Reaction mass of (E)-3-methylcyclotetradec-5-en-1-one and (Z)-3-methylcyclotetradec-5-en-1-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:

January 14 to February 03 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

conducted under GLP conditions

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)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(E)-3-methylcyclotetradec-5-en-1-one

Cas Number:

259854-70-1

Molecular formula:

C15H26O

IUPAC Name:

(E)-3-methylcyclotetradec-5-en-1-one

Constituent 2

Reference substance name:

(Z)-3-methylcyclotetradec-5-en-1-one

Cas Number:

259854-71-2

Molecular formula:

C15H26O

IUPAC Name:

(Z)-3-methylcyclotetradec-5-en-1-one

Test material form:

liquid

Specific details on test material used for the study:

Identity: KARMALONE
Batch No.: TQT0300497
Aggregate State at Room Temperature: liquid
Colour: colourless to pale yellow
Purity: 90.9 %
Stability in Solvent: not indicated by the sponsor
Storage: room temperature
Expiration Date: November 28, 2004

Method

Target gene:

histidine

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:

rat liver S9
Phenobarbital/ -Naphthoflavone induced rat liver S9 is used as the metabolic activation system. The S9 is 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 -Naphthoflavone p.o. (Aldrich, D-89555 Steinheim) each on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCI solution (1+3) followed by centrifugation at 9000 g. Aliquots of the supernatant are frozen and stored in ampoules at -80° C. Small numbers of the ampoules can be kept at -20°C for up to one week.

The protein concentration in the S9 preparation was 27.2 mg/ml (lot no. R 071103).

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% vlv 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.

During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al. .

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. Based on the toxic effects observed in strain TA 1537 at 2500 and 5000 µg/plate the same concentration range was chosen for the main experiments.

The concentration range included two logarithmic decades. The following concentrations were tested:
3; 1O; 33, 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

DMSO (purity > 99 %, MERCK, D-64293 Darmstadt)
The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
The test item precipitated in the overlay agar at 1000 µg/plate and above in experiment I, and at 333 µg/plate and above in experiment 11.
The undissolved particles of the test item had no influence on the data recording.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 4-NOPD and 2-aminoanthracene, 2-AA

Details on test system and experimental conditions:

Pre-experiment:
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre­ experiment were the same as described for the experiment I below (plate incorporation test).

Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

The pre-experiment is reported as main experiment I, since the following criteria are met: Evaluable plates (>O colonies) at five concentrations or more in all strains used.

Experimental performance:
For each strain and dose level, including the controls three plates were used.

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 µL S9 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.

After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.

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

Key result

Species / strain:

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

Reduced background growth was observed in the presence of metabolic activation from 1000 to 5000 µg/plate in experiment I and from 333 to 5000 µg/plate in experiment II.

Toxic effects; evident as a reduction in the number of revertants were observed at the following concentrations (µg/plate) (see table 1)
No toxic effects observed

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with KARMALONE 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.

The historical range of positive controls was exceeded in strains TA 1535 and TA 100 without metabolic activation in experiments I and II. This effect indicates the sensitivity of the strains rather than compromising the assay.

In experiment II, the data in the negative of strain TA 98 were slightly above our historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.

Appropriate reference mutagens were used as positive controls. They showed a distinct in­ crease in 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.

Any other information on results incl. tables

Table 1:

Strain	Experiment I		Experiment11
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	I	1000, 5000	I	333-5000     I
TA 1537	I	2500, 5000	I	2500,5000
TA98	I	I	I	I
TA 100	I	I	I	I
TA 102	I	I	I	5000

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 not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, KARMALONE is considered to be non-mutagenic in this Salmonella typhimu­ rium reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of KARMALONE 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, TA 98, TA 100, and TA102.

 

The assay was performed in two independent 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: 3; 1O;33, 100; 333; 1000; 2500; and 5000 µg/plate

 

Reduced background growth was observed in the presence of metabolic activation from 1000 to 5000 µg/plate in experiment I and from 333 to 5000 µg/plate in experiment11.

 

In the presence of metabolic activation, toxic effects, evident as a reduction in the number of revertants, were observed in strains TA 1535 and TA 1537 in experiment I and II, and in strainTA102inexperiment11.

 

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with KARMALONE 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, KARMALONE is considered to be non-mutagenic in this Salmonella typhimu­ rium reverse mutationassay.