Copper phtalocyanine monosulfonated and disulfonated

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23.08.2013 - 11.10.2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was carried out according to internationally valid test method in accordance with GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see Deviations in the Section: Additional information on results

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Copper phtalocyanine monosulfonated and disulfonated

EC Number:

700-870-8

Molecular formula:

C32H15N8Cu(HSO3)n where n=1.3-1.6

IUPAC Name:

Copper phtalocyanine monosulfonated and disulfonated

Constituent 2

Reference substance name:

Copper phtalocyanine mono- and disulphonated

IUPAC Name:

Copper phtalocyanine mono- and disulphonated

Constituent 3

Reference substance name:

Hysperse 12

IUPAC Name:

Hysperse 12

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Copper phtalocyanine mono- and disulphonated
- Molecular formula: C32H15N8Cu(SO3H)1.4x2.5 H2O
- Molecular weight: 733.3
- Smiles notation:
- InChl:
- Substance type: technical product
- Physical state: powder
- Lot/batch No.: 01
- Expiration date of the lot/batch:
- Stability under test conditions: stable
- Storage condition of test material: The substance was stored in closed vessel in dry room at the temperature bellow 25ºC in dark

Method

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:

other: histidine dependent strain

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: tryptophane dependent strain

Metabolic activation:

with and without

Metabolic activation system:

a supernatant of rat liver and a mixture of cofactors

Test concentrations with justification for top dose:

First round of experiments: 50, 150, 500, 1500 and 5000 µg per plate
Second round of experiments: 15, 50, 150, 500, 1500 µg per plate
In first round of experiments in 5000 µg per plate decrease of number of revertants occured.

Vehicle / solvent:

- Vehicle: water
- Justification for choice of vehicle: optimal solvent for test substance

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

sodium azide

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

other: 2-aminofluorene

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

other: 9-aminoacridine hydrochloride monohydrate

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

other: N-methyl-N´-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

THE BACTERIAL TESTER STRAINS:
- histidine dependent Salmonella typhimurium TA 98 (CCM 3811), TA 100 (CCM 3812), TA 1535 (CCM 3814), TA 1537 (CCM 3815) and tryptophan dependent strain Escherichia coli WP2 uvrA (CCM 4751) were obtained from Czech Collection of Microorganisms (CCM) of Masaryk University, Brno,

Strains TA 1537 and TA 98 detect frame shift mutations,
strains TA 100 and TA 1535 serve to detect base-pair substitution mutations,
strain E.coli WP2 uvrA detects cross-linking mutagens.

METHOD OF APPLICATION: in agar (plate incorporation)

Plate incorporation test
Test procedure:
100 µL of the test substance in required concentration, 0.1 mL 16-18 h culture of tester strain, 0.5 mL relevant buffer and 30 or 100 µL of S9 postmitochondrial fraction (in case of test with metabolic activation) were added to the 2 mL top agar (with trace of histidine or tryptophan) kept in a test tube at 45±3ºC. After shaking the mixture was poured into a minimal glucose agar plate. After incubation of 48 - 72 h at 37±1ºC, the number of revertant colonies on the plate was counted manually with exception of positive controls, which were counted automatically.
For an adequate estimate of variation, triplicate plating was used at each dose level.

Selection of doses/toxicity:
The test substance was suspended in water in concentration 2 500 microgram/0.1 mL. Starting suspension was diluted to concentration series (10 - 2500 µg per plate), which was tested for toxicity in strain TA 100 without metabolic activation. Besides, maximum dose recomended by guidelines - 5000 µg per plate - was dosed in volume 0.2 ml. Applications suspensions were shaken before withdrawal of aliquots for diluting as well as before
pouring of top agar with the test substances onto Petri dishes.
Precipitation was observed in dishes from the dose of 500 µg per plate, but no toxicity or difficulties with evaluation were observed during the toxicity experiment.

Dose of 5000 µg per plate was then used as maximum in the first mutagenicity experiments. The starting concentration was diluted according to guidelines (five different analysable concentrations with approximately half log (i.e. √10) intervals between test points). In the second experiments was used maximum dose 2500 µg per plate and additional dose of15 µg per plate in experiments.

Evaluation criteria:

The main criterion for evaluation of results was modified two-fold increase rule which is compatible with the application of statistical methods. After this rule the result is positive, if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached.
An increase is considered as "biologically relevant":
-if the number of reversions is at least twice as high as that in the solvent control for the strains having spontaneous reversions more than 10;
- if the number of reversions is at least three times as high as that in the solvent control for the strains having spontaneous reversion ≤ 10.
According to OECD TG 471, the biological relevance of the results is the criterion for the interpretation of resukts, a statistical evaluation of te results is noit regarded as necessary.

Statistics:

According to OECD TG 471, the biological relevance of the results is the criterion for the interpretation of resukts, a statistical evaluation of te results is not regarded as necessary.

Claxton L. D. et al. (1987): Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity, Mutat. Res. 189, 83 - 91

Results and discussion

Test results

Test results 1

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:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Deviations:
Two more experiments were performed in addition to the planned ones. In the second experiments in TA 98 (with and without metabolic activation), bacterial suspension used for testing was contaminated, so Petri dishes could not be evaluated. These experiments were repeated. This deviation had no impact onthe outcome of the study.

Any other information on results incl. tables

see attached background document

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation for all used strains
negative without metabolic activation for all used strains

Executive summary:

Test substance - Hysperse 12 - was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The test was performed according to EU method B.13/14 Mutagenicity - Reverse mutation test using bacteria, which is analogous to the OECD Test Guideline No. 471.

Four indicator Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and one indicator Escherichia coli WP2 uvrA strain were used. The test substance was dissolved in dimethylsulfoxide and assayed in doses of 15 - 2500 µg which were applied to plates in volume of 0.1 mL.

Two series of experiments were performed with each strain, without metabolic activation and with a supernatant of rat liver and a mixture of cofactors.

For all used strains was substance with and without metabolic activation non mutagenic.