Reaction mass of 2-[[[2-[(2-aminoethyl)amino]ethyl]amino]carbonyl]-benzoic acid and 2,2' -[iminobis(2,1-ethanediyliminocarbonyl)]bis-benzoic acid and 7,8,9,10,11,12,20,21,22,23,24,25-dodecahydrodibenzo[i,t] [1,4,7,12,15,18] hexaazacyclodocosine-5,13,18,26(6H,19H)-tetrone

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:

1977

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No GLP study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1977

Report date:

1977

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Saccharomyces cerevisiae used instead of E. coli

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-({2-[(2-aminoethyl)amino]ethyl}carbamoyl)benzoic acid; 2-{[2-({2-[(2-carboxyphenyl)formamido]ethyl}amino)ethyl]carbamoyl}benzoic acid; 3,6,9,18,21,24-hexaazatricyclo[24.4.0.0¹¹,¹⁶]triaconta-1(30),11,13,15,26,28-hexaene-2,10,17,25-tetrone

EC Number:

942-764-6

IUPAC Name:

2-({2-[(2-aminoethyl)amino]ethyl}carbamoyl)benzoic acid; 2-{[2-({2-[(2-carboxyphenyl)formamido]ethyl}amino)ethyl]carbamoyl}benzoic acid; 3,6,9,18,21,24-hexaazatricyclo[24.4.0.0¹¹,¹⁶]triaconta-1(30),11,13,15,26,28-hexaene-2,10,17,25-tetrone

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Description: Light green powder
- Date received: August 8, 1977

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):

N/A

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

N/A

Additional strain / cell type characteristics:

not applicable

Species / strain 3

Species / strain / cell type:

Saccharomyces cerevisiae

Details on mammalian cell type (if applicable):

N/A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Sprague-Dawley adult male rat liver induced by Aroclor1254

Test concentrations with justification for top dose:

With and without metabolic activation: 0.1/1.0/10/100/500 microgramme per plate.

Vehicle / solvent:

Either deionized water or dimethylsulfoxide (DMSO) was used to prepare stock solutions of solid materials. All dilutions of test materials were made in either deionized water or DMSO.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

other: Methylnitrosoguanidine (MNNG), Quinacrine (QM)

Remarks:

without metabolic activation.

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

other: 2-Anthramine (ANTH), 8-Aminoquilone (AMQ)

Remarks:

with metabolic activation.

Details on test system and experimental conditions:

Plate test: Overlay Method
Approximately 10E8 cells from an overnight culture of each indicator strain were added to separate test tubes containing 2.0 ml of molten agar supplemented with biotin and a trace of histidine. For non-activation test, at least four dose levels of the test compound were added to the contents of the appropraita tubes and poured over the surface of selective agar plates. in activation tests, a minimum of four different concentrations of the test chemical were added to the appropriate tubes with cells. Just prior to pouring, an aliquot of reaction mixture (0.5 ml containing the 9 000 xg liver hamogenate) was added to each of the activation overlay tubes, which where then mixed, and the contents poured over the surface of a minimal agar plate and allowed to solidify. The plates were incubated for 48 hours at 37°C, and scored for the number of colonie growing on each plate.
Positive and solvent controls using both directly active positives chemicals and those that require metabolic activation were run with each assay.

Evaluation criteria:

*Strains TA-1535, TA-1537, and TA-1538
If the solvent control value is within the normal range, a chemical that produces a positive dose response over three concentrations with the lowest increase equal to twice the solvent control value is considered to be mutagenic.
*Strains TA-98, TA-10Q, and D4
If the solvent control value is within the normal range, a chemical that produces a positive dose response over three concentrations with the highest increase equal to twice the solvent control value for TA-100 and two to three times the solvent control value for strains TA-98 and D4 is considered to be mutagenic.
For these strains, the dose response increase should start at approximately the solvent control value.
*Pattern
Because TA-1535 and TA-100 were both derived from the same parental strain (G-46) and because TA-1538 and TA-98 were both derived from the same parental strain (D3052), there is a built-in redundancy in the microbial assay. In general the two strains of a set respond to the same mutagen and such a pattern is sought. It is also anticipated that if a given strain, e.g. TA-1537, responds to a mutagen in nonactivation tests it will generally do so in activation . tests. (The converse of this relationship is not expected.) While similar response patterns are not required for all mutagens, they can be used to enhance the reliability of
an evaluation decision.
*Reproducibility
If a chemical produces a response in a single test that cannot be reproduced in one or more additional runs, the initial positive test data loses significance.
The preceding criteria are not absolute and other extenuating factors may enter into a final evaluation decision. However, these criteria are applied to the majority of situations and are presented to aid those individuals not familiar with this procedure. As the data base is increased, the criteria for evaluation can be more firmly established.

Statistics:

The numbers of colonies on each plate were counted and recorded on printed forms. these raw data were analyzed in a computer program and reported on a printout. the results are presented as revertants per plate for each indicator starin employed in the assay. The positive and teh sovent controls are provided as reference points. Other relevant data are provided on the computer printout.

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: N/A

Any other information on results incl. tables

The test material was examined for mutagenic activity in a series of in vitro microbial assays employing Salmonella and Saccharomyces indicator organisms. The compound was tested directly and in the presence of liver microsomal enzyme preparations from Aroclor-induced rats. The following results were obtained:

Toxicity test results:

The compound was tested over a series of concentrations such that there was either quantitative or qualitative evidence of some chemically-induced physiological effects at the high dose level. The low dose in all ceses was below a cocentration that demonstrated any toxic affect. The dose range employed for the evaluation of this compound was from 0.1 to 500 microgramme per plate.

Nonactivation test results:

The results of the tests conducted on the test material in the absence of the metabolic system were all negatice.

Activation test results:

The results of the tests conducted on the compound in the presence of the rat liver activation system were all negative.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test material did not demonstrate mutagenic activity in any of teh assay conducted in this evaluation and was considered not mutagenic under these test conditions.

Executive summary:

The objective of this study was to evaluate the test compound for genetic activity in microbial assays with and without the addition of mammalian metabolic activation preparations.

Salmonella typhimurium, strains: TA-1535, TA-98, TA-1537, TA-100, TA-1538 and Saccharomyces cerevisiae, strain: D4 were used to performed the test.

The metabolic activation system was S9 Homogenate:

A 9,000 x g supernatant was prepared from Sprague-Dawley adult male rat liver induced by Arocl.or 1254 five days prior to kill.

The test material was tested at different concentrations with and without metabolic activation: 0.1/1.0/10/100/500 microgramme per plate.

Positive control and solvent (DMSO or water) were used a s negative control.

Toxicity test results:

The compound was tested over a series of concentrations such that there was either quantitative or qualitative evidence of some chemically-induced physiological effects at the high dose level. The low dose in all ceses was below a cocentration that demonstrated any toxic affect. The dose range employed for the evaluation of this compound was from 0.1 to 500 microgramme per plate.

Nonactivation test results:

The results of the tests conducted on the test material in the absence of the metabolic system were all negatice.

Activation test results:

The results of the tests conducted on the compound in the presence of the rat liver activation system were all negative.

The test material did not demonstrate mutagenic activity in any of the assay conducted in this evaluation and was considered not mutagenic under these test conditions.