Reaction mass of 6-O-alfa-D-glucopyranosyl-D-fructofuranose and 1-O-alfa-D-glucopyranosyl-D-fructofuranoseand D-fructofuranose

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

Name "Isomaltulose Solution".
Trade name Isomaltulose Solution
Batch No. 03160483
Density 1.2 g/cm3 (20 °C)
Solubility in water unlimited.
Storage room temperature

Method

Target gene:

his-(S. typhimurium)

Species / strain

Species / strain / cell type:

other: S. typhimurium TA97a, TA98, TA100, TA102, TA1535

Metabolic activation:

with and without

Metabolic activation system:

S9 rat liver

Test concentrations with justification for top dose:

Test substance: 5000, 1667, 556, 185, and 62 ug/plate

Vehicle / solvent:

deionized water

Controls

Untreated negative controls:

yes

Remarks:

DI water

Positive controls:

yes

Remarks:

Varied based on strain

Details on test system and experimental conditions:

Positive controls were as follows without S9:
TA97a: 4NOPD, 10 ug
TA98: 2NF, 2 ug
TA100: sodium azide, 2 ug
TA102: tBHPO, 50 ug
TA1535: sodium azide, 1 ug

Positive controls were as follows with S9:
TA97a: DMBA, 10 ug
TA98: 2AA, 1 ug
TA100: 2AA, 2 ug
TA102: DHA, 50 ug
TA1535: 2AA, 2 ug

First experiment: Plate Incorporation Assay:
For each sample the following solutions were combined:
• 0.1 mL of the overnight culture of the bacteria,
• 0.5 mL of S9-mix (or phosphate buffered saline for samples without metabolic activation),
• 0.1 mL of the appropriate test- or reference substance solution and
• 2 mL of top agar.
The combined solutions were mixed and spread over a plate with minimal agar (9 cm diameter). After the top agar had solidified, the plates were incubated at 37 °C until the colonies were visible (2 days).

Second experiment: Preincubation Assay:
For each sample the following solutions were combined:
• 0.1 mL of the overnight culture of the bacteria,
• 0.5 mL of S9-mix (or phosphate buffered saline for samples without metabolic activation),
• 0.1 mL of the appropriate test- or reference substance solution.
The solutions were preincubated for 20 minutes at 37 °C using a shaker, afterwards combined with 2 mL of top agar and spread over a plate with minimal agar (9 cm diameter). After the top agar had solidified, the plates were incubated at 37 °C until the colonies were visible (2 days).

Evaluation criteria:

Means and standard deviations were calculated for the number of mutants in every concentration group.
The criteria for a positive result were:
A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
•For the strains with a low spontaneous revertant rate i.e. TA98 and TA1535: 250 % of the amount of the spontaneous revertants.
•For the strains with a high spontaneous revertant rate i.e. TA97a, TA100 and TA102: 167 % of the amount of the spontaneous revertants.

Results and discussion

Test results

Species / strain:

other: S typhimurium TA97a, TA98, TA100, TA102, TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Results for Experiment I (Mean Revertants/plate (SD)) – Without S9

Concentration (ug/plate)	TA 97a	TA 98	TA 100	TA 102	TA 1535
Solvent	81.7	12	52.2	135.3	11.5
5000	60	9.7	48	113.3	7.7
1667	81.7	12.3	54.7	124.7	12
556	97.5	9.3	64.3	142.7	12
185	52	10.7	75.3	129.3	7.7
62	88.7	12	62	152.3	12

Results for Experiment II (Mean Revertants/plate (SD)) – Without S9

Concentration (ug/plate)	TA 97a	TA 98	TA 100	TA 102	TA 1535
Solvent	107	12.8	72.3	274.7	11
5000	125	11.7	68	282.7	8.3
1667	123.5	10.3	77.7	304.7	12.3
556	110.5	11.3	74.7	251.7	7.7
185	101	8.3	85.3	269.7	7.3
62	131.5	11	76.7	271.3	10.3

Results for Experiment I (Mean Revertants/plate (SD)) – With S9

Concentration (ug/plate)	TA 97a	TA 98	TA 100	TA 102	TA 1535
Solvent	148.2	12	72.8	168.8	11
5000	150.7	14	69.7	146.7	9
1667	148.3	12.3	67.3	188	14.3
556	150	12.3	73.7	182.3	12.3
185	118.7	14.3	80.3	167.3	13.3
62	147	11.7	75.7	161.7	9.3

Results for Experiment II (Mean Revertants/plate (SD)) – With S9

Concentration (ug/plate)	TA 97a	TA 98	TA 100	TA 102	TA 1535
Solvent	195.2	13.2	76.8	308.5	11.3
5000	188.3	10.7	93.7	356	9
1667	177.7	14.7	98.3	363.7	13.3
556	200.3	13	89.3	345	7.3
185	189.3	13.3	103.7	319.7	10.3
62	182.3	13	83.7	321.7	11.3

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test substance is not mutagenic in either the presence or absence of metabolic activation.

Executive summary:

This study examined the mutagenic potential of the test substance. Salmonella typhimuirum strains TA1535, TA102, TA100, TA98, and TA97a, were exposed to concentrations of 62, 185, 556, 1667, and 5000 ug/plate of test substance in solvent in both the presence and absence of S9. Positive control substances were sodium azide or 2-aminoanthracene. Cultures were tested in duplicate for mutation frequency. No treatment cultures showed mutation frequencies significantly greater than negative controls. Positive controls showed significantly greater mutation frequencies over negative controls, therefore the test was valid. The test substance is not mutagenic in either the presence or absence of metabolic activation.