Fusanus spicatus, 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:

Marc - May 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

see Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (OJ L 142, 31.5.2008, p. 1) B.13/B.14

Test guideline 2

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:

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:

Farnesol

EC Number:

225-004-1

EC Name:

Farnesol

Cas Number:

4602-84-0

Molecular formula:

C15H26O

IUPAC Name:

3,7,11-trimethyldodeca-2,6,10-trien-1-ol

Constituent 4

Reference substance name:

(Z,6S)-2-methyl-6-(4-methylphenyl)hept-2-en-1-ol

Cas Number:

78339-53-4

Molecular formula:

C15H22O

IUPAC Name:

(Z,6S)-2-methyl-6-(4-methylphenyl)hept-2-en-1-ol

Constituent 5

Reference substance name:

(2R)-6-methyl-2-[(1S)-4-methylcyclohex-3-en-1-yl]hept-5-en-2-ol

Cas Number:

78148-59-1

Molecular formula:

C15H26O

IUPAC Name:

(2R)-6-methyl-2-[(1S)-4-methylcyclohex-3-en-1-yl]hept-5-en-2-ol

Constituent 6

Reference substance name:

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

Cas Number:

88034-74-6

Molecular formula:

C15H24O

IUPAC Name:

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

Constituent 7

Reference substance name:

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

Cas Number:

10067-28-4

Molecular formula:

C15H24O

IUPAC Name:

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

Constituent 8

Reference substance name:

(Z)-2-methyl-5-[(1R,2R,4S)-2-methyl-3-methylidene-2-bicyclo[2.2.1]heptanyl]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-methylidene-2-bicyclo[2.2.1]heptanyl]pent-2-en-1-ol

Test material form:

liquid

Method

Target gene:

Each strain derived from Salmonella typhimurium LT 2 contains one mutation in the histidine operon, resulting in a requirement for histidine.
In addition, to increase their sensitivity to mutagenic items, further mutations have been added:
- the rfa mutation causes partial loss of the lipopolysaccharide barrier that coats the surface of the bacteria and increases permeability to large molecules that do not penetrate the normal bacteria cell wall,
- the uvrB mutation is a deletion of a gene coding for the DNA excision repair system, which renders the bacteria unable to use this repair mechanism to remove the damaged DNA,
- the addition of the plasmid pKM 101 to strains TA 98, TA 100 and TA 102 enhances their sensitivity of detection to some mutagens,
- in case of TA 102 strain, the histidine mutation is located on the multicopy plasmid pAQ1.
Table: Genotype of the bacterial strains
Strains Main mutations Additional mutations
TA 1535 His G 46 rfa uvrB -
TA 100 His G 46 rfa uvrB pKM 101
TA 102 His G 428 (pQA1) rfa - pKM 101
TA 1537 His C 3076 rfa uvrB -
TA 98 His D 3052 rfa uvrB pKM 101
The TA 1535, TA 100 and TA 102 strains are reverted by base-pair substitution mutagens and the TA 1537 and TA 98 strains by frameshift mutagens. In addition, the TA 102 strain detects oxidative mutagens.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

The test item was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.
The S9 mix consists of induced enzymatic systems contained in rat liver post-mitochondrial fraction (S9 fraction) and the cofactors necessary for their function. S9 fraction was purchased from Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA) and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route. Each batch of S9 is tested and validated by Moltox for its ability to activate benzo(a)pyrene and 2-anthramine (also known as 2-amino anthracene) to mutagenic intermediates.
The S9 fraction was preserved in sterile tubes at -80 °C, until use. The S9 mix was prepared at +4 °C immediately before use and maintained at this temperature until added to the overlay agar.

Test concentrations with justification for top dose:

Since the test item was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.
Selected treatment-levels in experiments without S9 mix:
1st experiment: 9.77, 19.53, 39.06, 78.13 and 156.3 µg/plate, for the TA 1535 and TA 100, and 39.06, 78.13, 156.3, 312.5 and 625 µg/plate, for the TA 1537, TA 98 and TA 102 strains
2nd experiment: 4.88, 9.77, 19.54, 39.06, 78.13 and 156.3 µg/plate, for the TA 100 strain and 19.54, 39.06, 78.13, 156.3 and 312.5 µg/plate, for the TA 1535, TA 1537, TA 98 and TA 102 strains.
Moderate to severe toxicity was induced at dose-levels ≥ 78.13 µg/plate in the TA 100 and TA 102 strains and ≥ 156.3 µg/plate in the remaining tester strains. No significant increase in the number of revertants was noted in any of the five tester strains.
Selected treatment-levels in experiments with S9 mix:
1st experiment: 39.06, 78.13, 156.3, 312.5 and 625 µg/plate, for the TA 1535 and TA100 strains and 78.13, 156.3, 312.5, 625 and 1250 µg/plate, for the TA 102 strain and 78.13, 156.3, 312.5, 625 and 1250 µg/plate, for the TA 102 strain and 312.5, 625, 1250, 2500 and 5000 µg/plate, for the TA 1537 and TA 98 strains
2nd experiment: 19.54, 39.06, 78.13, 156.3, 312.5 and 625 µg/plate, for the TA 98 and TA 1537 strains and 39.06, 78.13, 156.3, 312.5 and 625 µg/plate, for the TA100 strain and 39.06, 78.13, 156.3, 312.5, 625 and 1250 µg/plate, for the TA 1535 strain and 78.13, 156.3, 312.5, 625, 1250 and 2500 µg/plate, for the TA 102 strain
A moderate emulsion was observed in the Petri plates when scoring the revertants at dose-levels ≥ 2500 µg/plate.
A moderate to severe toxicity was induced at dose-levels ≥ 156.3 µg/plate in the TA 100 and TA 1537 strains, ≥ 312.5 µg/plate in the TA 1535 strain and ≥ 625 µg/plate in the TA 98 and TA 102 strains.
No significant increase in the number of revertants was noted in any of the five tester strains.

Vehicle / solvent:

dimethylsulfoxide (DMSO), batch Nos. K32136150327 and K33208450435 (Merck Eurolab, Fontenay—Sous-Bois, France).

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

1 µg/plate NaN3 for TA1535 and TA100, without S9 mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

50 µg/plate 9AA for TA 1537, without S9 mix

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

0.5 µg/plate 2NF for TA98, without S9 mix

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

0.5 µg/plate for TA102, without S9 mix

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-anthramine

Remarks:

2 µg/plate for TA1537, 1537, 98 and 100, with S9 mix

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-anthramine

Remarks:

10 µg/plate for TA102, with S9 mix

Details on test system and experimental conditions:

Treatment: The test item was tested in a preliminary test and two mutagenicity experiments. The preliminary test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second experiment with S9 mix was performed according to the preincubation method.
The direct plate incorporation method was performed as follows: test item solution (0.05 mL), S9 mix when required or phosphate buffer pH 7.4 (0.5 mL) and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45 °C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.
The preincubation method was performed as follows: test item solution (0.05 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37 °C, under shaking, before adding the overlay agar and pouring onto the surface of a minimum agar plate. After 48 to 72 hours of incubation at 37 °C, revertants were scored with an automatic counter (Cardinal counter, Perceptive Instruments, Suffolk CB9 7 BN, UK).
Preliminary toxicity test: To assess the toxicity of the test item to the bacteria, six dose-levels (one plate/dose-level) were tested in the TA 98, TA 100 and TA 102 strains, with and without S9 mix.
The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

Rationale for test conditions:

In two independent experiments, using three plates/dose-level, each strain was tested, with and without S9 mix, with at least five dose-levels of the test item, the vehicle control, and the appropriate positive control.
The sterility of the S9 mix was checked before the beginning and at the end of each experiment and was found to be satisfactory.

Evaluation criteria:

Treatment of results: In each experiment, for each strain and for each experimental point, the number of revertants per plate was scored. The individual results and the mean number of revertants, with the corresponding standard deviation and ratio (mutants obtained in the presence of the test item/mutants obtained in the presence of the vehicle), were presented in tabular form.
Acceptance criteria: This study is considered valid if the following criteria are fully met:
- the number of revertants in the vehicle controls is consistent with the historical data of the testing facility,
-the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with the historical data of the testing facility.
Evaluation criteria
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST
The test item was freely soluble in the vehicle (DMSO) at 100 mg/mL. Consequently, with a treatment volume of 50 µL/plate, the dose-levels were 10, 100, 500, 1000, 2500 and 5000 µg/plate.
A moderate emulsion was observed in the Petri plates when scoring the revertants at dose-levels ≥ 2500 µg/plate.
Without S9 mix, a moderate to severe toxicity was induced at dose-levels ≥ 100 µg/plate in the TA 100 strain and ≥ 500 µg/plate in the TA 98 and TA 102 strains.
With S9 mix, a moderate to severe toxicity was noted at dose levels ≥ 500 µg/plate in the TA 100 strain, ≥ 1000 µg/plate in the TA 102 strain and at 5000 µg/plate in the TA 98 strain.

Any other information on results incl. tables

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

Since the test item was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.

The selected treatment-levels in experiments without S9 mix were:

9.77, 19.53, 39.06, 78.13 and 156.3µg/plate, for the TA 1535 and TA 100 strains in the first experiment, 39.06, 78.13, 156.3, 312.5 and 625µg/plate, for the TA 1537, TA 98 and TA 102 strains in the first experiment, 4.88, 9.77, 19.54, 39.06, 78.13 and 156.3µg/plate, for the TA 100 strain in the second experiment, 19.54, 39.06, 78.13, 156.3 and 312.5µg/plate, for the TA 1535, TA 1537, TA 98 and TA 102 strains in the second experiment.

A moderate to severe toxicity was induced at dose-levels≥78.13µg/plate in the TA 100 and TA 102 strains and≥156.3µg/plate in the remaining tester strains.

No significant increase in the number of revertants was noted in any of the five tester strains.

The selected treatment-levels in experiments with S9 mix were:

19.54, 39.06, 78.13, 156.3, 312.5 and 625µg/plate, for the TA 98 and TA 1537 strains in the second experiment, 39.06, 78.13, 156.3, 312.5 and 625µg/plate, for the TA 1535 strain in the first experiment as well as for the TA 100 strain in both experiments, 39.06, 78.13, 156.3, 312.5, 625 and 1250µg/plate, for the TA 1535 strain in the second experiment, 78.13, 156.3, 312.5, 625 and 1250µg/plate, for the TA 102 strain in the first experiment, 78.13, 156.3, 312.5, 625, 1250 and 2500µg/plate, for the TA 102 strain in the second experiment, 312.5, 625, 1250, 2500 and 5000µg/plate, for the TA 1537 and TA 98 strains in the first experiment.

A moderate emulsion was observed in the Petri plates when scoring the revertants at dose-levels≥2500µg/plate. Moderate to severe toxicity was induced at dose-levels≥156.3µg/plate in the TA 100 and TA 1537 strains,≥312.5µg/plate in the TA 1535 strain and≥625µg/plate in the TA 98 and TA 102 strains.

No significant increase in the number of revertants was noted in any of the five tester strains.

Thus, under the experimental conditions, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions, the test item HE de Bois de Santal Australie (Australian sandalwood oil) did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium (TA98, 100, 102, 1535, 1537).

Executive summary:

The objective of this study was to evaluate the potential of the test fusanus spicatus, ext. to induce reverse mutation in Salmonella typhimurium.

The study was performed according to the international guidelines (OECD 471, Commission Directive No. B13/14) and in compliance with the Principles of Good Laboratory Practice Regulations.

A preliminary toxicity test was performed to define the dose-levels of the test item to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37 °C).

Five strains of bacteria Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37 °C, the revertant colonies were scored.

 

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

The test item was dissolved in dimethylsulfoxide (DMSO). All strains were tested with appropriate positive controls, with and without metabolic activation.

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

Since the test item was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria Specified in the international guidelines.

The selected treatment-levels in experiments without S9 mix were:

9.77, 19.53, 39.06, 78.13 and 156.3µg/plate, for the TA 1535 and TA 100 strains in the first experiment, 39.06, 78.13, 156.3, 312.5 and 625µg/plate, for the TA 1537, TA 98 and TA 102 strains in the first experiment, 4.88, 9.77, 19.54, 39.06, 78.13 and 156.3µg/plate, for the TA 100 strain in the second experiment, 19.54, 39.06, 78.13, 156.3 and 312.5µg/plate, for the TA 1535, TA 1537, TA 98 and TA 102 strains in the second experiment.

A moderate to severe toxicity was induced at dose-levels≥78.13µg/plate in the TA 100 and TA 102 strains and≥156.3µg/plate in the remaining tester strains.

No significant increase in the number of revertants was noted in any of the five tester strains.

The selected treatment-levels in experiments with S9 mix were:

19.54, 39.06, 78.13, 156.3, 312.5 and 625µg/plate, for the TA 98 and TA 1537 strains in the second experiment, 39.06, 78.13, 156.3, 312.5 and 625µg/plate, for the TA 1535 strain in the first experiment as well as for the TA 100 strain in both experiments, 39.06, 78.13, 156.3, 312.5, 625 and 1250µg/plate, for the TA 1535 strain in the second experiment, 78.13, 156.3, 312.5, 625 and 1250µg/plate, for the TA 102 strain in the first experiment, 78.13, 156.3, 312.5, 625, 1250 and 2500µg/plate, for the TA 102 strain in the second experiment, 312.5, 625, 1250, 2500 and 5000µg/plate, for the TA 1537 and TA 98 strains in the first experiment.

A moderate emulsion was observed in the Petri plates when scoring the revertants at dose-levels≥2500µg/plate. Moderate to severe toxicity was induced at dose-levels≥156.3µg/plate in the TA 100 and TA 1537 strains,≥312.5µg/plate in the TA 1535 strain and≥625µg/plate in the TA 98 and TA 102 strains.

No significant increase in the number of revertants was noted in any of the five tester strains.

Thus, under the experimental conditions, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.