Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-01-28 to 2013-02-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study; GLP study without deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
OPPTS changed its name to OCSPP,
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
Characteristics: clear, colourless, viscous

Method

Target gene:
mutated gene loci resposible for histidine auxotropy
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
- obtained from Trinova Biochem according to Dr. Bruce N. AMES,
Additional strain / cell type characteristics:
other: histidine auxotroph
Metabolic activation:
with and without
Metabolic activation system:
Arochlor 1254 induced rat liver S9; male rats
Test concentrations with justification for top dose:
Plate incorporation test: 31.6, 100, 316, 1000, 3160 and 5000 µg per plate;
Preincubation test: 31.6, 100, 316, 1000, 3160 and 5000 µg per plate
Vehicle / solvent:
The test item was completely dissolved in dimethylsulfoxide (DMSO) . Fresh preparations of the test item were used for the treatment in all experimental parts.
Controls
Untreated negative controls:
no
Remarks:
solvent test will be used as negative reference item
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
for details see below
Positive control substance:
other: without metabolic activation: sodium azide in aqua ad iniectabilia for TA 1535 and TA 100, 2-nitroflurene in DMSO for TA 98, 9-amino-acridine in ethanol abs. for TA 1537, Mitomycin C in DMSO for TA 102
Remarks:
with metabolic activation: 2-aminoanthracene in DMSO for TA 100, TA 1535, Benzo(a)pyrene in DMSO for TA 98, TA 102, TA 1537
Details on test system and experimental conditions:
Bacterial Reverse Mutation Test
SYSTEM OF TESTING
- Pre-Experiment: plate incorporation cytotoxicity test (+/- metabolic activation) with strain TA 100,
The test item was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain
TA100. Ten concentrations ranging from 0.316 to 5000 µg test item/plate were tested. No signs of cytotoxicity were noted up to the top
concentration of 5000/plate.
Hence, 5000 µg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.
- Main test: 1st - Standard plate incorporation method, 2nd - Preincubation method
- Metabolic activation assay: Arochlor 1254 induced rat liver S9 fraction, the protein content of the S9 fraction was 33.1 mg/mL S9, cytochrome
P-450: 0.40 nmol/mg protein
ADMINISTRATION
- Dosing:
* Plate incorporation test: 31.6, 100, 316, 1000, 3160 and 5000 µg per plate
* Preincubation test: 31.6, 100, 316, 1000, 3160 and 5000 µg per plate
- Data : 2 independent experiments with and without metabolic activation
- Number of replicates: 3 per concentration and experiment
- Positive and negative control groups and treatment:
- without metabolic activation:
* sodium azide in aqua ad iniectabilia for TA 1535 and TA 100, 10 µg/plate
* 2-nitroflurene in DMSO for TA 98, 10 µg/plate
* 9-amino-acridine in ethanol abs. for TA 1537, 100 µg/plate
* Mitomycin C in DMSO for TA 102, 10 µg/plate
- with metabolic acivation
* 2-aminoanthracene in DMSO for TA 100 and TA 1535, 2 µg/plate
* Benzo(a)pyrene in DMSO for TA 98, TA 102 and 1537, 10 µg/plate
- negative control: the vehicle DMSO was used as negative reference item (all test strains).
- Incubation time: 48 h to 72 h at 37 °C in the dark
- Pre-incubation time: 20 min at 37 °C;

NUMBER OF REPLICATIONS: 3 per concentration and experiment

NUMBER OF CELLS EVALUATED: approximately 10E8 viable cells in the late exponential or early stationary phase

DETERMINATION OF CYTOTOXICITY
- Method: No signs of cytotoxicity were noted up to the top concentration of 5000 µg test item/plate in the plate incorporation test and in the
preincubation test, each carried out without and with metabolic activation in all test strains.
Evaluation criteria:
The statistical evaluation of the results of the AMES test is still under discussion. In our laboratory, a test item is considered to show a
positive response if
- at one or more concentrations the number of revertants is reproducibly increased in at least one strain with or without metabolic activation. A
2-fold increase in comparison to the solvent control is regarded as being relevant for a positive response in the strains TA98, TA100 and TA102. For the strains TA1535 and TA1537 a 3-fold increase represents a biological relevant effect. The Mann and Whitney test (p ≤ 0.05, see section 6,
reference 3.) may be used to determine statistical significance.
Or
- a concentration-related increase over the range tested in the number of the revertants per plate is observed. The Spearman's rank correlation
coefficient (section 6, reference 3.) may be applied.
Biological relevance of the results should be considered first.
Positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on
histidine-free agar plates.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.
Statistics:
According to the OECD Guideline 471, a statistical analysis of the data is not mandatory

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
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:
GENTOXIC EFFECTS:
- With metabolic activation: negativ
- Without metabolic activation: negativ

CYTOTOXICITY EFFECTS:
- plate incorporation test without and with metabolic activation: no signs of cytotoxicity
- preincubation test without metabolic activation: no signs of cytotoxicity
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

see attchached document

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, under the present test conditions the test item tested up to a concentration of 5000 µg/plate caused no mutagenic effect in the Salmonella typhimuriumstrains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.
Executive summary:

The purpose of this study was to evaluate the test item for mutagenic activity (gene mutation) in bacteria without and with the addition of a mammalian metabolic activation system as originally described by AMES et al. (1973, 1975) and revised by MARON and (1983).

The test substance was examined in the 5 Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test.

The test item wascompletely dissolved in dimethylsulfoxide (DMSO).The vehicle served as the negative control.

Preliminary test

The test item was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg test item/plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000/plate.

Hence, 5000 µg test item /plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Main study

Six concentrations ranging from 31.6 to 5000 µg test item /plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.

Cytotoxicity

No signs of cytotoxicity were noted up to the top concentration of 5000 µg test item/plate inthe plate incorporation test and in the preincubation test, each carried out without and with metabolic activation in all test strains.

Mutagenicity

No increase in revertant colony numbers as compared with control counts was observed for the test item, tested up to a concentration of 5000 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).

The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.

 

In conclusion, under the present test conditions the test substance tested up to a concentration of 5000 µg/plate caused no mutagenic effect in the Salmonella typhimuriumstrains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.