oligomeric Reaction products from Hydrogenation and Hydrolysation of Starch

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan 2004 - Mar 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

473-690-8

EC Name:

-

Cas Number:

738602-93-2

Molecular formula:

not applicable for UVCB

IUPAC Name:

(2R,3R,4S,5S,6R)-2-{[(2R,3R,4R,5S,6R)-5-{[(2R,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-{[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}oxan-2-yl]oxy}-3,4-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-6-(hydroxymethyl)oxane-3,4,5-triol; (2S,3R,5R)-4-{[(2R,3R,4R,5S,6R)-5-{[(2R,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-{[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}oxan-2-yl]oxy}-3,4-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}hexane-1,2,3,5,6-pentol

Test material form:

liquid

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: Hayashibara Biochemical laboratories, Inc. Lot 3I021
- Purity, including information on contaminants, isomers, etc.: solid content of 72.5 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: at preparation, reaction to the vehicle was confirmed
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: not stated
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Water soluble
- Reactivity of the test material with the incubation material used (e.g. plastic ware): not stated

Method

Target gene:

no single target gene but whole genome

Species / strain

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: CHL/IU cell line as frozen stock from Japan Health Sciences Foundation, Health Science Research Resources Bank
- Suitability of cells: The CHL cell line is a practical model because its chromosomes are small in number and comparatively large in size. additionally, CHL/IU is sensitive to chemicals that induce chromosomal aberration.
- Normal cell cycle time (negative control): 15 hours

For cell lines:
- Absence of Mycoplasma contamination: yes
- Number of passages if applicable: 7 at receipt, 8 at stock
- Methods for maintenance in cell culture: not stated
- Cell cycle length, doubling time or proliferation index : 16.7 h
- Modal number of chromosomes: 25 +-2
- Periodically checked for karyotype stability: not stated
- Periodically ‘cleansed’ of spontaneous mutants: not stated

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: 10% CS MEM

Metabolic activation:

with and without

Metabolic activation system:

S9 rat liver

Test concentrations with justification for top dose:

1250, 2500, and 5000 µg/mL

Vehicle / solvent:

water for injection

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments 2 (short-term treatment, contineous treatment)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1*20^4 cells/mL
- Test substance added in medium;

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 6 h /24 h
- Harvest time after the end of treatment (sampling/recovery times): 18 h / 0 h

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): iColcemid, 10 µg/mL

- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): after fixation with Carnoys fixative solution, staining with Giemsa
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 100 cells/dish
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification):
according to "Sofuni T. Production Mechanism, Classification, and Criteria of Chromosomal Aberrations. In: Environmetnal Mutagen Society of Japan, Mammalian Mutagenicity Study Group. the Atlas of Chromosomal Aberration on Chemical Commpounds: first ed. Tokyo: Asakura shoten; 1988. p. 16-35
- Determination of polyploidy: according to "Sofuni T. Production Mechanism, Classification, and Criteria of Chromosomal Aberrations. In: Environmetnal Mutagen Society of Japan, Mammalian Mutagenicity Study Group. the Atlas of Chromosomal Aberration on Chemical Commpounds: first ed. Tokyo: Asakura shoten; 1988. p. 16-35
- Determination of endoreplication:according to "Sofuni T. Production Mechanism, Classification, and Criteria of Chromosomal Aberrations. In: Environmetnal Mutagen Society of Japan, Mammalian Mutagenicity Study Group. the Atlas of Chromosomal Aberration on Chemical Commpounds: first ed. Tokyo: Asakura shoten; 1988. p. 16-35

Rationale for test conditions:

according to Guideline

Evaluation criteria:

Ishidate's method was used to determine the frequency of cells with chromosomal aberrations in the test article treatment group. The judgement criteria are shown below. Negative (-) frequency < 5 %, Equivocal(+-) 5% <= frequence < 10 %, Positve (+) 10% <= frequency

Statistics:

none applied

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Acceptance Criteria:
The frequencies of cells with chromosomal aberrations in the negative and positive control groups were within th erange (Mean +. 2 SD) of the background data. Accordingly, it was judged that th echromosomal aberration test was performed satisfactorily.

Changes in pH by Test Article and Test Article Precipitations
Color changes indicating changes in pH in the culture medium were not observed upon addition of the test article preparation. Test article precipitation in the culture medium was not observed at up to 5000 µg/mL at the start or end of test article treatment.

Applicant's summary and conclusion

Conclusions:

Under the conditions of hte study, MG-60 did not induce chromosomal aberrationsin CHL/IU cells, regardless of a metabolic activation system or treatment time length

Executive summary:

In order to evaluate th eclastogenicity of MG-60, an invitro chromosomal aberration test using Chinese hamster lung (CHL/IU) cells was performed, and the frequencies of cells having structural and numerical chromosomal aberrations were investigated. Three dose levels, 1250, 2500 and 5000 µg/mL, were set for short-term treatments without and with metabolic activation, and for continuous treatment for 24 hours.

1) in chort-treatments without and with metabolic activation, and in continuous treatment for 24 hours, the frequencies of cells having structural and numerical chromosomal aberrations were below 5%, and no devrease was noted in the cell proliferation ratio at up to the highest dose level, which was estimated from the viable cell count.

2) Color changes indicating changes in pH in the culture medium were not observed upon addition of the test article preparation. Test article precipitation i n the culture medium was not observed at up to 5000 µg/mL at the start or end of test article treatment.

3) The frequencies of cells havin gchromosomal aberrations in the negative and poitive control groups in short-term treatmnets without and with metabolic activation, and in continuous treatment for 24 hours, were within the range of the backgorund data. Accordingly, it was judged that thhe chromosomal aberration test was performed satisfactorily.