Registration Dossier

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:
14.09. - 10.10.2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to guideline under GLP
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
Not relevant
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Dihydro-C14-aldehyde
- Physical state: liquid
- Storage condition of test material: Room temperature, tightly closed, under nitrogen

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Pre-Experiment and Experiment 1: 3; 10;33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment 11: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II A: 0.3; 1; 3; 10; 33; 100; 333; and 1000 µg/plate
Vehicle / solvent:
DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Migrated to IUCLID6: and: 4-nitro-o-phenylene-diamine, NOPD; methyl methane sulfonate, MMS; 2-aminoanthracene, 2-AA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
In the pre-incubation assay 100 µL test solution, 500 µL S9 mix / S9 mix substitution buffer
and 100 µL bacterial suspension were mixed in a test tube and shaken at 37 °C for 60
minutes. After pre-incubation 2.0 mL overlay agar (45" C) was added to each tube. The
mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37°C in
the dark.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of
revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or
thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control
is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded
at more than one concentration .
An increase exceeding the threshold at only one concentration is judged as bioiogically
relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is
regarded as an indication of a mutagenic potential if reproduced in an independent second
experiment. However, whenever the colony counts remain within the historical range of
negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and 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
Species / strain:
E. coli WP2 uvr A
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
Additional information on results:
No substantial increase in revertant colony numbers of any of the five tester strains was
observed following treatment with Dihydro-C14-Aldehyde at any dose level, neither in the
presence nor absence of metabolic activation (S9 mix). There was also no tendency of
higher mutation rates with increasing concentrations in the range below the generally
acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase
of induced revertant colonies.
The laboratory's historical control range was exceeded in the untreated control of strains
TA 98 (without S9 mix), WP2 uvrA (with S9 mix) in experiment I, and in strains TA 1537
(with and without S9 mix) and WP2 uvrA (with S9 mix) in experiment II. These deviations
are judged to be based on biological fluctuations in the number of colonies and have no
impact on the outcome of the study.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

No remarks

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

in conclusion, it can be stated that during the described mutagenicity test and under the
experimental conditions reported, the test item did not induce gene mutations by base pair
changes or frameshifts in the genome of the strains used.
Therefore, Dihydro-C14-Aldehyde is considered to be non-mutagenic in this Salmonella
typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of Dihydro-C14-Aldehyde to induce

gene mutations in the plate incorporation test (experiment I) and the pre-incubation test

(experiment II and I1 A) using the Salmonella typhimurium strains TA 1535, TA 1537,

TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed with and without liver microsomal activation. Each

concentration, including the controls, was tested in triplicate. The test item was tested at

the following concentrations:

Pre-Experiment and Experiment 1: 3; 10;33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment ll A: 0.3; 1; 3; 10; 33; 100; 333; and 1000 µg/plate

Reduced background growth was observed at higher concentrations without metabolic

activation in nearly all strains used in experiment I1 and II A.

Toxic effects, evident as a reduction in the number of revertants, were observed in strain

TA 1537 without S9 mix (experiment I, II, and II A) and in strain TA 100 with S9 mix

(experiment I and 11).

No substantial increase in revertant colony numbers of any of the five tester strains was

observed following treatment with Dihydro-C14-Aldehyde at any dose level, neither in the

presence nor absence of metabolic activation (S9 mix). There was also no tendency of

higher mutation rates with increasing concentrations in the range below the generally

acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase

of induced revertant colonies.