Santalum austro-caledonicum, 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:

17 March to 17 April 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD 471 Guideline without any deviation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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)

Remarks:

landesamt für umwelt wasserwirtschaft und gewerbeaufsicht (inspected on 29-30 November 2010 / signed on 11 April 2011)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

5-(2,3-dimethyltricyclo[2.2.1.02,6]hept-3-yl)-2-methylpent-2-en-1-ol, stereoisomer

EC Number:

204-102-8

EC Name:

5-(2,3-dimethyltricyclo[2.2.1.02,6]hept-3-yl)-2-methylpent-2-en-1-ol, stereoisomer

Cas Number:

115-71-9

Molecular formula:

C15H24O

IUPAC Name:

5-(2,3-Dimethyltricyclo[2.2.1.0~2,6~]hept-3-yl)-2-methylpent-2-en-1-ol

Constituent 2

Reference substance name:

[1S-[1α,2α(Z),4α]]-2-methyl-5-(2-methyl-3-methylenebicyclo[2.2.1]hept-2-yl)-2-penten-1-ol

EC Number:

201-027-2

EC Name:

[1S-[1α,2α(Z),4α]]-2-methyl-5-(2-methyl-3-methylenebicyclo[2.2.1]hept-2-yl)-2-penten-1-ol

Cas Number:

77-42-9

Molecular formula:

C15H24O

IUPAC Name:

2-Methyl-5-(2-methyl-3-methylenebicyclo[2.2.1]hept-2-yl)pent-2-en-1-ol

Constituent 3

Reference substance name:

(2E)-2-methyl-6-[(1S)-4-methylcyclohex-3-en-1-yl]hepta-2,6-dien-1-ol

Cas Number:

942226-77-9

Molecular formula:

C15H24O

IUPAC Name:

(2E)-2-methyl-6-[(1S)-4-methylcyclohex-3-en-1-yl]hepta-2,6-dien-1-ol

Constituent 4

Reference substance name:

(Z)-5-(4,6-dimethyl-6-bicyclo[3.1.1]hept-3-enyl)-2-methylpent-2-en-1-ol

Cas Number:

88034-74-6

Molecular formula:

C15H24O

IUPAC Name:

(Z)-5-(4,6-dimethyl-6-bicyclo[3.1.1]hept-3-enyl)-2-methylpent-2-en-1-ol

Constituent 5

Reference substance name:

(Z)-2-Methyl-5-((1R,2R,4S)-2-methyl-3-methylenebicyclo[2.2.1]heptan-2-yl)pent-2-en-1-ol

Cas Number:

79081-90-6

Molecular formula:

C15H24O

IUPAC Name:

(Z)-2-Methyl-5-((1R,2R,4S)-2-methyl-3-methylenebicyclo[2.2.1]heptan-2-yl)pent-2-en-1-ol

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Robertet S.A. / 2259263
- Appearance: Colourless to pale yellow liquid / essential oil
- Production date: 18 December 2013
- Expiration date of the lot/batch: 18 December 2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (20 ± 5 °C); test item was stored in the test facility in a closed vessel at room temperature
- Stability under test conditions: Not stated

Method

Target gene:

Histidine gene for Salmonella typhimurium

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102 and TA 1535

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

4% S9; S9 fraction produced from the livers of male Sprague-Dawley rats which were treated with Aroclor 500 mg/kg bw intraperitoneally

Test concentrations with justification for top dose:

Cytotoxicity test: 150 μg/plate in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, with and without S9 mix using plate incorporation method
Experiment 1: 0.05, 0.15, 0.5, 1.5, 5 and 150 μg/plate in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, with and without S9 mix using plate incorporation method
Experiment 2: 5, 9, 19, 38, 75 and 150 μg/plate in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, with and without S9 mix using pre-incubation method
Experiment 3: 0.5, 1.5, 5, 15, 50 and 150 μg/plate in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, with and without S9 mix using plate incorporation method

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: DMSO was chosen as vehicle, because the test item was sufficiently soluble, and this solvent doesn’t have any effects on the viability of the bacteria or the number of spontaneous revertants.
- On the day of the start of the first experiment, a stock solution containing 15 g/L of the test item in DMSO was prepared. On the respective day of the start of the second resp. third experiment, a stock solution containing 1.5 g/L of the test item in DMSO was prepared. The stock solution was used to prepare the geometric series of the concentrations to be tested. Each solution was membrane filtrated to accomplish sterility.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-Nitro-1,2-phenylene Diamine

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Amino-Anthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: Salmonella typhimurium (all strains used) were obtained from TRINOVA BioChem (batch of the bacteria strains: TA 97a: 4488D, TA 98: 4789D; TA 100: 4569D; TA 102: 4712D; TA 1535: 4717D) and were stored as lyophilisate in the refrigerator at 2 - 8 °C.

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

DURATION
- Preincubation period: 37 °C for 20 min
- Exposure duration: 37 °C for 48 h

NUMBER OF REPLICATIONS:
- Cytotoxicity test: 4 plates/dose
- Experiment 1, 2 and 3: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: Evaluation of the toxicity was performed on the basis of growth of the bacterial background lawn.

Evaluation criteria:

A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor ≥ 2) in at least one strain can be observed.
A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.

Statistics:

None

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102 and TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: None

FIRST EXPERIMENT
- Toxicity: After addition of phosphate buffer to the test item solution, the resulting solution showed turbidity in the highest concentration: 150 μg/plate. No signs of toxicity towards the tested strains could be observed. The background lawn was visible and the number of revertant colonies was not reduced.
- Mutagenicity: No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found. To verify this result, a second experiment was performed using the pre-incubation method.

SECOND EXPERIMENT
- No signs of toxicity towards the tested strains could be observed. The background lawn was visible and the number of revertant colonies was not reduced.
- No significant increase of the number of revertant colonies in the treatments with and without metabolic activation was observed. No concentration-related increase over the tested range was found.

THIRD EXPERIMENT
- A third experiment was performed, because a repetition of the first experiment was necessary. In the first experiment, five concentrations of the test item dilutions had been lower than intended by a factor of ten, because, inadvertently, a calculation error was made when diluting the stock solution.
- No signs of toxicity towards the tested strains could be observed. The background lawn was visible and the number of revertant colonies was not reduced.
- No significant increase of the number of revertant colonies in the treatments with and without metabolic activation was observed. No concentration-related increase over the tested range was found.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation)
- Positive historical control data: 369-908 (577 ± 165), 109-542 (284 ± 172), 466-772 (595 ± 97), 540-976 (726 ± 137), 108-527 (252 ± 160) for TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains (without metabolic activation), respectively; 420-831 (607 ± 137), 95-195 (126 ± 29), 552-1080 (808 ± 155), 449-1332 (780 ± 284), 114-210 (148 ± 30) for TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains (with metabolic activation), respectively.
- Negative historical control data (Water): 99-150 (122 ± 14), 12-18 (14 ± 2), 115-154 (140 ± 12), 138-252 (205 ± 30), 13-23 (19 ± 3) for TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains (without metabolic activation), respectively; 106-152 (131 ± 18), 10-17 (14 ± 2), 119-153 (138 ± 14), 171-254 (207 ± 20), 15-25 (21 ± 3) for TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains (with metabolic activation), respectively.
- Vehicle historical control data (DMSO): 105-141 (127 ± 11), 10-17 (13 ± 2), 118-166 (141± 15), 136-250 (198 ± 28), 16-22 (19 ± 2) for TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains (without metabolic activation), respectively; 105-159 (128 ± 16), 12-18 (14 ± 2), 117-157 (139 ± 14), 183-245 (207 ± 21), 16-22 (19 ± 2) for TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains (with metabolic activation), respectively.

OTHERS:
- Confirmation of genotype is performed once a quarter. The last performance showed no abnormalities.
- Confirmation of the criteria and validity: The treatments for the sterility control and the determination of the titre didn’t show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range of the literature data. All positive controls (diagnostic mutagenes) showed mutagenic effects with and without metabolic activation.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

Under the test conditions, test substance is not considered as mutagenic in S. typhimurium (TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains).

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 97a, TA 98, TA 100, TA 102 and TA 1535) were exposed to the test item both in the presence and in the absence of a metabolic activation system (4%S9-mix, rat liver S9-mix induced by Aroclor 1254) for 48 h. 

Experiment 1: 0.05, 0.15, 0.5, 1.5, 5 and 150 μg/plate in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, with and without S9 mix using plate incorporation method

Experiment 2: 5, 9, 19, 38, 75 and 150 μg/plate in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, with and without S9 mix using pre-incubation method

Experiment 3: 0.5, 1.5, 5, 15, 50 and 150 μg/plate in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, with and without S9 mix using plate incorporation method 

 

Negative, vehicle and positive control groups were also included in mutagenicity tests. 

 

The sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation. Therefore, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

No signs of toxicity towards the tested strains could be observed. No visible reduction in the growth of the bacterial background lawn at any dose level. No significant increase of the number of revertant colonies in the treatments with and without metabolic activation was observed in any of the experiments. No concentration-related increase over the tested range was found.

 

Under the test condition, the test substance is not mutagenic with and without metabolic activation in Salmonella typhimurium, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535.