PALMARIA PALMATA EXTRACT

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:

January 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

N/A

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Enzymatic hydrolysis products of Palmaria palmata extract

EC Number:

607-053-4

Cas Number:

223751-74-4

IUPAC Name:

Enzymatic hydrolysis products of Palmaria palmata extract

Test material form:

liquid

Method

Target gene:

Salmonella typhimurium

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 applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 ( rat liver microsome fraction)

Test concentrations with justification for top dose:

Dose per plate: 5 μL, 1.5 μL, 0.5 μL, 0.15 μL, 0.05 μL with and without S9-mix in TA 98, TA 100, TA 102, TA 1535 , TA 1537
Confirmation test: same concentrations of test item with adding of PBS (-S9) or metabolic activation system mix (+S9)

Vehicle / solvent:

Water
Justification for choice of solvent/vehicle: The test substance was soluble in water.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

absolute negative control

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

Remarks:

Without S9

Controls 2

Untreated negative controls:

yes

Remarks:

absolute negative control

True negative controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With S9

Details on test system and experimental conditions:

Solubility test: Solubility was assessed :
Liquid: dilutions: 100%, 30%, 10%, 3%, 1%
Solid (100µl/plate): 50 mg/mL, 16.67 mg/mL, 5 mg/mL, 1.67 mg/mL, 0.5 mg/mL

Cytotoxicity test: A potential cytotoxicity effect of the test item that would interfere in the results was ruled out with the following test: 5 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 mineral agar and incubated at 37°C for 48hours.
Inhibition of growth by the test item suggests a cytotoxicity activity. A cytotoxicity effect at high concentrations only would require lower concentrations of the test item in the main test.
Cytotoxicity activity at lower concentrations could rule out the bacterial reverse mutation test for the evaluation of mutagenicity.

Test performance: Cultures of bacteria were incubated overnight with nutrient broth up to an absorbance (60 nm) of approximately 1.2-1.4 OD. This OD indicates that bacteria are growing in the late exponential or early stationary phase of growth (approx. 10^9 bacteria/ml).
Plates were prepared with minimal agar medium . Medium was mixed and preheated to about 45°C and then poured into 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 system mix (+S9) were mixed and tempered at about 37°C.

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 37°C 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
- positives control: control mutagens were used for each strain as detailled above

Rationale for test conditions:

Confirmation test:
An independant confirmation test was performed with the test item. After the bacterial suspension, the test item and PSB (-S9) or metabolic activation system mix (+S9) were mixed and the mixture was be incubated at about 37°C for about 20 minutes. Thereafter, the study was performed in the same was as the first test (described behind)

Evaluation criteria:

The number of colonies per plate was counted with an automatic colony counter. Data are presented as the number of colonies present per plate (mean +/- standard deviation). The ratio R 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 are used for determining a positive result: a dose-response in the range tested and/or a reproductible increase at one or more concentrations in the number of revertant colonies per plates in at least one strain with or without metabolic activation system.

Positive results from the bacterial reverse mutation test indicate taht a test induces point mutations or readings frame-shifts in the genome of the tested experimental system.

Negative results from the test indicates that under the test conditions, the test item is not mutagenic and pro-mutagenic in the tested species.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA98, TA100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The controls of the test were in accordance with the expected results:
- No cytotoxic effect was observed
- Sterility test showed no contamination during the study
- All positive controls performed showed absolute number 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 of without S9 metabolic activation
- No dose response was observed in none of the tested bacterial strains

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 bacterials strains.
Based on the results obtained in this study, the test item was found 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 Chemical (Bacterial Reverse Mutation Test, Adopted 21st, July 1997) and the TestMethod B13/B14 Of Commission Directive 2000/32/EC.

Suspension of 5 amino-acid requiring strains of Salmonella typhimurim (TA98, TA100, TA102, TA1535, TA 1537) 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 solvant 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 was found NON MUTAGENIC and NON PROMUTAGENIC under the test conditions