1,1,3,3-tetramethylguanidine

Genetic toxicity: in vitro

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 21st July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

dated 30th May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 4

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

Test material information

Constituent 1

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.