Reactive Orange 16

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:

2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of disodium 6-acetamido-4-hydroxy-3-[(4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)diazenyl]naphthalene-2-sulfonate and sodium 6-acetamido-4-hydroxy-3-{[4(vinylsulfonyl)phenyl]diazenyl}naphthalene-2-sulfonate

Molecular formula:

n.a.

IUPAC Name:

Reaction mass of disodium 6-acetamido-4-hydroxy-3-[(4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)diazenyl]naphthalene-2-sulfonate and sodium 6-acetamido-4-hydroxy-3-{[4(vinylsulfonyl)phenyl]diazenyl}naphthalene-2-sulfonate

Test material form:

solid: particulate/powder

Method

Target gene:

his+/- and trp-

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Rat Liver S9 Homogenate
Due to the limited capacity for metabolic activation of potential mutagens in in vitro methods an exogenous metabolic activation system is necessary.
Phenobarbital/-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared and stored according to the currently valid version of the SOP for rat liver S9 preparation. Each batch of S9 is routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test. The protein concentration of the S9 preparation was 30.9 mg/mL (Lot. No.: 080721K) in experiment I.

Rat S9 Mix
An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 10 % v/v in the S9 mix. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment, the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.

Hamster Liver S9 Homogenate
The S9 liver microsomal fraction was obtained from the liver of 7 - 8 weeks old male Syrian golden hamsters (not induced).
The S9 was prepared and stored according to the currently valid version of the SOP for hamster liver S9 preparation.
The protein concentration of the S9 preparation was 26.9 mg/mL (Lot. No.: 150621) in experiment II.
Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as congo red.

Hamster S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. The amount of S9 supernatant was 30% v/v. The concentrated cofactor solution yielded the following concentrations in the S9 mix:
8.0 mM MgCl2
33.0 mM KCl
20.0 mM Glucose-6-phosphate
2.8 units/ml Glucose-6-phosphate-dehydrogenase
4.0 mM NADP
2.0 mM NADH
2.0 mM FMN
in 100 mM Sodium-Ortho-Phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al (2) and Prival and Mitchell (1).

S9 Mix Substitution Buffer
The S9 mix substitution buffer contained per litre:
700 mL 100 mM sodium-ortho-phosphate-buffer pH 7.4
300 mL KCl solution 0.15 M
During the experiment, the S9 mix substitution buffer was stored in an ice bath

Test concentrations with justification for top dose:

3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate for the plate incorporation test (experiment I)

33; 100; 333; 1000; 2500; and 5000 µg/plate for the Prival modification (Experiment II)

The top dose was selected based on a preliminary toxicity test; In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The pre-experiment is reported as experiment I. Since no relevant toxic effects were observed 5000 µg/plate were chosen as maximal concentration.
The concentration range included two logarithmic decades.

Vehicle / solvent:

On the day of the experiment, the test item was dissolved in deionized water. The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
All formulations were prepared freshly before treatment and used within two hours of preparation. The formulation was assumed to be stable

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

congo red

Details on test system and experimental conditions:

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

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): not applicable
- Test substance added in medium; in agar (plate incorporation) for experiment I and preincubation for experiment II




METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition or reduction of spontaneous revertants

METHODS FOR MEASUREMENTS OF GENOTOXICIY: count of reversal bacteria coloniesa by a validated computer system , which was connected to a PC with printer to print out the individual values, the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates (see tables of results). Due to the intense color of the test item the colonies were partly counted manually

- OTHER:

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

not mandatory according to the guidelines

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

follogin Prival modification and pre-incubatio design

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No precipitation of the test item in the overlay agar was observed neither in the test tubes nor on the incubated agar plates. Strong coloring of the overlay agar was observed on the incubated agar plates at 5000 µg/plate.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.
A single minor toxic effect, evident as a reduction in the number of revertants (below the indication factor of 0.5), was observed in strain TA 98 with S9 mix at 5000 µg/plate in experiment I.

Any other information on results incl. tables

see attached file for individual tables of results

Applicant's summary and conclusion

Conclusions:

The substance was tested for in vitro gene mutation toxicity to bacteria following OECD 471. Under the experimental conditions the substance induced gene mutation based on the result for strain WP2 uvrA with non-induced Hamster liver S9 mix (Prival modification).

Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Experiment I was performed with induced rat liver S9 mix as an exogenous metabolic activation system and Experiment II was performed with non-induced hamster liver S9 mix. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:        3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                                 33; 100; 333; 1000; 2500; and 5000 µg/plate

No precipitation of the test item in the overlay agar was observed neither in the test tubes nor on the incubated agar plates. Strong coloring of the overlay agar was observed on the incubated agar plates at 5000 µg/plate.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

A single minor toxic effect, evident as a reduction in the number of revertants (below the indication factor of 0.5), was observed in strain TA 98 with S9 mix at 5000 µg/plate in experiment I.

In experiment I a minor increase in revertant colony count, neither reaching nor exceeding the threshold of twofold the revertant colony count of the corresponding solvent control, was observed following treatment with Reactive Orange 16 in strain WP2 uvrA in the presence of S9 mix. The historical control data of the negative and solvent control were exceeded from 2500 µg/plate onwards.
In experiment II a substantial increase in revertant colony numbers was observed following treatment with Reactive Orange 16 in strain WP2 uvrA in the presence S9 mix. The threshold of twofold the colony count of the corresponding solvent was exceeded at concentrations ranging from 1000 to 5000 µg/plate.

 

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease in induced revertant colonies