Oils, neroli

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:

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Essential oil of Citrus aurantium var. amara or Bigaradia (Rutaceae) obtained from the flowers by steam distillation

EC Number:

639-655-8

Cas Number:

8016-38-4

Molecular formula:

not applicable for a UVCB substance

IUPAC Name:

Essential oil of Citrus aurantium var. amara or Bigaradia (Rutaceae) obtained from the flowers by steam distillation

Test material form:

liquid

Details on test material:

Batch number : 1110104170100

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9, prepared from rat livers treated with an enzymatic inducer

Test concentrations with justification for top dose:

Based on the preliminary test results on TA 100, the following initial range concentrations where chosen: 1600, 500, 160, 50, 16 and 5 µg/plate.

Based on the results of the definitive test with the other strains, the following range concentrations where chosen:
- Test without pre-incubation without S9 mix:
TA 100, TA 98, TA 1535, TA 1537: 1600, 500, 160, 50, 16 and 5 µg/plate.
TA 102: 5000, 1600, 500, 160, 50, 16 and 5 µg/plate.

- Test without pre-incubation with S9 mix:
TA 100: 1600, 500, 160, 50, 16 and 5 µg/plate.
TA 102, TA 98, TA 1535, TA 1537: 5000, 1600, 500, 160, 50, 16 and 5 µg/plate.

- Test with pre-incubation without and without S9 mix:
all strains: 5000, 1600, 500, 160, 50, 16, 5 and 1.6 µg/plate.

Vehicle / solvent:

- Solvent used: ethanol
- Justification for choice of solvent: following a preliminary dissolution test, ethanol was found to be the most appropriate solvent: the test item showed no insolubility.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- First assay with and without S9 mix: by plate incorporation method.
- Second assay with and without S9 mix : with pre-incubation method.
- Bacterial culture: approximately 10E8 to 10E9 cells/ml.


DURATION
- Preincubation period: for test 2 : 20 to 30 min at 37°C ± 2°C
- Exposure duration: 48 to 72 hours at 37°C ± 2°C

NUMBER OF REPLICATES: 3/doses and assay

DETERMINATION OF CYTOTOXICITY
- Method: count number of revertant colonies and qualitative observation of the bottom bacterial layer reduction.

Evaluation criteria:

The results are expressed in number of revertants R = (Number of revertant colonies wiith the test substance) / (Number of revertant colonies in the absence of the test substance):

The test item is considered as mutagenic if at the end of the verifications steps, it has been obtained, in a reproducible way, a relation dose-effect on one or some of 5 strains with and/or without metabolic activation. The mutagenicity is taken into account for a given concentration only when the number of revertants is equal at least to the double of the spontaneous rate of reversion for TA98, TA100 and TA102 strains (R >= 2) and the triple of the spontaneous rate of reversion for TA1535 and TA1537 strains (R >= 3).

The test item is considered non-mutagenic if at the end of the verifications steps, the rate of revertants always remained lower than the double of the rate of spontaneous reversion for all the tested concentrations, for TA98, TA100 and TA102 strains (R<2) and lower than the triple of the spontaneous rate of reversion for TA1535 and TA 1537 strains (R<3), with and without metabolic activation, and on the condition of having made sure that the absence of mutagen effect is not bound to the toxicity of the tested concentrations.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

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:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: slight signs of precipitate have been observed on mix reagent at 5000 µg/plate with and without S9 mix.

RANGE-FINDING/SCREENING STUDIES: yes, preliminary cytotoxicity test.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: : count number of revertant colonies and qualitative observation of the bottom bacterial layer reduction.

Applicant's summary and conclusion

Conclusions:

Under these conditions, the test item Essential oil of Citrus aurantium var.amara or Bigaradia (Rutacea) obtained from the flowers by steam distillation does not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102, without or with metabolic activation, according to the OECD Guidelines n°471 and the method B13/B14 of the directive 2000/32/CE.

Executive summary:

The ability of the test item to induce mutation was assessed using the bacterial reverse mutation test (Ames test). The test was performed on five Salmonella typhimurium strains.

 

The test item dilutions were prepared in ethanol.

 

A preliminary cytotoxicity test was performed on S. typhimurium TA100 strain.

 

The test has been performed at the concentrations 5000, 1600, 500, 160 and 50 μg/plate, with and without metabolic activation.

 

As the preliminary experiment revealed cytotoxicity of the test item, the Study Director has chosen the following range of concentrations: 1600, 500, 160, 50, 16 and 5μg/plate. The Test 1 also includes the test on the strain TA100.

 

According to the results obtained in the Test 1, the Study Director decided to relaunch Test 1 at 5000μg/plate for strains TA102 without metabolic activation, and for strains TA98, TA102, TA1535 and TA1537 with metabolic activation, in order to verify cytotoxic activity of test item at this concentration.

Moreover, range of concentrations for Test 2 was the following: 5000, 1600, 500, 160, 50, 16, 5 and 1.6μg/plate.

 

The revertant analysis shows that:

·        A cytotoxic effect has been observed in the following conditions:

o   Without metabolic activation:

§ With direct incorporation (Test 1): Until 160μg/plate for strain TA100, until 500μg/plate for strains TA98, TA1535 and TA1537, at 5000μg/plate for strain TA102.

 

§ This cytotoxic effect has been confirmed with the pre-incubation method (Test 2) until 50μg/plate for all strains (TA98, TA100, TA102, TA1535 and TA1537).

 

o   With metabolic activation:

 

§ With direct incorporation (Test 1): Until 500μg/plate for strain TA100, at 5000μg/plate for strain TA98, TA102, TA1535 and TA1537.

 

§ This cytotoxic effect has been confirmed with the pre-incubation method (Test 2) until 160μg/plate for strain TA100, and until 500μg/plate for strains TA98, TA102, TA1535 and TA1537.

 

·        No concentration of the test item showed ratio R higher or equal at least to the double of the spontaneous rate of reversion for TA98, TA100 and TA102 strains and to the triple of the spontaneous rate of reversion for TA1535 and TA1537 strains, with and without metabolic activation.

·        No dose response was observed, whatever the test system or conditions of the test.

 

In addition, slight signs of precipitate have been observed on mix reagent at 5000μg/plate, with and without metabolic activation.

 

Based on the result of this study, the test itemwas found to be non mutagenic and non pro-mutagenic, but shows a cytotoxic activity, under the test conditions.