Registration Dossier

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:
2010-02-08/2010-03-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to the OECD Guideline and it is GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

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:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
Amines, C20-22-alkyldimethyl
EC Number:
296-866-4
EC Name:
Amines, C20-22-alkyldimethyl
Cas Number:
93164-85-3
Molecular formula:
CnH2nN(CH3)2 n: from 18 to 22
IUPAC Name:
Amines, C20-22-alkyldimethyl
Details on test material:
- Physical state: white solid
- Storage: at room temperature in the dark under nitrogen
- Stability: stable

Method

Target gene:
Histidine
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
other: sodium azide
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
other: 9-aminoacridine
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other: 2-nitrofluorene
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: methylmethanesulfonate
Species / strain / cell type:
other: WP2uvrA
Additional strain / cell type characteristics:
other: 4-nitroquinoline N-oxide
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
The substance was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the presence of S9-mix.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Details on test system and experimental conditions:
At least five different doses of the test substance were tested in triplicate in each strain. In the first experiment was tested both in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537, and TA98. In an independent repeat of the assay with additional parameters, the test susbtance was tested both in the absence and presence of 10% (v/v) S9-mix in all tester strains. An additonal third experiment with four dose levels was performed with the strains TA13535 and TA1537 in the absence and presence of S9-mix.
The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.

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
Vehicle controls validity:
not valid
Positive controls validity:
valid
Species / strain:
other: WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain

 Dose (µg/plate)  TA1535  TA1537  TA98  TA100  WP2uvrA
 without S9 -mix          
 positive control  834 ± 17  256 ± 72  999 ± 60  861 ± 9  925 ± 25
 solvent control  10 ± 4  3 ± 1  19 ±3  93 ± 9  24 ± 6
 3        83 ± 8  19 ± 3
 10        87 ± 3  15 ± 5
 33  10 ± 2  3 ± 2  21 ± 3  78 ± 3  20 ± 7
 100  8 ± 3  3 ± 1  17 ± 3  88 ± 8  16 ± 3
 333  11 ± 6  4 ± 1  22 ± 3  85 ± 5  19 ± 10
 1000  14 ± 6  10 ± 2  15 ± 4  94 ± 1  17 ± 2
 3330 SP  13 ± 7  13 ± 9  21 ± 2  53 ± 5  21 ± 6
 5000 SP        28 ± 14 s  24 ± 2
 with S9 -mix (1)          
 positive control  209 ± 33  305 ± 80  1420 ± 65 1187 ± 87  329 ± 8
 solvent control  7 ± 3  4 ± 1  29 ± 4  77 ± 19  18 ± 6
 3        62 ± 7  17 ± 4
 10        68 ± 4  14 ± 3
 33  6 ± 2  4 ± 1  20 ± 2  70 ± 15  17 ± 4
 100  5 ± 1  4 ± 2  32 ± 8  70 ± 3  16 ± 3
 333  7 ± 3  3 ± 1  16 ± 2  86 ± 14  18 ± 4
 1000  9 ± 5  6 ± 3  29 ± 5  94 ± 5  16 ± 8
3330 SP   11 ± 1  16 ± 2  32 ± 4  100 ± 48  18 ± 8
 5000 SP        55 ± 18s  12 ± 2

Solvent control: 0.1 mL ethanol

1 The S9 -mix contained 5% (v/v) S9 fraction

s Bacterial background lawn slightly reduced

sp Slight Precipitate

Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain

 Dose (µg/plate)  TA1535  TA1537  TA98  TA100  WP2uvrA
without S9 -mix          
positive control  785 ± 42  284 ±34  946 ±60  1280 ±8  1031 ±29
 solvent control  7 ± 1  6 ±1  20 ±1  76 ±10  19 ±4
 33  4 ±2  4 ±2  19 ±2  84 ±10  13 ±2
 100  7 ±5  6 ±2  20 ±3  80 ±5  19 ±2
 333  7 ±3  5 ±4  32 ±5  84 ±4  13 ±3
 1000  7 ±1  14 ±7  41 ±27  90 ±3  17 ±3
 3330 SP  22 ±5  32 ±2  31 ±6  112 ±5  25 ±3
 With S9 -mix (1)          
 positive control  149 ±5  367 ±40  537 ±10  977 ±37  296 ±16
solvent control   4 ±2 5 ±2  21 ±3  69 ±6  12 ±4
 33  7 ±2  4 ±1  21 ±1  62 ±4  9 ±5
 100  3 ±0  3 ±0  20 ±1  59 ±2  11 ±4
 333  7 ±3  5 ±2  15 ±3  71 ±3  14 ±2
 1000  8 ±6  12 ±4  30 ±11  71 ±3  19 ±9
 3330 SP  21 ±6 36 ±6  33 ±10 84 ±7  30 ±5

Solvent control: 0.1 mL ethanol

1: The S9 -mix contained 10% (v/v) S9 fraction

SP Slight Precipitate

Mean number of revertant colonies/3 replicate plates (±S.D.) with two Salmonella typhimurium strains

 Dose (µg/plate)  TA1535  TA1537
 without S9 -mix    
 positive control  894 ± 39  298 ± 12
 solvent control  13 ± 7  4 ± 2
 333  16 ± 4  5 ± 3
 1000  14 ± 3  4 ± 1
 1666 SP  22 ± 4  11 ± 4
 3330 SP  21 ± 2  3 ± 2
 with S9 -mix    
 positive control  143 ± 5  341 ± 94
 solvent control  11 ± 2  6 ± 3
 333  6 ± 1  4 ± 1
 1000  14 ± 3  7 ± 2
 1666 SP  16 ± 3  5 ± 1
 3330 SP  18 ± 4  3 ± 1

Solvent control: 0.1 mL ethanol

1 The S9 -mix contained 10% (v/v) S9 fraction

SP Slight Precipitate

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

The study procedures described in this report were based on the Guideline OECD 471 and B.13/14. The substance was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli. The test was performed in two independent experiments in the presence and absence of S9 -mix. To obtain more information about the possible mutagenicity of the susbtance, an additional experiment was performed with the tester strains TA1535 and TA1537 in the absence and presence of 10% (v/v) S9 -mix.

Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.