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Toxicological information

Genetic toxicity: in vitro

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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

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1980
Report date:
1980

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3,3'-[(2,5-dimethyl-p-phenylene)bis[imino(1-acetyl-2-oxoethylene)azo]]bis[4-chloro-N-(5-chloro-o-tolyl)benzamide]
EC Number:
226-107-4
EC Name:
3,3'-[(2,5-dimethyl-p-phenylene)bis[imino(1-acetyl-2-oxoethylene)azo]]bis[4-chloro-N-(5-chloro-o-tolyl)benzamide]
Cas Number:
5280-80-8
Molecular formula:
C44H38Cl4N8O6
IUPAC Name:
3,3'-{(2,5-dimethyl-1,4-phenylene)bis[imino(1,3-dioxobutane-2,1-diyl)diazene-2,1-diyl]}bis[4-chloro-N-(5-chloro-2-methylphenyl)benzamide]
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Batch: EN 44395

Method

Target gene:
his operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see Details on test system and conditions
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

Test results
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
(tested up to precipitating concentrations)
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
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