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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
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

Constituent 1
Chemical structure
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
Molecular formula:
C32H16CuN8
IUPAC Name:
[29H,31H-phthalocyaninato(2-)-kappa~2~N~29~,N~31~]copper
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
- 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 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.

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
Key result
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
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.

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      

Applicant's summary and conclusion

Conclusions:
No increase in the number of his+ revertants was seen in the standard plate test (with or without metabolic activation) in all tested S. typhimurium strains (TA1535, TA100, TA1537, TA98).
According to the results of the present study, the test substance is not mutagenic in the Ames test under the experimental conditions chosen here.