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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:
01. Dec. 2004 - 23. Dec. 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
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

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
480-680-7
EC Name:
-
Cas Number:
120128-90-7
Molecular formula:
Hill formula: C8H16N2O3 CAS formula: C8H16N2O3
IUPAC Name:
2-[(3-formamidopropyl)dimethylazaniumyl]acetate

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Mammalian liver post-mitochondrial fraction (S9)
Test concentrations with justification for top dose:
Experiment 1: 62, 185, 556, 1667, 5000 µg/plate
Experiment 2: 1000, 2000, 3000, 4000, 5000 µg/plate
Vehicle / solvent:
demineralized water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: without S9: Sodium azide, 9-Aminoacridine, 2-Nitrofluorene, Mitomycin C (MMC); with S9: 2-Aminoanthracene, Benzo[a]pyrene
Details on test system and experimental conditions:
Bacteria : Salmonella typhimurium
Strains : TA 1535, TA 1537, TA 98, TA 100, TA102
Date of receipt : 21 st September 1995, 3rd March 1998, 9th August 2002
Supplier : Fa. Boehringer Ingelheim Pharma GmbH & Co. KG, former Dr. Karl Thomae GmbH, Dept. of Exp. Pathology and Toxicology, 88397 Biberach an der Riß, Germany. Bruce Ames Laboratory, Department of Molecular and Cell Biology, 401 Barker Hall, Berkeley, California 94720-0001.
Storage : The Salmonella strain cultures are stored as stock cultures in ampoules with nutrient broth + 9% DMSO (Merck, 1.02950.0500) in liquid nitrogen.
Metabolic Activation : Mammalian liver post-mitochondrial fraction (S9)
Species : Rat
Enzyme-inducing agent : Aroclor 1254
Supplier : Molecular Toxicology, INC., Annapolis, USA

Bacteria were grown overnight for 10 hours in nutrient broth at 37°C / 110 rpm. For inoculation stock cultures, stored at -196°C (Salmonella typhimurium strains) were used. They had been checked for strain characteristics of rfa-character, UVrB-deletion, resistance to ampicillin and tetracycline. Checks were carried out according to Maron and Ames (3). All treatments were performed at the end of the incubation period.

The test was performed by direct plate incorporation method. The mutagenicity experiments with his- Salmonella typhimurium were performed with
minor modifications of the method described by Ames et al. (3). Modifications were as follows:
2 ml Top agar, 100 µL test substance, 100 µL bacterial suspension and 500 µL S9-Mix (or 500 µL phosphate buffer) were mixed in a test tube and poured on the surface of the Minimal-Glucose-Agar plates. For each strain and dose level three plates were used.
Each experiment contained positive controls to check the activity of the metabolizing system and the mutagenicity of the bacteria in triplicate as well as sixfold negative controls to check the spontaneous reversion rate of each used bacteria strain. After solidification the plates were incubated upside down for at least 48 hours at 3r C in the dark.
Evaluation criteria:
A test substance producing no biologically relevant positive response at anyone of the test points is considered to be non-mutagenic in this system. A biologically relevant response is described as follows: If the number of revertants is at least twice the spontaneous reversion rate in one of the
strains and if there is a concentration related, increasing number of revertants over the range tested.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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

Experiment 1:  

 

Dose [µg/plate]

Mean number of revertant colonies/3 replicates (±S.D.) with 5 different strains of Salmonella typhimurium

 

TA 1535

TA 1537

TA 98

TA 100

TA 102

 

Without S9

Positive control

548±42

> 1000

313±13

633±119

> 1000

Negative control

20±2

20±2

19±2

61±3

264±38

62

25±8

29±4

24±5

54±3

230±10

185

18±´4

28±5

24±8

52±5

248±27

556

26±3

22±3

19±4

56±7

296±58

1667

26±7

26±9

26±6

61±2

291±97

5000

21±2

27±7

17±4

45±6

266±22

 

With S9 *

Positive control

369±86

287±34

840±163

> 1000

> 1000

Negative control

23±6

32±3

27±4

72±12

338±48

62

23±7

29±4

24±2

81±10

432±72

185

21±3

35±10

33±6

76±7

370±61

556

23±3

33±5

40±4

85±6

351±43

1667

26±1

32±3

33±2

82±4

360±56

5000

23±2

24±3

24±4

82±6

312±51

T=Toxicity

P=Precipitation

C=Contamination

M=Mean

SD=Standard deviation

 


 

Experiment 2:  

 

Dose [µg/plate]

Mean number of revertant colonies/3 replicates (±S.D.) with 5 different strains of Salmonella typhimurium

 

TA 1535

TA 1537

TA 98

TA 100

TA 102

 

Without S9

Positive control

430±62

> 1000

328±2

883±62

> 1000

Solvent control

18±4

31±4

24±4

53±6

286±29

1000

16±2

26±1

22±3

62±5

285±37

2000

14±4

28±4

22±2

55±10

267±63

3000

19±4

28±3

24±2

50±13

345±14

4000

17±4

32±9

22±4

59±3

253±13

5000

20±5

26±4

22±0

50±6

329±64

 

With S9 *

Positive control

237±30

632±87

784±142

> 1000

> 1000

Solvent control

15±2

31±4

28±10

72±6

315±49

1000

18±5

32±5

22±8

54±8

391±26

2000

17±4

35±3

28±3

64±4

387±32

3000

21±6

26±7

22±2

71±9

381±53

4000

17±3

23±2

27±5

61±6

371±65

5000

19±4

30±3

30±8

65±9

323±54

T=Toxicity

P=Precipitation

C=Contamination

M=Mean

SD=Standard deviation

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Based on the results of this study it is concluded that Formamidopropyldimethylbetaine is not mutagenic in the Salmonella typhimurium reverse mutation assay.
Executive summary:

The purpose of the study was to investigate the potential of the test substance Formamidopropyldimethylbetaine to induce mutations using the bacterial reverse mutation assay.

The study was conducted in accordance with the OECD guidelines for testing of chemicals No. 471 "Bacterial reverse mutation test" (1997) and with the procedure described in the Commission Directive 2000/32/EC (2000).

The assay was performed in two independent experiments. Both took place as a plate incorporation test, using the Salmonella typhimurium strains T A 1535, TA 1537, TA 98, TA 100, TA 102. All experiments were in the absence and in the presence of a metabolic activation by an Aroclor 1254 induced rat liver post mitochondrial fraction (S9). The test substance and the positive controls were triple tested, the negative controls sixfold. Demineralized water (A. dem.) was used as vehicle for all dilutions of the test substance.

Five concentrations were tested in each experiment, using 0.1 ml for each plate. The test substance was tested in the following concentrations:

Experiment 1: 62, 185, 556, 1667, 5000 µg/plate

No mutagenic effects, evident as an elevation of the number of revertant colonies or toxic effects, evident as a reduction of the number of colonies occurred with or without metabolic activation with the test substance concentrations. Due to this, the concentrations of the second test were chosen as follows:

Experiment 2: 1000, 2000, 3000, 4000, 5000 µg/plate

No mutagenic or toxic effects occurred. Negative (solvent) and positive control treatments were included for all strains in both experiments. All mean numbers of revertant colonies on negative control plates fell within acceptable ranges and were significantly elevated by positive control treatments.

Based on the results of this study it is concluded that Formamidopropyldimethylbetaine is not mutagenic in the Salmonella typhimurium reverse mutation assay.