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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
other: read across from similar substance
Adequacy of study:
weight of evidence
Study period:
2020
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020

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

Constituent 1
Chemical structure
Reference substance name:
Monosodium aqua-[5-[[2,4-dihydroxy-5-[(2-hydroxy-3,5-dinitrophenyl)azo]phenyl]azo]-2-naphthalensulfonate], iron complex
EC Number:
400-720-9
EC Name:
Monosodium aqua-[5-[[2,4-dihydroxy-5-[(2-hydroxy-3,5-dinitrophenyl)azo]phenyl]azo]-2-naphthalensulfonate], iron complex
Cas Number:
126851-40-9
Molecular formula:
C22H11FeN6NaO10S.H2O
IUPAC Name:
Monosodium aqua-[(5-((E)-(5-((Z)-(3,5-dinitro-2-oxidophenyl)diazenyl)-2-hydroxy-4-oxidophenyl)diazenyl)naphthalene-2-sulfonate)ferrate(-)]

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 first experiment

A maximum concentration of 5000 µg/plate was selected for Mutation Experiment 1in 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:
Mutation Experiment 1 treatments were performed using a 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 resultsopen allclose all
Species / strain:
bacteria, other: TA 98NR
Metabolic activation:
without
Genotoxicity:
other: negative, except at the highest tested dose
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
bacteria, other: TA100 NR
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
bacteria, other: TA100 NR
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no 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 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.
The results shows that a clear direct acting mutagenicity activity is present when traditional strains are used for detecting gene mutation, while this properties is completely deleted (or markedly diminshed) when NR deficient strains are used. The results are therefore considered as sufficient for assessing the substance not mutagenic to bacteria ( see expert assessment in genetic toxicity).

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 Acid Black 210 at 5, 16, 50, 160, 500, 1600 and 5000 µg/plate, plus vehicle and positive controls.

No precipitation of test article was observed following any of the strain treatments, but the intensity of the test article colouration at the higher treatment concentrations meant that some of these plates had to be scored manually to obtain an accurate revertant count. No clear toxic effects were observed following any of the strain treatments.

 

Clear and concentration-related increases in revertant numbers were observed following treatments of strains TA100, TA1535, TA1537 and TA102 in the absence and presence of S-9. A small (1.9-fold) increase in revertant numbers was also observed in strain TA100NR in the absence of S-9 at the highest tested dose, and this corresponded to all the other tester strains where a greater magnitude of increase in revertant numbers was observed in the absence of S-9 compared to the those in the presence of S-9. These mutation data are sufficient to be considered as a clear indication of Acid Brown 434 mutagenic activity in strains TA100, TA1535, TA1537 and TA102 in this assay system. The reduced (or absent) increases in revertants in strain TA100NR compared to those seen with corresponding treatments in the parent strain TA100strain TA100NR suggests that nitroreduction plays a major role in this mutagenic activity.

Clear and concentration-related increases in revertant numbers that exceeded 2-fold the concurrent vehicle control level were observed in strain TA98  and TA98NR in the absence and presence of S-9. When the relative responses between the parent and NR strains were compared, the magnitude of the responses were greater and the responses started at lower concentrations in TA98 compared to TA98NR in both the absence and presence of S-9. These relative responses were considered to provide a clear indication that nitroreduction played a role in the mutagenicity observed in these strains.

 

Applicant's summary and conclusion

Conclusions:
The substance was tested for gene mutation in bacteria following OECD 471 with nitroreductase deficinet strains. Under the experimental conditions the mutagenicity of the substance was deleted or greatly reduced for the NR strains confirming that direct acting mutagenicity of the substances, based on the nitroreductase activity, plays a major role in the mutagenic properties of the substance