Melaleuca viridiflora extract

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames test: non mutagenic (OECD 471, GLP, K, rel. 2).

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:

25 October - 15 November 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study (restriction as no data on the protocol used for volatile compounds)

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:

yes

Remarks:

no data on the protocol used for volatile compounds

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

None

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: see table 7.6.1/1

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Main test: 0.06, 0.19, 0.56, 1.67 and 5.00 μL/plate, with and without S9 mix in all strains (plate incorporation method)
Confirmatory test: 0.06, 0.19, 0.56, 1.67 and 5.00 μL/plate, with and without S9 mix in all strains (pre-incubation method)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol 96 %
- Test item solubility: The test item was soluble in ethanol (96 %) at a concentration of 50.0 μL/mL.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol 96%

True negative controls:

no

Positive controls:

yes

Remarks:

See Table 7.6.1/2

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

Without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

See Table 7.6.1/2

Positive control substance:

other: 2-amino-anthracene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: Moltox; Bacterial strains used for the study were grown from controlled Working Banks obtained from Master Banks (generated in Vivotecnia) in nutrient broth supplemented with the corresponding antibiotics when required.

METHOD OF APPLICATION: In agar (direct plate incorporation and preincubation method)

DURATION
- Preincubation period: 20 minutes at 37 °C
- Incubation period: 48 h at 37 °C for both direct plate incorporation and preincubation methods

NUMBER OF REPLICATIONS:
-3 plates/dose for treatment, vehicle and positive controls

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity evaluation of the test item was based on the decrease in the number of revertant colonies, or a clearing or diminution of the background lawn.

OTHER:
- Number of revertant colonies per plate was counted and recorded by an automatic colony counter.
- The sterility of the test item was assayed by adding of 5 μL/plate to a minimal agar plate and incubating at 37 ºC for 48 h. No growth was observed in the minimal agar plate after incubation with the test item.

Evaluation criteria:

The criteria used for determining a positive result take into account a dose-response effect in the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.
A result is considered positive whenever the number of revertants of the test item treated plates is increased when compared to the solvent treated plates according to the following criteria:
- TA98, TA100 and WP2(pKM101): 2 fold; TA1535 and TA1537: 3 fold
Biological relevance of the results was also considered.

Statistics:

None

Species / strain:

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

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:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility: Solubility of the test item was evaluated in a standard solvent panel (milliQ water, ethanol 96 %, DMSO and corn oil). Observation of precipitation by the unaided eye indicated that the test item is not soluble. The test item was soluble in ethanol (96%) at a concentration of 50.0 μL/mL.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with historical data.

RANGE-FINDING/SCREENING STUDIES:
Cytotoxicity evaluation of the test item was performed in the S.typhimurium TA 100 strain by the direct incorporation procedure with 5 concentrations prepared by 1:3 serial dilutions starting at 50.0 μL/mL up to 0.6mg/mL, based on the solubility profile of the test item. No cytotoxic activity was observed at a test item concentration of 50.0 μL/mL

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

See attached Document for Tables of Results

Conclusions:

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

Under the test conditions, NIAOULI Essential Oil (Melaleuca quinquenervia) 021261 is not considered as mutagenic in S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli (WP2(pKM101)) strains, with and without 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 of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli (WP2(pKM101)) were exposed to NIAOULI Essential Oil (Melaleuca quinquenervia) 021261, with and without metabolic activation at the following concentrations:

 

Cytotoxicity test: Cytotoxicity evaluation of the test item was performed in the S.typhimurium TA 100 strain by the direct incorporation procedure with 5 concentrations prepared by 1:3 serial dilutions starting at 50.0 μL/mL up to 0.6mg/mL, based on the solubility profile of the test item.

Main test: 0.06, 0.19, 0.56, 1.67 and 5.00 μL/plate, with and without S9 mix in all strains (plate incorporation method)

Confirmatory test: 0.06, 0.19, 0.56, 1.67 and 5.00 μL/plate, with and without S9 mix in all strains (pre-incubation method)

 

Vehicle (Ethanol 96 %) and positive control groups were also included in mutagenicity tests.

 

No cytotoxic activity was observed at a test item concentration of 50.0 μL/mL. None of the concentrations assayed for the test item showed an increase in the R value either with or without S9 metabolic activation regardless of the procedure. No dose response for the test item NIAOULI Essential Oil (Melaleuca quinquenervia) 021261 was observed in any of the tested bacterial strains. The positive and vehicle controls induced the appropriate responses in the corresponding strains indicating the validity of the study.

 

Under the test conditions, NIAOULI Essential Oil (Melaleuca quinquenervia) 021261 is not considered as mutagenic in these bacterial systems.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Table 7.6/1: Summary of genotoxicity test

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1   García, 2010	Ames Test (OECD 471) K, rel. 2	Gene mutation	TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA-	-S9 +S9	Up to limit concentration	-S9 : non mutagenic +S9 : non mutagenic

 

 

A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD test guideline No 471 with Niaouli oil (Test No. 1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose, either in the presence or absence of metabolic activation. Niaouli oil does not induce gene mutations in bacteria under the test conditions whereas the positive control chemical (with and without metabolic activation) induced significant increase of colonies. Niaouli oil is therefore considered as non-mutagenic according to the Ames test.

Justification for selection of genetic toxicity endpoint
Only one study is available. The key study is GLP-compliant and of high quality (Klimisch score =2). The result being negative, no further tests are required for this REACH Annex VII substance.

Justification for classification or non-classification

Harmonized classification:

Niaouli oil has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available information, no additional classification is proposed.