D-cellobiose

Endpoint summary

Description of key information

Cellobiose was negative in GLP-compliant studies (h-CLAT according to OECD TG 442E and DPRA according to OECD TG 442C). These data were combined in the defined approach ITS DA as described in OECD GL 497. The sum score of the two results is 0. Regardless of the result of any in silico prediction (DEREK Nexus or OECD Toolbox), this corresponds to a GHS/CLP classification as not classified (NC) ( see Figure 3.1 of OECD GL 497).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 22, 2021, to January 13, 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the key event on activation of dendritic cells on the Adverse Outcome Pathway for skin sensitisation)

Version / remarks:

ANNEX I: IN VITRO SKIN SENSITISATION: HUMAN CELL LINE ACTIVATION TEST (H-CLAT) (25 JUNE 2018)

Deviations:

no

GLP compliance:

yes

Type of study:

human Cell Line Activation Test (h-CLAT)

Specific details on test material used for the study:

LOT No.: L1021174
Appearance: white, dry powder
Expiry date: 28 October 2023
Storage condition: at room temperature, protected from light
Safety precautions: according to MSDS
Purity: 99.54 %

Details of test system:

THP-1 cell line [442E]

Details on the study design:

442E

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: Saline was used to dissolve the test item for the stock solution (SS) in the preliminary and the main test. In the main test, the test item was first diluted to the concentration corresponding to the highest soluble concentration (HSC - 50 mg/mL) determined in the solubility and preliminary test

- Preparation of the test chemical serial dilutions: In the main test, for the master solutions (MS), 1.2-fold serial dilutions were made from the stock solution using saline (eight concentrations were prepared from the test item stock solution at the concentration of 50.6.0 mg/mL in the first test and at concentration of 50.1 mg/mL in the second test). The master solutions were then further diluted 50-fold into the culture medium to obtain the working solutions (WS). These working solutions were finally used for exposure with a further final 2-fold dilution factor in the plate.

- Preparation of the positive controls: DNCB was used as the positive control for CD86/CD54 expression measurement at a final nominal concentration of 4.0 μg/mL in the plate. To obtain a 4.0 μg/mL concentration a 2 mg/mL stock solution of DNCB in DMSO were prepared and further diluted 250-fold with culture medium to an 8.0 μg/mL working solution. The working solution then was diluted 2-fold when added to the cells.

- Preparation of the solvent, vehicle and negative controls: Culture medium was used as negative control for the test item and to assess the impact of DMSO. Moreover, DMSO was used as a solvent control for the positive control (DNCB) at a single final concentration in the plate of 0.2 %. To obtain a 0.2 % concentration, DMSO was diluted 250-fold with culture medium to a 0.4 % working solution. The working solution then was diluted 2-fold when added to the cells.

- Stable dispersion obtained: Yes
- Log Kow of the test chemical: < 3.5


DOSE RANGE FINDING ASSAY:
- Highest concentration used: 500.0 µg/mL
- Highest soluble concentration (in solvents and/or incubation medium): 500.0 µg/mL
- Results of selecting appropriate concentration and determination of cytotoxicity e.g. CV75: The highest soluble concentration of 500.0 µg/mL was not cytotoxic.
- Final concentration range selected on basis of: 139.5 to 500 µg/mL based on highest soluble concentration and dilution series with a dilution factor of 1/1.2.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: 3
- Number of repetitions: 2 (runs)
- Test chemical concentrations: 139.5, 167.5, 200.9, 241.1, 289.4, 347.2, 416.7, 500 µg/mL
- Application procedure: The culture medium or working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate.
- Exposure time: 24 ± 0.5 hours
- Study evaluation and decision criteria used: If the RFI of CD86 is equal to or greater than 150 % at any tested dose (>50 % of cell viability) in at least 2 independent runs, AND/OR if the RFI of CD54 is equal to or greater than 200 % at any tested dose (>50 % of cell viability) in at least 2 independent runs, the test item prediction is considered as positive. Otherwise, it is considered as a negative.
In case the first two independent runs are not concordant a third run needs to be performed and the final prediction will be based on the mode of conclusions from the three individual runs.

- Description on study acceptance criteria:
- The cell viabilities of medium and solvent/vehicle controls should be higher than 90 %.
- In the positive control (DNCB), relative fluorescence intensity (RFI) values of both CD86 and CD54 should be over the positive criteria (CD86 RFI ≥ 150 % and CD54 RFI ≥ 200 %) and cell viability should be more than 50 %.
- In the solvent/vehicle controls, RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI ≥ 150 % and
CD54 RFI ≥ 200 %)
- For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control should be >105 %.
- For the test chemical, the cell viability should be more than 50 % in at least four tested concentrations in each run.
- For the test chemical resulting in negative outcome, the cell viability at the 1.2 x CV75 should be less than 90 %. When the test item is tested 5000 µg/mL in saline (maintenance medium alternatively) or 1000 µg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration, a negative result is acceptable even if the cell viability is above 90 %.
- For the test chemical resulting in positive outcome, the cell viability at the 1.2 x CV75 is more than 90 %, the data is acceptable

SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density): THP-1 cells (passage 6) were seeded at a density of either 0.1 × 106 cells/mL or 0.2 × 106 cells/mL, and precultured in culture flasks for 72 hours or 48 hours respectively
- Incubation conditions: 37° C under 5 % CO2

MEASUREMENT OF CELL SURFACE EXPRESSION/LUCIFERASE ACTIVITY
For h-CLAT and USENS
- Flow cytometry used: Name: Beckman Coulter Flow Cytometer, Type: CytoFLEX System B4-R0-V0, Serial number: BC46360
- Plate used: 24 well flat-bottom plate
- Propidium iodide staining/cytotoxicity measurements: After 24 ± 0.5 hours of exposure, cells were transferred into sample tubes and 600 μL of FACS buffer was added to each sample. Cells were then collected by centrifugation (300 g,
5 min, 4 ºC). The supernatants were discarded and the remaining cells were washed again with 600 μL of FACS buffer. Finally, cells were resuspended in 400 μL of FACS buffer and 20 μL of 1 × PI solution was added for each sample. The PI uptake was analysed using flow cytometry with the acquisition channel FL-3. A total of minimum 10,000 living cells (PI negative) were acquired. Cell viability was analyzed by the CytExpert Software by gating out PI positive cells, and the calculated percentage of PI negative cells was displayed on the software.
- Preparation for CD54 and/or CD86 expression measurements/cell staining: After 24 ± 0.5 hours of exposure, cells were transferred from the 24-well plate into sample tubes, then 1 mL of FACS buffer was added to each sample and cells were collected by centrifugation (300 g, 5 min, 4 ºC). The washing step was repeated once more with 1 mL of FACS buffer. After washing, cells were blocked with 600 μL of 1 × blocking solution and incubated at approximately 4 °C for 15 min. After blocking, cells were split in three aliquots of 200 μL into sample tubes. After centrifugation (300 g, 5 min, 4 ºC), cells were stained with 50 μL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 (isotype) antibodies and incubated at approximately 4 °C for 30 min. The antibodies described in the h-CLAT DB-ALM protocol 158° were used. After washing twice with 150 μL of FACS buffer, cells were resuspended in 400 μL of FACS buffer and 20 μL of 1 × PI solution was added to each sample. The expression levels of CD86 and CD54, and cell viability were analysed using flow cytometry.

DATA EVALUATION
- Cytotoxicity assessment: Two independent runs for the dose finding assay were performed to determine the test item concentration that results in 75 % cell viability (CV75) compared to the solvent/vehicle control.
- Prediction model used: If the RFI of CD86 is equal to or greater than 150 % at any tested dose (>50 % of cell viability) in at least 2 independent runs, AND/OR if the RFI of CD54 is equal to or greater than 200 % at any tested dose (>50 % of cell viability) in at least 2 independent runs, the test item prediction is considered as positive. Otherwise, it is considered as a negative.

In case the first two independent runs are not concordant a third run needs to be performed and the final prediction will be based on the mode of conclusions from the three individual runs.

When the item is tested at 5000 µg/mL in saline, 1000 µg/ml in DMSO, or highest soluble dose as the maximal test concentration instead of CV75-based dose and does not meet the positive criteria above without affecting cytotoxicity at all tested doses, the test item prediction should be considered as negative.

Since EC150 and EC200 values are just optional for test items that are found to be sensitisers, the values are calculated only in cases where a firm dose response curve can be constructed.

Up to six runs, meeting requirements for qualified testing, are permitted to reach a conclusion for each test item. The six runs may include runs for which the data adoption criteria are not met for this test item. If no prediction can be made after the sixth run, the result is inconclusive.

Vehicle / solvent control:

saline [442E]

Negative control:

other: DMSO

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC200, CD54 [442E]

Cell viability:

at least 97.8%

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC150, CD86 [442E]

Cell viability:

at least 97.8%

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC200, CD54 [442E]

Cell viability:

at least 96.17%

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC150, CD86 [442E]

Cell viability:

at least 96.17%

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the test method described in Annex II of the Test Guideline 442E, TOXI-COOP ZRT. demonstrated technical proficiency by correctly obtaining the expected h-CLAT prediction for the 10 proficiency substances recommended and listed in APPENDIX II of the OECD TG 442E and by obtaining CV75, EC150 and EC200 values that fell within the respective reference ranges (Study Number: 392-442-5369). Moreover, a historical database of data generated with the reactivity checks and with the positive and solvent/vehicle controls is maintained over time to confirm the reproducibility of the test method in the laboratory.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Interpretation of results:

other: negative/non-sensitiser, i.e. no indication of skin sensitization potential (to be used in Defined Approach acc. to OECD GL 497)

Conclusions:

In a GLP-compliant study according to OECD TG 442E with the h-CLAT, cellobiose did not show any skin sensitization potential.

Executive summary:

The potential of cellobiose to induce skin sensitisation was investigated in a GLP-compliant study according to OECD TG 442E with the h-CLAT.

The extent of cytotoxicity induced on THP-1 cells by the test item was studied in two dose finding tests. The maximal final test item concentration dissolved in saline was 500.0 μg/mL in both runs. Since no cytotoxicity was observed in any of the runs, no CV75 value was calculated. Based on the results of the two dose finding tests, eight doses between 500.0 μg/mL – 139.5 μg/mL (nominal concentrations) were used for the main test in two independent runs and both runs were concluded as valid.

The increase in CD86 marker expression (RFI) was below 150 % at all tested doses compared to the respective negative controls in both independent runs. Also, no cytotoxicity was observed. Based on the two valid concordant negative results for CD86 marker expression, the main test was concluded to be negative.

The increase in CD54 marker expression (RFI) was below 200 % at all tested doses compared to the respective negative controls in both independent runs. Also, no cytotoxicity was observed. Based on the two valid concordant negative results for CD54 marker expression, the main test was concluded to be negative.

Since both CD86 and CD54 marker expressions showed negative results, the overall h-CLAT prediction is concluded to be negative.

To derive a final conclusion on the skin sensitisation potential in terms of GHS classification, the results of this study will be combined with other information in a defined appraoch according to OECD GL 497.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from November 30, 2021, to February 14, 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)

Version / remarks:

DPRA

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

LOT number: L1021174
CAS-number: 528-50-7
Appearance: white, dry powder
Expiry date: 28 October 2023
Storage condition: at room temperature, protected from light
Safety precautions: according to MSDS
Purity: 99.54 %
Molecular weight: 342.3 g/mol

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions:
Cysteine peptide stock solution, 0.667 mM, 0.501 mg/mL:
With 18 mL of estimated buffer volume, 0.00931 g of the cysteine peptide would be the target weight. 0.01075 g of the peptide was pre-weighted and 20.784 mL of pH 7.5 phosphate buffer was added, based on the second equation below, right before beginning the assay.

"estimated mL of phosphate buffer"×(0.501 mg/mL)/(purity of the peptide)= peptide target weight (mg)

"estimated mL of phosphate buffer"×(peptide actual weight (mg))/(peptide target weight (mg))=actual volume of buffer (mL)

Lysine peptide stock solution, 0.667 mM, 0.518 mg/mL:
With 18 mL of estimated buffer volume, 0.00985 g of the lysine peptide would be the target weight. 0.01085 g of the peptide was pre-weighted and 19.827 mL of pH 10.2 acetate buffer was added based on the second equation below, right before beginning of the assay.

"estimated mL of acetate buffer"×(0.518 mg/mL)/(purity of the peptide)= peptide target weight (mg)

"estimated mL of acetate buffer"×(peptide actual weight (mg))/(peptide target weight (mg))=actual volume of buffer (mL)


- Preparation of the test chemical solutions:
100 mM solutions of the test chemical in the appropriate solvent were prepared just before use. The needed amount of test chemical (0.0344 g) for 1 mL solvent was calculated based on the molecular weight and purity of the substance with the equations below. 0.0344 g test chemical was weighted for the stock solutions used for the cysteine peptide depletion determination.

In a 1 mL volumetric glass (for cysteine analysis):

(molecular weight)/(% purity) ×10=target weight of test chemical (mg)

The needed amount of test chemical (0.0688 g) for 2 mL solvent was calculated based on the molecular weight and purity of the substance with the equations below. 0.0688 g test chemical was weighted for the stock solution used for lysine peptide depletion determination.

In a 2 mL volumetric glass (for lysine analysis):

(molecular weight)/(% purity) ×20=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.

- Preparation of the positive controls, reference controls and co-elution controls:
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. 0.0666 g cinnamaldehyde was weighted for the positive stock solution used for the cysteine peptide depletion determination and 0.0663 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 were combined with acetonitrile. System suitability was checked by the use of the three replicates of reference control A.

Reference control B: Peptide stock solutions were combined with acetonitrile. Stability of the peptides were 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 were combined with the respective solvent of the test item (and acetonitrile in case of cysteine peptide). Three replicates of reference control C were used as a solvent control to which the peptide concentration/depletion of the reaction samples was compared. Since acetonitrile is not the chosen solvent for the test item, a reference control C with acetonitrile was prepared additionally as the solvent control for the positive control.

Co-elution controls: Test item stock solution (and acetonitrile in case of cysteine peptide) was combined with the respective buffer solutions in each run (see Table 3). Co-elution controls were used to check for test item and peptide co-elution.


INCUBATION
- Incubation conditions: 24 ± 2 h incubation at 22.5°C - 30°C in the dark.
- Precipitation noted: no

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys: 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.
- Verification of the suitability of the HPLC for test chemical and control substances: Reference control A replicates were included in the HPLC run sequence to verify the HPLC system suitability prior analysis. The mean peptide concentration of A reference control sample replicates was 0.50 mM for the cysteine and lysine peptides. A standard calibration curve was generated for both cysteine and lysine peptides using serial dilutions from the peptide stock solutions. Calibration standard points were analyzed by linear regression. Means of the peak areas versus the concentrations of both peptides showed good linearity with r2 = 0.9929 for cysteine peptide and r2 = 0.9998 for the lysine peptide, covering the concentration range from 0.534 mM to 0.0167 mM. All validity criteria were within acceptable limits and therefore the study can be considered valid.


DATA EVALUATION
- Cys and Lys peptide detection wavelength: 220 nm

Prediction 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. Before applying a prediction model, the experimental data regarding possible co-elution is evaluated and the appropriate approach is selected based on the below mentioned scenarios in Table 4.

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.

Note: A single HPLC analysis for both the cysteine and the lysine peptide should be sufficient for a test chemical when the result is unequivocal. However, in cases of results close to the threshold used to discriminate between positive and negative results (i.e. mean percent depletion falls in the range of 3 % to 10 % for the cysteine 1:10/lysine 1:50 prediction model or cysteine percent depletion falls in the range of 9 % to 17 % for the cysteine 1:10 prediction model), additional testing is recommended. In particular, in case of negative results in these ranges (i.e. 3 % to 6.38 % for the cysteine 1:10/lysine 1:50 prediction model or 9 % to 13.89 % for the cysteine 1:10 prediction model), a second run should be conducted, as well as a third one in case of discordant results between the first two runs.

Vehicle / solvent:

water

Positive control:

cinnamic aldehyde

Positive control results:

The acceptance criteria were met for the positive control with a cysteine peptide depletion value of 67.13 % ± 0.54 % and with lysine peptide depletion value of 46.27 % ± 2.85 %.

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

mean lysine depletion

Value:

3 %

At concentration:

25 mM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

standard deviation was 2.7%

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

mean cystein depletion

Value:

0 %

At concentration:

5 mM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

the three replicates were all negative (< 10%) and considered as 0%

Outcome of the prediction model:

no or minimal reactivity [in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method, TOXI-COOP ZRT. demonstrated technical proficiency in a separate study (Study number.: 392-442-2996) by correctly obtaining the expected DPRA prediction for 10 proficiency substances as recommended in the OECD TG 442C guideline.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for reference controls A to C: yes
- Acceptance criteria met for co-elution controls (Lysine and Cysteine): yes

Interpretation of results:

other: negative/non to minimally reactive, i.e. no indication of skin sensitization potential (to be used in Defined Approach acc. to OECD GL 497)

Conclusions:

In a GLP-compliant study according to OECD TG 442C with the DPRA, cellobiose was not to minimally reactive.

Executive summary:

In a GLP-compliant study according to OECD TG 442C, the skin sensitization potential of the test item “Cellobiose” was studied using theDirect Peptide Reactivity Assay (DPRA).

In order to derive a prediction for both, the test chemical and the positive control substance two independent tests were evaluated, one with cysteine and one with lysine peptides,.

 

Peptide depletion resulting from the positive control cinnamaldehyde was within the expected percentage range both with cysteine and lysine peptides.

The mean back-calculated peptide concentrations of the reference control replicates were within the expected molarity concentration range and the CV % values for the nine reference controls B and C in acetonitrile were also acceptable. For each peptide, all validity criteria were met, confirming the validity of the study.

The mean cysteine peptide depletion value of the test item was 0 % ± 1.81 % while the lysine peptide depletion value of the test item was 3.00 % ± 2.70 %. The overall mean peptide depletion of the test item was 1.50 %, which is clearly below the borderline range.

 

Based on these results and thecysteine 1:10 / lysine 1:50 prediction model, the test item “Cellobiose” was concluded to be negative and to show no or minimal reactivity towards the synthetic peptides. Thus, “Cellobiose” is not a potential skin sensitizer under the experimental conditions of the in chemico Direct Peptide Reactivity Assay (DPRA) method.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The study results were combined according to OECD TG 497 ITS DA.

Justification for classification or non-classification

Cellobiose was negative in GLP-compliant studies (h-CLAT according to OECD TG 442E and DPRA according to OECD TG 442C). These data were combined in the defined approach ITS DA as described in OECD GL 497. The sum score of the two results is 0. Regardless of the result of any in silico prediction (DEREK Nexus or OECD Toolbox), this corresponds to a GHS/CLP classification as not classified (NC) ( see Figure 3.1 of OECD GL 497).

In conclusion, cellobiose is not a skin sensitiser and does not need to be classified in this regard.