4-O-β-D-galactopyranosyl-D-glucitol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

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:

Mutagenicity Studies of Lactitol (NS-4)

Author:

Iwakura K, Tamura H, Watanabe M, and Sumi N

Year:

1994

Bibliographic source:

J Tox Sci, 19(Suppl. III):487–497

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Principles of method if other than guideline:

Lactitol was examined for mutagenicity in the reverse mutation test in bacteria (S. typhimurium TA1535, TA100, TA1537, TA98 and E. coli WP2wvrA).

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-O-β-D-galactopyranosyl-D-glucitol

EC Number:

209-566-5

EC Name:

4-O-β-D-galactopyranosyl-D-glucitol

Cas Number:

585-86-4

Molecular formula:

C12H24O11

IUPAC Name:

4-O-beta-D-galactopyranosyl-D-glucitol

Specific details on test material used for the study:

- NS-4
- Lot No. 12

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

- S-9 mix was constituted on 100 µL of S-9 fraction, MgCl2 8 µmoles, KCl 33 µmoles, glucose-6-phosphate 5 µmoles, NADPH 4 µmoles, NADH 4 µmoles, and Na-phosphate buffer (pH 7.4) 100 µmoles in 1 mL, and was prepared at time of use.
-S-9 fraction was liver homogenate 9000 X g supernatant fraction obtained by combined administration of phenobarbital and 5,6-benzoflavone as drug metabolizing enzyme inducers to male SD rats purchased from Oriental Yeast Co., Ltd., stored at –80°C, and used in testing after its activity was checked.

Test concentrations with justification for top dose:

Preliminary test: 1000, 500, 100, 50, and 10 µg/plate, with a maximum concentration of 5000 μg/plate
Main test: 500, 2500, 1250, 625, 313 and 156 µg/plate

Vehicle / solvent:

distilled water

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

distilled water

Controls 2

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene (all strains with S9) and 2-(2-furyl)-3-(5-nitr-2-furyl)-acryl-amide (TA100, TA98, and WP2urvA without S9)

Details on test system and experimental conditions:

0.1 mL of the test substance solution, 0.5 mL of S9 mix or 0.1 M Na-phosphate buffer (pH 7.4), and 0.1 mL of the preculture of the test strain were added to sterile test tubes, mixed, and then subjected to pre-incubation while shaking for 20 minutes at 37°C. Next, after adding and mixing 2 mL of top agar maintained at a temperature about 45°C, the resultant mixture was layered atop minimum glucose agar plate medium, and cultured at 37°C for 48 hours. After culturing was finished, the number of reverse-mutated colonies produced were counted, and a microscope was used to observe antimicrobial effects of the test substance, sediment formation, etc. Additionally, after adding and mixing 2 mL top agar to 0.1 mL test substance solution, and S9 mix or 0.1 M Na-phosphate buffer, the resultant mixtures were layered atop nutrient agar culture medium and cultured for 48 hours at 37°C, it was confirmed that no contamination was present in the test system. During testing, 2 plates were used for each concentration.

Evaluation criteria:

Results of the plates were determined to be positive when their average values were more than twice that of the negative control and dose-dependent.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test substance at any concentration did not increase the number of revertant colonies for all testing bacteria stocks relative to the negative control irrespective of whether metabolic activation was present, nor was any dose-dependency observed between processing concentrations and revertant colonies. Also, no precipitate formation was observed on the agar plates up to the maximum concentration, nor was any antibiotic action. On the other hand, all positive control substances caused a marked increase in the number of revertant colonies.

Applicant's summary and conclusion

Conclusions:

The test substance was negative with and without activation when tested in the reverse mutation assay using strains S. typhimurium TA1535, TA1537, TA98, TA100, and E. coli WP2 uvrA at concentrations up to 5000 µg/plate.

Executive summary:

Lactitol (NS-4) was examined for mutagenicity in the reverse mutation test in bacteria. In the reverse mutation test using Salmonella typhimurium (TA1535, TA100, TA1537, and TA98) and Escherichia coli (WP2wvrA), the drug did not significantly increase revertant colonies in any of the test strains with or without metabolic activation system (S-9 mix).