Disodium naphthalene-1,5-disulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

1,5-Naphthalenedisulfonic acid, disodium salt was investigated in two bacterial gene mutation tetst. The compound was reported to be negative in all tests with all bacterial strains (TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA) in the absence or presence of metabolic activation.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 29 August 2017 Experimental completion date: 15 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Identification: 1,5-Naphthalenedisulfonic acid, disodium salt monohydrate
CAS name: 1,5-Naphthalenedisulfonic acid, sodium salt (1:2) monohydrate
Physical State / Appearance: Solid, white*
Expiry Date: 01 March 2018
Purity Correction factor: 1.056
Storage Conditions: At room temperature, protected from moisture
The dose selection was adjusted to purity.

Target gene:

Histidine locus in S. typhimurium and tryptophan locus in E.coli.

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/B-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

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 relevant toxic effects were observed 5000 µg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades.
The following concentrations were tested in experiment II:
33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

The test item was insoluble in deionised water. The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Remarks:

10 µg/plate for TA1535 and TA 100

Positive control substance:

sodium azide

Remarks:

Absence of S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Remarks:

10 µg/plate for TA98 and 50 µg/plate for TA 1537

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

Absence of S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Remarks:

2.0 µg/plate for WP2 uvrA

Positive control substance:

methylmethanesulfonate

Remarks:

Absence of S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

2.5 µg/plate (10.0 µg/plate in WP2 uvrA)

Positive control substance:

other: 2-Aminoanthracene

Remarks:

Presence of S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

5 µg/plate for TA98

Positive control substance:

benzo(a)pyrene

Remarks:

Presence of S9-mix

Details on test system and experimental conditions:

Experimental Design and Study Conduct
Test Item Preparation and Analysis
All formulations were prepared freshly before treatment and used within two hours of preparation. The formulation was proven to be stable for this period based on Envigo Study no. JC81NN.

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 were 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.
Experiment I (Plate Incorporation)
The following materials were mixed in a test tube and poured onto the selective agar plates:
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.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twice (strains TA 98, TA 100, and WP2 uvrA) or of thrice (strains TA 1535 and TA 1537) the spontaneous mutation rate 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 of revertant colonies 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

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:

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

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

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

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:

The test item 1,5-Naphthalenedisulfonic acid, disodium salt monohydrate 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 the Escherichia coli strain WP2 uvrA.
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:
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 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 1,5-Naphthalenedisulfonic acid, disodium salt monohydrate 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.

Summary of Experiment I

Study Name: 1843501	Study Code: Envigo 1843501
Experiment: 1843501 VV Plate	Date Plated: 29.08.2017
Assay Conditions:	Date Counted: 04.09.2017

 

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			8 ± 3	8 ± 3	27 ± 9	147 ± 12	42 ± 1
	Untreated			11 ± 5	10 ± 2	26 ± 9	143 ± 12	36 ± 5
	1,5-Naphthalenedisulfonic	3 µg		11 ± 1	7 ± 3	31 ± 3	155 ± 9	36 ± 7
	acid, disodium salt	10 µg		10 ± 1	8 ± 2	23 ± 8	149 ± 10	41 ± 4
	monohydrate	33 µg		8 ± 4	6 ± 1	26 ± 3	150 ± 9	43 ± 8
		100 µg		11 ± 0	7 ± 3	29 ± 3	133 ± 8	41 ± 6
		333 µg		7 ± 2	7 ± 3	27 ± 5	155 ± 34	36 ± 6
		1000 µg		8 ± 2	9 ± 2	26 ± 5	144 ± 5	36 ± 8
		2500 µg		9 ± 3	8 ± 2	24 ± 3	139 ± 13	40 ± 5
		5000 µg		13 ± 2	9 ± 1	31 ± 1	158 ± 12	45 ± 5
	NaN3	10 µg		1037 ± 125			2084 ± 87
	4-NOPD	10 µg				399 ± 23
	4-NOPD	50 µg			76 ± 13
	MMS	2.0 µL						877 ± 21
With Activation	Deionised water			11 ± 2	11 ± 4	43 ± 11	141 ± 13	41 ± 1
	Untreated			13 ± 3	9 ± 4	42 ± 3	142 ± 8	47 ± 10
	1,5-Naphthalenedisulfonic	3 µg		9 ± 3	14 ± 7	43 ± 8	136 ± 15	51 ± 10
	acid, disodium salt	10 µg		14 ± 3	13 ± 2	53 ± 7	146 ± 1	47 ± 8
	monohydrate	33 µg		12 ± 3	14 ± 3	47 ± 2	143 ± 13	53 ± 8
		100 µg		10 ± 1	16 ± 6	49 ± 6	138 ± 14	55 ± 10
		333 µg		14 ± 0	15 ± 5	47 ± 6	153 ± 13	51 ± 11
		1000 µg		11 ± 3	9 ± 3	38 ± 6	125 ± 11	48 ± 6
		2500 µg		15 ± 2	13 ± 1	45 ± 5	143 ± 19	55 ± 5
		5000 µg		16 ± 2	13 ± 6	42 ± 7	123 ± 10	54 ± 8
	2-AA	2.5 µg		366 ± 18	222 ± 11	3561 ± 346	3716 ± 300
	2-AA	10.0 µg						416 ± 56

 

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

 

 

Summary of Experiment II

Study Name: 1843501	Study Code: Envigo 1843501
Experiment: 1843501 HV2 Pre	Date Plated: 12.09.2017
Assay Conditions:	Date Counted: 15.09.2017

 

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			12 ± 3	8 ± 2	28 ± 4	177 ± 18	38 ± 7
	Untreated			10 ± 5	11 ± 5	21 ± 7	176 ± 22	33 ± 2
	1,5-Naphthalenedisulfonic	33 µg		11 ± 2	7 ± 2	27 ± 1	190 ± 12	40 ± 5
	acid, disodium salt	100 µg		13 ± 3	9 ± 4	25 ± 5	198 ± 15	37 ± 6
	monohydrate	333 µg		11 ± 4	8 ± 2	27 ± 6	201 ± 26	34 ± 6
		1000 µg		12 ± 4	10 ± 0	26 ± 1	195 ± 8	35 ± 7
		2500 µg		11 ± 4	9 ± 5	27 ± 7	197 ± 3	35 ± 4
		5000 µg		12 ± 3	7 ± 2	25 ± 7	178 ± 13	40 ± 3
	NaN3	10 µg		1245 ± 43			2050 ± 63
	4-NOPD	10 µg				604 ± 25
	4-NOPD	50 µg			91 ± 2
	MMS	2.0 µL						798 ± 28
With Activation	Deionised water			17 ± 7	13 ± 3	36 ± 8	191 ± 10	51 ± 5
	Untreated			13 ± 3	15 ± 5	38 ± 6	169 ± 25	48 ± 8
	1,5-Naphthalenedisulfonic	33 µg		12 ± 3	9 ± 4	40 ± 0	187 ± 7	41 ± 9
	acid, disodium salt	100 µg		12 ± 3	10 ± 4	49 ± 9	185 ± 11	42 ± 2
	monohydrate	333 µg		20 ± 4	12 ± 4	40 ± 8	173 ± 11	47 ± 8
		1000 µg		14 ± 2	9 ± 1	40 ± 4	188 ± 10	45 ± 6
		2500 µg		15 ± 4	17 ± 3	37 ± 6	172 ± 26	50 ± 9
		5000 µg		10 ± 3	16 ± 4	46 ± 8	161 ± 5	41 ± 7
	2-AA	2.5 µg		437 ± 20	96 ± 11	5071 ± 104	3986 ± 243
	2-AA	10.0 µg						387 ± 36

 

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

 

 

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 1,5-Naphthalenedisulfonic acid, disodium salt monohydrate to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using theSalmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain 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:

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 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.

No substantial increase in revertant colony numbers of any of the fivetester strains was observed following treatment with 1,5-Naphthalenedisulfonic acid, disodium salt monohydrate 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, 1,5-Naphthalenedisulfonic acid, disodium salt monohydrate is considered to be non-mutagenic in thisSalmonella typhimurium and Escherichia coli reverse mutation assay.

Genetic toxicity in vivo

Description of key information

no data available

Justification for classification or non-classification

1,5-Naphthalenedisulfonic acid, disodium salt was investigated in two bacterial gene mutation tetst. The compound was reported to be negative in all tests with all bacterial strains (TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA) in the absence or presence of metabolic activation. No classifiaction is warranted.