Vetiveria zizanioides, ext.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation in bacteria (Ames): negative with and without metabolic activation (OECD 471, GLP)

Link to relevant study records

Reference

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.

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

Target gene:

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

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

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

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'.

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

An in vitro gene mutation test in bacteria was performed in accordance with OECD 471 and under GLP conditions. 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). 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.

Justification for selection of genetic toxicity endpoint
The selected study is the key and only study for this endpoint.

Justification for classification or non-classification

Based on the available information, Vetiver oil does not have to be classified as mutagenic in accordance with the criteria outlined in Annex I of 1272/2008/EC (CLP) and Annex VI of 67/548/EEC (DSD).