N,N'-(methylenedi-p-phenylene)bis[hexahydro-2-oxo-1H-azepine-1-carboxamide]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from September 4, 2012 to November 26, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

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:

Grilbond IL-6

IUPAC Name:

Grilbond IL-6

Constituent 2

Reference substance name:

N,N'-(methylenedi-p-phenylene)bis[hexahydro-2-oxo-1H-azepine-1-carboxamide]

EC Number:

258-981-8

EC Name:

N,N'-(methylenedi-p-phenylene)bis[hexahydro-2-oxo-1H-azepine-1-carboxamide]

Cas Number:

54112-23-1

Molecular formula:

C27H32N4O4

IUPAC Name:

2-oxo-N-[4-({4-[(2-oxoazepane-1-carbonyl)amino]phenyl}methyl)phenyl]azepane-1-carboxamide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Name: Grilbond IL-6 100%
Batch/Lot No.: 9114761/024
CAS No.: 54112-23-1
Manufacturer EMS-GRITECH CH-7013 Domat/Ems
Production date: 12 July 2012
Expiry date: 11 July 2013
Purity: 99 %
Molecular formula: C27H32N4O4
Appearance: White powder
Storage condition: Room temperature
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Method

Target gene:

selected loci of several strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, TA 1537) and at the tryptophan locus of Escherichia coli WP2 uvrA strain

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

16 November 2010, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

16 November 2010, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat Liver Enzymes inducted by phenobarbital (PB) and β-naphthoflavone (BNF)

Test concentrations with justification for top dose:

Concentrations were selected on the basis of the Preliminary Solubility Test and Preliminary Range Finding Test (Informatory Toxicity Test). In the Initial Mutation Test and Confirmatory Mutation Test the same concentrations were used.
Examined concentrations in the main tests were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/plate.

Vehicle / solvent:

1. DMSO
Supplier: Sigma-Aldrich Co.
Batch No.: SZBC0040V
Expiry date: 19 December 2014
Grade: puriss, p.a., ACS reagent, ≥ 99.9% (GC)
Purity: 100 %
2. Distilled water
Supplier: TEVA Hungary Co.
Batch No.: 3450611
Expiry date: 30 June 2014

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

without S9

Positive control substance:

other: 4-nitro-1,2-phenylene diamine (NPD) MERCK

Remarks:

4 µg /plate for TA 98

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

without s9

Positive control substance:

9-aminoacridine

Remarks:

2 µg /plate for TA 100 and TA 1535

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

without s9

Remarks:

50 µg /plate for TA 1537

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

without s9

Positive control substance:

methylmethanesulfonate

Remarks:

2 µl/plate for E.coli WP2 urvA

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

with s9

Positive control substance:

other: 2-aminoanthracene (2AA) SIGMA-ALDRICH

Remarks:

2 µg /plate for all Salmonella strains and 50 µg/plate for E.coli WP2 urvA

Details on test system and experimental conditions:

The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test and a Confirmatory Mutation Test. In the Range Finding Test as well as in the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method was used.

Preliminary Concentration Range Finding Test (Informatory Toxicity Test)
Based on the solubility test, 100 mg/mL stock formulation was prepared in DMSO which was diluted in 6 steps by factors of 2, 2.5 and approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98 and TA100) were determined at the concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item.
Strain-specific positive and negative (vehicle/solvent) controls, both with and without metabolic activation were included in each test. In addition, untreated control was used demonstrating that the chosen vehicle (solvent) induced no deleterious or mutagenic effects.
Procedure for Exposure in the Initial Mutation Test
A standard plate incorporation procedure was performed, as an Initial Mutation Test. Bacteria were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.
Procedure for Exposure in the Confirmatory Mutation Test
A pre-incubation procedure was performed as a Confirmatory Mutation Test since in the Initial Mutation Test no positive effect was observed.

Evaluation criteria:

Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.

Criteria for a Negative Response:
A test article was considered non-mutagenic if it produced neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (vehicle/solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analysable concentrations were presented in all strains of the main tests.

Statistics:

The colony numbers on the untreated / negative (vehicle/solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft Excel software.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

all strains/cell types tested

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

preliminary Range finding test:
The observed numbers of revertant colonies were mostly in the normal range. Slightly lower numbers of revertant colonies (compared to the vehicle control plates) were observed in some cases. However, they had no biological significance, and the revertant counts were in the historical control range in all cases; thus, they were considered as reflecting the variability of the test system.
Precipitate / slight precipitate was observed in the preliminary experiment in both tester strains at 5000, 2500, 1000 and 316 µg/plate concentration with or without metabolic concentration.
INITIAL AND CONFIRMATORY MUTATION TESTS
In the Initial Mutation Test and Confirmatory Mutation Test none of the observed revertant colony numbers were above the respective biological threshold value. There were no reproducible dose-related trends and no indication of any treatment effect.
Higher numbers of revertant colonies compared to the vehicle control plates were detected in the Initial Mutation Test and Confirmatory Mutation Test in some cases. However, no dose-dependence was observed, and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in all cases, so they were considered as reflecting the biological variability of the test.
Lower revertant counts compared to the vehicle control were observed in the Initial Mutation Test and Confirmatory Mutation Test at some concentrations. However, they had no biological significance and were considered as reflecting the variability of the test system.
Precipitate / slight precipitate was observed in the Initial Mutation Test and Confirmatory Mutation Test in all examined bacterial strains at 5000, 1581, 500 and 158.1 µg/plate concentrations with and without metabolic activation. The precipitate did not interfere with the scoring.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative with and without metabolic activation
Based on the results given in this study, the test article caused no mutagenic activity in bacterial reverse mutation system with and without metabolic activation (Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA).

Executive summary:

This study was performed to evaluate the potential mutagenic activity of test article with bacterial reverse mutation system. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavone-induced rats. The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test and a Confirmatory Mutation Test. In the Range Finding Test as well as in the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method was used. Based on the results of the Solubility Test and available information, the test item was dissolved in DMSO. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 µg/plate were examined in the Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the main tests were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/plate.

All available data showed that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.