D-cellobiose

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 7, 2016, to September 21, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

version of September 26, 2014

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

D-cellobiose

EC Number:

208-436-5

EC Name:

D-cellobiose

Cas Number:

528-50-7

Molecular formula:

C12H22O11

IUPAC Name:

(2R,3S,4S,5R,6S)-2-(hydroxymethyl)-6-[(2R,3S,4R,5R,6R)- 4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxane-3,4,5-triol

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Pfeifer & Langen GmbH & Co. KG, Batch no. CB 20.05.2016
- Purity, including information on contaminants, isomers, etc.: purity > 99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Method

Species / strain

Species / strain / cell type:

lymphocytes: human peripheral blood lymphocytes

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

S9: Post-mitochondrial fraction (S9 fraction) from rats treated with Aroclor 1254, prepared according to MARON and AMES (1983) was purchased from Trinova Biochem4. S9 was collected from male rats.

Test concentrations with justification for top dose:

preliminary experiment without and with metabolic: 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 μg/mL
main experiments:
4-h exposure and 24-h exposure without S9 as well as 4-h exposure with S9: 125, 250, 500, 1000 or 2000 μg/mL

The maximum concentration of 2000 μg/mL is the recommended limit concentration.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Well established solvent for this test

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

highly purified water

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- preliminary test (for cytotoxicity): 4 and 24 hours
- Exposure duration: main test: 4h (with and without S9) and 24h (without S9)

NUMBER OF REPLICATIONS: two cultures per concentration

NUMBER OF CELLS EVALUATED: 2000 binucleated cells per concentration, i.e. 1000 per culture

DETERMINATION OF CYTOTOXICITY
Cytokinesis- Block Proliferation Index (CBPI) or the Replicative Index (RI)

Cytokinesis was blocked with Cytochalasin B

Rationale for test conditions:

Standard conditions according to OECD Test Guideline 487

Evaluation criteria:

Only the frequencies of binucleate cells with micronuclei (independent of the number of micronuclei per cell) were used in the evaluation of micronucleus induction. Concurrent measures of cytotoxicity and/or cytostasis for all treated and vehicle control cultures were determined. Individual culture data were provided.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
• at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
• the increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test
• any of the results are outside the distribution of the historical negative control data (Poisson-based 95% control limits)
When all of these criteria are met, the test chemical is then considered able to induce chromosome breaks and/or gain or loss in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:
• none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
• there is no concentration-related increase when evaluated with an appropriate trend test,
• all results are inside the distribution of the historical negative control data (Poisson-based 95% control limits).
The test chemical is then considered unable to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification by additional testing of a clear positive or negative response.
Equivocal results may be clarified by analysis of another 1000 cells from all the cultures to avoid loss of blinding. If this approach does not resolve the result, further testing would be necessary. Modification of study parameters over an extended or narrowed range of conditions, as approp

Statistics:

according to OECD Test Guideline 487

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The results for the vehicle controls were within historical control range.
In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Therefore, the test is considered valid.

Any other information on results incl. tables

 

The concentration of 125 μg Cellobiose/mL was not analysed for micronucleus frequencies, as it was thought that it would provide no further information.

 

 

Applicant's summary and conclusion

Conclusions:

In a GLP-study according to OECD test guideline 487, cellobiose did not exhibit clastogenic or aneugenic activity in cultured human peripheral blood lymphocytes and is therefore considered negative.

Executive summary:

Test samples of Cellobiose were assayed in an in vitro micronucleus test using human peripheral lymphocytes both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals. The test was carried out employing two exposure times without S9 mix: 4 and 24 hours, and one exposure time with S9 mix: 4 hours. The harvesting time was 20 hours after the end of exposure. The cytokinesis-block technique was applied.The test item was completely dissolved in highly purified water. The vehicle highly purified water served as the negative control.

The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 μg Cellobiose/mL medium were employed. No signs of cytotoxicity were noted up to the top concentration of 2000 μg/mL medium. No relevant changes in pH or osmolality of the test item formulations at concentrations ranging from 3.16 to 2000 mg/mL medium were noted. Hence, 2000 μg/mL were employed as the top concentration for the genotoxicity tests without (4-hour or 24-hour exposure) and with metabolic activation (4-hour exposure).

No signs of cytotoxicity were noted in the main study up to the top concentration of 2000 μg Cellobiose/mL medium in the experiments without and with metabolic activation.

Under the present test conditions, Cellobiose tested up to a concentration of 2000 μg/ml medium in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of chromosomal damage in the in vitro micronucleus test.