Cymbopogon nardus, 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:

13 Feb 2018 - 28 Mar 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Cymbopogon nardus, ext.

EC Number:

289-753-6

EC Name:

Cymbopogon nardus, ext.

Cas Number:

89998-15-2

IUPAC Name:

Essential oil of Citronella obtained from the aerial parts of Cymbopogon nardus, (Poaceae), by steam distillation

Test material form:

liquid

Details on test material:

Citronella nardus oil
CAS no.: 8000-29-1

Method

Target gene:

histidine locus

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Trinova Biochem GmbH, Germany [Master culture from Dr. Bruce N. Ames (TA1535, TA1537, TA98, TA100; and Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK (WP2uvrA)]
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD, EC).
- Methods for maintenance in cell culture if applicable: The Salmonella typhimurium strains were checked at least every year to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.

MEDIA USED
- Properly maintained: Yes
- Periodically 'cleansed' against high spontaneous background: Yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 (Trinova Biochem GmbH, Giessen, Germany)

Test concentrations with justification for top dose:

Dose range finder (direct plate assay,with and without S9):
1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate (is reported as a part of the direct plate assay)

First experiment (direct plate assay,with and without S9):
5.4, 17, 52, 164 and 512 μg/plate

Second experiment (pre-incubation assay, with and without S9):
5.4, 17, 52, 164, 512 and 1600 µg/plate

Second experiment (additional experiment, pre-incubation assay, without S9):
0.55, 1.7, 5.4 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: commonly used vehicle, suitable for dissolving the test item

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:in agar (plate incorporation) and preincubation
- Cell density at seeding (if applicable): 10^9 cells/mL

DURATION
- Preincubation period: 30 ± 2 minutes by 70 rpm at 37 ± 1°C
- Exposure duration: 37.0 ± 1.0°C for 48 ± 4 h

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies.

Rationale for test conditions:

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Evaluation criteria:

any increase in the total number of revertants was evaluated for its biological relevance including a comparison of the results with the historical control data range.
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

direct plate assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

direct plate assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

direct plate assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

direct plate assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

direct plate assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

Pre-incubation assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

Pre-incubation assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 8

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Pre-incubation assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 9

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 10

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Direct plate assay: Precipitation of Citronella nardus oil on the plates was observed at the start of the incubation period at concentrations of 5000 µg/plate and at 512 µg/plate and above at the end of the incubation period.
Pre-incubation assay: Precipitation of Citronella nardus oil on the plates was not observed at the start of the incubation period, but was observed and at the concentration of 1600 µg/plate at the end of the incubation period.

RANGE-FINDING/SCREENING STUDIES: Based on the results of the dose-range finding test, the following dose-range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 5.4, 17, 52, 164 and 512 μg/plate. these results are reported as a part of the direct plate assay

HISTORICAL CONTROL DATA (refer to any other information on results)

Any other information on results incl. tables

Historical Control Data of the Solvent Control

	TA1535		TA1537		TA98		TA100		WP2uvrA
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	3 – 29	3 - 27	3 – 20	3 – 23	8 - 41	8 - 55	63 – 176	54 - 160	10 – 59	9 - 69
Mean	10	11	6	7	16	23	108	107	25	32
SD	3	4	2	3	5	7	19	20	7	8
n	2356	2336	2264	2235	2319	2360	2341	2336	2075	2078

Historical Control Data of the Positive Control Items

 

	TA1535		TA1537		TA98
S9-mix	-	+	-	+	-	+
Range	125 – 1248	73 – 1206	55 – 1353	54 – 1051	365 – 1995	250 – 1977
Mean	846	219	787	353	1406	887
SD	146	119	345	162	258	349
n	2348	2229	2003	2234	2200	2276

 

	TA100		WP2uvrA
S9-mix	-	+	-	+
Range	439 – 1848	408 - 2651	93 – 1951	111 - 1359
Mean	901	1232	1094	437
SD	168	343	477	149
n	2335	2327	2021	2085

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between Oct 2015 and Oct 2017. 

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, Citronella nardus oil does not need to be classified for genotoxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Executive summary:

The mutagenicity of Citronella nardus oil was examined in a  bacterial mutagenicity assay (Ames) in accordance with OECD TG 471, under GLP conditions.

Several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and the Escherichia coli (E. coli) strain WP2uvrA were exposed to the test substance (in DMSO), in the presence or absence of an exogenous mammalian metabolic activation system (S9).  The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay.

In the first mutation experiment, the test item was tested up to concentrations of 512 µg/plate in the strains TA1535, TA1537 and TA98 (dosed chosen based on cytotoxicity and precipitation observed in the dose-range finding study).  Citronella nardus oil precipitated on the plates at the top dose level of 512 μg/plates.  The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the second mutation experiment, the test item was tested up to concentrations of 1600 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay.  The test item precipitated on the plates at the top dose of 1600 μg/plate.  Cytotoxicity was observed in all tester strains in the absence and presence of S9-mix.  Due to the observed cytotoxicity, an additional pre-incubation experiment was performed up to a concentration of 5.4 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the absence of S9-mix.  The test item did not precipitate, and bacterial background lawn was not reduced at any of the concentrations tested. No biologically relevant decrease in the number of revertants was observed. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

In conclusion, based on the results of this study it is concluded that Citronella nardus oil is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay and need not to be classified for genotoxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).