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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria (OECD TG 471, Ames): Not mutagenic

Presence of the suspected carcinogen 4-allylveratrole (methyl eugenol, CAS #93-15-2) as a constituent.

Link to relevant study records
Reference
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
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
Target gene:
histidine locus
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
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.
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
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
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
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
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
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
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
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
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
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)

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. 

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).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

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.  

Justification for classification or non-classification

Based on the results, Citronella nardus oil does not need to be classified for mutagenicity. However due to the presence of the suspected carcinogen 4-allylveratrole (methyl eugenol, CAS #93-15-2) as a constituent up to 1%, Citronella nardus oil is classified as Carc. 2 in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC)

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