3,3'-[(2,5-dimethyl-p-phenylene)bis[imino(1-acetyl-2-oxoethylene)azo]]bis[4-chloro-N-(5-chloro-o-tolyl)benzamide]

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

(mostly due to limited documentation before GLP (only the arithmetic means were shown in tables; no data on test substance purity), only 4 S. typhimurium strains tested; the activation mixture was tested only with strain TA 1535).

Data source

Materials and methods

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Specific details on test material used for the study:

Batch: EN 44395

Method

Target gene:

his operon

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of liver from rats induced with Aroclor 1254

Test concentrations with justification for top dose:

15, 45, 135, 405 and 1215 µg/0.1 mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

In the experiments in which the substance was metabolically activated, activation mixture was added also. 1 mL activation mixture contains: 0.3 mL S9 fraction and 0.7 mL of a solution of co-factors.
The plates were incubated for about 48 hours at 37 °C in darkness.

NUMBER OF REPLICATIONS: triplicate

Positive controls:
1) for strain TA 98: daunorubicin-HCl (DAUNOBLASTIN), 5 and 10 µg/0.1 mL phosphate buffer;
2) for strain TA 100: 4-nitroquinoline-N-oxide, 0.125 and 0.25 µg/0.1 mL phosphate buffer;
3) for strain TA 1535: N-methyl-N'-nitro-N-nitrosoguanidine, 3 and 5 µg/0.1 mL phosphate buffer;
4) for strain TA 1537: 9(5) aminoacridine hydrochloride monohydrate, 50 and 100 µg/0.1 mL DMSO.

The activation mixture was tested with Strain TA 1535 and cyclophosphamide (ENDOXAN-ASTA), 250 µg/0.1 mL phosphate buffer.

Evaluation criteria:

A test substance is generally considered to be non-mutagenic if the colony count in relation to the negative control is not doubled at any concentration.

Statistics:

When the colonies had been counted, the arithmetic mean was calculated.

Results and discussion

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the concentrations of 45 µg/0.1 mL and above, the substance precipitated in soft agar.

Any other information on results incl. tables

Standard plate test (15 - 1215 µg/0.1 mL)
Strain	Metabolic activation system	mean his+-revertant colonies (negative control)	maximum revertant factor (conc. (µg/0.1 mL))	dose dependency	Assessment	maximum revertant factor (positive control)
TA 98	no	11	1.4 (45)	no	negative	17.5 / 34.1* (D-HCl)
	yes	29	1.1 (45 / 135)	no	negative	/
TA 100	no	127	1.2 (135)	no	negative	4.2 / 6.6** (NQNO)
	yes	140	1.1 (405)	no	negative	/
TA 1537	no	3	1.7 (135)	no	negative	12.8 / 138****(AA)
	yes	5	1.0 (135 / 405)	no	negative	/
TA 1535	no	5	2.2 (1215)	no	negative	158.8 / 173.8*** (MNNG)
	yes	6	1.8 (15)	no	negative	37.1 (CPP)
* 5 / 10 µg/0.1 mL
** 0.125 / 0.25 µg/0.1 mL
*** 3 / 5 µg/0.1 mL
**** 50 / 100 µg/0.1 mL
D-HCl = Daunorubicin
NQNO = 4-Nitroquinolin-N-oxide
MNNG = N-Methyl-N'-nitro-N-nitroso-guanidine
AA = 9 (5) Aminoacridine hydrochloride
CPP = Cyclophosphamide

A slight increase in the number of back-mutant colonies (from 5 to 8) was observed in the experiment without microsomal activation on strain TA 1535. This is attributed to a higher incidence of spontaneously occurring back-mutants.

Applicant's summary and conclusion

Conclusions:

negative