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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
QUALITY ASSURANCE ATTESTATION, G.L.P. COMPLIANCE STATEMENT & GLP certificates included in the full report. The study was conducted according to an internationally recognised method, and under GLP. No deviation was reported. The substance is adequately characterised. Therefore full validation applies.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
test Method B13/B14 of Commission Directive 2000/32/EC
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
6,8-dimethylnon-7-enal
EC Number:
486-670-9
Cas Number:
899810-84-5
Molecular formula:
C11H20O
IUPAC Name:
6,8-dimethylnon-7-enal
Test material form:
other: liquid
Details on test material:
Batch 862036
State Liquid
Solubility EtOH
Purity 97.79 %
Storage conditions Room temperature
Stability in assay conditions Room temperature
Expiration date 30.09.08

Method

Target gene:
His D 3052
His G 46
Trp
His C 3076
Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Five concentrations tested
0.05 / 0.15 / 0.5 / 1.5 / 5 μL (dose/plate)
Vehicle / solvent:
None
Controls
True negative controls:
yes
Remarks:
spontaneous reversion rate
Positive controls:
yes
Remarks:
Control mutagens were used for each strain and experimental condition - See full report for details
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-Aminoanthracene
Details on test system and experimental conditions:
Cultures of bacteria were incubated overnight with nutrient broth up to an absorbance (at
660nm) of approximately 1.2-1.4 OD. This OD indicates that bacteria are growing in the late
exponential or early stationary phase of growth (approximately 109 bacteria/mL).
Plates were prepared with minimum agar medium. Medium was mixed and preheated to
about 45ºC, and then poured over the plate and cooled at room temperature.
Each bacterial strain was tested by triplicate, both, in the presence and absence of the
metabolic system (S9). The bacterial suspension, the test item and PBS (-S9) or metabolic
activation system mix (+S9) were mixed and tempered at about 37ºC.
Evaluation criteria:
Readout and presentation of data
After an incubation of about 48 hours at about 37ºC, the number of colonies per plate was
counted.
Data are presented as the number of colonies present per plate (mean ± standard deviation)
per plate. 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

Interpretation of data
Several criteria were 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 reading frame shifts in the genome of either Salmonella Typhimurium and/or
Escherichia Coli.
Negative results from the test indicate that under the test conditions, the test item is not
mutagenic and non-promutagenic in the tested species

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
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The following conclusions can be inferred from the obtained results:
− No test item showed ratios (R) above 2.5 as compared to the negative control, either
with or without S9 metabolic activation.
− No dose response was observed in none of the tested bacterial strains.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The controls of the test were in concordance with the expected results:

− No cytotoxic effect was observed.

− Sterility test showed no contamination during the study.

− All positive controls performed showed valid ratios (R) above 2.5.

− Positive and negative controls showed absolute numbers of revertant colonies

comparable to historical data.

− 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 none of the tested bacterial strains.

Applicant's summary and conclusion

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

Based on the results obtained in this study, the test item NOREENAL was found to
be NON MUTAGENIC and NON-PROMUTAGENIC under the test conditions.
Executive summary:

The present Bacterial Reverse Mutation Test (Ames test) was performed in order to evaluate

the mutagenic potential of the test item. This study was conducted according to the

European Directive 2004/10/CE and the Good Laboratory Practice (GLP) principles of Spain

(Principios de Buenas Prácticas de Laboratorio: RD 1369/2000). 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.

Suspensions of 4 amino-acid requiring strains of salmonella typhimurium (TA98, TA100,

TA1535, TA1537) and one Escherichia coli WP2 strain (pKM 101) auxotroph for an amino

acid, were exposed to the test item in the presence and in the absence of an exogenous

metabolic activation system.

After incubation, revertant colonies due to point mutations were counted and compared to

the number of spontaneous revertant colonies on solvent control plate (negative control).

Similarly, specific standard mutagens were tested and used as positive controls.

Based on the results obtained in this study, the test item NOREENAL was found to

be NON MUTAGENIC and NON-PROMUTAGENIC under the test conditions.