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Toxicological information

Genetic toxicity: in vitro

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Administrative data

in vitro gene mutation study in bacteria
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-10-27 to 2008-11-03
1 (reliable without restriction)

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Countings for the negative control & test item in the repetition test without metabolic activation showed values about double than historical data. No contamination was detected and all the replicas showed similar values. the data is considered acceptable
Principles of method if other than guideline:
Only E.Coli stain
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): LCA08034
- Substance type: Lipoamino acid
- Physical state: White powder
- Lot/batch No.: 1549JG
- Storage condition of test material: Room temperature


Target gene:
Strain: E.Coli WP2
Target gene: Trp-
Species / strain
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 metabolic activation system
Test concentrations with justification for top dose:
1000μg, 333μg, 111μg, 37μg and 12μg/plate
Vehicle / solvent:
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
other: 2-Aminoanthracene
Details on test system and experimental conditions:
Test type Escherichia coli
Number of replicas Three
Length of the experimental part 8 days
Metabolic activation system S9 fraction from Aroclor induced rats (Moltox, USA)
Incubation time 72 hours
Sterility test Performed
Cytoxicity test Observation of dose-dependent effects
Repetition test Performed independently with the preincubation method
Counting Automatic colony counter
Guidelines OECD Guideline no.471 for the Testing of Chemicals Method B13/B14 of Commission Directive 2000/32/EC

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 45ºC and poured over minimal agar medium plates.
The plates were incubated for about 72 hours at about 37ºC. 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.

Cytotoxicity test:
A reduction in the number of colonies in a dose-dependent manner compared to negative control for any strain and condition might indicate

Test performance:
Cultures of bacteria were incubated overnight with nutrient broth up to an absorbance (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 minimal agar medium. Medium was mixed and preheated to about 45ºC, and then poured into the plate and cooled at room temperature.
Each bacterial strain was tested by triplicate 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.

The suspension was mixed with top agar and poured over minimal agar medium plate. The top agar was allowed to solidify at room temperature
before final incubation. Plates were incubated at about 37ºC for about 72 hours.
Two controls were included in the experiment:
 Negative control: Control cultures were treated with solvent.
 Positive control: Control mutagens were used for each strain and experimental
Without S9: 4-Nitroquinoline-N-Oxide 3.5µg/plate in DMSO
With S9: 2-Aminoanthracene 5µg/plate in DMSO

An independent confirmation test was performed with the test item according to the preincubation
procedure. After the bacterial suspension, the test item and PBS (-S9) or metabolic activation system mix (+S9) were mixed and the mixture was
incubated at about 37ºC for about 20 minutes. Thereafter, the study was performed in the same way as the first test.

Evaluation criteria:
The number of colonies per plate was counted with an automatic colony counter.
Data are presented in tables as the number of colonies present per plate (mean ± standard deviation). The ratio R is calculated as follows:
R = Number of revertant in presence of test item / Number of revertant in absence of test item

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.

Results and discussion

Test results
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
Vehicle controls validity:
Untreated negative controls validity:
not applicable
Positive controls validity:
Additional information on results:
The controls of the test were in concordance with the expected results:
 Sterility test showed no contamination during the study.
 No cytotoxic effect was observed.
 All positive controls performed showed values clearly different than negative values.
 Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data, excepting the negative control without metabolic activation.
 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
Remarks on result:
other: strain/cell type: E.Coli WP2 uvr A pKM 101
Migrated from field 'Test system'.

Applicant's summary and conclusion

Interpretation of results (migrated information):

The following conclusions can be inferred from the obtained results:
 No experiment with the 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.
Based on the results obtained in this study, the test item LCA08034 was found to be NON
MUTAGENIC and NON PRO-MUTAGENIC 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.

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.

Doses ranging from 1000μg to 12μg per plate were tested. No cytotoxicity was observed at any dose.

Suspensions an Escherichia coli WP2 strain (pKM 101) auxotroph for an amino acid were exposed by the direct plate incorporation method to five doses of the test item in the presence and in the absence of an exogenous metabolic activation system. Both tests were

repeated with the pre-incubation method. Revertant bacteria due to point or frameshift-mutations at specific locus are able to grow,

forming colonies. These colonies 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 LCA08034 was found to be NON MUTAGENIC and NON-PROMUTAGENIC under the test conditions.