Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance gave negative results in a reliable vitro bacterial mutation assay. A structural analogue of the substance (methyl trimethyl-3-[(1-oxododecyl)amino]propylammonium sulphate; CAS 10595-49-0) also gave negative results in three reliable in vitro assays - in vitro bacterial mutation, in vitro cytogenetics and in vitro gene mutation assays.


Justification for selection of genetic toxicity endpoint
All of the available in vitro studies were negative so no study has been selected.

Short description of key information:
The substance and/or a structural analogue gave negative results in four different in vitro genotoxicity assays.

Link to relevant study records

Referenceopen allclose all

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:
1999-04-13 to 1999-04-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study to GLP
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Additional strain / cell type characteristics:
other: trptophan auxotroph
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: histidin auxotroph
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (phenobarbital/ß-naphtoflavone induced), Lot-No. 070898, micorsomal protein-content: 24.7 mg/ml; purchased by CCR GmbH & Co. KG, 64380 Roßdorf, Germany
Test concentrations with justification for top dose:
Preliminary assay with TA 100 : 0.38, 0.76, 1.52, 3.03, 6.06, 12.13, 24.25, 48.5, 97.00 mg/plate
1st assay performed on all strains: 24.25, 76.69, 242.50, 766.85, 2425.00 µg/plate
2nd assay performed on all strains: 75.78, 151.56, 303.13, 606.25, 1212.5 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Aqua dest.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, 0.5 µg/plate for TA 100, TA 98, 2.0 µg/plate for TA 1535, TA 1537, and 1.0 µg/plate for E. coli with metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Migrated to IUCLID6: 12.9 µg/plate for E. coli without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Migrated to IUCLID6: 50 µg/plate for TA 1537, without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene, 0.2 µg/plate for TA 98 without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Migrated to IUCLID6: 0.25 µg/plate for TA 100, TA 1535 without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Exposure duration: 48 h

SELECTION AGENT (mutation assays): histidine for s.typhimurium strains, tryptophan for E. coli

NUMBER OF PLATES EVALUATED: 3 parallel plates were performed for each experimental point. Two indipendent experiments.

DETERMINATION OF CYTOTOXICITY
- Method: The highest concentration used in the test must either reduce the survival of the cells to at least 50 % or reduce the background growth of auxotrophic cells and the number of revertants.

OTHER: Because of the reduced background lawn in the 1st assay the 2nd assay was performed at a concentration range from 75.78 to 1212.5 1 µg/plate.
Evaluation criteria:
Selection of the test compound:
The highest concentration used in the test must either reduce the survival of the cells to at least 50 % or reduce the background growth of auxotrophic cells and the number of revertants. If no toxicity is observed, the survey is carried out with the highest soluble concentration of the test article, however, not exceeding 5 mg/plate for solids and 10 mM of Iiquids or 200 µl/plate of liquids or extracts. The concentration intervals between the test doses have maximum a factor of sqrt. 10,

Analysis of results:
A test compound is considered as mutagenic if there is a dose dependent increase in the number of revertant colonies of one or more strains. For the highest non-toxic concentration an increase should be by a factor of about 2 and deemed to be biologically relevant. Any evidence af mutagenic activity must be reproducible in an independent experiment.
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 1212.5 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
cytotoxicity
Remarks:
1212.5 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PRELIMINARY TOXICITY TEST:
The test substance reduced the survival at a concentration of 1.52 mg/plate to 46.3 % of the control value. The number of the his-cells of the strain TA 100 at the concentration of 1.52 mg/plate was reduced to 58.3 % of the control value. No precipitation could be observed up to 97 mg/plate.

MAIN TEST:
The results of the 1st assay and the 2nd assay indicate, that in the tested concentration range (1 st assay: 24.25, 76.69, 242.5, 766.85 and 2425.0 µg/plate, 2nd assay: 75.78, 151.56, 303.13, 606.25 and 1212.5 µg/plate) with and without metabolic activation no significant increase in the numbers of his+- or trp+-revertants over the spontaneous values could be detected with the Salmonella strains TA 100, TA 1535, TA 98, TA 1537
and E. coli WP2 uvrA pKM(101).

Because of the reduced background lawn in the 1st assay the 2nd assay was performed at a concentration range from 75.78 to 1212.5 1 µg/plate.

STERILITY CONTROLS:
The plates for the sterility control of top agar, S9-mix and test compound showed no growth.

NEGATIVE CONTROLS:
In all experiments the control plates without mutagen showed a normal number of spontaneous revertants.

POSITIVE CONTROLS:
The positive controls demonstrated the sensitivity of the indicator strains and the activity of the metabolizing system.
Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Evaluation of the results of the two independent plate incorporation experiments did not provide evidence of a mutagenic potency of the substance.
Up to 2425.0 µg/plate, the substance did not induce biologically relevant increases in revertants in any of the tested strains. This applied to both the activated and the non-activated assay system. The substance is therefore found not to be genetically active under the aforementioned test conditions.
Executive summary:

In a reverse gene mutation assay in bacteria according OECD guidelines 471/472, and EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria), 1992, four strains of S. typhimurium (TA 1535, TA 1537, TA 100 and TA 98) and E. coli WP2 uvr A pKM 101 were exposed to the substance (48.5 % a.i. in Aqua dest.), at concentrations of 24.25, 76.69, 242.50, 766.85, 2425.00 µg/plate (1st assay) and 75.78, 151.56, 303.13, 606.25, 1212.5 µg/plate (2nd assay) in the presence and absence of mammalian metabolic activation (plate incorporation). Concentration selection for the first assay was based on a preliminary toxicity study with and without metabolic activation on strain TA 100 in which the concentration of 1.52 mg/plate was cytotoxic. Because of an reduced background lawn in the 1st assay the 2nd assay was performed at the concentration range from 75.78 to 1212.5 µg/plate.

No evidence of biologically significant mutagenic activity of the test item was found in the presence and absence of metabolic activation, up to and including the highest tested concentration of 2425 µg/plate. The test material was tested up to its toxic limit. The positive controls induced the appropriate responses in the corresponding strains and thus demonstrated the sensitivity of the indicator strains and the activity of metabolizing system..

There was no evidence of induced mutant colonies over background.

The adopted OECD TG 471 (1997) requires at least 5 test strains. These should include four strains of S. typhimurium (TA1535; TA137 or TA97a or TA97; TA98; and TA 100 and in addition the use of E. coliWP2 strains or Salmonella typhimurium TA 102 to detect certain oxidizing mutagens, cross-linking agents and hydrazines. This requirement is fulfilled by this test performed according to former versions of OECD guidelines 471/472 and EU Method B.13/14 (Version Commission Directive 92/69/EEC) on S. typhimurium (TA 1535, TA 1537, TA 100 and TA 98) and E. coliWP2 uvr A pKM 101.

Thus, this GLP test is considered as sufficient to evaluate the mutagenic activity of the substance in this bacterial test system.

 

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: Recently conducted guideline study to GLP.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
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:
S9 Microsomal fraction from rats induced with Phenobarbital /beta-Naphthoflavone
Test concentrations with justification for top dose:
3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Vehicle / solvent:
Water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191 (Acridine mutagen)
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results (migrated information):
negative

The substance was considered to be non-mutagenic to bacteria in vitro.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Recently conducted guideline study to GLP.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes:
Metabolic activation:
with and without
Metabolic activation system:
S9 microsomal fraction from male rats which has been induced with phenobarbital/beta-naphthoflavone.
Test concentrations with justification for top dose:
0, 2, 4, 8, 16, 32, 48, 64, 128, 160 - Expt 1 (-S9)
0, 4, 8, 16, 32, 48, 64, 128, 160 - Expt 1 (+S9)
0, 2, 4, 8, 16, 32, 48, 64 - Expt 2 (-S9)
0, 8, 16, 32, 48, 64, 128 - Expt 2 (+S9)
The purity of the test item was 39.7% and was accounted for in the formulations.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Evaluation criteria:
A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results (migrated information):
negative

The substance was considered to be non-clastogenic to human lymphocytes in vitro.
Endpoint:
in vitro gene mutation study in mammalian cells
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: Recently conducted guideline study to GLP.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9 microsomal fraction from male rats which has been induced with phenobarbital/beta-naphthoflavone.
Test concentrations with justification for top dose:
0, 6.25, 12.5, 25, 50, 75, 100, 125, 150 ug/ml - Expt 1 (-/+S9)
0, 2.5, 5, 10, 15, 20, 25, 30, 35 - Expt 2 (-S9)
0, 3.13, 6.25, 12.5, 25, 50, 75, 100, 125 - Expt 2 (+S9)
The purity of the test item was 39.7% and was therefore accounted for when formulating the dosing solutions.


Vehicle / solvent:
R0 medium
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Evaluation criteria:
For a test item to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. Following discussions at an International Workshop on Genotoxicity Test Procedures in Plymouth, UK, 2002 (Moore et al 2003) it was felt that the IMF must exceed some value based on the global background MF for each method (agar or microwell). This Global Evaluation Factor (GEF) value was set following a further meeting of the International Workshop in Aberdeen, Scotland, 2003 (Moore et al 2006) at 126 x 10-6 for the microwell method. Therefore, any test item dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10-6 and demonstrates a positive linear trend will be considered positive. However, if a test item produces a modest increase in mutant frequency, which
only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test item induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.
Statistics:
The experimental data was analysed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al, 1989).
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results (migrated information):
negative

The substance was considered to be non-mutagenic to mouse lymphoma cells in vitro.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

The substance and/or a structural analogue gave negative results in four in vitro genotoxicity assays and therefore there is no justification for classification.