Disodium [6-hydroxy-5-[(2-hydroxy-5-sulphophenyl)azo]naphthalene-2-sulphonato(4-)]cuprate(2-)

Genetic toxicity: in vitro

Administrative data

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:

2016-04-19 till 2016-05-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline-conform study under GLP without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

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:

Disodium [6-hydroxy-5-[(2-hydroxy-5-sulphophenyl)azo]naphthalene-2-sulphonato(4-)]cuprate(2-)

EC Number:

276-423-1

EC Name:

Disodium [6-hydroxy-5-[(2-hydroxy-5-sulphophenyl)azo]naphthalene-2-sulphonato(4-)]cuprate(2-)

Cas Number:

72152-71-7

Molecular formula:

C16H8CuN2O8S2.2Na

IUPAC Name:

Cuprate(2-), [6-(hydroxy-kO)-5-[2-[2-(hydroxy-kO)-5-sulfophenyl]diazenyl-kN1]-2-naphthalenesulfonato(4-)]-, sodium

Method

Species / strain

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 in experiment I and not induced hamster S9

Test concentrations with justification for top dose:

experiment I: 3.49 ; 10.47; 34.91; 104.73; 349.11; 1047.23; 2618.35; 5236.69
experiment II: 3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Aqua deion.
- Justification for choice of solvent/vehicle:best suitable solvent

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

congo red

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

Details on test system and experimental conditions:

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


DURATION
- Preincubation period: 30 min; 30°C
- 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.

Results and discussion

Test results

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
- Effects of pH: none
- Water solubility: yes
- Precipitation:IIn Experiment II precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate in the absence of S9 mix and from 1000 to 5000 µg/plate in the presence of S9 mix. The undissolved particles had no influence on the data recording. In Experiment I the minimal agar plates were densely colored after treatment with the test items at concentrations ranging from 2618.35 to 5236.69 µg/plate. This had no impact on the automatical evaluation.
- Other confounding effects:
COMPARISON WITH HISTORICAL CONTROL DATA: In experiment II, the number of colonies exceeded the upper limit of our historical control data in strain TA 98 in the solvent control. Since this deviation is rather small, this effect is judged to be based upon statistical fluctuations and has no detrimental impact on the outcome of the study.
ADDITIONAL INFORMATION ON CYTOTOXICITY:TToxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):
Strain Experiment I Experiment II
without S9 mix with S9 mix without S9 mix with S9 mix
TA 1535 / / / /
TA 1537 / / / 5000
TA 98 / / / 5000
TA 100 / / / /
WP2 uvrA 5236.69 / 5000 /

Remarks on result:

other: other: reverse mutation assay

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table1            Summary of Experiment I

Study Name: 1763602	Study Code: Envigo CRS 1763602
Experiment: 1763602 VV Plate	Date Plated: 19/04/2016
Assay Conditions:	Date Counted: 22/04/2016

 

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	Deion.water			9 ± 4	10 ± 2	36 ± 4	151 ± 21	58 ± 0
	Untreated			11 ± 4	9 ± 4	30 ± 3	163 ± 11	56 ± 3
	Sanodal-	3.49 µg		11 ± 3	10 ± 3	27 ± 3	163 ± 23	58 ± 7
	Rot B3LW	10.47 µg		12 ± 2	10 ± 3	34 ± 4	176 ± 18	65 ± 14
		34.91 µg		13 ± 2	13 ± 3	32 ± 9	181 ± 18	56 ± 6
		104.73 µg		8 ± 2	13 ± 4	33 ± 3	175 ± 23	60 ± 8
		349.11 µg		7 ± 2	9 ± 3	29 ± 6	177 ± 13	44 ± 7
		1047.23 µg		12 ± 2	8 ± 3	31 ± 4	170 ± 14	53 ± 4
		2618.35 µg		9 ± 2^D	8 ± 3^D	31 ± 9^D	165 ± 9^D	32 ± 9^D
		5236.69 µg		10 ± 3^D	6 ± 1^D	26 ± 9^D	154 ± 14^D	26 ± 3^D
	NaN3	10 µg		1243 ± 62			2442 ± 141
	4-NOPD	10 µg				462 ± 12
	4-NOPD	50 µg			94 ± 2
	MMS	2.0 µL						1006 ± 47
With Activation	Deion.water			13 ± 4	13 ± 1	42 ± 2	168 ± 4	67 ± 3
	Untreated			12 ± 4	13 ± 4	40 ± 2	174 ± 19	58 ± 4
	Sanodal-	3.49 µg		10 ± 2	11 ± 5	37 ± 10	159 ± 13	63 ± 3
	Rot B3LW	10.47 µg		10 ± 3	13 ± 2	34 ± 4	179 ± 8	59 ± 3
		34.91 µg		12 ± 3	13 ± 1	36 ± 3	184 ± 14	69 ± 7
		104.73 µg		10 ± 3	16 ± 3	37 ± 6	161 ± 7	62 ± 7
		349.11 µg		11 ± 3	11 ± 2	33 ± 14	178 ± 3	69 ± 10
		1047.23 µg		11 ± 3	7 ± 1	32 ± 11	138 ± 3	57 ± 6
		2618.35 µg		12 ± 4^D	8 ± 1^D	20 ± 6^D	169 ± 10^D	49 ± 6^D
		5236.69 µg		9 ± 1^D	7 ± 1^D	27 ± 3^D	135 ± 2^D	43 ± 17^D
	2-AA	2.5 µg		394 ± 6	197 ± 22	5311 ± 166	4669 ± 88
	2-AA	10.0 µg						380 ± 42

 

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	D	Densely coloured plate

 

 

Table2            Summary of Experiment II

Study Name: 1763602	Study Code: Envigo CRS 1763602
Experiment: 1763602 HV2 Pre	Date Plated: 12.05.2016
Assay Conditions:	Date Counted: 18.05.2016

 

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	Deion. water		10 ± 2	10 ± 1	25 ± 4	172 ± 19	40 ± 7
	Untreated		13 ± 5	15 ± 5	20 ± 4	182 ± 17	42 ± 4
	Sanodal-Rot	3 µg	14 ± 3	13 ± 1	23 ± 2	171 ± 17	43 ± 2
	B3LW	10 µg	9 ± 2	11 ± 4	33 ± 7	169 ± 27	42 ± 5
		33 µg	11 ± 3	14 ± 2	33 ± 7	169 ± 16	41 ± 6
		100 µg	9 ± 3	11 ± 4	26 ± 1	157 ± 9	31 ± 5
		333 µg	10 ± 4	12 ± 4	27 ± 5	170 ± 5	30 ± 2
		1000 µg	11 ± 3	10 ± 4	19 ± 5	158 ± 10	24 ± 11
		2500 µg	14 ± 2^P	7 ± 2^P	20 ± 5^P	121 ± 15^P M	18 ± 3^M P
		5000 µg	9 ± 2^P	5 ± 1^P M	13 ± 3^P M	127 ± 16^P M	15 ± 4^M P
	NaN3	10 µg	1164 ± 65			2289 ± 68
	4-NOPD	10 µg			401 ± 15
	4-NOPD	50 µg		70 ± 15
	MMS	2 µL					863 ± 15
With Activation	Deion. water		18 ± 7	21 ± 1^B M	52 ± 11	124 ± 9	52 ± 9
	Untreated		17 ± 4	23 ± 4^B M	55 ± 8	109 ± 23	54 ± 4
	Sanodal-Rot	3 µg	14 ± 2	25 ± 3^B M	51 ± 3	108 ± 20	54 ± 4
	B3LW	10 µg	17 ± 5	23 ± 6^B M	60 ± 6	126 ± 14	57 ± 5
		33 µg	20 ± 5	21 ± 5^B M	58 ± 6	135 ± 14	54 ± 6
		100 µg	14 ± 6	18 ± 4^B M	54 ± 7	145 ± 8	54 ± 3
		333 µg	18 ± 2	12 ± 2^B M	53 ± 4	124 ± 4	43 ± 7
		1000 µg	14 ± 3^P	13 ± 3^P B M	45 ± 6^P	127 ± 20^P	29 ± 6^M P
		2500 µg	13 ± 3^P M	13 ± 1^P B M	25 ± 6^P	125 ± 10^P M	27 ± 2^M P
		5000 µg	14 ± 4^P M	9 ± 1^P B M	21 ± 3^P M	125 ± 5^P M	23 ± 5^M P
	2-AA	2.5 µg				1032 ± 68
	2-AA	2.5 µg	362 ± 28	114 ± 13^B M
	2-AA	10 µg					1089 ± 83
	Congo red	500 µg			322 ± 15

 

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 M B	Precipitate Manual count Extensive bacterial growth

 

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 the test item 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 the 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 in both experiments:

Experiment I:   3.49; 10.47; 34.91; 104.73; 349.11; 1047.23; 2618. 35; and 5236.69 µg/plate

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

In Experiment II precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate in the absence of S9 mix and from 1000 to 5000 µg/plate in the presence of S9 mix. The undissolved particles had no influence on the data recording. In Experiment I the minimal agar plates were densely colored after treatment with the test items at concentrations ranging from 2618.35 to 5236.69 µg/plate. This had no impact on the automatical evaluation.

The plates incubated with the test item showed normal background growth up to 5236.69 µg/plate with and without S9 mix in all strains used.

 

Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	/	/	/	/
TA 1537	/	/	/	5000
TA 98	/	/	/	5000
TA 100	/	/	/	/
WP2 uvrA	5236.69	/	5000	/

 

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Sanodal-Rot B3LW 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 in­crease in induced revertant colonies. In experiment II, the number of colonies exceeded the upper limit of our historical control data in strain TA 98 in the solvent control. Since this deviation is rather small, this effect is judged to be based upon statistical fluctuations and has no detrimental impact on the outcome of the study.