Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
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 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)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Designation in test facility : 15070801G
Date of receuip: 08/07/2015
Conditions at receipt : room temperature, proper conditions
Appearance Colourless liquid
Composition 2-(3,3-DIMETHYLCYCLOHEX-1-ENYL)-2,5,5-
TRIMETHYL-1,3-DIOXANE
Molecular formula C15H26O2
Molecular weight 238.37 g/mol
Purity Sum of isomers: 98.5%
Homogeneity Homogeneous
Stability H2O: not stated; EtOH: not stated; acetone: not stated;
CH3CN: not stated; DMSO: not stated
Solubility H2O: < 0.1 g/L; EtOH: > 1 g/L; acetone: > 1 g/L; CH3CN:
unknown; DMSO: < 0.1 g/L
Production date Mar. 2015
Expiry date Mar. 2016
Storage Room temperature: 20 ± 5 °C, keep away from light
Name : MADERAL ZA1076
Batch n°100026324

Method

Target gene:
TA97a : hisD6610 ; uvrB ; rfa ; pKM101
TA98 : hisD3052 ; uvrB ; rfa ; pKM101
TA100 : hisG46 ; uvrB ; rfa ; pKM101
TA102 : hisG428 ; rfa ; pKM101 ; pAQ1
TA1535 : hisG46 ; uvrB ; rfa
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 97
Remarks:
TA 97a
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
S. typhimurium TA 1535
Metabolic activation:
with and without
Metabolic activation system:
S9-mix, rat-liver S9-mix induced by Aroclor 1254
Test concentrations with justification for top dose:
The test item is only soluble in ethanol in a concentration of 50 g/L, the chosen concentrations are above.
- First experiment : 5 concentrations (50, 150, 500, 1500, 5000 µg/plate)
- Second experiment : 6 concentrations (156, 313, 625, 1250, 2500, 5000 µg/plate)
Vehicle / solvent:
ethanol. (the test item is only soluble in ethanol)
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 4-Nitro-1,2-phenylene Diamine, 2-Amino-Anthracene
Remarks:
3 replicates / Stock solutions of the substance diluted to effect an application volume of 0.1 mL/plate
Details on test system and experimental conditions:
First experiment : Plate incorporation method
Second experiment : pre-incubation method

Cell density : 10^9 cells/mL, correlating to 100 colonies/plate after dilution

Duration ???

Number of replications :
First test : 3 / strain and concentration
Second test: 3 / strain and concentration

Culture of bacteria :
All strain of Salmonella typhimurium were stored as lyophilisates in the fridge at 2-8 °C. 8 h before the start of each experiment, one lyophilisate per strain to be used was taken from the fridge to inoculate a culture vessel containing nutrient broth. After incubation overnight at 37 ± 1 °C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.

Prepation of media and solutions :
In the days before each test, the media and solutions were prepared.
Statistics:
Not specified

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 97
Remarks:
TA97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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
Vehicle controls validity:
valid
Untreated negative 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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative 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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative 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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative 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
Remarks:
at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative 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
Remarks:
at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Executive summary:

The genetic toxicity in vitro was performed according to the OECD Guideline 471 (Bacterial Reverse Mutation Assay). The test item was considered as not mutagenic and not pro-mutagenic.