Short Chain Fructo Oligosaccharides

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Clevenger et al. (1988) describes a series of in vitro genotoxicity studies on ScFOS. in this study, The genotoxicity of  scFOS (Neosugar®) has been evaluated iin vitro genotoxicity assays including a bacterial reverse mutation assay and an unscheduled DNA repair assay conducted in accordance with guidelines established by the Organization for Economic Cooperation and Development (OECD 471 and 482) and a mammalian cell mutation assay conducted according to recognized methods. As a results, the ScFOS was negative for genotoxicity in bacterial (Ames), mouse lymphoma, and unscheduled DNA synthesis assays, both with and without metabolic activation. In addition, a chromosome aberration assay on Chinese hamster ovary cells performed with oligofructose indicated an absence of toxicity and clastogenicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Data waiving:

study waived due to provisions of other regulation

Justification for data waiving:

other:

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline followed

Principles of method if other than guideline:

Study of the safety and metabolic handling of scFOS or gluco-mannan

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Test concentrations with justification for top dose:

313, 625, 1250, 2500, and 5000 μg oligofructose/ml

Details on test system and experimental conditions:

In the chromosome aberration assay, cultures of Chinese hamster ovary cells to concentrations of 313, 625, 1250, 2500, and 5000 μg oligofructose/ml for 4 hours and 20 hours in the absence of S9 metabolic activation and for 4 hours in its presence.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Additional information on results:

No statistically significant increases in structural or numerical chromosome aberrations were observed in either the activated or non-activated groups at any tested dose, indicating an absence of toxicity and clastogenicity.

In the initial and confirmatory assay no toxicity, as measured by a >50% reduction in cell growth relative to the solvent control, was
observed at any dose level tested in both the absence and presence of S9 activation. The dose levels selected for evaluation of chromosomal
aberrations in all treatment groups of the initial assay were 1250, 2500, and 5000 lg/mL. Either no statistically significant increases
in structural or numerical chromosome aberrations, relative to the solvent control, were observed in the non-activated or S9 activated groups, regardless of dose level.

Conclusions:

No statistically significant increases in structural or numerical chromosome aberrations were observed in either the activated or non-activated groups at any tested dose, indicating an absence of toxicity and clastogenicity.

Executive summary:

In the chromosome aberration assay, cultures of Chinese hamster ovary cells to concentrations of 313, 625, 1250, 2500, and 5000 μg oligofructose/ml for 4 hours and 20 hours in the absence of S9 metabolic activation and for 4 hours in its presence. No statistically significant increases in structural or numerical chromosome aberrations were observed in either the activated or non-activated groups at any tested dose, indicating an absence of toxicity and clastogenicity.

Reference 3

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

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

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.

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.

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.

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.

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.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

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.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

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 microbial tests, the S-9 mix contained 10% vol/vol s-9 fraction, 8 mM MgCI2, 33 mM KCI, 100 mM sodium phosphate buffer (pH 7.4), 5 mM glucose-6-phosphate, 4 mM NADPH, and 4 mM NADH; 0.5 ml of this mixture was added to 2 ml of top agar with 0.1 ml bacterial suspension and 0.1 ml test solution.

Test concentrations with justification for top dose:

Neosugar was tested in a standard plate incorporation assay at 0, 50, 150, 500, 1500, and 5000 µg/plate in each tester strain with and without metabolic activation.

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene

Details on test system and experimental conditions:

Neosugar was tested in a standard plate incorporation assay at 0, 50, 150, 500, 1500, and 5000 µg/plate in each tester strain with and without metabolic activation, using procedures complying with OECD and Japanese Ministry of Health and Welfare guidelines. Three test plates per strain per tireatment condition were used. Appropriate negative and positive controls were used with each strain.

Two independent trials were conducted, both with and without metabolic activation.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No signs of toxicity were seen at any of the doses of neosugar tested, and there was no increase in mutants per plate in any bacterial strain either with or without metabolic activation. The positive control chemicals produced the expected clear increases in mutation frequency.

Conclusions:

Test results gave no indication that neosugar possessed any genotoxic potential.

Executive summary:

A Microbial reverse mutation assays (Ames assay) was performed according the OECD 471. The study was carried out in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100 and Escherichia coli WP2 uvrA, with 0, 50, 150, 500, 1500, or 5000 μg/plate, with and without S9 metabolic activation. The study was replicated with three test plates per strain per treatment condition. 

The results of the mutagenicity study demonstrate that, over a wide range of dose levels, neosugar does not cause gene mutations in bacteria. No indication of mutagenicity was observed.

Reference 5

Endpoint:

in vitro DNA damage and/or repair study

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

GLP compliance:

not specified

Type of assay:

other: unscheduled DNA synthesis in mammalian cells in vitro

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.

Species / strain / cell type:

mammalian cell line, other: human epithelioid cells (HeLa S3)

Details on mammalian cell type (if applicable):

HeLa S3 epithelioid cells, originally derived from. a human cervical carcinoma, were obtained from Flow Laboratories, Ltd.,

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix was prepared by mixing 4 ml of S-9 fraction with 2 ml of 0.15 M KCI, 0.36 g glucose-6- phosphate, 0.05 g NADP, and 4 ml distilled water. Aliquots of 0.1 ml of that mixture were added to each 2 ml of medium in the tissue culture dishes containing cells.

Test concentrations with justification for top dose:

final concentration of 25, 50, 100, 200, 400, 800, 1600, 3200, 6400, 12,800, 25,600, or 51,200 µg/ml.

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

other: 2-aminoanthracene

Details on test system and experimental conditions:

HeLa S3 epithelioid cells, originally derived from. a human cervical carcinoma, were obtained from Flow Laboratories, Ltd., and cultured in Eagle’s Minimum Essential Medium (EMEM) with Earle’s salts and gentamicin at 50 pg/ml, plus 15% fetal calf serum. For the UDS assay, cells were grown to confluence on 22 mm coverslips in
multiwell tissue culture dishes in normal EMEM. The medium was then replaced with arginine-deficient medium containing 2.5% dialyzed fetal calf serum and kept at 37°C for an additional 72 hr to suppress normal scheduled DNA replication. 3H-Thymidine (20 Ci/mmole) was then added to a final concentration of 5 pCi/ml, together with 100 pl of an appropriate concentration of the test chemical to give a final concentration of 25, 50, 100, 200, 400, 800, 1600, 3200, 6400, 12,800, 25,600, or 51,200 µg/ml.
Appropriate negative (water and dimethylsulfoxide) and positive (4-nitroquinoline-1-oxide and 2-aminoanthracene) controls also were tested.“” After treatment for 3 hr, the coverslips and cells were removed, washed, fixed, stained in orcein, mounted on slides, and processed for autoradiography
using Kodak AR-I0 stripping film. After application of the photographic emulsion to the coverslips., they were kept in the dark for 13 days to expose the emulsion. The autoradiographs were then developed, fixed, rinsed, and air dried.
UDS was quantitated by counting silver grains overlying 100 non-S-phase cells and over equal
areas of cytoplasm (background counts).

Key result

Species / strain:

mammalian cell line, other: human epithelioid cells (HeLa S3)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Positive controls validity:

valid

Additional information on results:

Two independent trials were conducted, both with and without metabolic activation.
In the first trial, a statistically significant (P < 0.05, ANOVA) increase in net nuclear grainis occurred at a concentration of neosugar of 1600 pg/ml in the absence of metabolic activation.
There was no indication of a dose-related trend in grain counts, however, and the increase was
not reproduced in the second trial. In neither trial with metabolic activation was there any significant (effect on net nuclear grain counts.

Conclusions:

The results indicated no genotoxic potential from the use of scFOS.

Executive summary:

An OECD compliant assay for the induction of unscheduled DNA synthesis (UDS) in human epithelioid cells (HeLa S3) derived from a human cervical carcinoma was performed. A wide range of test doses was used for each assay (25, 50, 100, 200, 400, 800, 1600, 3200, 6400, 12,800, 25,600, 51,200 μg/ml), with and without metabolic activation. The results indicated no genotoxic potential from the use of scFOS.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The results of the mutagenicity studies demonstrated that, over a wide range of dose levels, the scFOS did not cause gene mutations in bacteria or mammalian cells and did not induce UDS in mammalian cells either in the absence or in the presence of a metabolic activation system. In addition, a chromosome aberration assay on Chinese hamster ovary cells performed with oligofructose indicated an absence of toxicity and clastogenicity.