reaction product of wheat aminoacids and lauryl chloride

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:

28.January.2008-04.February.2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline, GLP study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

acylation product between lauroyl chloride and amino acids

EC Number:

927-837-2

Molecular formula:

Not applicable

IUPAC Name:

acylation product between lauroyl chloride and amino acids

Test material form:

liquid

Specific details on test material used for the study:

Test item:
Name: LCE07106
Supplier SEPPIC
Batch: 0715200018
State: Liquid
Solvent: Milli-Q sterile water
Purity: 52.5 % (dry extract)
Storage conditions: Room temperature
Expiration date: 31.05.09

Reference items Supplier Cat# Batch
2-Nitrofluorene Sigma-Aldrich N1.1675-4 S08447-244
Sodium Azide Sigma-Aldrich S2002 034K0385
4-Nitroquinoline-N-Oxide Sigma-Aldrich N8141 034K1332
2-Aminoanthracene Sigma-Aldrich A3800-0 S17744-374
9-Aminoacridine Sigma-Aldrich A7295 106F0668I

Method

Target gene:

TA98 : Histidine D 3052
TA100: Histidine G 46
TA1535: Histidine G 46
TA1537: Histidine C 3076
E.Coli WP2 (pKM 101): Tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Strains of salmonella typhimurium and E.coli were obtained from Moltox. Stock plates were stored at about -80°C and master plates at about 4°C in Vivotecnia's facilities.

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Details on mammalian cell type (if applicable):

Strains of salmonella typhimurium and E.coli were obtained from Moltox. Stock plates were stored at about -80°C and master plates at about 4°C in Vivotecnia's facilities.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

The test was performed at concentrations of 5 / 1.5 / 0.5 / 0.15 and 0.05 µg/plate

Vehicle / solvent:

sterile water

Controls

Controls 1

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Positive control: 90 µg/plate TA98 without S9

Controls 2

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Positive control: 10 µg/plate TA100 / 30 µg/plate TA1535 without S9

Controls 3

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Positive control: 100 µg/plate TA1537 without S9

Controls 4

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Positive control: 7 µg/plate E.Coli WP2 without S9

Controls 5

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Positive control: 10 µg/plate TA 98 with S9

Controls 6

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Positive control: 10 µg/plate TA 100 with S9

Controls 7

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Positive control: 30 µg/plate TA 1535 with S9

Controls 8

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Positive control: 30 µg/plate TA 1537 with S9

Controls 9

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Positive control: 10 µg/plate E.Coli WP2 with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION / DURATION:
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.

DETERMINATION OF CYTOTOXICITY
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. The test item was mixed with top agar containing 2.5 mM
Histidine / Biotin. The solution was then added to a plate containing minimal agar and
incubated at about 37ºC for about 72 hours.
Inhibition of growth by the test item suggests a cytotoxic activity. A cytotoxic effect at high
concentrations only would require lower concentrations of the test item in the main test.
Cytotoxic activity at lower concentrations could rule out the bacterial reverse mutation test for
the evaluation of mutagenicity.

Evaluation criteria:

After an incubation of about 72 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
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:

other: all cell type tested

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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 LCE07106 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 LCE07106 was found to be NON

MUTAGENIC and NON-PROMUTAGENIC under the test conditions.