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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:
23 May 2018 to 26 October 2018
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

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)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Phosphoric acid, butyl ester
EC Number:
235-826-2
EC Name:
Phosphoric acid, butyl ester
Cas Number:
12788-93-1
Molecular formula:
C4H11PO4
IUPAC Name:
Phosphoric acid, butyl ester
Specific details on test material used for the study:
Batch 34493
Purity butyl dihydrogen phosphate 39 - 50%
dibutyl hydrogen phosphate 47-56%

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium, other: TA 97a
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Experiment 1: In the first experiment, the test item (dissolved in DMSO) was tested up to concentrations of 5 µL/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method. The test item showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation. The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

Experiment 2: Based on the non- toxic results of the first experiment, the test item was tested up to concentrations of 5 µL/plate in the absence and presence of S9-mix in all bacteria strains using the pre-incubation method.

The following nominal concentrations were prepared for the second experiment: 5 µL/plate, 2.5 µL/plate, 1.25 µL/plate, 0.63 µL/plate, 0.31 µL/plate, 0.16 µL/plate and 0.08 µL/plate.
Vehicle / solvent:
Based on the non-GLP pre-test, DMSO was chosen as vehicle, because the test item was sufficiently soluble and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 4-Nitro-1,2-phenylene Diamine, 2-Amino-Anthracene
Details on test system and experimental conditions:
Test System
Specification
Species Salmonella typhimurium LT2
Strains TA97a, TA98, TA100, TA102 and TA1535

Mutations of the strains are listed in the table below.

Origin and Culture
All Salmonella typhimurium strains were obtained from TRINOVA BioChem GmbH (batch: TA97a: 4997D, TA98: 5011D, TA100: 4996D, TA102: 4982D, TA1535: 5012D) and were stored as lyophilizates in the refrigerator at 2-8 °C. The lyophilizates were used to prepare permanent cultures which were filled into vials and stored at < - 75 °C.

Eight hours before the start of each experiment, an aliquot of a permanent culture per strain to be used was taken from the deep freezer to inoculate a culture vessel containing nutrient broth. After incubation overnight for eight hours at 37 ± 1 °C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.

PERFORMANCE OF THE STUDY
Culture of Bacteria
Eight hours before the start of each experiment, one vial permanent culture of each strain was taken from the deep freezer and an aliquot was put into a culture flask containing nutrient broth. After incubation for eight hours at 37 ±1 °C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.

Conduct of Experiment
Preparations
Different media and solutions were prepared preliminary (exact production dates are documented in the raw data). On the day of the test, the bacteria cultures were checked for growth visually. The incubation chambers were heated to 37 ±1 °C. The water bath was turned to 43 ±1 °C. The table surface was disinfected. The S9 mix was freshly prepared and stored at 0 °C.

Experimental Parameters
First Experiment
Date of treatment 23. May 2018
Concentrations tested 5 / 1.5 / 0.5 / 0.15 / 0.05 µL/plate
Incubation time 48 h
Incubation temperature 37 ±1 °C
Tested strains TA97a, TA98, TA100, TA102, TA1535
Method plate incorporation method

Second Experiment
Date of treatment 28. May 2018
Concentrations tested 5 / 2.5 / 1.25 / 0.63 / 0.31 / 0.16 / 0.08 µL/plate
Incubation time 48 h
Incubation temperature 37 ±1 °C
Tested strains TA97a, TA98, TA100, TA102, TA1535
Method pre-incubation method

Description of the Method
General preparation
Per bacteria strain and concentration, three plates with and three plates without metabolic activation (-S9) were used. For the top agar 100 mL agar basis was melted in a microwave oven, 10 mL of the histidine-biotin-solution 0.5 mM was added, then the mixture was placed in the water bath at 43 ±1 °C.

Plate incorporation method
The following materials were gently vortexed in a test tube and poured onto the selective agar plates:

-100 µL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
-500 µL S9 mix or phosphate buffer (for test without metabolic activation).
-100 µL bacteria suspension
-2000 µL overlay agar (top agar)

The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ±1 °C.

Pre-incubation method
The following materials were gently vortexed in a test tube and incubated at 37 ±1°C for 20 minutes:

-100 µL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
-500 µL S9 mix or phosphate buffer (for test without metabolic activation).
-100 µL bacteria suspension

After the pre-incubation for 20 minutes, 2000 µL top agar was added and the tube was gently vortexed. The mixture was poured onto the selective agar plate. The plates were closed and left to solidify for a few minutes, then inverted and placed in the incubator at 37 ±1 °C.
Evaluation criteria:
The colonies were counted visually and the numbers were recorded. A validated spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control. The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given. A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium, other: TA 97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In all experiments, no precipitation of the test item Phosphoric acid, butyl ester was observed at any of the tested concentrations up to 5 µL/plate. In the first and the second experiment, the test item caused no cytotoxicity towards all bacteria strains The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value of 109 bacteria/mL. All of the means of all replicates of the spontaneous revertants (in negative and solvent controls) were within the range of the historical data of the test facility. Nearly all numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory, but all were increased in comparison with the negative controls, which demonstrated the mutagenic potential of the diagnostic mutagens. Since all criteria for acceptability have been met, the study is considered valid.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study it is concluded that Phosphoric acid, butyl ester is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.
Executive summary:

The test item Phosphoric acid, butyl ester showed no increase in the number of revertants in all bacteria strains in both experiments. All negative and nearly all strain-specific positive control values (only one exception) were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that Phosphoric acid, butyl ester is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.

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