1-methylpiperazine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There is a Ames bacterial reverse mutation assays available, an in-vitro mammalian cell gene mutation assay, the (L5178 TK+/-) mouse lymphoma assay and a human lymphocyte chromosomal aberration assay, all are negative.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11. May 1997 - 01. Jul 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline Study (OECD 471/472)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

; OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidin operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from Aroclor 1254 treated male Sprague-Dawley rats.

Test concentrations with justification for top dose:

0, 20, 100, 500, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: with S-9 mix: all strains 2-aminoanthracene; without S-9 mix: strains TA100, TA1535: N-methyl-N'-nitro-N-nitrosoguanidine; TA98: 4-nitro-o-phenylendiamine; TA1537: 9-aminoacridine; E.coli WP2 uvrA: N-ethyl-N'-nitro-N-nitrosoguanidine.

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 h


NUMBER OF REPLICATIONS: 3

METHOD OF APPLICATION: standard plate test

DURATION
- Exposure duration: 48 - 72 h


NUMBER OF REPLICATIONS: 3

Evaluation criteria:

The test chemical is considered positive in this assay if the following criteria are met: A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if: The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Complete solubility of test substance in aqua dest.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Standart plate test:

Dose (µg/plate)

TA1535

TA100

TA1537

TA98

E. coli WP2 uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

0

20±2

20±1

153±17

158±9

13±4

12±3

31±3

45±3

37±7

36±5

20

18±1

17±1

147±12

161±9

13±2

12±2

34±3

42±1

31±1

26±3

100

17±1

16±1

163±5

157±12

11±1

16±2

32±2

37±2

34±4

30±2

500

14±3

20±1

153±7

157±16

11±2

14±2

34±3

38±6

34±3

32±3

2500

15±2

17±2

152±14

156±5

13±4

14±2

32±3

42±3

28±3

31±3

5000

14±2

17±3

154±6

163±5

11±2

12±1

33±2

40±2

29±1

34±7

2-AA

-

149±7

-

1474±62

-

179±2

-

1237±32

-

317±37

MNNG

955±36

-

858±42

-

-

-

-

-

-

-

AAC

-

-

-

-

750±6

-

-

-

-

-

NPD

-

-

-

-

-

-

1104±77

-

-

-

ENNG

-

-

-

-

-

-

-

-

1185±189

-

Mean ± SD

Preincubation-test:

Dose (µg/plate)

TA1535

TA100

TA1537

TA98

E. coli WP2 uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

0

20±2

20±2

129±2

124±19

11±2

11±2

30±1

41±3

29±1

35±3

20

21±3

17±1

146±16

151±21

8±2

10±1

26±7

39±6

25±3

29±4

100

17±1

17±2

143±11

195±18

10±1

10±1

25±3

41±6

25±6

30±4

500

17±3

18±1

150±20

153±7

9±1

9±1

21±1

30±10

26±3

32±3

2500

17±1

16±2

158±13

176±25

8±2

9±1

23±4

41±1

24±2

28±3

5000

12±1

14±3

130±4

173±25

7±2

7±2

20±1

35±6

19±8

33±3

2-AA

-

154±22

-

805±107

-

148±10

-

1173±41

-

242±8

MNNG

981±45

-

1232±70

-

971±34

-

-

-

-

-

AAC

-

-

-

-

-

-

-

-

-

-

NPD

-

-

-

-

-

-

1228±98

-

-

-

ENNG

-

-

-

-

-

-

-

-

875±45

-

Mean ± SD

2-AA: 2-aminoanthracene;

MNNG; N-methyl-N-nitro-N-nitrosoguanidine

ENNG; N-ethyl-N-nitro-N-nitrosoguanidine

NPD: 4-nitro-o-phenylendiamine

AAC: 9-aminoacridine chloride monohydrate

Under the conditions tested Diethylaminopropylamin is non mutagenic in the bacterial AMES-test with or without metabolic activation.

The positive control gave the expected values.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phases of the study were performed between 06 September 2011 and 13 December 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The work described was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). Date of certificate 31 August 2011. Date of Inspection 19-21 July 2011

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable.

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral
infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The average AGT for
the regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone and beta-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary toxicity test
The dose range for the Preliminary Toxicity Test was 3.91 to 1000 µg/ml. The maximum dose was the 10 mM concentration.
Chromosome Aberration Test - Experiment 1
The dose levels of the controls and the test item are given in the table below:
Group Final concentration of N-methylpiperazine (µg/ml)
4(20)-hour without S9 0*, 31.25, 62.5, 125*, 250*, 500*, 1000*, MMC 0.4*
4(20)-hour with S9 0*, 31.25, 62.5, 125*, 250*, 500*, 1000*, CP 5*

Chromosome Aberration Test - Experiment 2
The dose levels of the controls and the test item are given in the table below:
Group Final concentration of N-methylpiperazine (µg/ml)
24-hour without S9 0*, 62.5, 125, 250*, 375*, 500*, 1000*, MMC 0.2*
4(20)-hour with S9 0*, 31.25, 62.5, 125*, 250*, 500*, 1000*, CP 5*

* Dose levels selected for metaphase analysis
MMC = Mitomycin C
CP = Cyclophosphamide

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Eagle's minimal essential medium with HEPES buffer (MEM)
- Justification for choice of solvent/vehicle: MEM was selected as the solvent because the test material was readily soluble in it at the required
concentrations.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

MEM

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: cyclophosphamide

Remarks:

In the presence of S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

MEM

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

In the absence of S9 Migrated to IUCLID6: (MMC)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
in medium

DURATION
- Preincubation period:
48 hrs

- Exposure duration:
Experiment 1 - 4 hrs with and without S9. Experiment 2 - 24 hrs without S9, 4 hrs with S9.

- Expression time (cells in growth medium):
20 hrs for 4 hrs exposure.

- Selection time (if incubation with a selection agent):
Not applicable.

- Fixation time (start of exposure up to fixation or harvest of cells):
24 hrs.


SELECTION AGENT (mutation assays):
No selection agent.

SPINDLE INHIBITOR (cytogenetic assays):
Demecolcine

STAIN (for cytogenetic assays):
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and coverslipped using mounting medium.


NUMBER OF REPLICATIONS:
Duplicate cultures


NUMBER OF CELLS EVALUATED:
100/culture


DETERMINATION OF CYTOTOXICITY
- Method:
mitotic index - A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic
index and as a percentage of the vehicle control value.

-Scoring of Chromosome Damage:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted
according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing (Appendix 1). Cells with
chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

OTHER EXAMINATIONS:
- Determination of polyploidy:
Frequency of polyploid cells


OTHER:
None.

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and
appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Refer to information on results and attached tables.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test material was dosed into media.
- Effects of osmolality: The osmalality did not increase by more than 50 mOsm.
- Evaporation from medium: Not applicable.
- Water solubility: Not applicable, test material dissolved in MEM
RESULTS
Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 3.91 to 1000 µg/ml. The maximum dose was based on the 10 mM concentration. No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, in any of the three exposure groups.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 1000 µg/ml in the
4(20)-hour exposures in the presence and absence of metabolic activation (S9). The maximum dose with metaphases present in the 24-hour
continuous exposure was 500 µg/ml. The mitotic index data are presented in the attached Table 1. The test item induced some evidence of toxicity
in all of the exposure groups, however more toxicity was seen in the 24 hour continuous exposure group.
The selection of the maximum dose level was based on the 10 mM concentration and was 1000 µg/ml for the 4(20)-hour exposure groups and 1000 µg/ml for the continuous exposure group used in Experiment 2.
Chromosome Aberration Test - Experiment 1
The dose levels of the controls and the test item are given in the table below:
Group Final concentration of N-methylpiperazine (µg/ml)
4(20)-hour without S9 0*, 31.25, 62.5, 125*, 250*, 500*, 1000*, MMC 0.4*
4(20)-hour with S9 0*, 31.25, 62.5, 125*, 250*, 500*, 1000*, CP 5*
The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present at the maximum dose level of test item, 1000 µg/ml in the absence and presence of metabolic activation (S9). No precipitate of the test item was observed in either exposure group, however a pink colouration was observed in the 4 (20)-hour exposure group
in the absence of S9, at and above 250 µg/ml, and at and above 500 µg/ml in the 4 (20) hour exposure group in the presence of S9.
The mitotic index data are given in the attached Table 2. They confirm the qualitative observations in that a slight dose-related inhibition of mitotic
index was observed, and that 24% mitotic inhibition was achieved at 500 µg/ml in the absence of S9, however at 1000 µg/ml no mitotic inhibition was observed. In the presence of S9 only 15% mitotic inhibition was achieved at 1000 µg/ml.
The maximum dose level selected for metaphase analysis was the 10 mM concentration dose level (1000 µg/ml) for both exposure groups.
The chromosome aberration data are given in the attached Table 4 and Table 5. All of the vehicle control cultures had frequencies of cells with
chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells
with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of
metabolic activation.
The polyploid cell frequency data are given in the attached Tables 4 and 5. The test item did not induce any statistically significant increases in the
numbers of polyploid cells at any dose level in either of the exposure groups.
Chromosome Aberration Test - Experiment 2
The dose levels of the controls and the test item are given in the table below:
Group Final concentration of N-methylpiperazine (µg/ml)
24-hour without S9 0*, 62.5, 125, 250*, 375*, 500*, 1000*, MMC 0.2*
4(20)-hour with S9 0*, 31.25, 62.5, 125*, 250*, 500*, 1000*, CP 5*
The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test item dose level of
1000 µg/ml in the absence and presence of S9. No precipitate of the test item was observed in either exposure group, however a pink colouration
was observed in the 24-hour exposure group in the absence of S9, at and above 125 µg/ml, and at and above 250 µg/ml in the 4(20) hour exposure group in the presence of S9.
The mitotic index data are given in the attached Table 3. They confirm the qualitative observations in that a dose-related inhibition of mitotic index
was observed, and that 42% and 71% mitotic inhibition was achieved at 500 and 1000 µg/ml in the absence of S9. In the presence of S9 only 11%
mitotic inhibition was achieved at 1000 µg/ml.
The maximum dose level selected for metaphase analysis was the same as Experiment 1, and was the 10 mM concentration dose level (1000 µg/ml).
The chromosome aberration data are given in the attached Table 6 and Table 7. All of the vehicle control cultures had frequencies of cells with
chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells
with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or
presence of metabolic activation.
The polyploid cell frequency data are given in the attached Tables 6 and 7. The test item did not induce a statistically significant increase in the
numbers of polyploid cells at any dose level in either of the exposure groups. An increase in polyploid cells was recorded in the absence of S9 at
1000 µg/ml. However, the response was only seen in the more toxic of the duplicate cultures and in a dose level that markedly exceeded the
optimum toxicity of 50%. It was therefore considered to have no biological relevance.

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

For the tables and figures of resluts mentioned above, please refer to the attached background material section for the following tables:

Table 1 Mitotic Index - Preliminary Toxicity Test                       

Table 2  Mitotic Index - Experiment 1                                                                 

Table 3  Mitotic Index - Experiment 2                                                                  

Table 4  Results of Chromosome Aberration Test - Experiment 1 Without Metabolic Activation (S9)                                                                                          

Table 5 Results of Chromosome Aberration Test - Experiment 1With Metabolic Activation (S9)                                                                                                       

Table 6  Results of Chromosome Aberration Test - Experiment 2 Without Metabolic Activation (S9)                                                                                          

Table 7 Results of Chromosome Aberration Test - Experiment 2 With Metabolic Activation (S9)

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

Introduction. This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al, 1990). The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008. The study design was also compatible with the UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity.

Methods. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:

 

Group	Final concentration of N-methylpiperazine(µg/ml)
4(20)-hour without S9	0, 31.25, 62.5, 125, 250, 500, 1000
4(20)-hour with S9 (2%)	0, 31.25, 62.5, 125, 250, 500, 1000
24-hour without S9	0, 62.5, 125, 250, 375, 500, 1000
4(20)-hour with S9 (1%)	0, 31.25, 62.5, 125, 250, 500, 1000

 

Results. All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test item was moderately toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced some slight mitotic inhibition at the 10mM concentration.

Conclusion. The test item was considered to be non-clastogenic to human lymphocytes in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phases of the study were performed between 25 August 2011 and 06 December 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of therelevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of GLP inspection: 19 - 21 July 2011 Date of signature on GLP form: 31 August 2011

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Type and identity of media:
RPMI 1640 (R0)

Properly maintained:
Yes

Periodically checked for Mycoplasma contamination:
Yes

Periodically checked for karyotype stability:
No

Periodically "cleansed" against high spontaneous background:
Yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and beta-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

The maximum dose level used in the 4-hour exposure groups in Experiment 1 and 2 was the 10 mM limit dose of 1000 µg/ml, and limited by toxicity in the 24-hour exposure group of Experiment 2.

Vehicle and positive controls were used in parallel with the test item. Solvent (R0 medium) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS), Sigma batches 100930580 and BCBC4573V at 400 µg/ml and 150 µg/ml for Experiment 1 and Experiment 2, respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch A0164185 at 2 µg/ml was used as the positive control in the presence of metabolic activation.

Vehicle / solvent:

Vehicle(s)/solvent(s) used:
Solvent (R0 medium) treatment groups were used as the vehicle controls.

Justification for choice of solvent/vehicle:
Formed a solution suitable for dosing at the required concentration.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (R0 medium) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (R0 medium) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

Introduction.

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances.

Methods.

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at six dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at eight dose levels using a 4 hour exposure group in the presence of metabolic activation (1% S9) and a 24 hour exposure group in the absence of metabolic activation.

The dose range of test item was selected following the results of a preliminary toxicity test and was 62.5 to 1000 µg/ml in both the absence and presence of metabolic activation for Experiment 1. In Experiment 2 the dose range was 12.5 to 300 µg/ml in the absence of metabolic activation, and 15.63 to 1000 µg/ml in the presence of metabolic activation.

Results.

The maximum dose level used in the 4-hour exposure groups in Experiment 1 and 2 was the 10 mM limit dose of 1000 µg/ml, and limited by toxicity in the 24-hour exposure group of Experiment 2. Precipitate of test item was not observed at any of the dose levels.

The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus.

The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

Evaluation criteria:

Please see "Any other information on materials and methods incl. tables" section.

Statistics:

Please see "Any other information on materials and methods incl. tables" section.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

non-mutagenic

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test

The dose range of the test item used in the preliminary toxicity test was 3.91 to 1000 µg/ml. In the 4-hour exposure group in the presence of metabolic activation there was evidence of a modest dose-related reduction in the Relative Suspension Growth (%RSG) of cells treated with the test item at the 10 mM limit dose when compared to the concurrent vehicle control. More marked dose-related reductions were observed in the 4-hour and 24-hour exposure groups in the absence of metabolic activation with the greatest reductions observed in the 24-hour exposure group. Precipitate of the test item was not observed at any of the dose levels. Based on %RSG values observed, the maximum dose levels in the subsequent Mutagenicity Test was the 10 mM limit dose level for the 4-hour exposure groups, and limited by toxicity in the 24-hour exposure group.

Mutagenicity Test

A summary of the results from the test is presented in attached Table 1.

Experiment 1

The results of the microtitre plate counts and their analysis are presented in attached Tables 2 to 7.

There was once again evidence of dose-related toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the RTG and %RSG values, with the most marked reductions observed in the absence of metabolic activation (Tables 3 and 6). Acceptable levels of toxicity were seen with both positive control substances (Tables 3 and 6).

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 3 and 6).

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 3 and 6). Precipitate of test item was not observed at any of the dose levels.

The numbers of small and large colonies and their analysis are presented in Tables 4 and 7.

Experiment 2

The results of the microtitre plate counts and their analysis are presented in attached Tables 8 to 13.

As was seen previously, there was evidence of dose-related toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the RTG and %RSG values, with the most marked reductions observed in the absence of metabolic activation (Tables 9 and 12). There was also evidence of significant dose related reductions in viability (%V) in the absence of metabolic activation, therefore indicating that residual toxicity had occurred in this exposure group. Based on the %RSG and / or RTG values observed, it was considered that optimum levels of toxicity had been achieved in the absence of metabolic activation. Acceptable levels of toxicity were seen with both positive control substances (Tables 9 and 12).

The 24-hour exposure group without metabolic activation demonstrated that the extended time point had a marked effect on the toxicity of the test item. It should also be noted that the lowering of the S9 concentration to 1% in this second experiment resulted in slightly greater levels of toxicity being observed when compared to 4-hour exposure groups in the presence of 2% metabolic activation in the Preliminary Toxicity Test and Experiment 1.

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 9 and 12).

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 9 and 12). Precipitate of test item was not observed at any of the dose levels.

The numbers of small and large colonies and their analysis are presented in Tables 10 and 13.

Remarks on result:

other: strain/cell type: Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Remarks:

Migrated from field 'Test system'.

Please see Attached "Tables 1 to 13"

Due to the nature and quantity of tables it was not possible to insert them in this section.

Conclusions:

Interpretation of results (migrated information):
other: Non-mutagenic

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non mutagenic under the conditions of the test.

Executive summary:

Introduction. 

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. Thethod was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances.

Methods. 

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at six dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at eight dose levels using a 4‑hour exposure group in the presence of metabolic activation (1% S9) and a 24‑hour exposure group in the absence of metabolic activation.

The dose range of test item was selected following the results of a preliminary toxicity test and was 62.5 to 1000 µg/ml in both the absence and presence of metabolic activation for Experiment 1. In Experiment 2 the dose range was 12.5 to 300 µg/ml in the absence of metabolic activation, and 15.63 to 1000 µg/ml in the presence of metabolic activation.

Results. 

The maximum dose level used in the 4-hour exposure groups in Experiment 1 and 2 was the 10 mM limit dose of 1000 µg/ml, and limited by toxicity in the 24-hour exposure group of Experiment 2. Precipitate of test item was not observed at any of the dose levels.

The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

Conclusion. 

The test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

No further details.

Additional information

in vitro studies:

Bacterial systems:

There is an Ames test available, which is a Klimisch 1 study carried out to GLP and OECD guideline 471. This study included four strains of S. typhimurium, TA1535, TA1537, TA98 and TA100 and E. Coli WP2 uvrA. The study was negative with and without metabolic activation (BASF, 1997).

 

An other Ames test (according to OECD 471) is available, including the test strain S. typhimurium TA100, TA1535, TA98, TA1537 and E.coliWP2uvrA. The test show a positive result in the test strain TA100 with and without metabolic activation. Cytotoxic effects were also seen in the corresponding concentrations (MHO Japan, 2009).

 

Mammalian cell gene mutation tests:

1-methyl piperazine was also negative in the in vitro Mouse Lymphoma L5178Y TK +/- assay. No toxicologically significant dose-related increases in the mutant frequency were seen at any dose level, either with or without metabolic activation (Harlan, 2012b). 

 

Mammalian cell cytogenetic tests:

1 -methyl piperazinge was also negative for clastogenicity in the human lymphocyte chromosomal aberration study, where it was found to be moderately toxic. In this study the the test substance did not induce any significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced some slight mitotic inhibition at the 10mM concentration. 1-methyl piperazine was non-clastogenic in human lymphocytes (Harlan, 2011).

 

All three aspects of in vitro genetic toxicity testing are available covering gene mutation in bacterial and mammalian cells and clastogenicity in human lymphocytes. In the absence of any adverse effects it is clear that 1-methyl piperazine is not genotoxic and there are no effects in vitro requiring clarifications with in vivo genetic toxicology testing.

 

in vivo studies:

No studies available.

Justification for classification or non-classification

There is an Ames, bacterial reverse mutation studies, a Mouse lymphoma L5178Y (TK+/-) mammalian cell mutation assay and an in vitro chromosomal aberration assay in human lymphocytes. All these assays are Klimisch 1 for validity and were conducted to GLP and OECD guidelines.

These assays showed no evidence of mutagenicity or clastogenicty for 1-methyl piperazine, CAS No 109-01-3, therefore there is no data to indicate any requirement for classification for genetic toxicity under the EU CLP (GHS) criteria.