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Genetic toxicity: in vitro

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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
Study period:
25 Aug 2000 - 14 Nov 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD Guideline 471 and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Certificate of analysis included in report.
Name: Base 136
description: Sel thioclycolate dímidazoline
CASno:: 68991-84-4
Batch: Lot AB771
mw = 581 (pure salt)
Aspect: brown solid (@25ºC)
pH: 9,25 (5% inwater)
acid number: 122 mg KOH/g

Method

Target gene:
- S. typhimurium: Histidine gene
Species / strain
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9-fraction from Moltox (Molecular Toxicology, INC, Annapolis, MD 21401,USA); liver of rats treated with Aroclor 1254 (500 mg/kg) i.p.
Test concentrations with justification for top dose:
Without S9-mix:
- 12.5,25,50, 100 and 200 µg/plate: for all tester strains in the first experiment,
- 6.25, 12.5,25,50 and 100 µg/plate: for all tester strains in the second experiment,
- 1.5625, 3.125, 6.25, 12.5 and 25 µg/plate: for all tester strains in the third experiment.
With S9-mix:
- 12.5, 25, 50, 100 and 200 µg/plate: for all tester strains in both experiments, except for the TA 1537 strain in the second experiment,
- 6.25, 12.5,25,50 and 100 µg/plate: for the TA 1537 strain in the second experiment.
Vehicle / solvent:
DMSO
Controlsopen allclose all
Positive controls:
yes
Positive control substance:
other: Sodium-azide 1 µg/plate (TA100, TA1535); 2-Nitrofluorene 0.5µg/plate (TA98); 9-Aminoacridine 50 µg/plate (TA1537); Mitomycin C 0.5µg/plate (TA102).
Remarks:
Without metabolic activation
Positive controls:
yes
Positive control substance:
other: 2-Anthramine: 2 µg/plate (TA1535, TA1537, TA98, TA100), 10 µg/plate (TA102)
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation: (preliminary toxicity test, all experiments without 89 mix, first experiment with 89 mix); preincubation ((second experiment with 89 mix)

DURATION
- Exposure duration: 48 to 72 hours at 37 °C in the dark

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted on each strain with and without S9 mix, and a third experiment on TA98, TA100 and TA102 strains, without S9 mix.

DETERMINATION OF CYTOTOXICITY
Six dose-levels (one plate/dose-level) were tested in the TA 98, TAl00 and TA 102 strains, with and without 89 mix.
Based on decrease in the number ofrevertant colonies and/or a thinning ofthe bacterial lawn.

OTHER EXAMINATIONS:
In each experiment, the following controls were included using triplicate plates:
. vehicle controls: each bacterial tester strain treated with the vehicle,
. positive controls: each bacterial tester strain treated with appropriate reference mutagens.
The sterility of the 89 mix was checked before the beginning and at the end of each experiment and was found to be satisfactory.
Evaluation criteria:
A reproducible two-fold increase in the number ofrevertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation ofthe data obtained.
Statistics:
Not indicated.

Results and discussion

Test results
Species / strain:
other: All Salmonella strains
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
With S9 ≥ 500µg/plate; without S9 ≥ 100µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

Since the test substance was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.

Experiments without S9 mix:
- 12.5,25,50, 100 and 200 µg/plate: for all tester strains in the first experiment,
- 6.25, 12.5,25,50 and 100 µg/plate: for all tester strains in the second experiment,
- 1.5625, 3.125, 6.25, 12.5 and 25 µg/plate: for all tester strains in the third experiment.

In the first experiment, a slight to marked toxicity was noted in the tester strains generally at dose-levels ≥ 100 µg/plate.
In the second experiment, a slight toxicity was induced in the TA 1535 and TA 1537 strains at 100 µg/plate. In the three remaining tester strains, the toxicity induced was more important than the first experiment: a slight to strong toxicity, depending on the dose-levels and the tester strains. Due to the variability in toxicity and in order to avoid too toxic dose-levels, a third experiment was performed with these three tester strains at lower dose-levels.
In this third experiment, except for a slight thinning of the bacterial lawn noted in the TA 100 strain at dose-levels ≥ 12.5 µg/plate, no toxicity was observed.

In the three experiments, no noteworthy increase in the number of revertants was observed in all the tested strains.

Experiments with S9 mix:
- 12.5, 25, 50, 100 and 200 µg/plate: for all tester strains in both experiments, except for the TA 1537 strain in the second experiment,
- 6.25, 12.5,25,50 and 100 µg/plate: for the TA 1537 strain in the second experiment.

In the first experiment, except for a moderate toxicity noted in the TA 1537 and TA 98 strains at 200 µg/plate, no noteworthy toxicity was induced.
In the second experiment (preincubation method), a slight to moderate toxicity was noted in all tester strains, generally at dose-levels ≥ 100 µg/plate.

In both experiments, no noteworthy increase in the number of revertants was observed in all the tested strains.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The negative and strain-specific positive control values indicate that the test conditions were adequate and that the metabolic activation system functioned properly. Under the experimental conditions of this study, the test substance does not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.
Executive summary:

The objective of this study was to evaluate the potential of the test substance BASE 136 (batch No. AB771) to induce reverse mutation inSalmonella typhimurium.

 

Methods:

A preliminary toxicity test was performed to define the dose-levels of BASE 136 to be used for the mutagenicity study. The test substance was then tested in two independent experiments with and three independent experiments without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

 

All experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C).

 

Five strains of bacteriaSalmonella typhimurium:TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test substance (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

 

The test substance BASE 136 was dissolved in dimethylsulfoxide (DMSO).

 

The dose-levels of the positive controls were as follows:

Without S9 mix:

- 1 µg/plate of sodium azide (NaN3): TA 1535 and TA 100 strains,

- 50 µg/plate of 9-Aminoacridine (9AA): TA 1537 strain,

- 0.5 µg/plate of 2-Nitrofluorene (2NF): TA 98 strain,

- 0.5 µg/plate of Mitomycin C (MMC): TA 102 strain.

 

With S9mix:

- 2 µg/plate of2-Anthramine (2AM): TA 1535, TA 1537, TA 98 and TA 100 strains,

- 10 µg/plate of2-Anthramine (2AM): TA 102 strain.

 

Results

Since the test substance was toxic in the preliminary test, the choice of the highest dose-level for the main test was based on the level of toxicity, according to the criteria specified in the international guidelines.

 

Experiments without S9 mix:

- 12.5,25,50, 100 and 200 µg/plate: for all tester strains in the first experiment,

- 6.25, 12.5,25,50 and 100 µg/plate: for all tester strains in the second experiment,

- 1.5625, 3.125, 6.25, 12.5 and 25 µg/plate: for all tester strains in the third experiment.

 

A slight to marked toxicity was induced in the tester strains, depending on the dose-levels.

 

In the three experiments, no noteworthy increase in the number of revertants was observed in all the tested strains.

 

Experiments with S9 mix:

- 12.5, 25, 50, 100 and 200 µg/plate: for all tester strains in both experiments, except for the TA 1537 strain in the second experiment,

- 6.25, 12.5,25,50 and 100 µg/plate: for the TA 1537 strain in the second experiment.

 

A slight to moderate toxicity was induced, depending on the tester strain, the dose-levels and the experimental conditions.

 

In both experiments, no noteworthy increase in the number of revertants was observed in all the tested strains.

 

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

 

Conclusion

Under our experimental conditions, the test substance BASE 136 (batch No. AB771) does not show mutagenic activity in the bacterial reverse mutation test withSalmonella typhimurium.

These results on Fatty acids C16-18, C18 unsat reaction products with tetraethylenepentamine are fully valid for Tall oil, reaction products with tetraethylene-pentamine (Amidoamine/Imidazoline). Their respective fatty acid moieties are only marginally differing in chain lengths distribution, and it is considered that their reaction products with TEPA are principally the same when evaluating for genotoxicty.