Saccharides of mannose and galactose from Ceratonia Siliqua seed

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:

April 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

Saccharides of mannose and galactose from Ceratonia Siliqua seed

IUPAC Name:

Saccharides of mannose and galactose from Ceratonia Siliqua seed

Test material form:

liquid

Details on test material:

Clear yellow

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

N/A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

10% S9: S9 fraction from Aroclor induced rats (Moltox, USA)

Test concentrations with justification for top dose:

Main test : 5 μL, 1.5μL, 0.5 μL, 0.15μL and 0.05μL
Confirmation test: same concentrations of test item with adding of PBS with or without 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:

Control cultures were trated with solvent

Negative solvent / vehicle controls:

no

True negative 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:

Control cultures were treated with solvent

Negative solvent / vehicle controls:

no

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With S9

Details on test system and experimental conditions:

Solubility test: Solubility was assessed as precipiation in the final mixture under the actual test conditions. Observation of precipitation by naked eye indicates insolubilty.

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/Biotine. The solution was then added to a plate containing mineral agar and incubated at 37°C for 72hours.
Inhibition of growth by the test item suggests a cytotoxicity activity. A cytotoxicity effect a high concentrations only would require lower concentration of the test item in the main test.

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 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 suspension was mixed with top agar and poured over minimal agar medium plate. The to agar was allowed to solidify at room temperature before final incubation.
Plates were incubated at about 37°C for about 72 hours.
Two controls were included in the experiment:
- negative control: absolute negative control
- positives control: control mutagens were used for each strain and experimental conditions

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 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 in tables 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 areproductible 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 frame-shifts in the genome of the tested experimental system.
Historical negative (solvent/vehicle) and positive control values are represented in the final report. this table include rationR, maxima, mean values and standard deviations. The historical control values have at least an n of 50. The values are updated on regular intervals with data obtained throughout the performance of studies.

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

Positive controls validity:

valid

Additional information on results:

The following conclusions can be inferred from the obtained results:
- no experiment with the test item showed rations (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 was found NON MUTAGENIC and NON PRO-MUTAGENIC under the test conditions.

Applicant's summary and conclusion

Conclusions:

The following conclusions can be inferred from the obtained results:
no experiment with the test item showed rations (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 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 Test Method B13/B14 Of Commission Directive 2000/32/EC.

Doses ranging from 5μl to 0.05μl per plate were tested. No cytotoxicity was observed at any dose.

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.