A mixture of: 7,9,9-trimethyl-3,14-dioxa-4,13-dioxo-5,12-diazahexadecane-1,16-diylprop-2-enoate; 7,7,9-trimethyl-3,14-dioxa-4,13-dioxo-5,12-diazahexadecan-1,16-diylprop-2-enoate

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 March 04, 2004 to March 22, 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

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:

A mixture of: 7,9,9-trimethyl-3,14-dioxa-4,13-dioxo-5,12-diazahexadecane-1,16-diylprop-2-enoate; 7,7,9-trimethyl-3,14-dioxa-4,13-dioxo-5,12-diazahexadecan-1,16-diylprop-2-enoate

EC Number:

412-260-6

EC Name:

A mixture of: 7,9,9-trimethyl-3,14-dioxa-4,13-dioxo-5,12-diazahexadecane-1,16-diylprop-2-enoate; 7,7,9-trimethyl-3,14-dioxa-4,13-dioxo-5,12-diazahexadecan-1,16-diylprop-2-enoate

Cas Number:

52658-19-2

Molecular formula:

C21H34N2O8 (molecular formula of the two isomers constituting the substance)

IUPAC Name:

2-({[(2R)-2,4,4-trimethyl-6-({[2-(prop-2-enoyloxy)ethoxy]carbonyl}amino)hexyl]carbamoyl}oxy)ethyl prop-2-enoate; 2-({[(2S)-2,4,4-trimethyl-6-({[2-(prop-2-enoyloxy)ethoxy]carbonyl}amino)hexyl]carbamoyl}oxy)ethyl prop-2-enoate; 2-({[(3R)-3,5,5-trimethyl-6-({[2-(prop-2-enoyloxy)ethoxy]carbonyl}amino)hexyl]carbamoyl}oxy)ethyl prop-2-enoate; 2-({[(3S)-3,5,5-trimethyl-6-({[2-(prop-2-enoyloxy)ethoxy]carbonyl}amino)hexyl]carbamoyl}oxy)ethyl prop-2-enoate; 2-({[2,2,4-trimethyl-6-({[2-(prop-2-enoyloxy)ethoxy]carbonyl}amino)hexyl]carbamoyl}oxy)ethyl prop-2-enoate; 2-({[2,4,4-trimethyl-6-({[2-(prop-2-enoyloxy)ethoxy]carbonyl}amino)hexyl]carbamoyl}oxy)ethyl prop-2-enoate

Test material form:

liquid: viscous

Remarks:

clear yellowish liquid

Details on test material:

- Expiration date of the lot/batch: 30 November 2008
- Stability under test conditions: stable

Method

Target gene:

Histidine gene in S. typhimurium
Tryptophan gene in E. coli

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-1254 induced rat liver S9-mix

Test concentrations with justification for top dose:

Preliminary test: 3-10-33-100-333-1000-3330-5000 μg/plate
First and second test: 33, 100, 333, 1000, 3330 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

dimethyl sulfoxide

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 mix: 5 μg/plate in saline for TA1535

Controls 2

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix: 60 μg/plate in milli-Q water for TA1537

Controls 3

Positive control substance:

other: daunomycine 4 μg/plate in Saline for TA98

Remarks:

without S9 mix

Controls 4

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 mix: methylmethanesulfonate 650 μg/plate in DMSO for TA100

Controls 5

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix: 10 μg/plate in DMSO for WP2 uvr A

Controls 6

Positive control substance:

other: 2-aminoanthracene in DMSO for all tester strains

Remarks:

with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48h

NUMBER OF REPLICATIONS: doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

DETERMINATION OF CYTOTOXICITY
- Method: reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies

OTHER EXAMINATIONS:
- Other: precipitation of test substance

Evaluation criteria:

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 independently 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 independently repeated experiment.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitated in the top agar at concentrations of 1000 µg/plate and upwards. Precipitation of test substance on the plates was observed at the start and at the end of the incubation period at the concentration of 3330 µg/plate.

RANGE-FINDING/SCREENING STUDIES:
No toxicity and mutagenicity was observed up to concentrations of 3300 μg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance is not mutagenic in the S. typhimurium reverse mutation assay and in the E. coli reverse mutation assay.

Executive summary:

A study was conducted to determine the mutagenic activity of the test substance in theSalmonella typhimuriumandEscherichia colireverse mutation assay (with independent repeat) according to OECD Guideline 471 and EU Method B13/14, in compliance with GLP. The test substance was examined using four strains ofS. typhimurium(TA1535, TA1537, TA100 and TA98) and in WP2uvrA strain ofE. coli. The test was performed in two experiments in the presence and absence of S9-mix. The test substance was dissolved in dimethyl sulfoxide. Based on the results of the dose range finding study, the test substance was tested in the first mutation assay at a concentration range of 33 to 3330 µg/plate in the absence and presence of 5% v/v S9-mix in tester strains TA1535, TA1537 and TA98. In the second mutation assay, the same concentration range was tested in the absence and presence of 10% v/v S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Precipitation was observed on the plates at the top dose of 3330 µg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed. The test substance did not induce a dose-related increase in the number of revertant (His+) colonies in any of the four tester strains ofS.typhimuriumand in the number of revertant (Trp+) colonies in tester strain WP2uvrA ofE.coliboth in the absence and presence of metabolic activation. These results were confirmed in an independently repeated experiment. In this study, the negative and strain-specific control values were within the laboratory historical control values indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the study conditions, the test substance was not considered to be mutagenic in theS. typhimuriumandE. colireverse mutation assay (Verspeek-Rip, 2004).