Short Chain Fructo Oligosaccharides

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1988

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

Constituent 1

Reference substance name:

short chain Fructo-Oligosaccharides

EC Number:

908-300-1

Molecular formula:

C6H11O5(C6H10O5)nOH

IUPAC Name:

short chain Fructo-Oligosaccharides

Details on test material:

Neosugar is a mixture of fructooligosaccharides such as 1-kestose (GF2), nystose (GF3) and 1F-1-fructofuranosyl nystose (GF4). The sugar composition of Neosugar is approximately 37% GF2, 21% GF3 and 12% GF4.

Specific details on test material used for the study:

Neosugar is a mixture of fructooligosaccharides such as 1-kestose (GF2), nystose (GF3) and 1F-1-fructofuranosyl nystose (GF4). The sugar composition of Neosugar is approximately 37% GF2, 21% GF3 and 12% GF4.
Neosugar provided by Meiji Seika Kaisha Ltd.

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver microsomal metabolic activation system.
Metabolic activation system used consisting of liver microsomal fraction (S-9) from Charles River CD rats, 6-8 weeks old, given a single intraperitoneal injection of Aroclor 1254 at 500 mg/kg body weight 5 days before killing. The livers were removed and homogenized in three times their weight of ice-cold 0.15 MKCI. The homogenate was centrifuged at 900 g for 10 min, and the supernatant (S-9 fraction) was collected and stored at -70°C. The S-9 fraction was mixed with appropriate cofactors immediately before use.
In all cases, the functioning of the S-9 mix was tested by including positive control materials that required metabolic activation to express a genotoxic effect.

For the mouse lymphoma assay, the S-9 fraction was added to a threefold larger volume of icecold RPMI 1640 medium containing isocitric acid at 15 mg/ml and NADP at 8 mg/ml immediately before use, and 8 ml of that mixture was added to 12 ml of cell suspension with 0.2 ml of test solution.

Test concentrations with justification for top dose:

Mutagenicity was tested by treating populations of 12 x 10E6 cells for 3 hr at 37°C with neosugar at 2000, 3000,4000, and 5000 µg/ml.

Controls

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Preliminary toxicity tests:
Preliminary toxicity tests were conducted by treating cells in suspension with neosugar at 50, 100, 250, 1000, 2500, and 5000 pg/ml at 37°C for 3 hr. The highest dose used represented the maximum dose used routinely in this assay to avoid confounding physical effects. The cells were then washed and resuspended in normal growth medium and cultured at 37°C. Cell population growth was monitored at 24 and 48 hr using a Coulter counter.

Defintive toxiciy tests:
Mutagenicity was tested by treating populations of 12 x 10E6 cells for 3 hr at 37°C with neosugar at 2000, 3000, 4000, and 5000 pg/ml in the presence and absence of an Aroclor-induced rat liver microsomal
metabolic activation system. After treatment in serum-free medium, the cells were washed and grown for 48 hr in normal growth medium to allow expression of any induced mutations, then cloned in soft agar to test for viability (600 cells/plate) and mutations (lo6 cells/plate, plus trifluorothymidine at 4 pg/ml). The procedures used are based on those of Clive and Spectorl and Amacher et al.

Two independent trials were conducted both in the presence and in the absence of metabolic activation.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In both trials, no significant toxicity at any dose level was detected, as measured by cell population growth after treatment or colony-forming ability Also, no increase in mutation frequency was seen in either trial, either with or without metabolic activation.
The positive control chemicals produced the expected clear increases in mutation frequency.

Applicant's summary and conclusion

Conclusions:

No increase in mutation frequency was seen in either trial, either with or without metabolic activation.
No indication of mutagenicity was observed.

Executive summary:

A Mammalian cell mutation assay with mouse lymphoma L5178Y cells was performed. Preliminary toxicity was tested with scFOS concentrations of 50, 100, 250, 1000, 2500, or 5000 μg/ml. Mutagenicity was tested with concentrations of 2000, 3000, 4000, or 5000 μg/ml in the presence and absence of an Arochlor-induced rat liver microsomal metabolic activation system.

No indication of mutagenicity was observed.