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Genetic toxicity: in vitro

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in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 07 JUN 2016 to 17 JUN 2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21. July 1997
according to guideline
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
adopted 31. May 2008
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Trinonyl benzene-1,2,4-tricarboxylate
EC Number:
EC Name:
Trinonyl benzene-1,2,4-tricarboxylate
Cas Number:
Molecular formula:
trinonyl benzene-1,2,4-tricarboxylate
Test material form:


Species / strain
Species / strain / cell type:
other: S. typhimurium LT2 strains TA97a, TA98, TA100, TA102 and TA1535
Additional strain / cell type characteristics:
other: Genetic characteristics of each test bacteria strain such as amino acid requirement, UV sensitivity, membrane mutation and drug resistance were verified in advance and bacteria strains possessing the required characteristics were used.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced male rat liver S9 mix
Test concentrations with justification for top dose:
plate incorporation test (experiments 1 a and b): 0, 50, 150, 500, 1500, and 5000 µg/plate
pre-incubation (experiment 2): 0, 156, 313, 625, 1250, 2500, and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test item was sufficiently soluble in DMSO, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
sodium azide
other: 4- Nitro-1,2-phenylene diamine (CASRN 99-56-9) without S9 mix for strains TA97a, TA98, and TA102; 2-amino-anthracene (CASRN 613-13-8) with S9 mix for strains TA97a, TA100, TA102, and TA1535
4- Nitro-1,2-phenylene Diamine is not a standard positive control according to OECD guideline 471 but is recommended according to Ames et al. [D.M. Maron, B.N. Ames: “Revised methods...”, Mutation Research 113 (1983) 173- 215, Elsevier Biomedical Press].
Details on test system and experimental conditions:
experiment 1: plate incorporation
experiment 2: preincubation

- Preincubation period: only experimt 1 - 20 minutes
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 plates per concentration tested

- Method: other: Performed in experiment 1 only, analogously to the titre control (see below) with the maximum dose of test item with and without S9 mix on maximal-soft agar, two replicates with and without metabolic activation, incubation for 48 hours at 37 ±1°C.
- Titer control: The titre was determined by dilution of the overnight culture using sodium chloride solution and placing 0.1 mL on maximal-soft agar. Incubation for 48 hours at 37 ±1 °C followed. It should give a density of 109 cells/mL (at the least), two replicates with and without metabolic activation.
Rationale for test conditions:
The test material was sufficiently soluble in DMSO. Testing was performed to the maximal test concentration mentioned in the testing guideline and no relevant cytotxicity was observed. Based on this and in concordance with the previsions from the testing guideline at least four other (i.e. a total of at least 5 analysable) test concentrations were chosen.
Evaluation criteria:
A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.
The colonies were counted visually and the numbers were recorded. A spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium LT2 strains TA97a, TA98, TA100, TA102 and TA1535
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
Untreated negative controls validity:
not applicable
Positive controls validity:

Applicant's summary and conclusion

Trinonyl benzene-1,2,4-tricarboxylate did not exert mutagenic activity in the reverse bacterial mutation assay (AMES Test according to OECD TG471 and GLP; plate incorporation and pre-incubation method) with and without metabolic activation.
Executive summary:

Reverse mutation testing of the test material, trinonyl benzene-1,2,4 -tricarboxylate, was conducted according to OECD testing guideline 471 and GLP using 5 strains of bacteria: Salmonella typhimurium TA97a, TA98, TA100, TA102, and TA1535. The test material (dissolved in DMSO) was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix (0.74 % final concentration in the treatment) in two experiments (experiment 1: plate incorporation method; experiment 2: pre-incubation method).

In all experiments, no precipitation of the test item was observed at any of the tested concentrations up to 5000 μg/plate. In all three experiments, the test item showed no cytotoxicity towards all bacteria strains (no reduction of background lawn and number of revertants). The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value of 10EXP9 bacteria/mL. All of the means of all replicates of the spontaneous revertants (in negative and solvent controls) were within the range of the historical data of the test facility. Nearly all numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory, but all were increased in comparison with the negative controls, which demonstrated the mutagenic potential of the diagnostic mutagens. Thus indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Since all criteria for acceptability have been met, the study is considered valid.

The test item showed no increase in the number of revertants in all bacteria strains in both experiments. Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102, and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.