6,10,14-Trimethyl-5-Pentadecen-2-on

Genetic toxicity: in vitro

Administrative data

Endpoint:

genetic toxicity in vitro

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Principles of method if other than guideline:

Method: other: according to Gee P. et al. (1998) and to the "Users Manual" prepared by XENOMETRIX, Inc. Colorado, USA
Salmonella typhimurium reverse mutation assay (liquid fluctuation test - microtiter version)

PRINCIPLE
The "Ames II Assay" is a liquid version of the classical Ames test which is commonly employed as an initial screening method for detecting a point mutagenic activity of chemical substances and which has been extensively used in genetic toxicology testing to screen for possible mammalian mutagens and carcinogens. In contrast to the classical Ames test the new assay version is performed in microwell plates using a modified fluctuation test protocol; furthermore, besides using the traditional tester strain TA 98 for identifying mutagens that induce small frameshift mutations in the his operon, a new set of 6 his- mutant strains TA 7001 - TA 7006 (Ames II tester strains) was developed to revert by unique base-pair substitutions.
The principle of both assays is that mutations are detected by the reversion of mutations present in the amino acid requiring bacterial strains . This leads to a restoration of the functional capability of the bacteria to synthesize the essential amino acid and thus to the
ability to grow in the absence of the amino acid required by the parent strains. In the Ames II Assay this growth leads to an accumulation of catabolites from the metabolic activity of revertants dropping down the pH and, thus, turning the purple Ames II
Reversion Indicator medium into yellow.
Based on a validation study it was found that the Ames II Assay gave results comparable to those found in the NTP database with the classical Ames test. In an additional validation study with about 130 chemicals using the automated high throughput configuration
the high concordance of the two Ames test versions was also confirmed. This high concordance with the traditional Salmonella test demonstrates that the Ames II test procedure using the Ames II tester strains together with the traditional strain TA 98, is an effective screen for identifying Salmonella mutagens.
Most of the substances are not mutagenic or carcinogenic themselves, but only after metabolic transformation, which in vivo is catalyzed mainly by the enzyme systems of the liver. Therefore, the tests are carried out not only directly, but also in the presence of an
exogenous metabolic activation system. The most commonly used system is a cofactorsupplemented postmitochondrial fraction (S-9) obtained from livers of rats treated with an enzyme-inducing agent, e .g . Aroclor 1254.

GLP compliance:

yes

Remarks:

however, QAU did not inspect study but lab involved was subject to regular GLP inspections while study was being carried out

Type of assay:

other: Ames II (liquid fluctuation test)

Test material

Test material information

Constituent 1

Reference substance name:

6,10,14-Trimethyl-5-Pentadecen-2-on

EC Number:

609-306-4

Cas Number:

3689-69-8

Molecular formula:

C18H34O

IUPAC Name:

6,10,14-Trimethyl-5-Pentadecen-2-on

Details on test material:

Tetrahydrofarnesylaceton (THFAC)
- Test substance number: 01/0753-1
- Batch-No. 01rg11
- purity 99.48%
- date of manufacture: December 2001
- appearance, consistency: colorless liquid
- storage: refrigerator
- stability throughout the study period has not been verified by reanalysis

Specific details on test material used for the study:

purity: ca. 95%
batch no.: 01rg11

Method

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium: TA 98, TA mix containing the strains TA 7001 - TA 7006

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9-mix (Sprague-Dawley)

Test concentrations with justification for top dose:

0, 4, 20, 100, 500, 2500 and 5000 µg/ml

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA): 5.0 µg/ml

Remarks:

with S9-mix

Controls 2

Positive control substance:

2-nitrofluorene

Remarks:

without S9-mix Migrated to IUCLID6: 0.25 µg/ml

Controls 3

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9-mix Migrated to IUCLID6: 0.0625 µg/ml

Details on test system and experimental conditions:

- no precipitation of the test substance was found
- triplicate plates per dose

Strain Mutation Type Target Cell wall Repair pKM 101
TA 98 his D 3052 FS GC rfa uvrB +
TA Mix
TA 7001 his G 1775 BPS T:A → C:G rfa uvrB +
TA 7002 his C 9138 BPS T:A → A:T rfa uvrB +
TA 7003 his C 9074 BPS T:A → G:C rfa uvrB +
TA 7004 his G 9133 BPS C:G → T:A rfa uvrB +
TA 7005 his G 9130 BPS C:G → A:T rfa uvrB +
TA 7006 his G 9070 BPS C:G → G:C rfa uvrB +

FS = frameshift mutation
BPS = base-pair substitution

In addition to the his mutation, all strains carry different auxiliary features that enhance the mutability of the target his mutation . These include a deletion of the uvrB componen of excision repair leading to mutants which are deficient in excision repair of bulky lesions as measured by their lack of survival following UV254 irradiation . Furthermore, all strains carry a mutation (rfa) which affect the lipopolysaccharin (LPS) component of the cell envelope and, thus, have an increased permeability for bulky molecules .
Finally, all strains carry an R factor plasmid pKM 101 and, in addition to having genes resistant to antibiotics, they have a SOS-inducible mucAB system .

Evaluation criteria:

The test chemical is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of positive wells by a factor of about 2 (calculated primarily on the basis of baseline data) in at least one tester strain either without S-9 mix or after adding a metabolizing system
A test substance is generally considered non-mutagenic in this test if:
• The number of revertant wells for all tester strains were within the historical negative control range under all experimental conditions

Results and discussion

Test results

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: No bacteriotoxic effect observed

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

There were no increased numbers of his-revertants observed in any strain, neither with nor without metabolic activation.

exact result table in "executive summary" due to problems with inserting tables

into fields that have been used by IUC4 -> IUC5 migrated data

According to the results of the present  study, the test substance 
Tetrahydrofarnesylaceton (THFAC) is not mutagenic in  the Ames II Assay 
(Salmonella typhimurium reverse mutation assay) under the  experimental 
conditions chosen here.


    
    
    
    
    

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Executive summary:

TA 98

without S9-mix

dose [µg/ml]	plate 1	plate 2	plate 3	mean	SD
DMSO	5	2	2	3.0	1.41
4	1	4	2	2.3	1.25
20	1	4	2	2.3	1.25
100	4	3	1	2.7	1.25
500	3	2	4	3.0	0.82
2500	1	4	0	1.7	1.70
5000	1	1	0	0.7	0.47
4-NQO + 2-NF	20	21	20	20.3	0.47

with S9-mix

dose [µg/ml]	plate 1	plate 2	plate 3	mean	SD
DMSO	4	3	6	4.3	1.25
4	5	6	3	4.7	1.25
20	4	4	3	3.7	0.47
100	6	4	4	4.7	0.94
500	4	3	4	3.7	0.47
2500	5	4	2	3.7	1.25
5000	3	3	5	3.7	0.94
2-AA	48	48	48	48.0	0.00

TA-mix

without S9-mix

dose [µg/ml]	plate 1	plate 2	plate 3	mean	SD
DMSO	1	2	5	2.7	1.70
4	1	1	0	0.7	0.47
20	0	0	1	0.3	0.47
100	1	0	0	0.3	0.47
500	0	0	1	0.3	0.47
2500	2	1	1	1.3	0.47
5000	2	0	0	0.7	0.94
4-NQO + 2-NF	24	21	27	24.0	2.45

with S9-mix

dose [µg/ml]	plate 1	plate 2	plate 3	mean	SD
DMSO	2	0	1	1.0	0.82
4	1	1	0	0.7	0.47
20	0	1	1	0.7	0.47
100	0	0	0	0.0	0.00
500	1	0	1	0.7	0.47
2500	0	2	0	0.7	0.94
5000	0	2	1	1.0	0.82
2-AA	24	29	28	27.0	2.16

SD = standard deviation

4-NQO + 2-NF = 4-nitroquinoline + 2-nitrofluorene

2-AA = 2-aminoanthracene