Amyris balsamifera, ext.

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:

06 July 2016 - 22 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Amyris balsamifera, ext.

EC Number:

291-076-6

EC Name:

Amyris balsamifera, ext.

Cas Number:

90320-49-3

Molecular formula:

Not applicable due to the UVCB nature of the substance.

IUPAC Name:

Essential oil of Amyris obtained from the wood of Amyris balsamifera (Rutaceae) trees by steam distillation.

Test material form:

liquid: viscous

Details on test material:

For specific details on test material if available - See in RSS
Name of test material (as cited in study report or in reference): Amyris oil

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Obtained from Berjé batch R-87775
- Expiration date of the lot/batch: 23 June 2017, as stated on the Certificate of Analysis

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 11 to 25°C (ambient)
- Stability under test conditions: Not provided
- Solubility and stability of the test substance in the solvent/vehicle: not applicable as was made fresh

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A stock solution of Amyris Oil in DMSO was prepared and then diluted in the same solvent to prepare the required test solutions
- Final dilution of stock liquid: Diluted to 50, 16, 5, 1.6, 0.5, 0.16, 0.05, 0.016 mg/ml

Method

Target gene:

- S. typhimurium: Histidine gene
- Escherichia coli: Tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells:
TA1535, TA1537 and TA100 were originally obtained in 1995 by AZ Sweden, from Professor B N Ames, Biochemistry Department, University of California, Berkeley,
California, USA.
TA98 was originally obtained in 2008 by AZ Sweden, from BioReliance Corporation, Rockville, Maryland, USA.

- Checks: Before establishing cryopreserved working stock cultures and on the day of the first mutagenicity test, the strains were checked for the following characteristics (phenotype check): absence of contamination; spontaneous revertant frequency; pre-existing revertant numbers; presence of the deep rough (rfa) marker; sensitivity to UV light; presence of plasmid pKM101 ((TA98, TA100)


MEDIA USED
- Type and identity of media: Nutrient Broth No.2 (Oxoid Limited) supplemented with ampicillin, as required
- Properly maintained: yes

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells:
E. coli strain was originally obtained in 1995 by AZ Sweden, from Dr S Venitt, Institute of Cancer Research, Royal Marsden Hospital, Surrey, UK.

- Checks: Before establishing cryopreserved working stock cultures and on the day of the first mutagenicity test, the strains were checked for the following characteristics (phenotype check): absence of contamination; spontaneous revertant frequency; pre-existing revertant numbers; ; sensitivity to UV light; presence of plasmid pKM101


MEDIA USED
- Type and identity of media: Nutrient Broth No.2 (Oxoid Limited) supplemented with ampicillin, as required
- Properly maintained: yes

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

n/a

Metabolic activation:

with and without

Metabolic activation system:

The post-mitochondrial fraction (S9) of a rat liver homogenate (from Sprague-Dawley rats treated with Aroclor 1254)

Test concentrations with justification for top dose:

Concentration of test solutions:
The dose ranges used were:
Mutagenicity test 1 1.6 to 5000 μg per plate (Doses were chosen based on current OECD guidelines)
Mutagenicity test 2 1.6 to 500 μg per plated. (Based on toxicity findings in mutagenicity test 1)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Preferred vehicle. As solubility of the test item in DMSO was satisfactory, it was used as the solvent for the mutagenicity testing.

Details on test system and experimental conditions:

METHOD OF APPLICATION: ; in agar (plate incorporation)

DURATION
- Preincubation period: none
- Exposure duration: 3 days of incubation

- Expression time (cells in growth medium): cells were started up and incubated overnight (8-10 hours)
- Selection time (if incubation with a selection agent): for TA98, TA100 and uvrA/pKM101 the nutrient broth was supplemented with ampicillin at 25 μg/mL

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: reductions in the growth of the background lawns or in the incidence of spontaneous revertant colonies. Microscopic examination was used to check the condition of the histidine/tryptophan-requiring microcolonies that make up the ‘background lawns’.

SCORING:
Revertant (his+, trp+) colony numbers were scored using a Sorcerer Colony Counter (Perceptive Instruments, UK). In cases where the mean incidence of revertant colonies on the solvent control plates was 20 or less (typically with strains TA1535 and TA1537), or if cytotoxicity or precipitate interfered with image analysis, the plates were scored manually. All counts were manually entered into the results sheet.

Rationale for test conditions:

Following OECD TH 471

Evaluation criteria:

Solvent controls: The number of revertant colonies in the solvent control plates was measured for all strains and compared with published values and data generated in-house. The control mean value for each strain was required to be in the acceptable range, defined as within 1 and 99 percentiles of the historical mean.
Positive controls: An individual test was considered acceptable only if a clear response was seen in the positive control plates. For strains TA1535, TA1537, TA98, TA100 and E. coli uvrA/pKM101 minimum mean increases in revertant colonies of 3 times the concurrent solvent control were required.
Toxicity
A dose of the test item was judged to be toxic to a bacterial strain if the formation of microcolonies (background lawn) was reduced, or a decrease in the number of revertant colonies was seen.

Mutagenicity: A test item was considered to be mutagenic if the following criteria were satisfied.
1. For TA1535 or TA1537, the mean number of revertant colonies of one or more doses of the test item, with or without metabolic activation is equal to or greater than 2 times the concurrent solvent control mean value and the relevant historical mean value; for any other strain, the mean number of revertant colonies is equal to or greater than 2 times the concurrent solvent control mean value in the presence of one or more doses of the test item, with or without metabolic activation.
2. There was a dose-related increase in the number of revertant colonies.
3. Any increase was reproducible.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA98 and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above a dose of 50 μg per plate for all five strains in both the presence and absence of S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above a dose of 50 μg per plate for all five strains in both the presence and absence of S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: On both the day of the test and the day of scoring, precipitate was seen at and above 500 μg per
plate in both the presence and absence of S9 mix.


HISTORICAL CONTROL DATA
- Positive historical control data: Find attached
- Negative (solvent/vehicle) historical control data: Find attached

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, Amyris oil was determined to be not mutagenic and does not need to be classified for mutagenicity in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay in accordance with the criteria outline in Annex I of CLP (1272/2008/EC).

Executive summary:

Amyris Oil was tested for mutagenic activity using genetically modifiedSalmonella typhimuriumLT2 bacteria of strains TA1535, TA1537, TA98 and TA100, andEscherichia coliWP2 strainuvrA/pKM101 as indicator organisms. A preliminary assessment of solubility was performed to determine suitable dose levels of Amyris Oil for the mutagenicity tests. In the mutagenicity tests, Amyris Oil was tested using all five indicator strains in both the presence and absence of anin vitroactivation system based on S9 fraction obtained from Aroclor 1254-induced rat liver (S9 mix). The plate incorporation method was used for both tests.

 

The dose ranges used were:

Mutagenicity test 1 1.6 to 5000μg per plate

Mutagenicity test 2 1.6 to 500μg per plate

 

The test item showed evidence of cytotoxicity to the bacteria. The minimum dose showing evidence of cytotoxicity was 50μg per plate and the maximum dose level scored for revertant colonies was 600μg per plate. The minimum dose at which precipitate was seen on the day of scoring was 500μg per plate. No significant increase in numbers of revertant (histidine or tryptophan-independent) colonies was seen with any of the five indicator strains either in the presence or absence of S9 mix. It was concluded that Amyris Oil was not mutagenic using a plate incorporation method forSalmonella TyphimuriumLT2 strains TA1535, TA1537, TA98 and TA100, and Escherichia coliWP2 strainuvrA/pKM101, either in the presence or absence of S9 mix, when tested under the conditions used in this assay.