[29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]iron

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial Reverse mutation:

The test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02.05.2018 to 18.06.2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

This study was performed to investigate the potential of the test chemical to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Not applicalbe

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced S9 metabolic activation system

Test concentrations with justification for top dose:

0, 0.005, 0.016, 0.050, 0.158, 0.501 mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was solulble in DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-Nitro-o-phenylenediamine (TA 1537, TA 98, without S9); 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)

DURATION
- Preincubation period: Trial I: Not applicable Trial II: 60 min
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Each concentration, including the negative, vehicle and positive controls was tested in triplicate in two independent experiments performed

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data

Rationale for test conditions:

No data

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 TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding vehicle/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 only 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 of negative control and vehicle control such an increase is not considered biologically relevant.

Statistics:

No data

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations (0, 0.005, 0.0016, 0.050, 0.0158, 0.0501, 1.582 and 5 mg/plate ) were tested for toxicity and mutation induction with 3 plates each (triplicates). The experimental conditions in this pre-experiment were the same as described below for the Trial-I (Plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

In the pre-experiment, the concentration range of the test item was 0.002 – 5 mg/plate based on the solubility and precipitation test. There was no reduction in colony count but reduction in background lawn was observed in treated concentrations 5 mg/plate (T8), 1.582 mg/plate (T7) and no reduction in colony count as well as in background lawn in treated concentrations (0.158 (T6) mg/plate – 0.002 (T1) mg/plate) both in absence and in the presence of metabolic activation. Based on the results of pre-experiment following doses were selected for the main study trials: (0, 0.005, 0.016, 0.050, 0.158 and 0.0501 mg/plate , both in the absence (-S9) as well as in the presence of metabolic activation (+S9).

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data

Remarks on result:

other: No mutagenic potential

Table 1: Revertant count for pre-experiment

Dose (mg/plate)	R	Without metabolic activation (-S9)		With metabolic activation (+S9)
		TA 100	TA 98	TA 100	TA 98
NC (0.00)	R1	117	23	118	24
	R2	108	20	121	19
	R3	119	19	120	23
VC (0.00)	R1	121	25	125	26
	R2	124	22	123	22
	R3	117	21	121	24
T1 (0.002)	R1	99	17	100	19
	R2	108	19	106	21
	R3	102	20	110	20
T2 (0.005)	R1	111	22	113	18
	R2	109	20	108	22
	R3	104	20	111	21
T3 (0.016)	R1	112	18	113	19
	R2	108	20	109	23
	R3	110	17	117	21
T4 (0.050)	R1	111	19	114	18
	R2	106	23	109	23
	R3	108	21	116	20
T5 (0.158)	R1	110	21	114	21
	R2	116	19	119	22
	R3	109	18	116	22
T6 (0.501)	R1	108	21	109	23
	R2	110	23	118	20
	R3	112	18	113	19
T7 (1.582)	R1	116	23	119	24
	R2	113	21	121	18
	R3	121	21	120	20
T8 (5)	R1	119	24	124	24
	R2	123	21	118	24
	R3	108	22	121	25
PC	R1	1158	996	1458	1242
	R2	1162	1024	1432	1180
	R3	1148	1012	1488	1208

NC=negative control

VC=vehicle control

PC=positive control

R=replicate

T=test concentration (T8: highest, T1: Lowest)

4-Noytro-o-phenylenediamine (10µg/plate):TA98

Sodium azide (10µg/plate):TA100,

2-Aminoanthracene (2.5 µg/plate):TA98, TA 100

 

Table 2: Revertant count in plate incorporation method (Trial I)

Dose (mg/plate)	R	In the presence of metabolic activation (+S9)
		TA 1537	TA1535	TA 98	TA 100	TA 102
NC (0.00)	R1	4	10	24	118	250
	R2	6	11	19	121	236
	R3	5	10	23	120	246
VC (0.00)	R1	7	17	26	125	270
	R2	7	16	22	123	264
	R3	8	16	24	121	260
T1 (0.005)	R1	5	11	18	113	240
	R2	5	12	22	108	232
	R3	5	11	21	111	236
T2 (0.016)	R1	6	12	19	113	230
	R2	5	13	23	109	236
	R3	7	14	21	117	246
T3 (0.050)	R1	4	13	18	114	244
	R2	5	13	23	109	259
	R3	6	14	20	116	241
T4 (0.158)	R1	6	14	21	114	258
	R2	5	15	22	119	256
	R3	6	14	22	116	243
T5 (0.501)	R1	7	14	23	109	256
	R2	6	16	20	118	261
	R3	6	16	19	113	264
PC	R1	172	440	1242	1458	1384
	R2	186	408	1180	1432	1336
	R3	163	398	1208	1488	1312

 

Dose (mg/plate)	R	In the absence of metabolic activation (-S9)
		TA 1537	TA1535	TA 98	TA 100	TA 102
NC (0.00)	R1	5	10	23	117	248
	R2	6	10	20	108	230
	R3	4	11	19	119	232
VC (0.00)	R1	8	16	25	121	268
	R2	6	15	22	124	260
	R3	6	15	21	117	262
T1 (0.005)	R1	5	10	22	111	240
	R2	5	11	20	109	246
	R3	6	10	20	104	238
T2 (0.016)	R1	6	11	18	112	238
	R2	6	15	20	108	228
	R3	5	11	17	110	234
T3 (0.050)	R1	6	13	19	111	236
	R2	6	12	23	106	250
	R3	6	14	21	108	246
T4 (0.158)	R1	7	13	21	110	252
	R2	6	13	19	116	242
	R3	6	14	18	109	250
T5 (0.501)	R1	6	14	21	108	248
	R2	7	16	23	110	246
	R3	7	15	18	112	252
PC	R1	175	1170	996	1158	1864
	R2	183	1230	1024	1162	1624
	R3	166	1218	1012	1148	1688

 

NC=negative control

VC=vehicle control

PC=positive control

R=replicate

T=test concentration (T5: highest, T1: Lowest)

4-Nitro-o-phenylenediamine (10µg/plate):TA98; (50 µg/plate) TA1537

Sodium azide (10µg/plate):TA100, TA 1535

2-Aminoanthracene (10 µg/plate): TA 102

2-Aminoanthracene (2.5 µg/plate): TA 1537, TA1535, A98, TA 100

Methyl methanosulfonate (4 µg/plate): TA 102

 

Table 3: Revertant count in pre-incubation method (Trial II)

Dose (mg/plate)	R	In the presence of metabolic activation (+S9)
		TA 1537	TA1535	TA 98	TA 100	TA 102
NC (0.00)	R1	6	11	20	116	24
	R2	4	10	20	120	228
	R3	5	10	1	118	22
VC (0.00)	R1	6	16	25	124	268
	R2	7	13	22	126	256
	R3	8	15	23	122	272
T1 (0.005)	R1	5	10	20	116	234
	R2	5	12	18	112	248
	R3	6	11	20	116	230
T2 (0.016)	R1	6	12	22	110	242
	R2	6	13	17	122	254
	R3	6	12	21	117	240
T3 (0.050)	R1	7	14	22	121	248
	R2	6	13	20	126	240
	R3	7	13	18	118	256
T4 (0.158)	R1	5	14	21	124	244
	R2	6	14	20	126	252
	R3	7	13	22	120	262
T5 (0.501)	R1	7	14	24	116	236
	R2	6	13	22	127	248
	R3	6	12	20	120	260
PC	R1	156	340	1328	1448	1736
	R2	184	430	1360	1518	1712
	R3	179	376	1384	1480	1704

 

Dose (mg/plate)	R	In the absence of metabolic activation (-S9)
		TA 1537	TA1535	TA 98	TA 100	TA 102
NC (0.00)	R1	5	10	18	112	248
	R2	4	9	19	116	36
	R3	5	10	20	118	242
VC (0.00)	R1	8	15	24	120	262
	R2	7	13	22	124	270
	R3	7	12	22	122	262
T1 (0.005)	R1	5	10	16	112	252
	R2	6	10	18	116	248
	R3	4	11	16	120	250
T2 (0.016)	R1	7	12	18	104	248
	R2	5	10	20	102	246
	R3	6	13	18	100	262
T3 (0.050)	R1	7	13	20	108	264
	R2	5	10	16	120	254
	R3	4	11	18	118	250
T4 (0.158)	R1	7	13	20	114	266
	R2	6	12	22	108	266
	R3	6	12	20	110	246
T5 (0.501)	R1	6	12	22	120	270
	R2	7	14	20	120	262
	R3	7	12	22	118	264
PC	R1	178	1160	894	1128	1568
	R2	187	1224	928	1086	1608
	R3	182	1272	952	1164	1656

 

NC=negative control

VC=vehicle control

PC=positive control

R=replicate

T=test concentration (T5: highest, T1: Lowest)

4-Nitro-o-phenylenediamine (10µg/plate):TA98; (50 µg/plate) TA1537

Sodium azide (10µg/plate):TA100, TA 1535

2-Aminoanthracene (10 µg/plate): TA 102

2-Aminoanthracene (2.5 µg/plate): TA 1537, TA1535, A98, TA 100

Methyl methanosulfonate (4 µg/plate): TA 102

 

Table 4: Mean revertant count in plate incorporation method (Trial I)

Dose (mg/plate)	In the presence of metabolic activation (+S9)
	TA 157		TA 1535		TA 98		TA 100		TA 102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (0.00)	5.00	1.00	10.33	0.58	22.00	2.65	119.67	1.53	244.00	7.21
VC (0.00)	7.33	0.58	16.33	0.58	24.00	2.00	123.00	2.00	264.67	5.03
T1 (0.005)	5.00	0.00	11.33	0.58	20.33	2.08	110.67	2.52	236.00	4.00
T2 (0.016)	3.00	1.00	13.00	1.00	21.00	2.00	113.00	4.00	237.33	8.08
T3 (0.050)	5.00	1.00	13.33	0.58	20.33	2.52	113.00	3.61	248.00	9.64
T4 (0.158)	5.67	0.58	14.33	0.58	21.67	0.58	116.33	2.52	252.33	8.14
T5 (0.501)	6.33	0.58	15.33	1.15	20.67	2.08	113.33	4.51	260.33	4.04
PC	173.67	11.59	415.33	21.94	1210.00	31.05	1459.33	28.02	1344.00	36.66

 

Dose (mg/plate)	In the absence of metabolic activation (-S9)
	TA 157		TA 1535		TA 98		TA 100		TA 102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (0.00)	5.00	1.00	10.33	0.58	20.67	2.08	114.67	5.86	236.67	9.87
VC (0.00)	6.67	1.15	15.33	0.58	22.67	2.08	120.67	3.51	263.33	4.16
T1 (0.005)	5.33	0.58	10.33	0.58	20.67	1.15	108.00	3.61	241.33	4.16
T2 (0.016)	5.67	0.58	11.33	0.58	18.33	1.53	110.00	2.00	2233.33	5.03
T3 (0.050)	6.00	0.00	13.00	1.00	21.00	2.00	108.33	2.52	244.00	7.21
T4 (0.158)	6.33	0.58	13.33	0.58	19.33	1.53	111.67	3.79	248.00	5.29
T5 (0.501)	6.67	0.58	15.00	1.00	20.67	2.52	110.00	2.00	248.67	3.06
PC	174.67	8.50	1206.00	31.75	1010.67	14.05	1156.00	7.21	1725.33	124.28

 

NC=negative control

VC=vehicle control

PC=positive control

R=replicate

SD= Standard deviation

T=test concentration (T5: highest, T1: Lowest)

4-Nitro-o-phenylenediamine (10µg/plate):TA98; (50 µg/plate) TA1537

Sodium azide (10µg/plate):TA100, TA 1535

2-Aminoanthracene (10 µg/plate): TA 102

2-Aminoanthracene (2.5 µg/plate): TA 1537, TA1535, A98, TA 100

Methyl methanosulfonate (4 µg/plate): TA 102

 

 

 

 

Table 5: Mean revertant count in plate incorporation method (Trial II)

Dose (mg/plate)	In the presence of metabolic activation (+S9)
	TA 157		TA 1535		TA 98		TA 100		TA 102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (0.00)	5.00	1.00	10.33	0.58	19.67	0.58	118.00	2.00	253.33	9.45
VC (0.00)	7.00	1.00	14.67	1.53	23.33	1.53	124.00	2.00	265.33	8.33
T1 (0.005)	5.33	0.58	11.00	1.00	19.33	1.15	114.67	2.31	237.33	9.45
T2 (0.016)	6.00	0.00	12.33	0.58	20.00	2.65	116.33	6.03	245.33	7.57
T3 (0.050)	6.67	0.58	13.33	0.58	20.00	2.00	1221.67	4.04	248.00	8.00
T4 (0.158)	6.00	1.00	13.67	0.58	21.00	1.00	123.33	3.06	252.67	9.02
T5 (0.501)	6.33	0.58	13.00	1.00	22.00	2.00	121.00	5.57	248.00	12.00
PC	173.00	14.93	382.00	45.30	1357.33	28.10	1482.00	35.04	1717.33	16.65

 

Dose (mg/plate)	In the absence of metabolic activation (-S9)
	TA 157		TA 1535		TA 98		TA 100		TA 102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (0.00)	4.67	0.58	9.67	0.58	19.00	1.00	115.33	3.06	242.00	6.00
VC (0.00)	7.33	0.58	13.33	1.53	22.67	1.15	122.00	2.00	264.67	4.62
T1 (0.005)	5.00	1.00	10.33	0.58	16.67	1.15	116.00	4.00	250.00	2.00
T2 (0.016)	6.00	1.00	11.67	1.53	18.67	1.15	102.00	2.00	252.00	8.72
T3 (0.050)	5.33	1.53	11.33	1.53	18.00	2.00	115.33	6.43	256.00	7.21
T4 (0.158)	6.33	0.58	12.33	0.58	20.67	1.15	110.67	3.06	259.33	11.55
T5 (0.501)	6.67	0.58	12.67	1.15	21.33	1.15	119.33	1.15	265.33	4.16
PC	182.33	4.51	1218.67	56.19	924.67	29.14	1126.00	39.04	1610.67	44.06

 

NC=negative control

VC=vehicle control

PC=positive control

R=replicate

SD= Standard deviation

T=test concentration (T5: highest, T1: Lowest)

4-Nitro-o-phenylenediamine (10µg/plate):TA98; (50 µg/plate) TA1537

Sodium azide (10µg/plate):TA100, TA 1535

2-Aminoanthracene (10 µg/plate): TA 102

2-Aminoanthracene (2.5 µg/plate): TA 1537, TA1535, A98, TA 100

Methyl methanosulfonate (4 µg/plate): TA 102

Conclusions:

The test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Executive summary:

Ames assay was performed to investigate the potential of the test chemical to induce gene muta­tions in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0, 0.005, 0.0016, 0.050, 0.0158, 0.0501, 1.582 and 5 mg/plate were selected for pre-experiment.

Based on the pre-experiment results, the test item was tested with the following concentrations 0, 0.005, 0.016, 0.050, 0.158 and 0.0501 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9).

No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with test chemical at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.The spontaneous reversion rates in the negative, vehicle and positive controls are within the range of our historical data. The positive controls used for various strains showed a distinct increase in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method.

In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Bacterial reverse mutation:

Ames assay was performed to investigate the potential of the test chemical to induce gene muta­tions in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0, 0.005, 0.0016, 0.050, 0.0158, 0.0501, 1.582 and 5 mg/plate were selected for pre-experiment.

Based on the pre-experiment results, the test item was tested with the following concentrations 0, 0.005, 0.016, 0.050, 0.158 and 0.0501 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9).

No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with test chemical at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.The spontaneous reversion rates in the negative, vehicle and positive controls are within the range of our historical data. The positive controls used for various strains showed a distinct increase in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method.

In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.