Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May - June 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The only difference to guideline was that the repeat experiment was not performed by pre-incubation method but rather as a second plate-incorporation test. Study was performed under GLP-like quality control.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1985
Report date:
1985

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 1997
Deviations:
no
Remarks:
The only difference to guideline was that the repeat experiment was not performed by pre-incubation method but rather as a second plate-incorporation test. Study was performed under GLP-like quality control.
GLP compliance:
no
Remarks:
but performed under GLP like quality assurance with QAU statement included.
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(N,N-bis(2-ethylhexyl)aminomethyl)-1,2,4-triazole
EC Number:
401-280-0
EC Name:
1-(N,N-bis(2-ethylhexyl)aminomethyl)-1,2,4-triazole
Cas Number:
91273-04-0
Molecular formula:
C19 H38 N4
IUPAC Name:
1-(N,N-bis(2-ethylhexyl)aminomethyl)-1,2,4-triazole
Details on test material:
- Analytical purity: commercial grade

Method

Target gene:
his
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
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:
0.08 - 5000 µg/plate in the toxicity test.
20, 78, 313, 1250 and 5000 µg/plate in the mutagenicity test.
Vehicle / solvent:
Acetone
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: TA 98: daunorubicin-HCl; TA 100: 4-nitroquinoline-N-oxide; TA 102: mitomycin-C; TA 1535: sodium azide; TA 1537: 9(5)-aminoacridine hydrochloride monohydrate
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: TA 98, TA 100, TA 1537, TA 102: 2-aminoanthracene; TA 1535: cyclophosphamide
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

- Exposure duration: 48 h

NUMBER OF REPLICATIONS: without and with the addition of microsomal activation mixture three Petri dishes were prepared per strain and per group (i.e. per concentration or per control group). In order to confirm the results the experiments were repeated.

DETERMINATION OF CYTOTOXICITY
- Method: other: reduction in the colony count
Evaluation criteria:
The test substance is generally considered to be nonmutagenic 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:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was carried out with the concentrations ranging from 0.08 to 5000 µg/0.1 ml. Thereafter, the concentration of 5000 µg/0.1 ml was used as the highest in the mutagenicity test.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A growth-inhibiting effect of the test item occurred in the experiments without microsomal activation at the upper concentrations (cytotoxicity started to occur at 313 or 1250 µg/plate, depending on test strain). In the experiments with microsomal activation this effect was less pronounced (cytotoxicity started to occur at 1250 and/or 5000 µg/plate, depending on test strain).

Any other information on results incl. tables

Experiment I

TA 98 TA 100 TA 102 TA 1535 TA 1537
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
solvent control 22 37 185 204 230 263 16 18 10 15
20 25 36 176 191 186 264 15 19 4 11
78 28 41 166 251 178 257 14 18 7 11
313 23 44 171 192 71 225 8 15 6 16
1250 16 26 95 145 17 90 0 13 0 0
5000 0 0 0 0 0 0 0 4 0 0
solvent control 20 45 171 170 210 275 13 19 9 10
positive control A 264 1148 665 608 887 907 470 353 684 98
positive control B 668 1215 1255 387 1597

Experiment II

TA 98 TA 100 TA 102 TA 1535 TA 1537
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
solvent control 24 47 158 171 322 298 13 17 8 20
20 31 47 143 153 298 344 12 13 11 15
78 28 40 183 179 276 326 14 12 8 11
313 20 34 158 165 215 263 9 12 10 14
1250 5 29 92 172 0 52 0 3 0 1
5000 0 0 0 0 0 0 0 0 0 0
solvent control 20 45 160 157 256 332 12 16 7 15
positive control A 442 1451 670 1489 797 2170 246 300 558 262
positive control B 649 1286 992 528 1520

Positive Controls

Without S9 mix:

TA 98: daunorubicin-HCl, A: 5 and B: 10 µg/0.1 mL phosphate buffer

TA 100: 4-nitroquinoline-N-oxide, A: 0.125 and B: 0.25 µg/0.1 mL phosphate buffer

TA 102: mitomycin-C, A: 0.5 and B: 1.0 µg/0.1 mL bidistilled water

TA 1535: sodium azide, A: 2.5 and B: 5.0 µg/0.1 mL bidistilled water

TA 1537: 9(5)- aminoacridine hydrochloride monohydrate, A: 50 and B: 100 µg/0.1 mL DMSO

With S9-Mix:

TA 98, TA 100, TA 1537: 2-aminoanthracene, 5 µg/0.1 mL DMSO

TA 102: 2-aminoanthracene, 20 µg/0.1 mL DMSO

TA 1535: cyclophosphamide, 250 µg/0.1 mL phosphate buffer

Applicant's summary and conclusion

Conclusions:
Interpretation of results: Negative with and without metabolic activation

Based on the presented results and under the conditions employed, the test article did not induce point mutations in presence or absence of a metabolic activation system and is therefore regarded as not mutagenic in the Ames test.
Executive summary:

In order to investigate the test article's potential to cause point mutation in bacteria, an AMES test similar in design to the OECD guideline No. 471 was carried out with the tester strains Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537. The test article was applied by the plate incorporation method at concentrations of 20, 78, 313, 1250 and 5000 µg/0.1 ml either with or without a metabolic activation system (rat liver S9 mix). The experiment was performed in triplicates and repeated once for confirmation. Positive controls were performed in parallel to check the tester strains sensitivity. In none of the experiments did treatment with the test substance lead to an increase in the incidence of histidine-prototrophic mutants in comparison with the controls. A growth-inhibiting effect of the test item occurred in the experiments without microsomal activation at the upper concentrations (cytotoxicity started to occur at 313 or 1250 µg/plate, depending on test strain). In the experiments with microsomal activation this effect was less pronounced (cytotoxicity started to occur at 1250 and/or 5000 µg/plate, depending on test strain). In conclusion, no evidence of the induction of point mutations by the test substance or by its metabolites formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments. Therefore, the substance is considered as not mutagenic in this assay.