4-Piperidinecarboxylic acid, hydrochloride (1:1)

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:

2006-03-06 to 2006-03-22

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study conducted according to OECD Guideline 471 (Bacterial Reverse Mutation Assay) and EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria) with an acceptable deviation (only three tester strains were used). Expert statement is made available as described in attachment and in the field "overall remarks" to justify the fact that no further testing is necessary

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

: Only three tester strains were used.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

: Only three tester strains were used.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-piperidinecarboxylic acid hydrochloride

EC Number:

611-901-9

Cas Number:

5984-56-5

Molecular formula:

C6H11NO2.HCl

IUPAC Name:

4-piperidinecarboxylic acid hydrochloride

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study reports): JNJ-150098-AAC (T001310)
- Physical state: solid
- Appearance white powder

Specific details on test material used for the study:

Description: white solid
Batch number: 00465047 RT001310G4A851
Purity: 100 %
Stability in solvent: not indicated by sponsor
Storage conditions: Room temperature
Expiry date: 2003-06-30

Method

Target gene:

The histidine dependent strains are derived from S. typhimurium strain LT2 through a mutation in the histidine locus.

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA98, TA100 and TA102

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

- Pre-experiment and Experiment 1: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
- Experiment 2: 33, 100, 333, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9; at 10 µg/plate for TA100

Controls 2

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

without S9; at 10µg/plate for TA98

Controls 3

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9; at 4.0 µL/plate for TA102

Controls 4

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

with S9; at 2.5 µg/plate for TA98 and TA100 and 10.0 µg/plate for TA102

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- In Experiment 1 (plate incorporation), the following materials were mixed in a test tube and poured onto the selective agar plates: 100 µL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control), 500 µL S9 mix (for test with S9) or S9 mix substitution buffer (for test without S9), 100 µL bacteria suspension (cf. test system, pre-culture of the strains), and 2000 µL overlay agar.
- In Experiment 2 (pre-incubation), 100 µL test solution, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacteria suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on selective agar plates.
- After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

DURATION
- Preincubation period: 60 minutes (Experiment 2)
- Exposure duration: at least 48 hours at 37°C in the dark (both experiments)
- Selection time: 48h (simultaneous with exposure)

SELECTION AGENT (mutation assays): histidine (S. typhimurium strains)

NUMBER OF REPLICATIONS:
- Each concentration and the controls were tested in triplicate.

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn

Evaluation criteria:

- The test substance was considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control was observed.
- A dose dependent increase was considered biologically relevant if the threshold was exceeded at more than one concentration.
- An increase exceeding the threshold at only one concentration was judged as biologically relevant if reproduced in an independent second experiment.
- A dose dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remained within the historical range of negative and solvent controls such an increase was not considered biologically relevant.

Statistics:

- A statistical analysis of the data was not required.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA98, TA100 and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: no data
- Precipitation: none

RANGE-FINDING/SCREENING STUDIES:
- The pre-experiment was reported as main Experiment 1. See relevant sections for results.

COMPARISON WITH HISTORICAL CONTROL DATA:

- The positive controls showed a distinct increase in induced revertant colonies. The positive control mean revertant values for TA98 and TA102 in the presence of S9 mix and the vehicle and negative control mean revertant values for TA102 in the presence and absence of S9 mix in Experiment 1 were above the historical data range. The positive control mean revertant value for TA98 in the absence of S9 mix and the negative control mean revertant value for TA102 in the absence of S9 mix in experiment 2 were above the historical data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- The plates incubated with the test substance showed normal background growth up to the highest concentration. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups.

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative with and without metabolic activation

The test substance was evaluated for mutagenic potential in the bacterial reverse mutation assay in S. typhimurium strains TA98, TA100 and TA102 in the presence and absence of S9 metabolic activation. Under the conditions of the study, the test substance was determined to be negative for mutagenic potential in all tester strains in the absence and presence of metabolic activation.