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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Interpretation of results:


negative

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.Interpretation of results:


negative

On the basis of the results, interpreted according to OECD 417:1997 the test substance LCA08018 proved to be NON MUTAGENIC for the test trains, either in the presence or absence of metabolic activation.


Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-10-27 to 2008-11-03
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
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. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Strain: E.Coli WP2
Target gene: Trp-
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:
DMSO 1%
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
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
cytotoxicity.

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.
Species / strain:
E. coli WP2 uvr A pKM 101
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
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
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

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.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-07-31 to 2008-09-16
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
Only Salmonella stains
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
Before the test, the following genetic characteristics of the strains were verified
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from hepatic homogenate obtained from the liver of adult male rats which had previously been induced with "aroclor 1254" soybean oil solution.
Test concentrations with justification for top dose:
0.62 ; 1.86 ; 5.57 ; 16.67 ; 50 mg/mL
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
cyclophosphamide
mitomycin C
other: 2-Aminoantracene
Details on test system and experimental conditions:
preliminary tests:

- Sterility tests:
Aliquots of the assay sample were directly placed in Petri dishes ontaining the full medium and in Petri containing the basic medium. The development of any bacterial colonies will be observed after 48hours of incubation at 37°C+/-1°C

Determination of toxicity:
The sample toxicity assay for the mutnat Salmonella typhimurium strains xas evaluated by transplanting the bacteria in the basic medium and determining any reduction in culture growth and number of spontaneously reverting organisms caused by the assay sample.

positive controls:
concentrations expresed as µg/plate
NaN3: 5
9-AAc: 40
2-NF: 10
MMC: 50
2-AAn: 5
CP: 300

negative control:
In the presence and absence of solvent used for the preparation of the substance dilutions (DMSO 10 µL/plate)

Executuion of the assay:
The whole assay was performed twice

- Plate test without metabolic activation:
0.1mL of assay sample was added to 2.0 mL of top agar, which was kept fluid in sterile test tubes placed in a thermostatic bath at 45°C+/- 1°C.
Another 0.1mL of the bacterial cell suspensions and 0.5mL of PBS was then added. The cell density of the suspensions was 10e8 to 10e9 celles/mL in a stationary growth phase.
The sample was briefly stirred and then poured into plates containing the basic medium.
At the same time, negative control with the test substance at zero concentration and positive controls with reference mutagens at the proper concentrations were also prepared.
The plates were then incubated at 37°C +/- 1 °C for 48-72 hours.
After incubation, the reverted colonies of the assay sample at the different concentrations, as well as those of the negative control and positive controls, were counted in each plate.
Three replications were performed with the same sample, negative control and positive controls.

- Plate test with metabolic activation:
0.1mL of assay sample was added to 2.0 mL of top agar, which was kept fluid in sterile test tubes placed in a thermostatic bath at 45°C+/- 1°C.
Another 0.1mL of the bacterial cell suspensions and 0.5mL of the enzylmatic system for metabolism activation was then added.
The whole was stirred and then poured into plates containing the basic medium.
At the same time, negative control without test substance and positive controls with reference mutagens at the proper concentrations were also prepared.
The plates were then incubated at 37°C +/- 1 °C for 48-72 hours.
After incubation, the reverted colonies of the assay sample at the different concentrations, and those of the negative control and positive controls, were counted in each plate.
Three replications were performed with the same sample, negative control and positive controls.
Evaluation criteria:
Assay validity criteria:
In the sterility test performed at the end of the experiment, on plates with the full medium, S-9 Mix should not be contaminated by more than two colonies per 0.5 mL.
In the sterility test performed at the end of the experiment with the highest concentration tested, the assay sample should not be contaminated by more than two colonies per plate.
On average, for the TA1535, TA1537 and TA98 strains, the number of reverting colonies developped by positive control should be at least 300% higher than that of the respective negative control (lower limit for positive controle/negative control ratio for strains with low mutation frequency); for the TA100 and TA102 strains, the number of reverting colonies developped by the positive control should be at least 200% higher than of the respective negative control (a 20% of variation is accepted at the lower limit of the positive/negative control ratio in strains with high mutation frequency). These criteria refer to internal method validation because OECD 471 guideline doesn't give any reference thereof.
The number of spontaneously reverting colonies per plate in the negative controls should be included between the validated limits:
TA1535 30 +/-10
TA1537 15 +/-10
TA98 25 +/-10
TA100 120 +/-40
TA102 200 +/-50

Interpretation of results:
There are several criteria for determining a positive result, such as a concentration-related increased over 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.
A test substance for which the resulsts do not meet the above criteria is considered non mutagenic in this test.
Statistics:
Statistical t-tst I:Grouped data (Pharmacologic Calculation System-Version 4.2)
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
For the TA1535 strain, the product showed a cytotoxic effect at the highest test concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Mutagenic activity:
Neither a concentration-related increase over the range tested nor a reproductible increase at one more concentrations in the number of revertant colonies per plate in any strain with or without metabolic activation system was detected at the tested concentrations (50; 16.67; 5.57; 1.86 and 0.62 mg/mL).
Besides, the statistical test applied showed no significant difference between the average number of revertant colonies at the different dilutions of the test substance and the negative control.

Toxicity of test substance:
The product did not show any toxic effects either in the presence or absence of the enzymatic system for metabolism activation for the TA1537, TA98; TA10 and TA102 strains.
For TA1535 strain, the product showed a cytotoxic effect at the highest test concentration.

Negative control:
The number of spontaneous reverting colonies in the negative control plates did not exceeed the established limits.

Positive controls:
All positive controls caused a significant increase in the number of reverting organisms.

Sterility assay:
The sterility assay performed on the assay sample and S-9 mix did not show any contamination.
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

On the basis of the results, interpreted according to OECD 417:1997 the test substance LCA08018 proved to be NON MUTAGENIC for the test trains, either in the presence or absence of metabolic activation.
Executive summary:

On the test substance LCA08018 a toxicological study was carried out to evaluate the mutagenic effects. The following test was performed:

Salmonelle typhimurium-reverse mutation assay (Ames test)

The Salmonella typhimurium-reverse mutation assay was performed on the five mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100, TA102). The presumed mutagenic test substance activity was determined by comparing number of reverting colonies with the number of the reverting organisms in the control cultures.

The direct incorporation methode in a plate was used both in the presence of, and without, an enzymatic system for metabolic activation.

The test substance consisted of a powder, so it was prepared as DMSO solution with concentration equivalent to 50mg/mL (equivalent to 5 mg/plate) that is the maximum concentration recommended by OECD 471:1997 for soluble substances. Afterwards 4 following half log dilutions in DMSO were performed : 16,67; 5.57; 1.86 and 0.62 mg/mL. The assay was performed in two replicates

On the basis of the results, interpreted according to OECD 417:1997 the test substance LCA08018, at the concentration of 50 mg/mL, proved to be NON MUTAGENIC for the test trains, either in the presence or absence of metabolic activation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification