Amitrole

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24-29 June 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well conducted and well described study in accordance with GLP and OECD G9uideline 473 without any deviation

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

dated 21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated 30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

dated August 1998

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro cytogenicity / chromosome aberration study in mammalian cells

Test material

Test material information

Constituent 1

Reference substance name:

Amitrole

EC Number:

200-521-5

EC Name:

Amitrole

Cas Number:

61-82-5

Molecular formula:

C2H4N4

IUPAC Name:

amitrole

Test material form:

solid: crystalline

Remarks:

colourless

Details on test material:

- Manufacturing date: 04 February 2008
- Expiration date: 04 February 2010
- Purity test date: 02 July 2009
- Storage condition of test material: Room temperature

Specific details on test material used for the study:

Name as cited in the report: Amitrole Technical

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

lymphocytes: cultures prepared from the pooled blood of a single donor

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction from Wistar rats induced with Phenobarbital (80 mg/kg bw) and ß-Naphthoflavone (100 mg/kg bw) for 3 days by oral route

Test concentrations with justification for top dose:

Pre-experiment: 0, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2500 and 5000 µg/mL, with and without metabolic activation
Main experiments:
- Experiment 1: with and without metabolic activation, 4 h treatment, 24 h preparation interval: 125, 250, 500, 1000, 2500 and 5000 µg/mL
- Experiment 2:
without metabolic activation, 24 h treatment, 24 h preparation interval: 31.3, 62.5, 125, 250, 500, 1000, 2500 and 5000 µg/mL;
with metabolic activation, 4 h treatment, 24 h preparation interval: 500, 1000, 2000, 3000, 4000 and 5000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: The test item was dissolved and diluted in cell culture medium (RPMI 1640) prior to treatment.
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the cells and the S9 activity.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in RPMI 1640 medium

DURATION
- Exposure duration: 4 or 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid, 0.2 µg/mL

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: At least 2 h before harvesting, Colcemid was added to the cultures (final concentration 0.2 µg/mL). The cultures were harvested by centrifugation 24 h after beginning of treatment. The supernatant was discarded and the cells were resuspended in approximately 5 mL hypotonic solution (0.4% KCl). The cell suspension was allowed to stand at 37°C for 20 minutes. After removal of the hypotonic solution by centrifugation the cells were fixed with 3+1 methanol +glacial acetic acid. The fixation procedure was repeated twice. Slides were prepared by dropping the cell suspension onto a clean microscopic slide. The cells were stained with Giemsa and according to the Fluorescent plus Giemsa technique, respectively.

NUMBER OF CELLS EVALUATED: Per culture 100 metaphases were scored for structural chromosomal aberrations.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): At least, 200 well spread metaphases per concentration and control were scored for cytogenetic damage. Metaphases with 46 ± 2 centromere regions were included in the analysis.

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index
- Any supplementary information relevant to cytotoxicity: To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.

OTHER:
- Proliferation Index: The BrdU-technique was used to detect a possible cell cycle delay after treatment with the test item.
- Additionally the number of polyploid cells was scored.

Rationale for test conditions:

The test item could be dissolved and diluted in cell culture medium at a concentration of 5000 µg/mL, and no precipitation of the test item was indicated. Therefore, the highest recommended dose of 5000 µg/mL was used in the pre-experiment. The concentrations evaluated in the main experiment were based on the results obtained in the pre-experiment.

Evaluation criteria:

Criteria for determining a positive result are the following:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (up to 4.0% aberrant cells).
According to the OECD guidelines, the biological relevance of the results will be the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary. However, for the interpretation of the data both, biological and if evaluated, statistical significance should be considered together.
A test item is considered to be negative if there is no biologically relevant increase in the percentages of aberrant cells above concurrent control levels, at any dose group.

Statistics:

None

Results and discussion

Test results

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was noted with and without metabolic activation in experiments 1 and 2.
- Toxicity: In experiment 1 with and without metabolic activation, no biologically relevant decrease of the relative mitotic index (decrease below 70% rel. mitotic index) was noted in all dose groups evaluated. In experiment 2 without metabolic activation, a decrease of the relative mitotic index was observed at a concentration of 2500 µg/mL down to 44%. With metabolic activation no toxic effects of the test item were noted at the concentrations evaluated.
- Proliferation Index: In the experiment 1, the values of the proliferation index of the negative controls were 1.70 (without metabolic activation) and 1.25 (with metabolic activation). The proliferation index of the highest dose groups evaluated were 1.52 (5000 µg/mL, without metabolic activation) and 1.11 (5000 µg/mL, with metabolic activation). In the experiment 2, the values of the proliferation index of the negative controls were 1.62 (without metabolic activation) and 1.21 (with metabolic activation). The proliferation index of the highest dose groups evaluated without metabolic activation (1000 and 2500 µg/mL) were 1.63 and 1.52 respectively, with metabolic activation the proliferation index of the highest dose group evaluated (5000 µg/mL) was 1.22. No biologically relevant decrease of the proliferation index was indicated.

PRELIMINARY EXPERIMENT: No cytotoxicity or precipitation was seen up to the highest tested dose of 5000 µg/mL, with and without metabolic activation.

MAIN EXPERIMENTS:
- In experiment 1 without metabolic activation the aberration rates of the negative control (1.0%) and all dose groups treated with the test item [2.0%, (1000 µg/mL), 1.0% (2500 µg/mL) and 0.5% (5000 µg/mL)] were within the historical control data of the testing facility (0.0% - 4.0%). With metabolic activation the aberration rates of the negative control (1.0%) and all dose groups treated with the test item [0.0% (1000 µg/mL) and 1.5% (2500 and 5000 µg/mL)] were within the historical control data of the testing facility (0.0%- 4.0%). The number of aberrant cells found in the dose groups treated with the test item did not show a biologically relevant increase compared to the corresponding negative control. In addition, no dose-response relationship was observed.
- In experiment 2 without metabolic activation the aberration rates of the negative control (0.5%) and all dose groups treated with the test item [1.0% (500 µg/mL), 1.5% (1000 µg/mL) and 2.0% (2500 µg/mL] were within the historical control data of the testing facility (0.0% - 4.0%). With metabolic activation the aberration rates of the negative control (1.0%) and all dose groups treated with the test item [1.5% (3000 and 4000 µg/mL) and 3.0% (5000 µg/mL)] were within the historical control data of the testing facility (0.0% - 4.0%). The number of aberrant cells found in the dose groups treated with the test item did not show a biologically relevant increase compared to the corresponding negative control. In addition, no dose-response relationship was observed.
- No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item.
- The positive controls EMS and CPA induced distinct and biologically relevant increases in cells with structural chromosomal aberrations.
- Refer Tables 7.6.1/1 and 7.6.1/2 for more details.

COMPARISON WITH HISTORICAL CONTROL DATA: Proportion of cells with structural aberrations in negative control cultures fell within historical control (normal) ranges.

Any other information on results incl. tables

Table 7.6.1/1: Experiment 1: Summary of aberration results

Dose group	Mean % aberrant cells
	Incl. gaps	Excl. gaps
Without metabolic activation, 4 h treatment time, 24 h fixation interval
Negative control	3	1
1000 µg/mL	2	2
2500 µg/mL	1	1
5000 µg/mL	0.5	0.5
Positive control (ethylmethanesulphonate)	20	17
With metabolic activation, 4 h treatment time, 24 h fixation interval
Negative control	4.5	1
1000 µg/mL	1	0
2500 µg/mL	3	1.5
5000 µg/mL	3.5	1.5
Positive control (cyclophosphamide)	12.5	11

 

Table 7.6.1/2: Experiment 2: Summary of aberration results

Dose group	Mean % aberrant cells
	Incl. gaps	Excl. gaps
Without metabolic activation, 24 h treatment time, 24 h fixation interval
Negative control	1	0.5
500 µg/mL	3.5	1
1000 µg/mL	5.5	1.5
2500 µg/mL	4.5	2
Positive control (ethylmethanesulphonate)	24	17
With metabolic activation, 4 h treatment time, 24 h fixation interval
Negative control	4	1
3000 µg/mL	2	1.5
4000 µg/mL	3.5	1.5
5000 µg/mL	5	3
Positive control (cyclophosphamide)	24.5	18.5

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the test item did not induce structural chromosomal aberrations in human lymphocyte cells, and was not considered as clastogenic in human lymphocytes.

Executive summary:

In an in vitro chromosome aberration test performed according to OECD guideline 473 and in compliance with GLP, human primary lymphocyte cultures were exposed to the test item in the RPMI 1640 medium at concentration range of 7.8-5000 µg/mL without and with metabolic activation (S9 liver microsomal fraction of Phenobarbital and ß-Naphthoflavone induced Wistar rats) for a preliminary experiment. In the main test, two experiments were performed at concentrations up to 5000 µg/mL without and with S-9 and the following concentrations were selected for microscopic analysis:

- Experiment 1: with and without metabolic activation, 4 h treatment, 24 h preparation interval: 1000, 2500 and 5000 µg/mL

- Experiment 2: without metabolic activation, 24 h treatment, 24 h preparation interval: 500, 1000, and 2500 µg/mL; with metabolic activation, 4 h treatment, 24 h preparation interval: 3000, 4000 and 5000 µg/mL

No precipitation of the test item was noted with and without metabolic activation in experiments 1 and 2. Toxic effect of the test item was observed only in experiment 2 without metabolic activation (long time exposure) at a concentration of 2500 µg/mL. In both experiments, no biologically relevant increase of the aberration rates or in the frequency of polyploid cells was noted after treatment with the test item with and without metabolic activation as compared to the controls. The positive controls ethylmethanesulphonate and cyclophosphamide induced distinct and biologically relevant increases in cells with structural chromosomal aberrations.

Under the test conditions, the test item did not induce structural chromosomal aberrations in human lymphocyte cells, and was not considered as clastogenic in human lymphocytes.