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Genetic toxicity in vitro

Description of key information

Gene mutation toxicity study was performed for the test chemical 1-amino-4-methylpiperazine. The study was perfomed using Salmonella typhimurium strains TA1535, TA100 and TA98 both in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in suitable solvent at doses of 0, 1, 3, 10, 30, 100 or 300 µmoles/plate. The plates were incubated for 2 days before the observation of revertant colonies. 1-amino-4-methylpiperazine did not show any mutagenic nature in Salmonella typhimurium strain TA98 in the presence and absence of S9 and also in strains TA1535 and TA100 in the presence of S9. However in the absence of S9, 1-amino-4-methylpiperazine showed mutagenic behaviour in strains TA1535 and TA100. Based on the observations made, the test chemical is not likely to classify as a gene mutant in vitro.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed publication
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
The Ames mutagenicity test was conducted on salmonella typhirium of strains TA98, TA 100 and TA 1535 to study the mutagenicity of chemical 1-amino-4-methylpiperazine.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of the test material: 4-methylpiperazin-1-amine
- IUPAC name: 4-methylpiperazin-1-amine
- Molecular weight: 115.179 g/mol
- Molecular formula: C5H13N3
- Substance type: Organic
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium, other: TA98, TA100 and TA1535
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction was prepared from Aroclor 1254-induced rat-liver extract
Test concentrations with justification for top dose:
0, 1, 3, 10, 30, 100 or 300 µmoles/plate
Vehicle / solvent:
No data
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 4-nitro-o-phenylene (TA98, without S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: No data
- Exposure duration: 2 days
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: All doses were run in triplicate with concurrent solvent controls, and all tests were repeated at least once

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data
Rationale for test conditions:
No data
Evaluation criteria:
The plates were observed for a dose dependent increase in the number of revertants/plate
Statistics:
Mean plate count ± S.E.M. (3 plates)
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA1535 and TA100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA1535 and TA100
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
No data

TABLE 1: Mutagenicity of1-Amino-4-MethylpiperazineFor S. Typhimurium TA1535 In The Absence Of S9

Dose (µmoles/plate)

NPA

0

11±1

0.03

-

0.1

-

0.3

-

1

9±3

3

16±3

10

50±4

30

384±23

100

770±71

300

611±49

aMean plate count _+ S.E.M. (3 plates).

bNot done.

CToxic response; few or no his + revertants on plates.

Conclusions:
1-amino-4-methylpiperazine did not show any mutagenic nature in Salmonella typhimurium strain TA98 in the presence and absence of S9 and also in strains TA1535 and TA100 in the presence of S9. However in the absence of S9, 1-amino-4-methylpiperazine showed mutagenic behaviour in strains TA1535 and TA100. Based on the observations made, the test chemical is not likely to classify as a gene mutant in vitro.
Executive summary:

Gene mutation toxicity study was performed for the test chemical 1-amino-4-methylpiperazine. The study was perfomed using Salmonella typhimurium strains TA1535, TA100 and TA98 both in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in suitable solvent at doses of 0, 1, 3, 10, 30, 100 or 300 µmoles/plate. The plates were incubated for 2 days before the observation of revertant colonies. 1-amino-4-methylpiperazine did not show any mutagenic nature in Salmonella typhimurium strain TA98 in the presence and absence of S9 and also in strains TA1535 and TA100 in the presence of S9. However in the absence of S9, 1-amino-4-methylpiperazine showed mutagenic behaviour in strains TA1535 and TA100. Based on the observations made, the test chemical is not likely to classify as a gene mutant in vitro.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutation in vitro:

Prediction model based estimation for the target chemical and data from read across chemicals have been reviewed to determine the mutagenic nature of 4-Methylpiperazin-1-amine. The studies are as mentioned below:

Gene mutation toxicity study was performed by Zeiger and Guthrie (Mutation Research, 1981) for the test chemical 1-amino-4-methylpiperazine (CAS no 6928 -85 -4). The study was perfomed using Salmonella typhimurium strains TA1535, TA100 and TA98 both in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in suitable solvent at doses of 0, 1, 3, 10, 30, 100 or 300 µmoles/plate. The plates were incubated for 2 days before the observation of revertant colonies. 1-amino-4-methylpiperazine did not show any mutagenic nature in Salmonella typhimurium strain TA98 in the presence and absence of S9 and also in strains TA1535 and TA100 in the presence of S9. However in the absence of S9, 1-amino-4-methylpiperazine showed mutagenic behaviour in strains TA1535 and TA100. Based on the observations made, the test chemical is not likely to classify as a gene mutant in vitro.

Elespuru and Lijinsky ( Cancer Research, 1976) performed gene mutation toxicity study to determine the mutagenic nature of 87% structurally similar read across chemical 1-nitroso-4- methylpiperazine (RA CAS no 16339 -07 -4; IUPAC name: 1-methyl-4-nitrosopiperazine). Stationary overnight cultures of E. coli WU 3610 were diluted 20-fold into Difco nutrient broth and grown for 1 hr, to early log phase (1 to 2 x 108bacteria/mL). Twenty ml of bacterial suspension were pelleted, and the pellet was re-suspended in 10 to 20 ml of a solution of cofactors (1 ml contains 7.5 mg NADP, 10 mg glucose 6-phosphate, 10 mg nicotinamide, and 6.5 mg MgCl2 in 0.01 M sodium-potassium phosphate buffer, pH 7.4). The suspension containing bacteria and cofactors was warmed to 37°C, and glucose-6-phosphate dehydrogenase, 1µI/mI, was added. The solution was kept warm and was dispensed rapidly into tubes containing the test compound, in volumes appropriate to make 2 to 4 ml of 50 mM solution. When the test compound was dissolved, 0.5-mL aliquots were dispensed into 4 tubes, and volumes of freshly thawed microsomal suspension were added, ranging from 0.1 to 1.0 ml. Phosphate buffer was added to each of the solutions containing less than 1.5 ml so as to make the volumes equal. The volume in each tube was 1.5 ml and the concentration of the test compound was 16.7mM. The tubes were incubated with shaking at 37°C for 1 hr. Aliquots of 0.2 ml of each solution (containing 2 x 107bacteria) were plated on minimum medium E supplemented with 2.5% nutrient broth + 0.4% glucose + 20µg L-leucine per ml. Plates were incubated for 2 days at 37°C and then counted. Under these conditions, bacteria divided several times, allowing expression of mutations and the appearance of a background lawn of bacteria. 1-nitroso-4- methylpiperazine did not induce gene mutation in E. coli WU 3610 both in the presence and absence of S9 metabolic activation system and hence the test chemical is not likely to classify as a gene mutant in vitro.

Salmonella/microsome test in the absence of exogenous metabolic activation and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters was performed to evaluate the mutagenic nature of the 50 -60% structurally similar read across chemical N-Methyl diethanolamine (RA CAS no 105 -59 -9; IUPAC name: 2,2'-(methylimino)diethanol) using S. typhimurium tester strains TA1535, TA97, TA98 and TA100. The study was performed as per the preincubation assay and the preincubation time was 20 mins and the plates were incubated for 48 hrs. The test compound was used at a dosage level of 0, 33, 100, 333, 1000, 3333 or 10000 µg/plate in the preincubation assay of 48 hrs. Concurrent solvent and positive control chemicals were included in the study. N-Methyl diethanolamine did not induce a reproducible, dose-related increase in his+revertants over the corresponding solventin the S. typhimurium tester strains TA1535, TA1537, TA98 and TA100 in the presence and absence of S9 metabolic activation system and hence is negative for mutation in vitro.

Based on the data available for the target chemical and its read across, 4-Methylpiperazin-1-amine does not exhibit gene mutation in vitro. Thus the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available for the target chemical and its read across, 4-Methylpiperazin-1-amine (CAS no 6928 -85 -4) does not exhibit gene mutation in vitro. Thus the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.