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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-06-15 to 2009-07-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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)
Version / remarks:
, 21-July-1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
, 30-May-2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Amides, C6-18, N-[3-(dimethylamino)propyl]
IUPAC Name:
Amides, C6-18, N-[3-(dimethylamino)propyl]

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Mammalian liver post mitochondrial fraction (S9): obtained from livers of Arochlor 1254 induced male Sprague-Dawley rats; protein: 33,8-36,9 mg/ml
Test concentrations with justification for top dose:
First experiment: 62, 185, 556, 1667 and 5000 µg/plate
Second experiment: 1, 4, 11, 33 and 100 µg/plate,
Third experiment: 1, 2, 6, 17 and 50 µg/plate
(all strains)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Test substance is soluble in acetone
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: Sodium azide, 9-Aminoacridine, 2-Nitrofluorene, 4-Nitroquinoline 1-oxide, 2-Aminoanthracene, Benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 h

SELECTION AGENT (mutation assays): histidine

Bacteria
Bacteria were grown overnight for 10 hours (Test 1, 2, 3) in nutrient broth at 37°C / 110 rpm. For inoculation, stock cultures stored at -196°C were used. They had been checked for strain characteristics of rfa-character, uvrB/uvrA-deletion, resistance to ampicillin and tetracycline. Checks were carried out according to Maron and Ames.
Experiments
The test was performed by the direct plate incorporation method. The mutagenicity experiments with his' Salmonella typhimurium and trp' Escherichia coli were performed with minor modifications of the method described by Maron and Ames. Modifications were as follows: 2 mL Top agar, 100 µL test substance, 100 µL bacterial suspension and 500 µL S9-Mix (or 500 µL phosphate buffer) were mixed in a test tube and poured on the surface of the Minimal-Glucose-Agar plates. For each strain and concentration, three plates were used. Each experiment contained positive control substances to check the activity of the metabolizing system and the mutagenicity of the bacteria in triplicate as well as six fold negative controls to check the spontaneous reversion rate of each of the used bacteria strain. After solidification, the plates were incubated upside down for 48 hours at 37° C in the dark.

NUMBER OF REPLICATIONS: three
In the first experiment test substance was tested at concentrations of 62, 185, 556, 1667 and 5000 µg/plate. Toxicity was observed in both test systems beginning at the lowest concentration. Mutagenic effects, evident as an elevation of the number of revertant colonies cannot be estimated.
Based on these results, the concentrations of the second experiment were chosen as follows: 1, 4, 11, 33, 100 µg/plate. Toxicity was observed in the highest concentration in both test systems. No mutagenic effects occurred in the lower concentrations.
Based on these results, a third experiment was performed with the following concentrations: 1, 2, 6, 17 and 50 µg/plate. Toxicity was still observed in the highest concentration in both test systems.
Evaluation criteria:
The Salmonella Typhimurium reverse mutation assay is considered acceptable if it meets the following criteria:
- phenotypic strain characteristics of all strains fulfil the criteria
- normal background growth in the negative and solvent control
- the mean negative control counts are similar to those described by Maron and Ames and to the historical ranges described in the laboratory
- the positive control substances induce clear increases in relevant colonies (more than twice the spontaneous rate of the respective negative control, more than three times in case of TA 1537, respectively)

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:
cytotoxicity
Remarks:
at the concentration of >33 µg/plate with and >50 µg/plate without S9-mix. Precipitation of the test substance in the top agar mixture was observed from the concentration 62 µg/plate upwards.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
observed at the concentration of >100 µg/plate without and >185 µg/plate with S9-mix. Precipitation of the test substance in the top agar mixture was observed from the concentration 62 µg/plate upwards.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: not soluble
- Precipitation: observed from the concentration 62 µg/plate upwards
- Other confounding effects: no data
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Revertant counts higher than 1000 were counted and calculated as 1000. From the data it can be seen that the control plates without mutagens (negative control) showed numbers of spontaneous colonies within the ranges of the historical control data of the Laboratory for Toxicology and Ecology; the positive control chemicals induced without exceptions large increases in the revertant numbers in the corresponding strains. The strain characteristics of the stock cultures (rfa-character, uvrA/uvrB-deletion and resistance to ampicillin and tetracycline) had been confirmed.

Thus, the present study is valid without restrictions.

Precipitation of the test substance in the top agar mixture was observed from the concentration 62 μg/plate upwards. Compared to the negative control values, the test substance does not indicate an elevation of the revertant colonies neither in the absence nor in the presence of S9-Mix.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In the described mutagenicity study, under the experimental conditions reported, the test substance Amides, C6-C18, N-[3-(dimethylamino)propyl], (a.i. 98.4%) did not induce gene mutations by base-pair substitution or frame shifts in the genome of the used strains below its toxic concentration.
Therefore, the test substance is considered to be non-mutagenic in the bacterial reverse mutation assay under the conditions employed for this study.
Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471, 21 July 1997 and EU Method B.13/14, 30 May 2008, strains TA 1535, TA 1537, TA 98 and TA 100 of S.typhimurium and strain WP2 uvrA of E.coli were exposed to Amides, C6 -C18, N-[3 -(dimethylamino)propyl] (a.i. 98.4 %). Three independent experiments were performed at concentrations of 0 (control), 62, 185, 556, 1667 and 5000 µg/plate in the first experiment; 0 (control), 1, 4, 11, 33 and 100 µg/plate in the second experiment; 0 (control), 1, 2, 6, 17 and 30 µg/plate in the third experiment all in the presence and in the absence of mammalian activation.

No evidence of biologically significant mutagenic activity of the test item was found in the presence and in the absence of metabolic activation, up to and including the limit concentration of 5000 µg/plate. Biologically significant bacteriotoxic effects were observed at concentrations >33 µg/plate. Precipitation of the test substance in the top agar mixture was observed from the concentration 62 µg/plate upwards. The positive controls induced the appropriate responses in the corresponding strain and activity of metabolizing system was confirmed. There was no evidence of induced mutant colonies over background.

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