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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:
2016-08-30 to 2016-09-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 21, 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 30, 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-piperazine-1,4-diyldiethanesulfonic acid
EC Number:
227-057-6
Cas Number:
5625-37-6
Molecular formula:
C8H18N2O6S2
IUPAC Name:
2,2'-piperazine-1,4-diyldiethanesulfonic acid
Test material form:
solid: particulate/powder

Method

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:
- source of S9: S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH (Rathenau Str. 2.; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).
- method of preparation of S9 mix : The complete S9 Mix was freshly prepared containing components as follows:
Ice cold 0.2 M sodium phosphate-buffer, pH 7.4 500 mL
Rat liver homogenate (S9) 100 mL
Salt solution for S9 Mix 400 mL
The S9 Mix was kept in an ice bath before it was added to the culture medium.
- volume of S9 mix in the final culture medium: 0.5 mL
- quality controls of S9: enzymatic activity, sterility and metabolic capability were controlled by the manufacturer
Test concentrations with justification for top dose:
16, 50, 160, 500, 1600, 5000 µg/plate
At the concentration choice the guideline criterion for soluble, non-toxic test compounds was taken into consideration where the recommended maximum test concentration is 5 mg/plate.
Vehicle / solvent:
- Solvent used: ultrapure water
For facilitating the test item dissolution the pH of the ultrapure water vehicle was increased to pH 9. At 50 mg/L concentration after 5 min ultrasonic treatment a thick milky suspension was obtained. This suspension was further diluted and for complete dissolution additional 1N NaOH dispensed. The test item was completely dissolved at the concentration of 25 mg/mL. The pH of its stock solution (25 mg/mL) was measured after the experiment. The obtained pH at complete dissolution was 7.48.

- Justification for choice of solvent/vehicle: The vehicle is compatible with the survival of the bacteria and the S9 activity and was chosen based on the results of the preliminary Solubility Test.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-Nitro-1,2-phenylenediamine and 2-aminoanthracene (NPD) 2-aminoanthracene (2AA)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation) and preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The colony numbers on the controls (untreated, vehicle, positive) and the test plates were determined (counted manually), the mean values and appropriate standard deviations and mutation rates were calculated.
Rationale for test conditions:
Test condictions according to guideline were used.
Evaluation criteria:
A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Criteria for a Negative Response:
A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Statistics:
Not performed.

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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The pH of the test item stock solution (prepared for the highest concentration) and additionally the pH of the overlay (top agar-phosphate buffer-test item system (at the highest concentration of 25 mg/mL)) was checked and recorded. The pH values of the test item stock solutions (25 mg/mL) with the necessary pH setting were 6.62 and 7.02. The pH of the different overlays without test item stock solution was in the range of 7.29 - 7.41, and the pH of overlays completed with test item stock solution was in the range of 7.25 - 7.30.
Extremes of pH could have influencing effect on the mutagenicity results; however in this study the slightly acidic or rather neutral test item solutions (1N NaOH facilitated the test item dissolution) had only slight effect on the buffered overlay system. Unequivocal negative results were obtained and the additional information about the pH conditions was not involved into the result interpretation.
- Data on osmolality: not applicable
- Possibility of evaporation from medium: not specified
- Water solubility: Test item was sufficiently soluble in water using 1N NaOH for facilitating the test item dissolution.
- Precipitation and time of the determination: No precipitation of the test item was observed in the Initial and Confirmatory Mutation Tests on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix).
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES (if applicable):
A concentration range finding test (Informatory Toxicity Test) was conducted to determine the concentrations for the main tests. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 1600, 500, 160, 50, 16 and 5 μg/plate. In the Concentration Range Finding Test the plate incorporate procedure was applied. The revertant colony numbers of vehicle control plates in both strains with and without S9 Mix were in line with the corresponding historical control data ranges. The positive control treatments showed the expected, biological relevant increases in induced revertant colonies in both tester strains.
In the Informatory Toxicity Test inhibitory effect of the test item was not observed. The colony and background lawn development was not affected in any case; all of the obtained slight revertant colony number decreases or increases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.
No precipitation of the test item was observed on the plates in the above bacterial strains at any examined concentration level (±S9 Mix).

STUDY RESULTS
For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible: Not detected.

Ames test:
- Signs of toxicity: In the Initial and Confirmatory Mutation Tests, inhibitory effect of the test item on bacterial growth was not observed. All of the noticed lower revertant colony numbers (when compared to the revertant colony numbers of the corresponding vehicle control) remained in the range of the biological variability of the applied test system and the background lawn development was not affected in any case.
- For plate counts please refer to Any other information on results.

HISTORICAL CONTROL DATA
Please refer to Any other information on results.

Any other information on results incl. tables

Table 1: Summary Table of the Results of the Initial Mutation Test


Initial Mutation Test (Plate Incorporation Test)













































































































































































































































































































































































































Concentrations
(µg/plate)
Salmonella typhimurium tester strainsEscherichia coli 
TA 98TA100TA 1535TA 1537WP2 uvrA
-S9+S9-S9+S9-S9+S9-S9+S9-S9+S9
Mean values of revertants per plate Mutation rate (MR)MeanMRMeanMRMeanMRMeanMRMeanMRMeanMRMeanMRMeanMRMeanMRMeanMR
Untreated Control18.30.8515.70.841051.02106.31.07131.0512.31.066.71.185.30.94441.1491.04
DMSO Control14.71161--108.31--11.314.314.71--431
Ultrapure Water Control21.7118.71102.7199.3112.3111.715.715.7140147.31
500021.71211.1394.70.92890.911.30.929.30.84.30.7671.2444.71.12541.14
160021.7118.30.9888.70.86102.31.0311.30.92100.867.71.354.70.82421.0547.31
50018.70.8624.31.394.70.9295.70.9611.70.9511.7161.066.31.1240.31.01450.95
160200.9221.31.1497.70.9599111.30.9290.776.71.185.7142.71.0748.31.02
50200.92160.86980.9596.70.9712.31110.9461.067.71.35441.149.31.04
1618.70.8618.7187.30.851111.1211.30.9210.30.896.71.186.31.12471.1841.30.87
NPD (4 µg/plate)281.319.18------------------
SAZ (2 µg/plate)----1666.716.23--1560126.49----------
9AA (50 µg/plate)------------24255.85------
MMS (2 µL/plate)----------------837.320.93--
2AA (2 µg/plate)--1378.786.17--154414.25--11710.32--17437.29----
2AA (50 µg/plate)------------------183.3

4.26


 



Table 2: Summary Table of the Results of the Confirmatory Mutation Test


Confirmatory Mutation Test (Pre-incubation Test)




































































































































































































































































































































































































































Concentrations
(µg/plate)
Salmonella typhimurium tester strainsEscherichia coli
TA 98TA 100TA 1535TA 1537WP2 uvrA
-S9+S9-S9+S9-S9+S9-S9+S9-S9+S9
Mean values of revertauts per plate Mutation rate (MR)MeanMRMeanMRMeanMRMeanMRMeanMRMeanMRMeanMRMeanMRMeanMRMeanMR
Untreated Control19.31.3837.31.241040.961361.0614.31.268.30.9671.2460.7828.30.88380.86
DMSO control18119.31--104.71--917181--391
Ultrapure Water Control141221108.31128.3111.318.715.717.7132.31441
500018.31.3129.71.35980.9127.30.99110.9710.71.2350.888.31.0931.30.97350.8
160011.70.83281.27102.70.951220.959.70.85141.6261.067.30.96341.05430.98
50019.31.3825.31.151020.941170.918.70.769.71.1261.0681.0426.30.81390.89
16015.71.1230.71.39105.70.981240.9710.30.919.31.085.715.70.74270.8440.30.92
5024.31.74321.45110.31.021100.8610.30.9111.31.316.31.126.30.8330.70.9541.30.94
1620.31.45251.1494.70.871170.919.30.8212.31.426.31.128.310.9321.0135.30.8
NPD (4 µg/plate)307.317.07--------------7---
SAZ (2 µg/plate)----212019.57--865.376.35----------
9AA (50 µg/plate)------------1838.7262.67------
MMS (2 µL/plate)----------------128039.59--
2AA (2 µg/plate)--774.740.07--2634.725.17--13815.33--14217.75----
2AA (50 µg/plate)------------------167.74.3
                     

MR: Mutation Rate; NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoactidine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene


Remarks: Ultrapure water was applied as vehicle of the test item and the positive control substances: SAZ and MMS; and the DMSO was applied as vehicle for positive control substances: NPD, 9AA and 2AA. The mutation rate of the test item, SAZ, MMS and untreated control refers to the ultrapure water; the mutation rate of NPD, 9AA and 2AA refers to DMSO.


Table 3: Historical Control Values for Revertants Plate (for the Period of 2008-2015)

































































































































































































































































































































































































 Bacterial strains
Historical control data of untreated control  TA98TA100TA1535TA1537E. coli
 Average21.410610.48.125.6
-S9SD3.727.31.52.52.5
 Minimum9653211
 Maximum39157231945
  TA98TA100TA1535TA1537E. coli
 Average28117.111.9934.3
+S9SD4.219.41.525.4
 Minimum12754318
 Maximum48166242056
 Bacterial strains
Historical control data of DMSO control  TA98TA100TA1535TA1537E. coli
 Average20.9101.410.37.924.9
-S9SD3.526.21.42.54.9
 Minimum10653211
 Maximum39150232044
  TA98TA100TA1535TA1537E. coli
 Average27.1114.7128.834.2
+S9SD419.31.42.15.2
 Minimum15714316
 Maximum48161242056
 Bacterial strains
Historical control data of Water control  TA98TA100TA1535TA1537E. coli
 Average22.4105.510.47.526.3
-S9SD3.627.61.62.35.9
 Minimum12673213
 Maximum36156241547
  TA98TA100TA1535TA1537E. coli
 Average28117.411.58.735.2
+S9SD419.81.42.35.2
 Minimum15834418
 Maximum43166221656
 Bacterial strains
Historical control data of positive controls  TA98TA100TA1535TA1537E. coli
 Average255.6958.9842.1467.4712.3
-S9SD30.7149.9134105.757.5
 Minimum123522354109320
 Maximum6471927187114981283
  TA98TA100TA1535TA1537E. coli
 Average1224.81431.9165.4148264.7
+S9SD293.8339.935.121.374.2
 Minimum4095818568141
 Maximum25872923507407487

Abbreviations: TA98, TA100, TA1535, TA1537: Salmonella typhimurium TA98, TA100, TA1535, TA1537; E. coli: Escherichia coli WP2 uvrA

Applicant's summary and conclusion

Conclusions:
The test item is considered to be not mutagenic.
Executive summary:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD 471. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats. The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test). Based on the results of the Solubility and the Range Finding Tests the test item was dissolved in ultrapure water (ASTM Type 1) with appropriate pH setting performed with 1N NaOH. The applied system was compatible with the survival of the bacteria and the S9 activity. Based on the results of the preliminary Range Finding Test the concentrations 5000, 1600, 500, 160, 50 and 16 μg/plate of the test item were prepared and investigated in the Initial and Confirmatory Mutation Tests. In the preliminary experiment for facilitating the test item dissolution the pH of the vehicle was increased with addition of 1N NaOH. The pH of the obtained stock solution (25 mg/mL) at the complete dissolution was 7.48. Extremes of pH could have influencing effect on the mutagenicity results, therefore in the Initial and Confirmatory Mutation Tests the pH of the test item stock solution (prepared for the highest concentration of 25 mg/mL) and additionally the pH of the overlay (top agar-phosphate buffer-test item system (at 25 mg/mL)) was checked and recorded. The pH values of the test item stock solutions (25 mg/mL) were 6.62 and 7.02. The pH of the overlays was in the range of 7.25-7.30 in the main experiments. In this study the test item could be dissolved with additional pH setting, consequently the resulted slightly acidic but rather neutral pH of the test item solutions had only slight effect on the buffered overlay system. Unequivocal negative results were obtained and the additional information about the pH conditions was not involved into the mutagenicity result interpretation. No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study. The test item did not show inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system. The revertant colony numbers of vehicle control (ultrapure water) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the bacterium tester strains used. In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.