Reaction mass of Sodium, bis[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)]chromate(1-) and sodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1Hpyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)]hydroxychromate(1-)]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Non mutagenic

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

October 02, 1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine locus

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 rat liver fraction

Test concentrations with justification for top dose:

0.2, 2, 20, 200 and 2000 µg (or nl) per Petri dish

Vehicle / solvent:

Sterile water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-anthramine

Remarks:

with S9 mix

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

other: daunomycine, N-methyl-N'-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix

Details on test system and experimental conditions:

REPLICATES: 3

CHECKING OUT TESTER STRAINS
All strains were tested for the presence of their mutations.
a) The mutation in the histidine operon, basic to the test system was tested by checking for growth in the presence and absence of histidine on a minimal medium-agar base.
Bacteria of the nutrient agar plate were streaked on a minimal medium plate supplemented with 0.1 ml of 0.1 mM L-histidine and 0.1 ml of 0.5 mM biotin as a growth requirement.
The plates were incubated at 37 °C overnight. Growth, for all strains, was seen only where histidine and biotin were present.
b) Sensitivity to crystal violet is a check for the presence of the deep rough (rfa) mutation, the loss of the lipopolysaccharide coat on the bacterial surface.
Three drops of the broth culture were spread on the surface of a nutrient agar plate. A sterile filter paper disc containing crystal violet (10 µl of a I mg/ml solution) was placed on this surface. A zone of inhibition around the disc, after 24 hours incubation, shows the presence of the (rfa) mutation.
c) Two of the tested strains (TA 98 and TA 100) contain a plasmid expressing resistance to ampicillin (R factor). To check for the presence of the plasmid, a sterile filter paper disc containing Ampicillin (10 µl of 8 mg/ml in 0.02 N NaOH) was placed on a nutrient agar plate spread with the broth.
In strains TA 1535 and TA 1537 a zone of inhibition occurs around the disc, but for TA 98 and TA 100 where the R factor is present, no zone of inhibition is seen.
d) The uvrB deletion, the loss of the excision repair system, makes the bacteria sensitive to UV irradiation.
One drop of the broth was cross-streaked in a nutrient agar plate. One half of the plate was irradiated for 20 seconds under a 15 watt UV lamp at a distance of approximately 30 cm. After 24 hours' incubation, growth was found only on the unirradiated part of the streak for all strains.

Species / strain:

other: TA I535, TA I537, TA 98 and TA lOO

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

Non mutagenic

Executive summary:

Method

The substance was tested with the Salmonella typhimurium strains TA I535, TA I537, TA 98 and TA 100 at concentrations from 0.2 to 2000 µg per Petri dish both in the presence and absence of metabolic activation, according to a method similar to the OECD Guideline 471.

Results

No mutagenic effect was observed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

GERM CELL MUTAGENICITY

This hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class.

Category 1: Substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans. Substances known to induce heritable mutations in the germ cells of humans.

Categoty 2: Substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

Classification for heritable effects in human germ cells is made on the basis of well conducted, sufficiently validated tests as In vitro mutagenicity tests such as these indicated in 3.5.2.3.8:

- in vitro mammalian chromosome aberration test;

- in vitro mammalian cell gene mutation test;

- bacterial reverse mutation tests

The substance did not create gene mutations in the strains of Salmonella typhimurium under the performed test, therefore according to the 3.5. of the CLP Regulation EC n.1272/2008, it cannot be classified as mutagenic for germ cells.