Reaction products of diazotised 4-amino-5-hydroxy-2,7-naphthalenedisulfonic acid coupled with resorcinol, subsequently coupled with diazotized 2-amino-4,6-dinitrophenol and diazotized 4-nitrobenzenamine, chelated with iron (3+), sodium potassium salts

Genetic toxicity: in vitro

Administrative data

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:

August 2020 - September 2020

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:

OECD, 1997, as corrected in 2020

Deviations:

yes

Remarks:

due to the nature of the test article, a modified (reductive Prival) metabolic activation system was employed, and only a single experiment was performed. Use of NR-deficient strains.

Principles of method if other than guideline:

The traditional strains used for OECD TG 471 were checked in parallel with the same strains deficient in the nitroreductase enzyme (present only in bacteria) as to avoid the NO2 group reduction present in the test substance. The reduction of nitro groups in fact produces aromatic amines in the substance that typically give false positive results for the traditional tested strains.

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 #3

IUPAC Name:

Similar Substance #3

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:

TA 98NR, TA100 NR

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: reductive (Prival) metabolic activation system.
- Source of S9: uninduced male Golden Syrian hamsters.
- Method of preparation of S9 mix: the S9 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). Treatments were carried out both in the absence and presence of S9 by addition of either buffer solution or 30% reductive (Prival) S9 mix respectively. The composition of the mix and buffer solutions are reported in the section "Any other information on material and methods incl. tables".
- Concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL of 30% reductive S9 mix.
- Quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): 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:

- Test concentrations: 5, 16, 50, 160, 500, 1600 and 5000 µg/plate (final concentrations). 5000 µg/plate is the maximum recommended concentration according to current regulatory guidelines.

Vehicle / solvent:

Purified 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 (MTZ), 2-aminoanthracene (AAN)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate.
- Number of independent experiments: single experiment.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): not applicable.
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk: pre-incubation methodology.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 30 minutes at 37°C (for the treatments in the absence of S9) or 30°C (for treatments in the presence of S9).
- Exposure duration/duration of treatment: the plates were inverted and incubated protected from light for 3 days in an incubator set to 37°C.
- Harvest time after the end of treatment: not applicable.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: the background lawns of the plates were examined for signs of cytotoxicity. Revertant plate count data were also assessed, as a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response would also be considered as evidence of cytotoxicity.
- Any supplementary information relevant to cytotoxicity: not supplementary information reported.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as intensely coloured agar or bubbles/splits in the agar affected the accuracy of the automated counter.

OTHER: it should be noted that data from the initial treatments of strain TA98 and TA98NR in the absence and presence of S9, and data from initial repeat treatments of strain TA98NR, were invalidated due to a combination of unacceptable vehicle control counts and confounding contaminating colonies. A further repeat of these strain treatments was therefore performed in order to provide the Mutation Experiment data presented in this study. As comparisons between concurrent strain TA98 and TA98NR data are required in this study, strain TA98 and TA98NR treatments were performed in the initial repeat treatments, and data from these strain TA98 treatments were valid, but strain TA98 was also treated alongside strain TA98NR in the further repeat treatments. The strain TA98 data from the initial repeat treatments are therefore presented as further Mutation Experiment in this study.
As data from the Mutation Experiment showed clear positive responses in most tester strains, then further testing in a second experiment was not considered necessary, in accordance with current regulatory guidance.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
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.
The test article was considered positive in this assay if the above criterion was met.
The test article was considered negative in this assay if the above criterion was not
met.
Results which only partially satisfied the above criteria were dealt with on a case-bycase
basis. Biological relevance was taken into account, for example consistency of
response within and between concentrations.
Data from strain TA98 were compared (non-statistically) with that from TA98NR,
and data from strain TA100 were compared with that from TA100NR. Where a
mutagenic response was seen in one or both parent strains but was absent or much
reduced in the corresponding NR variant strain(s), this was considered to be indicative
that bacterial nitroreduction enzymes play a significant role in the mutagenicity of the
test compound as observed in this study.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

bacteria, other: TA 98NR

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

bacteria, other: TA 100NR

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 7

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Cytotoxicity, Solubility and Concentration Selection:
Mutation Experiment treatments of all the tester strains were performed using a pre-incubation methodology in the absence and presence of a modified (reductive) S9 mix using final concentrations of the test substance at 5, 16, 50, 160, 500, 1600 and 5000 μg/plate, plus vehicle and positive controls. Following these treatments, no evidence of cytotoxicity was observed, as would usually be indicated by a diminution of the background bacterial lawn and/or a marked reduction in revertant numbers.

Data Acceptability and Validity:
The individual mutagenicity plate counts were averaged to give mean values. From the data it can be seen that vehicle control counts fell within the laboratory’s historical ranges, with the exception of a few isolated vehicle control counts that fell slightly outside the laboratory control ranges. In each case, these counts were comparable to the other vehicle control replicate counts and the laboratory historical control ranges, and therefore these data were accepted as characteristic and valid. The positive control chemicals all induced increases in revertant numbers of ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle controls, and ≥2-fold with NQO, B[a]P and CR in strain TA98NR and NaN3, AAN and CR in strain TA100NR. The positive control treatments with 2NF in TA98NR and with MTZ in TA100NR each demonstrated a reduced response compared to the equivalent treatments in the parent strains TA98 and TA100, confirming the nitroreductase deficient status of strain TA98NR and TA100NR. The control treatments therefore confirmed discrimination between different strains, and an active S9 preparation, and the correct strain and assay functioning was demonstrated. The study data were therefore accepted as valid.

Mutation:
Following test substance treatments of all the test strains apart from TA100NR, clear and concentration-related increases in revertant numbers were observed in the absence and presence of S9.
These increases were ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98, TA98NR or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control, although it should be noted that in strain TA98NR in the absence of S9, the 2-fold threshold level was only achieved at the maximum treatment concentration of 5000 μg/plate. These increases were however all sufficient to be considered as clear evidence of the test substance mutagenic activity in this assay system. In strain TA100NR, a notable and concentration-related increase in revertant numbers was observed in the absence of S9 only, but this increase fell just below the 2-fold threshold for an increase to be considered as clear evidence of mutagenic activity in this strain. No notable increase in revertant numbers was observed following strain TA100NR treatments in the presence of S9. The mutagenic responses in strains TA98NR and TA100NR in the absence of S9 were markedly reduced when compared to the responses with their respective parent strain, both in terms of magnitude of increase in revertants, and lowest responding treatment concentration. In the presence of S9 this difference was again observed, particularly between strain TA100 and TA100NR, as no response was observed in the latter strain, but the difference was somewhat less pronounced between strains TA98 and TA98NR. Strains TA98NR and TA100NR still retain some low level residual nitroreductase activity (Rosenkranz and Mermelstein, 1983), and therefore complete elimination of any mutagenic response was not expected in these strains. Therefore, overall the data from this study with the nitroreductase deficient strains provided reduced responses compared to their nitroreductase proficient parent strains, indicating that nitroreduction plays a significant role in the mutagenic activity of the test substance.

Applicant's summary and conclusion

Conclusions:

It was concluded that the test substance induced mutation in histidine-requiring strains TA98, TA98NR, TA100, TA1535, TA1537 and TA102 of Salmonella typhimurium in the absence and in the presence of a reductive hamster liver metabolic activation system (S9), 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 reduction in (or absence of) mutagenic response 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.

Executive summary:

The test 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 (S9) 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 S9 in this study was performed using a modified reductive (Prival) S9 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) S9 mix using final concentrations of the test substance at 5, 16, 50, 160, 500, 1600 and 5000 µg/plate. Following these treatments, no clear evidence of cytotoxicity was observed, as would usually be indicated by a diminution of the background bacterial lawn and/or a marked reduction in revertant numbers.
It should be noted that due to a combination of uncharacteristic vehicle control data and confounding contaminant colonies on the test plates following the initial Mutation Experiment treatments of strain TA98 and TA98NR in the absence and presence of S9, these treatments were repeated to provide valid mutation data for these strain treatments. No valid strain TA98NR data were available from these initial repeat treatments due to confounding contaminant colonies on these test plates, but the strain TA98 mutation data were valid. In order that concurrent mutation data were available for direct comparison of any mutagenic response in the nitroreductase deficient strain and the parent strain, strain TA98 treatments were also included alongside the strain TA98NR repeat treatments, and the data from these further repeat treatments are presented in this report as the Mutation Experiment data for these strains. As mutation data from the initial repeat treatments in strain TA98 in the absence and presence of S9 were valid, these are reported as further Mutation Experiment data.

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 were elevated by positive control treatments. Following the test substance treatments of all the test strains apart from TA100NR, clear and concentration-related increases in revertant numbers were observed in the absence and presence of S9. These increases were ≥ 1.5-fold (in strain TA102), ≥ 2-fold (in strains TA98, TA98NR or TA100) or ≥ 3-fold (in strains TA1535 or TA1537) the concurrent vehicle control, although it should be noted that in strain TA98NR in the absence of S9, the 2-fold threshold level was only achieved at the maximum treatment concentration of 5000 µg/plate. These increase were however sufficient to be considered as clear evidence of the test substance mutagenic activity in this assay system. In strain TA100NR, a notable and concentration-related increase in revertant numbers was observed in the absence of S9 only, but this increase fell just below the 2-fold threshold for an increase to be considered as clear evidence of mutagenic activity in this strain.

The mutagenic responses in strains TA98NR and TA100NR in the absence of S9 were markedly reduced when compared to the responses with their respective parent strain, both in terms of magnitude of increase in revertants, and lowest responding treatment concentration. In the presence of S9 this difference was again observed, particularly between strain TA100 and TA100NR, as no response was observed in the latter strain, but the difference was somewhat less pronounced between strains TA98 and TA98NR. Strains TA98NR and TA100NR still retain some low level residual nitroreductase activity (Rosenkranz and Mermelstein, 1983), and therefore complete elimination of any mutagenic response was not expected in these strains. Therefore, overall the data from this study with the nitroreductase deficient strains provided reduced responses compared to their nitroreductase proficient parent strains, indicating that nitroreduction plays a significant role in the mutagenic activity of the test substance.

It was concluded that the test substance induced mutation in histidine-requiring strains TA98, TA98NR, TA100, TA1535, TA1537 and TA102 of Salmonella typhimurium in the absence and in the presence of a reductive hamster liver metabolic activation system (S9), 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 reduction in (or absence of) mutagenic response 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.