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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
Study period:
19. Jun. 2002 - 09. Jul. 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Name : Benzoesäure-isononylester
Purity / constituents : 99.9%
Stability : < 1 year
Homogeneity : clear liquid
Batch No : 1276/00576
Date of manufacture : 08.02.02
Solvent : DMSO

Method

Target gene:
His -
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital induced rat liver S9
Test concentrations with justification for top dose:
50 - 5000 µg/plate for plate incorporation test
50 - 1000 µg/plate for pre-incubation test
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Details on test system and experimental conditions:
Five dose levels of BENZOESÄURE-ISONONYLESTER in the absence and in the presence 01 metabolic activation were spaced at
half-log intervals. The test #AM-02/09.2 was performed with a concentration range based on the results 01 test #AM-02/09.1. In test #AM-02/09.1 the plate incorporation method, and in test #AM-02/09.2 the preincubation method was used.

Plate incorporation test
In a sterile tube,
- 0.1 ml of the appropriately diluted test material (or 0.1 ml of the solvent)
(or 0.1 ml [0.05 ml for TA 1535] of the strain specific positive control item)
(or 0.05 ml of the positive control items for proving metabolie activation)
- 0.5 ml phosphate buffer
(or 0.5 ml 89 mix in the experiment with metabolie activation)
- 2 ml of molten trace histidine supplemented top agar at approx. 45"C
- 0.1 ml of the bacterial overnight culture
were mixed. Mixing was done in triplicate, for each bacterial strain and for each concentration of the test material. The mixture was then poured onto the surface of minimal agar plates. These plates were incubated at 37 °C for 72 hours and then the number of revertant colonies was counted.

Preincubation test
In a sterile tube, a 0.1 ml aliquot of each one of the bacterial overnight cultures was mixed with a 0.5 ml volume of 89 mix (for tests with metabolie activation) or phosphate-buffer (for tests without metabolie activation). Then either 50 µl of the positive controls 2-Aminofluorene
and 2-Aminoanthracene (+89), 50 µl of the solvent, or 50 µl of the test item solution were added. The tubes were incubated at 30°C for 30 min with gentle agitation. At the end of the incubation period, 2 ml of molten trace histidine supplemented top agar was added to each tube, mixed briefly and poured onto minimal agar plates. These plates were incubated at 37 °C for 72 hours and then the nurnber of revertant colonies was counted.
Evaluation criteria:
For a test compound to be considered positive, it must (in two independent experiments) cause at least a doubling in the mean revertants per plate of at least one tester strain. This increase must be accompanied by a dose response towards increasing concentrations of the test article. A test article that does not meet these criteria will be called non-mutagenic in bacteria. Single increases in revertant frequencies, which are not dose-related and not reproducible in two independent tests are considered non-relevant. If however these increases do occur in both tests, this will be taken as an indication of a mutagenic effect.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
in TA 1537, thinning of bacterial lawn was observed
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
With two exceptions all five bacterial strains exhibited a positive mutagenic response with the positive controls tested both with and without metabolie activation by S9 mix. The strain TA 1537 showed a limited response to the presence of the positive control item 2-Aminoanthracene in case of the plate incorporation test with metabolic activation (#AM-02/09.1). But the parallel tested additional positive control item 2-Aminolluorene showed
enough activity. The strain TA 102 also showed a limited response to the positive control item 2-Aminoanthracene (tests #AM-02l09.1 and #AM-02/09.2 with metabolie aetivation), but 2-Aminofluorene showed enough activity.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Plate incorporation test # AM-02/09.1

 

µg/

plate

TA 98

TA 100

TA 102

TA 1535

TA 1537

 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Water (untreated)

 

1.2

1.4

1.0

1.1

1.0

1.0

0.9

1.4

1.0

0.8

DMSO

 

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

Test item

50

1.0

1.3

1.0

1.1

1.1

1.1

0.8

1.1

0.9

0.8

Test item

160

1.5

1.3

1.0

1.1

1.1

1.0

0.6

1.5

1.1

0.7

Test item

500

0.9

1.3

1.0

1.1

1.1

1.1

0.7

1.2

1.1

0.6

Test item

1600

1.1

1.3

1.0

1.3

1.1

0.9

0.8

0.9

0.9

0.9

Test item

5000

1.4

1.6

1.1

1.0

1.1

1.1

0.6

1.1

1.0

1.0

 

 

 

 

 

 

 

 

 

 

 

 

2-Nitrofluorene

2.5

2.5

 

 

 

 

 

 

 

 

 

Sodium azide

5.0

 

 

7.6

 

 

 

15.8

 

 

 

Sodium azide

2.5

 

 

 

 

 

 

 

 

 

 

Mitomycin C

2.5

 

 

 

 

3.5

 

 

 

 

 

9-Aminoacridine

40.0

 

 

 

 

 

 

 

 

2.1

 

2-Aminofluorene

100

 

102.2

 

17.6

 

2.0

 

2.0

 

2.5

2-Aminoanthracene

2.5

 

7.8

 

3.3

 

1.1

 

3.2

 

1.3

Preincubation test # AM-02/09.2

 

µg/

plate

TA 98

TA 100

TA 102

TA 1535

TA 1537

 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Water (untreated)

 

1.1

1.0

1.0

1.1

1.0

1.1

1.3

0.8

1.0

1.1

DMSO

 

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

Test item

50

1.1

0.7

1.0

0.9

1.0

1.0

1.1

0.7

1.2

1.5

Test item

160

1.0

0.7

1.0

1.0

1.0

1.0

0.8

0.9

1.1

1.1

Test item

500

0.9

1.0

0.9

0.9

1.0

1.0

1.4

0.9

1.2

1.5

Test item

750

1.2

0.9

1.0

0.9

1.0

1.0

0.9

0.7

1.0

1.1

Test item

1000

1.1

1.1

1.0

1.0

1.2

1.0

0.7

0.9

1.1

1.5

 

 

 

 

 

 

 

 

 

 

 

 

2-Nitrofluorene

2.5

3.9

 

 

 

 

 

 

 

 

 

Sodium azide

5.0

 

 

7.2

 

 

 

 

 

 

 

Sodium azide

2.5

 

 

 

 

 

 

38.1

 

 

 

Mitomycin C

2.5

 

 

 

 

3.5

 

 

 

 

 

9-Aminoacridine

40.0

 

 

 

 

 

 

 

 

3.1

 

2-Aminofluorene

100

 

60.2

 

16.9

 

2.1

 

2.6

 

4.2

2-Aminoanthracene

2.5

 

3.3

 

2.3

 

1.1

 

4.2

 

2.1

Applicant's summary and conclusion

Conclusions:
BENZOESÄURE-ISONONYLESTER did not induce a mutagenic effect in S. typhimurium. It is therefore not considered to be a bacterial mutagen.
Executive summary:

BENZOESÄURE-ISONONYLESTER was tested for its ability to induce reverse mutations in an in vitro bacterial system. Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 were treated with the test item by the Ames test plate incorporation (test #AM-02/09.1) as well as by the preincubation method (test #AM-02l09.2). Dose levels covering the range of 50 to 5000 µg/plate, in triplicate both with and without the addition of a metabolising system (Phenobarbital/-ß-Naphthoflavone co-induced rat liver S9 mix) were employed.

A reproducible mutagenic activity of the test compound to any of the tester strains TA 98, TA 100, TA 102, TA 1535 or TA 1537 was not observed with and without metabolic activation. It is therefore concluded, that Benzoesäure-isononylester is not a bacterial mutagen.