Trisodium 5-[[5-methyl-4-[(4-nitro-2-sulphonatophenyl)azo]-2-(3-sulphonatopropoxy)phenyl]azo]salicylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenicity was investigated in an Ames reverse mutation assay. No mutagenic effects were detected. The test item did not show mutagenic potential.

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:

2016 -01-19 till 2016-02-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations

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

Species / strain / cell type:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9 (experiment I) and non-induced hamster liver S9 (experiment II)

Test concentrations with justification for top dose:

3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I
1; 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / experiment II

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionized water
- Justification for choice of solvent/vehicle: best suitable solvent

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene; Congo red

Details on test system and experimental conditions:

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


DURATION
- Preincubation period: 30 minutes
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

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:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Water solubility: soluble
- Precipitation: The test item precipitated in the overlay agar in the test tubes from 10 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 100 to 5000 µg/plate. The undissolved particles had no influence on the data recording.
- Other confounding effects: none
COMPARISON WITH HISTORICAL CONTROL DATA: In experiment II, the data in solvent controls of strain TA 100 without S9 and strain 1535 with S9 mix and the untreated controls of strains TA 1537 and TA 100 with S9mix were slightly above our 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.
ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabol¬ic activation. Only in experiment II minor toxic effects were observed in strain TA 1535 with S9 mix at 5000 µg/plate.

Remarks on result:

other: other: reverse mutation assay

Remarks:

Migrated from field 'Test system'.

Summary Tabellen

Table1            Summary of Experiment I

Study Name: 1743802	Study Code: Envigo 1743802
Experiment: 1743802 VV Plate	Date Plated: 19/01/2016
Assay Conditions:	Date Counted: 22/01/2016

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	Deionised water			11 ± 5	9 ± 4	25 ± 8	181 ± 30	45 ± 9
	Untreated			8 ± 1	13 ± 4	26 ± 10	199 ± 7	49 ± 5
	Aluminium-	3 µg		11 ± 5	10 ± 4	29 ± 5	185 ± 22	45 ± 10
	Braun 1134 p	10 µg		12 ± 4	10 ± 4	21 ± 1	172 ± 12	50 ± 17
		33 µg		10 ± 3	10 ± 4	25 ± 5	179 ± 5	51 ± 7
		100 µg		10 ± 2^P	9 ± 2^P	26 ± 6^P	174 ± 13^P	48 ± 5^P
		333 µg		10 ± 4^P	8 ± 2^P	28 ± 2^P	196 ± 19^P	44 ± 12^P
		1000 µg		9 ± 4^P	6 ± 1^P	27 ± 8^P	194 ± 18^P	40 ± 6^P
		2500 µg		12 ± 2^P	7 ± 2^P	34 ± 6^P	185 ± 18^P	40 ± 6^P
		5000 µg		9 ± 4^P	8 ± 2^P	27 ± 4^P	172 ± 11^P	31 ± 3^P
	NaN3	10 µg		1171 ± 88			2083 ± 137
	4-NOPD	10 µg				302 ± 36
	4-NOPD	50 µg			63 ± 5
	MMS	2.0 µL						999 ± 76
With Activation	Deionised water			13 ± 3	12 ± 2	30 ± 5	185 ± 17	45 ± 4
	Untreated			10 ± 3	10 ± 3	28 ± 6	195 ± 29	59 ± 8
	Aluminium-	3 µg		13 ± 5	10 ± 5	28 ± 2	189 ± 15	51 ± 8
	Braun 1134 p	10 µg		12 ± 5	7 ± 2	25 ± 13	173 ± 25	52 ± 6
		33 µg		13 ± 3	8 ± 2	27 ± 6	186 ± 21	52 ± 10
		100 µg		10 ± 4^P	9 ± 3^P	24 ± 5^P	191 ± 5^P	62 ± 8^P
		333 µg		11 ± 4^P	11 ± 5^P	31 ± 5^P	202 ± 17^P	50 ± 3^P
		1000 µg		7 ± 1^P	7 ± 3^P	27 ± 4^P	200 ± 23^P	58 ± 9^P
		2500 µg		9 ± 1^P	10 ± 5^P	30 ± 4^P	176 ± 27^P	43 ± 3^P
		5000 µg		10 ± 5^P	10 ± 3^P	26 ± 6^P	147 ± 25^P	31 ± 7^P
	2-AA	2.5 µg		510 ± 32	107 ± 11	4431 ± 85	4790 ± 229
	2-AA	10.0 µg						359 ± 14

 

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

 

 

Table2            Summary of Experiment II

Study Name: 1743802	Study Code: Envigo 1743802
Experiment: 1743802 HV2 Pre	Date Plated: 12/02/2016
Assay Conditions:	Date Counted: 18/02/2016

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	Deionised water			14 ± 5	8 ± 1	33 ± 8	197 ± 5	52 ± 10
	Untreated			16 ± 5	7 ± 1	30 ± 4	206 ± 6	55 ± 5
	Aluminium-	1 µg		14 ± 2	8 ± 1	33 ± 9	247 ± 6	55 ± 7
	Braun 1134 p	3 µg		15 ± 5	9 ± 2	32 ± 3	259 ± 7	59 ± 7
		10 µg		13 ± 2	9 ± 3	29 ± 7	263 ± 24	49 ± 10
		33 µg		13 ± 4	9 ± 1	34 ± 10	267 ± 14	50 ± 8
		100 µg		14 ± 5^P	9 ± 1^P	33 ± 4^P	253 ± 35^P	51 ± 8^P
		333 µg		13 ± 3^P	8 ± 3^P	29 ± 7^P	271 ± 25^P	47 ± 2^P
		1000 µg		16 ± 4^P	15 ± 1^P	29 ± 2^P	249 ± 25^P	50 ± 7^P
		2500 µg		12 ± 4^P	8 ± 1^P	36 ± 6^P	252 ± 22^P	56 ± 2^P
		5000 µg		9 ± 4^P	8 ± 1^P	49 ± 6^P	246 ± 9^P	39 ± 11^P
	NaN3	10 µg		1421 ± 64			2394 ± 58
	4-NOPD	10 µg				371 ± 10
	4-NOPD	50 µg			72 ± 13
	MMS	2 µL						1169 ± 126
With Activation	Deionised water			22 ± 3	27 ± 4	49 ± 3	204 ± 4	55 ± 11
	Untreated			25 ± 4	27 ± 4	55 ± 1	216 ± 16	61 ± 2
	Aluminium-	1 µg		26 ± 3	26 ± 3	51 ± 2	225 ± 18	60 ± 12
	Braun 1134 p	3 µg		28 ± 2	32 ± 3	53 ± 2	230 ± 23	54 ± 11
		10 µg		28 ± 2	32 ± 1	56 ± 1	235 ± 25	56 ± 6
		33 µg		27 ± 5	30 ± 3	58 ± 11	242 ± 3	55 ± 1
		100 µg		25 ± 6^P	31 ± 3^P	54 ± 2^P	233 ± 5^P	64 ± 9^P
		333 µg		21 ± 3^P	32 ± 4^P	51 ± 10^P	240 ± 9^P	63 ± 6^P
		1000 µg		22 ± 1^P	25 ± 3^P	59 ± 11^P	250 ± 7^P	53 ± 4^P
		2500 µg		14 ± 4^P	20 ± 3^P	81 ± 7^P	231 ± 19^P	47 ± 2^P
		5000 µg		9 ± 1^P	20 ± 4^P	82 ± 6^P	242 ± 48^P	49 ± 13^P
	2-AA	2.5 µg					2251 ± 49
	2-AA	2.5 µg		395 ± 21	119 ± 12
	2-AA	10 µg						832 ± 103
	Congo red	500 µg				328 ± 11

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD Congo red MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine Congo red methyl methane sulfonate	P	Precipitate

 

 

Conclusions:

Interpretation of results (migrated information):
negative with and without rat- and hamster S9

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 the test substance to induce gene mutations according to the plate incorporation test (experiment I) with and without rat S9 mix and the pre-incubation test (experiment II) with and without hamster S9 mix using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia colistrain WP2 uvrA.

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 was tested at the following concentrations:

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

Experiment II:                                1; 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 10 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 100 to 5000 µg/plate. The undissolved particles had no influence on the data recording. 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 all strains with and without metabolic activation. Only in experiment II minor toxic effects were observed in strain TA 1535 with S9 mix at 5000 µg/plate. No substantial increase in revertant colony numbers of any of the fivetester strains was observed following treatment with Aluminium-Braun 1134 p at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

no mutagenic effects

Justification for classification or non-classification

No classification; no mutagenic effects were detected.