Reaction products from Nerolidol by dehydration

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:

January 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

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:

(3Z,6Z)-3,7,11-trimethyldodeca-1,3,6,10-tetraene

Cas Number:

28973-99-1

Molecular formula:

C15H24

IUPAC Name:

(3Z,6Z)-3,7,11-trimethyldodeca-1,3,6,10-tetraene

Constituent 2

Reference substance name:

(E)-1-methyl-4-(6-methylhepta-2,5-dien-2-yl)cyclohex-1-ene

Cas Number:

25532-79-0

Molecular formula:

C15H24

IUPAC Name:

(E)-1-methyl-4-(6-methylhepta-2,5-dien-2-yl)cyclohex-1-ene

Constituent 3

Reference substance name:

(Z)-1-methyl-4-(6-methylhepta-2,5-dien-2-yl)

Cas Number:

29837-07-8

Molecular formula:

C15H24

IUPAC Name:

(Z)-1-methyl-4-(6-methylhepta-2,5-dien-2-yl)

Constituent 4

Reference substance name:

(E)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene

EC Number:

242-582-0

EC Name:

(E)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene

Cas Number:

18794-84-8

Molecular formula:

C15H24

IUPAC Name:

7,11-dimethyl-3-methylenedodeca-1,6,10-triene

Constituent 5

Reference substance name:

(E)-1-methyl-4-(6-methylhept-5-en-2-ylidene)

Cas Number:

53585-13-0

Molecular formula:

C15H24

IUPAC Name:

(E)-1-methyl-4-(6-methylhept-5-en-2-ylidene)

Constituent 6

Reference substance name:

(Z)-1-methyl-4-(6-methylhept-5-en-2-ylidene)

Cas Number:

13062-00-5

Molecular formula:

C15H24

IUPAC Name:

(Z)-1-methyl-4-(6-methylhept-5-en-2-ylidene)

Constituent 7

Reference substance name:

1-methyl-4-(6-methylhepta-1,5-dien-2-yl)

Cas Number:

869843-05-0

Molecular formula:

C15H24

IUPAC Name:

1-methyl-4-(6-methylhepta-1,5-dien-2-yl)

Constituent 8

Reference substance name:

(3Z,6E)-3,7,11-trimethyldodeca-1,3,6,10-tetraene

Cas Number:

26560-14-5

Molecular formula:

C15H24

IUPAC Name:

(3Z,6E)-3,7,11-trimethyldodeca-1,3,6,10-tetraene

Constituent 9

Reference substance name:

(Z)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene

Cas Number:

28973-97-9

Molecular formula:

C15H24

IUPAC Name:

(Z)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene

Constituent 10

Reference substance name:

1-methyl-4-(6-methylhept-5-en-2-yl)cyclohexa-1,4-diene

Cas Number:

72345-84-7

Molecular formula:

C15H24

IUPAC Name:

1-methyl-4-(6-methylhept-5-en-2-yl)cyclohexa-1,4-diene

Constituent 11

Reference substance name:

2,6,10-trimethyldodeca-2,6,9,11-tetraene

EC Number:

207-948-6

EC Name:

2,6,10-trimethyldodeca-2,6,9,11-tetraene

Cas Number:

502-61-4

Molecular formula:

C15H24

IUPAC Name:

3,7,11-trimethyldodeca-1,3,6,10-tetraene

Constituent 12

Reference substance name:

Likely sesquiterpene hydrocarbons

Cas Number:

n/a

Molecular formula:

C15H24

IUPAC Name:

Likely sesquiterpene hydrocarbons

Test material form:

liquid

Details on test material:

UVCB substance

Specific details on test material used for the study:

Batch number: 9000468190
Storage: at approximately 4°C

Method

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:

S9 mix
S9 preparation: Phenobarbital/beta-Naphtoflavone induced rat liver S9 is used as the metabolic activation system. The S9 is prepared from 8-12 weeks old male Wistar HanIbm rats, weight approx. 220 - 320g induced by applications of 80 mg/kg b.w. Phenobarbital i.p. and beta-Naphtoflavone p.o. each on three consecutive days. The livers are prepared 24 hours after last treatment. The S9 fractions are produced by dilution of the liver homogenate with KCl solution followed by centrifugation at 9000g. Aliquotes of the supernatant are frozen and stored in ampoules at -80°C. Small numbers of the ampoules can be kept at -20X for up to one week.
The protein concentration in the S9 preparatuib was 38.2 mg/ml.
S9 Mix: Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15% v/v in the cultures. Cofactors are added to the S9 miy to reach the following concentrations in the S9 mix:
8mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed acording to Ames et al..

Test concentrations with justification for top dose:

33; 100; 333; 1000; 2500; and 5000 ug/plate

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Details on test system and experimental conditions:

The first experiment was performed as a plate incorporation assay and the second experiment was performed as pre-incubation assay.
Experimental performance: For each strain and dose level, including the controls of three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 microL of test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
500 microL of S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation)
100 microL of bacteria suspension
2000 microL of overlay agar

In the pre-incubation assay, 100 microL test solution, 500 microL S9 mix / S9 mix substitution buffer and 100 microL bacterial suspension were mixed in a test tube and incubated at 37°C for 60 minutes. After pre-incubation 2.0 ml overlay agar (45°C) was added to each tube. The mixture was poured on minimal agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark.
The colonies were counted using AUTOCOUNT. The counter was connected to an IBM AT compatible PC with printer which printed out both, the individual and mean values of the plates for each concentration together with standard deviations and enhancement factors compared to the spontaneous reversion rates.

Rationale for test conditions:

Test concentrations based on pre-incubation assay.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA98, TA100, and TA102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independant second experiment. however, whenever the colony counts remain within the historical range of negative and solvent controls such as an increase is not considered biologically relevant.

Statistics:

A statistical analysis of the data is not required.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium 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

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

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

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

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

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

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

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

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:

No toxic effects, evident as a reduction in the number of revertants, occured in the test groups with and witout metabolic activation in experiment I. in experiment II, slight toxic effects were observed in strain TA1535 from 333 up to 5000ug/plate with S9 mix and in strain TA 1537 at 2500ug/plate without S9 mix.

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did nto induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, Bisabolene is considered to be non-mutagenic in this Salmenella typhimurium reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of Bisabolene to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA98, TA 100 an TA 102.

The assay was performed in two independant experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.

The test item was tested at the following concentrations: 33, 100, 333, 1000, 2500 and 5000 micrograms per plate.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation in experiment I. In experiment II, slight toxic effects were observed in strain TA 1535 from 333 up to 5000 micrograms per plate with S9 mix and in strain TA 1537 at 2500 micrograms per plate without S9 mix.

The plates incubated with the test item showed normal background growth up to 5000 micrograms per plate with and without S9 mix in all strains used.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Bisabolee at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did nto induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, Bisabolene is considered to be non-mutagenic in this Salmenella typhimurium reverse mutation assay.