Essential oil of Cinnamomum camphora (Lauraceae) obtained from leaves by steam distillation

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:

14 June - 2 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

02 November 2017

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Pin-2(3)-ene

EC Number:

201-291-9

EC Name:

Pin-2(3)-ene

Cas Number:

80-56-8

Molecular formula:

C10H16

IUPAC Name:

2,6,6-trimethylbicyclo[3.1.1]hept-2-ene

Constituent 2

Reference substance name:

Pin-2(10)-ene

EC Number:

204-872-5

EC Name:

Pin-2(10)-ene

Cas Number:

127-91-3

Molecular formula:

C10H16

IUPAC Name:

6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane

Constituent 3

Reference substance name:

Thuj-4(10)-ene

EC Number:

222-212-4

EC Name:

Thuj-4(10)-ene

Cas Number:

3387-41-5

Molecular formula:

C10H16

IUPAC Name:

1-isopropyl-4-methylenebicyclo[3.1.0]hexane

Constituent 4

Reference substance name:

7-methyl-3-methyleneocta-1,6-diene

EC Number:

204-622-5

EC Name:

7-methyl-3-methyleneocta-1,6-diene

Cas Number:

123-35-3

Molecular formula:

C10H16

IUPAC Name:

7-methyl-3-methylideneocta-1,6-diene

Constituent 5

Reference substance name:

Cineole

EC Number:

207-431-5

EC Name:

Cineole

Cas Number:

470-82-6

Molecular formula:

C10H18O

IUPAC Name:

1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane

Constituent 6

Reference substance name:

p-mentha-1,4-diene

EC Number:

202-794-6

EC Name:

p-mentha-1,4-diene

Cas Number:

99-85-4

Molecular formula:

C10H16

IUPAC Name:

1-isopropyl-4-methylcyclohexa-1,4-diene

Constituent 7

Reference substance name:

p-menth-1-en-4-ol

EC Number:

209-235-5

EC Name:

p-menth-1-en-4-ol

Cas Number:

562-74-3

Molecular formula:

C10H18O

IUPAC Name:

1-isopropyl-4-methylcyclohex-3-en-1-ol

Constituent 8

Reference substance name:

p-menth-1-en-8-ol

EC Number:

202-680-6

EC Name:

p-menth-1-en-8-ol

Cas Number:

98-55-5

Molecular formula:

C10H18O

IUPAC Name:

alpha,alpha-4-trimethyl-3-cyclohexene-1-methanol

Test material form:

liquid

Details on test material:

Name: RAVINTSARA LEAF OIL
Other name: Essential oil obtained from leaves of Cinnamomum camphora (Lauraceae) by steam distillation
Batch no.: B790145
Appearance: clear, colorless liquid
Composition: see CoA (experimental report)
Related CAS No.: 92201-50-8 and 8008-51-3
Related EINECS-No.: 295-980-1
Purity: 100%wt (UVCB substance)
Production date: Jun. 2016
Expiry date: 31. Dec. 2020
Storage: Room Temperature (20 ± 5°C), keep away from light / humidity

Specific details on test material used for the study:

- Physical state: clear liquid
- Purity: 100%
- Lot/batch No.: B790145
- Date of receipt: 23 May 2017
- Expiration date of the lot/batch: 2020
- Stability: Stable under normal storage conditions (room temperature)

Method

Target gene:

Histidine and Tryptophane

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

10 % S9 mix; S9 fraction prepared from liver homogenates of male Sprague Dawley rats

Test concentrations with justification for top dose:

- TA1535, TA1537, TA98, TA100 and E. Coli WP2: 50, 150, 500, 1500, 3500 and 5000 μg/plate, with and without S9-mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Antramine (2µg/plate with S9 metablica activation), 9-Aminoacridine (50µg/plate without metabolic activation), cis-Platinum (II) Diammine dichloride (1µg/plate without metabolica activation)

Details on test system and experimental conditions:

SOURCE OF THE TEST SYSTEM: Strains were obtained from MOLTOX TM.

METHOD OF APPLICATION: In agar (plate incorporation); preincubation

NUMBER OF REPLICATES: Controls and treatment were performed in triplicate.

DURATION
- Preincubation period: 30 minutes at 37 °C, with shaking
- Incubation period: Plates were inverted and incubated at 37 °C in the dark for 48-72 hour in both direct plate and preincubation methods.

Evaluation criteria:

The following criteria were checked to validate the study:
- the bacteriostatic activity of the highest concentration tested shall be equal to or less than 75 %,
- the spontaneous reversion rate of the absolute negative control shall comply with the historical values of the laboratory,
- the spontaneous reversion rate of the solvent shall not be statistically different from absolute negative control,
- the mean number of revertant colonies obtained for each strain and the corresponding positive control, with and/or without metabolic activation shall comply with the historical values of the laboratory.
- Negative and positive values should not show significant difference with the historical values of the laboratory (± 2 standard deviations).

Statistics:

No data

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

Remarks:

light thinning of the background bacterial lawn at 5000µg/plate

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

Remarks:

light thinning of the background bacterial lawn at 5000µg/plate

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

Remarks:

light thinning of the background bacterial lawn at 5000µg/plate

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary cytotoxicity testing (Strain TA100):
Bacteriostatic test has been performed, in case of bacteriostatic activity. The highest concentration that will be retained for the study is the concentration that induices a bacteriostatic activity of 75% or less.
Bacteriostatic activity acceptable with thr maximum tolerated (75%) was observed at 5000 µg/plate.

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the test item is not considered as mutagenic in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2 (uvrA-) (pKM 101) strains without or with metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains ofSalmonella typhimurium(TA1535, TA1537, TA98, TA100) and E.coli WP2 were exposed to test item Ravintsara essential oil at the following concentrations: 

- TA1535, TA1537, TA98, TA100 and E.coli WP2: 50, 150, 500, 1500, 3500 and 5000 μg/plate, with and without S9-mix

 

Metabolic activation system used in this test 10 % S9 mix; S9 fraction prepared from liver homogenates of male Sprague Dawley rats . Vehicle, negative and positive control groups were also included in mutagenicity tests.

 

Following treatment, evidence of toxicity was observed at 5000 μg/plate in strains TA 98, TA1535 and TA 1537 in the absence and presence of S-9.

 

The mean numbers of revertant colonies felt within acceptable historical ranges for vehicle control treatments, and were significantly elevated by positive control treatments.

 

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation. 

 

Under the test conditions, the test item is not considered as mutagenic in S. typhimurium  TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2 (uvrA-) (pKM 101) strains without or with metabolic activation.