Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 January 2010 - 11 February 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test was conducted according to OECD guideline 471, and according to GLP described in Directive 2004/9/EC.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
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:
Not applicable
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
vetiveria zizanioides, ext.
IUPAC Name:
vetiveria zizanioides, ext.
Details on test material:
- Name of test material (as cited in study report) : vetiver zizanoides, ext.
- Physical state: Liquid
- Purity : 100%
- Lot/batch N° : confidential
- Stability under test conditions: no data
- Storage condition of test material: Room temperature (20 ± 5° C) - Keep away from light

Method

Target gene:
His-gene: Amino acid histidine (hisD, hisC, hisG) and Trp-gene: Amino acid tryptophan (trypE)

Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Details on mammalian cell type (if applicable):
Not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Metabolic activation:
with and without
Metabolic activation system:
Sprague Dawley rat liver S9 induced by Aroclor 1254
Test concentrations with justification for top dose:
All strains, ± S9 mix, plate incorporation and preincubation assay: 50, 150, 500, 1500, and 5000 µg/plate
Vehicle / solvent:
paraffin oil
Controls
Untreated negative controls:
yes
Remarks:
positive control solvent
Negative solvent / vehicle controls:
yes
Remarks:
paraffin oil
True negative controls:
yes
Remarks:
absolute negative control (spontaneous reversion rate)
Positive controls:
yes
Positive control substance:
other: - S9 mix: Strain TA1535 + TA100: Sodium-azide, Strain TA1537: 9-Aminoacridine, Strain TA98: 2-Nitrofluorene. + S9-mix: All S. typhimurium strains: 2-Anthramine. E.Coli WP2: - S9 mix: cis-Platinum (11), + S9 mix: Dimethylbenzanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: at least 20 minutes
- Selection time (if incubation with a selection agent): 48-72 hours

SELECTION AGENT (mutation assays): agar containing Histidine or Tryptophan

NUMBER OF REPLICATIONS: 3 plates per concentration

NUMBER OF CELLS EVALUATED: 10*9

DETERMINATION OF CYTOTOXICITY
- Method: other: bacteriostatic activity test with strain TA 100
Evaluation criteria:
The result of the test is considered as negative if the revertant number is below three fold the number of spontaneous reversions, for TA1535 and TA1537 strains, and below two fold the number of spontaneous reversions for TA98, TA100 and Escherichia coli WP2(uvrA-) (pKM 101)strains with and/or without metabolic activation. The validity criteria are as follow :
-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 account 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 TA98, TA100 and Escherichia coli WP2(uvrA-) (pKM 101), and three fold for TA1535 and TA1537.
Statistics:
Mean and Standard Deviation

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: In order to choose the range of concentration to be tested, the bacteriostatic activity of the test substance is evaluated with strain TA 100 in a preliminary cytotoxicity testing. The test substance is tested at the doses: 50, 150, 500, 1500 and 5000 µg/plate. There was no evidence of any bacteriostatic activity in the presence of the test substance.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and strain-specific positive control values were within the laboratory historical control data ranges.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The test substance does not induce reverse mutation on four Salmonella typhimurium strains and one Escherichia coli WP2(uvrA-) (pKM 101) strain according to the OECD Guidelines n°471 and to the method B13/B14 of the directive 2000/32/EC.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test substance, Vetiveria zizanioides, ext., does not induce any mutagenic change in Salmonella typhimurium TA1535, TA1537, TA98 and TA100 and Escherichia coli WP2 (uvrA- pKM101) without or with metabolic activation according to OECD Guidelines n° 471 and to the method B13/B14 of the Directive /32/EC. Based on these results it is concluded that Vetiveria zizanioides, ext. is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

Vetiveria zizanioides, ext. (vetivert oil) was tested in the Salmonella typhimurium reverse mutation assay with 4 strains of Salmonella typhimurium (TAI535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a strain of Escherichia coli (WP2 uvrA pKM 101). The test was performed in 2 independent experiments in the presence and absence of metabolic activation S9-mix (Sprague Dawley rat liver S9 induced by Aroclor 1254). The study procedures described in this report were based on OECD Guideline 471. In the dose range finding preliminary cytotoxicity test, Vetiveria zizanioides, ext. was tested at the doses: 50, 150, 500, 1500 and 5000 µg/plate in the strain TA100. There was no evidence of any bacteriostatic activity in the presence of the test substance. Based on the results of the dose range finding test, Vetiveria zizanioides, ext. was tested in all strains, without and with metabolic activation, in both a plate incorporation and a preincubation assay at 5 dilutions in paraffin oil (50, 150, 500, 1500, and 5000 µg/plate).

There was no evidence of any increase in the number of revertant colonies in the presence of the test substance without and with metabolic activation for all bacterial strains. The results were confirmed in two separate experiments. There was no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean of corresponding experimental historic values obtained in the laboratory.

The test substance, Vetiveria zizanioides, ext., does not induce any mutagenic change in Salmonella typhimurium TA1535, TA1537, TA98 and TA100 and Escherichia coli WP2 (uvrA- pKM101) without or with metabolic activation according to OECD Guidelines n° 471 and to the method B13/B14 of the Directive /32/EC. Based on the results of this study it is concluded that Vetiveria zizanioides, ext. is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.