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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 20 June 2017 to 31 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Reaction mass of Fatty acids, montan-wax and Fatty acids, montan-wax, ethylene esters and Fatty acids, montan wax, mixed esters with fatty acids C16-18 and ethylene glycol and Fatty acids, montan-wax, stearyl esters and Montan wax
EC Number:
911-428-0
IUPAC Name:
Reaction mass of Fatty acids, montan-wax and Fatty acids, montan-wax, ethylene esters and Fatty acids, montan wax, mixed esters with fatty acids C16-18 and ethylene glycol and Fatty acids, montan-wax, stearyl esters and Montan wax
Test material form:
solid: flakes
Details on test material:
Test item: WARADUR® LG
Purity (certified): > 99.5%
Water solubility: not specified
Stability under test conditions: Not specified
Appearance: Pale yellow, solid flakes
Expiry date: 2018-04-11

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 liver extract from phenobarbital (PB) and β-naphthoflavone (BNF) induced male Wistar rats
Test concentrations with justification for top dose:
Pre-Test: 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate
Main study: 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate, additionally 1.581 µg/plate in complementary confirmatory tests
Top dose according to guideline
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: polyethylene glycol PEG 400
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylenediamine (TA98), Sodium azide (TA100, TA1535), 9-aminoacridine (TA1537), Methyl-methanesulfonate (WP2 uvrA)
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (all strains)
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) in initial mutation test, preincubation in confirmatory mutation test and complementary confirmatory mutation test

DURATION
- Preincubation period: 20 min at 37 °C
- Exposure duration: 48 +/- 1 h
- Fixation time (start of exposure up to fixation or harvest of cells): 48 +/- 1 h

NUMBER OF REPLICATIONS: triplicates for each dose level

DETERMINATION OF CYTOTOXICITY
- Method: other: number of revertant colonies and inhibition of background lawn
Evaluation criteria:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- the number of reversions was 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 was more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.

The test item was considered to have shown no mutagenic activity in this study if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Statistics:
not performed

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: not in initial test, but in confirmatory test without metabolic activation at >/= 1581 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: not in initial test, but in confirmatory test without metabolic activation at >/= 1581 µg/plate µg/plate, with metabolic activation at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Summary tables are attached to his study record

Any other information on results incl. tables

Slight precipitation or precipitation of the test item was observed at concentrations >/= 500 µg/plate.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the test item was not mutagenic to bacteria.
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 (Escherichia coli WP2 uvrA) 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 (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method), a Confirmatory Mutation Test (Pre-Incubation Method). Due to excessive cytotoxicity in parts of the study by the Pre-Incubation Method, a Complementary Confirmatory Test was performed by the Plate Incorporation (Salmonella typhimurium TA100 and TA1537 without metabolic activation) or Pre-Incubation Method (Salmonella typhimurium TA1535 without metabolic activation).

Based on the results of the Compatibility Test, the test item was dissolved in PEG 400 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 tester strains Salmonella typhimurium TA100 and TA98 in the absence and presence of metabolic activation.

Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test and Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50 15.81 and 5 μg/plate; in the Complementary Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate in case of Salmonella typhimurium TA100 and TA1537 without metabolic activation and 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate in case of Salmonella typhimurium TA1535 without metabolic activation.

Precipitate/slight precipitate was detected on the plates in the Preliminary Concentration Range Finding Test at higher concentrations with and without metabolic activation in all examined bacterial strains.

Precipitate/slight precipitate/ was detected on the plates in the main tests at higher concentrations with and without metabolic activation in all examined bacterial strains with the exception of Escherichia coli WP2 uvrA strain with and without metabolic activation in the Initial Mutation Test. Inhibitory, cytotoxic effect of the test item was not detected in the Preliminary Concentration Range Finding Test and in the Initial Mutation Test. In the Confirmatory Mutation Test slightly reduced background lawn or lower numbers of revertant colonies was detected in Salmonella typhimurium TA98 strain with and without metabolic activation at higher concentrations. In the Complementary Confirmatory Mutation Test slightly reduced background lawn was observed in Salmonella typhimurium TA1535 strain without metabolic activation at higher concentrations.

In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no consistent dose-related trends and no indication of any treatment-related effect. No historical control database exists for PEG 400; however, the observed data for this solvent was confirmed to be comparable with the historical control range of other solvents, therefore it was considered to be acceptable. Some cytotoxicity was observed with the vehicle under some conditions, but sufficient data was generated within the study for the results to be fully valid.

The mean values of revertant colonies of the other solvent control plates were in harmony 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 tests were 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 WARADUR LG (Batch Number: W00004534) has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

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