Registration Dossier

Diss Factsheets

Administrative data

Description of key information

- QSAR DEREK: Inconclusive. DEREK did not yield any alerts for skin sensitization of the test item and predicted the query structure to be a non-sensitizer. However, the query structure contains a misclassified substructure, (furan-2-yl)methanol, present in two structures with strong resemblance to the test item. Both structurally similar substances were tested positive in the LLNA

- DPRA (OECD 442C): mean peptide depletion: 45.46 %, high reactivity, Sensitizer

- KeratinosensTM(OECD 442D): Positive

Cytotoxic: IC30 values of 1722 µg/mL and 1627 µg/mL and IC50 values of 1975 µg/mL and 1925 µg/mL

EC1.5 values 221 µg/mL and 262 µg/mL

Imax: 268.81 and 194.55 fold induction, Sensitizer

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation, other
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
25 June 2019
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
1. SOFTWARE: DEREX NEXUS

2. MODEL (incl. version number): DEREK NEXUS 6.0.1

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL: O1C(=CC=C1C=O)CO

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL: See the QMRF in the study document attached

5. APPLICABILITY DOMAIN (OECD principle 3):
Domains:
-Descriptor domain: The scope of the structure-activity relationship describing the skin sensitation endpoint are defined by the developer to be the applicability domain for the model.Therefore, if a chemical activates an alert describing a structure-activty for skin sensitation, it can be considered to be within the applicability domain. The applicability of potency predictions may be judged, and modified, by the user based on the displayed data for nearest neighbours. If a compound does not activate an alert or reasoning rule then Derek makes a negative prediction. The applicability of the negative prediction to the query compounds can be determined by an expert, if required, by investigating the presence (or absence) of misclassified and/or unclassified features.
-Structural fragment domain: For skin sensitization, which features multiple alerts believed to cover most of the mechanisms and chemical classes responsible for activity, “no alerts fired” may be extrapolated to a negative prediction. All structure fragments were found in the DEREK negative prediction database and consequently the structure falls within the applicability domain of DEREK’s skin sensitization endpoint.
-Mechanism domain:As the prediction indicated “no alerts fired” none of the mechanisms for skin sensitization is predicted to be applicable to this structure.
-Metabolic domain: not relevant

Uncertainty of the prediction (OECD 4):
DEREK NEXUS predictive performance against a combined human dataset had an accuracy of 76%.

The chemical and biological mechanisms according to the model underpinning the predicted result (OECD principle 5):
Not relevant, DEREK NEXUS did not yield any alerts for skin sensitization.

6 ADEQUACY OF THE RESULTS
- Regulatory purpose: The present prediction may be used for preparing the REACH Registration Dossier on the substance for submission to ECHA, as required by Regulation (EC) 1907/2006 and related amendments.
- Approach for regulatory interpretation of the model result: This result can be directly used within a weight-of-evidence approach to complete the endpoint skin sensitization.
Qualifier:
according to guideline
Guideline:
other: REACH Guidance on information requirements and chemical safety assessment Chapter R.6: QSARs and grouping of chemicals
Version / remarks:
May 2008
Deviations:
no
Principles of method if other than guideline:
- Software tool(s) used including version: DEREK NEXUS 6.0.1
- Knowledge Base: Derek KB 2018 1.1
- Model description: see field 'Justification for non-standard information', 'Attached justification'
- Justification of QSAR prediction: see field 'Justification for type of information', 'Attached
justification'
Specific details on test material used for the study:
SMILES: O1C(=CC=C1C=O)CO
Key result
Parameter:
other: alerts for skin sensitation
Remarks on result:
other: DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization of the test item and predicted the query structure to be a non-sensitizer.
Other effects / acceptance of results:
The query structure contains a misclassified substructure, (furan-2-yl)methanol, present in two structures with strong resemblance to the test item: (furan-2-yl)methanol (CAS 98-00-0; structural similarity 83%) and 5-(methoxymethyl)furan-2-carbaldehyde (CAS 1917-64-2; structural similarity 73%). Both structurally similar substances were tested positive in the LLNA (Appendix 2). As the mechanism of skin sensitization of these two analogues is not known, it cannot be stated whether the misclassified feature is contributory to the skin sensitizing properties.
Interpretation of results:
other: inconclusive
Conclusions:
In conclusion, based on the negative DEREK prediction from the alert database and the positive skin sensitization data of highly similar structures to the query structure, the results of the in silico assessment of the skin sensitization potential of 5-(Hydroxymethyl)furfural is considered to be inconclusive.
Executive summary:

The objective of this study was to obtain a prediction on the potential for skin sensitization of the test item (CAS No. 67-47-0) with the in silico model DEREK NEXUS. In this assessment version 6.0.1 of DEREK NEXUS was used.

The result as generated by DEREK NEXUS is presented in Appendix 2. The relevant QSAR Model Reporting Format (QMRF) and the QSAR Prediction Reporting Format (QPRF) are presented in Appendix 3.

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization of the test item and predicted the query structure to be a non-sensitizer. However, the query structure contains a misclassified substructure, (furan-2-yl)methanol, present in two structures with strong

resemblance to the test item: (furan-2-yl)methanol (CAS 98-00-0; structural similarity 83%) and 5-(methoxymethyl)furan-2-carbaldehyde (CAS 1917-64-2; structural similarity 73%). Both structurally similar substances were tested positive in the LLNA (Appendix 2). As the mechanism of skin sensitization of these two analogues is not known, it cannot be stated whether the misclassified feature is contributory to the skin sensitizing properties.

In conclusion, based on the negative DEREK prediction from the alert database and the positive skin sensitization data of highly similar structures to the query structure, the results of the in silico assessment of the skin sensitization potential of 5-(Hydroxymethyl)furfural is considered to be inconclusive.

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
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)
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
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..
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).
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
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

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
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.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
29 May 2019 - 05 August 2019
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:
2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Specific details on test material used for the study:
- Batch-No.: 1808A-RO-C-I1-FD-Chg#3
- Storage conditions: 2-8°C
Details on the study design:
- Test concentrations: Final test concentrations of 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.50, 125, 250, 500, 1000 and 2000 µM.
- Replicates: 3 replicates in two independent tests
- Positive control: The positive control used was Trans-Cinnamaldehyde for which a series of five 100 × master concentrations ranging from 0.4 to 6.4 mM were prepared in DMSO (from a 200 mM stock solution) and diluted as described for the 4 × master solutions. The final concentration of the positive control on the treated plates ranged from 4 to 64 µM.
- Negative Control: The negative (solvent) control used was DMSO, for which six wells per plate were prepared. It underwent the same dilution as described for the master and working solution concentrations in 6.3.1, so that the final negative (solvent) control concentration was 1 % DMSO in exposure medium on the treated plates.This DMSO concentration is known not to affect cell viability and corresponds to the same concentration of DMSO used in the tested chemical and in the positive control.

- Test system: Immortalised adherent cell line derived from HaCaT human keratinocytes stably transfected with a selectable plasmid. The cell line contains the luciferase gene under the transcriptional control of a constitutive promoter fused with an ARE element from a gene that is known to be up-regulated by skin sensitisers (KeratinoSens™ cell line). The KeratinoSens™ cell line is generated at and obtained by Givaudan Schweiz AG.

- Cell Culture:
* Maintenance (culture) medium: DMEM supplemented with 9.0 (v/v) % fetal bovine serum (FBS) and ~ 500 µg/mL G418.
* Thawing medium: DMEM containing 9.1 (v/v) % FBS without G418.
* Exposure medium: DMEM containing 1 (v/v) % FBS without G418

- Environmental Conditions: Controlled environment, 37 ± 1 °C in the presence of 5 % CO2.


EXPERIMENTAL DESIGN
- Preparation of cells:
Cells were subcultured upon reaching 80 - 90 % confluence and care was taken to ensure that cells were never grown to full confluence. One day prior to testing cells were harvested in thawing medium and distributed into 96-well plates (10 000 cells/well) homogenously. For each individual test in the study, three replicates were used for the luciferase activity measurements, and one parallel replicate for the cell viability assay. One well per plate was left empty to assess background values. Cells were grown for 24 ± 0.5 hours in 96-wells microplates at 37 ± 1 °C in the presence of 5 % CO2.
- Exposure:
After the 24-hour incubation time, thawing medium was replaced with fresh exposure medium. The 4 × master solutions of the test item and control substances were added to each well in a way that an additional 4-fold dilution was achieved on the plate for the final concentrations to be established (50 µL of 4× master solution to 150 µL of exposure medium). The treated plates were then incubated for about 48 ± 1 hours at 37 ± 1 °C in the presence of 5 % CO2. Care was taken to avoid cross-contamination between wells by covering the plates with a foil prior to the incubation with the test item.
- Luciferase activity measurement:
After the 48-hour exposure time with the test item and control substances, cells were washed with DPBS (270 µL), and 1× lysis buffer (20 µL) for luminescence readings was added to each well for 20 minutes at room temperature (on all three plates). Plates with the cell lysate were then placed in the luminometer for reading. First the luciferase substrate (50 µL) was added to each well and after one second, the luciferase activity was integrated for 2 seconds.
- Cytotoxicity:
For the cell viability assay, medium was replaced after the 48-hour exposure time with MTT working solution (200 µL) and cells were incubated for 4 hours at 37 ± 1 °C in the presence of 5 % CO2. The MTT working solution was then removed and cells were solubilised by the addition of isopropanol (50 µL). After shaking for 30 minutes the absorption was measured at 570 nm with a spectrophotometer.


ACCEPTANCE CRITERIA
For each test item and positive control substance, in order to derive a prediction, at least two independent tests, each containing three replicates for the luminescence measurements and one for viability measurement, were needed. In case of discordant results between the two independent tests, a third test should be performed. Each independent test was to be performed on a different day with fresh stock solution of test items and independently harvested cells. Cells may however have come from the same passage. KeratinoSens™ prediction should be considered in the framework of an IATA and in accordance with the limitations stated in the OECD test guideline.

The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations. The EC1.5 value of the positive control should be within two standard deviations of the historical mean of the testing facility or between 7 µM and 30 µM (based on the validation dataset). In addition, the average induction in the parallel plates for Trans-Cinnamaldehyde at 64 µM should be between 2 and 8. If the latter criterion is not fulfilled, the dose-response of Trans-Cinnamaldehyde should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.

Historical control mean for EC1.5 value of the positive control: 13.9 ± 8.2 µM
Acceptance range for EC1.5 value of the positive control: 5.7 µM – 22.0 µM

Finally, the average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO should be below 20 % in each test which consists of 6 wells tested in triplicate.

Furthermore, the cellular viability is measured at the lowest concentration leading to = 1.5-fold luciferase induction. This concentration should induce no significant cytotoxic effects and be below the IC30 value. In addition, viability should be > 70 % in at least two consecutive concentrations, otherwise the concentration range needs adjustment.



DATA EVALUATION

- The following parameters (endpoint values) are calculated in the KeratinoSens™ test method:
* Maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control;
* 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;
* IC50 and IC30 concentration values for 50 % and 30 % reduction of cellular viability.


FOLD INDUCTION AND MAXIMAL AVERAGE FOLD INDUCTION
Fold luciferase activity induction is calculated by the equation below. Maximal fold induction is determined in each individual test, while the overall maximal fold induction (Imax) is calculated as the average of the individual tests.

fold induction = (Lsample – Lblank) / (Lsolvent – Lblank)

where:
Lsample is the luminescence reading in the test item well
Lblank is the luminescence reading in the blank well containing no cells or treatment
Lsolvent is the average luminescence reading in the wells containing cells and solvent (negative) control


DETERMINATION OF EC1.5:
The concentrations of the test item needed for a 1.5-fold luciferase induction are calculated by linear interpolation according to the equation below, and the overall EC1.5 is calculated as the geometric mean of the individual tests.

EC1.5 = (Cb – Ca) × [(1.5 – Ia) / (Ib – Ia)] + Ca

where:
Ca is the lowest concentration in µM with > 1.5-fold induction
Cb is the highest concentration in µM with < 1.5-fold induction
Ia is the fold induction measured at the lowest concentration with > 1.5-fold induction (mean of three replicate wells)
Ib is the fold induction at the highest concentration with < 1.5-fold induction (mean of three replicate wells)


- CYTOTOXICITY (DETERMINATION OF IC50 AND IC30)
Viability is calculated by the equation below:

viability = [(Vsample – Vblank) / (Vsolvent – Vblank)] × 100

where:
Vsample is the MTT-absorbance reading in the test item well
Vblank is the MTT-absorbance reading in the blank well containing no cells and no treatment
Vsolvent is the average MTT-absorbance reading in the wells containing cells and solvent (negative) control

IC50 and IC30 are calculated by linear interpolation according to the equation below, and the overall IC50 and IC30 are calculated as the geometric mean of the individual tests.

ICx = (Cb – Ca) × [(100 – x) – Va) / (Vb – Va)] + Ca

where:
x is the % reduction at the concentration to be calculated (50 and 30)
Ca is the lowest concentration in µM with > x % reduction in viability
Cb is the highest concentration in µM with < x % reduction in viability
Va is the % viability at the lowest concentration with > x % reduction in viability
Vb is the % viability at the highest concentration with < x % reduction in viability

For each concentration showing a luciferase activity induction equal or higher than 1.5-fold, statistical significance was determined (e.g. using a two-tailed Student’s t-test) by comparing the luminescence values of the three replicate samples with the luminescence values in the solvent/vehicle control wells (p < 0.05).

PREDICTION MODEL
A KeratinoSens™ prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 tests, otherwise the KeratinoSens™ prediction is considered negative:
- the Imax is equal or higher than 1.5-fold and statistically significantly different as compared to the negative/solvent control (as determined by a two-tailed, unpaired Student’s T-test);
- the cellular viability is higher than 70 % at the lowest concentration with induction of luciferase activity = 1.5-fold;
- the EC1.5 value is less than 1000 µM
- there is an apparent overall dose-response for luciferase induction (or a biphasic response).
Positive control results:
- The coefficient of variation (CV%) of the luminescence reading for the negative control DMSO was below 20 % in both tests (19.13 % and 17.25 % respectively).
- The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde was statistically significant above the threshold of 1.5 at three concentrations in both tests. The EC1.5 values of the positive control were between 5.7 µM and 22.0 µM (12 µM and 16 µM in the first and second tests respectively).
Key result
Run / experiment:
other: 1
Parameter:
other: IC30
Value:
1 722
Vehicle controls validity:
not examined
Negative controls validity:
not examined
Positive controls validity:
not examined
Remarks on result:
other: Citotoxic
Key result
Run / experiment:
other: 1
Parameter:
other: IC50
Value:
1 975
Vehicle controls validity:
not examined
Negative controls validity:
not examined
Positive controls validity:
not examined
Remarks on result:
other: Citotoxic
Key result
Run / experiment:
other: 1
Parameter:
other: EC1.5 concentration for which induction of luciferase activity is above the 1.5 fold t hreshold
Value:
221
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Run / experiment:
other: 1
Parameter:
other: Imax (maximal average fold induction of luciferase activity)
Value:
268.81
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Run / experiment:
other: 2
Parameter:
other: IC30
Value:
1 627
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Citotoxic
Key result
Run / experiment:
other: 2
Parameter:
other: IC50
Value:
1 925
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: citotoxic
Key result
Run / experiment:
other: 2
Parameter:
other: EC1.5 concentration for which induction of luciferase activity is above the 1.5 fold t hreshold
Value:
262
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Run / experiment:
other: 2
Parameter:
other: Imax (maximal average fold induction of luciferase activity)
Value:
194.55
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
Acceptance criteria
- The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde was statistically significant above the threshold of 1.5 at three concentrations in both tests. The EC1.5 values of the positive control were between 5.7 µM and 22.0 µM (12 µM and 16 µM in the first and second tests respectively).
- The coefficient of variation (CV%) of the luminescence reading for the negative control DMSO was below 20 % in both tests (19.13 % and 17.25 % respectively).
-The average inductions in the parallel plates for Trans-Cinnamaldehyde at 64 µM were 39.5 fold and 3.30 fold in the first and second tests, respectively. Although the luciferase activity induction in the first test was is outside of the 2 – 8-fold induction range and the cell viability was below 70% at this concentration, there was a clear dose response relationship in the luciferase activity induction for the positive control, therefore it was accepted. In the second test, there was no cytotoxicity (cell viability lower than 70 %) induced by the positive control any of the tested concentrations.
- Both individual tests met the acceptance criteria for the negative and positive controls and were therefore considered valid under the conditions described.

Cytotoxic effects

Test 1: The lowest concentration (250 µM) leading to = 1.5-fold luciferase induction (significant), induced no significant cytotoxic effects (viability of 114 %) and this concentration is below the calculated IC30 value of 1722 µM.

Test 2: The lowest concentration (250 µM) leading to = 1.5-fold luciferase induction (significant), induced no significant cytotoxic effects and this concentration is below the calculated IC30 value of 1722 µM, meeting the evaluation criteria for a positive result.

No precipitation was observed at any point during both tests.

Summary of KeratinoSens™ results for the first test

  Statistically significant induction over 1.5 -fold  yes
 Viability > 70 % at lowest concentration with >1.5-fold  yes
 EC1.5 (µM)  221
 Clear dose response  yes
positive / negative   positive

Summary of KeratinoSens™ results for the second test

  Statistically significant induction over 1.5 -fold  yes
 Viability > 70 % at lowest concentration with >1.5-fold  yes
 EC1.5 (µM)  262
 Clear dose response  yes
positive / negative   positive

 

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
In the course of this study the skin sensitization potential of the test item “5-(Hydroxymethyl)furfural” was studied using the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

For the test item and positive control substance, in order to derive a prediction two independent tests were conducted.

The luciferase activity induction obtained with the test item was statistically significant above the threshold of 1.5 at 250 µM and above in the first test, meeting all acceptance criteria and the criteria for a positive response.

The luciferase activity induction obtained with the test item was statistically significant above the threshold of 1.5 at 500 µM and above in the second test, also meeting all acceptance criteria and the criteria for a positive response.

Since the results of the two tests were concordant, two out of the two valid tests were concluded positive for luciferase gene induction and met the acceptance criteria, no repeat tests were needed.

Based on these results and the KeratinoSens™ prediction model, the test item “5-(Hydroxymethyl)furfural was concluded positive under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

Based on the prediction model and the above described results, the test item was concluded positive, since the above-mentioned conditions for a positive result were all met in both tests
Executive summary:

In the course of this study the skin sensitization potential of the test item “5-(Hydroxymethyl)furfural” was studied using the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

For the test item and positive control substance, in order to derive a prediction two independent tests were conducted. The results of the two tests were concordant and met the acceptance criteria, therefore no repeat tests were needed.

The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde, was statistically significant above the threshold of 1.5-fold in all tests.

For the test item, twelve doses ranging from 2000 µM to 0.98 µM were used in two independent tests. The test item induced cytotoxicity (viability < 70 %) in KeratinoSens™ cells compared to the solvent/vehicle. In the first test the IC30 and IC50 values were 1722 µg/mL and 1975 µg/mL while in the second test 1627 µg/mL and 1925 µg/mL respectively. EC1.5 values were 221 µg/mL and 262 µg/mL in the first and second test respectively. Both tests were concluded positive for luciferase gene induction.

Based on these results and the KeratinoSens™ prediction model, the test item 5-(Hydroxymethyl)furfural was concluded positive under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

Table 1. Summary of the KeratinoSens™ results

 Number of tests

Significant induction above 1.5-fold

(yes/no)

Viability > 70 % at lowest concentration with = 1.5-fold

(yes/no)

EC1.5 < 1000 µM or 200 µg/ml

(yes/no)

 Showing clear dose response (yes/no)  
KeratinoSens™ result obtained (positive/ negative/inconclusive-repeat)
 1 yes  yes    yes   yes   yes
 2  yes    yes   yes   yes   yes
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization of the test item and predicted the query structure to be a non-sensitizer. However, the query structure contains a misclassified substructure, (furan-2-yl)methanol, present in two structures with strong resemblance to the test item: (furan-2-yl)methanol (CAS 98-00-0; structural similarity 83%) and 5 -(methoxymethyl)furan-2-carbaldehyde (CAS 1917-64-2; structural similarity 73%). Both structurally

similar substances were tested positive in the LLNA (Appendix 2). As the mechanism of skin sensitization of these two analogues is not known, it cannot be stated whether the misclassified feature is contributory to the skin sensitizing properties. In conclusion, based on the negative DEREK prediction from the alert database and the positive skin

sensitization data of highly similar structures to the query structure, the results of the in silico assessment of the skin sensitization potential of 5-(Hydroxymethyl)furfural is considered to be inconclusive.

A valid DPRA assay was performed according to OECD 442C and GLP principles. The test item was dissolved in ultrapure water. There was no evidence of co-elution of 5-(Hydroxymethyl)furfural with either Cysteine or Lysine peptide. Peptide depletion was calculated as 49.43% and 41.49% in Cysteine and Lysine Assays, respectively, resulting in a mean peptide depletion of 45.46%. This value places 5 -(Hydroxymethyl)furfural in the High Reactivity Class resulting in a DPRA prediction of sensitiser.

A valid Keratinosens assay was performed according to OECD 442D and GLP principles. The test item was dissolved in dimethyl sulfoxide at 200 mM. Twelve doses ranging from 2000 µM to 0.98 µM were used in two independent tests. No precipitate was observed at any dose level tested.

The test item showed toxicity (IC30 values of The test item showed toxicity (IC30 values of 1722 and 1625 µM and IC50 values of 1975 and 1925 µM in experiment 1 and 2, respectively). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 221 and 262 µM in experiment 1 and 2, respectively) was measured in both

experiments. The maximum luciferase activity induction (Imax) was 268.81-fold and 194.55-fold in experiment 1 and 2, respectively. 5 -(Hydroxymethyl)furfural is classified as positive in the KeratinoSens assay since positive results (>1.5-fold induction) were observed at test concentrations of <1000 µM with a cell viability of >70% compared to the vehicle control.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Considering the inconclusive outcome of the DEREK assessment, the positive outcome of the DPRA and KeratinoSens assay, 5-(Hydroxymethyl)furfural is considered to be a skin sensitizer. Performance of an in vitro assay assessing key event 3 (activation of dendritic cells) is not expected to give additional information as with a negative outcome 5-(Hydroxymethyl)furfural is still considered to be a skin sensitizer, while a positive outcome will only confirm that the test item is a skin sensitizer.

Based on the above data, 5-(Hydroxymethyl)furfural (CAS 67-47-0) is considered to be a skin sensitizer. The test item is classified for skin sensitization according to Regulation 1272/2008 and amendments with category 1A.