Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07.11.-18.12.2017
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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9
Test concentrations with justification for top dose:
Concentration range in Exp I: 3-5000 µg/plate
Concentration range in Exp II: 1.0-2500 µg/plate
According to the results of the pre-experiment
Vehicle / solvent:
Solvent: Dimethylsulfoxide
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
A. dest
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-NOPD
Remarks:
without metabolic activation
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabilic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION:
in agar (plate incorporation) in Experiment I and pre-incubation in
Experiment II.

DURATION
- Exposure duration: 48h at 37°C

NUMBER OF REPLICATIONS: 3

ACCEPTANCE CRITERIA:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100 and TA 102)
- the negative control plates (A. dest.) with and without S9 mix are within the historical control data
range of the test facility
- corresponding background growth on solvent control, negative control and test plates is observed.
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analyzable
Rationale for test conditions:
these are according to the OECD guideline 471
Evaluation criteria:
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean
values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one
tester strain with or without metabolic activation

A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher as co
mpared to the reversion rate of the solvent control
A test item producing neither a dose related increase in the number of revertants nor a reproducible
biologically relevant positive response at any of the dose groups is considered to be non-mutagenic
in this system.
Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the
interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No biologically relevant increases in revertant colony numbers of any of the five tester strains were
observed

Any other information on results incl. tables

Positive controls produced the expected increases in the number of revertants.

Applicant's summary and conclusion

Conclusions:
The test item is considered to be non-mutagenic in this bacterial reverse mutation assay
Executive summary:

In the current study the potential of the test item to induce gene mutations according to the plate

incorporation test (experiment I) and the pre-incubation test (experiment II) was assessed in the Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA102 with and without matabolic activation, and according to OECD TG 471 and in compliance to GLP.

In two independent experiments several concentrations of the test item were used.

The concentrations, including the controls, were tested in triplicate.

The following concentrations of the test item were prepared and used in the experiments:

Exp. I: 3.16, 10.1, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate

Exp. II: 1.0, 3.16, 10.0, 31.6, 100, 316, 1000 and 2500 μg/plate

No precipitation of the test item was observed in any tester strain used in experiment I and II (with

and without metabolic activation).

Toxic effects of the test item were noted in all of the five tester strains evaluated in experiment I and II.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were

observed at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

In experiment I in the tester strain TA98 a mutation factor of 2.0 was observed at a concentration of 3.16 μg/plate (with metabolic activation).

However, the correspondant revertant colony number was within the range of the historical negative control data and no dose-response relationship was observed. Thus, the effect was considered as not biologically relevant.

All criteria of validity were met.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.

Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.