Disodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

27-02-2018 - 23-03-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

This study was performed to investigate the potential of Disodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate (CAS no. 17372-87-1) to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate

EC Number:

241-409-6

EC Name:

Disodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate

Cas Number:

17372-87-1

Molecular formula:

C20H6Br4Na2O5

IUPAC Name:

disodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxo-3H-xanthen-9-yl)benzoate

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Eosine
- Molecular formula (if other than submission substance): C20H8Br4O5.2Na
- Molecular weight (if other than submission substance): 691.858 g/mol
- Smiles notation (if other than submission substance): c1(c2c(oc3c1cc(Br)c(c3Br)[O-])c(c(=O)c(c2)Br)Br)c1c(cccc1)C(=O)[O-].[Na+].[Na+]
- InChl (if other than submission substance): 1S/C20H8Br4O5.2Na/c21-11-5-9-13(7-3-1-2-4-8(7)20(27)28)10-6-12(22)17(26)15(24)19(10)29-18(9)14(23)16(11)25;;/h1-6,25H,(H,27,28);;/q;2*+1/p-2
- Substance type: Organic
- Physical state: Solid

Specific details on test material used for the study:

- Name of the test chemical: Disodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate
- IUPAC name: disodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxo-3H-xanthen-9-yl)benzoate
- Molecular formula: C20H8Br4O5.2Na
- Molecular weight: 691.858 g/mol
- Substance type: Organic

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other:

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced S9 metabolic activation system

Test concentrations with justification for top dose:

0.0, 0.050, 0.158, 0.501, 1.582 or 5.0 mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RO water
- Justification for choice of solvent/vehicle: The test chemical was solulble in RO water

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

RO water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-Nitro-o-phenylenediamine (TA 1537, TA 98, without S9); 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)

DURATION
- Preincubation period: Trial I: Not applicable Trial II: 60 min
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Each concentration, including the negative, vehicle and positive controls was tested in triplicate in two independent experiments performed

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data

Rationale for test conditions:

No data

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 TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding vehicle/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 control and vehicle control such an increase is not considered biologically relevant.

Statistics:

No data

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No precipitation was noted at a dose upto 5 mg/plate in the pre-experiment
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations (0.0, 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5.0 mg/plate) were tested for toxicity and mutation induction with 3 plates each (triplicates). The experimental conditions in this pre-experiment were the same as described for the Trial-I (Plate incorporation test). Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

In the pre-experiment, the concentration range of the test item was 0.002 – 5 mg/plate based on the solubility and precipitation test. There was no reduction in colony count as well as in background lawn in treated concentrations 5 (T8) mg/plate – 0.002 (T1) mg/plate) both in absence and in the presence of metabolic activation. Based on the results of pre-experiment following doses were selected for the main study trials: 0.050, 0.158, 0.501, 1.582 and 5 mg/plate, both in the absence (-S9) as well as in the presence of metabolic activation (+S9).

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data

Remarks on result:

other: No mutagenic potential

Any other information on results incl. tables

TABLE1- REVERTANT COUNT FOR PRE-EXPERIMENT

Dose (mg/plate)	R	Without metabolic activation (-S9)		With metabolic activation (+S9)
		TA100	TA 98	TA100	TA 98
NC (0.00)	R1	108	22	128	22
	R2	110	24	124	28
	R3	108	26	126	26
T1 (0.002)	R1	84	12	92	18
	R2	84	14	84	16
	R3	92	12	86	16
T2 (0.005)	R1	86	16	94	20
	R2	90	10	88	22
	R3	94	14	92	16
T3 (0.016)	R1	90	16	96	18
	R2	94	14	102	21
	R3	86	14	102	16
T4 (0.050)	R1	94	20	104	16
	R2	82	16	106	22
	R3	96	18	102	18
T5 (0.158)	R1	100	20	110	20
	R2	92	22	106	20
	R3	96	20	108	22
T6 (0.501)	R1	98	22	110	20
	R2	102	20	110	18
	R3	92	20	112	20
T7 (1.582)	R1	96	22	114	22
	R2	100	22	110	20
	R3	98	18	116	18
T8 (5)	R1	104	22	114	24
	R2	102	24	112	22
	R3	102	24	116	24
PC	R1	1312	1072	1696	968
	R2	1352	1024	1728	912
	R3	1376	1088	1640	1016

NC           =     Negative control

PC            =     Positive control             

R              =     Replicate

T              =     Test concentration (T8: Highest, T1: Lowest)

4-Nitro-o-phenylenediamine [10μg/plate]: TA 98

Sodium azide [10μg/plate]: TA 100,

2-Aminoanthracene [2.5μg/plate]: TA98, TA100

 

TABLE 2 - REVERTANT COUNT IN PLATE INCORPORATION METHOD
(TRIAL I)

Dose (mg/plate)	R	In the Presence of Metabolic Activation (+S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	7	12	22	128	294
	R2	6	12	28	124	284
	R3	8	14	26	126	276
T1 (0.050)	R1	5	10	16	104	230
	R2	6	9	22	106	244
	R3	5	10	18	102	238
T2 (0.158)	R1	7	10	20	110	232
	R2	5	10	20	106	252
	R3	5	10	22	108	246
T3 (0.501)	R1	5	10	20	110	256
	R2	7	12	18	110	232
	R3	6	10	20	112	244
T4 (1.582)	R1	6	10	22	114	266
	R2	7	14	20	110	272
	R3	6	10	18	116	260
T5 (5)	R1	7	12	24	114	282
	R2	6	10	22	112	270
	R3	7	14	24	116	266
PC	R1	150	436	968	1696	1242
	R2	166	452	912	1728	1226
	R3	174	448	1016	1640	1258

 

Dose (mg/plate)	R	In the Absence of Metabolic Activation (-S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	7	14	22	108	282
	R2	6	12	24	110	296
	R3	6	14	26	108	288
T1 (0.050)	R1	5	8	20	94	220
	R2	4	10	16	82	232
	R3	4	10	18	96	220
T2 (0.158)	R1	5	12	20	100	228
	R2	4	10	22	92	240
	R3	6	8	20	96	230
T3 (0.501)	R1	4	12	22	98	242
	R2	5	10	20	102	254
	R3	5	10	20	92	236
T4 (1.582)	R1	6	12	22	96	252
	R2	5	12	22	100	262
	R3	5	10	18	98	246
T5 (5)	R1	6	12	22	104	258
	R2	5	10	24	102	264
	R3	6	14	24	102	260
PC	R1	172	1210	1072	1312	1560
	R2	158	1230	1024	1352	1588
	R3	180	1224	1088	1376	1566

NC= Negative Control,T=Test concentration (T5: Highest, T1: Lowest),R= Replicate

PC= Positive control                                                     2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA 98, TA 100        
2- Aminoanthracene [10μg/plate]:TA 102                                           Sodium azide [10μg/plate]: TA 1535, TA 100                                              

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98[10μg/plate]        Methyl methanesulfonate [4μl/plate]: TA 102

 

TABLE 3 - REVERTANT COUNT IN PRE-INCUBATION METHOD (TRIAL II)

Dose (mg/plate)	R	In the Presence of Metabolic Activation (+S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	6	12	27	114	256
	R2	5	15	24	116	264
	R3	7	14	23	110	257
T1 (0.050)	R1	5	10	21	98	238
	R2	4	12	22	100	243
	R3	4	12	24	96	232
T2 (0.158)	R1	5	13	23	102	242
	R2	4	9	21	105	233
	R3	5	13	21	107	239
T3 (0.501)	R1	6	10	24	100	246
	R2	5	14	25	106	240
	R3	4	13	22	110	242
T4 (1.582)	R1	6	13	23	108	239
	R2	5	11	25	109	245
	R3	5	14	24	113	254
T5 (5)	R1	6	13	25	113	252
	R2	6	12	24	110	249
	R3	5	14	24	109	247
PC	R1	176	468	1406	1408	1712
	R2	188	456	1452	1386	1734
	R3	196	472	1436	1362	1728

 

 

Dose (mg/plate)	R	In the Absence of Metabolic Activation (-S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	6	14	25	109	248
	R2	6	12	24	118	254
	R3	7	14	26	112	262
T1 (0.050)	R1	5	10	20	98	232
	R2	4	10	22	114	240
	R3	4	10	24	106	245
T2 (0.158)	R1	5	10	23	101	238
	R2	4	10	21	110	242
	R3	5	12	23	113	236
T3 (0.501)	R1	6	12	24	100	244
	R2	4	12	22	116	239
	R3	5	10	22	115	236
T4 (1.582)	R1	5	12	23	108	245
	R2	6	10	21	116	237
	R3	5	14	25	110	250
T5 (5)	R1	6	12	24	112	252
	R2	5	12	23	109	246
	R3	6	14	25	115	240
PC	R1	182	1058	886	1202	1528
	R2	194	1112	904	1244	1536
	R3	206	1106	938	1228	1504

NC= Negative Control,T =Test concentration (T5: Highest, T1: Lowest), R= Replicate

PC= Positive control                                                                       2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA98, TA100        
2-Aminoanthracene [10μg/plate]:TA 102                                              Sodium azide [10μg/plate]: TA 1535, TA 100,                                            

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate]        Methyl methanesulfonate [4μl/plate]: TA 102

 

 

 

 

TABLE 4 -    MEAN REVERTANT COUNT IN PLATE INCORPORATION METHOD (TRIALI)

Dose (mg/plate)	In the presence of Metabolic Activation (+S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	7.00	1.00	12.67	1.15	25.33	3.06	126.00	2.00	284.67	9.02
T1 (0.050)	5.33	0.58	9.67	0.58	18.67	3.06	104.00	2.00	237.33	7.02
T2 (0.158)	5.67	1.15	10.00	0.00	20.67	1.15	108.00	2.00	243.33	10.26
T3 (0.501)	6.00	1.00	10.67	1.15	19.33	1.15	110.67	1.15	244.00	12.00
T4 (1.582)	6.33	0.58	11.33	2.31	20.00	2.00	113.33	3.06	266.00	6.00
T5 (5)	6.67	0.58	12.00	2.00	23.33	1.15	114.00	2.00	272.67	8.33
PC	163.33	12.22	445.33	8.33	965.33	52.05	1688.00	44.54	1242.00	16.00

 

Dose (mg/plate)	In the Absence of Metabolic Activation (-S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	6.33	0.58	13.33	1.15	24.00	2.00	108.67	1.15	288.67	7.02
T1 (0.050)	4.33	0.58	9.33	1.15	18.00	2.00	90.67	7.57	224.00	6.93
T2 (0.158)	5.00	1.00	10.00	2.00	20.67	1.15	96.00	4.00	232.67	6.43
T3 (0.501)	4.67	0.58	10.67	1.15	20.67	1.15	97.33	5.03	244.00	9.17
T4 (1.582)	5.33	0.58	11.33	1.15	20.67	2.31	98.00	2.00	253.33	8.08
T5 (5)	5.67	0.58	12.00	2.00	23.33	1.15	102.67	1.15	260.67	3.06
PC	170.00	11.14	1221.33	10.26	1061.33	33.31	1346.67	32.33	1571.33	14.74

NC= Negative Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100                  Methyl methanesulfonate [4μl/plate]: TA 102

2-Aminoanthracene [10μg/plate]:TA 102                                           

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98 [10μg/plate]

 

TABLE 5 -    MEAN REVERTANT COUNT IN PRE-INCUBATIONMETHOD
(TRIAL II)

Dose (mg/plate)	In the presence of Metabolic Activation (+S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	6.00	1.00	13.67	1.53	24.67	2.08	113.33	3.06	259.00	4.36
T1 (0.050)	4.33	0.58	11.33	1.15	22.33	1.53	98.00	2.00	237.67	5.51
T2 (0.158)	4.67	0.58	11.67	2.31	21.67	1.15	104.67	2.52	238.00	4.58
T3 (0.501)	5.00	1.00	12.33	2.08	23.67	1.53	105.33	5.03	242.67	3.06
T4 (1.582)	5.33	0.58	12.67	1.53	24.00	1.00	110.00	2.65	246.00	7.55
T5 (5)	5.67	0.58	13.00	1.00	24.33	0.58	110.67	2.08	249.33	2.52
PC	186.67	10.07	465.33	8.33	1431.33	23.35	1385.33	23.01	1724.67	11.37

 

Dose (mg/plate)	In the Absence of Metabolic Activation (-S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	6.33	0.58	13.33	1.15	25.00	1.00	113.00	4.58	254.67	7.02
T1 (0.050)	4.33	0.58	10.00	0.00	22.00	2.00	106.00	8.00	239.00	6.56
T2 (0.158)	4.67	0.58	10.67	1.15	22.33	1.15	108.00	6.24	238.67	3.06
T3 (0.501)	5.00	1.00	11.33	1.15	22.67	1.15	110.33	8.96	239.67	4.04
T4 (1.582)	5.33	0.58	12.00	2.00	23.00	2.00	111.33	4.16	244.00	6.56
T5 (5)	5.67	0.58	12.67	1.15	24.00	1.00	112.00	3.00	246.00	6.00
PC	194.00	12.00	1092.00	29.60	909.33	26.41	1224.67	21.20	1522.67	16.65

NC= Negative Control, T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100

2-Aminoanthracene [10μg/plate]: TA 102

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate]

Methyl methanesulfonate: [4μl/plate]: TA 102

Applicant's summary and conclusion

Conclusions:

Test substance did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Executive summary:

Ames assay was performed to investigate the potential of test substance to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0, 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5.0 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0.0, 0.050, 0.158, 0.501, 1.582, 5.0 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment test substance at any dose level in both the confirmatory trials, neither in the presence nor in the 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 spontaneous reversion rates in the negative, positive controls were within the range of our historical data. The positive controls used for various strains showed a distinct increase in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method.

Conclusion

In conclusion, it is 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 frame shifts in the genome of the strains used.