Cobalt Nickel Manganese Oxide

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:

8 June 2020 - 9 October 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD 471

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997, 9th addendum

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

impurity 1

Reference substance name:

Total unspecified impurities

IUPAC Name:

Total unspecified impurities

Constituent 1

Reference substance name:

Cobalt Nickel Manganese Oxide

Cas Number:

37348-84-8

Molecular formula:

Ni1-x-yMnxCoyOz with Me=Ni+Mn+Co=1 and Ni/Me= 0.45-0.98 Mn/Me= 0.01-0.35 Co/Me= 0.01-0.35 O/Me= 1.00-1.33

IUPAC Name:

Cobalt Nickel Manganese Oxide

Test material form:

solid

Specific details on test material used for the study:

Batch/Lot number: PVX-144A PVX14
Description: Grey Powder
Purity: 100% (7.44% Co ; 6.99% Mn ; 59.67% Ni)
Manufacturer: Umicore
Expiry date: 30 April 2021
Storage conditions: Controlled room temperature (15-25 °C, ≤70% relative humidity). Protected from light and humidity (store in a tightly closed container).
The Certificate of Analysis is attached in Appendix 1 of the study report.

Method

Target gene:

histidine, tryptophan

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 :livers ofphenobarbital/β-naphthoflavone-induced male Wistar rat
- method of preparation of S9 mix: Ames et al (1975), Maron and Ames (1983)
- concentration or volume of S9 mix and S9 in the final culture medium: The mean protein concentration of the S9 fraction used was determined to be 28.0 g/L; S9mix: containing 10% (v/v) of S9
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The sterility of the preparation was confirmed in each case. The biological activity in the Salmonella assay of S9 was characterized in each case using the two mutagens 2-Aminoanthracene and Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. The batches of S9 used in this study functioned appropriately.

Test concentrations with justification for top dose:

Concentrations for the main tests were selected on the basis of the Preliminary Compatibility Test and Preliminary Range Finding Test. In the Assay 1 and Assay 2, slightly different concentrations were used.
Based on the results of the preliminary test, a 100 mg/mL stock solution was prepared in 1% (w/v) methyl cellulose, which was diluted by serial dilutions in several steps to obtain the dosing formulations for lower doses. The maximum test concentration was 1581 μg test item/plate due to cytotoxicity at the higher concentrations.
Examined concentrations in Assay 1 were 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate.
Examined concentrations in Assay 2 were 1581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 1% (w/v) methyl cellulose

- Justification for choice of solvent/vehicle: 1% (w/v) methyl cellulose was used as vehicle (solvent) based on the available information

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water; DMSO, 1%(w/v) Methyl cellulose

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylene-diamine; 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation) (preliminary range finding test, assay 1); preincubation (assay 2);

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test, an Assay 1 and an Assay 2. In the Preliminary Range Finding Test and Assay 1, the plate incorporation method was used. In the Assay 2 the pre-incubation method was used.

Concentrations for the main tests were selected on the basis of the Preliminary Compatibility Test and Preliminary Range Finding Test. In the Assay 1 and Assay 2, slightly different concentrations were used.

Preliminary Compatibility test
Based on the available information 1% (w/v) methyl cellulose was selected as vehicle (solvent) of the study. The obtained stock formulation (50 μL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension.

Preliminary range finding test
100 mg/mL stock solution was prepared in 1% (w/v) methyl cellulose. Seven test concentrations were prepared by successive dilutions of the stock solution, spaced by factors of 2, 2.5 and approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98 and TA100) were determined at concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item, in the absence and presence of metabolic activation. In the Preliminary Range Finding Test the plate incorporation method was used.

Test item concentrationsin the mutagencity tests (assay 1/2)
Based on the results of the preliminary test, a 100 mg/mL stock solution was prepared in 1% (w/v) methyl cellulose, which was diluted by serial dilutions in several steps to obtain the dosing formulations for lower doses. The maximum test concentration was 1581 μg test item/plate due to cytotoxicity at the higher concentrations.
Examined concentrations in Assay 1 were 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate. Examined concentrations in Assay 2 were 1581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate.


OTHER:
The colony numbers on the untreated / negative (solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.
* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.

Evaluation criteria:

The colony numbers on the untreated / negative (solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.
* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.

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:

cytotoxicity

Remarks:

assay 1: at precipitating concentration 1581 µg/plate, and 500 µg/plate; assay 2: at precipitating concentration 1581 µg/plate, and 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

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:

cytotoxicity

Remarks:

assay 1: at 1581µm/plate (precipitate); assay 2: at 500µg/plate and 1581 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

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:

cytotoxicity

Remarks:

assay 1: at 1581 µg/plate (precipitate); assay 2: at 1581 µg/plate (precipitate) and 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

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:

cytotoxicity

Remarks:

assay 1: at 1581 µg/plate (precipitate), and 500 µg/plate; assay 2: at 1581 µg/plate (precipitate), 500 µg/plate, 158,1 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

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:

cytotoxicity

Remarks:

assay 1: at 1581 µg/plate (precipitate), and 500 µg/plate; assay 2: at 1581 µg/plate (precipitate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In Assay 1 (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA98 bacterial strain at 50 μg/plate concentration with metabolic activation (the observed mutation factor value was: MF: 1.33). However, there was no dose-response relationship, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and the number of revertant colonies was within the historical control range.
In Assay 2 (pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA98 bacterial strain at 15.81 μg/plate concentration with metabolic activation (the observed mutation factor value was: MF: 1.23). However, there was no dose-response relationship, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and the number of revertant colonies was within the historical control range.
Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.
Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.

Untreated, negative (vehicle/solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains. At least five analysable concentrations were presented in all strains with and without metabolic activation.
The reference mutagens showed a distinct increase of induced revertant colonies in each strain with and without metabolic activation. The viability of the bacterial cells was checked by a plating experiment in each test. The study was considered to be valid.

Any other information on results incl. tables

Assay 1 (Plate Incorporation Method)

		Reverant colony number
Concentration (µg/plate)		TA98    -S9 / +S9		TA100    -S9 / +S9		TA1535   -S9 / +S9		TA1537   -S9 / +S9		E.coliWP2uvrA   -S9 / +S9
1581	mean SD MF	* and ** 21.3 4.04 1.19	* and ** 25.7 4.04 1.26	* and **  93.0 4.00 1.03	* and ** 106.7 3.51 1.10	* and ** 13.3 0.58 1.00	* and ** 13.7 1.15 0.93	* and ** 11.0 1.00 1.03	* and ** 11.7 1.15 1.03	* and ** 37.7 5.13 0.87	* and ** 44.3 0.58 0.97
500	mean SD MF	18.7 3.79 1.04	20.7 4.51 1.02	*** 92.3 3.21 1.02	*** 98.3 2.08 1.02	*** 13.0 1.00 0.98	*** 12.3 0.58 0.84	10.7 3.21 1.00	11.0 2.00 0.97	***  40.7 3.06 0.94	*** 42.0 3.00 0.92
158.1	mean SD MF	21.0 2.65 1.17	23.0 2.00 1.13	92.3 4.04 1.02	99.7 1.53 1.03	15.3 1.15 1.15	12.0 0.00 0.82	10.7 2.52 1.00	11.3 2.52 1.00	40.0 3.61 0.92	43.0 1.73 0.94
50	mean SD MF	21.0 2.00 1.17	27.0 6.08 1.33	90.7 3.06 1.00	103.7 9.24 1.07	15.0 1.00 1.13	15.0 1.00 1.02	9.3 2.52 0.88	12.7 0.58 1.12	43.0 1.00 0.99	45.0 1.73 0.99
15.81	mean SD MF	20.0 1.00 1.11	24.7 8.02 1.21	92.0 2.65 1.02	102.3 3.06 1.06	15.7 0.58 1.18	15.3 1.15 1.05	9.3 1.53 0.88	11.0 1.73 0.97	40.7 2.89 0.94	45.7 0.58 1.00
5	mean SD MF	19.7 0.58 1.09	25.7 4.51 1.26	90.3 0.58 1.00	95.0 2.65 0.98	14.3 0.58 1.08	14.7 0.58 1.00	10.3 2.08 0.97	11.3 0.58 1.00	43.3 1.15 1.00	43.7 2.08 0.96
1.581	mean SD MF	21.0 2.00 1.17	23.3 3.51 1.15	92.3 1.53 1.02	97.0 4.36 1.00	14.3 0.58 1.08	12.3 0.58 0.84	8.3 2.52 0.78	8.7 1.53 0.76	41.3 1.15 0.95	44.3 0.58 0.97
Untreated control	mean SD MF	17.7 1.53 0.98	19.3 1.53 0.95	89.7 3.79 0.99	107.0 5.20 1.11	13.3 2.08 1.00	13.3 0.58 0.91	9.3 2.52 0.88	12.0 2.00 1.06	40.3 1.53 0.93	44.7 1.15 0.98
DMSO control	mean SD MF	17.3 0.58 0.96	19.3 0.58 0.95	95.3 16.17 1.06	92.7 3.06 0.96	13.7 0.58 1.03	13.0 1.00 0.89	9.0 1.73 0.84	12.0 1.73 1.06	42.7 1.15 0.98	44.7 0.58 0.98
Distilled water control	mean SD MF	-	-	89.0 2.00 0.99	101.7 7.37 1.05	14.0 0.00 1.05	13.7 1.15 0.93	-	-	41.3 1.15 0.95	44.0 2.00 0.96
1% (w/v) Methyl cellulose control	mean SD MF	18.0 1.00 1.00	20.3 3.06 1.00	90.3 3.06 1.00	96.7 7.64 1.00	13.3 0.58 1.00	14.7 0.58 1.00	10.7 2.31 1.00	11.3 1.53 1.00	43.3 3.06 1.00	45.7 0.58 1.00
Positive control	mean SD MF	NPD (4µg) 425.3 16.65 24.54	2AA (2µg) 2406.7 16.65 124.48	SAZ (2 µg) 1054.7 37.17 11.85	2AA (2 µg) 2466.7 16.65 26.62	SAZ (2µg) 1212.0 30.20 86.57	2AA (2µg) 212.3 7.02 16.33	9AA (50 µg) 416.0 22.27 46.22	2AA (2 µg) 203.7 7.51 16.97	MMS (2µl) 1104.0 32.00 26.71	2AA (50 µg) 250.7 14.05 5.61

* precipitate

** Reduced background lawn

*** Slightly reduced background lawn

Assay 2 (Pre-Incubation Method)

		Reverant colony number
Concentration (µg/plate)		TA98    -S9 / +S9		TA100   -S9 / +S9		TA1535   -S9 / +S9		TA1537     -S9 / +S9		E.coliWP2uvrA   -S9 / +S9
1581	mean SD MF	* and ** 19.3 1.15 0.97	* and ** 19.0 1.00 1.02	* and ** 87.7 13.05 0.96	* and ** 101.0 10.58 1.02	* and ** 11.3 3.06 0.92	* and **  9.7 1.53 0.74	* and ** 11.7 1.15 0.90	* and **  14.7 1.53 0.88	* and ** 42.3 0.58 1.01	* and ** 50.3 1.15 1.06
500	mean SD MF	***  20.0 0.00 1.00	*** 20.3 1.53 1.09	** 83.7 7.51 0.92	** 110.3 4.93 1.11	***  11.3 2.08 0.92	*** 7.7 4.51 0.59	*** 12.3 1.53 0.95	***  13.7 1.53 0.92	45.0 1.73 1.07	49.3 0.58 1.03
158.1	mean SD MF	19.0 1.73 0.95	21.0 1.00 1.13	***  84.3 4.04 0.93	*** 95.0 17.78 0.96	10.3 4.73 0.84	11.3 0.58 0.87	14.7 1.53 1.13	15.3 1.15 0.92	43.7 1.53 1.04	50.0 3.46 1.05
50	mean SD MF	18.3 1.53 0.92	20.3 1.53 1.09	92.0 9.17 1.01	107.0 2.65 1.08	13.7 2.08 1.11	11.0 5.29 0.85	13.3 2.52 1.03	16.0 1.73 0.96	45.7 0.58 1.09	54.0 2.00 1.13
15.81	mean SD MF	20.0 1.00 1.00	23.0 0.00 1.23	88.3 1.53 0.97	106.0 7.81 1.07	12.3 3.06 1.00	11.0 1.00 0.85	13.3 0.58 1.03	16.3 1.15 0.98	45.0 1.00 1.07	52.7 3.21 1.10
5	mean SD MF	19.7 0.58 0.98	21.0 3.46 1.13	89.0 2.65 0.98	104.0 7.55 1.05	13.0 1.73 1.05	13.3 2.89 1.03	10.7 0.58 0.82	15.3 1.53 0.92	44.7 2.31 1.06	50.7 1.53 1.06
1.581	mean SD MF	18.0 2.00 0.90	21.3 2.08 1.14	88.7 3.21 0.97	108.7 9.71 1.10	15.0 1.00 1.22	12.0 4.00 0.92	13.3 1.15 1.03	17.0 1.00 1.02	42.3 0.58 1.01	49.7 2.31 1.04
0.5	mean SD MF	19.0 1.00 0.95	20.0 1.73 1.07	88.0 6.08 0.97	104.0 6.24 1.05	13.0 2.00 1.05	12.7 2.08 0.97	13.3 1.15 1.03	14.3 0.58 0.86	44.7 2.31 1.06	46.7 2.08 0.98
Untreated control	mean SD MF	18.3 1.53 0.92	22.3 0.58 1.20	91.0 1.73 1.00	96.7 2.52 0.98	12.3 1.53 1.00	11.3 0.58 0.87	13.3 1.15 1.03	16.3 0.58 0.98	44.0 1.00 1.05	47.0 1.00 0.99
DMSO control	mean SD MF	19.0 1.73 0.95	23.3 0.58 1.25	93.0 3.00 1.02	102.7 2.08 1.04	13.3 2.08 1.08	13.0 1.73 1.00	15.3 1.15 1.18	15.7 1.53 0.94	43.7 2.08 1.04	48.0 1.00 1.01
Distilled water control	mean SD MF	-	-	95.0 4.36 1.04	102.3 2.52 1.03	13.0 1.73 1.05	13.7 1.53 1.05	-	-	42.7 1.15 1.02	52.3 1.53 1.10
1% (w/v) Methyl cellulose control	mean SD MF	20.0 1.0 1.0	18.7 1.53 1.00	91.0 5.00 1.00	99.0 5.57 1.00	12.3 2.08 1.00	13.0 1.00 1.00	13.0 1.00 1.00	16.7 0.58 1.00	42.0 1.00 1.00	47.7 0.58 1.00
Positive control	mean SD MF	NPD (4 µg) 406.7 16.65 21.40	2AA (2 µg) 2380.0 66.09 102.00	SAZ (2 µg) 1232.0 42.33 12.97	2AA (2 µg) 2412.0 36.66 23.49	SAZ (2 µg) 1117.3 14.05 85.95	2AA (2 µg) 204.7 9.71 15.74	9AA (50 µg) 417.3 28.38 27.22	2AA (2 µg) 214.7 9.02 13.70	MMS (2 µl) 1117.3 32.33 26.19	2AA (50 µg) 246.0 7.21 5.13

* precipitate

** Reduced background lawn

*** Slightly reduced background lawn

Applicant's summary and conclusion

Conclusions:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.
The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavone- induced rats.
The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Plate Incorporation Method), an Assay 1 (Plate Incorporation Method) and an Assay 2 (Pre- Incubation Method).
The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item pNMC oxide (8:1:1) (Batch Number: PVX- 144A PVX14) had no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (E.coli WP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test, an Assay 1 (Plate Incorporation Method) and an Assay 2 (Pre-Incubation Method).

Based on the available information, the test item was dissolved in 1% (w/v) methyl cellulose at a concentration of 100 mg/mL. Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate were examined in the Range Finding Test in Salmonella typhimurium TA98 and TA100 tester strains in the absence and presence of metabolic activation. Based on the results of the preliminary experiment, with excessive cytotoxicity at higher concentrations, the examined test concentrations in the Assay 1 were 1581, 500, 158.1, 50, 15.81, 5 and 1.581μg/plate and in the Assay 2 were 1581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate.

In the assays the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect.

Precipitate was detected on the plates in the main tests in all examined bacterial strains with and without metabolic activation at 1581 μg/plateconcentration. Inhibitory, cytotoxic effect of the test item was observed in the main tests in all examined bacterial strains with and without metabolic activation at higher concentrations.

In the assays the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect. The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test item pNMC oxide (8:1:1 ) had no mutagenic activity on the growths of the bacterial strains under the test conditions used in this study.