7,7'-(carbonyldiimino)bis[4-hydroxy-3-[(6-sulpho-2-naphthyl)azo]naphthalene-2-sulphonic] acid, sodium salt, compound with 2,2',2''-nitrilotriethanol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Includes the Prival and Mitchell (1982) modification to assess the mutagenic activity of azo dyes.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Including liquid pre-incubation assay with uninduced hamster liver S9 according to Prival et al.

Deviations:

not applicable

Remarks:

Not related to the experiment

Principles of method if other than guideline:

The purpose of this study is to establish the potential of the test item to induce gene mutations in bacteria by means of a S. typhimurium reverse mutation assay. For the confirmatory experiment modifications are carried out which include the performance of the liquid pre-incubation assay with uninduced hamster liver S9 according to Prival et al. Under these conditions reduction of azo-compounds to their constituent aromatic amines occurs to allow an evaluation of their mutagenic potential.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

7,7'-(carbonyldiimino)bis[4-hydroxy-3-[(6-sulpho-2-naphthyl)azo]naphthalene-2-sulphonic] acid, sodium salt, compound with 2,2',2''-nitrilotriethanol

EC Number:

279-255-7

EC Name:

7,7'-(carbonyldiimino)bis[4-hydroxy-3-[(6-sulpho-2-naphthyl)azo]naphthalene-2-sulphonic] acid, sodium salt, compound with 2,2',2''-nitrilotriethanol

Cas Number:

79770-29-9

Molecular formula:

C41H28N6O15S4.xC6H15NO3.xNa

IUPAC Name:

2-[bis(2-hydroxyethyl)amino]ethan-1-ol 4-hydroxy-7-[({5-hydroxy-7-sulfo-6-[(E)-2-(6-sulfonaphthalen-2-yl)diazen-1-yl]naphthalen-2-yl}carbamoyl)amino]-3-[(E)-2-(6-sulfonaphthalen-2-yl)diazen-1-yl]naphthalene-2-sulfonic acid sodium hydride

Test material form:

liquid

Specific details on test material used for the study:

A correction factor of 1.10 was applied for the purity of the test item.

Method

Target gene:

rfa mutation in histidine operon, uvrB deletion

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Tester strains TA 98, TA 1535 and TA 102 were obtained from MOLTOX, INC., NC 28607, USA. Tester strains TA 100 and TA 1537 were obtained from Xenometrix AG, Switzerland. They were stored as stock cultures in ampoules with nutrient broth (OXOID) supplemented with DMSO (approx. 8% v/v) over liquid nitrogen.
Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route. The hamster liver S9 fraction (uninduced male Syrian Hamster) was obtained from Trinova Biochem GmbH, Gießen, Germany.The S9 mix preparation is performed according to Prival et al.
The Vogel-Bonner Medium E agar plates with 2% glucose (Experiment I) and 0.5% in experiment II.

Metabolic activation:

with and without

Metabolic activation system:

Experiment I: Rat liver S9, Experiment II: Hamster liver S9

Test concentrations with justification for top dose:

0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate (according to results of the pre-experiment)

Vehicle / solvent:

Aqua destillata (purified water), substance soluble beyond limit dose.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Same as negative control

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

congo red

methylmethanesulfonate

other: 4-nitro-o-phenylenediamine(TA 98, TA 1537), 2-aminoanthracene (TA 98, TA 100, TA 1535, TA 1537 and TA 102)

Details on test system and experimental conditions:

METHOD OF APPLICATION: For the plate incorporation method with rat liver S9 the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 µL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control),
500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
100 µL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
2000 µL Overlay agar.
For the pre-incubation method with hamster liver S9 the following materials were mixed in a test tube and incubated for 30 min. at 30 °C:
100 µL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control),
500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
100 µL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
After the incubation period (30 min., 30 °C), the overlay agar (2000 µL) was added and poured onto the surface of a minimal agar plate.
For each strain and dose level, including the controls, three plates were used.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.
DETERMINATION OF CYTOTOXICITY
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as "N" or "B", respectively in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

Statistics:

No statistical evaluation necessary

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 98

Remarks:

Experiment II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 7

Species / strain:

S. typhimurium TA 100

Remarks:

Experiment II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 8

Species / strain:

S. typhimurium TA 1535

Remarks:

Experiment II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 9

Species / strain:

S. typhimurium TA 1537

Remarks:

Experiment II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 10

Species / strain:

S. typhimurium TA 102

Remarks:

Experiment II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item Direct Red 236 was investigated for its potential to induce gene mutations according to
the plate incorporation test with rat liver S9 (experiment I) and the pre-incubation test with hamster
liver S9 (experiment II) using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and
TA 102.
In two independent experiments several concentrations of the test item were used. Each assay was
conducted with and without metabolic activation. The concentrations, including the controls, were
tested in triplicate. The following concentrations of the test item were prepared and used in the
experiments:
0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate
No precipitation of the test item was observed in any tester strain used in experiment I and II (with
and without metabolic activation).
No toxic effects of the test item were noted in any of the five tester strains used up to the highest
dose group evaluated with and without metabolic activation in experiment I and II. The reductions
in the number of revertants down to a mutation factor of ≤ 0.5 found in experiment II in tester strain
TA 1535 at concentrations of 1.0 and 5.0 µL/plate (with metabolic activation) and in tester strain
TA 1537 at a concentration of 2.5 µL/plate (without metabolic activation) were regarded as not
biologically relevant due to lack of a dose-response relationship and lack of concomitant clearing of
the background lawn.
No biologically relevant increases in revertant colony numbers of any of the five tester strains were
observed following treatment with Direct Red 236 at any concentration level, neither in the presence
nor absence of metabolic activation in experiment I and II

Applicant's summary and conclusion

Conclusions:

The test item Direct Red 236 was investigated for its potential to induce gene mutations according to the plate incorporation test with rat liver S9 (experiment I) and the pre-incubation test with hamster liver S9 (experiment II) using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.
In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:
0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I and II. The reductions in the number of revertants down to a mutation factor of ≤ 0.5 found in experiment II in tester strain TA 1535 at concentrations of 1.0 and 5.0 µL/plate (with metabolic activation) and in tester strain TA 1537 at a concentration of 2.5 µL/plate (without metabolic activation) were regarded as not biologically relevant due to lack of a dose-response relationship and lack of concomitant clearing of the background lawn.
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Direct Red 236 at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. All criteria of validity were met.

Executive summary:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Direct Red 236 did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.

Therefore, Direct Red 236 is considered to be non-mutagenic in this bacterial reverse mutation assay.