Lupine, L. albus, ext.

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:

26 January- 11 February 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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:

Not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Lupine, L. albus, ext.

EC Number:

282-001-8

EC Name:

Lupine, L. albus, ext.

Cas Number:

84082-55-3

IUPAC Name:

Enzymatic hydrolysis products of Lupinus Albus, Fabaceae, seed

Test material form:

liquid

Details on test material:

Color Amber
Batch: 09 077 21
Storage conditions: room temperature
Expiry date: 18 September 2010

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):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

10% S9: S9-mix from the livers of male Sprague Dawley rats

Test concentrations with justification for top dose:

Preliminary cytotoxicity testing study: 0; 50;150; 500; 1500; 5000 μg/plate, with and without S9-mix in TA 100 strain
Main study - Direct plate incorporation method: 0; 50;150; 500; 1500; 5000 μg/plate, with and without S9-mix in TA 1535, TA 1537, TA 98, TA 100, TA 102 strains
Confirmatory assay - pre-incubation method: 0; 50;150; 500; 1500; 5000 μg/plate, with and without S9-mix in TA 1535, TA 1537, TA 98, TA 100 and TA 102 strains

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Sterile and apyrogen sodium chloride

Controls

Untreated negative controls:

yes

Remarks:

absolute negative control: spontaneous reversion rate

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-Anthramine

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM
- Bacteria used in the test was obtained from Unité de Programmation moléculaire et toxicologie génétiuqe CNRS UA 144 (Institut Pasteur)

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 37 °C for 20 minutes
- Exposure duration: Plates were incubated at 37 ± 1 °C for 48-72 h

NUMBER OF REPLICATIONS: Triplicate plates per dose level

Evaluation criteria:

The result of the test is considererd as negative if the revertant number is below three fold the number of spontaneous reversions for TA 1535 and TA 1537 strains, and below two fold the number of spontaneous reversions fo TA 98, TA 100 and TA 102 strains with and/or without metabolic activation.

The validity criteria are:
- bacteriostatic activity of the highest concentration shall be equal to or less than 75%
- the spontaneous reversion rate of the absolute negative control shall comply with the laboratory's historical control data.
- the mean number of revertant colonies obtained for each strain and the corresponding positive control, with and/or without metabolic activation shall comply with laboratory's historical control data

The result of the test is considered positive if a concentration- related increase is obtained in one, or several of the 5 strains, with and/or without metabolic activation; a mutagenic effect is taken into acount for a given concentration of the test substance if the number of revertant colonies is at least two fold that of spontaneous revertant colonies number for TA 98, TA 100 and TA 102 and 3 folds for TA 1535 and TA 1537.

All results must be confirmed in an ondependent experiment

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

Interpretation of results
negative with metabolic activation
negative without metabolic activation

Under the test conditions, test item there is no evidence of any increase of the number of revertant colonies in the presence of the test substance; without and with metabolic activation for bacterial strains in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102.

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100 and TA102) were exposed to test item, at the following concentrations: 

Range finding study: 0; 50;150; 500; 1500; 5000 μg/plate, with and without S9-mix in TA 1535, TA 1537, TA 98, TA 100 and TA 102 strains

Main study - Direct plate incorporation method: 0; 50;150; 500; 1500; 5000 μg/plate, with and without S9-mix in TA 1535, TA 1537, TA 98, TA 100 and TA 102 strains

Confirmatory assay - pre-incubation method: 0; 50;150; 500; 1500; 5000 μg/plate, with and without S9-mix in TA 1535, TA 1537, TA 98, TA 100 and TA 102 strains

 

Metabolic activation system used in this test 10 % S9 mix; S9 fraction prepared from liver homogenates of male Sprague Dawley rats. Vehicle and negative and positive control groups were also included in mutagenicity tests.

 

In a range-finding study, no cytotoxicity exhibited in terms of a clearance in the background lawn or reduction in the revertant colonies. No marked or consistent differences in the number of revertant colonies per plate was observed in various test concentrations and solvent controls of all the tester strains, both with (10% S9 mix) and without exogenous mammalian S9 activation component in the main assays.

 

The mean numbers of revertant colonies are fell within acceptable ranges for vehicle control treatments. The positive controls of the five tester strains with and without S9 mix exhibited a statistically significant increase in the number of histidine and tryptophan positive colonies, validating the sensitivity of the assay.

 

Under the test conditions, test item is not considered as mutagenic in this bacterial system.