2,9,16,23-tetrakis(2,5-di-tert-butyl-4-methoxyphenoxy)phthalocyanine

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:

12th February 2019 - 28th February 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Recommended test according to the OECD TG.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 21, 1997

Deviations:

yes

Remarks:

160 and 500 µL of a dilution of the substance in DMSO was added to the top agar to reach concentrations of 1600 and 5000 µg/plate, respectively. Due to the poor solubility of the substance. The amount of DMSO is of no influence on the results.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Official Journal of the European Union No. L142, 31 May 2008

Deviations:

yes

Remarks:

160 and 500 µL of a dilution of the substance in DMSO was added to the top agar to reach concentrations of 1600 and 5000 µg/plate, respectively. Due to the poor solubility of the substance. The amount of DMSO is of no influence on the results.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,9,16,23-tetrakis(2,5-di-tert-butyl-4-methoxyphenoxy)phthalocyanine

EC Number:

831-167-5

Cas Number:

2126827-44-7

Molecular formula:

C92H106N8O8

IUPAC Name:

2,9,16,23-tetrakis(2,5-di-tert-butyl-4-methoxyphenoxy)phthalocyanine

Test material form:

solid

Specific details on test material used for the study:

Batch (Lot) Number: 2715/18/01
Expiry date: 01 July 2020 (expiry date)
Physical Description: Dark green solid
Purity/Composition: 99%
Storage Conditions: At room temperature protected from light
Test item handling: Use amber glassware or wrap container in aluminum foil

Method

Target gene:

Strain: Histidine mutation: Mutation type:
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions
WP2uvrA - Excision repair system deletion

*: R-factor = plasmid pKM101 (increases error-prone DNA repair).

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: Each srtarain contains additional mutations: rfa: deep rough (defective lipopolysaccharide cellcoat) gal: mutation in the galactose metabolism chl: mutation in nitrate reductase bio: defective biotin synthesis uvrB: loss of the excision repair system

Metabolic activation:

with and without

Metabolic activation system:

Microsomal enzymes (S9 homogenate) obtained from male Sprague Dawley rat injected with Aroclor 1254 (500 mg/kg body weight).

Test concentrations with justification for top dose:

The substance initially tested in the dose-range finding study exposing TA100 and WP2uvrA tester strains (with and without S9) to the following substance dose: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate. Based on the results of the dose-range finding test, the following concentrations were chosen for the main experiment: 5.4, 17, 52, 164, 512 and 1600 μg/plate.

Vehicle / solvent:

DMSO and saline

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191

Controls 2

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

Experimental Design:

Dose-range Finding Test.

Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate. The highest concentration of the substance used in the subsequent mutation assays was the level at which the substance exhibited limited solubility.

Mutation Assay.

At least five different doses (increasing with approximately half-log steps) of the substance were tested in triplicate in each strain. The above mentioned dose-range finding study with the two tester strains TA100 and WP2uvrA, is reported as a part of the first mutation experiment. In the second part of this experiment, the substance was tested both in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. In a follow-up experiment with additional parameters, the substance was tested both in the absence and presence of 10% (v/v) S9-mix in all tester strains.
The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.
Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (109 cells/mL) of one of the tester strains, 0.1 to 0.5 mL of a dilution of the substanse in DMSO and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

Colony Counting.

The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient substance precipitate to interfere with automated colony counting were counted manually. Evidence of substance precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Evaluation criteria:

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated. All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

A substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In all the experiments the bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

Dose-range test: Precipitation of the substance on the plates was observed at the start of the incubation period at concentrations of 512 μg/plate and upwards. At the end of the incubation period the substance precipitated at 512 μg/plate and above in tester strains TA100 and WP2uvrA and at 164 μg/plate and above in tester strains TA98, TA1535 and TA1537.

Second mutation experiment: Precipitation of the substance on the plates was observed at the start and at the end of the incubation period at concentrations of 200 μg/plate and upwards.

Remarks on result:

other: Not mutagenic

Remarks:

up to 5000 ug/plate

Any other information on results incl. tables

Dose-Range Finding Test: Mutagenic Response of the test item in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay.

Without metabolic activation.

Substance concentration (ug/plate)	TA100	+/- SD	WP2uvrA	+/-SD
0 (DMSO 100 uL)	113	11	45	0
0 (DMSO 500 uL)	102 (nNP)	10	38 (nNP)	7
1.7^2	99	2	36	2
5.4^2	88	4	36	5
17^2	97	15	38	8
52^2	110	11	37	3
164^2	96  (NP)	15	36 (NP)	5
512^2	99 (SP)	6	37 (SP)	7
1600^3	103 (SP)	19	33 (SP)	2
5000^4	105 (nSP)	3	49 (nSP)	12
Positive control	848	40	1668	72

With metabolic activation¹.

Substance concentration (ug/plate)	TA100	+/- SD	WP2uvrA	+/-SD
0 (DMSO 100 uL)	103	10	38	8
0 (DMSO 500 uL)	113 (nNP)	13	44 (nNP)	10
1.7^2	97	1	35	3
5.4^2	107	13	48	9
17^2	105	9	44	13
52^2	100	3	48	5
164^2	113 (NP)	7	45 (NP)	7
512^2	105 (SP)	20	41 (SP)	6
1600^3	116 (SP)	28	48 (SP)	13
5000^4	108 (nSP)	17	51 (nSP)	6
Positive control	1374	430	472	27

1 Plate incorporation assay (5% S9)

2 100 μL DMSO

3 160 μL DMSO

4 500 μL DMSO

NP No precipitate

SP Slight Precipitate

n Normal bacterial background lawn

Experiment 1: Mutagenic Response of the test item in the Salmonella typhimurium Reverse Mutation Assay

Without metabolic activation.

Substance concentration (ug/plate)	TA1535	+/- SD	TA1537	+/-SD	TA98	+/-SD
0 (DMSO 100 uL)	10	5	5	2	11	3
0 (DMSO 160 uL)	9 (nNP)	4	5 (nNP)	2	12 (nNP)	2
5.4^2	12	5	4	1	10	1
17^2	15	3	5	0	14	2
52^2	11 (NP)	2	6 (NP)	3	16 (NP)	3
164^2	13 (SP)	8	5  (SP)	2	15 (SP)	4
512^2	14 (SP)	2	5 (SP)	3	16 (SP)	5
1600^3	15 (nSP)	3	6 (nSP)	3	12 (nSP)	4
Positive control	1009	31	1100	135	1317	95

With metabolic activation.

Substance concentration (ug/plate)	TA1535	+/- SD	TA1537	+/-SD	TA98	+/-SD
0 (DMSO 100 uL)	15	4	5	3	18	3
0 (DMSO 160 uL)	11 (nNP)	7	4 (nNP)	1	23 (nNP)	4
5.4^2	8	3	6	1	12	3
17^2	11	2	6	2	13	5
52^2	8 (NP)	3	4 (NP)	4	17 (NP)	4
164^2	9 (SP)	2	4 (SP)	1	15 (SP)	9
512^2	8 (SP)	4	7 (SP)	3	10 (SP)	5
1600^3	9 (nSP)	2	6 (nSP)	2	14 (nSP)	3
Positive control	296	12	210	20	1189	298

1 Plate incorporation assay (5% S9)

2 100 μL DMSO

3 160 μL DMSO

NP No precipitate

SP Slight Precipitate

n Normal bacterial background lawn

Experiment 2: Mutagenic Response of the test item in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay

Without metabolic activation.

Substance concentration (ug/plate)	TA1535 ( +/- SD)	TA1537   (+/- SD)	TA98 ( +/- SD)	TA100  ( +/- SD)	WP2uvrA   ( +/- SD)
0 (DMSO 100 uL)	11 (1)	3 (1)	6 (4)	91 (20)	30 (9)
5^2	10 (2)	5 (5)	10 (3)	101 (18)	34 (3)
10^2	9 (5)	1 (1)	8 (6)	96 (9)	29 (8)
50^2	12 (8) NP	3 (2) NP	16 (2) NP	118 (6) NP	28 (3) NP
200^2	6 (5) SP	3 (3) SP	19 (1) SP	100 (2) SP	28 (7) SP
500^2	9 (2) SP	4 (4) SP	10 (4) SP	101 (11) SP	31 (3) SP
1000^2	11 (6) nSP	2 (2) nSP	7 (4) nSP	93 (3) nSP	37 (5) nSP
Positive control	1013 (6)	1168 (58)	1153 (110)	913 (41)	1698 (54)

With metabolic activation.

Substance concentration (ug/plate)	TA1535 ( +/- SD)	TA1537   (+/- SD)	TA98 ( +/- SD)	TA100  ( +/- SD)	WP2uvrA   ( +/- SD)
0 (DMSO 100 uL)	14 (2)	9 (2)	20 (7)	98 (15)	46 (15)
5^2	12 (4)	3 (2)	19 (4)	79 (7)	35 (2)
10^2	13 (3)	4 (2)	17 (3)	85 (8)	35 (13)
50^2	14 (9) NP	7 (6)	15 (6) NP	78 (4) NP	33 (8) NP
200^2	12 (4) SP	9 (3) SP	22 (8) SP	75 (10) SP	30 (5) SP
500^2	14 (2) SP	6 (5) SP	18 (8) SP	85 (12) SP	37 (3) SP
1000^2	13 (4) nSP	6 (3) nSP	18 (4) nSP	84 (4) nSP	38 (0) nSP
Positive control	313 (16)	452 (16)	559 (22)	1512 (363)	340 (23)

1 Plate incorporation assay (10% S9)

2 100 μL DMSO

NP No precipitate

SP Slight Precipitate

n Normal bacterial background lawn

Applicant's summary and conclusion

Conclusions:

The substance is not mutagenic in the Salmonella typhimurium and the Escherichia coli reverse mutation assay in the presence and absence of metabolic activation.  

Executive summary:

The potential of the substance to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli strain WP2uvrA was evaluated in the presence or absence of an exogenous mammalian metabolic activation system (S9). The study was conducted in accordance with OECD TG 471 ‘Bacterial Reverse Mutation Assay’.

Prior to the main study a dose-range finding test was conducted on S. typhimurium TA100 and E.coli WP2urvA strains (with and without metabolic activation system), which were exposed to the following concentrations of the substance: 0, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate (in triplicate). The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

The first mutation assay was performed using the following concentrations of the substance 0, 5.4, 17, 52, 164, 512 and 1600 µg/plate (in triplicate) in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98.The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

The second mutation assay was performed using the following concentrations of the substance: 0, 5, 10, 50, 200, 500 and 1000 µg/plate (in triplicate) in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP₂uvrA. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertant colonies was observed.

Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium and the Escherichia coli reverse mutation assay in the presence and absence of metabolic activation.