Pentanediamide, N1,N5-dibutyl-2-[(2-ethyl-1-oxohexyl)amino]-, (2S)-

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:

27 February 2001 - 9 March 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

(2S)-N,N'-dibutyl-2-(2-ethylhexanamido)pentanediamide

EC Number:

700-664-8

Cas Number:

486455-65-6

Molecular formula:

C21H41N3O3

IUPAC Name:

(2S)-N,N'-dibutyl-2-(2-ethylhexanamido)pentanediamide

Method

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):

TA1537: his C 3076; rfa-; uvrB-: frame shift mutations
TA 98: his D 3052; rfa-; UvrB-; R-factor: frame shift mutations
TA1535: his G 46; rfa- uvrb-: base pair substitutions
TA 100: his G 46; rfa; uvrb-; R-factor: base pair substitutions

The histidine dependent strains are derived from S. typhimurium strain LT2 through a mutation in the histidine locus. Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA 98 and TA 100 the R-Factor plasmid pKM 101 carries the ampicillin resistance marker.

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):

trp-; uvrA-: base pair substitutions and others

Strain WP2 and its derivatives all carry the same defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent (Trp*) mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detescting all types of mutagen which substitute one base for another. Additionally, the uvrA derivative is deficient in the DNA repair process (excision repair damage). Such a repair-deficient strain may be more readily mutated by agents.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment I: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 156.25; 312.5; 625; 1250; 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: On the day of the experiment, the test item was dissolved in ethanol (MERCK, D-64293 Darmstadt; purity > 99 %). The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: see materials and methods for more information

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATIONS:
For each strain and dose level including controls, three plates ware used.

RANGE-FINDING/SCREENING STUDIES:
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as described in the main test.
Toxicity of the test item results in a reduction in the number at spontaneous revertants or a clearing of the bacterial background lawn.

The pre-experiment is reported as main experiment I, if the following criteria are met:
Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

Dose Selection
In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as experiment I since no relevant topic effects were observed and 5000 µg/plate were chosen as maximal concentration.

The concentration range included two logarithmic decades. The following concentrations were tested.
Experiment l: 33, 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 156.25; 312.5; 625; 1250; 2500 and 5000 µg/plate

Evaluation criteria:

The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

Evaluation of Results
- A test item is considered positive if either a dose related and reproducible increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced.

- A test item producing neither a reproducible and dose related increase in the number of revertants, nor a biologically relevant and reproducible positive response at any one of the test points is considered non-mutageoic in this system.

A biologically relevant response is described as follows:

- A test item is considered mutagenic if in the strains TA 98, TA100, and WP2uvrA the number of reversions will be at least twice as high and in the strains TA 1535 and TA 1537 at least three times higher as compared to the spontaneous reversion rate.

- Also a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test item regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

No statistical evaluation of the data is required

Results and discussion

Test results

Test results 1

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:

cytotoxicity

Remarks:

Reduction in the number of revertants, occurred in the test strain TA 1537 (without metabolic activation) at 2500 μg/plate and above and strain TA 98 with and without metabolic activation at 5000 μg/plate in experiment I.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

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

Additional information on results:

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.
Weak toxic effects, evident as a reduction in the number of revertants, occurred in the test strain TA 1537 (without metabolic activation) at 2500 µg/plate and above and strain TA 98 with and without metabolic activation at 5000 µg/plate in experiment I.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with EH at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test material is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coil reverse mutation assay.

Executive summary:

The test item was assessed for its potential to induce gene mutations according to the pre-incubation test (both experiments) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.

Weak toxic effects, evident as a reduction in the nurnber of revertants, occurred in the test strain TA 1537 (without metabolic activation) at 2500 µg/plate and above and strain TA 98 with and without metabolic activation at 5000 µg/plate in experiment I.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with EH at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.