Tetralithium 5,5'-[vinylenebis[(3-sulphonato-4,1-phenylene)azo]]bis[3-methylsalicylate]

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:

March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tetralithium 5,5'-[vinylenebis[(3-sulphonato-4,1-phenylene)azo]]bis[3-methylsalicylate]

EC Number:

258-605-2

EC Name:

Tetralithium 5,5'-[vinylenebis[(3-sulphonato-4,1-phenylene)azo]]bis[3-methylsalicylate]

Cas Number:

53523-90-3

Molecular formula:

C30H20Li4N4O12S2

IUPAC Name:

tetralithium 5,5'-[vinylenebis[(3-sulphonato-4,1-phenylene)azo]]bis[3-methylsalicylate]

impurity 1

Reference substance name:

Unknown impurities, none of which are present at ≥1%

Molecular formula:

Unknown impurities, none of which are present at ≥1%

IUPAC Name:

Unknown impurities, none of which are present at ≥1%

Test material form:

liquid

Specific details on test material used for the study:

Identification: Bayscript Gelbkomponente
CAS-No.: 53523-90-3
Chemical Name: Tetralithium 5,5'-[vinylenebis[(3-sulphonato-4,1-phenylene)azo]]bis[3-methylsalicylate]
Empirical Formula: C30H20N4O12S2Li4
Molecular Mass: 720.4 g/mol
Appearance: Red-brown powder
Storage Conditions: At room temperature, moisture protected
Stability in Solvent: Stable for 4 and 24 hours at room temperature in the light in deion. Water (based on analytical study number PV29HC)
Expiry Date: 27 February 2017

Method

Target gene:

histidine locus

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9 were used as the metabolic activation system

Test concentrations with justification for top dose:

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
with and without metabolic activation.

Since no toxic effects were observed, 5000 μg/plate were chosen as maximal concentration.

Vehicle / solvent:

Deionized water. The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

deionized water

True negative controls:

no

Positive controls:

yes

Remarks:

10 µg/plate, TA 1535, TA 100

Positive control substance:

sodium azide

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

deionized water

True negative controls:

no

Positive controls:

yes

Remarks:

10 µg/plate in strain TA 98. 50 µg/plater in strain TA 1537

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

without metabolic activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

2.0 µL/plate TA 102

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

2.5 µg/plate for all strains except TA 102 (10.0 µg/plate)

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Precultures
The thawed bacterial suspension was transferred into 250 mL Erlenmeyer flasks containing 50 mL nutrient medium. A solution of 50 μL ampicillin (25 μg/mL) was added to the strains TA 98, TA 100, and TA 102. This nutrient medium contains per litre:
8 g Nutrient Broth 5 g NaCl
The bacterial cultures were incubated in a shaking water bath for 4 hours at 37° C. The optical density of the bacteria was determined by absorption measurement and the obtained values indicated that the bacteria were harvested at the late exponential or early stationary phase (10^8-10^9 cells/mL).

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 experimental conditions in this pre-experiment were the same as described for the experiment I below (plate incorporation test).
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.
The pre-experiment is reported as main experiment I, since the following criteria are met:
Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

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:
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)
In the pre-incubation assay 100 μL test solution (solvent or reference mutagen solution (positive control)), 500 μL S9 mix / S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube. The 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 with the software program Ames Study Manager.

Evaluation criteria:

Evaluation of Results
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 102) 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.

Results and discussion

Test results

Test results 1

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 2

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

Test results 3

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 4

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 5

Species / strain:

S. typhimurium TA 102

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:

The test item Bayscript Gelbkomponente was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.
The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were 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 occurred up to the highest investigated dose.
The plates incubated with the test item showed an increasing dense color from 333 to 5000 μg/plate, which had no impact on evaluation of the plates.
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.
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.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Bayscript Gelbkomponente at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also 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 in-crease in induced revertant colonies.

Any other information on results incl. tables

            Summary of Experiment I

Study Name: 1747203	Study Code: Envigo 1747203
Experiment: 1747203 VV Plate	Date Plated: 11/03/2016
Assay Conditions:	Date Counted: 14/03/2016

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	TA 102
Without Activation	Deionised water			13 ± 3	7 ± 2	25 ± 5	160 ± 24	520 ± 8
	Untreated			12 ± 1	10 ± 5	23 ± 1	160 ± 7	506 ± 6
	Bayscript	3 µg		14 ± 2	9 ± 1	26 ± 6	170 ± 27	516 ± 12
	Gelbkomponente	10 µg		15 ± 5	7 ± 2	20 ± 7	144 ± 19	539 ± 23
		33 µg		13 ± 5	8 ± 1	21 ± 1	151 ± 16	511 ± 26
		100 µg		14 ± 2	7 ± 2	24 ± 10	149 ± 17	495 ± 78
		333 µg		13 ± 3^D	11 ± 2^D	24 ± 10^D	168 ± 8^D	519 ± 37^D
		1000 µg		12 ± 3^D	8 ± 1^D	25 ± 3^D	142 ± 10^D	539 ± 29^D
		2500 µg		13 ± 3^D	7 ± 1^D	20 ± 5^D	152 ± 12^D	530 ± 26^D
		5000 µg		10 ± 5^D	7 ± 2^D	21 ± 6^D	143 ± 16^D	503 ± 39^D
	NaN3	10 µg		1223 ± 29			2143 ± 132
	4-NOPD	10 µg				390 ± 31
	4-NOPD	50 µg			66 ± 12
	MMS	2.0 µL						5562 ± 1178
With Activation	Deionised water			10 ± 1	11 ± 4	34 ± 2	153 ± 35	624 ± 42
	Untreated			11 ± 4	16 ± 7	36 ± 7	119 ± 8	657 ± 12
	Bayscript	3 µg		10 ± 3	10 ± 2	27 ± 2	131 ± 8	762 ± 28
	Gelbkomponente	10 µg		9 ± 3	11 ± 4	27 ± 10	134 ± 26	754 ± 55
		33 µg		8 ± 1	7 ± 1	26 ± 6	142 ± 5	713 ± 63
		100 µg		13 ± 6	10 ± 4	32 ± 3	128 ± 5	733 ± 130
		333 µg		14 ± 2^D	10 ± 2^D	34 ± 4^D	132 ± 13^D	734 ± 15^D
		1000 µg		11 ± 4^D	6 ± 1^D	26 ± 7^D	122 ± 15^D	692 ± 48^D
		2500 µg		11 ± 5^D	10 ± 3^D	26 ± 4^D	129 ± 8^D	675 ± 82^D
		5000 µg		14 ± 2^D	10 ± 5^D	37 ± 1^D	143 ± 7^D	666 ± 103^D
	2-AA	2.5 µg		426 ± 17	112 ± 8	4516 ± 613	2495 ± 184
	2-AA	10.0 µg						1715 ± 215

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA MMS 4-NOPD	sodium azide 2-aminoanthracene methyl methane sulfonate 4-nitro-o-phenylene-diamine	D	Densely coloured plate

Summary of Experiment II

Study Name: 1747203	Study Code: Envigo 1747203
Experiment: 1747203 HV2 Pre	Date Plated: 24/03/2016
Assay Conditions:	Date Counted: 30/03/2016

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	TA 102
Without Activation	Deionised water			9 ± 2	8 ± 1	21 ± 2	192 ± 7	451 ± 31
	Untreated			12 ± 2	8 ± 2	24 ± 3	196 ± 14	471 ± 30
	Bayscript	33 µg		9 ± 3	8 ± 2	22 ± 6	215 ± 22	453 ± 25
	Gelbkomponente	100 µg		8 ± 3	8 ± 1	23 ± 4	177 ± 17	443 ± 11
		333 µg		10 ± 3^D	8 ± 2^D	24 ± 3^D	194 ± 9^D	451 ± 70^D
		1000 µg		8 ± 2^D	7 ± 2^D	23 ± 3^D	201 ± 4^D	453 ± 33^D
		2500 µg		9 ± 2^D	7 ± 1^D	19 ± 4^D	197 ± 12^D	395 ± 36^D
		5000 µg		10 ± 2^D	9 ± 4^D	25 ± 1^D	207 ± 21^D	351 ± 62^D
	NaN3	10 µg		1057 ± 47			1977 ± 31
	4-NOPD	10 µg				408 ± 30
	4-NOPD	50 µg			109 ± 15
	MMS	2.0 µL						5449 ± 739
With Activation	Deionised water			11 ± 2	10 ± 5	29 ± 2	199 ± 27	603 ± 18
	Untreated			7 ± 1	11 ± 4	38 ± 2	180 ± 18	643 ± 47
	Bayscript	33 µg		9 ± 3	10 ± 5	30 ± 1	185 ± 23	648 ± 49
	Gelbkomponente	100 µg		10 ± 3	9 ± 2	35 ± 8	198 ± 10	585 ± 5
		333 µg		12 ± 4^D	11 ± 1^D	34 ± 6^D	202 ± 15^D	563 ± 118^D
		1000 µg		11 ± 4^D	11 ± 2^D	25 ± 7^D	202 ± 15^D	583 ± 27^D
		2500 µg		14 ± 2^D	10 ± 2^D	26 ± 3^D	211 ± 11^D	551 ± 31^D
		5000 µg		15 ± 2^D	9 ± 3^D	28 ± 3^D	181 ± 8^D	449 ± 7^D
	2-AA	2.5 µg		417 ± 45	106 ± 13	4377 ± 111	2370 ± 210
	2-AA	10.0 µg						1872 ± 278

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA MMS 4-NOPD	sodium azide 2-aminoanthracene methyl methane sulfonate 4-nitro-o-phenylene-diamine	D	Densely coloured plate

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

This study was performed to investigate the potential of Bayscript Gelbkomponente 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 TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item, dissolved in deionized water, 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 occurred up to the highest investigated dose.

The plates incubated with the test item showed an increasing dense color from 333 to 5000 μg/plate, which had no impact on evaluation of the plates.

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.

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.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Bayscript Gelbkomponente at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also 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 and showed a distinct increase of induced revertant colonies.

Conclusion

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

Therefore, Bayscript Gelbkomponente is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.