Guanidine, N-phenyl-, compd. with carbonic acid, hydrate (2:1:2)

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:

April - October 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5375 (In Vitro Mammalian Chromosome Aberration)

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes

Remarks:

This study has been performed in compliance with GLP in Switzerland, Procedures and Principles, March 1986, issued by the Swiss federal Department of the Interior and the Intercantonal Office for the Control of Medicaments.

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

485-350-6

EC Name:

-

Cas Number:

405095-33-2

Molecular formula:

C15H20N6O3

IUPAC Name:

Carbonic acid - 1-phenylguanidine (1:2)

Test material form:

solid: bulk

Method

Target gene:

not applicable, examination of metaphase cells for the presence of structural chromosome aberrations

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Cell line: CHO CCL 61
- Type and identity of media: supplemented nutrient mixture F-12
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

Post mitochondrial fraction S9 from Aroclor 1254 induced rat liver.

Test concentrations with justification for top dose:

- Original study, first experiment, without metabolic activation: 8 concentrations, range: 25.78 - 3300 µg/ml
- Original study, second experiment, with metabolic activation: 8 concentrations, range: 25.78 - 3300 µg/ml
- Confirmatory study, first experiment, without metabolic activation: 5 concentrations, range: 25.78 - 412.5 µg/ml
- Confirmatory study, second experiment, with metabolic activation: 5 concentrations, range: 103.13 - 1650 µg/ml
- Confirmatory study, third experiment, without metabolic activation: 10 concentrations, range: 6.45 - 3300 µg/ml
- Confirmatory study, fourth experiment, with metabolic activation: 8 concentrations, range: 25.78 - 3300 µg/ml

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

two in each experiment: one supplemented with vehicle, one without any additions

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: ASTA-Werke, Germany, 20 µg/ml; with S9

Controls 2

Untreated negative controls:

yes

Remarks:

two in each experiment: one supplemented with vehicle, one without any additions

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Migrated to IUCLID6: KYOWA HAKKO KOGYO Ltd., Japan, 0.2 µg/ml; without S9

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

experiments without S9: cytotoxicity with final concentrations > 412.5 or 206.5 µg/ml of culture medium, cytotoxicity; experiments with S9: cytotoxicity with final concentrations > 1650 µg/ml of culture medium

Vehicle controls validity:

not applicable

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative

It is concluded that under the given experimental conditions no evidence of clastogenic effects was obtained in Chinese hamster ovary cells in vitro treated with CA 1139 A.

Executive summary:

CA 1139 A (Intermediate of CGA 219417), identified as a light beige crystalline powder, 76.2 % purity, batch no. P.201025, was investigated for clastogenic (chromosome-damaging) effects on Chinese Hamster ovary cells in vitro with and without extrinsic metabolic activation (S9). The compound was dissolved in culture medium (without metabolic activation) or in nutrient mixture F-12 (with metabolic activation) and tested at each of the following conditions:

 

Experiment without metabolic activation:

- 18 hours treatment time:

original experiment: 103.13, 206.25 and 412.5 µg/ml

confirmatory experiment: 103.13, 206.25 and 412.5 µg/ml

- 42 hours incubation time: 51.56, 103.13 and 206.65 µg/ml

Final concentrations higher than 412.5 or 206.65 µg/ml of culture medium could not be scored due to cytotoxicity. Cytotoxicity was observed at concentrations of 412.5 µg/ml and higher. Mitomycin C (0.2 µg/ml) was used as a positive control in the 18 hours experiments.

 

Experiment with metabolic activation:

- 3 hours treatment followed by 15 hours recovery period:

original experiment: 412.5, 825 and 1650 µg/ml;

confirmatory experiment: 412.5, 825 and 1650 µg/ml.

- 3 hours incubation followed by 39 hours recovery period: 825, 1650 and 3300 µg/ml.

Final concentrations greater than 3300 µg/ml of culture medium could not be achieved due to solubility limitations. In the 3h/15h experiments, concentrations greater than 1650 µg/ml of culture medium could not be scored due to cytotoxicity. Cyclophosphamide (20.0 µg/ml) was used as a positive control in the 3h/15h experiments.

 

In both the experiments performed according to OECD Guideline 473 without and with metabolic activation CA 1139 A did not induce a biologically significant increase in the number of metaphases containing specific chromosomal aberrations.