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Diss Factsheets

Toxicological information

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:
weight of evidence
Study period:
20 March 2012 - 18 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

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

Test material

Constituent 1
Reference substance name:
Amines, N-C16-22-alkyltrimethylenedi-
EC Number:
292-564-1
EC Name:
Amines, N-C16-22-alkyltrimethylenedi-
Cas Number:
90640-45-2
Molecular formula:
R-NH-(CH2)3-NH2 R = alkyl mainly C16-22-(even numbered)-alkyl
IUPAC Name:
Amines, N-C16-22-alkyltrimethylenedi-
Test material form:
solid
Details on test material:
Chemical registery number : 292-564-1
Chemical name : Amines, N-C16-22-alkyltrimethylenedi-

Based on the qualitative and quantitative information on the composition, the sample used are representative of the boundary composition shared and agree by each registrant.

Method

Target gene:
Histidine operon
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:
rat liver S9 mix
Test concentrations with justification for top dose:
In accordance with the OECD Testing Guideline 471
Vehicle / solvent:
- Vehicle used: ethanol
- Justification for choice: test item was soluble in the vehicle at 100 mg/mL
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-Anthramine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 to 72 hours.

DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertant colonies and/or thinning of the bacterial lawn
Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants 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.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
The test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, either in the presence or in the absence of a rat metabolizing system.
Executive summary:

The objective of this study was to evaluate the potential of the test item, DINORAM 42, to induce reverse mutation in Salmonella typhimurium.


 


The study was performed according to the international guidelines (OECD No. 471 and Commission Directive No. B13/14) and in compliance with the principles of Good Laboratory Practice.


 


Methods


A preliminary toxicity test was performed to define the dose-levels of DINORAM 42 to be used for the mutagenicity study. The test item was then tested in three independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.


 


All experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method, whereas the second and third tests with S9 mix were performed according to the pre-incubation method (60 minutes, 37°C).


 


Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to six dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at, 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.


 


Results


The test item DINORAM 42 was dissolved in ethanol.


Since the test item was found cytotoxic in the preliminary test towards some strains and since a strong precipitate observed in the Petri dishes interfered with the scoring of revertants, the selection of the highest dose-level for the main experiments was based on the level of toxicity and/or precipitate, according to the criteria specified in the international guidelines.


 


Experiments without S9 mix


The selected treatment-levels were as follows:


.           15.6, 31.3, 62.5, 125, 250 and 500 µg/plate for the strains TA 1535 and TA100 in the first experiment and for all the tester strains in the second experiment,


.           7.81, 15.6, 31.3, 62.5, 125 and 250 µg/plate for the strains TA 1537, TA 98 and TA102 in the first experiment,


.           31.3, 62.5, 93.75, 125, 187.5 and 250 µg/plate for the strain TA1535 in the third experiment.


 


A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels = 250 µg/plate in the first and second experiments. In the third experiment, no precipitate was observed at any tested dose-levels.


 


In the first experiment:


A moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose-levels >= 125 µg/plate in the strains TA 1537, TA 98, TA 102.


No noteworthy toxicity was noted towards the strains TA 1535 and TA 100.


 


In the second experiment:


A moderate to strong toxicity (thinning of the bacterial lawn) was noted at dose-levels >= 125 µg/plate in the strain TA 98, at dose-levels >= 250 µg/plate in the strains TA 1535 and TA 1537, and at 500 µg/plate for the TA 102 strain.


No noteworthy toxicity was noted towards the strain TA 100.


 


In the third experiment:


A moderate toxicity (thinning of the bacterial lawn) was noted at 250 µg/plate in the strain TA 1535.


 


No biologically relevant increase in thenumber of revertants was noted towards all the strains, in any experiment.


 


Experiments with S9 mix


The selected treatment-levels were as follows:


.           15.6, 31.3, 62.5, 125, 250 and 500 µg/plate in the first and second experiments for all the tester strains,


.           15.6, 31.3, 46.9, 62.5, 125 and 250 µg/plate for the TA 1535 strain in the third experiment.


 


A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels = 250 µg/plate in the first and second experiments. In the third experiment, no precipitate was observed at any tested dose-levels.


 


In the first experiment (performed using the direct plate incorporation method):


No noteworthy toxicity was noted towards the strains used.


 


In the second and third experiments (performed using the pre-incubation method):


A strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose-levels >= 125 µg/plate in the strain TA 1535 and at dose-levels >= 250 µg/plate in the strains TA 1537, TA 98, TA 100 and TA 102.


 


No biologically relevant increase in thenumber of revertantswas notedtowards all the strains, in any experiment.


 


Conclusion


The test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, either in the presence or in the absence of a rat metabolizing system.