Copper, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, aminosulfonyl sulfo derivs., ammonium 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 October 3rd to November 11th, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

ninth addendum, adopted July 21, 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Direct Blue 199_Na

IUPAC Name:

Direct Blue 199_Na

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

Characteristics of the strains was checked every 2 to 6 months.
Histidine-auxotrophy of the Salmonella strains was demonstrated by the requirement for L-histidine. The presence of the rfa character was assayed by the sensitivity for crystal-violet.
The deletion of the uvrB gene was demonstrated by the sensitivity for UV-light. Strains containing the R-factor: TA 98 and TA 100, were additionally checked for ampicillin resistance.
Tryptophan-auxotrophy of E. coli WP2 uvrA was demonstrated by the requirement for tryptophan.
The absence of the uvrA gene was demonstrated by the sensitivity of the strain to UV-Iight.
All strains were checked for their characteristic reversion properties with known mutagens (reference item).

The strain cultures were stored as stock cultures in ampoules with nutrient broth + DMSO (10 ml + l ml) in a deep freezer at about -80 °C.

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9

Test concentrations with justification for top dose:

312.5, 625.0, 1250.0, 2500.0 and 5000.0 µg/plate, both preincubation assay and standard plate incorporation assay.

Vehicle / solvent:

- Vehicle/solvent bidistilled water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

cyclophosphamide

other: 2-amonianthracene

Details on test system and experimental conditions:

TEST ITEM
On the day of the experiment, the test substance was dissolved in bidistilled water after warming up to 50°C in a water bath and strongly shaken. The test item was soluble up to the concentration of 50 mg/ml. Lower concentrations of the test item were obtained by serial dilution of the stock solution with bidistilled water. The stock solution was sterile filtered without residue.

PRE CULTURES
Aliquots from frozen stocks were grown in liquid nutrient broth medium (NB-medium, Oxoid, United Kingdom) for 8 hours and then used for the experiment. The bacterial cultures were incubated in a time-temperature controlled incubater at about 37 °C.

STANDARD PLATE INCORPORATION ASSAY - With and without metabolic activation
The plates with the selective agar (minimal agar plates) were made in-house. Each Petri dish contained about 20.0 ml of minimal agar (1.5 % agar supplemented with 2 % salts of the Vogel-Bonner Medium E and 2 % glucose). The agar used was Select AGAR, GIBCO BRL, Switzerland. Glucose, D (+) glucose, anhydrous. The Vogel-Bonner Medium E was prepared in-house.

Overlay Agar: the overlay agar contained per litre: 6.0 g of GIBCO BRL Select Agar and 6.0 g NaCl. Sterilisation was performed at 121 °C in an autoclave, cooled down to 50 °C and dispensed into glass bottles. On the day of test peformance, the agar was molten in a water bath and 10 % aliquots (v/v) of 0.5 mM L-histidine / 0.5 mM d-biotin for Salmonella strains or 0.5 mM tryptophan, dissolved in bidistilled water, for E. coli strains were added sterile filtered.

Plates: the following materials were mixed in a test tube and poured onto the minimal agar plates:
100 µI Test solution at each dose level, solvent (negative control) or reference item solution (positive control)
500 µ1 S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation)
100 µI Bacteria suspension (cf. test system, pre-culture of the strains)
2000 µI Overlay agar

PRE-INCUBATION ASSAY - With metabolic activation
In the pre-incubation assay 100 p1 test solution, 500 µl S9 mix and 100 µl bacterial suspension were mixed in a test tube and shaken at about 37 °C for 30 minutes. After pre-incubation 2.0 ml overlay agar (about 45 °C) was added to each tube and well mixed. The mixture was poured on minimal agar plates. In addition, the respective controls (solvent) and positive controls (reference item) were run together with each strain.
After solidification the plates were incubated upside down for at least 48 hours at 37° C ± 2 °C in the dark.

NUMBER OF REPLICATIONS: For each strain and dose level including the controls, three plates were used.

MAMMALIAN MICROSOMAL FRACTION S9 Mix
Rat-liver post mitochondrial supernatant (S9 fraction) was prepared in advance from male rats (HanBrl:WIST SPF), delivered by RCC Ltd, Animal Breeding and Biotechnology, Füllinsdorf, Switzerland. The animals were treated with Aroclor 1254 (Analabs Inc., delivered from Antechnika, Karslruhe, Germany), 500 mg/kg, i.p. 5 days prior to sacrifice. The livers were homogenized with 3 volumes of 150 mM KCI. The homogenate was centrifuged for 15 minutes at 9000 × g and the resulting supernatant (S9 fraction) was stored at approximately -80 °C for no longer than one year. The protein content of the S9 fraction was 42.5 mg/ml.

On the day of the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 10 % v/v in the mixture. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCI2
33 mM KCI
5 mM Glucose-6-phosphate
4 mM NADP
in 100 mM sodium phosphate-buffer, pH 7.4.
Before starting the experiment the S9 mix was sterile filtered and stored in a refrigerator. The S9 mix preparation was performed according to Ames et al, 1983.

RANGE FINDING STUDY
To evaluate the toxicity of the test item a range finding test was carried out with strains S. typhimurium TA 100 and E. coli WP2 uvrA with and without metabolic activation at six concentrations of the test item. The concentrations applied were 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000.0 µg/plate. One plate was prepared per test item concentration, negative and positive controls. The plates were inverted and incubated for about 48 hours at 37 ± 2 °C in darkness. Thereafter, they were evaluated by counting the colonies and determining the background lawn. Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn. The range finding test is reported separately.

DATA RECORDING
Colonies were counted electronically using an Accucount 1000 (Biologics, Gainsville, Virginia, USA), or manually where minor agar damage or test chemical precipitates or strong coloration of the agar plates might have interfered with automating counting. The results were sent on line to a computer. The operator checked them on a random basis. Observations indicating precipitates of the test item in the top agar or a reduced or absent bacterial background lawn were registered additionally.

ACCEPTABILITY of the ASSAY
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if the mean colony counts of the negative contro) values of all strains are within the acceptable ranges listed in the historical data and if the results of the positive controls meet the criteria for a positive response. Normal bacterial background lawn shall be visible in the solvent control. In either case the final decision is based on the scientific judgement of the Study Director.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at oniy one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range solvent controls such an increase is not considered biologically relevant.

Statistics:

A statistical analysis was not required.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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

Additional information on results:

The plates incubated with the test item showed normal background growth up to 5000 pg/plate with and without S9 mix in both experiments. No reduction in the growth of the bacterial background lawn was observed. No precipitation of the test item was visible on the surface of the agar plates.
No substantial increase in revertant colony numbers of any of the five tester strains was observed at any concentration level, neither in the presence nor in the absence of an metabolic activation system (S9). There was also no tendency of higher mutation rates with increasing concentrations in the range beiow the generally acknowledged border of biological relevance.

Appropriate reference items were used as positive controls. They showed a distinct increase of induced revertant coionies.

Any other information on results incl. tables

Summary of the First Mutagenicity Test

Experiment with metabolic activation

Strain number		TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537
	µg/plate	Mean	Mean	Mean	Mean	Mean
Bidist. water		122	22	35	31	17
	312.5	106	23	38	29	11
	625.0	107	22	34	30	8
	1250.0	102	19	34	36	13
	2500.0	100	22	31	30	15
	5000.0	94	22	33	31	10
2-A-anthracene	1.5	1548			1084	264
2-A-anthracene	20.0			858
CPA	200.0		324

Experiment without metabolic activation

Strain number		TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537
	µg/plate	Mean	Mean	Mean	Mean	Mean
Bidist. water		122	23	30	23	10
	312.5	119	28	26	28	13
	625.0	114	25	29	25	11
	1250.0	129	26	30	22	8
	2500.0	124	31	26	19	9
	50000.0	114	25	24	23	8
2-Nitrofluorene	5.0				583
4-Nitroquinoline	2.0			865
9-A-acridine	80.0					1191
Sodium azide	2.0	862	720

Summary of the Second Mutagenicity Test

Experiment with metabolic activation

Strain number		TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537
	µg/plate	Mean	Mean	Mean	Mean	Mean
Bidist. water		101	16	36	40	14
	312.5	107	20	29	34	8
	625.0	104	19	40	25	15
	1250.0	106	22	39	28	13
	2500.0	101	16	32	20	12
	5000.0	107	21	36	17	14
2-A-anthracene	1.5	1171			925	255
2-A-anthracene	20.0			471
CPA	200.0		367

Experiment without metabolic activation

Strain number		TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537
	µg/plate	Mean	Mean	Mean	Mean	Mean
Bidist. water		108	24	20	18	8
	312.5	106	20	23	15	6
	625.0	109	21	28	16	7
	1250.0	102	21	25	22	9
	2500.0	100	23	26	19	10
	5000.0	105	17	20	20	5
2-Nitrofluorene	5.0				725
4-Nitroquinoline	2.0			779
9-A-acridine	80.0					1014
Sodium azide	2.0	874	683

Applicant's summary and conclusion

Conclusions:

The test substance did not induce gene mutation by base-pair changes or frameshifts, in the Salmonella typhimurium and Escherichia coli reverse mutation assay, under the experimental conditions reported.

Executive summary:

The test item was assessed for its potential to induce gene mutations according to the plate incorporation test ( first experiment with and without metabolic activation, second experiment without activation) using Salmonella typhimurium strains TA 100, TA 1535, TA 98, TA 1537, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, the negative (solvent) and the positive controls (reference items) were tested in triplicate. The test item was tested at the following concentrations: 312.5, 625.0, 1250.0, 2500.0 and 5000.0 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments. No reduction in the growth of the bacterial background lawn was observed. No precipitation of the test item was visible on the surface of the agar plates. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment at any concentration level, neither in the presence nor in the absence of an metabolic activation system (S9). There was asso no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference items were used as positive controls. They showed a distinct increase of induced revertant coionies.

Conclusion

Under the test conditions, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.