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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:
30 September - 30 October 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

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 (incl. QA statement)
Remarks:
(Landesamt für Umweltschutz und Gewerbeaufsicht , Rheinallee 97-101 , D-55118 Mainz , Germany)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
4-morpholinopropanesulphonic acid
EC Number:
214-478-5
EC Name:
4-morpholinopropanesulphonic acid
Cas Number:
1132-61-2
Molecular formula:
C7H15NO4S
IUPAC Name:
3-morpholin-4-ylpropane-1-sulfonic acid
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): MOPS HIGH PURITY
- Composition of test material : 3-(N-Morpholino)-propane sulfonic acid
- Substance type: white powder
- Analytical purity: 100,3%
- Purity test date : not stated
- Lot/batch No.: 1190567/006
- Expiration date of the lot/batch : 2004-08-31
- Storage condition of test material: stored in a closed polyethylene vessel at room temperature and kept away from ignition sources.

Method

Target gene:
his
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 97a, TA 98, TA 100, TA 102
Details on mammalian cell type (if applicable):
TA97a: hisD6610, TA 98: hisD3052 TA 100 und TA 1535 hisG46, TA102:hisG428, TA97, TA98 and TA100 contain uvrB; TA97, TA98, TA100 and TA102 contain pKM 101 and rfa.
Metabolic activation:
with and without
Metabolic activation system:
S9 ; produced from the livers of male Wistar rats which were treated with 80 mg Phenobarbital/kg body weight intraperitoneally and, on the following day, with 80 mg bNaphthoflavon/kg body weight orally.
Test concentrations with justification for top dose:
First experiment : 5, 1.5, 0.5, 0.15 and 0.05 mg/plate (Plate incorporation method)
Second experiment : 5, 2.5 and 1.25 mg/plate (pre-incubation method)
Vehicle / solvent:
- Solvent used: deionised water
- Justification for choice of solvent : deionised water was chosen as solvent, because the test item was completely soluble, and this solvent doesn’t have any effects on the viability of the bacteria or the number of spontaneous revertants.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 4-Nitro-1,2-phenylene diamine ; 2-Aminoanthracene
Remarks:
See -Any other information on materials and methods incl. tables- for details
Details on test system and experimental conditions:
Bacteria :
12 hours before the start of each experiment, a stock culture of each strain was thawed and placed into a culture flask containing 70 ml nutrient broth. The flasks were incubated at 37°C for 12 hours.

Description of the Method
Preparations :
In the days before each test, the media and solutions were prepared. Two days before the test, the plates were sterilized and the first batches poured. On the day before the test, the remaining plates were poured. On the day of the test, the overnight cultures were checked for growth. The incubation chamber was heated to 37°C. The water bath and the heating block were turned to 45°C. The table surface was disinfected. The S9 mix was freshly prepared and stored at 0°C.

Test Data
First Experiment :
date of treatment : Sep. 30th, 2003
concentrations tested : 5 / 1,5 / 0,5 / 0,15 / 0,05 mg/plate
incubation time : 48 hours
incubation temperature : 37°C
tester strains : TA97a, TA98, TA100, TA102, TA1535
method : Plate incorporation method

Second Experiment
date of treatment : Oct. 28th, 2003
concentrations tested : 5 / 2,5 / 1,25 mg/plate
incubation time : 48 hours
incubation temperature : 37°C
tester strains : TA97a, TA98, TA100, TA102, TA1535
method : pre-incubation method

Description of the Method
Plate incorporation method :
Per strain and dose, four plates with and four plates without S9 mix were used. 10 ml of the test solution of the appropriate concentration were membrane filtrated into sterile vessels. Top agar basis was melted in a microwave oven, after melting, 10 ml of histidin-biotin-solution 0,5 mMol per 100 ml basis was added and the bottle was placed in the water bath at 45°C. 0,1 ml of the appropriate solution of the test item were given into a sterile tube. After mixing with 2 ml Top-Agar, 0,1 ml overnight culture of the respective strain and 0,5 ml phosphate buffer (only for treatments without S9) or 0,5 ml S9 mix were added. The mixture was gently vortexed, then poured on a minimal glucose plate and evenly distributed, using a Drigalski
spatula. The plates were closed, covered with brown paper and left to harden for a few minutes,then inverted and placed in the dark incubator at 37°C.

Pre-incubation method
Per strain and dose, four plates with and four plates without S9 mix were used. 10 ml of the test solution of the appropriate concentration were membrane filtrated into sterile vessels. Top agar basis was melted in a microwave oven, after melting, 10 ml of histidin-biotin-solution 0,5 mMol per 100 ml basis was added and the bottle was placed in the water bath at 45°C. 0,1 ml of the appropriate solution of the test item were given into a sterile tube. After mixing with 0,1 ml overnight culture of the respective strain, 0,5 ml phosphate buffer (only for treatments without S9) or 0,5 ml S9 mix were added. The mixture was incubated in a incubation chamber at 37°C for 20 minutes. During this time the vessels were aerated through careful shaking. Then 2 ml top agar were added. The mixture was vortexed gently, then poured on a minimal glucose plate and evenly distributed, using a Drigalski spatula. The plates were closed,covered with brown paper and left to harden for a few minutes,then inverted and placed in the dark incubator at 37°C.

References
Genotype Confirmation
-Histidine requirement : Each strain was streaked on a biotin and a histidin-biotin-plate, using a sterilized wire loop.
-Ampicilline-Resistance (pKM 101) resp. ampicilline-tetracycline-resistance (pAQ1) The strains were streaked on ampicilline agar, TA102 on ampicilline-tetracycline agar. TA1535 was taking the function of control strain, since it is not ampicilline resistant.
-UV-sensitivity (uvrB) : Two plates were streaked with the five strains, and one half of the plate covered with aluminium foil so that one half of each streak was protected against light. The plates were irradiated for 8 seconds with a germicidal lamp (254 nm, 30W), keeping a distance of 33 cm. Incubation over night at 37°C followed.
-Crystal violet sensitivity (deep rough) : For each strain, two plates were used. 0,1 ml of bacteria suspension were mixed with 2 ml Top-Agar and poured on nutrient agar. Sterile paper discs (9 mm diameter), each soaked with 10 μl of crystal violet solution (0,1%) were placed into the middle of each plate, followed by incubation over night.
-Spontaneous revertants : Four replicates, with/without S9, for each solvent which was used in the test.
-Determination of Titre : The titre was determined by dilution of the overnight culture using sodium chloride solution and placing 0,1 ml on maximal-soft agar. It should give a density of 109 cells/ml (at the least).
-Sterility Control : Performed analogously to the test with solvent only and S9 (without adding bacteria) on top agar.
-Toxicity Control : Each strain was incubated with the maximum dose of the test item on maximal soft agar.
-Positive Controls : Using diagnostic mutagens, four replicates were prepared. The stock solutions of the substances were diluted to effect an application volume of 0,1 ml/plate.
Evaluation criteria:
The colonies were counted by eye, the numbers were recorded. A spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations as well as the increase factor of revertant induction.
A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor ≥ 2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA 1535, TA 97a, TA 98, TA 100, TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no 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

FINDINGS

First Experiment

Confirmation of the criteria :

The treatments for the confirmation of the genotype, the sterility control and the determination of the titre didn’t show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagenes) showed mutagenic effects with and without metabolic activation.

Solubility and Toxicity :

The test item was dissolved in deionised water. A stock solution containing 50 g/L was prepared. No signs of toxicity towards the tested strains could be observed. The determined values for the toxicity control were in the range of the titre. The background lawn was visible and the number of revertant colonies was not reduced.

Mutagenicity :

No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.

Therefore the test item is stated as not mutagenic under the test conditions.

Survey of the findings

strain     97a     98     100     102     1535    
induction     -S9  +S9  -S9  +S9  -S9  +S9  -S9  +S9  -S9  +S9 
water  mean  83  66  19  23  144  145  144  131  14  17 
  sd  43,3  52,2  11,8  3,5  17  11  28,1  29,1  7,2  5,0 
DMSO  mean  78  112  18  21  126  130  123  134  12  11 
   sd 66,9  24,5  2,2  4,5  26  6,8  12,4  2,8  3,9 
pos. contr.  mean  924  560  1038  70  633  754  656  717  590  439 
   sd 103  126  221  25  163  256  32  184  76,1  49 
   f(l) 11,9  5,0  57,7  3,4  4,4  5,8  5,3  5,4  42,9  40,8 
5 mg/pl.  mean  79  116  13  16  136  148  129  141  12  12 
   sd 86  78  28  18  11  13 
   f(l) 0,95  1,75  0,69  0,70  0,94  1,02  0,90  1,07  0,89  0,70 
1,5mg/pl.  mean  61  62  16  13  121  104  101  81  10  13 
   sd 50  48  14  20  11  10 
   f(l) 0,73  0,94  0,83  0,58  0,84  0,72  0,70  0,62  0,73  0,77 
0,5 mg/pl.  mean  91  52  14  13  129  83  102  65  14  18 
   sd 11  53  23  25  10 
   f(l) 1,10  0,78  0,73  0,59  0,90  0,57  0,71  0,49  1,00  1,03 
0,15 mg/pl.  mean  73  59  18  16  71  74  107  57  11  12 
   sd 37  39  14  11  20  10 
   f(l) 0,88  0,90  0,95  0,69  0,49  0,51  0,74  0,43  0,76  0,67 
0,05 mg/pl.  mean  90  87  15  16  127  101  117  72  14  16 
   sd 17  24  41  21  11  17 
   f(l) 1,08  1,32  0,75  0,72  0,88  0,70  0,82  0,54  1,02  0,90 

Second Experiment

Confirmation of the Criteria :

The treatments for the confirmation of the genotype, the sterility control and the determination of the titre didn’t show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls showed mutagenic effects with and without metabolic activation.

Solubility and Toxicity :

The test item was dissolved in deionised water. A stock solution containing 50 g/L was prepared. No signs of toxicity towards the tested strains could be observed. The determined values for the toxicity control were in the range of the titre. The background lawn was visible and the number of revertant colonies was not significantly reduced.

Mutagenicity :

No significant increase of the number of revertant colonies in the treatments with and without metabolic activation was observed. No concentration-related increase over the tested range was found.

Therefore the test item is stated as not mutagenic under the test conditions.

Survey of the findings

strain     97a     98     100     102     1535    
induction     -S9  +S9  -S9  +S9  -S9  +S9  -S9  +S9  -S9  +S9 
water  mean  149  117  10  11  131  120  128  119  10  10 
  sd  55,1  67,9  2,1  2,6  22  18  16,8  7,5  2,6  2,2 
DMSO  mean  93  118  11  93  96  119  116  11 
   sd 67,8  18,6  2,5  4,1  28  36,8  11,9  1,7  2,6 
pos. contr.  mean  571  721  604  61  948  575  847  678  1014  223 
   sd 275  198  121  153  236  62  173  157  11 
   f(l) 6,17  6,14  65,3  5,40  7,24  5,98  7,12  5,84  98,9  19,8 
5 mg/pl.  mean  93  110  122  129  131  121  12 
   sd 44  25  24  20  20 
   f(l) 0,62  0,94  0,64  0,74  0,93  1,08  1,02  1,02  1,20  0,85 
2,5mg/pl.  mean  112  103  10  98  120  135  111  11 
   sd 30  64  13  35 
   f(l) 0,75  0,88  0,74  0,95  0,75  1,00  1,05  0,93  1,02  0,85 
1,25 mg/pl.  mean  108  71  97  95  102  94  10 
   sd 49  70  16 
   f(l) 0,72  0,60  0,69  0,76  0,74  0,79  0,80  0,79  0,78  0,95

Detailed data in the attached full study report .

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The study was performed according to the OECD TG471 without deviations and therefore considered to be of the highest quality (reliability Klimisch 1). The validity criteria of the test system are fulfilled. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains , with any dose of the test material , either with or without metabolic activation . The test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

The test substance MOPS high purity was investigated according to OECD TG471 for its potential to cause gene mutation in Salmonella typhimurium strains (TA97a, TA98, TA100, TA102 and TA1535). Two valid experiments were performed. In the first experiment five concentrations of the test item, dissolved in deionized water (from 5 to 0,05 mg/plate) were used. Five genetically manipulated strains of Salmonella typhimurium (TA 97a, TA 98, TA 100, TA 102 and TA 1535) were exposed to the test item both in the presence and in the absence of a metabolic activation system (S9) for 48 hours, using the plate incorporation method. None of the concentrations caused an increase of the revertants in the tested strains. The test item didn't show any mutagenic effects in the first experiment. No signs of toxicity towards the bacteria could be observed. The treatments for the confirmation of the genotype, the sterility control and the determination of the titre didn't show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

To verify the results of the first experiment, a second experiment was performed, using three concentrations of the test item (5 to 1,25 mg/plate) and a modification in study performance (pre-incubation method). The test item didn't show mutagenic effects in the second experiment. No signs of toxicity towards the bacteria could be observed. The treatments for the confirmation of the genotype, the sterility control and the determination of the titre didn't show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation. Under the conditions of the test, the test item didn't show mutagenic effects towards Salmonella typhimurium, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535. Therefore, no concentration-effect relationship could be determined.

Therefore the test item MOPS high purity can be stated as not mutagenic under the conditions of the test.