Hydrogen [29H,31H-phthalocyaninesulphonato(3-)-N29,N30,N31,N32]cuprate(1-), compound with dodecylamine (1:1)

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 from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD guideline 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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

; 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 according to OECD guideline 471. 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:

108300-90-9

Cas Number:

108300-90-9

IUPAC Name:

108300-90-9

Details on test material:

- Test Substance No. 85/565
- Analytical purity: > 95 %
- Batch No. Partie 1 + 2 / 1985
- Appearance consistency: Blue powder
- Storage: + 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:

1st experiment: 0, 20, 100, 500, 2500 and 5000 µg/plate
2nd experiment: with S9-mix: 0, 4, 20, 100, 500 and 1500 µg/plate; without S9-mix: 0, 1, 5, 10, 50 and 100 µg/plate

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 test 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.
Composition of the minimal glucose agar:
980 ml aqua dest.
20 ml Vogel-Bonner E medium
15 g Difco bacto agar
20 g D-glucose monohydrate.
After incubation at 37 °C for 48 hours in the dark, the bacterial colonies (his+ revertants) are counted.

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 amounts of 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.

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

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

bacteriotoxic effect >=100 µg/plate without S9-mix and >=1500 µg/plate with S9-mix

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, 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 at concentrations of > 2500 µg/plate.

TOXICITY:
A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ revertants) was observed > 100 µg per plate onward (without S9-mix) or at doses > 1500 µg/plate (with S9-mix).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: in standard plate test (SPT)

Any other information on results incl. tables

Table 1: Maximum revertants/plate and corresponding test concentrations in the standard plate test:
Strain	Tested compound	Maximum revertants/plate [corresponding dose unit in µg/plate]
		without S9-mix	with S9-mix
S. typhimurium TA1535	DMSO	18 ± 5	19 ± 2
	Test substance	21 ± 2 [5]	18 ± 4 [100]
	Positive Control	2017 ± 176 [5; MNNG]	526 ± 26 [10; 2-AA]
S. typhimurium TA100	DMSO	115 ± 10	109 ± 3
	Test substance	113 ± 7 [10]	133 ± 10 [100]
	Positive Control	2050 ± 150 [5; MNNG]	1857 ± 145 [10; 2-AA]
S. typhimurium TA1537	DMSO	10 ± 1	12 ± 1
	Test substance	13 ± 4 [1]	12 ± 3 [500]
	Positive Control	716 ± 106 [10; NOPD]	205 ± 23 [10; 2-AA]
S. typhimurium TA98	DMSO	10 ± 1	35 ± 3
	Test substance	13 ± 4 [1]	38 ± 3 []
	Positive Control	1167 ± 76 [100; AAC]	1537 ± 153 [10; 2-AA]
2-AA = 2-aminoanthracene
MNNG = N-methyl-N'-nitro-N-nitrosoguanidine
NOPD = 4-nitro-o-phenylenediamine
AAC = 9-aminoacridine

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative