Reaction mass of butyl palmitate and butyl oleate and butyl (9Z,12Z)-octadeca-9,12-dienoate and 482-680-2

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-05-15 to 2006-05-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

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 reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of butyl palmitate and butyl oleate and butyl (9Z,12Z)-octadeca-9,12-dienoate and 482-680-2

EC Number:

944-892-8

Molecular formula:

not applicable

IUPAC Name:

Reaction mass of butyl palmitate and butyl oleate and butyl (9Z,12Z)-octadeca-9,12-dienoate and 482-680-2

Test material form:

liquid: viscous

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-fraction (10% v/v) ; MgCl2 (0.4M) / KCl (1.65M) (2% v/v) ; Glucose-6-phosphate (1M) (0.45% v/v) ; NaDPH (0.2mM) (2% v/v) ; Phosphate Buffer (0.2M) (50% v/v)

Test concentrations with justification for top dose:

0.05 µL ; 0.15 µL ; 0.5 µL ; 1.5 µL ; 5 µL

Vehicle / solvent:

undiluted

Controls

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

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) in five salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537) strains by incubation with five concentrations of the test item. These strains have several features that make them more sensitive for the detection of mutations. The mutagenic effect was analyzed in the presence and in the absence of a metabolic activation system, namely rat liver microsome fraction (S9).

Although the bacterial reverse mutation test employs prokaryotic cells that differ from mammalian cells in several factors, this test is a first screening of mutagenic potential of a wide range of products. The test consists in the exposure of the five types of bacteria strains to the test item, both in the presence and in the absence of an exogenous metabolic activation system (S9). The exogenous activation system allows to distinguish between promutagens and direct mutagens. After about 48 hours of incubation, revertant colonies are counted by means of an automatic colony counter and compared to the number of spontaneous revertant colonies on control plates.

The solution was mixed with top agar and poured over the 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 48 hours. The number of colonies per plate was then counted.
Two controls were included in the experiment:
− Negative control: Absolute negative control (spontaneous reversion rate)
− Positive control: Control mutagens were used for each strain and experimental condition.

Rationale for test conditions:

All the strains scored positively when checked with a reference item in accordance with provider information at the time of arrival to Vivotecnia.
The sterility of the test item and the metabolic activation system (S9) were tested.
A potential cytotoxic effect of the test item that would interfere in the results was ruled out with the following test. Five concentrations and a negative control of test item were tested in salmonella typhimurium TA100.
A solubilit test was performed.

Evaluation criteria:

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 salmonella typhimurium. Negative results from the test indicate that under the test conditions, the test item is not mutagenic and non-promutagenic in the tested species.

Statistics:

After an incubation of about 48 hours at about 37°C, 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

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 8

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 9

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 10

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Non-mutagenic and non-promutagenic

Applicant's summary and conclusion

Conclusions:

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.

Based on the results obtained in this study, the test item 5 ALPHA AVOCUTA 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 5 amino-acid requiring strains of salmonella typhimurium (TA98, TA100, TA102, TA1535, TA1537) 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 5 ALPHA AVOCUTA was found to be NON MUTAGENIC and NON-PROMUTAGENIC under the test conditions.