Disodium 3-[(2,4-dimethyl-5-sulphonatophenyl)azo]-4-hydroxynaphthalene-1-sulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The end point for the genetic toxicity test was found to be negative (with and without) when treated with Ponceau SX (4548-53-2).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Data is from peer reviwed journal

Qualifier:

according to guideline

Guideline:

other: refer below principle

Principles of method if other than guideline:

The purpose of this study is to present the results and data from the test chemical for their ability to induce mutations in tester strains of Salmonella typhimurium.

GLP compliance:

not specified

Type of assay:

bacterial gene mutation assay

Species / strain / cell type:

S. typhimurium, other: TA100, TA98, TA97 and TA 1535

Details on mammalian cell type (if applicable):

No data available

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9

Test concentrations with justification for top dose:

0.00,1.000, 3.000, 10.00, 33.00, 100.00,333.00,1000.00,3333.00,10000.00 µg/Plate

Vehicle / solvent:

Solvent : DMSOJustification for choice of solvent/vehicle: No data available

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

mitomycin C

other: 4-nitro-o phenylenediamine

Details on test system and experimental conditions:

METHOD OF APPLICATION: pre incubationDURATION- Pre incubation period: 20 min- Exposure duration: No data available- Expression time (cells in growth medium): 2 days- Selection time (if incubation with a selection agent): No data available- Fixation time (start of exposure up to fixation or harvest of cells): No data availableSELECTION AGENT (mutation assays): No data availableSPINDLE INHIBITOR (cytogenetic assays): No data availableSTAIN (for cytogenetic assays): No data availableNUMBER OF REPLICATIONS: No data availableNUMBER OF CELLS EVALUATED: No data availableDETERMINATION OF CYTOTOXICITY- Method: No data availableOTHER EXAMINATIONS:- Determination of polyploidy: No data available- Determination of endoreplication: No data available- Other: No data available

Evaluation criteria:

Histidine- independent (his+) colonies arising on the plates were counted

Statistics:

No data available

Species / strain:

S. typhimurium, other: TA100, TA98, TA97 and TA 1535

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:

not specified

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: No data available- Effects of osmolality: No data available- Evaporation from medium: No data available- Water solubility: No data available- Precipitation: No data available- Other confounding effects: No data availableRANGE-FINDING/SCREENING STUDIES: yes,each chemical was initially tested in the preincubation test at half-log dose intervals up to a dose that elicited toxicity, or to a dose immediately below one that was toxic in the preliminary toxicity procedure.COMPARISON WITH HISTORICAL CONTROL DATA: No data availableADDITIONAL INFORMATION ON CYTOTOXICITY: No data available

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Dose	TA100
	NA (-)                                    NA (-)
µg/plate	MEAN	SEM	MEAN	SEM
0.00	107	4.6	95	6.8
1.000
3.000
10.00
33.00
100.00	90	25.0	104	8.4
333.00	117	2.4	113	3.3
1000.00	133	3.7	100	3.2
3333.00	109	8.2	121	7.9
10000.00	116	9.0	96	4.6
POS	397	8.0	502	9.2

 

 

Dose	TA100
	10% HLI (-)		30% HLI (?)		30% HLI (-)
µg/plate	MEAN	SEM	MEAN	SEM	MEAN	SEM
0.00	107	1.2	121	4.2	110	9.4
1.000					113	3.5
3.000					100	3.8
10.00					93	3.2
33.00					115	3.7
100.00	83	4.0	163	2.9	112	2.3
333.00	104	6.8	154	9.9	120	3.2
1000.00	106	6.2	154	10.8	122	1.9
3333.00	89	5.2	150	11.2	138	6.1
10000.00	117	8.4	162	3.0	114	8.2
POS	688	6.3	702	25.8	735	106.9

 

Dose	TA100
	10% RLI (-)                30% RLI (-)
µg/plate	MEAN	SEM	MEAN	SEM
0.00	106	12.0	144	5.0
1.000
3.000
10.00
33.00
100.00	97	5.7	131	2.2
333.00	94	8.5	155	5.2
1000.00	79	4.6	155	6.3
3333.00	102	13.9	146	1.9
10000.00	94	5.6	145	7.4
POS	691	152.3	967	21.5

 

 

Dose	TA1535
	NA (-)		10% HLI (-)		30% HLI (-)		10%RLI (-)		30%RLI (-)
µg/plate	MEAN	SEM	MEAN	SEM	MEAN	SEM	MEAN	SEM	MEAN	SEM
0.00	27	2.6	23	3.3	14	2.9	18	3.5	16	2.0
1.000
3.000
10.00
33.00
100.00	30	1.2	17	1.3	14	3.4	25	1.8	13	3.2
333.00	24	5.5	15	2.2	12	1.2	17	4.4	18	3.2
1000.00	27	0.9	17	2.0	12	1.2	19	0.6	13	1.2
3333.00	30	5.4	22	1.0	18	1.2	17	3.0	13	1.9
10000.00	30	3.5	13	0.9	17	0.3	22	1.5	12	2.0
POS	327	20.3	145	2.8	188	12.2	1612x		183	4.0

 

 

Dose	TA97
	NA (-)		10% HLI (-)		30% HLI (-)		10%RLI (-)		30%RLI (-)
µg/plate	MEAN	SEM	MEAN	SEM	MEAN	SEM	MEAN	SEM	MEAN	SEM
0.00	113	4.7	145	10.4	141	8.4	153	0.7	163	1.7
1.000
3.000
10.00
33.00
100.00	118	8.2	129	7.6	146	9.2	135	3.8	146	4.1
333.00	122	10.7	137	2.3	136	6.7	143	10.3	144	2.3
1000.00	104	6.1	140	4.7	160	2.1	136	1.2	139	7.7
3333.00	89	25.0	146	0.9	150	7.6	134	5.3	135	4.4
10000.00	116	1.2	159	4.6	131	10.8	130	4.7	119	3.8
POS	582	16.5	932	175.2	994	65.9	2046	86.0	699	14.4

 

 

Dose	TA98
	NA (-)				10% HLI (-)		30%HLI (-)		10%RLI (-)		30 % RLI(-)
µg/plate	MEAN	SEM	MEAN	SEM	MEAN	SEM	MEAN	SEM	MEAN	SEM	MEAN	SEM
0.00	20	2.5	18	3.0	29	0.9	25	4.6	29	3.2	22	0.7
1.000
3.000
10.00
33.00
100.00	13	4.1	14	1.5	34	3.5	40	7.5	27	2.7	23	3.4
333.00	20	2.0	19	1.7	32	1.5	36	4.9	33	7.3	24	1.2
1000.00	17	2.2	22	3.7	41	3.5	36	1.5	31	0.7	22	1.2
3333.00	19	0.0	20	3.3	39	3.2	39	3.3	33	1.5	23	2.0
10000.00	14	1.2	23	1.9	28	2.0	28	3.3	25	3.2	14	2.7
POS	355	21.4	425	11.6	664	42.2	372	8.4	460	164.9	374	18.8

Conclusions:

Interpretation of results (migrated information):negativeThe end point for the genetic toxicity test was found to be negative (with and without) when treated with Ponceau SX (4548-53-2).

Executive summary:

Genetic toxicity test was performed by Ponceau SX onS typhimurium(Strain TA100, TA98, TA97 and TA 1535).Initial testing was in strain without activation and with 30% rat and hamster S-9. If a positive response was obtained in one or both strains, only the positive test condition was repeated. Each chemical was initially tested in the preincubation test at half-log dose intervals up to a dose that elicited toxicity, or to a dose immediately below one that was toxic in the preliminary toxicity procedure.The test chemical (0.05 ml), overnight culture of Salmonella (0.10 ml at CWR and MIC, and 0.05 ml at SRI), and S-9 mix or buffer (0.50 ml), were incubated at 37◦C, without shaking, for 20 min.At least five doses of each chemical were tested in triplicate, and repeat experiments were performed at least one week following the initial trial.A maximum of 0.05 ml solvent was added to each plate.Different concentrations used are 0, 1, 3.10, 33, 100, 333, 1000, 3333, 10000, µg/plates.X

 

The positive controls in the absence of metabolic activation were sodium azide (TA1535 and TA100), 9-aminoacridine (TA97 and TA 1537), 4-nitro-o phenylenediamine (TA98 and TA1538), mitomycin C (TA102), and methyl methane sulfonate (TA 104) and the positive control for metabolic activation with all strains was 2-aminoanthracene, and either sterigmatocystin or 2 aminoanthracene were used.

 

 A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increases in his⁺revertants did not meet the criteria for a “+W” response, or if only single doses produced increases in his⁺revertants in repeat trials. Chemicals were judged nonmutagenic (-) if they did not meet the criteria for a mutagenic or questionable response.

 

From the experiment onS typhimurium(Strain TA100, TA98, TA97 and TA 1535)it was concluded that thePonceau SX(CAS No 4548-53-2) was genetically non-toxic chemical with and without activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Gene toxicity in vitro:

Peer reviewed articles were viewed to determine the mutagenic nature of the test compound FD and C red 4 (CAS no 4548 -53 -2). The studies are summarized as below:

Genetic toxicity test was performed by Zeiger et al, 1992 for Ponceau SX on S typhimurium (Strain TA100, TA98, TA97 and TA 1535). Initial testing was in strain without activation and with 30% rat and hamster S-9. If a positive response was obtained in one or both strains, only the positive test condition was repeated. Each chemical was initially tested in the preincubation test at half-log dose intervals up to a dose that elicited toxicity, or to a dose immediately below one that was toxic in the preliminary toxicity procedure.The test chemical (0.05 ml), overnight culture of Salmonella (0.10 ml at CWR and MIC, and 0.05 ml at SRI), and S-9 mix or buffer (0.50 ml), were incubated at 37◦C, without shaking, for 20 min.At least five doses of each chemical were tested in triplicate, and repeat experiments were performed at least one week following the initial trial.A maximum of 0.05 ml solvent was added to each plate.Different concentrations used are 0, 1, 3.10, 33, 100, 333, 1000, 3333, 10000, µg/plates.The positive controls in the absence of metabolic activation were sodium azide (TA1535 and TA100), 9-aminoacridine (TA97 and TA 1537), 4-nitro-o phenylenediamine (TA98 and TA1538), mitomycin C (TA102), and methyl methane sulfonate (TA 104) and the positive control for metabolic activation with all strains was 2-aminoanthracene, and either sterigmatocystin or 2 aminoanthracene were used. A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increases in his⁺revertants did not meet the criteria for a “+W” response, or if only single doses produced increases in his⁺revertants in repeat trials. Chemicals were judged nonmutagenic (-) if they did not meet the criteria for a mutagenic or questionable response. From the experiment onS typhimurium(Strain TA100, TA98, TA97 and TA 1535)it was concluded that thePonceau SX(CAS No 4548-53-2) was genetically non-toxic chemical with and without activation.

Genetic toxicity test was performed on Salmonella typhimurium strains (TA100, TA98) was also performed by Zeiger et al, 1992 by using FMN protocol (some modification in standard preincubation assay). In which the 30% liver S-9 was supplemented, per ml, with 0.2 M MgCL₂/0.825 M KCI, 0.04 ml; 0.02 M NADH, 0.10 ml; 0.04 M NADP, 0.1 ml; 0.02 M FMN, 0.10 ml; 0.2 M glucose-6-P0₄, 0.10 ml; glucose-6-P0₄dehydrogenase,

2.8 U/0. 10 ml; and 1 .0 M NaH₂PO₄/K₂HPO₄, pH 7.4,0. 10 ml.The dye, congo red, was used as the positive control chemical.

A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increases in his⁺revertants did not meet the criteria for a “+W” response, or if only single doses produced increases in his⁺revertants in repeat trials. Chemicals were judged nonmutagenic (-) if they did not meet the criteria for a mutagenic or questionable response. From the experiment onS typhimurium(Strain TA100, TA98)it was concluded that the Ponceau SX (CAS No 4548-53-2) was genetically non-toxic chemical with and without activation.

Salmonella-mammalian microsome mutagenicity test was performed by King-Thom (1981) on strains TA1535, TA1537, TA1538, TA98, or TA100. Two assays were performed a) plate incorporation and b) Preincubation assay.The plate incorporation assays were performed as described by Ames et al.The liquid preincubation assays were timed for 30 min at 37°C in a Dri-block.(S9) were prepared from male Sprague-Dawley rats stimulated with Aroclor 1254 (500 mg/kg intraperitoneally 5 days before sacrifice).Dimethyl sulfoxide is the solvent used.The positive control chemicals sodium azide, 9-aminoacridine, 2- nitrofluorene, and 2-aminoanthracene were used with the tester strains. The dose-response curves for the mutagenic compounds used the tester strain which showed maximum mutagenicity and covered a range of 1 to 1,000 or 5 to 5,000 µg. From the experiment it was found that the maximum non-toxic dose for plate incorporation was found to be 5000 µg and for liquid preincubation assay was found to be 500 µg. Therefore,genetic toxicity for the Salmonella-mammalian microsome mutagenicity test was found to be negative (with and without S9 activation) on using Ponceau SX (CAS no 4548-53-2).

The in vitro genetic toxicity test was performed by Kornburst (1985) on rat hepatocytes with following concentrations (1X 10-^{3, 1X}10-^{4, 1X}10-^5,and 2X 10-⁶⁾. Rat hepatocytes were isolated and cultured by the two-step in situ liver perfusion method.2 X 10⁵ viable hepatocytes were seeded into 25-mm wells and were allowed to attach to plastic cover slips for 2 hr. The cells were then incubated for 4 hr with [³H]-thymidine. Net nuclear grains (NNG) were determined by counting the number of grains in each nuclei and subtracting the average number of grains present in three equal-size adjacent cytoplasmic areas. Therefore, the genetic toxicity for In Vitro Rat Hepatocyte Primary Culture/ DNA Repair Assays test was found to be negative by using Food red 1 (CAS no 4548-53-2).

Genetic toxicity test were performed by Price et al (1978) on 500 Fisher rat embryo cells with different concentrations of dyes as0.1, 1,10,100,1000 µg ml. The rat cells were plated on medium without food colourings and incubate at 37◦C in humidified 5% CO₂, in air for 2 h. The medium was then decanted and replaced by a medium containing the test dyes at the various dilutions (three plates per dilution). After 48 h the medium was replaced with a freshly prepared medium. The dishes were fixed and stained on day 5 and toxicity was determined by reduction in cloning efficiency relative to a control medium and maximum non-toxic dose were determined. From the study it was found that the genetic toxicity result on the bases of maximum non-toxic dose (MND) was found to be positive for FD&C Red No. 4 (4548-53-2) at 10 µg per ml.

Based on the majority of studies reviewed, the test material Ponceau SX is not mutagenic in vitro.

Justification for selection of genetic toxicity endpoint
The test compound Ponceau SX is not mutagenic in the in vitro test performed with and without metabolic activation system.

Justification for classification or non-classification

Gene toxicity in vitro:

Based on the key study used and its relative supporting data, the test material Ponceau SX is not mutagenic in vitro.