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:
From 02-17-1988 to 03-03-1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study comparable to OECD guideline 471

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report date:
1988

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Reliability scoring based on 1997 guideline for test n°471
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Genamin SH 301
IUPAC Name:
Genamin SH 301
Constituent 2
Reference substance name:
Amines, di-C14-18-alkylmethyl
EC Number:
266-923-8
EC Name:
Amines, di-C14-18-alkylmethyl
Cas Number:
67700-99-6
IUPAC Name:
N-hexadecyl-N-methylhexadecan-1-amine
Details on test material:
- Name of test material (as cited in study report): Genamin SH 301
- A statement of identity for Genamin SH 301 was provided on june 2009 by the sponsor Clariant Produkte GmbH (see attached background material). The statement specifies that the following chemical names, CAS registration and EC numbers are appropriate synonyms to describe GENAMIN SH 301:
-> Amines, bis(hydrogentated tallow alkyl)methyl CAS 61788-63-4 EC 262-991-8
-> Amines, di-C14-18-alkylmethyl CAS 67700-99-6 EC 266-923-8
- Appearance: brownish resin
- Analytical purity: 100%
- Lot/batch No.: E 06206493 dated december 16, 1987
- Storage conditions: dark at 22 °C

Method

Target gene:
In Salmonella typhimurium: ability to synthetise histidine after mutation
in E. Coli WP2uvra: ability to synthetise Tryptophane after mutation
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium, other: TA1535, TA1537, TA1538, TA98 and TA100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (liver post mitochondrial fraction of rats induced with Aroclor 1254)
Test concentrations with justification for top dose:
- First mutagenicity experiment with and without S9 mix: 4, 20, 100, 500, 2500 and 5000 µg/plate
- Second mutagenicity experiment with and without S9 mix: 4, 20, 100, 500, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle used: DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: see table 1
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- In agar (plate incorporation): first mutagenicity and second mutagenicity tests with or without S9 mix
- Exposure duration: 48h to 72h

SELECTION AGENT (mutation assays): agar containing traces of histidine and biotin, maintained at 45°C for Salmonella strains.
agar containing traces of tryptophan and biotin, maintained at 45°C for Escherichia coli.

NUMBER OF REPLICATIONS: two independent mutagenicity experiments each using three plates/dose-level

DETERMINATION OF CYTOTOXICITY
- Method: the evaluation of the toxicity was based on the decrease in the number of revertant colonies and/or thinning of the bacterial lawn.
Evaluation criteria:
After incubation for 48 to 72 hour at 37 °C in the dark, colonies were counted
Statistics:
No statistical analysis performed

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Tested up to limit concentrations recommended by the test guideline
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537, TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Tested up to limit concentrations recommended by the test guideline
Vehicle controls validity:
valid
Untreated negative controls validity:
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

- The number of revertants for the vehicle and positive controls were as specified in the acceptance criteria. The study was therefore considered as valid.

 

- Since the test item was freely soluble and non-toxic in the preliminary test, the highest dose-level was 5000 µg/plate, according to the criteria specified in the international guidelines.

 

- No precipitate was observed in the petri plates when scoring the revertants at any of the tested dose-levels.

 

- No toxicity was noted at any dose-level in any strain.

 

- The test item did not induce any noteworthy increase in the number of revertants, in any of the strains, either with or without S9 mix.

- Table: Detailed results for the two mutagenicity test performed with six bacterial strains for six concentrations of the test Genamin SH301

as well as the vehicle and positive control.

Strain Compound First mutagenicity test Second mutagenicity test
Dose level (µg/plate) S9 mix Revertant colony count Dose level (µg/plate) S9 mix Revertant colony count
Mean   Mean  
TA 98 DMSO 0 without 20 0 without 23
Genamin SH301 4 without 20  4 without 25
 Genamin SH301 20 without 24  20 without 23
 Genamin SH301 100 without 20  100 without 20
 Genamin SH301 500 without 23 500 without 23
 Genamin SH301 2500 without 21 2500 without 21
 Genamin SH301 5000 without 25 5000 without 29
2 -nitrofluorene 2.5 without 391  2.5 without 357
 DMSO 0 with 31 0 with 29
 Genamin SH301 4 with 38  4 with 25
 Genamin SH301 20 with 31 20 with 25 
 Genamin SH301 100 with 34 100 with 29
 Genamin SH301 500 with 31 500 with 20
 Genamin SH301 2500 with 30 2500 with 29
    Genamin SH301  5000   with  35   5000   with 36
   2 -aminoanthracene  0.5   with 390    0.5   with 441
   Benzo(a)pyrene  10   with 682  10   with 689

Strain Compound First mutagenicity test Second mutagenicity test
Dose level (µg/plate) S9 mix Revertant colonycount Dose level (µg/plate) S9 mix Revertant colonycount
Mean   Mean  
TA 100 DMSO 0 without 155 0 without 180
Genamin SH301 4 without 173 4 without 170
 Genamin SH301 20 without 174 20 without 172
 Genamin SH301 100 without 156 100 without 167
 Genamin SH301 500 without 162 500 without 187 
 Genamin SH301 2500 without 173 2500 without 200
 Genamin SH301 5000 without 155 5000 without 194
Sodium azide 1 without 613 1 without 620
 DMSO 0 with 182 0 with 194
 Genamin SH301 4 with 168 4 with 182
 Genamin SH301 20 with 171 20 with 171
 Genamin SH301 100 with 165 100 with 165
 Genamin SH301 500 with 168 500 with 172
 Genamin SH301 2500 with 172 2500 with 214
    Genamin SH301  5000   with   232  5000   with 230
   2 -aminoanthracene  0.5   with 584 0.5   with 760
   Benzo(a)pyrene  10   with  621    10   with 630

Strain Compound First mutagenicity test Second mutagenicity test
Dose level (µg/plate) S9 mix Revertant colony count Dose level (µg/plate) S9 mix Revertant colony count
Mean   Mean  
TA 1537 DMSO 0 without 0 without 8
Genamin SH301 4 without 11 4 without 8
 Genamin SH301 20 without 10 20 without 7
 Genamin SH301 100 without 8 100 without 6
 Genamin SH301 500 without 7 500 without 9
 Genamin SH301 2500 without 10 2500 without 5
 Genamin SH301 5000 without 10 5000 without 9
9-aminoacridine 50 without 146 50 without 184
 DMSO 0 with 9 0 with 11
 Genamin SH301 4 with 9   4 with
 Genamin SH301 20 with 10 20 with
 Genamin SH301 100 with 10 100 with 15
 Genamin SH301 500 with 9 500 with 5  
 Genamin SH301 2500 with 7 2500 with 8
    Genamin SH301  5000   with   13  5000   with 10
   2 -aminoanthracene  1   with 98   1   with 64
   Benzo(a)pyrene  10   with  122   10   with 116

Strain Compound First mutagenicity test Second mutagenicity test
Dose level (µg/plate) S9 mix Revertant colony count Dose level (µg/plate) S9 mix Revertant colony count
Mean   Mean  
TA 1538 DMSO 0 without 10 0 without 10
Genamin SH301 4 without 11 4 without 12
 Genamin SH301 20 without 16 20 without 16
 Genamin SH301 100 without 12 100 without 9
 Genamin SH301 500 without 12 500 without 12
 Genamin SH301 2500 without 13 2500 without 14
 Genamin SH301 5000 without 15 5000 without 15
2 -nitrofluorene 2.5 without 468 2.5 without 397 
 DMSO 0 with 15 0 with 20
 Genamin SH301 4 with 15   4 with 17
 Genamin SH301 20 with 17 20 with 22  
 Genamin SH301 100 with 22 100 with 17
 Genamin SH301 500 with 12 500 with 21  
 Genamin SH301 2500 with 20 2500 with 20
    Genamin SH301  5000   with   21    5000   with 20
   2 -aminoanthracene 0.5   with 488   0.5   with 453
   Benzo(a)pyrene  10   with  212   10   with 194

Strain Compound First mutagenicity test Second mutagenicity test
Dose level (µg/plate) S9 mix Revertant colony count Dose level (µg/plate) S9 mix Revertant colony count
Mean   Mean  
TA 1535 DMSO 0 without 16  0 without 11
Genamin SH301 4 without 13  4 without 16
 Genamin SH301 20 without 17  20 without 18
 Genamin SH301 100 without 15  100 without 15
 Genamin SH301 500 without 10 500 without 19 
 Genamin SH301 2500 without 13 2500 without 17 
 Genamin SH301 5000 without 11 5000 without 20
Sodium azide 1 without 388  1 without 401 
 DMSO 0 with 15 0 with
 Genamin SH301 4 with 4 with
 Genamin SH301 20 with 12 20 with 5  
 Genamin SH301 100 with 12 100 with 11
 Genamin SH301 500 with 8 500 with 6  
 Genamin SH301 2500 with 12 2500 with 10
    Genamin SH301  5000   with  15   5000   with 12 
   2 -aminoanthracene  1   with 169      1   with 139 
   Benzo(a)pyrene  10   with  19     10   with 17 

Strain Compound First mutagenicity test Second mutagenicity test
Dose level (µg/plate) S9 mix Revertant colony count Dose level (µg/plate) S9 mix Revertant colony count
Mean   Mean  
WP2uvrA DMSO 0 without 80 0 without 99
Genamin SH301 4 without 87  4 without 99
 Genamin SH301 20 without 80 20 without 93
 Genamin SH301 100 without 81 100 without 106
 Genamin SH301 500 without 73 500 without 112
 Genamin SH301 2500 without 73 2500 without 110
 Genamin SH301 5000 without 89 5000 without 117
MNNG 2.5 without 277 2.5 without 316
 DMSO 0 with 89 0 with 90
 Genamin SH301 4 with 85 4 with 91
 Genamin SH301 20 with 90 20 with 87 
 Genamin SH301 100 with 85 100 with 86
 Genamin SH301 500 with 93 500 with 104
 Genamin SH301 2500 with 81 2500 with 88
    Genamin SH301  5000   with  86  5000   with 99
   2 -aminoanthracene  10   with 458    10   with 318
   Benzo(a)pyrene  10   with 125  10   with 159

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under these experimental conditions, no noteworthy increase in the number of revertants was observed towards all the strains used, both with and without S9 mix. Genamin SH301 did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia Coli WP2uvrA.
Executive summary:

The potential of Genamin SH 301 to induce reverse mutation in bacteria was assessed using five strains of Salmonella typhimurium and Escherichia coli Wp2uvrA according to a method comparable to OECD guideline 471. The study was conducted in compliance with the principles of Good Laboratory Practice.

A preliminary toxicity test was performed to define the dose-levels of Genamin SH 301 to be used for the mutagenicity study. The test item was then tested in two independent experiments, both 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.

Both experiments were performed according to the direct plate incorporation method.

 

The five strains of bacteriaSalmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 1538 were exposed to the following dose-levels of Genamin SH 301 (three plates/dose-level):

- 4, 20, 100, 500, 2500 and 5000 µg/plate, for the first and the second mutagenicity experiments with and without S9 mix,

After 48 to 72 hours of incubation at 37°C, 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.

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

No precipitate was observed in the petri plates when scoring the revertants at all dose-levels.

No toxicity was noted towards all the strains used, both with and without S9 mix.

The test item did not induce any significant increase in the number of revertants, both with or without S9 mix, in any of the five strains of Salmonella typhimurium and Escherichia coli WP2uvrA.

Under these experimental conditions Genamin SH 301 did not show any mutagenic activity in the bacterial reverse mutation test .