Copper dioleate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

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:

19. April-12.May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
Name: Copper dioleate
Batch no.: 21092015
Appearance: Green, solid/paste
Composition: Cu-dioleate
Purity: > 99 %, IR
Homogeneity: homogeneous
Expiry date: 21. Sep. 2018
Storage: Room Temperature: (20 ± 5°C)


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was stored in the test facility in a closed vessel at room temperature (20±5°C).
Ethanol was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.

On the day of the start of the first experiment, a stock solution containing 51.8 g/L of the test item in ethanol was prepared. The test item solution was not sterile filtrated before use. The stock solution was used to prepare the geometric series of the concentrations to be tested. The following nominal concentrations were prepared for the first experiment: 5000 μg/plate, 1500 μg/plate, 500 μg/plate, 150 μg/plate, 50 μg/plate and 15 μg/plate.

On the day of the start of the second experiment, a stock solution containing 15.9 g/L of the test item in ethanol was prepared. The test item solution was not sterile filtrated before use. The following nominal concentrations were prepared for the second experiment: 1500 μg/plate, 750 μg/plate, 375 μg/plate, 188 μg/plate, 94 μg/plate, 47 μg/plate and 23 μg/plate. For both experiments the test item stock solution was kept in the ultrasonic bath for 5 min.

Species / strain / cell type:

S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

In the first experiment, Copper dioleate (dissolved in ethanol) was tested up to concentra-tions of 5000 µg/plate in the absence and presence of S9-mix (0.74 % final concentration in the treatment) in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method. Copper dioleate showed no precipitates on the plates at any of the concentrations.
The bacterial background lawn was reduced at the highest concentrations and a relevant decrease in the number of revertants was observed in all bacteria strains. The test item Copper dioleate showed signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.
The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

On the base of the first experiment, Copper dioleate was tested up to concentrations of 1500 µg/plate in the absence and presence of S9-mix (0.74% final concentration in the treatment) in all bacteria strain using the pre-incubation method. Copper dioleate showed no precipitates on the plates at any of the concentrations.
The bacterial background lawn was partly reduced at the two highest concentrations (1500 and 750 µg/plate) and a decrease in the number of revertants was observed in all bacteria strains.
The results of this experiments showed that the test item Copper dioleate caused no in-crease in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test item Copper dioleate did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.

Vehicle / solvent:

Ethanol

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-1,2-phenylene Diamine, 2-Amino-Anthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Preparations
In the days before each test, the media and solutions were prepared. On the day of the test, the bacteria cultures were checked for growth. The incubation chambers were heated to 37 ±1 °C. The water bath was turned to 43 ±1 °C. The table surface was disinfected. The S9 mix was freshly prepared and stored at 0 °C.

General preparation
Per strain and dose, 3 plates with and 3 plates without S9 mix were used. The test item solutions were prepared according to chapter 6.1.3.
Top agar basis was melted in a microwave oven, after melting, 10 mL of histidine-biotinsolution 0.5 mM per 100 mL basis was added and the bottle was placed in the water bath at 43 ±1 °C.

Plate incorporation method
The following materials were gently vortexed in a test tube and poured onto the selective agar plates:
-100 μL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
-500 μL S9 mix or phosphate buffer (for test without metabolic activation).
-100 μL bacteria suspension
-2000 μL overlay agar (top agar)
The plates were closed and left to harden for a few minutes, then inverted and placed in the dark incubator at 37 ±1 °C.

Pre-incubation method
The following materials were gently vortexed in a test tube and incubated at 37 ±1°C for 20 min:
-100 μL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
- 500 μL S9 mix (for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
- 100 μL bacteria suspension

After pre-incubation, 2000 μL overlay agar (top agar) was added, the tube was gently vortexed and the mixture was poured onto the selective agar plate. The plates were closed and left to harden for a few minutes, then inverted and placed in the dark incubator at 37 ±1 °C.

Genotype Confirmation
Genotype confirmation is performed for each batch of lyophilized bacteria before stock culture preparation.

Histidine requirement
Each strain was streaked on a biotin and a histidine-biotin-plate, using a sterilized wire loop. The plates were incubated for 24 h at 37 ±1 °C.

Ampicillin/Tetracycline-Resistance (pKM101, pAQ1) Each strain was streaked on an ampicillin agar plate and on an ampicillin-tetracycline agar
plate. TA1535 was taking the function of control strain, since it is not ampicillin resistant. The plates were incubated for 24 h at 37 ±1 °C.

UV-sensitivity (uvrB)
Each strain was streaked on a plate, and one half of the plate covered with aluminium foil so that one half of each streak was protected against light. The plates for the strain TA97a, TA100 and TA102 were irradiated for 8 sec, the plates for the strain TA98 were irradiated for 10 sec and the plates for the strain TA1535 were irradiated for 6 sec with a germicidal lamp (254 nm, 30W). Keeping a distance of 33 cm for the strains TA97a, TA102 and TA1535. Keeping a distance of 66 cm for the following strains: TA98, TA100. Incubation 24 h at 37 ±1 °C followed.

Crystal violet sensitivity (deep rough)
For each strain, 2 plates were used. 0.1 mL of bacteria suspension were mixed with 2 mL. Top-Agar and poured on nutrient agar. Sterile paper discs ( 9 mm), each soaked with 10 μL of crystal violet solution (0.1%) were placed into the middle of each plate, followed by incubation 24 h at 37 ±1°C.

Spontaneous Revertants
3 replicates, with/without S9, for each solvent which was used in the test, incubation for 48 h at 37 ±1°C.

Determination of Titre
The titre was determined by dilution of the overnight culture using sodium chloride solution and placing 0.1 mL on maximal-soft agar. Incubation for 48h at 37 ±1 °C followed. It should give a density of 109 cells/mL (at the least), 2 replicates with and without metabolic activation.

Toxicity Control
Performed in experiment 1 only analogously to the titre control with the maximum dose of test item with and without S9 on maximal-soft agar, 2 replicates with and without metabolic activation, incubation for 48h at 37 ±1°C.

Sterility Control
Performed analogously to the test with solvent only and S9 (without adding bacteria) on top agar, incubation for 48h at 37 ±1°C, 4 replicates.

Solubility
Plates were checked for precipitation of test item at the end of the incubation by visual inspection.

Positive Controls
Using diagnostic mutagens (see chapter 6.2, page 12), 3 replicates were prepared. The stock solutions of the substances were diluted to achieve an application volume of 0.1 mL/plate, incubation for 48h at 37 ±1°C.

Rationale for test conditions:

In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 g/L in demineralised H2O, dimethyl sulfoxide (DMSO) and ethanol. Ethanol was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.

Evaluation criteria:

The colonies were counted visually and the numbers were recorded. A spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control. The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given. A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Statistics:

Mean, SD

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Signs of toxicity towards all the bacteria strains could be observed in the two highest concentrations (1500 and 750 μg/plate)

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 97

Remarks:

TA97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Signs of toxicity towards all the bacteria strains could be observed in the two highest concentrations (1500 and 750 μg/plate)

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Signs of toxicity towards all the bacteria strains could be observed in the two highest concentrations (1500 and 750 μg/plate)

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Signs of toxicity towards all the bacteria strains could be observed in the two highest concentrations (1500 and 750 μg/plate)

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Signs of toxicity towards all the bacteria strains could be observed in the two highest concentrations (1500 and 750 μg/plate)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 g/L in demineralised H2O, dimethyl sulfoxide (DMSO) and ethanol.
Ethanol was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations. The stock solution was used to prepare the geometric series of the concentrations to be tested. The following nominal concentrations were prepared for the first experiment: 5000 μg/plate, 1500 μg/plate, 500 μg/plate, 150 μg/plate, 50 μg/plate and 15 μg/plate.

HISTORICAL CONTROL DATA
Positive historical control data: All values within the historical data


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Signs of toxicity towards all the bacteria strains could be observed in the two highest concentrations (1500 and 750 μg/plate). The number of revertants was decreased and the bacterial background lawn was slightly visible. In the lower concentrations the bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.

Experiment 1

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
H2O	Mean	95	103	13	16	98	88	221	196	21	16
	sd	4.0	5.8	3.6	4.0	15.6	7.0	28.4	10.6	2.0	1.0
DMSO	Mean	94	98	12	12	86	93	229	205	17	18
	sd	2.6	15.7	3.1	1.5	4.6	9.1	34.0	32.6	1.2	1.2
Ethanol	Mean	88	99	11	11	87	80	268	199	20	15
	sd	10.3	17.5	1.2	3.2	11.2	6.0	59.7	12.2	3.5	3.2
Positive Controls*	Mean	623	331	351	49	779	632	1085	1127	267	96
	sd	12.9	44.1	87.1	6.4	124.5	127.0	43.1	56.0	23.0	7.0
	f(I)	6.63	3.38	29.25	4.08	7.95	6.80	4.74	5.50	12.71	5.33

5000 µg/plate	Mean	0	0	0	0	0	0	77	75	3	4
	sd	0.0	0.0	0.0	0.0	0.0	0.0	7.0	5.0	1.7	1.7
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.29	0.38	0.15	0.27
1500 µg/plate	Mean	83	78	12	12	75	78	192	169	14	11
	sd	11.9	2.1	0.6	0.6	5.3	9.9	58.9	24.4	4.6	1.2
	f(I)	0.94	0.79	1.09	1.09	0.86	0.98	0.72	0.85	0.70	0.73
500 µg/plate	Mean	90	103	12	10	87	105	248	155	17	13
	sd	9.0	12.6	0.0	2.5	5.5	12.3	58.9	35.9	5.5	1.5
	f(I)	1.02	1.04	1.09	0.91	1.00	1.31	0.93	0.78	0.85	0.87
150 µg/plate	Mean	87	95	12	13	91	94	148	208	13	14
	sd	5.3	5.1	1.5	2.6	8.5	11.1	8.0	42.1	3.6	3.5
	f(I)	0.99	0.96	1.09	1.18	1.05	1.18	0.55	1.05	0.65	0.93
50 µg/plate	Mean	89	98	13	18	85	92	184	253	13	19
	sd	2.1	11.4	3.1	4.7	11.9	8.4	4.0	11.5	2.6	3.0
	f(I)	1.01	0.99	1.18	1.64	0.98	1.15	0.69	1.27	0.65	1.27
15 µg/plate	Mean	100	91	12	15	100	82	241	220	17	12
	sd	7.8	1.2	3.1	3.2	8.1	6.0	27.2	21.2	2.1	2.1
	f(I)	1.14	0.92	1.09	1.36	1.15	1.03	0.90	1.11	0.85	0.80

Second Experiment

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
H2O	Mean	118	86	12	17	96	100	372	380	21	25
	sd	3.6	7.0	4.6	6.9	7.9	7.5	20.8	63.5	4.5	3.2
DMSO	Mean	113	112	15	16	81	93	331	304	23	25

	sd	15.0	7.8	4.2	2.6	5.7	3.5	56.2	28.0	4.9	3.1
Ethanol	Mean	111	124	12	16	75	91	295	336	24	24
	sd	14.2	7.5	1.5	2.9	4.0	17.1	42.7	41.8	6.1	2.6
Positive Controls*	Mean	459	505	343	83	329	861	960	1147	342	133
	sd	178.5	29.5	6.1	2.0	61.1	176.4	185.4	20.5	30.0	32.6
	f(I)	4.06	4.51	22.87	5.19	3.43	9.26	2.90	3.77	16.29	5.32
1500 µg/plate	Mean	0	0	0	1	0	20	14	45	0	0
	sd	0.0	0.0	0.0	0.0	0.0	3.0	1.5	27.6	0.0	0.0
	f(I)	0.00	0.00	0.00	0.06	0.00	0.22	0.05	0.13	0.00	0.00
750 µg/plate	Mean	10	11	5	2	0	11	70	80	0	0
	sd	1.0	2.1	3.5	1.2	0.0	5.9	17.3	8.5	0.0	0.0
	f(I)	0.09	0.09	0.42	0.13	0.00	0.12	0.24	0.24	0.00	0.00
375 µg/plate	Mean	112	105	12	12	115	81	201	234	14	14
	sd	20.1	7.5	0.6	0.0	17.5	12.9	26.9	82.1	3.2	2.5
	f(I)	1.01	0.85	1.00	0.75	1.53	0.89	0.68	0.70	0.58	0.58
188 µg/plate	Mean	90	90	15	14	104	104	236	248	20	13
	sd	1.5	4.2	3.1	3.2	13.8	25.6	57.2	39.9	3.2	4.2
	f(I)	0.81	0.73	1.25	0.88	1.39	1.14	0.80	0.74	0.83	0.54
94 µg/plate	Mean	101	95	11	11	102	84	337	243	15	12
	sd	1.2	3.8	1.2	1.7	9.0	14.3	44.6	7.0	1.5	0.0
	f(I)	0.91	0.77	0.92	0.69	1.36	0.92	1.14	0.72	0.63	0.50
47 µg/plate	Mean	100	110	11	14	99	89	221	269	25	19
	sd	0.6	5.5	2.1	2.6	2.0	3.5	4.6	28.7	5.0	8.7
	f(I)	0.90	0.89	0.92	0.88	1.32	0.98	0.75	0.80	1.04	0.79
23 µg/plate	Mean	109	106	11	10	89	85	241	277	14	19
	sd	4.0	2.5	1.5	2.5	5.5	13.3	32.1	13.0	2.5	3.6
	f(I)	0.98	0.85	0.92	0.63	1.19	0.93	0.82	0.82	0.58	0.79

Conclusions:

Copper dioleate is not mutagenic. Based on the results of this study it is concluded that Copper dioleate is not mutagenic in the Salmonella typhimuriumstrains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.

Executive summary:

The test item Copper dioleate was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535). The test was performed in two experiments in the presence and absence of S9- mix (rat liver S9-mix induced by Aroclor 1254). In the first experiment, Copper dioleate (dissolved in ethanol) was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix (0.74 % final concentration in the treatment) in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method. Copper dioleate showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was reduced at the highest concentrations and a relevant decrease in the number of revertants was observed in all bacteria strains. The test item Copper dioleate showed signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation. The results of this experiment showed that none of the tested concentrations showed a

significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

On the base of the first experiment, Copper dioleate was tested up to concentrations of 1500 μg/plate in the absence and presence of S9-mix (0.74% final concentration in the treatment) in all bacteria strains using the pre-incubation method. Copper dioleate showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was partly reduced at the two highest concentrations (1500 and 750 μg/plate) and a decrease in the number of revertants was observed in all bacteria strains.

The results of this experiments showed that the test item Copper dioleate caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test item Copper dioleate did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.

Based on the results of this study it is concluded that Copper dioleate is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental

conditions in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the results of this study it is concluded that Copper dioleate is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental

conditions in this study.