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Genetic toxicity: in vitro

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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:
1981-06-30
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable and well documented report which meets basic scientific principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1981
Report Date:
1981

Materials and methods

Principles of method if other than guideline:
Method: Ames et al. (1975), Mutation Research 31, 347-364
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: Morpholine
- Analytical purity: 99.8 %

Method

Target gene:
In the Salmonella typhimurium strains (TA 1535, TA 100, TA 1537, TA 98) the amino acid histidine locus is the target gene, in which induced back mutations will transform the histidine auxotrophy (his-) to histidine prototrophy (his+).
The Salmonella typhimurium histidine (his) reversion system measures his- => his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations.
Species / strainopen allclose all
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 mix (from liver homogenate of Aroclor 1254 treated rats )
Test concentrations with justification for top dose:
0, 15.8, 50, 158, 500, 1580, 5000, 15800, 31,600 and 50000 µg/plate. Higher doses were tested but were too cytotoxic.
Desiccator method: 0.5, 1.0, 1.5 and 2.5 mg/20 L
Vehicle / solvent:
Vehicle used: water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine and benzo(a)pyrene 4,5-oxide were used in the experiments for direct mutagenicity and 3-methylcholanthrene, benzo(a)pyrene, 2-aminoanthracene and benzo(e)pyrene in the experiments with S9 mix.
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation and desiccator method.


DURATION
- Exposure duration: 2-3 days



Evaluation criteria:
No data
Statistics:
Not indicated

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:
ambiguous
Remarks:
slight increase at high doses
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Remarks:
slight increase at high doses
Cytotoxicity / choice of top concentrations:
cytotoxicity
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

Plate incorporation with S-9 mix: experiment 1.

 Dose (nL/per plate)  TA 100  TA 1537  TA 1537  TA 98   WP2 uvrA                        
 0  146  15  29  23  29                        
 15.8  136  16  33  20  35                        
 50  144 16  37  25  27                        
 158  128  12  31  25  29                        
 500  140  19  41  28  24                        
 1580  142  16  39  29  30                        
 5000  162  16  40  27  26                        
 15800  136  14  34  27  25                        
 50000  167x  14  27  24  60x                        
 10 µg B(a)P  1020  25  172  365  105                        
 50 µg B(e)P  303  15  66  61  41                        
 10 µg 2-AA  4200  371  181  3050  428                        
 90 µg 3-MC  4000  10  114  1385  39                        

Plate incorporation without S-9 mix: experiment 1.

 Dose (nL/per plate)  TA 100  TA 1537  TA 1537  TA 98  WP2 uvrA                   
 0 94 13 14 15 21                
 15.8 83 9 9 15 30                
 50 96 10 13 18 24                
 158 107 9 14 15 29                
 500 109 15 11 14 32                
 1580 104 13 14 16 28                
 5000 97 10 13 17 28                
 15800 112 11 13 16 31                
 50000 148x 16 4 12 29                
 1 µg B(a)P 3300 11 689 3200 93                
 10 µg MNNG 25500 32500 98 48 441                

Plate incorporation with S-9 mix: experiment 2.

 Dose (nL/per plate)  TA 100  TA 1537  TA 1537  TA 98   WP2 uvrA                              
 0 113 19 21 33 30                              
 15800 129 17 16 34 26                              
 50000 204 16 13 23 38x                              
 100000 29 4 0 3 12                              
 200000 0 0 0 0 0                              
 10 µg B(a)P 1189 30 189 565 74                              
 50 µg B(e)P 256 35 42 81 33                              
 10 µg 2-AA 2500 284 137 3150 363                              
 90 µg 3-MC 3150 10 107 2700 43                              

Plate incorporation without S-9 mix: experiment 2.

 Dose (nL/per plate)  TA 100  TA 1537  TA 1537  TA 98     WP2 uvrA                            
 0 85 12 8 20 24                          
 15800 108x 15 7 22 28                          
 50000 154x 14 8 18 28                          
 100000 0 0 0 0 5                          
 200000 0 0 0 0 0                          
 1 µg B(a)P 1310 27 439 1550 48                          
 10 µg MNNG 22000 32500 91 41 380                          

Plate incorporation with S-9 mix: experiment 3.

 Dose (nL/per plate)  TA 100  TA 1537  WP2 uvrA                                     
 0 146 12 37                                  
 15800 172 12 34                                  
 31600 194 12 55x                                  
 50000 237 13 71x                                  
 10 µg B(a)P 1315 27 65                                  
 50 µg BPO 352 14 40                                  
 10 µg 2-AA 2600 338 398                                  
 90 µg 3-MC 2950 6 57                                

Desiccator method:

 Dose (ml/per 20 L)  TA 100  TA 100  TA 1535  TA1535 TA1537  TA1537  TA 98  TA 98    WP2 uvrA   WP2 uvrA                    
+ S9 -S9 +S9 -S9 +S9 -S9  +S9  -S9  +S9  -S9                    
0 182 145 21 16 20 7  29  16  42 25                    
0.5 152 133 30 17 26 8  28  17 40   20                    
1 166 131 27 24 18 9  39  24 46   24                    
1.5 159 136 26 22 15 13  36  23  36  20                    
2.5 156 39 27 18 22 9 30   14  44  23                    
10 µg B(a)P 1030 - 40 - 136 - 384   - 95   -                    
10 µg 2-AA 3200 - 256 - 185 -  2600  - 423   -                    
90 µg 3-MC  3300  -  9  - 111   -  1700  -  32  -                    
1 µg BP0  -  1950  -  18  327  -  2250  -  93                    
10 µg MNNG  -  46500  -  38500  -  34  -  76  -  412                    

B(a)P: Benzo(a)pyrene

2-AA: 2-Aminoanthracene

3-MC: 3-Methylcholanthrene

BPO: Benzo(a)pyrene 4,5- oxide

MNNGG: N-Methyl-N'-nitro-N-nitrosoguanidine .


In the 1st experiment, a slight increase in the number of revertants was found at the 50000 µg dose in TA 100 without metabolic activation (increased by a factor of 1.57) and was reproduced in the 2nd experiment (factor: 1.8). Using metabolic activation, a slight increase in WP2 uvrA revertants was seen (factor: 2) in the 1st experiment; it was not reproduced in the 2nd experiment, but in a 3rd experiment it was only marginally increased (factor: 1.9) at the 50000 µg dose. Results from the dessicator method showed no increase in revertants.

 


 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
ambiguous
Executive summary:

In a reverse gene mutation assay (Glatt & Oesch, 1981), Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 were exposed to Morpholine (99.8 %) at concentrations of 0, 15.8, 50, 158, 500, 1580, 5000, 15800, 31600 or 50000 µg/plate (vehicle: water) in the presence and absence of mammalian metabolic activation (S9 mix) in the standard plate test (plate incorporation method). Higher doses were tested, but were too cytotoxic. Slight increases in the number of revertants were observed at 50000 µg/plate with TA100 in the presence and absence of S9 mix and with WP2 uvrA only in its presence. Morpholine exposure at 0.5, 1.0, 1.5 or 2.5 mL/20 L by means of the desiccator method did not reveal mutagenicity of Morpholine at any dose. Morpholine (or possibly an impurity) was weakly mutagenic in the Ames test at a concentration of 50000 µg/plate. However, the observed increase in the number of mutants at this relatively high dose was not more than 2 -fold. Therefore this test result is considered as ambiguous. The positive controls induced the appropriate responses in the corresponding strains.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.