[29H,31H-phthalocyaninato(2-)-kappa4N29,N30,N31,N32]copper, sulfonated, condensed with 2,4- diaminobenzenesulfonic acid and ammonia, converted with sodium hydroxide 2,4,6-trichloro-1,3,5-triazine and ammonium chloride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-02-05 to 2014-03-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of [29H,31H-phthalocyaninato(2-)-k4N29,N30,N31,N32]copper, thionyl dichloride, and sulfurochloridic acid, further condensed with 2,4-diaminobenzenesulfonic acid and ammonia, further converted with sodium hydroxide 2,4,6-trichloro-1,3, -triazine, and ammonium chloride

EC Number:

942-002-2

Molecular formula:

C42.53H20.97Cl1.17N15.41Na3.62O13.95S5.17

IUPAC Name:

Reaction products of [29H,31H-phthalocyaninato(2-)-k4N29,N30,N31,N32]copper, thionyl dichloride, and sulfurochloridic acid, further condensed with 2,4-diaminobenzenesulfonic acid and ammonia, further converted with sodium hydroxide 2,4,6-trichloro-1,3, -triazine, and ammonium chloride

Test material form:

solid: particulate/powder

Remarks:

powder

Method

Target gene:

hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

-Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

Pre-experiment for experiment I (with and without metabolic activation):
5, 10, 25, 50, 100, 250, 500, 1000, 2500, 5000 µg/mL
Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay):
5, 10, 25, 50, 100, 200, 350, 500, 750, 1000 µg/mL
Experiment I
without metabolic activation: 25, 50, 75, 100, 125, 150, 200, 225 and 250 µg/mL
and with metabolic activation: 5, 10, 25, 50, 100, 250, 500 and 1000 µg/mL

Experiment II
without metabolic activation: 10, 20, 50, 100, 250, 500, 750, 1000, 1250 and 1500 µg/mL
and with metabolic activation: 70, 150, 300, 400, 500, 600, 700, 800 and 900 µg/mL

Vehicle / solvent:

Vehicle (Solvent) used: cell culture medium (MEM + 0% FBS 4 h treatment; MEM + 10% FBS 20 h treatment).

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation; 300 µg/mL

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation; 0.8 and 1.0 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: dissolved in medium
DURATION: 4 h (short-term exposure), 20 h (long-term exposure)
Expression time (cells in growth medium): 5 days
Selection time (if incubation with selection agent): about one week

SELECTION AGENT ( mutation assay) 11 µg/mL 6-thioguanine (TG)
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; 5 individual flasks were seeded and evaluated
NUMBER OF CELLS EVALUATED: 400000 cells per flask
DETERMINATION OF CYTOTOXICITY: Method: relative growth

Evaluation criteria:

A test is considered to be negative if there is no biologically relevant increase in the number of mutants.
There are several criteria for determining a positive result:
- a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
- a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of
the mutant frequency is not observed;
- if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I without S9: ≥ 50 μg/mL; experiment I with S9: ≥ 500 μg/mL; Experiment II without S9: ≥ 250 μg/mL; Experiment II with S9:≥ 400 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Precipitation:

No precipitation of the test item was noted in any of the experiments.

 

Toxicity:

A biologically relevant growth inhibition (reduction of relative growth below 70 %) was observed after the treatment with the test item in experiment I and II with and without metabolic activation.

In experiment I without metabolic activation the relative growth was 11.5 % for the highest concentration (250 μg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 1000 μg/mL with a relative growth of 10.6 %.

In experiment II without metabolic activation the relative growth was 12.6 % for the highest concentration (1500 μg/mL) evaluated. The highest concentration evaluated with metabolic activation was 900 μg/mL with a relative growth of 11.5 %.

 

Mutagenicity:

In experiment I without metabolic activation all mutant values of the negative controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-43 mutants per 10E6 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls.

Mutation frequencies with the negative control were found to be 18.35 and 20.81 mutants/10E6 cells and in the range of 4.71 to 29.49 mutants/10E6 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 1.51 was found at a concentration of 225 μg/mL with a relative growth of 11.1 %.

With metabolic activation all mutant values of the negative controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-44 mutants per 10E6 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls.

Mutation frequencies with the negative control were found to be 16.30 and 11.84 mutants/10E6 cells and in the range of 6.29 to 18.71 mutants/10E6 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 1.33 was found at a concentration of 10 μg/mL with a relative growth of 113.8 %.

In experiment II without metabolic activation all mutant values of the negative controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-43 mutants per 10E6 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls.

Mutation frequencies with the negative control were found to be 26.82 and 29.28 mutants/10E6 cells and in the range of 12.74 to 40.26 mutants/10E6 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 1.44 was found at a concentration of 50 μg/mL with a relative growth of 90.4 %.

In experiment II with metabolic activation all mutant values of the negative controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-44 mutants per 10E6 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls.

Mutation frequencies with the negative control were found to be 23.10 and 13.15 mutants/10E6 cells and in the range of 13.89 to 42.28 mutants/10E6 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 2.33 was found at a concentration of 300 μg/mL with a relative growth of 77.4 %.

 

DMBA (0.8 and 1.0 μg/mL) and EMS (300 μg/mL) were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

Applicant's summary and conclusion

Conclusions:

FAT 40045/Z is considered to be not mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

In this mammalian cell gene mutation assay (HPRT locus), V79 cells cultured in vitro were exposed to FAT 40045/Z at concentrations of

- 25, 50, 75, 100, 125, 150, 200, 225 and 250 µg/mL (without metabolic activation, Experiment I)

- 5, 10, 25, 50, 100, 250, 500 and 1000 µg/mL (with metabolic activation, Experiment I)

- 10, 20, 50, 100, 250, 500, 750, 1000, 1250 and 1500 µg/mL (without metabolic activation, Experiment II)

- 70, 150, 300, 400, 500, 600, 700, 800 and 900 µg/mL (with metabolic activation, Experiment II).

FAT 40045/Z TE was tested up to cytotoxic concentrations.

Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 11.5 % for the highest concentration (250 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 1000 µg/mL with a relative growth of 10.6 %. In experiment II without metabolic activation the relative growth was 12.6 % for the highest concentration (1500 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 900 µg/mL with a relative growth of 11.5 %.

In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 1.51 was found at a concentration of 225 µg/mL with a relative growth of 11.1 %.

In experiment I with metabolic activation the highest mutation rate (compared to the negative control values) of 1.33 was found at a concentration of 10 µg/mL with a relative growth of 113.8 %. In experiment II without metabolic activation the highest mutation rate (compared to the negative control values) of 1.44 was found at a concentration of 50 µg/mL with a relative growth of 90.4 %. In experiment II with metabolic activation the highest mutation rate (compared to the negative control values) of 2.33 was found at concentrations of 300 µg/mL with a relative growth of 77.4 %.

The positive controls did induce the appropriate response. 

There was no evidence of a concentration related positive response of induced mutant colonies over background. Hence FAT 40045/Z is considered to be not mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.