[2,9,16,23-tetrachloro-29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]copper

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented study with the following restriction: Only 4 strains of bacteria (S. typhimurium TA1535, TA1537, TA100 and TA98) were tested, 5 strains are recommended in OECD guideline 471 (July 1997). No E. coli strain was tested.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

; the test substance was not tested in an additional E. coli strain or in S. typhimurium TA102, as recommended in the latest version of OECD guideline 471 (July 1997).

Principles of method if other than guideline:

The rate of induced back mutations was tested in the S. typhimurium indicator organisms TA1535, TA1537, TA98 and TA100. However, the mutagenic potential of the test substance was not tested in an additional E. coli strain or in S. typhimurium TA102, as recommended in the latest version of OECD guideline 471 (July 1997).

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32 copper

EC Number:

205-685-1

EC Name:

29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32 copper

Cas Number:

147-14-8

IUPAC Name:

[29H,31H-phthalocyaninato(2-)-kappa~2~N~29~,N~31~]copper

Details on test material:

- Name of test material (as cited in study report): Heliogen Blau K 7080
- Analytical purity: ca. 98 %
- Impurities (identity and concentrations): no data given
- Storage condition of test material: 4 °C

Method

Target gene:

Determination of the rate of induced back mutations of several bacteria mutants from histidine auxotrophy (his-) to histidine prototrophy (his+).

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from the liver of male Sprague-Dawley rats (treated with a single dose of 500 mg/kg bw Aroclor 1254 five days before sacrifice) and mixed with a series of cofactors (MgCl2, KCl, glucose-6-phosphate, NADP, phosphate buffer pH 7.4).

Test concentrations with justification for top dose:

- Standard plate test:
1st experiment:
0, 20, 100, 500, 2500 and 5000 µg/plate (tested in the strains S. typhimurium T98, TA100, TA1535 and TA1537), with and without metabolic activation.
2nd experiment:
0, 100, 500, 2500, 5000 and 10000 µg/plate (tested in the strains S. typhimurium T98, TA100, TA1535 and TA1537) with and without metabolic activation.

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see details on test system

Details on test system and experimental conditions:

METHOD OF APPLICATION:
A standard plate was conducted, with and without metabolic activation (S9-mix). Each test was conducted in triplicates.

STANDARD PLATE TEST:
The experimental procedure is based on the method of Ames et al. (Mut. Res. 31: 347-364, 1975):
Test tubes containing 2 ml portions of soft agar which consists of 100 ml agar (0.6 % agar + 0.6 % NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at 45 °C and the remaining components are added in the following order:
0.1 ml test solution
0.1 ml bacterial suspension
0.5 ml S-9 mix (in tests with metabolic activation)
or
0.5 ml phosphate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.

CONTROLS:
- Negative control:
Parallel with each experiment with and without S-9 mix, a negative control (solvent control with DMSO) is carried out for each tester strain in order to determine the spontaneous mutation rate.
- Positive controls:
The following positive control substances are used to check tIhe mutability of the bacteria and the activity of the S-9 mix:
with S-9 mix: 10 µg 2-aminoanthracene (dissolved in DMSO) for the strains TA 100, TA 98, TA 1537 and TA 1535.
without S-9 mix: 5 µg N-methyl-N´-nitro-N-nitrosoguanidine (MNNG) (dissolved in DMSO) for the strains TA 100 and TA 1535; 10 µg 4-nitro-o-phenylendiamine (dissolved in DMSO) for the strain TA 98; 100 µg 9-aminoacridine chloride monohydrate (dissolved in DMSO) for the strain TA 1537.

TITER DETERMINATION:
In general, the titer is determined only in the experiments with S-9 mix both without test substance (solvent only) and after adding the two highest doses of test substance. For this purpose, 0.1 ml of the overnight cultures is diluted to 1 x 10exp-6 in each case. Test tubes containing 2 ml portions of soft agar containing maximal amino acid solution (5 mM histidine + 0.5 mM biotin) are kept in a water bath at 45 °C, and the remaining components are added in the following order:
0.1 ml solvent (without and with test substance)
0.1 ml bacterial suspension (dilution: 1 x 10exp-6)
0.5 ml S-9 mix
After mixing, the samples are poured onto the Vogel-Bonner agar plates within approx. 30 seconds. After incubation at 37°C for 48 hours in the dark, the bacterial colonies are counted.

OTHER EXAMINATIONS:
The Salmonella strains are checked for the following characteristics at regular intervals: deep rough character, UV sensitivity, ampicillin resistance. Histidine auxotrophy is automatically checked in each experiment via the spontaneous rate.

Evaluation criteria:

In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

in the standard plate test

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

no bacteriotoxic effect was observed

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

MUTAGENICITY:
No increase in the number of his+ revertants was observed in the standard plate test, with or without the addition of S9 mix in all S. typhimurium strains tested (TA1535, TA100, TA1537, TA98).
The results of the negative and positive controls were as expected and confirmed the validity and sensitivity of the test system used.

SOLUBILITY:
The test substance was incompletely soluble in DMSO from about 500 µg/plate onward.

TOXICITY:
No bacteriotoxic effect (reduced his- background growth) was observed in the standard plate test with and without S9-mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Maximum revertants/plate and corresponding test concentrations in the preincubation test:

1st experiment:
Strain	Tested compound	Maximum revertants/plate [corresponding dose unit in µg/plate]
		without S9-mix	with S9-mix
S. typhimurium TA1535	DMSO	14 ± 2	16 ± 4
	Test substance	28 ± 22 [5000]	15 ± 1 [20]
	Positive Control	1983 ± 231 [5; MNNG]	526 ± 22 [10; 2 -AA]
S. typhimurium TA100	DMSO	115 ± 20	97 ± 8
	Test substance	104 ± 15 [2500]	116 ± 11 [100]
	Positive Control	1717 ± 35 [5; MNNG]	1905 ±  65 [10; 2 -AA]
S. typhimurium TA1537	DMSO	6 ± 2	7 ± 3
	Test substance	8 ± 2 [2500]	10 ± 3 [20]
	Positive Control	654 ± 235 [10; NOPD]	141 ± 20 [10; 2 -AA]
S. typhimurium TA98	DMSO	17 ± 2	33 ± 7
	Test substance	23 ± 1 [500]	37 ± 4 [20]
	Positive Control	789 ± 200 [100; AAC]	1537 ± 93 [10; 2 -AA]
2nd experiment
Strain	Tested compound	Maximum revertants/plate [corresponding dose unit in µg/plate]
		without S9-mix	with S9-mix
S. typhimurium TA1535	DMSO	15 ± 2	18 ± 3
	Test substance	14 ± 2 [2500]	18 ± 2 [500]
	Positive Control	1260 ± 122 [5; MNNG]	236 ± 26  [10; 2 -AA]
S. typhimurium TA100	DMSO	120 ± 5	118 ± 14
	Test substance	116 ± 14 [100]	117 ± 12 [500]
	Positive Control	1407 ± 70 [5; MNNG]	1433 ± 61 [10; 2 -AA]
S. typhimurium TA1537	DMSO	9 ± 2	10 ± 2
	Test substance	10 ± 3 [5000]	11 ± 4 [500]
	Positive Control	557 ± 100 [10; NOPD]	131 ± 18 [10; 2 -AA]
S. typhimurium TA98	DMSO	25 ± 1	37 ± 4
	Test substance	24 ± 3 [100]	39 ± 7 [2500]
	Positive Control	665 ± 88 [100; AAC]	743 ± 95 [10; 2 -AA]
2-AA = 2-aminoanthracene
MNNG = N-methyl-N'-nitro-N-nitrosoguanidine
NOPD = 4-nitro-o-phenylenediamine
AAC = 9-aminoacridine