Dilithium peroxide

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:

21 JUL 2021 - 12 OCT 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1998

Deviations:

yes

Remarks:

see explanation below

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated May 30, 2008

Deviations:

yes

Remarks:

see explanation below

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21st July, 1997, corrected 26th June 2020

Deviations:

yes

Remarks:

see explanation below

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use

Version / remarks:

November 2011

Deviations:

not specified

Principles of method if other than guideline:

Concerning: The results of concentration range finding test (informatory toxicity test).
According to the Study Plan: Beside the absent or decreased revertant colony counts affected background lawn development: absent, reduced or slightly reduced background lawn was mentioned as indication of test item inhibitory effect.
Deviation: Only two grade of affected background lawn development was noticed: absent or reduced background lawn.
Reason for this change: Technical, more precise description of these details.
Effect on the Study: This deviation is not considered to have affected the integrity and validity of the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dilithium peroxide

EC Number:

234-758-0

EC Name:

Dilithium peroxide

Cas Number:

12031-80-0

Molecular formula:

Li2O2

IUPAC Name:

Dilihtium peroxide

Test material form:

solid: particulate/powder

Method

Target gene:

his

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : The S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH (Rathenau Str. 2; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).
- method of preparation of S9 mix : The complete S9 Mix was freshly prepared containing components as follows: Ice cold 0.2 M sodium phosphate-buffer, pH 7.4 500 mL, Rat liver homogenate (S9) 100 mL, Salt solution for S9 Mix 400 mL
- concentration or volume of S9 mix and S9 in the final culture medium : 500 μL
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The Quality Control & Production Certificate of each lot of S9 was obtained from the supplier. The original Quality Control & Production Certificates of rat liver S9 are stored in the Laboratory of TOXI-COOP ZRT. The copies of the quality control certificates of the used S9 lots are given in Appendix VIII.
The following lots of the S9 were applied:
Lot Number: 4335; Expiry date: October 06, 2022; Protein content: 38.6 mg/mL (used in the informatory toxicity and initial mutation tests);
Lot Number: 4344; Expiry date: October 15, 2022; Protein content: 33.5 mg/mL (used in the confirmatory mutation test);
Lot Number: 4399; Expiry date: January 27, 2023; Protein content: 37.3 mg/mL (used in the initial mutation and confirmatory mutation tests).

Test concentrations with justification for top dose:

in the case of Salmonella typhimurium strains:
-S9: 500, 250, 160, 50, 16, 5 and 1.6 μg/plate
+S9: 1200, 800, 500, 160, 50, 16 and 5 μg/plate
in the case of Escherichia coli WP2 uvrA:
±S9: 1600, 500, 160, 50, 16, 5 and 1.6 μg/plate

Based on the solubility test, a test item suspension with concentration of 50 mg/mL was prepared in dimethyl sulfoxide (DMSO) and diluted in 6 steps by a factor of approximately √10.
At the chosen top concentrations no precipitate was expected on the minimal glucose agar plates.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (Water content: < 0.1 %), DMSO anhydrous (Water content: 0.003 %)
- Justification for choice of solvent/vehicle: DMSO (water content: ≤ 0.1 %) was used as vehicle for the positive control items (NPD, 9AA and 2AA) throughout the study; furthermore, as vehicle for the test item in the solubility and informatory toxicity tests. Because of the specific test item characteristic (rapid hydrolysis was expected) anhydrous DMSO (water content: ≤ 0.005 %) was used for the test item in the main tests.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 2-Aminoanthracene, 2AA, 4-Nitro-1,2-phenylenediamine, NPD

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate

FOR GENE MUTATION:
Bacteria (cultured in Nutrient Broth No.2. (Section: 5.4.2)) were exposed to the test item both in the presence and absence of rat liver S9 as metabolic activation system. Molten top agar was prepared and kept at 45°C. Two mL of top agar was aliquoted into individual test tubes. This solution was mixed and poured on the surface of the properly labeled minimal agar plates. For incubations with metabolic activation, instead of phosphate buffer, 0.5 mL of the S9 Mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions and each with the addition of negative and positive controls. The plates were incubated at 37°C for about 48 hours.
- Criteria for small (slow growing) and large (fast growing) colonies: An increase is considered biologically relevant if: in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control, in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: The cytotoxicity was indicated by affected background lawn development (absent, reduced, or slightly reduced background lawn) and/or decreased revertant colony counts (absent revertants or revertants below the historical control data ranges and/or corresponding vehicle control data ranges).

Rationale for test conditions:

Selection of the concentration range was done on the basis of solubility test and concentration range finding test (informatory toxicity test).
The cytotoxicity was indicated by affected background lawn development (absent, reduced, or slightly reduced background lawn) and/or decreased revertant colony counts (absent revertants or revertants below the historical control data ranges and/or corresponding vehicle control data ranges).

Evaluation criteria:

A test item is 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 concentration groups occurs in at least one strain with or without metabolic activation.
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

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (± S9) throughout the study.

STUDY RESULTS
No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. The revertant colony numbers were higher, or slightly higher than the actual vehicle control revertant colony numbers. In the initial and confirmatory mutation tests inhibitory effect of the test item on bacterial growth was observed in the examined strains, in the absence and presence of exogenous metabolic activation (±S9).

For all test methods and criteria for data analysis and interpretation: All criteria for the validity of the performed experiments have therefore been met.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: The positive control reference items (diagnostic mutagens) induced the expected, biological relevant increases (more than 3-fold increase) in revertant colonies and the number of revertant colonies was within the historical control data range per strain.
- Negative (solvent/vehicle): The spontaneous revertant colony numbers of the dimethyl sulfoxide (DMSO) vehicle control plates showed characteristic mean numbers agreed with the actual historical control data ranges in all strains in the main experiments

Applicant's summary and conclusion

Conclusions:

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 neither in the presence nor in the absence of a metabolic activation. In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

The test was performed according to the OECD Guideline 471, EU Method B13/14, EPA OPPTS 870.5100 and ICH Guidance S2(R1) under GLP compliance. The test item was tested with regard to a 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 post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats. The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations in the genome of the strains of Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and of Escherichia coli WP2 uvrA,neither with nor without metabolic activation. In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.