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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:
key study
Study period:
24 Sep 2009 to 06 Nov 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-(2-heptadec-8-enyl-2-imidazolin-1-yl)ethanol
EC Number:
202-414-9
EC Name:
2-(2-heptadec-8-enyl-2-imidazolin-1-yl)ethanol
Cas Number:
95-38-5
Molecular formula:
C18H34N2O - C24H46N2O
IUPAC Name:
2-(2-heptadec-8-en-1-yl-4,5-dihydro-1H-imidazol-1-yl)ethanol
Constituent 2
Chemical structure
Reference substance name:
(Z)-2-(8-heptadecenyl)-4,5-dihydro-1H-imidazole-1-ethanol
EC Number:
244-501-4
EC Name:
(Z)-2-(8-heptadecenyl)-4,5-dihydro-1H-imidazole-1-ethanol
Cas Number:
21652-27-7
Molecular formula:
C22H42N2O
IUPAC Name:
2-(2-heptadec-8-en-1-yl-4,5-dihydro-1H-imidazol-1-yl)ethanol
Specific details on test material used for the study:
- Name of test material (as cited in study report): Amine O
- Analytical purity: 96.6%
- Lot/batch No.: 05774HP8
- Expiration date of the lot/batch: 04 May 2012
- Substance type: organic
- Physical state: Liquid, yellow to brown, clear
- Stability under test conditions: Stable
- Storage condition of test material: Room temperature

Method

Target gene:
S. typhimurium: his
E. coli: trp
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
1st Experiment, SPT: 0; 22; 110; 550; 2750 and 5500 μg/plate, all strains

2nd Experiment, SPT: 0; 0.2; 1; 5; 25 and 50 μg/plate (TA 1535 ± S9 mix and E. coli - S9 mix); 0; 0.1; 0.5; 2.5; 12.5 and 25 μg/plate (TA 100, TA 1537, TA 98 ± S9 mix and E. coli + S9 mix). Due to strong bacteriotoxicity observed in the 1st experiment, the experimental part was repeated with lower dose groups.

3rd Experiment, PIT: 0; 0.2; 1; 5; 25 and 50 μg/plate (TA 1535 ± S9 mix and E. coli - S9 mix); 0; 0.1; 0.5; 2.5; 12.5 and 25 μg/plate (E. coli + S9 mix); 0; 0.05; 0.25; 1.25; 6.25 and 12.5 μg/plate (TA 100, TA 1537, TA 98 ± S9 mix)
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle: Due to the limited solubility of the test item in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Controls
Untreated negative controls:
yes
Remarks:
Sterility control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-aminoanthracene (2-AA), 4-nitro-o-phenylenediamine (NOPD)
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation; preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: at 37°C for 48 – 72 hours in the dark

NUMBER OF REPLICATIONS: 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn, reduction in the titer
Evaluation criteria:
ACCEPTANCE CRITERIA
Generally, the experiment is considered valid if the following criteria are met:
- The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain
- The sterility controls revealed no indication of bacterial contamination.
- The positive control items both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
- The titer of viable bacteria was ≥ 108/mL.

ASSESSMENT CRITERIA
The test item is considered positive in this assay if the following criteria are met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test item is generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.
Statistics:
none

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium, other: 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:
valid
Positive controls validity:
valid
Key result
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:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test item precipitation was found with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
In addition, the positive control items both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A strong bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed in the standard plate test depending on the strain and test conditions from about 12.5 μg/plate onward.
In the preincubation assay clear bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed depending on the strain and test conditions from about 6.25 μg/plate onward.

Any other information on results incl. tables

1st Experiment (SPT)

  Mean revertants per plate
  TA 98 TA 100 TA 1535 TA 1537 WP2uvrA
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
solvent control 31 32 101 102 15 14 8 9 47 48
22 9 12 67 76 14 14 3 2 51 15
110 0 0 0 0 0 0 0 0 0 0
550 0 0 0 0 0 0 0 0 0 0
2750 0 0 0 0 0 0 0 0 0 0
5500 0 0 0 0 0 0 0 0 0 0
positive control 502 836 724 973 775 184 351 176 863 213

2nd Experiment (SPT)

  Mean revertants per plate
  TA 98 TA 100 TA 1535 TA 1537 WP2uvrA
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
solvent control         18 17     41  
0.2         18 17     44  
1         16 17     37  
5         19 17     39  
25         15 15     41  
50         3 6     8  
positive control         1013 175     827  

  Mean revertants per plate
  TA 98 TA 100 TA 1535 TA 1537 WP2uvrA
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
solvent control 29 36 130 134     10 12   45
0.1 31 37 123 125     10 10   43
0.5 26 32 138 100     11 10   42
2.5 27 34 130 111     12 9   46
12.5 19 31 82 96     12 7   43
25 5 5 8 15     2 2   5
positive control 426 831 831 874     395 179   230

3rd Experiment (PIT)

  Mean revertants per plate
  TA 98 TA 100 TA 1535 TA 1537 WP2uvrA
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
solvent control         16 15     33  
0.2         14 18     34  
1         11 15     42  
5         16 14     30  
25         11 13     14  
50         0 2     9  
positive control         605 137     606  

  Mean revertants per plate
  TA 98 TA 100 TA 1535 TA 1537 WP2uvrA
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
solvent control 25 27 98 108     8 7    
0.05 20 27 101 107     7 7    
0.25 20 31 97 120     7 7    
1.25 22 28 92 104     7 8    
6.25 18 25 72 102     2 4    
12.5 0 7 0 15     0 3    
positive control 494 834 882 995     356 145    

  Mean revertants per plate
  TA 98 TA 100 TA 1535 TA 1537 WP2uvrA
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
solvent control                   36
0.1                   31
0.5                   31
2.5                   33
12.5                   24
25                   16
positive control                   234

Applicant's summary and conclusion

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