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Toxicological information

Genetic toxicity: in vitro

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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Octa-1,7-diene
EC Number:
223-054-9
EC Name:
Octa-1,7-diene
Cas Number:
3710-30-3
Molecular formula:
C8H14
IUPAC Name:
octa-1,7-diene
Test material form:
liquid

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (rat)
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I:
3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II:
Salmonella strains: 1; 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
WP2 uvrA: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Vehicle / solvent:
DMSO (>99%)
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
10 μg/plate
Positive control substance:
sodium azide
Remarks:
TA 1535, TA 100 - Without metabolic activation
Positive controls:
yes
Remarks:
10 μg/plate in strain TA 98, 50 μg/plate in strain TA 1537
Positive control substance:
other: 4-nitro-o-phenylene-diamine, 4-NOPD
Remarks:
TA 1537, TA 98 - Without metabolic activation
Positive controls:
yes
Remarks:
2.0 μL/plate
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA - Without metabolic activation
Positive controls:
yes
Remarks:
2.5 μg/plate (10.0 μg/plate in WP2 uvrA)
Positive control substance:
other: 2-aminoanthracene, 2-AA
Remarks:
TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA - With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- 1st main experiment: plate incorporation
- 2nd main experiment: preincubation

DURATION:
- 1st main experiment: 48 hours incubation (37°C)
- 2nd main experiment: 30 min preincubation (30°C), 48 hours incubation (37°C)

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY:
Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
Evaluation criteria:
VALIDITY CRITERIA:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies
- a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the
number of revertants below the indication factor of 0.5.

CRITERIA FOR POSITIVE RESPONSE:
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 biologically 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 results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The plates incubated with the test item showed reduced background growth at the following concentrations (μg/plate):

 Strain  Experiment I   Experiment II
   without S9 mix  with S9 Mix  without S9 mix   with S9 Mix
 TA 1535  333 - 5000  333 - 5000  100 - 5000  333 - 5000
 TA 1537  333 - 5000  1000 - 5000  333 - 5000  333 - 5000
 TA 98  333 - 5000  1000 - 5000  100 - 5000  333 - 5000
 TA 100  333 - 5000  333 - 5000  1000 - 5000  333 - 5000
 WP2 uvrA  2500 - 5000   2500 - 5000   5000  2500 - 5000 

Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (μg/plate):

 Strain  Experiment I   Experiment II
   without S9 mix  with S9 Mix  without S9 mix   with S9 Mix
 TA 1535  5000  /  2500 - 5000  1000 - 5000
 TA 1537  333 - 5000  1000 - 5000  333 - 5000  333 - 5000
 TA 98  2500 - 5000  5000  1000 - 5000  2500 - 5000
 TA 100  333 - 5000  333 - 5000  1000 - 5000  333 - 5000
WP2 uvrA  /  /  /  5000 

/ = no toxic effects evident as a reduction in the number of revertants (below the induction factor of 0.5)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the bacterial strains used.