Octanal, 7-hydroxy-3,7-dimethyl-, reaction products with 1H-indole

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

η-1H-indol-3-yl-α,α,ε-trimethyl-1H-indole-3-heptanol

EC Number:

267-434-2

EC Name:

η-1H-indol-3-yl-α,α,ε-trimethyl-1H-indole-3-heptanol

Cas Number:

67860-00-8

Molecular formula:

C26H32N2O

IUPAC Name:

8,8-di(1H-indol-3-yl)-2,6-dimethyloctan-2-ol

Constituent 2

Reference substance name:

Indole

EC Number:

204-420-7

EC Name:

Indole

Cas Number:

120-72-9

Molecular formula:

C8H7N

IUPAC Name:

1H-indole

Constituent 3

Reference substance name:

7-hydroxycitronellal

EC Number:

203-518-7

EC Name:

7-hydroxycitronellal

Cas Number:

107-75-5

Molecular formula:

C10H20O2

IUPAC Name:

7-hydroxy-3,7-dimethyloctanal

Constituent 4

Reference substance name:

1-(1H-indol-1-yl)-3,7-dimethyloctane-1,7-diol

Cas Number:

1642782-49-7

Molecular formula:

C18H27NO2

IUPAC Name:

1-(1H-indol-1-yl)-3,7-dimethyloctane-1,7-diol

Constituent 5

Reference substance name:

Unknown constituents

IUPAC Name:

Unknown constituents

Specific details on test material used for the study:

Identification: Indolene
CAS Number: 68908-82-7
Description: Green to yellow viscous liquid
Batch: 9000490905
Test substance storage: At room temperature in the dark
Stability under storage conditions: stable
Expiry date: 03 March 2003

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Selection of an adequate range of doses was based on dose range finding tests with the strains TA 100 and TA 98 both with an without S9 mix. Eight concentrations of Indolene, 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate were tested in triplicate. These dose range finding tests were reported as a part of the direct plate assay and the preincubation assay respectively. The highest concentration of Indolene used in the subsequent mutation assay was the level at which the test substance exhibited limited solubility.

Vehicle / solvent:

DMSO (Dimethyl sulfoxide)

Controls

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

cumene hydroperoxide

methylmethanesulfonate

other: Daunomycin (TA 98 without metabolic activation), 2-aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 with metabolic activation), 1,8-dihydroxyanthraquinone (TA 102 with metabolic activation)

Details on test system and experimental conditions:

At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain.
The test substance was tested both in the absence and presence of S9-mix in each strain. The first experiment was a direct plate assay and the second experiment was a preincubation assay.

Evaluation criteria:

A Salmonella typhimurium reverse mutation assay is considered acceptable if it meets the following criteria:
a) the negative control data (number of spontaneous revertants per plate) should be within the laboratory background historical range for each tester strain.
b) the positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance.
c) the selected dose range should include a clearly toxic concentration or shuld exhibit linited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

Statistics:

No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) the total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) the negative response should be reproducible in at least one repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) it induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation.
However, any mean plate count of less than 20 is considered to be not significant.
b) the positive response should be reproducible in at least one repeat experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

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 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

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Indolene induced an up to 2.0 increase in the number of revertant colonies compared to the sovent control in the presence of S9 mix.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

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 6

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed at dose levels 3330 and 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 7

Key result

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

Test results 8

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 9

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 10

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed at dose levels 3330 and 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the direct plate assay, at first Indolene was tested in a dose range finding study up to concentrations of 5000‎ μg/plate in the strains TA98 and TA100. Indolene precipitated on the plates at dose levels of 3330 and 5000 ‎μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.
Secondly, Indolene was tested up to concentrations of 333 ‎μg/plate in the strains TA1535, TA1537 and TA102. Indolene precipitated on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the preincubation assay, at first Indolene was tested in a dose range finding study up to concentrations of 5000 μg/plate in the strains TA98 and TA100. Indolene precipitated on the plates at dose levels of 3330 and 5000 μg/plate. In both tester strains, toxicity was observed at dose levels 3330 and 5000 μg/plate in the absence and presence of S9-mix.

After that, Indolene was tested up to concentrations of 3330 μg/plate n the strains TA1535, TA1537 and TA102 . Indolene precipitated on the plates at this dose level. Toxicity was observed in all tester strains.

Indolene induced an up to 2.0-fold increase in the number of revertant colonies compared to the solvent control in the teter strain TA1537 in the presence of S9-mix in the preincubation assay. However, this increase was observed only at an intermediate dose level (1000 μg/plate). Furthermore, the number of revertants was not higher than 20 and within the historical control data range. Therefore, this increase is considered to be not biologically relevant and Indolene is considered not to be mutagenic.

All other bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two separate experiments.

Remarks on result:

other:

Remarks:

Direct plate assay (based on the results of the first dose range finding test)

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that Indolene is not mutagenic in the Salmonella typhimurium reverse mutation assay.‎

Executive summary:

The negative and strain-specific positive control values were within the laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Indolene induced an up to 2.0-fold increase in the number of revertant colonies compared to the solvent control in tester strain TA 1537 in the presence of S9-mix in the preincubation assay. However, this increase was observed only at an intermediate dose level (1000 ‎μg/plate).

Furthermore, the number of revertants was not higher than 20 and within the historical control data range. Therefore, this increase is considered to be not biologically relevant and Indolene is considered to be not mutagenic.