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Toxicological information

Genetic toxicity: in vitro

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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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
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.