Propanoic acid, 2-hydroxy-, compd. with 7-[[4,6-bis[[3-(diethylamino)propyl]amino]-1,3,5-triazin-2-yl]amino]-3-[2-(2,3-dihydro-2-oxo-1H-benzimidazol-6-yl)diazenyl]-4-hydroxy-2-naphthalenesulfonic acid (1:1)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance registered does not induce structural chromosome aberrations determined by the chromosome aberration test in V79 cells in vitro. During the described mutagenicity test (AMES) and under the experimental conditions reported, the substance registered did not induce gene mutations. Under the experimental conditions reported for a valid HRPT test the registration substance did not induce gene mutations at the HPRT locus in V79 cells.

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:

2005

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other:

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: given in table below

Metabolic activation:

with and without

Metabolic activation system:

S9 liver fractions of rats, induced with phenobarbital/beta-naphthoflavone

Test concentrations with justification for top dose:

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

Vehicle / solvent:

Solvent: Deionised water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: Sodium azide (TA1535, TA100), 4-nitro-o-phenylene-diamine (TA1537, TA98), methyl methane sulfonate (WP2uvrA); 2-aminoanthracene (all strains with metabolic activation)

Details on test system and experimental conditions:

Concentration of the test substance resulting in precipitation: 5000 µg/plate

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 TA98, TA100, and WP2uvrA) or thrice (strains TA1535 and TA1537) 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.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the pre-experiment the concentration range of the test item was 3-5000 µg/plate. The pre-experiment is reported as experiment I since no toxic effects were observed and 5000 µg/plate were chosen as maximal concentration.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

No toxic effects, evident as a reduction in the number of revertants, were observed with and without metabolic activation in experiment I. In experiment II, minor toxic effects were observed in the presence of metabolic activation in strain TA1537 at 5000 µg/plate and in strain TA100 from 1000 up to 5000 µg/plate.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Red LF 6382/18L-R 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.
The number of colonies exceeded the laboratory's historical control range slightly in the negative control of strain WP2 uvrA with and without metabolic activation in experiment I. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies and has no impact on the outcome of the study.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

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.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 2: Summary of Results Pre-Experiment and Experiment I

Metabolic activation	Test group	Dose level [µg/plate]	Revertant colony counts (Mean±SD)
			TA1535	TA1537	TA98	TA100	WP2uvrA
Without	Water		23±1	10±3	32±3	134±7	60±7
	Untreated		29±2	14±3	25±5	128±6	64±13
	Red LF6382/18L-R	3	24±3	10±3	25±3	120±2	59±3
		10	21±1	14±1	24±11	138±13	64±9
		33	19±7	8±6	23±1	128±5	79±18
		100	22±4	13±6	24±6	129±9	67±7
		333	27±7	10±2	28±8	135±10	60±5
		1000	23±3	9±3	24±8	141±3	58±9
		2500	16±3	7±1	26±7	134±7	56±4
		5000	17±3	5±1	17±2	123±12	46±8
	Sodium azide	10	1715±36			2381±161
	4-NOPD	10			369±23
	4-NOPD	50		83±9
	MMS	4 µL					1556±63
With	Water		34±12	16±5	38±15	151±7	62±6
	Untreated		30±10	13±4	36±4	166±8	69±13
	Red LF6382/18L-R	3	30±1	11±3	47±5	155±7	68±10
		10	30±6	13±5	40±4	168±2	61±11
		33	32±3	12±2	31±3	159±2	83±26
		100	28±7	12±2	36±2	154±12	70±5
		333	29±2	14±6	40±7	149±10	61±8
		1000	28±2	12±4	33±10	130±18	57±7
		2500	24±2	9±2	31±4	128±9	61±6
		5000	21±3	7±2	20±5	123±14	50±10
	2-AA	2.5	378±5	317±13	2196±27	1323±325
	2-AA	10					222±14

4-NOPD        4-nitro-o-phenylene-diamine

MMS              methyl methane sulfonate

2-AA              2-aminoanthracene

 

Table 3: Summary of Results Experiment II

Metabolic activation	Test group	Dose level [µg/plate]	Revertant colony counts (Mean±SD)
			TA1535	TA1537	TA98	TA100	WP2uvrA
Without	Water		21±1	14±6	26±6	161±25	47±10
	Untreated		18±3	17±4	25±2	154±14	44±5
	Red LF6382/18L-R	33	19±4	19±4	29±7	154±21	54±10
		100	19±8	17±2	30±6	161±20	54±9
		333	24±4	21±4	29±11	157±25	51±10
		1000	26±5	17±3	27±10	145±15	44±3
		2500	23±4	11±1	18±3	134±6	37±4
		5000	20±4	10±3	22±2	112±10	35±3
	Sodium azide	10	1531±67			2192±80
	4-NOPD	10			377±26
	4-NOPD	50		84±6
	MMS	4 µL					315±32
With	Water		23±2	24±3	41±1	195±22	50±1
	Untreated		27±4	29±2	48±8	162±1	54±8
	Red LF6382/18L-R	33	22±8	26±5	44±7	186±24	52±8
		100	29±5	24±3	50±9	184±11	53±13
		333	31±6	27±4	50±1	150±12	45±3
		1000	29±6	26±3	45±1	79±7	56±6
		2500	23±3	23±4	31±6	82±9	37±5
		5000	24±3	10±2	28±2	76±7	37±5
	2-AA	2.5	397±24	269±5	2065±206	2559±334
	2-AA	10					319±26

4-NOPD        4-nitro-o-phenylene-diamine

MMS              methyl methane sulfonate

2-AA              2-aminoanthracene

 

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

not relevant for classification and labelling;

Executive summary:

This study was performed according to OECD Guideline 471 to investigate the potential of the substance to induce gene mutations in the plate incorporation test and the pre-incubation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The substance was incubated in concentrations from 3-5000 µg/plate (experiment I) and from 33-5000 µg/plate (experiment II) either with and without metabolic activation (rat S9 mix). Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants, were observed with and without metabolic activation in experiment I. In experiment II, minor toxic effects were observed in the presence of metabolic activation in strain TA 1537 at 5000 µg/plate and in strain TA 100 from 1000 up to 5000 µg/plate.

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, the substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Three valid in vitro mutagenicity tests with the registration substance are available.

OECD 471 Ames test

In order to investigate the potential of the substance to induce gene mutations, a valid Ames test was performed, where the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA were tested. The substance was incubated in concentrations from 3-5000 µg/plate (experiment I) and from 33-5000 µg/plate (experiment II) either with and without metabolic activation (rat S9 mix). Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the substance registered did not induce gene mutations.

OECD 473 Chromosome Aberration test

Under the experimental conditions reported, the substance registered did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.Therefore, the substance is considered to be non-clastogenic in this chromosome aberration test with and without S9 mix when tested up to precipitating concentrations (4 hrs treatment with and without S9 mix) or up to the highest scorable concentrations (18 hrs and 28 hrs continuous treatment without S9 mix).

OECD 476 HPRT

Under the experimental conditions reported the substance registered did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance registered is considered to be non-mutagenic.

Based on the negative results from three valid in vitro mutagenicity tests, it is considered that the test substance registered has no mutagenic potential.

Justification for classification or non-classification

The available three in vitro genotoxicity studies on the substance registered revealed all negative results. On this basis the substance registered is not to be classified as to its genotoxic properties.