1-Octadecanaminium,N,N-Dimethyl-N-Octadecyl-,(Sp-4-2)-[29h,31h-Phthalocyanine2-Sulfonato(3-)-N 29,N 30,N 31,N 32]Cuprate(1-)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: L5178Y TK +/- Mouse lymphoma Assay

Test material

Test material information

Constituent 1

Details on test material:

- Molecular weight (if other than submission substance): 1079
- Analytical purity: 97.5 %

Method

Target gene:

Thymidine kinase, TK +/-

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

PB/βNF S9 prepared in house from the livers of male Sprague-Dawley rats that received orally for three consecutive daily does of Phenobarbital/β-naphthoflavone (80/100 mg/per kg per day)

Test concentrations with justification for top dose:

Preliminary toxicity test concentrations were 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500, and 5000 ug/l in presence and absence of S9
Experiment 1 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 9.37.5 ug/mL in the absence and presence of S9
Experiment 2 4.88, 9.77, 19.53, 39.06, 78.13, 156.25, 234.38, and 312.5 in absence of S9 and 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 468.75, and 625 ug/mL in presence of S9

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Vehicle R0 Medium;
Remarks: Solvent (R0 medium) treatment groups were used as vehicle controls

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: Cyclophosphamide and Ethylmethansulphonate

Details on test system and experimental conditions:

DURATION
- Exposure duration: Preliminary experiment – 4 hour with and without S9 ; and 24 hour without S9 ; Experiment 1 -4 hours with and without S9; Experiment 2 4 hour with and 24 hours without S9
- Expression time (cells in growth medium): Preliminary experiment 5x 10 ^5 cells/ml (4 hour exposure) and 1.5 x 10^ 5 cells/ml (24 hour exposure); Experiment 1 1 x 10^6 cells/ml; Experiment 2 1 x 10^6 cells/ml (4 hour exposure) and 0.3 x 10^6 (24 hour exposure)
- Fixation time (start of exposure up to fixation or harvest of cells): The cultures were incubated and sub-cultured every 24 hours for the expression period of two days.
STAIN (for cytogenetic assays): To assist the scoring of the TFT mutant colonies 0.025 ml of MTT solution (2.5 mg/ml in PBS) was added to each well of the mutation plates.
NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: On day 2 of the experiment the cells were counted, diluted to 10^4 cells/ml and plated for mutant frequency (2000 cells/well) in selective medium containing 4 ug/ml 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/ml and plated (2cells/well) for viability in non-selective medium

DETERMINATION OF CYTOTOXICITY
-Method: mitotic index; cloning efficiency; relative total growth; other: The daily counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability data a Relative Total Growth (RTG) value



OTHER:
Microtitre plates were scored after ten to fourteen days incubation. The number of positive wells and total number of scorable wells were recorded. The number of small and large colonies seen in the TFT mutation plates were also recorded. The plates were incubated for approximately two hours.

Statistics:

The experimental data was analyzed using a dedicated computer program which follows the statistical guidelines recommended by the UKEMS.

Results and discussion

Test results

Species / strain:

other: L5178Y TK +/- Mouse Lymphoma Assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

> 19.53 ug/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary Toxicity Test

The results for the Relative Suspension Growth (%RSG) were as follows:

 

Dose ug/ml)	%RSG (-S9) 4-hour exposure	%RSG (+S9) 4-hour exposure	%RSG (-S9) 24-hour exposure
0	100	100	100
19.53	104	97	63
39.06	93	100	44
78.13	78	77	34
156.25	55	64	28
312.5	28	32	7
625	17	19	0
1250	4	7	0
2500	0	0	0
5000	0	0	0

In all three of the exposure groups there was a clear and marked reduction in the Relative Suspension Growth (%RSG) of cells treated with the test materials when compared to the concurrent vehicle controls. A precipitate of the test material was observed at and above 19.53 ug/ml. In the subsequent mutagenicity experiments the maximum dose was limited by toxicity.

  

Experiment 1

There was evidence of toxicity following exposure to the test material in both the absence and presence of metabolic activation. There was evidence of a modest reduction in viability, therefore indicating some residual toxicity had occurred in both the absence and presence of metabolic activation. Optimum levels of toxicity were achieved in both the absence and presence of metabolic activation. 

 

A precipitate of the test material was observed at and above 9.77 ug/ml.

 

Treatment (ug/ml)	4-Hours - S-9			Treatment (ug/ml)	4-Hours + S-9
	%RSG	RTG	MFδ		%RSG	RTG	MFδ
0	100	1.0	88.16	0	100	1.0	80.93
9.77 Ø	96			9.77 Ø	100
19.53 Ø	95			19.53 Ø	106
39.06	83	1.08	82.46	39.06	94	1.15	72.15
78.13	79	0.94	89.18	78.13	86	0.94	85.01
156.25	57	0.78	77.79	156.25	68	0.85	82.72
312.5	34	0.43	73.39	312.5	42	0.51	91.28
625	30	0.18	112.39	625	20	0.13	110.71
937.5 X	14	0.06	163.30	937.5 X	11	0.05	195.34
Linear Trend			NS	Linear Trend			NS
				CP
400	70	0.26	805.87	2	55	0.26	825.04

%RSG = Relative Suspension Growth

RTG =Relative Total Growth

CP = Cyclophosphamide

= Ethylmethanesulphonate

NS = Not significant

Ø= Not plated for viability or TFT resistance

X= Treatment excluded from test statistics due to toxicity

δ=Positive wells per tray, 96 wells plated unless otherwise stated in parenthesis

MF = 5-TFT resistant mutants/10^6 viable cells 2 days after treatment

 

Experiment 2

There was evidence of a dose-related reduction in % RSG and RTG values in cultures dosed with the test materials in both the absence and presence of metabolic activation. There was evidence of a modest reduction in viability in the presence of metabolic activation only. The 24 hour exposure without metabolic activation treatment demonstrated that the extended time point had a marked effect on the toxicity of the test material. A precipitate of the test material was observed at and above 4.88 ug/ml.

 

 

Treatment (ug/ml)	24-Hours - S-9			Treatment (ug/ml)	4-Hours + S-9
	%RSG	RTG	MFδ		%RSG	RTG	MFδ
0	100	1.0	90.63	0	100	1.0	121.67
4.88	94	0.89	94.02	9.77 Ø	91
9.77	73	0.72	99.21	19.53 Ø	103
19.53	72	0.76	88.62	39.06	92	0.94	121.29
39.06	55	0.55	86.47	78.13	73	0.84	123.09
78.13	43	0.52	102.34	156.25	55	0.64	80.22
156.25	27	0.41	67.09	312.5	27	0.33	141.29
234.38	24	0.29	95.19	468.75	22	0.23	149.12
312.5 X	13	0.09	149.78	625	20	0.15	136.18
Linear Trend			NS	Linear Trend			NS
				CP
150	51	0.37	662.16	2	62	0.38	633.01

%RSG = Relative Suspension Growth

RTG =Relative Total Growth

CP = Cyclophosphamide

= Ethylmethanesulphonate

NS = Not significant

Ø= Not plated for viability or TFT resistance

X= Treatment excluded from test statistics due to toxicity

δ=Positive wells per tray, 96 wells plated unless otherwise stated in parenthesis

MF = 5-TFT resistant mutants/10^6 viable cells 2 days after treatment

  

In both experiment 1 and 2 acceptable levels of toxicity were seen with both positive control substances.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Executive summary:

The maximum dose level tested was limited by test material induced toxicity. A precipitate of the test material was observed at and above 9.77 and 4.88 ug/ml in Experiment 1 and 2 respectively. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases it the mutant frequency indicating the satisfactory performance of the test and the activity of the metabolizing system. The test material did not induce any statistically significant or dose-related increases in the mutant frequency at any dose level either with or without metabolic activation, in either the first or the second experiment using a dose range that induced optimum or near optimum levels of toxicity.