Reaction mass of 2-Piperazin-1-ylethanol and Piperazine-1,4-diethanol and Piperazine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro transformation study in mammalian cells

Remarks:

Type of genotoxicity: genome mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

30 sep 1980 - 2 Dec 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study performed in accordance with EU guideline B21. No deviations from protocol or GLP. Results of negative control differ significantly from the historical value. However, sensitivity of the assay was normal. Piperazine is one constituent (ca. 15%) of the test substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1980

Report date:

1980

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.21 (In Vitro Mammalian Cell Transformation Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

Litton Bionetics, Inc

Type of assay:

in vitro mammalian cell transformation assay

Test material

Test material information

Constituent 1

Reference substance name:

Piperazine

EC Number:

203-808-3

EC Name:

Piperazine

Cas Number:

110-85-0

Molecular formula:

C4H10N2

IUPAC Name:

piperazine

Details on test material:

- Name of test material (as cited in study report): 4236-32-15
- Physical state: solid

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

mammalian cell line, other: BALB/3T3 mouse cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle's Minimum Essential Medium (EMEM) supplemented with 10 % fetal bovine serum
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

other: selected for low spontaneous frequencies of foci formation

Metabolic activation:

not specified

Test concentrations with justification for top dose:

1.0, 15.0, 75.0, 150.0, 300.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none (culture medium)

Controls

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

no

Remarks:

not applicable

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

Migrated to IUCLID6: 5 µg/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24h
- Exposure duration: 3d
- Expression time (cells in growth medium): 4 weeks (refeeding twice a week)
- Fixing the cell monolayers with methanol
- Staining with Giemsa

NUMBER OF REPLICATIONS: 15 dishes per dose, 15 for negative control, 15 for positive control

Examined by eye and by microscope to determine the number of foci of transformed cells.

Evaluation criteria:

- Negative control flasks consist of a contiguous monolayer of cells which may or may not contain transformed foci. The lack of contiguous sheet of cells indicates growth conditions too poor to allow the reliable detection of weak transforming agents.
- The negative control transformation frequency does not exceed an average of 2 foci/flask. Attempts are made to isolate and maintain cell stocks (subclones of Balb/3T3 I13) with a very low spontaneous frequency of transformation.
- Positive control yields an average number of foci/flask that is significantly different from the negative control at the 99% CL.
- A minimum of 8 flasks per test condition are available for analysis. At least 4 dose levels of test substance are assayed.
- The dose range of test substance assayed falls within the 50-100% survival range as determined by the preliminary toxicity test, which measures relative cloning efficiencies.

Statistics:

The statistical tables provided by Kastenbaum and Bowman are used to determine whether the results at each dose level are significantly different from the historical control at the 95% or 99% confidence levels (Kastenbaum and Bowman (1970) Tables for determining the statistical significance of mutation frequencies. Mut. Res. 9, 527-549).
The test compares variables distributed according to Poissonian expectations by summing the probabilities in the tails of two binomial distributions. The 95% confidence level must be met to consider the test substance active at a particular dose level.

Results and discussion

Test results

Species / strain:

mammalian cell line, other: BALB/3T3 cells

Metabolic activation:

not specified

Genotoxicity:

negative

Remarks:

concentration range: 1.0 µg/mL to 300.0 µg/mL

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 1000 µg/mL and higher

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Fifteen dose levels of the test compound are chosen (max 1000 µg/mL - min 0.061 µg/mL)(decreasing in two-fold dilution steps).
Each dose is applied to 3 culture dishes seeded 24 h earlier with 200 cells per dish. After an exposure period of 3 days, the cells are washed and incubated in growth medium for an additional 4 days. Surviving colonies are stained and counted. A relative survival for each dose is obtained by comparing the number of colonies surviving treatment to the colony counts in negative control dishes.
The relative survivals ranged between 41.0% at 500.0 µg/mL and approx. 90-95% at concentrations between 0.977 µg/mL and 0.061 µg/mL.
The highest dose chosen for subsequent transformation assays should normally cause no more than a 50% reduction in colony forming ability and is best located near 30% reduction. Four lower doses (including 1 or 2 doses with low or no apparent toxicity) are also selected for the transformation assay.

COMPARISON WITH HISTORICAL CONTROL DATA:
The historical negative control for the subclone of 3T3 cells used in this assay consists of 147 flasks containing a total of 17 transformed foci for an average of 0.12 foci / flask. In this assay, a total of 5 transformed foci were observed among the 15 negative control flasks for an average of 0.33 foci/flask.
The present result was significantly different from the historical value using the Kastenbaum-Bowman tables. Therefore, the experimental results were evaluated independently.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Negative control differs significantly from historical data. Sensitivity of the assay was normal.
Significant numbers of transformed foci were observed only among flasks treated with 150.0 µg/mL of test material. This increase represented a 3-fold elevation in transformation frequency relative to the negative control value and was just significant at the 95% confidence level. However, no clear evidence of a dose-related response was observed.
Therefore, it was concluded that test material did not induce a significant and dose-related number of transformed foci over the concentration range of 1.0 µg/mL to 300.0 µg/mL. This concentration range corresponds to a survival range of approx. 90% to 50 % in the preliminary cytotoxicity test. The test material is considered to be inactive in the Balb/3T3 in vitro transformation assay.

Executive summary:

In a study performed according to procedures similar to EU Method B.21 (In Vitro Mammalian Cell Transformation Test), piperazine did not induce a significant and dose-related number of transformed foci over the concentration range of 1.0 µg/mL to 300.0 µg/mL. This concentration range corresponds to a survival range of approx. 90% to 50 % in the preliminary cytotoxicity test. The test material is considered to be inactive in the Balb/3T3 in vitro transformation assay.