α-D-glucose pentaacetate

Endpoint summary

Description of key information

- QSAR DEREK: No alert for skin sensitization

- DPRA (OECD 442C): mean peptide depletion: 26%, moderate reactivity, Sensitizer

- Keratinosens^TM(OECD 442D): Positive (Cytotoxic: IC30 values of 280 and 728 µM and IC50 values of 347 and 824 µM; Imax: 118 and 190 fold induction), Sensitizer

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation, other

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

29 August 2017

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 5.0.2.
3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL:[C@H]1([C@@H]([C@H](O[C@H]([C@@H]1OC(C)=O)OC(C)=O)COC(C )=O)OC(C)=O)OC(C)=O
4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL: See the QMRF in the study document attached
5. APPLICABILITY DOMAIN (OECD principle 3)

- Descriptor domain:The scopes of the structure-activity relationships describing the skin sensitisation endpoint are defined by the developer to be the applicability domain for the model. Therefore, if a chemical activates an alert describing a structure-activity for skin sensitisation 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 in Derek, a result of ‘no alerts fired’ is presented to the user. This can be interpreted as a negative prediction or that the query compound is outside the domain of the model. Which of these is more appropriate may depend on the endpoint of interest. For the endpoint of skin sensitisation, 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

- Structural fragment domain: DEREK NEXUS is applicable to organic structures including their salts, e.g. sodium, potassium. For skin sensitisation, which features 80 alerts believed to cover most of the mechanisms and chemical classes responsible for activity, ’no alerts fired’ may be extrapolated to a negative prediction. The alpha-D-glucose pentaacetate (CAS Nr. 604 -68 -2) has no extraordinary features and is considered to fall within the applicability domain.

- Mechanism domain:as the prediction is ‘no alerts fired’ none of the mechanisms predicted in the 80 skin sensitisation alerts is applicable to this structure.

-Metabolic domain: no evident metabolism that might lead to skin sensitization is predicted for this structure

6. ADEQUACY OF THE RESULT
-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.

Guideline:

other: REACH Guidance on QSARs R.6

Version / remarks:

Prediction on the potential for skin sensitization with the in silico model DEREK NEXUS, version 5.0.2.

Principles of method if other than guideline:

- Software tool(s) used including version: in silico model DEREK NEXUS version 5.0.2
- Knowledge Base: Derek KB 2015 2.0
- Model description: see field 'Justification for non-standard information', 'Attached justification'
- Justification of QSAR prediction: see field 'Justification for type of information', 'Attached justification'

GLP compliance:

no

Specific details on test material used for the study:

SMILES: C([C@@H]1[C@@H](O)[C@@H](O)[C@@H](O1)N2C=NC=3C(N)=NC=NC2=3)OP(=O)(O)O

Key result

Parameter:

other: alerts for skin sensitization

Remarks on result:

other: DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitization for the test item.Test item is predicted to be not sensitizing to the skin.

Interpretation of results:

other: Non-Sensitizer

Conclusions:

DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitization for the test item. Alpha-D-glucose pentaacetate (CAS Nr. 604-68-2) is predicted to be not sensitizing to the skin.

Executive summary:

The objective of this study was to obtain a prediction on the potential for skin sensitization of alpha-D-glucose pentaacetate (CAS Nr. 604-68-2) with the in-silico model DEREK NEXUS. In this assessment version 5.0.2 of DEREK NEXUS was used.

DEREK NEXUS is a knowledge-based system that contains 80 alerts for skin sensitization based on the presence of molecular substructures. LHASA has inserted validation comments for the skin sensitization alerts: The DEREK NEXUS system has been designed for the qualitative prediction of the possible toxicity of chemicals. The predictions made by DEREK NEXUS are intended as an aid to toxicological assessment and, where appropriate, should be used in conjunction with other methods. “No alerts fired” may be extrapolated to a negative prediction.

DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitization for the test item alpha-D-glucose pentaacetate (CAS Nr. 604 -68 -2).

Therefore, substance should not be classified according to DEREK NEXUS; however, this (Q)SAR prediction cannot be used as stand-alone for classification purposes or for covering the endpoint skin sensitization for registration under REACH.

The result is adequate to be used in a weight-of-evidence approach together within chemico/in vitro studies to complete the endpoint skin sensitization.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

22 May-14 June 2017

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:

04 February 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

SUMMARY: DPRA measures the reaction of the test item with synthetic peptides containing cysteine (Ac-RFAACAA-COOH) or lysine (Ac-RFAAKAA-COOH). The custom peptides contained cysteine or lysine as the nucleophilic reaction centres and phenylalanine to facilitate HPLC detection. Test item and peptide were combined and incubated together for 24 h at room temperature. Following this incubation, the concentration of free (i.e. unreacted) peptide remaining was measured by HPLC immediately prior to the lysine peptide assay.


EXPERIMENTAL PROCEDURES

PEPTIDES:
Source: RS Synthesis
Batch:
- Cys: No. P170419-LC180433
- Lys: No. P161108-LC107617
Purity:
-Cys: 96.47%
-Lys: 98.14%

BUFFERS USED:
- Phosphate buffer: ca 100 mM, pH 7.47
- Ammonium acetate buffer: ca 100 mM, pH 10.18

SOLUBILITY ASSESSMENT:
- acetonitrile was selected as the most suitable solvent for the test material

PREPARATION PEPTIDE STOCK SOLUTIONS:
- CYSTEINE: stock solution of 0.501 mg/mL (0.667 mM) in phosphate buffer
- LYSINE: stock solution of 0.517 mg/mL (0.666 mM) in ammonium acetate buffer

CYSTEINE PEPTIDE ASSAY:
-PREPARATION: test item was dissolved in acetronile. The vial was sonicated until fully in solution.. The concentration of the test solution corrected for purity, was 39.0 mg/mL (100 mM, 100% of target). Cinnamic aldehyde was dissolved in acetonitrile with a concentration of 13.20 mg/mL (100 mM, 99.9% of the target). All test item and control solutions were prepared immediately prior to use.
-PREPARATION OF THE SANDARD CURVE: Dilution buffer was prepared by mixing phosphate buffer (pH 7.47, 8 mL) with acetonitrile (2 mL). Standard 1 (STD1) was prepared by mixing peptide stock solution (1600 µL) with acetonitrile (400 µL). Serial dilutions (1:1, v/v) were prepared from this to make a standard curve (from 0.534 to 0.0167 mM). An additional sample containing only dilution buffer was included as a blank (0 mM) standard. The standard curve was analysed by HPLC immediately prior to the cysteine peptide assay.
-REFERENCE CONTROL: Acetonitrile (250 µL) was mixed with peptide stock solution (750 µL). Three replicates of this were produced for Reference Control A. Reference Control B was prepared as described for Reference Control A. Three replicates were analyzed at the beginning of the testing run, and three at the end of the testing run, to demonstrate peptide stability over the analysis time. Reference Control C samples were prepared containing acetonitrile (250 µL) and cysteine peptide stock (750 µL), three replicated distributed throughout the run. These samples were included in every assay run together with the samples containing test item to verify that the solvent does not impact upon peptide stability during the assay, and to calculate percentage peptide depletion.
- PEPTIDE ASSAY METHOD: The assay contained a 1:10 molar ratio of peptide to test item. Positive control or test item (50 µL) was mixed with acetonitrile (200 µL) and the peptide solution (750 µL). The vials were mixed by inversion. Co-elution controls were prepared by mixing together acetonitrile (200 µL), phosphate buffer (750 µL) and test item (50 µL). All test items and positive control samples were prepared in triplicate. All vials were stored in the dark at ambient temperature for ca 24 h until analyzed by HPLC.

LYSINE PEPTIDE ASSAY:
-PREPARATION: test item was dissolved in acetronile and mixed by inversion until fully in solution. The concentration of the test solution corrected for purity, was 39.0 mg/mL (100 mM, 100.0% of target). Cinnamic aldehyde was dissolved in acetonitrile with concentration of 13.24 mg/mL (100 mM, 100.1% of target). All tets item and control solutions were prepared immediately prior to use.
- PREPARATION OF THE STANDARD CURVE: Dilution buffer was prepared by mixing ammonium acetate buffer (pH 10.18, 8 mL) with acetonitrile (2 mL). Standard 1 (STD1) was prepared by mixing peptide stock solution (1600 µL) with acetonitrile (400 µL). Serial dilutions (1:1, v/v) were prepared from this to make a standard curve (from 0.534 to 0.0167 mM). An additional sample containing only dilution buffer was included as a blank (0 mM) standard. The standard curve was analyzed by HPLC.
- REFERENCE CONTROL: like for cysteine
- PEPTIDE ASSAY METHOD: The assay contained a 1:50 molar ratio of peptide to test item. Cinnamic aldehyde or test item (250 µL) were mixed with peptide solution (750 µL). The vials were mixed by inversion and vortex. Co-elution controls were prepared by mixing together ammonium acetate buffer (750 µL) and test item (250 µL). All vials were stored in the dark at ambient temperature for ca 24 h until analysed by HPLC.

CHROMATOGRAPHIC AND DETECTOR PARAMETERS
- Column: Phenomenex Luna C18 (2) (2 x 100 mm, 3 µm)
- Run Time: 20 min
- Mobile Phase Conditions: Mobile Phase A: trifluoracetic acid (0.1%, v/v) in Milli-Q H2O
Mobile Phase B: trifluoracetic acid (0.085%, v/v) in acetonitrile
- Flow Rate: 0.35 mL/min
- Column Temperature: 30°C
- Auto Sampler Temperature: Room temperature
- Injection Volume: 7 µL
- UV Wavelength: 220 nm
- HPLC Gradient: see below

CALCULATIONS:
The concentration of peptide remaining in each sample following incubation was calculated from integrated peak area, with reference to the peptide standard curve. Percent peptide depletion was calculated from the following formula:
Peptide Depletion (%) = 1 – ( Peak Area (Sample) / Mean Peak Area (Reference Control C)) x 100

Positive control results:

The mean depletion value for the positive control was 69.8% showing a high reactivity (Sensitizer)

Key result

Run / experiment:

other: DPRA cysteine and lysine prediction model

Parameter:

other: Mean Depletion Value

Value:

26.8

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

Cinnamic aldehyde

Remarks on result:

other:

Remarks:

Moderate Reactivity (Sensitizer)

Other effects / acceptance of results:

No co-elution of the test item with either peptide was observed.

SYSTEM SUITABILITY FOR THE CYSTEINE ASSAY
The calibration linearity, r2, for the cysteine standard curve was 0.9994. This met the acceptance criteria for r2 which was >0.990.
The mean peptide concentration of Reference Control A was 0.509 ± 0.003 mM (mean ± SD). The calculated peptide concentration in the Reference Control C samples was 0.495 ± 0.005 mM. These controls met the acceptance criteria (0.5 ± 0.05 mM).

For the six Reference Control B and three Reference Control C samples in acetonitrile, the coefficient of variation (CV) was 1.5% (acceptance criteria for CV was <15%).

The mean percentage peptide depletion value of the three replicates for cinnamic aldehyde fell within the lower bound and upper bound values of 60.8% and 100.0% for cysteine, with a peptide depletion value of 78.0 ± 0.3% (mean ± SD).

Finally, the standard deviation of replicate test item samples was <14.9% for a-D-Glucose Pentaacetate (actual standard deviation = 1.6%).


SYSTEM SUITABILITY FOR THE LYSINE ASSAY
The calibration linearity, r2, for the lysine standard curve was 0.9999. This met the acceptance criteria for r2 which was >0.990.

The mean peptide concentration of Reference Control A was 0.495 ± 0.002 mM (mean ± SD). The calculated peptide concentration in the Reference Control C samples was 0.495 ± 0.001 mM. These controls met the acceptance criteria (0.5 ± 0.05 mM).

For the six Reference Control B and three Reference Control C samples in acetonitrile, the coefficient of variation (CV) was 0.6% (acceptance criteria for CV was <15%).

The mean percentage peptide depletion value of the three replicates for cinnamic aldehyde fell within the lower bound and upper bound values of 40.2% and 69.0% for lysine, with the SD <11.6%. The actual percentage peptide depletion value reported for cinnamic aldehyde was 61.5% ± 4.6% (mean ± SD).

Finally, the standard deviation of replicate test item samples was <11.6% for the test item (actual standard deviation = 1.6%).

PROTOCOL DEVIATIONS
The study was performed in accordance with the protocol with the following deviation:
- the protocol states that prior to HPLC analysis samples will be visually inspected. This was not done for the lysine or cysteine assay. As there was no evidence of a precipitate forming (i.e. the needle would have blocked), and all replicates of the same conditions are consistent with each other, there is no impact on study integrity.


DEMONSTRATION OF TECHNICAL PROFICIENCY
Prior to use, Charles River Laboratories demonstrated technical proficiency in the DPRA test, using the panel of proficiency chemicals listed in OECD 442C (Toner, F, 2015).

Test Item	% Peptide Depletion Cysteine (Mean  ± SD)	% Peptide Depletion Lysine (Mean ± SD)	Mean of Cysteine and Lysine	DPRA Classification (Cysteine and Lysine Prediction Model)
alpha-D-Glucose Pentaacetate	22.4 ± 1.6	31.2 ± 1.6	26.8	Moderate Reactivity (Sensitizer)
Cinnamic Aldehyde (Positive Control)	78.0 ± 0.3	61.5 ± 4.6	69.8	High Reactivity (Sensitizer)

Using the cysteine and lysine prediction model (see Table below) the test material was categorised as moderately reactive and a sensitiser.

Mean depletion values (Cys Lys)	Mean Depletion values (cys only)	Reactivity classification	DPRA Prediction
<6.38 %	<13.89%	Minimal	Non Sensitizer
6.38 -22.62%	13.89 -23.09%	Low	Sensitizer
22.62 -42.47%	23.09%-98.24%	Moderate	Sensitizer
>42.47	>98.24%	High	Sensitizer

Interpretation of results:

other: moderately reactive: sensitizer

Conclusions:

In conclusion, according to the DPRA cysteine and lysine prediction model, alpha-D-Glucose Pentaacetate (CAS No. 604-68-2) was classified as moderately reactive and was, therefore, a sensitiser

Executive summary:

Skin sensitisation is a type IV (delayed) hypersensitivity reaction that results from the interaction of a sensitising agent with host proteins to form an immunogenic complex.

Small molecules that can interact with proteins in this way are referred to as haptens, and are generally not immunogenic in isolation. Hapten-modified proteins are recognised as foreign by antigen presenting cells, leading to T-cell activation and localised inflammation at the site of all subsequent exposures to the hapten.

Most skin sensitising agents are electrophiles, i.e. will accept an electron pair from a nucleophile to form a covalent bond.  The amino acids cysteine and lysine are thought to be the nucleophiles most frequently modified in proteins during sensitisation, and the ability of small molecules to react with these amino acids forms the basis of the Direct Peptide Reactivity Assay (DPRA).

The objective of this study was to assess the peptide binding capability of alpha-D-Glucose Pentaacetate (CAS No. 604-68-2) using synthetic cysteine and lysine peptides and to classify the test item to one of the four reactivity classes leading to a DPRA prediction according to the following prediction model

Mean depletion values (Cys Lys)	Mean Depletion values (cys only)	Reactivity classification	DPRA Prediction
<6.38 %	<13.89%	Minimal	Non Sensitizer
6.38 -22.62%	13.89 -23.09%	Low	Sensitizer
22.62 -42.47%	23.09%-98.24%	Moderate	Sensitizer
>42.47	>98.24%	High	Sensitizer

The reaction of the test item with synthetic peptides containing cysteine (Ac-RFAACAA-COOH) or lysine (Ac-RFAAKAA-COOH) was performed.  The custom peptides contained cysteine or lysine as the nucleophilic reaction centres and phenylalanine to facilitate detection by HPLC analysis.

The solubility of the test item was assessed and acetonitrile was selected as the appropriate solvent.  For each peptide assay, the test item was prepared at a concentration of 100 mM in acetonitrile.  The test item and peptides were combined and incubated together for ca 24 h at room temperature.  Following this incubation, the concentration of free (i.e. unreacted) peptide remaining was measured by HPLC.  From the results obtained, a reactivity class was assigned and a DPRA prediction was made according to the above criteria.  The validity criteria were fulfilled.

The results obtained are presented in the following table:

Test Item	% Peptide Depletion Cysteine	% Peptide Depletion Lysine	Mean % peptide depletion	DPRA Classification
alpha-D-Glucose Pentaacetate	22.4 ± 1.6	31.2 ± 1.6	26.8	Moderate Reactivity (sensitizer)
Cinnamic Aldehyde (positive control)	78.0  ± 0.3	61.5 ± 4.6	69.8	High Reactivity (Sensitizer)

In conclusion, according to the DPRA cysteine and lysine prediction model, alpha-D-Glucose Pentaacetate (CAS No. 604-68-2) was classified as moderately reactive and was, therefore, a sensitiser.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13 October - 03 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: final test concentrations of 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 µM (final concentration vehicle DMSO of 1%)
- All concentrations of the test item were tested in triplicate.
- Positive control: Ethylene dimethacrylate glycol, final concentration 7.8 to 250 µM (final concentration DMSO of 1%)
- 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). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switzerland).

- Cell culture:
Basic medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.
Manteinance Medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 µg/ml).
Exposure medium: Dulbecco’s minimal supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.

- Environmental conditions:
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 28 – 99 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.6 – 37.4 °C).

EXPEIMENTAL DESIGN
- Plating of cells
For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each test item, one plate was used for the luciferase activity measurements, and one parallel plate was used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was p+17 in experiment 1 and p+3 in experiment 2.
- Treatment of cells
The medium was removed and replaced with fresh culture medium (150 µL culture medium containing serum but without Geneticin) to which 50 µL of the 25-fold diluted test chemical and control substances were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about 48 hours at 37±1.0 °C in the presence of 5% CO2. In total 2 experiments were performed.

- Luciferase activity measurement
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the luminometer to assess the quantity of luciferase (integration time one second).
- Cytotoxicity assessment
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured with the TECAN Infinite® M200 Pro Plate Reader.

ACCEPTABILITY CRITERIA
The KeratinoSensTM test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 µM).
b) The EC1.5 should be between 5 and 125 µM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 µM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol 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.
c) Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

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.

- Data interpretation
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 Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s t-test)
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration)
3. The EC1.5 value is less than (<) 1000 µM (or < 200 µg/mL for test chemicals with no defined MW)
4. There is an apparent overall dose-response for luciferase induction

Positive control results:

• Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.29 and the EC1.5 127 µM.
• Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.74 and the EC1.5 46 µM.

Key result

Run / experiment:

other: 1

Parameter:

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

Value:

118

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

Imax: 2.29

Key result

Run / experiment:

other: 2

Parameter:

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

Value:

190

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

Imax: 2.74

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)

Remarks:

IC30: 280 µM and IC50: 347 µM

Value:

12

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

EC1.5: 127 µM

Remarks on result:

other: positive in the KeratinoSensTM assay

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)

Remarks:

IC30: 728 µM and IC50: 824 µM

Value:

27

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

EC1.5 46 µM

Remarks on result:

other: positive in the KeratinoSensTM assay

Other effects / acceptance of results:

Acceptance criteria:
• The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
• The EC1.5 of the positive control was between 5 and 125 µM (127 µM and 46 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.29-fold and 2.74-fold in experiment 1 and 2, respectively).
• Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (9.7% and 5.4% in experiment 1 and 2, respectively).

alpha-D-glucose penataacetate showed toxicity (IC30 values of 280 µM and 728 µM and IC50 values of 347 µM and 824 µM in experiment 1 and 2, respectively). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 12 µM and 27 µM in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 118-fold and 190-fold in experiment 1 and 2 respectively. a-D-glucose penataacetate (CAS 604-68-2) is classified as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed at test concentrations < 1000 µM with a cell viability of >70% compared to the vehicle control.

Table1          
Overview Luminescence Induction and Cell Viability of alpha-D-glucose penataacetate in Experiment 1 and 2

Concentration (µM)	0.98	2.0	3.9	7.8	16	31	63	125	250	500	1000	2000
Exp 1 luminescence	1.08	1.18	1.20	1.40	1.61^***	1.84^***	3.23^***	12.38^***	118^***	0.18	0.01	0.01
Exp 1 viability (%)	100.1	97.1	103.7	101.9	105.7	114.8	110.3	101.9	79.2	3.6	1.1	0.6
Exp 2 luminescence	1.02	1.05	1.11	1.20	1.36	1.55^**	2.09^***	3.75^***	7.68^***	190^***	7.95	0.03
Exp 2 viability (%)	109.8	106.1	103.6	113.4	110.9	120.7	123.9	125.6	140.4	117.0	13.7	5.7

^**p < 0.05,^***p<0.001 Student’s t test

 

Table2          
Overview Luminescence Induction and Cell Viability Positive Control EDMG in Experiment 1 and 2

Concentration (µM)	7.8	16	31	63	125	250
Exp 1 luminescence	1.22	1.18	1.31	1.27	1.49	2.29^***
Exp 1 viability (%)	102.5	102.6	96.8	98.1	94.7	97.5
Exp 2 luminescence	1.07	1.16	1.36	1.67^***	2.24^***	2.74^***
Exp 2 viability (%)	100.3	99.6	100.7	110.0	112.6	108.2

^***p<0.001 Student’s t test

 

Table3          
Overview EC_1.5, I_max, IC₃₀and IC₅₀Values

	EC1.5(µM)	Imax	IC30(µM)	IC50(µM)
Test item Experiment 1	12	118	280	347
Test item Experiment 2	27	190	728	824
Pos Control Experiment 1	127	2.29	NA	NA
Pos Control Experiment 2	46	2.74	NA	NA

NA = Not applicable

Interpretation of results:

other: activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes

Remarks:

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

Conclusions:

In conclusion, the KeratinoSensTM assay is valid and alpha-D-glucose penataacetate (CAS 604-68-2) is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in
this report.

Executive summary:

The objective of this study was to evaluate the ability of alpha-D-glucose penataacetate (CAS 604-68-2) to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSensTM assay.

The study procedures described in this report were based on the most recent OECD guideline.

Batch 14126200 of the test item was a white crystalline powder. A correction factor of 1.112 was used to correct for the purity (89.9%). The test item was dissolved in dimethyl sulfoxide at 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000 µM (2-fold dilution series). The highest test concentration was the highest dose required in the current guideline. No precipitate was observed at any dose level tested. Two independent experiments were performed.

Both experiments were considered acceptable:

- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.

- The EC1.5 of the positive control was 127 µM (see study plan deviation) and 46 µM in experiment 1 and 2, respectively. A dose response was observed and the induction at 250 µM was higher than 2-fold (2.29-fold and 2.74-fold in experiment 1 and 2, respectively).

- Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (9.7% and 5.4% in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

The test item showed toxicity (IC30 values of 280 µM and 728 µM and IC50 values of 347 µM and 824 µM in experiment 1 and 2, respectively). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 12 µM and 27 µM in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 118-fold and 190-fold in experiment 1 and 2 respectively. alpha-D-glucose penataacetate is classified as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed at test concentrations < 1000 µM with a cell viability of >70% compared to the vehicle control.

In conclusion, the KeratinoSensTM assay is valid and alpha-D-glucose penataacetate (CAS 604-68-2) is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitization for the test item. Alpha-D-glucose pentaacetate is predicted to be not sensitizing to the skin.

A valid DPRA assay was performed according to OECD 442C and GLP principles. The test item was dissolved in acetonitrile at 100 mM. There was no evidence of co-elution of ATP, Di-Na with either Cysteine or Lysine peptide. Peptide depletion was calculated as 22.4% and 31.2% in Cysteine and Lysine Assays, respectively, resulting in a mean peptide depletion of 26.8%. This value places alpha-D-glucose pentaacetate in the Moderate 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. From this stock 11 spike solutions were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.977 – 2000 µM (2-fold dilution series). No precipitate was observed at any dose level tested. Two independent experiments were performed.

The test item showed toxicity (IC30 values of 280 and 728 µM and IC50 values of 347 and 824 µM in experiment 1 and 2, respectively). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 12 and 27 µM in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 118-fold and 190 -fold in experiment 1 and 2, respectively. Alpha-D-glucose pentaacetate 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 negative outcome of the DEREK assessment, the positive outcome of the DPRA and KeratinoSens assay, alpha-D-glucose pentaacetate 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 alpha-D-glucose pentaacetate is still considered to be a skin sensitizer, while a positive outcome will only confirm that the test item is a skin sensitizer.

Acetate esters as present in alpha-D-glucose pentaacetate are known to be not entirely stable in water, which may be the initiating reaction in skin sensitization; the anomeric acetate group can be removed by water, upon which a hemi-acetal can be formed, which is in equilibrium with the open ring aldose form of glucose. The aldehyde of the aldose form may react with thiols/amines and thus be skin sensitizing. Therefore, in absence of potency data a worst case classification of alpha-D-glucose pentaacetate with category 1A is considered appropriate.

Based on the above data, alpha-D-glucose pentaacetate (CAS 604-68-2) 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.