[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16.04.2009 - 20.05.2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1998

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1998

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Annex 4A-B10, No. L136

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: JMAFF Notification No. 12 –Nousan-8147 (Guideline No. 2-1-19-2)

Version / remarks:

November 24, 2000 and later revisions

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

2-Amino-5-cyano-N,3-dimethylbenzamide

Cas Number:

890707-29-6

Molecular formula:

C10H11N3O

IUPAC Name:

2-Amino-5-cyano-N,3-dimethylbenzamide

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain / cell type:

lymphocytes: from human donor (HPBL)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary toxicity assay: Concentrations of 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/mL were evaluated in the presence and absence of S9 activation.

Chromosome aberration assay: Concentrations of 62.5, 125, 250, 500, 750, 100 and 1250 μg/mL were evaluated in duplicate in the presence and absence of S9 activation.
The highest dose level was set based on reduction in mitotic index relative to the solvent control.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO;
- Justification for choice of solvent/vehicle: Dimethyl sulfoxide (DMSO) was used as the solvent based on the solubility of the test substance and compatibility with the target cells.

Controls

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Positive control for S9 activated studies: cyclophosphamide
Positive control for non-activated studies: mitomycin C

Details on test system and experimental conditions:

Preliminary cytotoxicity assay:
Evaluation of test substance effect on mitotic index (MI, number of dividing cells/500 cells counted). The cells were exposed to solvent alone and to concentrations of the test substance ranging from 0.5 to 5000 μg/mL for 4 hours in both the presence and absence of S9 activation and for 20 hours continuously in the absence of S9 activation.

Cytogenetic Assay:
Cell treatment: Cells were exposed to test compound, solvent or positive control for 4 h or 20 h (non-activated) or 4 h (activated).

Spindle inhibition:
Two hours prior to the scheduled cell harvest at 20 h after treatment initiation, Colcemid® was added to the cell cultures at a final concentration of 0.1 μg/mL.

Cell harvest:
Two hours after the addition of Colcemid®, metaphase cells were harvested for both the activated and non-activated studies.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human peripheral blood lymphocytes (HPBL)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Substantial toxicity was observed at dose levels  1500 μg/mL in all three treatment groups, at the dose selection test.

Any other information on results incl. tables

Table 1: Summary of chromosome aberration data

Treatment (a)	Conc. μg/mL	% of cells with structural aberrations			% of cells with polyploidy:
		S9+	S9-		S9+	S9-
		4h	4h	20 h	4h	4h	20 h
DMSO (vehicle)	0	0	0	0	0	0	0
Test substance	250	0	0	0	0	0	0
	500	0	0	0	0	0	0
	1000	0	0	0	0	0	0
MMC 0.6		ne	18.0 (b)	ne	ne	0	ne
MMC 0.3		ne	ne	16.0 (b)	ne	ne	0
CP 20		16.0 (b)	ne	ne	0	ne	ne

 

MMC:  mitomycin C in water at 0.3 or 0.6 μg/mL

CP: cyclophosphamide in water at 20 μg/mL

ne: not evaluated

a: Cells from all treatment conditions were harvested at 20 hours after the initiation of the treatments.

b: statistically significant (p≤0.01; Fisher’s exact test)

 

Applicant's summary and conclusion

Conclusions:

Based on the findings of this study, the test substance was concluded to be negative for the induction of structural and numerical chromosome aberrations in both non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using human peripheral blood lymphocytes.

Executive summary:

The clastogenic potential of the test substance based upon its ability to induce chromosome aberrations in human peripheral blood lymphocytes was evaluated according to OECD guideline 473.

The test substance was tested in the in vitro mammalian chromosome aberration test using human peripheral blood lymphocytes (HPBL) both with and without an exogenous metabolic activation system (Aroclor-induced rat liver S9). Doses in the chromosome aberration assay were 62.5, 125, 250, 500, 750, 1000, and 1250 μg/mL. The highest dose level was set based on reduction in mitotic index relative to the solvent control. Visible precipitate was observed in treatment medium at dose levels ≥ 1500 μg/mL and dose levels ≤ 500 μg/mL were soluble in treatment medium at the beginning and conclusion of the treatment period. Evaluations were necessary only at 250, 500, and 1000 μg/mL. The test substance was administered to the test system as a solution in dimethyl sulfoxide (DMSO). HPBL were treated for 4 hours (activated test system), and 4 and 20 hours (non activated test system). After exposure to Colcemid®, metaphase cells were harvested about 20 hours following the initiation of treatment. Cells were evaluated for toxicity (mitotic inhibition) then structural and numerical chromosome aberrations. The positive and solvent controls fulfilled the requirements for a valid test.
No statistically significant increases in structural chromosome aberrations were observed at any of the concentrations evaluated. In addition, no statistically significant increases in polyploidy were observed. Positive controls induced the appropriate response. Toxicity (mitotic inhibition) in excess of 50%, relative to the solvent control, was observed at concentrations ≥1000 μg/mL in the non activated and S9 activated 4 h exposure groups and at concentrations ≥1000 μg/mL in the non-activated 20 h exposure group.
Based on the findings of this study, the test substance was concluded to be  negative for the induction of structural and numerical chromosome aberrations in cultured human peripheral blood lymphocytes with and without an exogenous metabolic activation system.