Piperidine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria
(1) Ames test with S. typhimurium TA 100, TA 1537 and TA 98 with and without metabolic activation: negative up to 2129 µg/mL (non-GLP, non-Guideline; Riebe et al., 1982)
(2) Ames test with S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 with and without metabolic activation: negative up to 255 µg/plate (non-GLP, non-Guideline; Florin et al. 1980)
(3) Ames test with E.coli WP2 uvr A without metabolic activation: negative up to 10 mg/plate, non-GLP, non-Guideline (Pia et al., 1977).

Gene mutation in mammalian cells
Mammalian cell gene mutation assay with mouse lymphoma L5178Y cells with and without metabolic activation: positive without metabolic activation, ambiguous with S9 activation up to 688 µg/mL (non-GLP, non-Guideline; Wangenheim and Bolcsfoldi, 1988).

Cytogenicity in mammalian cells
DNA alkaline unwinding and hydroxyapatite elution assay with mouse lymphoma L5178Y cells: ambiguous at the highest dose level of 8.02E-03 mmol/L exhibiting cytotoxicity with metabolic activation and negative without metabolic activation up to 6.01E-03 mmol/L (non-GLP, non-Guideline; Garberg et al. 1988).

Link to relevant study records

Reference

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

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

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).

Target gene:

HPRT locus

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

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.

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).

Conclusions:

Interpretation of results:
negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Gene mutation
no data

Cytogenicity
Micronucleus test, mouse, i.p. injections: negative up to 400 mg/kg bw/d (GLP, OECD 474; CCR 1989)

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(First Addendum to the OECD Guideline for Testing of Chemicals, Section 4, No. 474, adopted May 26, 1983, "Micronucleus Test")

Deviations:

yes

Remarks:

Deviation to updated version of OECD TG 474 (1997): 1000 polychromatic erythrocytes analysed per animal (instead of 2000 PCEs)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

- Name of test material (as cited in study report): Piperidin
- Physical state: liquid, colourless/yellowish
- Analytical purity: 99.3 %
- Lot/batch No.: 459-60 17 FA. 10000 GR 1 FA. GEZ
- Expiration date of the lot/batch: June 27, 1990
- Stability under test conditions: not indicated
- Storage condition of test material: 4°C, closed container

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: minimum 10 weeks
- Weight at study initiation: approximately 30 g
- Fasting period before study: 18 hours
- Housing: single, Makrolon Type I, with wire mesh top (EBECO, Castrop-Rauxel, Germany)
- Diet (ad libitum): pelleted standard diet (ALTROMIN, Lage/Lippe, Germany)
- Water (ad libitum): tap water
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): not regulated
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

VEHICLE
- Vehicle(s)/solvent(s) used: water
- Amount of vehicle (if gavage): 10 mL/kg body weight orally
- Justification for choice of vehicle: non-toxicity for the animals.

Duration of treatment / exposure:

single dose

Frequency of treatment:

single dose

Post exposure period:

Bone marrow preparation intervals: 24, 48, 72 h

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Remarks:

24 hours

Dose / conc.:

120 mg/kg bw/day (actual dose received)

Remarks:

24 hours

Dose / conc.:

400 mg/kg bw/day (actual dose received)

Remarks:

24 hours, 48 hours and 72 hours

No. of animals per sex per dose:

6 animals/sex/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamid
- Route of administration: gavage
- Doses / concentrations: 30 mg/kg bw in 10 mL physiological saline /kg bw

Tissues and cell types examined:

Polychromatic erythrocytes (PCE) in the bone marrow of the mouse

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A preliminary study on acute toxicity was performed with the same strain and under identical conditions as in the mutagenicity study.
The maximum tolerated dose level was determined to be the dose that caused toxic reactions without having major effects on survival within 72 hours.
Three adequate spaced dose levels extending over a single log range were applied at the central sampling interval 24 h after treatment. The highest dose level used was the maximum tolerated dose or that producing some indication of cytotoxicity. For this dose level additional samples were taken at 48 h and 72 h after treatment.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Preparation of the Animals:
The animais were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with 2.0 mL fetal calf serum, using a 5 mL syringe, into 1 mL fetal calf serum. The cell suspension was centrifuged at 1000 rpm for 5 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide.

DETAILS OF SLIDE PREPARATION:
The smear was air-dried and then stained with May-Gruenwald/Giemsa. Cover slips were mounted with EUKITT (KINDLER, D-7800 Freiburg F.R.G.). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed as normochromatic erythrocytes per 1000 PCEs. The analysis was performed with coded slides.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points.
A test article producing neither a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant and reproducible positive response at anyone of the test points is considered nonmutagenic in this system.
This can be confirmed by means of the nonparametric Mann-Whitney test. However, both biological and statistical significance was considered together.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

After treatment with 400 mg/kg bw piperidine 5 out of 36 animals died in the micronucleus assay. One male and one female died at preparation intervals 24 and 48 h, respectively, and one male animal at preparation interval 72 h.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
1. In a first pre-experiment 6 animals (3 males, 3 females) received orally a single dose of 500 mg/kg b.w. piperidine dissolved in aqua dest.. The volume administered was 10 mL/kg b.w..
All treated animals expressed toxic reactions: eyelid closure, apathy, tremor, dyspnoea.
Lethalities: one female within 24 hours, one male and one female within 48 hours.
2. In a second pre-experiment 6 animals (3 males, 3 females) per dose level received orally a single dose of 300 or 400 mg/kg b.w., respectively, piperidine dissolved in aqua dest.. The volume administered was 10 mL/kg b.w..
300 mg/kg b.w.:
All treated animals expressed toxic reactions: eyelid closure, apathy. None of the treated animals died.
400 mg/kg b.w.:
All treated animals expressed toxic reactions: eyelid closure, apathy. None of the treated animals died.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: In comparison with the corresponding negative controls there was no substantial enhancement in the freguency of the detected micronuclei at any preparation interval or dose level of the test article. The mean values of micronuclei observed after treatment with piperidine were in the same range as compared to the negative control groups.
- Ratio of PCE/NCE: As compared to the corresponding negative controls the mean number of normochromatic erythrocytes was enhanced after the treatment with highest dose of piperidine, indicating that the test article had cytotoxic properties.
- Positive control: 30 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a distinct increase in induced micronucleus frequency.

Test group	Dose [mg/kg bw]	Sampling time [h]	PCEs with micronuclei [%]	Range	PCE/NCE
solvent	0	24	0.12	0-3	1000/496
test article	40	24	0.12	0-4	1000/441
test article	120	24	0.08	0-3	1000/612
test article	400	24	0.13	0-4	1000/1001
cyclophosphamide	30	24	1.21	4-24	1000/769
solvent	0	48	0.09	0-2	1000/630
test article	400	48	0.14	0-3	1000/1181
solvent	0	72	0.14	0-3	1000/605
test article	400	72	0.14	0-3	1000/435

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Conclusions:

Interpretation of results: negative
Under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Executive summary:

In a NMRI mouse bone marrow micronucleus assay, performed according to GLP and OECD 474, five animals per sex and per dose were treated orally at a single time point with the test item (99.3 % a.i.) at doses of 0, 40, 120, and 400 mg/kg bw (CCR/BG Chemie, 1989). Bone marrow cells were harvested at 24, 48 and 72 h post-treatment. The vehicle was aqua dest..

In the highest dose of 400 mg/kg bw all treated animals expressed toxic reactions such as eyelid closure and apathy. None of the treated animals died. The mean number of normochromatic erythrocytes was enhanced after the treatment with highest dose of the test item as compared to the corresponding negative control, indicating that the test article had cytotoxic properties. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time or dose level. The positive control induced the appropriate response. The study is classified as acceptable (reliability 1) and satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenetic mutagenicity data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro

Gene mutation in bacteria

In a non-GLP, non-Guideline bacterial reverse gene mutation assay, strains TA 98, TA 100, TA 1537 of S. typhimurium were exposed to the test item (purity unknown), using water as solvent, at concentrations of 21, 106, 532, 1064, 2129 µg/mL (1.25–25 mM) in the presence and absence of mammalian metabolic activation (liver fraction (S-9) from Phenobarbital induced mice) using the pre-incubation method (1 hour, 37 °C) (Riebe et al., 1982). The test item was tested up to cytotoxic concentrations. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background. The study is classified as acceptable, but does not satisfy the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data (limited documentation, only one positive control substance in the presence of metabolic activation, reduced number of bacterial strains).

The test item was negative in Salmonella typhimurium assay using strains TA98, TA100, and TA1537 with and without metabolic activation with S9 from phenobarbital-induced mouse liver, when tested at concentrations of 1.25–25 mM.

 

In a non-GLP, non-Guideline bacterial reverse gene mutation screening assay for tobacco smoke constituents, strains TA 1535, TA 1537, TA 98 and TA 100 of S. typhimurium were exposed to the test item (≥ 97 % a.i.), diluted by the vehicle ethanol at a concentration of 255 µg/plate in the presence and absence of mammalian metabolic activation (liver fraction (S-9) from Aroclor 1254 induced male Sprague-Dawley rats) using a standard plate incubation test (Florin et al. 1980). The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background. The study is classified as acceptable (reliability 2), but does not satisfy the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data (limited documentation, only one concentration tested, only one positive control substance in the presence of metabolic activation).

The test item was negative in Salmonella typhimurium assay using strains TA 1535, TA 1537, TA 98 and TA 100 with and without metabolic activation with S9 from Aroclor 1254 induced rat liver, when tested at a concentrations of 255 µg/plate.

 

In a non-GLP, non-Guideline bacterial reverse gene mutation assay (spot test), E. coli WP2 uvr A were exposed to the undiluted test item (commercial samples purified by standard techniques, a.i.) at concentrations of 5 to 10 mg/plate in the absence of mammalian metabolic activation (Pia et al., 1977). There were no data about positive controls included. There was no evidence of induced mutant colonies over background. The study was classified as acceptable (reliability 2), but does not satisfy the requirement for OECD 471 for in vitro bacterial reverse gene mutation assays (limited documentation, no positive controls, and only one tested strain).

The test item was negative in the E. coli assay using E. coli WP2 uvr A with and without metabolic activation with S9 from Aroclor 1254 induced rat liver, when tested at concentrations up to 10 mg/plate.

 

In summary, the test item was tested in all bacterial strains required according to the OECD Guideline 471 and reported as negative.

 

Gene mutation in mammalian cells

In a GLP conform in vitro mammalian cell gene mutation test (HPRT-Test) according to OECD Guideline 476 (Harlan 1473904 / BASF, 50M0108/09X106, 2012) the test substance was investigated for its potential to induce gene mutations at the HPRT locus in V79 cells. The assay was performed in two independent experiments, using two parallel cultures each. In the first main experiment cells were exposed for 4 hours to the test item at concentrations of 53.8; 107.5; 215.0; 430.0; 860.0 μg/mL with and without liver microsomal activation. The second experiment was performed with a treatment time of 4 hours with metabolic activation (53.8; 107.5; 215.0; 430.0; 860.0 μg/mL) and a treatment time of 24 hours without metabolic activation (53.8; 107.5; 215.0; 430.0 μg/mL). The maximum concentration of the pre-experiment and the main experiments was 860 μg/mL or approximately 10 mM, respectively. The test item was dissolved in deionized water.

Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Relevant cytotoxic effects indicated by a relative cloning efficiency I (survival 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).

No substantial and reproducible dose dependent increase in mutation frequency was observed up to the maximum concentration with and without metabolic activation.

All mutant frequencies remained well within the historical range of solvent controls.

The study is classified as acceptable (reliability 1) and satisfies the requirement for OECD 476 for in vitro gene mutation in mammalian cells.

According to the results of the study, the test substance did not induce gene mutations in the in vitro mammalian cell gene mutation test under the experimental conditions chosen.

 

In a mammalian cell gene mutation assay (Thymidine kinase locus), mouse lymphoma L5178Y cells cultured in vitro were exposed to the test item (highest available purity), using DMSO or ethanol as solvent at concentrations of 258, 344, 430, 516, 602, 688 µg/mL (3.03, 4.04, 5.05, 6.06, 7.07, 8.08 mM) in the presence and absence of mammalian metabolic activation (liver fraction (S-9) from Aroclor 1254 induced male Sprague-Dawley rats) for 4 hours in triplicate in suspension (Wangenheim and Bolcsfoldi, 1988). The positive controls did induce the appropriate response.

The mutation frequency was significantly and dose-dependently increased without metabolic activation at 5, 6 and 7 mM (mutation index: 1.6, 3.4 and 11.0, respectively); relative total growth was reduced at these concentration levels to 53, 24 and 4 % of the control, respectively. After addition of Aroclor induced rat liver S-9, only slight, but significant increased mutation frequencies were observed at 6 and 8 mM without showing dose-dependency (mutation index 1.4).

The study is classified as acceptable (reliability 2), but does not satisfy the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data (limited documentation).

The test item was mutagenic in the mouse lymphoma assay without metabolic activation at cytotoxic concentrations, but due to the absence of dose dependency ambiguous with S9 activation.

 

Cytogenicity in mammalian cells / DNA damage and/or repair in mammalian cells

In a non-GLP and non-Guideline DNA alkaline unwinding and hydroxyapatite elution assay, mouse lymphoma L5178Y cells were exposed to the test item (analytical grade), at concentrations of 2 - 8 mmol/L (170.3 - 681.2 µg/mL) for 3 hours (Garberg et al. 1988) in the presence and absence of mammalian metabolic activation (Rat liver homogenate (S9) from male Sprague-Dawley rats pretreated with Aroclor 1254). The test item was tested up to cytotoxic concentrations. There were no data about positive controls. Without metabolic activation piperidine was negative under the experimental conditions of this assay. With metabolic activation an increase in the relative fraction of ssDNA occurred at a relative toxicity of ≥ 5%. Since such an increase was observed only at the highest cytotoxic concentration, the result was regarded as equivocal.

Cytotoxicity was observed at a concentration of 6.01E-03 mmol/L with and 8.02E-03 mmol/L without metabolic activation. The study is classified as acceptable (reliability 2).

There was no unambiguous evidence for induction of DNA strand breaks, as determined by an increase in the relative fraction of ssDNA, in mouse lymphoma cells in the presence of metabolic activation.

 

In vivo

Chromosome aberration

In a NMRI mouse bone marrow micronucleus assay, performed according to GLP and OECD 474, five animals per sex and per dose were treated orally at a single time point with the test item (99.3 % a.i.) at doses of 0, 40, 120, and 400 mg/kg bw (CCR/BG Chemie, 1989). Bone marrow cells were harvested at 24, 48 and 72 h post-treatment. The vehicle was aqua dest..

In the highest dose of 400 mg/kg bw all treated animals expressed toxic reactions such as eyelid closure and apathy. None of the treated animals died. The mean number of normochromatic erythrocytes was enhanced after the treatment with highest dose of the test item as compared to the corresponding negative control, indicating that the test article had cytotoxic properties. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time or dose level. The positive control induced the appropriate response. The study is classified as acceptable (reliability 1) and satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenetic mutagenicity data.

Under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

 

Summary: In genotoxicity studies, the test item was shown not to be mutagenic in Salmonella with or without metabolic activation with either the Ames or the host-mediated assay. The test item also was negative in the E. coli recombination assay. The test item was mutagenic in the mouse lymphoma assay without metabolic activation at cytotoxic concentrations, but due to the absence of dose dependency ambiguous with S9 activation. In mouse lymphoma cells the test item induced DNA strand breaks at cytotoxic concentrations in the presence of metabolic activation, but not without metabolic activation. In a HPRT gene mutation test in mammalian V79 cells the test item did not induce substantial and reproducible dose dependent increase in mutation frequency up to the maximum concentration with and without metabolic activation. In a NMRI mouse bone marrow micronucleus assay no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time or dose level were observed. In the highest dose there was evidence for cytotoxic properties.

 

Conclusion: The test item showed negative effects in bacterial test systems and in addition to ambiguous effects in different in-vitro test systems using mouse lymphoma cells, clear negative effects in the gene mutation assay in V79 cells. Furthermore, no mutagenic effects were observed in an in vivo mammalian test system. Moreover, different studies concerning carcinogenicity gave no indication on a carcinogenic potential of the test item.

 

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data for genetic mutagenicity are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. As a result, the substance is not warranted to be classified for mutagenicity, under Regulation (EC) No.1272/2008.