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Genetic toxicity in vitro

Description of key information

Ames test (OECD471): not mutagenic

Link to relevant study records
Reference
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 15-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
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
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
Target gene:
S. typhimurium: Histidine gene
E. coli: Tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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 f3-naphthoflavone
Test concentrations with justification for top dose:
Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 pg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 10, 33, 100, 333, 1000 and 2000 pg/plate
Experiment 2:
Without and with S9-mix: 10, 33, 100, 333, 1000 and 2000 pg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test compound was stable and soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines
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 tester strains.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)

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 tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester 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 tester strains, the positive response should be reproducible in at least one independently repeated experiment.
Statistics:
Mean and standard deviation
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
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 1000 pg/plate and above in the absence and presence of S9-mix. In test strain WP2uvrA, toxicity was observed at dose levels of 3330 pg/plate and above in the absence and presence of S9-mix.

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: 1000 pg/plate and above and with S9: 2000 pg/plate
TA1537: without S9: 2000 pg/plate and with S9: 2000 pg/plate
TA98: without S9: 1000 pg/plate and above and with S9: 2000 pg/plate
TA100: without S9: 1000 pg/plate and above and with S9: 333 pg/plate and above WP2uvrA: without S9: 2000 pg/plate and with S9: 2000 pg/plate
Conclusions:
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 Cinnamon Leaf Oil is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

Cinnamon Leaf Oil was tested in the Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Ecoli (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). The study procedures described in this report were based on the most recent OECD and EC guidelines. In the dose range finding test, Cinnamon Leaf Oil was tested up to concentrations of 5000 pg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. Cinnamon Leaf Oil did not precipitate on the plates at this dose level. In test strain TA100, toxicity was observed at dose levels of 1000 pg/plate and above in the absence and presence of S9-mix. In test strain WP2uvrA, toxicity was observed at dose levels of 3330 and 5000 pg/plate in the absence of and presence S9-mix. Results of this dose range finding test were reported as part of the first experiment of the mutation assay. Based on the results of the dose range finding test, Cinnamon Leaf Oil was tested in the first mutation assay at a concentration range of 10 to 2000 pg/plate in the absence and presence of 5% (v/v) S9-mix in test strains TA1535, TA1 537 and TA98. In an independent repeat of the assay with additional parameters, Cinnamon Leaf Oil was tested at the same concentration range as the first assay in the absence and presence of 10% (v/v) S9-mix in test strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Toxicity was observed in all test strains. Cinnamon Leaf Oil did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, 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. In this study, 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 Cinnamon Leaf Oil is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Additional information

An Ames test in accordance with OECD471 was performed to determine the mutagenic potential of cinnamon leaf oil to cause gene mutations in bacteria. Cinnamon Leaf Oil did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, 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. Furthermore, 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 Cinnamon Leaf Oil is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

An additional study is included that was disregarded. In this study, cinnamon leaf oil was tested for mutagenicity in the Ames test without metabolic activation in four S. typhimurium strains (TA98, TA1535, TA1537 and TA1538) at two concentrations (5 and 10 pl/plate). Cinnamon leaf exhibited mutagenicity in the Ames test at the lowest dose (5 pl/plate) in two of the four strains tested, i.e. S. typhimurium TA98 (weak mutagen – colonies more than control) and TA1537 (mutagen - colonies more than 70% of control). As the effects were observed at the lowest dose only, and no independent repeat was performed, no conclusion can be drawn based on this study.

Based on the findings in the key study, in which six doses were tested, with and without metabolic activation, and an independent repeat was performed, it can be concluded that cinnamon leaf oil does not cause gene mutations in bacteria.


Short description of key information:
In vitro gene mutation in bacteria: not mutagenic (OECD471)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available information for Cinnamon leaf oil, the substance can be considered not mutagenic and should therefore not need to be classified for mutagenicity according to the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC). However, based on the Safrole content in Cinnamon leaf oil the substance does need to be classified as genotoxic.

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