Reaction mass of Cobaltate(1-), bis[6-(amino-κN)-5-[2-[2-(hydroxy-κO)-4-nitrophenyl]diazenyl-κN1]-N-methyl-2-naphthalenesulfonamidato(2-)]-, sodium (1:1) and disodium [6-amino-5-[(2-hydroxy-4-nitrophenyl)azo]-N-methylnaphthalene-2-sulphonamidato(2-)][6-amino-5-[(2-hydroxy-4-nitrophenyl)azo]naphthalene-2-sulphonato(3-)]cobaltate(2-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

FAT 20036/F was found to induce gene mutations in bacterial cells when tested in vitro, however it failed to produce the mutations in the mammalian cells in vitro. Further it did not produce clastogenic effects in the in vitro chromosomal aberration assay. Hence, it can be concluded that FAT 20036/F is neither mutagenic nor clastogenic, hence not genotoxic.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 8 May 1995 to 19 October 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA 52 FR 19072; 798.5300

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC Directive 87/302

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

Identification: IRGALAN BLAU 3GL ROH FEUCHT (FAT 20'036/F)

Batch Number: 1

Purity / Formulation: approx. 97.7%

Expiration date: 07/99

Storage Conditions: at room temperature.

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: The cells were cultured in Ham's F10 medium supplemented with 10% pre-tested foetal calf serum, 100 U/ml penicillin
and 100 microg/ml streptomycin in tissue culture (plastic) flasks.
- 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:

Cytotoxicity test with metabolic activation: 500, 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.9063, 1.9531, 0.9766, 0.4883, 0.2441 microgr/ml
Cytotoxicity test without metabolic activation: 500, 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.9063, 1.9531, 0.9766, 0.4883, 0.2441 microgr/ml

Mutagenic experiment:
Range with metabolic activation: 500, 166.6667, 55.5556, 18.5185 microg/ml
Range without metabolic activation: 100, 33.3333, 11.1111, 3.7037 microg/ml

Confirmatory experiment:
Range with metabolic activation: 500, 250,125, 62.5 microg/ml
Range without metabolic activation: 100, 50, 25, 12.5 microg/ml

Vehicle / solvent:

-Vehicle (s)/solvent(s) used: DMSO

- Solubilisation of the test substance:

The test item was insoluble in all common vehicles it had to be applied as a suspension.

FAT 20036/F was suspended in DMSO at slightly elevated temperature (40 °C). The highest suitable concentration of FAT 20036/F was determined to be 50.0 mg/ml suspended in DMSO. Lower concentrations of the test substance were obtained by appropriate dilution of the stock solution with DMSO. The respective solutions were added 1:100 to the cell culture medium. The final concentration of DMSO in the culture medium was 1%. The test substance suspensions were prepared immediately before the start of the test.

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

used at 1.0 microl/ml

Positive control substance:

other: N-Nitrosodimethylamine

Remarks:

with metabolic activation

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

used at 0. microl/ml

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Details on test system and experimental conditions:

TREATMENT TIME:
4 hours (Experiment I with and without metabolic activation, experiment II with metabolic activation)
20 hours (Experiment II without metabolic activation)

FIXATION INTERVAL: 20 hours (Experiment I and II with and without metabolic activation)
NUMBER OF REPLICATIONS: 2 independent experiments
NUMBER OF CELLS SEEDED: 1 x 10^4 - 5 x 10^4 cells
NUMBER OF CULTURES: two cultures per concentration
NUMBER OF CELLS SCORED: 200 cells per concentration (100 cells per culture)
DETERMINATION OF CYTOTOXICITY: Mitotic index, cell count

Medium durig treatment: During the treatment period cells were maintained in growth medium in which the foetal calf serum was reduced to 3%. Antibiotics were omitted.

Selection medium: The selection medium was growth medium to which 6- thioguanine (6-TG) was added to a final concentration of 8 microg/ml.

Evaluation criteria:

All mutant frequencies are normalized to a virtual cloning efficiency of 100% at the end of the expression period. If the cloning efficiency of the viability cultures is lower than 15 %, the corresponding mutant frequency is usually not calculated, owing to the high statistical insignificance of the result. For every concentration a mean mutant factor, which is defined as the ratio of the mean mutant frequencies of the treated cultures with the mean mutant frequencies of the solvent control cultures will be calculated.

Criteria for a positive response:
The test substance will be considered to be mutagenic if:
• The assay is valid (see assay acceptance criteria)
• The mutant frequency at one or more concentrations is significantly greater than that of the negative control and the number of normalized mutant clones in the treated and untreated cultures differs by more than 20.
• There is a significant dose-relationship as indicated by the linear trend analysis.
• The effects described above are reproducible.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Toxicity

In the part with metabolic activation the highest concentration produced an acute growth inhibition of 70.78%. In the part without metabolic activation FAT 20036/F exerted a complete growth inhibitory effect down to the concentration of 250.0 ug/ml. The next lower concentration revealed an acute growth inhibitory effect of 97.44 %.

Accordingly, four concentrations were selected for the original experiment ranging from 18.52 to 500.0 ug/ml and from 3.71 to 100.0 ug/ml in the presence and absence of metabolic activation, respectively.

In the part with metabolic activation, the growth inhibition determined after treatment at the highest concentration of 500.0 ug/ml showed a mean value of 68.46 %. After expression this concentration revealed a mean inhibitory value of 4.33 %.

In the absence of metabolic activation the mean growth inhibitory effect determined after treatment was 90.87 % at the highest concentration. After the expression period the determined cytotoxicity revealed a mean value of 25.76 %.

In the confirmatory experiment with metabolic activation concentrations ranging from 62.5 to 500.0 ug/ml were used. In the part without metabolic activation a range of 12.5 to 100.0 ug/ml was selected. In the presence of metabolic activation the mean acute cytotoxicity at the highest concentration was 78.0%. After expression there was again only a weak cytotoxicity (9.94 %). In the part without activation, the mean growth inhibition at the highest concentration was 78.23% and after the expression period the value of 29.94% was observed.

Mutagenicity

In the presence and absence of metabolic activation, no significant increase in mutant frequency was observed at any concentration level of FAT 20036/F tested in the original or the confirmatory experiment in comparison with the negative control.

The positive controls induced a clear increase in mutant frequency.

Conclusions:

FAT 20036/F and its metabolites did not show any mutagenic activity in this forward mutation system.

Executive summary:

FAT 20036/F, identified as black crystalls, ca. 97.7% purity, batch no. 1 was tested for mutagenic effects on V79 Chinese hamster cells in vitro. The test substance was dissolved in DMSO. The cells were treated in the experiments with metabolic activation for 5 hours and in the experiments without metabolic activation for 21 hours. The results of each experiment were confirmed in a second and independent experiment (confirmatory experiment).

Cytotoxicity test

A preliminary range finding test was run assessing cytotoxicity. FAT 20036/F was tested at concentrations up to 500 ug/ml. Higher concentrations could not be applied due to solubility limitations in the culture vehicle. In the part with metabolic activation, at the highest concentration of 500 microg/ml an acute growth inhibiting effect of 70.78 % could be seen, while the next lower concentration inhibited 30.56%. Without metabolic activation treatment with FAT 20036/F proved fully growth inhibiting down to the concentration of 250.0 microg/ml. The next lower concentration revealed an acute inhibition of growth of 97.44 %. Accordingly, 500.0 microg/ml with and 100.0 microg/ml without metabolic activation were chosen as highest concentrations for the first mutagenicity assay.

Mutagenicity test with metabolic activation

The original experiment was performed at the following concentrations: 18.52, 55.56, 166.67 and 500.0 microg/ml. 500.0 microg/ml is above the solubility limit of the test chemical and represents the highest concentration which could be applied in the test. The mean growth inhibiting values found at the highest concentration after treatment and expression were 68.46%* and 4.33%* respectively. In the confirmatory experiment the concentrations applied were 62.5, 125.0, 250.0 and 500.0 microg/ml. The highest concentration revealed a mean acute growth inhibition of 78 0%. The mean growth inhibitory effect after the expression period was 9.94 %*. N-Nitrosodimethylamine (DMN, 1.0 ul/ml) was used as positive control.

In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-Thioguanine (6-TG).

Mutagenicity test without metabolic activation

The original experiment was performed at the following concentrations: 3.70, 11.11, 33.33 and 100.0 microg/ml. The mean growth inhibition values found at the highest concentration after treatment and expression were 90.87 %* and 25.76 %* respectively. In the confirmatory experiment the concentrations applied were 12.5, 25.0, 50.0 and 100.0 microg/ml. The highest concentration revealed a mean acute growth inhibitory effect of 78.23 %*. The mean growth inhibition after the expression period was 29.94 %*. Ethylmethansulfonate (EMS, 0.3 ul/ml) was used as positive control.

In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-TG.

Based on the results of two independently performed experiments and under the given experimental conditions, it is concluded that FAT 20036/F and its metabolites did not show any mutagenic activity in this forward mutation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Several mutagenicity studies have been conducted with FAT 20036/F in in-vitro test systems which all turned out to be negative excepted for the reverse mutation assay on S. typhimurium which is positive.

Bacterial reverse mutation assay:

This study was performed to investigate the potential of FAT 20036/F; Irgalan Blau 3GL roh feucht to induce gene mutations according to the plate incorporation test (OECD test guideline 471) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100 with and without liver microsomal activation. The test article was tested at the following concentrations: 33.3, 100, 333.3, 1000, 2500 and 5000 microg/plate (active ingredient). Toxic effects, evidenced by a reduction in the number of revertants, occurred at the higher concentrations in the strains TA1535 with S9 mix at 2500 and 5000 microg/plate in experiment I and at 5000 microg/plate without S9 mix in experiment II. The plates incubated with the test article showed normal background growth up to 5000 microg/plate with and without S9 mix in all strains used. Dose-dependent increases in revertant colony numbers were observed following treatment with FAT 20'036/F in the strains TA 1537, TA 98 with and without S9 mix in experiment I and II and in strain TA 100 without S9 mix in experiment I. However the increase with TA 100 witghout S9 mix could not be reproduced in the independent experiment.

Based on the above findings, it can be concluded that under the experimental conditions reported, the test article induced gene mutations by frameshifts in the genome of the strains TA 1537 and TA 98. Therefore, FAT 20'036/F is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

In vitro mammalian cell gene mutation assay:

FAT 20036/F, identified was tested for mutagenic effects on V79 Chinese hamster cells in vitro. The test substance was dissolved in DMSO. The cells were treated in the experiments with metabolic activation for 5 hours and in the experiments without metabolic activation for 21 hours. The results of each experiment were confirmed in a second and independent experiment (confirmatory experiment).

Mutagenicity test with metabolic activation

The original experiment was performed at the following concentrations: 18.52, 55.56, 166.67 and 500.0 microg/ml. 500.0 microg/ml is above the solubility limit of the test chemical and represents the highest concentration which could be applied in the test. The mean growth inhibiting values found at the highest concentration after treatment and expression were 68.46%* and 4.33%* respectively. In the confirmatory experiment the concentrations applied were 62.5, 125.0, 250.0 and 500.0 microg/ml. The highest concentration revealed a mean acute growth inhibition of 78 0%. The mean growth inhibitory effect after the expression period was 9.94%*. N-Nitrosodimethylamine (DMN, 1.0 ul/ml) was used as positive control.

In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-Thioguanine (6-TG).

Mutagenicity test without metabolic activation

The original experiment was performed at the following concentrations: 3.70, 11.11, 33.33 and 100.0 microg/ml. The mean growth inhibition values found at the highest concentration after treatment and expression were 90.87%* and 25.76%* respectively. In the confirmatory experiment the concentrations applied were 12.5, 25.0, 50.0 and 100.0 microg/ml. The highest concentration revealed a mean acute growth inhibitory effect of 78.23%*. The mean growth inhibition after the expression period was 29.94%*. Ethylmethansulfonate (EMS, 0.3 ul/ml) was used as positive control.

In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-TG.

Based on the results of two independently performed experiments and under the given experimental conditions, it is concluded that FAT 20036/F and its metabolites did not show any mutagenic activity in this forward mutation system.

In vitro chromosomal aberration assay:

FAT 20036/F was assessed for its potential to induce structural chromosome aberrations in the V79 cell line of the Chinese hamster according to the OECD Guideline 473 and EU Method B.10. In the absence and the presence of S9 mix the highest possible test article concentrations were evaluated. Per culture 100 metaphase plates were scored for structural chromosome aberrations. The highest concentration used in a pre-test on toxicity (5000 ug/ml) was chosen with regard to the current OECD-Guideline for in vitro mammalian cytogenetic tests. Dose selection of the cytogenetic experiments was performed considering the toxicity data of the pre-test.

In the main experiment, reduced cell numbers below 50 % of control were observed in the absence of S9 mix after continuous treatment and in the presence of S9 mix after 4 h treatment. No reduction of the mitotic indices was observed. Neither a significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test article. No increase in the frequencies of polyploid metaphases was found after treatment with the test article as compared to the frequencies of the controls.

Based on the above findings, it was concluded that the test article did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, FAT 20036/F is considered to be non clastogenic in this chromosome aberration test.

Conclusion on genotoxicity:

FAT 20036/F was found to induce gene mutations in bacterial cells when tested in vitro, however it failed to produce the mutations in the mammalian cells in vitro. Further it did not produce clastogenic effects in the in vitro chromosomal aberration assay. Hence, it can be concluded that FAT 20036/F is neither mutagenic nor clastogenic, hence not genotoxic.

Justification for classification or non-classification

Based on the above mentioned results, FAT 20036/F does not need to be classified according to CLP regulation (Regulation EC No. 1272/2008) and DSD (Directive 67/548/EEC).