1-Piperazinecarboxylic acid, 4-(6-amino-3-pyridinyl)-, 1,1-dimethylethyl ester

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

This study was performed following the SOP’s for this test. However, the experiments do not comply with GLP regulations, since they were not controlled by GLP-QA

Data source

Reference

Reference Type:

other company data

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The objective of the present study was to assess the mutagenic potential of the test item using Salmonella typhimurium in vitro, with and without the addition of a mammalian
metabolizing system.
The Salmonella/liver homogenate assay (Ames test) is the most widely used test system to assess mutagenicity. It has proved to be a highly sensitive and reliable test to detect
mutagenicity and to predict the carcinogenicity of chemicals.
In this test, histidine auxotrophic strains of Salmonella typhimurium are plated on minimal agar together with the test item, both in the presence and absence of a rat-liver S9 mix for metabolic activation. The medium contains traces of histidine, which allow the bacteria to make a few cell divisions. After exhaustion of this histidine, only prototrophic, i.e. mutant, bacteria are able to grow. Thus, only histidine prototrophic bacteria are able to form colonies during an incubation
time of 3 days. The number of colonies on the tester wells is therefore directly proportional to the number of mutant bacteria. A clear increase in the colony numbers on the treated wells above the corresponding negative control values indicates a mutagenic potential of the test article.

Remarks: This study was performed following the SOP’s for this test. However, the experiments do not comply with GLP regulations, since they were not controlled by GLP-QA.

GLP compliance:

no

Remarks:

This study was performed following the SOP’s for this test. However, the experiments do not comply with GLP regulations, since they were not controlled by GLP-QA

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

tert-butyl 4-(6-aminopyridin-3-yl)piperazine-1-carboxylate

Cas Number:

571188-59-5

Molecular formula:

C14H22N4O2

IUPAC Name:

tert-butyl 4-(6-aminopyridin-3-yl)piperazine-1-carboxylate

Test material form:

solid: bulk

Method

Target gene:

In this test, histidine auxotrophic strains of Salmonella typhimurium are plated on minimal agar together with the test item, both in the presence and absence of a rat-liver S9 mix for metabolic activation. The medium contains traces of histidine, which allow the bacteria to make a few cell divisions. After exhaustion of this histidine, only prototrophic, i.e. mutant, bacteria are able to grow. Thus, only histidine prototrophic bacteria are able to form colonies during an incubation time of 3 days. The number of colonies on the tester wells is therefore directly proportional to the number of mutant bacteria. A clear increase in the colony numbers on the treated wells above the corresponding negative control values indicates a mutagenic potential of the test article.

Species / strain

Species / strain / cell type:

other: Strains of Salmonella typhimurium

Details on mammalian cell type (if applicable):

Strains of Salmonella typhimurium TA1535, TA98, TA100, TA97a and TA102

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 mix from male rats, Aroclor 1254-pretreated. Per plate, 25 μl S9 was added.

Test concentrations with justification for top dose:

Concentrations tested:
• 1.6, 8, 40, 200, 1000, 5000 μg/plate (experiment 1 using strains TA98 and TA100)
• 1.6, 8, 40, 200, 1000, 5000 μg/plate (experiment 2 using strains TA1535, TA97a and TA102)
Metabolic activation system: Liver S9 mix from male rats, Aroclor 1254-pretreated. Per plate, 25 μl S9 was added.

Positive controls used:
All positive controls were dissolved in DMSO and used at one concentration level per strain only at 0.1 mL/plate.
2-Aminoanthracene: This is an indirect-acting mutagen. At 3 μg/plate it is mutagenic for strains TA1535, TA98 and TA100. At 10 μg/plate it is mutagenic for strains TA97a and TA102.
Benzo(a)pyrene: This is an indirect-acting frame-shift mutagen. At the concentration level used (3 μg/plate) it is mutagenic for strain TA98.
Sodium azide: This is a direct-acting agent which induces base-pair substitutions. At 3 μg/plate it is mutagenic for strains TA1535 and TA100.
9-Aminoacridine: This is a direct-acting frame-shift mutagen. At the concentration level used (100 μg/plate) it is mutagenic for strain TA97a.
2-Nitrofluorene: This is a direct-acting frame-shift mutagen. At the concentration level used (2 μg/plate) it is mutagenic for the strain TA98.
Mitomycin C: This is a direct acting agent which is mutagenic for strain TA102 at the concentration level used (0.5 μg/plate).

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: TA98, TA100,TA1535, TA97a and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

The mean mutant numbers of negative control plates lay within the range of acceptable negative control values except for strain TA1535 +S9 in experiment 2 where the values were found to be above our historical control ranges. However, since the deviation was small, this was not considered to influence the results of the present study.

Applicant's summary and conclusion

Conclusions:

Under the testing conditions used and applying standard mutagenicity criteria, LEE011-A2 did not show evidence of a mutagenic potential.