Disodium 2-[4-[[1-[[(2-methoxy-5-methyl-4-sulphonatophenyl)amino]carbonyl]-2-oxopropyl]azo]phenyl]-6-methylbenzothiazole-7-sulphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

other: experimental data on similar substance

Adequacy of study:

key study

Study period:

1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

only four strains tested

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted: 26 May 1983

Deviations:

yes

Remarks:

only four strains

Principles of method if other than guideline:

References:
Ames BN, Durson WE, Yamasaki E, and Lee FD (1970). Proc. Nat. Acad. Sci. USA, 70, 2285.
Ames BN, McCann J, and Yamasaki E (1975) Mutation Research 31, 347.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium sodium 2-[4-[[1-[[(2-methoxy-5-methyl-4-sulphonatophenyl)amino]carbonyl]-2-oxopropyl]azo]phenyl]-6-methylbenzothiazole-7-sulphonate

EC Number:

276-770-9

EC Name:

Ammonium sodium 2-[4-[[1-[[(2-methoxy-5-methyl-4-sulphonatophenyl)amino]carbonyl]-2-oxopropyl]azo]phenyl]-6-methylbenzothiazole-7-sulphonate

Cas Number:

72705-24-9

Molecular formula:

C26H24N4O9S3.H3N.Na

IUPAC Name:

ammonium sodium 2-[4-({1-[(2-methoxy-5-methyl-4-sulfonatophenyl)carbamoyl]-2-oxopropyl}diazenyl)phenyl]-6-methyl-1,3-benzothiazole-7-sulfonate

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S-9 Mix; Microsomal Enzyme Fraction prepared from animals pre-treated with Arochlor 1254.

Test concentrations with justification for top dose:

20, 100, 500, 2500, 12500 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Good solubility of the test substance, readily soluble in DMSO.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO alone

True negative controls:

no

Positive controls:

yes

Remarks:

Furthermore, 2-amino-anthracene which does not revert in the absence of metabolic activation was added at a dose of 10 µg/plate (activity check of liver enzyme preparation)

Positive control substance:

other: see remarks

Remarks:

M-Methyl-N-Nitro-N-Nitrosoguanidine (MNNG) 2 µg/plate (TA1535 & TA100); 9 Amino Acridine (9AA) 100 µg/plate (TA 1537); 4-Nitro-O-phenyldiamine (4NOPD) 10 µg/plate (TA 98).

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Test tubes containing 9 mL portions of molten histidine deficient top agar at 45°C.
- Addition of 0.1 mL test solution or negative/positive control solution, 0.1 mL bacterial suspension, and optional, 0.5 mL S-9 Mix.
- Pouring of samples of 3 mL onto Vogel-Bonner agar plates (minimal medium).
- A solution containing 0.5 mM Histidine and 0.5 mM Biotin was added to top agar to enable that a background lawn of growth was possible in the test.
- Growth indicates that a reverse mutation has reverted the his- gene back to an active form.

DURATION
- Preincubation period: no
- Exposure duration: Incubation at 37°C °C for 48 hours

NUMBER OF REPLICATIONS: 3 (Petri dishes prepared per strain and per group)

CELLS EVALUATED: The bacterial colonies (his+ revertants) are counted.

DETERMINATION OF CYTOTOXICITY
- Method: bacterial growth (colony formation)

OTHER INFORMATION
Microsomal Enzyme Fraction: A commercial preparation was obtained from Uniscience Limited, 8 Jesus Lane, Cambridge, CBS 8BA, UK. This fraction is obtained from animals pre-treated with Arochlor 1254 (a mixture of polychlorinated biphenyls) and was stated to be from batch no. 03300 with a protein level of 40 mg/mL (Aryl hydrocarbon hydroxylase activity: 6.4 nmol hydroxy benzo(a)pyrene/20 minutes/mg protein).
Reference:
SPECIFICATIONS FOR RAT LIVER S-9 PRODUCT, AROCLOR 1254 INDUCED, CATOLOG NOS. 8360-01 AND 8360-03. Date prepared: 1984-04-25, Uniscience Limited, UK.

Evaluation criteria:

Positive result: Dose-related and statistically significant increase in mutation rate (of at least twice the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence of the S-9 Mix.

Negative result: The number of induced revertants compared to spontaneous revertants should be less than two fold at all dose levels employed, the intervals of which should be between 3 and 5 fold and extend to the limits imposed by toxicity or solubility.

Statistics:

The arithmetic mean was calculated.

Results and discussion

Test results

Test results 1

Key result

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:

not examined

Positive controls validity:

valid

Test results 2

Key result

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:

not examined

Positive controls validity:

valid

Test results 3

Key result

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:

not examined

Positive controls validity:

valid

Test results 4

Key result

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:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
No inhibition was observed at any of the concentrations of the test substance employed in this test and the main mutation study was set up using test substance at concentrations in the range 20 to 12500 µg/plate.


ADDITIONAL INFORMATION:
No significant increase in the number of revertant colonies was recorded for any of the strains at any of the dose levels employed in this study, either with or without metabolic activation.
All counts of revertant colonies were similar to those recorded for the negative control plates and were within the range expected for spontaneous reversion for each strain.
The positive control substances all produced marked increases in the number of revertant colonies and the activity of the S9 fraction was found to be satisfactory.

Any other information on results incl. tables

 

Plate incorporation with S-9 Mix (colony number = mean values):

Dose (µg/per 0.1 mL [plate])	TA 98	TA 100	TA 1535	TA1537
0 (DMSO)	27	102	19	7
20	25	104	20	7
100	24	109	25	8
500	25	109	26	6
2500	26	103	24	8
12500	28	112	22	8
----------
Positive controls:
4-NOP (10 µg/plate)	850
MNNG (2 µg/plate)		> 1000	797
9-AA (100 µg/plate)				880
----------
9-AA (10 µg/plate)	> 1000	> 1000	487	617
Three plates per tester strain were evaluated.

 

 

Plate incorporation without S-9 Mix (colony number = mean values):

Dose (µg/per 0.1 mL [plate])	TA 98	TA 100	TA 1535	TA1537
0 (DMSO)	24	93	21	8
20	25	100	22	8
100	22	113	25	6
500	25	101	26	6
2500	26	107	28	6
12500	25	107	32	8
----------
Positive controls:
4-NOP (10 µg/plate)	917
MNNG (2 µg/plate)		> 1000	683
9-AA (100 µg/plate)				> 1000
----------
9-AA (10 µg/plate)	28	103	20	6
Three plates per tester strain were evaluated.

 

 

VIABILITY AND SPONTANEOUS REVERSION TESTS

Strain                Salmonella typhimurium	Total Counts on Histidine deficient agar (x 10E7)	Minimal agar with overlay of Histidine/Biotin top agar
	1 mL of overnight nutrient broth
TA 98	21	26	28	28
TA 100	30	97	98	101
TA 1535	15	18	18	17
TA 1537	19	6	6	8

 

 

STERILITY CHECKS

Sample	Count
Top Agar + His/Biotin	0	0	0
Top Agar only	0	0	0
Top Agar + His/Biotin + S9	0	0	0

 

Applicant's summary and conclusion

Conclusions:

Not mutagenic in Ames test