Isoleucine, N-(1-oxohexadecyl)-

Genetic toxicity: in vitro

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

Study period:

2009-05-18 to 2009-05-25

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

LCA09005

IUPAC Name:

LCA09005

Constituent 2

Reference substance name:

DL-Isoleucine, N-(1-oxohexadecyl)

IUPAC Name:

DL-Isoleucine, N-(1-oxohexadecyl)

Details on test material:

- Name of test material (as cited in study report): LCA09005
- Substance type: Lipoamino acid
- Physical state: Solid
- Analytical purity: 100% Dry matter
- Lot/batch No.: 1893JG
- Expiration date of the lot/batch: May 2010
- Storage condition of test material: Room temparature

Method

Target gene:

Strain Target mutation Excision repair Plasmid Cell wall Mutation type
TA98 His D 3052 uvrB pKM101 rfa Frameshift
TA100 His G 46 uvrB pKM101 rfa Base-pair substitution
TA1535 His G 46 uvrB - rfa Base-pair substitution
TA1537 His C 3076 uvrB - rfa Frameshift
WP2 Trp uvrA pKM101 - Base-pair substitution

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

5000μg, 1667μg, 556μg, 185μg and 62μg

Vehicle / solvent:

Ethanol :
Solubility was assessed as precipitation in the final mixture under the actual test conditions.
Observation of precipitation by naked eye indicates insolubility.
The test item was dissolved in ethanol and no insolubility was detected. The solution was maintained over ice in order to minimize the ethanol evaporation.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

The purpose of this assay was to test whether the test item is mutagenic or pro-mutagenic.
The assay is based on the detection of point mutations (substitution, addition or deletion of one or a few DNA base pairs) or frameshift-mutations in four Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) strains and one Escherichia coli WP2 strain (pKM 101) by incubation with five concentrations of the test item. The mutagenic effect was analyzed in the presence and in the absence of S9 microsome fraction of Aroclor induced rat liver. An independent confirmation test was performed with the test item according to the preincubation procedure.
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

I- Method:
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
STRAIN ref Without S9 µg/plate Solvent ref With S9 µg/plate Solvent
S. typhimurium TA98 2-Nitrofluorene 90 DMSO 2-Aminoanthracene 5 DMSO
S. typhimurium TA100 Sodium Azide 10 H2O 2-Aminoanthracene 10 DMSO
S. typhimurium TA1535 Sodium Azide 30 H2O 2-Aminoanthracene 30 DMSO
S. typhimurium TA1537 9-Aminoacridine 50 DMSO 2-Aminoanthracene 10 DMSO
E. Coli WP2 (pKM101) 4-Nitroquinoline-N-Oxide 3.5 DMSO 2-Aminoanthracene 5 DMSO

I-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.

II-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. No cytotoxic effect was observed for this test item.

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:

Data are presented in tables as the number of colonies present per plate (mean ± standard
deviation). The ratio R is calculated as follows:

Results and discussion

Test results

Species / strain:

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:

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 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.

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

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 LCA09005 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 5000μg to 62μg per plate were tested. No cytotoxicity was observed at any dose. 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 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 LCA09005 was found to be NON MUTAGENIC and NON-PROMUTAGENIC under the test conditions.