Myristica fragrans, ext.

Genetic toxicity: in vitro

Administrative data

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:

30-Jun-2010 to 12-jul-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EU guidelines and according to GLP principles

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

84082-68-8 (8008-45-5)

IUPAC Name:

84082-68-8 (8008-45-5)

Constituent 2

Reference substance name:

Nutmeg Oil, Myristica fragrance oil

IUPAC Name:

Nutmeg Oil, Myristica fragrance oil

Details on test material:

- Name of test material (as cited in study report): Nutmeg Oil, Myristica fragrance oil
- Substance type: Clear colourless to yellowish liquid
- Physical state: liquid
- Composition of test material, percentage of components: confidential
- Lot/batch No.: confidential
- Expiration date of the lot/batch: confidential
- Stability under test conditions: stable
- Storage condition of test material: At room temperature in the dark

Method

Target gene:

S. typhimurium: Histidine gene
E. coli: Tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and B-naphthoflavone

Test concentrations with justification for top dose:

Preliminary test (without and with S9) TA100 and W P2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 pg/plate.

Main study: TA1535, TA1537 and TA98:
Without S9-mix: 1, 3, 10, 20, 30 and 100 pg/plate
With S9-mix: 3, 10, 33, 100, 200 and 333 pg/plate
Experiment 2:
TAI535, TA1537, TA98 and TA100
Without S9-mix: 0.3, 1, 3, 10, 33 and 100 pg/plate
With S9-mix: 1, 3, 10, 33, 100 and 200 pg/plate
WP2uvrA
Without S9-mix: 0.3, 1, 3, 10, 33 and 100 pg/plate
With S9-mix: 1, 3, 10, 33, 100 and 333 pg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Test compound was stable and soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9: sodium azide (TA1535); 9-aminoacridine (TA1537); 2-nitrofluorene (TA98); methylmethanesulfonate (TA100); 4-nitroquinoline-N-oxide (WP2uvrA). With S9: 2-aminoanthracene for all test strains.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (preincubation, 30 minutes at 20° C)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in test strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in test strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the test strains, the positive response should be reproducible in at least one independently repeated experiment.

Statistics:

Mean and standard deviation

Results and discussion

Test results

Test results 1

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:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 pg/plate

RANGE-FINDING/SCREENING STUDIES: In test strain TA100, toxicity was observed at dose levels of 33 and 100 pg/plate and above in the absence and presence of S9-mix, respectively. In test strain WP2uvrA, toxicity was observed at dose levels of 33 and 333 pg/plate and above in the absence and presence of S9-mix, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
TA1535: without S9: 30 pg/plate and above and with S9: 200 pg/plate and above
TA1537: without S9: 100 pg/plate and above and with S9: 200 pg/plate and above
TA98: without S9: 100 pg/plate and above and with S9: 200 pg/plate and above
TA100: without S9: 33 pg/plate and above and with S9: 100 pg/plate and above
WP2uvrA: without S9: 33 pg/plate and above and with S9: 333 pg/plate and above

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

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. Based on the results of this study it is concluded that Nutmeg Oil, Myristica fragrance oil is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The study procedures described in this report were based on the most recent OECD and EC guidelines. Nutmeg Oil, Myristica fragrance oil was tested in the Salmonella typhimurium reverse mutation assay with 4 histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and B-naphthoflavone) with the inclusion of a preincubation step.

Overview of experiments is as follows: Preliminary test (without and with S9 mix) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 pg/plate. Main study: TA1535, TA1537 and TA98: without S9-mix: 1, 3, 10, 20, 30 and 100 pg/plate; with S9-mix: 3, 10, 33, 100, 200 and 333 pg/plate. Experiment 2: TA1535, TA1537, TA98 and TA100: without S9-mix: 0.3, 1, 3, 10, 33 and 100 pg/plate; with S9-mix: 1, 3, 10, 33, 100 and 200 pg/plate. WP2uvrA: without S9-mix: 0.3, 1, 3, 10, 33 and 100 pg/plate; with S9-mix: 1, 3, 10, 33, 100 and 333 pg/plate. Nutmeg Oil, Myristica fragrance oil did not precipitate on the plates at the top dose of 5000 pg/plate. In the dose range finding test, in test strain TA100, toxicity was observed at dose levels of 33 and 100 pg/plate and above in the absence and presence of S9-mix, respectively. In test strain WP2uvrA, toxicity was observed at dose levels of 33 and 333 pg/plate and above in the absence and presence of S9-mix, respectively. Results of this dose range finding test were reported as part of the first experiment of the mutation assay. In the mutation experiments, cytotoxicity, as evidenced by a decrease of the bacterial background lawn and/or a decrease in the number of revertants was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA in the presence of S9-mix. 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. Nutmeg Oil, Myristica fragrance oil did not induce a dose-related increase in the number of revertant (His+) colonies in each of the four test strains (TA1535, TAI537, TA98 and TA100) and in the number of revertant (Trp+) colonies in test strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. Based on the results of this study it is concluded that Nutmeg Oil, Myristica fragrance oil is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.