Benzenecarboximidamide, 4-amino-N-hydroxy-

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted as Ames II test under non GLP-conditions. The Ames II assay is a high throughput version showing a high correlation with the traditional Salmonella assay. At the date of this dossier the method still is not included in the OECD 471 guideline. The documentation is sufficient for evaluation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

The Ames II assay is a high throughput version showing a high correlation with the traditional Salmonella assay. Concurrently with positive and negative (vehicle) controls, the test article was tested over a concentration range from 1 to 5000 μg/mL medium with and without microsomal rat liver enzymes (Aroclor 1254-induced). The bacterial tester strains S. typhimurium TA Mix (TA 7001, TA 7002, TA 7003, TA 7004, TA 7005, TA 7006 sensitive to base-pair substitution) and TA 98 (susceptible to frameshift mutagens) are histidine-auxotrophic and were exposed for 90 minutes. Using the liquid fluctuation technique revertant growth was quantified colorimetrically in 384-well plates after 48 hrs at 37°C.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-amino-N-hydroxybenzene-1-carboximidamide

EC Number:

664-406-5

Cas Number:

277319-62-7

Molecular formula:

C7 H9 N3 O

IUPAC Name:

4-amino-N-hydroxybenzene-1-carboximidamide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: TA Mix (TA 7001- 7006)

Additional strain / cell type characteristics:

other: sensitive to base-pair substitution

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: susceptible to frameshift mutagens

Metabolic activation:

with and without

Metabolic activation system:

S9 rat liver

Test concentrations with justification for top dose:

1 to 5000 μg/mL

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

other: 2-aminoanthracene

Details on test system and experimental conditions:

The assay was performed according to the instruction manual for the Ames II (Xenometrix, Boulder/USA). Vehicle, test substance or positive
control in a volume of 0.01 mL were incubated with 0.24 mL bacterial overnight culture (ca 107/mL)/exposure medium in 24-well plates for 90 min at 37°C and 250 rpm. With metabolic activation 0.2 mL strain mixture and 0.04 mL S9-mix (30%) were used. After 90 min the exposed cultures
were diluted with pH indicator medium lacking histidine and aliquoted into 48 wells of a 384-well plate (3 replicates) using an 8-channel pipettor.
The plates were incubated for 48 hrs at 37°C. To confirm the sensitivity of the tester strains and the metabolic capacity of the S9 fractions, the
diagnostic mutagens 2-nitrofluorene (2-NF), 4-nitroquinoline-N-oxide (4-NQO) and 2-aminoanthracene (2-AA) were used, respectively.

Evaluation criteria:

The pH indicator bromocresol purple turns the colour of the cultures from blue to yellow as the pH drops due to the accumulation of catabolites
from the metabolic activity of revertant cells. The number of positive wells (yellow) out of a total of 48 wells is an indication of the frequency of
reversion per replicate per dose and was compared to the number of spontaneous revertant wells of the solvent control. Each test point contains
48 wells of a 384-well plate. In each 48-well section, the wells were scored for the number of revertant wells (yellow) and the mean value of the
triplicates was calculated.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA Mix (TA 7001- 7006)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

The test substance did neither precipitate nor show bacteriotoxicity up to the highest concentration.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation.

BIBR 1048 ABA did not consistently increase the number of positive wells in the different tester strains neither in presence nor absence of a
metabolic system as compared to the vehicle control. The negative control showed a mean number of positive wells for S. typhimurium TA Mix
(TA 7001-7006) and TA 98 similar to those described in the literature (≤8/48 wells).
As expected, the positive controls (2-NF, 4-NQO and 2-AA, respectively) showed a clear mutagenic response demonstrating the validity of the study.