Barium bis[2-[(2-hydroxy-1-naphthyl)azo]benzoate]

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:

2014-11-07 to 2014-11-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Barium bis[2-[(2-hydroxy-1-naphthyl)azo]benzoate]

EC Number:

228-906-3

EC Name:

Barium bis[2-[(2-hydroxy-1-naphthyl)azo]benzoate]

Cas Number:

6372-81-2

Molecular formula:

C17H12N2O3.1/2Ba

IUPAC Name:

barium bis{2-[(2-hydroxy-1-naphthyl)diazenyl]benzoate}

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 non-induced hamster S9 in experiment II

Test concentrations with justification for top dose:

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

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO resulting a homogeneous suspension

Controls

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: 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.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

minor

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Water solubility: unsoluble
- Precipitation:
The test item precipitated in the overlay agar in the test tubes from 33 to 5000 µg/plate in experiment I and from 333 to 5000 µg/plate in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 1000 to 5000 µg/plate in experiment II. The undissolved particles had no influence on the data recording.
- Other confounding effects:
COMPARISON WITH HISTORICAL CONTROL DATA: performed
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 nearly all test groups. A reduction in the number of revertants (below the indication factor of 0.5), was observed in the presence of metabolic activation at 5000 µg/plate in experiment I in strains TA 1535 and TA 98 and in experiment II in strains TA 1535 and WP2 uvrA.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table1     Summary of Experiment I

Study Name: 1664301	Study Code: Harlan CCR 1664301
Experiment: 1664301 VV Plate	Date Plated: 07/11/2014
Assay Conditions:	Date Counted: 10/11/2014

 

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	DMSO			12 ± 2	11 ± 3	19 ± 5	165 ± 13	35 ± 2
	Untreated			11 ± 3	11 ± 4	19 ± 4	181 ± 3	39 ± 2
	Pigment Red	3 µg		10 ± 4	9 ± 1	22 ± 4	156 ± 6	34 ± 1
	50:1	10 µg		12 ± 3	11 ± 1	21 ± 7	154 ± 10	27 ± 3
		33 µg		11 ± 2	13 ± 3	21 ± 6	145 ± 9	28 ± 9
		100 µg		12 ± 3	12 ± 2	21 ± 3	151 ± 13	32 ± 4
		333 µg		10 ± 3	12 ± 3	23 ± 4	163 ± 14	29 ± 6
		1000 µg		11 ± 2^D	11 ± 3^D	19 ± 5^D	147 ± 13^D	27 ± 1^D
		2500 µg		10 ± 1^D M	8 ± 1^D M	21 ± 2^D M	151 ± 13^D M	22 ± 4^D M
		5000 µg		10 ± 2^D R M	7 ± 2^D M	12 ± 2^D M R	137 ± 13^D M	24 ± 3^D M
	NaN3	10 µg		1110 ± 130			1742 ± 116
	4-NOPD	10 µg				323 ± 36
	4-NOPD	50 µg			85 ± 8
	MMS	2.0 µL						965 ± 7
With Activation	DMSO			11 ± 2	12 ± 4	27 ± 8	140 ± 16	43 ± 5
	Untreated			10 ± 0	13 ± 1	29 ± 2	176 ± 14	46 ± 4
	Pigment Red	3 µg		11 ± 2	11 ± 3	29 ± 6	153 ± 29	39 ± 7
	50:1	10 µg		9 ± 2	12 ± 2	30 ± 4	158 ± 7	40 ± 6
		33 µg		9 ± 2^R	10 ± 2	25 ± 2	158 ± 14	41 ± 5
		100 µg		11 ± 3^R	11 ± 4	26 ± 8	141 ± 9	39 ± 5
		333 µg		11 ± 2^R	11 ± 3	25 ± 8	146 ± 3	36 ± 6
		1000 µg		11 ± 1^R D	9 ± 1^D	26 ± 6^D	130 ± 6^D	22 ± 2^D
		2500 µg		10 ± 3^R D M	8 ± 2^D M	21 ± 4^D M	137 ± 14^D M	23 ± 3^D M
		5000 µg		2 ± 1^R D M	7 ± 1^D M	10 ± 2^D M	134 ± 9^D M	24 ± 3^D M
	2-AA	2.5 µg		406 ± 25	292 ± 48	3275 ± 632	4099 ± 56
	2-AA	10.0 µg						264 ± 25

 

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 M R	Densely coloured plate Manual count Reduced background growth

 

Table2     Summary of Experiment II

Study Name: 1664301	Study Code: Harlan CCR 1664301
Experiment: 1664301 HV2 Pre	Date Plated: 19/11/2014
Assay Conditions:	Date Counted: 24/11/2014

 

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	DMSO			11 ± 3	11 ± 3	24 ± 5	173 ± 11	35 ± 2
	Untreated			13 ± 1	10 ± 3	22 ± 2	195 ± 4	39 ± 2
	Pigment Red	10 µg		11 ± 1	11 ± 4	20 ± 7	153 ± 18	34 ± 6
	50:1	33 µg		12 ± 4	10 ± 3	21 ± 5	135 ± 6	34 ± 3
		100 µg		13 ± 3	9 ± 1	20 ± 1	162 ± 12	37 ± 6
		333 µg		14 ± 2	12 ± 4	20 ± 4	165 ± 9	28 ± 6
		1000 µg		11 ± 4^P	12 ± 3^P	23 ± 8^P	136 ± 25^P	28 ± 5^P
		2500 µg		10 ± 2^P M	11 ± 2^P M	23 ± 1^P M	151 ± 19^P M	34 ± 2^P M
		5000 µg		6 ± 1^P M	5 ± 2^P M	17 ± 4^P M	153 ± 12^P M	25 ± 2^P M
	NaN3	10 µg		1043 ± 4			1997 ± 183
	4-NOPD	10 µg				300 ± 16
	4-NOPD	50 µg			72 ± 2
	MMS	2 µL						918 ± 34
With Activation	DMSO			26 ± 5	29 ± 2	43 ± 6	142 ± 21	49 ± 4
	Untreated			13 ± 1	29 ± 1	44 ± 11	160 ± 16	48 ± 9
	Pigment Red	10 µg		28 ± 4	32 ± 6	41 ± 7	136 ± 12	49 ± 6
	50:1	33 µg		24 ± 4	30 ± 8	39 ± 4	148 ± 31	53 ± 5
		100 µg		24 ± 6	28 ± 6	45 ± 3	153 ± 36	49 ± 6
		333 µg		18 ± 5	24 ± 2	43 ± 2	154 ± 17	32 ± 3
		1000 µg		16 ± 1^P	26 ± 3^P	42 ± 9^P	155 ± 19^P	32 ± 1^P
		2500 µg		14 ± 3^P M	21 ± 2^P M	35 ± 3^P M	146 ± 17^P M	23 ± 1^P M
		5000 µg		10 ± 1^P M	14 ± 4^P M	26 ± 3^P M	127 ± 7^P M	21 ± 1^P M
	2-AA	2.5 µg					3058 ± 178
	2-AA	2.5 µg		398 ± 1	308 ± 16
	2-AA	10 µg						728 ± 90
	Congo red	500 µg				542 ± 94

 

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	Precipitate Manual count

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without hamster and rat 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 Pigment Red 50:1 to induce gene mutations according to the plate incorporation assay with rat liver S9 (experiment I), and the pre-incubation test with hamster liver S9 (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the Escherichia coli strain WP2uvrA.

 

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

The test item precipitated in the overlay agar in the test tubes from 33 to 5000 µg/plate in experiment I and from 333 to 5000 µg/plate in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 1000 to 5000 µg/plate in experiment II. 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 nearly all strains used. Reduced background growth was observed in experiment I at 5000 µg/plate in strains TA 1535 and TA 98 in the absence of metabolic activation and from 1000 to 5000 µg/plate in strain TA 1535 in the presence of metabolic activation.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in nearly all test groups. A reduction in the number of revertants (below the indication factor of 0.5), was observed in the presence of metabolic activation at 5000 µg/plate in experiment I in strains TA 1535 and TA 98 and in experiment II in strains TA 1535 and WP2uvrA.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Pigment Red 50:1 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.