Reaction mass of isomers 4-[[2-[(Aminocarbonyl)amino]-4-[(2,6-difluoro-4-pyrimidyl)-amino]phenyl]azo]-1,3-benzenedisulfonic acid, sodium salt and 4-[[2-[(Aminocarbonyl)amino]-4-[(4,6-difluoro-2-pyrimidyl)-amino]phenyl]azo]-1,3-benzenedisulfonic acid, sodium salt

Genetic toxicity: in vitro

Administrative data

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:

2013-03-20 to 2013-05-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

tetrasodium 4-[(E)-2-[2-(carbamoylamino)-4-[(2,6-difluoropyrimidin-4-yl)amino]phenyl]diazen-1-yl]benzene-1,3-disulfonate 4-[(E)-2-[2-(carbamoylamino)-4-[(4,6-difluoropyrimidin-2-yl)amino]phenyl]diazen-1-yl]benzene-1,3-disulfonate

EC Number:

700-936-6

Molecular formula:

C17H11F2N7Na2O7S2

IUPAC Name:

tetrasodium 4-[(E)-2-[2-(carbamoylamino)-4-[(2,6-difluoropyrimidin-4-yl)amino]phenyl]diazen-1-yl]benzene-1,3-disulfonate 4-[(E)-2-[2-(carbamoylamino)-4-[(4,6-difluoropyrimidin-2-yl)amino]phenyl]diazen-1-yl]benzene-1,3-disulfonate

Details on test material:

Name: Reactive Golden Yellow HF-RN 1331
Batch No.: Kilo 10&12
Chemical Name: 4-[[2-[(Aminocarbonyl)amino]-4-[(2,6-difluoro-4 pyrimidinyl)-amino]phenyl]azo]-1,3-benzenedisulfonic acid, sodium salt
Formula: C17H11F2N7Na2O7S2
Physical State: solid (powder)
Colour: reddish orange
pH: 4.5 at 5%
Molecular Weight: 573.42 g/mol
Purity: 100 % (composition see AZ. No. 12-1327/2473 v.2, dated 2012-10-08)
Storage Conditions: room temperature
Expiry Date: 17.01.2014

Method

Target gene:

His

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat S9 liver microsomal fraction (Experiment I) and hamster S9 liver microsomal fraction Experiment II)

Test concentrations with justification for top dose:

31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

yes

Remarks:

A. dest., BSL Lot No. 130301, 130306, 130408

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, AppliChem Lot No. 2M005967 and 2K005471

Positive controls:

yes

Positive control substance:

other:

Remarks:

Positive control -S9: sodium azide for tester strains TA100, TA1535; 4-nitro-o-phenylene-diamine for TA98, TA1537; methylmethanesulfonate for TA102. Positive control +S9: 2-aminoanthracene for all strains except Congo Red TA 98, TA 100 +S9 (hamster)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min; 100 µL of the test item preparation was pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
- Exposure duration: at least 48 h; After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.


NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.


DETERMINATION OF CYTOTOXICITY
- Method: 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 through 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 E.coli 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.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Additional information on results:

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 with two exceptions: in experiment I toxic effects of the test item were observed in tester strain TA 1537 at a concentration of 5000 µg/plate (without metabolic activation) and in experiment II toxic effects of the test item were noted in tester strain TA 100 at a concentration of 5000 µg/plate (without metabolic activation).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Reactive Golden Yellow HF-RN 1331 did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, Reactive Golden Yellow HF-RN 1331 is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535, TA1537 of S. typhimurium and E.coli WP2 uvrA were exposed to Reactive Golden Yellow HF-RN 1331 at concentrations of 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (experiment I and II), in the presence and absence of mammalian metabolic activation according to the plate incorporation method (experiment I) and the pre-incubation method (experiment II).

Reactive Golden Yellow HF-RN 1331 was tested up to the limit concentration of 5000 µg/plate in all tester strains used.

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5100; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.