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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of the experimentsperformed according to the OECD guideline 471and on standard evaluation criteria, it is concluded that the test material and its metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli used.

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

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5375 (In Vitro Mammalian Chromosome Aberration)

Qualifier:

according to guideline

Guideline:

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

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

Target gene:

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

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

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

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

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

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.

 

Reference 2

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:

June - August 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

other: MHW Japan 1984

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Qualifier:

according to guideline

Guideline:

other: EPA U.S.A. 1987

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:

bacterial reverse mutation assay

Target gene:

- base-pair substitution: S. typhimurium TA 100, TA 1535 and E. coli WP 2 uvrA
- frame-shift: S. typhimurium TA 98, TA 1537

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

other: histidine-requiring

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

other: tryptophan-requiring

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:

- Range in the cytotoxicity test: 20.6 - 5000 µg/plate
- Range in the mutagenicity test:
* without metabolic activation: 312.5 - 5000 g/plate
* with metabolic activation: 312.5 - 5000 g/plate

Untreated negative controls:

yes

Remarks:

solvent dimethylsulfoxide

Negative solvent / vehicle controls:

other: sovent /vehicle dimethylsulfoxide is negative control

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: experiment with metabolic activation: sodium azide, 4-nitroquinoline-N-oxide, 2-nitrofluorene, 9(5)-aminoacridine; experiments without metabolic activation: 2-aminoanthracene, cyclophosphamide*H2O

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:

not specified

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Conclusions:

Interpretation of results:
negative with and without metabolic activation

Based on the results of these experiments and on standard evaluation criteria, it is concluded that CA 1139 A and its metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli used.

Executive summary:

Salmonella and Escherichia / Liver-Microsome Test on the Test Substance CA 1139 A.

The test was performed according to the OECD guideline 471.

 

The concentration range of the test material to be tested in the mutagenicity test was determined in a preliminary toxicity test. Thus, the test material was tested for mutagenic effects without metabolic activation at five concentrations in the range of 312.5 - 5000 µg/plate. In the experiment carried out with metabolic activation five concentrations in the range of 312.5 - 5000 µg/plate were tested. In order to confirm the results, the experiments were repeated with the same concentration ranges.

 

Mutagenicity test, original experiment

In the original experiment performed without and with metabolic activation, none of the tested concentrations of the test material led to an increase in the incidence of either histidine- or tryptophan-prototrophic mutants by comparison with the negative control.

 

Mutagenicity test, confirmatory experiment

In the confirmatory experiment performed without and with metabolic activation, again, the tested concentrations of the test material did not lead to an increase in the incidence of either histidine- or tryptophan-prototrophic mutants by comparison with the negative control.

 

Based on the results of the experiments performed according to the OECD guideline 471 and on standard evaluation criteria, it is concluded that the test material and its metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli used.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the results of the experimentsperformed according to the OECD guideline 471and on standard evaluation criteria, it is concluded that the test material and its metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli used.

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

Based on the studies no classification is necessary.