5,5'-sulfonylbis(2-benzofuran-1,3-dione)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay/Ames test: negative (OECD 471; GLP compliant)

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-11-20 to 2014-12-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 1997-07-21

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2013-04-11

Type of assay:

bacterial reverse mutation assay

Target gene:

not applicable

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9 were used as the metabolic activation system. Protein concentration of the S9 prepartion was 30.9 mg/mL in experiment I and 34.0 mg/mL in experiment II.

Test concentrations with justification for top dose:

Pre-experiment/Experiment I (plate incorporation assay): 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate (with and without metabolic activation)
Experiment II (pre-incubation assay): 33, 100, 333, 1000, 2500, and 5000 µg/plate (with and without metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

The pH-value was 5 in experiment I. The test item was not neutralized with NaOH because it precipitated after neutralization. Therefore the pH-value was not determined in experiment II.
All formulations were prepared freshly before treatment and used within two hours of preparation. The formulation was assumed to be stable for this period unless specified otherwise by the Sponsor.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Positive control without metabolic activation: dissolved in deionised water; concentration: 10 μg/plate (strains TA 1535, TA 100)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

Positive control without metabolic activation: dissolved in DMSO; concentration: 10 μg/plate (strain TA 98); 50 μg/plate (strain TA 1537)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Positive control without metabolic activation: dissolved in deionised water; concentration: 2.0 μL/plate (strain WP2 uvrA)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Positive control with metabolic activation: dissolved in DMSO; concentration: 2.5 μg/plate (strains TA 1535, TA 1537, TA 98, TA 100); 10.0 μg/plate in WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation, Pre-experiment/Experiment I) and preincubation (Experiment II)

PRE-EXPERIMENT FOR TOXICITY:
To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The pre-experiment was reported as experiment I.
Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

DOSE SELECTION:
In the pre-experiment/Experiment I the concentration range of the test item was 3 – 5000 µg/plate. Since no relevant toxic effects were observed 5000 µg/plate were chosen as maximal concentration.

EXPERIMENTAL PERFORMANCE:
For each strain and dose level, including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:

Pre-experiment/Experiment I (Plate Incorporation):
- 100 µL: test solution at each dose level (solvent or reference mutagen solution (positive control)),
- 500 µL: S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
- 100 µL: bacteria suspension (cf. test system, pre-culture of the strains),
- 2000 µL: overlay agar

Experiment II (Pre-Incubation):
- 100 µL test solution (solvent or reference mutagen solution (positive control)), 500 µL S9 mix / S9 mix substitution buffer, 100 µL bacterial suspension were mixed in a test tube
- incubation at 37 °C for 60 minutes.
- after pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube.
- mixture was poured on minimal agar plates.

After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

In parallel to each test a sterile control of the test item was performed and documented in the raw data. Therefore, 100 µL of the stock solution, 500 µl S9 mix / S9 mix substitution buffer were mixed with 2.0 mL overlay agar and poured on minimal agar plates.

DATA RECORDING:
The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB9 7BN, UK) with the software program Ames Study Manager (v.1.21). The individual values and mean values of the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates were recorded. Due to reduced background growth the colonies were partly counted manually.

ACCEPTABILITY OF THE ASSAY:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of the historical data
- the positive control substances should produce a significant increase in mutant colony frequencies
- a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.

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:

A statistical analysis of the data is not mandatory.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

please refer to the field "Additional information on results" below.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

please refer to the field "Additional information on results" below.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
The test item precipitated in the overlay agar in the test tubes at 5000 µg/plate. The undissolved particles had no influence on the data recording.
No precipitation of the test item in the overlay agar on the incubated agar plates was observed up to the highest investigated dose.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- experiment I: plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.
- experiment II: plates incubated with the test item showed reduced background growth at 5000 µg/plate in TA 1535, TA 1537 and TA 98 with and without S9 mix and in strain WP2 uvrA with S9 mix.
- no toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

EXPERIMENT I AND II:
- no substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item any concentration level, neither in the presence nor absence of metabolic activation (S9 mix).
- no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
- appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.
- experiment II: data in the solvent control of strain TA 98 without S9 mix and strain WP2 uvrA with S9 mix were slightly under the historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies and had no detrimental impact on the outcome of the study.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
According to the EC-Commission directive 67/548/EEC and its subsequent amendments, the test substance is not mutagenic.
According to the EC-Regulation 1272/2008 and subsequent regulations, the test item is not classified as mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The GLP conform bacterial reverse mutation assay (k_Sokolowski_2015) performed according to OECD 471 showed that the test item did not induce gene mutations.

Justification for selection of genetic toxicity endpoint
GLP guideline study conducted with the test item

Justification for classification or non-classification

Based on the guideline-conform study conducted under GLP, the test substance should be considered void of genotoxicity. Thus, according to Directive 67/548 /EEC and its subsequent amendments and according to Regulation (EC) No 1272/2008 and subsequent regulations, the test substance should not be considered to have a mutagenic potential, and hence no classification or labelling is required.