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Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-12-04 to 2009-12-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP, OECD Guideline 471

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4-dimethyl-2-undecyl-4,5-dihydro-1,3-oxazole
EC Number:
686-853-5
Cas Number:
46921-17-9
Molecular formula:
C16H31NO
IUPAC Name:
4,4-dimethyl-2-undecyl-4,5-dihydro-1,3-oxazole
impurity 1
Reference substance name:
Unknown impurities
IUPAC Name:
Unknown impurities
Test material form:
liquid
Details on test material:
Other name of 4,4-Dimethyl-2-undecyl-2-oxazoline : cycloceramide
Specific details on test material used for the study:
Physical state : Liquid
Purity : 96%
Storage conditions : 4°C, keep away from daylight and moisture

Method

Target gene:
All the bacterial strains used in the Ames test carry a mutant gene that prevents them from synthetizing an essential amino acid (histidine in the case of S.typhimurium).
These strains carry additional mutations which increase their sensitivity to different types of mutagens. All S.typhimurium strains used in the test carry the rfa mutation.
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:
Chemical. Without metabolic activation S9(-): TA98 : 2-nitrofluorene; TA100 : Sodium Azide; TA102 : Mitomycin C; TA1535 : Sodium Azide; TA1537 : 9-aminoacridine With metabolic activation S9(+): For all Strains : 2-aminoanthracene
Test concentrations with justification for top dose:
5 concentrations (F1 to F5) starting at 5.00µL/plate (F1), based on the solubility profile of the test item (The test item was found to be soluble in corn oil at a concentration of 50µL/mL).
Concentrations F2 to F5 were prepared by 1:3 serial dilutions from the selected F1 concentration.
Vehicle / solvent:
Without metabolic activation :
TA98 : DMSO
TA100 : H2O
TA102 : H2O
TA1535 : H2O
TA1537 : DMSO

With metabolic activation :

For all Strains : DMSO
Controls
Untreated negative controls:
yes
Remarks:
with vehicle as negative control
Negative solvent / vehicle controls:
yes
Remarks:
Corresponding to the negative control
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
Details on test system and experimental conditions:
Number of replicate : 3
Number of doses : 5 concentrations (µL/plate) (F1 to F5). F1 = 5,00; F2 = 1,67; F3 = 0,56; F4 = 0,19; F5 = 0,06 and reference item controls.
Negative control : Yes, with vehicle as negative control (H2O or DMSO).
Metabolic activation system : with and without (S9) under the direct incorporation (main study) and the pre-incubation (confirmatory study) procedures.

In the direct incorporation procedure the mixture was immediately poured over a minimal agar medium plate and incubated at 37ºC for 48 hours. Whereas in the pre-incubation procedure, the mixture was incubated for 20 min at 37ºC prior to be poured over the minimal agar medium plate.
Rationale for test conditions:
The test item was found to be soluble in corn oil at a concentration of 50uL/mL.
5 concentrations (F1 to F5) starting at 5.00uL/plate, based on the solubility profile of the test item.
Concentrations F2 to F5 were prepared by 1:3 serial dilutions from the selected F1 concentration.
Evaluation criteria:
A result is considered positive whenever the number of revertants of the test item treated plates is increased when compared to the solvent treated plates according to the following criteria:

(See table below in the section "Any other information on materials and methods incl. tables

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
Direct incorporation (main test)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Remarks:
as negative controls
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
Pre-incubation (confirmatory test)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Remarks:
as negative controls
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
Direct incorporation (main test)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Remarks:
as negative controls
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
Pre-incubation (confirmatory test)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Remarks:
as negative controls
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Remarks:
Direct incorporation (main test)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Remarks:
as negative controls
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Remarks:
Pre-incubation (confirmatory test)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Remarks:
as negative controls
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
Direct incorporation (main test)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Remarks:
as negative controls
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
Pre-incubation (confirmatory test)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Remarks:
as negative controls
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
Direct incorporation (main test)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Remarks:
as negative controls
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
Pre-incubation (confirmatory test)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Remarks:
as negative controls
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
Direct incorporation (main test)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Remarks:
as negative controls
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
Pre-incubation (confirmatory test)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Remarks:
as negative controls
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
Direct incorporation (main test)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Remarks:
as negative controls
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
Pre-incubation (confirmatory test)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Remarks:
as negative controls
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Remarks:
Direct incorporation (main test)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Remarks:
as negative controls
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Remarks:
Pre-incubation (confirmatory test)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Remarks:
as negative controls
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
Direct incorporation (main test)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Remarks:
as negative controls
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
Pre-incubation (confirmatory test)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Remarks:
as negative controls
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
Direct incorporation (main test)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Remarks:
as negative controls
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
Pre-incubation (confirmatory test)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Remarks:
as negative controls
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The bacterial reverse mutation test for the test item Cycloceramide® was considered valid as the following criteria were met:
- The mean solvent control counts complied with Vivotecnia historical data for each strain.
- The mean reference item control counts complied with Vivotecnia historical data for each strain.
Remarks on result:
other:
Remarks:
NON MUTAGENIC / NON PRO-MUTAGENIC

Any other information on results incl. tables

A result is considered positive whenever the number of revertants of the test item treated plates is increased when compared to the solvent treated plates according to the following criteria:

 species strain  criteria 
 S. typhimurium TA98  2 fold 
   S. typhimurium  TA100  2 fold
   S. typhimurium  TA102  2 fold
   S. typhimurium  TA1535  3 fold
   S. typhimurium  TA1537  3 fold

Applicant's summary and conclusion

Conclusions:
Based on the results obtained in this study, it can be concluded that the test item does not induce point mutations or frame-shifts in the genome of the bacterial strains with or without metabolic activation regardless of the procedure. None of the concentrations assayed for the test item showed an increase in the R value either with or without S9 metabolic activation regardless of the procedure.

Therefore, the test item Cycloceramide® is considered to be NON MUTAGENIC / NON PRO-MUTAGENIC under the experimental conditions assayed.
Executive summary:

The bacterial reverse mutation test (Ames test) assesses the mutagenic or promutagenic potential of the test item Cycloceramide® in the Salmonella typhimurium TA 1535, 1537, 98, 100 and 102 bacterial strains. The test was performed in accordance with OECD Guideline 471 for the Testing of Chemicals (Bacterial Reverse Mutation Test. Adopted 21st July 1997) and the test Method B13/B14 of Commission Directive 2000/32/EC.

No cytotoxic activity was observed by the test item in the bacterial system.

Five test item concentrations ranged between 5.00 and 0.06 uL/plate were assayed.

None of the concentrations assayed for the test item showed an increase in the R value either with or without S9 metabolic activation regardless of the procedure.

No dose response for the test item Cycloceramide® was observed in none of the tested bacterial strains.

Based on the results obtained in this study, it can be concluded that the test item does not induce point mutations or frame-shifts in the genome of the bacterial strains with or without metabolic activation regardless of the procedure.

Therefore, the test item Cycloceramide® is considered to be NON MUTAGENIC / NON PRO-MUTAGENIC

under the experimental conditions assayed.