Disodium 5,5'-(2-(1,3-dihydro-3-oxo-2H-indazol-2-ylidene)-1,2-dihydro-3H-indol-3-one)disulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance C.I. Acid blue 74 is considered to be not mutagenic in Salmonella typhimurium strain TA1535, TA1537, TA1538, TA98 and TA100 with and without metabolic activation.

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)

Qualifier:

according to guideline

Guideline:

other: as per mentioned below

Principles of method if other than guideline:

Evaluate mutagenicity of C.I. Acid blue 74 by the standard plate-incorporation assay (Ames et al. 1975).

GLP compliance:

no

Type of assay:

bacterial gene mutation assay

Target gene:

Standard Plate-incorporation

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Test concentrations with justification for top dose:

3333, 1000, 6666 and 10000 µg/plate

Vehicle / solvent:

Vehicle- Vehicle(s)/solvent(s) used: Yes- Justification 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:

other: sodium azide for strains TA1535 & TA100:(1.0/1µg/plate), 9-aminoacridine for TA1537 (50.0/µg/plate), and 2-nitrofluorene for TA1538 &TA98 (1.0, 2.0 or 5.0 /µg/plate) 2-aminoanthracene (2.5/µg/plate for TA1535 and TA1537, 1.0/µg/plate for TA1538 and TA98

Details on test system and experimental conditions:

Details on test system and conditionsMETHOD OF APPLICATION: in agar (plate incorporation)DURATION- Preincubation period: No data available- Exposure duration: No data available- Expression time (cells in growth medium): No data available- Selection time (if incubation with a selection agent): No data available- Fixation time (start of exposure): 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:

Dose-related increase in the number of revertants above spontaneous solvent controls, with a 2-fold increase for strains TA1535, TA1538, TA98 and TA100, and a 3-fold increase for TA1537.

Statistics:

No data available

Species / strain:

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

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:

No data available

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Aroclor 1254-induced male Fischer 344 rats and Syrian golden hamsters

MUTAGENICITY OF MISCELLANEOUS DYE IN THE S. typhimurium ASSAY:

Revertants per plate:

µg/plate	TA1535			TA1537
	(-)S9	(R)S9	(H)S9	(-)S9	(R)S9	(H)S9
Solvent control	23±3	9±5	15±5	9±2	10±3	9±4
Positive control	814±42	156±17	171±14	269±28	294±14	157±11
333	23±2	13±4	11±3	7±1	7±4	Toxic
1000	17±3	10±4	13±5	4±1	8±4	6±3
3333	21±2	11±2	9±3	3±2	7±3	3±1
6666	19±7	12±0	14±3	5±1	5±2	6±1
10000	19±3	22±4	10±4	3±1	Toxic	4±2

 

µg/plate	TA1538			TA98			TA100
	(-)S9	(R)S9	(H)S9	(-)S9	(R)S9	(H)S9	(-)S9	(R)S9	(H)S9
Solvent control	20±6	19±6	22±7	20±4	33±7	30±6	161±26	160±17	183±4
Positive control	677±27	750±33	449±23	269±12	1725±66	775±60	1104±36	2818±14	1469±35
333	10±2	17±3	27±7	18±2	23±6	32±4	160±7	147±20	171±17
1000	6±5	15±5	19±6	17±1	22±6	28±3	158±10	161±7	187±2
3333	5±3	13±3	13±3	13±6	16±7	22±3	158±16	169±11	205±48
6666	7±3	9±2	18±2	13±3	20±5	15±4	193±28	182±15	246±46
10000	7±1	7±5	13±3	23±11	17±2	15±7	167±31	198±17	201±11

Conclusions:

Interpretation of results (migrated information):negative Negative (with and without)The substance C.I. Acid blue 74 is considered to be not mutagenic in Salmonella typhimurium strain TA1535, TA1537, TA1538, TA98 and TA100 with and without metabolic activation.

Executive summary:

Plate-incorporation was carried out according to the method of Prival and Mitchell, 1982; Prival et al., 1984, to observe genetic effect of C.I. Acid blue 74 in S.typhimurium strainTA1535, TA1537, TA1538, TA98 and TA100. All strain were tested at dose 3333, 1000, 6666 and 10000 µg/plate both with and without metabolic activation (Aroclor 1254-induced male Fischer). Positive control and solvent control as used. There is no dose-related increase in the number of revertants above spontaneous solvent controls, with a 2-fold increase for strains TA1535, TA1538, TA98 and TA100, and a 3-fold increase for TA1537.Hence, the substanceC.I. Acid blue 74is considered to be not mutagenic inSalmonella typhimurium strain TA1535, TA1537, TA1538, TA98 and TA100 with and without metabolic 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 C.I. Acid blue 74 (CAS no 860-22-0). The studies are summarized as below:

 

Genetic toxicity test was performed by T.P. Cameron et al, 1987 for Standard Plate-incorporation onS typhimurium(Strain TA1535, TA1537, TA1538, TA98 and TA100).All strain were tested at dose 3333, 1000, 6666 and 10000 µg/plate both with and without metabolic activation (Aroclor 1254-induced male Fischer). Positive control and solvent control as used. There is no dose-related increase in the number of revertants above spontaneous solvent controls, with a 2-fold increase for strains TA1535, TA1538, TA98 and TA100, and a 3-fold increase for TA1537.Hence, the substanceC.I. Acid blue 74is considered to be not mutagenic inSalmonella typhimurium strain TA1535, TA1537, TA1538, TA98 and TA100 with and without metabolic activation.

 

Mutagenicity testing of C.I. Acid blue 74 was performed by T.P. Cameron et al, 1987 by using the FMN preincubation modification of the Salmonella assay. Salmonella typhimurium strain TA98 and TA100 was exposed to C.I. Acid blue 74 at 33, 100, 333, 1000 and 3333 µg/plate. For metabolic activation Hamster liver $9 (30% V/V), plus cofactors (FMN, NADH, G6PD, G6P) was used and Trypan blue (228 /µg/plate) used as a positive control substance. No positive mutagenic response obtained at any tested concentration. Hence,the substance is considered to be not mutagenic inSalmonella typhimurium strain TA98 and TA100 with metabolic activation.

 

C.I. Acid blue 74 tested for mutagenicity was performed by T.P. Cameron et al, 1987, in mouse lymphoma TK⁺′⁻assay. toxicity of test chemical was determined both with and without S9 prepared from Aroclor-1254-induced male Fischer 344 rats. L5178Y TK +/- mouse lymphoma cells was exposed to Without S9: 971, 971, 1229, 1229, 1486, 1486, 1743, 1743, 2000 and 2000 µg/ml and positive control is Ethyl methanesulfonate at 0.5 µl/ml. With S9: 92, 206, 332, 439, 439, 556 and 556 µg/ml concentration of test chemical and positive control is 3-methylcholanthrene at 5.0 or 10.0 µg/ml. There was no dose-related increase in the mutant frequency above the spontaneous control frequency, no 2-fold increase at more than 1 dose and not relative total growth greater than 10% at any exposed concentration.Hence, the substance is considered to be not mutagenic inL5178Y TK +/- mouse lymphoma cells with and without metabolic activation.

 

Mutagenicity of FD&C Blue no. 2 was determined by the Angela E. Auletta et al. 1977 with Salmonella typhimurium strain TA1535, TA1537, TA1538, TA98 and TA100. All strain were tested at dose 1, 10, 100, 1000 and 10000 µg/plate both with and without metabolic activation (Aroclor 1254-induced male Fischer). Positive control and solvent control as used. There is no dose-related increase in the number of revertants colonies per plate either with or without metabolic activation with either of these strains. Therefore,the substanceFD&C Blue no. 2is considered to be not mutagenic inSalmonella typhimurium strain TA1535, TA1537, TA1538, TA98 and TA100 with and without metabolic activation.

 

Reverse mutation assay was of Food blue 2 (Indigo carmine) y the M. ISHIDATE et al 1984, carried out using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 by method of Ames, McCann & Yamasaki (1975). Duplicate plates were used for each of six different concentrations of the sample. All strain were tested at dose 3333, 1000, 6666 and 10000 µg/plate both with and without metabolic activation (Aroclor 1254-induced male Fischer). All trains were exposed to six different concentrations of the chemical having 5000 µg/plate (5 mg/plate) is highest concentration. No significant increases in the number of revertant colonies were detected in any S. typhimurium strains at the maximum dose.  Therefore, the substanceFood blue 2 (Indigo carmine)is considered to be not mutagenic inSalmonella typhimurium strain TA92, TA1535, TA100, TA1537, TA94 and TA98 with and without metabolic activation.

 

Chromosomal aberration tests were performed by M. ISHIDATE et al (1984) carried out on Food blue 2 using a Chinese hamster fibroblast cell line. The cells were exposed to three different concentration having highest concentration is 12000 µg/ml (12 mg/ml). Test substance induced polyploid cells at 48 hr after treatment. On the basis of this, the substance is considered to be mutagenic in Chinese hamster fibroblast cell line without metabolic activation.

 

Mutagenicity of Food blue 2 (Indigo carmine) performed by Joseph P. Brown et al (1978) was evaluate in S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 by method of by Ames et al. All strain were tested at dose 50, 250 and 1000 µg/plate both with and without metabolic activation). No significant increases in the number of revertant colonies were detected in any S. typhimurium strains with and without metabolic activation.Therefore, the substanceFood blue 2 (Indigo carmine)is considered to be not mutagenic inSalmonella typhimurium strain TA92, TA1535, TA100, TA1537, TA94 and TA98 with and without metabolic activation.

 

Mutagenicity test was conducted to observe genetic effect ofC.I. Acid blue 74 by H. E. Seifried (2006) in S.typhimurium strainTA98, TA100, TA1535, TA1537, and TA1538 by plate Incorporation methodology. All strain were tested at dose Five doses (100-10000 µg/plate) both with and without metabolic activation (Aroclor 1254-induced male Fischer). Positive control and solvent control as used. There is no dose-related increase in the number of revertants.Hence,the substance C.I. Acid blue 74is considered to be not mutagenic inSalmonella typhimurium/TA98, TA100, TA1535, TA1537, and TA1538with and without metabolic activation.

 

C.I. Acid blue 74 tested for mutagenicity in L5178Y TK+/- 3.7.Cby H. E. Seifried (2006) inmouse lymphoma cells. Toxicity of test chemical was determined both with and without S9 prepared from Aroclor-1254-induced male Fischer 344 rats. L5178Y TK +/- mouse lymphoma cells was exposed to 92-2000 µg/ml concentration of test chemical and positive control for without metabolic activation: ethyl methylsulfonate at 4.7Χ10-6 M (or methyl methanesulfonate at 10-20 µg/ mL), for metabolic activation: 3-methylcholanthrene at 1.86 Χ 10-5 M (or dimethylbenz[a]- anthracene at 0.5-4 µg/mL). There is dose related 2-fold increase in mutant frequency was observed at 92 µg/ml with S9.Hence, the substanceC.I. Acid blue 74is considered to be mutagenic inL5178Y TK +/- mouse lymphoma cells with metabolic activation. And, not mutagenic without activation.

 

From the available studies, though there was presence of two positive results but based on the majority of studies reviewed, the test material C.I. Acid blue 74 is not mutagenic in vitro.

Justification for selection of genetic toxicity endpoint
Data is from peer reveiw publications.

Justification for classification or non-classification

Gene toxicity in vitro:

Based on the key study used and its relative supporting data, the test materialC.I. Acid blue 74is not mutagenic in vitro.