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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:
12 December 2007 - 11 January 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997-07-21
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2000-05-19
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): 1-(2-Hydroxy-3-sulfopropyl)-pyridinium-betain
- Substance type: white powder
- Storage condition of test material: room temperature 20 ±- 5°C. The test item was stored in a dark closed vessel at room temperature.

Method

Target gene:
his
Species / strain
Species / strain / cell type:
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 [obtained by TRINOVA Biochem GmbH, Gießen, Germany (Batch nos: 2081, 2113, 2118)] Specification: produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254 per kg body weight intraperitoneally.
Test concentrations with justification for top dose:
First experiment : 5010, 1503, 501, 150 and 50 µg/plate (Plate incorporation method)
Second experiment : 5002, 2501 and 1251 µg/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 an aliquot was 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 43°C. The table surface was disinfected. The S9 mix was freshly prepared and stored at 0°C.

Experimental Parameters
First Experiment :
Date of treatment : 12 December 2007
Concentrations tested : 5010 / 1503 / 501 / 150 / 50 µg/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 : 09 January 2008
Concentrations tested : 5002 / 2501 / 1251 µg/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 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, 2 mL Top-Agar were added. The mixture was gently vortexed, then poured an a minimal glucose plate and distributed evenly, 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 histidine-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 an 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 distributed evenly, 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 10E9 cells/mL (at the least).
-Toxicity Control : Performed analogously to the titre control with the maximum dose of test item with and without S9 on maximal-soft agar.
-Sterility Control : Performed analogously to the test with solvent only and S9 (without adding bacteria) on top 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 visually, 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
Key result
Species / strain:
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

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

The study is regarded as a valid guideline study with certificated GLP compliance. The test item didn't show mutagenic effects in both experiments. The number of revertant colonies was not significantly increased in comparison with the spontaneous revertants (solvent only), as the increase factor never reached 2.0. In the first experiment, a weak dose-response correlation could be observed in TA97, whereas in the second experiment, this relation was not verified.
Cytotoxicity of the test item was not detected. The background lawn was visible and the number of revertants was not significantly decreased.
Therefore it can be stated, that under the conditions of the test, the test item 1-(2-Hydroxy-3-sulfopropyl)-pyridinium-betain is not mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535.
Executive summary:

This study was performed in order to evaluate the mutagenic potential of 1-(2-Hydroxy-3-sulfopropyl)-pyridinium-betain.

The study was conducted in accordance with the following guidelines:

-OECD Guidelines for the Testing of Chemicals Part 471, adopted July 21st, 1997 „Bacterial Reverse Mutation Test"

-EU-Guideline 67/548 EWG, last changed by 29th amendment, Annex V, Method B.13/14 "Mutagenicity - Salmonella typhimurium, reverse mutation

assay", adopted May 19th, 2000

Two valid experiments were performed.

 

First Experiment:

Five concentrations of the test item, dissolved in deionised water (ranging from 5010 to 50 µg/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 in the number of revertant colonies 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 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.

Second experiment:

To verify the results of the first experiment, a second experiment was performed, using three concentrations of the test item (ranging from 5002 to 1251 µg/plate) and a modification in study performance (pre-incubation method).

The test item didn't show mutagenic effects in the second experiment, either. No signs of toxicity towards the bacteria could be observed.

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.

The test item 1-(2-Hydroxy-3-sulfopropyI)-pyridinium-betain is considered as "not mutagenic under the conditions of the test".