N-nitro-N-(3-methyl-3,6-dihydro-2H-1,3,5-oxadiazin-4-yl)amine

Genetic toxicity: in vitro

Administrative data

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:

February - April 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study; documentation sufficient for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5375 (In Vitro Mammalian Chromosome Aberration)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes

Remarks:

This study was performed in compliance with Good Laboratory Practice (GLP) in Switzerland, Procedures and Principles, March 1986, issued by the Federal Department of the Interior and the Intercantonal Office for the Control of Medicaments, Switzerland.

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

431-060-1

EC Name:

-

Cas Number:

153719-38-1

Molecular formula:

C4H8N4O3

IUPAC Name:

3-methyl-N-nitro-3,6-dihydro-2H-1,3,5-oxadiazin-4-amine

Details on test material:

- Name of test material (as cited in study report): CA 2343 A (Intermediate of CGA 293343)
- Physical state: fine, white powder
- Analytical purity: 96.7 %
- Batch No.: P.503005
- Stability under test conditions: stable
- Storage condition of test material: room temperature
- Reanalysis date: December 31, 1996

Method

Target gene:

ATCC (American Type Culture Collection) CCL 61 (ovary, Chiense hamster, CHO K1)

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Test organism: ATCC (American Type Culture Collection) CCL 61 (ovary, Chiense hamster, CHO K1)
- Type and identity of media: culture medium consisting of Nutrient Mixture F-12 supplemented with 10 % fetal calf serum + Penicillin/Streptomycin 100 units/ml/ 100 µg/ml (Gibco AG, Basle, Switzerland) in 75 cm² tissue-culture (plastic) flasks; the cultures were incubated at 37 °C in a humidified atmosphere containing 5 % CO2; the cells were passaged twice weekly
- duration of normal cell cycle: 12 - 13 hours
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

post mitochondrial supernatant (S9 fraction) from Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

Experiment without metabolic activation:
21 hours treatment time:
original experiment: 312.5, 625, and 1250.0 µg/ml
confirmatory experiment: 625, 937.5, and 1250.0 µg/ml

Experiment with metabolic activation:
3 hours treatment followed by 18 hours recovery period:
original experiment: 1250.0, 2500.0 and 5000 µg/ml
confirmatory experiment: 2500.0, 3750.0 and 5000 µg/ml

Vehicle / solvent:

Solvent used:
- culture medium without activation
- nutrient mixture F-12 (HAM) with activation

Controls

Controls 1

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Migrated to IUCLID6: (KYOWA KAKKO KOGYO CO. Ltd., Japan), 0.2 µg/ml

Controls 2

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Migrated to IUCLID6: (ASTA-Werke, Germany), 20.0 µg/ml

Controls 3

Untreated negative controls:

yes

Remarks:

vehicle

Remarks:

set in each experiment

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Preincubation period: 24 hours
- Exposure duration: Experiment without metabolic activation: 21 hours treatment time; Experiment with metabolic activation: 3 hours treatment followed by 18 hours recovery period



SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays): orcein


NUMBER OF INDEPENDENT EXPERIMENTS:
- three experiments without metabolic activation (twice 21 hours and once 45 hours)
- three trials with metabolic activationm (twice 21 hours and once 45 hours)


NUMBER OF REPLICATES:
- 2 cultures evaluated per concentration


NUMBER OF CELLS EVALUATED (per concentration):
- 200 (whenever possible; except positive controls)



DETERMINATION OF CYTOTOXICITY
- Method: mitotic index


OTHER EXAMINATIONS:
- Determination of polyploidy: yes;
- Determination of endoreplication: yes;


RAT-LIVER POST MITOCHONDRIAL SUPERNATANT:
- male RAI rats (Tif: RaIf[SPF]) treated with Aroclor 1254 5 days prior to sacrifice
- livers were homogenized with 3 volumes of 150 ml KCl
- homogenate was centrifuged at 9000x g for 15 min
- S9 protein content: 36.80 mg/ml
- concentrations in S9 mixture used: rat liver S9 fraction 150 µl/ml, NADP 31.4 µmol/ml, Isocitric acid (trisodium salt) 153 µmol/ml
- sterilasation: filtration through a 0.22 µm filter
- final S9 concentration during treatment: 1.5 %

Evaluation criteria:

Criteria for positive reponse:
- the percentage of metaphases containing specific aberrations in a treatment group is higher than 6.0 (based on historical negative control range) and differs statistically significant from the respective value of the negative control
- a concentration-related response should be demonstrable

Criteria for negative response:
- the percentage of metaphases containing specific aberrations in a treatment group is less than or equal 6.0 (based on historical negative control range) and does not differ statistically significant from the respective value of the negative control

Exceptions:
At the limits of the criteria for a positive or for a negative response or if the criteria for a positive response are only partially fulfilled or if effects are obtained at extremely high concentrations or in the toxic range of the test substance only, the Study Director will decide by experience about the interpretation of the results.

Assay acceptance criteria:
- The results of the experiments should not be influenced by a technical error, contamination or a recognized artefact
- The quality of the slide should allow, at least to a large extent, the chromosomes to be easily identifiable
- In the negative controls the percentage of metaphases showing specific chromosomal aberrations should be less than 6.0 (based on historical negative control range).
- The results of the positive control experiments should meet the criteria for a positive response.
- The highest concentration to which cells were exposed in the mutagenicity test should exert sufficient toxicity (suppression of miotic activity by 50 % or more), represent the limit of solubility of the test material, or be at least 5 mg/ml (or 10 mMol/l).

Statistics:

The evaluated numbers of specific aberrations were subjected to statistical analysis. In the preliminary tests the data were assessed for flask effects (dependence of cells within each culture) using chi-squared test. The nonsignificant result of this test means there is no substantial evidence to conclude a flask effect (although a flask effect still might exist). Accordingly, a chi-squared test for trend was performed modelling all cells in a given experiment as independent. That is, the individual cell is taken as the experimental unit. Consequently the power of the test is substantially increased, resulting in a rather safe judgement of the observed effects.

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

no statistically significant increase in the number of metaphases containing specific chromosomal aberrations was observed; the incidence of aberrant cells was within the historical control range at all doses assessed

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The highest conc. of 1250.0 µg/ml selected for chromosome analysis in the first experiment caused 21.9% suppression of mitotic activity. The next highest conc. of 2500.0 µg/ml could not be scored due to toxicity.

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

a statistically significant and concentration-dependant increase in the numbers of metaphases containing specific chromosomal aberrations was observed

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The highest concentration of 5000.0 µg/ml selected for chromosome analysis in the second experiment caused 38.62 % suppression of mitotic activity.

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 3

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

no statistically significant increase in the number of metaphases containing specific chromosomal aberrations was observed; the incidence of aberrant cells was within the historical control range at all doses assessed

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The highest conc. of 1250.0 µg/ml selected for chromosome analysis in the first experiment (confirmatory exp.) caused 2.68% suppression of mitotic activity. The next highest conc. of 1875.0 µg/ml could not be scor

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 4

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

a statistically significant and concentration-dependant increase in the numbers of metaphases containing specific chromosomal aberrations was observed

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The highest conc. of 1250.0 µg/ml selected for chromosome analysis in the second experiment (confirmatory experiment) caused 60.61 % suppression of mitotic activity.

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 5

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No chromosome aberrations were scored in the experiments of the confirmatory studies due to the clear clastogenic eff. observed in the experiments with metab. activ. using the short treatment time (3hrs/15 hrs recovery).

Cytotoxicity / choice of top concentrations:

other: clear clastogenic effects => no slides were selected for scoring

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ANALYTICAL RESULTS:
- concentration values found: 108.6 % and 105.6%
- the test substance is sufficiently stable in the vehicle


POSITIVE CONTROLS:
- the treatment of the cultures with mitomycin-C, 0.2 µg/ml and cyclophosphamide, 20.0 µg/ml, respectively, was followed by a high incidence of specific chromosomal aberrations in the experiments of the original study (60.0% and 64.0%) and in the experiments of the confirmatory study (64.0% and 68.0%)


COMPARISON WITH HISTORICAL CONTROL DATA: yes

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: 21 hours treatment (original study)

Any other information on results incl. tables

Original mutagenicity study:

-        21 hours treatment, without metabolic activation: 2.5 % metaphases with specific chromosomal aberrations were detected in the negative control; at the concentrations of 312.5 , 625.0 and 1250.0 µg/ml 3.5%, 0.5% and 3.0 % of cells with specific chromosomal aberrations were found

-        3 hours treatment / 18 hours recovery, with metabolic activation: 2.0 % metaphases with specific chromosomal aberrations were detected in the negative control; at the concentrations of 1250.0, 2500.0 and 5000.0 µg/ml 3.5%, 8.5% and 44 % of cells with specific chromosomal aberrations were found

 

Confirmatory mutagenicity study:

-        21 hours treatment, without metabolic activation: 1.5 % metaphases with specific chromosomal aberrations were detected in the negative control; at the concentrations of 625.0, 937.5 and 1250.0 µg/ml 2.5%, 0.5% and 1.5 % of cells with specific chromosomal aberrations were found

-        3 hours treatment / 18 hours recovery, with metabolic activation: 2.5 % metaphases with specific chromosomal aberrations were detected in the negative control; at the concentrations of 2500.0, 3750.0 and 5000.0 µg/ml 7.5%, 18.5% and 66 % of cells with specific chromosomal aberrations were found

 

In the original experiment with metabolic activation the numbers of cells containing specific chromosomal aberrations obtained at the concentrations of 2500.0 and 5000.0 µg/ml showed a statistically significant and concentration-dependent increase. In the confirmatory experiment with metabolic activation, a similar effect was registered at the concentrations of 2500, 3750 and 5000 µg/ml. In both experiments performed with metabolic activation, also increased frequencies of endoreduplications and polyploidy metaphases were observed at subtoxic concentrations.

 

Unspecific chromosomal aberrations in the form of chromatid gaps found in all experiments were within the frequency generally observed.

 

Applicant's summary and conclusion

Conclusions:

It is concluded that under the given experimental conditions evidence of clastogenic effects was obtained in Chinese hamster ovary cells in vitro treated with CA 2343 A (Intermediate of CGA293343) in the presence and absence of metabolic activation. No effect occurred in the absence of metabolic activation.

Executive summary:

CA 2343 A (Intermediate of CGA 293343), identified as a fine white powder, 96.7 % purity, batch no. P.503005, was investigated for clastogenic (chromosome-damaging) effects on Chinese Hamster ovary cells in vitro with and without metabolic activation (S9). The compound was dissolved in culture medium and tested at each of the following conditions:

 

Experiment without metabolic activation:

21 hours treatment time:

original experiment: 312.5, 625, and 1250.0 µg/ml

confirmatory experiment: 625, 937.5, and 1250.0 µg/ml

Final concentrations higher than 1250 µg/ml of culture medium could not be scored due to cytotoxicity. Mitomycin C (0.2 µg/ml) was used as a positive control in the 21 hours experiments.

 

Experiment with metabolic activation:

3 hours treatment followed by 18 hours recovery period:

original experiment: 1250.0, 2500.0 and 5000 µg/ml

confirmatory experiment: 2500.0, 3750.0 and 5000 µg/ml

Slight cytotoxicity was observed at the concentration of 5000 µg/ml. Cyclophosphamide (20.0 µg/ml) was used as a positive control in the 3 hours experiments.

 

In the two experiments performed without metabolic activation no statistically significant increase in the number of metaphases containing specific chromosomal aberrations was observed. The incidence of aberrant cells was within the historical control range at all doses assessed.

 

In the two experiments performed with metabolic activation a statistically significant and concentration-dependant increase in the numbers of metaphases containing specific chromosomal aberrations was observed.

 

No chromosome aberrations were scored in the experiments of the confirmatory studies (without and with metabolic activation, 45 hours treatment and 3 hours treatment/ 42 hours recovery) due to the clear clastogenic effects observed in the experiments with metabolic activation using the short treatment time (3 hours / 15 hours recovery).