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Toxicological information

Genetic toxicity: in vitro

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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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The experiment was done following the protocol corresponding to the Ames test. However, in the article it is not mentioned if the GLP rules were observed. The study does not meet modern standards where a limit of 5 mg/plate is required. The difference between 3.6 and 5.0 mg/plate is small and I it would be highly unusual to see something positive at 5.0 and negative at 3.6. Therefore the study is considered as acceptable.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1982

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
tested up to 3.6 mg/plate instead of 5 mg/plate
GLP compliance:
not specified
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): alpha-Hexylcinnamaldehyde
- Molecular formula (if other than submission substance): Not applicable
- Molecular weight (if other than submission substance): Not applicable
- Smiles notation (if other than submission substance): Not applicable
- InChl (if other than submission substance): Not applicable
- Structural formula attached as image file (if other than submission substance): see Fig.
- Substance type: Not reported
- Physical state: Not reported
- Analytical purity: Not reported
- Impurities (identity and concentrations): Not reported
- Composition of test material, percentage of components: Not reported
- Isomers composition: Not reported
- Purity test date: Not reported
- Lot/batch No.: Not reported
- Expiration date of the lot/batch: Not reported
- Radiochemical purity (if radiolabelling): Not reported
- Specific activity (if radiolabelling): Not applicable
- Locations of the label (if radiolabelling): Not applicable
- Expiration date of radiochemical substance (if radiolabelling): Not applicable
- Stability under test conditions: Not reported
- Storage condition of test material: Not reported
- Other: Not reported

Method

Target gene:
Histidine encoding gene (his) for Salmonella.
Species / strain
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA1538, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
other: including a deletion through the excision repair gene (uvrB-) which renders the capability of DNA exision repair and deep rough mutation (rfa)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix, Aroclor 1254 (500 mg/Kg i.p.) pretreated rat liver adjusted to 25 mg protein/L
Test concentrations with justification for top dose:
5 different doses were applied up to 3.6 mg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water and DMSO in cases of not solubile in water.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water and DMSO in cases of not solubile in water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
0.5 µg/plate, without S9mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water and DMSO in cases of not solubile in water
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
5 µg/plate, with S9mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: standard plate procedure (Ames, McCann & Yamazaki, 1975)
Overnight bacterial cultures had cell titres of at least 10(sup9 )cells/mL. S9 liver fractions were prepared from Aroclor-pretreated rats (Aroclor 1254, 500 mg/kg i.p.) and ajusted to 25 mg protein/mL; 0.5mL S9mix, equivalent to 50 µL S9, was incorporated into the plates. Vogel-Bonner medium (Vogel & Bonner, 1956) was used throughout; plates were incubated for 48 hr.


DURATION
- Preincubation period: 0 min
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 48hrs


SELECTION AGENT (mutation assays): Not applicable
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Not applicable


NUMBER OF REPLICATIONS: three or four plates for each dose (test was repeated at least twice)


NUMBER OF CELLS EVALUATED: Not applicable


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.


OTHER EXAMINATIONS:
- Determination of polyploidy: Not applicable
- Determination of endoreplication: Not applicable
- Other: Not applicable


OTHER: overnight cultures were used
Evaluation criteria:
The results that met the following criteria were regarded as positive: a reproducible, dose-related and at least two-fold elevation of the spontaneous revertant requency. Producing reproducible, dose-related and significant (P<0.01) but less than two-fold elevation were classifed as marginally mutagenic under the experimental conditions.
Statistics:
Statistical significance was determined according to the methods of Kastenbaum & Bowman (1970).

Results and discussion

Test results
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:
not examined
Positive controls validity:
valid
Additional information on results:
Not reported revertant colony numbers.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Not applicable

Applicant's summary and conclusion

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

Under the test conditions HCA showed no mutagenic activity in bacteria system in the absence and presence of metabolic activation.
Executive summary:

In a bacterial reverse mutation test, alpha-Hexylcinnamaldehyde (HCA) was tested for toxicity similarly to OECD Guideline 471 (Bacterial Reverse Mutation Assay).

The strains S. typhimurium TA1535, TA1537, TA1538, TA98 and TA100 were treated with HCA at 5 concentrations up to 3.6 mg/plate (if not toxic, toxicity not reported) in DMSO, with and without activation with liver preparations (S9 mix) from rats treated with Aroclor 1254.This study was carried out using the standard plate incorporation method.Vehicle and positive controls were performed in both tests.

No biologically significant increase in the number of revertants was noted in any strain, either with or without metabolic activation.

The study does not meet modern standards where a limit of 5 mg/plate is required. The difference between 3.6 and 5.0 mg/plate is small and I it would be highly unusual to see something positive at 5.0 and negative at 3.6. Therefore the study is considered as acceptable.

Under the test conditions HCA showed no mutagenic activity in bacteria system in the absence and presence of metabolic activation.