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Diss Factsheets

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: GLP Study, only 4 Salmonella strains used

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Principles of method if other than guideline:
Salmonella/microsome test, plate incorporation assay, as described by Ames et al., (1973a, 1975) and Maron and Ames (1983); only 4 Salmonella strains used
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4',4''-methylidynetrianiline
EC Number:
208-952-0
EC Name:
4,4',4''-methylidynetrianiline
Cas Number:
548-61-8
Molecular formula:
C19H19N3
IUPAC Name:
4,4',4''-methylidynetrianiline

Method

Target gene:
mutant histidine gene
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 mix was made from the livers of male Sprague Dawley rats, which received a single intraperitoneal injection of 500 mg/kg bw Aroclor 1254, dissolved in corn oil, 5 days prior to sacrifice. The S9 mix comprised 30 % S9 fraction.
Test concentrations with justification for top dose:
plate incorporation assay:
0, 8, 40, 200, 1000, and 5000 µg/plate with and without S9 mix (first and second trial)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
The highest dose of Delta-R-Base was applied as suspension.
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
Na-azide (only TA 1535), nitrofurantoin (only TA 100), 4-nitro-1,2-phenylene diamine (TA 1537 and TA 98), 2-aminoanthracene
Evaluation criteria:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgment.
In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.
Statistics:
no statistics performed; evaluation based on criteria mentioned above

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
On TA 100 and TA 98 a biologically relevant increase was found in the mutant count compared to the corresponding negative control. Positive response was found only with S9 mix at 40 µg/plate (TA 100) and 8 µg/plate (TA 98).
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 5000 µg/plate the substance had a weak, strain specific bacteriotoxic effect, so that dose could nevertheless be used for assessment purposes. Substance precipitation occurred at 5000 µg/plate.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Two of the five strains (TA 98 and TA 100) in the plate incorporation test with S9 mix showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls. For TA 98 this increase was even higher than that of the positive control 2-aminoanthracene. TA 98 counts for frameshift effects and TA 100 for base-pair substitution.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary and mean values with S9 mix

Table and group µg/plate

Strain

TA 1535

Strain

TA 100

Strain

TA 1535

Strain

TA 98

+ S9 (30 %)

 

plates 1-4 / 5-8

0 (Ethanol)

20 / 14

147 / 146

10 / 9

53 / 53

8

20 /15

188 /143

14 / 14

242 / 291

40

21 /16

257 / 292

23 / 20

521 / 485

200

24 /13

379 / 413

17 / 19

502 / 638

1000

18 / 17

426 / 370

26 / 24

499 / 601

5000

20 / 16

444 / 432

23 / 25

P / P

2-AA (pos. control)

204 / 84

758 / 902

81 / 81

437 / 381

P = precipitation
numbers in bold show values which led to a statistical significance in the respective quotients

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation
Executive summary:

The test substance was investigated in the Ames Test using Salmonella typhimurium strains TA 1535, TA 100, TA 1537, and TA 98 for point mutagenic effects in doses of 8 up to 5000 µg/plate. At 5000 µg/plate a weak, strain specific bacteriotoxic effect and precipitation of the test substance appeared. Clear evidence of mutagenic activity of the test substance was seen in cultures with S9-mix at doses of >= 8 µg/plate for TA 98 and at >= 40 µg/plate for TA 100. For TA 98 the dose-dependent increase reached higher mutant frequencies than the positive control 2-aminoanthacene. TA 98 and TA 100 cover different mutagenic effects, i.e. frameshift effects and base-pair substitution, respectively. Thus, the test substance should be regarded as a potent and definite mutagen under the conditions of the Salmonella/microsome assay.