Cuprate(4-), [C-(aminosulfonyl)-C-[[[2-[[4-chloro-6-[(2,5-disulfophenyl)amino]-1,3,5-triazin-2-yl]amino]ethyl]amino]sulfonyl]-29H,31H-phthalocyanine-C,C-disulfonato(6-)-N29,N30,N31,N32]-, sodium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 April 1993 to 17 June 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guidelines for the Testing of Chemicals, Protocol No. 471

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

Method B14 in Commission Directive 92/69/EEC

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Cuprate(4-), [C-(aminosulfonyl)-C-[[[2-[[4-chloro-6-[(2,5-disulfophenyl)amino]-1,3,5-triazin-2-yl]amino]ethyl]amino]sulfonyl]-29H,31H-phthalocyanine-C,C-disulfonato(6-)-N29,N30,N31,N32]-, sodium salts

EC Number:

689-188-9

Cas Number:

149343-84-0

Molecular formula:

C32H24N12O8S4Cu to C76H56N28O32S12Cl4Na8Cu

IUPAC Name:

Cuprate(4-), [C-(aminosulfonyl)-C-[[[2-[[4-chloro-6-[(2,5-disulfophenyl)amino]-1,3,5-triazin-2-yl]amino]ethyl]amino]sulfonyl]-29H,31H-phthalocyanine-C,C-disulfonato(6-)-N29,N30,N31,N32]-, sodium salts

Test material form:

solid: particulate/powder

Details on test material:

Identification: JPR Blue 100
Description: Blue powder
Chemical Name: Cuprate(2-),[2-[[4-chloro-6-[[2-[(29H,31H-phthalocyaninylsulphonyl)amino]ethyl]amino]-1,3,5-triazin-2-yl]amino]-1,4-benzenedisulphonato(4-)-N29,N30,N31,N32]-mono(or bis)aminosulphonyl mono (or di)sulpho derivs., sodium salts.
Lot Number: 303001
Purity: 95.2%
Major Impurities: H2o, NaCl, Na2SO4
Date Received: 30 March 1993
Container: Opaque plastic jar x 7
Storage conditions: Room temperature

Specific details on test material used for the study:

No further details specified in the study report.

Method

Target gene:

histidine and tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Preliminary Toxicity Study: 0, 312.5, 625, 1250, 2500, 5000 μg/plate
Experiment 1: 0, 8.0, 40, 200, 1000, 5000 μg/plate
Experiment 2: 0, 312.5, 625, 1250, 2500, 5000 μg/plate
Appropriate dose levels used in the main study set following the preliminary toxicity study and in accordance with the test guidelines.

Vehicle / solvent:

JPR Blue 100 was accurately weighed (with an allowance made for purity) and dissolved in sterile distilled water (lot number 301005 108 Exp. 2/96) and appropriate dilutions made.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene (2AA)

Details on test system and experimental conditions:

Microsomal Enzyme Fraction
Lot No. Aro. S9/10/05/93, prepared on 10/05/93 and Aro. S9/23/04/ 93, prepared on 23/04/93, were obtained from the British Industrial Biological Research Association on 11/ 05/93. They were prepared from the livers of male Sprague-Dawley rats weighing ~ 200 g. These had received a single i.p. injection of Aroclor 1254 at 500 mg/kg, 5 days before S9 preparation.
The S9 was stored at -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.

S9 Mix and Agar
The S9 Mix was prepared at 4 °C as follows:
S9: 5.0 ml
1.65 M KCl / 0.4 M MgCl2: 1.0 ml
0.1 M Glucose-6-phosphate: 2.5 ml
0.1 M NADP: 2.0 ml
0.2 M Sodium phosphate buffer (pH 7.4): 25.0 ml
Sterile distilled water: 14.5 ml

Top agar was prepared using 0.6% Difco Bacto agar (lot number 801679 10/ 96) and 0.5% sodium chloride. For the Salmonella strains 5 ml of 1.0 mM histidine/1.0 mM biotin solution was added to each 100 ml of top agar and for the Escherichia coli strain 5 ml of 1.0 mM tryptophan was added to each 100 ml of top agar. Base agar plates were prepared using 1.2% Oxoid Agar Technical No.3 (lot number 225 62262 7/97 experiment 1 and lot number 336 66640 10/97 experiment 2) with Vogel-Bonner Medium E and 20 mg/ ml 0-glucose.

Test Procedure
a) Preliminary Toxicity Study
In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material to the tester organisms. 0.1 ml of bacterial suspension (TA100 or WP2uvrA-), 2 ml of molten, trace histidine or tryptophan supplemented media (histidine/ biotin or tryptophan and top agar), 0.1 ml of test solution and 0.5 ml of phosphate buffer were overlayed onto sterile plates of Vogel -Bonner minimal agar (30 ml / plate). Five doses of the test material and a solvent control (sterile distilled water) were tested in duplicate. After 48 hours incubation at 37 °C the plates were scored for revertant colonies and examined for a thinning of the background lawn.

b) Mutation Study
EXPERIMENT 1
Five concentrations of the test material were assayed in triplicate against each tester strain using the direct plate incorporation method in accordance with the standard methods for mutagenicity tests using bacteria.

Test Material and Negative Controls
0.1 ml aliquots of one of the bacterial suspensions were dispensed into sets of sterile test tubes followed by 2.0 ml of molten trace histidine (or tryptophan in the case of WP2uvrA-) supplemented top agar at 45 °C. These sets comprised two test tubes for each bacterial tester strain. 0.1 ml of the appropriately diluted test material or negative control solution was also added to each of the two tubes, followed by either 0.5 ml of the S9 liver microsome mix or 0.5 ml of pH 7.4 buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material.

Positive Controls
i) Without Activation
0.1 ml of one of the positive control solutions (ENNG, 9AA or 4NQO) was added to a test tube containing 2.0 ml of molten, trace histidine or tryptophan supplemented top agar and 0.1 ml of the appropriate bacterial suspension. Finally, 0.5 ml of pH 7.4 buffer was added to the tube, the contents mixed and poured onto agar plates. This procedure was then repeated in triplicate, for each of the positive controls.
ii) With Activation
0.1 ml of 2AA or BP solution was added to a test tube containing 2.0 ml of molten, trace histidine or tryptophan supplemented top agar and 0.1 ml of one of the test bacterial suspensions. Finally, 0.5 ml of S9 mix was added to the test tube, the contents mixed and poured onto an agar plate. This procedure was then repeated, in triplicate, for each tester strain.

The plates were incubated at 37 °C for 48 hours and the number of revertant colonies counted.

EXPERIMENT 2
The second experiment was performed using fresh bacterial cultures, test material and control solutions in triplicate.

Rationale for test conditions:

In accordance with test guidelines

Evaluation criteria:

For a substance to be considered positive in this test system, it should have induced a dose-related and significant increase in mutation rate (of at least twice the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels. If the two experiments give conflicting results then a third experiment may be used to confirm the correct response. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity or solubility or up to the maximum recommended dose of 5000 μg/plate.
In this case the limiting factor was the maximum recommended dose.

Statistics:

Not specified

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Study
The dose range of JPR Blue 100 used in the preliminary toxicity study was 0, 312 .5, 625, 1250, 2500 and 5000 μg/plate. JPR Blue 100 was non-toxic in the strains of bacteria used (TA100 and WP2uvrA-).

Mutation Study
The results of the checks for characteristics, viability and spontaneous reversion rate for each tester strain were all found to be satisfactory.
No toxicity was exhibited to any of the strains of bacteria used.
No significant increases in the numbers of revertant colonies of bacteria were recorded for any of the strains of bacteria used, at any dose level, either with or without metabolic activation.
The positive control substances all produced marked increases in the number of revertant colonies and the activity of the S9 fraction was found to be satisfactory.

Any other information on results incl. tables

 Preliminary Toxicity Study

The mean number of revertant colonies for the toxicity assay were:

Strain	Dose (μg/plate)
	0	312.5	625	1250	2500	5000
TA100 WP2uvrA-	158.5 38.5	176.5 38.0	170.5 35.5	175.5 36.0	172.5 36.0	163.0 33.5

 

KEY TO TABLES OF TEST RESULTS

NOTES:

1. When bacterial growth inhibition is found, the applicable value is marked with an asterisk.

2. The average number of colonies for each concentration is recorded in parenthesis.

3. “Number of revertants” – The observed values and average value are shown in order, beginning with the low concentration of the test substance.

4. The following postfixes are used when required:-

C = contaminated

P = precipitate

X = plate unscorable

 

ENNG = N-ethyl-N’-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-Aminoacridine

2AA = 2-Aminoanthracene

BP = Benzo(a)pyrene

 

TABLE OF TEST RESULTS: EXPERIMENT 1

NAME OF TEST SUBSTANCE: JPR BLUE 100

WITH (+) OR WITHOUT (-) S9 MIX	TEST SUBSTANCE CONCENTRATION (μg/plate)	NUMBER OF REVERTANTS (number of colonies/plate)
		BASE-PAIR SUBSTITUTION TYPE						FRAMESHIFT TYPE
		TA100		TA1535		WP2uvrA-		TA98		TA1537
-	0	144 144 166	(151.3)	14 9 13	(12.0)	10 14 9	(11.0)	32 27 24	(27.7)	5 12 9	(8.7)
-	8.0	143 167 167	(159.0)	10 11 9	(10.0)	9 14 13	(12.0)	16 22 26	(21.3)	7 7 6	(6.7)
-	40	145 120 152	(139.0)	10 10 10	(10.0)	11 9 13	(11.0)	26 22 22	(23.3)	7 5 6	(6.0)
-	200	135 166 144	(148.3)	11 14 14	(13.0)	9 12 15	(12.0)	21 24 24	(23.0)	6 6 9	(7.0)
-	1000	145 166 161	(157.3)	13 10 9	(10.7)	13 13 18	(14.7)	28 22 19	(23.0)	8 7 7	(7.3)
-	5000	141 160 163	(154.7)	10 13 10	(11.0)	16 14 13	(14.3)	22 26 20	(22.7)	9 8 5	(7.3)
POSITIVE CONTROL NOT REQUIRING S9 MIX	NAME	ENNG		ENNG		ENNG		4NQO		9AA
	CONCENTRATION (μg/plate)	3.0		5.0		2.0		0.2		80.0
-	NUMBER OF COLONIES/PLATE	508 531 533	(557.3)	149 152 139	(146.7)	276 254 266	(265.3)	134 129 170	(144.3)	1072 1003 1064	(1046.3)

 

TABLE OF TEST RESULTS: EXPERIMENT 1 (contd)

NAME OF TEST SUBSTANCE: JPR BLUE 100

WITH (+) OR WITHOUT (-) S9 MIX	TEST SUBSTANCE CONCENTRATION (μg/plate)	NUMBER OF REVERTANTS (number of colonies/plate)
		BASE-PAIR SUBSTITUTION TYPE						FRAMESHIFT TYPE
		TA100		TA1535		WP2uvrA-		TA98		TA1537
+	0	156 158 176	(163.3)	11 11 17	(13.0)	11 19 9	(13.0)	27 23 18	(22.7)	9 9 7	(8.3)
+	8.0	149 162 167	(159.3)	11 16 11	(12.7)	20 15 13	(16.0)	19 22 23	(21.3)	14 9 14	(12.3)
+	40	145 141 140	(142.0)	15 15 10	(13.3)	16 13 10	(13.0)	25 23 20	(22.7)	6 10 7	(7.7)
+	200	136 162 133	(143.7)	10 10 16	(12.0)	13 14 13	(13.3)	29 24 24	(25.7)	13 5 10	(9.3)
+	1000	148 158 161	(155.7)	11 14 14	(13.0)	15 19 16	(16.7)	19 19 25	(21.0)	6 10 9	(8.3)
+	5000	149 155 171	(158.3)	13 9 13	(11.7)	13 12 17	(14.0)	20 23 23	(22.0)	8 10 8	(8.7)
POSITIVE CONTROL REQUIRING S9 MIX	NAME	BP		2AA		2AA		BP		BP
	CONCENTRATION (μg/plate)	5.0		2.0		10.0		5.0		5.0
+	NUMBER OF COLONIES/PLATE	401 446 420	(422.3)	183 132 172	(162.3)	210 211 204	(208.3)	181 157 156	(164.7)	99 107 110	(105.3)

 

 

TABLE OF TEST RESULT: EXPERIMENT 2

NAME OF TEST SUBSTANCE: JPR BLUE 100

WITH (+) OR WITHOUT (-) S9 MIX	TEST SUBSTANCE CONCENTRATION (μg/plate)	NUMBER OF REVERTANTS (number of colonies/plate)
		BASE-PAIR SUBSTITUTION TYPE						FRAMESHIFT TYPE
		TA100		TA1535		WP2uvrA-		TA98		TA1537
-	0	179 146 184	(169.7)	10 16 11	(12.3)	26 16 15	(19.0)	20 16 15	(17.0)	9 9 10	(9.3)
-	312.5	175 150 160	(161.7)	15 11 11	(12.3)	15 13 11	(13.0)	15 16 21	(17.3)	9 10 11	(10.0)
-	625	150 170 162	(160.7)	10 12 13	(11.7)	19 18 20	(19.0)	16 15 18	(16.3)	8 9 14	(10.3)
-	1250	145 146 166	(152.3)	11 9 15	(11.7)	19 14 11	(14.7)	17 15 22	(18.0)	9 11 9	(9.7)
-	2500	150 146 171	(155.7)	16 12 16	(14.7)	17 17 15	(16.3)	16 18 21	(18.3)	9 12 11	(10.7)
-	5000	151 169 144	(154.7)	11 14 14	(13.0)	9 15 18	(14.0)	19 14 19	(17.3)	10 7 12	(9.7)
POSITIVE CONTROL NOT REQUIRING S9 MIX	NAME	ENNG		ENNG		ENNG		4NQO		9AA
	CONCENTRATION (μg/plate)	3.0		5.0		2.0		0.2		80.0
-	NUMBER OF COLONIES/PLATE	751 707 709	(722.3)	179 161 136	(158.7)	421 427 462	(436.7)	154 174 153	(160.3)	691 701 755	(715.7)

 

TABLE OF TEST RESULTS: EXPERIMENT 2 (contd)

NAME OF TEST SUBSTANCE: JPR BLUE 100

WITH (+) OR WITHOUT (-) S9 MIX	TEST SUBSTANCE CONCENTRATION (μg/plate)	NUMBER OF REVERTANTS (number of colonies/plate)
		BASE-PAIR SUBSTITUTION TYPE						FRAMESHIFT TYPE
		TA100		TA1535		WP2uvrA-		TA98		TA1537
+	0	184 174 124	(160.7)	15 13 14	(14.0)	20 16 11	(15.7)	26 23 23	(24.0)	13 14 11	(12.7)
+	312.5	144 132 165	(147.0)	9 15 15	(13.0)	C 18 13	(15.5)	23 20 19	(20.7)	13 11 8	(10.7)
+	625	148 137 148	(144.3)	14 11 18	(14.3)	18 19 13	(16.7)	25 23 22	(23.3)	14 7 7	(9.3)
+	1250	159 149 144	(150.7)	18 19 14	(17.0)	10 16 14	(13.3)	16 17 23	(18.7)	7 8 7	(7.3)
+	2500	145 161 158	(154.7)	11 15 16	(14.0)	18 18 19	(18.3)	19 24 25	(22.7)	9 11 15	(11.7)
+	5000	170 155 161	(162.0)	10 14 15	(13.0)	16 14 18	(16.0)	22 24 19	(21.7)	7 8 12	(9.0)
POSITIVE CONTROL REQUIRING S9 MIX	NAME	BP		2AA		2AA		BP		BP
	CONCENTRATION (μg/plate)	5.0		2.0		10.0		5.0		5.0
+	NUMBER OF COLONIES/PLATE	457 458 395	(436.7)	161 178 161	(166.7)	137 141 133	(137.0)	161 178 187	(175.3)	98 119 120	(112.3)

 

Applicant's summary and conclusion

Conclusions:

The test material, JPR Blue 100, was found to be non-mutagenic under the conditions of this test.

Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with JPR Blue 100 by the Ames plate incorporation method at five dose levels, in triplicate both with and without the addition of a rat liver homogenate metabolizing system at 10% in standard co-factors. This method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHW, MOL and MAFF. It also meets the requirements of the OECD, EEC and USA, EPA (TSCA) guidelines. The dose range was determined in a preliminary toxicity assay and was 8 to 5000μg/platein the first experiment. The experiment was repeated on a separate day using different cultures of the bacterial strains and fresh chemical formulations. In this case the dose range of JPR Blue 100 was 312.5 to 5000μg/plate.

 

The solvent (sterile distilled water) control plates gave counts of revertant colonies within the normal range.

 

All positive control chemicals produced marked increases in the numbers of revertant colonies, both with and without the metabolising system.

 

JPR Blue 100 caused no reduction in the growth of the bacterial lawn at any dose level either with or without metabolic activation. JPR Blue 100 was therefore tested up to the maximum recommended dose level of 5000μg/ plate.

 

No significant increase in the numbers of revertant colonies was recorded for any of the bacterial strains with any dose of JPR Blue 100, either with or without metabolic activation. JPR Blue 100 was found to be non-mutagenic under the conditions of this test.