N,N,N-tributylbutan-1-aminium ({[(2S,5R)-2-carbamoyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-6-yl]oxy}sulfonyl)oxidanide

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:

21 October 2005 - 09 November 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This Ames study was performed in accordance with guidelines and GLP principles, however no E.coli strain was included.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(21 July 1997)

Deviations:

yes

Remarks:

(No E.coli strain included)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guideline (including recommendations for adoption at Step 4 of the ICH Process on 19 July 1995 and 16 July 1997 by the ICH Steering Committee).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd. 09 May 2005

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

NXL 104

IUPAC Name:

NXL 104

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): NXL104
- Substance type: Organic
- Physical state: Almost white crystalline powder
- Expiration date of the batch: July 2006
- Storage condition of test material: at +4°C and protected from humidity

Method

Target gene:

Histidine gene

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 mix obtained from the liver of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Preliminary test: 10, 100, 500, 1000, 2500 and 5000 µg/plate;
Main experiment 1: 0.78, 1.56, 3.13, 6.25, 12.5, 25 µg/plate (TA100, TA 1535, with and without S9-mix); 1.56, 3.13, 6.25, 12.5, 25, 50 µg/plate (TA98, TA 1537and TA102, without S9-mix); 1.56, 3.13, 6.25, 12.5, 25, 50 µg/plate (TA98, TA 1537, with S9-mix); 3.13, 6.25, 12.5, 25, 50, 100 µg/plate (TA102, with S9-mix)
Main experiment 2: 1.56, 3.13, 6.25, 12.5, 25, 50 µg/plate (TA98, TA100, TA1535, TA1537, with S9-mix); 3.13, 6.25, 12.5, 25, 50 µg/plate (TA102, with S9-mix)

Vehicle / solvent:

- Vehicle used: water (the test item was freely soluble in water at 50 mg/mL.).

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

NAN3 at 1 µg/plate (TA 1535 and TA 100); 9AA at 50 µg/plate (TA1537); 2NF at 0.5 µg/plate (TA98); MMC at 0.5 µg/plate (TA 102).

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

Remarks:

Without S9 mix

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

at 2 µg/plate (TA1535, TA 1537, TA 98, TA 100); at 10 µg/plate (TA102)

Positive control substance:

other: 2-Anthramine (2AM)

Remarks:

With S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: direct plate incorporation method (preliminary test, both experiments without S9 mix and the first experiment with S9 mix ) and preincubation method (experiment with S9 mix).

All the concentrations and dose-levels were expressed as active item taking into account the declared content of 86.44%.

Evaluation criteria:

The study was considered valid if the following criteria were fully met:
- the number of revertants in the vehicle controls was consistent with the historical data of the testing facility,
- the number of revertants in the positive controls was higher than that of the vehicle controls and consistent with the historical data of the testing facility.

A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was seen without S9 mix at ≥ 50 μg/plate and in presence of S9-mix at ≥ 25 μg/plate (except for TA102: at ≥ 50 μg/plate).

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed at any dose level.

RANGE-FINDING/SCREENING STUDIES:
The test item was strongly toxic at dose-levels ≥100 μg/plate in the three tested strains (TA98, TA100 and TA102), with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A slight to marked toxicity (decrease in the number of revertants) was noted in the tester strains generally at 50 μg/plate in absence of S9 mix. In presence of S9-mix, a slight to marked toxicity was noted in the TA 102 strain at dose-levels ≥ 50 μg/plate and in the remaining tester strains generally at dose-levels ≥ 25 μg/plate.

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

In an Ames test performed according to OECD guideline and GLP principles (although no E.coli strain was included), NXL104 was found not to be mutagenic with or without metabolic activation when tested up to cytotoxic concentrations.

Executive summary:

An AMES test was performed with NXL104 according to OECD guideline and GLP principles, although no E.coli strain was included. The test item was shown to induce cytotoxicity at 50 μg/plate and above in absence of S9 mix. In presence of S9-mix, a slight to marked cytotoxicity was noted in the TA 102 strain at dose-levels ≥ 50 μg/plate and in the remaining tester strains generally at dose-levels ≥ 25 μg/plate. Up to cytotoxic level, all bacterial strains (TA98, TA100, TA 1535, TA 1537 and TA 102) showed negative responses, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. No precipitation of the test substance was observed at any dose level. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that NXL104 is not mutagenic in the Salmonella typhimurium reverse mutation assay with or without metabolic activation.