5,6,7-TRIMETHYLOCTA-2,5-DIEN-4-ONE

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:

From 14 November 2003 to 27 January 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

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:

(2E,5Z)-5,6,7-trimethylocta-2,5-dien-4-one

Cas Number:

357650-26-1

Molecular formula:

C11H18O

IUPAC Name:

(2E,5Z)-5,6,7-trimethylocta-2,5-dien-4-one

Constituent 2

Reference substance name:

(2E,5E)-5,6,7-trimethylocta-2,5-dien-4-one

Cas Number:

847144-75-6

Molecular formula:

C11H18O

IUPAC Name:

(2E,5E)-5,6,7-trimethylocta-2,5-dien-4-one

Test material form:

liquid

Specific details on test material used for the study:

Batch No. 9000530064
Aspect: Slightly yellow liquid
Purity: 96.7%
Expiry date: 12-Sept-2004

Method

Target gene:

Histidine locus

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : prepared from 8 - 12 weeks old male Wistar Hanlbm rats, weight approx. 220 - 320 g induced by applications of 80 mg/kg b.w. Phenobarbital i.p. (Desitin; D-22335 Hamburg) and P-Naphthoflavone p.o. (Aldrich, D-89555 Steinheim) each on three consecutive days. The livers are prepared 24 hours after the last treatment.

- method of preparation of 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 mix to reach the following concentrations in the S9 mix:

8 mM MgCl2
33 mM KCI
5 mM Glucose-6-phosphate
5 mM NADP

in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

- concentration or volume of S9 mix and S9 in the final culture medium : 500 µL S9 mix (for test with metabolic activation)

Test concentrations with justification for top dose:

In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as experiment I since relevant toxic effects were observed the concentrations in experiment II were reduced.

The concentration range included two logarithmic decades. The following concentrations were tested:

Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II
strain TA 1535: 0.1; 0.3; 1; 3; 1O; 33; 100; 333; 1000; and 2500 µg/plate

strains TA 98, TA 102 without S9 mix: 1; 3; 10; 33; 100; 250; 333; and 500 µg/plate
with S9 mix:1; 3; 10; 33; 100; 333; 500 and 1000 µg/plate

strains TA 1537, TA 100:
1; 3; 10; 33; 100; 333; 1000; and 2500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol 99%

- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine for TA 1537 and TA 98 without S9; 2-aminoanthracene for all strains with S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS: Triplicate


METHOD OF APPLICATION: In agar (plate incorporation method)

The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 µLTest solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
- 500 µLS9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
- 100 µL Bacteria suspension (cf. test system, pre-culture of the strains),
- 2000 µL Overlay agar

In the pre-incubation assay 100 µL test solution, 500 µL S9 mix/ S9 mix substitution buffer and 100 µL 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.

DURATION
- Incubation period: ca. 48 hours at 37 °C

Rationale for test conditions:

In accordance with the relevant guidelines.

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 TA 98, TA 100, and TA 102) 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 independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such 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

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

True 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:

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with GR-85-2517 at any concentration 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.

Any other information on results incl. tables

The plates incubated with the test item showed irregular background growth at the following concentrations (µg / plate)

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	2500 - 5000	2500 - 5000	1000 - 2500	2500
TA 1537	2500 - 5000	2500- 5000	1000 - 2500	1000 - 2500
TA98	2500- 5000	1000- 5000	I	500 - 1000
TA 100	2500 - 5000	1000- 5000	333- 2500	1000 - 2500
TA 102	2500 - 5000	2500 - 5000	333-500	500 - 1000

I= no reduced back ground growth observed

 Toxic effects, evident as a reduction in the number of revertants were observed at the following concentrations ( µg/plate):

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	5000	2500 -5000	1000 - 2500	2500
TA 1537	2500 - 5000	2500- 5000	1000 - 2500	2500
TA98	5000	2500- 5000	500	500 - 1000
TA 100	2500 - 5000	1000 - 5000	333- 2500	1000 - 2500
TA 102	333- 5000	1000 - 5000	333- 500	500 - 1000

Applicant's summary and conclusion

Conclusions:

It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

This study was performed to investigate the potential of GR-85-2517 to induce gene muta­ tions according to the plate incorporation test (experiment I) and the pre-incubation test (experiment 11) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.

 

The assay was performed in four independent experiments all with and without liver microsomal activation. Due to strong toxicity in the second experiment performed as pre­ incubation assay (data not shown) this test had to be repeated as Experiment III (reported as exp. 11). Since in the third experiment in strain TA 1535 no toxic effects were observed, the pre-incubation assay was repeated again with strain TA 1535 with higher concentrations (data reported as exp. II). Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

 

Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II

strain TA 1535: 0.1; 0.3; 1; 3; 10; 33; 100; 333; 1000; and 2500 µg/plate

 

strains TA 98, TA 102

without S9 mix: 1; 3; 10; 33; 100; 250; 333; and 500 µg/plate

with S9 mix: 1; 3; 10; 33; 100; 333; 500; and 1000 µg/plate

strains TA 1537, TA 100: 1; 3; 10; 33; 100; 333; 1000; and 2500 µg/plate

 

At higher concentrations, irregular background growth was observed with and without metabolic activation in all strains used.

 

Strong toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation at higherconcentrations.

 

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with GR-85-2517 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 in­ crease 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 not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

 Therefore, GR-85-2517 is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.