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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:
1981-06-30
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

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

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
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

Constituent 1
Chemical structure
Reference substance name:
Morpholine
EC Number:
203-815-1
EC Name:
Morpholine
Cas Number:
110-91-8
Molecular formula:
C4H9NO
IUPAC Name:
morpholine
Test material form:
liquid

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:
Type and composition of metabolic activation system: S9 mix
- source of S9
Male Sprague Dawley rats (200-300 g) received a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight, dissolved in sunflower oil 1:5 v/v) 6 days before sacrifice. The liver was homogenized in three volumes of sterile, cold 150 mM KCl buffered with 10 mM Na phosphate at pH 7.4 . The homogenate was centrifuged at 10,000 g for 10 minutes. One volume of the resulting supernatant fraction was mixed with one volume of 24 mM MgCl2 containing 100 mM KCl and one volume of a solution which contained 12 mM NADP, 15 mM glucose-6-phosphate, and 150 mM Na phosphate buffer, pH 7.4 .
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
Controlsopen allclose all
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
Remarks:
without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: benzo(a)pyrene 4,5-oxide
Remarks:
without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
with S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: benzo(e)pyrene
Remarks:
with S9 mix
Details on test system and experimental conditions:
UMBER OF REPLICATIONS:
- Number of cultures per concentration duplicate
- Number of independent experiments: 4

METHOD OF TREATMENT/ EXPOSURE:
- Test substance in preincubation and desiccator method.

DURATION
- Exposure duration: 2-3 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition



Evaluation criteria:
No data
Statistics:
Not indicated

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
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
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
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
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
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
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
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
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium 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
True negative controls validity:
not examined
Positive controls validity:
valid

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