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Toxicological information

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to the OECD guideline 471 and GLP compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
N-(3-aminopropyl)iminodiethanol
EC Number:
225-642-0
EC Name:
N-(3-aminopropyl)iminodiethanol
Cas Number:
4985-85-7
Molecular formula:
C7H18N2O2
IUPAC Name:
2-[(3-aminopropyl)(2-hydroxyethyl)amino]ethan-1-ol
Constituent 2
Reference substance name:
N-(3-Aminopropyl)diethanolamine
IUPAC Name:
N-(3-Aminopropyl)diethanolamine
Test material form:
other: viscous liquid
Details on test material:
- Name of test material (as cited in study report): N-(3-Aminopropyl) Diethanolamine (APDEA)
- Physical state: Clear colourless viscous liquid
- Analytical purity: 92.1% by GC method, 99.6% by titration method
- Impurities (identity and concentrations): no data
- Purity test date: 02 March 2009
- Lot/batch No.: FHM02
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: Room temperature in the dark over silica gel

Method

Target gene:
Histidine operon for S. typhimurium and tryptophan operon for E. coli
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate metabolising system (1 0% liver 89 in standard co-factors)
Test concentrations with justification for top dose:
Preliminary study: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Main study: 50, 150, 500, 1500 and 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile distilled water
- Justification for choice of solvent/vehicle: solubility
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene (2AA)
Details on test system and experimental conditions:
Preliminary study: in order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
The test was performed by mixing 0.1 ml of bacterial culture (TA100 or WP2uvrA), 2 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of test material formulation and 0.5 ml of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). Ten concentrations of the test material formulation and a vehicle control (sterile distilled water) were tested.
ln addition, 0.1 ml of the maximum concentration of the test material and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Nutrient agar plate in arder to assess the sterility of the test material. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: none
- Exposure duration: at 37°C for approximately 48 hour

DETERMINATION OF CYTOTOXICITY
- Method: growth of the bacterial background lawn

Evaluation criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM1 01 plasmid R-factor etc.
All tester strain cultures should be in the range of 1 to 9.9 x 10e9 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-taxie test material dose levels.
There should be no evidence of excessive contamination.

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEM can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in sorne instances the data generated will prohibit a definitive judgement about the test material activity.
Results of this type will be reported as equivocal.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate.
No test material precipitate was observed on the plates of any of the doses tested in either the presence or absence of S9-mix.

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: yes
- Precipitation: no

RANGE-FINDING/SCREENING STUDIES: The test material was non-toxic to the strains of bacteria used (TA100 and WP2 uvrA). The test material formulation and S9-mix used in this experiment were both shown to be sterile.
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 test material was considered to be non-mutagenic the conditions of this test.
Executive summary:

The method meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", and No. 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay).

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2 uvrA were treated with N-(3-Aminopropyl) Diethanolamine using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (1 0% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

N-(3-Aminopropyl) Diethanolamine caused no visible reduction in the growth of the bacterial background lawn at any dose level. N-(3-Aminopropyl) Diethanolamine was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

N-(3-Aminopropyl) Diethanolamine was considered to be non-mutagenic the conditions of this test.

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