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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 09 October 2012 to 09 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
(adopted July 21, 1997)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
dated May 30, 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Constituent 1
Chemical structure
Reference substance name:
Piperidine
EC Number:
203-813-0
EC Name:
Piperidine
Cas Number:
110-89-4
Molecular formula:
C5H11N
IUPAC Name:
piperidine
Specific details on test material used for the study:
- Name of test material (as cited in study report): Piperidine
- Physical state: Liquid, colorless, clear
- Analytical purity: 99.7 corr. area-%
- Lot/batch No.: 000STD77L0
- Expiration Date: May 16, 2013
- Storage condition of test material: Room temperature
- Stability in Solvent: Not indicated
- On the day of the experiment (immediately before treatment), the test item was dissolved in deionised water. The solvent was chosen based on solubility properties and its relative non-toxicity to the cell cultures. The final concentration of deionised water in the culture medium was 10% (v/v).
- The osmolarity and pH-value was determined in the solvent control and in the highest concentration of the pre-experiment without metabolic activation: solvent control: Osmolarity mOsm: 276 / pH-value: 7.36; Piperidine (860 µg/mL): Osmolarity mOsm 303 / pH-value: 7.57 (pH was adjusted with 2 N HCl).

Method

Target gene:
HPRT locus

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts, 10% foetal bovine serum, neomycin (5 μg/mL) and amphotericin B (1 %). For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes, before freezing, the level of spontaneous mutants was depressed by treatment with HAT medium.
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of 8 - 12 weeks old male Wistar rats [Hsd Cpb: WU], treated i.p. with 80 mg/kg b.w. phenobarbital and orally with β-naphthoflavone each on three consecutive days.
Test concentrations with justification for top dose:
Range finding pre-experiment: 6.7 to 860 μg/mL (≈10 mM), in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation.
Main experiment: 1st Experiment: with and without S9 mix (4-hour exposure period): 26.9; 53.8; 107.5; 215.0; 430.0; 860.0 μg/mL;
2nd Experiment: without S9 mix (24-hour exposure period): 53.8; 107.5; 215.0; 430.0; 645.0; 860.0 μg/mL; with S9 mix (4-hour exposure period): 26.9; 53.8; 107.5; 215.0; 430.0; 860.0 μg/mL.
In experiment I with and without metabolic activation the cultures at the lowest concentration were not continued since a minimum of only four analysable concentrations is required by the guidelines. In experiment II the cultures at the lowest concentration with metabolic activation were not continued for the same reason. The cultures at the two highest concentrations of experiment II without metabolic activation were not continued due to exceedingly severe cytotoxic effects.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(water)
True negative controls:
no
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
ethylmethanesulphonate
Remarks:
0.150 mg/mL (1.2 mM); dissolved in nutrient medium; dilutions of the stock solution were prepared on the day of the experiment and used immediately.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(water)
True negative controls:
no
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
1.1 µg/mL (4.3 µM); dissolved in Dimethylsulfoxide; dilutions of the stock solution were prepared on the day of the experiment and used immediately; final concentration in nutrient medium 0.5%.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;

DURATION
- Exposure duration:
1st experiment: 4 h exposure with and without S9 mix
2nd experiment: 4 h exposure with S9 mix and 24 h without S9 mix
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 to 10 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 to 17 days

SELECTION AGENT (mutation assays): 6-thioguanine (10 μg/mL)

NUMBER OF REPLICATIONS: duplicates

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER:
- In the range finding pre-experiment relevant cytotoxic effects were solely observed at 860 μg/mL following 24 hours treatment without metabolic activation.
Evaluation criteria:
A test item was classified as positive if it induced either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points was considered non-mutagenic in the system.
A positive response was described as follows:
- A test item was classified as mutagenic if it reproducibly induced a mutation frequency that was three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
- The test item was classified as mutagenic if there was a reproducible concentration-related increase of the mutation frequency. Such evaluation was considered also in the case that a threefold increase of the mutant frequency was not observed.
- However, in a case by case evaluation this decision depend on the level of the corresponding solvent control data. If there was by chance a low spontaneous mutation rate within the laboratory's historical control data range, a concentration-related increase of the mutations within this range was discussed. The variability of the mutation rates of solvent controls within all experiments of the study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend was judged as significant whenever the p-value (probability value) was below 0.05. However, both, biological and statistical significance was considered together.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
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
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item. The pH was adjusted with 2 N hydrochloric acid at the two highest concentrations in the pre-experiment and in both main experiments.
- Precipitation: The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. No precipitation or phase separation was observed up to the maximum concentrations with and without metabolic activation following 4 and 24 hours treatment.

RANGE-FINDING/SCREENING STUDIES:
- Based on the results of the pre-experiment, the individual concentrations of the main experiments were selected. A series of concentrations spaced by a factor of 2 was applied. Narrower spacing was used at high concentrations of the second experiment without metabolic activation to cover the toxic range more closely.

ADDITIONAL INFORMATION ON GENOTOXICIY AND COMPARISON WITH HISTORICAL CONTROL DATA:
- The highest solvent control (42.5 colonies per 10⁶ cells) exceeded the historical range of solvent controls (3.4 - 36.6 colonies per 10⁶ cells). However, this effect was judged as irrelevant since the solvent control of the parallel culture (17.9 colonies per 106 cells) and the mean of both cultures (42.5 and 17.9 equal to a mean of 30.2 colonies per 10⁶ cells) remained well within the range of historical controls.
- There was no relevant and reproducible increase in mutant colony numbers/10⁶ cells in the main experiments up to the maximum concentration. An isolated increase of the induction factor exceeding the induction factor of three times the mutation frequency of the corresponding solvent control was observed in the second culture of the second experiment without metabolic activation at 107.5 μg/mL (24 hours treatment).
At this data point the absolute value of the mutation frequency (34.5 mutant colonies per 10⁶ cells) did not exceed the historical range of solvent controls (2.6 - 40.3 mutant colonies/10⁶ cells). Therefore, the increase was judged to be based upon the rather low solvent control of 8.3 mutant colonies/10⁶ cells.
- A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequency. A single significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the second experiment at culture II with metabolic activation (see above). However, the trend was judged as biologically irrelevant as it was not reproduced in the parallel culture and the induction factor did not reach or exceed the threshold of three times the mutation frequency of the corresponding solvent control.
- In both experiments of the study (with and without S9 mix) the range of the solvent controls was from 8.3 up to 42.5 mutants per 10⁶ cells; the range of the groups treated with the test item was from 4.4 up to 38.7 mutants per 10⁶ cells.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Relevant cytotoxic effects indicated by a relative cloning efficiency I (survival, cloning efficiency determined immediately after treatment to measure toxicity) and/or a relative cell density below 50% were observed in the second experiment at 430.0 μg/mL without metabolic activation (24 hours treatment).

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative