Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro reliable, key studies have been identified: one bacterial reverse mutation test and one mammalian cell gene mutation test, performed with the test substance dimethyl piperazine and one chromosome aberration test performed with the analogue related substance N-methyl piperazine.
The bacterial reverse mutation assay was performed according to a method equivalent to OECD Guideline 471 in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA (Furusawa, 1996). According to the results of the study, the test substance is not mutagenic in the Ames test with and without metabolic activation.
The mammalian cell gene mutation test was performed according to a method similar to OECD Guideline 476 in mouse lymphoma L5178Y cells (Myhr, 1980). The results of the test indicate that, under the conditions of this study, the test substance is negative with and without metabolic activation.
No chromosome aberration test is available for the test substance but data was generated with the analogue source substance for read-across N-methylpiperazine. The test was performed with and without metabolic activation according to OECD Guideline 473, EU Method B.10 and the UK Department of Health Guidelines for Testing of chemicals for Mutagenicity (Lacey, 2011). According to the results of the study, N-methylpiperazine is not mutagenic in the chromosome aberration test with and without metabolic activation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996-01-19 - 1996-02-16
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Only two replicates per group were used, method for measuring toxicity was not specified, no historical control data provided, and the procedure (preincubation or plate incorporation) used for the preliminary reverse mutation test was not provided. However, all other parts of OECD guideline 471 were followed.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Only two replicates per group were used, method for measuring toxicity was not specified, no historical control data provided, and procedure (preincubation or plate incorporation) used for the preliminary reverse mutation test was not provided.
GLP compliance:
yes
Remarks:
Japanese Good Laboratory Practice
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): N,N'-dimethylpiperazine (DMP)
- Molecular weight (if other than submission substance): 114.19
- Physical state: colorless liquid
- Analytical purity: 99.0 % or more
- Impurities (identity and concentrations): water, 0.1 %
- Lot/batch No.: U3-1-1294
- Stability under test conditions: stable at room temperature; stability in solvent: stable in distilled water
- Storage condition of test material: at room temperature, shield under from light
Target gene:
Histidine locus (Salmonella strains) and tryptophan locus (E. coli strain)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: TA100 and TA1535 are base pair substitution detecting strains and TA98 and TA1537 are frameshift detecting strains
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: base pair substitution detecting strain
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and 5,6-benzoflavone induced rat liver S9
Test concentrations with justification for top dose:
313, 625, 1250, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: the test substance was soluble in water at concentrations of 100 g/L or more. From these results, distilled water was used as the solvent for the test substance.
Untreated negative controls:
yes
Remarks:
distilled water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide-used for strains TA100 and WP2 uvrA at 0.01 ug/plate and TA98 at 0.1 ug/plate
Remarks:
without activation
Untreated negative controls:
yes
Remarks:
distilled water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without activation used for strain TA1535 at 0.5 ug/plate
Untreated negative controls:
yes
Remarks:
distilled water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without activation used for strain TA1537 at 80 ug/ plate
Untreated negative controls:
yes
Remarks:
distilled water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene-used for strain TA1535 at 2 ug/plate and WP2 uvrA at 10 ug/plate
Remarks:
with activation
Untreated negative controls:
yes
Remarks:
distilled water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with activation used for strains TA100, TA98 and TA1537 at 5 ug/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: pre-incubation


DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48 hours
- Selection time (if incubation with a selection agent): 48 hours (simultaneous with exposure)


SELECTION AGENT (mutation assays): L-histidine (Salmonella strains), L-tryptophan (E.coli strain)


NUMBER OF REPLICATIONS: 2


DETERMINATION OF CYTOTOXICITY
- Method: cytotoxicity was determined but no information was given on how it was determined.


Evaluation criteria:
The number of revertant colonies at each dose was compared with that of the solvent control group for each bacterial strain with and without metabolic activation. The test substance was considered as positive when significant dose-related and more than two-fold increases in the number of revertant colonies were observed with sufficient reproducibility.
Statistics:
No statistical analysis was performed for evaluation of the test results.
Key result
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:
not applicable
Positive controls validity:
valid
Key result
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:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: A preliminary reverse mutation test was performed in each of the strains listed above with and without metabolic activation to determine the suitable dosage of the test substance to be applied in the reverse mutation test. The maximum dose level for the preliminary test was set at 5000 µg/plate and six dose levels (10, 50, 100, 500, 1000 and 5000 µg/plate) were employed.

Neither toxic effect nor more than two-fold increases in the number of revertant colonies were observed in any test substance treated groups with and without metabolic activation, while the dose related increase in the number of revertant colonies was observed in TA100 with and without metabolic activation. Considering these results, the maximum dose level for the reverse mutation test was set at 5000 µg/plate, and five different amounts of the test substance were prepared by serial dilution with a factor of 2 from the maximum dose.




Remarks on result:
other: strain/cell type: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Neither toxic nor more than two-fold increase in the number of revertant colonies were observed in any test substance treated groups with and without metabolic activation, while the dose related increase in the number of revertant colonies was observed in TA100 with and without metabolic activation. On the other hand, all of the positive controls produced more than two-fold increases in the number of the revertant colonies in comparison with that of the solvent control with all bacterial strains with and without metabolic activation. These results indicated that the test had been properly carried out.

Conclusions:
The test substance was evaluated for mutagenic potential in the reverse mutation test in the presence and absence of metabolic activation using S. typhimurium strains TA100, TA98, TA1535, TA1537 and E. coli strain WP2 uvrA. Based on the results of the assay, the test substance was considered to be negative under the test conditions employed up to a dose level of 5000 µg/plate. According to the criteria laid down in the CLP Regulation, the substance is considered not classified as mutagenic.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1980-09-08 - 1980-09-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): 4236-30-10
- Substance type: Clear, light-yellow liquid
- Physical state: liquid
Target gene:
thymidine kinase locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
* maintained in Fischer's mouse leukemia medium suplemented with L-glutamine, sodium pyruvate and horse serum (10% by volume).
* cloning medium: preceding growth medium with the addition of agar to a final concentration of 0.35% to achieve a semisolid state.
* selection medium: cloning medium containing 100 µg/ml of BrdU or 3 µg/ml of TFT.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9
Test concentrations with justification for top dose:
93.8, 188.0, 375.0, 750.0, 1500.0 nl/ml
Vehicle / solvent:
vehicle(s)/solvent(s) used: water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
0.5 µl/ml without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
Remarks:
0.3 µl/ml with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2 or 3 days
- Selection time (if incubation with a selection agent): 10 days


SELECTION AGENT (mutation assays): 5-bromo-2'deoxyuridine or 5-trifluorothymidine


NUMBER OF REPLICATIONS: 3 selection plates per test concentration


NUMBER OF CELLS EVALUATED: mutant frequency is calculated as the ratio of mutant colonies to viable colonies times 1E-04.


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:
The minimum condition for mutagenesis is a mutant frequency that exceeds 150% of the concurrent background frequency by at least 10 x 1E-06. The background frequency is defined as the average mutant frequency of the solvent and untreated negative controls. The observation of a mutant frequency that meets the minimum criterion for a single treated culture within a range of assayed concentrations is not sufficient evidence to evaluate a test material as a mutagen. The following test results must be obtained to reach this conclusion for either activation/nonactivation conditions:
- Dose-related or toxicity-related increase in mutant frequency: it is desirable to obtain this relation for at least 3 doses.
- Increase in mutant frequency may be followed by only small or no further increases at higher concentrations or toxicities. However, a decrease in mutant frequency to values below the minimum criterion is not acceptable in a single assay. If the mutagenic activity at lower concentrations or toxicities was large, a repeat assay will be performed to confirm the mutagenic activity.
- If an increase of about 2 times the minimum criterion or greater is observed for a single dose near the highest testable toxicity, the test material will be considered mutagenic. Smaller increases at a single dose near the highest testable toxicity will require confirmation by a repeat assay.
- A negative correlation with dose is acceptable only if a positive correlation with toxicity exists. An apparent increase in mutagenic activity as a function of decreasing toxicity is not acceptable evidence for mutagenicity.

A test material will be evaluated as nonmutagenic in a single assay only if the minimum increase in mutant frequency is not observed for a range of applied concentrations that extends to toxicity causing 5% to 10% relative suspension growth.
If a repeat assay does not confirm an earlier, minimal response, the test material will be evaluated as nonmutagenic in this assay system.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Range of toxicities from moderate at 93.8 and 188 nL/mL (approximately 50% relative growth) to high at 1500 nl/mL (13.9% relative growth). Treatment with 3000 nL/mL was completely lethal to the cells
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Range of toxicities: no observable effect at 188 nL/mL to moderate toxicity at 1500 nL/mL. Treatment with 3000 nL/mL: completely lethal. Reduction in toxicity in 93.8 to 1500 nL/mL concentration range: possibility of interaction with activation system
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
In the preliminary cytotoxicity test, the 24-hour cell growth was reduced by exposures to concentrations of test material up to 1250 nl/ml. Treatment with 2500 nl/ml was almost completely lethal. Therefore, the mutation assay was initiated with a series of concentrations from 3000 nl/ml to 5.86 nl/ml in two-fold dilution steps in an effort to cover a wide range of toxic action.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Concentrations up to 1500 nL/mL became very toxic without metabolic activation and moderately toxic with rat liver S9 metabolic activation.
Treatment with 3000 nL/mL was lethal under both conditions.
Remarks on result:
other: all strains/cell types tested
Conclusions:
The test material did not induce significant changes in the mutant frequency at the TK locus in L5178Y mouse lymphoma cells.
Concentrations up to 1500 nl/ml were assayed and became very toxic without activation and moderately toxic with rat liver S9 metabolic activation. Treatment with 3000 nl/ml was lethal under both conditions. The test material was therefore considered to be inactive in the Mouse Lymphoma Forward Mutation Assay. According to the criteria laid down in the CLP Regulation, the test substance is not to be classified.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2011-09-06 - 2011-12-13
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
A read-across strategy for chromosome aberration testing with the analogue substance 1-methylpiperazine was followed. Justification of this approach is included in section 13.
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: based on the results of 1-methylpiperazine
Remarks:
the test substance 1,4-dimethylpiperazine is not expected to exhibit in vitro toxicity
Conclusions:
No reliable chromosome aberration study with the test substance is available. Data generated with the analogue substance 1-methylpiperazine is used for endpoint coverage. This substance did not induce 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 seperate experiments. The analogue test item is therefore considered to be non-clastogenic to human lymphocytes in vitro. The same is assumed to be correct for the test substance.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-09-06 - 2011-12-13
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
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
Specific details on test material used for the study:
Sponsor's identification : N-methylpiperazine
Description : Clear colourless liquid
Chemical name : N-methylpiperazine
Purity : Min 99.9%
Batch number : 101124-11
Label : N-METHYLPIPERAZINE 101124 NMP F-5711
Date received : 19 August 2011
Expiry date : Not supplied
Storage conditions : Room temperature in the dark over silica gel
A copy of the Certificate of analysis is attached
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 exposure without S9 mix 0*, 31.25, 62.5, 125*, 250*, 500*, 1000*, MMC 0.4*
4(20)-hour exposure with S9 mix 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 exposure without S9 mix 0*, 62.5, 125, 250*, 375*, 500*, 1000*, MMC 0.2*
4(20)-hour exposure with S9 mix 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 (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.
Key result
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.


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:
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.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2006-11-1 - 2006-11-22
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Although sufficient strains and doses are tested in presence and absence of metabolic activation and positive control substances were valid, it is difficult to judge the reliability based on a report in Japanese. Therefore a klimisch score of 4 was assigned.
Qualifier:
according to guideline
Guideline:
other: ISHL methods= CSCL methods (no OECD method)
Deviations:
no
GLP compliance:
no
Remarks:
CSCL & MHLW
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): N,N'-dimethylpiperazine
- Analytical purity: 100 wt%
- Lot/batch No.: PFW050197
Target gene:
Histidine locus (Salmonella)
Tryptophan locus (E. coli)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
- S9 mix: 313, 625, 1250, 2500, 5000 µg/plate
+ S9 mix: 313, 625, 1250, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Remarks:
0.01 µg/plate for TA100 and WP2uvrA, without S9; 0.1 µg/plate for TA98 without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
0.5 µg/plate for TA1535 without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
1.0 µg/plate for TA1537 without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
5.0 µg/plate for TA100, TA98 and TA1537 with S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
2 µg/plate for TA1535 and 10 µg/plate for WP2uvrA with metabolic activation
Details on test system and experimental conditions:
No data
Evaluation criteria:
No data
Statistics:
No data
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:
not specified
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:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No increase in colonies equal to or more than twice negative control at all doses with and without S9.
Remarks on result:
other: all strains/cell types tested
Conclusions:
The substance was not considered to be mugatenic in the Ames test under the conditions of the current test with and without metabolic activation.
Endpoint:
in vitro transformation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1980-09-17 - 1980-11-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.21 (In Vitro Mammalian Cell Transformation Test)
Deviations:
no
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell transformation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): 4236-30-10
- Physical state: liquid
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:
0.01, 0.3, 3.0, 150.0, 300.0 nl/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: none (culture medium)
Untreated negative controls:
yes
Remarks:
culture medium
Negative solvent / vehicle controls:
other: not applicable
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
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 2-3 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 signicficantly 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.
Key result
Species / strain:
mammalian cell line, other: BALB/3T3 cells
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations of 1000 nl/ml and higher
Vehicle controls validity:
not applicable
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 nl/ml - min 0.061 nl/ml)(decreasing in two-fold dilution steps).
Each dose is applied to 3 culture dishes seeded 24h 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 highest dose chosen for subsequent transformation assays should normally cause no more than an 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, one transformed focus was observed among the 15 negative control flasks for an average of 0.07 foci/flask. Sets of 15 flasks containing one focus are included in the historical database, so the effect of the test material could be evaluated by comparison with the historical spontaneous transformation frequency.
Remarks on result:
other: all strains/cell types tested
Conclusions:
Concentrations of the test material from 0.01 nl/ml to 300 nl/ml were not detectably transforming 3T3 cells. The positive control results showed that the sensitivity of the assay was normal. The test material is considered to be inactive in the Balb/3T3 in vitro transformation assay.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No in vivo genetic toxicity study has been conducted with the test substance. However, no positive result was obtained in the in vitro genotoxicity tests with this substance. Therefore, it is not necessary to perform an in vivo genetic toxicity test (column 2 adaptation, Annex IX, section 8.4).

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic toxicity: in vitro

Three reliable in vitro studies were selected as key studies: a bacterial reverse mutation assay (Ames test), an in vitro mammalian cell gene mutation test and an in vitro chromosome aberration test.

Bacterial reverse mutation assay (Ames test):

The results of an Ames Assay with the test substance (conducted according to a method equivalent to OECD Guideline 471) were negative in strains TA1535, TA1537, TA98 and TA100 of Salmonella typhimurium and E. coli strain WP2 uvrA both with and without metabolic activation at following concentrations: 313, 625, 1250, 2500 and 5000 µg/plate. No cytotoxicity has been observed. The substance is considered not mutagenic.

 

In vitro mammalian cell gene mutation test:

In a study conducted according to a method equivalent to OECD Guideline 476, the test substance did not produce any statistically significant increases in the frequency of mutations of mouse lymphoma L5178Y cells at following concentrations: 93.8, 188.0, 375,0, 750.0 and 1500 nL/mL in tests with and without an S9 metabolic activation system (K1; Myhr, 1980). Concentrations up to 1500 nL/mL became very toxic without metabolic activation and moderately toxic with rat liver S9 metabolic activation. Treatment with 3000 nL/mL was lethal under both conditions. The test material was therefore considered to be inactive in the Mouse Lymphoma Forward Mutation Assay.

 

In vitro chromosome aberration test:

An in vitro chromosome aberration test with the read-across substance N-methylpiperazine was conducted according to a method equivalent to OECD Guideline 473, EU Method B.10 and UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity at following concentration:

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

 

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 metabolizing system in either of two separate experiments. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro. 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.

 

A reliable, supporting cell transformation assay was also identified. This in vitro mammalian cell transformation assay in BALB/3T3 cells has been performed according to a method equivalent to EU Method B.21. Concentrations of the test material from 0.01 nL/mL to 300 nL/mL were not detectably transforming 3T3 cells. The positive control results showed that the sensitivity of the assay was normal. The test material is considered to be inactive in the Balb/3T3 in vitro transformation assay.

 

Genetic toxicity: in vivo

No in vivo genetic toxicity study has been conducted with the test substance. However, no positive result was obtained in the in vitro genotoxicity tests with this substance. Therefore, it is not necessary to perform an in vivo genetic toxicity test (column 2 adaptation, Annex IX, section 8.4).




Justification for classification or non-classification

Based on the available data from the test substance and from the structural analogue substance, and according to the criteria of the CLP Regulation (EC) 1272/2008, the test substance is considered not to be classified for mutagenicity.