Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-11-11 to 2015-02-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
07-1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Method

Target gene:
HIS operon (S. thyphimurium)
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:
Liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors
Test concentrations with justification for top dose:
1st series: 5, 16, 50, 160, 500, 1600 and 5000 µg/plate
2nd series: 20.48, 51.2, 128, 320, 800, 2000 and 5000 µg/plate
In the two series with S9 mix, 10 % S9 in the S9 mix were used.
Vehicle / solvent:
DMSO
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-aminoanthracene
Remarks:
with S9 mix
Details on test system and experimental conditions:
Acceptance Criteria
The assay was to be considered valid if all the following criteria were met:
The vehicle control counts fell within the laboratory’s historical control ranges.
The positive control chemicals induced increases in revertant numbers of >= 1.5-fold (in strain TA102), >= 2-fold (in strains TA98 and TA100) or >= 3-fold (in strains TA1535 and TA1537) the concurrent vehicle control confirming discrimination between different strains, and an active S-9 preparation.
Evaluation criteria:
For valid data, the test article was considered to be mutagenic if:
A concentration related increase in revertant numbers was >= 1.5-fold (in strain TA102), >= 2-fold (in strains TA98 or TA100) or >= 3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values.
The positive trend/effects described above were reproducible.
The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if neither of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.
Statistics:
n.a.

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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True 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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True 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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True 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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.
Executive summary:

Study design

The mutagenic potential of the test item was examined in a GLP study according to OECD GL 471 using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. In the two series with S9 mix, 10 % S9 in the S9 mix was used. The test item was dissolved in anhydrous analytical grade dimethyl sulphoxide (DMSO) and tested at concentrations ranging from 5 - 5000 μg/plate.

Results

In Experiment 1 treatments of all the tester strains were performed in the absence and presence of S-9, using final concentrations at 5, 16, 50, 160, 500, 1600 and 5000 μg/plate, plus vehicle and positive controls. Following these treatments, evidence of toxicity was observed at 5000 μg/plate in strain TA1537 in the absence of S-9 and strain TA98 in the presence of S-9.

Experiment 2 treatments of all the tester strains were performed in the absence and presence of S-9. The maximum test concentration of 5000 μg/plate was retained for all strains. Narrowed concentration intervals were employed covering the range 20.48 to 5000 μg/plate, in order to examine more closely those concentrations of the test material approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments, no evidence of toxicity was observed.

Precipitation was observed on the test plates at concentrations of 800 μg/plate and above.

Vehicle and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies all fell within acceptable ranges for vehicle control treatments, and were elevated by positive control treatments.

Following test material treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were ≥1.5 -fold (in strain TA102), ≥2 -fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control. This study was considered therefore to have provided no evidence of any test material mutagenic activity in this assay system.

Conclusion

With and without addition of S9 mix as the extemal metabolizing system, the test item was not mutagenic under the experimental conditions described.