Trisodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)][2-hydroxy-3-[(2-hydroxy-1-naphthyl)azo]-5-nitrobenzenesulphonato(3-)]chromate(3-)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

key study

Study period:

2020 - 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

nitroreductase-deficient strains TA98NR and TA100NR

Principles of method if other than guideline:

The objective of this study was to evaluate the ability of test item to induce reverse mutations in histidine-requiring strains of Salmonella typhimurium in the absence and presence of a reductive hamster liver metabolising system (S-9). By assessing the mutagenicity of test item in nitroreductase deficient strains (TA98NR and TA100NR) alongside parent nitroreductase competent strains (TA98 and TA100), the role of nitroreduction in any test article related mutagenic activity could be determined.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 01

IUPAC Name:

Similar Substance 01

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

nitroreductase deficient

Remarks:

TA98NR and TA100NR

Metabolic activation:

with and without

Metabolic activation system:

The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it was prepared from uninduced male Golden Syrian hamsters. The S-9 was stored frozen at <-50°C, and thawed prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P 450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities

Test concentrations with justification for top dose:

5, 16, 50, 160, 500, 1600 and 5000 µg/plate

A maximum concentration of 5000 µg/plate was selected for Mutation Experiment treatmenty, in order that treatments were performed up to this maximum recommended concentration according to current regulatory guidelines OECD 471.

Vehicle / solvent:

water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

congo red

mitomycin C

other: Metronidazole, 2-aminoanthracene

Details on test system and experimental conditions:

pre-incubation methodology in the absence and presence of a modified (reductive) S-9 mix. These platings were achieved by the following sequence of additions to sterile pre incubation tubes:
• 0.1 mL of bacterial culture
• 0.1 mL of test article solution/vehicle control or 0.05 mL of positive control
• 0.5 mL of 30% reductive S-9 mix or buffer solution
Quantities of test article or control solution, bacteria and S-9 mix or buffer solution detailed above, were mixed together and placed in an orbital incubator set to either 37°C (for the treatments in the absence of S-9) or 30°C (for treatments in the presence of S 9) for 30 minutes, before the addition of 2 mL of supplemented molten agar at 45±1°C followed by rapid mixing and pouring on to Vogel-Bonner E agar plates.
When set, the plates were inverted and incubated protected from light for 3 days in an incubator set to 37°C. Following incubation, these plates were examined for evidence of cytotoxicity to the background lawn, and where possible revertant colonies were counted.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98, TA98NR, TA100 or TA100NR) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values
2. The positive trend/effects described above were reproducible.
The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if neither of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and between experiments

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

bacteria, other: TA 98NR

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

bacteria, other: TA 98NR

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 7

Species / strain:

bacteria, other: TA100NR

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 8

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 9

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 10

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 11

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 12

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The objective of this study was to evaluate the ability of test item to induce reverse mutations in histidine-requiring strains of Salmonella typhimurium in the absence and presence of a reductive hamster liver metabolising system (S-9). By assessing the mutagenicity of the test substance in nitroreductase deficient strains (TA98NR and TA100NR) alongside parent nitroreductase competent strains (TA98 and TA100), the role of nitroreduction in any test article related mutagenic activity could be determined.
The results shows that in the absence of S-9, as a clear mutagenic response was observed in strain TA98 but not in strain TA98NR, nitroreduction appears to be playing a key role in the mutagenicity of test item.

Any other information on results incl. tables

Mutation Experiment 1 treatments of all the tester strains were performed using a pre incubation methodology in the absence and presence of a modified (reductive) S 9 mix using final concentrations of test item at 5, 16, 50, 160, 500, 1600 and 5000 µg/plate, plus vehicle and positive controls. Following these treatments, no clear evidence of cytotoxicity was observed, as would usually be manifest by a diminution of the background bacterial lawn and/or a marked reduction in revertant numbers. Reductions in what appeared to be mutagenic responses at the higher concentrations in some strains were however considered likely to have been due to test article-related cytotoxicity.

 

Following test item treatments of all the tester strains, notable increases in revertant numbers were observed in strains TA98, TA100 and TA1537 in the absence of S-9 and in strains TA98NR in the presence of S-9 only. These increase all approached or exceeded 2-fold the concurrent vehicle control level, and provided at least some evidence of a concentration-relationship, although in almost all cases the maximum increase was observed below the maximum treatment concentration, but it was considered that this was likely due to test article-related cytotoxicity. By far the largest magnitude of increases occurred in strain TA98NR in the presence of S-9, and but further experimentation is required to understand the reason behind this finding.

 

Clear mutagenic responses in the absence of S-9 were observed in strain TA98, TA100 and TA537 with no clear responses observed following comparable treatments with the corresponding nitroreductase deficient strain TA98NR and TA100NR, this is considered indicative that nitroreduction is involved in the observed mutagenicity in the absence of S-9. However, in the presence of S-9 a much larger magnitude of increase was observed in strain TA98NR compared to comparable treatments in strain TA98. It may be that for treatments performed in the presence of S-9, the enzymes in the S-9 are affecting the impact that nitroreduction has on the mutagenic activity. It is therefore considered that the involvement of nitroreduction in the mutagenicity of the test substance in the presence of S-9 is unclear, and further experimentation is required to elucidate the mechanism of mutagenicity under these treatment conditions.

 

Finally, all the data can be considered Acceptable and Valide According to the criteria mentioned above.

Applicant's summary and conclusion

Conclusions:

It was concluded that the tested substance induced mutation in histidine-requiring Salmonella typhimurium strain TA98NR in the presence of a reductive hamster liver metabolic activation system (S-9), and in strains TA98, TA100 and TA1537 in the absence of S-9, when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 μg/plate (the maximum recommended concentration according to current regulatory guidelines). The relative reduced mutagenic responses in the absence of S-9 with the nitroreductase deficient strains compared to those with the parent strains indicate that nitroreduction plays a significant role in the mutagenic activity of the tested substance, but the mechanism of mutagenicity in the presence of S-9 remains unclear.

Executive summary:

The substance was assayed for mutation in seven histidine-requiring strains (TA98, TA100, TA1535, TA1537, TA102, TA98NR and TA100NR) of Salmonella typhimurium, both in the absence and in the presence of a reductive hamster liver metabolising system (S-9) in a single experiment. All treatments in this study were performed using formulations prepared in purified water. As the test substance is an azo compound, testing in the presence of S-9 in this study was performed using a modified reductive (Prival) S-9 pre-incubation methodology, as it is known that azo compounds can be reduced to free aromatic amines, which can be mutagenic.
Mutation Experiment treatments of all the tester strains were performed using a pre-incubation methodology in the absence and presence of a modified (reductive) S-9 mix using final concentrations of the test substance at 5, 16, 50, 160, 500, 1600 and 5000 μg/plate. Following these treatments, clear evidence of cytotoxicity was observed at 5000 μg/plate in strain TA102 in the absence of S-9 only.
It should be noted that due to uncharacteristic vehicle control data following the initial Mutation Experiment treatments of strain TA98NR in the absence and presence of S-9, these treatments were repeated to provide valid mutation data for this strain. In order that concurrent mutation data were available for direct comparison of any mutagenic response in this strain with the parent strain, strain TA98 treatments were also repeated alongside the strain TA98NR repeat treatments. As mutation data from the initial strain TA98 treatments in the absence and presence of S-9 were valid, data from these further TA98 treatments are reported as further Mutation Experiment data.
The test article was completely soluble in the aqueous assay system at all concentrations treated.
Vehicle and positive control treatments were included for all strains. The mean numbers of revertant colonies were comparable with acceptable ranges for vehicle control treatments, and the responses with the positive control treatments were sufficient to confirm the correct strain and assay functioning.
Following treatments of all the test strains with the test substance, notable increases in revertant numbers were observed in strain TA98 in the absence and presence of S-9, in strains TA100 and TA1537 in the absence of S-9 only and in strain TA98NR in the presence of S-9 only. In strains TA100 and TA1537 in the absence of S-9 and in strain TA98NR in the presence of S-9, concentration-related increases that exceeded 2-fold (strains TA100, TA98 and TA98NR) or 3-fold (strain TA1537) the vehicle control level were observed, although in some cases the responses ‘tailed off’ at the highest concentration(s), which was considered likely to have been due to test article related cytotoxicity. All these increases were sufficient to be considered as clear evidence of test substance mutagenic activity in these strains. In strain TA98 in the presence of S-9, concentration-related increases that ‘tailed off’ at the highest concentration(s) were observed, but the increase only exceeded 2-fold the concurrent vehicle control level with the further Mutation Experiment treatments. In strain TA98NR in the absence of S-9, an increase in revertant numbers was observed that provided at least some evidence of a concentration-relationship. This increase fell slightly below the 2-fold threshold for an increase to be considered as clear evidence of mutagenic activity in this strain, and therefore along with the increases seen in strain TA98 in the presence of S-9 may have been further evidence of the mutagenic activity described previously. In strain TA102 in the presence of S-9, a 1.4-fold increase was seen. This increase was only evident at a single intermediate treatment concentration, and was therefore not considered to be a clearly concentration-related effect. Together with the relatively small magnitude of the increase (below 1.5-fold the concurrent vehicle control level), this increase was not sufficient to be considered as clear evidence of mutagenic activity.
When the relative magnitude of the responses in the absence of S-9 between the nitroreductase deficient strains and their respective parent strains were assessed, there was a markedly greater increase in strain TA100 than in TA100NR, as no notable increase was observed with the nitroreductase deficient strain. The increases in strain TA98 (in both the initial and further treatments) were slightly greater in magnitude, and occurred at a lower treatment concentration, than the increase observed in strain TA98NR. As there is still some low level residual nitroreductase activity present in strains TA98NR and TA100NR (Rosenkranz and Mermelstein (1983)), complete elimination of any mutagenic response was not expected in these strains, although this did appear to occur with strain TA100NR in the absence of S-9. These data were considered to indicate that nitroreduction played at least some part in the mutagenic activity observed in the absence of S-9.
In the presence of S-9, no notable increases were observed in strains TA100 or TA100NR, so only the relative responses in strains TA98 and TA98NR provided an indication of the role that nitroeduction may play in the observed mutagenic activity. A clear mutagenic response was only observed in strain TA98NR, with only small increases seen in strain TA98. It is unclear why an increased response should occur in the absence of nitroreduction, but it is known that the enzymes present in S-9 can result in confounding effects when trying to assess the role of an individual enzyme (such as nitroreductase) in the mutagenic activity of a compound. It is therefore considered that the involvement of nitroreduction in the mutagenicity of the test substance in the presence of S-9 is unclear, and further experimentation is required to elucidate the mechanism of mutagenicity under these treatment conditions. It was concluded that the test substance induced mutation in histidine-requiring Salmonella typhimurium strain TA98NR in the presence of a reductive hamster liver metabolic activation system (S-9), and in strains TA98, TA100 and TA1537 in the absence of S-9, when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 μg/plate (the maximum recommended concentration according to current regulatory guidelines). The relative reduced mutagenic responses in the absence of S-9 with the nitroreductase deficient strains compared to those with the parent strains indicate that nitroreduction plays a significant role in the mutagenic activity of the test substance, but the mechanism of mutagenicity in the presence of S-9 remains unclear.