3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The Ames Salmonella/mammalian-microsome assay was used to evaluate the bacterial mutagenicity of 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (Phloxine B) (CAS Number: 18472-87-2) .3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (Phloxine B) did not showed any mutagenic activity at concentrations up to 100 μg/plate in S.typhimurium strains TA1535, TA100, TA1537, TA1538 and TA98 in the presence and absence of S9 metabolic activation system and hence, according to CLP criteria, it can be concluded that 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (Phloxine B) is non genotoxic.

Chromosomal Aberration study:

C.I Pigment red 23 (30 to microgram / ml) was found to be negative for induction of chromosomal aberrations in Chinese hamster ovary cells, with and without metabolic activation.

Gene Mutation Studies in Mammalian cells

Erythrosine failed to induce gene mutation in Bacillus subtilis H17 (Rec+) and M45 (Rec-) in the rec assay.

Under the test conditions the test item 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (Acid red 92) (CAS No. 18472-87-2) was considered to be non mutagenic in this assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Data is from peer-reviewed scientific journal

Principles of method if other than guideline:

Genetic toxicity were performed for erythrosine in Bacillus subtilis/: H17(rec+)and M45(Rec-) using Bacillus subtilis recombination assay.

GLP compliance:

not specified

Type of assay:

Bacillus subtilis recombination assay

Species / strain / cell type:

other: Bacillus subtilis/: H17(rec+)and M45(Rec-)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

not specified

Test concentrations with justification for top dose:

10µM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: as impregnation on paper disk
DURATION
- Exposure duration: 24 hrs
- Expression time (cells in growth medium): 24 hrs

Evaluation criteria:

The length of growth inhibition of the bacterial streak was measured

Statistics:

No data available

Species / strain:

other: Bacillus subtilis/: H17(rec+)and M45(Rec-)

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Erythrosine failed to induce gene mutation in Bacillus subtilis H17 (Rec+) and M45 (Rec-) in the rec assay.

Executive summary:

Erythrosine was tested for gene mutation in vitro in the bacterium Basillus subtilis H17 (Rec⁺) and M45 (Rec^-) in the rec assay performed.

 

Agar plates were streaked with inocula of two strains and paper disk 8 mm of diameter, immersed with 10 µM of dye in DMSO solution, was placed on the surface so as to cover the beginning of the bacterial streaks. After incubation for 24 hrs at 37^◦C, the length of growth inhibition of the bacterial streak was measured.

 

The length of growth inhibition was found to be 2mm in Rec⁺and 3mm in Rec^-strains.

 

Erythrosine failed to induce gene mutation in Bacillus subtilis H17 (Rec⁺) and M45 (Rec^-) in the rec assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

Data is from secondary source

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Fuchsia Red
Molecular formula: C20H2Br4Cl4O5Na2
- Molecular weight: 829.66 g/mol
- Substance type: Organic
- Physical state: Red powder
- Purity: 99.5 area % (HPLC), 85 % (w/w) (NMR, calculated as sodium salt).

Target gene:

TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Naphthoflavone treated rat liver homogenate (S 9)

Test concentrations with justification for top dose:

Exp.1: -S9: 5, 10, 20, 30, 40 μg/ml
+S9: 5, 10, 20, 40, 60 μg/ml

Exp.2: -S9: 19.5, 39, 78, 156 μg/ml

Vehicle / solvent:

No data available.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Exp.1: (-S9): 4 hours
(+S9): 4 hours
Exp.2: -(S9): 24 hours

NUMBER OF REPLICATIONS: Yes, two

Evaluation criteria:

No data available.

Statistics:

No data available.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Toxicity: in a preliminary experiment a relevant toxicity was observed at 39.1 μg/ml in all conditions at 4 h of treatment; in the 24 h of treatment a severe toxicity was observed at 312.5 μg/ml. The doses for the main experiments were chosen on the base of a preliminary toxicity experiment.

Mutagenicity:
A) positive controls
MMS (-S9): 4 h: 1st culture: small colonies: 164 (51); large colonies:114 (51); 2nd culture: small colonies: 225(53),large colonies:157 (49).(in brackets the untreated control values)

24 h: small colonies: 1247 (61); large colonies: 346 (43)
3-MC (+S9):4 h.1st culture: small colonies: 260 (67); large colonies: 311 (67);

2nd culture: small colonies: 137 (44); large colonies: 121 (40).

The results obtained with the positive controls make acceptable the study.

B) Treated cells
A mutagenic effect observed in one culture was not repeated in the replicate culture.

Conclusions:

Under the test conditions the test item 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (Acid red 92) (CAS No. 18472-87-2) was considered to be non mutagenic in this assay.

Executive summary:

In vitro Mammalian Cell Gene Mutation assay was performed to evaluate the mutagenic potential of the test compound 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (Acid red 92) (CAS No. 18472-87-2) in the Mouse lymphoma L5178 cells (TK locus).

 

Two experiments were performed: at different concentration and for different time duration

 Exp.1:-S9: 5, 10, 20, 30, 40 μg/ml (4 hours)

            +S9: 5, 10, 20, 40, 60 μg/ml (4 hours)

 Exp.2:-S9: 19.5, 39, 78, 156 μg/ml (24 hours).

 

In a preliminary experiment a relevant toxicity was observed at 39.1 μg/ml in all conditions at 4 h of treatment; in the 24 h of treatment a severe toxicity was observed at 312.5 μg/ml. The doses for the main experiments were chosen on the base of a preliminary toxicity experiment. In treated cell a mutagenic effect observed in one culture was not repeated in the replicate culture. Therefore under the test conditions tested the test item 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (Acid red 92) (CAS No. 18472-87-2) was considered to be non mutagenic in this assay. 

 

In accordance with the CLP classification, the test material does not classify as a gene mutant.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Data is from peer-reviewed journal.

Qualifier:

no guideline available

Principles of method if other than guideline:

The Ames Salmonella/mammalian-microsome assay was used to evaluate the bacterial mutagenicity of 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo- 6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (Phloxine B) (CAS Number: 18472-87-2) .

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Phloxine B
- Molecular formula: C20H2Br4Cl4O5Na2
- Molecular weight: 829.66 g/mol
- Substance type: Organic
- Physical state: Red powder
- Purity: 7-9%

Target gene:

Histidine operon

Species / strain / cell type:

S. typhimurium, other: TA1535, TA100, TA1537, TA1538, TA98

Metabolic activation:

with and without

Metabolic activation system:

Liver-microsome preparation from rats injected with Aroclor 1254, 0.25 ml "S-9 mix"-plate with 0.2 ml S9/ml 'S9 mix'.

Test concentrations with justification for top dose:

10,50 and 100 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

ethylmethanesulphonate

methylmethanesulfonate

other:

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar

DURATION
- Preincubation period: No data available
- Exposure duration:3 days

Evaluation criteria:

Mutations in Salmonella typhimurium i.e. Number of His + Revertants/plate

Statistics:

Rounded mean ± standard deviation

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other:

Remarks:

slight toxicity was observed at top dose

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other:

Remarks:

slight toxicity was observed at top dose

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other:

Remarks:

slight toxicity was observed at top dose

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other:

Remarks:

slight toxicity was observed at top dose

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other:

Remarks:

slight toxicity was observed at top dose

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA: Yes, historical negative control were used.

Remarks on result:

other: no mutagenic potential

Table: Mutagenicity Testing Of D And C Red No. 27 With The Salmonella/Microsome Test

Chemical	Dose	Microsome activation	Number of His^+Revertants/plate
			TA1535	TA100	TA1537	TA1538	TA98
Negative controls
A)		-	35±22 (265)	98 ± 26 (271)	8 ± 5 (297)	13 ± 5 (297)	33 ± 20 (282)
		±	20 ± 19 (239)	90 ± 25 (252)	11 ± 4 (283)	34 ± 15 (280)	27 ± 13 (308)
B) This study		-	42 ± 14 (9)	126 ± 24 (12)	11 ± 5 (9)	13 ± 2 (9)	24 ± 8 (9)
		±	20 ± 19 (8)	104 ± 27 (11)	12 ± 4 (9)	24 ± 6 (12)	28 ± 10 (12)
Positive controls
EMS	2µL	-	440	-	-	-	-
MMS	1µL	-	-	4860	-	-	-
9 AA	100	-	-	-	103	-	-
Anthragallol	50	-	-	-	-	47	81
2- Anthramine	1	±	824	7600	488	6400	7600
D and C Red No. 27	10	-	43	113	8	12	14^h
		±	7	114	16^h	30	24
	50	-	51	30	9	18	11
		±	6	98	12	19	23
	100	-	41	109	7	11	11^h
		±	16	90	5	19	24

 

Conclusions:

The substance 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxo xanthen-9-yl)benzoic acid (Phloxine B) did not showed any mutagenic activity at concentrations up to 100 μg/plate in S.typhimurium strains TA1535, TA100, TA1537, TA1538 and TA98 in the presence and absence of S9 metabolic activation system and hence, it can be concluded that 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (Phloxine B) is non genotoxic.

Executive summary:

The Ames Salmonella/mammalian-microsome assay was used to evaluate the bacterial mutagenicity of 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (Phloxine B) (CAS Number: 18472-87-2) .3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (Phloxine B) did not showed any mutagenic activity at concentrations up to 100 μg/plate in S.typhimurium strains TA1535, TA100, TA1537, TA1538 and TA98 in the presence and absence of S9 metabolic activation system and hence, according to CLP criteria, it can be concluded that 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (Phloxine B) is non genotoxic.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Remarks:

Read across based on category approach

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

The study contains experimental data of a read-across analogue

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was conducted to assess the mutagenicity of C.I. Pigment Red 23. To access mutagenicity, chromosomal aberration test was conducted.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Name- C.I Pigment Red
Physical form- Bluish red powder
Purity- > 96%
Solubility- It is insoluble in water, poorly soluble in ethanol or xylene, and highly soluble in 5% sodium carbonate solution or in oleic acid

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (metabolic activation enzymes and cofactors from Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster liver)

Test concentrations with justification for top dose:

100 microgram/ml

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

no

Details on test system and experimental conditions:

In the Abs test without S9, cells were incubated in McCoy's 5A medium with C.1. Pigment Red 23 for 10 hours; Colcemid was added and incubation continued for 2 to 3 hours. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa. For the Abs test with S9, cells were treated with C.1. Pigment Red 23 and S9 for 2 hours, after which the treatment medium was removed and the cells incubated for 11 hours in fresh medium, with Colcemid present for the final 2 hours. Cells were harvested in the same manner as for the treatment without S9.
The harvest time for the Abs test was based on the cell cycle information obtained in the
SCE test; if cell cycle delay was anticipated, the incubation period was extended.

Evaluation criteria:

Cells were selected for scoring on the basis of good morphology and completeness of karyotype
(21 ± 2 chromosomes). All slides were scored blind and those from a single test were read by the same person. For the SCE test, usually 50 second-division metaphase cells were scored for frequency of SCEs per cell from each dose level; 200 first-division metaphase cells were scored at each dose level for the Abs test. Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations).

Statistics:

Abs data are presented as percentage of cells with aberrations. As with the SCE assay, both the dose response curve and individual dose points were statistically analyzed. A statistically significant (P

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Remarks on result:

other: Non Mutagenic

Conclusions:

C.I Pigment red 23 (30 to microgram / ml) was found to be negative for induction of chromosomal aberrations in Chinese hamster ovary cells, with and without metabolic activation.

Executive summary:

The study was conducted to access the mutagenicity of C.1. Pigment Red 23. Chromosomal aberration was assessed in Chinese hamster ovary cells, with and without metabolic activation. In the Abs test without S9, cells were incubated in McCoy's 5A medium with C.1. Pigment Red 23 for 10 hours; Colcemid was added, and incubation continued for 2 to 3 hours. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa. For the Abs test with S9, cells were treated with C.1. Pigment Red 23 and S9 for 2 hours, after which the treatment medium was removed and the cells incubated for 11 hours in fresh medium, with Colcemid present for the final 2 hours. Cells were harvested in the same manner as for the treatment without S9. The harvest time for the Abs test was based on the cell cycle information obtained in the SCE test; if cell cycle delay was anticipated, the incubation period was extended.

Statistical analyses were conducted on both the slopes of the dose-response curves and the individual dose points. Abs data are presented as percentage of cells with aberrations. As with the SCE assay, both the dose response curve and individual dose points were statistically analyzed. A statistically significant (P < O.05) difference for one dose point was considered weak evidence for a positive response; significant differences for two or more doses indicated the trial was positive.

C.I Pigment red 23 was found to be negative for induction of chromosomal aberrations in Chinese hamster ovary cells, with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The substance 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (Acid Red 92) (CAS Number: 18472-87-2) is non mutagenic in the In vivo Mammalian Erythrocyte Micronucleus Test performed.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

Data is from secondary source

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

In vivo Mammalian Erythrocyte Micronucleus Test of Acid Red 92 in NMRI mouse was conducted.

GLP compliance:

not specified

Type of assay:

micronucleus assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Fuchsia Red WR 802177
- Molecular formula: C20H2Br4Cl4O5Na2
- Molecular weight: 829. 6398 g/mol
- Substance type: Organic
- Physical state: Red powder
- Purity: 99.5 area % (HPLC), 85 % (w/w) (NMR, calculated as sodium salt).

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

No data available.

Route of administration:

intraperitoneal

Vehicle:

No data available.

Details on exposure:

No data available

Duration of treatment / exposure:

Exp.1-
24 hrs- 25, 50, 100 mg/kg
48 hrs-100 mg/kg
Exp.2-100 mg/kg
Exp.3- 200 mg/kg
Exp.4-150 mg/kg

Remarks:

Doses / Concentrations:Exp.1- 24 hrs- 25, 50, 100 mg/kg 48 hrs-100 mg/kg Exp.3- 200 mg/kg Exp.4-150 mg/kg Basis:

No. of animals per sex per dose:

Total 24 animals-12 male and 12 female
Exp.1-25 mg/kg: 2male and 2 female
50 mg/kg: 2male and 2 female
100 mg/kg: 2male and 2 female
Exp.2-100 mg/kg: 2male and 2 female
Exp.3- 200 mg/kg: 2male and 2 female
Exp.4- 150 mg/kg: 2male and 2 female

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive controls: Cyclophosphamide
- Justification for choice of positive control(s): No data available
- Route of administration: No data available
- Doses / concentrations: No data available

Tissues and cell types examined:

Micronucleates (MN) produced in the polychromatic erythrocytes were observed

Details of tissue and slide preparation:

No data available

Evaluation criteria:

Frequency of MN was noted

Statistics:

No data available

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: No data available
- Solubility: No data available
- Clinical signs of toxicity in test animals: No data available
- Evidence of cytotoxicity in tissue analyzed: No data available
- Rationale for exposure: No data available
- Harvest times: No data available
- High dose with and without activation: No data available
- Other: No data available

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): No data available
- Induction of micronuclei (for Micronucleus assay): In the treated animals with 100 mg/kg a percentage of MN 0.150 (24h) and 0.110 (48h) was observed: these values, although higher than the control, had a p > 0.34. reduction of PCE was observed, thus indicating a cytotoxic effect of the test item in the bone marrow cells.
- Ratio of PCE/NCE (for Micronucleus assay): No data available
- Appropriateness of dose levels and route: No data available
- Statistical evaluation: No data available

Conclusions:

The substance 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (Acid Red 92) (CAS Number: 18472-87-2) is non mutagenic in the In vivo Mammalian Erythrocyte Micronucleus Test performed.

Executive summary:

Genetic toxicity in vivo study was performed for the test chemical3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (Acid Red 92) (CAS Number: 18472-87-2) on male and female NMRI mice. Acid red 92 was injected intraperitoneally in mice and the number of micronucleus formed in the polychromatic erythrocytes was noted. 

The positive control (CPA) induced 2.030 % of MN in PCEs (0.08 % in the control animals: significance p 0.0040). 

In the treated animals with 100 mg/kg a percentage of MN 0.150 (24h) and 0.110 (48h) was observed: these values, although higher than the control, had a p > 0.34. A reduction of PCE was observed, thus indicating a cytotoxic effect of the test item in the bone marrow cells. 

Acid Red 92 is non mutagenicintheIn vivo Mammalian Erythrocyte Micronucleus Test performed. 

In accordance with the CLP classification, 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (Acid Red 92) (CAS Number: 18472-87-2)is non mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The potential genotoxicity of 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (CAS Number: 18472-87-2) was evaluated using in vitro and in vivo assays. Various peer reviewed publications for target substance have been reviewed and are summarized to determine the mutagenic potential of 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid:

In Vitro

The Ames Salmonella/mammalian-microsome assay was conducted by J.P. Brown et al. (Mutation Research, 66 ,181-185, 1979) to evaluate the bacterial mutagenicity of 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (Phloxine B) (CAS Number: 18472-87-2) .3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (Phloxine B) did not showed any mutagenic activity at concentrations up to 100 μg/plate in S.typhimurium strains TA1535, TA100, TA1537, TA1538 and TA98 in the presence and absence of S9 metabolic activation system.

 

Further, gene toxicity study was published in a SCCNFP report (COLIPA n° C53, 2004). The study was performed using reverse mutation assay on Salmonella typhimurium strains TA 1535; TA 1537; TA 98; TA 100; TA 102 to evaluate the mutagenic potential of the test compound 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid at dosage levels of 1, 10, 100, 1000, 5000 μg/plate. 

The result was found to be as follows:

TA 98: no increase in the no. mutants in all conditions;

TA 100:no increase in the no. mutants in all conditions; 5000 μg/plate toxic ± S 9;

TA 1535: a weak mutagenic activity in the presence of S9 of no relevance; toxic effect at the maximum dose with S9.

TA 1537: no increase in mutants in all conditions

TA 102: some increase with the dose in all conditions, but never reaching the double.

There is a very weak indication of induced mutagenicity. The study as such can be considered as negative. The test item is not mutagenic in this assay.

 

In addition, the in vitro Mammalian Cell Gene Mutation assay was performed to evaluate the mutagenic potential of the test compound 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (Acid red 92) (CAS No. 18472-87-2) in the Mouse lymphoma L5178 cells (TK locus). The study was published in a SCCNFP report (COLIPA n° C53, 2004). 

Two experiments were performed: at different concentration and for different time duration

 Exp.1:-S9: 5, 10, 20, 30, 40 μg/ml (4 hours)

            +S9: 5, 10, 20, 40, 60 μg/ml (4 hours)

 Exp.2:-S9: 19.5, 39, 78, 156 μg/ml (24 hours).  

In a preliminary experiment a relevant toxicity was observed at 39.1 μg/ml in all conditions at 4 h of treatment; in the 24 h of treatment a severe toxicity was observed at 312.5 μg/ml. The doses for the main experiments were chosen on the base of a preliminary toxicity experiment. In treated cell a mutagenic effect observed in one culture was not repeated in the replicate culture. Therefore under the test conditions tested the test item 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (Acid red 92) (CAS No. 18472-87-2) was considered to be non mutagenic in this assay. 

 

Also, the gene toxicity study was conducted by K. LAURIE MAUS et al. (Mutation Research, 91 (1981) 315 -320) for the test compound 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (Phloxine B) in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 by the Salmonella/microsome test with preincubation.  The assay was carried out at 1000 µg/µmoles in duplicate and the number of His+revertants was calculated. Phloxine B was found to be negative (with and without S9 metabolic activation system) in this assay.

 

Moreover, K. LAURIE MAUS et al. (Mutation Research, 91 (1981) 315 -320) conducted gene toxicity study for the test compound Phloxine B in Escherichia coli strain WP2 by E. coli suspension test and by microtitre fluctuation assay.

The E. coli suspension assay was carried out at a concentration of 15,30 and 45 µg/ml in duplicate and the number of prototrophs /plate was calculated. Phloxine B was found to be negative in Escherichia coli strain WP2 by suspension test.

Whereas Escherichia coli microtitre fluctuation assay was carried out at a concentration range in duplicate and positive wells were scored on the basis of turbidity. The test compound Phloxine B is negative in Escherichia coli strain WP2 by microtitre fluctuation assay. Hence, it can be concluded that 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9 -yl)benzoic acid (Phloxine B) (CAS No. 18472-87-2) is non genotoxic.

 

Based on the data summarized above, the test material does not classify as a gene mutant in vitro.

 

In Vivo

The genetic toxicity in vivo study was performed for the test chemical3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (Acid Red 92) (CAS Number: 18472-87-2) on male and female NMRI mice.(published in a SCCNFP report (2004)). Acid red 92 was injected intraperitoneally in mice and the number of micronucleus formed in the polychromatic erythrocytes was noted. 

The positive control (CPA) induced 2.030 % of MN in PCEs (0.08 % in the control animals: significance p 0.0040). 

In the treated animals with 100 mg/kg a percentage of MN 0.150 (24h) and 0.110 (48h) was observed: these values, although higher than the control, had a p > 0.34. A reduction of PCE was observed, thus indicating a cytotoxic effect of the test item in the bone marrow cells. 

3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (Acid Red 92) is non mutagenic in the In vivo Mammalian Erythrocyte Micronucleus Test performed. 

 

Based on the above mentioned in vitro and in vivo studies for target substance, by applying weight of evidence approach and according to CLP classification criteria, it can be concluded that 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (CAS Number: 18472-87-2) is non genotoxic.

Justification for classification or non-classification

The results of several mutagenicity studies in vitro and in vivo shows that no classification for mutagenicity according to CLP regulation is warranted for 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (CAS Number: 18472-87-2).