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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 to 26 November 2007.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP study conducted in compliance with OECD Guideline No. 471 with acceptable restriction. The substance is adequately identified, but details on composition are missing. Therefore validation applies with restrictions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997.
Deviations:
yes
Remarks:
2-Aminoanthracene was used as the sole indicator of the efficacy of the S9-mix. No information on the characterisation of the batch of S9 used with a mutagen that requires metabolic activation by microsomal enzymes has been included in the report.
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Directive 2000/32/EC.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Inspected on 18- 07-2007 / Signed on 2007-07-26.
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
7-nonenal, 6,8-dimethyl-, (6S)
Cas Number:
899810-99-2
Molecular formula:
C11 H20 O
IUPAC Name:
7-nonenal, 6,8-dimethyl-, (6S)
Constituent 2
Chemical structure
Reference substance name:
7-nonenal, 6,8-dimethyl-,(6R)-
Cas Number:
899810-91-4
Molecular formula:
C11 H20 O
IUPAC Name:
7-nonenal, 6,8-dimethyl-,(6R)-
Test material form:
liquid
Details on test material:
- Physical state: colourless translucent liquid
- Storage condition of test material: Room temperature
Specific details on test material used for the study:
Stability of the test substance in the solvent/vehicle: Stable in ethanol at room temperature

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10% v/v S9 fraction; rat liver microsome fraction (S9)
Test concentrations with justification for top dose:
Mutagenicity test:
Main test: 0.05, 0.15, 0.5, 1.5 and 5 μL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) with and without S9 under the direct plate incorporation method.
Confirmation test: 0.05, 0.15, 0.5, 1.5 and 5 μL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) with and without S9 under the pre-incubation method.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: none.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
Remarks:
without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 20 min at 37 °C
- Exposure duration: 37 °C for 48 h

NUMBER OF REPLICATIONS: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: A reduction in the number of colonies in a dose-dependent manner compared to negative control for any strain and condition might indicate cytotoxicity.

OTHER:
- After an incubation of about 48 hours at about 37 ºC, the number of colonies per plate was counted.
Data are presented as the number of colonies present per plate (mean ± standard deviation). The R ratio is calculated as follows:
R = Number of revertant colonies in the presence of the test item / Number of revertant colonies in the absence of the test item
- Sterility test: The sterility of the test item and the metabolic activation system (S9) were tested. For this purpose, the highest concentration of test item and a sample of the S9 mix were added respectively to top agar preheated at about 45 ºC and poured over minimal agar medium plates. The plates were incubated for about 48 hours at about 37 ºC. Presence or absence of colonies was observed. Bacterial growth would be an indication of microbiological contamination of the test item or S9 mix respectively.
- Solubility test: Solubility was assessed as precipitation in the final mixture under the actual test conditions. Observation of precipitation by naked eye indicates insolubility.
Evaluation criteria:
Several criteria are used for determining a positive result: a dose-response in the range tested and / or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.
Positive results from the bacterial reverse mutation test indicate that a test item induces point mutations or frame-shifts in the genome of the tested bacterial strains.
Negative results from the test indicate that under the test conditions, the test item neither mutagenic nor-pro-mutagenic in the tested experimental system.
Statistics:
None

Results and discussion

Test results
Key result
Species / strain:
bacteria, other: 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:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: None
- Precipitation: None
- Other confounding effects: None

CYTOTOXICITY TEST:
No cytotoxic effect was observed for this test item.

MUTAGENICITY TEST:
- No concentration of the test item showed a biological significant increase (R ≥ 2.5) of the number of revertant either with or without S9 metabolic activation.
− No dose response was observed in any of the tested bacterial strains.

HISTORICAL CONTROL DATA
- Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data of the test facility.

OTHERS:
- Sterility test showed no contamination during the study.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Under the test condition, the test material is not mutagenic in presence and absence of metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coli WP2uvrA.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli WP2(pKM101) were exposed to the undiluted test item at the following concentrations both in the presence and absence of metabolic activation system (10% v/v S9).


 


Main test: 0.05, 0.15, 0.5, 1.5 and 5 μL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) with and without S9 under the direct plate incorporation method.


Confirmation test: 0.05, 0.15, 0.5, 1.5 and 5 μL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) with and without S9 under the pre-incubation method.


 


Negative and positive control groups were also included in mutagenicity tests.


 


Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data. All positive controls showed valid ratios (R) above 2.5.


 


No cytotoxic effect was observed. No concentration of the test item showed a biological significant increase (R ≥ 2.5) of the number of revertant either with or without S9 metabolic activation. No dose response was observed in any of the tested bacterial strains.


 


Under the test condition, the test material is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coli WP2uvrA.


This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.