATION TEST STOCK SOLUTIONS
100 mM solutions of the test chemical in the appropriate solvent were prepared just before use. The needed amount of test chemical was calculated (0.0738 g ± 10 %) and weighted based on the molecular weight and purity. 0.0755 g test chemical was weighted for the stock solution used for the cysteine peptide depletion determination and 0.0759 g test chemical was weighted for the stock solution used for lysine peptide depletion determination in the runs.

In a 5 mL volumetric glass: [(molecular weight) / % purity] × 50 = target weight of test chemical (mg)

When the test item stock solution was combined with the cysteine and lysine stock solutions (reaction samples) or the phosphate and ammonium acetate buffer (co-elution controls), the samples appeared to be clear and homogenous.

STANDARD CURVE
Six calibration standard points were prepared by serial dilution of the peptide stock solutions with the following nominal molarities: STD 1 = 0.534 mM, STD 2 = 0.267 mM, STD 3 = 0.1335 mM, STD 4 = 0.0667 mM, STD 5 = 0.0334 mM and STD 6 = 0.0167 mM. As dilution buffer a 20% acetonitrile:buffer solution (phosphate or ammonium acetate) was used. For the zero standard point (STD 7 = 0 mM) dilution buffer was used.

REFERENCE CONTROL
100 mM solutions of the positive control chemical in acetonitrile were prepared just before use. The needed amount of test chemical was calculated (0.0664 g ± 10%) based on the molecular weight and purity of the substance with the equation below. 0.0670 g cinnamaldehyde was weighted for the positive stock solution used for the cysteine peptide depletion determination and 0.0698 g cinnamaldehyde was weighted for the stock solution used for lysine peptide depletion determination in the runs.

In a 5 mL volumetric glass: [(molecular weight) / % purity] × 50 = target weight of cinnamaldehyde (mg)

- Reference control A: Peptide stock solutions are combined with acetonitrile (see Table 3). System suitability is checked by the use of the three replicates of reference control A.
- Reference control B: Peptide stock solutions are combined with acetonitrile (see Table 3). Stability of the peptides are checked by the use of the three replicates of reference control B, measured before and after of the reaction samples.
- Reference control C: Peptide stock solutions are combined with the respective solvent of the test item (and acetonitrile in case of cysteine peptide). Three replicates of reference control C are used as a solvent control to which the peptide concentration/depletion of the reaction samples is compared. In case acetonitrile is not the chosen solvent for the test item, a reference control C with acetonitrile is prepared additionally as the solvent control for the positive control.
- Co-elution controls: Test item stock solution (and acetonitrile in case of cysteine peptide) is combined with the respective buffer solutions in each run (see Table 3). Co-elution controls are used to check for test item and peptide co-elution.

PEPTIDE ASSAY METHOD
Assembly of reaction controls:
* 1:10 ratio cysteine peptide (0.5mM peptide, 5mM test item): 750 µL cysteine peptide stock solution (or phosphate buffer for the co-elution control and for the positive control); 200 µL acetonitrile; 50 µL 100mM test item solution (or solvent for the reference controls A,B,C or 100 mM positive control solution for the positive control)
* 1:50 Lysine peptide (0.5 mM peptide, 25 mM test item): 750 µL lysine peptide stock solution (or ammonium acetate buffer for the co-elution control and for the positive control); 250 µL 100mM test item solution (or solvent for the reference controls A,B,C or 100 mM positive control solution for the positive control)
* The vials were capped, vortexed to mix and placed to the HPLC autosampler for 24 ± 2 h incubation at 25 ± 2.5 °C in the dark. HPLC analysis of the batch of reaction samples started 24 ± 2 h hours after the test chemical was added to the peptide solution. The batches were consisted of 2 parts: one part with the reference controls A, the calibration standards and the co-elution controls. These samples could be run before the 24 ± 2 h incubation time ends and right before the other part started or right after the other part. The other part contained the reference controls B and C, the positive controls and the reaction samples and these samples were run right after the 24 ± 2 h incubation time ended.

CHROMATOGRAPHIG AN DETECTOR PARAMETERS
HPLC: Shimadzu LC-2030i Prominence
Detector: D2 lamp (220 nm)
Column: Zorbax SB-C18 (2.1 x 100 mm, 3.5 µm)
Column temperature: 30°C
Sample temperature: 25°C
Injection volume: 7µL
System equilibration: Running mobile phase A and mobile phase B in a ratio of 1:1 for 2 hours at 30°C column temperature and running the gradient twice before injecting the first sample
Run time: 20 min
Flow conditions: gradient flow
*Note: For the analysis with the lysine peptide, system equilibration with the 1:1 ratio A and B phases occurred for 1 hour instead of 2 hours.
Mobile phases for HPLC:
Mobile Phase A – 0.1 % (v/v) trifluoroacetic acid in ultra-pure water
Mobile Phase B – 0.085 % (v/v) trifluoroacetic acid in acetonitrile

CALCULATIONS
The concentration of the peptide was determined in each reaction sample from absorbance at 220 nm, measuring the peak area of the appropriate peaks and calculating the concentration of the peptide using the linear calibration curves derived from the standards.

The percent peptide depletion was determined in each reaction sample and positive control (pc) sample measuring the quotient of the peak area and the mean respective reference control C peak area, according to the formula described below.

peptide percent depletion = [1-((peak area of the reaction or pc sample) / (mean peak area of reference controls C))] × 100


PREDICITON MODEL
The mean percent cysteine and percent lysine depletion value is calculated for each test chemical. Negative depletion is considered as “0” when calculating the mean. By using the cysteine 1:10/lysine 1:50 prediction model (Table 5.), the threshold of 6.38 % average peptide depletion is used to support the discrimination between skin sensitisers and non-sensitisers.

Application of the prediction models assigns a test chemical to a reactivity class (minimal, low, moderate or high reactivity). Chemicals assigned to the minimal reactivity category should be classified as non-sensitisers whereas chemicals assigned to the low, moderate or high reactivity categories should be classified as sensitisers under these test conditions.

Before applying the cysteine 1:10 lysine/1:50 or the cysteine 1:10 prediction model, the experimental data regarding possible co-elution is evaluated and the appropriate approach is selected based on the below mentioned scenarios..

Results and discussion

Positive control results:

The mean percent cysteine peptide depletion for the positive control cinnamic aldehyde was 72.63 % ± 0.59 % and the mean percent lysine peptide depletion for the positive control cinnamic aldehyde was 51.83 % ± 0.14%. Thus, this validity criteria concerning the positive control was met (high reactivity).

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: DPRA cysteine prediciton model

Parameter:

other: Mean depletion value

Value:

49.43

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

high reactivity

Results 2

Key result

Run / experiment:

other: DPRA lysine prediction model

Parameter:

other: Mean depletion value

Value:

41.49

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

Moderate reactivity

Other effects / acceptance of results:

CO-ELUTION
The test chemical did not absorb at 220 nm significantly (> 10 % compared to the respective reference control) when tested with the cysteine and lysine peptides (see representative chromatograms in Appendix I). Therefore, no co-elution was observed with either of the peptides.


SYSTEM SUITABILITY FOR THE PEPTIDE ASSAY
Reference control A replicates were included in the HPLC run sequence to verify the HPLC system suitability prior analysis. The mean peptide concentration of reference control A sample replicates was 0.49 mM and 0.50 mM for the cysteine and the lysine peptides respectively.

A standard calibration curve was generated for both cysteine and lysine peptides using serial dilutions standards from the peptide stock solutions. Calibration standard points were analysed by linear regression.

Means of the peak areas versus the concentrations of both peptides showed good linearity with r^2 = 0.9958 for the cysteine peptide and r^2 = 1 for the lysine peptide, covering the concentration range from 0.534 mM to 0.0167 mM. Validity criteria concerning the calibration curve and reference control A were within acceptable limits and therefore the study can be considered valid.


ANALYSIS SEQUENCES
Reference control B replicates were included in the sequence to verify the stability of the peptide over time and reference control C replicates were used to verify that the solvent of the test item did not impact the percent peptide depletion. The mean cysteine peptide concentration of the reference control C (solvent – ultrapure water - upw) replicates was 0.48 mM, and the mean lysine peptide concentration of the reference control C (solvent – ultrapure water - upw) replicates was 0.50 mM, which were both within the acceptable range of 0.50 ± 0.05 mM.

Moreover the CV % for the nine reference control B and C replicates in acetonitrile (acn) were much lower than the maximum acceptable coefficient of variation of 15 % for both peptides, since it was 3.1 % for cysteine and 0.4 % for lysine peptides. Validity criteria concerning reference controls B and C were within acceptable limits and therefore the study can be considered valid.

STANDARD DEVIATION FOR TEST ITEM Cysteine, lysine and mean peptide depletion
Standard deviation cysteine peptide depletion test item: 1.21%
Standard deviation lysine peptide depletion test item: 0.14%
Validity criteria concerning the standard deviation for the test item was also met.

DEVIATIONS FROM THE STUDY PLAN
Deviation 1
- Concerning: HPLC analysis
- According to the Study Plan: System equilibration: 50% phase A and 50% phase B in a ratio of 1:1 for 2 hours at 30°C
- Deviation: System equilibration: 50% phase A and 50% phase B in a ratio of 1:1 for 1 hour at 30°C
- Reason: Unexpected delay in analysis schedule with lysine peptide
- Presumed effect on the study: None

Deviation 2
- Concerning: Vehicles
- According to the Study Plan: Name: Acetonitrile
- Batch: 17J231486
- Deviation: Name: Acetonitrile
- Lot: 17J231486
- Reason: Typing error
- Presumed effect on the study: None

Deviation 3
- Concerning: Archiving
- According to the Study Plan: For the first 5 years archiving is included in the study price, thereafter archiving occurs at additional costs of the Sponsor
- Deviation: Thereafter, the archiving time could be prolonged on behalf of the sponsor if agreed by a contract.
- Reason: mutual agreement with the Sponsor
- Presumed effect on the study: None

Any other information on results incl. tables

Mean peptide depletion values for the positive control and the test chemical

Name, replicate No.	Obtained mean % cystein peptide depletion	Obtained mean % lysine peptide depletion	Mean % obtained peptide depletion
5-(Hydroxymethyl)furfural	49.43	41.49	45.46
CINNAMALDEHYDE	72.63	51.83	62.23

The average percent peptide depletion was calculated for the test item. Since no co-elution was observed, the cysteine 1:10 / lysine 1:50 prediction model was applied and the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer. The mean percent peptide depletion value was 45.46 %. Thus, the test item is considered to be positive in the DPRA and classified in the high reactivity class when using the cysteine 1:10 / lysine 1:50 prediction model.

Cysteine 1:10 / Lysine 1:50 prediction model

mean percent peptide depletion	Reactiviry class	DPRA prediction
0% = % depletion = 6.38 %	no or minimal reactivity	negative
6.38 % < % depletion = 22.62%	low reactivity	positive
22.62 % < % depletion = 42.47 %	moderate reactivity	positive
42.47 % < % depletion = 100%	high reactivity	positive

Applicant's summary and conclusion

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Conclusions:

the test item is considered to be positive in the DPRA and classified in the high reactivity class when using the cysteine 1:10 / lysine 1:50 prediction model.

Executive summary:

In the course of this study the skin sensitization potential of the test item “5-(Hydroxymethyl)furfural” was studied using the Direct Peptide Reactivity Assay (DPRA).

For the test chemical and positive control substance, in order to derive a prediction two independent tests were conducted, one with cysteine and lysine peptides each. The results of the two runs were used for the classification of the test item.

Peptide depletion resulted from the positive control cinnamaldehyde was 72.63 % with cysteine peptide and 51.83 % with the lysine peptide. The back-calculated values of the reference control replicates were within the expected molarity concentration range for the cysteine (0.49 – 0.48 mM) and lysine peptides (0.50 mM) and the CV % for the for the nine reference controls B and C in acetonitrile were 3.1 % and 0.4 % percentages for the cysteine and lysine peptides. For each peptide all validity criteria were met, confirming the validity of the assay.

The percent cysteine peptide depletion value of the test item was 49.43 % while the percent lysine peptide depletion was 41.49 %. The mean depletion value of the peptides was used to categorize the test chemical in one of the four classes of reactivity. No co-elution was observed with either cysteine or lysine peptides; therefore the cysteine 1:10 / lysine 1:50 prediction model was used for the discrimination between sensitizers and non-sensitizers. The mean peptide depletion of the test item was 45.46 %, which exceeded the 6.38 % threshold of the applicable prediction model and fell into the high reactivity class.

Based on these results and the cysteine 1:10 / lysine 1:50 prediction model, the test item “5-(Hydroxymethyl)furfural” was concluded to be positive and to show high reactivity towards the synthetic peptides thus is a potential skin sensitizer under the experimental conditions of the in chemico Direct Peptide Reactivity Assay (DPRA) method.