2-(4-(diethylaminopropylcarbamoyl)phenylazo)-3-oxo-N-(2,3-dihydro-2-oxobenzimidazol-5-yl)butyramide

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:

Aug - Oct 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted 26 May 1983

Deviations:

no

Remarks:

E. coli or Salmonella th. TA102 is missing

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-(4-(diethylaminopropylcarbamoyl)phenylazo)-3-oxo-N-(2,3-dihydro-2-oxobenzimidazol-5-yl)butyramide

EC Number:

404-910-2

EC Name:

2-(4-(diethylaminopropylcarbamoyl)phenylazo)-3-oxo-N-(2,3-dihydro-2-oxobenzimidazol-5-yl)butyramide

Cas Number:

164578-14-7

Molecular formula:

C25H31N7O4

IUPAC Name:

N-[3-(diethylamino)propyl]-4-[(1E)-2-{2-oxo-1-[(2-oxo-2,3-dihydro-1H-1,3-benzodiazol-5-yl)carbamoyl]propyl}diazen-1-yl]benzamide

Test material form:

solid: bulk

Method

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:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

rat and hamster S9 mix

Test concentrations with justification for top dose:

4, 20, 100, 500, 2500 and 5000 µg/plate (with 5000 µg/plate as highest test concentration required by guideline)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

congo red

other: 2-Aminoanthracene (Ames/Prival Test), Benzidine (Prival)

Details on test system and experimental conditions:

The mutagenicity tests were conducted in the standard plate test (Ames Test) and in a modified preincubation test (Prival Test). The studies were performed in the absence and presence of a metabolising system derived from rat or hamster liver homogenate. A dose range from 8 different doses from 4 to 5000 µg/plate was used.

Two independent experiments for each test protocol (Ames; Prival; 3 plates per dose) were performed.

METHOD OF APPLICATION:
1. Ames Test:
in agar (plate incorporation)
2. Prival Test:
preincubation;
- Cell density at seeding (if applicable): 0.1 mL of an overnight broth culture of bacterial tester strain

DURATION
- Preincubation period: 30 min (Prival Test)

- Exposure duration: 48-72 h

SELECTION AGENT (mutation assays): Histidine

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: surviving fraction
- Any supplementary information relevant to cytotoxicity:
preliminary toxicity tests were performed with five or four tester strains using three plates per dose. A reduced rate of spontaeously occuring colonies as well as visible thinning of the bacterial lawn were used as indicator for toxicity.

Rationale for test conditions:

based on pretest

Evaluation criteria:

according to OECD guideline

Statistics:

none

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

incomplete bacterial at 500 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

incomplete bacterial lawn at 500 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

incomplete bacterial lawn at 500 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

incomplete bacterial lawn at 300 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

incomplete bacterial lawn

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: observed at 2500 µg/plate and above

Remarks on result:

other:

Remarks:

only tested with rat S9 mix

Applicant's summary and conclusion

Conclusions:

The test item increased the number of revertant colonies in tester strains Salmonella thyphimurium TA 98 and TA 1538 with an without metabolic activation (10% rat S9 mix): An increase in the number of revertants was also reported in Salmonella thyphimurium TA 98 with metabolic activation (30% rat S9 mix and 30% hamster S9 mix). Therefore, the test substance is considered to be mutagenic under the conditions described. However, based on the detailed results the biological relevance is unclear.

Executive summary:

The test substance was tested for mutagenicity with Salmonella thyphimurium strains TA 100, TA 1535, TA 1537, TA 1538 and TA 98. The mutagenicity studies were conducted in the standard plate (Ames Test) and in a modified preincubation test (Prival Test). The studies were performed in the absence and in the presence of a metabolising system derived from rat or hamster liver homogenate. A dose range of 8 different doses from 4 to 5000 µg/plate was used. Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All positive control compounds gave the expected increase in the number of revertant colonies.

- Toxicity:

The test compound proved to be toxic to the bacterial strains at 2500 µg/plate. 5000 µg/plate was chosen as top dose level for the mutagencity study.

- Ames Test:

In the absence of metabolic activation the test item gave a dose dependent increase in the number of revertant colonies with the bacterial strains TA 1538 and TA 98. In the presence of metabolic activation (rat S9 mix, 10%) treatment of the cells with the test substance results in relevant increases in the number of revertant colonies with TA 98 and TA 1538.

- Prival Test:

In the presence of hamster liver S9 mix the preincubation method according to Prival and in the presence of rat liver S9 mix (30%) using the standard plate test the test compound resulted in increases in the number of revertant colonies with tester strain TA 98 (TA 1538 was not tested).

However, in case of TA 98 the increase in the number of revertants reached only barlely twice the value of the negative control while the revertant number in case of the positive coontrols was approx. 30fold (in case of 10% rat S9 mix) to 5.5 (in case of hamster S9 mix) depending on utilised positive control compound increased. Furthermore, it should be considered that the tester strain TA 98 contains the R-factor, the plasmid pKM 101, which increases error-prone DNA repair and therefore, artificially increases the susceptibility to DNA damages regardless if their origin. In contrast, no changes were observed in the number revertant colonies in case of the corresponding tester strain TA 1537 not containing the plasmid. Taking into account the before mentioned, the biological relevance and additionally the relevance for humans of the observed tendency to an increasing number of revertant colonies as reported for tester strains TA 98 and 1538 is unclear.