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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:
21 November 2017 to 12 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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

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

Method

Target gene:
Histidine
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):
CELLS USED
- Source of cells: TA 98, TA 1535 and TA 102 MOLTOX, INC., NC 28607, USA; TA 100 and TA 1537 Xenometrix AG, Switzerland.
- Methods for maintenance in cell culture if applicable: stock cultures stored with nutrient broth (OXOID) supplemented with DMSO (approx. 8% v/v) over liquid nitrogen
Metabolic activation:
with and without
Metabolic activation system:
liver microsomal fraction from rats treated with phenobarbital / β-naphthoflavone
Test concentrations with justification for top dose:
exp 1 :
0.100, 0.316, 1.00, 3.16, 10.0, 31.6, 100 and 316 nL/plate or TA 100, TA 1535 and TA 1537 (with and without metabolic activation),for TA 98 and 102 (without metabolic activation)
3.16, 10.0, 31.6, 100, 316, 100, 316 and 1000 nL/plate for TA98 and TA 102 with metabolic activation

exp 2
0.050, 0.158, 0.50, 1.58, 5.0, 15.8, 50.0, 158 and 500 nL/plate for all strains with and without metabolic activation

Top doses limited by cytotoxicity (reduction of bacterial background lawn or reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 of solvent controls)
Vehicle / solvent:
Aqua destillata (purified water)
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine; 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation (Due to severe toxicity of the test item, the confirmatory experiment is carried out according to the plate incorporation method with a different spacing between dose levels)

DURATION
- Preincubation period: NA
- Incubation time: at 37 °C for at least 48 h in the dark

NUMBER OF REPLICATIONS: 3/concentration

DETERMINATION OF CYTOTOXICITY
- Method: reduction of bacterial background lawn or reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 of solvent control

Evaluation criteria:
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
than the reversion rate of the solvent control
Statistics:
NA

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 100 nL/plate and 15.8 nL/plate in exp 1 and 2
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 31.6 and 50 nL/plate in exp 1 and 2
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 100 and 15.8 nL/plate in exp 1 and 2
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 100 and 158 nL/plate in exp 1 and 2
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 316 and 15.8 nL/plate in exp 1 and 2
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 100 nL/plate and 500 nL/plate in exp 1 and 2
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 100 and 158 nL/plate in exp 1 and 2
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 100 and 50 nL/plate in exp 1 and 2
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 100 and 158 nL/plate in exp 1 and 2
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 15.8 and 500 nL/plate in exp 1 and 2
Vehicle controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
The substance is considered non-mutagenic under the conditions of the test.
Executive summary:

The substance was tested in an Ames test in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 with and without metabolic activation. The maximum concentration tested was limited mainly by a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 of solvent control. In two independent experiments no biologically relevant increase in revertant colony numbers of any of the five tester strains was observed. Therefore the substance is considered non-mutagenic.