Amines, rosin, compds. with 9-(2-carboxyphenyl)-3,6-bis(diethylamino)xanthylium chloride and disodium hydrogen bis[4-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-3-hydroxy-1-naphthalenesulfonato(3-)]chromate(3-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In two independently performed Ames tests, the test item led to a slight increase in the number of revertant for two (TA 98 and TA 1538) of the five tester strains in the absence of metabolic activation only. Thus two further genotoxicity tests were conducted. The test item was not mutagenic in an in vitro mammalian cell gene mutation test (HPRT) performed with 79V cells. Also no clastogenicity was observed in an in vitro mammalian bone marrow chromosome aberration test with human lymphocytes.

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:

1985-11-12 to 1986-04-21

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

- Purity: Commercial grade

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Hams's F10 tissue culture medium plus 3 % FCS
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (aroclor1254 induced)

Test concentrations with justification for top dose:

Preliminary cytotoxixity test:
31 ng/mL to 2000 mg/mL

Main mutagenicity test
without metabolic activation: 0.5, 1, 2, 4, 8, 12, 16 and 20 µg/mL
with metabolic activation: 12.5, 25, 50, 100, 200, 300, 400 and 500 µg/mL

Vehicle / solvent:

The test item was dissolved in DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without metabolic activation: ethylmethanesulphonate; with metabolic activation: N-dimethylnitrosamine

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 h (with and without S9 mix)
- Expression time (cells in growth medium): 6 days
- Selection time (if incubation with a selection agent): 7 days



SELECTION AGENT: 6-thioguanine (6 TG) and 8-azaguanine (8 TG)
STAIN: Giemsa

NUMBER OF REPLICATIONS: 2
NUMBER OF EXPERIMENTS: 1

DETERMINATION OF CYTOTOXICITY
- Method: survivng colonies determined with the aid of an electric colony counter

Evaluation criteria:

A test substance is considered mutagenic in this test system, if the analysis of the data demonstrates either:
- a dose-dependent increase in the mutant frequency and
an increase in the highest mutant frequency with respect to the negative control by a factor of at least 2.5;
or
- an increase in any mutant frequency by a factor of 3.0 or more with respect to the negative control at any concentration tested and an absolute difference of at least 20 clones per 10 cells plated between the negative control and substance treated dishes.

Statistics:

All mutant frequencies are normalized to a virtual cloning efficiency of 100% at the end of the expression period. If the cloning efficiency is lower than 15%, the corresponding mutant frequency is usually not calculated, owing to the high statistical insignificance of the result.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

90 % reduction in viability was observed between 125 - 250 µg/mL (with metabolic activation) and between 7.813 and 15.625(without metabolic activation).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: In the preliminary toxicity tests, a 90% reduction in the viability of the cells treated with the test item was obtained between the concentrations of 125 and 250 µg/mL in the experiment with microsomal activation and between the concentrations of 7.813 and 15.625 µg/mL in the experiment without microsomal activation.

COMPARISON WITH HISTORICAL CONTROL DATA: The mutant frequencies of the negative and positive controls were in the range of the historical control.

Table: Mutation frequencies without metabolic activation

Treatment conc. (µg/mL)	Mutant frequency (x10^-6)
	Selection Agent 8-AG	Selection Agent 6-TG
0.5	< 4.0	6.0
1.0	4.8	4.8
2.0	< 4.0	6.6
4.0	< 4.0	< 4.0
8.0	5.2	13.9
12.0	--	--
16.0	--	--
20.0	--	--
Solvent control	< 4.0	< 4.0
300 nL/mL EMS	3157.3	3712.0

 

Table: Mutation frequencies with metabolic activation

Treatment conc. (µg/mL)	Mutant frequency (x10^-6)
	Selection Agent 8-AG	Selection Agent 6-TG
13	< 4.0	5.1
25	< 4.0	< 4.0
50	< 4.0	4.7
100	15.7	15.7
200	< 4	<4
300	--	--
400	--	--
500	--	--
Solvent control	< 4.0	< 7.1
1 µL/mL DMN	800.0	683.0

 

Executive summary:

The test material did not induce gene mutations in a mammalian cell line.

Additional information

The test item was evaluated for its in vitro genotoxicity potential via three different test methods. In two independently performed Ames tests, a slight increase in the number of revertants for two (TA 98 and TA1538) of the five tester strains was observed in the absence of metabolic activation only. For the remaining strains (TA 100, TA 1535 and TA 1537) no increase in mutation frequency, neither in the presence nor in the absence of metabolic activation was observed. To clarify this result, two further tests, an in vitro mammalian cell mutation assay (HPRT) and an in vitro chromosome aberration were conducted. The test item did not exert a mutagenic or clastogenic potential in these two tests.

Mammalien cell gene muttaion test (HPRT)

The test item was tested for mutagenic effects on V79 Chinese hamster cells in vitro. Following a preliminary range finding test with a concentration range of 31 ng/mL to 2000 mg/mL the main experiments were performed without microsomal activation at concentrations of 0.5, 1, 2, 4, 8, 12, 16 and 20 µg/mL and with microsomal activation at concentrations of 12.5, 25, 50,100, 200, 300 , 400 and 500 µg/mL. Both, 6-thioguanine (6-TG) and 8-azaguanine (8-AG) were used as selection agents.

Treatment with the test item, either without or with microsomal activation, did not lead to a mutant factor greater than 3.0 and there was also no indication of any concentration mutant-frequency relation. The mutation frequencies of the test item treated cultures remained in the range of those observed for the negative controls. The positive control chemicals induced the expected increase in mutation frequency. Therefore, it was concluded, that under the given experimental conditions the test item did not induce mutagenic effects.

Chromosome abberation test

The test item was tested for mutagenic effects on human lymphocytes using an in vitro chromosome aberration test which was conducted in similar to the OECD 473 guideline. Following a preliminary range finding test, the experiments were performed without metabolic activation at concentrations of 3.75, 7.5, 15.0, 30.0 and 60.0 µg/mL and with microsomal activation at concentrations of 8.75, 17.5, 35.0, 70.0 and 140.0 µg/mL. One hundred metaphase plates from duplicate cultures of the vehicle control, from the test item treated cultures and from the positive controls were examined (without metabolic activation mitomycin-C at 0.8 µg/ml and with metablic activation cyclophosphamide at 10 µg/mL). For the test item treated cells the incidence of specific chromosomal aberrations was within the frequency observed generally in control cultures and can therefore be considered spontaneous in origin. The treatment of the cultures with the positive control chemicals was followed by a high incidence of specific chromosomal aberrations (27% and 30%). Overall, it can be concluded that under the given experimental conditions, no evidence of mutagenic effects was obtained in human lymphocytes in vitro treated with the test item.

Bacterial reverse mutation test

In a study with the bacterial reverse mutation test (Ciba-Geigy Ltd., 1984) five bacterial strains, Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538 were used to investigate the mutagenic potential of the test item in two independent experiments conducted similar to OECD guideline No. 471. Each assay was conducted with and without metabolic activation (S9 Mix). The following test item concentrations were used: 20, 80, 320, 1280 and 5120 µg/0.1 mL. In order to confirm the results, the experiments were repeated. In addition, experiments were performed on strains TA 98 and TA 1538 with the concentrations of 160, 226, 320, 452 and 640 µg/0.1 mL. In the experiments performed without microsomal activation treatment with the test item led to a slight increase (approx. 3-fold) in the number of revertants for TA 98 and TA 1538 at the concentrations of 160 to 452 µg/0.1 mL. The experiments performed without and with microsomal activation on strains TA 98, TA 100, TA 1535 and TA 1537, did not lead to an increase in revertant frequency. For all strains a growth-inhibiting effect of the test item at the highest concentration was observed. The test item thus displayed a weak mutagenic effect only for TA 98 and TA 1538 without metabolic activation in this bacterial reverse mutation assay.

Similarly, in a second study with the bacterial reverse mutation test (Ciba-Geigy Ltd., 1980) five bacterial strains, Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538 were used to investigate the mutagenic potential of the test item in two independent experiments conducted similar to OECD guideline No. 471. Each assay was conducted with and without metabolic activation (S9 Mix). The following test item concentrations were used: 25, 75, 225, 675 and 2025 µg/0.1 mL. In order to confirm the results, the experiments were repeated. In addition, experiments were performed on strains TA 98 and TA 1538 with the concentrations of 250, 500, 1000 and 2000 µg/0.1 mL. In the experiments performed without microsomal activation treatment with the test item, a slight increase in the number of revertants for TA 98 at concentrations of 225 µg/0.1 ml and above, and for TA 1538 at the concentrations of 500 µg/0.1 ml and above were observed. The experiments performed without and with microsomal activation on strains TA 98, TA 100, TA 1535 and TA 1537, did not lead to an increase in revertant frequency. For all strains a growth-inhibiting effect of the test item at the highest concentration was observed. The test item thus displayed a weak mutagenic effect only for TA 98 and TA 1538 without metabolic activation in this bacterial reverse mutation assay.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

In the absence of in-vivo data, the substance cannot be classified for genotoxicity.