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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

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

Target gene:

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

Species / strain / cell type:

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

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

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

Positive control substance:

9-aminoacridine

Remarks:

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

Positive control substance:

other: daunomycine 4 μg/plate in Saline for TA98

Remarks:

without S9 mix

Positive control substance:

methylmethanesulfonate

Remarks:

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

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

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

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.

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

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

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).

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity assay: micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Information from migrated NONS file, as per inquiry number 06-0000014943-66-0000, permission to refer granted by ECHA

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

no data

Route of administration:

oral: unspecified

Details on exposure:

Sesame oil

Duration of treatment / exposure:

no data

Frequency of treatment:

no data

Post exposure period:

no data

Remarks:

Doses / Concentrations:
2000 mg/l
Basis:
no data

No. of animals per sex per dose:

5

Control animals:

not specified

Positive control(s):

no data

Tissues and cell types examined:

no data

Details of tissue and slide preparation:

no data

Evaluation criteria:

no data

Statistics:

no data

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Doses producing toxicity > 2000 mg/kg b.w.

Conclusions:

Under the test conditions the test substance was not mutagenic in Mammalian Erythrocyte Micronucleus Test.

Executive summary:

A study was conducted to determine the genotoxic potential of the test substance according to OECD Guideline 474, in compliance with GLP. No detailed information about the test method employed and results obtained were provided. The test was conducted in NMRI mice at the test substance concentration of 2000 mg/L. Under the test conditions, the test substance was not considered to be genotoxic in the mammalian erythrocyte micronucleus test (Anonymous, 1993).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro:

A study was conducted to determine the mutagenic activity of the test substance in the Salmonella typhimurium and Escherichia coli reverse 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 of S.typhimurium and in the number of revertant (Trp+) colonies in tester strain WP2uvrA of E.coli both 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 the S. typhimurium and E. coli reverse mutation assay (Verspeek-Rip, 2004).

Genetic toxicity in vivo:

A study was conducted to determine the genotoxic potential of the test substance according to OECD Guideline 474, compliance with GLP. No detailed information about the test method employed and results obtained were provided. The test was conducted in NMRI mice at the test substance concentration of 2000 mg/L. Under the test conditions, the test substance was not considered to be genotoxic in the mammalian erythrocyte micronucleus test (Anonymous, 1993).

Justification for classification or non-classification

Based on the results of in vitro and in vivo studies, the test substance does not warrant classification for genotoxicity according to EU CLP (1272/2008/EC) criteria.