Copper, [[[[3-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]phenyl]amino]sulfonyl]-29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, aminosulfonyl sulfo derivs., sodium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 14th to April 5th, 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The study was conducted with the old version of guideline, which did not include the use of Escherichia Coli

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reference Substance 02

IUPAC Name:

Reference Substance 02

Test material form:

solid

Remarks:

blue powder

Method

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:

Type and composition of metabolic activation system:
- source of S9 : liver microsomal fraction was obtained from the liver of 8-12 weeks old male Wistar rats
- method of preparation of S9 mix: Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution in a ratio 3:7.
- concentration or volume of S9 mix: 500 microliters

Test concentrations with justification for top dose:

Pre-experiment for toxicity: the plates with the test article showed normal background growth up to 5000 microliters/plate in strain TA 98 and TA 100, respectively.
According to the dose selection criteria, the test article was tested at the following concentrations:
10.0, 100.0, 333.3, 1000.0, 5000.0 microliters/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water bidest
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylene-diamine / 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration : for each strain and dose level, including the controls, a minimum of three plates were used
- Number of independent experiments : two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 10 hours
- Exposure duration/duration of treatment: 48 hours

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

not determined

Remarks:

Old guideline, E.coli not included

Cytotoxicity / choice of top concentrations:

not determined

Remarks:

Old guideline, E.coli not included

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

only at 5000 µg/plate in exp. II

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Remarks:

exp. I and II

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 5

Species / strain:

S. typhimurium TA 98

Remarks:

exp. I and II

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 6

Species / strain:

S. typhimurium TA 100

Remarks:

Exp. I and II

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

Any other information on results incl. tables

Without S9 mix

Revertants/plate mean from three plates
Dose μg/plate	TA 1535 Experiment I	TA 1535 Experiment II	TA 1537 Experiment I	TA 1537 Experiment II	TA 98 Experiment I	TA 98 Experiment II	TA 100 Experiment I	TA 100 Experiment II
Neg. Contr.	14	11	11	7	29	21	100	80
Solv. Contr.	15	15	11	7	27	15	92	87
10	21	10	10	8	33	17	106	71
100	16	9	12	8	26	16	103	81
333.3	15	9	12	4	28	12	93	84
1000	13	10	11	4	29	17	88	75
5000	15	8	10	4	27	13	77	76
Positive controls
Sodium azide (10 μg/plate)	1180	830					1172	675
4-Nitro-o-phenylene-diamine (50μg/plate)			281	195	2394	1474

With S9 mix

Revertants/plate mean from three plates
Dose μg/plate	TA 1535 Experiment I	TA 1535 Experiment II	TA 1537 Experiment I	TA 1537 Experiment II	TA 98 Experiment I	TA 98 Experiment II	TA 100 Experiment I	TA 100 Experiment II
Neg. Contr.	15	15	11	6	49	25	146	99
Solv. Contr.	18	12	15	5	44	30	131	105
10	19	15	15	6	52	36	142	101
100	15	16	14	8	43	62	134	106
333.3	18	13	12	7	47	36	132	106
1000	18	10	12	5	42	21	123	114
5000	15	12	8	7	31	18	116	93
Positive controls
2-amino-anthracene (2.5 μg/plate)	362	306	314	255	2156	1623	2158	1644

Applicant's summary and conclusion

Conclusions:

It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test substance did not induce point mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore the test substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of the test substance to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100.

The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test substance was tested as the following concentrations: 10.0, 100.0, 333.3, 1000.0 and 5000.0 µg/plate.

Toxic effects, evidenced by a reduction in the number of revertants, occured only in strain TA 1535 at 5000.0 µg/plate withoutn metabolic activation in experiment II.

The plates incubated with the test substance showed normal back-ground growth up to 5000.0 µg/plate with and without S9 mix in all strains used.

Up to the highest investigated dose, neither a significant and reproducibile increase of the number of revertants was found in any strain as compared to the solvent control nor a concentration-dependent enhancement of the revrtant number exists. The presence of liver microsomal activation did not influence the findings.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore the test substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.