lithium nickel dioxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames (OECD 471): negative (BASF SE, 2021)

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:

30.Nov.2020 - 03.Mar.2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 Jul. 1997

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

Incl. certificate from the competent authority

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch number of test material: 8800315
- Purity: Approx. 99.8 /100 g
- Water content: 0.1 g/100 g


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Expiry date: August 12, 2021
- Storage stability: The stability of the test substance under storage conditions
is guaranteed until 12 Aug 2021
- Homogeneity: The test item was homogeneous by visual inspection.
- Stability and homogeneity of the test material in the vehicleunder test conditions: The stability of the test substance in the vehicle DMSO was not determined analytically, because the test substance was administered immediately after preparation and is usually stable.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. The test substance was suspended in dimethyl sulfoxide (DMSO). To achieve a homogeneity of the test substance in the vehicle, the test substance preparation was treated with ultrasonic waves and was shaken thoroughly. The further concentrations were diluted according to the planned doses. To keep the test substance homogeneously in the vehicle, the test substance preparation was carefully pipetted before removal. All test substance formulations were prepared immediately before use.

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: The S9 fraction was prepared at BASF SE in an AAALAC approved
laboratory in accordance with the German Animal Welfare Act and the effective
European Council Directive.
- method of preparation of S9 mix: At least 5 male Wistar rats [Crl:WI(Han)] (200 - 300 g; Charles River Laboratories Germany GmbH) received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally (both supplied by Sigma-Aldrich, 82024 Taufkirchen, Germany) each on three consecutive days. 24 hours after the last administration, the rats were sacrificed, and the livers were prepared
using sterile solvents and glassware at a temperature of +4°C. The livers were weighed and washed in a weight-equivalent volume of a 150 mM KCl solution and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, 5 mL portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.
- volume of S9 mix and S9 in the final culture medium: 0.5 ml
- quality controls of S9: Sterility and test for metabolic capability using a standard plate test with benzo[a]pyrene and tester strains TA100 and TA98

Test concentrations with justification for top dose:

0; 33; 100; 333; 1000; 2500 and 5000 μg/plate

In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose was tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be tested in repeat experiments for further clarification/substantiation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene (2-AA) (96%) N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (97%) 4-nitro-o-phenylenediamine (NOPD) (98%)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate plating
- Number of independent experiments: Three

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 0.5 ml of culture solution containing approx. 10exp9 cells per ml
- Test substance added in agar (standard plate test) and by preincubation (preincubation test)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 min
- Exposure duration/duration of treatment: 48 - 72 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition; decrease in the number of revertants (factor <0.6)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were determined for all dose and test groups

Rationale for test conditions:

Doses were chosen in agreement with the recommendations of current guidelines.
Test substance precipitation was observed in the preincubation assay at a concentration of 5000 μg/plate both with and without S9 mix. If precipitation of the test material was observed, it would be recorded. As long as precipitation did not interfere with the colony scoring, 5 mg/plate was generally selected and analyzed as the maximum dose (at least in the 1st Experiment) even in the case of relatively insoluble test compounds to detect possible mutagenic impurities.

Evaluation criteria:

The test substance was considered positive in this assay if the following criteria were met: A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Occasionally at and above 100 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Occasionally at and above concentrations of 2.5 mg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Occasionally at concentrations at and above 100 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Remarks:

Standard plate test and preincubation assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 5 mg/plate in the preincubation assay

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Remarks:

Standard plate test and preincubation assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the 1. experiment with the TA100 strain with S9 mix, a slight increase in the number of revertant colonies was observed (below a factor of 2). This effect was due to a low number of revertant colonies in the vehicle control and could not be confirmed in a 2. experiment. Therefore, this finding was considered to be not biologically relevant.

Conclusions:

Under the experimental conditions chosen here, it is concluded that the test substance is not mutagenic in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The test substance Lithium nickel dioxide was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay (OECD 471, BASF SE, 2021).

STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
DOSE RANGE: 33 μg - 5000 μg/plate (SPT), 33 μg - 5000 μg/plate (PIT)
TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) with and without metabolic activation (liver S9 mix from induced rats).
SOLUBILITY: Precipitation of the test substance was observed in the preincubation test at 5000 μg/plate with and without S9 mix.
TOXICITY: A weak bacteriotoxic effect was observed depending on the strain and test conditions at and above 100 μg/plate

A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.
Under the experimental conditions of this study, the test substance Lithium nickel dioxide is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Justification for classification or non-classification

Under the experimental conditions of the chosen key study, it is concluded that the test substance is not mutagenic in the bacterial reverse mutation test in the absence and the presence of metabolic activation. The present data on genetic toxicity do not fulfill the criteria laid down in Regulation (EC) No 1272/2008 (CLP Regulation) and therefore, a non-classification is warranted.