Bis(N,N-dimethylpropane-1,3-diamine-N)[29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]cobalt(1+) chloride

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:

2016-10-28 to 2016-11-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 471, "Bacterial Reverse Mutation Test", adopted 21st July, 1997.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30, 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

EPA 712-C-98-247, August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Bis(N,N-dimethylpropane-1,3-diamine-N)[29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]cobalt(1+) chloride

EC Number:

284-943-5

EC Name:

Bis(N,N-dimethylpropane-1,3-diamine-N)[29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]cobalt(1+) chloride

Cas Number:

84989-53-7

Molecular formula:

C42H44CoN12.Cl

IUPAC Name:

hydroxylamine

Test material form:

solid: particulate/powder

Method

Target gene:

histidine

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from rat liver

Test concentrations with justification for top dose:

Pre-experiment:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Experiment I:
0.316, 1.00, 3.16, 10.0, 31.6, 100, 316 and 1000 μg/plate (all tester strains except TA98 and TA100 with metabolic activation)
3.16, 10.0, 31.6, 100, 316 and 1000 μg/plate (only TA98 and TA100 with metabolic activation)

Experiment II:
0.158, 0.50, 1.58, 5.0, 15.8, 50, 158 and 500 μg/plate
The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. 1000 µg/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Aqua Dest.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

S. typhimurium: TA 100, TA 1535 without metabolic activation dissolved in Aqua dest.

Positive control substance:

sodium azide

Controls 2

Positive controls:

yes

Remarks:

S. typhimurium: TA 98, TA 1537 without metabolic activation dissolved in DMSO

Positive control substance:

other: 4-nitro-o-phenylene-diamine (4-NOPD) 10 μg/plate for TA 98, 40 μg/plate for TA 1537

Controls 3

Positive controls:

yes

Remarks:

S. typhimurium: TA 102 without metabolic activation dissolved in Aqua dest.

Positive control substance:

methylmethanesulfonate

Controls 4

Positive controls:

yes

Remarks:

S. typhimurium: TA 98, TA 100, TA 1535, TA 1537 and TA 102 with metabolic activation dissolved in DMSO

Positive control substance:

other: 2-aminoanthracene (2-AA) 2.5 μg/plate; 10 μg/plate for TA 102

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment I and II: in agar (plate incorporation)

DURATION
- Preincubation period: No
- Exposure duration: at least 48 h in the dark at 37 °C
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): at least 48 h in the dark at 37 °C
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable

SELECTION AGENT (mutation assays): histidine (overlay agar)

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Experiment I and II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp I without S9 toxicity from 316 µg/plate; Exp II without S9 toxicity from 50 µg/plate, with S9 toxicity from 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Experiment I and II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp I: without S9 toxicity from 100 µg/plate, with S9 toxicity from 1000 µg/plate; Exp II without S9 toxicity from 50 µg/plate,with S9 toxicity from 158 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

Experiment I and II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp I: without S9 toxicity from 100 µg/plate, with S9 toxicity from 100 µg/plate; Exp II: without S9 toxicity from 50µg/plate, with S9 toxicity from 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

Experiment I and II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp I: without S9 toxicity from 100 µg/plate, with S9 toxicity from 1000 µg/plate; Exp II: without S9 toxicity from 50 µg/plate, with S9 toxicity from 158 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Remarks:

Experiment I and II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp I: without S9 toxicity from 100 µg/plate, with S9 toxicity from 316 µg/plate; Exp II: without S9 toxicity from 50 µg/plate, with S9 toxicity from 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test item was observed in all tester strains used in experiment I (with and without metabolic activation) from 316 µg/plate. No precipitation of the test item was observed in any tester strain used in experiment II (with and without metabolic activation).

RANGE-FINDING STUDY:
Based on the observations in the range-finding study, the maximum concentration for the main experiments was set at 1000 µg/plate (see detailed results in section any other information on results)

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Negative (solvent/vehicle) historical control data: The negative control plates (A. dest.) with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range (2013 -2015)):

-S9 + S9 (rat liver)
min max min max
TA 98 13 54 13 61
TA 100 49 139 67 162
TA 1535 4 39 4 32
TA 1537 2 35 3 36
TA 102 141 472 91 586


ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Toxicity may be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.
Toxic effects of the test item were noted in all tester strains used in experiment I and II:
- In experiment I toxic effects of the test item were observed at concentrations of 100 µg/plate and higher (with and without metabolic activation), depending on the particular tester strain.
- In experiment II toxic effects of the test item were noted at concentrations of 50 µg/plate and higher (without metabolic activation) and at concentrations of 158 µg/plate and higher (with metabolic activation), depending on the particular tester strain.

Any other information on results incl. tables

Dose-range finder experiment:

Substance	Dose (μg/plate)	TA 98 Mutation Factor [toxicity]*		TA 100 Mutation Factor [toxicity]*
		without S9	with S9	without S9	with S9
Solvent Control (DMSO)		1.0	1.0	1.0	1.0
4-NOPD	10.0	11.3	-	-	-
NaN3	10.0	-	-	5.4	-
2-AA	2.50	-	64.7	-	10.9
Test Item	3.16	0.7	1.1	1.4	0.8
	10.0	0.8	1.1	1.6	0.9
	31.6	0.7	1.1	1.6	0.8
	100	0.7	1.1	1.2	1.3
	316	0.0 [B,P]	0.9 [P]	0.0 [B,P]	1.5 [P]
	1000	0.0 [B,P]	0.9 [P]	0.0 [B,P]	0.7 [B,P]
	2500	0.0 [B,P]	0.0 [B,P]	0.0 [B,P]	0.0 [B,P]
	5000	0.0 [B,P]	0.0 [B,P]	0.0 [B,P]	0.0 [B,P]

B = Background lawn reduced; P = Precipitation

 

Experiment I:

		TA 98				TA 100				TA 1535				TA 1537				TA 102
	Dose (µg)/plate	Revertant colonies per plate		Mutation factor		Revertant colonies per plate		Mutation factor		Revertant colonies per plate		Mutation factor		Revertant colonies per plate		Mutation factor		Revertant colonies per plate		Mutation factor
Treatment		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
A. Dest		22	26	0.9	1.4	114	138	1.1	1	24	21	1.1	1.1	13	9	1.3	1	420	495	1	1
DMSO		24	19	1	1	109	143	1	1	22	20	1	1	10	9	1	1	413	520	1	1
Test item	0.316	21	-	0.9	-	89	-	0.8	-	26	18	1.2	0.9	8	9	0.8	1	356	447	0.9	0.9
Test item	1	23	-	1	-	92	-	0.8	-	24	17	1.1	0.9	8	11	0.8	1.2	382	472	0.9	0.9
Test item	3.16	17	21	0.7	1.1	91	116	0.8	0.8	23	17	1.1	0.9	9	12	0.9	1.3	339	482	0.8	0.9
Test item	10	19	22	0.8	1.1	86	124	0.8	0.9	23	19	1.1	1	8	12	0.8	1.3	331	459	0.8	0.9
Test item	31.6	18	22	0.7	1.1	106	114	1	0.8	23	14	1.1	0.7	7	9	0.7	0.9	395	453	1	0.9
Test item	100	15	20	0.5	1.1	B	180	0	1.3	B	8	0	0.4	B	9	0	1	B	450	0	0.9
Test item	316	BP	17	0	0.9	BP	210	0	1.5	BP	BP	0	0	BP	11	0	1.1	BP	BP	0	0
Test item	1000	BP	18	0	0.9	BP	99	0	0.7	BP	BP	0	0	BP	BP	0	0	BP	BP	0	0
4-NOPD/NaN3/MMS	10	573	-	23.9	-	891	-	8.2	-	974	-	45	-	104	-	10.1	-	2264	-	5.5	-
2-AA	2.5	-	1252	-	64.7	-	1569	-	10.9	-	221	-	11	-	324	-	34.7	-	1149	-	2.2

P: Precipitation

B: Background lawn reduced 

Experiment II:

		TA 98				TA 100				TA 1535				TA 1537				TA 102
	Dose (µg)/plate	Revertant colonies per plate		Mutation factor		Revertant colonies per plate		Mutation factor		Revertant colonies per plate		Mutation factor		Revertant colonies per plate		Mutation factor		Revertant colonies per plate		Mutation factor
Treatment		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
A. Dest		14	24	0.9	1.2	103	114	0.9	0.9	6	13	0.7	1.2	12	11	1.3	0.9	227	276	1.1	1.1
DMSO		16	20	1	1	114	125	1	1	9	11	1	1	9	12	1	1	255	257	1	1
Test item	0.158	19	20	1.2	1	84	99	0.7	0.8	6	11	0.7	1	8	14	0.9	1.2	216	275	0.8	1.1
Test item	0.5	21	24	1.3	1.2	79	93	0.7	0.7	5	11	0.6	1	9	13	1	1.1	245	302	1	1.2
Test item	1.58	16	19	1	0.9	74	111	0.6	0.9	8	14	0.9	1.3	7	14	0.7	1.2	224	274	0.9	1.1
Test item	5	15	18	0.9	0.9	83	117	0.7	0.9	5	13	0.6	1.2	8	13	0.9	1.1	229	247	0.9	1
Test item	15.8	16	17	1	0.9	67	101	0.6	0.8	7	14	0.7	1.3	7	11	0.8	0.9	172	233	0.7	0.9
Test item	50	7	23	0.4	1.2	51	109	0.4	0.9	B	13	0	1.2	B	14	0	1.2	119	243	0.5	0.9
Test item	158	3	18	0.2	0.9	4	59	0	0.5	1	8	0.1	0.8	B	5	0	0.4	21	265	0.1	1
Test item	500	B	B	0	0	B	28	0	0.2	B	6	0	0.5	B	0	0	0	B	162	0	0.6
4-NOPD/NaN3/MMS	10	214	-	13.1	-	769	-	6.7	-	595	-	66.1	-	87	-	9.7	-	1436	-	5.6	-
2-AA	2.5	-	1707	-	85.4	-	1531	-	12.2	-	239	-	21.7	-	335	-	28.7	-	622	-	2.4

P: Precipitation

B: Background lawn reduced 

Applicant's summary and conclusion

Conclusions:

The test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

In order to investigate the potential of the test item for its ability to induce gene mutations, a bacterial reverse mutation assay (Ames) was performed according to OECD 471. The plate incorporation test (experiment I and II) was performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation (S9 mix from rat liver).

A pre-experiment was performed in order to determine the toxicity of the substance as well as that the concentration of the main test is based on this pre-experiment results. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:

Experiment I:

0.316, 1.00, 3.16, 10.0, 31.6, 100, 316 and 1000 μg/plate (all tester strains except TA98 and TA100 with metabolic activation)

3.16, 10.0, 31.6, 100, 316 and 1000 μg/plate (only TA98 and TA100 with metabolic activation)

Experiment II:

0.158, 0.50, 1.58, 5.0, 15.8, 50, 158 and 500 μg/plate

Precipitation was observed in all tester strains used in experiment I (with and without metabolic activation). Toxic effects of the test item were noted in all tester strains used in experiment I and II:

- In experiment I toxic effects of the test item were observed at concentrations of 100 µg/plate and higher (with and without metabolic activation), depending on the particular tester strain.

- In experiment II toxic effects of the test item were noted at concentrations of 50 µg/plate and higher (without metabolic activation) and at concentrations of 158 µg/plate and higher (with metabolic activation), depending on the particular tester strain.

However, no biologically relevant increases in revertant colony numbers of any of the five tester strains were observed at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.

Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.