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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:
3 June 2015 - 11 June 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted in 21st July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
dated 30th May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use, 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Gyemszi, Budapest, Hungary
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1,1,3,3-tetramethylguanidine
EC Number:
201-302-7
EC Name:
1,1,3,3-tetramethylguanidine
Cas Number:
80-70-6
Molecular formula:
C5H13N3
IUPAC Name:
N,N,N',N'-tetramethylguanidine
Details on test material:
- Name of test material (as cited in section 1): 1,1,3,3-Tetramethylguanidine

Method

Target gene:
his and trp operon
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:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and β-naphthoflavone
Test concentrations with justification for top dose:
16, 50, 160, 500, 1600 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water (ultrapure)
- Justification for choice of solvent/vehicle: ultrapure water was found as suitable vehicle for preparing the test item solutions in a preliminary solubility test
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9: 9-aminoacridine (50 µg/plate) for TA1537; 4-nitro-1,2-phenylenediamine (4 µg/plate) for TA98; sodium azide (2 µg/plate) for TA100 and TA1535; methyl methanesulfonate (2 µL/plate) for E. coli; +S9: 2-aminoanthracene (2 or 50 µg/plate) for all strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation; initial mutation test) and preincubation (confirmatory mutation test)

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: decreased or absent revertant colony counts and affected background lawn development

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 TA98, TA100 and E. coli WP2 uvrA the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain TA1535, TA1537 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

Results and discussion

Test results
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:
cytotoxicity
Remarks:
in the confirmatory mutation test at 5000 μg/plate ±S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: soluble
- Precipitation: no precipitation of the test item was observed in the initial and confirmatory mutation tests on the plates in the examined bacterial strains at any examined concentration level (±S9 mix)

RANGE-FINDING/SCREENING STUDIES: based on the results of the solubility and the range finding tests the vehicle (ultrapure water) and the test item concentrations for both mutation tests were determined (16, 50, 160, 500, 1600 and 5000 µg/plate)

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: in the confirmatory mutation test, following the pre-incubation procedure an inhibitory effect of the test item on bacterial growth was observed in all examined strains at the highest examined concentration of 5000 μg/plate in absence and also in the presence of exogenous metabolic activation (±S9 mix)
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Test Results of Experiment 1

EXPERIMENT 1 (plate incorporation test; initial mutation test)

S9-Mix

Without

 

Test item (µg/plate)

TA 98

TA 100

TA 1535

TA 1537

E. coli

NC (DMSO)

18.3±1.53

-

-

9.7±3.06

-

NC (water)

20.7±4.73

93.0±5.29

11.7±4.93

8.3±3.79

20.3±2.52

5000

13.3±2.31

87.7±15.14

12.3±4.73

9.0±1.73

19.0±6.24

1600

22.3±3.51

95.3±12.01

11.0±3.61

7.7±1.15

16.7±4.51

500

17.0±2.65

93.3±10.69

10.3±3.21

7.3±3.21

17.3±1.53

160

16.0±3.61

86.7±7.77

14.7±2.08

9.0±1.73

15.7±3.21

50

17.0±2.65

83.0±3.61

9.0±3.00

10.3±2.31

23.0±6.93

16

16.7±2.89

91.7±2.08

10.3±1.53

9.7±3.51

15.3±0.58

NPD

269.3±21.57

-

-

-

-

SAZ

-

1089.3±55.47

900.0±203.45

-

-

9AA

-

-

-

734.0±104.10

-

MMS

-

-

-

-

587.3±17.24

2AA

-

--

-

-

-

S9-Mix

With

 

 

 

 

 

 

 

Test item (µg/plate)

TA 98

TA 100

TA 1535

TA 1537

E. coli

NC (DMSO)

24.3±2.89

101.0±7.94

12.7±4.04

7.7±2.52

20.7±5.86

NC (water)

27.3±5.13

100.3±15.57

8.3±2.89

10.7±3.79

24.0±3.61

5000

21.7±2.31

86.0±21.93

12.7±1.15

11.0±3.46

23.3±2.52

1600

27.0±4.58

106.3±6.81

9.3±0.58

8.7±5.51

22.0±2.00

500

32.0±10.00

111.0±13.53

10.0±3.00

9.0±3.00

20.7±4.51

160

28.0±5.29

114.0±3.46

9.0±3.00

10.3±0.58

22.7±4.93

50

25.3±8.09

98.7±5.51

7.0±1.00

8.7±4.04

22.3±4.62

16

21.0±6.24

102.0±11.53

10.0±5.20

11.3±5.77

21.0±5.00

NPD

-

-

-

-

-

SAZ

-

-

-

-

-

9AA

-

-

-

-

-

MMS

-

-

-

-

-

2AA

1232.0±215.70

1669.3±224.19

163.3±54.17

103.30±5.03

336.0±45.08

NC = Negative/Vehicle Control

PC = Respective positive control substances (for details see method description)

Ultrapure water was applied as vehicle for the test item and the positive control substances SAZ and MMS; DMSO was applied as vehicle for the positive control substances NPD, 9AA and 2AA.

 

Table 2: Test Results of Experiment 2

EXPERIMENT 2 (preincubation; confirmatory mutation test)

S9-Mix

Without

 

Test item (µg/plate)

TA 98

TA 100

TA 1535

TA 1537

E. coli

NC (DMSO)

15.7±0.58

-

-

13.0±3.00

-

NC (water)

18.3±5.13

85.7±5.13

11.0±3.61

14.0±2.00

20.3±2.52

5000

0.0±0.00B

0.0±0.00B

0.0±0.00B

0.0±0.00B

8.0±2.65SB

1600

15.7±8.33

94.0±12.17

13.7±3.06

8.3±2.08

13.7±3.79

500

22.7±1.15

84.7±16.29

13.7±3.79

13.0±5.29

20.3±4.93

160

22.3±7.57

87.0±5.57

13.0±3.61

12.3±4.51

21.7±0.58

50

13.7±0.58

89.3±1.15

16.0±2.65

15.0±2.65

21.0±1.00

16

19.3±8.39

95.0±7.81

9.7±1.53

13.0±1.73

18.0±4.36

NPD

249.3±23.18

-

-

-

-

SAZ

-

1241.3±231.80

1456.0±169.33

-

-

9AA

-

-

-

1933.3±59.28

-

MMS

-

-

-

-

752.0±124.96

2AA

-

--

-

-

-

S9-Mix

With

 

 

 

 

 

 

 

Test item (µg/plate)

TA 98

TA 100

TA 1535

TA 1537

E. coli

NC (DMSO)

24.3±2.52

94.0±19.16

10.7±3.51

10.7±1.15

20.0±4.36

NC (water)

30.3±2.52

94.7±6.11

14.0±2.00

11.3±3.79

21.0±4.00

5000

3.7±6.35B

0.0±0.00B

0.0±0.00B

0.0±0.00B

0.0±0.00B

1600

27.0±7.94

113.3±9.87

9.0±5.00

7.7±4.73

17.3±2.89

500

24.3±6.11

96.7±12.86

14.0±1.73

9.7±1.53

21.7±1.15

160

26.0±4.36

119.3±2.31

13.0±4.36

12.0±1.00

21.0±2.65

50

19.3±4.51

115.7±7.64

14.3±4.62

12.0±5.57

20.0±4.36

16

21.7±8.33

108.0±5.29

11.7±4.62

10.7±2.52

20.0±2.00

NPD

-

-

-

-

-

SAZ

-

-

-

-

-

9AA

-

-

-

-

-

MMS

-

-

-

-

-

2AA

1280.0±42.33

1636.0±130.05

135.7±15.95

102.0±4.36

118.0±31.05

NC = Negative/Vehicle Control

PC = Respective positive control substances (for details see method description)

B = Reduced background lawn development

SB =Slightly reduced background lawn development

Ultrapure water was applied as vehicle for the test item and the positive control substances SAZ and MMS; DMSO was applied as vehicle for the positive control substances NPD, 9AA and 2AA.

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test item LZ 754 has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.
Executive summary:

The study was performed in year 2015 according to GLP and OECD Guideline 471. No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with LZ 754 at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. The reported data of this mutagenicity assay shows that 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 LZ 754 has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.