Bis[N-(2-hydroxyethyl)-N-methylglycinato-N,O,ON]copper

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:

2016-10-27 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-(2-hydroxyethyl)-N-methylglycinato-N,O,ON]copper

EC Number:

300-491-4

EC Name:

Bis[N-(2-hydroxyethyl)-N-methylglycinato-N,O,ON]copper

Cas Number:

93940-93-3

Molecular formula:

C10H20CuN2O6

IUPAC Name:

bis[N-(2-hydroxyethyl)-N-methylglycinato-N,O,ON]copper

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:

rat liver S9 mix

Test concentrations with justification for top dose:

Pre-experiment (plate incorporation): 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
Experiment I (plate incorporation test with and without rat liver S9): 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
Experiment II (pre-incubation test with and without rat liver S9):3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. 5000 μg/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Aqua dest.
- 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 control =solvent control

Negative solvent / vehicle controls:

yes

Remarks:

Aqua dest.; negative control =solvent control

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)

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)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment I: in agar (plate incorporation)
Experiment II: pre-incubation

DURATION
Experiment I:
- 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

Experiment II:
- Preincubation period: 60 min at 37°C
- Exposure duration: 60 min at 37°C and 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 (in two cases only two plates were evaluated)

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 100

Remarks:

Experiment I

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

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Experiment I

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

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

Experiment I

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

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

Experiment I

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

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Remarks:

Experiment I

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

Test results 6

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Experiment II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 1000 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate and higher (with metabolic activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Experiment II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 1000 µg/plate and higher (with metabolic activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 8

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

Experiment II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 316 µg/plate and higher (with metabolic activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 9

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

Experiment II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 316 µg/plate and higher (with metabolic activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 10

Key result

Species / strain:

S. typhimurium TA 102

Remarks:

Experiment II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at a concentration of 5000 µg/plate (without metabolic activation) and at concentrations of 2500 µg/plate and higher (with metabolic activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

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

RANGE-FINDING/SCREENING STUDIES:
see 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: Cytotoxicity can 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.
- Other observations when applicable: Toxic effects of the test item were noted in all tester strains used in experiment II at concentrations of 1000 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate and higher (with metabolic activation), depending on the particular tester strain.

Any other information on results incl. tables

Pre-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 (A. dest)		1.0	1.0	1.0	1.0
4-NOPD	10.0	25.8	-	-	-
NaN3	10.0	-	-	6.1	-
2-AA	2.50	-	53.2	-	11.6
Test Item	3.16	1.1	0.9	0.9	0.9
	10.0	1.1	0.8	0.9	0.8
	31.6	0.9	0.7	1.0	1.1
	100	1.1	0.7	1.2	1.1
	316	1.2	0.8	1.1	1.1
	1000	1.3	0.9	1.2	1.1
	2500	1.0	1.1	1.2	1.3
	5000	1.3	0.8	1.3	1.2

* [toxicity parameter]: B = Background lawn reduced; N = No background lawn

 

Experiment I

		TA 98				TA 100				TA 1535				TA 1537				TA 102
		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	Dose (µg)/plate	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
A. Dest		22	31	1	1	128	128	1	1	20	17	1	1	7	9	1	1	297	336	1	1
Test item	31.6	20	22	0.9	0.7	128	135	1	1.1	19	26	0.9	1.6	9	8	1.2	0.9	244	363	0.8	1.1
Test item	100	25	21	1.1	0.7	148	143	1.2	1.1	46	28	2.2	1.7	10	6	1.4	0.6	260	403	0.9	1.2
Test item	316	326	26	1.2	0.8	137	135	1.1	1.1	24	36	1.2	2.1	8	6	1	0.6	307	372	1	1.1
Test item	1000	28	28	1.3	0.9	158	141	1.2	1.1	46	28	2.2	1.7	11	10	1.5	1.1	278	327	0.9	1
Test item	2500	22	33	1	1.1	153	165	1.2	1.3	25	26	1.2	1.5	9	9	1.3	1	288	372	1	1.1
Test item	5000	30	24	1.3	0.8	164	148	1.3	1.2	24	26	1.2	1.6	8	7	1	0.7	317	404	1.1	1.2
4-NOPD/NaN3/MMS	10	576	-	25.8	-	776	-	6.1	-	1009	-	49.6	-	91	-	12.4	-	1303	-	4.4	-
2-AA	2.5	-	1632	-	53.2	-	1488	-	11.6	-	211	-	12.7	-	223	-	24.7	-	757	-	2.3

No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I.

Experiment II:

		TA 98				TA 100				TA 1535				TA 1537				TA 102
		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	Dose (µg)/plate	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
A. Dest		25	29	1	1	133	95	1	1	18	19	1	1	11	10	1	1	267	255	1	1
Test item	31.6	26	21	1.1	0.7	126	122	1	1.3	25	29	1.4	1.5	12	8	1.1	0.8	298	377	1.1	1.5
Test item	100	22	35	0.9	1.2	149	124	1.1	1.3	27	20	1.5	1	12	8	1.1	0.7	253	338	0.9	1.3
Test item	316	26	19	1	0.7	149	112	1.1	1.2	20	22	1.1	1.2	10	5	0.9	0.5	267	190	1	0.7
Test item	1000	23	29	0.9	1	90	106	0.7	1.1	31	18	1.7	0.9	7	7	0.6	0.7	266	258	1	1
Test item	2500	18	14	0.7	0.5	123	103	0.9	1.1	22	7	1.2	0.4	9	7	0.8	0.7	277	183	1	0.7
Test item	5000	25	3	1	0.1	79	39	0.6	0.4	19	5	1.1	0.3	7	3	0.6	0.3	155	149	0.6	0.6
4-NOPD/NaN3/MMS	10	545	-	22.1	-	890	-	6.7	-	947	-	52.6	-	128	-	11.7	-	1257	-	4.7	-
2-AA	2.5	-	125	-	4.3	-	1032	-	10.9	-	82	-	4.2	-	103	-	9.9	-	710	-	2.8

Due to more sensitive pre-incubation method, in experiment II toxic effects of the test item were seen in most tester strains.

- In tester strain TA 98 toxic effects of the test item were observed at concentrations of 1000 µg/plate and higher (with metabolic activation).

- In tester strain TA 100 toxic effects of the test item were noted at concentrations of 1000 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate and higher (with metabolic activation).

- In tester strains TA 1535 and TA 1537 toxic effects of the test item were seen at concentrations of 316 µg/plate and higher (with metabolic activation).

- In tester strain TA 102 toxic effects of the test item were observed at a concentration of 5000 µg/plate (without metabolic activation) and at concentrations of 2500 µg/plate and higher (with metabolic activation).

Applicant's summary and conclusion

Conclusions:

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.

Executive summary:

In order to investigate the ability of the test substance to induce gene mutations, a bacterial reverse mutation assay (Ames) according to OECD 471 was performed. Two experiments were ran: the plate incorporation test (experiment I) and the pre-incubation test (experiment II) 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 ). The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in both experiments: 31.6, 100, 316, 1000, 2500 and 5000 μg/plate

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation). No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I.

Due to more sensitive pre-incubation method, in experiment II toxic effects of the test item were seen in most tester strains. In tester strain TA 98 toxic effects of the test item were observed at concentrations of 1000 µg/plate and higher (with metabolic activation). In tester strain TA 100 toxic effects of the test item were noted at concentrations of 1000 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate and higher (with metabolic activation). In tester strains TA 1535 and TA 1537 toxic effects of the test item were seen at concentrations of 316 µg/plate and higher (with metabolic activation). In tester strain TA 102 toxic effects of the test item were observed at a concentration of 5000 µg/plate (without metabolic activation) and at concentrations of 2500 µg/plate and higher (with metabolic activation).

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

All criteria of validity were met.

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.