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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AMES Assay

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.

In vitro chromosome aberration study in mammalian cell

The test chemical did not induce chromosomal aberrations in the cultured in the mammalian cell line with and without metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Link to relevant study records

Reference

Reference 1

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

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

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

Target gene:

Histidine

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

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

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

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

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.

 

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on the data from various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

WoE derived based on the experimental data from various test chemicals publication.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: McCoy's 5A (modified) medium buffered with 20 mM HEPES and supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 IU/ml penicillin, and 50 µg/ml streptomycin
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

The rat liver microsomal fraction was prepared from Aroclor 1254-induced male Sprague-Dawley rats and was combined with cofactors and culture medium to form the metabolic activation system.

Test concentrations with justification for top dose:

1,Without S9: 0.0, 49.8000, 150.0000, 499.0000 µg/mL

With S9: 0.0, 500.0000, 1500.0000, 5000.0000 µg/mL

2,125,400 and 1250 µg/mL

Vehicle / solvent:

1,- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: The test chemical was soluble in water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

1,METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data available
- Exposure duration:
Without S9: 8 hrs
With S9: 2 hrs
- Expression time (cells in growth medium):
Without S9: 10-10.5 hrs
With S9: 12 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): Standard harvest time: 10-14 hr after addition of BrdUrd

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: Only one trial was performed

NUMBER OF CELLS EVALUATED: 100-200 cells were scored

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available

OTHER: No data available

2,Details on test system and conditions
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: The chemical treatment periods were appoximately 25 hr without S9 and 2 hr with S9.
- Expression time (cells in growth medium): 25-26 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): 14 hour

SELECTION AGENT (mutation assays): After staining for 10 min in “concentrated” Hoechst 33258 (5 pg/ml in pH 6.8 buffer) and exposure to “black light” at 55 to 60°C for about 5 min, slides were stained in Giemsa
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: 50 cells per dose were scored from the three highest doses

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: Cells were collected by mitotic shake off for evaluation
All types of aberrations were recorded separately, but for data analysis they were grouped into categories of “simple” (breaks and terminal deletions), “complex” (exchanges and rearrangements), “other” (includes pulverized chromosomes), and “total. ” Gaps and endoreduplications were recorded but were not included in the totals. We did not score aberrations in polyploidy cells but used metaphases with 19-23 chromosomes (the modal number being 21).

Rationale for test conditions:

No data

Evaluation criteria:

Selection of cells for scoring was based on well-spread chromosomes with good morphology and a chromosome number of 21 ± 2. Cells were analyzed for the following categories of chromosomal aberrations: “simple,” defined as a chromatid gap, break, fragment, and deletion or chromosome gap,break, or double minutes; “complex,” defined as interstitial deletions, triradials, quadriradials, rings, and dicentric chromosomes; and “other,” defined as pulverized chromosomesor cells with greater than 10 aberrations. Chromatid and chromosome gaps were recorded but were not used in the analysis.

A positive response at a single dose was designated “ + W”, weak evidence for clastogenicity. If there was a strong trend as the result of a large increase in ABs at a single dose only, we designated the result ‘‘ + W*”. A test was designated “ + ” if at least two doses gave significantly increased responses.

Statistics:

A binomial sampling assumption as described by Margolin et al. was used to examine absolute increases in ABs over solvent control levels at each dose. The P values were adjusted by Dunnett’s method to take into account the multiple dose comparisons. Only the “total” percent cells with aberrations were analyzed, and a positive response was defined as one for which the adjusted P value was <0.05.

Species / strain:

Chinese hamster Ovary (CHO)

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

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

(CHO-W-B1)

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:

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 data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: Test concentrations for the AB assays were empirically chosen based on toxicity and cell cycle delay as noted in the SCE experiments. At least five
concentrations of the test chemical were selected; the concentrations were spaced using two merged half-log scales (e.g., 1,000, 500, 300, 150, 100, etc.), and the highest concentrations analyzed were those yielding a sufficient number of suitable metaphase cells. The concentrations analyzed generally covered a one-log range.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce chromosomal aberrations in the cultured in the mammalian cell line with and without metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Data available for the various test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. Approximately 24 hr before chemical treatment, cultures were initiated at a density of 1.75 X 10⁶cells/75 cm²flask. In the AB trials without S9, the cultures were treated with the test chemical in medium for 8 hr, washed to remove the test chemical, and treated with colcemid M) for 2-2.5 hr before cell harvest. In the experiments with activation, cultures were exposed to the test chemical in serum free medium with S9 and cofactors for 2 hr, washed to remove the test chemical and S9, and incubated at 37°C with fresh medium for 8 hr. Colcemid was then added, andthe cells were harvested 2 hr later. Thus the total durations of the nonactivated and activated AB experiments were 10hr and 12 hr, respectively, to give 10 hr growth in medium with serum for each experiment. For ABs, slides were stained in 5% Giemsa for 5 min. In early studies, one hundred cells were scored for each ofthree concentrations: the highest test concentration in whichsufficient metaphase cells could be scored and the next two lower concentrations, covering a one-log range. For later studies, 200 cells per dose were scored; however, fewer cells were scored if a test chemical produced a strong positive response or the chemical was toxic. Cells were analyzed for the following categories of chromosomal aberrations: “simple,” defined as a chromatid gap, break, fragment, and deletion or chromosome gap,break, or double minutes; “complex,” defined as interstitial deletions, triradials, quadriradials, rings, and dicentric chromosomes; and “other,” defined as pulverized chromosomesor cells with greater than 10 aberrations. Chromatid and chromosome gaps were recorded but were not used in the analysis. The test chemical did not induce chromosome aberrations in the CHO-LB cell line in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In another study, in vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The test chemical was studied at a dose level of 0, 125, 400 and 1250 µg/mL in the absence and presence of S9 using Chinese hamster ovary cells (CHO-W-B1).Cells were exposed to the test chemical for 2 hr in the presence of S9 or throughout the incubation period without S9. 100 cells were scored from each of the three highest dose groups having sufficient metaphases for analysis. All types of aberrations were recorded separately, but for data analysis they were grouped into categories of “simple” (breaks and terminal deletions), “complex” (exchanges and rearrangements), “other” (includes pulverized chromosomes), and “total”. Gaps and endo-reduplications were recorded but were not included in the totals. Polyploid cells were not scored but used metaphases with 19-23 chromosomes (the modal number being 21). Based on the results noted, the test chemical did not induce chromosome aberrations in the Chinese hamster ovary cells (CHO-W-B1) in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Based on the data available, the test chemical did not induce chromosomal aberrations in the cultured in the mammalian cell line with and without metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Data for test chemicals was reviewed to determine the mutagenic nature of disodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxo-3H-xanthen-9-yl)benzoate (17372-87-1). The studies are as mentioned below:

AMES Assay

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.

 

Test substance was tested for mutagenic potential with the Salmonella/ mammalian-microsome test. The 2 ml of liquid top agar was cooled to 45°C and 0.1 ml of a broth culture of microorganism and test substance at a dose level of 10 -250 mg in volumes of ≤ 0.4 ml of DMSO was added prior to placing on minimal agar plates. After 48 h incubation at 37°C, the colonies which reverted to the prototroph were counted and compared to counts on the control plate (containing no test substance) to demonstrate mutagenicity or toxicity. Materials which caused a 2-fold increase of revertants, as compared to the number of spontaneous revertants on the control plates, were denoted as mutagens. Those which reduced the number of revertants were considered inhibitory. The test compound did not induce mutation in Salmonella typhimurium TA98, TA1537, TA100, TA1535 and hence is negative for gene mutation in vivo.

 

Salmonella/ mammalian-microsome test (Spot test) was performed to evaluate the mutagenic nature of the test compound. The spot test was used to screen the test material for potential mutagenicity. The test material was placed in the center of the plate. Eosin was tested with and without the S9 mix. Inhibition of the bacterium was indicated by a clearing of the background lawn in a zone surrounding the test material. Mutagenicity was indicated by a clustering of revertant colonies directly around the test material or at the edge of the inhibitory zone. A known mutagen, Captan, was used as a reference mutagen. The test compound did not induce mutation in Salmonella typhimurium TA98, TA1537, TA100, TA1535) in the spot test performed and hence is negative for gene mutation in vitro.

Gene mutation toxicity study was performed to determine the mutagenic nature of test substance. The study was performed using Salmonella typhimurium strains TA98, TA97, TA100, TA1537 and TA1535 in the presence and absence of S9 metabolic activation system. The chemical was dissolved in water as solvent and used at dose levels 0, 100, 333, 666, 1000, 1666, 3333, 6666, 10000, 15000 µg/plate by the preincubation method. The doses were selected on the basis of preliminary dose range finding study and concurrent solvent and positive controls were included in the study. The test compound did not induce mutation in the Salmonella typhimurium tester strains TA100, TA1537 and TA98 in the presence and absence of S9 metabolic activation system. It however induced gene mutation in the tester strains TA1535 and TA97 in the presence of S9 metabolic activation system.

Salmonella/mammalian microsome assay was performed to evaluate the mutagenic nature of the test compound. The dye was dissolved in test substance and up to 0.2 ml was introduced into 2.5 ml of the tempered top agar together with 0.1 ml Salmonella typhimurium broth suspension and 0.25 ml Aroclor 1254 induced rat liver S9. The mixtures was plated on 20 ml of Vogel-Bonner E bottom agar in the usual fashion and incubated for 3 days at 35°. Each agent was tested with all 5 basic tester strains (TA1535, TA100, TA1537, TA1538, TA98) with and without microsomal activation at 3 or more concentrations (0, 50, 100 or 500 µg/plate). The test compound did not show any mutagenic activity in the Salmonella typhimurium tester strains TA1535, TA100, TA1537, TA1538, TA98 in the presence and absence of S9 metabolic activation system and hence is not mutagenic in vitro.

In vitro gene mutation assay in mammalian cell

In vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Mouse lymphoma L5178Y cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in ethanol and used at dose level of 0, 5, 10, 20, 30 , 40 or 60 nL/mL (trial 1), 0, 20, 30, 40, 50, 60 or 80 nL/mL (trial 2), 0, 10, 20, 30, 40, 60 or 80 nL/mL (trial 3) or 0, 10, 20, 30 or 40 nL/mL (trial 4) without S9 and 0, 30, 40, 50, 60, 80 or 100 nL/mL (trial 1) and 0, 30, 40, 50, 60, 80 or 100 nL/mL (trial 2) with S9. Mouse lymphoma L5178Y cells were maintained at 37˚C as suspension cultures in supplemented Fischer's medium; normal cycling time was approximately 10 hours. To reduce the number of spontaneously occurring trifluorothymidine-resistant cells, subcultures were exposed to medium containing THMG (thymidine, hypoxanthine, methotrexate, and glycine) for 1 day, to medium containing THG (thymidine, hypoxanthine, and glycine) for 1 day, and to normal medium for 3 to 5 days. For cloning, the horse serum content was increased and Noble agar was added. All treatment levels within an experiment, including concurrent positive and solvent controls, were replicated. Treated cultures contained 6 x 106cells in 10 mL medium. This volume included the S9 fraction in those experiments performed with metabolic activation. Incubation with butyl benzyl phthalate continued for 4 hours, at which time the medium plus butyl benzyl phthalate was removed and the cells were resuspended in fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was monitored so that log phase growth was maintained. After the 48-hour expression period, 3 x 106cells were plated in medium and soft agar supplemented with trifluorothymidine (TFT) for selection of TFT-resistant (TK-1) cells; 600 cells were plated in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37" C in 5% CO2 for 10 to 12 days. The test was initially performed without 59.' If a clearly positive response was not obtained, the test was repeated using freshly prepared S9 from the livers of Aroclor 1254 induced maleFischer344/N rats. Based on the observations made, thetest chemical did not induce gene mutation inMouse lymphoma L5178Y cells in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.

 

In vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Mouse lymphoma L5178Y cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in ethanol and used at dose level of 0, 1.25, 2.5, 3.75, 5.0 or 7.5µg/mL (trial 1), 0, 2, 3, 4, 5, 6 or 8µg/mL (trial 2), 0, 2, 3, 4, 5, 6, 8 or 10µg/mL (trial 3) without S9 and 0, 2.5, 5.0, 7.5, 10 or 15µg/mL (trial 1) with S9. Mouse lymphoma L5178Y cells were maintained at 37˚C as suspension cultures in supplemented Fischer's medium; normal cycling time was approximately 10 hours. To reduce the number of spontaneously occurring trifluorothymidine-resistant cells, subcultures were exposed to medium containing THMG (thymidine, hypoxanthine, methotrexate, and glycine) for 1 day, to medium containing THG (thymidine, hypoxanthine, and glycine) for 1 day, and to normal medium for 3 to 5 days. For cloning, the horse serum content was increased and Noble agar was added. All treatment levels within an experiment, including concurrent positive and solvent controls, were replicated. Treated cultures contained 6 x 106cells in 10 mL medium. This volume included the S9 fraction in those experiments performed with metabolic activation. Incubation with butyl benzyl phthalate continued for 4 hours, at which time the medium plus butyl benzyl phthalate was removed and the cells were resuspended in fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was monitored so that log phase growth was maintained. After the 48-hour expression period, 3 x 106cells were plated in medium and soft agar supplemented with trifluorothymidine (TFT) for selection of TFT-resistant (TK-1) cells; 600 cells were plated in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37˚C in 5% CO2 for 10 to 12 days. Based on the observations made, thetest chemical did not induce gene mutation in Mouse lymphoma L5178Y cells in the presence S9 metabolic activation system. It however induced gene mutation I the cell line in the absence of S9 metabolic activation system.

Based on the data summarized disodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxo-3H-xanthen-9-yl)benzoate (17372-87-1) is expected to not induce gene mutation In vitro. Hence it is not likely to be mutagenic in vitro.

Justification for classification or non-classification

Thus based on the above annotation and CLP criteria for target substance disodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxo-3H-xanthen-9-yl)benzoate (17372-87-1) does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro.