Nickel Cobalt Manganese Hydroxide

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

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, Ninth Addendum

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

Nickel Cobalt Manganese Hydroxide

EC Number:

839-353-8

Cas Number:

189139-63-7

Molecular formula:

Molecular formula: Ni1-x-yMnxCoy(OH)2, Stoechiometry of (Me = Ni + Co + Mn) equals 1, ranges of the elements approx. as: Ni/Me = 0.25 – 0.95 Mn/Me = 0.02 – 0.40 Co/Me = 0.02 – 0.40 OH = 2.00

IUPAC Name:

Nickel Cobalt Manganese Hydroxide

Constituent 1

Reference substance name:

Manganese dihydroxide

Cas Number:

18933-05-6

Molecular formula:

Mn(OH)2

IUPAC Name:

Manganese dihydroxide

Constituent 2

Reference substance name:

Cobalt dihydroxide

EC Number:

244-166-4

EC Name:

Cobalt dihydroxide

Cas Number:

21041-93-0

Molecular formula:

CoH2O2

IUPAC Name:

cobalt(2+) dihydroxide

Constituent 3

Reference substance name:

Nickel dihydroxide

EC Number:

235-008-5

EC Name:

Nickel dihydroxide

Cas Number:

12054-48-7

Molecular formula:

H2NiO2

IUPAC Name:

nickel(2+) dihydroxide

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Batch/Lot number: PVX-089 PVX 14
Description: Dark Grey Powder
Purity: 100% (6.31% Co ; 5.96% Mn ; 50.87% 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:

Mammalian microsomal enzyme activation mixture (liver extract, S9 fraction), rat: pre-treated with phenobarbital and ß-naphthoflavone

The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory of Charles River Laboratories Hungary Kft. according to Ames et al. () and Maron and Ames(). The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly.

Induction of Liver Enzymes
Male Wistar rats (433-642 g, animals were 13-17 weeks old) were treated with phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days. Rats were given drinking water and food ad libitum until 12 h before sacrifice when food was removed. Sacrifice was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels. Initiation of the induction of liver enzymes used for preparation S9 used in this study was 13 January 2020.

Preparation of Rat Liver Homogenate S9 Fraction
The mean protein concentration of the S9 fraction used was determined to be 28.0 g/L.
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.

S9 Mix: containing 10% (v/v) of S9

Test concentrations with justification for top dose:

Assay 1:
concentration of the test item (mg/ml): 31.62, 10, 3.162, 1, 0.3162, 0.1, 0.03162
concentration (µg/plate): 1581, 500, 158.1, 50, 15.81, 5, 1.581

Assay 2:
concentration of the test item (mg/ml): 31.62, 10, 3.162, 1, 0.3162, 0.1, 0.03162, 0.01
concentration (µg/plate): 1581, 500, 158.1, 50, 15.81, 5, 1.581, 0.5

Vehicle / solvent:

1% (w/v) methyl cellulose (based on available information)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

1% (w/v) methyl cellulose; DMSO; Distilled water

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:

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.

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

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 Concentrations in the Mutagenicity Test (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.

Control Groups Used
Strain-specific positive and negative (solvent) controls, both with and without metabolic activation were included in each test. In addition, an untreated control was used demonstrating that the chosen vehicle induced no deleterious or mutagenic effects.

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.

Criteria for a Positive Response
A test item was considered mutagenic if:
• a concentration-related increase in the number of revertants occurs and/or;
• a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
• the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
• the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.

Criteria for a negative Response
A test article is considered non-mutagenic if it produces neither a concentration-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the concentration groups, with or without metabolic activation.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

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 1581 µg/plate (precipitate), 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:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

assay 1: at 1581 g/plate, with metabolic activation: MF = 1.38

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

assay 2: 1.581 µg/plate, with metabolic activation, MF = 1.25

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

assay 1: at 1581 µg/plate (precipitate), 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:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary range finding test
In the Preliminary Range Finding Test, the plate incorporation method was used. The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (solvent) and positive controls. Each sample (including the controls) was tested in triplicate.
Following concentrations were examined: 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate.
No precipitate was detected on the plates in the preliminary experiment in both examined bacterial strains with and without metabolic activation.
Inhibitory, cytotoxic effect of the test item (slight reduced / reduced background lawn development) was observed in the preliminary experiment in Salmonella typhimurium TA98 bacterial strains with and without metabolic activation at 5000 and 2500μg/plate concentrations and in the Salmonella typhimurium TA100 bacterial strains with and without metabolic activation at 5000, 2500 and 1000 μg/plate concentrations.

Mutagenicity tests
The assays were performed in the presence and absence of a metabolic activation system. Each test was performed with appropriate untreated, negative (solvent) and positive controls. In the main tests each sample (including the controls) was tested in triplicate.
In the main 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/plate concentration.
Assay 1 (plate incorporation method)
Based on the results of the preliminary experiment, the examined test concentrations in the Assay 1 were 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate
Inhibitory, cytotoxic effect: in all Salmonella typhimurium bacterial strains with and without metabolic activation at 1581 and 500 μg/plate concentrations; in the Escherichia coli WP2 uvrA bacterial strains with and without metabolic activation at 1581 μg/plate concentration
The highest revertant rate was observed in Salmonella typhimurium TA100 bacterial strain at 1581 μg/plate concentration with metabolic activation (the observed mutation factor value was: MF: 1.38). 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.
Assay 2 (pre-incubation method)
Based on the results of the preliminary experiment, the examined test concentrations in the Assay 2 were 1581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plateInhibitory, cytotoxic effect: in Salmonella typhimurium TA98 and TA1535 bacterial strains with and without metabolic activation at 1581 and 500μg/plate concentrations; in Salmonella typhimurium TA100 and TA1537 bacterial strains with and without metabolic activation at 1581, 500 and 158.1 μg/plate concentrations; in Escherichia coli WP2 uvrA bacterial strain with and without metabolic activation at 1581 μg/plate concentration
The highest revertant rate was observed in Salmonella typhimurium TA98 bacterial strain at 1.581 μg/plate concentration with metabolic activation (the observed mutation factor value was: MF: 1.25). 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.

Validity of the tests
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.coli WP2 uvrA   -S9 /+S9
1581*	mean SD MF	18.0  1.0 1.0	21.7 2.08 1.07	105.7 17.21 1.17	133.0 10.58 1.38	11.3 0.58 0.85	11.7 2.52 0.80	11.0 1.00 1.03	12.3 1.15 1.09	34.0 7.00 0.78	43.3 0.58 0.95
500 **	mean SD MF	18.7 2.08 1.04	23.0 1.73 1.13	95.3 6.51 1.06	106.7 7.02 1.10	10.3 1.53 0.78	12.3 1.53 0.84	8.0 1.00 0.73	11.3 0.58 1.00	38.7 3.06 0.98	43.0 1.00 0.94
158.1	mean SD MF	19.3 1.53 1.07	21.7 2.31 1.07	98.0 9.64 1.08	104.3 8.96 1.08	10.7 1.15 0.80	10.7 3.06 0.73	10.3 0.58 0.97	10.3 1.15 1.91	41.0 1.73 0.95	43.7 1.15 0.96
50	mean SD MF	16.7 0.58 0.93	23.7 0.58 1.16	108.3 12.74 1.20	113.0 9.17 1.17	12.0 2.00 0.90	8.3 0.58 0.57	9.3 0.58 0.88	8.3 1.53 0.74	41.7 0.58 0.96	42.3 2.52 0.93
15.81	mean SD MF	17.3 0.58 0.96	23.3 0.58 1.15	92.7 2.89 1.03	104.0 4.36 1.08	11.0 3.00 0.83	12.7 3.21 0.86	8.3 0.58 0.78	10.3 0.58 0.91	41.3 1.15 0.95	44.7 1.15 0.98
5	mean SD MF	18.7 1.53 1.04	23.3 1.15 1.15	93.0 11.27 1.03	98.7 1.15 1.02	10.3 3.51 0.78	10.7 1.15 0.73	8.0 1.00 0.75	10.3 1.15 0.91	42.7 1.15 0.98	45.3 3.06 0.99
1.581	mean SD MF	16.0 1.00 0.89	22.3 0.58 1.10	95.0 10.82 1.05	96.7 0.58 1.00	12.0 2.65 0.90	13.7 1.15 0.93	8.0 1.00 0.75	10.0 1.73 0.88	40.7 1.15 0.94	45.7 0.58 1.00
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 0.53 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 3.03 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 (2 µg) 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 (2 µg) 1212.0 30.20 86.57	2AA (2 µg) 212.3 7.02 16.33	9AA (50µg) 416.0 22.27 46.22	2AA (2 µg) 203.7 7.51 16.97	MMS (2 µl) 1104.0 32.00 26.71	2AA (2 µg) 250.7 14.05 5.61

* Precipitate and reduced background lawn

** Slightly reduced background lawn (except for  E.coliWP2uvrA)

Assay 2 (Pre-Incubation Method)

		Reverant colony number
Concentration (µg/plate)		TA98     -S9 /+S9		TA100     -S9 /+S9		TA1535     -S9 /+S9		TA1537   -S9 /+S9		E.coli WP2 uvrA    -S9 /+S9
1581	mean SD MF	* and**  14.7 3.57 0.73	* and**  20.7 2.08 1.11	* and**  67.3 10.69 0.74	* and** 100.0 20.07 1.01	* and** 11.0 2.65 0.89	* and** 12.0 0.00 0.92	* and** 12.7 1.53 0.97	* and** 15.0 3.00 0.90	* and ** 45.3 1.15 1.08	* and ** 48.3 0.58 1.01
500	mean SD MF	*** 17.3 0.58 0.87	*** 19.3 0.58 1.04	**  95.0 8.00 1.04	** 96.0 7.21 0.97	**  15.0 2.65 1.22	** 13.3 1.53 1.03	**  12.7 1.15 0.97	** 13.0 2.00 0.78	45.3 0.58 1.08	47.7 1.53 1.00
158.1	mean SD MF	18.7 1.53 0.93	20.7 0.58 1.11	***  88.7 7.23 0.97	*** 118.0 2.00 1.19	13.7 1.53 1.11	13.0 1.00 1.00	***  11.3 1.15 0.97	*** 15.7 0.58 0.94	45.3 1.15 1.08	48.7 2.08 1.02
50	mean SD MF	20.3 1.15 1.02	21.0 2.00 1.13	92.7 10.02 1.02	114.7 8.39 1.16	14.3 0.58 1.16	13.7 1.53 1.05	12.7 1.15 0.97	13.0 3.61 0.78	45.7 0.58 1.09	47.7 0.58 1.00
15.81	mean SD MF	18.7 2.08 0.93	20.7 0.58 1.11	85.3 5.51 0.94	111.3 9.07 1.12	13.0 1.00 1.05	14.3 1.15 1.10	13.0 1.00 1.00	16.0 1.00 0.96	45.7 0.58 1.09	47.0 1.73 0.99
5	mean SD MF	20.7 2.31 1.03	21.0 3.61 1.13	85.0 3.00 0.95	111.7 7.02 1.13	13.7 1.53 1.11	13.7 0.58 1.05	13.7 0.58 1.05	13.3 3.06 0.80	45.0 1.00 1.07	47.3 1.15 0.99
1.581	mean SD MF	19.0 1.00 0.95	23.3 2.08 1.25	86.0 3.61 0.95	101.0 1.00 1.02	13.3 1.15 1.08	13.0 1.00 1.00	12.3 2.08 0.95	12.7 2.08 0.76	45.3 1.15 1.08	46.3 1.53 0.97
0.5	mean SD MF	17.3 0.58 0.87	21.0 1.00 1.13	85.7 4.93 0.94	108.0 9.54 1.09	12.3 1.53 1.00	13.7 1.53 1.05	11.0 3.61 0.85	13.0 1.73 0.78	44.0 2.00 1.05	48.0 1.00 1.01
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	47.7 0.58 1.00
1% (w/v) Methyl cellulose control	mean SD MF	20.0 1.00 1.00	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 (4µg)  406.7 16.65 21.40	2AA (2µg) 2380.0 66.09 102.00	SAZ (2µg)  1232.0 42.33 12.97	2AA (2µg)  2412.0 36.66 23.49	SAZ (2µg) 1117.3 14.05 85.95	2AA (2µg) 204.7 9.71 15.74	9AA (50 µg) 417.3 28.38 27.22	2AA (2µg) 214.7 9.02 13.70	MMS (2 µl) 1117.3 32.33 26.19	2AA (50µg) 246.0 7.21 5.13

* Precipitate

** Reduced background lawn

*** Slightly reduced background lawn (except for  E.coliWP2uvrA)

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, pNMC-hydroxide had no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

Executive summary:

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (salmonella typhimurium TA98, TA100, TA 1535 and TA 1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (WP2urvA) 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 1.581 μg/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/plate concentration. 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-hydroxide (8:1:1) had no mutagenic activity on the growth of the bacterial strains under the test conditions in this study.