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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 September 2012 to 13 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2009
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1-Propanesulfonic acid, 3-(cyclohexylamino)-2-hydroxy-, monosodium salt
EC Number:
700-631-8
Cas Number:
102601-34-3
Molecular formula:
C9 H19 N O4 S . Na
IUPAC Name:
1-Propanesulfonic acid, 3-(cyclohexylamino)-2-hydroxy-, monosodium salt

Method

Target gene:
All Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium. They contain the deep rough (rfa) mutation, which deletes the polysaccharide side chain of the lipopolysaccharides of the bacterial cell surface. This increases cell permeability of larger substances. The other mutation is a deletion of the uvrB gene coding for a protein of the DNA nucleotide excision repair system resulting in an increased sensitivity in detecting many mutagens. This deletion also includes the nitrate reductase (chi) and biotin (bio) genes (bacteria require biotin for growth).The tester strains TA 98 and TA 100 contain the R-factor plasmid, pl(M101. These strains are reverted by a number of mutagens that are detected weakly or not at all with the non R-factor parent strains. plcM101 increases chemical and spontaneous mutagenesis by enhancing an error-prone DNA repair system which is normally present in these organisms.The tester strain E. coli WP2 uvrA carries the defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagens which substitute one base for another. Additionally, the strain is deficient in the DNA nucleotide excision repair system.
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:
Due to migration, the value was transferred to one of the current document's attachments
Test concentrations with justification for top dose:
5000; 1581; 500; 158; 50 and 15.8 μg/plate
Choice of the concentrations was done on the basis of a Solubility Test and a concentration Range Finding Test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water (test item; sodium azide, Methyl-methanesulfonate); DMSO (4-nitro-1,2-phenylene-diamine; 9-aminoacridine; 2-aminoanthracene)
- Justification for choice of solvent/vehicle: water is an established solvent
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-nitro-1,2-phenylene-diamine; 2-aminoanthracene
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation, Initial Mutation Test) and preincubation (Confirmatory Mutation Test).
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min
- Exposure duration: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition of two of the tester strains (Salmonella typhimurium TA98, TA100).

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The colony numbers on the control, positive control and the test plates were determined (counted manually), the mean values and appropriate standard deviations and mutation rates were calculated.
Evaluation criteria:
A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control;
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Criteria for a Negative Response:
A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Statistics:
According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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
Additional information on results:
RANGE-FINDING/SCREENING STUDY
In the Informatory Toxicity Test for technical reason, the incubation period after the plate incorporation procedure was exceptionally about 72 hours. The laboratory’s historical database is generated from data originated from the results obtained after about 48-hour incubation time (laboratory’s expertise). Therefore, the obtained values were not compared with the actual historical control data ranges. In comparison with the revertant colony numbers of the vehicle control plates, at the treatments revertant colony number increases or decreases were not noticed. The slight changes remained in the 0.80-1.20 mutation rate range .

STUDY RESULTS
- Concurrent vehicle negative and positive control data :
The spontaneous revertant colony numbers of the ultrapure water vehicle control plates showed mostly the characteristic mean numbers agreed with the actual historical control data ranges. In the Initial and Confirmatory Mutation Tests in the case of Salmonella typhimurium TA100 lower revertant colony numbers (below the corresponding historical control data ranges) were obtained at the corresponding ultrapure water vehicle controls, without metabolic activation (-S9 Mix). The observed lower values were considered as acceptable as acceptable without any influence on the final conclusion of the study. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains.
The revertant colony numbers of the untreated and DMSO control plates in the different experimental phases were slightly higher or lower than the vehicle control plates, but these higher or lower revertant counts of these controls remained mostly in the corresponding historical control data ranges (in the Confirmatory Mutation Test in the case of S. typhimurium TA100 (-S9 Mix) lower counts were obtained at the untreated controls, however without any biological significance).

Ames test:
- Signs of toxicity : No
- Individual plate counts /Mean number of revertant colonies per plate and standard deviation :
Please refer to table 2 and 3 under "An other information on results"

HISTORICAL CONTROL DATA
Please refer to table 1 under "An other information on results"

Any other information on results incl. tables

Table 1: Historical Control Values for Revertants/Plate

 

Bacterial strains

Historical control data of untreated control

- S9

 

TA98

TA100

TA1535

TA1537

E.coli

Average

22.3

126.6

10.9

8.0

24.9

SD

2.9

22.6

0.3

3.3

3.0

Minimum

11

72

3

2

12

Maximum

38

189

26

21

42

+ S9

 

TA98

TA100

TA1535

TA1537

E.coli

Average

29.5

126.7

12.9

9.1

34.7

SD

3.7

22.9

0.8

2.8

3.7

Minimum

14

79

5

3

20

Maximum

46

189

24

21

54

 

Bacterial strains

Historical control data of DMSO control

- S9

 

TA98

TA100

TA1535

TA1537

E.coli

Average

21.9

122.0

10.6

7.8

24.2

SD

2.9

20.1

0.3

3.4

2.9

Minimum

12

72

3

2

11

Maximum

38

185

24

19

40

+ S9

 

TA98

TA100

TA1535

TA1537

E.coli

Average

28.1

127.0

13.0

8.9

34.5

SD

3.3

20.9

1.0

2.7

4.1

Minimum

17

79

4

2

17

Maximum

48

185

26

20

56

 

Bacterial strains

Historical control data of Water control

- S9

 

TA98

TA100

TA1535

TA1537

E.coli

Average

24.0

126.1

10.7

6.7

25.1

SD

6.5

24.4

0.4

3.7

3.5

Minimum

9

78

3

1

12

Maximum

43

184

25

16

45

+ S9

 

TA98

TA100

TA1535

TA1537

E.coli

Average

29.2

128.9

12.7

8.0

36.4

SD

6.8

21.7

0.5

3.7

4.8

Minimum

11

84

3

2

21

Maximum

48

194

24

20

55

 

Bacterial strains

Historical control data of positive controls

- S9

 

TA98

TA100

TA1535

TA1537

E.coli

Average

263.1

955.0

850.1

469.7

713.1

SD

40.7

96.4

71.8

137.7

66.4

Minimum

115

505

390

123

387

Maximum

709

2018

1359

1593

1283

+ S9

 

TA98

TA100

TA1535

TA1537

E.coli

Average

1328.0

1346.7

186.3

160.3

304.5

SD

399.4

448.4

40.8

25.2

91.7

Minimum

490

532

86

67

160

Maximum

2732

3077

785

514

550

 

Table 2: Summary Table of the Results of the Initial Mutation Test

Initial Mutation Test (Plate Incorporation Test)

Concentrations (μg/plate)

Salmonella typhimuriumtester strains

Escherichia coli

 

WP2uvrA

TA 98

TA 100

TA 1535

TA 1537

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean values of revertants per plate Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

30.0

1.08

29.3

0.86

76.0

1.00

91.7

0.92

8.0

1.20

8.0

0.86

5.0

0.79

6.0

1.00

17.0

0.81

27.7

0.91

DMSO Control

21.7

1.00

34.3

1.00

93.3

1.00

8.7

1.00

6.0

1.00

5.0

1.00

25.0

1.00

Ultrapure Water Control

27.7

1.00

34.0

1.00

76.0

1.00

99.3

1.00

6.7

1.00

9.3

1.00

6.3

1.00

6.0

1.00

21.0

1.00

30.3

1.00

5000

27.3

0.99

33.7

0.99

82.0

1.08

100.0

1.01

6.7

1.00

11.7

1.25

5.0

0.79

4.3

0.72

20.3

0.97

28.7

0.95

1581

27.7

1.00

33.0

0.97

72.3

0.95

95.3

0.96

8.7

1.30

9.3

1.00

5.3

0.84

7.3

1.22

17.7

0.84

26.0

0.86

500

26.3

0.95

29.7

0.87

76.0

1.00

96.7

0.97

7.0

1.05

7.7

0.82

4.7

0.74

5.7

0.94

19.0

0.90

29.0

0.96

158

25.3

0.92

30.0

0.88

73.3

0.96

95.0

0.96

7.7

1.15

8.0

0.86

4.0

0.63

5.0

0.83

17.0

0.81

35.7

1.18

50

21.0

0.76

28.3

0.83

74.7

0.98

85.0

0.86

6.7

1.00

10.0

1.07

6.3

1.00

6.3

1.06

20.7

0.98

33.7

1.11

15.8

30.0

1.08

31.3

0.92

68.7

0.90

91.7

0.92

7.0

1.05

10.3

1.11

4.3

0.68

5.7

0.94

17.7

0.84

26.0

0.86

NPD (4 μg)

228.3

10.54

SAZ (2 μg)

644.7

8.48

581.3

87.20

9AA (50 μg)

356.0

59.33

MMS (2μL)

633.3

30.16

2AA (2 μg)

853.3

24.85

1254.7

13.44

175.7

20.27

100.3

20.07

2AA (50 μg)

164.3

6.57

 

MR:Mutation Rate

Remarks:Ultrapure water control was applied as vehicle of the test item, for SAZ and MMS and the DMSO was applied as vehicle of the positive control substances: 9AA, NPD and 2AA. The mutation rate of the untreated control, test item SAZ and MMS is given referring to the ultrapure water, the mutation rate of the 9AA, NPD and 2AA is given referring to the DMSO.

 

 

Table 3: Summary Table of the Results of the Confirmatory Mutation Test

Confirmatory Mutation Test (Pre-Incubation Test)

Concentrations (μg/plate)

Salmonella typhimuriumtester strains

Escherichia coli

 

WP2uvrA

TA 98

TA 100

TA 1535

TA 1537

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean values of revertants per plate Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

30.7

1.18

26.7

0.83

60.3

0.88

105.0

1.09

4.0

0.86

10.0

1.15

6.0

1.38

7.3

0.96

19.3

1.21

33.0

1.16

DMSO Control

30.7

1.00

29.3

1.00

93.3

1.00

7.3

1.00

3.0

1.00

8.0

1.00

29.3

1.00

Ultrapure Water Control

26.0

1.00

32.0

1.00

68.3

1.00

96.3

1.00

4.7

1.00

8.7

1.00

4.3

1.00

7.7

1.00

16.0

1.00

28.3

1.00

5000

27.0

1.04

33.3

1.04

71.3

1.04

90.7

0.94

6.7

1.43

9.3

1.08

4.7

1.08

9.7

1.26

19.3

1.21

36.0

1.27

1581

24.3

0.94

29.7

0.93

71.3

1.04

88.7

0.92

6.7

1.43

7.0

0.81

5.0

1.15

5.3

0.70

21.0

1.31

33.3

1.18

500

26.0

1.00

30.3

0.95

67.7

0.99

99.0

1.03

5.0

1.07

9.7

1.12

5.0

1.15

7.0

0.91

17.0

1.06

37.7

1.33

158

22.3

0.86

20.0

0.63

80.3

1.18

81.3

0.84

6.7

1.43

11.0

1.27

6.3

1.46

7.0

0.91

22.0

1.38

34.7

1.22

50

22.0

0.85

29.3

0.92

63.7

0.93

96.3

1.00

5.7

1.21

8.7

1.00

5.7

1.31

5.7

0.74

20.0

1.25

31.7

1.12

15.8

26.3

1.01

26.3

0.82

61.3

0.90

88.7

0.92

5.0

1.07

8.7

1.00

4.0

0.92

9.0

1.17

22.7

1.42

38.0

1.34

NPD (4 μg)

275.7

8.99

SAZ (2 μg)

722.0

10.57

585.3

125.43

9AA (50 μg)

289.0

96.33

MMS (2μL)

746.7

46.67

2AA (2 μg)

1214.0

41.39

1520.0

16.29

132.0

18.00

124.3

15.54

2AA (50 μg)

208.7

7.11

 

MR:Mutation Rate

Remarks:Ultrapure water control was applied as vehicle of the test item, for SAZ and MMS and the DMSO was applied as vehicle of the positive control substances: 9AA, NPD and 2AA. The mutation rate of the untreated control, test item SAZ and MMS is given referring to the ultrapure water, the mutation rate of the 9AA, NPD and 2AA is given referring to the DMSO.

Applicant's summary and conclusion

Conclusions:
In a GLP study according to OECD Test Guideline 471 (Ames test), the test item was not mutagenic.
Executive summary:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay (acc. to OECD TG 471). The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats. The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test). Based on the results of the preliminary Range Finding Test the following concentrations of the test item were prepared and used in the Initial and Confirmatory Mutation Tests: 5000; 1581; 500; 158; 50 and 15.8 μg/plate. The revertant colony numbers of vehicle control (ultrapure water control) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants was in line with the corresponding historical control data ranges. The obtained slightly lower values in Salmonella typhimurium TA100 were considered as acceptable without any effect on the final conclusion of the study. No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

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